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Sample records for virus-like particles expressing

  1. Capsid protein expression and adeno-associated virus like particles assembly in Saccharomyces cerevisiae

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    Backovic Ana

    2012-09-01

    Full Text Available Abstract Background The budding yeast Saccharomyces cerevisiae supports replication of many different RNA or DNA viruses (e.g. Tombusviruses or Papillomaviruses and has provided means for up-scalable, cost- and time-effective production of various virus-like particles (e.g. Human Parvovirus B19 or Rotavirus. We have recently demonstrated that S. cerevisiae can form single stranded DNA AAV2 genomes starting from a circular plasmid. In this work, we have investigated the possibility to assemble AAV capsids in yeast. Results To do this, at least two out of three AAV structural proteins, VP1 and VP3, have to be simultaneously expressed in yeast cells and their intracellular stoichiometry has to resemble the one found in the particles derived from mammalian or insect cells. This was achieved by stable co-transformation of yeast cells with two plasmids, one expressing VP3 from its natural p40 promoter and the other one primarily expressing VP1 from a modified AAV2 Cap gene under the control of the inducible yeast promoter Gal1. Among various induction strategies we tested, the best one to yield the appropriate VP1:VP3 ratio was 4.5 hour induction in the medium containing 0.5% glucose and 5% galactose. Following such induction, AAV virus like particles (VLPs were isolated from yeast by two step ultracentrifugation procedure. The transmission electron microscopy analysis revealed that their morphology is similar to the empty capsids produced in human cells. Conclusions Taken together, the results show for the first time that yeast can be used to assemble AAV capsid and, therefore, as a genetic system to identify novel cellular factors involved in AAV biology.

  2. Expression and purification of human papillomavirus 18 L1 virus-like particle from saccharomyces cerevisiae.

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    Woo, Mi-Kyung; An, Jung-Mo; Kim, Jun-Dong; Park, Sue-Nie; Kim, Hong-Jin

    2008-02-01

    Cervical cancer caused by human papillomavirus (HPV) might be successfully prevented by HPV vaccination and screening. HPV vaccination and HPV serology assays have been investigated using HPV virus-like particles (VLPs). In this study we produced HPV18 L1 VLPs in Saccharomyces cerevisiae and purified them. The HPV18 L1 gene was cloned into the yeast expression vector YEGalpha-HIR525, and transformed into Saccharomyces cerevisiae. Expression of HPV18 L1 protein was demonstrated by Western blotting. The HPV18 L1 protein was purified by ultracentrifugation, size-exclusion chromatography and cation-exchange chromatography, and was up to 95% pure. We showed by transmission electron microscopy that the purified protein self-assembled into VLPs. These findings should be useful for establishing vaccine efficacy as well as characterizing vaccine candidates, and may provide an international reference standard for HPV serology assays.

  3. Immunogenic virus-like particles continuously expressed in mammalian cells as a veterinary rabies vaccine candidate.

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    Fontana, Diego; Kratje, Ricardo; Etcheverrigaray, Marina; Prieto, Claudio

    2015-08-20

    Rabies is one of the most lethal infectious diseases in the world, with a mortality approaching 100%. There are between 60,000 and 70,000 reported annual deaths, but this is probably an underestimation. Despite the fact that there are vaccines available for rabies, there is a real need of developing more efficacious and cheaper vaccines. This is particularly true for veterinary vaccines because dogs are still the main vector for rabies transmission to human beings. In a previous work, we described the development and characterization of rabies virus-like particles (RV-VLPs) expressed in HEK293 cells. We showed that RV-VLPs are able to induce a specific antibodies response. In this work, we show that VLPs are able to protect mice against virus challenge. Furthermore, we developed a VLPs expressing HEK-293 clone (sP2E5) that grows in serum free medium (SFM) reaching high cell densities. sP2E5 was cultured in perfusion mode in a 5 L bioreactor for 20 days, and the RV-VLPs produced were capable of triggering a protective immune response without the need of concentration or adjuvant addition. Further, these VLPs are able to induce the production of rabies virus neutralizing antibodies. These results demonstrate that RV-VLPs are a promising rabies vaccine candidate. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Method for rapid optimization of recombinant GPCR protein expression and stability using virus-like particles.

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    Ho, Thao T; Nguyen, Jasmine T; Liu, Juping; Stanczak, Pawel; Thompson, Aaron A; Yan, Yingzhuo G; Chen, Jasmine; Allerston, Charles K; Dillard, Charles L; Xu, Hao; Shoger, Nicholas J; Cameron, Jill S; Massari, Mark E; Aertgeerts, Kathleen

    2017-05-01

    Recent innovative approaches to stabilize and crystallize GPCRs have resulted in an unprecedented breakthrough in GPCR crystal structures as well as application of the purified receptor protein in biophysical and biochemical ligand binding assays. However, the protein optimization process to enable these technologies is lengthy and requires iterative overexpression, solubilization, purification and functional analysis of tens to hundreds of protein variants. Here, we report a new and versatile method to screen in parallel hundreds of GPCR variants in HEK293 produced virus-like particles (VLPs) for protein yield, stability, functionality and ligand binding. This approach reduces the time and resources during GPCR construct optimization by eliminating lengthy protein solubilization and purification steps and by its adaptability to many binding assay formats (label or label-free detection). We exemplified the robustness of our VLP method by screening 210 GALR3-VLP variants in a radiometric agonist-based binding assay and a subset of 88 variants in a label-free antagonist-based assay. The resulting GALR3 agonist or antagonist stabilizing variants were then further used for recombinant protein expression in transfected insect cells. The final purified protein variants were successfully immobilized on a biosensor chip and used in a surface plasmon resonance binding assay. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Assembly of SIV virus-like particles containing envelope proteins using a baculovirus expression system.

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    Yamshchikov, G V; Ritter, G D; Vey, M; Compans, R W

    1995-12-01

    The requirements for SIV particle assembly and envelope incorporation were investigated using a baculovirus expression system. The Pr56gag precursor protein expressed under control of the polyhedrin promoter (pPolh) produced high levels of immature retrovirus-like particles (VLP) upon expression in Sf9 insect cells. To determine the optimal conditions for envelope protein (Env) incorporation into VLP, two recombinant baculoviruses expressing the SIV envelope protein under control of a very late pPolh or a hybrid late/very late capsid/polyhedrin (Pcap/polh) promoter and a recombinant expressing a truncated form of the SIV envelope protein (Envt) under the hybrid Pcap/polh promoter were compared. We have observed that utilization of the earlier hybrid promoter resulted in higher levels of Env expression on the cell surface and its incorporation into budding virus particles. We have also found that the Envt protein is transported to the cell surface of insect cells and incorporated into VLP more efficiently than full-length Env. In addition, we examined the effect of coexpression of the protease furin, which has been implicated in the proteolytic cleavage of the Env precursor gp160 in mammalian cells. Coexpression of furin in insect cells resulted in more efficient proteolytic cleavage into gp120 and gp41, and the cleaved proteins were incorporated into VLP.

  6. Canine parvovirus VP2 protein expressed in silkworm pupae self-assembles into virus-like particles with high immunogenicity.

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    Feng, Hao; Hu, Gui-qiu; Wang, Hua-lei; Liang, Meng; Liang, Hongru; Guo, He; Zhao, Pingsen; Yang, Yu-jiao; Zheng, Xue-xing; Zhang, Zhi-fang; Zhao, Yong-kun; Gao, Yu-wei; Yang, Song-tao; Xia, Xian-zhu

    2014-01-01

    The VP2 structural protein of parvovirus can produce virus-like particles (VLPs) by a self-assembly process in vitro, making VLPs attractive vaccine candidates. In this study, the VP2 protein of canine parvovirus (CPV) was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells and pupae. Electron micrographs of VLPs showed that they were very similar in size and morphology when compared to the wild-type parvovirus. The immunogenicity of the VLPs was investigated in mice and dogs. Mice immunized intramuscularly with purified VLPs, in the absence of an adjuvant, elicited CD4(+) and CD8(+) T cell responses and were able to elicit a neutralizing antibody response against CPV, while the oral administration of raw homogenates containing VLPs to the dogs resulted in a systemic immune response and long-lasting immunity. These results demonstrate that the CPV-VLPs stimulate both cellular and humoral immune responses, and so CPV-VLPs may be a promising candidate vaccine for the prevention of CPV-associated disease.

  7. Canine parvovirus VP2 protein expressed in silkworm pupae self-assembles into virus-like particles with high immunogenicity.

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    Hao Feng

    Full Text Available The VP2 structural protein of parvovirus can produce virus-like particles (VLPs by a self-assembly process in vitro, making VLPs attractive vaccine candidates. In this study, the VP2 protein of canine parvovirus (CPV was expressed using a baculovirus expression system and assembled into parvovirus-like particles in insect cells and pupae. Electron micrographs of VLPs showed that they were very similar in size and morphology when compared to the wild-type parvovirus. The immunogenicity of the VLPs was investigated in mice and dogs. Mice immunized intramuscularly with purified VLPs, in the absence of an adjuvant, elicited CD4(+ and CD8(+ T cell responses and were able to elicit a neutralizing antibody response against CPV, while the oral administration of raw homogenates containing VLPs to the dogs resulted in a systemic immune response and long-lasting immunity. These results demonstrate that the CPV-VLPs stimulate both cellular and humoral immune responses, and so CPV-VLPs may be a promising candidate vaccine for the prevention of CPV-associated disease.

  8. Norovirus Narita 104 Virus-Like Particles Expressed in Nicotiana benthamiana Induce Serum and Mucosal Immune Responses

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    Lolita George Mathew

    2014-01-01

    Full Text Available Narita 104 virus is a human pathogen belonging to the norovirus (family Caliciviridae genogroup II. Noroviruses cause epidemic gastroenteritis worldwide. To explore the potential of developing a plant-based vaccine, a plant optimized gene encoding Narita 104 virus capsid protein (NaVCP was expressed transiently in Nicotiana benthamiana using a tobacco mosaic virus expression system. NaVCP accumulated up to approximately 0.3 mg/g fresh weight of leaf at 4 days postinfection. Initiation of hypersensitive response-like symptoms followed by tissue necrosis necessitated a brief infection time and was a significant factor limiting expression. Transmission electron microscopy of plant-derived NaVCP confirmed the presence of fully assembled virus-like particles (VLPs. In this study, an optimized method to express and partially purify NaVCP is described. Further, partially purified NaVCP was used to immunize mice by intranasal delivery and generated significant mucosal and serum antibody responses. Thus, plant-derived Narita 104 VLPs have potential for use as a candidate subunit vaccine or as a component of a multivalent subunit vaccine, along with other genotype-specific plant-derived VLPs.

  9. Expression and assembly of Norwalk virus-like particles in plants using a viral RNA silencing suppressor gene.

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    Souza, Ana Cláudia; Vasques, Raquel Medeiros; Inoue-Nagata, Alice Kazuko; Lacorte, Cristiano; Maldaner, Franciele Roberta; Noronha, Eliane Ferreira; Nagata, Tatsuya

    2013-10-01

    Binary vector-based transient expression of heterologous proteins in plants is a very attractive strategy due to the short time required for proceeding from planning to expression. However, this expression system is limited by comparatively lower yields due to strong post-transcriptional gene silencing (PTGS) in the host plants. The aim of this study was to optimize a procedure for expression of norovirus virus-like particles (VLPs) in plants using a binary vector with co-expression of a PTGS suppressor to increase the yield of the target protein. The effects of four plant viral PTGS suppressors on protein expression were evaluated using green fluorescent protein (GFP) as a reporter. Constructs for both GFP and PTGS suppressor genes were co-infiltrated in Nicotiana benthamiana plants, and the accumulation of GFP was evaluated. The most effective PTGS suppressor was the 126K protein of Pepper mild mottle virus. Therefore, this suppressor was selected as the norovirus capsid gene co-expression partner for subsequent studies. The construct containing the major (vp1) and minor capsid (vp2) genes with a 3'UTR produced a greater amount of protein than the construct with the major capsid gene alone. Thus, the vp1-vp2-3'UTR and 126K PTGS suppressor constructs were co-infiltrated at middle scale and VLPs were purified by sucrose gradient centrifugation. Proteins of the expected size, specific to the norovirus capsid antibody, were observed by Western blot. VLPs were observed by transmission electron microscopy. It was concluded that protein expression in a binary vector co-expressed with the 126K PTGS suppressor protein enabled superior expression and assembly of norovirus VLPs.

  10. Self-assembly of virus-like particles of porcine circovirus type 2 capsid protein expressed from Escherichia coli

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    Cai Xuepeng

    2010-07-01

    Full Text Available Abstract Background Porcine circovirus 2 (PCV2 is a serious problem to the swine industry and can lead to significant negative impacts on profitability of pork production. Syndrome associated with PCV2 is known as porcine circovirus closely associated with post-weaning multisystemic wasting syndrome (PMWS. The capsid (Cap protein of PCV2 is a major candidate antigen for development of recombinant vaccine and serological diagnostic method. The recombinant Cap protein has the ability to self-assemble into virus-like particles (VLPs in vitro, it is particularly opportunity to develop the PV2 VLPs vaccine in Escherichia coli,(E.coli , because where the cost of the vaccine must be weighed against the value of the vaccinated pig, when it was to extend use the VLPs vaccine of PCV2. Results In this report, a highly soluble Cap-tag protein expressed in E.coli was constructed with a p-SMK expression vector with a fusion tag of small ubiquitin-like modifiers (SUMO. The recombinant Cap was purified using Ni2+ affinity resins, whereas the tag was used to remove the SUMO protease. Simultaneously, the whole native Cap protein was able to self-assemble into VLPs in vitro when viewed under an electron microscope. The Cap-like particles had a size and shape that resembled the authentic Cap. The result could also be applied in the large-scale production of VLPs of PCV2 and could be used as a diagnostic antigen or a potential VLP vaccine against PCV2 infection in pigs. Conclusion we have, for the first time, utilized the SUMO fusion motif to successfully express the entire authentic Cap protein of PCV2 in E. coli. After the cleavage of the fusion motif, the nCap protein has the ability to self-assemble into VLPs, which can be used as as a potential vaccine to protect pigs from PCV2-infection.

  11. In Vivo siRNA Delivery Using JC Virus-like Particles Decreases the Expression of RANKL in Rats

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    Daniel B Hoffmann

    2016-01-01

    Full Text Available Bone remodeling requires a precise balance between formation and resorption. This complex process involves numerous factors that orchestrate a multitude of biochemical events. Among these factors are hormones, growth factors, vitamins, cytokines, and, most notably, osteoprotegerin (OPG and the receptor activator for nuclear factor-kappaB ligand (RANKL. Inflammatory cytokines play a major role in shifting the RANKL/OPG balance toward excessive RANKL, resulting in osteoclastogenesis, which in turn initiates bone resorption, which is frequently associated with osteoporosis. Rebalancing RANKL/OPG levels may be achieved through either upregulation of OPG or through transient silencing of RANKL by means of RNA interference. Here, we describe the utilization of a viral capsid-based delivery system for in vivo and in vitro RNAi using synthetic small interfering RNA (siRNA molecules in rat osteoblasts. Polyoma JC virus-derived virus-like particles are capable of delivering siRNAs to target RANKL in osteoblast cells both in vitro and in a rat in vivo system. Expression levels were monitored using quantitative real-time polymerase reaction and enzyme-linked immunosorbent assay after single and repeated injections over a 14-day period. Our data indicate that this is an efficient and safe route for in vivo delivery of gene modulatory tools to study important molecular factors in a rat osteoporosis model.

  12. Characterization of chikungunya virus-like particles.

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    Nitchakarn Noranate

    Full Text Available Chikungunya virus (CHIKV is becoming a global concern due to the increasing number of outbreaks throughout the world and the absence of any CHIKV-specific vaccine or treatment. Virus-like particles (VLPs are multistructured proteins that mimic the organization and conformation of native viruses but lack the viral genome. They are noninfectious and potentially safer vaccine candidates. Recent studies demonstrated that the yield of CHIKV VLPs varies depending on the strains, despite the 95% amino acid similarity of the strains. This might be due to the codon usage, since protein expression is differently controlled by different organisms. We optimized the region encoding CHIKV structural proteins, C-E3-E2-6k-E1, inserted it into a mammalian expression vector, and used the resulting construct to transfect 293 cells. We detected 50-kDa proteins corresponding to E1 and/or E2 in the cell lysate and the supernatant. Transmission electron microscopy revealed spherical particles with a 50- to 60-nm diameter in the supernatant that resembled the native CHIKV virions. The buoyant density of the VLPs was 1.23 g/mL, and the yield was 20 µg purified VLPs per 108 cells. The VLPs aggregated when mixed with convalescent sera from chikungunya patients, indicating that their antigenicity is similar to that of native CHIKV. Antibodies elicited with the VLPs were capable of detecting native CHIKV, demonstrating that the VLPs retain immunogenicity similar to that of the native virion. These results indicated that CHIKV VLPs are morphologically, antigenically, and immunologically similar to the native CHIKV, suggesting that they have potential for use in chikungunya vaccines.

  13. Enveloped virus-like particles as vaccines against pathogenic arboviruses

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    Pijlman, G.P.

    2015-01-01

    Arthropod-borne arboviruses form a continuous threat to human and animal health, but few arboviral vaccines are currently available. Advances in expression technology for complex, enveloped virus-like particles (eVLPs) create new opportunities to develop potent vaccines against pathogenic

  14. Expression and purification of virus like particles (VLPs) of foot-and-mouth disease virus in Eri silkworm (Samia cynthia ricini) larvae

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    Kumar, Manoj; Saravanan, P.; S.K.Jalali

    2015-01-01

    Foot-and-mouth disease (FMD) is a highly contagious viral disease, which causes severe economic loss to livestock. Virus like particles (VLPs) produced by recombinant DNA technology are gaining importance because of their immunogenic properties and safety in developing a new vaccine for FMD. In the present study, a practical and economically feasible approach of expression, purification and characterization of VLPs of FMDV in Eri silkworm (Samia cynthia ricini) larvae was described. Although ...

  15. Construction, expression and immunogenicity of a novel anti-hypertension angiotensin II vaccine based on hepatitis A virus-like particle

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    Ou, Xia; Guo, Lili; Wu, Jinyuan; Mi, Kai; Yin, Na; Zhang, Guangming; Li, Hongjun; Sun, Maosheng

    2013-01-01

    Hypertension is a serious worldwide public health problem. The aim of this study is to design anti-hypertension angiotensin II (Ang II) vaccine using molecular biology and immunological method. This novel anti-hypertension vaccine, which is a chimeric protein named pHAV–4Ang IIs, presents four successive repeated Ang IIs as the functional epitope on the surface of the hepatitis A virus-like particle(HAVLP). In this study, pHAV–4Ang IIs was expressed using Bac-to-Bac Baculovirus Expression Sys...

  16. Biochemical composition of haemagglutinin-based influenza virus-like particle vaccine produced by transient expression in tobacco plants.

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    Le Mauff, François; Mercier, Geneviève; Chan, Philippe; Burel, Carole; Vaudry, David; Bardor, Muriel; Vézina, Louis-Philippe; Couture, Manon; Lerouge, Patrice; Landry, Nathalie

    2015-06-01

    Influenza virus-like particles (VLPs) are noninfectious particles resembling the influenza virus representing a promising vaccine alternative to inactivated influenza virions as antigens. Medicago inc. has developed a plant-based VLP manufacturing platform allowing the large-scale production of GMP-grade influenza VLPs. In this article, we report on the biochemical compositions of these plant-based influenza candidate vaccines, more particularly the characterization of the N-glycan profiles of the viral haemagglutinins H1 and H5 proteins as well as the tobacco-derived lipid content and residual impurities. Mass spectrometry analyses showed that all N-glycosylation sites of the extracellular domain of the recombinant haemagglutinins carry plant-specific complex-type N-glycans having core α(1,3)-fucose, core β(1,2)-xylose epitopes and Lewis(a) extensions. Previous phases I and II clinical studies have demonstrated that no hypersensibility nor induction of IgG or IgE directed against these glycans was observed. In addition, this article showed that the plant-made influenza vaccines are highly pure VLPs preparations while detecting no protein contaminants coming either from Agrobacterium or from the enzymes used for the enzyme-assisted extraction process. In contrast, VLPs contain few host cell proteins and glucosylceramides associated with plant lipid rafts. Identification of such raft markers, together with the type of host cell impurity identified, confirmed that the mechanism of VLP formation in planta is similar to the natural process of influenza virus assembly in mammals. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  17. Virus-like particles as nanovaccine candidates

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    Guillen, G.; Aguilar, J. C.; Dueñas, S.; Hermida, L.; Iglesias, E.; Penton, E.; Lobaina, Y.; Lopez, M.; Mussachio, A.; Falcon, V.; Alvarez, L.; Martinez, G.; Gil, L.; Valdes, I.; Izquierdo, A.; Lazo, L.; Marcos, E.; Guzman, G.; Muzio, V.; Herrera, L.

    2013-03-01

    The existing vaccines are mainly limited to the microorganisms we are able to culture and produce and/or to those whose killing is mediated by humoral response (antibody mediated). It has been more difficult to develop vaccines capable of inducing a functional cellular response needed to prevent or cure chronic diseases. New strategies should be taken into account in the improvement of cell-based immune responses in order to prevent and control the infections and eventually clear the virus. Preclinical and clinical results with vaccine candidates developed as a vaccine platform based on virus-like particles (VLPs) evidenced their ability to stimulate mucosal as well as systemic immunity. Particles based on envelope, membrane or nucleocapsid microbial proteins induce a strong immune response after nasal or parenteral administration in mice, non-human primates and humans. In addition, the immune response obtained was modulated in a Th1 sense. The VLPs were also able to immunoenhance the humoral and cellular immune responses against several viral pathogens. Studies in animals and humans with nasal and systemic formulations evidenced that it is possible to induce functional immune response against HBV, HCV, HIV and dengue virus. Invited talk at the 6th International Workshop on Advanced Materials Science and Nanotechnology, 30 October - 2 November 2012, Ha Long, Vietnam.

  18. Co-expression of HIV-1 virus-like particles and granulocyte-macrophage colony stimulating factor by GEO-D03 DNA vaccine.

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    Hellerstein, Michael; Xu, Yongxian; Marino, Tracie; Lu, Shan; Yi, Hong; Wright, Elizabeth R; Robinson, Harriet L

    2012-11-01

    Here, we report on GEO-D03, a DNA vaccine that co-expresses non-infectious HIV-1 virus-like particles (VLPs) and the human cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). The virus-like particles display the native gp160 form of the HIV-1 Envelope glycoprotein (Env) and are designed to elicit antibody against the natural form of Env on virus and virus-infected cells. The DNA-expressed HIV Gag, Pol and Env proteins also have the potential to elicit virus-specific CD4 and CD8 T cells. The purpose of the co-expressed GM-CSF is to target a cytokine that recruits, expands and differentiates macrophages and dendritic cells to the site of VLP expression. The GEO-D03 DNA vaccine is currently entered into human trials as a prime for a recombinant modified vaccinia Ankara (MVA) boost. In preclinical studies in macaques using an SIV prototype vaccine, this vaccination regimen elicited both anti-viral T cells and antibody, and provided 70% protection against acquisition during 12 weekly rectal exposures with a heterologous SIV. Higher avidity of the Env-specific Ab for the native form of the Env in the challenge virus correlated with lower likelihood of SIV infection.

  19. Human papillomavirus L1 protein expressed in Escherichia coli self-assembles into virus-like particles that are highly immunogenic.

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    Chen, Yumei; Liu, Yunchao; Zhang, Gaiping; Wang, Aiping; Dong, Ziming; Qi, Yanhua; Wang, Jucai; Zhao, Baolei; Li, Ning; Jiang, Min

    2016-07-15

    HPV vaccines based on L1 virus-like particles (VLPs) provided a high degree of protection against HPVs infection. In this study, the codon optimized HPV16 L1 gene were sub-cloned into five procaryotic expression vectors (pET-28a, pET-32a, pGEX-4T-2, pE-sumo and pHSIE), and fused with different protein tags. No recombinant proteins were expressed in pET-28a-L1 and pHSIE-L1, and the proteins expressed by pET-32a-L1 plasmid with TRX-tag were in the form of inclusion body. Only SUMO-tagged and GST-tagged L1 proteins expressed by pE-Sumo-L1 or pGEX-4T-L1 were soluble. The yield of SUMO-L1 protein reached 260mg/L fermentation medium in shake flask. After SUMO tags were eliminated, a 90% purity of L1 proteins was generated by ion-exchange and Ni-NTA affinity chromatography. The purified HPV16 L1 protein self-assembled into virus-like particles (VLPs) and showed a haemagglutination activity. High titers specific and neutralizing antibodies were detected in HPV 16 L1VLPs vaccinated mice. Cytokines such as IFN-γ and IL-2 showed significant higher in VLPs vaccinated mice compared with negative control (p<0.05, p=0.055). Thus, the expression of recombinant HPV16 L1 VLPs in Escherichia coli was feasible, which could potentially be used for a VLP-based HPV vaccine. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose

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    Ju, Huanyu; Wei, Na; Wang, Qian; Wang, Chunyuan; Jing, Zhiqiang; Guo, Lu; Liu, Dapeng; Gao, Mingchun; Ma, Bo [College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030 (China); Wang, Junwei, E-mail: jwwang@neau.edu.cn [College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang 150030 (China)

    2011-05-27

    Highlights: {yields} All three capsid proteins can be expressed in insect cells in baculovirus expression system. {yields} All three recombinant proteins were spontaneously self-assemble into virus-like particles whose size and appearance were similar to those of native purified GPV virions. {yields} The immunogenicity of GPV-VLPs was better than commercial inactivated vaccine and attenuated vaccine. -- Abstract: Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs.

  1. Heterologous prime-boost-boost immunisation of Chinese cynomolgus macaques using DNA and recombinant poxvirus vectors expressing HIV-1 virus-like particles.

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    Bridge, Simon H; Sharpe, Sally A; Dennis, Mike J; Dowall, Stuart D; Getty, Brian; Anson, Donald S; Skinner, Michael A; Stewart, James P; Blanchard, Tom J

    2011-09-07

    There is renewed interest in the development of poxvirus vector-based HIV vaccines due to the protective effect observed with repeated recombinant canarypox priming with gp120 boosting in the recent Thai placebo-controlled trial. This study sought to investigate whether a heterologous prime-boost-boost vaccine regimen in Chinese cynomolgus macaques with a DNA vaccine and recombinant poxviral vectors expressing HIV virus-like particles bearing envelopes derived from the most prevalent clades circulating in sub-Saharan Africa, focused the antibody response to shared neutralising epitopes. Three Chinese cynomolgus macaques were immunised via intramuscular injections using a regimen composed of a prime with two DNA vaccines expressing clade A Env/clade B Gag followed by boosting with recombinant fowlpox virus expressing HIV-1 clade D Gag, Env and cholera toxin B subunit followed by the final boost with recombinant modified vaccinia virus Ankara expressing HIV-1 clade C Env, Gag and human complement protein C3d. We measured the macaque serum antibody responses by ELISA, enumerated T cell responses by IFN-γ ELISpot and assessed seroneutralisation of HIV-1 using the TZM-bl β-galactosidase assay with primary isolates of HIV-1. This study shows that large and complex synthetic DNA sequences can be successfully cloned in a single step into two poxvirus vectors: MVA and FPV and the recombinant poxviruses could be grown to high titres. The vaccine candidates showed appropriate expression of recombinant proteins with the formation of authentic HIV virus-like particles seen on transmission electron microscopy. In addition the b12 epitope was shown to be held in common by the vaccine candidates using confocal immunofluorescent microscopy. The vaccine candidates were safely administered to Chinese cynomolgus macaques which elicited modest T cell responses at the end of the study but only one out of the three macaques elicited an HIV-specific antibody response. However, the

  2. Heterologous prime-boost-boost immunisation of Chinese cynomolgus macaques using DNA and recombinant poxvirus vectors expressing HIV-1 virus-like particles

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    Anson Donald S

    2011-09-01

    Full Text Available Abstract Background There is renewed interest in the development of poxvirus vector-based HIV vaccines due to the protective effect observed with repeated recombinant canarypox priming with gp120 boosting in the recent Thai placebo-controlled trial. This study sought to investigate whether a heterologous prime-boost-boost vaccine regimen in Chinese cynomolgus macaques with a DNA vaccine and recombinant poxviral vectors expressing HIV virus-like particles bearing envelopes derived from the most prevalent clades circulating in sub-Saharan Africa, focused the antibody response to shared neutralising epitopes. Methods Three Chinese cynomolgus macaques were immunised via intramuscular injections using a regimen composed of a prime with two DNA vaccines expressing clade A Env/clade B Gag followed by boosting with recombinant fowlpox virus expressing HIV-1 clade D Gag, Env and cholera toxin B subunit followed by the final boost with recombinant modified vaccinia virus Ankara expressing HIV-1 clade C Env, Gag and human complement protein C3d. We measured the macaque serum antibody responses by ELISA, enumerated T cell responses by IFN-γ ELISpot and assessed seroneutralisation of HIV-1 using the TZM-bl β-galactosidase assay with primary isolates of HIV-1. Results This study shows that large and complex synthetic DNA sequences can be successfully cloned in a single step into two poxvirus vectors: MVA and FPV and the recombinant poxviruses could be grown to high titres. The vaccine candidates showed appropriate expression of recombinant proteins with the formation of authentic HIV virus-like particles seen on transmission electron microscopy. In addition the b12 epitope was shown to be held in common by the vaccine candidates using confocal immunofluorescent microscopy. The vaccine candidates were safely administered to Chinese cynomolgus macaques which elicited modest T cell responses at the end of the study but only one out of the three macaques

  3. Porcine circovirus type 2 protective epitope densely carried by chimeric papaya ringspot virus-like particles expressed in Escherichia coli as a cost-effective vaccine manufacture alternative.

    Science.gov (United States)

    Aguilera, Brenda Eugenia; Chávez-Calvillo, Gabriela; Elizondo-Quiroga, Darwin; Jimenez-García, Mónica Noemí; Carrillo-Tripp, Mauricio; Silva-Rosales, Laura; Hernández-Gutiérrez, Rodolfo; Gutiérrez-Ortega, Abel

    2017-05-01

    Porcine circovirus type 2 (PCV2) still represents a major problem to the swine industry worldwide, causing high mortality rates in infected animals. Virus-like particles (VLPs) have gained attention for vaccine development, serving both as scaffolds for epitope expression and immune response enhancers. The commercial subunit vaccines against PCV2 consist of VLPs formed by the self-assembly of PCV2 capsid protein (CP) expressed in the baculovirus vector system. In this work, a PCV2 protective epitope was inserted into three different regions of papaya ringspot virus (PRSV) CP, namely, the N- and C-termini and a predicted antigenic region located near the N-terminus. Wild-type and chimeric CPs were modeled in silico, expressed in Escherichia coli, purified, and visualized by transmission electron microscopy. This is the first report that shows the formation of chimeric VLPs using PRSV as epitope-presentation scaffold. Moreover, it was found that PCV2 epitope localization strongly influences VLP length. Also, the estimated yields of the chimeric VLPs at a small-scale level ranged between 65 and 80 mg/L of culture medium. Finally, the three chimeric VLPs induced high levels of immunoglobulin G against the PCV2 epitope in immunized BALB/c mice, suggesting that these chimeric VLPs can be used for swine immunoprophylaxis against PCV2. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  4. Goose parvovirus structural proteins expressed by recombinant baculoviruses self-assemble into virus-like particles with strong immunogenicity in goose.

    Science.gov (United States)

    Ju, Huanyu; Wei, Na; Wang, Qian; Wang, Chunyuan; Jing, Zhiqiang; Guo, Lu; Liu, Dapeng; Gao, Mingchun; Ma, Bo; Wang, Junwei

    2011-05-27

    Goose parvovirus (GPV), a small non-enveloped ssDNA virus, can cause Derzsy's disease, and three capsid proteins of VP1, VP2, and VP3 are encoded by an overlapping nucleotide sequence. However, little is known on whether recombinant viral proteins (VPs) could spontaneously assemble into virus-like particles (VLPs) in insect cells and whether these VLPs could retain their immunoreactivity and immunogenicity in susceptible geese. To address these issues, genes for these GPV VPs were amplified by PCR, and the recombinant VPs proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures, immunoreactivity, and immunogenicity. The rVP1, rVP2, and rVP3 expressed in Sf9 cells were detected by anti-GPV sera, anti-VP3 sera, and anti-His antibodies, respectively. Electron microscopy revealed that these rVPs spontaneously assembled into VLPs in insect cells, similar to that of the purified wild-type GPV virions. In addition, vaccination with individual types of VLPs, particularly with the rVP2-VLPs, induced higher titers of antibodies and neutralized different strains of GPVs in primary goose and duck embryo fibroblast cells in vitro. These data indicated that these VLPs retained immunoreactivity and had strong immunogenicity in susceptible geese. Therefore, our findings may provide a framework for development of new vaccines for the prevention of Derzsy's disease and vehicles for the delivery of drugs. Copyright © 2011 Elsevier Inc. All rights reserved.

  5. Expression and purification of virus like particles (VLPs) of foot-and-mouth disease virus in Eri silkworm (Samia cynthia ricini) larvae.

    Science.gov (United States)

    Kumar, Manoj; Saravanan, P; Jalali, S K

    2016-03-01

    Foot-and-mouth disease (FMD) is a highly contagious viral disease, which causes severe economic loss to livestock. Virus like particles (VLPs) produced by recombinant DNA technology are gaining importance because of their immunogenic properties and safety in developing a new vaccine for FMD. In the present study, a practical and economically feasible approach of expression, purification and characterization of VLPs of FMDV in Eri silkworm (Samia cynthia ricini) larvae was described. Although three lepidopteran insect larvae (Helicoverpa armigera, Spodoptera litura and Samia cynthia ricini) were tested for production of VLPs, expression was obtained only in Eri silkworm larvae. High titred recombinant baculovirus encoding the polyprotein P1-2A-3C of FMDV was prepared in Sf9 cells. Injection of recombinant baculovirus into hemocoel of Eri silkworm larvae resulted in increasing levels of expression of VLPs in the hemolymph from 3 to 7 days post infection (dpi) compared to low level expression by oral feeding. The VLPs reacted in Sandwich ELISA with serum raised against whole virus particles of FMDV type O/IND/R2/75 and protein banding pattern of 26, 37 and 47 kDa in Western blotting demonstrated their antigenic resemblance to native virus. Sucrose density gradient purified VLPs were used for immunization of rabbits and guinea pigs for assessing immunogenicity. Further, the reactivity of serum samples of rabbits and guinea pigs in Indirect-ELISA with titres (1.30-2.81 Log10) indicated that the VLPs were antigenic and immunogenic in nature. We demonstrate that Eri silkworm larvae could be used for production of VLPs of FMDV type O/IND/R2/75 for the first time. This approach could be useful for large scale production of recombinant VLPs for vaccine or diagnostic use in FMD control programme.

  6. Novel immunogenic baculovirus expressed virus-like particles of foot-and-mouth disease (FMD) virus protect guinea pigs against challenge.

    Science.gov (United States)

    Bhat, S A; Saravanan, P; Hosamani, M; Basagoudanavar, S H; Sreenivasa, B P; Tamilselvan, R P; Venkataramanan, R

    2013-12-01

    Vaccination is a well accepted strategy for control of foot-and-mouth disease (FMD) in endemic countries. Currently, chemically inactivated virus antigens are used for preparation of FMD vaccine. To develop a non-infectious and safe recombinant vaccine, we expressed structural polypeptide of FMDV (O/IND/R2/75) using baculovirus expression system. We show that inclusion of mutated viral 3C protease in frame with the polypeptide (P1-2A), enhanced the yield of structural proteins. The structural proteins retained antigenicity and assembled into empty virus-like particles (VLPs). Immunization of guinea pigs with purified fractions of the VLPs resulted in humoral and cell mediated immune response by 4 weeks. The VLPs elicited comparable humoral immune response and relatively higher cell mediated immune response, when compared to conventional vaccine in guinea pigs. Further, up to 70% of the VLP immunized guinea pigs were protected against challenge with homologous guinea pig adapted virus. Our results highlight the application of recombinant FMDV VLPs in FMD vaccination. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Encapsulation of phthalocyanine supramolecular stacks into virus-like particles

    NARCIS (Netherlands)

    Brasch, M.; de la Escosura, Andrés; Ma, Y.; Uetrecht, Charlotte; Heck, Albert J.R.; Torres, Tomás; Cornelissen, Jeroen Johannes Lambertus Maria

    2011-01-01

    We report herein the encapsulation of a water-soluble phthalocyanine (Pc) into virus-like particles (VLPs) of two different sizes, depending on the conditions. At neutral pH, the cooperative encapsulation/templated assembly of the particles induces the formation of Pc stacks instead of Pc dimers,

  8. Virus-Like Particles That Can Deliver Proteins and RNA | NCI Technology Transfer Center | TTC

    Science.gov (United States)

    The present invention describes novel virus-like particles (VLPs) that are capable of binding to and replicating within a target mammalian cell, including human cells. The claimed VLPs are safer than viral delivery because they are incapable of re-infecting target cells. The National Cancer Institute's Protein Expression Laboratory seeks parties interested in licensing the novel delivery of RNA to mammalian cells using virus-like particles.

  9. Progress in Developing Virus-like Particle Influenza Vaccines

    Science.gov (United States)

    Quan, Fu-Shi; Lee, Young-Tae; Kim, Ki-Hye; Kim, Min-Chul; Kang, Sang-Moo

    2016-01-01

    Summary Recombinant vaccines based on virus-like particles (VLPs) or nanoparticles have been successful in their safety and efficacy in preclinical and clinical studies. The technology of expressing enveloped VLP vaccines has combined with molecular engineering of proteins in membrane-anchor and immunogenic forms mimicking the native conformation of surface proteins on the enveloped viruses. This review summarizes recent developments in influenza VLP vaccines against seasonal, pandemic, and avian influenza viruses from the perspective of use in humans. The immunogenicity and efficacies of influenza VLP vaccine in the homologous and cross-protection were reviewed. Discussions include limitations of current influenza vaccination strategies and future directions to confer broadly cross protective new influenza vaccines as well as vaccination. PMID:27058302

  10. Cryo-electron microscopy study of insect cell-expressed enterovirus 71 and coxsackievirus a16 virus-like particles provides a structural basis for vaccine development.

    Science.gov (United States)

    Gong, Minqing; Zhu, Hongtao; Zhou, Jun; Yang, Chunting; Feng, Jing; Huang, Xiaojun; Ji, Gang; Xu, Honglin; Zhu, Ping

    2014-06-01

    Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two most common etiological agents responsible for the epidemics of hand, foot, and mouth disease (HFMD), a childhood illness with occasional severe neurological complications. A number of vaccine candidates against EV71 or CA16 have been reported; however, no vaccine is currently available for clinical use. Here, we generated a secreted version of EV71 and CA16 virus-like particles (VLPs) using a baculovirus-insect cell expression system and reconstructed the three-dimensional (3D) structures of both VLPs by cryo-electron microscopy (cryo-EM) single-particle analysis at 5.2-Å and 5.5-Å resolutions, respectively. The reconstruction results showed that the cryo-EM structures of EV71 and CA16 VLPs highly resemble the recently published crystal structures for EV71 natural empty particles and CA16 135S-like expanded particles, respectively. Our cryo-EM analysis also revealed that the majority of previously identified linear neutralizing epitopes are well preserved on the surface of EV71 and CA16 VLPs. In addition, both VLPs were able to induce efficiently neutralizing antibodies against various strains of EV71 and CA16 viruses in mouse immunization. These studies provide a structural basis for the development of insect cell-expressed VLP vaccines and for a potential bivalent VLP vaccine against both EV71- and CA16-associated HFMD. The recent outbreaks of hand, foot, and mouth disease (HFMD) in the Asia Pacific region spurred the search for effective vaccines against EV71 and CA16 viruses, the two most common etiological agents responsible for HFMD. In this paper, we show that secreted versions of EV71 and CA16 VLPs generated in the baculovirus-insect cell expression system highly resemble the crystal structures of their viral conterparts and that the majority of previously identified linear neutralizing epitopes are well preserved on the VLP surfaces. In addition, the generated VLPs can efficiently induce

  11. Cell wall biochemical alterations during Agrobacterium-mediated expression of haemagglutinin-based influenza virus-like vaccine particles in tobacco.

    Science.gov (United States)

    Le Mauff, François; Loutelier-Bourhis, Corinne; Bardor, Muriel; Berard, Caroline; Doucet, Alain; D'Aoust, Marc-André; Vezina, Louis-Philippe; Driouich, Azeddine; Couture, Manon M-J; Lerouge, Patrice

    2017-03-01

    Influenza virus-like particles (VLPs) have been shown to induce a safe and potent immune response through both humoral and cellular responses. They represent promising novel influenza vaccines. Plant-based biotechnology allows for the large-scale production of VLPs of biopharmaceutical interest using different model organisms, including Nicotiana benthamiana plants. Through this platform, influenza VLPs bud from the plasma membrane and accumulate between the membrane and the plant cell wall. To design and optimize efficient production processes, a better understanding of the plant cell wall composition of infiltrated tobacco leaves is a major interest for the plant biotechnology industry. In this study, we have investigated the alteration of the biochemical composition of the cell walls of N. benthamiana leaves subjected to abiotic and biotic stresses induced by the Agrobacterium-mediated transient transformation and the resulting high expression levels of influenza VLPs. Results show that abiotic stress due to vacuum infiltration without Agrobacterium did not induce any detectable modification of the leaf cell wall when compared to non infiltrated leaves. In contrast, various chemical changes of the leaf cell wall were observed post-Agrobacterium infiltration. Indeed, Agrobacterium infection induced deposition of callose and lignin, modified the pectin methylesterification and increased both arabinosylation of RG-I side chains and the expression of arabinogalactan proteins. Moreover, these modifications were slightly greater in plants expressing haemagglutinin-based VLP than in plants infiltrated with the Agrobacterium strain containing only the p19 suppressor of silencing. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  12. Pichia pastoris-expressed dengue 3 envelope-based virus-like particles elicit predominantly domain III-focused high titer neutralizing antibodies.

    Science.gov (United States)

    Tripathi, Lav; Mani, Shailendra; Raut, Rajendra; Poddar, Ankur; Tyagi, Poornima; Arora, Upasana; de Silva, Aravinda; Swaminathan, Sathyamangalam; Khanna, Navin

    2015-01-01

    Dengue poses a serious public health risk to nearly half the global population. It causes ~400 million infections annually and is considered to be one of the fastest spreading vector-borne diseases. Four distinct serotypes of dengue viruses (DENV-1, -2, -3, and -4) cause dengue disease, which may be either mild or extremely severe. Antibody-dependent enhancement (ADE), by pre-existing cross-reactive antibodies, is considered to be the major mechanism underlying severe disease. This mandates that a preventive vaccine must confer simultaneous and durable immunity to each of the four prevalent DENV serotypes. Recently, we used Pichia pastoris, to express recombinant DENV-2 E ectodomain, and found that it assembled into virus-like particles (VLPs), in the absence of prM, implicated in the elicitation of ADE-mediating antibodies. These VLPs elicited predominantly type-specific neutralizing antibodies that conferred significant protection against lethal DENV-2 challenge, in a mouse model. The current work is an extension of this approach to develop prM-lacking DENV-3 E VLPs. Our data reveal that P. pastoris-produced DENV-3 E VLPs not only preserve the antigenic integrity of the major neutralizing epitopes, but also elicit potent DENV-3 virus-neutralizing antibodies. Further, these neutralizing antibodies appear to be exclusively directed toward domain III of the DENV-3 E VLPs. Significantly, they also lack discernible ADE potential toward heterotypic DENVs. Taken together with the high productivity of the P. pastoris expression system, this approach could potentially pave the way toward developing a DENV E-based, inexpensive, safe, and efficacious tetravalent sub-unit vaccine, for use in resource-poor dengue endemic countries.

  13. Virus-like particles derived from Pichia pastoris-expressed dengue virus type 1 glycoprotein elicit homotypic virus-neutralizing envelope domain III-directed antibodies.

    Science.gov (United States)

    Poddar, Ankur; Ramasamy, Viswanathan; Shukla, Rahul; Rajpoot, Ravi Kant; Arora, Upasana; Jain, Swatantra K; Swaminathan, Sathyamangalam; Khanna, Navin

    2016-06-14

    Four antigenically distinct serotypes (1-4) of dengue viruses (DENVs) cause dengue disease. Antibodies to any one DENV serotype have the potential to predispose an individual to more severe disease upon infection with a different DENV serotype. A dengue vaccine must elicit homotypic neutralizing antibodies to all four DENV serotypes to avoid the risk of such antibody-dependent enhancement in the vaccine recipient. This is a formidable challenge as evident from the lack of protective efficacy against DENV-2 by a tetravalent live attenuated dengue vaccine that has completed phase III trials recently. These trial data underscore the need to explore non-replicating subunit vaccine alternatives. Recently, using the methylotrophic yeast Pichia pastoris, we showed that DENV-2 and DENV-3 envelope (E) glycoproteins, expressed in absence of prM, implicated in causing severe dengue disease, self-assemble into virus-like particles (VLPs), which elicit predominantly virus-neutralizing antibodies and confer significant protection against lethal DENV challenge in an animal model. The current study extends this work to a third DENV serotype. We cloned and expressed DENV-1 E antigen in P. pastoris, and purified it to near homogeneity. Recombinant DENV-1 E underwent post-translational processing, namely, signal peptide cleavage and glycosylation. Purified DENV-1 E self-assembled into stable VLPs, based on electron microscopy and dynamic light scattering analysis. Epitope mapping with monoclonal antibodies revealed that the VLPs retained the overall antigenic integrity of the virion particles despite the absence of prM. Subtle changes accompanied the efficient display of E domain III (EDIII), which contains type-specific neutralizing epitopes. These VLPs were immunogenic, eliciting predominantly homotypic EDIII-directed DENV-1-specific neutralizing antibodies. This work demonstrates the inherent potential of P. pastoris-expressed DENV-1 E glycoprotein to self-assemble into VLPs

  14. Baculovirus-expressed virus-like particle vaccine in combination with DNA encoding the fusion protein confers protection against respiratory syncytial virus.

    Science.gov (United States)

    Lee, Jong Seok; Kwon, Young-Man; Hwang, Hye Suk; Lee, Yu-Na; Ko, Eun-Ju; Yoo, Si-Eun; Kim, Min-Chul; Kim, Ki-Hye; Cho, Min Kyoung; Lee, Young-Tae; Lee, You Ri; Quan, Fu-Shi; Kang, Sang-Moo

    2014-10-07

    Respiratory syncytial virus (RSV) is a major viral agent causing significant morbidity and mortality in young infants and the elderly. There is no licensed vaccine against RSV and it is a high priority to develop a safe RSV vaccine. We determined the immunogenicity and protective efficacy of combined virus-like particle and DNA vaccines presenting RSV glycoproteins (Fd.VLP) in comparison with formalin inactivated RSV (FI-RSV). Immunization of mice with Fd.VLP induced higher ratios of IgG2a/IgG1 antibody responses compared to those with FI-RSV. Upon live RSV challenge, Fd.VLP and FI-RSV vaccines were similarly effective in clearing lung viral loads. However, FI-RSV immunized mice showed a substantial weight loss and high levels of T helper type 2 (Th2) cytokines as well as extensive lung histopathology and eosinophil infiltration. In contrast, Fd.VLP immunized mice did not exhibit Th2 type cytokines locally and systemically, which might contribute to preventing vaccine-associated RSV lung disease. These results indicate that virus-like particles in combination with DNA vaccines represent a potential approach for developing a safe and effective RSV vaccine. Copyright © 2014 Elsevier Ltd. All rights reserved.

  15. Pichia pastoris-expressed dengue 2 envelope forms virus-like particles without pre-membrane protein and induces high titer neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Shailendra Mani

    Full Text Available Dengue is a mosquito-borne viral disease with a global prevalence. It is caused by four closely-related dengue viruses (DENVs 1-4. A dengue vaccine that can protect against all four viruses is an unmet public health need. Live attenuated vaccine development efforts have encountered unexpected interactions between the vaccine viruses, raising safety concerns. This has emphasized the need to explore non-replicating dengue vaccine options. Virus-like particles (VLPs which can elicit robust immunity in the absence of infection offer potential promise for the development of non-replicating dengue vaccine alternatives. We have used the methylotrophic yeast Pichia pastoris to develop DENV envelope (E protein-based VLPs. We designed a synthetic codon-optimized gene, encoding the N-terminal 395 amino acid residues of the DENV-2 E protein. It also included 5' pre-membrane-derived signal peptide-encoding sequences to ensure proper translational processing, and 3' 6× His tag-encoding sequences to facilitate purification of the expressed protein. This gene was integrated into the genome of P. pastoris host and expressed under the alcohol oxidase 1 promoter by methanol induction. Recombinant DENV-2 protein, which was present in the insoluble membrane fraction, was extracted and purified using Ni(2+-affinity chromatography under denaturing conditions. Amino terminal sequencing and detection of glycosylation indicated that DENV-2 E had undergone proper post-translational processing. Electron microscopy revealed the presence of discrete VLPs in the purified protein preparation after dialysis. The E protein present in these VLPs was recognized by two different conformation-sensitive monoclonal antibodies. Low doses of DENV-2 E VLPs formulated in alum were immunogenic in inbred and outbred mice eliciting virus neutralizing titers >1,1200 in flow cytometry based assays and protected AG129 mice against lethal challenge (p<0.05. The formation of immunogenic DENV-2 E

  16. Zika virus-like particle (VLP) based vaccine

    Science.gov (United States)

    Boigard, Hélène; Alimova, Alexandra; Martin, George R.; Katz, Al; Gottlieb, Paul

    2017-01-01

    The newly emerged mosquito-borne Zika virus poses a major public challenge due to its ability to cause significant birth defects and neurological disorders. The impact of sexual transmission is unclear but raises further concerns about virus dissemination. No specific treatment or vaccine is currently available, thus the development of a safe and effective vaccine is paramount. Here we describe a novel strategy to assemble Zika virus-like particles (VLPs) by co-expressing the structural (CprME) and non-structural (NS2B/NS3) proteins, and demonstrate their effectiveness as vaccines. VLPs are produced in a suspension culture of mammalian cells and self-assembled into particles closely resembling Zika viruses as shown by electron microscopy studies. We tested various VLP vaccines and compared them to analogous compositions of an inactivated Zika virus (In-ZIKV) used as a reference. VLP immunizations elicited high titers of antibodies, as did the In-ZIKV controls. However, in mice the VLP vaccine stimulated significantly higher virus neutralizing antibody titers than comparable formulations of the In-ZIKV vaccine. The serum neutralizing activity elicited by the VLP vaccine was enhanced using a higher VLP dose and with the addition of an adjuvant, reaching neutralizing titers greater than those detected in the serum of a patient who recovered from a Zika infection in Brazil in 2015. Discrepancies in neutralization levels between the VLP vaccine and the In-ZIKV suggest that chemical inactivation has deleterious effects on neutralizing epitopes within the E protein. This along with the inability of a VLP vaccine to cause infection makes it a preferable candidate for vaccine development. PMID:28481898

  17. T-body formation precedes virus-like particle maturation in S. cerevisiae

    DEFF Research Database (Denmark)

    Malagon, Francisco; Jensen, Torben Heick

    2011-01-01

    T-bodies are localized S. cerevisiae RNPs containing Ty1 retroviral components and speculated to play a role in the assembly of virus-like particles (VLPs). Mapping requirements for T-body formation, we demonstrate that ectopic expression of immature TyA1/Gag (Gag-p49), a structural component of ...

  18. Hantavirus Gn and Gc glycoproteins self-assemble into virus-like particles.

    Science.gov (United States)

    Acuña, Rodrigo; Cifuentes-Muñoz, Nicolás; Márquez, Chantal L; Bulling, Manuela; Klingström, Jonas; Mancini, Roberta; Lozach, Pierre-Yves; Tischler, Nicole D

    2014-02-01

    How hantaviruses assemble and exit infected cells remains largely unknown. Here, we show that the expression of Andes (ANDV) and Puumala (PUUV) hantavirus Gn and Gc envelope glycoproteins lead to their self-assembly into virus-like particles (VLPs) which were released to cell supernatants. The viral nucleoprotein was not required for particle formation. Further, a Gc endodomain deletion mutant did not abrogate VLP formation. The VLPs were pleomorphic, exposed protrusions and reacted with patient sera.

  19. Recent advances in mucosal immunization using virus-like particles.

    Science.gov (United States)

    Vacher, Gaëlle; Kaeser, Matthias D; Moser, Christian; Gurny, Robert; Borchard, Gerrit

    2013-05-06

    Mucosal immunization offers the promises of eliciting a systemic and mucosal immune response, as well as enhanced patient compliance. Mucosal vaccination using defined antigens such as proteins and peptides requires delivery systems that combine good safety profiles with strong immunogenicity, which may be provided by virus-like particles (VLP). VLP are assembled from viral structural proteins and thus are devoid of any genetic material. They excel by mimicking natural pathogens, therefore providing antigen-protecting particulate nature, inherent immune-cell stimulatory mechanisms, and tissue-specific targeting depending on their parental virus. Nevertheless, despite of promising preclinical results, VLP remain rarely investigated in clinical studies. This review is intended to give an overview of obstacles and promises of VLP-based mucosal immunization as well as to identify strategies to further improve VLP while maintaining a good safety and tolerability profile.

  20. The generation of Turnip crinkle virus-like particles in plants by the transient expression of wild-type and modified forms of its coat protein

    Directory of Open Access Journals (Sweden)

    Keith eSaunders

    2015-12-01

    Full Text Available Turnip crinkle virus (TCV, a member of the genus carmovirus of the Tombusviridae family, has a genome consisting of a single positive-sense RNA molecule that is encapsidated in an icosahedral particle composed of 180 copies of a single type of coat protein. We have employed the CPMV-HT transient expression system to investigate the formation of TCV-like particles following the expression of the wild-type coat protein or modified forms of it that contain either deletions and/or additions insertions. Transient expression of the coat protein in plants results in the formation of capsid structures that morphologically resemble TCV virions (T=3 structure but encapsidate heterogeneous cellular RNAs, rather than the specific TCV coat protein messenger RNA. Expression of an amino-terminal deleted form of the coat protein resulted in the formation of smaller T=1 structures that are free of RNA. The possibility of utilising TCV as a carrier for the presentation of foreign proteins on the particle surface was also explored by fusing the sequence of GFP to the C-terminus of the coat protein. The expression of coat protein-GFP hybrids permitted the formation of VLPs but the yield of particles is diminished compared to the yield obtained with unmodified coat protein. Our results confirm the importance of the N-terminus of the coat protein for the encapsidation of RNA and show that the coat protein’s exterior P domain plays a key role in particle formation.

  1. Recombinant expression and purification of 'virus-like' bacterial encapsulin protein cages

    NARCIS (Netherlands)

    Rurup, W.F.; Cornelissen, Jeroen Johannes Lambertus Maria; Koay, M.S.T.; Orner, Brendan P.

    2014-01-01

    Ultracentrifugation, particularly the use of sucrose or cesium chloride density gradients, is a highly reliable and efficient technique for the purification of virus-like particles and protein cages. Since virus-like particles and protein cages have a unique size compared to cellular macromolecules

  2. Recombinant expression and purification of 'virus-like' bacterial encapsulin protein cages

    NARCIS (Netherlands)

    Rurup, W.F.; Cornelissen, Jeroen Johannes Lambertus Maria; Koay, M.S.T.; Orner, Brendan P.

    2015-01-01

    Ultracentrifugation, particularly the use of sucrose or cesium chloride density gradients, is a highly reliable and efficient technique for the purification of virus-like particles and protein cages. Since virus-like particles and protein cages have a unique size compared to cellular macromolecules

  3. A novel recombinant virus-like particle vaccine for prevention of porcine parvovirus-induced reproductive failure

    NARCIS (Netherlands)

    Antonis, A.F.G.; Bruschke, C.J.M.; Rueda, P.; Maranga, L.; Casal, J.; Vela, C.; Hilgers, L.A.T.; Belt, P.B.G.M.; Weerdmeester, K.; Carrondo, M.J.; Langeveld, J.P.M.

    2006-01-01

    A novel vaccine against porcine parvovirus (PPV), composed of recombinant virus-like particles (PPV-VLPs) produced with the baculovirus expression vector system (BEVS) at industrial scale, was tested for its immunogenicity and protective potency. A formulation of submicrogram amounts of PPV-VLPs in

  4. Anti-tumor effect of the alphavirus-based virus-like particle vector expressing prostate-specific antigen in a HLA-DR transgenic mouse model of prostate cancer.

    Science.gov (United States)

    Riabov, V; Tretyakova, I; Alexander, R B; Pushko, P; Klyushnenkova, E N

    2015-10-05

    The goal of this study was to determine if an alphavirus-based vaccine encoding human Prostate-Specific Antigen (PSA) could generate an effective anti-tumor immune response in a stringent mouse model of prostate cancer. DR2bxPSA F1 male mice expressing human PSA and HLA-DRB1(*)1501 transgenes were vaccinated with virus-like particle vector encoding PSA (VLPV-PSA) followed by the challenge with Transgenic Adenocarcinoma of Mouse Prostate cells engineered to express PSA (TRAMP-PSA). PSA-specific cellular and humoral immune responses were measured before and after tumor challenge. PSA and CD8 reactivity in the tumors was detected by immunohistochemistry. Tumor growth was compared in vaccinated and control groups. We found that VLPV-PSA could infect mouse dendritic cells in vitro and induce a robust PSA-specific immune response in vivo. A substantial proportion of splenic CD8 T cells (19.6 ± 7.4%) produced IFNγ in response to the immunodominant peptide PSA(65-73). In the blood of vaccinated mice, 18.4 ± 4.1% of CD8 T cells were PSA-specific as determined by the staining with H-2D(b)/PSA(65-73) dextramers. VLPV-PSA vaccination also strongly stimulated production of IgG2a/b anti-PSA antibodies. Tumors in vaccinated mice showed low levels of PSA expression and significant CD8+ T cell infiltration. Tumor growth in VLPV-PSA vaccinated mice was significantly delayed at early time points (p=0.002, Gehan-Breslow test). Our data suggest that TC-83-based VLPV-PSA vaccine can efficiently overcome immune tolerance to PSA, mediate rapid clearance of PSA-expressing tumor cells and delay tumor growth. The VLPV-PSA vaccine will undergo further testing for the immunotherapy of prostate cancer. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Bacterially produced recombinant influenza vaccines based on virus-like particles.

    Directory of Open Access Journals (Sweden)

    Andrea Jegerlehner

    Full Text Available Although current influenza vaccines are effective in general, there is an urgent need for the development of new technologies to improve vaccine production timelines, capacities and immunogenicity. Herein, we describe the development of an influenza vaccine technology which enables recombinant production of highly efficient influenza vaccines in bacterial expression systems. The globular head domain of influenza hemagglutinin, comprising most of the protein's neutralizing epitopes, was expressed in E. coli and covalently conjugated to bacteriophage-derived virus-like particles produced independently in E.coli. Conjugate influenza vaccines produced this way were used to immunize mice and found to elicit immune sera with high antibody titers specific for the native influenza hemagglutinin protein and high hemagglutination-inhibition titers. Moreover vaccination with these vaccines induced full protection against lethal challenges with homologous and highly drifted influenza strains.

  6. Novel adenovirus encoded virus-like particles displaying the placental malaria associated VAR2CSA antigen

    DEFF Research Database (Denmark)

    Andersson, Anne-Marie C; dos Santos Marques Resende, Mafalda; Salanti, Ali

    2017-01-01

    and the CSA binding region of VAR2CSA has been identified as a promising vaccine target against placental malaria. Here we designed adenovirus encoded virus-like particles (VLP) by co-encoding Simian Immunodeficiency Virus (SIV) gag and VAR2CSA. The VAR2CSA antigen was fused to the transmembrane (TM......The malaria parasite Plasmodium falciparum presents antigens on the infected erythrocyte surface that bind human receptors expressed on the vascular endothelium. The VAR2CSA mediated binding to a distinct chondroitin sulphate A (CSA) is a crucial step in the pathophysiology of placental malaria......CSA fused to HA TM-CT was significantly superior in inducing ID1-ID2a specific antibodies after the first immunization. A sequential study was performed to include a comparison to the soluble VAR2CSA protein vaccine, which has entered a phase I clinical trial (NCT02647489). The results revealed...

  7. Human Norovirus Detection and Production, Quantification, and Storage of Virus-Like Particles

    Science.gov (United States)

    Debbink, Kari; Costantini, Veronica; Swanstrom, Jesica; Agnihothram, Sudhakar; Vinjé, Jan; Baric, Ralph

    2014-01-01

    Human noroviruses constitute a significant worldwide disease burden. Each year noroviruses cause over 267 million infections, deaths in over 200,000 children under the age of five, and over 50% of U.S. food borne illness. Due to the absence of a tissue culture model or small animal model to study human norovirus, virus-like particles (VLPs) and ELISA-based biological assays have been used to answer questions about norovirus evolution and immunity as well provide a potential vaccine platform. This chapter outlines the protocols on norovirus detection in stool and norovirus VLP design, production, purification, and storage using a Venezuelan equine encephalitis virus (VEE)-based VRP expression system. PMID:24510290

  8. Effective chikungunya virus-like particle vaccine produced in insect cells.

    Directory of Open Access Journals (Sweden)

    Stefan W Metz

    Full Text Available The emerging arthritogenic, mosquito-borne chikungunya virus (CHIKV causes severe disease in humans and represents a serious public health threat in countries where Aedes spp mosquitoes are present. This study describes for the first time the successful production of CHIKV virus-like particles (VLPs in insect cells using recombinant baculoviruses. This well-established expression system is rapidly scalable to volumes required for epidemic responses and proved well suited for processing of CHIKV glycoproteins and production of enveloped VLPs. Herein we show that a single immunization with 1 µg of non-adjuvanted CHIKV VLPs induced high titer neutralizing antibody responses and provided complete protection against viraemia and joint inflammation upon challenge with the Réunion Island CHIKV strain in an adult wild-type mouse model of CHIKV disease. CHIKV VLPs produced in insect cells using recombinant baculoviruses thus represents as a new, safe, non-replicating and effective vaccine candidate against CHIKV infections.

  9. A virus-like particle vaccine for epidemic Chikungunya virus protects nonhuman primates against infection.

    Science.gov (United States)

    Akahata, Wataru; Yang, Zhi-Yong; Andersen, Hanne; Sun, Siyang; Holdaway, Heather A; Kong, Wing-Pui; Lewis, Mark G; Higgs, Stephen; Rossmann, Michael G; Rao, Srinivas; Nabel, Gary J

    2010-03-01

    Chikungunya virus (CHIKV) has infected millions of people in Africa, Europe and Asia since this alphavirus reemerged from Kenya in 2004. The severity of the disease and the spread of this epidemic virus present a serious public health threat in the absence of vaccines or antiviral therapies. Here, we describe a new vaccine that protects against CHIKV infection of nonhuman primates. We show that selective expression of viral structural proteins gives rise to virus-like particles (VLPs) in vitro that resemble replication-competent alphaviruses. Immunization with these VLPs elicited neutralizing antibodies against envelope proteins from alternative CHIKV strains. Monkeys immunized with VLPs produced high-titer neutralizing antibodies that protected against viremia after high-dose challenge. We transferred these antibodies into immunodeficient mice, where they protected against subsequent lethal CHIKV challenge, indicating a humoral mechanism of protection. Immunization with alphavirus VLP vaccines represents a strategy to contain the spread of CHIKV and related pathogenic viruses in humans.

  10. Virus-like particles in cystic mammary adenoma of a snow leopard.

    Science.gov (United States)

    Chandra, S; Laughlin, D C

    1975-11-01

    Virus-like particles were observed in the giant cells of a mammary adenoma of a snow leopard kept in captivity. Particles that measured 115 to 125 nm in diameter budded from the lamella of endoplasmic reticulum and were studded on their inner surfaces with dense granules (approximately 12 nm) that gave them their unique ultrastructural morphology. Such particles were not observed extracellularly. Type B or type C particles were not seen in the tumor tissue.

  11. High-throughput characterization of virus-like particles by interlaced size-exclusion chromatography.

    Science.gov (United States)

    Ladd Effio, Christopher; Oelmeier, Stefan A; Hubbuch, Jürgen

    2016-03-04

    The development and manufacturing of safe and effective vaccines relies essentially on the availability of robust and precise analytical techniques. Virus-like particles (VLPs) have emerged as an important and valuable class of vaccines for the containment of infectious diseases. VLPs are produced by recombinant protein expression followed by purification procedures to minimize the levels of process- and product-related impurities. The control of these impurities is necessary during process development and manufacturing. Especially monitoring of the VLP size distribution is important for the characterization of the final vaccine product. Currently used methods require long analysis times and tailor-made assays. In this work, we present a size-exclusion ultra-high performance liquid chromatography (SE-UHPLC) method to characterize VLPs and quantify aggregates within 3.1min per sample applying interlaced injections. Four analytical SEC columns were evaluated for the analysis of human B19 parvo-VLPs and murine polyoma-VLPs. The optimized method was successfully used for the characterization of five recombinant protein-based VLPs including human papillomavirus (HPV) VLPs, human enterovirus 71 (EV71) VLPs, and chimeric hepatitis B core antigen (HBcAg) VLPs pointing out the generic applicability of the assay. Measurements were supported by transmission electron microscopy and dynamic light scattering. It was demonstrated that the iSE-UHPLC method provides a rapid, precise and robust tool for the characterization of VLPs. Two case studies on purification tools for VLP aggregates and storage conditions of HPV VLPs highlight the relevance of the analytical method for high-throughput process development and process monitoring of virus-like particles. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Quantum dot encapsulation in virus-like particles with tuneable structural properties and low toxicity

    NARCIS (Netherlands)

    Tagit, O.; De Ruiter, M. V.; Brasch, M.; Ma, Y.; Cornelissen, J. J.L.M.

    2017-01-01

    A simple method for the encapsulation of quantum dots (QDs) in virus-like particle (VLP) nanoassemblies with tuneable structural properties and enhanced biocompatibility is presented. Cowpea chlorotic mottle virus-based capsid proteins assemble around the carboxylated QDs to form QD/VLP

  13. Proliferative pododermatitis associated with virus-like particles in a northern gannet.

    Science.gov (United States)

    Daoust, P Y; Wadowska, D; Kibenge, F; Campagnoli, R P; Latimer, K S; Ritchie, B W

    2000-04-01

    Small multifocal lesions of proliferative pododermatitis were observed in an emaciated adult male northern gannet (Morus bassanus). Ultrastructurally, these lesions were associated with numerous virus-like particles with a size and morphology suggestive of Papovaviridae. DNA in situ hybridization with probes for avian polyomaviral and papillomaviral nucleic acid and an immunohistochemical test for the presence of papillomaviral antigen failed to identify this virus further. To our knowledge, papovavirus-like particles have not been recognized previously in this avian species.

  14. Robust production of virus-like particles and monoclonal antibodies with geminiviral replicon vectors in lettuce.

    Science.gov (United States)

    Lai, Huafang; He, Junyun; Engle, Michael; Diamond, Michael S; Chen, Qiang

    2012-01-01

    Pharmaceutical protein production in plants has been greatly promoted by the development of viral-based vectors and transient expression systems. Tobacco and related Nicotiana species are currently the most common host plants for the generation of plant-made pharmaceutical proteins (PMPs). Downstream processing of target PMPs from these plants, however, is hindered by potential technical and regulatory difficulties owing to the presence of high levels of phenolics and toxic alkaloids. Here, we explored the use of lettuce, which grows quickly yet produces low levels of secondary metabolites and viral vector-based transient expression systems to develop a robust PMP production platform. Our results showed that a geminiviral replicon system based on the bean yellow dwarf virus permits high-level expression in lettuce of virus-like particles (VLP) derived from the Norwalk virus capsid protein and therapeutic monoclonal antibodies (mAbs) against Ebola and West Nile viruses. These vaccine and therapeutic candidates can be readily purified from lettuce leaves with scalable processing methods while fully retaining functional activity. Furthermore, this study also demonstrated the feasibility of using commercially produced lettuce for high-level PMP production. This allows our production system to have access to unlimited quantities of inexpensive plant material for large-scale production. These results establish a new production platform for biological pharmaceutical agents that are effective, safe, low cost, and amenable to large-scale manufacturing. © 2011 The Authors. Plant Biotechnology Journal © 2011 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  15. Human transbodies to VP40 inhibit cellular egress of Ebola virus-like particles.

    Science.gov (United States)

    Teimoori, Salma; Seesuay, Watee; Jittavisutthikul, Surasak; Chaisri, Urai; Sookrung, Nitat; Densumite, Jaslan; Saelim, Nawannaporn; Chulanetra, Monrat; Maneewatch, Santi; Chaicumpa, Wanpen

    2016-10-14

    A direct acting anti-Ebola agent is needed. VP40, a conserved protein across Ebolavirus (EBOV) species has several pivotal roles in the virus life cycle. Inhibition of VP40 functions would lessen the virion integrity and interfere with the viral assembly, budding, and spread. In this study, cell penetrable human scFvs (HuscFvs) that bound to EBOV VP40 were produced by phage display technology. Gene sequences coding for VP40-bound-HuscFvs were subcloned from phagemids into protein expression plasmids downstream to a gene of cell penetrating peptide, i.e., nonaarginine (R9). By electron microscopy, transbodies from three clones effectively inhibited egress of the Ebola virus-like particles from human hepatic cells transduced with pseudo-typed-Lentivirus particles carrying EBOV VP40 and GP genes. Computerized simulation indicated that the effective HuscFvs bound to multiple basic residues in the cationic patch of VP40 C-terminal domain which are important in membrane-binding for viral matrix assembly and virus budding. The transbodies bound also to VP40 N-terminal domain and L domain peptide encompassed the PTAPPEY (WW binding) motif, suggesting that they might confer VP40 function inhibition through additional mechanism(s). The generated transbodies are worthwhile tested with authentic EBOV before developing to direct acting anti-Ebola agent for preclinical and clinical trials. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Vaccination with dengue virus-like particles induces humoral and cellular immune responses in mice

    Directory of Open Access Journals (Sweden)

    Zhang Quanfu

    2011-06-01

    Full Text Available Abstract Background The incidence of dengue, an infectious disease caused by dengue virus (DENV, has dramatically increased around the world in recent decades and is becoming a severe public health threat. However, there is currently no specific treatment for dengue fever, and licensed vaccine against dengue is not available. Vaccination with virus-like particles (VLPs has shown considerable promise for many viral diseases, but the effect of DENV VLPs to induce specific immune responses has not been adequately investigated. Results By optimizing the expression plasmids, recombinant VLPs of four antigenically different DENV serotypes DENV1-4 were successfully produced in 293T cells. The vaccination effect of dengue VLPs in mice showed that monovalent VLPs of each serotype stimulated specific IgG responses and potent neutralizing antibodies against homotypic virus. Tetravalent VLPs efficiently enhanced specific IgG and neutralizing antibodies against all four serotypes of DENV. Moreover, vaccination with monovalent or tetravalent VLPs resulted in the induction of specific cytotoxic T cell responses. Conclusions Mammalian cell expressed dengue VLPs are capable to induce VLP-specific humoral and cellular immune responses in mice, and being a promising subunit vaccine candidate for prevention of dengue virus infection.

  17. Particle size effects on protein and virus-like particle adsorption on perfusion chromatography media.

    Science.gov (United States)

    Wu, Yige; Abraham, Dicky; Carta, Giorgio

    2015-01-02

    The resin structure, chromatographic behavior, and adsorption kinetics of proteins and virus-like-particles (VLPs) are studied for POROS HS 20 and POROS HS 50 (23 and 52 μm mean diameter, respectively) to determine the effects of particle size on perfusion chromatography and to determine the predictive ability of available models. Transmission electron microscopy (TEM) and inverse size-exclusion chromatography (iSEC) show similar structures for the two resins, both containing 200-1000 nm pores that transect a network of much smaller pores. For non-binding conditions, trends of the height equivalent to a theoretical plate (HETP) as a function of reduced velocity are consistent with perfusion. The estimated intraparticle flow fractions for these conditions are 0.0018 and 0.00063 for POROS HS 20 and HS 50, respectively. For strong binding conditions, confocal laser scanning microscopy (CLSM) shows asymmetrical intraparticle concentrations profiles and enhanced rates of IgG adsorption on POROS HS 20 at 1000 cm/h. The corresponding effective diffusivity under flow is 2-3 times larger than for non-flow conditions and much larger than observed for POROS HS 50, consistent with available models. For VLPs, however, adsorption is confined to a thin layer near the particle surface for both resins, suggesting that the bound VLPs block the pores. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Prospective on multiscale simulation of virus-like particles: Application to computer-aided vaccine design.

    Science.gov (United States)

    Abi Mansour, Andrew; Sereda, Yuriy V; Yang, Jing; Ortoleva, Peter J

    2015-11-04

    Simulations of virus-like particles needed for computer-aided vaccine design highlight the need for new algorithms that accelerate molecular dynamics. Such simulations via conventional molecular dynamics present a practical challenge due to the millions of atoms involved and the long timescales of the phenomena of interest. These phenomena include structural transitions, self-assembly, and interaction with a cell surface. A promising approach for addressing this challenge is multiscale factorization. The approach is distinct from coarse-graining techniques in that it (1) avoids the need for conjecturing phenomenological governing equations for coarse-grained variables, (2) provides simulations with atomic resolution, (3) captures the cross-talk between disturbances at the atomic and the whole virus-like particle scale, and (4) achieves significant speedup over molecular dynamics. A brief review of multiscale factorization method is provided, as is a prospective on its development. Copyright © 2015 Elsevier Ltd. All rights reserved.

  19. Production of FMDV virus-like particles by a SUMO fusion protein approach in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Liang Shu-Mei

    2009-08-01

    Full Text Available Abstract Virus-like particles (VLPs are formed by the self-assembly of envelope and/or capsid proteins from many viruses. Some VLPs have been proven successful as vaccines, and others have recently found applications as carriers for foreign antigens or as scaffolds in nanoparticle biotechnology. However, production of VLP was usually impeded due to low water-solubility of recombinant virus capsid proteins. Previous studies revealed that virus capsid and envelope proteins were often posttranslationally modified by SUMO in vivo, leading into a hypothesis that SUMO modification might be a common mechanism for virus proteins to retain water-solubility or prevent improper self-aggregation before virus assembly. We then propose a simple approach to produce VLPs of viruses, e.g., foot-and-mouth disease virus (FMDV. An improved SUMO fusion protein system we developed recently was applied to the simultaneous expression of three capsid proteins of FMDV in E. coli. The three SUMO fusion proteins formed a stable heterotrimeric complex. Proteolytic removal of SUMO moieties from the ternary complexes resulted in VLPs with size and shape resembling the authentic FMDV. The method described here can also apply to produce capsid/envelope protein complexes or VLPs of other disease-causing viruses.

  20. Immunological response to parenteral vaccination with recombinant hepatitis B virus surface antigen virus-like particles expressing Helicobacter pylori KatA epitopes in a murine H. pylori challenge model.

    Science.gov (United States)

    Kotiw, Michael; Johnson, Megan; Pandey, Manisha; Fry, Scott; Hazell, Stuart L; Netter, Hans J; Good, Michael F; Olive, Colleen

    2012-02-01

    Virus-like particles (VLPs) based on the small envelope protein of hepatitis B virus (HBsAg-S) are immunogenic at the B- and T-cell level. In this study, we inserted overlapping sequences encoding the carboxy terminus of the Helicobacter pylori katA gene product into HBsAg-S. The HBsAg-S-KatA fusion proteins were able to assemble into secretion-competent VLPs (VLP-KatA). The VLP-KatA proteins were able to induce KatA-specific antibodies in immunized mice. The mean total IgG antibody titers 41 days post-primary immunization with VLP-KatA (2.3 × 10(3)) were significantly greater (P < 0.05) than those observed for vaccination with VLP alone (5.2 × 10(2)). Measurement of IgG isotypes revealed responses to both IgG1 and IgG2a (mean titers, 9.0 × 10(4) and 2.6 × 10(4), respectively), with the IgG2a response to vaccination with VLP-KatA being significantly higher than that for mice immunized with KatA alone (P < 0.05). Following challenge of mice with H. pylori, a significantly reduced bacterial load in the gastric mucosa was observed (P < 0.05). This is the first report describing the use of VLPs as a delivery vehicle for H. pylori antigens.

  1. Immunological Response to Parenteral Vaccination with Recombinant Hepatitis B Virus Surface Antigen Virus-Like Particles Expressing Helicobacter pylori KatA Epitopes in a Murine H. pylori Challenge Model

    Science.gov (United States)

    Johnson, Megan; Pandey, Manisha; Fry, Scott; Hazell, Stuart L.; Netter, Hans J.; Good, Michael F.; Olive, Colleen

    2012-01-01

    Virus-like particles (VLPs) based on the small envelope protein of hepatitis B virus (HBsAg-S) are immunogenic at the B- and T-cell level. In this study, we inserted overlapping sequences encoding the carboxy terminus of the Helicobacter pylori katA gene product into HBsAg-S. The HBsAg-S–KatA fusion proteins were able to assemble into secretion-competent VLPs (VLP-KatA). The VLP-KatA proteins were able to induce KatA-specific antibodies in immunized mice. The mean total IgG antibody titers 41 days post-primary immunization with VLP-KatA (2.3 × 103) were significantly greater (P < 0.05) than those observed for vaccination with VLP alone (5.2 × 102). Measurement of IgG isotypes revealed responses to both IgG1 and IgG2a (mean titers, 9.0 × 104 and 2.6 × 104, respectively), with the IgG2a response to vaccination with VLP-KatA being significantly higher than that for mice immunized with KatA alone (P < 0.05). Following challenge of mice with H. pylori, a significantly reduced bacterial load in the gastric mucosa was observed (P < 0.05). This is the first report describing the use of VLPs as a delivery vehicle for H. pylori antigens. PMID:22205658

  2. Three-dimensional visualization of forming Hepatitis C virus-like particles by electron-tomography

    Energy Technology Data Exchange (ETDEWEB)

    Badia-Martinez, Daniel; Peralta, Bibiana [Structural Biology Unit, CIC bioGUNE, CIBERehd, 48160 Derio (Spain); Andres, German; Guerra, Milagros [Electron Microscopy Unit, Centro de Biologia Molecular Severo Ochoa, CSIC-UAM, Campus Cantoblanco, 28049 Madrid (Spain); Gil-Carton, David [Structural Biology Unit, CIC bioGUNE, CIBERehd, 48160 Derio (Spain); Abrescia, Nicola G.A., E-mail: nabrescia@cicbiogune.es [Structural Biology Unit, CIC bioGUNE, CIBERehd, 48160 Derio (Spain); IKERBASQUE, Basque Foundation for Science, 48011 Bilbao (Spain)

    2012-09-01

    Hepatitis C virus infects almost 170 million people per year but its assembly pathway, architecture and the structures of its envelope proteins are poorly understood. Using electron tomography of plastic-embedded sections of insect cells, we have visualized the morphogenesis of recombinant Hepatitis C virus-like particles. Our data provide a three-dimensional sketch of viral assembly at the endoplasmic reticulum showing different budding stages and contiguity of buds. This latter phenomenon could play an important role during the assembly of wt-HCV and explain the size-heterogeneity of its particles.

  3. Infection of naive target cells with virus-like particles: implications for the function of ebola virus VP24.

    Science.gov (United States)

    Hoenen, Thomas; Groseth, Allison; Kolesnikova, Larissa; Theriault, Steven; Ebihara, Hideki; Hartlieb, Bettina; Bamberg, Sandra; Feldmann, Heinz; Ströher, Ute; Becker, Stephan

    2006-07-01

    Infectious virus-like particle (iVLP) systems have recently been established for several negative-strand RNA viruses, including the highly pathogenic Zaire ebolavirus (ZEBOV), and allow study of the viral life cycle under biosafety level 2 conditions. However, current systems depend on the expression of viral helper nucleocapsid proteins in target cells, thus making it impossible to determine whether ribonucleoprotein complexes transferred by iVLPs are able to facilitate initial transcription, an indispensable step in natural infection. Here we describe a ZEBOV iVLP system which overcomes this limitation and show that VP24 is essential for the formation of a functional ribonucleoprotein complex.

  4. Stabilization of human papillomavirus virus-like particles by non-ionic surfactants.

    Science.gov (United States)

    Shi, Li; Sanyal, Gautam; Ni, Alex; Luo, Zheng; Doshna, Sarah; Wang, Bei; Graham, Tammy L; Wang, Ning; Volkin, David B

    2005-07-01

    Human papillomavirus (HPV) virus-like-particles (VLPs) produced by recombinant expression systems are promising vaccine candidates for prevention of cervical cancers as well as genital warts. At high protein concentrations, HPV VLPs, comprised of the viral capsid protein L1 and expressed and purified from yeast, are protected against detectable aggregation during preparation and storage by high concentrations of NaCl. At low protein concentrations, however, high salt concentration alone does not fully protect HPV VLPs from aggregation. Moreover, the analytical analysis of HPV VLPs proved to be a challenge due to surface adsorption of HPV VLPs to storage containers and cuvettes. The introduction of non-ionic surfactants into HPV VLP aqueous solutions provides significantly enhanced stabilization of HPV VLPs against aggregation upon exposure to low salt and protein concentration, as well as protection against surface adsorption and aggregation due to heat stress and physical agitation. The mechanism of non-ionic surfactant stabilization of HPV VLPs was extensively studied using polysorbate 80 (PS80) as a representative non-ionic surfactant. The results suggest that PS80 stabilizes HPV VLPs mainly by competing with the VLPs for various container surfaces and air/water interfaces. No appreciable binding of PS80 to intact HPV VLPs was observed although PS80 does bind to the denatured HPV L1 protein. Even in the presence of stabilizing level of PS80, however, an ionic strength dependence of HPV VLP stabilization against aggregation is observed indicating optimization of both salt and non-ionic surfactant levels is required for effective stabilization of HPV VLPs in solution. (c) 2005 Wiley-Liss, Inc.

  5. Characterization of human enterovirus71 virus-like particles used for vaccine antigens.

    Directory of Open Access Journals (Sweden)

    Dandan Zhao

    Full Text Available Human enterovirus 71 (EV71 is a major causative pathogen of hand, foot and mouth disease (HFMD and has caused outbreaks with significant mortality among young children in the Asia-Pacific region in recent years. Towards developing a vaccine for this disease, we have expressed and purified EV71 virus-like particles (VLPs, which resemble the authentic virus in appearance, capsid structure and protein sequence, from insect cells (Sf9 using a multistep chromatography process. We demonstrated intracellular localization of the VLPs in host cells by in situ immunogold detection, electron microscopy and immunofluorescence. Characteristics of these EV71 VLPs were studied using a variety of immunological and physicochemical techniques, which aimed to reveal that the purified EV71 VLPs have good morphology and structure consistent with natural EV71 empty capsids. Results of the amino acid analysis, SDS-PAGE, Western blotting and high-performance liquid chromatography confirmed the high purity of the EV71 VLPs. However the sedimentation coefficient of the VLPs showed that they were smaller than that of secreted EV71 VLPs purified by discontinuous cesium chloride density gradients, they were similar to the empty capsids of natural EV71 virions reported previously. Combined with the previous study that EV71 VLPs purified by a multistep chromatography process were able to elicit strong humoral immune responses in mice, our results further supported the conclusion that our EV71 VLPs had well-preserved molecular and structural characteristics. The EV71 VLPs produced from the baculovirus expression system and purified by a multistep chromatography process displayed key structural and immunological features, which would contribute to their efficacy as a HFMD vaccine.

  6. ESCRT-independent budding of HIV-1 gag virus-like particles from Saccharomyces cerevisiae spheroplasts.

    Directory of Open Access Journals (Sweden)

    Andrew P Norgan

    Full Text Available Heterologous expression of HIV-1 Gag in a variety of host cells results in its packaging into virus-like particles (VLPs that are subsequently released into the extracellular milieu. This phenomenon represents a useful tool for probing cellular factors required for viral budding and has contributed to the discovery of roles for ubiquitin ligases and the endosomal sorting complexes required for transport (ESCRTs in viral budding. These factors are highly conserved throughout eukaryotes and have been studied extensively in the yeast Saccharomyces cerevisiae, a model eukaryote previously utilized as a host for the production of VLPs. We used heterologous expression of HIV Gag in yeast spheroplasts to examine the role of ESCRTs and associated factors (Rsp5, a HECT ubiquitin ligase of the Nedd4 family; Bro1, a homolog of Alix; and Vps4, the AAA-ATPase required for ESCRT function in all contexts/organisms investigated in the generation of VLPs. Our data reveal: 1 characterized Gag-ESCRT interaction motifs (late domains are not required for VLP budding, 2 loss of function alleles of the essential HECT ubiquitin ligase Rsp5 do not display defects in VLP formation, and 3 ESCRT function is not required for VLP formation from spheroplasts. These results suggest that the egress of HIV Gag from yeast cells is distinct from the most commonly described mode of exit from mammalian cells, instead mimicking ESCRT-independent VLP formation observed in a subset of mammalian cells. As such, budding of Gag from yeast cells appears to represent ESCRT-independent budding relevant to viral replication in at least some situations. Thus the myriad of genetic and biochemical tools available in the yeast system may be of utility in the study of this aspect of viral budding.

  7. Comparative immunogenicity in mice of rotavirus VP6 tubular structures and virus-like particles.

    Science.gov (United States)

    Lappalainen, Suvi; Tamminen, Kirsi; Vesikari, Timo; Blazevic, Vesna

    2013-09-01

    Rotavirus (RV) is the most important cause of severe gastroenteritis in children worldwide. Current live RV vaccines are efficacious but show lower efficacy in developing countries, as well as a low risk of intussusception. This has led to the development of parenteral non-live candidate vaccines against RV. RV capsid VP6 protein is highly conserved and the most abundant RV protein forming highly immunogenic oligomeric structures with multivalent antigen expression. Both recombinant VP6 (rVP6) or double-layered (dl) 2/6-virus-like particles (VLPs), might be considered as the simplest RV subunit vaccine candidates. Human rVP6 protein and dl2/6-VLPs were produced in Sf9 insect cells by baculovirus expression system. Formation of rVP6 tubules and VLPs were confirmed by electron microscopy. BALB/c mice were immunized intramuscularly, and immune responses were analyzed. Both rVP6 and dl2/6-VLPs induced a balanced Th1-type and Th2-type response and high levels of serum IgG antibodies with cross-reactivity against different RV strains (Wa, SC2, BrB, 69M, L26, WC3, and RRV). In addition, mucosal VP6-specific IgG and IgA antibodies were detected in feces and vaginal washes (VW) of immunized animals. Importantly, VWs of immunized mice inhibited RV Wa and RRV infection in vitro. Immunization with either protein preparation induced a similar level of VP6-specific, interferon-γ secreting CD4(+) T cells in response to different RVs or the 18-mer peptide (AA 242-259), a VP6-specific CD4(+) T cell epitope. RV rVP6 and dl2/6-VLPs induced equally strong humoral and cellular responses against RV in mice and therefore, may be considered as non-live vaccine candidates against RV.

  8. Self-adjuvanting modular virus-like particles for mucosal vaccination against group A streptococcus (GAS).

    Science.gov (United States)

    Rivera-Hernandez, Tania; Hartas, Jon; Wu, Yang; Chuan, Yap P; Lua, Linda H L; Good, Michael; Batzloff, Michael R; Middelberg, Anton P J

    2013-04-08

    Group A streptococcus (GAS) causes a wide range of diseases, some of them related to autoimmune diseases triggered by repeated GAS infections. Despite the fact that GAS primarily colonizes the mucosal epithelium of the pharynx, the main mechanism of action of most vaccine candidates is based on development of systemic antibodies that do not cross-react with host tissues, neglecting the induction of mucosal immunity that could potentially block disease transmission. Peptide antigens from GAS M-surface protein can confer protection against infection; however, translation of such peptides into immunogenic mucosal vaccines that can be easily manufactured remains a challenge. In this work, a modular murine polyomavirus (MuPyV) virus-like particle (VLP) was engineered to display a GAS antigenic peptide, J8i. Heterologous modules containing one or two J8i antigen elements were integrated with the MuPyV VLP, and produced using microbial protein expression, standard purification techniques and in vitro VLP assembly. Both modular VLPs, when delivered intranasally to outbred mice without adjuvant, induced significant titers of J8i-specific IgG and IgA antibodies, indicating significant systemic and mucosal responses, respectively. GAS colonization in the throats of mice challenged intranasally was reduced in these immunized mice, and protection against lethal challenge was observed. This study shows that modular MuPyV VLPs prepared using microbial synthesis have potential to facilitate cost-effective vaccine delivery to remote communities through the use of mucosal immunization. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Immunization against active ghrelin using virus-like particles for obesity treatment.

    Science.gov (United States)

    Andrade, Sara; Pinho, Filipa; Ribeiro, Andreia M; Carreira, Marcos; Casanueva, Felipe F; Roy, Polly; Monteiro, Mariana P

    2013-01-01

    Ghrelin is a gut hormone that stimulates food intake. In physiological conditions, ghrelin plasma levels rise with fasting and decrease after meals. Obese individuals have low fasting ghrelin levels that rise after food restriction, which is pointed out as a reason for the difficulty in maintaining weight loss. Some bariatric surgery procedures prevent rise in ghrelin levels with weight loss and this has been hypothesised to contribute to the long-term success of the treatment. The main goal of this study was to develop a safe and effective anti-ghrelin vaccine for obesity, through the chemical conjugation of ghrelin with a virus like particle, namely NS1 protein tubules from the Bluetongue Virus (BTV) using a hetero-bifunctional cross linker. Male adult C57BL/6 mice, with a normal weight and with diet-induced obesity (DIO), were randomized into six weight matched groups (n=6/group) and each group of mice received three intra-peritoneal injections with two weeks intervals, containing either 75 μg of ghrelin- NS1 immunoconjugate, 75 μg of NS1 or PBS. Our data show that immunized animals present increasing titres of anti-ghrelin antibodies, while their cumulative food intake significantly decreased and energy expenditure was significantly enhanced, although there were no significative changes in body weight.Vaccinated DIO mice also displayed significant decrease of NPY gene expression in the basal hypothalamus reflecting a decrease in central orexigenic signals. This study suggests that this anti-ghrelin vaccine has a positive impact on energy homeostasis and may be an additional therapeutical tool to be used with diet and exercise for obesity treatment.

  10. Intracellular delivery of antibodies by chimeric Sesbania mosaic virus (SeMV) virus like particles

    OpenAIRE

    Ambily Abraham; Usha Natraj; Anjali A. Karande; Ashutosh Gulati; Murthy, Mathur R. N.; Sathyabalan Murugesan; Pavithra Mukunda; Handanahal S. Savithri

    2016-01-01

    The therapeutic potential of antibodies has not been fully exploited as they fail to cross cell membrane. In this article, we have tested the possibility of using plant virus based nanoparticles for intracellular delivery of antibodies. For this purpose, Sesbania mosaic virus coat protein (CP) was genetically engineered with the B domain of Staphylococcus aureus protein A (SpA) at the beta H-beta I loop, to generate SeMV loop B (SLB), which self-assembled to virus like particles (VLPs) with 4...

  11. Crystallization and preliminary X-ray diffraction analysis of recombinant hepatitis E virus-like particle

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Che-Yen [Molecular and Cellular Biology, University of California, Davis, CA 95616 (United States); Karolinska Institute Structural Virology, F68 Karolinska University Hospital, SE-14186 Stockholm (Sweden); Institute of Public Health, National Yang-Ming University, 112 Taipei,Taiwan (China); Miyazaki, Naoyuki [Molecular and Cellular Biology, University of California, Davis, CA 95616 (United States); Karolinska Institute Structural Virology, F68 Karolinska University Hospital, SE-14186 Stockholm (Sweden); Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Yamashita, Tetsuo [Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Institute for Microbial Diseases, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Higashiura, Akifumi; Nakagawa, Atsushi [Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Li, Tian-Cheng; Takeda, Naokazu [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan); Xing, Li [Molecular and Cellular Biology, University of California, Davis, CA 95616 (United States); Karolinska Institute Structural Virology, F68 Karolinska University Hospital, SE-14186 Stockholm (Sweden); Hjalmarsson, Erik; Friberg, Claes [Crystal Research AB, 22370 Lund (Sweden); Liou, Der-Ming [Institute of Public Health, National Yang-Ming University, 112 Taipei,Taiwan (China); Sung, Yen-Jen [Institute of Public Health, National Yang-Ming University, 112 Taipei,Taiwan (China); Institute of Anatomy and Cell Biology, National Yang-Ming University, 112 Taipei,Taiwan (China); Tsukihara, Tomitake [Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Matsuura, Yoshiharu [Institute for Microbial Diseases, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Miyamura, Tatsuo [Department of Virology II, National Institute of Infectious Diseases, Tokyo (Japan); Cheng, R. Holland, E-mail: rhch@ucdavis.edu [Molecular and Cellular Biology, University of California, Davis, CA 95616 (United States); Karolinska Institute Structural Virology, F68 Karolinska University Hospital, SE-14186 Stockholm (Sweden)

    2008-04-01

    A recombinant virus-like particle that is a potential oral hepatitis E vaccine was crystallized. Diffraction data were collected to 8.3 Å resolution and the X-ray structure was phased with the aid of a low-resolution density map determined using cryo-electron microscopy data. Hepatitis E virus (HEV) accounts for the majority of enterically transmitted hepatitis infections worldwide. Currently, there is no specific treatment for or vaccine against HEV. The major structural protein is derived from open reading frame (ORF) 2 of the viral genome. A potential oral vaccine is provided by the virus-like particles formed by a protein construct of partial ORF3 protein (residue 70–123) fused to the N-terminus of the ORF2 protein (residues 112–608). Single crystals obtained by the hanging-drop vapour-diffusion method at 293 K diffract X-rays to 8.3 Å resolution. The crystals belong to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 337, b = 343, c = 346 Å, α = β = γ = 90°, and contain one particle per asymmetric unit.

  12. Enterovirus 71 virus-like particle vaccine: improved production conditions for enhanced yield.

    Science.gov (United States)

    Chung, Cheng-Yu; Chen, Chi-Yuan; Lin, Shih-Yeh; Chung, Yao-Chi; Chiu, Hsin-Yi; Chi, Wei-Kuang; Lin, Yu-Li; Chiang, Bor-Luen; Chen, Wei-Jheng; Hu, Yu-Chen

    2010-10-08

    To develop the enterovirus 71 (EV71) vaccine, we previously constructed a recombinant baculovirus (Bac-P1-3CD) co-expressing EV71 P1 (under polyhedrin promoter) and 3CD (under p10 promoter) proteins, which caused P1 cleavage by 3CD protease and self-assembly of virus-like particles (VLPs) in Sf-9 cells. Assuming that reducing the 3CD expression can alleviate the competition with P1 expression and elevate the VLPs yield, hereby we constructed Bac-P1-C3CD and Bac-P1-I3CD expressing 3CD under weaker CMV and IE-1 promoters, respectively. Western blot and ELISA analyses revealed that Bac-P1-C3CD and Bac-P1-I3CD led to the VLPs release into the supernatant and enhanced the extracellular VLPs yield in Sf-9 cells, but gave poor VLPs production in High Five™ (Hi-5) cells. By optimizing the process parameters including host cells, cell density, culture mode and dissolved oxygen (DO), the best extracellular VLPs yield was achieved by infecting Sf-9 cells (4 × 10(6)cells/mL) cultured in the bioreactor (DO=30%) with Bac-P1-C3CD, which approached ≈64.3mg/L and represented a ≈43-fold increase over the yield (1.5mg/L) attained using the old process (Bac-P1-3CD infection of Sf-9 cells in the spinner flasks). The resultant VLPs not only resembled the VLPs produced from Bac-P1-3CD infection in density, size and shape, but also induced potent antibody responses in mouse models. The antibodies neutralized EV71 strains of homologous and heterologous genogroups, implicating the potential of the VLPs to confer cross-protection for the prevention of future epidemics. Altogether, Bac-P1-C3CD and the bioprocess render mass production more economical, obviate the need for cell lysis and hold promise for future industrial vaccine production. Copyright © 2010 Elsevier Ltd. All rights reserved.

  13. αEnv-decorated phosphatidylserine liposomes trigger phagocytosis of HIV-virus-like particles in macrophages.

    Science.gov (United States)

    Gramatica, Andrea; Petazzi, Roberto A; Lehmann, Maik J; Ziomkowska, Joanna; Herrmann, Andreas; Chiantia, Salvatore

    2014-07-01

    Macrophages represent an important cellular target of HIV-1. Interestingly, they are also believed to play a potential role counteracting its infection. However, HIV-1 is known to impair macrophage immune functions such as antibody-mediated phagocytosis. Here, we present immunoliposomes that can bind HIV-1 virus-like particles (HIV-VLPs) while being specifically phagocytosed by macrophages, thus allowing the co-internalization of HIV-VLPs. These liposomes are decorated with anti-Env antibodies and contain phosphatidylserine (PS). PS mediates liposome internalization by macrophages via a mechanism not affected by HIV-1. Hence, PS-liposomes mimic apoptotic cells and are internalized into the macrophages due to specific recognition, carrying the previously bound HIV-VLPs. With a combination of flow cytometry, confocal live-cell imaging and electron microscopy we demonstrate that the PS-immunoliposomes presented here are able to elicit efficient HIV-VLPs phagocytosis by macrophages and might represent a new nanotechnological approach to enhance HIV-1 antigen presentation and reduce the ongoing inflammation processes. This team of authors demonstrate that specific phosphatidylserin immunoliposomes are able to elicit efficient phagocytosis of HIV-virus-like particle by macrophages and might represent a new nanomedicine approach to enhance HIV-1 antigen presentation and reduce ongoing inflammation processes. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. Protection conferred by virus-like particle vaccines against respiratory syncytial virus infection in mice by intranasal vaccination.

    Science.gov (United States)

    Gu, Hongjing; Li, Tieling; Han, Lina; Zhu, Ping; Zhang, Peirui; Zhang, Shaogeng; Sun, Sujing; Duan, Yueqiang; Xing, Li; Zhao, Zhongpeng; Lai, Chengcai; Wen, Bohai; Wang, Xiliang; Yang, PengHui

    2015-01-01

    Respiratory syncytial virus (RSV) is a major pathogen in infants and the elderly, causing pneumonia and bronchiolitis. Despite decades of research, to date there is still no approved RSV vaccine available. In this study, we developed RSV virus-like particle (VLP) vaccines containing an RSV fusion (F) and/or attachment (G) protein with Newcastle disease virus (NDV) as the platform. The VLPs were expressed in a baculovirus system and purified by sucrose gradient centrifugation. BALB/c mice immunized intranasally (i.n.) with rNDV/RSV/F plus rNDV/RSV/G developed robust humoral, mucosal RSV-specific antibodies and cellular immune responses. Furthermore, rNDV/RSV/F plus rNDV/RSV/G provided better protection than did rNDV/RSV/F or rNDV/RSV/G alone, as shown by an obvious decrease in viral replication together with alleviation of histopathological changes in the lungs of the challenged mice. Our data demonstrate that the intranasal vaccination of combined RSV virus-like particle vaccine candidates has great potential for protection against RSV infection.

  15. Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus.

    Science.gov (United States)

    Garg, Himanshu; Sedano, Melina; Plata, Gabrielle; Punke, Erin B; Joshi, Anjali

    2017-10-15

    Recent worldwide outbreaks of Zika virus (ZIKV) infection and the lack of an approved vaccine raise serious concerns regarding preparedness to combat this emerging virus. We used a virus-like particle (VLP)-based approach to develop a vaccine and a microneutralization assay for ZIKV. A synthetic capsid-premembrane-envelope (C-prM-E) gene construct of ZIKV was used to generate reporter virus particles (RVPs) that package a green fluorescent protein (GFP) reporter-expressing West Nile virus (WNV) replicon. The assay was adapted to a 96-well format, similar to the plaque reduction neutralization test (PRNT), and showed high reproducibility with specific detection of ZIKV neutralizing antibodies. Furthermore, C-prM-E and prM-E VLPs were tested as vaccine candidates in mice and compared to DNA vaccination. While the ZIKV prM-E construct alone was sufficient for generating VLPs, efficient VLP production from the C-prM-E construct could be achieved in the presence of the WNV NS2B-3 protease, which cleaves C from prM, allowing virus release. Immunization studies in mice showed that VLPs generated higher neutralizing antibody titers than those with the DNA vaccines, with C-prM-E VLPs giving slightly higher titers than those with prM-E VLPs. The superiority of C-prM-E VLPs suggests that inclusion of capsid may have benefits for ZIKV and other flaviviral VLP vaccines. To facilitate the VLP platform, we generated a stable cell line expressing high levels of ZIKV prM-E proteins that constitutively produce VLPs as well as a cell line expressing ZIKV C-prM-E proteins for RVP production. While several vaccine platforms have been proposed for ZIKV, this study describes a safe, effective, and economical VLP-based vaccine against ZIKV.IMPORTANCE To address the growing Zika virus epidemic, we undertook this study with two objectives: first, to develop a safe, effective, and economical vaccine for ZIKV, and second, to develop a rapid and versatile assay to detect the anti-ZIKV immune

  16. Comparison of human papillomavirus type 16 L1 chimeric virus-like particles versus L1/L2 chimeric virus-like particles in tumor prevention.

    Science.gov (United States)

    Wakabayashi, Mark T; Da Silva, Diane M; Potkul, Ronald K; Kast, W Martin

    2002-01-01

    Chimeric human papillomavirus (HPV) virus-like particles (cVLPs) with the HPV16 E7 antigen fused to either the major capsid protein, L1, or the minor capsid protein, L2, have been used independently to protect against the formation of HPV-induced tumors in animal models. However, the advantages and disadvantages of both types of particles with respect to production and vaccine efficacy have never been analyzed. Therefore, in this study, we compared cVLPs with the HPV16 E7 antigen fused to L1 versus cVLPs with E7 fused to L2 with respect to their ability to protect mice from tumor challenge. The first 57 amino acids of E7 were used to overcome the size limitation and limited VLP production imposed by inserting polypeptides into L1 cVLPs. C57BL/6 mice were immunized with the above cVLPs at various doses. Tumor challenge was then performed with HPV16 E7-positive TC-1 cells. HPV16 L1-E7((1-57)) was superior to HPV16 L1/L2-E7((1-57)) in eliciting tumor protection at equivalent doses, although both types of particles were able to protect mice. Both cVLPs induced a specific cytotoxic T lymphocyte (CTL) response to the H2-D(b)-restricted E7 peptide (E7(49-57)) as determined by an ELISPOT assay and tetramer staining; however, immunization with the L1-E7((1-57)) cVLPs resulted in twofold higher CTL precursor frequencies. Our results demonstrate that cVLPs with the antigen fused to L1 are a more efficient vaccine with respect to tumor prevention than cVLPs with the antigen fused to L2. At the same time, however, L1 cVLPs are limited by the size of the antigen that can be incorporated and in the amount of cVLP that can be obtained from cultures when compared to L1/L2 cVLPs. This balances out their superior ability to induce protective immunity. Copyright 2002 S. Karger AG, Basel

  17. Formation of virus-like particles from O-type foot-and-mouth disease virus in insect cells using codon-optimized synthetic genes.

    Science.gov (United States)

    Cao, Yimei; Sun, Pu; Fu, Yuanfang; Bai, Xingwen; Tian, Feipen; Liu, Xiangtao; Lu, Zengjun; Liu, Zaixin

    2010-09-01

    A recombinant baculovirus was constructed to simultaneously express codon-optimized virus-like particles (VLP), A VP1-2A-VP3 and VP0 of serotype O foot-and-mouth disease virus (FMDV), from individual promoters. The target proteins were expressed in insect cells at high level, as shown by indirect sandwich ELISA; and the expressed VP1-2A-VP3 could autocatalytically be cleaved into the individual proteins, VP1-2A and VP3, as shown by Western-blot analyses. In addition, in the insect cells, the structural proteins, VP0, VP3 and VP1-2A, self-assembled into virus-like particles resembling the authentic FMDV particles. This information should prove useful for the development of more efficient VLP assembly using shorter genes.

  18. Chimeric L2-Based Virus-Like Particle (VLP) Vaccines Targeting Cutaneous Human Papillomaviruses (HPV)

    Science.gov (United States)

    Huber, Bettina; Schellenbacher, Christina; Shafti-Keramat, Saeed; Jindra, Christoph; Christensen, Neil

    2017-01-01

    Common cutaneous human papillomavirus (HPV) types induce skin warts, whereas species beta HPV are implicated, together with UV-radiation, in the development of non-melanoma skin cancer (NMSC) in immunosuppressed patients. Licensed HPV vaccines contain virus-like particles (VLP) self-assembled from L1 major capsid proteins that provide type-restricted protection against mucosal HPV infections causing cervical and other ano-genital and oro-pharyngeal carcinomas and warts (condylomas), but do not target heterologous HPV. Experimental papillomavirus vaccines have been designed based on L2 minor capsid proteins that contain type-common neutralization epitopes, to broaden protection to heterologous mucosal and cutaneous HPV types. Repetitive display of the HPV16 L2 cross-neutralization epitope RG1 (amino acids (aa) 17–36) on the surface of HPV16 L1 VLP has greatly enhanced immunogenicity of the L2 peptide. To more directly target cutaneous HPV, L1 fusion proteins were designed that incorporate the RG1 homolog of beta HPV17, the beta HPV5 L2 peptide aa53-72, or the common cutaneous HPV4 RG1 homolog, inserted into DE surface loops of HPV1, 5, 16 or 18 L1 VLP scaffolds. Baculovirus expressed chimeric proteins self-assembled into VLP and VLP-raised NZW rabbit immune sera were evaluated by ELISA and L1- and L2-based pseudovirion (PsV) neutralizing assays, including 12 novel beta PsV types. Chimeric VLP displaying the HPV17 RG1 epitope, but not the HPV5L2 aa53-72 epitope, induced cross-neutralizing humoral immune responses to beta HPV. In vivo cross-protection was evaluated by passive serum transfer in a murine PsV challenge model. Immune sera to HPV16L1-17RG1 VLP (cross-) protected against beta HPV5/20/24/38/96/16 (but not type 76), while antisera to HPV5L1-17RG1 VLP cross-protected against HPV20/24/96 only, and sera to HPV1L1-4RG1 VLP cross-protected against HPV4 challenge. In conclusion, RG1-based VLP are promising next generation vaccine candidates to target cutaneous

  19. Chimeric L2-Based Virus-Like Particle (VLP Vaccines Targeting Cutaneous Human Papillomaviruses (HPV.

    Directory of Open Access Journals (Sweden)

    Bettina Huber

    Full Text Available Common cutaneous human papillomavirus (HPV types induce skin warts, whereas species beta HPV are implicated, together with UV-radiation, in the development of non-melanoma skin cancer (NMSC in immunosuppressed patients. Licensed HPV vaccines contain virus-like particles (VLP self-assembled from L1 major capsid proteins that provide type-restricted protection against mucosal HPV infections causing cervical and other ano-genital and oro-pharyngeal carcinomas and warts (condylomas, but do not target heterologous HPV. Experimental papillomavirus vaccines have been designed based on L2 minor capsid proteins that contain type-common neutralization epitopes, to broaden protection to heterologous mucosal and cutaneous HPV types. Repetitive display of the HPV16 L2 cross-neutralization epitope RG1 (amino acids (aa 17-36 on the surface of HPV16 L1 VLP has greatly enhanced immunogenicity of the L2 peptide. To more directly target cutaneous HPV, L1 fusion proteins were designed that incorporate the RG1 homolog of beta HPV17, the beta HPV5 L2 peptide aa53-72, or the common cutaneous HPV4 RG1 homolog, inserted into DE surface loops of HPV1, 5, 16 or 18 L1 VLP scaffolds. Baculovirus expressed chimeric proteins self-assembled into VLP and VLP-raised NZW rabbit immune sera were evaluated by ELISA and L1- and L2-based pseudovirion (PsV neutralizing assays, including 12 novel beta PsV types. Chimeric VLP displaying the HPV17 RG1 epitope, but not the HPV5L2 aa53-72 epitope, induced cross-neutralizing humoral immune responses to beta HPV. In vivo cross-protection was evaluated by passive serum transfer in a murine PsV challenge model. Immune sera to HPV16L1-17RG1 VLP (cross- protected against beta HPV5/20/24/38/96/16 (but not type 76, while antisera to HPV5L1-17RG1 VLP cross-protected against HPV20/24/96 only, and sera to HPV1L1-4RG1 VLP cross-protected against HPV4 challenge. In conclusion, RG1-based VLP are promising next generation vaccine candidates to target

  20. Surface-enhanced Raman Scattering from Virus-like Particle Crystals

    Science.gov (United States)

    Dufort, Christopher; Dragnea, Bogdan

    2008-03-01

    Recently, a method for the encapsidation of gold nanoparticules by an icosahedral virus protein coat, termed a virus-like particle (VLP), has been developed. Of particular interest is in observing their spectroscopic properties upon arrangement into a three-dimensional crystal lattice. Here we present the surface-enhanced Raman scattering spectrum of such an assembly. This is made possible by the plasmonic coupling of adjacent gold nanoparticules when excited near their plasmon resonant frequency. To determine whether the SERS effect is arising from isolated hot spots or a large number of junctions acting in unison we employed scanning confocal Raman spectroscopy. This seems to indicate the latter, as a uniform Raman intensity is observed across entire crystals.

  1. Characterization of virus-like particles by atomic force microscopy in ambient conditions

    Science.gov (United States)

    Oropesa, Reinier; Ramos, Jorge R.; Falcón, Viviana; Felipe, Ariel

    2013-06-01

    Recombinant virus-like particles (VLPs) are attractive candidates for vaccine design since they resemble native viroids in size and morphology, but they are non-infectious due to the absence of a viral genome. The visualization of surface morphologies and structures can be used to deepen the understanding of physical, chemical, and biological phenomena. Atomic force microscopy (AFM) is a useful tool for the visualization of soft biological samples in a nanoscale resolution. In this work we have investigated the morphology of recombinant surface antigens of hepatitis B (rHBsAg) VLPs from Cuban vaccine against hepatitis B. The rHBsAg VLPs sizes estimated by AFM between 15 and 30 nm are similar to those reported on previous transmission electron microscopy (TEM) studies.

  2. Epitope-Specific Anti-hCG Vaccines on a Virus Like Particle Platform.

    Science.gov (United States)

    Caldeira, Jerri; Bustos, Jeremiah; Peabody, Julianne; Chackerian, Bryce; Peabody, David S

    2015-01-01

    The possibility of a contraceptive vaccine targeting human chorionic gonadotropin has long been recognized, but never fully realized. Here we describe an epitope-specific approach based on immunogenic display of hCG-derived peptides on virus-like particles of RNA bacteriophage. A number of recombinant VLPs were constructed, each displaying a different hCG-derived peptide. Some were taken from the disordered C-terminal tail of the hormone, another came from an internal loop, and yet another was an epitope mimic produced by affinity-selection on an hCG-neutralizing antibody target. Immunization of mice with some VLPs yielded antisera that bound the hormone and neutralized hCG biological activity.

  3. Bacterial superglue enables easy development of efficient virus-like particle based vaccines

    DEFF Research Database (Denmark)

    Thrane, Susan; Janitzek, Christoph M; Matondo, Sungwa

    2016-01-01

    BACKGROUND: Virus-like particles (VLPs) represent a significant advance in the development of subunit vaccines, combining high safety and efficacy. Their particulate nature and dense repetitive subunit organization makes them ideal scaffolds for display of vaccine antigens. Traditional approaches...... vaccine antigens fused to SpyCatcher or SpyTag resulted in formation of antigen-VLP complexes with coupling efficiencies (% occupancy of total VLP binding sites) ranging from 22-88 %. In mice, spy-VLP vaccines presenting the malaria proteins Pfs25 or VAR2CSA markedly increased antibody titer, affinity......, longevity and functional efficacy compared to corresponding vaccines employing monomeric proteins. The spy-VLP vaccines also effectively broke B cell self-tolerance and induced potent and durable antibody responses upon vaccination with cancer or allergy-associated self-antigens (PD-L1, CTLA-4 and IL-5...

  4. Quantitative analysis of Nipah virus proteins released as virus-like particles reveals central role for the matrix protein

    Directory of Open Access Journals (Sweden)

    Eaton Bryan T

    2007-01-01

    Full Text Available Abstract Background Nipah virus (NiV is an emerging paramyxovirus distinguished by its ability to cause fatal disease in both animal and human hosts. Together with Hendra virus (HeV, they comprise the genus Henipavirus in the Paramyxoviridae family. NiV and HeV are also restricted to Biosafety Level-4 containment and this has hampered progress towards examining details of their replication and morphogenesis. Here, we have established recombinant expression systems to study NiV particle assembly and budding through the formation of virus-like particles (VLPs. Results When expressed by recombinant Modified Vaccinia virus Ankara (rMVA or plasmid transfection, individual NiV matrix (M, fusion (F and attachment (G proteins were all released into culture supernatants in a membrane-associated state as determined by sucrose density gradient flotation and immunoprecipitation. However, co-expression of F and G along with M revealed a shift in their distribution across the gradient, indicating association with M in VLPs. Protein release was also altered depending on the context of viral proteins being expressed, with F, G and nucleocapsid (N protein reducing M release, and N release dependent on the co-expression of M. Immunoelectron microscopy and density analysis revealed VLPs that were similar to authentic virus. Differences in the budding dynamics of NiV proteins were also noted between rMVA and plasmid based strategies, suggesting that over-expression by poxvirus may not be appropriate for studying the details of recombinant virus particle assembly and release. Conclusion Taken together, the results indicate that NiV M, F, and G each possess some ability to bud from expressing cells, and that co-expression of these viral proteins results in a more organized budding process with M playing a central role. These findings will aid our understanding of paramyxovirus particle assembly in general and could help facilitate the development of a novel vaccine

  5. A method for rapid production of heteromultimeric protein complexes in plants: assembly of protective bluetongue virus-like particles.

    Science.gov (United States)

    Thuenemann, Eva C; Meyers, Ann E; Verwey, Jeanette; Rybicki, Edward P; Lomonossoff, George P

    2013-09-01

    Plant expression systems based on nonreplicating virus-based vectors can be used for the simultaneous expression of multiple genes within the same cell. They therefore have great potential for the production of heteromultimeric protein complexes. This work describes the efficient plant-based production and assembly of Bluetongue virus-like particles (VLPs), requiring the simultaneous expression of four distinct proteins in varying amounts. Such particles have the potential to serve as a safe and effective vaccine against Bluetongue virus (BTV), which causes high mortality rates in ruminants and thus has a severe effect on the livestock trade. Here, VLPs produced and assembled in Nicotiana benthamiana using the cowpea mosaic virus-based HyperTrans (CPMV-HT) and associated pEAQ plant transient expression vector system were shown to elicit a strong antibody response in sheep. Furthermore, they provided protective immunity against a challenge with a South African BTV-8 field isolate. The results show that transient expression can be used to produce immunologically relevant complex heteromultimeric structures in plants in a matter of days. The results have implications beyond the realm of veterinary vaccines and could be applied to the production of VLPs for human use or the coexpression of multiple enzymes for the manipulation of metabolic pathways. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  6. Thermal Stability of RNA Phage Virus-Like Particles Displaying Foreign Peptides

    Directory of Open Access Journals (Sweden)

    Peabody David S

    2011-05-01

    Full Text Available Abstract Background To be useful for genetic display of foreign peptides a viral coat protein must tolerate peptide insertions without major disruption of subunit folding and capsid assembly. The folding of the coat protein of RNA phage MS2 does not normally tolerate insertions in its AB-loop, but an engineered single-chain dimer readily accepts them as long as they are restricted to one of its two halves. Results Here we characterize the effects of peptide insertions on the thermal stabilities of MS2 virus-like particles (VLPs displaying a variety of different peptides in one AB-loop of the coat protein single-chain dimer. These particles typically denature at temperatures around 5-10°C lower than unmodified VLPs. Even so, they are generally stable up to about 50°C. VLPs of the related RNA phage PP7 are cross-linked with intersubunit disulfide bonds and are therefore significantly more stable. An AB-loop insertion also reduces the stability of PP7 VLPs, but they only begin to denature above about 70°C. Conclusions VLPs assembled from MS2 single-chain dimer coat proteins with peptide insertions in one of their AB-loops are somewhat less stable than the wild-type particle, but still resist heating up to about 50°C. Because they possess disulfide cross-links, PP7-derived VLPs provide an alternate platform with even higher stability.

  7. Mannosylation of virus-like particles enhances internalization by antigen presenting cells.

    Directory of Open Access Journals (Sweden)

    Farah Al-Barwani

    Full Text Available Internalization of peptides by antigen presenting cells is crucial for the initiation of the adaptive immune response. Mannosylation has been demonstrated to enhance antigen uptake through mannose receptors, leading to improved immune responses. In this study we test the effect of surface mannosylation of protein-based virus-like particles (VLP derived from Rabbit hemorrhagic disease virus (RHDV on uptake by murine and human antigen presenting cells. A monomannoside and a novel dimannoside were synthesized and successfully conjugated to RHDV VLP capsid protein, providing approximately 270 mannose groups on the surface of each virus particle. VLP conjugated to the mannoside or dimannoside exhibited significantly enhanced binding and internalization by murine dendritic cells, macrophages and B cells as well as human dendritic cells and macrophages. This uptake was inhibited by the inclusion of mannan as a specific inhibitor of mannose specific uptake, demonstrating that mannosylation of VLP targets mannose receptor-based uptake. Consistent with mannose receptor-based uptake, partial retargeting of the intracellular processing of RHDV VLP was observed, confirming that mannosylation of VLP provides both enhanced uptake and modified processing of associated antigens.

  8. Virus-Like Particle Vaccination Protects Nonhuman Primates from Lethal Aerosol Exposure with Marburgvirus (VLP Vaccination Protects Macaques against Aerosol Challenges)

    OpenAIRE

    John M. Dye; Warfield, Kelly L; Jay B. Wells; Unfer, Robert C.; Sergey Shulenin; Hong Vu; Donald K. Nichols; M Javad Aman; Sina Bavari

    2016-01-01

    Marburg virus (MARV) was the first filovirus to be identified following an outbreak of viral hemorrhagic fever disease in Marburg, Germany in 1967. Due to several factors inherent to filoviruses, they are considered a potential bioweapon that could be disseminated via an aerosol route. Previous studies demonstrated that MARV virus-like particles (VLPs) containing the glycoprotein (GP), matrix protein VP40 and nucleoprotein (NP) generated using a baculovirus/insect cell expression system could...

  9. Infection of Naïve Target Cells with Virus-Like Particles: Implications for the Function of Ebola Virus VP24

    OpenAIRE

    Hoenen, Thomas; Groseth, Allison; Kolesnikova, Larissa; Theriault, Steven; Ebihara, Hideki; Hartlieb, Bettina; Bamberg, Sandra; Feldmann, Heinz; Ströher, Ute; Becker, Stephan

    2006-01-01

    Infectious virus-like particle (iVLP) systems have recently been established for several negative-strand RNA viruses, including the highly pathogenic Zaire ebolavirus (ZEBOV), and allow study of the viral life cycle under biosafety level 2 conditions. However, current systems depend on the expression of viral helper nucleocapsid proteins in target cells, thus making it impossible to determine whether ribonucleoprotein complexes transferred by iVLPs are able to facilitate initial transcription...

  10. Protein-based polymers that bond to DNA : design of virus-like particles and supramolecular nanostructures

    NARCIS (Netherlands)

    Hernandez Garcia, A.

    2014-01-01

     In this thesis it is demonstrated that it is possible to use Protein-based Polymers (PbPs) as synthetic binders of DNA (or any other negatively charged polyelectrolyte). The PbPs co-assemble with their DNA templates to form highly organized virus-like particles and supramolecular structures. A

  11. 78 FR 18359 - Prospective Grant of Exclusive License: Papilloma Pseudovirus and Virus-Like Particles as a...

    Science.gov (United States)

    2013-03-26

    ... HUMAN SERVICES National Institutes of Health Prospective Grant of Exclusive License: Papilloma Pseudovirus and Virus-Like Particles as a Delivery System for Human Cancer Therapeutics and Diagnostics AGENCY... of human papillomavirus pseudoviruses (PsV) as a cancer diagnostic and therapeutic. Preliminary...

  12. Selection and optimization of transfection enhancer additives for increased virus-like particle production in HEK293 suspension cell cultures.

    Science.gov (United States)

    Cervera, Laura; Fuenmayor, Javier; González-Domínguez, Irene; Gutiérrez-Granados, Sonia; Segura, Maria Mercedes; Gòdia, Francesc

    2015-12-01

    The manufacturing of biopharmaceuticals in mammalian cells typically relies on the use of stable producer cell lines. However, in recent years, transient gene expression has emerged as a suitable technology for rapid production of biopharmaceuticals. Transient gene expression is particularly well suited for early developmental phases, where several potential therapeutic targets need to be produced and tested in vivo. As a relatively new bioprocessing modality, a number of opportunities exist for improving cell culture productivity upon transient transfection. For instance, several compounds have shown positive effects on transient gene expression. These transfection enhancers either facilitate entry of PEI/DNA transfection complexes into the cell or nucleus or increase levels of gene expression. In this work, the potential of combining transfection enhancers to increase Gag-based virus-like particle production levels upon transfection of suspension-growing HEK 293 cells is evaluated. Using Plackett-Burman design of experiments, it is first tested the effect of eight transfection enhancers: trichostatin A, valproic acid, sodium butyrate, dimethyl sulfoxide (DMSO), lithium acetate, caffeine, hydroxyurea, and nocodazole. An optimal combination of compounds exhibiting the highest effect on gene expression levels was subsequently identified using a surface response experimental design. The optimal consisted on the addition of 20 mM lithium acetate, 3.36 mM valproic acid, and 5.04 mM caffeine which increased VLP production levels 3.8-fold, while maintaining cell culture viability at 94%.

  13. Protein and virus-like particle adsorption on perfusion chromatography media.

    Science.gov (United States)

    Wu, Yige; Simons, Jared; Hooson, Sarah; Abraham, Dicky; Carta, Giorgio

    2013-07-05

    The structural and protein adsorption characteristics of the perfusion chromatography matrix POROS(®) HS 50 are determined. Transmission electron microscopy shows a broad distribution of pore sizes with 100-500nm through-pores transecting a network of much smaller pores formed by aggregates of microgranules about 100nm in size. Dextran standards, proteins, and virus-like particles (VLPs) show size-exclusion behavior consistent with such a bimodal distribution of pore sizes. For non-binding conditions, the trends in height equivalent to a theoretical plate (HETP) as a function of mobile phase velocity and molecular size are consistent with perfusion suggesting that a fraction of the mobile phase between 0.0005 and 0.0008 flows through the particles. This small fraction provides little or no enhancement of intraparticle mass transfer for relatively small proteins (lysozyme and IgG) even at 1000cm/h, but can contribute substantially to transport for large proteins (thyroglobulin) and VLPs. Intraparticle concentration profiles during transient adsorption are determined by confocal microscopy in batch and flow systems. The profiles are spherically symmetrical indicating a dominance of diffusion for smaller proteins in both batch and flow systems but become highly asymmetrical and skewed in the direction of flow for thyroglobulin at 1000cm/h. Estimates of the convective enhancement of intraparticle transport for these conditions based on the confocal measurements are consistent with estimates of the intraparticle Peclet number and previously published models. Adsorption of VLPs, however, was found to be confined to a thin layer on the outer surface of the particles indicting that bound VLPs block access to the underlying pore network and suggesting that pores larger than those present on the resin studies are needed to take advantage of the effects of perfusion for the adsorption of large VLPs. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Changes in human Langerhans cells following intradermal injection of influenza virus-like particle vaccines.

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    Marc Pearton

    Full Text Available There is a significant gap in our fundamental understanding of early morphological and migratory changes in human Langerhans cells (LCs in response to vaccine stimulation. As the vast majority of LCs studies are conducted in small animal models, substantial interspecies variation in skin architecture and immunity must be considered when extrapolating the results to humans. This study aims to determine whether excised human skin, maintained viable in organ culture, provides a useful human model for measuring and understanding early immune response to intradermally delivered vaccine candidates. Excised human breast skin was maintained viable in air-liquid-interface organ culture. This model was used for the first time to show morphological changes in human LCs stimulated with influenza virus-like particle (VLP vaccines delivered via intradermal injection. Immunohistochemistry of epidermal sheets and skin sections showed that LCs in VLP treated skin lost their typical dendritic morphology. The cells were more dispersed throughout the epidermis, often in close proximity to the basement membrane, and appeared vertically elongated. Our data provides for increased understanding of the complex morphological, spatial and temporal changes that occur to permit LC migration through the densely packed keratinocytes of the epidermis following exposure to vaccine. Significantly, the data not only supports previous animal data but also provides new and essential evidence of host response to this vaccination strategy in the real human skin environment.

  15. The Rationale for a Preventative HCV Virus-Like Particle (VLP Vaccine

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    Joseph Torresi

    2017-11-01

    Full Text Available HCV represents a global health problem with ~200 million individuals currently infected, worldwide. With the high cost of antiviral therapies, the global burden of chronic hepatitis C infection (CHCV infection will be substantially reduced by the development of an effective vaccine for HCV. The field of HCV vaccines is generally divided into proponents of strategies to induce neutralizing antibodies (NAb and those who propose to elicit cell mediated immunity (CMI. However, for a hepatitis C virus (HCV vaccine to be effective in preventing infection, it must be capable of generating cross-reactive CD4+, CD8+ T cell, and NAb responses that will cover the major viral genotypes. Simulation models of hepatitis C have predicted that a vaccine of even modest efficacy and coverage will significantly reduce the incidence of hepatitis C. A HCV virus like particle (VLP based vaccine would fulfill the requirement of delivering critical conformational neutralizing epitopes in addition to providing HCV specific CD4+ and CD8+ epitopes. Several approaches have been reported including insect cell-derived genotype 1b HCV VLPs; a human liver-derived quadrivalent genotype 1a, 1b, 2, and 3a vaccine; a genotype 1a HCV E1 and E2 glycoprotein/MLV Gag pseudotype VLP vaccine; and chimeric HBs-HCV VLP vaccines. All to result in the production of cross-NAb and/or T cell responses against HCV. This paper summarizes the evidence supporting the development of a HCV VLP based vaccine.

  16. Intracellular delivery of antibodies by chimeric Sesbania mosaic virus (SeMV) virus like particles.

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    Abraham, Ambily; Natraj, Usha; Karande, Anjali A; Gulati, Ashutosh; Murthy, Mathur R N; Murugesan, Sathyabalan; Mukunda, Pavithra; Savithri, Handanahal S

    2016-02-24

    The therapeutic potential of antibodies has not been fully exploited as they fail to cross cell membrane. In this article, we have tested the possibility of using plant virus based nanoparticles for intracellular delivery of antibodies. For this purpose, Sesbania mosaic virus coat protein (CP) was genetically engineered with the B domain of Staphylococcus aureus protein A (SpA) at the βH-βI loop, to generate SeMV loop B (SLB), which self-assembled to virus like particles (VLPs) with 43 times higher affinity towards antibodies. CP and SLB could internalize into various types of mammalian cells and SLB could efficiently deliver three different monoclonal antibodies-D6F10 (targeting abrin), anti-α-tubulin (targeting intracellular tubulin) and Herclon (against HER2 receptor) inside the cells. Such a mode of delivery was much more effective than antibodies alone treatment. These results highlight the potential of SLB as a universal nanocarrier for intracellular delivery of antibodies.

  17. Production of highly immunogenic virus-like particles of bovine papillomavirus type 6 in silkworm pupae.

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    Watanabe, Satoko; Iizuka, Tetsuya; Hatama, Shinichi; Kanno, Toru; Mase, Masaji; Shibahara, Tomoyuki

    2017-10-13

    Bovine papillomaviruses (BPVs) are the causative agent of bovine teat papillomatosis, which can lead to severe economic losses in dairy cattle. Among the 14 identified BPV genotypes, BPV type 6 (BPV6) is the most frequently detected in teat papilloma lesions, and is therefore thought to play a major role in teat papillomatosis. To develop an effective vaccine against BPV6 infection, we produced virus-like particles of BPV6 (BPV6-VLP) in silkworm (Bombyx mori) pupae and purified these by heparin affinity chromatography using a single column. About 0.7mg purified BPV6-VLP was obtained from one pupa. BPV6-VLP-immunized mice produced a specific IgG to BPV6 that recognized BPV6 antigen with high sensitivity in an immunohistochemical analysis. Thus, silkworm pupae are a useful bioreactor for the production of BPV6-VLP, which can potentially be used as a vaccine for bovine teat papillomatosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Effects of adjuvants on IgG subclasses elicited by virus-like Particles

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    Visciano Maria Luisa

    2012-01-01

    Full Text Available Abstract Background Virus-Like Particles (VLPs represent an efficient strategy to present and deliver conformational antigens to the immune system, inducing both arms of the adaptive immune response. Moreover, their particulate structure surrounded by cell membrane provides an adjuvanted effect to VLP-based immunizations. In the present study, the elicitation of different patterns of IgG subclasses by VLPs, administered in CpG ODN1826 or poly(I:C adjuvants, has been evaluated in an animal model. Results Adjuvanted VLPs elicited a higher titer of total specific IgG compared to VLPs alone. Furthermore, while VLPs alone induced a balanced TH2 pattern, VLPs formulated with either adjuvant elicited a TH1-biased IgG subclasses (IgG2a and IgG3, with poly(I:C more potent than CpG ODN1826. Conclusions The results confirmed that adjuvants efficiently improve antigen immunogenicity and represent a suitable strategy to skew the adaptive immune response toward the differentiation of the desired T helper subset, also using VLPs as antigen.

  19. Tetraspanins displayed in retrovirus-derived virus-like particles and their immunogenicity.

    Science.gov (United States)

    Soares, H R; Castro, R; Tomás, H A; Rodrigues, A F; Gomes-Alves, P; Bellier, B; Klatzmann, D; Carrondo, M J T; Alves, P M; Coroadinha, A S

    2016-03-18

    Virus-like particles (VLPs) are a particular subset of subunit vaccines which are currently explored as safer alternatives to live attenuated or inactivated vaccines. VLPs derived from retrovirus (retroVLPs) are commonly used as scaffolds for vaccine candidates due to their ability to incorporate heterologous envelope proteins. Pseudotyping retroVLPs is however not a selective process therefore, host cellular proteins such as tetraspanins are also included in the membrane. The contribution of these host-proteins to retrovirus immunogenicity remains unclear. In this work, human cells silenced and not silenced for tetraspanin CD81 were used to produce CD81(-) or CD81(+) retroVLPs. We first analyzed mice immune response against human CD81. Despite effective silencing of CD81 in retroVLP producing cells, both humoral and cellular immune responses showed persistent anti-CD81 immunogenicity, suggesting cross reactivity to related antigens. We thus compared the incorporation of related tetraspanins in retroVLPs and showed that decreased CD81 incorporation in CD81(-) retro-VLPs is compensated by an increased incorporation of CD9 and CD63 tetraspanins. These results highlight the dynamic nature of host-derived proteins incorporation in retroVLPs membrane, which should be considered when retrovirus-based biopharmaceuticals are produced in xenogeneic cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

  20. Virus-like particle display of HER2 induces potent anti-cancer responses.

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    Palladini, Arianna; Thrane, Susan; Janitzek, Christoph M; Pihl, Jessica; Clemmensen, Stine B; de Jongh, Willem Adriaan; Clausen, Thomas M; Nicoletti, Giordano; Landuzzi, Lorena; Penichet, Manuel L; Balboni, Tania; Ianzano, Marianna L; Giusti, Veronica; Theander, Thor G; Nielsen, Morten A; Salanti, Ali; Lollini, Pier-Luigi; Nanni, Patrizia; Sander, Adam F

    2018-01-01

    Overexpression of human epidermal growth factor receptor-2 (HER2) occurs in 20-30% of invasive breast cancers. Monoclonal antibody therapy is effective in treating HER2-driven mammary carcinomas, but its utility is limited by high costs, side effects and development of resistance. Active vaccination may represent a safer, more effective and cheaper alternative, although the induction of strong and durable autoantibody responses is hampered by immune-tolerogenic mechanisms. Using a novel virus-like particle (VLP) based vaccine platform we show that directional, high-density display of human HER2 on the surface of VLPs, allows induction of therapeutically potent anti-HER2 autoantibody responses. Prophylactic vaccination reduced spontaneous development of mammary carcinomas by 50%-100% in human HER2 transgenic mice and inhibited the growth of HER2-positive tumors implanted in wild-type mice. The HER2-VLP vaccine shows promise as a new cost-effective modality for prevention and treatment of HER2-positive cancer. The VLP platform may represent an effective tool for development of vaccines against other non-communicable diseases.

  1. Rotavirus virus-like particles (RV-VLPs) vaccines: An update.

    Science.gov (United States)

    Changotra, Harish; Vij, Avni

    2017-10-19

    Rotaviruses (RVs) cause over 0.2 million deaths annually and are reported to be the foremost cause of gastroenteritis in infants and children worldwide. Vaccination against RVs is the most successful and unsurpassed strategy to combat infection to date. Although the 2 current vaccines, Rotarix and RotaTeq, have dramatically reduced the disease burden, still there is a need for new vaccines. In this context, RV virus-like particles (RV-VLPs) represent potential vaccine candidates as they are noninfectious and effective nonreplicating immunogens that may reduce the risk of side effects related to the conventional vaccines. VLPs being conformationally similar to the parent virus are highly immunogenic and hence provide enhanced protection and better serotype coverage. In this review, we have highlighted the various advantages and the implications of RV-VLPs, discussed the general strategies employed for their production, and talked about the recent developments made in this regard. Overall, the review emphasizes the probable utility of RV-VLPs in eradicating the highly widespread RVs. Copyright © 2017 John Wiley & Sons, Ltd.

  2. Display of single-chain variable fragments on bacteriophage MS2 virus-like particles.

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    Lino, Christopher A; Caldeira, Jerri C; Peabody, David S

    2017-02-13

    Virus-like particles (VLPs) of the RNA bacteriophage MS2 have many potential applications in biotechnology. MS2 VLPs provide a platform for peptide display and affinity selection (i.e. biopanning). They are also under investigation as vehicles for targeted drug delivery, using display of receptor-specific peptides or nucleic acid aptamers to direct their binding to specific cell-surface receptors. However, there are few molecules more suited to the precise targeting and binding of a cellular receptor than antibodies. Here we describe a strategy for display of four different functional single-chain variable fragments (scFvs) on the surface of the MS2 VLP. Each scFv is validated both for its presence on the surface of the VLP and for its ability to bind its cognate antigen. This work demonstrates the suitability of the MS2 VLP platform to display genetically fused scFvs, allowing for many potential applications of these VLPs and paving the way for future work with libraries of scFvs displayed in a similar manner on the VLP surface. These libraries can then be biopanned and novel scFv binders to targets can be readily discovered.

  3. Immunoreactivity and trypsin sensitivity of recombinant virus-like particles of foot-and-mouth disease virus.

    Science.gov (United States)

    Basagoudanavar, S H; Hosamani, M; Tamil, R P; Sreenivasa, B P; Chandrasekhar, B K; Venkataramanan, R

    2015-03-01

    Foot-and-mouth disease (FMD) is an important infection affecting the health and productivity of cloven-hoofed livestock. Development of improved vaccines and diagnostic reagents is being explored to facilitate the disease control. There is an emerging interest in virus-like particles (VLPs), as their constituent structural proteins are the major immunogens. The VLPs are similar to natural virus particles but lack viral nucleic acid. The objective of the present study was to express the VLPs of FMD virus (FMDV) serotype Asia-1 (IND 63/72), using baculovirus system and characterize them for antigenic structure. The VLPs expressed in insect cells showed immunoreactivity similar to inactivated cell culture FMDV. Further they possess similar sensitivity to trypsin as the inactivated cell culture FMDV, suggesting that trypsin-sensitive antigenic sites could be similarly arranged. Our findings suggest that the FMD VLPs have similar antigenic conformational feature like the wild type virus, thus supporting their utility in development of non-infectious FMD vaccines and/or diagnostic assays.

  4. Physicochemical characterization and immunological properties of Pichia pastoris based HPV16L1 and 18L1 virus like particles.

    Science.gov (United States)

    Gupta, Gaurav; Glueck, Reinhard; Rishi, Narayan

    2017-03-01

    There continues to be an urgent need for cost-effective prophylaxis for HPV-associated cancers in socio-economically underdeveloped nations. Presently HPV vaccines, which are commercially available, are adjuvanted virus-like particles (VLPs) expressed from various recombinant expression systems. They have been characterized by different methods as safe, pure, and potent HPV vaccine antigens. We cloned and expressed L1 proteins of HPV16 & 18 in Pichia pastoris and tested their immunogenicity. We observed that HPVL1 proteins (16L1 and 18L1) are expressed in Pichia pastoris at high levels. Critical physicochemical parameters of these HPV recombinant L1 proteins were characterized by SDS PAGE, western blotting, peptide mapping, glycosylation pattern, mass spectrometry, host cell DNA and protein analysis, electron microscopy, and immunogenicity analysis. These data establish a blueprint of HPV recombinant protein antigens for standardizing & developing an alternative high-quality, cost-effective vaccine for HPV as well as similar recombinant protein-based vaccines. Copyright © 2016 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  5. Binding of virus-like particles of Norwalk virus to romaine lettuce veins.

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    Gandhi, Kamal M; Mandrell, Robert E; Tian, Peng

    2010-12-01

    Noroviruses (NoV) annually cause millions of cases of gastrointestinal disease in the United States. NoV are associated with raw shellfish outbreaks, particularly oysters, which are thought to bioaccumulate NoV particles during the filter-feeding process. NoV outbreaks, however, have also been known to occur from other common-source food-borne vehicles, such as lettuce, frozen raspberries, and salad. In this study, we evaluated romaine lettuce as a potential vehicle for NoV transmission by testing the binding and distribution of NoV to the surface of romaine. Recombinant Norwalk virus-like particles (rNVLP) applied to the surface of romaine lettuce localized as large clusters primarily on the leaf veins. An extract of romaine lettuce leaves in phosphate-buffered saline (PBS) (romaine extract [RE]) bound rNVLP in a dose-dependent manner. RE did not bind rNVLP by histo-blood group antigens (HBGA), nor was RE competitive with rNVLP binding to porcine gastric mucin. These results suggested that non-HBGA molecules in RE bind rNVLP by a binding site(s) that is different from the defined binding pocket on the virion. Extracts of cilantro, iceberg lettuce, spinach, and celery also bound rNVLP. Samples of each of the vegetables spiked with rNVLP and tested with anti-NVLP antibody revealed by confocal microscopy the presence of rNVLP not only on the veins of cilantro but also throughout the surface of iceberg lettuce.

  6. Exploiting fluorescent polymers to probe the self-assembly of virus-like particles.

    Science.gov (United States)

    Cadena-Nava, Ruben D; Hu, Yufang; Garmann, Rees F; Ng, Benny; Zelikin, Alexander N; Knobler, Charles M; Gelbart, William M

    2011-03-17

    The inside surfaces of the protein shells of many viruses are positively charged, thereby enhancing the self-assembly of capsid proteins around their (oppositely charged) RNA genome. These proteins have been shown to organize similarly around a variety of nonbiological, negatively charged, polymers, for example, poly(styrene sulfonate) (PSS), forming virus-like particles (VLPs). We have demonstrated recently that the VLPs formed from cowpea chlorotic mottle virus (CCMV) capsid protein increase in size (from T=2 to T=3 structures) upon increase in PSS molecular weight (from 400 kDa to 3.4 MDa), and that the total charge on the PSS exceeds that of the capsid protein by as much as a factor of 9. Here, we extend studies of this kind to PSS molecules that are sufficiently small that two or more can be packaged into VLPs. The use of 38 kDa PSS polymers that have been fluorescently labeled with Rhodamine B allows us to determine the number of PSS molecules per capsid. Electron micrographs of the VLPs show a bimodal distribution of particle diameters, with one peak centered around 19 nm, typical of a T=1 triangulation number, and the other around 21 nm, consistent with a pseudo T=2 structure; increasing the molar ratio of protein to PSS in the reaction mix shifts the VLP distribution from T=1 to T=2 structures. By combining fluorescence and gel electrophoresis measurements, it is determined that, on average, there are two polymers in each T=1 capsid and three in each T=2, with the PSS charge less than that of the capsid protein by as much as a factor of 2. VLPs of this kind provide a versatile model system for determining the principles underlying self-assembly of controlled numbers of cargo molecules in nanocontainers of increasing size. © 2011 American Chemical Society

  7. Comparison of perfusion media and monoliths for protein and virus-like particle chromatography.

    Science.gov (United States)

    Wu, Yige; Abraham, Dicky; Carta, Giorgio

    2016-05-20

    Structural and performance characteristics of perfusion chromatography media (POROS HS 20 and 50) and those of a polymethacrylate monolith (CIM SO3-1 tube monolith column) are compared for protein and virus-like particle chromatography using 1mL columns. Axial flow columns are used for POROS while the monolith has a radial flow configuration, which provides comparable operating pressures. The POROS beads contain a bimodal distribution of pore sizes, some as large as 0.5μm, which allow a small fraction of the mobile phase to flow within the particles, while the monolith contains 1-2μm flow channels. For proteins (lysozyme and IgG), the dynamic binding capacity of the POROS columns is more than twice that of the monolith at longer residence times. While the DBC of the POROS HS 50 column decreases at shorter residence times, the DBC of the POROS HS 20 column for IgG remains nearly twice that of the monolith at residence times at least as low as 0.2min as a result of intraparticle convection. Protein recoveries are comparable for all three columns. For VLPs, however, the eluted peaks are broader and recovery is lower for the monolith than for the POROS columns and is dependent on the direction of flow in the monolith, which is attributed to denser layer observed by SEM at the inlet surface of the monolith that appears to trap VLPs when loading in the normal flow direction. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Evidences of Changes in Surface Electrostatic Charge Distribution during Stabilization of HPV16 Virus-Like Particles.

    Science.gov (United States)

    Vega, Juan F; Vicente-Alique, Ernesto; Núñez-Ramírez, Rafael; Wang, Yang; Martínez-Salazar, Javier

    2016-01-01

    The stabilization of human papillomavirus type 16 virus-like particles has been examined by means of different techniques including dynamic and static light scattering, transmission electron microscopy and electrophoretic mobility. All these techniques provide different and often complementary perspectives about the aggregation process and generation of stabilized virus-like particles after a period of time of 48 hours at a temperature of 298 K. Interestingly, static light scattering results point towards a clear colloidal instability in the initial systems, as suggested by a negative value of the second virial coefficient. This is likely related to small repulsive electrostatic interactions among the particles, and in agreement with relatively small absolute values of the electrophoretic mobility and, hence, of the net surface charges. At this initial stage the small repulsive interactions are not able to compensate binding interactions, which tend to aggregate the particles. As time proceeds, an increase of the size of the particles is accompanied by strong increases, in absolute values, of the electrophoretic mobility and net surface charge, suggesting enhanced repulsive electrostatic interactions and, consequently, a stabilized colloidal system. These results show that electrophoretic mobility is a useful methodology that can be applied to screen the stabilization factors for virus-like particles during vaccine development.

  9. Evidences of Changes in Surface Electrostatic Charge Distribution during Stabilization of HPV16 Virus-Like Particles.

    Directory of Open Access Journals (Sweden)

    Juan F Vega

    Full Text Available The stabilization of human papillomavirus type 16 virus-like particles has been examined by means of different techniques including dynamic and static light scattering, transmission electron microscopy and electrophoretic mobility. All these techniques provide different and often complementary perspectives about the aggregation process and generation of stabilized virus-like particles after a period of time of 48 hours at a temperature of 298 K. Interestingly, static light scattering results point towards a clear colloidal instability in the initial systems, as suggested by a negative value of the second virial coefficient. This is likely related to small repulsive electrostatic interactions among the particles, and in agreement with relatively small absolute values of the electrophoretic mobility and, hence, of the net surface charges. At this initial stage the small repulsive interactions are not able to compensate binding interactions, which tend to aggregate the particles. As time proceeds, an increase of the size of the particles is accompanied by strong increases, in absolute values, of the electrophoretic mobility and net surface charge, suggesting enhanced repulsive electrostatic interactions and, consequently, a stabilized colloidal system. These results show that electrophoretic mobility is a useful methodology that can be applied to screen the stabilization factors for virus-like particles during vaccine development.

  10. The large-scale production of an artificial influenza virus-like particle vaccine in silkworm pupae.

    Science.gov (United States)

    Nerome, Kuniaki; Sugita, Shigeo; Kuroda, Kazumichi; Hirose, Toshiharu; Matsuda, Sayaka; Majima, Kei; Kawasaki, Kazunori; Shibata, Toshikatsu; Poetri, Okti Nadia; Soejoedono, Retno D; Mayasari, Ni L P Ika; Agungpriyono, Srihadi; Nerome, Reiko

    2015-01-01

    We successfully established a mass production system for an influenza virus-like particle (VLP) vaccine using a synthetic H5 hemagglutinin (HA) gene codon-optimized for the silkworm. A recombinant baculovirus containing the synthetic gene was inoculated into silkworm pupae. Four days after inoculation, the hemagglutination titer in homogenates from infected pupae reached a mean value of 0.8 million hemagglutination units (HAU), approximately 2,000 μg HA protein per pupa, more than 50-fold higher than that produced with an embryonated chicken egg. VLPs ranging from 30 nm to 300 nm in diameter and covered with a large number of spikes were detected in the homogenates. The spikes were approximately 14 nm long, similar to an authentic influenza HA spike. Detailed electron micrographs indicated that the VLP spike density was similar to that of authentic influenza virus particles. The results clearly show that the expression of a single HA gene can efficiently produce VLPs in silkworm pupae. When chickens were immunized with the pupae homogenate, the hemagglutination inhibition titer in their sera reached values of 2,048-8,192 after approximately 1 month. This is the first report demonstrating that a large amount of VLP vaccine could be produced by single synthetic HA gene in silkworm pupae. Our system might be useful for future vaccine development against other viral diseases. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Biomimetic structural engineering of P22 virus-like particles for catalysis and immune modulation

    Science.gov (United States)

    Schwarz, Benjamin

    Within biology molecules are arranged in hierarchical structures that coordinate and control the many processes that allow for complex organisms to exist. Proteins and other functional macromolecules are often studied outside their natural nanostructural context because it remains difficult to create controlled arrangements of proteins at this size scale. Viruses are elegantly simple nano-systems that exist at the interface of living organisms and non-living biological machines. Studied and viewed primarily as pathogens to be combatted, viruses have emerged as models of structural efficiency at the nanoscale and have spurred the development of biomimetic nanoparticle systems. Virus-like particles (VLPs) are noninfectious protein cages derived from viruses or other cage-forming systems. VLPs provide incredibly regular scaffolds for building at the nanoscale. In this work I have utilized the VLP derived from the bacteriophage P22 as a platform for the organization of enzymes, antigens, and immune-stimulating proteins inside and outside the capsid through purely genetic means. In the case of enzymes, encapsulation of a two-enzyme pathway has led to the development of metabolic nanoparticle catalysts and an expanded understanding of the control that structure exerts on metabolic flux. These same structural elements applied to the delivery of protein subunit antigens directed at cytotoxic T cell immunity result in drastically enhanced antigen processing and lasting immunological memory. Lastly, presentation of immune-stimulating proteins from the Tumor Necrosis Factor Super Family on the surface of the P22 VLP enhances the cell signaling efficiency of these compounds 50-fold and provides strategies for the application of these proteins as immune modulatory oncology therapeutics. In all of these cases, the reintroduction of nanostructure to these protein systems, reminiscent of their natural environment, has led to both new technologies and a better understanding of the

  12. Variability in bacteria and virus-like particle abundances during purging of unconfined aquifers.

    Science.gov (United States)

    Roudnew, Ben; Lavery, Trish J; Seymour, Justin R; Jeffries, Thomas C; Mitchell, James G

    2014-01-01

    Standard methodologies for sampling the physicochemical conditions of groundwater recommend purging a bore for three bore volumes to avoid sampling the stagnant water within a bore and instead gain samples representative of the aquifer. However, there are currently no methodological standards addressing the amount of purging required to gain representative biological samples to assess groundwater bacterial and viral abundances. The objective of this study was to examine how bacterial and viral abundances change during the purging of bore volumes. Six bores infiltrating into unconfined aquifers were pumped for five or six bore volumes each and bacteria and virus-like particles (VLPs) were enumerated from each bore volume using flow cytometry. In examination of the individual bores trends in bacterial abundances were observed to increase, decrease, or remain constant with each purged bore volume. Furthermore, triplicates taken at each bore volume indicated substantial variations in VLP and bacterial abundances that are often larger than the differences between bore volumes. This indicates a high level of small scale heterogeneity in microbial community abundance in groundwater samples, and we suggest that this may be an intrinsic feature of bore biology. The heterogeneity observed may be driven by bottom up processes (variability in the distribution of organic and inorganic nutrients), top-down processes (grazing and viral lysis), physical heterogeneities in the bore, or technical artifacts associated with the purging process. We suggest that a more detailed understanding of the ecology underpinning this variability is required to adequately describe the microbiological characteristics of groundwater ecosystems. © 2013, National Ground Water Association.

  13. Immunogenicity of Virus Like Particle Forming Baculoviral DNA Vaccine against Pandemic Influenza H1N1.

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    Yong-Dae Gwon

    Full Text Available An outbreak of influenza H1N1 in 2009, representing the first influenza pandemic of the 21st century, was transmitted to over a million individuals and claimed 18,449 lives. The current status in many countries is to prepare influenza vaccine using cell-based or egg-based killed vaccine. However, traditional influenza vaccine platforms have several limitations. To overcome these limitations, many researchers have tried various approaches to develop alternative production platforms. One of the alternative approach, we reported the efficacy of influenza HA vaccination using a baculoviral DNA vaccine (AcHERV-HA. However, the immune response elicited by the AcHERV-HA vaccine, which only targets the HA antigen, was lower than that of the commercial killed vaccine. To overcome the limitations of this previous vaccine, we constructed a human endogenous retrovirus (HERV envelope-coated, baculovirus-based, virus-like-particle (VLP-forming DNA vaccine (termed AcHERV-VLP against pandemic influenza A/California/04/2009 (pH1N1. BALB/c mice immunized with AcHERV-VLP (1×107 FFU AcHERV-VLP, i.m. and compared with mice immunized with the killed vaccine or mice immunized with AcHERV-HA. As a result, AcHERV-VLP immunization produced a greater humoral immune response and exhibited neutralizing activity with an intrasubgroup H1 strain (PR8, elicited neutralizing antibody production, a high level of interferon-γ secretion in splenocytes, and diminished virus shedding in the lung after challenge with a lethal dose of influenza virus. In conclusion, VLP-forming baculovirus DNA vaccine could be a potential vaccine candidate capable of efficiently delivering DNA to the vaccinee and VLP forming DNA eliciting stronger immunogenicity than egg-based killed vaccines.

  14. Characterizing Enterovirus 71 and Coxsackievirus A16 virus-like particles production in insect cells.

    Science.gov (United States)

    Somasundaram, Balaji; Chang, Cindy; Fan, Yuan Y; Lim, Pei-Yin; Cardosa, Jane; Lua, Linda

    2016-02-15

    Enterovirus 71 (EV71) and Coxsackievirus A16 (CVA16) are two viruses commonly responsible for hand, foot and mouth disease (HFMD) in children. The lack of prophylactic or therapeutic measures against HFMD is a major public health concern. Insect cell-based EV71 and CVA16 virus-like particles (VLPs) are promising vaccine candidates against HFMD and are currently under development. In this paper, the influence of insect cell line, incubation temperature, and serial passaging effect and stability of budded virus (BV) stocks on EV71 and CVA16 VLP production was investigated. Enhanced EV71 and CVA16 VLP production was observed in Sf9 cells compared to High Five™ cells. Lowering the incubation temperature from the standard 27°C to 21°C increased the production of both VLPs in Sf9 cells. Serial passaging of CVA16 BV stocks in cell culture had a detrimental effect on the productivity of the structural proteins and the effect was observed with only 5 passages of BV stocks. A 2.7× higher production yield was achieved with EV71 compared to CVA16. High-resolution asymmetric flow field-flow fractionation couple with multi-angle light scattering (AF4-MALS) was used for the first time to characterize EV71 and CVA16 VLPs, displaying an average root mean square radius of 15±1nm and 15.3±5.8 nm respectively. This study highlights the need for different approaches in the design of production process to develop a bivalent EV71 and CVA16 vaccine. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Tracking the virus-like particles of Macrobrachium rosenbergii nodavirus in insect cells

    Science.gov (United States)

    Hanapi, Ummi Fairuz; Yong, Chean Yeah; Goh, Zee Hong; Alitheen, Noorjahan Banu; Yeap, Swee Keong

    2017-01-01

    Macrobrachium rosenbergii nodavirus (MrNv) poses a major threat to the prawn industry. Currently, no effective vaccine and treatment are available to prevent the spread of MrNv. Its infection mechanism and localisation in a host cell are also not well characterised. The MrNv capsid protein (MrNvc) produced in Escherichia coli self-assembled into virus-like particles (VLPs) resembling the native virus. Thus, fluorescein labelled MrNvc VLPs were employed as a model to study the virus entry and localisation in Spodoptera frugiperda, Sf9 cells. Through fluorescence microscopy and sub-cellular fractionation, the MrNvc was shown to enter Sf9 cells, and eventually arrived at the nucleus. The presence of MrNvc within the cytoplasm and nucleus of Sf9 cells was further confirmed by the Z-stack imaging. The presence of ammonium chloride (NH4Cl), genistein, methyl-β-cyclodextrin or chlorpromazine (CPZ) inhibited the entry of MrNvc into Sf9 cells, but cytochalasin D did not inhibit this process. This suggests that the internalisation of MrNvc VLPs is facilitated by caveolae- and clathrin-mediated endocytosis. The whole internalisation process of MrNvc VLPs into a Sf9 cell was recorded with live cell imaging. We have also identified a potential nuclear localisation signal (NLS) of MrNvc through deletion mutagenesis and verified by classical-NLS mapping. Overall, this study provides an insight into the journey of MrNvc VLPs in insect cells. PMID:28194311

  16. Biomedical and Catalytic Opportunities of Virus-Like Particles in Nanotechnology.

    Science.gov (United States)

    Schwarz, B; Uchida, M; Douglas, T

    2017-01-01

    Within biology, molecules are arranged in hierarchical structures that coordinate and control the many processes that allow for complex organisms to exist. Proteins and other functional macromolecules are often studied outside their natural nanostructural context because it remains difficult to create controlled arrangements of proteins at this size scale. Viruses are elegantly simple nanosystems that exist at the interface of living organisms and nonliving biological machines. Studied and viewed primarily as pathogens to be combatted, viruses have emerged as models of structural efficiency at the nanoscale and have spurred the development of biomimetic nanoparticle systems. Virus-like particles (VLPs) are noninfectious protein cages derived from viruses or other cage-forming systems. VLPs provide incredibly regular scaffolds for building at the nanoscale. Composed of self-assembling protein subunits, VLPs provide both a model for studying materials' assembly at the nanoscale and useful building blocks for materials design. The robustness and degree of understanding of many VLP structures allow for the ready use of these systems as versatile nanoparticle platforms for the conjugation of active molecules or as scaffolds for the structural organization of chemical processes. Lastly the prevalence of viruses in all domains of life has led to unique activities of VLPs in biological systems most notably the immune system. Here we discuss recent efforts to apply VLPs in a wide variety of applications with the aim of highlighting how the common structural elements of VLPs have led to their emergence as paradigms for the understanding and design of biological nanomaterials. © 2017 Elsevier Inc. All rights reserved.

  17. Application of virus-like particles (VLP) to NMR characterization of viral membrane protein interactions

    Energy Technology Data Exchange (ETDEWEB)

    Antanasijevic, Aleksandar; Kingsley, Carolyn [University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics (United States); Basu, Arnab; Bowlin, Terry L. [Microbiotix Inc. (United States); Rong, Lijun [University of Illinois at Chicago, Department of Microbiology and Immunology (United States); Caffrey, Michael, E-mail: caffrey@uic.edu [University of Illinois at Chicago, Department of Biochemistry and Molecular Genetics (United States)

    2016-03-15

    The membrane proteins of viruses play critical roles in the virus life cycle and are attractive targets for therapeutic intervention. Virus-like particles (VLP) present the possibility to study the biochemical and biophysical properties of viral membrane proteins in their native environment. Specifically, the VLP constructs contain the entire protein sequence and are comprised of native membrane components including lipids, cholesterol, carbohydrates and cellular proteins. In this study we prepare VLP containing full-length hemagglutinin (HA) or neuraminidase (NA) from influenza and characterize their interactions with small molecule inhibitors. Using HA-VLP, we first show that VLP samples prepared using the standard sucrose gradient purification scheme contain significant amounts of serum proteins, which exhibit high potential for non-specific interactions, thereby complicating NMR studies of ligand-target interactions. We then show that the serum contaminants may be largely removed with the addition of a gel filtration chromatography step. Next, using HA-VLP we demonstrate that WaterLOGSY NMR is significantly more sensitive than Saturation Transfer Difference (STD) NMR for the study of ligand interactions with membrane bound targets. In addition, we compare the ligand orientation to HA embedded in VLP with that of recombinant HA by STD NMR. In a subsequent step, using NA-VLP we characterize the kinetic and binding properties of substrate analogs and inhibitors of NA, including study of the H274Y-NA mutant, which leads to wide spread resistance to current influenza antivirals. In summary, our work suggests that VLP have high potential to become standard tools in biochemical and biophysical studies of viral membrane proteins, particularly when VLP are highly purified and combined with control VLP containing native membrane proteins.

  18. SARS-CoV envelope protein palmitoylation or nucleocapid association is not required for promoting virus-like particle production.

    Science.gov (United States)

    Tseng, Ying-Tzu; Wang, Shiu-Mei; Huang, Kuo-Jung; Wang, Chin-Tien

    2014-04-27

    Coronavirus membrane (M) proteins are capable of interacting with nucleocapsid (N) and envelope (E) proteins. Severe acute respiratory syndrome coronavirus (SARS-CoV) M co-expression with either N or E is sufficient for producing virus-like particles (VLPs), although at a lower level compared to M, N and E co-expression. Whether E can release from cells or E/N interaction exists so as to contribute to enhanced VLP production is unknown. It also remains to be determined whether E palmitoylation or disulfide bond formation plays a role in SARS-CoV virus assembly. SARS-CoV N is released from cells through an association with E protein-containing vesicles. Further analysis suggests that domains involved in E/N interaction are largely located in both carboxyl-terminal regions. Changing all three E cysteine residues to alanines did not exert negative effects on E release, E association with N, or E enhancement of VLP production, suggesting that E palmitoylation modification or disulfide bond formation is not required for SARS-CoV virus assembly. We found that removal of the last E carboxyl-terminal residue markedly affected E release, N association, and VLP incorporation, but did not significantly compromise the contribution of E to efficient VLP production. The independence of the SARS-CoV E enhancement effect on VLP production from its viral packaging capacity suggests a distinct SARS-CoV E role in virus assembly.

  19. Rotavirus Recombinant VP6 Nanotubes Act as an Immunomodulator and Delivery Vehicle for Norovirus Virus-Like Particles

    Directory of Open Access Journals (Sweden)

    Maria Malm

    2016-01-01

    Full Text Available We have recently shown that tubular form of rotavirus (RV recombinant VP6 protein has an in vivo adjuvant effect on the immunogenicity of norovirus (NoV virus-like particle (VLP vaccine candidate. In here, we investigated in vitro effect of VP6 on antigen presenting cell (APC activation and maturation and whether VP6 facilitates NoV VLP uptake by these APCs. Mouse macrophage cell line RAW 264.7 and dendritic cell line JAWSII were used as model APCs. Internalization of VP6, cell surface expression of CD40, CD80, CD86, and major histocompatibility class II molecules, and cytokine and chemokine production were analyzed. VP6 nanotubes were efficiently internalized by APCs. VP6 upregulated the expression of cell surface activation and maturation molecules and induced secretion of several proinflammatory cytokines and chemokines. The mechanism of VP6 action was shown to be partially dependent on lipid raft-mediated endocytic pathway as shown by methyl-β-cyclodextrin inhibition on tumor necrosis factor α secretion. These findings add to the understanding of mechanism by which VP6 exerts its immunostimulatory and immunomodulatory actions and further support its use as a part of nonlive RV-NoV combination vaccine.

  20. Virus-like particles that display Zika virus envelope protein domain III induce potent neutralizing immune responses in mice.

    Science.gov (United States)

    Yang, Ming; Lai, Huafang; Sun, Haiyan; Chen, Qiang

    2017-08-09

    Several Zika virus (ZIKV) vaccine candidates have recently been described which use inactivated whole virus, DNA or RNA that express the virus' Envelope (E) glycoprotein as the antigen. These were successful in stimulating production of virus-targeted antibodies that protected animals against ZIKV challenges, but their use potentially will predispose vaccinated individuals to infection by the related Dengue virus (DENV). We have devised a virus like particle (VLP) carrier based on the hepatitis B core antigen (HBcAg) that displays the ZIKV E protein domain III (zDIII), and shown that it can be produced quickly and easily purified in large quantities from Nicotiana benthamiana plants. HBcAg-zDIII VLPs are shown to be highly immunogenic, as two doses elicited potent humoral and cellular responses in mice that exceed the threshold correlated with protective immunity against multiple strains of Zika virus. Notably, HBcAg-zDIII VLPs-elicited antibodies did not enhance the infection of DENV in Fc gamma receptor-expressing cells, offsetting the concern of ZIKV vaccines inducing cross-reactive antibodies and sensitizing people to subsequent DENV infection. Thus, our zDIII-based vaccine offers improved safety and lower cost production than other current alternatives, with equivalent effectiveness.

  1. Virus-like particles activate type I interferon pathways to facilitate post-exposure protection against Ebola virus infection.

    Directory of Open Access Journals (Sweden)

    Natarajan Ayithan

    Full Text Available Ebola virus (EBOV causes a severe hemorrhagic disease with high fatality. Virus-like particles (VLPs are a promising vaccine candidate against EBOV. We recently showed that VLPs protect mice from lethal EBOV infection when given before or after viral infection. To elucidate pathways through which VLPs confer post-exposure protection, we investigated the role of type I interferon (IFN signaling. We found that VLPs lead to accelerated induction of IFN stimulated genes (ISGs in liver and spleen of wild type mice, but not in Ifnar-/- mice. Accordingly, EBOV infected Ifnar-/- mice, unlike wild type mice succumbed to death even after VLP treatment. The ISGs induced in wild type mice included anti-viral proteins and negative feedback factors known to restrict viral replication and excessive inflammatory responses. Importantly, proinflammatory cytokine/chemokine expression was much higher in WT mice without VLPs than mice treated with VLPs. In EBOV infected Ifnar-/- mice, however, uninhibited viral replication and elevated proinflammatory factor expression ensued, irrespective of VLP treatment, supporting the view that type I IFN signaling helps to limit viral replication and attenuate inflammatory responses. Further analyses showed that VLP protection requires the transcription factor, IRF8 known to amplify type I IFN signaling in dendritic cells and macrophages, the probable sites of initial EBOV infection. Together, this study indicates that VLPs afford post-exposure protection by promoting expeditious initiation of type I IFN signaling in the host.

  2. Virus-Like Particles Activate Type I Interferon Pathways to Facilitate Post-Exposure Protection against Ebola Virus Infection

    Science.gov (United States)

    Ayithan, Natarajan; Bradfute, Steven B.; Anthony, Scott M.; Stuthman, Kelly S.; Bavari, Sina; Bray, Mike; Ozato, Keiko

    2015-01-01

    Ebola virus (EBOV) causes a severe hemorrhagic disease with high fatality. Virus-like particles (VLPs) are a promising vaccine candidate against EBOV. We recently showed that VLPs protect mice from lethal EBOV infection when given before or after viral infection. To elucidate pathways through which VLPs confer post-exposure protection, we investigated the role of type I interferon (IFN) signaling. We found that VLPs lead to accelerated induction of IFN stimulated genes (ISGs) in liver and spleen of wild type mice, but not in Ifnar-/- mice. Accordingly, EBOV infected Ifnar-/- mice, unlike wild type mice succumbed to death even after VLP treatment. The ISGs induced in wild type mice included anti-viral proteins and negative feedback factors known to restrict viral replication and excessive inflammatory responses. Importantly, proinflammatory cytokine/chemokine expression was much higher in WT mice without VLPs than mice treated with VLPs. In EBOV infected Ifnar-/- mice, however, uninhibited viral replication and elevated proinflammatory factor expression ensued, irrespective of VLP treatment, supporting the view that type I IFN signaling helps to limit viral replication and attenuate inflammatory responses. Further analyses showed that VLP protection requires the transcription factor, IRF8 known to amplify type I IFN signaling in dendritic cells and macrophages, the probable sites of initial EBOV infection. Together, this study indicates that VLPs afford post-exposure protection by promoting expeditious initiation of type I IFN signaling in the host. PMID:25719445

  3. Virus-Like Particles of Chimeric Recombinant Porcine Circovirus Type 2 as Antigen Vehicle Carrying Foreign Epitopes

    Directory of Open Access Journals (Sweden)

    Huawei Zhang

    2014-12-01

    Full Text Available Virus-like particles (VLPs of chimeric porcine circovirus type 2 (PCV2 were generated by replacing the nuclear localization signal (NLS; at 1–39 aa of PCV2 capsid protein (Cap with classical swine fever virus (CSFV T-cell epitope (1446–1460 aa, CSFV B-cell epitope (693–716 aa and CSFV T-cell epitope conjugated with B-cell epitope. The recombinant proteins were expressed using the baculovirus expression system and detected by immunoblotting and indirect immunofluorescence assay. The abilities to form PCV2 VLPs were confirmed by transmission electron microscopy. Immunogenicities of the three recombinant proteins were evaluated in mice. Our Results indicated that Cap protein NLS deletion or substitution with CSFV epitopes did not affect the VLPs assembly. Three chimeric Cap proteins could form VLPs and induce efficient humoral and cellular immunity against PCV2 and CSFV in mice. Results show that PCV2 VLPs can be used as an efficient antigen carrier for delivery of foreign epitopes, and a potential novel vaccine.

  4. Immunogenicity and performance of an enterovirus 71 virus-like-particle vaccine in nonhuman primates.

    Science.gov (United States)

    Lim, Pei-Yin; Hickey, Andrew C; Jamiluddin, Mohamad F; Hamid, Sharifah; Kramer, Joshua; Santos, Rosemary; Bossart, Katharine N; Cardosa, M Jane

    2015-11-04

    A vaccine against human enterovirus 71 (EV-A71) is urgently needed to combat outbreaks of EV-A71 and in particular, the serious neurological complications that manifest during these outbreaks. In this study, an EV-A71 virus-like-particle (VLP) based on a B5 subgenogroup (EV-A71-B5 VLP) was generated using an insect cell/baculovirus platform. Biochemical analysis demonstrated that the purified VLP had a highly native procapsid structure and initial studies in vivo demonstrated that the VLPs were immunogenic in mice. The impact of VLP immunization on infection was examined in non-human primates using a VLP prime-boost strategy prior to EV-A71 challenge. Rhesus macaques were immunized on day 0 and day 21 with VLPs (100 μg/dose) containing adjuvant or with adjuvant alone (controls), and were challenged with EV-A71 on day 42. Complete blood counts, serum chemistry, magnetic resonance imaging (MRI) scans, and histopathology results were mostly normal in vaccinated and control animals after virus challenge demonstrating that the fatal EV-A71-B3 clinical isolate used in this study was not highly virulent in rhesus macaques. Viral genome and/or infectious virus were detected in blood, spleen or brain of two of three control animals, but not in any specimens from the vaccinated animals, indicating that VLP immunization prevented systemic spread of EV-A71 in rhesus macaques. High levels of IgM and IgG were detected in VLP-vaccinated animals and these responses were highly specific for EV-A71 particles and capsid proteins. Serum from vaccinated animals also exhibited similar neutralizing activity against different subgenogroups of EV-A71 demonstrating that the VLPs induced cross-neutralizing antibodies. In conclusion, our EV-A71-B5 VLP is safe, highly immunogenic, and prevents systemic EV-A71-B3 infection in nonhuman primates making it a viable attractive vaccine candidate for EV-A71. Copyright © 2015 Elsevier Ltd. All rights reserved.

  5. Evidence for novel viruses by analysis of nucleic acids in virus-like particle fractions from Ambrosia psilostachya.

    Science.gov (United States)

    Melcher, Ulrich; Muthukumar, Vijay; Wiley, Graham B; Min, Byoung Eun; Palmer, Michael W; Verchot-Lubicz, Jeanmarie; Ali, Akhtar; Nelson, Richard S; Roe, Bruce A; Thapa, Vaskar; Pierce, Margaret L

    2008-09-01

    To test the hypothesis that many viruses remain to be discovered in plants, a procedure was developed to sequence nucleic acids cloned randomly from virus-like particle fractions of plant homogenates. As a test of the efficiency of the procedure we targeted Ambrosia psilostachya, western ragweed, plants growing at the Tallgrass Prairie Preserve of northeastern Oklahoma. Amplifiable nucleic acid was found in the fractions from six of twelve specimens and sequences were characterized from four of them. Evidence was obtained for the presence of viruses belonging to two families (Caulimoviridae, Flexiviridae). Multiple viral species were found in two of the four specimens and their level within the isolated nucleic acid population varied from less than 1-37%. None of the sequences were derived from reported sequences of known viruses. Thus, the analysis of nucleic acid from virus-like particles is a useful tool to expand our knowledge of the universe of viruses to non-cultivated species.

  6. Morphotypes of virus-like particles in two hydrothermal vent fields on the East Scotia Ridge, Antarctia

    OpenAIRE

    Millard, Andrew D.; Hands-Portman, Ian; Zwirglmaier, Katrin

    2014-01-01

    Viruses from extreme environments are still largely unexplored and may harbor unseen genetic potential. Here, we present a first glance at the morphological diversity of virus like particles (VLPs) from an environment that is extreme in more than one respect: two recently discovered hydrothermal vent fields on the East Scotia Ridge in the Southern Ocean near Antarctica. They are the southernmost hydrothermal sites found to date and have been shown to present a new biogeographic province, cont...

  7. Construction of target-specific virus-like particles for the delivery of algicidal compounds to harmful algae.

    Science.gov (United States)

    Kang, Beom Sik; Eom, Chi-Yong; Kim, Wonduck; Kim, Pyoung Il; Ju, Sun Yi; Ryu, Jaewon; Han, Gui Hwan; Oh, Jeong-Il; Cho, Hoon; Baek, Seung Ho; Kim, Gueeda; Kim, Minju; Hyun, Jaekyung; Jin, EonSeon; Kim, Si Wouk

    2015-04-01

    Harmful algal blooms (HABs) can lead to substantial socio-economic losses and extensive damage to aquatic ecosystems, drinking water sources and human health. Common algicidal techniques, including ozonation, ultrasonic treatment and dispersion of algae-killing chemicals, are unsatisfactory both economically and ecologically. This study therefore presents a novel alternative strategy for the efficient control of deleterious algae via the use of host-specific virus-like particles (VLPs) combined with chemically synthesized algicidal compounds. The capsid protein of HcRNAV34, a single-stranded RNA virus that infects the toxic dinoflagellate, Heterocapsa circularisquama, was expressed in and purified from Escherichia coli and then self-assembled into VLPs in vitro. Next, the algicidal compound, thiazolidinedione 49 (TD49), was encapsidated into HcRNAV34 VLPs for specific delivery to H. circularisquama. Consequently, HcRNAV34 VLPs demonstrated the same host selectivity as naturally occurring HcRNAV34 virions, while TD49-encapsidated VLPs showed a more potent target-specific algicidal effect than TD49 alone. These results indicate that target-specific VLPs for the delivery of cytotoxic compounds to nuisance algae might provide a safe, environmentally friendly approach for the management of HABs in aquatic ecosystems. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.

  8. Additive protection induced by mixed virus-like particles presenting respiratory syncytial virus fusion or attachment glycoproteins.

    Science.gov (United States)

    Lee, Sujin; Quan, Fu-Shi; Kwon, Youngman; Sakamoto, Kaori; Kang, Sang-Moo; Compans, Richard W; Moore, Martin L

    2014-11-01

    Respiratory syncytial virus (RSV) is the most important pathogen for lower respiratory tract illness in infants and a high priority for vaccine development. We previously reported that RSV virus-like particles (VLPs) expressing either the fusion (F) or attachment (G) glycoprotein could confer protection against RSV challenge in BALB/c mice. Here, we tested the hypothesis that RSV VLP vaccine efficacy can be enhanced by mixing RSV VLP F and RSV VLP G, and we analyzed host responses to these RSV VLPs. Mice were immunized with VLP F, VLP G, or VLP F+VLP G. Lung viral loads in BALB/c mice following RSV strain A2-line19F challenge were lower in mice vaccinated with RSV VLP F+VLP G compared to VLP F- or VLP G-vaccinated mice. Vaccination with VLP F or VLP F+VLP G induced similar levels of neutralizing antibodies. The enhanced protection against RSV challenge induced by vaccination with RSV VLP F+VLP G correlated with CD8 T cells producing T helper type 1 cytokines. VLP G vaccination alone followed by challenge resulted in immunopathology similar to formalin-inactivated RSV vaccination and RSV challenge. Taken together, mixed VLP F+VLP G provided a high level of protection against RSV without vaccine-induced immunopathology, but VLP G vaccination enhanced disease when used alone. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Purification of recombinant virus-like particles of porcine circovirus type 2 capsid protein using ion-exchange monolith chromatography.

    Science.gov (United States)

    Zaveckas, Mindaugas; Snipaitis, Simas; Pesliakas, Henrikas; Nainys, Juozas; Gedvilaite, Alma

    2015-06-01

    Diseases associated with porcine circovirus type 2 (PCV2) infection are having a severe economic impact on swine-producing countries. The PCV2 capsid (Cap) protein expressed in eukaryotic systems self-assemble into virus-like particles (VLPs) which can serve as antigens for diagnostics or/and as vaccine candidates. In this work, conventional adsorbents as well as a monolithic support with large pore sizes were examined for the chromatographic purification of PCV2 Cap VLPs from clarified yeast lysate. Q Sepharose XL was used for the initial separation of VLPs from residual host nucleic acids and some host cell proteins. For the further purification of PCV2 Cap VLPs, SP Sepharose XL, Heparin Sepharose CL-6B and CIMmultus SO3 monolith were tested. VLPs were not retained on SP Sepharose XL. The purity of VLPs after chromatography on Heparin Sepharose CL-6B was only 4-7% and the recovery of VLPs was 5-7%. Using ion-exchange chromatography on the CIMmultus SO3 monolith, PCV2 Cap VLPs with the purity of about 40% were obtained. The recovery of VLPs after chromatography on the CIMmultus SO3 monolith was 15-18%. The self-assembly of purified PCV2 Cap protein into VLPs was confirmed by electron microscopy. Two-step chromatographic purification procedure of PCV2 Cap VLPs from yeast lysate was developed using Q Sepharose XL and cation-exchange CIMmultus SO3 monolith. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Synthetic biology design to display an 18 kDa rotavirus large antigen on a modular virus-like particle.

    Science.gov (United States)

    Lua, Linda H L; Fan, Yuanyuan; Chang, Cindy; Connors, Natalie K; Middelberg, Anton P J

    2015-11-04

    Virus-like particles are an established class of commercial vaccine possessing excellent function and proven stability. Exciting developments made possible by modern tools of synthetic biology has stimulated emergence of modular VLPs, whereby parts of one pathogen are by design integrated into a less harmful VLP which has preferential physical and manufacturing character. This strategy allows the immunologically protective parts of a pathogen to be displayed on the most-suitable VLP. However, the field of modular VLP design is immature, and robust design principles are yet to emerge, particularly for larger antigenic structures. Here we use a combination of molecular dynamic simulation and experiment to reveal two key design principles for VLPs. First, the linkers connecting the integrated antigenic module with the VLP-forming protein must be well designed to ensure structural separation and independence. Second, the number of antigenic domains on the VLP surface must be sufficiently below the maximum such that a "steric barrier" to VLP formation cannot exist. This second principle leads to designs whereby co-expression of modular protein with unmodified VLP-forming protein can titrate down the amount of antigen on the surface of the VLP, to the point where assembly can proceed. In this work we elucidate these principles by displaying the 18.1 kDa VP8* domain from rotavirus on the murine polyomavirus VLP, and show functional presentation of the antigenic structure. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Protection against Multiple Subtypes of Influenza Viruses by Virus-Like Particle Vaccines Based on a Hemagglutinin Conserved Epitope

    Directory of Open Access Journals (Sweden)

    Shaoheng Chen

    2015-01-01

    Full Text Available We selected the conserved sequence in the stalk region of influenza virus hemagglutinin (HA trimmer, the long alpha helix (LAH, as the vaccine candidate sequence, and inserted it into the major immunodominant region (MIR of hepatitis B virus core protein (HBc, and, by using the E. coli expression system, we prepared a recombinant protein vaccine LAH-HBc in the form of virus-like particles (VLP. Intranasal immunization of mice with this LAH-HBc VLP plus cholera toxin B subunit with 0.2% of cholera toxin (CTB* adjuvant could effectively elicit humoral and cellular immune responses and protect mice against a lethal challenge of homologous influenza viruses (A/Puerto Rico/8/1934 (PR8 (H1N1. In addition, passage of the immune sera containing specific antibodies to naïve mice rendered them resistant against a lethal homologous challenge. Immunization with LAH-HBc VLP vaccine plus CTB* adjuvant could also fully protect mice against a lethal challenge of the 2009 pandemic H1N1 influenza virus or the avian H9N2 virus and could partially protect mice against a lethal challenge of the avian H5N1 influenza virus. This study demonstrated that the LAH-HBc VLP vaccine based on a conserved sequence of the HA trimmer stalk region is a promising candidate vaccine for developing a universal influenza vaccine against multiple influenza viruses infections.

  12. Protection against multiple subtypes of influenza viruses by virus-like particle vaccines based on a hemagglutinin conserved epitope.

    Science.gov (United States)

    Chen, Shaoheng; Zheng, Dan; Li, Changgui; Zhang, Wenjie; Xu, Wenting; Liu, Xueying; Fang, Fang; Chen, Ze

    2015-01-01

    We selected the conserved sequence in the stalk region of influenza virus hemagglutinin (HA) trimmer, the long alpha helix (LAH), as the vaccine candidate sequence, and inserted it into the major immunodominant region (MIR) of hepatitis B virus core protein (HBc), and, by using the E. coli expression system, we prepared a recombinant protein vaccine LAH-HBc in the form of virus-like particles (VLP). Intranasal immunization of mice with this LAH-HBc VLP plus cholera toxin B subunit with 0.2% of cholera toxin (CTB(*)) adjuvant could effectively elicit humoral and cellular immune responses and protect mice against a lethal challenge of homologous influenza viruses (A/Puerto Rico/8/1934 (PR8) (H1N1)). In addition, passage of the immune sera containing specific antibodies to naïve mice rendered them resistant against a lethal homologous challenge. Immunization with LAH-HBc VLP vaccine plus CTB(*) adjuvant could also fully protect mice against a lethal challenge of the 2009 pandemic H1N1 influenza virus or the avian H9N2 virus and could partially protect mice against a lethal challenge of the avian H5N1 influenza virus. This study demonstrated that the LAH-HBc VLP vaccine based on a conserved sequence of the HA trimmer stalk region is a promising candidate vaccine for developing a universal influenza vaccine against multiple influenza viruses infections.

  13. In Vivo Packaging of mRNA in Yeast-Produced Bacteriophage GA Derived Virus-Like Particles

    Directory of Open Access Journals (Sweden)

    Ārgule Dagnija

    2017-08-01

    Full Text Available Bacteriophage GA coat protein formed self-assembly competent virus-like particles (VLPs have been expressed previously in bacterial and yeast cells. On the basis of our previous experiments on the yeast vector pESC-URA / S. cerevisiae system containing two oppositely oriented promoters, new constructions were created with point-mutations in coat protein to mimic phage MS2-like RNA binding characteristics. Simultaneously, the MS2 operator sequence was added to mRNA desired for packaging. After the introduction of single-point mutations (S87N, K55N, R43K and double-point mutations (S87N + K55N and S87N + R43K, the coat protein’s ability to form VLPs was retained, but yield from cells was decreased. Exchange of the 87th Ser to Asn in coat protein sequence in combination with bacteriophage MS2 translational operator provided specific packaging of the gene of interest (GFP. Although non-specific nucleic acid sequences were packaged, the remarkable specificity for packaging of the gene of interest can be achieved using the described approach.

  14. Structure determination of feline calicivirus virus-like particles in the context of a pseudo-octahedral arrangement.

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    Wim P Burmeister

    Full Text Available The vesivirus feline calicivirus (FCV is a positive strand RNA virus encapsidated by an icosahedral T=3 shell formed by the viral VP1 protein. Upon its expression in the insect cell - baculovirus system in the context of vaccine development, two types of virus-like particles (VLPs were formed, a majority built of 60 subunits (T=1 and a minority probably built of 180 subunits (T=3. The structure of the small particles was determined by x-ray crystallography at 0.8 nm resolution helped by cryo-electron microscopy in order to understand their formation. Cubic crystals belonged to space group P213. Their self-rotation function showed the presence of an octahedral pseudo-symmetry similar to the one described previously by Agerbandje and co-workers for human parvovirus VLPs. The crystal structure could be solved starting from the published VP1 structure in the context of the T=3 viral capsid. In contrast to viral capsids, where the capsomers are interlocked by the exchange of the N-terminal arm (NTA domain, this domain is disordered in the T=1 capsid of the VLPs. Furthermore it is prone to proteolytic cleavage. The relative orientation of P (protrusion and S (shell domains is alerted so as to fit VP1 to the smaller T=1 particle whereas the intermolecular contacts around 2-fold, 3-fold and 5-fold axes are conserved. By consequence the surface of the VLP is very similar compared to the viral capsid and suggests a similar antigenicity. The knowledge of the structure of the VLPs will help to improve their stability, in respect to a use for vaccination.

  15. Effect of HIV-1 envelope cytoplasmic tail on adenovirus primed virus encoded virus-like particle immunizations

    DEFF Research Database (Denmark)

    Andersson, Anne Marie C; Ragonnaud, Emeline; Seaton, Kelly E.

    2016-01-01

    The low number of envelope (Env) spikes presented on native HIV-1 particles is a major impediment for HIV-1 prophylactic vaccine development. We designed virus-like particle encoding adenoviral vectors utilizing SIVmac239 Gag as an anchor for full length and truncated HIV-1 M consensus Env...... were found between the different priming regimens as both induced high titered tier 1 neutralizing antibodies, but no tier 2 antibodies, possibly reflecting the similar presentation of trimer specific antibody epitopes. The described vaccine regimens provide insight into the effects of the HIV-1 Env...

  16. Efficient self-assembly of human papillomavirus type 16 L1 and L1-L2 into virus-like particles.

    OpenAIRE

    Kirnbauer, R; Taub, J; Greenstone, H; Roden, R; Dürst, M; Gissmann, L; Lowy, D R; Schiller, J T

    1993-01-01

    The L1 genes of two human papillomavirus type 16 (HPV16) isolates derived from condylomata acuminata were used to express the L1 major capsid protein in insect cells via recombinant baculoviruses. Both L1 major capsid proteins self-assembled into virus-like particles (VLP) with high efficiency and could be purified in preparative amounts on density gradients. The yield of VLP was 3 orders of magnitude higher than what has been obtained previously, using L1 derived from the prototype HPV16. DN...

  17. A molecular assembly system for presentation of antigens on the surface of HBc virus-like particles

    Energy Technology Data Exchange (ETDEWEB)

    Blokhina, Elena A.; Kuprianov, Victor V. [Centre ' Bioengineering' , Russian Academy of Sciences, 117312 Prosp. 60-letya Oktyabrya 7-1, Moscow (Russian Federation); Stepanova, Ludmila A.; Tsybalova, Ludmila M. [Research Institute of Influenza, Russian Federation Ministry of Health and Social Development, St. Petersburg (Russian Federation); Kiselev, Oleg I. [Research Institute of Influenza, Russian Federation Ministry of Health and Social Development, St. Petersburg (Russian Federation); GenNanotech Ltd, St. Petersburg (Russian Federation); Ravin, Nikolai V., E-mail: nravin@biengi.ac.ru [Centre ' Bioengineering' , Russian Academy of Sciences, 117312 Prosp. 60-letya Oktyabrya 7-1, Moscow (Russian Federation); GenNanotech Ltd, St. Petersburg (Russian Federation); Skryabin, Konstantin G. [Centre ' Bioengineering' , Russian Academy of Sciences, 117312 Prosp. 60-letya Oktyabrya 7-1, Moscow (Russian Federation); GenNanotech Ltd, St. Petersburg (Russian Federation)

    2013-01-20

    Hepatitis B virus-like particles, icosahedral structures formed by multiple core protein dimers, are promising immune-enhancing vaccine carriers for foreign antigens. Insertions into the surface-exposed immunodominant loop are especially immunogenic. However, the need to conserve the particulate structure to ensure high immunogenicity imposes restraints on the nature of the heterologous sequence that can be inserted. We propose a new approach to constructing HBc particles linked to the target epitopes that relies on non-covalent interactions between the epitope and pre-assembled unmodified HBc particles. Interaction was enabled by fusion of the epitope to the GSLLGRMKGA peptide, binding to the spike tips. This peptide may be used as a 'binding tag' allowing in vitro construction of HBc particles carrying the target peptide. Such virus-like particles carrying multiple copies of the extracellular domain of the M2 protein of different influenza strains appeared to be highly immunogenic and protected immunised mice against a lethal influenza challenge.

  18. Mutations in the Transmembrane Domain and Cytoplasmic Tail of Hendra Virus Fusion Protein Disrupt Virus-Like-Particle Assembly.

    Science.gov (United States)

    Cifuentes-Muñoz, Nicolás; Sun, Weina; Ray, Greeshma; Schmitt, Phuong Tieu; Webb, Stacy; Gibson, Kathleen; Dutch, Rebecca Ellis; Schmitt, Anthony P

    2017-07-15

    Hendra virus (HeV) is a zoonotic paramyxovirus that causes deadly illness in horses and humans. An intriguing feature of HeV is the utilization of endosomal protease for activation of the viral fusion protein (F). Here we investigated how endosomal F trafficking affects HeV assembly. We found that the HeV matrix (M) and F proteins each induced particle release when they were expressed alone but that their coexpression led to coordinated assembly of virus-like particles (VLPs) that were morphologically and physically distinct from M-only or F-only VLPs. Mutations to the F protein transmembrane domain or cytoplasmic tail that disrupted endocytic trafficking led to failure of F to function with M for VLP assembly. Wild-type F functioned normally for VLP assembly even when its cleavage was prevented with a cathepsin inhibitor, indicating that it is endocytic F trafficking that is important for VLP assembly, not proteolytic F cleavage. Under specific conditions of reduced M expression, we found that M could no longer induce significant VLP release but retained the ability to be incorporated as a passenger into F-driven VLPs, provided that the F protein was competent for endocytic trafficking. The F and M proteins were both found to traffic through Rab11-positive recycling endosomes (REs), suggesting a model in which F and M trafficking pathways converge at REs, enabling these proteins to preassemble before arriving at plasma membrane budding sites. IMPORTANCE Hendra virus and Nipah virus are zoonotic paramyxoviruses that cause lethal infections in humans. Unlike that for most paramyxoviruses, activation of the henipavirus fusion protein occurs in recycling endosomal compartments. In this study, we demonstrate that the unique endocytic trafficking pathway of Hendra virus F protein is required for proper viral assembly and particle release. These results advance our basic understanding of the henipavirus assembly process and provide a novel model for the interplay between

  19. Chimeric rabies virus-like particles containing membrane-anchored GM-CSF enhances the immune response against rabies virus.

    Science.gov (United States)

    Kang, Hongtao; Qi, Yinglin; Wang, Hualei; Zheng, Xuexing; Gao, Yuwei; Li, Nan; Yang, Songtao; Xia, Xianzhu

    2015-03-11

    Rabies remains an important public health threat in most developing countries. To develop a more effective and safe vaccine against rabies, we have constructed a chimeric rabies virus-like particle (VLP), which containing glycoprotein (G) and matrix protein (M) of rabies virus (RABV) Evelyn-Rokitnicki-Abelseth (ERA) strain, and membrane-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF), and it was named of EVLP-G. The immunogenicity and protective efficacy of EVLP-G against RABV were evaluated by intramuscular administration in a mouse model. The EVLP-G was successfully produced in insect cells by coinfection with three recombinant baculoviruses expressing G, M, and GM-CSF, respectively. The membrane-anchored GM-CSF possesses a strong adjuvant activity. More B cells and dendritic cells (DCs) were recruited and/or activated in inguinal lymph nodes in mice immunized with EVLP-G. EVLP-G was found to induce a significantly increased RABV-specific virus-neutralizing antibody and elicit a larger and broader antibody subclass responses compared with the standard rabies VLP (sRVLP, consisting of G and M). The EVLP-G also elicited significantly more IFN-γ- or IL-4-secreting CD4+ and CD8+ T cells than the sRVLP. Moreover, the immune responses induced by EVLP-G protect all vaccinated mice from lethal challenge with RABV. These results suggest that EVLP-G has the potential to be developed as a novel vaccine candidate for the prevention and control of animal rabies.

  20. Mammalian Cell-Derived Respiratory Syncytial Virus-Like Particles Protect the Lower as well as the Upper Respiratory Tract.

    Directory of Open Access Journals (Sweden)

    Pramila Walpita

    Full Text Available Globally, Respiratory Syncytial Virus (RSV is a leading cause of bronchiolitis and pneumonia in children less than one year of age and in USA alone, between 85,000 and 144,000 infants are hospitalized every year. To date, there is no licensed vaccine. We have evaluated vaccine potential of mammalian cell-derived native RSV virus-like particles (RSV VLPs composed of the two surface glycoproteins G and F, and the matrix protein M. Results of in vitro testing showed that the VLPs were functionally assembled and immunoreactive, and that the recombinantly expressed F protein was cleaved intracellularly similarly to the virus-synthesized F protein to produce the F1 and F2 subunits; the presence of the F1 fragment is critical for vaccine development since all the neutralizing epitopes present in the F protein are embedded in this fragment. Additional in vitro testing in human macrophage cell line THP-1 showed that both virus and the VLPs were sensed by TLR-4 and induced a Th1-biased cytokine response. Cotton rats vaccinated with RSV VLPs adjuvanted with alum and monophosphoryl lipid A induced potent neutralizing antibody response, and conferred protection in the lower as well as the upper respiratory tract based on substantial virus clearance from these sites. To the best of our knowledge, this is the first VLP/virosome vaccine study reporting protection of the lower as well as the upper respiratory tract: Prevention from replication in the nose is an important consideration if the target population is infants < 6 months of age. This is because continued virus replication in the nose results in nasal congestion and babies at this age are obligate nose breathers. In conclusion, these results taken together suggest that our VLPs show promise to be a safe and effective vaccine for RSV.

  1. Modulation of the immunogenicity of virus-like particles composed of mutant hepatitis B virus envelope subunits.

    Science.gov (United States)

    Cheong, Wan-Shoo; Hyakumura, Michiko; Yuen, Lilly; Warner, Nadia; Locarnini, Stephen; Netter, Hans J

    2012-02-01

    Virus-like particles (VLPs) are non-infectious subviral protein complexes, which possess structural features identical or closely related to infectious virions. They are utilized as delivery tools for immunologically relevant antigenic sequences. In order to investigate whether mutant subunits can modulate the VLP immunogenicity, comparative immunization studies with wild-type and non-native VLPs were performed. To determine whether disulfide bonding impacts on the immunogenicity of hepatitis B virus envelope proteins (HBsAg), mutant HBsAg subunits with single, double and triple cysteine residue substitutions were generated. The mutant proteins were expressed in cell culture, secretion competent non-native VLPs generated, followed by immunization studies in mice to measure the cellular immune response. The reduced ability of mutant HBsAg proteins to form disulfide bonds does not interfere with their ability to assemble into secretion competent VLPs. Depending on specific cysteine to alanine changes, VLPs could be generated with or without an increased ratio of monomeric versus dimeric/oligomeric subunits compared to wild-type VLPs. The utilization of non-native VLPs resulted in enhanced cellular immune responses and does not seem to depend on the ratio between monomeric or dimeric/oligomeric subunits. Comparative immunization studies strongly indicate that changes in the disulfide bonding modulate the VLP immunogenicity most likely due to structural changes. We hypothesize that structural features have evolved with reduced immunogenicity to evade the constraints imposed by the immune system. Altering VLP conformation may represent an attractive strategy to modulate antigen processing resulting in an enhanced immune response and/or a changed hierarchy of epitope presentation. Copyright © 2011 Elsevier B.V. All rights reserved.

  2. A trivalent virus-like particle vaccine elicits protective immune responses against seasonal influenza strains in mice and ferrets.

    Directory of Open Access Journals (Sweden)

    Ted M Ross

    Full Text Available There is need for improved human influenza vaccines, particularly for older adults who are at greatest risk for severe disease, as well as to address the continuous antigenic drift within circulating human subtypes of influenza virus. We have engineered an influenza virus-like particle (VLP as a new generation vaccine candidate purified from the supernatants of Sf9 insect cells following infection by recombinant baculoviruses to express three influenza virus proteins, hemagglutinin (HA, neuraminidase (NA, and matrix 1 (M1. In this study, a seasonal trivalent VLP vaccine (TVV formulation, composed of influenza A H1N1 and H3N2 and influenza B VLPs, was evaluated in mice and ferrets for the ability to elicit antigen-specific immune responses. Animals vaccinated with the TVV formulation had hemagglutination-inhibition (HAI antibody titers against all three homologous influenza virus strains, as well as HAI antibodies against a panel of heterologous influenza viruses. HAI titers elicited by the TVV were statistically similar to HAI titers elicited in animals vaccinated with the corresponding monovalent VLP. Mice vaccinated with the TVV had higher level of influenza specific CD8+ T cell responses than a commercial trivalent inactivated vaccine (TIV. Ferrets vaccinated with the highest dose of the VLP vaccine and then challenged with the homologous H3N2 virus had the lowest titers of replicating virus in nasal washes and showed no signs of disease. Overall, a trivalent VLP vaccine elicits a broad array of immunity and can protect against influenza virus challenge.

  3. Chimeric Rabies Virus-Like Particles Containing Membrane-Anchored GM-CSF Enhances the Immune Response against Rabies Virus

    Directory of Open Access Journals (Sweden)

    Hongtao Kang

    2015-03-01

    Full Text Available Rabies remains an important public health threat in most developing countries. To develop a more effective and safe vaccine against rabies, we have constructed a chimeric rabies virus-like particle (VLP, which containing glycoprotein (G and matrix protein (M of rabies virus (RABV Evelyn-Rokitnicki-Abelseth (ERA strain, and membrane-anchored granulocyte-macrophage colony-stimulating factor (GM-CSF, and it was named of EVLP-G. The immunogenicity and protective efficacy of EVLP-G against RABV were evaluated by intramuscular administration in a mouse model. The EVLP-G was successfully produced in insect cells by coinfection with three recombinant baculoviruses expressing G, M, and GM-CSF, respectively. The membrane-anchored GM-CSF possesses a strong adjuvant activity. More B cells and dendritic cells (DCs were recruited and/or activated in inguinal lymph nodes in mice immunized with EVLP-G. EVLP-G was found to induce a significantly increased RABV-specific virus-neutralizing antibody and elicit a larger and broader antibody subclass responses compared with the standard rabies VLP (sRVLP, consisting of G and M. The EVLP-G also elicited significantly more IFN-γ- or IL-4-secreting CD4+ and CD8+ T cells than the sRVLP. Moreover, the immune responses induced by EVLP-G protect all vaccinated mice from lethal challenge with RABV. These results suggest that EVLP-G has the potential to be developed as a novel vaccine candidate for the prevention and control of animal rabies.

  4. Mammalian Cell-Derived Respiratory Syncytial Virus-Like Particles Protect the Lower as well as the Upper Respiratory Tract

    Science.gov (United States)

    Walpita, Pramila; Johns, Lisa M.; Tandon, Ravi; Moore, Martin L.

    2015-01-01

    Globally, Respiratory Syncytial Virus (RSV) is a leading cause of bronchiolitis and pneumonia in children less than one year of age and in USA alone, between 85,000 and 144,000 infants are hospitalized every year. To date, there is no licensed vaccine. We have evaluated vaccine potential of mammalian cell-derived native RSV virus-like particles (RSV VLPs) composed of the two surface glycoproteins G and F, and the matrix protein M. Results of in vitro testing showed that the VLPs were functionally assembled and immunoreactive, and that the recombinantly expressed F protein was cleaved intracellularly similarly to the virus-synthesized F protein to produce the F1 and F2 subunits; the presence of the F1 fragment is critical for vaccine development since all the neutralizing epitopes present in the F protein are embedded in this fragment. Additional in vitro testing in human macrophage cell line THP-1 showed that both virus and the VLPs were sensed by TLR-4 and induced a Th1-biased cytokine response. Cotton rats vaccinated with RSV VLPs adjuvanted with alum and monophosphoryl lipid A induced potent neutralizing antibody response, and conferred protection in the lower as well as the upper respiratory tract based on substantial virus clearance from these sites. To the best of our knowledge, this is the first VLP/virosome vaccine study reporting protection of the lower as well as the upper respiratory tract: Prevention from replication in the nose is an important consideration if the target population is infants virus replication in the nose results in nasal congestion and babies at this age are obligate nose breathers. In conclusion, these results taken together suggest that our VLPs show promise to be a safe and effective vaccine for RSV. PMID:26172453

  5. Long-Term Protective Immunity from an Influenza Virus-Like Particle Vaccine Administered with a Microneedle Patch

    OpenAIRE

    Quan, Fu-Shi; Kim, Yeu-Chun; Song, Jae-Min; Hwang, Hye Suk; Compans, Richard W.; Prausnitz, Mark R.; Kang, Sang-Moo

    2013-01-01

    Skin vaccination with influenza virus-like particles (VLPs) using microneedles has been shown to induce protection similar to or better than that induced by intramuscular immunization. In this study, we examined the long-term protective efficacy of influenza (H1N1 A/PR/8/34) VLPs after skin vaccination using microneedle patches coated with the vaccine. Microneedle vaccination of mice in the skin induced 100% protection against lethal challenge infection with influenza A/PR/8/34 virus 14 month...

  6. Synthetic virus-like particles prepared via protein corona formation enable effective vaccination in an avian model of coronavirus infection.

    Science.gov (United States)

    Chen, Hui-Wen; Huang, Chen-Yu; Lin, Shu-Yi; Fang, Zih-Syun; Hsu, Chen-Hsuan; Lin, Jung-Chen; Chen, Yuan-I; Yao, Bing-Yu; Hu, Che-Ming J

    2016-11-01

    The ongoing battle against current and rising viral infectious threats has prompted increasing effort in the development of vaccine technology. A major thrust in vaccine research focuses on developing formulations with virus-like features towards enhancing antigen presentation and immune processing. Herein, a facile approach to formulate synthetic virus-like particles (sVLPs) is demonstrated by exploiting the phenomenon of protein corona formation induced by the high-energy surfaces of synthetic nanoparticles. Using an avian coronavirus spike protein as a model antigen, sVLPs were prepared by incubating 100 nm gold nanoparticles in a solution containing an optimized concentration of viral proteins. Following removal of free proteins, antigen-laden particles were recovered and showed morphological semblance to natural viral particles under nanoparticle tracking analysis and transmission electron microscopy. As compared to inoculation with free proteins, vaccination with the sVLPs showed enhanced lymphatic antigen delivery, stronger antibody titers, increased splenic T-cell response, and reduced infection-associated symptoms in an avian model of coronavirus infection. Comparison to a commercial whole inactivated virus vaccine also showed evidence of superior antiviral protection by the sVLPs. The study demonstrates a simple yet robust method in bridging viral antigens with synthetic nanoparticles for improved vaccine application; it has practical implications in the management of human viral infections as well as in animal agriculture. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Enhanced production of Chikungunya virus-like particles using a high-pH adapted spodoptera frugiperda insect cell line.

    Directory of Open Access Journals (Sweden)

    James M Wagner

    Full Text Available Chikungunya virus-like particles (VLPs have potential to be used as a prophylactic vaccine based on testing in multiple animal models and are currently being evaluated for human use in a Phase I clinical trial. The current method for producing these enveloped alphavirus VLPs by transient gene expression in mammalian cells presents challenges for scalable and robust industrial manufacturing, so the insect cell baculovirus expression vector system was evaluated as an alternative expression technology. Subsequent to recombinant baculovirus infection of Sf21 cells in standard culture media (pH 6.2-6.4, properly processed Chikungunya structural proteins were detected and assembled capsids were observed. However, an increase in culture pH to 6.6-6.8 was necessary to produce detectable concentrations of assembled VLPs. Since this elevated production pH exceeds the optimum for growth medium stability and Sf21 culture, medium modifications were made and a novel insect cell variant (SfBasic was derived by exposure of Sf21 to elevated culture pH for a prolonged period of time. The high-pH adapted SfBasic insect cell line described herein is capable of maintaining normal cell growth into the typical mammalian cell culture pH range of 7.0-7.2 and produces 11-fold higher Chikungunya VLP yields relative to the parental Sf21 cell line. After scale-up into stirred tank bioreactors, SfBasic derived VLPs were chromatographically purified and shown to be similar in size and structure to a VLP standard derived from transient gene expression in HEK293 cells. Total serum anti-Chikungunya IgG and neutralizing titers from guinea pigs vaccinated with SfBasic derived VLPs or HEK293 derived VLPs were not significantly different with respect to production method, suggesting that this adapted insect cell line and production process could be useful for manufacturing Chikungunya VLPs for use as a vaccine. The adaptation of Sf21 to produce high levels of recombinant protein and

  8. Chimaeric virus-like particles derived from consensus genome sequences of human rotavirus strains co-circulating in Africa.

    Directory of Open Access Journals (Sweden)

    Khuzwayo C Jere

    Full Text Available Rotavirus virus-like particles (RV-VLPs are potential alternative non-live vaccine candidates due to their high immunogenicity. They mimic the natural conformation of native viral proteins but cannot replicate because they do not contain genomic material which makes them safe. To date, most RV-VLPs have been derived from cell culture adapted strains or common G1 and G3 rotaviruses that have been circulating in communities for some time. In this study, chimaeric RV-VLPs were generated from the consensus sequences of African rotaviruses (G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes characterised directly from human stool samples without prior adaptation of the wild type strains to cell culture. Codon-optimised sequences for insect cell expression of genome segments 2 (VP2, 4 (VP4, 6 (VP6 and 9 (VP7 were cloned into a modified pFASTBAC vector, which allowed simultaneous expression of up to four genes using the Bac-to-Bac Baculovirus Expression System (BEVS; Invitrogen. Several combinations of the genome segments originating from different field strains were cloned to produce double-layered RV-VLPs (dRV-VLP; VP2/6, triple-layered RV-VLPs (tRV-VLP; VP2/6/7 or VP2/6/7/4 and chimaeric tRV-VLPs. The RV-VLPs were produced by infecting Spodoptera frugiperda 9 and Trichoplusia ni cells with recombinant baculoviruses using multi-cistronic, dual co-infection and stepwise-infection expression strategies. The size and morphology of the RV-VLPs, as determined by transmission electron microscopy, revealed successful production of RV-VLPs. The novel approach of producing tRV-VLPs, by using the consensus insect cell codon-optimised nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed-up vaccine research and development by by-passing the need to adapt rotaviruses to cell culture. Other problems associated with cell culture adaptation, such as possible changes in epitopes, can also be

  9. Chimaeric virus-like particles derived from consensus genome sequences of human rotavirus strains co-circulating in Africa.

    Science.gov (United States)

    Jere, Khuzwayo C; O'Neill, Hester G; Potgieter, A Christiaan; van Dijk, Alberdina A

    2014-01-01

    Rotavirus virus-like particles (RV-VLPs) are potential alternative non-live vaccine candidates due to their high immunogenicity. They mimic the natural conformation of native viral proteins but cannot replicate because they do not contain genomic material which makes them safe. To date, most RV-VLPs have been derived from cell culture adapted strains or common G1 and G3 rotaviruses that have been circulating in communities for some time. In this study, chimaeric RV-VLPs were generated from the consensus sequences of African rotaviruses (G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes) characterised directly from human stool samples without prior adaptation of the wild type strains to cell culture. Codon-optimised sequences for insect cell expression of genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBAC vector, which allowed simultaneous expression of up to four genes using the Bac-to-Bac Baculovirus Expression System (BEVS; Invitrogen). Several combinations of the genome segments originating from different field strains were cloned to produce double-layered RV-VLPs (dRV-VLP; VP2/6), triple-layered RV-VLPs (tRV-VLP; VP2/6/7 or VP2/6/7/4) and chimaeric tRV-VLPs. The RV-VLPs were produced by infecting Spodoptera frugiperda 9 and Trichoplusia ni cells with recombinant baculoviruses using multi-cistronic, dual co-infection and stepwise-infection expression strategies. The size and morphology of the RV-VLPs, as determined by transmission electron microscopy, revealed successful production of RV-VLPs. The novel approach of producing tRV-VLPs, by using the consensus insect cell codon-optimised nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed-up vaccine research and development by by-passing the need to adapt rotaviruses to cell culture. Other problems associated with cell culture adaptation, such as possible changes in epitopes, can also be circumvented

  10. Chimaeric Virus-Like Particles Derived from Consensus Genome Sequences of Human Rotavirus Strains Co-Circulating in Africa

    Science.gov (United States)

    Jere, Khuzwayo C.; O'Neill, Hester G.; Potgieter, A. Christiaan; van Dijk, Alberdina A.

    2014-01-01

    Rotavirus virus-like particles (RV-VLPs) are potential alternative non-live vaccine candidates due to their high immunogenicity. They mimic the natural conformation of native viral proteins but cannot replicate because they do not contain genomic material which makes them safe. To date, most RV-VLPs have been derived from cell culture adapted strains or common G1 and G3 rotaviruses that have been circulating in communities for some time. In this study, chimaeric RV-VLPs were generated from the consensus sequences of African rotaviruses (G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes) characterised directly from human stool samples without prior adaptation of the wild type strains to cell culture. Codon-optimised sequences for insect cell expression of genome segments 2 (VP2), 4 (VP4), 6 (VP6) and 9 (VP7) were cloned into a modified pFASTBAC vector, which allowed simultaneous expression of up to four genes using the Bac-to-Bac Baculovirus Expression System (BEVS; Invitrogen). Several combinations of the genome segments originating from different field strains were cloned to produce double-layered RV-VLPs (dRV-VLP; VP2/6), triple-layered RV-VLPs (tRV-VLP; VP2/6/7 or VP2/6/7/4) and chimaeric tRV-VLPs. The RV-VLPs were produced by infecting Spodoptera frugiperda 9 and Trichoplusia ni cells with recombinant baculoviruses using multi-cistronic, dual co-infection and stepwise-infection expression strategies. The size and morphology of the RV-VLPs, as determined by transmission electron microscopy, revealed successful production of RV-VLPs. The novel approach of producing tRV-VLPs, by using the consensus insect cell codon-optimised nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed-up vaccine research and development by by-passing the need to adapt rotaviruses to cell culture. Other problems associated with cell culture adaptation, such as possible changes in epitopes, can also be circumvented

  11. Production in yeast of pseudotype virus-like particles harboring functionally active antibody fragments neutralizing the cytolytic activity of vaginolysin

    Directory of Open Access Journals (Sweden)

    Pleckaityte Milda

    2011-12-01

    Full Text Available Abstract Background Recombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY, the main virulence factor of Gardnerella vaginalis. Results The scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody. Conclusions Recombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the sc

  12. Synthetic virus-like particles target dendritic cell lipid rafts for rapid endocytosis primarily but not exclusively by macropinocytosis.

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    Rajni Sharma

    Full Text Available DC employ several endocytic routes for processing antigens, driving forward adaptive immunity. Recent advances in synthetic biology have created small (20-30 nm virus-like particles based on lipopeptides containing a virus-derived coiled coil sequence coupled to synthetic B- and T-cell epitope mimetics. These self-assembling SVLP efficiently induce adaptive immunity without requirement for adjuvant. We hypothesized that the characteristics of DC interaction with SVLP would elaborate on the roles of cell membrane and intracellular compartments in the handling of a virus-like entity known for its efficacy as a vaccine. DC rapidly bind SVLP within min, co-localised with CTB and CD9, but not caveolin-1. In contrast, internalisation is a relatively slow process, delivering SVLP into the cell periphery where they are maintained for a number of hrs in association with microtubules. Although there is early association with clathrin, this is no longer seen after 10 min. Association with EEA-1(+ early endosomes is also early, but proteolytic processing appears slow, the SVLP-vesicles remaining peripheral. Association with transferrin occurs rarely, and only in the periphery, possibly signifying translocation of some SVLP for delivery to B-lymphocytes. Most SVLP co-localise with high molecular weight dextran. Uptake of both is impaired with mature DC, but there remains a residual uptake of SVLP. These results imply that DC use multiple endocytic routes for SVLP uptake, dominated by caveolin-independent, lipid raft-mediated macropinocytosis. With most SVLP-containing vesicles being retained in the periphery, not always interacting with early endosomes, this relates to slow proteolytic degradation and antigen retention by DC. The present characterization allows for a definition of how DC handle virus-like particles showing efficacious immunogenicity, elements valuable for novel vaccine design in the future.

  13. Recombinant norovirus-specific scFv inhibit virus-like particle binding to cellular ligands

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    Hardy Michele E

    2008-01-01

    Full Text Available Abstract Background Noroviruses cause epidemic outbreaks of gastrointestinal illness in all age-groups. The rapid onset and ease of person-to-person transmission suggest that inhibitors of the initial steps of virus binding to susceptible cells have value in limiting spread and outbreak persistence. We previously generated a monoclonal antibody (mAb 54.6 that blocks binding of recombinant norovirus-like particles (VLP to Caco-2 intestinal cells and inhibits VLP-mediated hemagglutination. In this study, we engineered the antigen binding domains of mAb 54.6 into a single chain variable fragment (scFv and tested whether these scFv could function as cell binding inhibitors, similar to the parent mAb. Results The scFv54.6 construct was engineered to encode the light (VL and heavy (VH variable domains of mAb 54.6 separated by a flexible peptide linker, and this recombinant protein was expressed in Pichia pastoris. Purified scFv54.6 recognized native VLPs by immunoblot, inhibited VLP-mediated hemagglutination, and blocked VLP binding to H carbohydrate antigen expressed on the surface of a CHO cell line stably transfected to express α 1,2-fucosyltransferase. Conclusion scFv54.6 retained the functional properties of the parent mAb with respect to inhibiting norovirus particle interactions with cells. With further engineering into a form deliverable to the gut mucosa, norovirus neutralizing antibodies represent a prophylactic strategy that would be valuable in outbreak settings.

  14. An automated microscale chromatographic purification of virus-like particles as a strategy for process development.

    Science.gov (United States)

    Wenger, Marc D; Dephillips, Peter; Price, Colleen E; Bracewell, Daniel G

    2007-06-01

    The development of fermentation processes for recombinant vaccines requires optimizing expression while maintaining high product quality. Changes to cell fermentation conditions are typically evaluated following cell disruption, with expression levels quantified by immunoassay, liquid chromatography or enzyme activity. However, assay titres do not always predict the effects that intracellular aggregation, proteolysis, post-translational modifications and differences in relative impurity levels can have on purification yield and product purity. Furthermore, heterogeneity in the size and surface properties inherent in viral particles makes unit operations such as chromatography less predictable. In these cases, the purification procedure (or a mimic thereof) must be carried out to give accurate information on the impact of changes in fermentation conditions on purification process performance. This was demonstrated for the development of a recombinant vaccine against human papillomavirus produced in Saccharomyces cerevisiae, where the most informative feedback on fermentation variables was obtained by completing a multistep chromatographic purification to evaluate process yield and product purity. To increase the purification throughput and reduce labour, the chromatography was miniaturized 1000-fold from the laboratory scale using microlitre volumes of adsorbent in a pipette tip and automated on a robotic workstation. The microscale purification is shown to be predictive of the laboratory-scale purification in terms of yield and purity, while providing over a 10-fold increase in throughput and allowing for increased monitoring of fermentation processes. In addition, by reducing the volume of cells needed for this assessment, the fermentation can be correspondingly reduced in scale and carried out in parallel for additional throughput gains.

  15. A virus-like particle vaccine candidate for influenza A virus based on multiple conserved antigens presented on hepatitis B tandem core particles.

    Science.gov (United States)

    Ramirez, Alejandro; Morris, Stephen; Maucourant, Sophie; D'Ascanio, Isabella; Crescente, Vincenzo; Lu, I-Na; Farinelle, Sophie; Muller, Claude P; Whelan, Michael; Rosenberg, William

    2018-02-01

    Existing Influenza A virus (IAV) vaccines target variable parts of the virus that may change between seasons. Vaccine design relies on predicting the predominant circulating influenza strains but when there is a mismatch between vaccine and circulating strains, efficacy is sub-optimal. Furthermore, current approaches provide limited protection against emerging influenza strains that may cause pandemics. One solution is to design vaccines that target conserved protein domains of influenza, which remain largely unchanged over time and are likely to be found in emergent variants. We present a virus-like particle (VLP), built using the hepatitis B virus tandem core platform, as an IAV vaccine candidate containing multiple conserved antigens. Hepatitis B core protein spontaneously assembles into a VLP that is immunogenic and confers immunogenicity to proteins incorporated into the major insertion region (MIR) of core monomers. However, insertion of antigen sequences may disrupt particle assembly preventing VLP formation or result in unstable particles. We have overcome these problems by genetically manipulating the hepatitis B core to express core monomers in tandem, ligated with a flexible linker, incorporating different antigens at each of the MIRs. Immunisation with this VLP, named Tandiflu1, containing 4 conserved antigens from matrix protein 2 ectodomain and hemagglutinin stalk, leads to production of cross-reactive and protective antibodies. The polyclonal antibodies induced by Tandiflu1 can bind IAV Group 1 hemagglutinin types H1, H5, H11, H9, H16 and a conserved epitope on matrix protein 2 expressed by most strains of IAV. Vaccination with Tandiflu1 results in 100% protection from a lethal influenza challenge with H1N1 IAV. Serum transfer from vaccinated animals is sufficient to confer protection from influenza-associated illness in naïve mice. These data suggest that a Tandem Core based IAV vaccine might provide broad protection against common and emergent H1

  16. Morphotypes of virus-like particles in two hydrothermal vent fields on the East Scotia Ridge, Antarctica.

    Science.gov (United States)

    Millard, Andrew D; Hands-Portman, Ian; Zwirglmaier, Katrin

    2014-01-01

    Viruses from extreme environments are still largely unexplored and may harbor unseen genetic potential. Here, we present a first glance at the morphological diversity of virus like particles (VLPs) from an environment that is extreme in more than one respect: two recently discovered hydrothermal vent fields on the East Scotia Ridge in the Southern Ocean near Antarctica. They are the southernmost hydrothermal sites found to date and have been shown to present a new biogeographic province, containing several new macrofaunal species and associated microbial organisms. Transmission electron microscopy revealed a range of tailed and untailed VLPs of various morphologies as well as an unusual long rod-shaped VLP with three long filaments. Based on its distant similarity with several known archaeal viruses, we hypothesize that this presents a new viral morphology that most likely infects an archaeon. Notably absent in the samples we analyzed were lemon- or spindle-shaped VLPs that have previously been described in other hydrothermal vent settings.

  17. Incorporation of GM-CSF or CD40L Enhances the Immunogenicity of Hantaan Virus-Like Particles

    Science.gov (United States)

    Cheng, Lin-Feng; Wang, Fang; Zhang, Liang; Yu, Lan; Ye, Wei; Liu, Zi-Yu; Ying, Qi-Kang; Wu, Xing-An; Xu, Zhi-Kai; Zhang, Fang-Lin

    2016-01-01

    A safe and effective Hantaan virus (HTNV) vaccine is highly desirable because HTNV causes an acute and often fatal disease (hemorrhagic fever with renal syndrome, HFRS). Since the immunity of the inactivated vaccine is weak and the safety is poor, HTNV virus-like particles (VLPs) offer an attractive and safe alternative. These particles lack the viral genome but are perceived by the immune system as virus particles. We hypothesized that adding immunostimulatory signals to VLPs would enhance their efficacy. To accomplish this enhancement, we generated chimeric HTNV VLPs containing glycosylphosphatidylinositol (GPI)-anchored granulocyte macrophage colony-stimulating factor (GM-CSF) or CD40 ligand (CD40L) and investigated their biological activity in vitro. The immunization of mice with chimeric HTNV VLPs containing GM-CSF or CD40L induced stronger humoral immune responses and cellular immune responses compared to the HTNV VLPs and Chinese commercial inactivated hantavirus vaccine. Chimeric HTNV VLPs containing GM-CSF or CD40L also protected mice from an HTNV challenge. Altogether, our results suggest that anchoring immunostimulatory molecules into HTNV VLPs can be a potential approach for the control and prevention of HFRS. PMID:28066721

  18. Silica nanoparticles as the adjuvant for the immunisation of mice using hepatitis B core virus-like particles.

    Directory of Open Access Journals (Sweden)

    Dace Skrastina

    Full Text Available Advances in nanotechnology and nanomaterials have facilitated the development of silicon dioxide, or Silica, particles as a promising immunological adjuvant for the generation of novel prophylactic and therapeutic vaccines. In the present study, we have compared the adjuvanting potential of commercially available Silica nanoparticles (initial particles size of 10-20 nm with that of aluminium hydroxide, or Alum, as well as that of complete and incomplete Freund's adjuvants for the immunisation of BALB/c mice with virus-like particles (VLPs formed by recombinant full-length Hepatitis B virus core (HBc protein. The induction of B-cell and T-cell responses was studied after immunisation. Silica nanoparticles were able to adsorb maximally 40% of the added HBc, whereas the adsorption capacity of Alum exceeded 90% at the same VLPs/adjuvant ratio. Both Silica and Alum formed large complexes with HBc VLPs that sedimented rapidly after formulation, as detected by dynamic light scattering, spectrophotometry, and electron microscopy. Both Silica and Alum augmented the humoral response against HBc VLPs to the high anti-HBc level in the case of intraperitoneal immunisation, whereas in subcutaneous immunisation, the Silica-adjuvanted anti-HBc level even exceeded the level adjuvanted by Alum. The adjuvanting of HBc VLPs by Silica resulted in the same typical IgG2a/IgG1 ratios as in the case of the adjuvanting by Alum. The combination of Silica with monophosphoryl lipid A (MPL led to the same enhancement of the HBc-specific T-cell induction as in the case of the Alum and MPL combination. These findings demonstrate that Silica is not a weaker putative adjuvant than Alum for induction of B-cell and T-cell responses against recombinant HBc VLPs. This finding may have an essential impact on the development of the set of Silica-adjuvanted vaccines based on a long list of HBc-derived virus-like particles as the biological component.

  19. Characterization of self-assembled virus-like particles of Merkel cell polyomavirus.

    Science.gov (United States)

    Li, Tian-Cheng; Iwasaki, Kenji; Katano, Harutaka; Kataoka, Michiyo; Nagata, Noriyo; Kobayashi, Kazumi; Mizutani, Tetsuya; Takeda, Naokazu; Wakita, Takaji; Suzuki, Tetsuro

    2015-01-01

    In our recombinant baculovirus system, VP1 protein of merkel cell polyomavirus (MCPyV), which is implicated as a causative agent in Merkel cell carcinoma, was self-assembled into MCPyV-like particles (MCPyV-LP) with two different sizes in insect cells, followed by being released into the culture medium. DNA molecules of 1.5- to 5-kb, which were derived from host insect cells, were packaged in large, ~50-nm spherical particles but not in small, ~25-nm particles. Structure reconstruction using cryo-electron microscopy showed that large MCPyV-LPs are composed of 72 pentameric capsomeres arranged in a T = 7 icosahedral surface lattice and are 48 nm in diameter. The MCPyV-LPs did not share antigenic determinants with BK- and JC viruses (BKPyV and JCPyV). The VLP-based enzyme immunoassay was applied to investigate age-specific prevalence of MCPyV infection in the general Japanese population aged 1-70 years. While seroprevalence of MCPyV increased with age in children and young individuals, its seropositivity in each age group was lower compared with BKPyV and JCPyV.

  20. Characterization of self-assembled virus-like particles of Merkel cell polyomavirus.

    Directory of Open Access Journals (Sweden)

    Tian-Cheng Li

    Full Text Available In our recombinant baculovirus system, VP1 protein of merkel cell polyomavirus (MCPyV, which is implicated as a causative agent in Merkel cell carcinoma, was self-assembled into MCPyV-like particles (MCPyV-LP with two different sizes in insect cells, followed by being released into the culture medium. DNA molecules of 1.5- to 5-kb, which were derived from host insect cells, were packaged in large, ~50-nm spherical particles but not in small, ~25-nm particles. Structure reconstruction using cryo-electron microscopy showed that large MCPyV-LPs are composed of 72 pentameric capsomeres arranged in a T = 7 icosahedral surface lattice and are 48 nm in diameter. The MCPyV-LPs did not share antigenic determinants with BK- and JC viruses (BKPyV and JCPyV. The VLP-based enzyme immunoassay was applied to investigate age-specific prevalence of MCPyV infection in the general Japanese population aged 1-70 years. While seroprevalence of MCPyV increased with age in children and young individuals, its seropositivity in each age group was lower compared with BKPyV and JCPyV.

  1. Isolated Potato Virus A coat protein possesses unusual properties and forms different short virus-like particles.

    Science.gov (United States)

    Ksenofontov, Alexander L; Dobrov, Eugeny N; Fedorova, Natalia V; Serebryakova, Marina V; Prusov, Andrei N; Baratova, Ludmila A; Paalme, Viiu; Järvekülg, Lilian; Shtykova, Eleonora V

    2017-06-08

    In our previous study, we have observed that the isolated coat proteins (CP) of the Potyvirus Potato Virus A (PVA) virions exhibit an intrinsic tendency to self-associate into various multimeric forms containing some fractions of cross-β-structure. In this report, we studied the effect of solution conditions on the structure and dissociation of isolated PVA CP using a number of complementary physicochemical methods. Analysis of the structure of PVA CP in solution was performed by limited proteolysis with MALDI-TOF mass spectrometry analysis, transmission electron microscopy, intrinsic fluorescence spectroscopy, and synchrotron small angle X-ray scattering (SAXS). Overall structural characteristics of PVA CP obtained by combination of these methods and ab initio shape reconstruction by SAXS show that PVA CP forms large multi-subunit particles. We demonstrate that a mixture of compact virus-like particles (VLP) longer than 30 nm is assembled on dialysis of isolated CP into neutral pH buffer (at low ionic strength). Under conditions of high ionic strength (0.5 M NaCl) and high pH (pH 10.5), PVA dissociates into low compactness oval-shaped particles of approximately 30 subunits (20-30 nm). The results of limited trypsinolysis of these particles (enzyme/substrate ratio 1:100, 30 min) showed the existence of non-cleavable core-fragment, consisting of 137 amino acid residues. Trypsin treatment removed only a short N-terminal fragment in the intact virions. These particles are readily reassembled into regular VLPs by changing pH back to neutral. It is possible that these particles may represent some kind of intermediate in PVA assembly in vitro and in vivo.

  2. HIV/AIDS Vaccine Candidates Based on Replication-Competent Recombinant Poxvirus NYVAC-C-KC Expressing Trimeric gp140 and Gag-Derived Virus-Like Particles or Lacking the Viral Molecule B19 That Inhibits Type I Interferon Activate Relevant HIV-1-Specific B and T Cell Immune Functions in Nonhuman Primates

    Science.gov (United States)

    García-Arriaza, Juan; Perdiguero, Beatriz; Heeney, Jonathan L.; Seaman, Michael S.; Montefiori, David C.; Yates, Nicole L.; Tomaras, Georgia D.; Ferrari, Guido; Foulds, Kathryn E.; Roederer, Mario; Self, Steven G.; Borate, Bhavesh; Gottardo, Raphael; Phogat, Sanjay; Tartaglia, Jim; Barnett, Susan W.; Burke, Brian; Cristillo, Anthony D.; Weiss, Deborah E.; Lee, Carter; Kibler, Karen V.; Jacobs, Bertram L.; Wagner, Ralf; Ding, Song; Pantaleo, Giuseppe

    2017-01-01

    ABSTRACT The nonreplicating attenuated poxvirus vector NYVAC expressing clade C(CN54) HIV-1 Env(gp120) and Gag-Pol-Nef antigens (NYVAC-C) showed limited immunogenicity in phase I clinical trials. To enhance the capacity of the NYVAC vector to trigger broad humoral responses and a more balanced activation of CD4+ and CD8+ T cells, here we compared the HIV-1-specific immunogenicity elicited in nonhuman primates immunized with two replicating NYVAC vectors that have been modified by the insertion of the K1L and C7L vaccinia virus host range genes and express the clade C(ZM96) trimeric HIV-1 gp140 protein or a Gag(ZM96)-Pol-Nef(CN54) polyprotein as Gag-derived virus-like particles (termed NYVAC-C-KC). Additionally, one NYVAC-C-KC vector was generated by deleting the viral gene B19R, an inhibitor of the type I interferon response (NYVAC-C-KC-ΔB19R). An immunization protocol mimicking that of the RV144 phase III clinical trial was used. Two groups of macaques received two doses of the corresponding NYVAC-C-KC vectors (weeks 0 and 4) and booster doses with NYVAC-C-KC vectors plus the clade C HIV-1 gp120 protein (weeks 12 and 24). The two replicating NYVAC-C-KC vectors induced enhanced and similar HIV-1-specific CD4+ and CD8+ T cell responses, similar levels of binding IgG antibodies, low levels of IgA antibodies, and high levels of antibody-dependent cellular cytotoxicity responses and HIV-1-neutralizing antibodies. Small differences within the NYVAC-C-KC-ΔB19R group were seen in the magnitude of CD4+ and CD8+ T cells, the induction of some cytokines, and the neutralization of some HIV-1 isolates. Thus, replication-competent NYVAC-C-KC vectors acquired relevant immunological properties as vaccine candidates against HIV/AIDS, and the viral B19 molecule exerts some control of immune functions. IMPORTANCE It is of special importance to find a safe and effective HIV/AIDS vaccine that can induce strong and broad T cell and humoral immune responses correlating with HIV-1

  3. HIV/AIDS Vaccine Candidates Based on Replication-Competent Recombinant Poxvirus NYVAC-C-KC Expressing Trimeric gp140 and Gag-Derived Virus-Like Particles or Lacking the Viral Molecule B19 That Inhibits Type I Interferon Activate Relevant HIV-1-Specific B and T Cell Immune Functions in Nonhuman Primates.

    Science.gov (United States)

    García-Arriaza, Juan; Perdiguero, Beatriz; Heeney, Jonathan L; Seaman, Michael S; Montefiori, David C; Yates, Nicole L; Tomaras, Georgia D; Ferrari, Guido; Foulds, Kathryn E; Roederer, Mario; Self, Steven G; Borate, Bhavesh; Gottardo, Raphael; Phogat, Sanjay; Tartaglia, Jim; Barnett, Susan W; Burke, Brian; Cristillo, Anthony D; Weiss, Deborah E; Lee, Carter; Kibler, Karen V; Jacobs, Bertram L; Wagner, Ralf; Ding, Song; Pantaleo, Giuseppe; Esteban, Mariano

    2017-05-01

    The nonreplicating attenuated poxvirus vector NYVAC expressing clade C(CN54) HIV-1 Env(gp120) and Gag-Pol-Nef antigens (NYVAC-C) showed limited immunogenicity in phase I clinical trials. To enhance the capacity of the NYVAC vector to trigger broad humoral responses and a more balanced activation of CD4+ and CD8+ T cells, here we compared the HIV-1-specific immunogenicity elicited in nonhuman primates immunized with two replicating NYVAC vectors that have been modified by the insertion of the K1L and C7L vaccinia virus host range genes and express the clade C(ZM96) trimeric HIV-1 gp140 protein or a Gag(ZM96)-Pol-Nef(CN54) polyprotein as Gag-derived virus-like particles (termed NYVAC-C-KC). Additionally, one NYVAC-C-KC vector was generated by deleting the viral gene B19R, an inhibitor of the type I interferon response (NYVAC-C-KC-ΔB19R). An immunization protocol mimicking that of the RV144 phase III clinical trial was used. Two groups of macaques received two doses of the corresponding NYVAC-C-KC vectors (weeks 0 and 4) and booster doses with NYVAC-C-KC vectors plus the clade C HIV-1 gp120 protein (weeks 12 and 24). The two replicating NYVAC-C-KC vectors induced enhanced and similar HIV-1-specific CD4+ and CD8+ T cell responses, similar levels of binding IgG antibodies, low levels of IgA antibodies, and high levels of antibody-dependent cellular cytotoxicity responses and HIV-1-neutralizing antibodies. Small differences within the NYVAC-C-KC-ΔB19R group were seen in the magnitude of CD4+ and CD8+ T cells, the induction of some cytokines, and the neutralization of some HIV-1 isolates. Thus, replication-competent NYVAC-C-KC vectors acquired relevant immunological properties as vaccine candidates against HIV/AIDS, and the viral B19 molecule exerts some control of immune functions.IMPORTANCE It is of special importance to find a safe and effective HIV/AIDS vaccine that can induce strong and broad T cell and humoral immune responses correlating with HIV-1 protection

  4. Antiviral Activity of Gold/Copper Sulfide Core/Shell Nanoparticles against Human Norovirus Virus-Like Particles.

    Directory of Open Access Journals (Sweden)

    Jessica Jenkins Broglie

    Full Text Available Human norovirus is a leading cause of acute gastroenteritis worldwide in a plethora of residential and commercial settings, including restaurants, schools, and hospitals. Methods for easily detecting the virus and for treating and preventing infection are critical to stopping norovirus outbreaks, and inactivation via nanoparticles (NPs is a more universal and attractive alternative to other physical and chemical approaches. Using norovirus GI.1 (Norwalk virus-like particles (VLPs as a model viral system, this study characterized the antiviral activity of Au/CuS core/shell nanoparticles (NPs against GI.1 VLPs for the rapid inactivation of HuNoV. Inactivation of VLPs (GI.1 by Au/CuS NPs evaluated using an absorbance-based ELISA indicated that treatment with 0.083 μM NPs for 10 min inactivated ~50% VLPs in a 0.37 μg/ml VLP solution and 0.83 μM NPs for 10 min completely inactivated the VLPs. Increasing nanoparticle concentration and/or VLP-NP contact time significantly increased the virucidal efficacy of Au/CuS NPs. Changes to the VLP particle morphology, size, and capsid protein were characterized using dynamic light scattering, transmission electron microscopy, and Western blot analysis. The strategy reported here provides the first reported proof-of-concept Au/CuS NPs-based virucide for rapidly inactivating human norovirus.

  5. ALIX/AIP1 is required for NP incorporation into Mopeia virus Z-induced virus-like particles.

    Science.gov (United States)

    Shtanko, Olena; Watanabe, Shinji; Jasenosky, Luke D; Watanabe, Tokiko; Kawaoka, Yoshihiro

    2011-04-01

    During virus particle assembly, the arenavirus nucleoprotein (NP) associates with the viral genome to form nucleocapsids, which ultimately become incorporated into new virions at the cell membrane. Virion release is facilitated by the viral matrix Z protein through its interaction with the cellular endosomal sorting complex required for transport (ESCRT) machinery. However, the mechanism of nucleocapsid incorporation into virions is not well understood. Here, we demonstrate that ALIX/AIP1, an ESCRT-associated host protein, is required for the incorporation of the NP of Mopeia virus, a close relative of Lassa virus, into Z-induced virus-like particles (VLPs). Furthermore, we show that the Bro1 domain of ALIX/AIP1 interacts with the NP and Z proteins simultaneously, facilitating their interaction, and we identify residues 342 to 399 of NP as being necessary for its interaction with ALIX/AIP1. Our observations suggest a potential role for ALIX/AIP1 in linking Mopeia virus NP to Z and the budding apparatus, thereby promoting NP incorporation into virions.

  6. An Envelope-Modified Tetravalent Dengue Virus-Like-Particle Vaccine Has Implications for Flavivirus Vaccine Design.

    Science.gov (United States)

    Urakami, Akane; Ngwe Tun, Mya Myat; Moi, Meng Ling; Sakurai, Atsuko; Ishikawa, Momoko; Kuno, Sachiko; Ueno, Ryuji; Morita, Kouichi; Akahata, Wataru

    2017-12-01

    Dengue viruses (DENV) infect 50 to 100 million people each year. The spread of DENV-associated infections is one of the most serious public health problems worldwide, as there is no widely available vaccine or specific therapeutic for DENV infections. To address this, we developed a novel tetravalent dengue vaccine by utilizing virus-like particles (VLPs). We created recombinant DENV1 to -4 (DENV1-4) VLPs by coexpressing precursor membrane (prM) and envelope (E) proteins, with an F108A mutation in the fusion loop structure of E to increase the production of VLPs in mammalian cells. Immunization with DENV1-4 VLPs as individual, monovalent vaccines elicited strong neutralization activity against each DENV serotype in mice. For use as a tetravalent vaccine, DENV1-4 VLPs elicited high levels of neutralization activity against all four serotypes simultaneously. The neutralization antibody responses induced by the VLPs were significantly higher than those with DNA or recombinant E protein immunization. Moreover, antibody-dependent enhancement (ADE) was not observed against any serotype at a 1:10 serum dilution. We also demonstrated that the Zika virus (ZIKV) VLP production level was enhanced by introducing the same F108A mutation into the ZIKV envelope protein. Taken together, these results suggest that our strategy for DENV VLP production is applicable to other flavivirus VLP vaccine development, due to the similarity in viral structures, and they describe the promising development of an effective tetravalent vaccine against the prevalent flavivirus.IMPORTANCE Dengue virus poses one of the most serious public health problems worldwide, and the incidence of diseases caused by the virus has increased dramatically. Despite decades of effort, there is no effective treatment against dengue. A safe and potent vaccine against dengue is still needed. We developed a novel tetravalent dengue vaccine by using virus-like particles (VLPs), which are noninfectious because they lack

  7. [Generation of Japanese Encephalitis Virus-like Particle Vaccine and Preliminary Evaluation of Its Protective Efficiency].

    Science.gov (United States)

    Zhang, Yanfang; Du, Ruikun; Huang, Shaomei; Zhang, Tao; Liu, Jinliang; Zhu, Bibo; Wang, Hualin; Deng, Fei; Cao, Shengbo

    2016-03-01

    The cDNA fragment of JEV prME gene was cloned into the baculovirus shuttle vector (bacmid) to construct a recombinant baculovirus vector, defined as AcBac-prME. Then the recombinant baculovirus Ac-prME was obtained by transfecting Sf9 cells with AcBac-prME. Western blot analysis and immunofluorescence results indicated that both prM and E proteins were efficiently expressed in Sf9 cells. Electron microscopy suggested that prME was assembled into JEV-VLPs. To further evaluate the potential of JEV-VLPs as vaccine, the mice were immunized with JEV-VLPs and then challenged with lethal JEV. The results of mice survival and pathological changes demonstrated that the JEV-VLPs performed complete protection against JEV-P3 strain and relieved pathological changes in the mice brain significant. This study suggest that JEV-VLPs would be a potential vaccine for Japanese encephalitis virus.

  8. Preparation of quadri-subtype influenza virus-like particles using bovine immunodeficiency virus gag protein

    Energy Technology Data Exchange (ETDEWEB)

    Tretyakova, Irina; Hidajat, Rachmat; Hamilton, Garrett; Horn, Noah; Nickols, Brian; Prather, Raphael O. [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD (United States); Tumpey, Terrence M. [Influenza Division, Centers for Disease Control and Prevention, 1600 Clifton Road N.E., Atlanta, GA (United States); Pushko, Peter, E-mail: ppushko@medigen-usa.com [Medigen, Inc., 8420 Gas House Pike, Suite S, Frederick, MD (United States)

    2016-01-15

    Influenza VLPs comprised of hemagglutinin (HA), neuraminidase (NA), and matrix (M1) proteins have been previously used for immunological and virological studies. Here we demonstrated that influenza VLPs can be made in Sf9 cells by using the bovine immunodeficiency virus gag (Bgag) protein in place of M1. We showed that Bgag can be used to prepare VLPs for several influenza subtypes including H1N1 and H10N8. Furthermore, by using Bgag, we prepared quadri-subtype VLPs, which co-expressed within the VLP the four HA subtypes derived from avian-origin H5N1, H7N9, H9N2 and H10N8 viruses. VLPs showed hemagglutination and neuraminidase activities and reacted with specific antisera. The content and co-localization of each HA subtype within the quadri-subtype VLP were evaluated. Electron microscopy showed that Bgag-based VLPs resembled influenza virions with the diameter of 150–200 nm. This is the first report of quadri-subtype design for influenza VLP and the use of Bgag for influenza VLP preparation. - Highlights: • BIV gag protein was configured as influenza VLP core component. • Recombinant influenza VLPs were prepared in Sf9 cells using baculovirus expression system. • Single- and quadri-subtype VLPs were prepared by using BIV gag as a VLP core. • Co-localization of H5, H7, H9, and H10 HA was confirmed within quadri-subtype VLP. • Content of HA subtypes within quadri-subtype VLP was determined. • Potential advantages of quadri-subtype VLPs as influenza vaccine are discussed.

  9. Prevalence of virus-like particles within a staghorn scleractinian coral ( Acropora muricata) from the Great Barrier Reef

    Science.gov (United States)

    Patten, N. L.; Harrison, P. L.; Mitchell, J. G.

    2008-09-01

    Transmission electron microscopy (TEM) was used to determine whether Acropora muricata coral colonies from the Great Barrier Reef (GBR), Australia, harboured virus-like particles (VLPs). VLPs were present in all coral colonies sampled at Heron Island (southern GBR) and in tagged coral colonies sampled in at least two of the three sampling periods at Lizard Island (northern GBR). VLPs were observed within gastrodermal and epidermal tissues, and on rarer occasions, within the mesoglea. These VLPs had similar morphologies to known prokaryotic and eukaryotic viruses in other systems. Icosahedral VLPs were observed most frequently, however, filamentous VLPs (FVLPs) and phage were also noted. There were no clear differences in VLP size, morphology or location within the tissues with respect to sample date, coral health status or site. The most common VLP morphotype exhibited icosahedral symmetry, 120-150 nm in diameter, with an electron-dense core and an electronlucent membrane. Larger VLPs of similar morphology were also common. VLPs occurred as single entities, in groups, or in dense clusters, either as free particles within coral tissues, or within membrane-bound vacuoles. VLPs were commonly observed within the perinuclear region, with mitochondria, golgi apparatus and crescent-shaped particles frequently observed within close proximity. The host(s) of these observed VLPs was not clear; however, the different sizes and morphologies of VLPs observed within A. muricata tissues suggest that viruses are infecting either the coral animal, zooxanthellae, intracellular bacteria and/or other coral-associated microbiota, or that the one host is susceptible to infection from more than one type of virus. These results add to the limited but emerging body of evidence that viruses represent another potentially important component of the coral holobiont.

  10. Energetic changes caused by antigenic module insertion in a virus-like particle revealed by experiment and molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Lin Zhang

    Full Text Available The success of recombinant virus-like particles (VLPs for human papillomavirus and hepatitis B demonstrates the potential of VLPs as safe and efficacious vaccines. With new modular designs emerging, the effects of antigen module insertion on the self-assembly and structural integrity of VLPs should be clarified so as to better enabling improved design. Previous work has revealed insights into the molecular energetics of a VLP subunit, capsomere, comparing energetics within various solution conditions known to drive or inhibit self-assembly. In the present study, molecular dynamics (MD simulations coupled with the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA method were performed to examine the molecular interactions and energetics in a modular capsomere of a murine polyomavirus (MPV VLP designed to protect against influenza. Insertion of an influenza antigenic module is found to lower the binding energy within the capsomere, and a more active state is observed in Assembly Buffer as compared with that in Stabilization Buffer, which has been experimentally validated through measurements using differential scanning calorimetry. Further in-depth analysis based on free-energy decomposition indicates that destabilized binding can be attributed to electrostatic interaction induced by the chosen antigen module. These results provide molecular insights into the conformational stability of capsomeres and their abilities to be exploited for antigen presentation, and are expected to be beneficial for the biomolecular engineering of VLP vaccines.

  11. Chimeric virus-like particles for the delivery of an inserted conserved influenza A-specific CTL epitope.

    Science.gov (United States)

    Cheong, Wan-Shoo; Reiseger, Jessica; Turner, Stephen John; Boyd, Richard; Netter, Hans-Jürgen

    2009-02-01

    The small hepatitis B virus surface antigens (HBsAg-S) have the ability to self-assemble with host-derived lipids into empty non-infectious virus-like particles (VLPs). HBsAg-S VLPs are the sole component of the licensed hepatitis B vaccine, and they are a useful delivery platform for foreign epitopes. To develop VLPs capable of transporting foreign cytotoxic T lymphocyte (CTL) epitopes, HBsAg-S specific CTL epitopes at various sites were substituted with a conserved CTL epitope derived from the influenza matrix protein. Depending on the insertion site, the introduction of the MHC class I A2.1-restricted influenza epitope was compatible with the secretion competence of HBsAg-S indicating that chimeric VLPs were assembled. Immunizations of transgenic HHDII mice with chimeric VLPs induced anti-influenza CTL responses proving that the inserted foreign epitope can be correctly processed and cross-presented. Chimeric VLPs in the absence of adjuvant were able to induce memory T cell responses, which could be recalled by influenza virus infections in the mouse model system. The ability of chimeric HBsAg-S VLPs to induce anti-foreign CTL responses and also with the proven ability to induce humoral immune responses constitute a highly versatile platform for the delivery of selected multiple epitopes to target disease associated infectious agents.

  12. Protection induced by virus-like particle vaccine containing tandem repeat gene of respiratory syncytial virus G protein.

    Science.gov (United States)

    Kim, Ah-Ra; Lee, Dong-Hun; Lee, Su-Hwa; Rubino, Ilaria; Choi, Hyo-Jick; Quan, Fu-Shi

    2018-01-01

    Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants, young children and the elderly. However, there is no licensed vaccine available against RSV infection. In this study, we generated virus-like particle (VLP) vaccine and investigated the vaccine efficacy in a mouse model. For VLP vaccines, tandem gene (1-780 bp) for V1 VLPs and tandem repeat gene (repeated 450-780 bp) for V5 VLPs were constructed in pFastBacTM vectors, respectively. Influenza matrix protein 1 (M1) was used as a core protein in the VLPs. Notably, upon challenge infection, significantly lower virus loads were measured in the lung of mice immunized with V1 or V5 VLPs compared to those of naïve mice and formalin-inactivated RSV immunized control mice. In particular, V5 VLPs immunization showed significantly lower virus titers than V1 VLPs immunization. Furthermore, V5 VLPs immunization elicited increased memory B cells responses in the spleen. These results indicated that V5 VLP vaccine containing tandem repeat gene protein provided better protection than V1 VLPs with significantly decreased inflammation in the lungs. Thus, V5 VLPs could be a potential vaccine candidate against RSV.

  13. Co-immunization with virus-like particle and DNA vaccines induces protection against respiratory syncytial virus infection and bronchiolitis.

    Science.gov (United States)

    Hwang, Hye Suk; Kwon, Young-Man; Lee, Jong Seok; Yoo, Si-Eun; Lee, Yu-Na; Ko, Eun-Ju; Kim, Min-Chul; Cho, Min-Kyoung; Lee, Young-Tae; Jung, Yu-Jin; Lee, Ji-Yun; Li, Jian-Dong; Kang, Sang-Moo

    2014-10-01

    This study demonstrates that immunization with non-replicating virus-like particle (FFG VLP) containing RSV F and G glycoproteins together with RSV F DNA induced T helper type 1 antibody responses to RSV F similar to live RSV infection. Upon RSV challenge 21weeks after immunization, FFG VLP vaccination induced protection against RSV infection as shown by clearance of lung viral loads, and the absence of eosinophil infiltrates, and did not cause lung pathology. In contrast, formalin-inactivated RSV (FI-RSV) vaccination showed significant pulmonary eosinophilia, severe mucus production, and extensive histopathology resulting in a hallmark of pulmonary pathology. Substantial lung pathology was also observed in mice with RSV re-infections. High levels of systemic and local inflammatory cytokine-secreting cells were induced in mice with FI-RSV but not with FFG VLP immunization after RSV challenge. Therefore, the results provide evidence that recombinant RSV FFG VLP vaccine can confer long-term protection against RSV without causing lung pathology. Copyright © 2014 Elsevier B.V. All rights reserved.

  14. Immunogenicity and specificity of norovirus Consensus GII.4 virus-like particles in monovalent and bivalent vaccine formulations.

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    Parra, Gabriel I; Bok, Karin; Taylor, Ross; Haynes, Joel R; Sosnovtsev, Stanislav V; Richardson, Charles; Green, Kim Y

    2012-05-21

    Noroviruses, a major cause of acute gastroenteritis worldwide, present antigenic diversity that must be considered for the development of an effective vaccine. In this study, we explored approaches to increase the broad reactivity of virus-like particle (VLP) norovirus vaccine candidates. The immunogenicity of a GII.4 "Consensus" VLP that was engineered from sequences of three genetically distinct naturally occurring GII.4 strains was examined for its ability to induce cross-reactive immune responses against different clusters of GII.4 noroviruses. Rabbits immunized with GII.4 Consensus VLPs developed high serum antibody titers against VLPs derived from a number of distinct wild-type GII.4 viruses, including some that had been circulating over 30 years ago. Because the sera exhibited low cross-reactivity with antigenically distinct GI norovirus strains, we investigated the serum antibody response to a bivalent vaccine formulation containing GI.1 (Norwalk virus) and GII.4 Consensus VLPs that was administered to animals under varying conditions. In these studies, the highest homologous and heterologous antibody titers to the bivalent vaccine were elicited following immunization of animals by the intramuscular route using Alhydrogel (Al(OH)(3)) as adjuvant. Our data indicate that the use of both genetically engineered norovirus VLPs that incorporate relevant epitopes from multiple strains and multivalent vaccine formulations increase the breadth of the immune response to diverse variants within a genotype and, thus, prove helpful in the rational design of VLP-based vaccines against human noroviruses. Published by Elsevier Ltd.

  15. Long-term protective immunity from an influenza virus-like particle vaccine administered with a microneedle patch.

    Science.gov (United States)

    Quan, Fu-Shi; Kim, Yeu-Chun; Song, Jae-Min; Hwang, Hye Suk; Compans, Richard W; Prausnitz, Mark R; Kang, Sang-Moo

    2013-09-01

    Skin vaccination with influenza virus-like particles (VLPs) using microneedles has been shown to induce protection similar to or better than that induced by intramuscular immunization. In this study, we examined the long-term protective efficacy of influenza (H1N1 A/PR/8/34) VLPs after skin vaccination using microneedle patches coated with the vaccine. Microneedle vaccination of mice in the skin induced 100% protection against lethal challenge infection with influenza A/PR/8/34 virus 14 months after a single vaccine dose. Influenza virus-specific total IgG response and hemagglutination inhibition (HAI) titers were maintained at high levels for over 1 year after microneedle vaccination. Microneedle vaccination also induced substantial levels of lung IgG and IgA antibody responses, and antibody-secreting plasma cells from spleen and bone marrow, as well as conferring effective control of lung viral loads, resulting in complete protection 14 months after vaccination. These strong and long-lasting immune responses were enabled in part by stabilization of the vaccine by formulation with trehalose during microneedle patch fabrication. Administration of the stabilized vaccine using microneedles was especially effective at enabling strong recall responses measured 4 days after lethal virus challenge, including increased HAI and antibody-secreting cells in the spleen and reduced viral titer and inflammatory response in the lung. The results in this study indicate that skin vaccination with VLP vaccine using a microneedle patch provides long-term protection against influenza in mice.

  16. Effect of HPV16 L1 virus-like particles on the aggregation of non-functionalized gold nanoparticles.

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    Palomino-Vizcaino, Giovanni; Valencia Reséndiz, Diana Gabriela; Benítez-Hess, María Luisa; Martínez-Acuña, Natalia; Tapia-Vieyra, Juana Virginia; Bahena, Daniel; Díaz-Sánchez, Mauricio; García-González, Octavio Patricio; Alvarez-Sandoval, Brenda Arizaí; Alvarez-Salas, Luis Marat

    2018-02-15

    Colorimetric assays based on gold nanoparticles (GNPs) are of considerable interest for diagnostics because of their simplicity and low-cost. Nevertheless, a deep understanding of the interaction between the GNPs and the intended molecular target is critical for the development of reliable detection technologies. The present report describes the spontaneous interaction between HPV16 L1 virus-like particles (VLPs) and non-functionalized GNPs (nfGNPs) resulting in the inhibition of nfGNPs salt-induced aggregation and the stabilization of purified VLPs. Ionic-competition experiments suggested that the nature of nfGNPs-VLPs interaction is non-covalent. Adsorption of an RNA aptamer on nfGNPs surface showed an additive aggregation-inhibitory effect. The use of mutant VLPs confirmed that the interaction nfGNPs-VLPs is not mediated by the opposing superficial electrostatic charges, suggesting that non-electrostatic forces participate in the arrangement of nfGNPs on the VLPs surface. Competition experiments using increasing ethanol concentrations on nfGNPs-VLPs complexes suggested hydrophobic interactions as the main stabilizing force. Therefore, the nfGNPs-VLPs interaction described here should facilitate the development of adsorption assays based on nfGNPs for HPV detection and cervical cancer prevention. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Time-controlled phagocytosis of asymmetric liposomes: Application to phosphatidylserine immunoliposomes binding HIV-1 virus-like particles.

    Science.gov (United States)

    Petazzi, Roberto Arturo; Gramatica, Andrea; Herrmann, Andreas; Chiantia, Salvatore

    2015-11-01

    Macrophage immune functions such as antibody-mediated phagocytosis are strongly impaired in individuals affected by HIV-1. Nevertheless, infected macrophages are still able to phagocytose apoptotic cells. For this reason, we recently developed antibody-decorated phosphatidylserine (PS)-containing liposomes that bind HIV-1 virus-like particles and, by mimicking apoptotic cells, are efficiently internalized by macrophages. In the context of an in vivo application, it would be extremely important to initially protect immunoliposomes from macrophages, in order to provide enough time to redistribute through the body and achieve maximum virus binding. To this end, we have designed asymmetric immunoliposomes in which the PS is initially confined to the inner leaflet and thus cannot be recognized by macrophages. Spontaneous PS flip-flop to the outer surface leads to a time-delay in internalization by macrophages in vitro. Such a delay can be fine-tuned by altering the molecular composition of the immunoliposomes. In the fight against HIV-1, macrophage plays an important role. Ironically, the phagocytic functions of these cells are often impaired by HIV-1. In this interesting article, the authors described the development of asymmetric liposomes, which would bind HIV-1 with prolonged systemic circulation, such that the clearance of virus by macrophages is enhanced. This system represents a promising effective approach to utilize the phagocytic capability of macrophages. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Enterovirus type 71 neutralizing antibodies in the serum of macaque monkeys immunized with EV71 virus-like particles.

    Science.gov (United States)

    Lin, Yu-Li; Yu, Chun-I; Hu, Yu-Chen; Tsai, Tze-Jiun; Kuo, Yin-Chieh; Chi, Wei-Kuang; Lin, Ae-Ning; Chiang, Bor-Luen

    2012-02-08

    Enterovirus type 71 (EV71) is a virulent form of enteroviruses causing hospitalizations for children less than three years of age. Currently there are no anti-viral therapies or vaccines available for EV71. Due to the high risk of poliomyelitis-like paralysis and fatal encephalitis, an effective vaccine to EV71 could potentially prevent virus-induced morbidity and mortality. In this study, we first tested a potential EV71 vaccine candidate based on virus-like particles (VLP). We vaccinated macaque monkeys to validate the immunogenicity of the VLP vaccine to EV71. We detected the VLP or EV71-specific antibodies, neutralization titers, ELISPOT, and T cell response to find their immune responses to EV71. When the VLP vaccine adjuvanted with alum was given to macaque monkeys, these monkeys developed both specific humoral and cellular immune responses to EV71. Despite lower neutralizing antibodies to EV71 were found in sera of VLP-immunized monkeys than monkeys vaccinated with inactivated EV71, VLP-based vaccine generated a memory immune response to EV71. Hence, VLP-based EV71 vaccine is a potential vaccine against EV71 infection. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. Induction of long-term protective immune responses by influenza H5N1 virus-like particles.

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    Sang-Moo Kang

    Full Text Available Recurrent outbreaks of highly pathogenic H5N1 avian influenza virus pose a threat of eventually causing a pandemic. Early vaccination of the population would be the single most effective measure for the control of an emerging influenza pandemic.Influenza virus-like particles (VLPs produced in insect cell-culture substrates do not depend on the availability of fertile eggs for vaccine manufacturing. We produced VLPs containing influenza A/Viet Nam1203/04 (H5N1 hemagglutinin, neuraminidase, and matrix proteins, and investigated their preclinical immunogenicity and protective efficacy. Mice immunized intranasally with H5N1 VLPs developed high levels of H5N1 specific antibodies and were 100% protected against a high dose of homologous H5N1 virus infection at 30 weeks after immunization. Protection is likely to be correlated with humoral and cellular immunologic memory at systemic and mucosal sites as evidenced by rapid anamnestic responses to re-stimulation with viral antigen in vivo and in vitro.These results provide support for clinical evaluation of H5N1 VLP vaccination as a public health intervention to mitigate a possible pandemic of H5N1 influenza.

  20. Virus-like particle vaccine primes immune responses preventing inactivated-virus vaccine-enhanced disease against respiratory syncytial virus.

    Science.gov (United States)

    Hwang, Hye Suk; Lee, Young-Tae; Kim, Ki-Hye; Ko, Eun-Ju; Lee, Youri; Kwon, Young-Man; Kang, Sang-Moo

    2017-11-01

    Formalin inactivated respiratory syncytial virus (FI-RSV) vaccination caused vaccine-enhanced respiratory disease (ERD) upon exposure to RSV in children. Virus-like particles presenting RSV F fusion protein (F VLP) are known to increase T helper type-1 (Th1) immune responses and avoid ERD in animal models. We hypothesized that F VLP would prime immune responses preventing ERD upon subsequent exposure to ERD-prone FI-RSV. Here, we demonstrated that heterologous F VLP priming and FI-RSV boosting of mice prevented FI-RSV vaccine-enhanced lung inflammation and eosinophilia upon RSV challenge. F VLP priming redirected pulmonary T cells toward effector CD8 T cells producing Th1 cytokines and significantly suppressed pulmonary Th2 cytokines. This study suggests that RSV F VLP priming would modulate and shift immune responses to subsequent exposure to ERD-prone FI-RSV vaccine and RSV infection, suppressing Th2 immune-mediated pulmonary histopathology and eosinophilia. Copyright © 2017. Published by Elsevier Inc.

  1. On the effect of thermodynamic equilibrium on the assembly efficiency of complex multi-layered virus-like particles (VLP: the case of rotavirus VLP.

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    António Roldão

    Full Text Available Previous studies have reported the production of malformed virus-like-particles (VLP in recombinant host systems. Here we computationally investigate the case of a large triple-layered rotavirus VLP (RLP. In vitro assembly, disassembly and reassembly data provides strong evidence of microscopic reversibility of RLP assembly. Light scattering experimental data also evidences a slow and reversible assembly untypical of kinetic traps, thus further strengthening the fidelity of a thermodynamically controlled assembly. In silico analysis further reveals that under favourable conditions particles distribution is dominated by structural subunits and completely built icosahedra, while other intermediates are present only at residual concentrations. Except for harshly unfavourable conditions, assembly yield is maximised when proteins are provided in the same VLP protein mass composition. The assembly yield decreases abruptly due to thermodynamic equilibrium when the VLP protein mass composition is not obeyed. The latter effect is more pronounced the higher the Gibbs free energy of subunit association is and the more complex the particle is. Overall this study shows that the correct formation of complex multi-layered VLPs is restricted to a narrow range of association energies and protein concentrations, thus the choice of the host system is critical for successful assembly. Likewise, the dynamic control of intracellular protein expression rates becomes very important to minimize wasted proteins.

  2. Efficient self-assembly of human papillomavirus type 16 L1 and L1-L2 into virus-like particles.

    Science.gov (United States)

    Kirnbauer, R; Taub, J; Greenstone, H; Roden, R; Dürst, M; Gissmann, L; Lowy, D R; Schiller, J T

    1993-12-01

    The L1 genes of two human papillomavirus type 16 (HPV16) isolates derived from condylomata acuminata were used to express the L1 major capsid protein in insect cells via recombinant baculoviruses. Both L1 major capsid proteins self-assembled into virus-like particles (VLP) with high efficiency and could be purified in preparative amounts on density gradients. The yield of VLP was 3 orders of magnitude higher than what has been obtained previously, using L1 derived from the prototype HPV16. DNA sequence comparison identified a single nonconserved amino acid change to be responsible for the inefficient self-assembly of the prototype L1. VLP were also obtained by expressing L1 of HPV6, HPV11, and cottontail rabbit papillomavirus, indicating that L1 from a variety of papillomaviruses has the intrinsic capacity to self-assemble into VLP. Coexpression of HPV16 L1 plus L2 by using a baculovirus double-expression vector also resulted in efficient self-assembly of VLP, and the average particle yield increased about fourfold in comparison to when L1 only was expressed. Coimmunoprecipitation of L1 and L2 and cosedimentation of the two proteins in a sucrose gradient demonstrated that L2 was incorporated into the particles. The ability to generate preparative amounts of HPV16 L1 and L1-L2 VLP may have implications for the development of a serological assay to detect anti-HPV16 virion immune responses to conformational epitopes and for immunoprophylaxis against HPV16 infection.

  3. A leucine residue in the C terminus of human parainfluenza virus type 3 matrix protein is essential for efficient virus-like particle and virion release.

    Science.gov (United States)

    Zhang, Guangyuan; Zhang, Shengwei; Ding, Binbin; Yang, Xiaodan; Chen, Longyun; Yan, Qin; Jiang, Yanliang; Zhong, Yi; Chen, Mingzhou

    2014-11-01

    Paramyxovirus particles, like other enveloped virus particles, are formed by budding from membranes of infected cells, and matrix (M) proteins are critical for this process. To identify the M protein important for this process, we have characterized the budding of the human parainfluenza virus type 3 (HPIV3) M protein. Our results showed that expression of the HPIV3 M protein alone is sufficient to initiate the release of virus-like particles (VLPs). Electron microscopy analysis confirmed that VLPs are morphologically similar to HPIV3 virions. We identified a leucine (L302) residue within the C terminus of the HPIV3 M protein that is critical for M protein-mediated VLP production by regulating the ubiquitination of the M protein. When L302 was mutated into A302, ubiquitination of M protein was defective, the release of VLPs was abolished, and the membrane binding and budding abilities of M protein were greatly weakened, but the ML302A mutant retained oligomerization activity and had a dominant negative effect on M protein-mediated VLP production. Furthermore, treatment with a proteasome inhibitor also inhibited M protein-mediated VLP production and viral budding. Finally, recombinant HPIV3 containing the M(L302A) mutant could not be rescued. These results suggest that L302 acts as a critical regulating signal for the ubiquitination of the HPIV3 M protein and virion release. Human parainfluenza virus type 3 (HPIV3) is an enveloped virus with a nonsegmented negative-strand RNA genome. It can cause severe respiratory tract diseases, such as bronchiolitis, pneumonia, and croup in infants and young children. However, no valid antiviral therapy or vaccine is currently available. Thus, further elucidation of its assembly and budding will be helpful in the development of novel therapeutic approaches. Here, we show that a leucine residue (L302) located at the C terminus of the HPIV3 M protein is essential for efficient production of virus-like particles (VLPs). Furthermore

  4. Inhibition of cervical cancer cell growth by human papillomavirus virus-like particles packaged with human papillomavirus oncoprotein short hairpin RNAs.

    Science.gov (United States)

    Bousarghin, Latifa; Touze, Antoine; Gaud, Guillaume; Iochmann, Sophie; Alvarez, Eva; Reverdiau, Pascale; Gaitan, Julien; Jourdan, Marie-Lise; Sizaret, Pierre-Yves; Coursaget, Pierre L

    2009-02-01

    Overexpression of human papillomavirus (HPV E6 and HPV E7) oncogenes in human cervical cells results in the development of cancer, and E6 and E7 proteins are therefore targets for preventing cervical cancer progression. Here, we describe the silencing of E6 and E7 expression in cervical carcinoma cells by RNA interference. In order to increase the efficacy of the RNA interference, HPV pseudovirions coding for a short hairpin RNA (shRNA) sequence were produced. The results indicated the degradation of E6 and E7 mRNAs when shRNA against E6 or E7 were delivered by pseudovirions in HPV-positive cells (CaSki and TC1 cells). E6 silencing resulted in the accumulation of cellular p53 and reduced cell viability. More significant cell death was observed when E7 expression was suppressed. Silencing E6 and E7 and the consequences for cancer cell growth were also investigated in vivo in mice using the capacity of murine TC1 cells expressing HPV-16 E6 and E7 oncogenes to induce fast-growing tumors. Treatment with lentiviruses and HPV virus-like particle vectors coding for an E7 shRNA sequence both resulted in dramatic inhibition of tumor growth. These results show the ability of pseudovirion-delivered shRNA to produce specific gene suppression and provide an effective means of reducing HPV-positive tumor growth.

  5. High-yield production of recombinant virus-like particles of enterovirus 71 in Pichia pastoris and their protective efficacy against oral viral challenge in mice.

    Science.gov (United States)

    Zhang, Chao; Ku, Zhiqiang; Liu, Qingwei; Wang, Xiaoli; Chen, Tan; Ye, Xiaohua; Li, Dapeng; Jin, Xia; Huang, Zhong

    2015-05-11

    Enterovirus 71 (EV71) is one of the major causative pathogens of hand, foot and mouth disease (HFMD), which is highly prevalent in the Asia-Pacific regions. Severe HFMD cases with neurological complications and even death are often associated with EV71 infections. However, no licensed EV71 vaccine is currently available. Recombinant virus-like particles (VLPs) of EV71 have been produced and shown to be a promising vaccine candidate in preclinical studies. However, the performance of current recombinant expression systems for EV71 VLP production remains unsatisfactory with regard to VLP yield and manufacturing procedure, and thus hinders further product development. In this study, we evaluated the expression of EV71 VLPs in Pichia pastoris and determined their protective efficacy in mouse models of EV71 infections. We showed that EV71 VLPs could be produced at high levels up to 4.9% of total soluble protein in transgenic P. pastoris yeast co-expressing P1 and 3CD proteins of EV71. The resulting yeast-produced VLPs potently induced neutralizing antibodies against homologous and heterologous EV71 strains in mice. More importantly, maternal immunization with VLPs protected neonatal mice in both intraperitoneal and oral challenge experiments. Collectively, these results demonstrated the success of simple, high-yield production of EV71 VLPs in transgenic P. pastoris, thus lifting the major roadblock in commercial development of VLP-based EV71 vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Design strategies to address the effect of hydrophobic epitope on stability and in vitro assembly of modular virus-like particle.

    Science.gov (United States)

    Tekewe, Alemu; Connors, Natalie K; Middelberg, Anton P J; Lua, Linda H L

    2016-08-01

    Virus-like particles (VLPs) and capsomere subunits have shown promising potential as safe and effective vaccine candidates. They can serve as platforms for the display of foreign epitopes on their surfaces in a modular architecture. Depending on the physicochemical properties of the antigenic modules, modularization may affect the expression, solubility and stability of capsomeres, and VLP assembly. In this study, three module designs of a rotavirus hydrophobic peptide (RV10) were synthesized using synthetic biology. Among the three synthetic modules, modularization of the murine polyomavirus VP1 with a single copy of RV10 flanked by long linkers and charged residues resulted in the expression of stable modular capsomeres. Further employing the approach of module titration of RV10 modules on each capsomere via Escherichia coli co-expression of unmodified VP1 and modular VP1-RV10 successfully translated purified modular capomeres into modular VLPs when assembled in vitro. Our results demonstrate that tailoring the physicochemical properties of modules to enhance modular capsomeres stability is achievable through synthetic biology designs. Combined with module titration strategy to avoid steric hindrance to intercapsomere interactions, this allows bioprocessing of bacterially produced in vitro assembled modular VLPs. © 2016 The Protein Society.

  7. Self-assembly and release of peste des petits ruminants virus-like particles in an insect cell-baculovirus system and their immunogenicity in mice and goats.

    Directory of Open Access Journals (Sweden)

    Wenchao Li

    Full Text Available Peste des petits ruminants (PPR is an acute, febrile, viral disease of small ruminants that has a significant economic impact. For many viral diseases, vaccination with virus-like particles (VLPs has shown considerable promise as a prophylactic approach; however, the processes of assembly and release of peste des petits ruminants virus (PPRV VLPs are not well characterized, and their immunogenicity in the host is unknown. In this study, VLPs of PPRV were generated in a baculovirus system through simultaneous expression of PPRV matrix (M protein and hemaglutin in (H or fusion (F protein. The released VLPs showed morphology similar to that of the native virus particles. Subcutaneous injection of these VLPs (PPRV-H, PPRV-F into mice and goats elicited PPRV-specific IgG production, increased the levels of virus neutralizing antibodies, and promoted lymphocyte proliferation. Without adjuvants, the immune response induced by the PPRV-H VLPs was comparable to that obtained using equivalent amounts of PPRV vaccine. Thus, our results demonstrated that VLPs containing PPRV M protein and H or F protein are potential "differentiating infected from vaccinated animals" (DIVA vaccine candidates for the surveillance and eradication of PPR.

  8. Generation and characterization of a trackable plant-made influenza H5 virus-like particle (VLP) containing enhanced green fluorescent protein (eGFP).

    Science.gov (United States)

    Young, Katie R; Arthus-Cartier, Guillaume; Yam, Karen K; Lavoie, Pierre-Olivier; Landry, Nathalie; D'Aoust, Marc-André; Vézina, Louis-Philippe; Couture, Manon M-J; Ward, Brian J

    2015-09-01

    Medicago, Inc. has developed an efficient virus-like particle (VLP) vaccine production platform using the Nicotiana benthamiana expression system, and currently has influenza-based products targeting seasonal/pandemic hemagglutinin (HA) proteins in advanced clinical trials. We wished to generate a trackable HA-based VLP that would allow us to study both particle assembly in plants and VLP interactions within the mammalian immune system. To this end, a fusion protein was designed, composed of H5 (from influenza A/Indonesia/05/2005 [H5N1]) with enhanced green fluorescent protein (eGFP). Expression of H5-eGFP in N. benthamiana produced brightly fluorescent ∼160 nm particles resembling H5-VLPs. H5-eGFP-VLPs elicited anti-H5 serologic responses in mice comparable to those elicited by H5-VLPs in almost all assays tested (hemagglutination inhibition/IgG(total)/IgG1/IgG2b/IgG2a:IgG1 ratio), as well as a superior anti-GFP IgG response (mean optical density = 2.52 ± 0.16 sem) to that elicited by soluble GFP (mean optical density = 0.12 ± 0.06 sem). Confocal imaging of N. benthamiana cells expressing H5-eGFP displayed large fluorescent accumulations at the cell periphery, and draining lymph nodes from mice given H5-eGFP-VLPs via footpad injection demonstrated bright fluorescence shortly after administration (10 min), providing proof of concept that the H5-eGFP-protein/VLPs could be used to monitor both VLP assembly and immune trafficking. Given these findings, this novel fluorescent reagent will be a powerful tool to gain further fundamental insight into the biology of influenza VLP vaccines. © FASEB.

  9. Hepatitis B virus-like particles access major histocompatibility class I and II antigen presentation pathways in primary dendritic cells.

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    Moffat, Jessica M; Cheong, Wan-Shoo; Villadangos, José A; Mintern, Justine D; Netter, Hans J

    2013-04-26

    Virus-like particles (VLPs) represent high density displays of viral proteins that efficiently trigger immunity. VLPs composed of the small hepatitis B virus envelope protein (HBsAgS) are useful vaccine platforms that induce humoral and cellular immune responses. Notably, however, some studies suggest HBsAgS VLPs impair dendritic cell (DC) function. Here we investigated HBsAgS VLP interaction with DC subsets and antigen access to major histocompatibility complex (MHC) class I and II antigen presentation pathways in primary DCs. HBsAgS VLPs impaired plasmacytoid DC (pDC) interferon alpha (IFNα) production in response to CpG in vitro, but did not alter conventional DC (cDC) or pDC phenotype when administered in vivo. To assess cellular immune responses, HBsAgS VLPs were generated containing the ovalbumin (OVA) model epitopes OVA(257-264) and OVA(323-339) to access MHCI and MHCII antigen presentation pathways, respectively; both in vitro and following immunisation in vivo. HBsAgS VLP-OVA(257-264) elicited CTL responses in vivo that were not enhanced by inclusion of an additional MHCII helper epitope. HBsAgS VLP-OVA(257-264) administered in vivo was cross-presented by CD8(+) DCs, but not CD8(-) DCs. Therefore, HBsAgS VLPs can deliver antigen to both MHCI and MHCII antigen presentation pathways in primary DCs and promote cytotoxic and helper T cell priming despite their suppressive effect on pDCs. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Rational development of two flowthrough purification strategies for adenovirus type 5 and retro virus-like particles.

    Science.gov (United States)

    Nestola, Piergiuseppe; Peixoto, Cristina; Villain, Louis; Alves, Paula M; Carrondo, Manuel J T; Mota, José P B

    2015-12-24

    We report on the rational design and implementation of flowthrough (FT) platforms for purification of virus vectors (VVs) and virus-like particles (VLPs), combining anion-exchange polyallylamine membranes (Sartobind STIC) and core-shell octylamine resins (CaptoCore 700). In one configuration, the VV bulk is concentrated and conditioned with appropriate buffer in a ultra/diafiltration (UF/DF) unit prior to injection into the STIC chromatography membrane. The FT pool and an intermediate cut of the elution pool of the STIC membrane are admixed and directed to a second UF/DF. Finally, the retentate is injected into a CC700 packed bed adsorber where the purified VVs are collected in the FT pool, whereas the residual amount of DNA and host cell protein (HCP) are discarded in the eluate. The experimental recovery achieved with this downstream processing (DSP) platform is close to 100%, the DNA clearance is roughly a 4-log reduction, and the HCP level is reduced by 5 logs. The platform developed for VLP purification is simpler than the previous one, as the STIC membrane adsorber and CC700 bed are connected in series with no UF/DF unit in between. Experimentally, the FT scheme for VLP purification gave a recovery yield of 45% in the chromatography train; the experimental log reduction of DNA and HCP were 2.0 and 3.5, respectively. These results are in line with other purification strategies in the specific field of enveloped VLPs. Both DSP platforms were successfully developed from an initial design space of the binding of the major contaminant (DNA) to the two ligands, determined by surface plasmon resonance, which was subsequently scaled up and confirmed experimentally. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Identifying SARS-CoV membrane protein amino acid residues linked to virus-like particle assembly.

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    Ying-Tzu Tseng

    Full Text Available Severe acute respiratory syndrome coronavirus (SARS-CoV membrane (M proteins are capable of self-assembly and release in the form of membrane-enveloped vesicles, and of forming virus-like particles (VLPs when coexpressed with SARS-CoV nucleocapsid (N protein. According to previous deletion analyses, M self-assembly involves multiple M sequence regions. To identify important M amino acid residues for VLP assembly, we coexpressed N with multiple M mutants containing substitution mutations at the amino-terminal ectodomain, carboxyl-terminal endodomain, or transmembrane segments. Our results indicate that a dileucine motif in the endodomain tail (218LL219 is required for efficient N packaging into VLPs. Results from cross-linking VLP analyses suggest that the cysteine residues 63, 85 and 158 are not in close proximity to the M dimer interface. We noted a significant reduction in M secretion due to serine replacement for C158, but not for C63 or C85. Further analysis suggests that C158 is involved in M-N interaction. In addition to mutations of the highly conserved 107-SWWSFNPE-114 motif, substitutions at codons W19, W57, P58, W91, Y94 or F95 all resulted in significantly reduced VLP yields, largely due to defective M secretion. VLP production was not significantly affected by a tryptophan replacement of Y94 or F95 or a phenylalanine replacement of W19, W57 or W91. Combined, these results indicate the involvement of specific M amino acids during SARS-CoV virus assembly, and suggest that aromatic residue retention at specific positions is critical for M function in terms of directing virus assembly.

  12. Inactivation of a Human Norovirus Surrogate, Human Norovirus Virus-Like Particles, and Vesicular Stomatitis Virus by Gamma Irradiation ▿

    Science.gov (United States)

    Feng, Kurtis; Divers, Erin; Ma, Yuanmei; Li, Jianrong

    2011-01-01

    Gamma irradiation is a nonthermal processing technology that has been used for the preservation of a variety of food products. This technology has been shown to effectively inactivate bacterial pathogens. Currently, the FDA has approved doses of up to 4.0 kGy to control food-borne pathogens in fresh iceberg lettuce and spinach. However, whether this dose range effectively inactivates food-borne viruses is less understood. We have performed a systematic study on the inactivation of a human norovirus surrogate (murine norovirus 1 [MNV-1]), human norovirus virus-like particles (VLPs), and vesicular stomatitis virus (VSV) by gamma irradiation. We demonstrated that MNV-1 and human norovirus VLPs were resistant to gamma irradiation. For MNV-1, only a 1.7- to 2.4-log virus reduction in fresh produce at the dose of 5.6 kGy was observed. However, VSV was more susceptible to gamma irradiation, and a 3.3-log virus reduction at a dose of 5.6 kGy in Dulbecco's modified Eagle medium (DMEM) was achieved. We further demonstrated that gamma irradiation disrupted virion structure and degraded viral proteins and genomic RNA, which resulted in virus inactivation. Using human norovirus VLPs as a model, we provide the first evidence that the capsid of human norovirus has stability similar to that of MNV-1 after exposure to gamma irradiation. Overall, our results suggest that viruses are much more resistant to irradiation than bacterial pathogens. Although gamma irradiation used to eliminate the virus contaminants in fresh produce by the FDA-approved irradiation dose limits seems impractical, this technology may be practical to inactivate viruses for other purposes, such as sterilization of medical equipment. PMID:21441330

  13. Binding of Human GII.4 Norovirus Virus-Like Particles to Carbohydrates of Romaine Lettuce Leaf Cell Wall Materials

    Science.gov (United States)

    Esseili, Malak A.

    2012-01-01

    Norovirus (NoV) genogroup II genotype 4 (GII.4) strains are the dominant cause of the majority of food-borne outbreaks, including those that involve leafy greens, such as lettuce. Since human NoVs use carbohydrates of histo-blood group antigens as receptors/coreceptors, we examined the role of carbohydrates in the attachment of NoV to lettuce leaves by using virus-like particles (VLPs) of a human NoV/GII.4 strain. Immunofluorescence analysis showed that the VLPs attached to the leaf surface, especially to cut edges, stomata, and along minor veins. Binding was quantified using enzyme-linked immunosorbent assay (ELISA) performed on cell wall materials (CWM) from innermost younger leaves and outermost lamina of older leaves. The binding to CWM of older leaves was significantly (P carbohydrates of CWM or porcine gastric mucin (PGM) (a carbohydrate control) using 100 mM sodium periodate (NaIO4) significantly decreased the binding an average of 17% in younger leaves, 43% in older leaves, and 92% for PGM. In addition, lectins recognizing GalNAc, GlcNAc, and sialic acid at 100 μg/ml significantly decreased the binding an average of 41%, 33%, and 20% on CWM of older leaves but had no effect on younger leaves. Lectins recognizing α-d-Gal, α-d-Man/α-d-Glc, and α-l-Fuc showed significant inhibition on CWM of older leaves as well as that of younger leaves. All lectins, except for the lectin recognizing α-d-Gal, significantly inhibited NoV VLP binding to PGM. Collectively, our results indicate that NoV VLPs bind to lettuce CWM by utilizing multiple carbohydrate moieties. This binding may enhance virus persistence on the leaf surface and prevent effective decontamination. PMID:22138991

  14. Novel Respiratory Syncytial Virus-Like Particle Vaccine Composed of the Postfusion and Prefusion Conformations of the F Glycoprotein.

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    Cimica, Velasco; Boigard, Hélène; Bhatia, Bipin; Fallon, John T; Alimova, Alexandra; Gottlieb, Paul; Galarza, Jose M

    2016-06-01

    Respiratory syncytial virus (RSV) is the leading cause of severe respiratory disease in infants and children and represents an important global health burden for the elderly and the immunocompromised. Despite decades of research efforts, no licensed vaccine for RSV is available. We have developed virus-like particle (VLP)-based RSV vaccines assembled with the human metapneumovirus (hMPV) matrix protein (M) as the structural scaffold and the RSV fusion glycoprotein (F) in either the postfusion or prefusion conformation as its prime surface immunogen. Vaccines were composed of postfusion F, prefusion F, or a combination of the two conformations and formulated with a squalene-based oil emulsion as adjuvant. Immunization with these VLP vaccines afforded full protection against RSV infection and prevented detectable viral replication in the mouse lung after challenge. Analyses of lung cytokines and chemokines showed that VLP vaccination mostly induced the production of gamma interferon (IFN-γ), a marker of the Th1-mediated immune response, which is predominantly required for viral protection. Conversely, immunization with a formalin-inactivated RSV (FI-RSV) vaccine induced high levels of inflammatory chemokines and cytokines of the Th2- and Th17-mediated types of immune responses, as well as severe lung inflammation and histopathology. The VLP vaccines showed restricted production of these immune mediators and did not induce severe bronchiolitis or perivascular infiltration as seen with the FI-RSV vaccine. Remarkably, analysis of the serum from immunized mice showed that the VLP vaccine formulated using a combination of postfusion and prefusion F elicited the highest level of neutralizing antibody and enhanced the Th1-mediated immune response. Copyright © 2016 Cimica et al.

  15. Toll-like receptor agonist augments virus-like particle-mediated protection from Ebola virus with transient immune activation.

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    Karen A O Martins

    Full Text Available Identifying safe and effective adjuvants is critical for the advanced development of protein-based vaccines. Pattern recognition receptor (PRR agonists are increasingly being explored as potential adjuvants, but there is concern that the efficacy of these molecules may be dependent on potentially dangerous levels of non-specific immune activation. The filovirus virus-like particle (VLP vaccine protects mice, guinea pigs, and nonhuman primates from viral challenge. In this study, we explored the impact of a stabilized dsRNA mimic, polyICLC, on VLP vaccination of C57BL/6 mice and Hartley guinea pigs. We show that at dose levels as low as 100 ng, the adjuvant increased the efficacy of the vaccine in mice. Antigen-specific, polyfunctional CD4 and CD8 T cell responses and antibody responses increased significantly upon inclusion of adjuvant. To determine whether the efficacy of polyICLC correlated with systemic immune activation, we examined serum cytokine levels and cellular activation in the draining lymph node. PolyICLC administration was associated with increases in TNFα, IL6, MCP1, MIP1α, KC, and MIP1β levels in the periphery and with the activation of dendritic cells (DCs, NK cells, and B cells. However, this activation resolved within 24 to 72 hours at efficacious adjuvant dose levels. These studies are the first to examine the polyICLC-induced enhancement of antigen-specific immune responses in the context of non-specific immune activation, and they provide a framework from which to consider adjuvant dose levels.

  16. The C-terminal end of parainfluenza virus 5 NP protein is important for virus-like particle production and M-NP protein interaction.

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    Schmitt, Phuong Tieu; Ray, Greeshma; Schmitt, Anthony P

    2010-12-01

    Enveloped virus particles are formed by budding from infected-cell membranes. For paramyxoviruses, viral matrix (M) proteins are key drivers of virus assembly and budding. However, other paramyxovirus proteins, including glycoproteins, nucleocapsid (NP or N) proteins, and C proteins, are also important for particle formation in some cases. To investigate the role of NP protein in parainfluenza virus 5 (PIV5) particle formation, NP protein truncation and substitution mutants were analyzed. Alterations near the C-terminal end of NP protein completely disrupted its virus-like particle (VLP) production function and significantly impaired M-NP protein interaction. Recombinant viruses with altered NP proteins were generated, and these viruses acquired second-site mutations. Recombinant viruses propagated in Vero cells acquired mutations that mainly affected components of the viral polymerase, while recombinant viruses propagated in MDBK cells acquired mutations that mainly affected the viral M protein. Two of the Vero-propagated viruses acquired the same mutation, V/P(S157F), found previously to be responsible for elevated viral gene expression induced by a well-characterized variant of PIV5, P/V-CPI(-). Vero-propagated viruses caused elevated viral protein synthesis and spread rapidly through infected monolayers by direct cell-cell fusion, bypassing the need to bud infectious virions. Both Vero- and MDBK-propagated viruses exhibited infectivity defects and altered polypeptide composition, consistent with poor incorporation of viral ribonucleoprotein complexes (RNPs) into budding virions. Second-site mutations affecting M protein restored interaction with altered NP proteins in some cases and improved VLP production. These results suggest that multiple avenues are available to paramyxoviruses for overcoming defects in M-NP protein interaction.

  17. Integrated molecular and bioprocess engineering for bacterially produced immunogenic modular virus-like particle vaccine displaying 18 kDa rotavirus antigen.

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    Tekewe, Alemu; Fan, Yuanyuan; Tan, Emilyn; Middelberg, Anton P J; Lua, Linda H L

    2017-02-01

    A high global burden of rotavirus disease and the unresolved challenges with the marketed rotavirus vaccines, particularly in the developing world, have ignited efforts to develop virus-like particle (VLP) vaccines for rotavirus. While rotavirus-like particles comprising multiple viral proteins can be difficult to process, modular VLPs presenting rotavirus antigenic modules are promising alternatives in reducing process complexity and cost. In this study, integrated molecular and bioprocess engineering approaches were used to simplify the production of modular murine polyomavirus capsomeres and VLPs presenting a rotavirus 18 kDa VP8* antigen. A single construct was generated for dual expression of non-tagged murine polyomavirus capsid protein VP1 and modular VP1 inserted with VP8*, for co-expression in Escherichia coli. Co-expressed proteins assembled into pentameric capsomeres in E. coli. A selective salting-out precipitation and a polishing size exclusion chromatography step allowed the recovery of stable modular capsomeres from cell lysates at high purity, and modular capsomeres were successfully translated into modular VLPs when assembled in vitro. Immunogenicity study in mice showed that modular capsomeres and VLPs induced high levels of VP8*-specific antibodies. Our results demonstrate that a multipronged synthetic biology approach combining molecular and bioprocess engineering enabled simple and low-cost production of highly immunogenic modular capsomeres and VLPs presenting conformational VP8* antigenic modules. This strategy potentially provides a cost-effective production route for modular capsomere and VLP vaccines against rotavirus, highly suitable to manufacturing economics for the developing world. Biotechnol. Bioeng. 2017;114: 397-406. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  18. Evaluation of Trichodysplasia Spinulosa-Associated Polyomavirus Capsid Protein as a New Carrier for Construction of Chimeric Virus-Like Particles Harboring Foreign Epitopes.

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    Gedvilaite, Alma; Kucinskaite-Kodze, Indre; Lasickiene, Rita; Timinskas, Albertas; Vaitiekaite, Ausra; Ziogiene, Danguole; Zvirbliene, Aurelija

    2015-07-29

    Recombinant virus-like particles (VLPs) represent a promising tool for protein engineering. Recently, trichodysplasia spinulosa-associated polyomavirus (TSPyV) viral protein 1 (VP1) was efficiently produced in yeast expression system and shown to self-assemble to VLPs. In the current study, TSPyV VP1 protein was exploited as a carrier for construction of chimeric VLPs harboring selected B and T cell-specific epitopes and evaluated in comparison to hamster polyomavirus VP1 protein. Chimeric VLPs with inserted either hepatitis B virus preS1 epitope DPAFR or a universal T cell-specific epitope AKFVAAWTLKAAA were produced in yeast Saccharomyces cerevisiae. Target epitopes were incorporated either at the HI or BC loop of the VP1 protein. The insertion sites were selected based on molecular models of TSPyV VP1 protein. The surface exposure of the insert positions was confirmed using a collection of monoclonal antibodies raised against the intact TSPyV VP1 protein. All generated chimeric proteins were capable to self-assemble to VLPs, which induced a strong immune response in mice. The chimeric VLPs also activated dendritic cells and T cells as demonstrated by analysis of cell surface markers and cytokine production profiles in spleen cell cultures. In conclusion, TSPyV VP1 protein represents a new potential carrier for construction of chimeric VLPs harboring target epitopes.

  19. Virus-Like Particle Vaccination Protects Nonhuman Primates from Lethal Aerosol Exposure with Marburgvirus (VLP Vaccination Protects Macaques against Aerosol Challenges).

    Science.gov (United States)

    Dye, John M; Warfield, Kelly L; Wells, Jay B; Unfer, Robert C; Shulenin, Sergey; Vu, Hong; Nichols, Donald K; Aman, M Javad; Bavari, Sina

    2016-04-08

    Marburg virus (MARV) was the first filovirus to be identified following an outbreak of viral hemorrhagic fever disease in Marburg, Germany in 1967. Due to several factors inherent to filoviruses, they are considered a potential bioweapon that could be disseminated via an aerosol route. Previous studies demonstrated that MARV virus-like particles (VLPs) containing the glycoprotein (GP), matrix protein VP40 and nucleoprotein (NP) generated using a baculovirus/insect cell expression system could protect macaques from subcutaneous (SQ) challenge with multiple species of marburgviruses. In the current study, the protective efficacy of the MARV VLPs in conjunction with two different adjuvants: QS-21, a saponin derivative, and poly I:C against homologous aerosol challenge was assessed in cynomolgus macaques. Antibody responses against the GP antigen were equivalent in all groups receiving MARV VLPs irrespective of the adjuvant; adjuvant only-vaccinated macaques did not demonstrate appreciable antibody responses. All macaques were subsequently challenged with lethal doses of MARV via aerosol or SQ as a positive control. All MARV VLP-vaccinated macaques survived either aerosol or SQ challenge while animals administered adjuvant only exhibited clinical signs and lesions consistent with MARV disease and were euthanized after meeting the predetermined criteria. Therefore, MARV VLPs induce IgG antibodies recognizing MARV GP and VP40 and protect cynomolgus macaques from an otherwise lethal aerosol exposure with MARV.

  20. Single Dose of Consensus Hemagglutinin-Based Virus-Like Particles Vaccine Protects Chickens against Divergent H5 Subtype Influenza Viruses

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    Peipei Wu

    2017-11-01

    Full Text Available The H5 subtype highly pathogenic avian influenza (HPAI virus is one of the greatest threats to global poultry industry. To develop broadly protective H5 subunit vaccine, a recombinant consensus HA sequence (rHA was constructed and expressed in virus-like particles (rHA VLPs in the baculovirus-insect cell system. The efficacy of the rHA VLPs vaccine with or without immunopotentiator (CVCVA5 was assessed in chickens. Compared to the commercial Re6 or Re6-CVCVA5 vaccines, single dose immunization of chickens with rHA VLPs or rHA-CVCVA5 vaccines induced higher levels of serum hemagglutinin inhibition titers and neutralization titers, mucosal antibodies, IFN-γ and IL-4 cytokines in sera, and cytotoxic T lymphocyte responses. The rHA VLPs vaccine was superior to the commercial Re6 vaccine in conferring cross-protection against different clades of H5 subtype viruses. This study reports that H5 subtype consensus HA VLP single dose vaccination provides broad protection against HPAI virus in chickens.

  1. Multiple heterologous M2 extracellular domains presented on virus-like particles confer broader and stronger M2 immunity than live influenza A virus infection.

    Science.gov (United States)

    Kim, Min-Chul; Lee, Jong-Seok; Kwon, Young-Man; O, Eunju; Lee, Youn-Jeong; Choi, Jun-Gu; Wang, Bao-Zhong; Compans, Richard W; Kang, Sang-Moo

    2013-09-01

    The influenza M2 ectodomain (M2e) is poorly immunogenic and has some amino acid changes among isolates from different host species. We expressed a tandem repeat construct of heterologous M2e sequences (M2e5x) derived from human, swine, and avian origin influenza A viruses on virus-like particles (M2e5x VLPs) in a membrane-anchored form. Immunization of mice with M2e5x VLPs induced protective antibodies cross-reactive to antigenically different influenza A viruses and conferred cross protection. Anti-M2e antibodies induced by heterologous M2e5x VLPs showed a wider range of cross reactivity to influenza A viruses at higher levels than those by live virus infection, homologous M2e VLPs, or M2e monoclonal antibody 14C2. Fc receptors were found to be important for mediating protection by immune sera from M2e5x VLP vaccination. The present study provides evidence that heterologous recombinant M2e5x VLPs can be more effective in inducing protective M2e immunity than natural virus infection and further supports an approach for developing an effective universal influenza vaccine. Copyright © 2013 Elsevier B.V. All rights reserved.

  2. Virus-Like Particle Vaccination Protects Nonhuman Primates from Lethal Aerosol Exposure with Marburgvirus (VLP Vaccination Protects Macaques against Aerosol Challenges

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    John M. Dye

    2016-04-01

    Full Text Available Marburg virus (MARV was the first filovirus to be identified following an outbreak of viral hemorrhagic fever disease in Marburg, Germany in 1967. Due to several factors inherent to filoviruses, they are considered a potential bioweapon that could be disseminated via an aerosol route. Previous studies demonstrated that MARV virus-like particles (VLPs containing the glycoprotein (GP, matrix protein VP40 and nucleoprotein (NP generated using a baculovirus/insect cell expression system could protect macaques from subcutaneous (SQ challenge with multiple species of marburgviruses. In the current study, the protective efficacy of the MARV VLPs in conjunction with two different adjuvants: QS-21, a saponin derivative, and poly I:C against homologous aerosol challenge was assessed in cynomolgus macaques. Antibody responses against the GP antigen were equivalent in all groups receiving MARV VLPs irrespective of the adjuvant; adjuvant only-vaccinated macaques did not demonstrate appreciable antibody responses. All macaques were subsequently challenged with lethal doses of MARV via aerosol or SQ as a positive control. All MARV VLP-vaccinated macaques survived either aerosol or SQ challenge while animals administered adjuvant only exhibited clinical signs and lesions consistent with MARV disease and were euthanized after meeting the predetermined criteria. Therefore, MARV VLPs induce IgG antibodies recognizing MARV GP and VP40 and protect cynomolgus macaques from an otherwise lethal aerosol exposure with MARV.

  3. The concentration of carbon source in the medium affects the quality of virus-like particles of human papillomavirus type 16 produced in Saccharomyces cerevisiae.

    Science.gov (United States)

    Kim, Hyoung Jin; Jin, Yingji; Kim, Hong-Jin

    2014-01-01

    There is accumulating evidence that virus-like particles (VLPs) recombinantly produced in Saccharomyces cerevisiae (S. cerevisiae) are characterized by low structural stability, and that this is associated with reduced antigenicity and immunogenicity. However, little attention has been devoted to methods of improving the quality of the VLPs. Here, we investigated the effect of carbon source concentration in the medium on the antigenicity and immunogenicity of human papillomavirus (HPV) type 16 L1 VLPs expressed in S. cerevisiae from the galactose promoter. Media containing 2, 4, 6, and 8% carbon source, composed of both glucose and galactose in equal proportion, were used. VLP antigenicity was enhanced in cultures grown on media with 6 or 8% carbon source, compared to those from cultures with less than 6% carbon source. Moreover, the VLPs obtained from these cultures induced higher anti-HPV16 L1 IgG titers and neutralizing antibody titers in immunized mice than those purified from cultures with less than 6% carbon source. Our results indicate that the concentration of the carbon source in the medium plays a crucial role in determining the antigenicity and immunogenicity of HPV type16 L1 VLPs.

  4. A heterologous prime-boosting strategy with replicating Vaccinia virus vectors and plant-produced HIV-1 Gag/dgp41 virus-like particles.

    Science.gov (United States)

    Meador, Lydia R; Kessans, Sarah A; Kilbourne, Jacquelyn; Kibler, Karen V; Pantaleo, Giuseppe; Roderiguez, Mariano Esteban; Blattman, Joseph N; Jacobs, Bertram L; Mor, Tsafrir S

    2017-07-01

    Showing modest efficacy, the RV144 HIV-1 vaccine clinical trial utilized a non-replicating canarypox viral vector and a soluble gp120 protein boost. Here we built upon the RV144 strategy by developing a novel combination of a replicating, but highly-attenuated Vaccinia virus vector, NYVAC-KC, and plant-produced HIV-1 virus-like particles (VLPs). Both components contained the full-length Gag and a membrane anchored truncated gp41 presenting the membrane proximal external region with its conserved broadly neutralizing epitopes in the pre-fusion conformation. We tested different prime/boost combinations of these components in mice and showed that the group primed with NYVAC-KC and boosted with both the viral vectors and plant-produced VLPs have the most robust Gag-specific CD8 T cell responses, at 12.7% of CD8 T cells expressing IFN-γ in response to stimulation with five Gag epitopes. The same immunization group elicited the best systemic and mucosal antibody responses to Gag and dgp41 with a bias towards IgG1. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  5. Optimization of norovirus virus-like particle production in Pichia pastoris using a real-time near-infrared bioprocess monitor.

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    Parker, Stephanie A; Maloy, Mitchell H; Tome-Amat, Jaime; Bardliving, Cameron L; Batt, Carl A; Lanz, Kaylee J; Olesberg, Jonathon T; Arnold, Mark A

    2016-03-01

    The production of norovirus virus-like particles (NoV VLPs) displaying NY-ESO-1 cancer testis antigen in Pichia pastoris BG11 Mut(+) has been enhanced through feed-strategy optimization using a near-infrared bioprocess monitor (RTBio(®) Bioprocess Monitor, ASL Analytical, Inc.), capable of monitoring and controlling the concentrations of glycerol and methanol in real-time. The production of NoV VLPs displaying NY-ESO-1 in P. pastoris has potential as a novel cancer vaccine platform. Optimization of the growth conditions resulted in an almost two-fold increase in the expression levels in the fermentation supernatant of P. pastoris as compared to the starting conditions. We investigated the effect of methanol concentration, batch phase time, and batch to induction transition on NoV VLP-NY-ESO-1 production. The optimized process included a glycerol transition phase during the first 2 h of induction and a methanol concentration set point of 4 g L(-1) during induction. Utilizing the bioprocess monitor to control the glycerol and methanol concentrations during induction resulted in a maximum NoV VP1-NY-ESO-1 yield of 0.85 g L(-1) . © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:518-526, 2016. © 2016 American Institute of Chemical Engineers.

  6. Phase II studies to select the formulation of a multivalent HPV L1 virus-like particle (VLP) vaccine.

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    Luxembourg, Alain; Brown, Darron; Bouchard, Celine; Giuliano, Anna R; Iversen, Ole-Erik; Joura, Elmar A; Penny, Mary E; Restrepo, Jaime A; Romaguera, Josefina; Maansson, Roger; Moeller, Erin; Ritter, Michael; Chen, Joshua

    2015-01-01

    Our objective was to develop a multivalent prophylactic HPV vaccine that protects against infection and disease caused by HPV16/18 (oncogenic types in existing prophylactic vaccines) plus additional oncogenic types by conducting 3 Phase II studies comparing the immunogenicity (i.e., anti-HPV6/11/16/18 geometric mean titers [GMT]) and safety of 7 vaccine candidates with the licensed quadrivalent HPV6/11/16/18 vaccine (qHPV vaccine) in young women ages 16-26. In the first study (Study 1), subjects received one of 3 dose formulations of an 8-valent HPV6/11/16/18/31/45/52/58 vaccine or qHPV vaccine (control). In Study 2, subjects received one of 3 dose formulations (termed low-, mid-, and high-dose formulations, respectively) of a 9-valent HPV6/11/16/18/31/33/45/52/58 vaccine (9vHPV vaccine) or qHPV vaccine (control). In Study 3, subjects concomitantly received qHPV vaccine plus 5-valent HPV31/33/45/52/58 or qHPV vaccine plus placebo (control). All vaccines were administered at day 1/month 2/month 6. In studies 1 and 3, anti-HPV6/11/16/18 GMTs at month 7 were non-inferior in the experimental arms compared with the control arm; however, there was a trend for lower antibody responses for all 4 HPV types. In Study 2, this immune interference was overcome with the mid- and high-dose formulations of the 9vHPV vaccine by increasing antigen and adjuvant doses. In all 3 studies, all vaccine candidates were strongly immunogenic with respect to HPV31/33/45/52/58 and were well tolerated. Based on the totality of the results, the middle dose formulation of the 9vHPV vaccine was selected for Phase III evaluation. Each 0.5mL dose contains 30μg/40μg/60μg/40μg/20μg/20μg/20μg/20μg/20μg of HPV6/11/16/18/31/33/45/52/58 virus-like particles, and 500μg of amorphous aluminum hydroxyphosphate sulfate adjuvant.ClinicalTrials.gov numbers NCT00260039, NCT00543543, and NCT00551187.

  7. Disassembly and reassembly of human papillomavirus virus-like particles produces more virion-like antibody reactivity

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    Zhao Qinjian

    2012-02-01

    Full Text Available Abstract Background Human papillomavirus (HPV vaccines based on major capsid protein L1 are licensed in over 100 countries to prevent HPV infections. The yeast-derived recombinant quadrivalent HPV L1 vaccine, GARDASIL(R, has played an important role in reducing cancer and genital warts since its introduction in 2006. The L1 proteins self-assemble into virus-like particles (VLPs. Results VLPs were subjected to post-purification disassembly and reassembly (D/R treatment during bioprocessing to improve VLP immunoreactivity and stability. The post-D/R HPV16 VLPs and their complex with H16.V5 neutralizing antibody Fab fragments were visualized by cryo electron microscopy, showing VLPs densely decorated with antibody. Along with structural improvements, post-D/R VLPs showed markedly higher antigenicity to conformational and neutralizing monoclonal antibodies (mAbs H16.V5, H16.E70 and H263.A2, whereas binding to mAbs recognizing linear epitopes (H16.J4, H16.O7, and H16.H5 was greatly reduced. Strikingly, post-D/R VLPs showed no detectable binding to H16.H5, indicating that the H16.H5 epitope is not accessible in fully assembled VLPs. An atomic homology model of the entire HPV16 VLP was generated based on previously determined high-resolution structures of bovine papillomavirus and HPV16 L1 pentameric capsomeres. Conclusions D/R treatment of HPV16 L1 VLPs produces more homogeneous VLPs with more virion-like antibody reactivity. These effects can be attributed to a combination of more complete and regular assembly of the VLPs, better folding of L1, reduced non-specific disulfide-mediated aggregation and increased stability of the VLPs. Markedly different antigenicity of HPV16 VLPs was observed upon D/R treatment with a panel of monoclonal antibodies targeting neutralization sensitive epitopes. Multiple epitope-specific assays with a panel of mAbs with different properties and epitopes are required to gain a better understanding of the immunochemical

  8. A Novel Virus-Like Particle Based Vaccine Platform Displaying the Placental Malaria Antigen VAR2CSA.

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    Susan Thrane

    Full Text Available Placental malaria caused by Plasmodium falciparum is a major cause of mortality and severe morbidity. Clinical testing of a soluble protein-based vaccine containing the parasite ligand, VAR2CSA, has been initiated. VAR2CSA binds to the human receptor chondroitin sulphate A (CSA and is responsible for sequestration of Plasmodium falciparum infected erythrocytes in the placenta. It is imperative that a vaccine against malaria in pregnancy, if administered to women before they become pregnant, can induce a strong and long lasting immune response. While most soluble protein-based vaccines have failed during clinical testing, virus-like particle (VLP based vaccines (e.g., the licensed human papillomavirus vaccines have demonstrated high efficacy, suggesting that the spatial assembly of the vaccine antigen is a critical parameter for inducing an optimal long-lasting protective immune response. We have developed a VLP vaccine display platform by identifying regions of the HPV16 L1 coat protein where a biotin acceptor site (AviTagTM can be inserted without compromising VLP-assembly. Subsequent biotinylation of Avi-L1 VLPs allow us to anchor monovalent streptavidin (mSA-fused proteins to the biotin, thereby obtaining a dense and repetitive VLP-display of the vaccine antigen. The mSA-VAR2CSA antigen was delivered on the Avi-L1 VLP platform and tested in C57BL/6 mice in comparison to two soluble protein-based vaccines consisting of naked VAR2CSA and mSA-VAR2CSA. The mSA-VAR2CSA Avi-L1 VLP and soluble mSA-VAR2CSA vaccines induced higher antibody titers than the soluble naked VAR2CSA vaccine after three immunizations. The VAR2CSA Avi-L1 VLP vaccine induced statistically significantly higher endpoint titres compared to the soluble mSA-VAR2CSA vaccine, after 1st and 2nd immunization; however, this difference was not statistically significant after 3rd immunization. Importantly, the VLP-VAR2CSA induced antibodies were functional in inhibiting the binding of

  9. Conformational and thermal stability improvements for the large-scale production of yeast-derived rabbit hemorrhagic disease virus-like particles as multipurpose vaccine.

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    Erlinda Fernández

    Full Text Available Recombinant virus-like particles (VLP antigenically similar to rabbit hemorrhagic disease virus (RHDV were recently expressed at high levels inside Pichia pastoris cells. Based on the potential of RHDV VLP as platform for diverse vaccination purposes we undertook the design, development and scale-up of a production process. Conformational and stability issues were addressed to improve process control and optimization. Analyses on the structure, morphology and antigenicity of these multimers were carried out at different pH values during cell disruption and purification by size-exclusion chromatography. Process steps and environmental stresses in which aggregation or conformational instability can be detected were included. These analyses revealed higher stability and recoveries of properly assembled high-purity capsids at acidic and neutral pH in phosphate buffer. The use of stabilizers during long-term storage in solution showed that sucrose, sorbitol, trehalose and glycerol acted as useful aggregation-reducing agents. The VLP emulsified in an oil-based adjuvant were subjected to accelerated thermal stress treatments. None to slight variations were detected in the stability of formulations and in the structure of recovered capsids. A comprehensive analysis on scale-up strategies was accomplished and a nine steps large-scale production process was established. VLP produced after chromatographic separation protected rabbits against a lethal challenge. The minimum protective dose was identified. Stabilized particles were ultimately assayed as carriers of a foreign viral epitope from another pathogen affecting a larger animal species. For that purpose, a linear protective B-cell epitope from Classical Swine Fever Virus (CSFV E2 envelope protein was chemically coupled to RHDV VLP. Conjugates were able to present the E2 peptide fragment for immune recognition and significantly enhanced the peptide-specific antibody response in vaccinated pigs

  10. Virus-Like Particle (VLP Plus Microcrystalline Tyrosine (MCT Adjuvants Enhance Vaccine Efficacy Improving T and B Cell Immunogenicity and Protection against Plasmodium berghei/vivax

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    Gustavo Cabral-Miranda

    2017-05-01

    Full Text Available Vaccination is the most effective prophylactic tool against infectious diseases. Despite continued efforts to control malaria, the disease still generally represents a significant unmet medical need. Microcrystalline tyrosine (MCT is a well described depot used in licensed allergy immunotherapy products and in clinical development. However, its proof of concept in prophylactic vaccines has only recently been explored. MCT has never been used in combination with virus-like particles (VLPs, which are considered to be one of the most potent inducers of cellular and humoral immune responses in mice and humans. In the current study we assessed the potential of MCT to serve as an adjuvant in the development of a vaccine against malaria either alone or combined with VLP using Plasmodium vivax thrombospondin-related adhesive protein (TRAP as a target antigen. We chemically coupled PvTRAP to VLPs derived from the cucumber mosaic virus fused to a universal T-cell epitope of the tetanus toxin (CMVtt, formulated with MCT and compared the induced immune responses to PvTRAP formulated in PBS or Alum. The protective capacity of the various formulations was assessed using Plasmodium berghei expressing PvTRAP. All vaccine formulations using adjuvants and/or VLP increased humoral immunogenicity for PvTRAP compared to the antigen alone. The most proficient responder was the group of mice immunized with the vaccine formulated with PvTRAP-VLP + MCT. The VLP-based vaccine formulated in MCT also induced the strongest T cell response and conferred best protection against challenge with recombinant Plasmodium berghei. Thus, the combination of VLP with MCT may take advantage of the properties of each component and appears to be an alternative biodegradable depot adjuvant for development of novel prophylactic vaccines.

  11. Overcoming antigen masking of anti-amyloidbeta antibodies reveals breaking of B cell tolerance by virus-like particles in amyloidbeta immunized amyloid precursor protein transgenic mice

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    Ugen Kenneth E

    2004-06-01

    Full Text Available Abstract Background In prior work we detected reduced anti-Aβ antibody titers in Aβ-vaccinated transgenic mice expressing the human amyloid precursor protein (APP compared to nontransgenic littermates. We investigated this observation further by vaccinating APP and nontransgenic mice with either the wild-type human Aβ peptide, an Aβ peptide containing the "Dutch Mutation", E22Q, or a wild-type Aβ peptide conjugated to papillomavirus virus-like particles (VLPs. Results Anti-Aβ antibody titers were lower in vaccinated APP than nontransgenic mice even when vaccinated with the highly immunogenic Aβ E22Q. One concern was that human Aβ derived from the APP transgene might mask anti-Aβ antibodies in APP mice. To test this possibility, we dissociated antigen-antibody complexes by incubation at low pH. The low pH incubation increased the anti-Aβ antibody titers 20–40 fold in APP mice but had no effect in sera from nontransgenic mice. However, even after dissociation, the anti-Aβ titers were still lower in transgenic mice vaccinated with wild-type Aβ or E22Q Aβ relative to non-transgenic mice. Importantly, the dissociated anti-Aβ titers were equivalent in nontransgenic and APP mice after VLP-based vaccination. Control experiments demonstrated that after acid-dissociation, the increased antibody titer did not cross react with bovine serum albumin nor alpha-synuclein, and addition of Aβ back to the dissociated serum blocked the increase in antibody titers. Conclusions Circulating human Aβ can interfere with ELISA assay measurements of anti-Aβ titers. The E22Q Aβ peptide vaccine is more immunogenic than the wild-type peptide. Unlike peptide vaccines, VLP-based vaccines against Aβ abrogate the effects of Aβ self-tolerance.

  12. Antibodies against Lewis antigens inhibit the binding of human norovirus GII.4 virus-like particles to saliva but not to intestinal Caco-2 cells.

    Science.gov (United States)

    Carmona-Vicente, Noelia; Allen, David J; Rodríguez-Díaz, Jesús; Iturriza-Gómara, Miren; Buesa, Javier

    2016-05-21

    Human noroviruses (NoVs) are the main cause of gastroenteritis worldwide. The most commonly detected NoV strains belong to the genetically diverse GII.4 genotype, with new pandemic variants emerging periodically. Despite extensive efforts, NoV investigation has been hampered by the lack of an effective in vitro cell culture system. However, NoV-derived recombinant virus-like particles (VLPs) resembling empty capsids are good surrogates for analysing NoV antigenicity and virus-ligand interactions. NoV VLPs have been reported to bind to histo-blood group antigens (HBGAs). We have analysed the ability of NoV VLPs derived from GI.1 genotype and from three GII.4 genotype variants, GII.4-1999, GII.4-2004 and GII.4-2006b, to bind to porcine gastric mucin (PGM), human saliva and differentiated human intestinal Caco-2 cells (D-Caco-2 cells). Distinct patterns of saliva binding with the NoV GII.4 variant VLPs were observed, although they bound to D-Caco-2 cells independently of the expression of HBGAs. Monoclonal antibodies against Lewis antigens were able to block the binding of NoV VLPs to saliva, but not to D-Caco-2 cells. Blocking HBGAs on the surface of D-Caco-2 cells with specific monoclonal antibodies did not affect NoV VLP binding to cellular membranes. Co-localisation of Lewis y (Le(y)) and H-type 2 antigens with NoV VLPs was not observed by immunofluorescence assays. Although the binding of NoV VLPs of GII.4 genotype variants to human saliva samples occur with distinct HBGA binding patterns and can be blocked by antibodies against Lewis antigens, their attachment to D-Caco-2 cells can be mediated by other receptors, which still need further investigation.

  13. Stability studies of HIV-1 Pr55gag virus-like particles made in insect cells after storage in various formulation media

    Directory of Open Access Journals (Sweden)

    Lynch Alisson

    2012-09-01

    Full Text Available Abstract Background HIV-1 Pr55gag virus-like particles (VLPs expressed by baculovirus in insect cells are considered to be a very promising HIV-1 vaccine candidate, as they have been shown to elicit broad cellular immune responses when tested in animals, particularly when used as a boost to DNA or BCG vaccines. However, it is important for the VLPs to retain their structure for them to be fully functional and effective. The medium in which the VLPs are formulated and the temperature at which they are stored are two important factors affecting their stability. Findings We describe the screening of 3 different readily available formulation media (sorbitol, sucrose and trehalose for their ability to stabilise HIV-1 Pr55gag VLPs during prolonged storage. Transmission electron microscopy (TEM was done on VLPs stored at two different concentrations of the media at three different temperatures (4°C, –20°C and −70°C over different time periods, and the appearance of the VLPs was compared. VLPs stored in 15% trehalose at −70°C retained their original appearance the most effectively over a period of 12 months. VLPs stored in 5% trehalose, sorbitol or sucrose were not all intact even after 1 month storage at the temperatures tested. In addition, we showed that VLPs stored under these conditions were able to be frozen and re-thawed twice before showing changes in their appearance. Conclusions Although the inclusion of other analytical tools are essential to validate these preliminary findings, storage in 15% trehalose at −70°C for 12 months is most effective in retaining VLP stability.

  14. Impact of Internal RNA on Aggregation and Electrokinetics of Viruses: Comparison between MS2 Phage and Corresponding Virus-Like Particles

    OpenAIRE

    Dika, C.; Duval, J.F.L.; Ly-Chatain, H. M.; C. Merlin; Gantzer, C.

    2011-01-01

    We compare for the first time the electrokinetic and aggregation properties of MS2 phage (pH 2.5 to 7, 1 to 100 mM NaNO3 electrolyte concentration) with those of the corresponding virus-like particles (VLPs), which lack entirely the inner viral RNA component. In line with our previous work (J. Langlet, F. Gaboriaud, C. Gantzer, and J. F. L. Duval, Biophys. J. 94:3293-3312, 2008), it is found that modifying the content of RNA within the virus leads to very distinct electrohydrodynamic and aggr...

  15. Divergence of Primary Cognate B- and T-Cell Proliferative Responses to Subcutaneous and Intravenous Immunization with Virus-Like Particles

    OpenAIRE

    Temchura, Vladimir; Kalinin, Svetlana; Nabi, Ghulam; Tippler, Bettina; Niezold, Thomas; Überla, Klaus

    2014-01-01

    A major advantage of virus-like particle (VLP) vaccines against HIV is their structural identity to wild-type viruses, ensuring that antigen-specific B-cells encounter the envelope protein in its natural conformation. For the induction of affinity-matured antibodies, the B-cells must also obtain help from T-cells that are restricted by linear epitopes. Using B- and T-cell transgenic mouse models, we compared the efficacy of modified HIV-VLPs delivered by subcutaneous and intravenous immuniz...

  16. Human papillomavirus 16L1-58L2 chimeric virus-like particles elicit durable neutralizing antibody responses against a broad-spectrum of human papillomavirus types

    OpenAIRE

    Chen, Xue; Liu, Hongyang; Wang, Zhirong; Wang, Shuo; Zhang, Ting; Hu, Meili; Qiao, Liang; Xu, Xuemei

    2017-01-01

    The neutralizing antibodies elicited by human papillomavirus (HPV) major capsid protein L1 virus-like particle (VLP)-based vaccines are largely type-specific. An HPV vaccine inducing cross-neutralizing antibodies broadly will be cost-effective and of great value. To this end, we constructed HPV16L1-58L2 chimeric VLP (cVLP) by displaying HPV58 L2 aa.16-37 on the DE surface region of HPV16 L1. We found that vaccination with the HPV16L1-58L2 cVLP formulated with alum plus monophosphoryl lipid A ...

  17. HIV-1 and MLV Gag proteins are sufficient to recruit APOBEC3G into virus-like particles.

    Science.gov (United States)

    Douaisi, Marc; Dussart, Sylvie; Courcoul, Marianne; Bessou, Gilles; Vigne, Robert; Decroly, Etienne

    2004-08-27

    The cytidine deaminase hAPOBEC3G is an antiviral human factor that counteracts the replication of HIV-1 in absence of the Vif protein. hAPOBEC3G is packaged into virus particles and lethally hypermutates HIV-1. In this work, we examine the mechanisms governing hAPOBEC3G packaging. By GST pull-down and co-immunoprecipitation assays, we show that hAPOBEC3G binds to HIV-1 Pr55 Gag and its NC domain and to the RT and IN domains contained in Pr160 Gag-Pol. We demonstrate that the expression of HIV-1 Gag is sufficient to induce the packaging of hAPOBEC3G into Gag particles. Gag-Pol polypeptides containing RT and IN domains, as well as HIV-1 genomic RNA, seem not to be necessary for hAPOBEC3G packaging. Lastly, we show that hAPOBEC3G and its murine ortholog are packaged into HIV-1 and MLV Gag particles. We conclude that the Gag polypeptides from distant retroviruses have conserved domains allowing the packaging of the host antiviral factor APOBEC3G.

  18. Immunization with an HPV-16 L1-based chimeric virus-like particle containing HPV-16 E6 and E7 epitopes elicits long-lasting prophylactic and therapeutic efficacy in an HPV-16 tumor mice model.

    Science.gov (United States)

    Monroy-García, Alberto; Gómez-Lim, Miguel Angel; Weiss-Steider, Benny; Hernández-Montes, Jorge; Huerta-Yepez, Sara; Rangel-Santiago, Jesús F; Santiago-Osorio, Edelmiro; Mora García, María de Lourdes

    2014-02-01

    HPV L1-based virus-like particles vaccines (VLPs) efficiently induce temporary prophylactic activity through the induction of neutralizing antibodies; however, VLPs that can provide prophylactic as well as therapeutic properties for longer periods of time are needed. For this purpose, we generated a novel HPV 16 L1-based chimeric virus-like particle (cVLP) produced in plants that contains a string of T-cell epitopes from HPV 16 E6 and E7 fused to its C-terminus. In the present study, we analyzed the persistence of specific IgG antibodies with neutralizing activity induced by immunization with these cVLPs, as well as their therapeutic potential in a tumor model of C57BL/6 mice. We observed that these cVLPs induced persistent IgG antibodies for over 12 months, with reactivity and neutralizing activity for VLPs composed of only the HPV-16 L1 protein. Efficient protection for long periods of time and inhibition of tumor growth induced by TC-1 tumor cells expressing HPV-16 E6/E7 oncoproteins, as well as significant tumor reduction (57 %), were observed in mice immunized with these cVLPs. Finally, we discuss the possibility that chimeric particles of the type described in this work may be the basis for developing HPV prophylactic and therapeutic vaccines with high efficacy.

  19. Virus-like particle production with yeast: ultrastructural and immunocytochemical insights into Pichia pastoris producing high levels of the Hepatitis B surface antigen

    Directory of Open Access Journals (Sweden)

    Adnan Ahmad

    2011-06-01

    Full Text Available Abstract Background A protective immune response against Hepatitis B infection can be obtained through the administration of a single viral polypeptide, the Hepatitis B surface antigen (HBsAg. Thus, the Hepatitis B vaccine is generated through the utilization of recombinant DNA technology, preferentially by using yeast-based expression systems. However, the polypeptide needs to assemble into spherical particles, so-called virus-like particles (VLPs, to elicit the required protective immune response. So far, no clear evidence has been presented showing whether HBsAg assembles in vivo inside the yeast cell into VLPs or later in vitro during down-stream processing and purification. Results High level production of HBsAg was carried out with recombinant Pichia pastoris using the methanol inducible AOX1 expression system. The recombinant vaccine was isolated in form of VLPs after several down-stream steps from detergent-treated cell lysates. Search for the intracellular localization of the antigen using electron microscopic studies in combination with immunogold labeling revealed the presence of HBsAg in an extended endoplasmic reticulum where it was found to assemble into defined multi-layered, lamellar structures. The distance between two layers was determined as ~6 nm indicating that these lamellas represent monolayers of well-ordered HBsAg subunits. We did not find any evidence for the presence of VLPs within the endoplasmic reticulum or other parts of the yeast cell. Conclusions It is concluded that high level production and intrinsic slow HBsAg VLP assembly kinetics are leading to retention and accumulation of the antigen in the endoplasmic reticulum where it assembles at least partly into defined lamellar structures. Further transport of HBsAg to the Golgi apparatus is impaired thus leading to secretory pathway disfunction and the formation of an extended endoplasmic reticulum which bulges into irregular cloud-shaped formations. As VLPs were

  20. Gene Therapy for Human Lung Adenocarcinoma Using a Suicide Gene Driven by a Lung-Specific Promoter Delivered by JC Virus-Like Particles.

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    Chun-Nun Chao

    Full Text Available Lung adenocarcinoma, the most commonly diagnosed type of lung cancer, has a poor prognosis even with combined surgery, chemotherapy, or molecular targeted therapies. Most patients are diagnosed with an in-operable advanced or metastatic disease, both pointing to the necessity of developing effective therapies for lung adenocarcinoma. Surfactant protein B (SP-B has been found to be overexpressed in lung adenocarcinoma. In addition, it has also been demonstrated that human lung adenocarcinoma cells are susceptible to the JC polyomavirus (JCPyV infection. Therefore, we designed that the JCPyV virus-like particle (VLP packaged with an SP-B promoter-driven thymidine kinase suicide gene (pSPB-tk for possible gene therapy of human lung adenocarcinoma. Plasmids expressing the GFP (pSPB-gfp or thymidine kinase gene (pSPB-tk under the control of the human SP-B promoter were constructed. The promoter's tissue specificity was tested by transfection of pSPB-gfp into A549, CH27, and H460 human lung carcinoma cells and non-lung cells. The JCPyV VLP's gene transfer efficiency and the selective cytotoxicity of pSPB-tk combined with ganciclovir (GCV were tested in vitro and in a xenograft mouse model. In the current study, we found that SP-B promoter-driven GFP was specifically expressed in human lung adenocarcinoma (A549 and large cell carcinoma (H460 cells. JCPyV VLPs were able to deliver a GFP reporter gene into A549 cells for expression. Selective cytotoxicity was observed in A549 but not non-lung cells that were transfected with pSPB-tk or infected with pSPB-tk-carrying JCPyV VLPs. In mice injected with pSPB-tk-carrying JCPyV VLPs through the tail vein and treated with ganciclovir (GCV, a potent 80% inhibition of growth of human lung adenocarcinoma nodules resulted. The JCPyV VLPs combined with the use of SP-B promoter demonstrates effectiveness as a potential gene therapy against human lung adenocarcinoma.

  1. Gene Therapy for Human Lung Adenocarcinoma Using a Suicide Gene Driven by a Lung-Specific Promoter Delivered by JC Virus-Like Particles.

    Science.gov (United States)

    Chao, Chun-Nun; Lin, Mien-Chun; Fang, Chiung-Yao; Chen, Pei-Lain; Chang, Deching; Shen, Cheng-Huang; Wang, Meilin

    2016-01-01

    Lung adenocarcinoma, the most commonly diagnosed type of lung cancer, has a poor prognosis even with combined surgery, chemotherapy, or molecular targeted therapies. Most patients are diagnosed with an in-operable advanced or metastatic disease, both pointing to the necessity of developing effective therapies for lung adenocarcinoma. Surfactant protein B (SP-B) has been found to be overexpressed in lung adenocarcinoma. In addition, it has also been demonstrated that human lung adenocarcinoma cells are susceptible to the JC polyomavirus (JCPyV) infection. Therefore, we designed that the JCPyV virus-like particle (VLP) packaged with an SP-B promoter-driven thymidine kinase suicide gene (pSPB-tk) for possible gene therapy of human lung adenocarcinoma. Plasmids expressing the GFP (pSPB-gfp) or thymidine kinase gene (pSPB-tk) under the control of the human SP-B promoter were constructed. The promoter's tissue specificity was tested by transfection of pSPB-gfp into A549, CH27, and H460 human lung carcinoma cells and non-lung cells. The JCPyV VLP's gene transfer efficiency and the selective cytotoxicity of pSPB-tk combined with ganciclovir (GCV) were tested in vitro and in a xenograft mouse model. In the current study, we found that SP-B promoter-driven GFP was specifically expressed in human lung adenocarcinoma (A549) and large cell carcinoma (H460) cells. JCPyV VLPs were able to deliver a GFP reporter gene into A549 cells for expression. Selective cytotoxicity was observed in A549 but not non-lung cells that were transfected with pSPB-tk or infected with pSPB-tk-carrying JCPyV VLPs. In mice injected with pSPB-tk-carrying JCPyV VLPs through the tail vein and treated with ganciclovir (GCV), a potent 80% inhibition of growth of human lung adenocarcinoma nodules resulted. The JCPyV VLPs combined with the use of SP-B promoter demonstrates effectiveness as a potential gene therapy against human lung adenocarcinoma.

  2. Detection of norovirus virus-like particles using a surface plasmon resonance-assisted fluoroimmunosensor optimized for quantum dot fluorescent labels.

    Science.gov (United States)

    Ashiba, Hiroki; Sugiyama, Yuki; Wang, Xiaomin; Shirato, Haruko; Higo-Moriguchi, Kyoko; Taniguchi, Koki; Ohki, Yoshimichi; Fujimaki, Makoto

    2017-07-15

    A highly sensitive biosensor to detect norovirus in environment is desired to prevent the spread of infection. In this study, we investigated a design of surface plasmon resonance (SPR)-assisted fluoroimmunosensor to increase its sensitivity and performed detection of norovirus virus-like particles (VLPs). A quantum dot fluorescent dye was employed because of its large Stokes shift. The sensor design was optimized for the CdSe-ZnS-based quantum dots. The optimal design was applied to a simple SPR-assisted fluoroimmunosensor that uses a sensor chip equipped with a V-shaped trench. Excitation efficiency of the quantum dots, degree of electric field enhancement by SPR, and intensity of autofluorescence of a substrate of the sensor chip were theoretically and experimentally evaluated to maximize the signal-to-noise ratio. As the result, an excitation wavelength of 390nm was selected to excite SPR on an Al film of the sensor chip. The sandwich assay of norovirus VLPs was performed using the designed sensor. Minimum detectable concentration of 0.01ng/mL, which corresponds to 100 virus-like particles included in the detection region of the V-trench, was demonstrated. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  3. Lassa virus-like particles displaying all major immunological determinants as a vaccine candidate for Lassa hemorrhagic fever

    Directory of Open Access Journals (Sweden)

    Cashman Kathleen A

    2010-10-01

    Full Text Available Abstract Background Lassa fever is a neglected tropical disease with significant impact on the health care system, society, and economy of Western and Central African nations where it is endemic. Treatment of acute Lassa fever infections has successfully utilized intravenous administration of ribavirin, a nucleotide analogue drug, but this is not an approved use; efficacy of oral administration has not been demonstrated. To date, several potential new vaccine platforms have been explored, but none have progressed toward clinical trials and commercialization. Therefore, the development of a robust vaccine platform that could be generated in sufficient quantities and at a low cost per dose could herald a subcontinent-wide vaccination program. This would move Lassa endemic areas toward the control and reduction of major outbreaks and endemic infections. To this end, we have employed efficient mammalian expression systems to generate a Lassa virus (LASV-like particle (VLP-based modular vaccine platform. Results A mammalian expression system that generated large quantities of LASV VLP in human cells at small scale settings was developed. These VLP contained the major immunological determinants of the virus: glycoprotein complex, nucleoprotein, and Z matrix protein, with known post-translational modifications. The viral proteins packaged into LASV VLP were characterized, including glycosylation profiles of glycoprotein subunits GP1 and GP2, and structural compartmentalization of each polypeptide. The host cell protein component of LASV VLP was also partially analyzed, namely glycoprotein incorporation, though the identity of these proteins remain unknown. All combinations of LASV Z, GPC, and NP proteins that generated VLP did not incorporate host cell ribosomes, a known component of native arenaviral particles, despite detection of small RNA species packaged into pseudoparticles. Although VLP did not contain the same host cell components as the native

  4. Immunogenicity and protective efficacy of virus-like particles and recombinant fiber proteins in broiler-breeder vaccination against fowl adenovirus (FAdV)-8b.

    Science.gov (United States)

    Gupta, Ashish; Ahmed, Khawaja Ashfaque; Ayalew, Lisanework E; Popowich, Shelly; Kurukulasuriya, Shanika; Goonewardene, Kalhari; Gunawardana, Thushari; Karunarathna, Ruwani; Ojkic, Davor; Tikoo, Suresh K; Willson, Philip; Gomis, Susantha

    2017-05-09

    Inclusion body hepatitis (IBH) is an economically important diseases in broiler chicken industry. Several serotypes of fowl adenovirus (FAdV) can cause IBH, among them, serotype FAdV-8b is associated with the majority of the IBH cases in Canada. Here, we evaluated FAdV-8b virus-like particles (VLPs) and recombinant FAdV-8b fiber proteins (expressed in E. coli) as potential broiler-breeder vaccines against IBH. For assessing the immunogenicity of vaccines, we investigated both humoral and cellular immunity. The humoral immune response was evaluated by determining total IgY and virus-neutralizing antibody in serum at 14, 28, 35 and 60days post-immunization (dpi). We examined cellular immunity using flow cytometry by determining CD4:CD8 ratio change in peripheral blood after the booster vaccination. The protective effect of vaccines was tested by challenging 14day-old progeny (n=30/group) carrying maternal antibodies (MtAb) by challenging with virulent FAdV-8b virus (1×107 TCID50, FAdV-8b-SK). Although total IgY levels were comparable in all groups, the neutralizing antibody response in broiler-breeders at 35 and 60 dpi was significantly (pbroiler-breeders four days after the booster vaccination. Unlike FAdV-8b fiber-knob, FAdV-8b VLPs, and FAdV-8b fiber vaccinated broiler-breeders were able to transfer a substantial amount (28.4±9%) of MtAb to their progeny. Challenge revealed that MtAb provided 100% and 82.7% protection in progeny hatched from FAdV-8b VLPs, and FAdV-8b fiber vaccinated broiler-breeders, respectively. Collectively, our data suggest that FAdV-8b subunit vaccine-induced MtAb efficiently protected progeny against clinical IBH and broiler-breeder vaccination with subunit vaccines is a potential approach to protect against IBH. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Phase I trial of an alhydrogel adjuvanted hepatitis B core virus-like particle containing epitopes of Plasmodium falciparum circumsporozoite protein.

    Directory of Open Access Journals (Sweden)

    Aric L Gregson

    2008-02-01

    Full Text Available The objectives of this non-randomized, non-blinded, dose-escalating Phase I clinical trial were to assess the safety, reactogenicity and immunogenicity of ICC-1132 formulated with Alhydrogel (aluminum hydroxide in 51 healthy, malaria-naive adults aged 18 to 45 years. ICC-1132 (Malariavax is a recombinant, virus-like particle malaria vaccine comprised of hepatitis core antigen engineered to express the central repeat regions from Plasmodium falciparum circumsporozoite protein containing an immunodominant B [(NANP(3] epitope, an HLA-restricted CD4 (NANPNVDPNANP epitope and a universal T cell epitope (T* (amino acids 326-345, NF54 isolate. We assessed an Alhydrogel (aluminum hydroxide-adjuvanted vaccine formulation at three ICC-1132 dose levels, each injected intramuscularly (1.0 mL on study days 0, 56 and 168. A saline vaccine formulation was found to be unstable after prolonged storage and this formulation was subsequently removed from the study. Thirty-two volunteers were followed for one year. Local and systemic adverse clinical events were measured and immune responses to P. falciparum and hepatitis B virus core antigens were determined utilizing the following assays: IgG and IgM ELISA, indirect immunofluorescence against P. falciparum sporozoites, circumsporozoite precipitin (CSP and transgenic sporozoite neutralization assays. Cellular responses were measured by proliferation and IL-2 assays. Local and systemic reactions were similarly mild and well tolerated between dose cohorts. Depending on the ICC-1132 vaccine concentration, 95 to 100% of volunteers developed antibody responses to the ICC-1132 immunogen and HBc after two injections; however, only 29-75% and 29-63% of volunteers, respectively, developed malaria-specific responses measured by the malaria repeat synthetic peptide ELISA and IFA; 2 of 8 volunteers had positive reactions in the CSP assay. Maximal transgenic sporozoite neutralization assay inhibition was 54%. Forty-seven to

  6. Silica Nanoparticles as the Adjuvant for the Immunisation of Mice Using Hepatitis B Core Virus-Like Particles: e114006

    National Research Council Canada - National Science Library

    Dace Skrastina; Ivars Petrovskis; Ilva Lieknina; Janis Bogans; Regina Renhofa; Velta Ose; Andris Dishlers; Yuri Dekhtyar; Paul Pumpens

    2014-01-01

      Advances in nanotechnology and nanomaterials have facilitated the development of silicon dioxide, or Silica, particles as a promising immunological adjuvant for the generation of novel prophylactic...

  7. Soluble F proteins exacerbate pulmonary histopathology after vaccination upon respiratory syncytial virus challenge but not when presented on virus-like particles.

    Science.gov (United States)

    Lee, Youri; Lee, Young-Tae; Ko, Eun-Ju; Kim, Ki-Hye; Hwang, Hye Suk; Park, Soojin; Kwon, Young-Man; Kang, Sang Moo

    2017-08-30

    Respiratory syncytial virus (RSV) fusion (F) protein is suggested to be a protective vaccine target although its efficacy and safety concerns remain not well understood. We investigated immunogenicity, efficacy, and safety of F proteins in a soluble form or on virus-like particle (F-VLP). F VLP preferentially elicited IgG2a antibody and T helper type 1 (Th1) immune responses whereas F protein induced IgG1 isotype and Th2 responses. Despite lung viral clearance after prime or prime-boost and then RSV challenge, F protein immune mice displayed weight loss and lung histopathology and high mucus production and eosinophils. In contrast, prime or prime-boost vaccination of F VLP induced effective protection, prevented infiltration of eosinophils, and vaccine- enhanced disease after challenge. This study provides insight into developing an effective and safe RSV vaccine candidate.

  8. Bacterial superglue generates a full-length circumsporozoite protein virus-like particle vaccine capable of inducing high and durable antibody responses

    DEFF Research Database (Denmark)

    Janitzek, Christoph M; Matondo, Sungwa; Thrane, Susan

    2016-01-01

    BACKGROUND: Malaria, caused by Plasmodium falciparum, continues to have a devastating impact on global health, emphasizing the great need for a malaria vaccine. The circumsporozoite protein (CSP) is an attractive target for a malaria vaccine, and forms a major component of RTS,S, the most...... clinically advanced malaria vaccine. The clinical efficacy of RTS,S has been moderate, yet has demonstrated the viability of a CSP-based malaria vaccine. In this study, a vaccine comprised of the full-length CSP antigen presented on a virus-like particle (VLP) is produced using a split-intein conjugation...... to a control vaccine consisting of soluble CSP plus AP205 VLPs. The SpyTag-VLP platform utilized in this study constitutes a versatile and rapid method to develop highly immunogenic vaccines. It might serve as a generic tool for the cost-effective development of effective VLP-vaccines, e.g., against malaria....

  9. Affinity selection of Nipah and Hendra virus-related vaccine candidates from a complex random peptide library displayed on bacteriophage virus-like particles

    Energy Technology Data Exchange (ETDEWEB)

    Peabody, David S.; Chackerian, Bryce; Ashley, Carlee; Carnes, Eric; Negrete, Oscar

    2017-01-24

    The invention relates to virus-like particles of bacteriophage MS2 (MS2 VLPs) displaying peptide epitopes or peptide mimics of epitopes of Nipah Virus envelope glycoprotein that elicit an immune response against Nipah Virus upon vaccination of humans or animals. Affinity selection on Nipah Virus-neutralizing monoclonal antibodies using random sequence peptide libraries on MS2 VLPs selected peptides with sequence similarity to peptide sequences found within the envelope glycoprotein of Nipah itself, thus identifying the epitopes the antibodies recognize. The selected peptide sequences themselves are not necessarily identical in all respects to a sequence within Nipah Virus glycoprotein, and therefore may be referred to as epitope mimics VLPs displaying these epitope mimics can serve as vaccine. On the other hand, display of the corresponding wild-type sequence derived from Nipah Virus and corresponding to the epitope mapped by affinity selection, may also be used as a vaccine.

  10. An investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins.

    Science.gov (United States)

    Windram, Oliver P; Weber, Brandon; Jaffer, Mohamed A; Rybicki, Edward P; Shepherd, Dionne N; Varsani, Arvind

    2008-01-01

    Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size ( approximately 55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6-8 kb.

  11. Inhibition of virus-like particle release of Sendai virus and Nipah virus, but not that of mumps virus, by tetherin/CD317/BST-2.

    Science.gov (United States)

    Kong, Weng-Sheng; Irie, Takashi; Yoshida, Asuka; Kawabata, Ryoko; Kadoi, Takahiro; Sakaguchi, Takemasa

    2012-09-01

    Tetherin (also known as BST-2 or CD317) has recently been identified as a potent IFN-induced anti-viral protein that inhibits the release of diverse enveloped virus particles from infected cells. The anti-viral activity of tetherin on a number of enveloped viruses, including retroviruses, filoviruses and arenaviruses, has been examined. Here, we show that tetherin is also capable of blocking the release of virus-like particles (VLPs) driven by the matrix protein of Sendai virus. Together with inhibition of Nipah virus VLP release by tetherin, these results indicate that paramyxoviruses are to be added to the list of viruses that are susceptible to tetherin inhibition. Tetherin co-localized with Nipah virus matrix proteins and accumulated in cells, indicating that it is present at, or recruited to, sites of particle assembly. It should be noted, however, that tetherin was not effective against the release of paramyxovirus mumps VLPs, indicating that certain enveloped viruses may not be sensitive to tetherin activity.

  12. M2e-displaying virus-like particles with associated RNA promote T helper 1 type adaptive immunity against influenza A.

    Directory of Open Access Journals (Sweden)

    Lorena Itatí Ibañez

    Full Text Available The ectodomain of influenza A matrix protein 2 (M2e is a candidate for a universal influenza A vaccine. We used recombinant Hepatitis B core antigen to produce virus-like particles presenting M2e (M2e-VLPs. We produced the VLPs with and without entrapped nucleic acids and compared their immunogenicity and protective efficacy. Immunization of BALB/c mice with M2e-VLPs containing nucleic acids induced a stronger, Th1-biased antibody response compared to particles lacking nucleic acids. The former also induced a stronger M2e-specific CD4(+ T cell response, as determined by ELISPOT. Mice vaccinated with alum-adjuvanted M2e-VLPs containing the nucleic acid-binding domain were better protected against influenza A virus challenge than mice vaccinated with similar particles lacking this domain, as deduced from the loss in body weight following challenge with X47 (H3N2 or PR/8 virus. Challenge of mice that had been immunized with M2e-VLPs with or without nucleic acids displayed significantly lower mortality, morbidity and lung virus titers than control-immunized groups. We conclude that nucleic acids present in M2e-VLPs correlate with improved immune protection.

  13. Papillomavirus L1 major capsid protein self-assembles into virus-like particles that are highly immunogenic.

    OpenAIRE

    Kirnbauer, R.; Booy, F; Cheng, N.; Lowy, D R; Schiller, J T

    1992-01-01

    Infection by certain human papillomavirus types is regarded as the major risk factor in the development of cervical cancer, one of the most common cancers of women worldwide. Analysis of the immunogenic and structural features of papillomavirus virions has been hampered by the inability to efficiently propagate the viruses in cultured cells. For instance, it has not been established whether the major capsid protein L1 alone is sufficient for virus particle assembly. In addition, it is not kno...

  14. Antiviral effect of gold/copper sulfide core-shell nanoparticles on GI.1 human norovirus virus like particles (VLPS)

    Science.gov (United States)

    Alston, Brittny C.

    This research studied the effects of the Au/CuS core shell nanoparticles on norovirus (NoV) VLPs in efforts to disrupt the capsids and ultimately inactivate the virus. The results of the study showed that treatment of the GI.1 norovirus VLP ranging from 0.37-5.6ug/mL5.6 microg/mL with Au/CuS core shell nanoparticle concentrations ranging from 1%-25% (v/v) was effective in altering and completely inactivating the viral capsid of the VLP. The likely mechanism of action of the nanoparticles was that the particles degraded the capsid protein and disrupted the viral capsids. This mechanism of action has been supported by the TEM imaging results and Western blotting analysis of capsid protein which showed that the viral capsids were compromised and the major capsid protein degraded.

  15. Generation of porcine reproductive and respiratory syndrome (PRRS) virus-like-particles (VLPs) with different protein composition.

    Science.gov (United States)

    García Durán, Marga; Costa, Sofia; Sarraseca, Javier; de la Roja, Nuria; García, Julia; García, Isabel; Rodríguez, Maria José

    2016-10-01

    The causative agent of Porcine Reproductive and Respiratory Syndrome (PRRS) is an enveloped ssRNA (+) virus belonging to the Arteriviridae family. Gp5 and M proteins form disulfide-linked heterodimers that constitute the major components of PRRSV envelope. Gp2, Gp3, Gp4 and E are the minor structural proteins, being the first three incorporated as multimeric complexes in the virus surface. The disease has become one of the most important causes of economic losses in the swine industry. Despite efforts to design an effective vaccine, the available ones allow only partial protection. In the last years, VLPs have become good vaccine alternatives because of safety issues and their potential to activate both branches of the immunological response. The characteristics of recombinant baculoviruses as heterologous expression system have been exploited for the production of VLPs of a wide variety of viruses. In this work, two multiple baculovirus expression vectors (BEVs) with PRRS virus envelope proteins were engineered in order to generate PRRS VLPs: on the one hand, Gp5 and M cDNAs were cloned to generate the pBAC-Gp5M vector; on the other hand, Gp2, Gp3, Gp4 and E cDNAs have been cloned to generate the pBAC-Gp234E vector. The corresponding recombinant baculoviruses BAC-Gp5M and BAC-Gp234E were employed to produce two types of VLPs: basic Gp5M VLPs, by the simultaneous expression of Gp5 and M proteins; and complete VLPs, by the co-expression of the six PRRS proteins after co-infection. The characterization of VLPs by Western blot confirmed the presence of the recombinant proteins using the available specific antibodies (Abs). The analysis by Electron microscopy showed that the two types of VLPs were indistinguishable between them, being similar in shape and size to the native PRRS virus. This system represents a potential alternative for vaccine development and a useful tool to study the implication of specific PRRS proteins in the response against the virus. Copyright

  16. Structure of the hepatitis E virus-like particle suggests mechanisms for virus assembly and receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Guu, Tom S.Y.; Liu, Zheng; Ye, Qiaozhen; Mata, Douglas A.; Li, Kunpeng; Yin, Changcheng; Zhang, Jingqiang; Tao, Yizhi Jane; (Sun Yat-Sen); (Rice); (Peking)

    2009-08-25

    Hepatitis E virus (HEV), a small, non-enveloped RNA virus in the family Hepeviridae, is associated with endemic and epidemic acute viral hepatitis in developing countries. Our 3.5-{angstrom} structure of a HEV-like particle (VLP) shows that each capsid protein contains 3 linear domains that form distinct structural elements: S, the continuous capsid; P1, 3-fold protrusions; and P2, 2-fold spikes. The S domain adopts a jelly-roll fold commonly observed in small RNA viruses. The P1 and P2 domains both adopt {beta}-barrel folds. Each domain possesses a potential polysaccharide-binding site that may function in cell-receptor binding. Sugar binding to P1 at the capsid protein interface may lead to capsid disassembly and cell entry. Structural modeling indicates that native T = 3 capsid contains flat dimers, with less curvature than those of T = 1 VLP. Our findings significantly advance the understanding of HEV molecular biology and have application to the development of vaccines and antiviral medications.

  17. Impact of internal RNA on aggregation and electrokinetics of viruses: comparison between MS2 phage and corresponding virus-like particles.

    Science.gov (United States)

    Dika, C; Duval, J F L; Ly-Chatain, H M; Merlin, C; Gantzer, C

    2011-07-01

    We compare for the first time the electrokinetic and aggregation properties of MS2 phage (pH 2.5 to 7, 1 to 100 mM NaNO(3) electrolyte concentration) with those of the corresponding virus-like particles (VLPs), which lack entirely the inner viral RNA component. In line with our previous work (J. Langlet, F. Gaboriaud, C. Gantzer, and J. F. L. Duval, Biophys. J. 94:3293-3312, 2008), it is found that modifying the content of RNA within the virus leads to very distinct electrohydrodynamic and aggregation profiles for MS2 and MS2 VLPs. Under the given pH and concentration conditions, MS2 VLPs exhibit electrophoretic mobility larger in magnitude than that of MS2, and both have similar isoelectric point (IEP) values (∼4). The electrokinetic results reflect a greater permeability of MS2 VLPs to electroosmotic flow, developed within/around these soft particles during their migration under the action of the applied electrical field. Results also support the presence of some remaining negatively charged component within the VLPs. In addition, MS2 phage systematically forms aggregates at pH values below the IEP, regardless of the magnitude of the solution ionic strength, whereas MS2 VLPs aggregate under the strict condition where the pH is relatively equal to the IEP at sufficiently low salt concentrations (electrokinetics of MS2 and corresponding VLPs conform to recently developed formalisms for the stability and electrohydrodynamics of soft multilayered particles. The differences between the surface properties of these two kinds of particles reported here suggest that VLPs may not be appropriate for predicting the behavior of pathogenic viruses in aqueous media.

  18. Impact of Internal RNA on Aggregation and Electrokinetics of Viruses: Comparison between MS2 Phage and Corresponding Virus-Like Particles

    Science.gov (United States)

    Dika, C.; Duval, J. F. L.; Ly-Chatain, H. M.; Merlin, C.; Gantzer, C.

    2011-01-01

    We compare for the first time the electrokinetic and aggregation properties of MS2 phage (pH 2.5 to 7, 1 to 100 mM NaNO3 electrolyte concentration) with those of the corresponding virus-like particles (VLPs), which lack entirely the inner viral RNA component. In line with our previous work (J. Langlet, F. Gaboriaud, C. Gantzer, and J. F. L. Duval, Biophys. J. 94:3293-3312, 2008), it is found that modifying the content of RNA within the virus leads to very distinct electrohydrodynamic and aggregation profiles for MS2 and MS2 VLPs. Under the given pH and concentration conditions, MS2 VLPs exhibit electrophoretic mobility larger in magnitude than that of MS2, and both have similar isoelectric point (IEP) values (∼4). The electrokinetic results reflect a greater permeability of MS2 VLPs to electroosmotic flow, developed within/around these soft particles during their migration under the action of the applied electrical field. Results also support the presence of some remaining negatively charged component within the VLPs. In addition, MS2 phage systematically forms aggregates at pH values below the IEP, regardless of the magnitude of the solution ionic strength, whereas MS2 VLPs aggregate under the strict condition where the pH is relatively equal to the IEP at sufficiently low salt concentrations (electrokinetics of MS2 and corresponding VLPs conform to recently developed formalisms for the stability and electrohydrodynamics of soft multilayered particles. The differences between the surface properties of these two kinds of particles reported here suggest that VLPs may not be appropriate for predicting the behavior of pathogenic viruses in aqueous media. PMID:21622784

  19. Incorporation of membrane-anchored flagellin or Escherichia coli heat-labile enterotoxin B subunit enhances the immunogenicity of rabies virus-like particles in mice and dogs

    Directory of Open Access Journals (Sweden)

    Yinglin eQi

    2015-03-01

    Full Text Available Rabies remains an important worldwide public health threat, so safe, effective and affordable vaccines are still being sought. Virus-like particle (VLP-based vaccines targeting various viral pathogens have been successfully produced, licensed and commercialized. Here, we designed and constructed two chimeric rabies virus-like particles (cRVLPs containing rabies virus (RABV glycoprotein (G, matrix (M protein, and membrane-anchored flagellin (EVLP-F or Escherichia coli heat-labile enterotoxin B subunit (EVLP-L as molecular adjuvants to enhance the immune response against rabies. The immunogenicity and potential of cRVLPs as novel rabies vaccine were evaluated by intramuscular vaccination in mouse and dog models. Mouse studies demonstrated that both EVLP-F and EVLP-L induced faster and larger virus-neutralizing antibodies (VNA responses and elicited greater numbers of CD4+ and CD8+ T cells secreting IFN-γ or IL-4 compared with a standard rabies VLP (sRVLP containing only G and M. Moreover, cRVLPs recruited and/or activated more B cells and dendritic cells in inguinal lymph nodes. EVLP-F induced a strong, specific IgG2a response but not an IgG1 response, suggesting the activation of Th1 class immunity; in contrast, Th2 class immunity was observed with EVLP-L. The significantly enhanced humoral and cellular immune responses induced by cRVLPs provided complete protection against lethal challenge with RABV. Most importantly, dogs vaccinated with EVLP-F or EVLP-L exhibited increased VNA titers in sera and enhanced IFN-γ and IL-4 secretion from peripheral blood mononuclear cells. Taken together, these results illustrate that when incorporated into sRVLP, membrane-anchored flagellin and LTB possess strong adjuvant activity. EVLP-F and EVLP-L induce significantly enhanced RABV-specific humoral and cellular immune responses in both mouse and dog. Therefore, these cRVLPs may be developed as safe and more efficacious rabies vaccine candidate for animals.

  20. Vaccination with virus-like particles containing H5 antigens from three H5N1 clades protects chickens from H5N1 and H5N8 influenza viruses

    Science.gov (United States)

    Highly pathogenic avian influenza (HPAI) viruses, especially H5N1 strains, represent a public health threat and cause widespread morbidity and mortality in domestic poultry. Recombinant virus-like particles (VLPs) represent a promising novel vaccine approach to control avian influenza including HPAI...

  1. A Modular Vaccine Development Platform Based on Sortase-Mediated Site-Specific Tagging of Antigens onto Virus-Like Particles.

    Science.gov (United States)

    Tang, Shubing; Xuan, Baoqin; Ye, Xiaohua; Huang, Zhong; Qian, Zhikang

    2016-05-12

    Virus-like particles (VLPs) can be used as powerful nanoscale weapons to fight against virus infection. In addition to direct use as vaccines, VLPs have been extensively exploited as platforms on which to display foreign antigens for prophylactic vaccination and immunotherapeutic treatment. Unfortunately, fabrication of new chimeric VLP vaccines in a versatile, site-specific and highly efficient manner is beyond the capability of traditional VLP vaccine design approaches, genetic insertion and chemical conjugation. In this study, we described a greatly improved VLP display strategy by chemoenzymatic site-specific tailoring antigens on VLPs surface with high efficiency. Through the transpeptidation mediated by sortase A, one protein and two epitopes containing N-terminal oligoglycine were conjugated to the LPET motif on the surface of hepatitis B virus core protein (HBc) VLPs with high density. All of the new chimeric VLPs induced strong specific IgG responses. Furthermore, the chimeric VLPs with sortase A tagged enterovirus 71 (EV71) SP70 epitope could elicit effective antibodies against EV71 lethal challenging as well as the genetic insertion chimeric VLPs. The sortase A mediated chemoenzymatic site-specific tailoring of the HBc VLP approach shows great potential in new VLP vaccine design for its simplicity, site specificity, high efficiency, and versatility.

  2. Virus-like particle vaccines containing F or F and G proteins confer protection against respiratory syncytial virus without pulmonary inflammation in cotton rats.

    Science.gov (United States)

    Hwang, Hye Suk; Kim, Ki-Hye; Lee, Youri; Lee, Young-Tae; Ko, Eun-Ju; Park, SooJin; Lee, Jong Seok; Lee, Byung-Cheol; Kwon, Young-Man; Moore, Martin L; Kang, Sang-Moo

    2017-05-04

    Vaccine-enhanced disease has been a major obstacle in developing a safe vaccine against respiratory syncytial virus (RSV). This study demonstrates the immunogenicity, efficacy, and safety of virus-like particle (VLP) vaccines containing RSV F (F VLP), G (G VLP), or F and G proteins (FG VLP) in cotton rats. RSV specific antibodies were effectively induced by vaccination of cotton rats with F VLP or FG VLP vaccines. After challenge, lung RSV clearance was observed with RSV F, G, FG VLP, and formalin inactivated RSV (FI-RSV) vaccines. Upon RSV infection, cotton rats with RSV VLP vaccines were protected against airway hyper-responsiveness and weight loss, which are different from FI-RSV vaccination exhibiting vaccine-enhanced disease of airway obstruction, weight loss, and severe histopathology with eosinophilia and mucus production. FG VLP and F VLP vaccines did not cause pulmonary inflammation whereas G VLP induced moderate lung inflammation with eosinophilia and mucus production. In particular, F VLP and FG VLP vaccines were found to be effective in inducing antibody secreting cell responses in bone marrow and lymphoid organs as well as avoiding the induction of T helper type 2 cytokines. These results provide further evidence to develop a safe RSV vaccine based on VLP platforms.

  3. Combined virus-like particle and fusion protein-encoding DNA vaccination of cotton rats induces protection against respiratory syncytial virus without causing vaccine-enhanced disease.

    Science.gov (United States)

    Hwang, Hye Suk; Lee, Young-Tae; Kim, Ki-Hye; Park, Soojin; Kwon, Young-Man; Lee, Youri; Ko, Eun-Ju; Jung, Yu-Jin; Lee, Jong Seok; Kim, Yu-Jin; Lee, Yu-Na; Kim, Min-Chul; Cho, Minkyoung; Kang, Sang-Moo

    2016-07-01

    A safe and effective vaccine against respiratory syncytial virus (RSV) should confer protection without causing vaccine-enhanced disease. Here, using a cotton rat model, we investigated the protective efficacy and safety of an RSV combination vaccine composed of F-encoding plasmid DNA and virus-like particles containing RSV fusion (F) and attachment (G) glycoproteins (FFG-VLP). Cotton rats with FFG-VLP vaccination controlled lung viral replication below the detection limit, and effectively induced neutralizing activity and antibody-secreting cell responses. In comparison with formalin inactivated RSV (FI-RSV) causing severe RSV disease after challenge, FFG-VLP vaccination did not cause weight loss, airway hyper-responsiveness, IL-4 cytokines, histopathology, and infiltrates of proinflammatory cells such as eosinophils. FFG-VLP was even more effective in preventing RSV-induced pulmonary inflammation than live RSV infections. This study provides evidence that FFG-VLP can be developed into a safe and effective RSV vaccine candidate. Copyright © 2016. Published by Elsevier Inc.

  4. Microneedle patch delivery to the skin of virus-like particles containing heterologous M2e extracellular domains of influenza virus induces broad heterosubtypic cross-protection.

    Science.gov (United States)

    Kim, Min-Chul; Lee, Jeong Woo; Choi, Hyo-Jick; Lee, Yu-Na; Hwang, Hye Suk; Lee, Jongsang; Kim, Cheol; Lee, Jong Seok; Montemagno, Carlo; Prausnitz, Mark R; Kang, Sang-Moo

    2015-07-28

    A broadly cross-protective influenza vaccine that can be administrated by a painless self-immunization method would be a value as a potential universal mass vaccination strategy. This study developed a minimally-invasive microneedle (MN) patch for skin vaccination with virus-like particles containing influenza virus heterologous M2 extracellular (M2e) domains (M2e5x VLPs) as a universal vaccine candidate without adjuvants. The stability of M2e5x VLP-coated microneedles was maintained for 8weeks at room temperature without losing M2e antigenicity and immunogenicity. MN skin immunization induced strong humoral and mucosal M2e antibody responses and conferred cross-protection against heterosubtypic H1N1, H3N2, and H5N1 influenza virus challenges. In addition, M2e5x VLP MN skin vaccination induced T-helper type 1 responses such as IgG2a isotype antibodies and IFN-γ producing cells at higher levels than those by conventional intramuscular injection. These potential immunological and logistic advantages for skin delivery of M2e5x VLP MN vaccines could offer a promising approach to develop an easy-to-administer universal influenza vaccine. Copyright © 2015. Published by Elsevier B.V.

  5. Neutralizing antibodies induced by recombinant virus-like particles of enterovirus 71 genotype C4 inhibit infection at pre- and post-attachment steps.

    Directory of Open Access Journals (Sweden)

    Zhiqiang Ku

    Full Text Available BACKGROUND: Enterovirus 71 (EV71 is a major causative agent of hand, foot and mouth disease, which has been prevalent in Asia-Pacific regions, causing significant morbidity and mortality in young children. Antibodies elicited by experimental EV71 vaccines could neutralize infection in vitro and passively protect animal models from lethal challenge, indicating that neutralizing antibodies play an essential role in protection. However, how neutralizing antibodies inhibit infection in vitro remains unclear. METHODS/FINDINGS: In the present study, we explored the mechanisms of neutralization by antibodies against EV71 virus-like particles (VLPs. Recombinant VLPs of EV71 genotype C4 were produced in insect cells using baculovirus vectors. Immunization with the VLPs elicited a high-titer, EV71-specific antibody response in mice. Anti-VLP mouse sera potently neutralized EV71 infection in vitro. The neutralizing antibodies in the anti-VLP mouse sera were found to target mainly an extremely conserved epitope (FGEHKQEKDLEYGAC located at the GH loop of the VP1 protein. The neutralizing anti-VLP antisera were able to inhibit virus binding to target cells efficiently. In addition, post-attachment treatment of virus-bound cells with the anti-VLP antisera also neutralized virus infection, although the antibody concentration required was higher than that of the pre-attachment treatment. CONCLUSIONS: Collectively, our findings represent a valuable addition to the understanding of mechanisms of EV71 neutralization and have strong implications for EV71 vaccine development.

  6. Antibody Persistence in Adults Two Years after Vaccination with an H1N1 2009 Pandemic Influenza Virus-Like Particle Vaccine

    Science.gov (United States)

    Villasís-Keever, Miguel Ángel; Núñez-Valencia, Adriana; Boscó-Gárate, Ilka; Lozano-Dubernard, Bernardo; Lara-Puente, Horacio; Espitia, Clara; Alpuche-Aranda, Celia; Bonifaz, Laura C.; Arriaga-Pizano, Lourdes; Pastelin-Palacios, Rodolfo; Isibasi, Armando; López-Macías, Constantino

    2016-01-01

    The influenza virus is a human pathogen that causes epidemics every year, as well as potential pandemic outbreaks, as occurred in 2009. Vaccination has proven to be sufficient in the prevention and containment of viral spreading. In addition to the current egg-based vaccines, new and promising vaccine platforms, such as cell culture-derived vaccines that include virus-like particles (VLPs), have been developed. VLPs have been shown to be both safe and immunogenic against influenza infections. Although antibody persistence has been studied in traditional egg-based influenza vaccines, studies on antibody response durations induced by VLP influenza vaccines in humans are scarce. Here, we show that subjects vaccinated with an insect cell-derived VLP vaccine, in the midst of the 2009 H1N1 influenza pandemic outbreak in Mexico City, showed antibody persistence up to 24 months post-vaccination. Additionally, we found that subjects that reported being revaccinated with a subsequent inactivated influenza virus vaccine showed higher antibody titres to the pandemic influenza virus than those who were not revaccinated. These findings provide insights into the duration of the antibody responses elicited by an insect cell-derived pandemic influenza VLP vaccine and the possible effects of subsequent influenza vaccination on antibody persistence induced by this VLP vaccine in humans. PMID:26919288

  7. Protection induced by virus-like particles containing Toxoplasma gondii microneme protein 8 against highly virulent RH strain of Toxoplasma gondii infection.

    Science.gov (United States)

    Lee, Su-Hwa; Kim, Ah-Ra; Lee, Dong-Hun; Rubino, Ilaria; Choi, Hyo-Jick; Quan, Fu-Shi

    2017-01-01

    Toxoplasma gondii (T. gondii) microneme protein 8 (MIC8) represents a novel, functional distinct invasion factor. In this study, we generated virus-like particles (VLPs) targeting Toxoplasma gondii MIC8 for the first time, and investigated the protection against highly virulent RH strain of T. gondii in a mouse model. We found that VLP vaccination induced Toxoplasma gondii-specific IgG and IgG1 antibody responses in the sera. Upon challenge infection with RH strain of T. gondii tachyzoites, vaccinated mice showed a significant increase of both IgG antibodies in sera and IgA antibodies in feces compared to those before challenge, and a rapid expansion of both germinal center B cell (B220+, GL7+) and T cell (CD4+, CD8+) populations. Importantly, intranasally immunized mice showed higher neutralizing antibodies and displayed no proinflammatory cytokine IFN-γ in the spleen. Mice were completely protected from a lethal challenge infection with the highly virulent T. gondii (RH) showing no body weight loss (100% survival). Our study shows the effective protection against T. gondii infection provided by VLPs containing microneme protein 8 of T. gondii, thus indicating a potential T. gondii vaccine candidate.

  8. Novel chimeric foot-and-mouth disease virus-like particles harboring serotype O VP1 protect guinea pigs against challenge.

    Science.gov (United States)

    Li, Haitao; Li, Zhiyong; Xie, Yinli; Qin, Xiaodong; Qi, Xingcai; Sun, Peng; Bai, Xingwen; Ma, Youji; Zhang, Zhidong

    2016-02-01

    Foot-and-mouth disease is a highly contagious, acute viral disease of cloven-hoofed animal species causing severe economic losses worldwide. Among the seven serotypes of foot-and-mouth disease virus (FMDV), serotype O is predominant, but its viral capsid is more acid sensitive than other serotypes, making it more difficult to produce empty serotype O VLPs in the low pH insect hemolymph. Therefore, a novel chimeric virus-like particle (VLP)-based candidate vaccine for serotype O FMDV was developed and characterized in the present study. The chimeric VLPs were composed of antigenic VP1 from serotype O and segments of viral capsid proteins from serotype Asia1. These VLPs elicited significantly higher FMDV-specific antibody levels in immunized mice than did the inactivated vaccine. Furthermore, the chimeric VLPs protected guinea pigs from FMDV challenge with an efficacy similar to that of the inactivated vaccine. These results suggest that chimeric VLPs have the potential for use in vaccines against serotype O FMDV infection. Copyright © 2015 Elsevier B.V. All rights reserved.

  9. Antibody Persistence in Adults Two Years after Vaccination with an H1N1 2009 Pandemic Influenza Virus-Like Particle Vaccine.

    Directory of Open Access Journals (Sweden)

    Nuriban Valero-Pacheco

    Full Text Available The influenza virus is a human pathogen that causes epidemics every year, as well as potential pandemic outbreaks, as occurred in 2009. Vaccination has proven to be sufficient in the prevention and containment of viral spreading. In addition to the current egg-based vaccines, new and promising vaccine platforms, such as cell culture-derived vaccines that include virus-like particles (VLPs, have been developed. VLPs have been shown to be both safe and immunogenic against influenza infections. Although antibody persistence has been studied in traditional egg-based influenza vaccines, studies on antibody response durations induced by VLP influenza vaccines in humans are scarce. Here, we show that subjects vaccinated with an insect cell-derived VLP vaccine, in the midst of the 2009 H1N1 influenza pandemic outbreak in Mexico City, showed antibody persistence up to 24 months post-vaccination. Additionally, we found that subjects that reported being revaccinated with a subsequent inactivated influenza virus vaccine showed higher antibody titres to the pandemic influenza virus than those who were not revaccinated. These findings provide insights into the duration of the antibody responses elicited by an insect cell-derived pandemic influenza VLP vaccine and the possible effects of subsequent influenza vaccination on antibody persistence induced by this VLP vaccine in humans.

  10. Passive Transfer of Immune Sera Induced by a Zika Virus-Like Particle Vaccine Protects AG129 Mice Against Lethal Zika Virus Challenge.

    Science.gov (United States)

    Espinosa, Diego; Mendy, Jason; Manayani, Darly; Vang, Lo; Wang, Chunling; Richard, Tiffany; Guenther, Ben; Aruri, Jayavani; Avanzini, Jenny; Garduno, Fermin; Farness, Peggy; Gurwith, Marc; Smith, Jon; Harris, Eva; Alexander, Jeff

    2017-12-12

    Zika virus (ZIKV) poses a serious public health threat due to its association with birth defects in developing fetuses and Guillain-Barré Syndrome in adults. We are developing a ZIKV vaccine based on virus-like particles (VLPs) generated in transiently transfected HEK293 cells. The genetic construct consists of the prM and envelope structural protein genes of ZIKV placed downstream from a heterologous signal sequence. To better understand the humoral responses and correlates of protection (CoP) induced by the VLP vaccine, we evaluated VLP immunogenicity with and without alum in immune-competent mice (C57Bl/6 x Balb/c) and observed efficient induction of neutralizing antibody as well as a dose-sparing effect of alum. To assess the efficacy of the immune sera, we performed passive transfer experiments in AG129 mice. Mice that received the immune sera prior to ZIKV infection demonstrated significantly reduced viral replication as measured by viral RNA levels in the blood and remained healthy, whereas control mice succumbed to infection. The results underscore the protective effect of the antibody responses elicited by this ZIKV VLP vaccine candidate. These studies will help define optimal vaccine formulations, contribute to translational efforts in developing a vaccine for clinical development, and assist in the definition of immunologic CoP. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  11. Immunogenic Display of Diverse Peptides, Including a Broadly Cross-Type Neutralizing Human Papillomavirus L2 epitope, on Virus-like Particles of the RNA Bacteriophage PP7

    Science.gov (United States)

    Caldeira, Jerri do Carmo; Medford, Alexander; Kines, Rhonda C.; Lino, Christopher A.; Schiller, John T.; Chackerian, Bryce; Peabody, David S.

    2010-01-01

    The immunogenicity of an antigen can be dramatically increased by displaying it in a dense, multivalent context, such as on the surface of a virus or virus-like particle (VLP). Here we describe a highly versatile VLP platform for peptide display based on VLPs of the RNA bacteriophage PP7. We show that this platform can be used for the engineered display of specific peptide sequences as well as for the construction of random peptide libraries. Peptides representing the FLAG epitope, the V3 loop of HIV gp120, and a broadly cross-type neutralizing epitope from L2, the minor capsid protein of Human Papillomavirus type 16 (HPV16), were inserted into an exposed surface loop of a form of PP7 coat protein in which the two identical polypeptides of coat were fused together to form a single-chain dimer. The recombinant proteins assembled into VLPs, displayed these peptides on their surfaces, and induced high titer antibody responses. The single-chain dimer was also highly tolerant of random 6-, 8-, and 10-amino acid insertions. PP7 VLPs displaying the HPV16 L2 epitope generated robust anti-HPV16 L2 serum antibodies after intramuscular injection that protected mice from genital infection with HPV16 pseudovirus as well as a heterologous HPV pseudovirus type, HPV45. Thus, PP7 VLPs are well-suited for the display of a wide diversity of peptides in a highly immunogenic format. PMID:20434554

  12. Influenza M2 virus-like particles confer a broader range of cross protection to the strain-specific pre-existing immunity.

    Science.gov (United States)

    Kim, Min-Chul; Lee, Yu-Na; Hwang, Hye Suk; Lee, Young-Tae; Ko, Eun-Ju; Jung, Yu-Jin; Cho, Min Kyoung; Kim, Yu-Jin; Lee, Jong Seok; Ha, Suk-Hoon; Kang, Sang-Moo

    2014-10-07

    Immunity in humans with annual vaccination does not provide effective protection against antigenically distinct strains. As an approach to improve cross-protection in the presence of pre-existing strain-specific immunity, we investigated the efficacy of heterologous and heterosubtypic protection in previously vaccinated mice at earlier times after subsequent immunization with conserved-antigenic target influenza M2 ectodomain (M2e) virus-like particle vaccine (M2e5× VLP). Immunization of mice with H1N1 split vaccine induced virus specific antibodies to homologous influenza virus but did not provide heterosubtypic hemagglutination inhibiting antibody responses and cross-protection. However, subsequent M2e5× VLP immunization induced an M2e specific antibody response as well as interferon-γ (IFN-γ) producing cells in systemic and mucosal sites. Upon lethal challenge with H3N2 or H5N1 subtype influenza viruses, subsequently immunized mice with M2e5× VLP were well protected against heterosubtypic influenza viruses. These results provide evidence that non-seasonal immunization with M2e5× VLP, an experimental candidate for universal vaccine, is a promising approach for broadening the cross-protection even in the presence of strain-specific immunity. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Heterologous prime-boost immunization regimens using adenovirus vector and virus-like particles induce broadly neutralizing antibodies against H5N1 avian influenza viruses.

    Science.gov (United States)

    Lin, Shih-Chang; Liu, Wen-Chun; Lin, Yu-Fen; Huang, Yu-Hsuan; Liu, Jin-Hwang; Wu, Suh-Chin

    2013-11-01

    Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to trigger severe diseases in poultry and humans, prompting efforts to develop an effective vaccine. Toward that goal, we constructed a recombinant adenovirus vector encoding influenza hemagglutin (rAd-HA) and a flagellin-containing virus-like particle (FliC-VLP). Using a murine model, we investigated a heterologous prime-boost vaccination regimen combining these two vectors. Our results indicate that priming with the rAd-HA vector followed by a FliC-VLP booster induced the highest HA-specific total IgG, IgG1and IgG2a. Maximum neutralizing antibody titers against homologous and heterologous clades of H5N1 virus strains and hemagglutination inhibition resulted from the heterologous vaccination strategy. Our results are likely to contribute to the development of more effective H5N1 vaccines. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Development and application of a reversed-phase high-performance liquid chromatographic method for quantitation and characterization of a Chikungunya virus-like particle vaccine.

    Science.gov (United States)

    Shytuhina, Anastasija; Pristatsky, Pavlo; He, Jian; Casimiro, Danilo R; Schwartz, Richard M; Hoang, Van M; Ha, Sha

    2014-10-17

    To effectively support the development of a Chikungunya (CHIKV) virus-like particle (VLP) vaccine, a sensitive and robust high-performance liquid chromatography (HPLC) method that can quantitate CHIKV VLPs and monitor product purity throughout the manufacturing process is needed. We developed a sensitive reversed-phase HPLC (RP-HPLC) method that separates capsid, E1, and E2 proteins in CHIKV VLP vaccine with good resolution. Each protein component was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-ToF) mass spectrometry (MS). The post-translational modifications on the viral glycoproteins E1 and E2 were further identified by intact protein mass measurements with liquid chromatography-mass spectrometry (LC-MS). The RP-HPLC method has a linear range of 0.51-12 μg protein, an accuracy of 96-106% and a precision of 12% RSD, suitable for vaccine product release testing. In addition, we demonstrated that the RP-HPLC method is useful for characterizing viral glycoprotein post-translational modifications, monitoring product purity during process development and assessing product stability during formulation development. Published by Elsevier B.V.

  15. Virus-Like Particles Derived from HIV-1 for Delivery of Nuclear Proteins: Improvement of Production and Activity by Protein Engineering.

    Science.gov (United States)

    Robert, Marc-André; Lytvyn, Viktoria; Deforet, Francis; Gilbert, Rénald; Gaillet, Bruno

    2017-01-01

    Virus-like particles (VLPs) derived from retroviruses and lentiviruses can be used to deliver recombinant proteins without the fear of causing insertional mutagenesis to the host cell genome. In this study we evaluate the potential of an inducible lentiviral vector packaging cell line for VLP production. The Gag gene from HIV-1 was fused to a gene encoding a selected protein and it was transfected into the packaging cells. Three proteins served as model: the green fluorescent protein and two transcription factors-the cumate transactivator (cTA) of the inducible CR5 promoter and the human Krüppel-like factor 4 (KLF4). The sizes of the VLPs were 120-150 nm in diameter and they were resistant to freeze/thaw cycles. Protein delivery by the VLPs reached up to 100% efficacy in human cells and was well tolerated. Gag-cTA triggered up to 1100-fold gene activation of the reporter gene in comparison to the negative control. Protein engineering was required to detect Gag-KLF4 activity. Thus, insertion of the VP16 transactivation domain increased the activity of the VLPs by eightfold. An additional 2.4-fold enhancement was obtained by inserting nuclear export signal. In conclusion, our platform produced VLPs capable of efficient protein transfer, and it was shown that protein engineering can be used to improve the activity of the delivered proteins as well as VLP production.

  16. Combined virus-like particle and fusion protein-encoding DNA vaccination of cotton rats induces protection against respiratory syncytial virus without causing vaccine-enhanced disease

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Hye Suk; Lee, Young-Tae; Kim, Ki-Hye; Park, Soojin; Kwon, Young-Man; Lee, Youri; Ko, Eun-Ju; Jung, Yu-Jin [Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences and Department of Biology, Georgia State University, Atlanta, GA (United States); Lee, Jong Seok [Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences and Department of Biology, Georgia State University, Atlanta, GA (United States); National Institute of Biological Resources, Incheon (Korea, Republic of); Kim, Yu-Jin [Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences and Department of Biology, Georgia State University, Atlanta, GA (United States); Lee, Yu-Na; Kim, Min-Chul [Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences and Department of Biology, Georgia State University, Atlanta, GA (United States); Animal and Plant Quarantine Agency, Gyeonggi-do, Gimcheon, Gyeongsangbukdo (Korea, Republic of); Cho, Minkyoung [Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences and Department of Biology, Georgia State University, Atlanta, GA (United States); Kang, Sang-Moo, E-mail: skang24@gsu.edu [Center for Inflammation, Immunity & Infection, Institute for Biomedical Sciences and Department of Biology, Georgia State University, Atlanta, GA (United States)

    2016-07-15

    A safe and effective vaccine against respiratory syncytial virus (RSV) should confer protection without causing vaccine-enhanced disease. Here, using a cotton rat model, we investigated the protective efficacy and safety of an RSV combination vaccine composed of F-encoding plasmid DNA and virus-like particles containing RSV fusion (F) and attachment (G) glycoproteins (FFG-VLP). Cotton rats with FFG-VLP vaccination controlled lung viral replication below the detection limit, and effectively induced neutralizing activity and antibody-secreting cell responses. In comparison with formalin inactivated RSV (FI-RSV) causing severe RSV disease after challenge, FFG-VLP vaccination did not cause weight loss, airway hyper-responsiveness, IL-4 cytokines, histopathology, and infiltrates of proinflammatory cells such as eosinophils. FFG-VLP was even more effective in preventing RSV-induced pulmonary inflammation than live RSV infections. This study provides evidence that FFG-VLP can be developed into a safe and effective RSV vaccine candidate. - Highlights: • Combined RSV FFG VLP vaccine is effective in inducing F specific responses. • FFG VLP vaccine confers RSV neutralizing activity and viral control in cotton rats. • Cotton rats with RSV FFG VLP vaccination do not show vaccine-enhanced disease. • Cotton rats with FFG VLP vaccine induce F specific antibody secreting cell responses. • Cotton rats with FFG VLP do not induce lung cellular infiltrates and Th2 cytokine.

  17. Scalable chromatography-based purification of virus-like particle carrier for epitope based influenza A vaccine produced in Escherichia coli.

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    Lagoutte, Priscillia; Mignon, Charlotte; Donnat, Stéphanie; Stadthagen, Gustavo; Mast, Jan; Sodoyer, Régis; Lugari, Adrien; Werle, Bettina

    2016-06-01

    Virus-like particles (VLPs) are promising molecular structures for the design and construction of novel vaccines, diagnostic tools, and gene therapy vectors. Size, oligomer assembly and repetitiveness of epitopes are optimal features to induce strong immune responses. Several VLP-based vaccines are currently licensed and commercialized, and many vaccine candidates are now under preclinical and clinical studies. In recent years, the development of genetically engineered recombinant VLPs has accelerated the need for new, improved downstream processes. In particular, a rapid low cost purification process has been identified as a remaining key challenge in manufacturing process development. In the present study we set up a size-exclusion chromatography-based, scalable purification protocol for the purification of a VLP-based influenza A vaccine produced in Escherichia coli. Recombinant VLPs derived from the RNA bacteriophage MS2 displaying an epitope from the ectodomain of Matrix 2 protein from influenza A virus were produced and purified. The 3 steps purification protocol uses a recently developed multimodal size-exclusion chromatography medium (Capto™ Core 700) in combination with detergent extraction and size-exclusion polishing to reach a 89% VLP purity with a 19% yield. The combination of this downstream strategy following production in E. coli would be suited for production of VLP-based veterinary vaccines targeting livestock and companion animals where large amounts of doses must be produced at an affordable price. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Characterization of protection afforded by a bivalent virus-like particle vaccine against bluetongue virus serotypes 1 and 4 in sheep.

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    Ana Cristina Pérez de Diego

    Full Text Available BACKGROUND: Bluetongue virus (BTV is an economically important, arthropod borne, emerging pathogen in Europe, causing disease mainly in sheep and cattle. Routine vaccination for bluetongue would require the ability to distinguish between vaccinated and infected individuals (DIVA. Current vaccines are effective but are not DIVA. Virus-like particles (VLPs are highly immunogenic structural mimics of virus particles, that only contain a subset of the proteins present in a natural infection. VLPs therefore offer the potential for the development of DIVA compatible bluetongue vaccines. METHODOLOGY/PRINCIPAL FINDINGS: Merino sheep were vaccinated with either monovalent BTV-1 VLPs or a bivalent mixture of BTV-1 VLPs and BTV-4 VLPs, and challenged with virulent BTV-1 or BTV-4. Animals were monitored for clinical signs, antibody responses, and viral RNA. 19/20 animals vaccinated with BTV-1 VLPs either alone or in combination with BTV-4 VLPs developed neutralizing antibodies to BTV-1, and group specific antibodies to BTV VP7. The one animal that showed no detectable neutralizing antibodies, or group specific antibodies, had detectable viral RNA following challenge but did not display any clinical signs on challenge with virulent BTV-1. In contrast, all control animals' demonstrated classical clinical signs for bluetongue on challenge with the same virus. Six animals were vaccinated with bivalent vaccine and challenged with virulent BTV-4, two of these animals had detectable viral levels of viral RNA, and one of these showed clinical signs consistent with BTV infection and died. CONCLUSIONS: There is good evidence that BTV-1 VLPs delivered as monovalent or bivalent immunogen protect from bluetongue disease on challenge with virulent BTV-1. However, it is possible that there is some interference in protective response for BTV-4 in the bivalent BTV-1 and BTV-4 VLP vaccine. This raises the question of whether all combinations of bivalent BTV vaccines are

  19. Co-translational localization of an LTR-retrotransposon RNA to the endoplasmic reticulum nucleates virus-like particle assembly sites.

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    Jung H Doh

    2014-03-01

    Full Text Available The transcript of retrovirus-like transposons functions as an mRNA for synthesis of capsid and replication proteins and as the genomic RNA of virus-like particles (VLPs, wherein the genome is replicated. Retrotransposon RNA and proteins coalesce in a cytoplasmic focus, or retrosome, to initiate VLP assembly, but it is not known how the retrosome is nucleated. We determined how the RNA and Gag protein of the Saccharomyces cerevisiae Ty1 retrotransposon are directed to the retrosome. We found that Ty1 RNA is translated in association with signal recognition particle (SRP, a universally conserved chaperone that binds specific ribosome-nascent chain (RNC complexes and targets the nascent peptide to the endoplasmic reticulum (ER. Gag is translocated to the ER lumen; yet, it is also found in the cytoplasm, associated with SRP-RNC complexes. In the absence of ER translocation, Gag is synthesized but rapidly degraded, and Ty1 RNA does not coalesce in retrosomes. These findings suggest that Gag adopts a stable conformation in the ER lumen, is retrotranslocated to the cytoplasm, binds to Ty1 RNA on SRP-RNC complexes and multimerizes to nucleate retrosomes. Consistent with this model, we show that slowing the rate of co-translational ER translocation by limiting SRP increases the prevalence of retrosomes, while suppressing the translocation defect of srp hypomorphs by slowing translational elongation rapidly decreases retrosome formation. Thus, retrosomes are dynamic foci of Ty1 RNA-RNC complexes whose formation is modulated by the rate of co-translational ER translocation. Together, these findings suggest that translating Ty1 mRNA and the genomic RNA of VLPs originate in a single pool and moreover, that co-translational localization of Ty1 RNA nucleates the presumptive VLP assembly site. The separation of nascent Gag from its RNA template by transit through the ER allows Gag to bind translating Ty1 RNA without displaying a cis-preference for its encoding

  20. Characterization of Protection Afforded by a Bivalent Virus-Like Particle Vaccine against Bluetongue Virus Serotypes 1 and 4 in Sheep

    Science.gov (United States)

    Pérez de Diego, Ana Cristina; Athmaram, Thimmasandra N.; Stewart, Meredith; Rodríguez-Sánchez, Belén; Sánchez-Vizcaíno, José Manuel; Noad, Robert; Roy, Polly

    2011-01-01

    Background Bluetongue virus (BTV) is an economically important, arthropod borne, emerging pathogen in Europe, causing disease mainly in sheep and cattle. Routine vaccination for bluetongue would require the ability to distinguish between vaccinated and infected individuals (DIVA). Current vaccines are effective but are not DIVA. Virus-like particles (VLPs) are highly immunogenic structural mimics of virus particles, that only contain a subset of the proteins present in a natural infection. VLPs therefore offer the potential for the development of DIVA compatible bluetongue vaccines. Methodology/Principal Findings Merino sheep were vaccinated with either monovalent BTV-1 VLPs or a bivalent mixture of BTV-1 VLPs and BTV-4 VLPs, and challenged with virulent BTV-1 or BTV-4. Animals were monitored for clinical signs, antibody responses, and viral RNA. 19/20 animals vaccinated with BTV-1 VLPs either alone or in combination with BTV-4 VLPs developed neutralizing antibodies to BTV-1, and group specific antibodies to BTV VP7. The one animal that showed no detectable neutralizing antibodies, or group specific antibodies, had detectable viral RNA following challenge but did not display any clinical signs on challenge with virulent BTV-1. In contrast, all control animals' demonstrated classical clinical signs for bluetongue on challenge with the same virus. Six animals were vaccinated with bivalent vaccine and challenged with virulent BTV-4, two of these animals had detectable viral levels of viral RNA, and one of these showed clinical signs consistent with BTV infection and died. Conclusions There is good evidence that BTV-1 VLPs delivered as monovalent or bivalent immunogen protect from bluetongue disease on challenge with virulent BTV-1. However, it is possible that there is some interference in protective response for BTV-4 in the bivalent BTV-1 and BTV-4 VLP vaccine. This raises the question of whether all combinations of bivalent BTV vaccines are possible, or if

  1. An HPV 16 L1-based chimeric human papilloma virus-like particles containing a string of epitopes produced in plants is able to elicit humoral and cytotoxic T-cell activity in mice

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    Weiss-Steider Benny

    2009-01-01

    Full Text Available Abstract Background Even though two prophylactic vaccines against HPV are currently licensed, infections by the virus continue to be a major health problem mainly in developing countries. The cost of the vaccines limits wide-scale application in poor countries. A promising strategy for producing affordable and efficient vaccines involves the expression of recombinant immunogens in plants. Several HPV genes have been expressed in plants, including L1, which can self-assemble into virus-like particles. A plant-based, dual prophylactic/therapeutic vaccine remains an attractive possibility. Results We sought to express in tomato plants chimeric HPV 16 VLPs containing L1 fused to a string of epitopes from HPV 16 E6 and E7 proteins. The L1 employed had been modified to eliminate a strong inhibitory region at the 5' end of the molecule to increase expression levels. Several tomato lines were obtained expressing either L1 alone or L1-E6/E7 from 0.05% to 0.1% of total soluble protein. Stable integration of the transgenes was verified by Southern blot. Northern and western blot revealed successful expression of the transgenes at the mRNA and protein level. The chimeric VLPs were able to assemble adequately in tomato cells. Intraperitoneal administration in mice was able to elicit both neutralizing antibodies against the viral particle and cytotoxic T-lymphocytes activity against the epitopes. Conclusion In this work, we report for the first time the expression in plants of a chimeric particle containing the HPV 16 L1 sequence and a string of T-cell epitopes from HPV 16 E6 and E7 fused to the C-terminus. The particles were able to induce a significant antibody and cytotoxic T-lymphocytes response. Experiments in vivo are in progress to determine whether the chimeric particles are able to induce regression of disease and resolution of viral infection in mice. Chimeric particles of the type described in this work may potentially be the basis for developing

  2. Modification of Asparagine-Linked Glycan Density for the Design of Hepatitis B Virus Virus-Like Particles with Enhanced Immunogenicity.

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    Hyakumura, Michiko; Walsh, Renae; Thaysen-Andersen, Morten; Kingston, Natalie J; La, Mylinh; Lu, Louis; Lovrecz, George; Packer, Nicolle H; Locarnini, Stephen; Netter, Hans J

    2015-11-01

    The small envelope proteins (HBsAgS) derived from hepatitis B virus (HBV) represent the antigenic components of the HBV vaccine and are platforms for the delivery of foreign antigenic sequences. To investigate structure-immunogenicity relationships for the design of improved immunization vectors, we have generated biochemically modified virus-like particles (VLPs) exhibiting glycoengineered HBsAgS. For the generation of hypoglycosylated VLPs, the wild-type (WT) HBsAgS N146 glycosylation site was converted to N146Q; for constructing hyperglycosylated VLPs, potential glycosylation sites were introduced in the HBsAgS external loop region at positions T116 and G130 in addition to the WT site. The introduced T116N and G130N sites were utilized as glycosylation anchors resulting in the formation of hyperglycosylated VLPs. Mass spectroscopic analyses showed that the hyperglycosylated VLPs carry the same types of glycans as WT VLPs, with minor variations regarding the degree of fucosylation, bisecting N-acetylglucosamines, and sialylation. Antigenic fingerprints for the WT and hypo- and hyperglycosylated VLPs using a panel of 19 anti-HBsAgS monoclonal antibodies revealed that 15 antibodies retained their ability to bind to the different VLP glyco-analogues, suggesting that the additional N-glycans did not shield extensively for the HBsAgS-specific antigenicity. Immunization studies with the different VLPs showed a strong correlation between N-glycan abundance and antibody titers. The T116N VLPs induced earlier and longer-lasting antibody responses than did the hypoglycosylated and WT VLPs. The ability of nonnative VLPs to promote immune responses possibly due to differences in their glycosylation-related interaction with cells of the innate immune system illustrates pathways for the design of immunogens for superior preventive applications. The use of biochemically modified, nonnative immunogens represents an attractive strategy for the generation of modulated or enhanced

  3. Broad Cross-Protection Is Induced in Preclinical Models by a Human Papillomavirus Vaccine Composed of L1/L2 Chimeric Virus-Like Particles

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    Boxus, Mathieu; Fochesato, Michel; Miseur, Agnès; Mertens, Emmanuel; Dendouga, Najoua; Brendle, Sarah; Balogh, Karla K.; Christensen, Neil D.

    2016-01-01

    ABSTRACT At least 15 high-risk human papillomaviruses (HPVs) are linked to anogenital preneoplastic lesions and cancer. Currently, there are three licensed prophylactic HPV vaccines based on virus-like particles (VLPs) of the L1 major capsid protein from HPV-2, -4, or -9, including the AS04-adjuvanted HPV-16/18 L1 vaccine. The L2 minor capsid protein contains HPV-neutralizing epitopes that are well conserved across numerous high-risk HPVs. Therefore, the objective of our study was to assess the capacity to broaden vaccine-mediated protection using AS04-adjuvanted vaccines based on VLP chimeras of L1 with one or two L2 epitopes. Several chimeric VLPs were constructed by inserting L2 epitopes within the DE loop and/or C terminus of L1. Based on the shape, yield, size, and immunogenicity, one of seven chimeras was selected for further evaluation in mouse and rabbit challenge models. The chimeric VLP consisted of HPV-18 L1 with insertions of HPV-33 L2 (amino acid residues 17 to 36; L1 DE loop) and HPV-58 L2 (amino acid residues 56 to 75; L1 C terminus). This chimeric L1/L2 VLP vaccine induced persistent immune responses and protected against all of the different HPVs evaluated (HPV-6, -11, -16, -31, -35, -39, -45, -58, and -59 as pseudovirions or quasivirions) in both mouse and rabbit challenge models. The degree and breadth of protection in the rabbit were further enhanced when the chimeric L1/L2 VLP was formulated with the L1 VLPs from the HPV-16/18 L1 vaccine. Therefore, the novel HPV-18 L1/L2 chimeric VLP (alone or in combination with HPV-16 and HPV-18 L1 VLPs) formulated with AS04 has the potential to provide broad protective efficacy in human subjects. IMPORTANCE From evaluations in human papillomavirus (HPV) protection models in rabbits and mice, our study has identified a prophylactic vaccine with the potential to target a wide range of HPVs linked to anogenital cancer. The three currently licensed vaccines contain virus-like particles (VLPs) of the L1 major

  4. Characterization of Humoral Responses Induced by an H7N9 Influenza Virus-Like Particle Vaccine in BALB/C Mice

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    Li Zhang

    2015-08-01

    Full Text Available In April 2013, human infections with a novel avian influenza (H7N9 virus emerged in China. It has caused serious concerns for public health throughout the world. However, there is presently no effective treatment, and an A (H7N9 H7 subtype influenza vaccine is not available. Vaccination with virus-like particles (VLPs has showed considerable promise for many other subtype influenza viruses. To produce H7N9 VLPs, full length, unmodified hemagglutinin (HA, neuraminidase (NA, and matrix1 (M1 genes from the A/Wuxi/1/2013(H7N9 were cloned into a pCDNA5.1 FRT vector. By co-transfection, VLPs containing HA, NA, and M1 were secreted by 293T cells. VLPs were purified by ultracentrifugation and injected into mice by the intramuscular route. In animal experiments, humoral and cellular immunoresponse were all triggered by H7N9 VLPs. High levels of specific antibodies and the isotypes of IgG were detected by ELISA. Anamnestic cellular immune responses were examined by detecting specific cytotoxic T cell for IFN-Υ production in ELISPOT assay. The hemagglutination-inhibition (HAI against the homologous virus was more than 1:64, and cross-reactive HAI titers against the heterologous virus (H1N1 and H3N2 were more than 1:16. Moreover, VLPs immunized mice showed a rapid increase of neutralizing antibodies, with neutralizing antibody titers more than 1:8, which increased four-fold against PBS immunized mice in week four. By week six, the mice had high neutralization ability against the given strain and held a potent homologous virus neutralizing capacity. Thus, VLPs represent a potential strategy for the development of a safe and effective vaccine against novel avian influenza (H7N9 virus.

  5. Chimeric SV40 virus-like particles induce specific cytotoxicity and protective immunity against influenza A virus without the need of adjuvants

    Energy Technology Data Exchange (ETDEWEB)

    Kawano, Masaaki [Department of Allergy and Immunology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Morikawa, Katsuma [Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501 (Japan); Suda, Tatsuya [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Laboratory for Immunopharmacology of Microbial Products, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Ohno, Naohito [Laboratory for Immunopharmacology of Microbial Products, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Matsushita, Sho [Department of Allergy and Immunology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Allergy Center, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Akatsuka, Toshitaka [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Handa, Hiroshi, E-mail: handa.h.aa@m.titech.ac.jp [Solutions Research Laboratory, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8503 (Japan); Matsui, Masanori, E-mail: mmatsui@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

    2014-01-05

    Virus-like particles (VLPs) are a promising vaccine platform due to the safety and efficiency. However, it is still unclear whether polyomavirus-based VLPs are useful for this purpose. Here, we attempted to evaluate the potential of polyomavirus VLPs for the antiviral vaccine using simian virus 40 (SV40). We constructed chimeric SV40-VLPs carrying an HLA-A{sup ⁎}02:01-restricted, cytotoxic T lymphocyte (CTL) epitope derived from influenza A virus. HLA-A{sup ⁎}02:01-transgenic mice were then immunized with the chimeric SV40-VLPs. The chimeric SV40-VLPs effectively induced influenza-specific CTLs and heterosubtypic protection against influenza A viruses without the need of adjuvants. Because DNase I treatment of the chimeric SV40-VLPs did not disrupt CTL induction, the intrinsic adjuvant property may not result from DNA contaminants in the VLP preparation. In addition, immunization with the chimeric SV40-VLPs generated long-lasting memory CTLs. We here propose that the chimeric SV40-VLPs harboring an epitope may be a promising CTL-based vaccine platform with self-adjuvant properties. - Highlights: • We constructed chimeric SV40-VLPs carrying an influenza virus-derived CTL epitope. • Chimeric SV40-VLPs induce influenza-specific CTLs in mice without adjuvants. • Chimeric SV40-VLPs induce heterosubtypic protection against influenza A viruses. • Chimeric SV40-VLPs induce long-lasting memory CTLs. • Chimeric SV40-VLPs is a promising vaccine platform with self-adjuvant properties.

  6. A single intranasal administration of virus-like particle vaccine induces an efficient protection for mice against human respiratory syncytial virus.

    Science.gov (United States)

    Jiao, Yue-Ying; Fu, Yuan-Hui; Yan, Yi-Fei; Hua, Ying; Ma, Yao; Zhang, Xiu-Juan; Song, Jing-Dong; Peng, Xiang-Lei; Huang, Jiaqiang; Hong, Tao; He, Jin-Sheng

    2017-08-01

    Human respiratory syncytial virus (RSV) is an important pediatric pathogen causing acute viral respiratory disease in infants and young children. However, no licensed vaccines are currently available. Virus-like particles (VLPs) may bring new hope to producing RSV VLP vaccine with high immunogenicity and safety. Here, we constructed the recombinants of matrix protein (M) and fusion glycoprotein (F) of RSV, respectively into a replication-deficient first-generation adenoviral vector (FGAd), which were used to co-infect Vero cells to assemble RSV VLPs successfully. The resulting VLPs showed similar immunoreactivity and function to RSV virion in vitro. Moreover, Th1 polarized response, and effective mucosal virus-neutralizing antibody and CD8+ T-cell responses were induced by a single intranasal (i.n.) administration of RSV VLPs rather than intramuscular (i.m.) inoculation, although the comparable RSV F-specific serum IgG and long-lasting RSV-specific neutralizing antibody were detected in the mice immunized by both routes. Upon RSV challenge, VLP-immunized mice showed increased viral clearance but decreased signs of enhanced lung pathology and fewer eosinophils compared to mice immunized with formalin-inactivated RSV (FI-RSV). In addition, a single i.n. RSV VLP vaccine has the capability to induce RSV-specific long-lasting neutralizing antibody responses observable up to 15 months. Our results demonstrate that the long-term and memory immune responses in mice against RSV were induced by a single i.n. administration of RSV VLP vaccine, suggesting a successful approach of RSV VLPs as an effective and safe mucosal vaccine against RSV infection, and an applicable and qualified platform of FGAd-infected Vero cells for VLP production. Copyright © 2017. Published by Elsevier B.V.

  7. Generation and immunogenicity of porcine circovirus type 2 chimeric virus-like particles displaying porcine reproductive and respiratory syndrome virus GP5 epitope B.

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    Hu, Gaowei; Wang, Naidong; Yu, Wanting; Wang, Zhanfeng; Zou, Yawen; Zhang, Yan; Wang, Aibing; Deng, Zhibang; Yang, Yi

    2016-04-07

    Virus-like particles (VLPs) can be used as transfer vehicles carrying foreign proteins or antigen epitopes to produce chimeric VLPs for bivalent or multivalent vaccines. Based on the crystal structure of porcine circovirus type 2 (PCV2) capsid protein (Cap), in addition to alignment of the Cap sequences collected from various isolates of PCV2 and PCV1, we predicted that Loop CD of the PCV2 Cap should tolerate insertion of foreign epitopes, and furthermore that such an insertion could be presented on the surface of PCV2 VLPs. To validate this, the GP5 epitope B of porcine reproductive and respiratory syndrome virus (PRRSV) was inserted into Loop CD of the PCV2 Cap. The 3D structure of the recombinant PCV2 Cap (rCap) was simulated by homology modeling; it appeared that the GP5 epitope B was folded as a relatively independent unit, separated from the PCV2 Cap backbone. Furthermore, based on transmission electron microscopy, the purified PCV2 rCap self-assembled into chimeric VLPs which entered PK-15 cells. In addition, PCV2 chimeric VLPs induced strong humoral (neutralizing antibodies against PCV2 and PRRSV) and cellular immune responses in mice. We concluded that the identified insertion site in the PCV2 Cap had great potential to develop PCV2 VLPs-based bivalent or multivalent vaccines; furthermore, it would also facilitate development of a nano-device to present a functional peptide on the surface of the VLPs that could be used for therapeutic purposes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. Enterovirus71 virus-like particles produced from insect cells and purified by multistep chromatography elicit strong humoral immune responses in mice.

    Science.gov (United States)

    Zhao, D; Sun, B; Jiang, H; Sun, S; Kong, F T; Ma, Y; Jiang, L; Bai, L; Chen, X; Yang, P; Liu, C; Xu, Y; Su, W; Kong, W; Xu, F; Jiang, C

    2015-10-01

    The study aims to develop a novel multistep chromatographic purification process for human enterovirus71 virus-like particles (VLPs) produced from insect cells (Sf9) infected with recombinant baculovirus. Sf9 cells were maintained in the Wave Bioreactor system 20/50, and harvested when the viability decreased to 75% after infected with Bac-P1-3CD at the multiplicity of infection (MOI) of 1. After sonication and centrifugation, EV71 VLPs were purified with Capto(™) Core 700, Capto(™) adhere and Capto(™) butyl. The purity was then determined by SDS-PAGE, Western blotting and high-performance liquid chromatography (HPLC), while the diameter of purified EV71 VLPs was analysed by Dynamic Light Scattering (DLS) and Transmission electron microscopy (TEM). Immunization of BALB/c mice and serum collection were performed after contamination analysis, and neutralization antibodies were then analysed by pseudovirus-based microneutralization assay. Results showed that these purified EV71 VLPs can be successfully purified with ~31·52% yield and >95% purity. They could elicit stronger neutralization antibodies in mice compared with those produced from formalin-inactivated EV71 virus. Our results demonstrated that EV71 VLPs can be purified with the multistep chromatographic protocol. This work presents a novel multistep chromatographic technique, an effective way of purifying EV71 VLPs with high purity. This purification process can thus serve as foundation for further development of industrial-scale production process of EV71 VLP vaccine candidates. © 2015 The Society for Applied Microbiology.

  9. Divergence of primary cognate B- and T-cell proliferative responses to subcutaneous and intravenous immunization with virus-like particles.

    Science.gov (United States)

    Temchura, Vladimir; Kalinin, Svetlana; Nabi, Ghulam; Tippler, Bettina; Niezold, Thomas; Uberla, Klaus

    2014-08-22

    A major advantage of virus-like particle (VLP) vaccines against HIV is their structural identity to wild-type viruses, ensuring that antigen-specific B-cells encounter the envelope protein in its natural conformation. For the induction of affinity-matured antibodies, the B-cells must also obtain help from T-cells that are restricted by linear epitopes. Using B- and T-cell transgenic mouse models, we compared the efficacy of modified HIV-VLPs delivered by subcutaneous and intravenous immunization to stimulate primary B- and T-cell proliferative responses in different lymphoid organs. VLPs containing an influenza virus hemagglutinin epitope within the HIV-Gag protein induced comparable primary cognate T-cell proliferative responses in the draining lymph node and the spleen, irrespective of the delivery route. In contrast, after subcutaneous immunization with HIV-Gag VLPs containing hen egg lysozyme (HEL) on their surface, the proliferative response of transgenic HEL-specific B-cells was restricted to the draining lymph nodes, while intravenous VLP immunization primarily induced a B-cell proliferative response in the spleen. In vitro co-culture experiments further revealed that the presentation of VLP-associated surface antigens by dendritic cells to cognate B-cells is inefficient. This is consistent with a direct triggering of the B-cell proliferative response by the VLPs and suggests that HIV VLPs may indeed be suitable to directly promote the expansion of B-cells specific for conformational epitopes that are unique to functionally-active Env spikes on the virion. Further investigations are warranted to explore potential differences in the quality and protective potency of HIV-specific antibody responses induced by the two routes.

  10. Divergence of Primary Cognate B- and T-Cell Proliferative Responses to Subcutaneous and Intravenous Immunization with Virus-Like Particles

    Directory of Open Access Journals (Sweden)

    Vladimir Temchura

    2014-08-01

    Full Text Available A major advantage of virus-like particle (VLP vaccines against HIV is their structural identity to wild-type viruses, ensuring that antigen-specific B-cells encounter the envelope protein in its natural conformation. For the induction of affinity-matured antibodies, the B-cells must also obtain help from T-cells that are restricted by linear epitopes. Using B- and T-cell transgenic mouse models, we compared the efficacy of modified HIV-VLPs delivered by subcutaneous and intravenous immunization to stimulate primary B- and T-cell proliferative responses in different lymphoid organs. VLPs containing an influenza virus hemagglutinin epitope within the HIV-Gag protein induced comparable primary cognate T-cell proliferative responses in the draining lymph node and the spleen, irrespective of the delivery route. In contrast, after subcutaneous immunization with HIV-Gag VLPs containing hen egg lysozyme (HEL on their surface, the proliferative response of transgenic HEL-specific B-cells was restricted to the draining lymph nodes, while intravenous VLP immunization primarily induced a B-cell proliferative response in the spleen. In vitro co-culture experiments further revealed that the presentation of VLP-associated surface antigens by dendritic cells to cognate B-cells is inefficient. This is consistent with a direct triggering of the B-cell proliferative response by the VLPs and suggests that HIV VLPs may indeed be suitable to directly promote the expansion of B-cells specific for conformational epitopes that are unique to functionally-active Env spikes on the virion. Further investigations are warranted to explore potential differences in the quality and protective potency of HIV-specific antibody responses induced by the two routes.

  11. The YLDL sequence within Sendai virus M protein is critical for budding of virus-like particles and interacts with Alix/AIP1 independently of C protein.

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    Irie, Takashi; Shimazu, Yukie; Yoshida, Tetsuya; Sakaguchi, Takemasa

    2007-03-01

    For many enveloped viruses, cellular multivesicular body (MVB) sorting machinery has been reported to be utilized for efficient viral budding. Matrix and Gag proteins have been shown to contain one or two L-domain motifs (PPxY, PT/SAP, YPDL, and FPIV), some of which interact specifically with host cellular proteins involved in MVB sorting, which are recruited to the viral budding site. However, for many enveloped viruses, L-domain motifs have not yet been identified and the involvement of MVB sorting machinery in viral budding is still unknown. Here we show that both Sendai virus (SeV) matrix protein M and accessory protein C contribute to virus budding by physically interacting with Alix/AIP1. A YLDL sequence within the M protein showed L-domain activity, and its specific interaction with the N terminus of Alix/AIP1(1-211) was important for the budding of virus-like particles (VLPs) of M protein. In addition, M-VLP budding was inhibited by the overexpression of some deletion mutant forms of Alix/AIP1 and depletion of endogenous Alix/AIP1 with specific small interfering RNAs. The YLDL sequence was not replaceable by other L-domain motifs, such as PPxY and PT/SAP, and even YPxL. C protein was also able to physically interact with the N terminus of Alix/AIP1(212-357) and enhanced M-VLP budding independently of M-Alix/AIP1 interaction, although it was not released from the transfected cells itself. Our results suggest that the interaction of multiple viral proteins with Alix/AIP1 may enhance the efficiency of the utilization of cellular MVB sorting machinery for efficient SeV budding.

  12. Specificity of L1 Peptides versus Virus-Like Particles for Detection of Human Papillomavirus-Positive Cervical Lesions in Females Attending Engativa Hospital, Bogota, Colombia▿

    Science.gov (United States)

    Urquiza, Mauricio; Sánchez, Ricardo; Amaya, Jairo; León, Sandra; Acosta, Jenny; Patarroyo, Manuel A.; Camargo, Milena; Patarroyo, Manuel E.

    2008-01-01

    A serological test for the detection of human papillomavirus (HPV) infection in females at risk of developing cervical cancer could be based on conserved L1 peptides with low levels of antigenicity specifically recognized by antibodies from patients with cervical lesions infected with high-risk HPV (HR-HPV) types. The aim was to assess the ability of L1 peptides 18283, 18294, and 18301 compared with the ability of virus-like particles (VLPs) to identify these infections in females. A total of 391 HPV-infected female volunteers were interviewed, and peripheral blood and cervical cells were obtained for detection of anti-HPV antibodies and HPV DNA; all of the patients had a Pap smear test; 287 patients were referred for colposcopy or biopsy, according to gynecological criteria. The level of agreement, as determined by the use of the Lin coefficient (rho value), showed that 75 to 83% of females with HR-HPV DNA-positive cervical lesions had antibodies that recognized VLPs and peptide 18283, 18294, or 18301, while 15 to 23% of the HPV DNA-negative females with a normal cytology had antibodies that recognized these three peptides and 45% had antibodies that recognized VLPs. The rate of agreement between peptides and VLPs for antibody detection was higher for patients with HPV DNA-positive cervical lesions. Peptides 18283, 18294, and 18301 showed similar sensitivities for the detection of HR-HPV DNA-positive cervical lesions and were more specific than VLPs. Peptide 18301 might be detecting protective antibodies in HPV DNA-negative females with atypical squamous cells of undetermined significance. These peptides could be useful for the design of a serology test for the detection of HR-HPV infection in females with cervical lesions and at risk of cervical cancer. PMID:18799706

  13. Specificity of L1 peptides versus virus-like particles for detection of human papillomavirus-positive cervical lesions in females attending Engativa Hospital, Bogota, Colombia.

    Science.gov (United States)

    Urquiza, Mauricio; Sánchez, Ricardo; Amaya, Jairo; León, Sandra; Acosta, Jenny; Patarroyo, Manuel A; Camargo, Milena; Patarroyo, Manuel E

    2008-11-01

    A serological test for the detection of human papillomavirus (HPV) infection in females at risk of developing cervical cancer could be based on conserved L1 peptides with low levels of antigenicity specifically recognized by antibodies from patients with cervical lesions infected with high-risk HPV (HR-HPV) types. The aim was to assess the ability of L1 peptides 18283, 18294, and 18301 compared with the ability of virus-like particles (VLPs) to identify these infections in females. A total of 391 HPV-infected female volunteers were interviewed, and peripheral blood and cervical cells were obtained for detection of anti-HPV antibodies and HPV DNA; all of the patients had a Pap smear test; 287 patients were referred for colposcopy or biopsy, according to gynecological criteria. The level of agreement, as determined by the use of the Lin coefficient (rho value), showed that 75 to 83% of females with HR-HPV DNA-positive cervical lesions had antibodies that recognized VLPs and peptide 18283, 18294, or 18301, while 15 to 23% of the HPV DNA-negative females with a normal cytology had antibodies that recognized these three peptides and 45% had antibodies that recognized VLPs. The rate of agreement between peptides and VLPs for antibody detection was higher for patients with HPV DNA-positive cervical lesions. Peptides 18283, 18294, and 18301 showed similar sensitivities for the detection of HR-HPV DNA-positive cervical lesions and were more specific than VLPs. Peptide 18301 might be detecting protective antibodies in HPV DNA-negative females with atypical squamous cells of undetermined significance. These peptides could be useful for the design of a serology test for the detection of HR-HPV infection in females with cervical lesions and at risk of cervical cancer.

  14. Chitosan microparticles loaded with yeast-derived PCV2 virus-like particles elicit antigen-specific cellular immune response in mice after oral administration.

    Science.gov (United States)

    Bucarey, Sergio A; Pujol, Myriam; Poblete, Joaquín; Nuñez, Ignacio; Tapia, Cecilia V; Neira-Carrillo, Andrónico; Martinez, Jonatán; Bassa, Oliver

    2014-08-20

    Porcine circovirus type 2 (PCV2)-associated diseases are a major problem for the swine industry worldwide. In addition to improved management and husbandry practices, the availability of several anti-PCV2 vaccines provides an efficient immunological option for reducing the impact of these diseases. Most anti-PCV2 vaccines are marketed as injectable formulations. Although these are effective, there are problems associated with the use of injectable products, including laborious and time-consuming procedures, the induction of inflammatory responses at the injection site, and treatment-associated stress to the animals. Oral vaccines represent an improvement in antigen delivery technology; they overcome the problems associated with injection management and facilitate antigen boosting when an animals' immunity falls outside the protective window. Chitosan microparticles were used as both a vehicle and mucosal adjuvant to deliver yeast-derived PCV2 virus-like particles (VLPs) in an attempt to develop an oral vaccine. The physical characteristics of the microparticles, including size, Zeta potential, and polydispersity, were examined along with the potential to induce PCV2-specific cellular immune responses in mice after oral delivery. Feeding mice with PCV2 VLP-loaded, positively-charged chitosan microparticles with an average size of 2.5 μm induced the proliferation of PCV2-specific splenic CD4+/CD8+ lymphocytes and the subsequent production of IFN-γ to levels comparable with those induced by an injectable commercial formulation. Chitosan microparticles appear to be a safe, simple system on which to base PCV2 oral vaccines. Oral chitosan-mediated antigen delivery is a novel strategy that efficiently induces anti-PCV2 cellular responses in a mouse model. Further studies in swine are warranted.

  15. Foot-and-mouth disease virus-like particles produced by a SUMO fusion protein system in Escherichia coli induce potent protective immune responses in guinea pigs, swine and cattle.

    Science.gov (United States)

    Guo, Hui-Chen; Sun, Shi-Qi; Jin, Ye; Yang, Shun-Li; Wei, Yan-Quan; Sun, De-Hui; Yin, Shuang-Hui; Ma, Jun-Wu; Liu, Zai-Xin; Guo, Jian-Hong; Luo, Jian-Xun; Yin, Hong; Liu, Xiang-Tao; Liu, Ding Xiang

    2013-07-04

    Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. The format of FMD virus-like particles (VLP) as a non-replicating particulate vaccine candidate is a promising alternative to conventional inactivated FMDV vaccines. In this study, we explored a prokaryotic system to express and assemble the FMD VLP and validated the potential of VLP as an FMDV vaccine candidate. VLP composed entirely of FMDV (Asia1/Jiangsu/China/2005) capsid proteins (VP0, VP1 and VP3) were simultaneously produced as SUMO fusion proteins by an improved SUMO fusion protein system in E. coli. Proteolytic removal of the SUMO moiety from the fusion proteins resulted in the assembly of VLP with size and shape resembling the authentic FMDV. Immunization of guinea pigs, swine and cattle with FMD VLP by intramuscular inoculation stimulated the FMDV-specific antibody response, neutralizing antibody response, T-cell proliferation response and secretion of cytokine IFN-γ. In addition, immunization with one dose of the VLP resulted in complete protection of these animals from homologous FMDV challenge. The 50% protection dose (PD50) of FMD VLP in cattle is up to 6.34. These results suggest that FMD VLP expressed in E. coli are an effective vaccine in guinea pigs, swine and cattle and support further development of these VLP as a vaccine candidate for protection against FMDV.

  16. Foot-and-mouth disease virus-like particles produced by a SUMO fusion protein system in Escherichia coli induce potent protective immune responses in guinea pigs, swine and cattle

    Science.gov (United States)

    2013-01-01

    Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. The format of FMD virus-like particles (VLP) as a non-replicating particulate vaccine candidate is a promising alternative to conventional inactivated FMDV vaccines. In this study, we explored a prokaryotic system to express and assemble the FMD VLP and validated the potential of VLP as an FMDV vaccine candidate. VLP composed entirely of FMDV (Asia1/Jiangsu/China/2005) capsid proteins (VP0, VP1 and VP3) were simultaneously produced as SUMO fusion proteins by an improved SUMO fusion protein system in E. coli. Proteolytic removal of the SUMO moiety from the fusion proteins resulted in the assembly of VLP with size and shape resembling the authentic FMDV. Immunization of guinea pigs, swine and cattle with FMD VLP by intramuscular inoculation stimulated the FMDV-specific antibody response, neutralizing antibody response, T-cell proliferation response and secretion of cytokine IFN-γ. In addition, immunization with one dose of the VLP resulted in complete protection of these animals from homologous FMDV challenge. The 50% protection dose (PD50) of FMD VLP in cattle is up to 6.34. These results suggest that FMD VLP expressed in E. coli are an effective vaccine in guinea pigs, swine and cattle and support further development of these VLP as a vaccine candidate for protection against FMDV. PMID:23826638

  17. Chimeric severe acute respiratory syndrome coronavirus (SARS-CoV) S glycoprotein and influenza matrix 1 efficiently form virus-like particles (VLPs) that protect mice against challenge with SARS-CoV

    Science.gov (United States)

    Liu, Ye V.; Massare, Michael J.; Barnard, Dale L.; Kort, Thomas; Nathan, Margret; Wang, Lei; Smith, Gale

    2011-01-01

    SARS-CoV was the cause of the global pandemic in 2003 that infected over 8000 people in 8 months. Vaccines against SARS are still not available. We developed a novel method to produce high levels of a recombinant SARS virus-like particles (VLPs) vaccine containing the SARS spike (S) protein and the influenza M1 protein using the baculovirus insect cell expression system. These chimeric SARS VLPs have a similar size and morphology to the wild type SARS-CoV. We tested the immunogenicity and protective efficacy of purified chimeric SARS VLPs and full length SARS S protein vaccines in a mouse lethal challenge model. The SARS VLP vaccine, containing 0.8 μg of SARS S protein, completely protected mice from death when administered intramuscular (IM) or intranasal (IN) routes in the absence of an adjuvant. Likewise, the SARS VLP vaccine, containing 4 μg of S protein without adjuvant, reduced lung virus titer to below detectable level, protected mice from weight loss, and elicited a high level of neutralizing antibodies against SARS-CoV. Sf9 cell-produced full length purified SARS S protein was also an effective vaccine against SARS-CoV but only when co-administered IM with aluminum hydroxide. SARS-CoV VLPs are highly immunogenic and induce neutralizing antibodies and provide protection against lethal challenge. Sf9 cell-based VLP vaccines are a potential tool to provide protection against novel pandemic agents. PMID:21762752

  18. Novel Epstein-Barr virus-like particles incorporating gH/gL-EBNA1 or gB-LMP2 induce high neutralizing antibody titers and EBV-specific T-cell responses in immunized mice.

    Science.gov (United States)

    Perez, Elizabeth M; Foley, Joslyn; Tison, Timelia; Silva, Rute; Ogembo, Javier Gordon

    2017-03-21

    Previous Epstein-Barr virus (EBV) prophylactic vaccines based on the major surface glycoprotein gp350/220 as an immunogen have failed to block viral infection in humans, suggesting a need to target other viral envelope glycoproteins. In this study, we reasoned that incorporating gH/gL or gB, critical glycoproteins for viral fusion and entry, on the surface of a virus-like particle (VLP) would be more immunogenic than gp350/220 for generating effective neutralizing antibodies to prevent viral infection of both epithelial and B cell lines. To boost the humoral response and trigger cell-mediated immunity, EBV nuclear antigen 1 (EBNA1) and latent membrane protein 2 (LMP2), intracellular latency proteins expressed in all EBV-infected cells, were also included as critical components of the polyvalent EBV VLP. gH/gL-EBNA1 and gB-LMP2 VLPs were efficiently produced in Chinese hamster ovary cells, an FDA-approved vehicle for mass-production of biologics. Immunization with gH/gL-EBNA1 and gB-LMP2 VLPs without adjuvant generated both high neutralizing antibody titers in vitro and EBV-specific T-cell responses in BALB/c mice. These data demonstrate that will be invaluable not only in preventing EBV infection, but importantly, in preventing and treating the 200,000 cases of EBV-associated cancers that occur globally every year.

  19. A Bivalent Heterologous DNA Virus-Like-Particle Prime-Boost Vaccine Elicits Broad Protection against both Group 1 and 2 Influenza A Viruses.

    Science.gov (United States)

    Jiang, Wenbo; Wang, Shuangshuang; Chen, Honglin; Ren, Huanhuan; Huang, Xun; Wang, Guiqin; Chen, Ze; Chen, Ling; Chen, Zhiwei; Zhou, Paul

    2017-05-01

    Current seasonal influenza vaccines are efficacious when vaccine strains are matched with circulating strains. However, they do not protect antigenic variants and newly emerging pandemic and outbreak strains. Thus, there is a critical need for developing so-called "universal" vaccines that protect against all influenza viruses. In the present study, we developed a bivalent heterologous DNA virus-like particle prime-boost vaccine strategy. We show that mice immunized with this vaccine were broadly protected against lethal challenge from group 1 (H1, H5, and H9) and group 2 (H3 and H7) viruses, with 94% aggregate survival. To determine the immune correlates of protection, we performed passive immunizations and in vitro assays. We show that this vaccine elicited antibody responses that bound HA from group 1 (H1, H2, H5, H6, H8, H9, H11, and H12) and group 2 (H3, H4, H7, H10, H14, and H15) and neutralized homologous and intrasubtypic H5 and H7 and heterosubtypic H1 viruses and hemagglutinin-specific CD4 and CD8 T cell responses. As a result, passive immunization with immune sera fully protected mice against H5, H7, and H1 challenge, whereas with both immune sera and T cells the mice survived heterosubtypic H3 and H9 challenge. Thus, it appears that (i) neutralizing antibodies alone fully protect against homologous and intrasubtypic H5 and H7 and (ii) neutralizing and binding antibodies are sufficient to protect against heterosubtypic H1, (iii) but against heterosubtypic H3 and H9, binding antibodies and T cells are required for complete survival. We believe that this vaccine regimen could potentially be a candidate for a "universal" influenza vaccine. IMPORTANCE Influenza virus infection is global health problem. Current seasonal influenza vaccines are efficacious only when vaccine strains are matched with circulating strains. However, these vaccines do not protect antigenic variants and newly emerging pandemic and outbreak strains. Because of this, there is an urgent

  20. A chimeric 18L1-45RG1 virus-like particle vaccine cross-protects against oncogenic alpha-7 human papillomavirus types.

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    Bettina Huber

    Full Text Available Persistent infection with oncogenic human papillomaviruses (HPV types causes all cervical and a subset of other anogenital and oropharyngeal carcinomas. Four high-risk (hr mucosal types HPV16, 18, 45, or 59 cause almost all cervical adenocarcinomas (AC, a subset of cervical cancer (CxC. Although the incidence of cervical squamous cell carcinoma (SCC has dramatically decreased following introduction of Papanicolaou (PAP screening, the proportion of AC has relatively increased. Cervical SCC arise mainly from the ectocervix, whereas AC originate primarily from the endocervical canal, which is less accessible to obtain viable PAP smears. Licensed (bivalent and quadrivalent HPV vaccines comprise virus-like particles (VLP of the most important hr HPV16 and 18, self-assembled from the major capsid protein L1. Due to mainly type-restricted efficacy, both vaccines do not target 13 additional hr mucosal types causing 30% of CxC. The papillomavirus genus alpha species 7 (α7 includes a group of hr types of which HPV18, 45, 59 are proportionally overrepresented in cervical AC and only partially (HPV18 targeted by current vaccines. To target these types, we generated a chimeric vaccine antigen that consists of a cross-neutralizing epitope (homologue of HPV16 RG1 of the L2 minor capsid protein of HPV45 genetically inserted into a surface loop of HPV18 L1 VLP (18L1-45RG1. Vaccination of NZW rabbits with 18L1-45RG1 VLP plus alum-MPL adjuvant induced high-titer neutralizing antibodies against homologous HPV18, that cross-neutralized non-cognate hr α7 types HPV39, 45, 68, but not HPV59, and low risk HPV70 in vitro, and induced a robust L1-specific cellular immune response. Passive immunization protected mice against experimental vaginal challenge with pseudovirions of HPV18, 39, 45 and 68, but not HPV59 or the distantly related α9 type HPV16. 18L1-45RG1 VLP might be combined with our previously described 16L1-16RG1 VLP to develop a second generation bivalent

  1. Adsorption of virus-like particles on ion exchange surface: Conformational changes at different pH detected by dual polarization interferometry.

    Science.gov (United States)

    Yang, Yanli; Mengran Yu; Zhang, Songping; Ma, Guanghui; Su, Zhiguo

    2015-08-21

    Disassembling of virus-like particles (VLPs) like hepatitis B virus surface antigen (HB-VLPs) during chromatographic process has been identified as a major cause of loss of antigen activity. In this study, dual polarization interferometry (DPI) measurement, together with chromatography experiments, were performed to study the adsorption and conformational change of HB-VLPs on ion exchange surface at three different pHs. Changes in pH values of buffer solution showed only minimal effect on the HB-VLPs assembly and antigen activity, while significantly different degree of HB-VLPs disassembling was observed after ion exchange chromatography (IEC) at different pHs, indicating the conformational change of HB-VLPs caused mainly by its interactions with the adsorbent surface. By creating an ion exchange surface on chip surface, the conformational changes of HB-VLPs during adsorption to the surface were monitored in real time by DPI for the first time. As pH increased from 7.0 to 9.0, strong electrostatic interactions between oppositely charged HB-VLPs and the ion exchange surface make the HB-VLPs spread thinly or even adsorbed in disassembled formation on the surface as revealed by significant decrease in thickness of the adsorbed layer measured by DPI. Such findings were consistent with the results of IEC experiments operated at different pHs, that more disassembled HB-VLPs were detected in the eluted proteins at pH 9.0. At low pH like pH 5.0, however, possible bi-layer adsorption was involved as evidenced by an adsorbed layer thickness higher than average diameter of the HB-VLPs. The "lateral" protein-protein interactions might be unfavorable and would make additional contribution to the disassembling of HB-VLPs besides the primary mechanism related to the protein-surface interactions; therefore, the lowest antigen activity was observed after IEC at pH 5.0. Such real-time information on conformational change of VLPs is helpful for better understanding the real mechanism

  2. A Novel Prime and Boost Regimen of HIV Virus-Like Particles with TLR4 Adjuvant MPLA Induces Th1 Oriented Immune Responses against HIV.

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    Ethan Poteet

    Full Text Available HIV virus-like particles (VLPs present the HIV envelope protein in its native conformation, providing an ideal vaccine antigen. To enhance the immunogenicity of the VLP vaccine, we sought to improve upon two components; the route of administration and the additional adjuvant. Using HIV VLPs, we evaluated sub-cheek as a novel route of vaccine administration when combined with other conventional routes of immunization. Of five combinations of distinct prime and boost sequences, which included sub-cheek, intranasal, and intradermal routes of administration, intranasal prime and sub-cheek boost (IN+SC resulted in the highest HIV-specific IgG titers among the groups tested. Using the IN+SC regimen we tested the adjuvant VesiVax Conjugatable Adjuvant Lipid Vesicles (CALV + monophosphoryl lipid A (MPLA at MPLA concentrations of 0, 7.5, 12.5, and 25 μg/dose in combination with our VLPs. Mice that received 12.5 or 25 μg/dose MPLA had the highest concentrations of Env-specific IgG2c (20.7 and 18.4 μg/ml respectively, which represents a Th1 type of immune response in C57BL/6 mice. This was in sharp contrast to mice which received 0 or 7.5 μg MPLA adjuvant (6.05 and 5.68 μg/ml of IgG2c respectively. In contrast to IgG2c, MPLA had minor effects on Env-specific IgG1; therefore, 12.5 and 25 μg/dose of MPLA induced the optimal IgG1/IgG2c ratio of 1.3. Additionally, the percentage of germinal center B cells increased significantly from 15.4% in the control group to 31.9% in the CALV + 25 μg MPLA group. These mice also had significantly more IL-2 and less IL-4 Env-specific CD8+ T cells than controls, correlating with an increased percentage of Env-specific central memory CD4+ and CD8+ T cells. Our study shows the strong potential of IN+SC as an efficacious route of administration and the effectiveness of VLPs combined with MPLA adjuvant to induce Env specific Th1-oriented HIV-specific immune responses.

  3. Plant-based chimeric HPV-virus-like particles bearing amyloid-β epitopes elicit antibodies able to recognize amyloid plaques in APP-tg mouse and Alzheimer's disease brains.

    Science.gov (United States)

    Gonzalez-Castro, R; Acero Galindo, G; García Salcedo, Y; Uribe Campero, L; Vazquez Perez, V; Carrillo-Tripp, M; Gevorkian, G; Gomez Lim, M A

    2017-11-01

    The main amyloid-beta (Aβ) variants detected in the human brain are full-length Aβ1-40 and Aβ1-42 peptides; however, a significant proportion of AD brain Aβ consists also of N-terminal truncated/modified species. The majority of the previous immunotherapeutic strategies targeted the N-terminal immunodominant epitope of the full-length Aβ; however, most of the pathological N-truncated forms of Aβ lack this critical B cell epitope. Recently, virus-like particles (VLPs), self-assembled structures with highly ordered repetitive patterns on their surface and capable of inducing robust immune responses, were applied as a promising platform for various antigen expressions. In this study, we expressed in plants two chimeric HPV16 L1 capsid proteins obtained by introduction of the β-amyloid 11-28 epitope (Aβ 11-28) into the h4 helix or into the coil regions of the L1 protein. The Aβ 11-28 epitope was chosen because it is present in the full-length Aβ 1-42 as well as in the truncated/modified amyloid peptide species. After expression, we assembled the chimerical L1/Aβ 11-28 into a VLP in which the Aβ 11-28 epitope is exposed at very high density (360 times) on the surface of the VLP. The chimeric VLPs elicited in mice Aβ-specific antibodies binding to β-amyloid plaques in APP-tg mouse and AD brains. Our study is the first to demonstrate a successful production in plants and immunogenic properties in mice of chimeric HPV16 L1 VLPs bearing Aβ epitope that may be of potential relevance for the development of multivalent vaccines for a multifactorial disease such as AD.

  4. Involvement of an Arginine Triplet in M1 Matrix Protein Interaction with Membranes and in M1 Recruitment into Virus-Like Particles of the Influenza A(H1N1pdm09 Virus.

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    Adeline Kerviel

    Full Text Available The influenza A(H1N1pdm09 virus caused the first influenza pandemic of the 21st century. In this study, we wanted to decipher the role of conserved basic residues of the viral M1 matrix protein in virus assembly and release. M1 plays many roles in the influenza virus replication cycle. Specifically, it participates in viral particle assembly, can associate with the viral ribonucleoprotein complexes and can bind to the cell plasma membrane and/or the cytoplasmic tail of viral transmembrane proteins. M1 contains an N-terminal domain of 164 amino acids with two basic domains: the nuclear localization signal on helix 6 and an arginine triplet (R76/77/78 on helix 5. To investigate the role of these two M1 basic domains in influenza A(H1N1pdm09 virus molecular assembly, we analyzed M1 attachment to membranes, virus-like particle (VLP production and virus infectivity. In vitro, M1 binding to large unilamellar vesicles (LUVs, which contain negatively charged lipids, decreased significantly when the M1 R76/77/78 motif was mutated. In cells, M1 alone was mainly observed in the nucleus (47% and in the cytosol (42%. Conversely, when co-expressed with the viral proteins NS1/NEP and M2, M1 was relocated to the cell membranes (55%, as shown by subcellular fractionation experiments. This minimal system allowed the production of M1 containing-VLPs. However, M1 with mutations in the arginine triplet accumulated in intracellular clusters and its incorporation in VLPs was strongly diminished. M2 over-expression was essential for M1 membrane localization and VLP production, whereas the viral trans-membrane proteins HA and NA seemed dispensable. These results suggest that the M1 arginine triplet participates in M1 interaction with membranes. This R76/77/78 motif is essential for M1 incorporation in virus particles and the importance of this motif was confirmed by reverse genetic demonstrating that its mutation is lethal for the virus. These results highlight the

  5. Involvement of an Arginine Triplet in M1 Matrix Protein Interaction with Membranes and in M1 Recruitment into Virus-Like Particles of the Influenza A(H1N1)pdm09 Virus.

    Science.gov (United States)

    Kerviel, Adeline; Dash, Shantoshini; Moncorgé, Olivier; Panthu, Baptiste; Prchal, Jan; Décimo, Didier; Ohlmann, Théophile; Lina, Bruno; Favard, Cyril; Decroly, Etienne; Ottmann, Michèle; Roingeard, Philippe; Muriaux, Delphine

    2016-01-01

    The influenza A(H1N1)pdm09 virus caused the first influenza pandemic of the 21st century. In this study, we wanted to decipher the role of conserved basic residues of the viral M1 matrix protein in virus assembly and release. M1 plays many roles in the influenza virus replication cycle. Specifically, it participates in viral particle assembly, can associate with the viral ribonucleoprotein complexes and can bind to the cell plasma membrane and/or the cytoplasmic tail of viral transmembrane proteins. M1 contains an N-terminal domain of 164 amino acids with two basic domains: the nuclear localization signal on helix 6 and an arginine triplet (R76/77/78) on helix 5. To investigate the role of these two M1 basic domains in influenza A(H1N1)pdm09 virus molecular assembly, we analyzed M1 attachment to membranes, virus-like particle (VLP) production and virus infectivity. In vitro, M1 binding to large unilamellar vesicles (LUVs), which contain negatively charged lipids, decreased significantly when the M1 R76/77/78 motif was mutated. In cells, M1 alone was mainly observed in the nucleus (47%) and in the cytosol (42%). Conversely, when co-expressed with the viral proteins NS1/NEP and M2, M1 was relocated to the cell membranes (55%), as shown by subcellular fractionation experiments. This minimal system allowed the production of M1 containing-VLPs. However, M1 with mutations in the arginine triplet accumulated in intracellular clusters and its incorporation in VLPs was strongly diminished. M2 over-expression was essential for M1 membrane localization and VLP production, whereas the viral trans-membrane proteins HA and NA seemed dispensable. These results suggest that the M1 arginine triplet participates in M1 interaction with membranes. This R76/77/78 motif is essential for M1 incorporation in virus particles and the importance of this motif was confirmed by reverse genetic demonstrating that its mutation is lethal for the virus. These results highlight the molecular

  6. Detection of serum anti-NV-F antibodies in the convalescent phase of severe hepatitis in patients positive for tissue NV-F antigen and novel virus-like particles.

    Science.gov (United States)

    Yeh, Chau-Ting; Tsao, Mei-Lin

    2015-10-01

    Previously, a non-human DNA fragment named NV-F was isolated from a patient with non-A-E fulminant hepatitis. This sequence encoded an incomplete open reading frame (the NV-F antigen). In this study, we developed a western blot assay to detect serum anti-NV-F antibodies. Serum samples from 347 patients with severe hepatitis (ALT > fivefold ULN) were analyzed to understand the prevalence and distribution of the NV-F associated virus (HnFV) infection. Of these patients, acute HnFV infection was diagnosed (by positive serum NV-F DNA) in 34 patients (9.8%). However, none of these 34 serum samples were positive for serum anti-NV-F antibodies. In the remaining patients negative for serum NV-F DNA, 62 (17.9%) were positive for serum anti-NV-F antibodies. Liver biopsy samples from 35 severe hepatitis patients were submitted for immunohistochemistry and electron microscopy examination. Of them, seven were positive for hepatic NV-F antigen expression. Electron microscopy identified a novel virus-like particle in all of the seven NV-F antigen-positive liver tissues but not in the remaining 28 NV-F antigen-negative liver tissues. Longitudinal serum sample analysis revealed transient positivity of serum NV-F DNA in three of the seven patients during the clinical courses. Seroconversion of anti-NV-F antibody from negative to positive was found in four of the seven patients and all positive anti-NV-F antibodies were detected in the convalescent phases. In conclusion, in patients with severe hepatitis, a novel hepatotropic virus, temporarily named HnFV, was found in liver tissues expressing the NV-F antigen. Serum anti-NV-F antibodies were detected in the convalescent serum samples. © 2015 Wiley Periodicals, Inc.

  7. Co-delivery of GPI-anchored CCL28 and influenza HA in chimeric virus-like particles induces cross-protective immunity against H3N2 viruses.

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    Mohan, Teena; Kim, Jongrok; Berman, Zachary; Wang, Shelly; Compans, Richard W; Wang, Bao-Zhong

    2016-07-10

    Influenza infection typically initiates at respiratory mucosal surfaces. Induction of immune responses at the sites where pathogens initiate replication is crucial for the prevention of infection. We studied the adjuvanticity of GPI-anchored CCL28 co-incorporated with influenza HA-antigens in chimeric virus-like particles (cVLPs), in boosting strong protective immune responses through an intranasal (i.n.) route in mice. We compared the immune responses to that from influenza VLPs without CCL28, or physically mixed with soluble CCL28 at systemic and various mucosal compartments. The cVLPs containing GPI-CCL28 showed in-vitro chemotactic activity towards spleen and lung cells expressing CCR3/CCR10 chemokine receptors. The cVLPs induced antigen specific endpoint titers and avidity indices of IgG in sera and IgA in tracheal, lung, and intestinal secretions, significantly higher (4-6 fold) than other formulations. Significantly higher (3-5 fold) hemagglutination inhibition titers and high serum neutralization against H3N2 viruses were also detected with CCL28-containing VLPs compared to other groups. The CCL28-containing VLPs showed complete and 80% protection, when vaccinated animals were challenged with A/Aichi/2/1968/H3N2 (homologous) and A/Philippines/2/1982/H3N2 (heterologous) viruses, respectively. Thus, GPI-anchored CCL28 in influenza VLPs act as a strong immunostimulator at both systemic and mucosal sites, boosting significant cross-protection in animals against heterologous viruses across a large distance. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. A tetravalent virus-like particle vaccine designed to display domain III of dengue envelope proteins induces multi-serotype neutralizing antibodies in mice and macaques which confer protection against antibody dependent enhancement in AG129 mice.

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    Viswanathan Ramasamy

    2018-01-01

    Full Text Available Dengue is one of the fastest spreading vector-borne diseases, caused by four antigenically distinct dengue viruses (DENVs. Antibodies against DENVs are responsible for both protection as well as pathogenesis. A vaccine that is safe for and efficacious in all people irrespective of their age and domicile is still an unmet need. It is becoming increasingly apparent that vaccine design must eliminate epitopes implicated in the induction of infection-enhancing antibodies.We report a Pichia pastoris-expressed dengue immunogen, DSV4, based on DENV envelope protein domain III (EDIII, which contains well-characterized serotype-specific and cross-reactive epitopes. In natural infection, <10% of the total neutralizing antibody response is EDIII-directed. Yet, this is a functionally relevant domain which interacts with the host cell surface receptor. DSV4 was designed by in-frame fusion of EDIII of all four DENV serotypes and hepatitis B surface (S antigen and co-expressed with unfused S antigen to form mosaic virus-like particles (VLPs. These VLPs displayed EDIIIs of all four DENV serotypes based on probing with a battery of serotype-specific anti-EDIII monoclonal antibodies. The DSV4 VLPs were highly immunogenic, inducing potent and durable neutralizing antibodies against all four DENV serotypes encompassing multiple genotypes, in mice and macaques. DSV4-induced murine antibodies suppressed viremia in AG129 mice and conferred protection against lethal DENV-4 virus challenge. Further, neither murine nor macaque anti-DSV4 antibodies promoted mortality or inflammatory cytokine production when passively transferred and tested in an in vivo dengue disease enhancement model of AG129 mice.Directing the immune response to a non-immunodominant but functionally relevant serotype-specific dengue epitope of the four DENV serotypes, displayed on a VLP platform, can help minimize the risk of inducing disease-enhancing antibodies while eliciting effective tetravalent

  9. Plant-made virus-like particle vaccines bearing the hemagglutinin of either seasonal (H1) or avian (H5) influenza have distinct patterns of interaction with human immune cells in vitro.

    Science.gov (United States)

    Hendin, Hilary E; Pillet, Stéphane; Lara, Amanda N; Wu, Cheng-Ying; Charland, Nathalie; Landry, Nathalie; Ward, Brian J

    2017-05-02

    The recent emergence of avian influenza strains has fuelled concern about pandemic preparedness since vaccines targeting these viruses are often poorly immunogenic. Weak antibody responses to vaccines have been seen across multiple platforms including plant-made VLPs. To better understand these differences, we compared the in vitro responses of human immune cells exposed to plant-made virus-like particle (VLP) vaccines targeting H1N1 (H1-VLP) and H5N1 (H5-VLP). Peripheral blood mononuclear cells (PBMC) from healthy adults were stimulated ex vivo with 2-5µg/mL VLPs bearing the hemagglutinin (HA) of either H1N1 (A/California/7/2009) or H5N1 (A/Indonesia/5/05). VLP-immune cell interactions were characterized by confocal microscopy and flow cytometry 30min after stimulation with dialkylaminostyryl dye-labeled (DiD) VLP. Expression of CD69 and pro-inflammatory cytokines were used to assess innate immune activation 6h after stimulation. H1- and H5-VLPs rapidly associated with all subsets of human PBMC but exhibited unique binding preferences and frequencies. The H1-VLP bound to 88.7±1.6% of the CD19 + B cells compared to only 21.9±1.8% bound by the H5-VLP. At 6h in culture, CD69 expression on B cells was increased in response to H1-VLP but not H5-VLP (22.79±3.42% vs. 6.15±0.82% respectively: pvaccines. Plant-made VLP vaccines bearing H1 or H5 rapidly elicit immune activation and cytokine production in human PBMC. Differences in the VLP-immune cell interactions suggest that features of the HA proteins themselves, such as receptor specificity, influence innate immune responses. Although not generally considered for inactivated vaccines, the distribution and characteristics of influenza receptor(s) on the immune cells themselves may contribute to both the strength and pattern of the immune response generated. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. The inhibition of assembly of HIV-1 virus-like particles by 3-O-(3',3'-dimethylsuccinyl betulinic acid (DSB is counteracted by Vif and requires its Zinc-binding domain

    Directory of Open Access Journals (Sweden)

    Bouaziz Serge

    2008-12-01

    Full Text Available Abstract Background DSB, the 3-O-(3',3'dimethylsuccinyl derivative of betulinic acid, blocks the last step of protease-mediated processing of HIV-1 Gag precursor (Pr55Gag, which leads to immature, noninfectious virions. When administered to Pr55Gag-expressing insect cells (Sf9, DSB inhibits the assembly and budding of membrane-enveloped virus-like particles (VLP. In order to explore the possibility that viral factors could modulate the susceptibility to DSB of the VLP assembly process, several viral proteins were coexpressed individually with Pr55Gag in DSB-treated cells, and VLP yields assayed in the extracellular medium. Results Wild-type Vif (Vifwt restored the VLP production in DSB-treated cells to levels observed in control, untreated cells. DSB-counteracting effect was also observed with Vif mutants defective in encapsidation into VLP, suggesting that packaging and anti-DSB effect were separate functions in Vif. The anti-DSB effect was abolished for VifC133S and VifS116V, two mutants which lacked the zinc binding domain (ZBD formed by the four H108C114C133H139 coordinates with a Zn atom. Electron microscopic analysis of cells coexpressing Pr55Gag and Vifwt showed that a large proportion of VLP budded into cytoplasmic vesicles and were released from Sf9 cells by exocytosis. However, in the presence of mutant VifC133S or VifS116V, most of the VLP assembled and budded at the plasma membrane, as in control cells expressing Pr55Gag alone. Conclusion The function of HIV-1 Vif protein which negated the DSB inhibition of VLP assembly was independent of its packaging capability, but depended on the integrity of ZBD. In the presence of Vifwt, but not with ZBD mutants VifC133S and VifS116V, VLP were redirected to a vesicular compartment and egressed via the exocytic pathway.

  11. Immunization with DNA plasmids coding for crimean-congo hemorrhagic fever virus capsid and envelope proteins and/or virus-like particles induces protection and survival in challenged mice

    DEFF Research Database (Denmark)

    Hinkula, Jorma; Devignot, Stéphanie; Åkerström, Sara

    2017-01-01

    Crimean-Congo hemorrhagic fever virus (CCHFV) is a bunyavirus causing severe hemorrhagic fever disease in humans, with high mortality rates. The requirement of a high-containment laboratory and the lack of an animal model hampered the study of the immune response and protection of vaccine...... transcriptionally competent virus-like particles (tc-VLPs). In contrast to most studies that focus on neutralizing antibodies, we measured both humoral and cellular immune responses. We demonstrated a clear and 100% efficient preventive immunity against lethal CCHFV challenge with the DNA vaccine. Interestingly......, there was no correlation with the neutralizing antibody titers alone, which were higher in the tc-VLP-vaccinated mice. However, the animals with a lower neutralizing titer, but a dominant cell-mediated Th1 response and a balanced Th2 response, resisted the CCHFV challenge. Moreover, we found that in challenged mice...

  12. Virus-like-vaccines against HIV

    DEFF Research Database (Denmark)

    Andersson, Anne Marie C.; Schwerdtfeger, Melanie; Holst, Peter J.

    2018-01-01

    Protection against chronic infections has necessitated the development of ever-more potent vaccination tools. HIV seems to be the most challenging foe, with a remarkable, poorly immunogenic and fragile surface glycoprotein and the ability to overpower the cell immune system. Virus-like-particle (......Protection against chronic infections has necessitated the development of ever-more potent vaccination tools. HIV seems to be the most challenging foe, with a remarkable, poorly immunogenic and fragile surface glycoprotein and the ability to overpower the cell immune system. Virus...... of HIV. Such vaccines are immunologically perceived as viruses, as they infect cells and produce VLPs in situ, but they only resemble viruses, as the replication defective vectors and VLPs cannot propagate an infection. The inherent safety of such a platform, despite robust particle production...

  13. A novel HPV 16 L1-based chimeric virus-like particle containing E6 and E7 seroreactive epitopes permits highly specific detection of antibodies in patients with CIN 1 and HPV-16 infection.

    Science.gov (United States)

    Monroy-García, Alberto; Gómez-Lim, Miguel A; Weiss-Steider, Benny; la Rosa, Georgina Paz-de; Hernández-Montes, Jorge; Pérez-Saldaña, Karyna; Tapia-Guerrero, Yessica S; Toledo-Guzmán, Mariel E; Santiago-Osorio, Edelmiro; Sanchez-Peña, Héctor I; Mora-García, María de Lourdes

    2011-02-09

    The presence of IgG antibodies to HPV-16 L1-virus like particles (VLPs) in serum has been reported as a result of persistent exposure to the virus and as a marker of disease progression. However, detection of VLP-specific antibodies in sera does not always indicate a malignant lesion as positive results may also be due to a nonmalignant viral infection. Furthermore, malignant lesions are associated with an increased antibody titer for E6 and E7 proteins. The aim of this study was to develop an ELISA using a novel chimeric virus-like particle (cVLP) encoding an L1 protein fused with a string of HPV-16 E6 and E7 seroreactive epitopes to its C-terminus to be used for detection of HPV-16 specific antibodies in patients with cervical intraepithelial lesion grade 1 (CIN 1). The sera of 30 patients with CIN 1 who also tested positive for HPV-16 DNA and of 30 age-matched normal donors negative for HPV infection were tested for the presence of IgG antibodies specific for either VLP-L1 (HPV-16 L1), gVLP (derived from Gardasil), or cVLP by ELISA. The cVLP-reactive sera yielded two distinct groups of results: (H) reactivity levels that presented very strong cVLP-specific titers, and (L) reactivity levels with significantly lower titers similar to those obtained with VLP-L1 and gVLP antigens. Additionally, the sera that presented the higher cVLP titers closely matched those that had significantly stronger reactivity to E6 and E7 epitopes. Interestingly, the samples with the highest titers corresponded to patients with the higher numbers of sexual partners and pregnancies. On the other hand only 4 out of the 12 sera that harbored antibodies with VLP neutralizing ability corresponded to the group with high cVLP antibody titers. We report for the first time that chimeric particles containing HPV-16 L1 protein fused with E6 and E7 seroreactive epitopes enable much better detection of IgG antibodies in the sera of CIN 1 patients positive for HPV-16 infection than those obtained with

  14. Detection of viruses and virus-like particles in four species of wild and farmed bivalve molluscs in Alaska, U.S.A., from 1987 to 2009.

    Science.gov (United States)

    Meyers, Theodore R; Burton, Tamara; Evans, Wally; Starkey, Norman

    2009-12-22

    The U.S. Alaska Department of Fish and Game has regulatory oversight of the mariculture industry that is partially administered through a statewide shellfish health policy. Possession and transport of bivalve molluscs require development of indigenous pathogen histories from diagnostic examinations of wild and farmed populations. These examinations have resulted in the detection of various infectious agents and parasites including viruses: an aquareovirus and aquabirna-like virus isolated by fish cell culture, and papilloma- or polyoma- and herpes-like virus particles within bivalve cell intranuclear inclusion bodies observed by electron microscopy. This study summarizes these results in samples examined from 1987 to 2009 and is the first description of poikilothermic viruses from Alaskan waters isolated from or observed within the tissues of 4 species of bivalve molluscs: geoduck clam Panope abrupta, native littleneck clam Protothaca staminea, purple-hinged rock scallop Crassadoma gigantea and Pacific oyster Crassostrea gigas.

  15. Virus-Like Particle Vaccine Containing the F Protein of Respiratory Syncytial Virus Confers Protection without Pulmonary Disease by Modulating Specific Subsets of Dendritic Cells and Effector T Cells.

    Science.gov (United States)

    Kim, Ki-Hye; Lee, Young-Tae; Hwang, Hye Suk; Kwon, Young-Man; Kim, Min-Chul; Ko, Eun-Ju; Lee, Jong Seok; Lee, Youri; Kang, Sang-Moo

    2015-11-01

    There is no licensed vaccine against respiratory syncytial virus (RSV) since the failure of formalin-inactivated RSV (FI-RSV) due to its vaccine-enhanced disease. We investigated immune correlates conferring protection without causing disease after intranasal immunization with virus-like particle vaccine containing the RSV fusion protein (F VLP) in comparison to FI-RSV and live RSV. Upon RSV challenge, FI-RSV immune mice showed severe weight loss, eosinophilia, and histopathology, and RSV reinfection also caused substantial RSV disease despite their viral clearance. In contrast, F VLP immune mice showed least weight loss and no sign of histopathology and eosinophilia. High levels of interleukin-4-positive (IL-4(+)) and tumor necrosis factor alpha-positive (TNF-α(+)) CD4(+) T cells were found in FI-RSV immune mice, whereas gamma interferon-positive (IFN-γ(+)) and TNF-α(+) CD4(+) T cells were predominantly detected in live RSV-infected mice. More importantly, in contrast to FI-RSV and live RSV that induced higher levels of CD11b(+) dendritic cells, F VLP immunization induced CD8α(+) and CD103(+) dendritic cells, as well as F-specific IFN-γ(+) and TNF-α(+) CD8(+) T cells. These results suggest that F VLP can induce protection without causing pulmonary RSV disease by inducing RSV neutralizing antibodies, as well as modulating specific subsets of dendritic cells and CD8 T cell immunity. It has been a difficult challenge to develop an effective and safe vaccine against respiratory syncytial virus (RSV), a leading cause of respiratory disease. Immune correlates conferring protection but preventing vaccine-enhanced disease remain poorly understood. RSV F virus-like particle (VLP) would be an efficient vaccine platform conferring protection. Here, we investigated the protective immune correlates without causing disease after intranasal immunization with RSV F VLP in comparison to FI-RSV and live RSV. In addition to inducing RSV neutralizing antibodies responsible for

  16. Glutamic acid at residue 125 of the prM helix domain interacts with positively charged amino acids in E protein domain II for Japanese encephalitis virus-like-particle production.

    Science.gov (United States)

    Peng, Jia-Guan; Wu, Suh-Chin

    2014-08-01

    Interaction between E and prM proteins in flavivirus-infected cells is a major factor for virus-like particle (VLP) production. The prM helical (prM-H) domain is topologically close to and may interact with domain II of the E protein (EDII). In this study, we investigated prM-H domain amino acid residues facing Japanese encephalitis virus EDII using site-directed mutagenesis to determine their roles in prM-E interaction and VLP production. Our results indicate that negatively charged prM-E125 residue at the prM-H domain affected VLP production via one or more interactions with positively charged E-K93 and E-H246 residues at EDII. Exchanges of oppositely charged residue side chains at prM-E125/E-K93 and prM-E125/E-H246 are recoverable for VLP production. The prM-E125 and E-H246 residues are conserved and that the positive charge of the E-K93 residue is preserved in different flavivirus groups. These findings suggest that the electrostatic attractions of prM-E125, E-K93, and E-H246 residues are important to flavivirus VLP production and that inhibiting these interactions is a potential strategy for blocking flavivirus infections. Molecular interaction between E and prM proteins of Japanese encephalitis virus is a major driving force for virus-like particle (VLP) production. The current high-resolution structures available for prM-E complexes do not include the membrane proximal stem region of prM. The prM stem region contains an N-terminal loop and a helix domain (prM-H). Since the prM-H domain is topologically close to domain II of the E protein (EDII), this study was to determine molecular interactions between the prM-H domain and EDII. We found that the molecular interactions between prM-E125 residue and positively charged E-K93 and E-H246 residues at EDII are critical for VLP production. More importantly, the prM-E125 and E-H246 residues are conserved and the positive charge of the E-K93 residue is preserved in different flavivirus groups. Our findings help

  17. Studies towards the potential of poliovirus as a vector for the expression of HPV 16 virus-like-particles.

    NARCIS (Netherlands)

    Kuppeveld, F.J.M. van; Jong, A.S. de; Dijkman, H.B.P.M.; Andino, R.; Melchers, W.J.G.

    2002-01-01

    Development of human cervical carcinomas is associated with infection by certain human papillomavirus (HPV) types. Thus, protection against HPV infection through vaccination may prevent development of cervical cancer. The purpose of this study was to investigate the possibility of using a poliovirus

  18. Virus-like particles displaying H5, H7, H9 hemagglutinins and N1 neuraminidase elicit protective immunity to heterologous avian influenza viruses in chickens.

    Science.gov (United States)

    Pushko, Peter; Tretyakova, Irina; Hidajat, Rachmat; Zsak, Aniko; Chrzastek, Klaudia; Tumpey, Terrence M; Kapczynski, Darrell R

    2017-01-15

    Avian influenza (AI) viruses circulating in wild birds pose a serious threat to public health. Human and veterinary vaccines against AI subtypes are needed. Here we prepared triple-subtype VLPs that co-localized H5, H7 and H9 antigens derived from H5N1, H7N3 and H9N2 viruses. VLPs also contained influenza N1 neuraminidase and retroviral gag protein. The H5/H7/H9/N1/gag VLPs were prepared using baculovirus expression. Biochemical, functional and antigenic characteristics were determined including hemagglutination and neuraminidase enzyme activities. VLPs were further evaluated in a chicken AI challenge model for safety, immunogenicity and protective efficacy against heterologous AI viruses including H5N2, H7N3 and H9N2 subtypes. All vaccinated birds survived challenges with H5N2 and H7N3 highly pathogenic AI (HPAI) viruses, while all controls died. Immune response was also detectable after challenge with low pathogenicity AI (LPAI) H9N2 virus suggesting that H5/H7/H9/N1/gag VLPs represent a promising approach for the development of broadly protective AI vaccine. Copyright © 2016. Published by Elsevier Inc.

  19. Chimeric virus-like particles containing a conserved region of the G protein in combination with a single peptide of the M2 protein confer protection against respiratory syncytial virus infection.

    Science.gov (United States)

    Qiao, Lei; Zhang, Yuan; Chai, Feng; Tan, Yiluo; Huo, Chunling; Pan, Zishu

    2016-07-01

    To investigate the feasibility and efficacy of a virus-like particle (VLP) vaccine composed of the conserved antigenic epitopes of respiratory syncytial virus (RSV), the chimeric RSV VLPs HBcΔ-tG and HBcΔ-tG/M282-90 were generated based on the truncated hepatitis B virus core protein (HBcΔ). HBcΔ-tG consisted of HBcΔ, the conserved region (aa 144-204) of the RSV G protein. HBcΔ-tG was combined with a single peptide (aa 82-90) of the M2 protein to generate HBcΔ-tG/M282-90. Immunization of mice with the HBcΔ-tG or HBcΔ-tG/M282-90 VLPs elicited RSV-specific IgG and neutralizing antibody production and conferred protection against RSV infection. Compared with HBcΔ-tG, HBcΔ-tG/M282-90 induced decreased Th2 cytokine production (IL-4 and IL-5), increased Th1 cytokine response (IFN-γ, TNF-α, and IL-2), and increased ratios of IgG2a/IgG1 antibodies, thereby relieving pulmonary pathology upon subsequent RSV infection. Our results demonstrated that chimeric HBcΔ-tG/M282-90 VLPs represented an effective RSV subunit vaccine candidate. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. Intranasal Immunization with Influenza Virus-Like Particles Containing Membrane-Anchored Cholera Toxin B or Ricin Toxin B Enhances Adaptive Immune Responses and Protection against an Antigenically Distinct Virus

    Directory of Open Access Journals (Sweden)

    Xianliang Ji

    2016-04-01

    Full Text Available Vaccination is the most effective means to prevent influenza virus infection, although current approaches are associated with suboptimal efficacy. Here, we generated virus-like particles (VLPs composed of the hemagglutinin (HA, neuraminidase (NA and matrix protein (M1 of A/Changchun/01/2009 (H1N1 with or without either membrane-anchored cholera toxin B (CTB or ricin toxin B (RTB as molecular adjuvants. The intranasal immunization of mice with VLPs containing membrane-anchored CTB or RTB elicited stronger humoral and cellular immune responses when compared to mice immunized with VLPs alone. Administration of VLPs containing CTB or RTB significantly enhanced virus-specific systemic and mucosal antibody responses, hemagglutination inhibiting antibody titers, virus neutralizing antibody titers, and the frequency of virus-specific IFN-γ and IL-4 secreting splenocytes. VLPs with and without CTB or RTB conferred complete protection against lethal challenge with a mouse-adapted homologous virus. When challenged with an antigenically distinct H1N1 virus, all mice immunized with VLPs containing CTB or RTB survived whereas mice immunized with VLPs alone showed only partial protection (80% survival. Our results suggest that membrane-anchored CTB and RTB possess strong adjuvant properties when incorporated into an intranasally-delivered influenza VLP vaccine. Chimeric influenza VLPs containing CTB or RTB may represent promising vaccine candidates for improved immunological protection against homologous and antigenically distinct influenza viruses.

  1. Membrane fusion-competent virus-like proteoliposomes and proteinaceous supported bilayers made directly from cell plasma membranes.

    Science.gov (United States)

    Costello, Deirdre A; Hsia, Chih-Yun; Millet, Jean K; Porri, Teresa; Daniel, Susan

    2013-05-28

    Virus-like particles are useful materials for studying virus-host interactions in a safe manner. However, the standard production of pseudovirus based on the vesicular stomatitis virus (VSV) backbone is an intricate procedure that requires trained laboratory personnel. In this work, a new strategy for creating virus-like proteoliposomes (VLPLs) and virus-like supported bilayers (VLSBs) is presented. This strategy uses a cell blebbing technique to induce the formation of nanoscale vesicles from the plasma membrane of BHK cells expressing the hemagglutinin (HA) fusion protein of influenza X-31. These vesicles and supported bilayers contain HA and are used to carry out single particle membrane fusion events, monitored using total internal reflection fluorescence microscopy. The results of these studies show that the VLPLs and VLSBs contain HA proteins that are fully competent to carry out membrane fusion, including the formation of a fusion pore and the release of fluorophores loaded into vesicles. This new strategy for creating spherical and planar geometry virus-like membranes has many potential applications. VLPLs could be used to study fusion proteins of virulent viruses in a safe manner, or they could be used as therapeutic delivery particles to transport beneficial proteins coexpressed in the cells to a target cell. VLSBs could facilitate high throughput screening of antiviral drugs or pathogen-host cell interactions.

  2. Chikungunya virus-like particle vaccine

    NARCIS (Netherlands)

    Metz, S.W.H.

    2013-01-01

      Chikungunya virus (CHIKV) is an arthropod-borne alphavirus (family Togaviridae) and is the causative agent of chikungunya fever. This disease is characterised by the sudden onset of high fever and long-lasting arthritic disease. First identified in Tanzania in 1952, CHIKV has re-emerged in

  3. A novel H6N1 virus-like particle vaccine induces long-lasting cross-clade antibody immunity against human and avian H6N1 viruses.

    Science.gov (United States)

    Yang, Ji-Rong; Chen, Chih-Yuan; Kuo, Chuan-Yi; Cheng, Chieh-Yu; Lee, Min-Shiuh; Cheng, Ming-Chu; Yang, Yu-Chih; Wu, Chia-Ying; Wu, Ho-Sheng; Liu, Ming-Tsan; Hsiao, Pei-Wen

    2016-02-01

    Avian influenza A(H6N1) virus is one of the most common viruses isolated from migrating birds and domestic poultry in many countries. The first and only known case of human infection by H6N1 virus in the world was reported in Taiwan in 2013. This led to concern that H6N1 virus may cause a threat to public health. In this study, we engineered a recombinant H6N1 virus-like particle (VLP) and investigated its vaccine effectiveness compared to the traditional egg-based whole inactivated virus (WIV) vaccine. The H6N1-VLPs exhibited similar morphology and functional characteristics to influenza viruses. Prime-boost intramuscular immunization in mice with unadjuvanted H6N1-VLPs were highly immunogenic and induced long-lasting antibody immunity. The functional activity of the VLP-elicited IgG antibodies was proved by in vitro seroprotective hemagglutination inhibition and microneutralization titers against the homologous human H6N1 virus, as well as in vivo viral challenge analyses which showed H6N1-VLP immunization significantly reduced viral load in the lung, and protected against human H6N1 virus infection. Of particular note, the H6N1-VLPs but not the H6N1-WIVs were able to confer cross-reactive humoral immunity; antibodies induced by H6N1-VLP vaccine robustly inhibited the hemagglutination activities and in vitro replication of distantly-related heterologous avian H6N1 viruses. Furthermore, the H6N1-VLPs were found to elicit significantly greater anti-HA2 antibody responses in immunized mice than H6N1-WIVs. Collectively, we demonstrated for the first time a novel H6N1-VLP vaccine that effectively provides broadly protective immunity against both human and avian H6N1 viruses. These results, which uncover the underlying mechanisms for induction of wide-range immunity against influenza viruses, may be useful for future influenza vaccine development. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Immunization with DNA Plasmids Coding for Crimean-Congo Hemorrhagic Fever Virus Capsid and Envelope Proteins and/or Virus-Like Particles Induces Protection and Survival in Challenged Mice.

    Science.gov (United States)

    Hinkula, Jorma; Devignot, Stéphanie; Åkerström, Sara; Karlberg, Helen; Wattrang, Eva; Bereczky, Sándor; Mousavi-Jazi, Mehrdad; Risinger, Christian; Lindegren, Gunnel; Vernersson, Caroline; Paweska, Janusz; van Vuren, Petrus Jansen; Blixt, Ola; Brun, Alejandro; Weber, Friedemann; Mirazimi, Ali

    2017-05-15

    Crimean-Congo hemorrhagic fever virus (CCHFV) is a bunyavirus causing severe hemorrhagic fever disease in humans, with high mortality rates. The requirement of a high-containment laboratory and the lack of an animal model hampered the study of the immune response and protection of vaccine candidates. Using the recently developed interferon alpha receptor knockout (IFNAR(-/-)) mouse model, which replicates human disease, we investigated the immunogenicity and protection of two novel CCHFV vaccine candidates: a DNA vaccine encoding a ubiquitin-linked version of CCHFV Gc, Gn, and N and one using transcriptionally competent virus-like particles (tc-VLPs). In contrast to most studies that focus on neutralizing antibodies, we measured both humoral and cellular immune responses. We demonstrated a clear and 100% efficient preventive immunity against lethal CCHFV challenge with the DNA vaccine. Interestingly, there was no correlation with the neutralizing antibody titers alone, which were higher in the tc-VLP-vaccinated mice. However, the animals with a lower neutralizing titer, but a dominant cell-mediated Th1 response and a balanced Th2 response, resisted the CCHFV challenge. Moreover, we found that in challenged mice with a Th1 response (immunized by DNA/DNA and boosted by tc-VLPs), the immune response changed to Th2 at day 9 postchallenge. In addition, we were able to identify new linear B-cell epitope regions that are highly conserved between CCHFV strains. Altogether, our results suggest that a predominantly Th1-type immune response provides the most efficient protective immunity against CCHFV challenge. However, we cannot exclude the importance of the neutralizing antibodies as the surviving immunized mice exhibited substantial amounts of them.IMPORTANCE Crimean-Congo hemorrhagic fever virus (CCHFV) is responsible for hemorrhagic diseases in humans, with a high mortality rate. There is no FDA-approved vaccine, and there are still gaps in our knowledge of the immune

  5. Intranasal immunization of pigs with porcine reproductive and respiratory syndrome virus-like particles plus 2', 3'-cGAMP VacciGrade™ adjuvant exacerbates viremia after virus challenge.

    Science.gov (United States)

    Van Noort, Alexandria; Nelsen, April; Pillatzki, Angela E; Diel, Diego G; Li, Feng; Nelson, Eric; Wang, Xiuqing

    2017-04-12

    Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure in pregnant sows and acute respiratory disease in young pigs. It is a leading infectious agent of swine respiratory complex, which has significant negative economic impact on the swine industry. Commercial markets currently offer both live attenuated and killed vaccines; however, increasing controversy exists about their efficacy providing complete protection. Virus-like particles (VLPs) possess many desirable features of a potent vaccine candidate and have been proven to be highly immunogenic and protective against virus infections. Here we explored the efficacy of PRRSV VLPs together with the use of a novel 2', 3'-cGAMP VacciGrade™ adjuvant. Animals were immunized twice intranasally with phosphate buffered saline (PBS), PRRSV VLPs, or PRRSV VLPs plus 2', 3'-cGAMP VacciGrade™ at 2 weeks apart. Animals were challenged with PRRSV-23983 at 2 weeks post the second immunization. PRRSV specific antibody response and cytokines were measured. Viremia, clinical signs, and histological lesions were evaluated. PRRSV N protein specific antibody was detected in all animals at day 10 after challenge, but no significant difference was observed among the vaccinated and control groups. Surprisingly, a significantly higher viremia was observed in the VLPs and VLPs plus the adjuvant groups compared to the control group. The increased viremia is correlated with a higher interferon-α induction in the serum of the VLPs and the VLPs plus the adjuvant groups. Intranasal immunizations of pigs with PRRSV VLPs and VLPs plus the 2', 3'-cGAMP VacciGrade™ adjuvant exacerbates viremia. A higher level of interferon-α production, but not interferon-γ and IL-10, is correlated with enhanced virus replication. Overall, PRRSV VLPs and PRRSV VLPs plus the adjuvant fail to provide protection against PRRSV challenge. Different dose of VLPs and alternative route of vaccination such as intramuscular

  6. Enhancement of humoral and cell mediated immune response to HPV16 L1-derived peptides subsequent to vaccination with prophylactic bivalent HPV L1 virus-like particle vaccine in healthy females.

    Science.gov (United States)

    Yokomine, Masato; Matsueda, Satoko; Kawano, Kouichiro; Sasada, Tetsuro; Fukui, Akimasa; Yamashita, Takuto; Komatsu, Nobukazu; Shichijo, Shigeki; Tasaki, Kazuto; Matsukuma, Ken; Itoh, Kyogo; Kamura, Toshiharu; Ushijima, Kimio

    2017-04-01

    Currently prophylactic HPV16/18 L1 virus-like particle (VLP) vaccines are employed with great success for the prevention of HPV infection. However, limited information is available regarding the immune responses against human papillomavirus (HPV) 16/18 L1 subsequent to HPV16/18 L1 VLP vaccination, primarily due to the lack of widely used assays for immune monitoring. The aim of the present study was to identify HPV16 L1-derived B and T cell epitopes for monitoring the immune responses after HPV16/18 L1 VLP vaccination in healthy females. The levels of immunoglobulin G (IgG), IgE, IgA and IgM reactive to HPV16 L1-derived peptides were measured by multiplex bead suspension assay. Following detailed B cell epitope mapping, T cell responses specific to HPV16 L1-derived peptides were evaluated by an IFN-γ ELISPOT assay. The levels of IgG, IgM and IgA reactive to 20-mer peptides (PTPSGSMVTSDAQIFNKPYW) at positions 293-312 and 300-319 of HPV16 L1 were significantly increased in the plasma after 2, 7, and 12 months after first vaccination. Detailed epitope mapping identified the amino acid sequence (TSDAQIFNKP) at position 301-310 of HPV16 L1 as an immunogenic B cell epitope. In addition, T cell responses to an HLA-A2- and HLA-A24-restricted epitope (QIFNKPYWL) at position 305-313 of HPV16 L1 were increased following immunization, suggesting that the HPV16/18 L1-VLP vaccination as able to induce specific immune responses in T and B cells simultaneously. The identified B and T cell epitopes may be useful as a biomarker for monitoring the immune responses subsequent to HPV16/18 L1 VLP vaccination. Thus, the present study may provide novel information to improve the understanding of the immune responses to HPV16 L1.

  7. Point mutation in calcium-binding domain of mouse polyomavirus VP1 protein does not prevent virus-like particle formation, but changes VP1 interactions with Saccharomyces cerevisiae cell structures

    Czech Academy of Sciences Publication Activity Database

    Adamec, T.; Palková, Zdena; Velková, K.; Štokrová, Jitka; Forstová, J.

    2005-01-01

    Roč. 5, 4-5 (2005), s. 331-340 ISSN 1567-1356 R&D Projects: GA ČR GA204/03/0593 Institutional research plan: CEZ:AV0Z5052915 Keywords : polyomavirus VP1 * Saccharomyces cerevisiae * heterologous expression Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.477, year: 2005

  8. Impact of an HPV6/11/16/18 L1 virus-like particle vaccine on progression to cervical intraepithelial neoplasia in seropositive women with HPV16/18 infection

    DEFF Research Database (Denmark)

    Haupt, Richard M; Wheeler, Cosette M; Brown, Darron R

    2011-01-01

    prevaccination. Incidence is expressed as the number of women with an endpoint per 100 person-years-at-risk. In total, 419 vaccine and 446 placebo recipients were both seropositive and DNA positive to HPV16 or HPV18 prevaccination and had at least one follow-up visit. In Protocol 013, the incidence of HPV16...

  9. Alphavirus vector-based replicon particles expressing multivalent cross-protective Lassa virus glycoproteins.

    Science.gov (United States)

    Wang, Min; Jokinen, Jenny; Tretyakova, Irina; Pushko, Peter; Lukashevich, Igor S

    2018-01-29

    Lassa virus (LASV) is the most prevalent rodent-borne arenavirus circulated in West Africa. With population at risk from Senegal to Nigeria, LASV causes Lassa fever and is responsible for thousands of deaths annually. High genetic diversity of LASV is one of the challenges for vaccine R&D. We developed multivalent virus-like particle vectors (VLPVs) derived from the human Venezuelan equine encephalitis TC-83 IND vaccine (VEEV) as the next generation of alphavirus-based bicistronic RNA replicon particles. The genes encoding VEEV structural proteins were replaced with LASV glycoproteins (GPC) from distantly related clades I and IV with individual 26S promoters. Bicistronic RNA replicons encoding wild-type LASV GPC (GPCwt) and C-terminally deleted, non-cleavable modified glycoprotein (ΔGPfib), were encapsidated into VLPV particles using VEEV capsid and glycoproteins provided in trans. In transduced cells, VLPVs induced simultaneous expression of LASV GPCwt and ΔGPfib from 26S alphavirus promoters. LASV ΔGPfib was predominantly expressed as trimers, accumulated in the endoplasmic reticulum, induced ER stress and apoptosis promoting antigen cross-priming. VLPV vaccines were immunogenic and protective in mice and upregulated CD11c + /CD8 + dendritic cells playing the major role in cross-presentation. Notably, VLPV vaccination resulted in induction of cross-reactive multifunctional T cell responses after stimulation of immune splenocytes with peptide cocktails derived from LASV from clades I-IV. Multivalent RNA replicon-based LASV vaccines can be applicable for first responders, international travelers visiting endemic areas, military and lab personnel. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

  10. Filovirus-Like Particles as Vaccines and Discovery Tools

    Science.gov (United States)

    2005-06-01

    correct structural proteins is sufficient for forming VLPs. This is true for both nonenveloped viruses , such as parvovirus, papilloma - virus , rotavirus... viruses , HIV, parvoviruses and human papillomavirus , by expressing select viral proteins in insect or mammalian cells [72]. Since VLPs are morphologically...antibodies, cytotoxic T-lymphocytes, Ebola, filovirus, Marburg, protective immunity, vaccines, virus -like particles Ebola and Marburg viruses are members of

  11. Analytical expressions of the deformation limit of fluid particles

    Science.gov (United States)

    Fang, L.; Wang, S. L.; Hong, J. Y.

    2017-12-01

    The deformation of fluid particles is usually directly related to various Lagrangian phenomena in fluids. We show typical analytical expressions under external strain rate fields by defining the deformation limit for small fluid particles, aiming at providing analytical tools for supporting future investigations on the Lagrangian phenomena in fluids. In particular, when particles are driven by a constant strain rate which is greater or equal to the rotation rate, the orientation of fluid elements aligns with strain tensor in short times and then decouples with them in long times, while the fluid particles are flattened in most cases, which are similar to previous observations in turbulent flows. This supports the simplifications in previous Lagrangian models (e.g., Fang et al., 2015 [20]), where the strain rates were assumed to be locally homogeneous.

  12. Complete genome sequence of a novel extrachromosomal virus-like element identified in planarian Girardia tigrina

    Directory of Open Access Journals (Sweden)

    Vagner Loura L

    2002-06-01

    Full Text Available Abstract Background Freshwater planarians are widely used as models for investigation of pattern formation and studies on genetic variation in populations. Despite extensive information on the biology and genetics of planaria, the occurrence and distribution of viruses in these animals remains an unexplored area of research. Results Using a combination of Suppression Subtractive Hybridization (SSH and Mirror Orientation Selection (MOS, we compared the genomes of two strains of freshwater planarian, Girardia tigrina. The novel extrachromosomal DNA-containing virus-like element denoted PEVE (Planarian Extrachromosomal Virus-like Element was identified in one planarian strain. The PEVE genome (about 7.5 kb consists of two unique regions (Ul and Us flanked by inverted repeats. Sequence analyses reveal that PEVE comprises two helicase-like sequences in the genome, of which the first is a homolog of a circoviral replication initiator protein (Rep, and the second is similar to the papillomavirus E1 helicase domain. PEVE genome exists in at least two variant forms with different arrangements of single-stranded and double-stranded DNA stretches that correspond to the Us and Ul regions. Using PCR analysis and whole-mount in situ hybridization, we characterized PEVE distribution and expression in the planarian body. Conclusions PEVE is the first viral element identified in free-living flatworms. This element differs from all known viruses and viral elements, and comprises two potential helicases that are homologous to proteins from distant viral phyla. PEVE is unevenly distributed in the worm body, and is detected in specific parenchyma cells.

  13. High-Level Systemic Expression of Conserved Influenza Epitope in Plants on the Surface of Rod-Shaped Chimeric Particles

    Directory of Open Access Journals (Sweden)

    Natalia V. Petukhova

    2014-04-01

    Full Text Available Recombinant viruses based on the cDNA copy of the tobacco mosaic virus (TMV genome carrying different versions of the conserved M2e epitope from influenza virus A cloned into the coat protein (CP gene were obtained and partially characterized by our group previously; cysteines in the human consensus M2e sequence were changed to serine residues. This work intends to show some biological properties of these viruses following plant infections. Agroinfiltration experiments on Nicotiana benthamiana confirmed the efficient systemic expression of M2e peptides, and two point amino acid substitutions in recombinant CPs significantly influenced the symptoms and development of viral infections. Joint expression of RNA interference suppressor protein p19 from tomato bushy stunt virus (TBSV did not affect the accumulation of CP-M2e-ser recombinant protein in non-inoculated leaves. RT-PCR analysis of RNA isolated from either infected leaves or purified TMV-M2e particles proved the genetic stability of TMV‑based viral vectors. Immunoelectron microscopy of crude plant extracts demonstrated that foreign epitopes are located on the surface of chimeric virions. The rod‑shaped geometry of plant-produced M2e epitopes is different from the icosahedral or helical filamentous arrangement of M2e antigens on the carrier virus-like particles (VLP described earlier. Thereby, we created a simple and efficient system that employs agrobacteria and plant viral vectors in order to produce a candidate broad-spectrum flu vaccine.

  14. Minor displacements in the insertion site provoke major differences in the induction of antibody responses by chimeric parvovirus-like particles

    DEFF Research Database (Denmark)

    Rueda, P.; Hurtado, A.; del Barrio, M.

    1999-01-01

    An antigen-delivery system based on hybrid virus-like particles (VLPs) formed by the self-assembly of the capsid VP2 protein of canine parvovirus (CPV) and expressing foreign peptides was investigated. In this report, we have studied the effects of inserting the poliovirus C3:B epitope in the four...

  15. A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine

    OpenAIRE

    Chen, Yang; Guo, Wanzhu; Xu, Zhiwen; Yan, Qigui; Luo, Yan; Shi, Qian; Chen, Dishi; Zhu, Ling; Wang, Xiaoyu

    2011-01-01

    Abstract Background Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. Methods A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and...

  16. A Biocatalytic Nanomaterial for the Label-Free Detection of Virus-Like Particles.

    Science.gov (United States)

    Sykora, Sabine; Correro, M Rita; Moridi, Negar; Belliot, Gaël; Pothier, Pierre; Dudal, Yves; Corvini, Philippe F-X; Shahgaldian, Patrick

    2017-06-01

    The design of nanomaterials that are capable of specific and sensitive biomolecular recognition is an on-going challenge in the chemical and biochemical sciences. A number of sophisticated artificial systems have been designed to specifically recognize a variety of targets. However, methods based on natural biomolecular detection systems using antibodies are often superior. Besides greater affinity and selectivity, antibodies can be easily coupled to enzymatic systems that act as signal amplifiers, thus permitting impressively low detection limits. The possibility to translate this concept to artificial recognition systems remains limited due to design incompatibilities. Here we describe the synthesis of a synthetic nanomaterial capable of specific biomolecular detection by using an internal biocatalytic colorimetric detection and amplification system. The design of this nanomaterial relies on the ability to accurately grow hybrid protein-organosilica layers at the surface of silica nanoparticles. The method allows for label-free detection and quantification of targets at picomolar concentrations. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Virus-like particle display of HER2 induces potent anti-cancer responses

    DEFF Research Database (Denmark)

    Palladini, Arianna; Thrane, Susan; Janitzek, Christoph M

    2018-01-01

    Overexpression of human epidermal growth factor receptor-2 (HER2) occurs in 20-30% of invasive breast cancers. Monoclonal antibody therapy is effective in treating HER2-driven mammary carcinomas, but its utility is limited by high costs, side effects and development of resistance. Active vaccinat......Overexpression of human epidermal growth factor receptor-2 (HER2) occurs in 20-30% of invasive breast cancers. Monoclonal antibody therapy is effective in treating HER2-driven mammary carcinomas, but its utility is limited by high costs, side effects and development of resistance. Active...

  18. Real-time investigation of the assembly dynamics of artificial virus-like particles

    NARCIS (Netherlands)

    Marchetti, M.; Kamsma, D.; De Vries, R.; Roos, W.H.; Wuite, G.J.L.

    2017-01-01

    Artificial viruses are model systems for the understanding of natural viruses and potential vehicles for genetic material delivery. It is still a challenge to fully reproduce the natural viral cooperativity behavior during the self-assembly process[1], therefore we are working with simplified model

  19. Low Temperature-Dependent Salmonid Alphavirus Glycoprotein Processing and Recombinant Virus-Like Particle Formation

    NARCIS (Netherlands)

    Metz, S.W.H.; Feenstra, F.; Villoing, S.; Hulten, van M.C.; Lent, van J.W.M.; Koumans, J.; Vlak, J.M.; Pijlman, G.P.

    2011-01-01

    Pancreas disease (PD) and sleeping disease (SD) are important viral scourges in aquaculture of Atlantic salmon and rainbow trout. The etiological agent of PD and SD is salmonid alphavirus (SAV), an unusual member of the Togaviridae (genus Alphavirus). SAV replicates at lower temperatures in fish.

  20. Exploiting Fluorescent Polymers To Probe the Self-Assembly of Virus-like Particles

    DEFF Research Database (Denmark)

    Caden-Nava, Ruben D.; Hu, Yufang; Garmann, Rees F.

    2011-01-01

    ), and that the total charge on the PSS exceeds that of the capsid protein by as much as a factor of 9. Here, we extend studies of this kind to PSS molecules that are sufficiently small that two or more can be packaged into VLPs. The use of 38 kDa PSS polymers that have been fluorescently labeled with Rhodamine B...... the molar ratio of protein to PSS in the reaction mix shifts the VLP distribution from T = 1 to T = 2 structures. By combining fluorescence and gel electrophoresis measurements, it is determined that, on average, there are two polymers in each T = 1 capsid and three in each T = 2, with the PSS charge less...

  1. Immunization Against Active Ghrelin Using Virus-Like Particles for Obesity Treatment

    OpenAIRE

    Andrade, Sara; Pinho, Filipa; Ribeiro, AndreiaM; Carreira, Marcos; Casanueva, FelipeF; Roy, Polly; Monteiro, MarianaP

    2013-01-01

    Ghrelin is a gut hormone that stimulates food intake. In physiological conditions, ghrelin plasma levels rise with fasting and decrease after meals. Obese individuals have low fasting ghrelin levels that rise after food restriction, which is pointed out as a reason for the difficulty in maintaining weight loss. Some bariatric surgery procedures prevent rise in ghrelin levels with weight loss and this has been hypothesised to contribute to the long-term success of the treatment. The main goal ...

  2. Development of a New Zealand database of plant virus and virus-like organisms

    NARCIS (Netherlands)

    Fletcher, J.D.; Lister, R.A.; Clover, G.R.G.; Horner, M.B.; Thomas, J.E.; Vlugt, van der R.A.A.; MacDiarmid, R.M.

    2009-01-01

    The recent 8th Australasian plant virology workshop in Rotorua, New Zealand, discussed the development of a New Zealand database of plant virus and virus-like organisms. Key points of discussion included: (i) the purpose of such a database; (ii) who would benefit from the information in a database;

  3. Identification of a pegivirus (GB virus-like virus) that infects horses

    DEFF Research Database (Denmark)

    Kapoor, Amit; Simmonds, Peter; Cullen, John M

    2013-01-01

    The recent identification of nonprimate hepaciviruses in dogs and then in horses prompted us to look for pegiviruses (GB virus-like viruses) in these species. Although none were detected in canines, we found widespread natural infection of horses by a novel pegivirus. Unique genomic features...

  4. Fabrication and characterization of gold nano-wires templated on virus-like arrays of tobacco mosaic virus coat proteins

    Science.gov (United States)

    Wnęk, M.; Górzny, M. Ł.; Ward, M. B.; Wälti, C.; Davies, A. G.; Brydson, R.; Evans, S. D.; Stockley, P. G.

    2013-01-01

    The rod-shaped plant virus tobacco mosaic virus (TMV) is widely used as a nano-fabrication template, and chimeric peptide expression on its major coat protein has extended its potential applications. Here we describe a simple bacterial expression system for production and rapid purification of recombinant chimeric TMV coat protein carrying C-terminal peptide tags. These proteins do not bind TMV RNA or form disks at pH 7. However, they retain the ability to self-assemble into virus-like arrays at acidic pH. C-terminal peptide tags in such arrays are exposed on the protein surface, allowing interaction with target species. We have utilized a C-terminal His-tag to create virus coat protein-templated nano-rods able to bind gold nanoparticles uniformly. These can be transformed into gold nano-wires by deposition of additional gold atoms from solution, followed by thermal annealing. The resistivity of a typical annealed wire created by this approach is significantly less than values reported for other nano-wires made using different bio-templates. This expression construct is therefore a useful additional tool for the creation of chimeric TMV-like nano-rods for bio-templating.

  5. Virus-like nanoparticle and DNA vaccination confers protection against respiratory syncytial virus by modulating innate and adaptive immune cells.

    Science.gov (United States)

    Ko, Eun-Ju; Kwon, Young-Man; Lee, Jong Seok; Hwang, Hye Suk; Yoo, Si-Eun; Lee, Yu-Na; Lee, Young-Tae; Kim, Min-Chul; Cho, Min Kyoung; Lee, You Ri; Quan, Fu-Shi; Song, Jae-Min; Lee, Sujin; Moore, Martin L; Kang, Sang-Moo

    2015-01-01

    Respiratory syncytial virus (RSV) is an important human pathogen. Expression of virus structural proteins produces self-assembled virus-like nanoparticles (VLP). We investigated immune phenotypes after RSV challenge of immunized mice with VLP containing RSV F and G glycoproteins mixed with F-DNA (FdFG VLP). In contrast to formalin-inactivated RSV (FI-RSV) causing vaccination-associated eosinophilia, FdFG VLP immunization induced low bronchoalveolar cellularity, higher ratios of CD11c(+) versus CD11b(+) phenotypic cells and CD8(+) T versus CD4(+) T cells secreting interferon (IFN)-γ, T helper type-1 immune responses, and no sign of eosinophilia upon RSV challenge. Furthermore, RSV neutralizing activity, lung viral clearance, and histology results suggest that FdFG VLP can be comparable to live RSV in conferring protection against RSV and in preventing RSV disease. This study provides evidence that a combination of recombinant RSV VLP and plasmid DNA may have a potential anti-RSV prophylactic vaccine inducing balanced innate and adaptive immune responses. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Respiratory syncytial virus-like nanoparticle vaccination induces long-term protection without pulmonary disease by modulating cytokines and T-cells partially through alveolar macrophages.

    Science.gov (United States)

    Lee, Young-Tae; Ko, Eun-Ju; Hwang, Hye Suk; Lee, Jong Seok; Kim, Ki-Hye; Kwon, Young-Man; Kang, Sang-Moo

    2015-01-01

    The mechanisms of protection against respiratory syncytial virus (RSV) are poorly understood. Virus-like nanoparticles expressing RSV glycoproteins (eg, a combination of fusion and glycoprotein virus-like nanoparticles [FG VLPs]) have been suggested to be a promising RSV vaccine candidate. To understand the roles of alveolar macrophages (AMs) in inducing long-term protection, mice that were 12 months earlier vaccinated with formalin-inactivated RSV (FI-RSV) or FG VLPs were treated with clodronate liposome prior to RSV infection. FI-RSV immune mice with clodronate liposome treatment showed increases in eosinophils, plasmacytoid dendritic cells, interleukin (IL)-4(+) T-cell infiltration, proinflammatory cytokines, chemokines, and, in particular, mucus production upon RSV infection. In contrast to FI-RSV immune mice with severe pulmonary histopathology, FG VLP immune mice showed no overt sign of histopathology and significantly lower levels of eosinophils, T-cell infiltration, and inflammatory cytokines, but higher levels of interferon-γ, which are correlated with protection against RSV disease. FG VLP immune mice with depletion of AMs showed increases in inflammatory cytokines and chemokines, as well as eosinophils. The results in this study suggest that FG nanoparticle vaccination induces long-term protection against RSV and that AMs play a role in the RSV protection by modulating eosinophilia, mucus production, inflammatory cytokines, and T-cell infiltration.

  7. Nanoparticle Vaccines Adopting Virus-like Features for Enhanced Immune Potentiation

    Science.gov (United States)

    Chattopadhyay, Saborni; Chen, Jui-Yi; Chen, Hui-Wen; Hu, Che-Ming Jack

    2017-01-01

    Synthetic nanoparticles play an increasingly significant role in vaccine design and development as many nanoparticle vaccines show improved safety and efficacy over conventional formulations. These nanoformulations are structurally similar to viruses, which are nanoscale pathogenic organisms that have served as a key selective pressure driving the evolution of our immune system. As a result, mechanisms behind the benefits of nanoparticle vaccines can often find analogue to the interaction dynamics between the immune system and viruses. This review covers the advances in vaccine nanotechnology with a perspective on the advantages of virus mimicry towards immune potentiation. It provides an overview to the different types of nanomaterials utilized for nanoparticle vaccine development, including functionalization strategies that bestow nanoparticles with virus-like features. As understanding of human immunity and vaccine mechanisms continue to evolve, recognizing the fundamental semblance between synthetic nanoparticles and viruses may offer an explanation for the superiority of nanoparticle vaccines over conventional vaccines and may spur new design rationales for future vaccine research. These nanoformulations are poised to provide solutions towards pressing and emerging human diseases. PMID:29071191

  8. Characterization of the size distribution and aggregation of virus-like nanoparticles used as active ingredients of the HeberNasvac therapeutic vaccine against chronic hepatitis B

    Science.gov (United States)

    Lopez, Matilde; Rodriguez, Elias Nelson; Lobaina, Yadira; Musacchio, Alexis; Falcon, Viviana; Guillen, Gerardo; Aguilar, Julio C.

    2017-06-01

    The use of virus-like particles (VLPs) as antigens constitutes a well established strategy in preventive vaccination. These non-infective particles have a composition, size, and structure favoring their interaction and processing by the immune system. Recombinant viral nucleocapsids encapsulating bacterial nucleic acids result in potent Th1-driving immunogens. Several antigens have been coadministered with VLPs or conjugated to them to further increase their immunogenicity. In the present work we characterize the size distribution of two different recombinant VLPs obtained as components of HeberNasvac, a novel therapeutic vaccine recently registered to treat chronic hepatitis B. The vaccine ingredients, hepatitis B virus surface and nucleocapsid antigens (HBsAg and HBcAg, respectively) and the vaccine formulation, were evaluated using dynamic light scattering (DLS), transmission electron microscopy (TEM) and light obscuration technology. The results demonstrate that both antigens are nanoparticles with sizes ranging between 20-30 nm, in line with reports in the literature. In addition, DLS studies evidenced the capacity of both antigens to form homologous and heterologous aggregates, both as active ingredients as well as being part of the final product. The evaluation of subvisible particles in HeberNasvac formulation fulfills the requirements in terms of quantity and size established for parenteral pharmaceutical compositions. Invited talk at 8th Int. Workshop on Advanced Materials Science and Nanotechnology (IWAMSN2016) (Ha Long City, Vietnam, 8-12 November 2016)

  9. Gene Expression Profiling in Lung Tissues from Rat Exposed to Lunar Dust Particles

    Science.gov (United States)

    Zhang, Ye; Lam, Chiu-Wing; Zalesak, Selina M.; Kidane, Yared H.; Feiveson, Alan H.; Ploutz-Snyder, Robert; Scully, Robert R.; Williams, Kyle; Wu, Honglu; James, John T.

    2014-01-01

    The Moon's surface is covered by a layer of fine, reactive dust. Lunar dust contain about 1-2% of very fine dust (gene expression changes in lung tissues from rats exposed to lunar dust particles. F344 rats were exposed for 4 weeks (6h/d; 5d/wk) in nose-only inhalation chambers to concentrations of 0 (control air), 2.1, 6.8, 21, and 61 mg/m(exp 3) of lunar dust. Five rats per group were euthanized 1 day, and 3 months after the last inhalation exposure. The total RNAs were isolated from lung tissues after being lavaged. The Agilent Rat GE v3 microarray was used to profile global gene expression (44K). The genes with significant expression changes are identified and the gene expression data were further analyzed using various statistical tools.

  10. Cytokine expression in mice exposed to diesel exhaust particles by inhalation. Role of tumor necrosis factor

    Directory of Open Access Journals (Sweden)

    Loft Steffen

    2006-02-01

    Full Text Available Abstract Background Particulate air pollution has been associated with lung and cardiovascular disease, for which lung inflammation may be a driving mechanism. The pro-inflammatory cytokine, tumor necrosis factor (TNF has been suggested to have a key-role in particle-induced inflammation. We studied the time course of gene expression of inflammatory markers in the lungs of wild type mice and Tnf-/- mice after exposure to diesel exhaust particles (DEPs. Mice were exposed to either a single or multiple doses of DEP by inhalation. We measured the mRNA level of the cytokines Tnf and interleukin-6 (Il-6 and the chemokines, monocyte chemoattractant protein (Mcp-1, macrophage inflammatory protein-2 (Mip-2 and keratinocyte derived chemokine (Kc in the lung tissue at different time points after exposure. Results Tnf mRNA expression levels increased late after DEP-inhalation, whereas the expression levels of Il-6, Mcp-1 and Kc increased early. The expression of Mip-2 was independent of TNF if the dose was above a certain level. The expression levels of the cytokines Kc, Mcp-1 and Il-6, were increased in the absence of TNF. Conclusion Our data demonstrate that Tnf is not important in early DEP induced inflammation and rather exerts negative influence on Mcp-1 and Kc mRNA levels. This suggests that other signalling pathways are important, a candidate being one involving Mcp-1.

  11. Virus like particle based strategy to elicit HIV-protective antibodies to the alpha-helic regions of gp41.

    Science.gov (United States)

    Pastori, C; Tudor, D; Diomede, L; Drillet, A S; Jegerlehner, A; Röhn, T A; Bomsel, M; Lopalco, L

    Natural antibodies to gp41 inhibit HIV-1 replication through the recognition of two different regions, corresponding to the leucine zipper motif in the HR1 alpha-helix and to another motif within HR2 region, hosting 2F5 and 4E10 epitope. This study aimed at reproducing such protective responses through VLP vaccination. Six regions covering the alpha-helical regions of gp41 were conjugated to the surface of AP205 phage-based VLPs. Once administered in mice via systemic or mucosal route, these immunogens elicited high titers of gp41-specific IgG. Immunogenicity and HIV infectivity reduction were obtained either with HR2 regions or with peptides where aminoacid strings were added to either the C-terminus or N-terminus of core epitope in HR1 region. Antibody-dependent cell cytotoxicity (ADCC) activity was induced by one of the HR2 epitopes only. These results may have relevant implications for the development of new vaccinal approaches against HIV infection. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. A novel virus-like particle based vaccine platform displaying the placental malaria antigen VAR2CSA

    DEFF Research Database (Denmark)

    Thrane, Susan; Janitzek, Christoph M; Agerbæk, Mette Ø

    2015-01-01

    Placental malaria caused by Plasmodium falciparum is a major cause of mortality and severe morbidity. Clinical testing of a soluble protein-based vaccine containing the parasite ligand, VAR2CSA, has been initiated. VAR2CSA binds to the human receptor chondroitin sulphate A (CSA) and is responsible...... for sequestration of Plasmodium falciparum infected erythrocytes in the placenta. It is imperative that a vaccine against malaria in pregnancy, if administered to women before they become pregnant, can induce a strong and long lasting immune response. While most soluble protein-based vaccines have failed during...

  13. Monovalent Virus-Like Particle Vaccine Protects Guinea Pigs and Nonhuman Primates Against Infection with Multiple Marburg Viruses

    Science.gov (United States)

    2008-05-01

    RIBI* iMARV + RIBI* RIBI* Musoke‡ Ravn‡ Ci67‡ Musoke‡ Ravn‡ Ci67‡ Musoke‡ Ravn‡ Ci67‡ Brain Fibrin thrombi - - - - - - - - ++ Liver Hepatitis, mixed...Fibrin thrombi - - - - - - - - ++/++ Mesenteric lymph node Hemorrhage - - - - - - - - + Lymphoid depletion/lymphocytolysis...Fibrin thrombi - - - - - - - ++/++ - Peyer’s patches Lymphoid depletion/lymphocytolysis

  14. Inactivation of a Human Norovirus Surrogate, Human Norovirus Virus-Like Particles, and Vesicular Stomatitis Virus by Gamma Irradiation ▿

    OpenAIRE

    Feng, Kurtis; Divers, Erin; Ma, Yuanmei; Li, Jianrong

    2011-01-01

    Gamma irradiation is a nonthermal processing technology that has been used for the preservation of a variety of food products. This technology has been shown to effectively inactivate bacterial pathogens. Currently, the FDA has approved doses of up to 4.0 kGy to control food-borne pathogens in fresh iceberg lettuce and spinach. However, whether this dose range effectively inactivates food-borne viruses is less understood. We have performed a systematic study on the inactivation of a human nor...

  15. Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

    OpenAIRE

    Hoenen, Thomas; Watt, Ari; Mora, Anita; Feldmann, Heinz

    2014-01-01

    Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been lim...

  16. Monovalent Virus-Like Particle Vaccine Protects Guinea Pigs and Nonhuman Primates Against Infection with Multiple Marburg Viruses

    National Research Council Canada - National Science Library

    Swenson, Dana L; Warfield, Kelly L; Larsen, Tom; Alves, D. A; Coberley, Sadie S; Bavari, Sina

    2008-01-01

    .... Guinea pigs vaccinated with marburgvirus (m)VLPs or inactivated MARV (iMARV) develop homologous humoral and T cell responses and are completely protected from a lethal homologous MARV challenge...

  17. Microscopic derivation of particle-based coarse-grained dynamics: Exact expression for memory function

    Science.gov (United States)

    Izvekov, Sergei

    2017-03-01

    We consider the generalized Langevin equations of motion describing exactly the particle-based coarse-grained dynamics in the classical microscopic ensemble that were derived recently within the Mori-Zwanzig formalism based on new projection operators [S. Izvekov, J. Chem. Phys. 138(13), 134106 (2013)]. The fundamental difference between the new family of projection operators and the standard Zwanzig projection operator used in the past to derive the coarse-grained equations of motion is that the new operators average out the explicit irrelevant trajectories leading to the possibility of solving the projected dynamics exactly. We clarify the definition of the projection operators and revisit the formalism to compute the projected dynamics exactly for the microscopic system in equilibrium. The resulting expression for the projected force is in the form of a "generalized additive fluctuating force" describing the departure of the generalized microscopic force associated with the coarse-grained coordinate from its projection. Starting with this key expression, we formulate a new exact formula for the memory function in terms of microscopic and coarse-grained conservative forces. We conclude by studying two independent limiting cases of practical importance: the Markov limit (vanishing correlations of projected force) and the limit of weak dependence of the memory function on the particle momenta. We present computationally affordable expressions which can be efficiently evaluated from standard molecular dynamics simulations.

  18. Particle Radiation signals the Expression of Genes in stress-associated Pathways

    Science.gov (United States)

    Blakely, E.; Chang, P.; Bjornstad, K.; Dosanjh, M.; Cherbonnel, C.; Rosen, C.

    The explosive development of microarray screening methods has propelled genome research in a variety of biological systems allowing investigators to examine large-scale alterations in gene expression for research in toxicology pathology and therapy The radiation environment in space is complex and encompasses a variety of highly energetic and charged particles Estimation of biological responses after exposure to these types of radiation is important for NASA in their plans for long-term manned space missions Instead of using the 10 000 gene arrays that are in the marketplace we have chosen to examine particle radiation-induced changes in gene expression using a focused DNA microarray system to study the expression of about 100 genes specifically associated with both the upstream and downstream aspects of the TP53 stress-responsive pathway Genes that are regulated by TP53 include functional clusters that are implicated in cell cycle arrest apoptosis and DNA repair A cultured human lens epithelial cell model Blakely et al IOVS 41 3808 2000 was used for these studies Additional human normal and radiosensitive fibroblast cell lines have also been examined Lens cells were grown on matrix-coated substrate and exposed to 55 MeV u protons at the 88 cyclotron in LBNL or 1 GeV u Iron ions at the NASA Space Radiation Laboratory The other cells lines were grown on conventional tissue culture plasticware RNA and proteins were harvested at different times after irradiation RNA was isolated from sham-treated or select irradiated populations

  19. Silencing Bcl-2 Expression in Epithelial Cancer Cells Using “Smart” Particles

    Directory of Open Access Journals (Sweden)

    Yen-Ling Lin

    2014-09-01

    Full Text Available Short interfering RNA (siRNA targeted against anti-apoptotic Bcl-2 protein proved to knockdown its expression and trigger cancer cell death. We used degradable, pH-sensitive, comb-like [P(EAA-co-BMA-b-PNASI-g-P(HMA-co-TMAEMA] polymer to condense anti-Bcl-2 siRNA into “smart” particles, which proved to shuttle their cargo past the endosomal membrane and into the cytoplasm of HeLa and UM-SCC-17B cancer cells. HeLa and UM-SCC-17B cancer cells were treated with anti-Bcl-2 particles followed by quantifying Bcl-2 mRNA and protein levels using qRT-PCR and western blotting, respectively. “Smart” anti-Bcl-2 particles selectively suppress Bcl-2 mRNA and protein levels in HeLa cells by 50%–60% and 79%–81%, respectively. Similarly, “smart” anti-Bcl-2 particles inhibited Bcl-2 mRNA levels by 30%, 40%, and 20% upon incubation with UM-SCC-17B cancer cells for 48, 72, and 96 h, respectively. Bcl-2 protein expression in UM-SCC-17B cancer cells was inhibited by 30% after treatment for 72 h. Results show that pH-sensitive comb-like polymer complex anti-Bcl-2 siRNA forming “smart” nanoparticles that deliver their cargo into the cytoplasm of HeLa and UM-SCC-17B cancer cells causing Bcl-2 knockdown at the mRNA and protein levels.

  20. Changes in gene expression in the rat hippocampus following exposure to 56 fe particles and protection by berry diets

    Science.gov (United States)

    Exposing young rats to particles of high energy and charge (HZE particles), such as 56Fe, enhances indices of oxidative stress and inflammation and disrupts behavior, including spatial learning and memory. In the present study, we examined whether gene expression in the hippocampus, an area of the b...

  1. Gene Expression Profiling of Lung Tissue of Rats Exposed to Lunar Dust Particles

    Science.gov (United States)

    Zhang, Ye; Feiveson, Alan H.; Lam, Chiu-Wing; Kidane, Yared H.; Ploutz-Snyder Robert; Yeshitla, Samrawit; Zalesak, Selina M.; Scully, Robert R.; Wu, Honglu; James, John T.

    2014-01-01

    The purpose of the study is to analyze the dynamics of global gene expression changes in the lung tissue of rats exposed to lunar dust particles. Multiple pathways and transcription factors were identified using the Ingenuity Pathway Analysis tool, showing the potential networks of these signaling regulations involved in lunar dust-induced prolonged proflammatory response and toxicity. The data presented in this study, for the first time, explores the molecular mechanisms of lunar dust induced toxicity. This work contributes not only to the risk assessment for future space exploration, but also to the understanding of the dust-induced toxicity to humans on earth.

  2. Down-Regulation of Small Rubber Particle Protein Expression Affects Integrity of Rubber Particles and Rubber Content in Taraxacum brevicorniculatum

    Science.gov (United States)

    Hillebrand, Andrea; Post, Janina J.; Wurbs, David; Wahler, Daniela; Lenders, Malte; Krzyzanek, Vladislav; Prüfer, Dirk; Gronover, Christian Schulze

    2012-01-01

    The biosynthesis of rubber is thought to take place on the surface of rubber particles in laticifers, highly specialized cells that are present in more than 40 plant families. The small rubber particle protein (SRPP) has been supposed to be involved in rubber biosynthesis, and recently five SRPPs (TbSRPP1–5) were identified in the rubber-producing dandelion species Taraxacum brevicorniculatum. Here, we demonstrate by immunogold labeling that TbSRPPs are localized to rubber particles, and that rubber particles mainly consist of TbSRPP3, 4 and 5 as shown by high-resolution two-dimensional gel electrophoresis and mass spectrometric analysis. We also carried out an RNA-interference approach in transgenic plants to address the function of TbSRPPs in rubber biosynthesis as well as rubber particle morphology and stability. TbSRPP-RNAi transgenic T. brevicorniculatum plants showed a 40–50% reduction in the dry rubber content, but neither the rubber weight average molecular mass nor the polydispersity of the rubber were affected. Although no phenotypical differences to wild-type particles could be observed in vivo, rubber particles from the TbSRPP-RNAi transgenic lines were less stable and tend to rapidly aggregate in expelling latex after wounding of laticifers. Our results prove that TbSRPPs are very crucial for rubber production in T. brevicorniculatum, probably by contributing to a most favourable and stable rubber particle architecture for efficient rubber biosynthesis and eventually storage. PMID:22911861

  3. Down-regulation of small rubber particle protein expression affects integrity of rubber particles and rubber content in Taraxacum brevicorniculatum.

    Directory of Open Access Journals (Sweden)

    Andrea Hillebrand

    Full Text Available The biosynthesis of rubber is thought to take place on the surface of rubber particles in laticifers, highly specialized cells that are present in more than 40 plant families. The small rubber particle protein (SRPP has been supposed to be involved in rubber biosynthesis, and recently five SRPPs (TbSRPP1-5 were identified in the rubber-producing dandelion species Taraxacum brevicorniculatum. Here, we demonstrate by immunogold labeling that TbSRPPs are localized to rubber particles, and that rubber particles mainly consist of TbSRPP3, 4 and 5 as shown by high-resolution two-dimensional gel electrophoresis and mass spectrometric analysis. We also carried out an RNA-interference approach in transgenic plants to address the function of TbSRPPs in rubber biosynthesis as well as rubber particle morphology and stability. TbSRPP-RNAi transgenic T. brevicorniculatum plants showed a 40-50% reduction in the dry rubber content, but neither the rubber weight average molecular mass nor the polydispersity of the rubber were affected. Although no phenotypical differences to wild-type particles could be observed in vivo, rubber particles from the TbSRPP-RNAi transgenic lines were less stable and tend to rapidly aggregate in expelling latex after wounding of laticifers. Our results prove that TbSRPPs are very crucial for rubber production in T. brevicorniculatum, probably by contributing to a most favourable and stable rubber particle architecture for efficient rubber biosynthesis and eventually storage.

  4. Alphavirus replicon particles expressing TRP-2 provide potent therapeutic effect on melanoma through activation of humoral and cellular immunity.

    Directory of Open Access Journals (Sweden)

    Francesca Avogadri

    2010-09-01

    Full Text Available Malignant melanoma is the deadliest form of skin cancer and is refractory to conventional chemotherapy and radiotherapy. Therefore alternative approaches to treat this disease, such as immunotherapy, are needed. Melanoma vaccine design has mainly focused on targeting CD8+ T cells. Activation of effector CD8+ T cells has been achieved in patients, but provided limited clinical benefit, due to immune-escape mechanisms established by advanced tumors. We have previously shown that alphavirus-based virus-like replicon particles (VRP simultaneously activate strong cellular and humoral immunity against the weakly immunogenic melanoma differentiation antigen (MDA tyrosinase. Here we further investigate the antitumor effect and the immune mechanisms of VRP encoding different MDAs.VRP encoding different MDAs were screened for their ability to prevent the growth of the B16 mouse transplantable melanoma. The immunologic mechanisms of efficacy were investigated for the most effective vaccine identified, focusing on CD8+ T cells and humoral responses. To this end, ex vivo immune assays and transgenic mice lacking specific immune effector functions were used. The studies identified a potent therapeutic VRP vaccine, encoding tyrosinase related protein 2 (TRP-2, which provided a durable anti-tumor effect. The efficacy of VRP-TRP2 relies on a novel immune mechanism of action requiring the activation of both IgG and CD8+ T cell effector responses, and depends on signaling through activating Fcγ receptors.This study identifies a VRP-based vaccine able to elicit humoral immunity against TRP-2, which plays a role in melanoma immunotherapy and synergizes with tumor-specific CD8+ T cell responses. These findings will aid in the rational design of future immunotherapy clinical trials.

  5. Alphavirus replicon particles expressing TRP-2 provide potent therapeutic effect on melanoma through activation of humoral and cellular immunity.

    Science.gov (United States)

    Avogadri, Francesca; Merghoub, Taha; Maughan, Maureen F; Hirschhorn-Cymerman, Daniel; Morris, John; Ritter, Erika; Olmsted, Robert; Houghton, Alan N; Wolchok, Jedd D

    2010-09-10

    Malignant melanoma is the deadliest form of skin cancer and is refractory to conventional chemotherapy and radiotherapy. Therefore alternative approaches to treat this disease, such as immunotherapy, are needed. Melanoma vaccine design has mainly focused on targeting CD8+ T cells. Activation of effector CD8+ T cells has been achieved in patients, but provided limited clinical benefit, due to immune-escape mechanisms established by advanced tumors. We have previously shown that alphavirus-based virus-like replicon particles (VRP) simultaneously activate strong cellular and humoral immunity against the weakly immunogenic melanoma differentiation antigen (MDA) tyrosinase. Here we further investigate the antitumor effect and the immune mechanisms of VRP encoding different MDAs. VRP encoding different MDAs were screened for their ability to prevent the growth of the B16 mouse transplantable melanoma. The immunologic mechanisms of efficacy were investigated for the most effective vaccine identified, focusing on CD8+ T cells and humoral responses. To this end, ex vivo immune assays and transgenic mice lacking specific immune effector functions were used. The studies identified a potent therapeutic VRP vaccine, encoding tyrosinase related protein 2 (TRP-2), which provided a durable anti-tumor effect. The efficacy of VRP-TRP2 relies on a novel immune mechanism of action requiring the activation of both IgG and CD8+ T cell effector responses, and depends on signaling through activating Fcγ receptors. This study identifies a VRP-based vaccine able to elicit humoral immunity against TRP-2, which plays a role in melanoma immunotherapy and synergizes with tumor-specific CD8+ T cell responses. These findings will aid in the rational design of future immunotherapy clinical trials.

  6. Apolipoprotein E expression and behavioral toxicity of high charge, high energy (HZE) particle radiation

    Science.gov (United States)

    Higuchi, Yoshinori; Nelson, Gregory A.; Vazquez, Marcelo; Laskowitz, Daniel T.; Slater, James M.; Pearlstein, Robert D.

    2002-01-01

    Apolipoprotein E (apoE) is a lipid binding protein that plays an important role in tissue repair following brain injury. In the present studies, we have investigated whether apoE affects the behavioral toxicity of high charge, high energy (HZE) particle radiation. METHODS: Sixteen male apoE knockout (KO) mice and sixteen genetically matched wild-type (WT) C57BL mice were used in this experiment. Half of the KO and half of the WT animals were irradiated with 600 MeV/amu iron particles (2 Gy whole body). The effect of irradiation on motor coordination and stamina (Rotarod test), exploratory behavior (open field test), and spatial working and reference memory (Morris water maze) was assessed. ROTAROD TEST: Performance was adversely affected by radiation exposure in both KO and WT groups at 30 d after irradiation. By 60 d after radiation, the radiation effect was lost in WT, but still apparent in irradiated KO mice. OPEN FIELD TEST: Radiation reduced open field exploratory activity 14, 28, 56, 84, and 168 d after irradiation of KO mice, but had no effect on WT mice. MORRIS WATER MAZE: Radiation adversely affected spatial working memory in the KO mice, but had no discernible effect in the WT mice as assessed 180 d after irradiation. In contrast, irradiated WT mice showed marked impairment of spatial reference memory in comparison to non-irradiated mice, while no effect of radiation was observed in KO mice. CONCLUSIONS: These studies show that apoE expression influences the behavioral toxicity of HZE particle radiation and suggest that apoE plays a role in the repair/recovery from radiation injury of the CNS. ApoE deficiency may exacerbate the previously reported effects of HZE particle radiation in accelerating the brain aging process.

  7. Global Gene Expression Profiling in Lung Tissues of Rat Exposed to Lunar Dust Particles

    Science.gov (United States)

    Yeshitla, Samrawit A.; Lam, Chiu-Wing; Kidane, Yared H.; Feiveson, Alan H.; Ploutz-Snyder, Robert; Wu, Honglu; James, John T.; Meyers, Valerie E.; Zhang, Ye

    2014-01-01

    The Moon's surface is covered by a layer of fine, potential reactive dust. Lunar dust contain about 1-2% respirable very fine dust (less than 3 micrometers). The habitable area of any lunar landing vehicle and outpost would inevitably be contaminated with lunar dust that could pose a health risk. The purpose of the study is to analyze the dynamics of global gene expression changes in lung tissues of rats exposed to lunar dust particles. F344 rats were exposed for 4 weeks (6h/d; 5d/wk) in nose-only inhalation chambers to concentrations of 0 (control air), 2.1, 6.8, 21, and 61 mg/m3 of lunar dust. Animals were euthanized at 1 day and 13 weeks after the last inhalation exposure. After being lavaged, lung tissue from each animal was collected and total RNA was isolated. Four samples of each dose group were analyzed using Agilent Rat GE v3 microarray to profile global gene expression of 44K transcripts. After background subtraction, normalization, and log transformation, t tests were used to compare the mean expression levels of each exposed group to the control group. Correction for multiple testing was made using the method of Benjamini, Krieger, and Yekuteli (1) to control the false discovery rate. Genes with significant changes of at least 1.75 fold were identified as genes of interest. Both low and high doses of lunar dust caused dramatic, dose-dependent global gene expression changes in the lung tissues. However, the responses of lung tissue to low dose lunar dust are distinguished from those of high doses, especially those associated with 61mg/m3 dust exposure. The data were further integrated into the Ingenuity system to analyze the gene ontology (GO), pathway distribution and putative upstream regulators and gene targets. Multiple pathways, functions, and upstream regulators have been identified in response to lunar dust induced damage in the lung tissue.

  8. Distinct Particle Morphologies Revealed through Comparative Parallel Analyses of Retrovirus-Like Particles.

    Science.gov (United States)

    Martin, Jessica L; Cao, Sheng; Maldonado, Jose O; Zhang, Wei; Mansky, Louis M

    2016-09-15

    The Gag protein is the main retroviral structural protein, and its expression alone is usually sufficient for production of virus-like particles (VLPs). In this study, we sought to investigate-in parallel comparative analyses-Gag cellular distribution, VLP size, and basic morphological features using Gag expression constructs (Gag or Gag-YFP, where YFP is yellow fluorescent protein) created from all representative retroviral genera: Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lentivirus, and Spumavirus. We analyzed Gag cellular distribution by confocal microscopy, VLP budding by thin-section transmission electron microscopy (TEM), and general morphological features of the VLPs by cryogenic transmission electron microscopy (cryo-TEM). Punctate Gag was observed near the plasma membrane for all Gag constructs tested except for the representative Beta- and Epsilonretrovirus Gag proteins. This is the first report of Epsilonretrovirus Gag localizing to the nucleus of HeLa cells. While VLPs were not produced by the representative Beta- and Epsilonretrovirus Gag proteins, the other Gag proteins produced VLPs as confirmed by TEM, and morphological differences were observed by cryo-TEM. In particular, we observed Deltaretrovirus-like particles with flat regions of electron density that did not follow viral membrane curvature, Lentivirus-like particles with a narrow range and consistent electron density, suggesting a tightly packed Gag lattice, and Spumavirus-like particles with large envelope protein spikes and no visible electron density associated with a Gag lattice. Taken together, these parallel comparative analyses demonstrate for the first time the distinct morphological features that exist among retrovirus-like particles. Investigation of these differences will provide greater insights into the retroviral assembly pathway. Comparative analysis among retroviruses has been critically important in enhancing our understanding of

  9. Protein transfer-mediated surface engineering to adjuvantate virus-like nanoparticles for enhanced anti-viral immune responses.

    Science.gov (United States)

    Patel, Jaina M; Kim, Min-Chul; Vartabedian, Vincent F; Lee, Yu-Na; He, Sara; Song, Jae-Min; Choi, Hyo-Jick; Yamanaka, Satoshi; Amaram, Nikhil; Lukacher, Anna; Montemagno, Carlo D; Compans, Richard W; Kang, Sang-Moo; Selvaraj, Periasamy

    2015-07-01

    Recombinant virus-like nanoparticles (VLPs) are a promising nanoparticle platform to develop safe vaccines for many viruses. Herein, we describe a novel and rapid protein transfer process to enhance the potency of enveloped VLPs by decorating influenza VLPs with exogenously added glycosylphosphatidylinositol-anchored immunostimulatory molecules (GPI-ISMs). With protein transfer, the level of GPI-ISM incorporation onto VLPs is controllable by varying incubation time and concentration of GPI-ISMs added. ISM incorporation was dependent upon the presence of a GPI-anchor and incorporated proteins were stable and functional for at least 4weeks when stored at 4°C. Vaccinating mice with GPI-granulocyte macrophage colony-stimulating factor (GM-CSF)-incorporated-VLPs induced stronger antibody responses and better protection against a heterologous influenza virus challenge than unmodified VLPs. Thus, VLPs can be enriched with ISMs by protein transfer to increase the potency and breadth of the immune response, which has implications in developing effective nanoparticle-based vaccines against a broad spectrum of enveloped viruses. The inherent problem with current influenza vaccines is that they do not generate effective cross-protection against heterologous viral strains. In this article, the authors described the development of virus-like nanoparticles (VLPs) as influenza vaccines with enhanced efficacy for cross-protection, due to an easy protein transfer modification process. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Successful expression in pollen of various plant species of in vitro synthesized mRNA introduced by particle bombardment.

    Science.gov (United States)

    Tanaka, T; Nishihara, M; Seki, M; Sakamoto, A; Tanaka, K; Irifune, K; Morikawa, H

    1995-05-01

    Gold particles coated with beta-glucuronidase (GUS) mRNA with a 5' cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.

  11. Regulation of human hepatic drug transporter activity and expression by diesel exhaust particle extract.

    Directory of Open Access Journals (Sweden)

    Marc Le Vee

    Full Text Available Diesel exhaust particles (DEPs are common environmental air pollutants primarily affecting the lung. DEPs or chemicals adsorbed on DEPs also exert extra-pulmonary effects, including alteration of hepatic drug detoxifying enzyme expression. The present study was designed to determine whether organic DEP extract (DEPe may target hepatic drug transporters that contribute in a major way to drug detoxification. Using primary human hepatocytes and transporter-overexpressing cells, DEPe was first shown to strongly inhibit activities of the sinusoidal solute carrier (SLC uptake transporters organic anion-transporting polypeptides (OATP 1B1, 1B3 and 2B1 and of the canalicular ATP-binding cassette (ABC efflux pump multidrug resistance-associated protein 2, with IC50 values ranging from approximately 1 to 20 μg/mL and relevant to environmental exposure situations. By contrast, 25 μg/mL DEPe failed to alter activities of the SLC transporter organic cation transporter (OCT 1 and of the ABC efflux pumps P-glycoprotein and bile salt export pump (BSEP, whereas it only moderately inhibited those of sodium taurocholate co-transporting polypeptide and of breast cancer resistance protein (BCRP. Treatment by 25 μg/mL DEPe was next demonstrated to induce expression of BCRP at both mRNA and protein level in cultured human hepatic cells, whereas it concomitantly repressed mRNA expression of various transporters, including OATP1B3, OATP2B1, OCT1 and BSEP. Such changes in transporter expression were found to be highly correlated to those caused by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, a reference activator of the aryl hydrocarbon receptor (AhR pathway. This suggests that DEPe, which is enriched in known ligands of AhR like polycyclic aromatic hydrocarbons, alters drug transporter expression via activation of the AhR cascade. Taken together, these data established human hepatic transporters as targets of organic chemicals containing in DEPs, which may contribute

  12. Packaging HIV- or FIV-based lentivector expression constructs and transduction of VSV-G pseudotyped viral particles.

    Science.gov (United States)

    Mendenhall, Amy; Lesnik, Jacob; Mukherjee, Chandreyee; Antes, Travis; Sengupta, Ranjita

    2012-04-08

    As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction. There are three main components necessary for efficient lentiviral packaging. 1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin

  13. Recent exposure to ultrafine particles in school children alters miR-222 expression in the extracellular fraction of saliva.

    Science.gov (United States)

    Vriens, Annette; Nawrot, Tim S; Saenen, Nelly D; Provost, Eline B; Kicinski, Michal; Lefebvre, Wouter; Vanpoucke, Charlotte; Van Deun, Jan; De Wever, Olivier; Vrijens, Karen; De Boever, Patrick; Plusquin, Michelle

    2016-07-26

    Ultrafine particles (children. This study examined whether exposure to fine and ultrafine particulate matter (PM) is associated with changes in the extracellular content of miR-222 and miR-146a of children. Saliva was collected from 80 children at two different time points, circa 11 weeks apart and stabilized for RNA preservation. The extracellular fraction of saliva was obtained by means of differential centrifugation and ultracentrifugation. Expression levels of miR-222 and miR-146a were profiled by qPCR. We regressed the extracellular miRNA expression against recent exposure to ultrafine and fine particles measured at the school site using mixed models, while accounting for sex, age, BMI, passive smoking, maternal education, hours of television use, time of the day and day of the week. Exposure to ultrafine particles (UFP) at the school site was positively associated with miR-222 expression in the extracellular fraction in saliva. For each IQR increase in particles in the class room (+8504 particles/cm(3)) or playground (+28776 particles/cm(3)), miR-222 was, respectively 23.5 % (95 % CI: 3.5 %-41.1 %; p = 0.021) or 29.9 % (95 % CI:10.6 %-49.1 %; p = 0.0027) higher. No associations were found between miR-146a and recent exposure to fine and ultrafine particles. Our results suggest a possible epigenetic mechanism via which cells respond rapidly to small particles, as exemplified by miR-222 changes in the extracellular fraction of saliva.

  14. A mouse mammary tumor virus-like long terminal repeat superantigen in human breast cancer.

    Science.gov (United States)

    Wang, Yue; Jiang, Jian-Dong; Xu, Dongping; Li, Yan; Qu, Chunfeng; Holland, James F; Pogo, Beatriz G-T

    2004-06-15

    We previously reported a 660-bp mouse mammary tumor virus (MMTV)-like env gene sequence in approximately 38% of human breast cancer DNA, but not in normal breasts or other tumors. This MMTV-like env gene sequence was expressed in 66% of the env gene-positive human breast cancers. An entire proviral structure was identified in human breast cancer DNA with high homology to MMTV and low homology to known human endogenous retrovirus. MMTV-like long terminal repeat (LTR) sequences were also detected in 41.5% of human breast cancers. They contain hormone-responsive elements, TEF-1 family elements, and the open reading frame for the superantigen (SAg). We have now amplified and sequenced MMTV-like sag sequences from 10 human breast cancers, and we found that they are highly homologous to those of MMTV. However, deletions and insertions at the COOH-terminal of sag were observed. The immune function of the human MMTV-like LTR SAg was also investigated. The sag gene was cloned and expressed in a human B-cell line (Ramos). T-cell proliferation and cytokine releasing assays were performed after cocultivation of T cells with irradiated Ramos SAg-expressing cells. The results indicate that expression of the human SAg stimulates T-cell activation in vitro, as the mouse SAg does. Because the T-cell responses in vitro are considered similar to those in vivo, these results suggest that the human LTR SAg might also play a role in human breast carcinogenesis.

  15. Production of Hybrid Chimeric PVX Particles Using a Combination of TMV and PVX-Based Expression Vectors.

    Science.gov (United States)

    Dickmeis, Christina; Honickel, Mareike Michaela Antonia; Fischer, Rainer; Commandeur, Ulrich

    2015-01-01

    We have generated hybrid chimeric potato virus X (PVX) particles by coexpression of different PVX coat protein fusions utilizing tobacco mosaic virus (TMV) and PVX-based expression vectors. Coinfection was achieved with a modified PVX overcoat vector displaying a fluorescent protein and a TMV vector expressing another PVX fluorescent overcoat fusion protein. Coexpression of the PVX-CP fusions in the same cells was confirmed by epifluorescence microscopy. Labeling with specific antibodies and transmission electron microscopy revealed chimeric particles displaying green fluorescent protein and mCherry on the surface. These data were corroborated by bimolecular fluorescence complementation. We used split-mCherry fragments as PVX coat fusions and confirmed an interaction between the split-mCherry fragments in coinfected cells. The presence of assembled split-mCherry on the surface confirmed the hybrid character of the chimeric particles.

  16. INCREASED IL-8 AND IL-6 EXPRESSION IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES

    Science.gov (United States)

    INCREASED IL-6 AND IL-8 EXPRESSION IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO CARBON ULTRAFINE PARTICLES.R Silbajoris1, A G Lenz2, I Jaspers3, J M Samet1. 1NHEERL, USEPA, RTP, NC, USA; 2GSF-Institute for Inhalation Biology, Neuherberg, Germany; 3 CEMLB, UNC-CH, Chapel Hill, ...

  17. Replication, gene expression and particle production by a consensus Merkel Cell Polyomavirus (MCPyV genome.

    Directory of Open Access Journals (Sweden)

    Friederike Neumann

    Full Text Available Merkel Cell Polyomavirus (MCPyV genomes are clonally integrated in tumor tissues of approximately 85% of all Merkel cell carcinoma (MCC cases, a highly aggressive tumor of the skin which predominantly afflicts elderly and immunosuppressed patients. All integrated viral genomes recovered from MCC tissue or MCC cell lines harbor signature mutations in the early gene transcript encoding for the large T-Antigen (LT-Ag. These mutations selectively abrogate the ability of LT-Ag to support viral replication while still maintaining its Rb-binding activity, suggesting a continuous requirement for LT-Ag mediated cell cycle deregulation during MCC pathogenesis. To gain a better understanding of MCPyV biology, in vitro MCPyV replication systems are required. We have generated a synthetic MCPyV genomic clone (MCVSyn based on the consensus sequence of MCC-derived sequences deposited in the NCBI database. Here, we demonstrate that transfection of recircularized MCVSyn DNA into some human cell lines recapitulates efficient replication of the viral genome, early and late gene expression together with virus particle formation. However, serial transmission of infectious virus was not observed. This in vitro culturing system allows the study of viral replication and will facilitate the molecular dissection of important aspects of the MCPyV lifecycle.

  18. Coronavirus-like particles in laboratory rabbits with different syndromes in The Netherlands (Coronavirus-like particles in rabbits).

    NARCIS (Netherlands)

    A.D.M.E. Osterhaus (Albert); J.S. Teppema; G. van Steenis (Bert)

    1982-01-01

    textabstractVirus-like particles were identified from the plasma of rabbits which developed pleural effusion disease after inoculation with different strains of Treponema pallidum. These particles were considered coronavirus-like on the basis of their size, morphology, and buoyant density. Clinical

  19. A Comparitive Assessement of Cytokine Expression in Human-Derived Cell Lines Exposed to Alpha Particles and X-Rays

    Directory of Open Access Journals (Sweden)

    Vinita Chauhan

    2012-01-01

    Full Text Available Alpha- (α- particle radiation exposure has been linked to the development of lung cancer and has been identified as a radiation type likely to be employed in radiological dispersal devices. Currently, there exists a knowledge gap concerning cytokine modulations associated with exposure to α-particles. Bio-plex technology was employed to investigate changes in proinflammatory cytokines in two human-derived cell lines. Cells were irradiated at a dose of 1.5 Gy to either α-particles or X-rays at equivalent dose rates. The two cell lines exhibited a unique pattern of cytokine expression and the response varied with radiation type. Of the 27 cytokines assessed, only vascular endothelin growth factor (VEGF was observed to be modulated in both cell lines solely after α-particle exposure, and the expression of VEGF was shown to be dose responsive. These results suggest that certain proinflammatory cytokines may be involved in the biological effects related to α- particle exposure and the responses are cell type and radiation type specific.

  20. Cell viability, collagen synthesis and cytokine expression in human osteoblasts following incubation with generated wear particles using different bone cements.

    Science.gov (United States)

    Schulze, Christoph; Lochner, Katrin; Jonitz, Anika; Lenz, Robert; Duettmann, Oliver; Hansmann, Doris; Bader, Rainer

    2013-07-01

    In total hip arthroplasty, wear particles generated at articulating surfaces and interfaces between bone, cement and implants have a negative impact on osteoblasts, leading to osteolysis and implant loosening. The aim of this experimental study was to determine the effects of particulate wear debris generated at the interface between straight stainless steel hip stems (Exeter(®)) and three different bone cements (Palacos(®) R, Simplex™ P and Cemex(®) Genta) on cell viability, collagen synthesis and cytokine expression in human osteoblasts. Primary osteoblasts were treated with various concentrations of wear particles. The synthesis of procollagen type I and different cytokines was analysed, and markers for apoptosis and necrosis were also detected. The cytokine synthesis rates in the osteoblasts were initially increased and varied, depending on incubation time and particle concentration. Specific differences in the synthesis rates of interleukin (IL)‑6, IL-8, vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1) were observed with the different bone cements examined. The negative effect of the particles on the synthesis of procollagen type I and increased rates of cell apoptosis and necrosis were observed with all three cements analysed. Our present data suggest that wear particles from the interface between the total hip stem and bone cement have a significant effect on viability, cytokine expression and collagen synthesis in human osteoblasts, depending on the bone cement used.

  1. A novel method to produce Influenza A virus matrix protein M1 Capsid Like Particles (CLPs).

    Science.gov (United States)

    Baniasadi, Vahid; Lal, Sunil K

    2014-09-01

    Avian influenza viruses represent a growing threat for an influenza pandemic. The currently licensed influenza vaccines have inherent drawbacks which has led many research groups to explore different approaches of vaccine development among which Virus Like particles (VLPs) seem like a promising alternative in the near future. Although it is known that the Matrix 1 protein (M1) of influenza plays an essential role in VLP formation and it is documented that M1 is able to form dimers, it is not clear if M1 is capable of forming higher order structures without the interference of other influenza proteins or cell derived envelope. Here, for the first time we have demonstrated that expression of M1 alone is enough to form a Capsid Like Particle (CLP) without the requirement of any other external factor. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Induction of innate immunity in lungs with virus-like nanoparticles leads to protection against influenza and Streptococcus pneumoniae challenge.

    Science.gov (United States)

    Mathieu, Claudia; Rioux, Gervais; Dumas, Marie-Christine; Leclerc, Denis

    2013-10-01

    Nanoparticles composed of the coat protein of a plant virus (papaya mosaic virus; PapMV) and a single-stranded RNA (ssRNA) trigger a strong innate immune stimulation in the lungs of the animals a few hours following instillation. A rapid recruitment of neutrophils, monocytes/macrophages and lymphocytes follows. This treatment was able to provide protection to an influenza challenge that lasts at least 5 days. Protection could be recalled for longer periods by repeating the instillations once per week for more than 10 weeks. The treatment also conferred protection to a lethal challenge with Streptococcus pneumoniae--the major cause of bacterial pneumonia. Finally, we also showed that the nanoparticles could be used to treat mice infected with influenza and significantly decrease morbidity. These data strengthen the potential for using PapMV nanoparticles as non-specific inducers of the innate immune response in lungs during viral pandemics or to combat bioterrorist attack. In this study, virus-like nanoparticles were utilized to induce innate immune responses in a mouse model. They were also demonstrated to provide enhanced immune responses during actual pneumonia and ongoing viral infection. Strategies like this may become very helpful in human applications, including bioterrorism countermeasures. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Transcriptome Profiling of the Lungs Reveals Molecular Clock Genes Expression Changes after Chronic Exposure to Ambient Air Particles

    Directory of Open Access Journals (Sweden)

    Pengcheng Song

    2017-01-01

    Full Text Available The symptoms of asthma, breathlessness, insomnia, etc. all have relevance to pulmonary rhythmic disturbances. Epidemiology and toxicology studies have demonstrated that exposure to ambient air particles can result in pulmonary dysfunction. However, there are no data directly supporting a link between air pollution and circadian rhythm disorder. In the present study, we found that breathing highly polluted air resulted in changes of the molecular clock genes expression in lung by transcriptome profiling analyses in a rodent model. Compared to those exposed to filtered air, in both pregnant and offspring rats in the unfiltered group, key clock genes (Per1, Per2, Per3, Rev-erbα and Dbp expression level decreased and Bmal1 expression level increased. In both rat dams and their offspring, after continuous exposure to unfiltered air, we observed significant histologic evidence for both perivascular and peribronchial inflammation, increased tissue and systemic oxidative stress in the lungs. Our results suggest that chronic exposure to particulate matter can induce alterations of clock genes expression, which could be another important pathway for explaining the feedbacks of ambient particle exposure in addition to oxidative stress and inflammation.

  4. Identification of Retroviral Late Domains as Determinants of Particle Size

    OpenAIRE

    Garnier, Laurence; Parent, Leslie J.; Rovinski, Benjamin; Cao, Shi-Xian; Wills, John W.

    1999-01-01

    Retroviral Gag proteins, in the absence of any other viral products, induce budding and release of spherical, virus-like particles from the plasma membrane. Gag-produced particles, like those of authentic retrovirions, are not uniform in diameter but nevertheless fall within a fairly narrow distribution of sizes. For the human immunodeficiency virus type 1 (HIV-1) Gag protein, we recently reported that elements important for controlling particle size are contained within the C-terminal region...

  5. Lipid-soluble cigarette smoking particles induce expression of inflammatory and extracellular-matrix-related genes in rat cerebral arteries

    DEFF Research Database (Denmark)

    Vikman, Petter; Xu, Cang-Bao; Edvinsson, Lars

    2009-01-01

    AIMS: Cigarette smoking is one of the strongest risk factors for stroke. However, the underlying molecular mechanisms that smoke leads to the pathogenesis of stroke are incompletely understood. METHODS: Dimethyl sulfoxide (DMSO)-soluble (lipid-soluble) cigarette smoking particles (DSP) were...... extracted from cigarette smoke (0.8 mg nicotine per cigarette; Marlboro). Rat cerebral arteries were isolated and organ cultured in the presence of DSP (0.2 microl/ml, equivalent to the plasma level in smokers) for 24 h. The expression of matrix metalloproteinase 9 and 13 (MMP9 and MMP13), angiotensin...

  6. Reconstruction of putative DNA virus from endogenous rice tungro bacilliform virus-like sequences in the rice genome: implications for integration and evolution

    Directory of Open Access Journals (Sweden)

    Kishima Yuji

    2004-10-01

    Full Text Available Abstract Background Plant genomes contain various kinds of repetitive sequences such as transposable elements, microsatellites, tandem repeats and virus-like sequences. Most of them, with the exception of virus-like sequences, do not allow us to trace their origins nor to follow the process of their integration into the host genome. Recent discoveries of virus-like sequences in plant genomes led us to set the objective of elucidating the origin of the repetitive sequences. Endogenous rice tungro bacilliform virus (RTBV-like sequences (ERTBVs have been found throughout the rice genome. Here, we reconstructed putative virus structures from RTBV-like sequences in the rice genome and characterized to understand evolutionary implication, integration manner and involvements of endogenous virus segments in the corresponding disease response. Results We have collected ERTBVs from the rice genomes. They contain rearranged structures and no intact ORFs. The identified ERTBV segments were shown to be phylogenetically divided into three clusters. For each phylogenetic cluster, we were able to make a consensus alignment for a circular virus-like structure carrying two complete ORFs. Comparisons of DNA and amino acid sequences suggested the closely relationship between ERTBV and RTBV. The Oryza AA-genome species vary in the ERTBV copy number. The species carrying low-copy-number of ERTBV segments have been reported to be extremely susceptible to RTBV. The DNA methylation state of the ERTBV sequences was correlated with their copy number in the genome. Conclusions These ERTBV segments are unlikely to have functional potential as a virus. However, these sequences facilitate to establish putative virus that provided information underlying virus integration and evolutionary relationship with existing virus. Comparison of ERTBV among the Oryza AA-genome species allowed us to speculate a possible role of endogenous virus segments against its related disease.

  7. Pulmonary exposure to diesel exhaust particles induces airway inflammation and cytokine expression in NC/Nga mice

    Energy Technology Data Exchange (ETDEWEB)

    Inoue, Ken-ichiro; Yanagisawa, Rie [National Institute for Environmental Studies, Inhalation Toxicology and Pathophysiology Research Team, Tsukuba (Japan); Takano, Hirohisa [National Institute for Environmental Studies, Inhalation Toxicology and Pathophysiology Research Team, Tsukuba (Japan); Kyoto Prefectural University of Medicine, Inflammation and Immunology, Graduate School of Medical Science, Kyoto (Japan); Ichinose, Takamichi [Oita University of Nursing and Health Science, Department of Health Science, Oita (Japan); Shimada, Akinori [Tottori University, Department of Veterinary Pathology, Faculty of Agriculture, Tottori (Japan); Yoshikawa, Toshikazu [Kyoto Prefectural University of Medicine, Inflammation and Immunology, Graduate School of Medical Science, Kyoto (Japan)

    2005-10-01

    Although several studies have reported that diesel exhaust particles (DEP) affect cardiorespiratory health in animals and humans, the effect of DEP on animal models with spontaneous allergic disorders has been far less intensively studied. The Nc/Nga mouse is known to be a typical animal model for human atopic dermatitis (AD). In the present study, we investigated the effects of repeated pulmonary exposure to DEP on airway inflammation and cytokine expression in NC/Nga mice. The animals were randomized into two experimental groups that received vehicle or DEP by intratracheal instillation weekly for six weeks. Cellular profiles of bronchoalveolar lavage (BAL) fluid and expressions of cytokines and chemokines in both the BAL fluid and lung tissues were evaluated 24 h after the last instillation. The DEP challenge produced an increase in the numbers of total cells, neutrophils, and mononuclear cells in BAL fluid as compared to the vehicle challenge (P<0.01). DEP exposure significantly induced the lung expressions of interleukin (IL)-4, keratinocyte chemoattractant (KC), and macrophage inflammatory protein (MIP)-1{alpha} when compared to the vehicle challenge. These results indicate that intratracheal exposure to DEP induces the recruitment of inflammatory cells, at least partially, through the local expression of IL-4 and chemokines in NC/Nga mice. (orig.)

  8. Analytical expressions for noise and crosstalk voltages of the High Energy Silicon Particle Detector

    Science.gov (United States)

    Yadav, I.; Shrimali, H.; Liberali, V.; Andreazza, A.

    2018-01-01

    The paper presents design and implementation of a silicon particle detector array with the derived closed form equations of signal-to-noise ratio (SNR) and crosstalk voltages. The noise analysis demonstrates the effect of interpixel capacitances (IPC) between center pixel (where particle hits) and its neighbouring pixels, resulting as a capacitive crosstalk. The pixel array has been designed and simulated in a 180 nm BCD technology of STMicroelectronics. The technology uses the supply voltage (VDD) of 1.8 V and the substrate potential of ‑50 V. The area of unit pixel is 250×50 μm2 with the substrate resistivity of 125 Ωcm and the depletion depth of 30 μm. The mathematical model includes the effects of various types of noise viz. the shot noise, flicker noise, thermal noise and the capacitive crosstalk. This work compares the results of noise and crosstalk analysis from the proposed mathematical model with the circuit simulation results for a given simulation environment. The results show excellent agreement with the circuit simulations and the mathematical model. The average relative error (AVR) generated for the noise spectral densities with respect to the simulations and the model is 12% whereas the comparison gives the errors of 3% and 11.5% for the crosstalk voltages and the SNR results respectively.

  9. Regulation of early signaling and gene expression in the α-particle and bystander response of IMR-90 human fibroblasts

    Directory of Open Access Journals (Sweden)

    Hei Tom K

    2010-07-01

    Full Text Available Abstract Background The existence of a radiation bystander effect, in which non-irradiated cells respond to signals from irradiated cells, is well established. To understand early signaling and gene regulation in bystander cells, we used a bio-informatics approach, measuring global gene expression at 30 minutes and signaling pathways between 30 minutes and 4 hours after exposure to α-particles in IMR-90 fibroblasts. Methods We used whole human genome microarrays and real time quantitative PCR to measure and validate gene expression. Microarray analysis was done using BRB-Array Tools; pathway and ontology analyses were done using Ingenuity Pathway Analysis and PANTHER, respectively. We studied signaling in irradiated and bystander cells using immunoblotting and semi-quantitative image analysis. Results Gene ontology suggested signal transduction and transcriptional regulation responding 30 minutes after treatment affected cell structure, motility and adhesion, and interleukin synthesis. We measured time-dependent expression of genes controlled by the NF-κB pathway; matrix metalloproteinases 1 and 3; chemokine ligands 2, 3 and 5 and interleukins 1β, 6 and 33. There was an increased response of this set of genes 30 minutes after treatment and another wave of induction at 4 hours. We investigated AKT-GSK3β signaling and found both AKT and GSK3β are hyper-phosphorylated 30 minutes after irradiation and this effect is maintained through 4 hours. In bystander cells, a similar response was seen with a delay of 30 minutes. We proposed a network model where the observed decrease in phosphorylation of β-catenin protein after GSK3β dependent inactivation can trigger target gene expression at later times after radiation exposure Conclusions These results are the first to show that the radiation induced bystander signal induces a widespread gene expression response at 30 minutes after treatment and these changes are accompanied by modification of

  10. Synthesis and assembly of infectious bovine papillomavirus particles in vitro.

    Science.gov (United States)

    Zhou, J; Stenzel, D J; Sun, X Y; Frazer, I H

    1993-04-01

    Bovine papillomavirus type 1 (BPV-1) virions were produced in vitro using vaccinia virus (VV) recombinants expressing the BPV-1 L1 and L2 capsid proteins. Particles morphologically resembling papillomaviruses were observed in the nucleus of cells infected with a VV recombinant for the BPV-1 L1 protein, and greater numbers of similar particles were seen in the nuclei of cells infected with a VV double recombinant for L1 and L2. Virus-like particles (VLPs) assembled in cells infected with the VV double recombinant for BPV-1 L1 and L2, and not those assembled in cells infected with the VV recombinant for BPV-1 L1 alone, were able to package BPV-1 DNA. Transcription of the BPV-1 E1 viral open reading frame was observed after a mouse fibroblast cell line was exposed to VLPs produced using a BPV-1 L1/L2 VV recombinant in a cell line containing episomal BPV-1 DNA. E1 transcription was not observed when the VLPs were pre-incubated with antibodies to the capsid protein of BPV-1. This system should allow an in vitro approach to the definition of the BPV-1 cellular receptor.

  11. Histamine treatment induces rearrangements of orthogonal arrays of particles (OAPs) in human AQP4-expressing gastric cells

    Science.gov (United States)

    Carmosino, Monica; Procino, Giuseppe; Nicchia, Grazia Paola; Mannucci, Roberta; Verbavatz, Jean-Marc; Gobin, Renèe; Svelto, Maria; Valenti, Giovanna

    2001-01-01

    To test the involvement of the water channel aquaporin (AQP)-4 in gastric acid physiology, the human gastric cell line (HGT)-1 was stably transfected with rat AQP4. AQP4 was immunolocalized to the basolateral membrane of transfected HGT-1 cells, like in native parietal cells. Expression of AQP4 in transfected cells increased the osmotic water permeability coefficient (Pf) from 2.02 ± 0.3 × 10−4 to 16.37 ± 0.5 × 10−4 cm/s at 20°C. Freeze-fracture EM showed distinct orthogonal arrays of particles (OAPs), the morphological signature of AQP4, on the plasma membrane of AQP4-expressing cells. Quantitative morphometry showed that the density of OAPs was 2.5 ± 0.3% under basal condition and decreased by 50% to 1.2 ± 0.3% after 20 min of histamine stimulation, mainly due to a significant decrease of the OAPs number. Concomitantly, Pf decreased by ∼35% in 20-min histamine-stimulated cells. Both Pf and OAPs density were not modified after 10 min of histamine exposure, time at which the maximal hormonal response is observed. Cell surface biotinylation experiments confirmed that AQP4 is internalized after 20 min of histamine exposure, which may account for the downregulation of water transport. This is the first evidence for short term rearrangement of OAPs in an established AQP4-expressing cell line. PMID:11564760

  12. Lipid-soluble cigarette smoking particles induce expression of inflammatory and extracellular-matrix-related genes in rat cerebral arteries

    DEFF Research Database (Denmark)

    Vikman, Petter; Xu, Cang-Bao; Edvinsson, Lars

    2009-01-01

    AIMS: Cigarette smoking is one of the strongest risk factors for stroke. However, the underlying molecular mechanisms that smoke leads to the pathogenesis of stroke are incompletely understood. METHODS: Dimethyl sulfoxide (DMSO)-soluble (lipid-soluble) cigarette smoking particles (DSP) were...... receptor 1 and 2 (AT(1) and AT(2)), interleukin 6 and inducible nitric oxide synthase (iNOS) were investigated at mRNA level by real-time PCR and/or at protein level by immunohistochemistry. In addition, the activity of three mitogen-activated protein kinases (p38, ERK 1/2 and SAPK....../JNK) and their downstream transcription factors (ATF-2, Elk-1 and c-Jun) were examined. RESULTS: We observed that compared with control (DMSO-treated cerebral arteries), the cerebral arteries treated by DSP exhibited enhanced expression of MMP13 and AT(1) receptors, but not of AT(2) receptors, at both mRNA and protein...

  13. Global gene expression changes in human embryonic lung fibroblasts induced by organic extracts from respirable air particles

    Directory of Open Access Journals (Sweden)

    Líbalová Helena

    2012-01-01

    Full Text Available Abstract Background Recently, we used cell-free assays to demonstrate the toxic effects of complex mixtures of organic extracts from urban air particles (PM2.5 collected in four localities of the Czech Republic (Ostrava-Bartovice, Ostrava-Poruba, Karvina and Trebon which differed in the extent and sources of air pollution. To obtain further insight into the biological mechanisms of action of the extractable organic matter (EOM from ambient air particles, human embryonic lung fibroblasts (HEL12469 were treated with the same four EOMs to assess changes in the genome-wide expression profiles compared to DMSO treated controls. Method For this purpose, HEL cells were incubated with subtoxic EOM concentrations of 10, 30, and 60 μg EOM/ml for 24 hours and global gene expression changes were analyzed using human whole genome microarrays (Illumina. The expression of selected genes was verified by quantitative real-time PCR. Results Dose-dependent increases in the number of significantly deregulated transcripts as well as dose-response relationships in the levels of individual transcripts were observed. The transcriptomic data did not differ substantially between the localities, suggesting that the air pollution originating mainly from various sources may have similar biological effects. This was further confirmed by the analysis of deregulated pathways and by identification of the most contributing gene modulations. The number of significantly deregulated KEGG pathways, as identified by Goeman's global test, varied, depending on the locality, between 12 to 29. The Metabolism of xenobiotics by cytochrome P450 exhibited the strongest upregulation in all 4 localities and CYP1B1 had a major contribution to the upregulation of this pathway. Other important deregulated pathways in all 4 localities were ABC transporters (involved in the translocation of exogenous and endogenous metabolites across membranes and DNA repair, the Wnt and TGF-β signaling pathways

  14. Virus-like particles comprising H5, H7 and H9 hemagglutinins elicit protective immunity to heterologous avian influenza viruses in chickens

    Science.gov (United States)

    Avian influenza (AI) viruses circulating in wild birds pose a serious threat to public health. Human and veterinary vaccines against AI subtypes are needed. Here we prepared triple-subtype VLPs that co-localized H5, H7 and H9 antigens derived from H5N1, H7N3 and H9N2 viruses. VLPs also contained inf...

  15. Immunization of early adolescent females with human papillomavirus type 16 and 18 L1 virus-like particle vaccine containing AS04 adjuvant

    DEFF Research Database (Denmark)

    Pedersen, Court; Petaja, Tiina; Strauss, Gitte

    2007-01-01

    PURPOSE: In female individuals 15-25-years of age, the AS04-containing human papillomavirus (HPV)-16/18 vaccine is highly immunogenic and provides up to 100% protection against HPV-16/18 persistent infection and associated cervical lesions up to 4.5 years. Optimal cervical cancer prevention...... will require prophylactic vaccination against oncogenic HPV 16 and 18 before the onset of sexual activity in early adolescent girls. To establish the feasibility of vaccination in girls 10-14 years of age, we compared the immunogenicity and safety in early adolescent female individuals to those 15-25 years...... in whom vaccine efficacy has been demonstrated. METHODS: We enrolled 773 female participants aged 10-14 years and 15-25 years to receive the HPV-16/18 L1 VLP AS04 vaccine, which was administered at months 0, 1, and 6. Serum samples were collected at months 0 and 7; antibodies to HPV 16 and 18 VLPs were...

  16. Immunization of early adolescent females with human papillomavirus type 16 and 18 L1 virus-like particle vaccine containing AS04 adjuvant.

    NARCIS (Netherlands)

    Pedersen, C.; Petaja, T.; Strauss, G.; Rumke, H.C.; Poder, A.; Richardus, J.H.; Spiessens, B.; Descamps, D.; Hardt, K.; Lehtinen, M.; Dubin, G.

    2007-01-01

    PURPOSE: In female individuals 15-25-years of age, the AS04-containing human papillomavirus (HPV)-16/18 vaccine is highly immunogenic and provides up to 100% protection against HPV-16/18 persistent infection and associated cervical lesions up to 4.5 years. Optimal cervical cancer prevention will

  17. Reactivity to human papillomavirus type 16 Ll virus-like particles in sera from patients with genital cancer and patients with carcinomas at five different extragenital sites

    NARCIS (Netherlands)

    G.J.J. van Doornum (Gerard); C.M. Korse (Catharina); J.C.G.M. Buning-Kager (J. C G M); J.M. Bonfrer (Hans); S. Horenblas (Simon); B. Taal (Babs); J. Dillner (Joakim)

    2003-01-01

    textabstractA retrospective seroepidemiologic study was performed to examine the association between human papillomaviruses (HPV) 16 infection and carcinomas of the oropharynx, the oesophagus, penis and vagina. Sera were selected from the serum bank from the Antoni van Leeuwenhoek Hospital

  18. The impact of a quadrivalent human papillomavirus (types 6, 11, 16, 18) virus-like particle vaccine in European women aged 16 to 24

    DEFF Research Database (Denmark)

    Majewski, S; Bosch, F X; Dillner, J

    2009-01-01

    BACKGROUND: Quadrivalent human papillomavirus (HPV types 6/11/16/18) L1 VLP vaccine is highly effective in preventing HPV 6/11/16/18-related cervical and external genital disease. Herein, we evaluated the impact of the quadrivalent HPV 6/11/16/18 L1 VLP vaccine on prevention of HPV......-associated cervico-genital lesions in a broad population of sexually active European women. METHODS: Female subjects (N = 9265) aged 16-24 with four or fewer lifetime sexual partners were enrolled and randomized to quadrivalent HPV vaccine or placebo. Subjects underwent cervicovaginal sampling for HPV DNA detection....... Papanicolaou testing and anti-HPV 6/11/16/18 serology testing was also performed. RESULTS: Vaccine efficacy against lesions representing immediate cervical cancer precursors (cervical intraepithelial neoplasia grade 2/3 or adenocarcinoma in situ) related to HPV 6/11/16/18 in the per-protocol population was 100...

  19. Development of a Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS) Method for the Quantitation of Viral Envelope Glycoprotein in Ebola Virus-Like Particle Vaccine Preparations

    Science.gov (United States)

    2016-09-05

    the combined digest dried to completion by speedvac. 307 308 Animals , Vaccinations, and Viral challenge 309 Research was conducted under an...temperature. Since GP1,2 is a heavily 392 glycosylated membrane embedded protein , we performed PNGase deglycosylation prior to 393 digestion in the...excised, trypsin digested , and analyzed with long-gradient 469 CID survey runs as well as targeted LC-HRMS MS to identify any GP protein fragments 470

  20. Chitosan microparticles loaded with yeast-derived PCV2 virus-like particles elicit antigen-specific cellular immune response in mice after oral administration

    OpenAIRE

    Bucarey, Sergio A.; Pujol, Myriam; Poblete, Joaquín; Nuñez, Ignacio; Tapia, Cecilia V.; Neira-Carrillo, Andrónico; Martinez, Jonatán; Bassa, Oliver

    2014-01-01

    Background Porcine circovirus type 2 (PCV2)-associated diseases are a major problem for the swine industry worldwide. In addition to improved management and husbandry practices, the availability of several anti-PCV2 vaccines provides an efficient immunological option for reducing the impact of these diseases. Most anti-PCV2 vaccines are marketed as injectable formulations. Although these are effective, there are problems associated with the use of injectable products, including laborious and ...

  1. Chimeric virus-like particles containing influenza HA antigen and GPI-CCL28 induce long-lasting mucosal immunity against H3N2 viruses.

    Science.gov (United States)

    Mohan, Teena; Berman, Zachary; Luo, Yuan; Wang, Chao; Wang, Shelly; Compans, Richard W; Wang, Bao-Zhong

    2017-01-09

    Influenza virus is a significant cause of morbidity and mortality, with worldwide seasonal epidemics. The duration and quality of humoral immunity and generation of immunological memory to vaccines is critical for protective immunity. In the current study, we examined the long-lasting protective efficacy of chimeric VLPs (cVLPs) containing influenza HA and GPI-anchored CCL28 as antigen and mucosal adjuvant, respectively, when immunized intranasally in mice. We report that the cVLPs induced significantly higher and sustainable levels of virus-specific antibody responses, especially IgA levels and hemagglutination inhibition (HAI) titers, more than 8-month post-vaccination compared to influenza VLPs without CCL28 or influenza VLPs physically mixed with sCCL28 (soluble) in mice. After challenging the vaccinated animals at month 8 with H3N2 viruses, the cVLP group also demonstrated strong recall responses. On day 4 post-challenge, we measured increased antibody levels, ASCs and HAI titers with reduced viral load and inflammatory responses in the cVLP group. The animals vaccinated with the cVLP showed 20% cross-protection against drifted (Philippines) and 60% protection against homologous (Aichi) H3N2 viruses. Thus, the results suggest that the GPI-anchored CCL28 induces significantly higher mucosal antibody responses, involved in providing long-term cross-protection against H3N2 influenza virus when compared to other vaccination groups.

  2. Development and evaluation of a replicon particle vaccine expressing the E2 glycoprotein of bovine viral diarrhea virus (BVDV in cattle

    Directory of Open Access Journals (Sweden)

    Loy John Dustin

    2013-01-01

    Full Text Available Abstract Background Bovine viral diarrhea virus is one of the most significant and costly viral pathogens of cattle worldwide. Alphavirus-derived replicon particles have been shown to be safe and highly effective vaccine vectors against a variety of human and veterinary pathogens. Replicon particles are non-propagating, DIVA compatible, and can induce both humoral and cell mediated immune responses. This is the first experiment to demonstrate that Alphavirus-based replicon particles can be utilized in a standard prime/boost vaccination strategy in calves against a commercially significant bovine pathogen. Findings Replicon particles that express bovine viral diarrhea virus sub-genotype 1b E2 glycoprotein were generated and expression was confirmed in vitro using polyclonal and monoclonal antibodies specific to E2. Vaccine made from particles was generated in Vero cells and administered to BVDV free calves in a prime/boost regimen at two dosage levels. Vaccination resulted in neutralizing antibody titers that cross-neutralized both type 1 and type 2 BVD genotypes following booster vaccination. Additionally, high dose vaccine administration demonstrated some protection from clinical disease and significantly reduced the degree of leukopenia caused by viral infection. Conclusions Replicon particle vaccines administered in a prime/boost regimen expressing BVDV E2 glycoprotein can induce cross-neutralizing titers, reduce leukopenia post challenge, and mitigate clinical disease in calves. This strategy holds promise for a safe and effective vaccine to BVDV.

  3. Dense Array of Spikes on HIV-1 Virion Particles.

    Science.gov (United States)

    Stano, Armando; Leaman, Daniel P; Kim, Arthur S; Zhang, Lei; Autin, Ludovic; Ingale, Jidnyasa; Gift, Syna K; Truong, Jared; Wyatt, Richard T; Olson, Arthur J; Zwick, Michael B

    2017-07-15

    HIV-1 is rare among viruses for having a low number of envelope glycoprotein (Env) spikes per virion, i.e., ∼7 to 14. This exceptional feature has been associated with avoidance of humoral immunity, i.e., B cell activation and antibody neutralization. Virus-like particles (VLPs) with increased density of Env are being pursued for vaccine development; however, these typically require protein engineering that alters Env structure. Here, we used instead a strategy that targets the producer cell. We employed fluorescence-activated cell sorting (FACS) to sort for cells that are recognized by trimer cross-reactive broadly neutralizing antibody (bnAb) and not by nonneutralizing antibodies. Following multiple iterations of FACS, cells and progeny virions were shown to display higher levels of antigenically correct Env in a manner that correlated between cells and cognate virions (P = 0.027). High-Env VLPs, or hVLPs, were shown to be monodisperse and to display more than a 10-fold increase in spikes per particle by electron microscopy (average, 127 spikes; range, 90 to 214 spikes). Sequencing revealed a partial truncation in the C-terminal tail of Env that had emerged in the sort; however, iterative rounds of "cell factory" selection were required for the high-Env phenotype. hVLPs showed greater infectivity than standard pseudovirions but largely similar neutralization sensitivity. Importantly, hVLPs also showed superior activation of Env-specific B cells. Hence, high-Env HIV-1 virions, obtained through selection of producer cells, represent an adaptable platform for vaccine design and should aid in the study of native Env.IMPORTANCE The paucity of spikes on HIV is a unique feature that has been associated with evasion of the immune system, while increasing spike density has been a goal of vaccine design. Increasing the density of Env by modifying it in various ways has met with limited success. Here, we focused instead on the producer cell. Cells that stably express HIV

  4. Transient Bluetongue virus serotype 8 capsid protein expression in Nicotiana benthamiana

    Directory of Open Access Journals (Sweden)

    Albertha R. van Zyl

    2016-03-01

    Full Text Available Bluetongue virus (BTV causes severe disease in domestic and wild ruminants, and has recently caused several outbreaks in Europe. Current vaccines include live-attenuated and inactivated viruses; while these are effective, there is risk of reversion to virulence by mutation or reassortment with wild type viruses. Subunit or virus-like particle (VLP vaccines are safer options: VLP vaccines produced in insect cells by expression of the four BTV capsid proteins are protective against challenge; however, this is a costly production method. We investigated production of BTV VLPs in plants via Agrobacterium-mediated transient expression, an inexpensive production system very well suited to developing country use. Leaves infiltrated with recombinant pEAQ-HT vectors separately encoding the four BTV-8 capsid proteins produced more proteins than recombinant pTRA vectors. Plant expression using the pEAQ-HT vector resulted in both BTV-8 core-like particles (CLPs and VLPs; differentially controlling the concentration of infiltrated bacteria significantly influenced yield of the VLPs. In situ localisation of assembled particles was investigated by using transmission electron microscopy (TEM and it was shown that a mixed population of core-like particles (CLPs, consisting of VP3 and VP7 and VLPs were present as paracrystalline arrays in the cytoplasm of plant cells co-expressing all four capsid proteins.

  5. Structure and Immunogenicity of Alternative Forms of the Simian Immunodeficiency Virus Gag Protein Expressed Using Venezuelan Equine Encephalitis Virus Replicon Particles

    OpenAIRE

    Cecil, Chad; West, Ande; Collier, Martha; Jurgens, Christy; Madden, Victoria; Whitmore, Alan; Johnston, Robert; Moore, Dominic T.; Swanstrom, Ronald; Davis, Nancy L.

    2007-01-01

    Venezuelan equine encephalitis virus replicon particles (VRP) were engineered to express different forms of SIV Gag to compare expression in vitro, formation of intra- and extracellular structures and induction of humoral and cellular immunity in mice. The three forms examined were full-length myristylated SIV Gag (Gagmyr+), full-length Gag lacking the myristylation signal (Gagmyr-), or a truncated form of Gagmyr- comprising only the matrix and capsid domains (MA/CA). Comparison of VRP-infect...

  6. APOBEC3G ubiquitination by Nedd4-1 favors its packaging into HIV-1 particles.

    Science.gov (United States)

    Dussart, Sylvie; Douaisi, Marc; Courcoul, Marianne; Bessou, Gilles; Vigne, Robert; Decroly, Etienne

    2005-01-21

    APOBEC3G is a cytidine deaminase that limits the replication of many retroviruses. This antiviral host factor is packaged into retrovirus particles, where it targets single-stranded DNA generated during reverse transcription and induces up to 2% of G-to-A mutations, which are lethal for the HIV-1 provirus. Vif protein counteracts this antiviral factor by decreasing its packaging into lentivirus particles. Here, we demonstrate that Nedd4-1, an HECT E3 ubiquitin ligase, interacts with APOBEC3G, through its WW2 and WW3 domains. As a result of this interaction, APOBEC3G undergoes post-translational modification by addition of ubiquitin moieties. Accordingly, we demonstrate that the dominant negative Nedd4-1 C/S form prevents APOBEC3G ubiquitination. Moreover, the packaging of APOBEC3G into Pr55 Gag virus-like particles and into HIV-1 virions is reduced when Nedd4-1 C/S is expressed. During HIV-1 viral production in the presence of APOBEC3G, Nedd4-1 C/S restores partially the infectivity of Deltavif HIV-1. We conclude that the ubiquitination of APOBEC3G by Nedd4-1 favors its targeting to the virus assembly site where APOBEC3G interacts with Gag and is packaged into HIV-1 particles in the absence of Vif.

  7. Rotavirus-Like Particles: A Novel Nanocarrier for the Gut

    Directory of Open Access Journals (Sweden)

    Naima G. Cortes-Perez

    2010-01-01

    Full Text Available The delivery of bioactive molecules directly to damaged tissues represents a technological challenge. We propose here a new system based on virus-like particles (VLP from rotavirus, with a marked tropism for the gut to deliver bio-active molecules to intestinal cells. For this, nonreplicative VLP nanoparticles were constructed using a baculovirus expression system and used to deliver an exogenous biomolecule, the green fluorescent protein (GFP, into either MA104 cells or intestinal cells from healthy and 2,4,6-trinitrobenzene sulfonic acid (TNBS-treated mice. Our results show that expression of rotavirus capsid proteins in baculovirus led to the auto assembly of VLP that display similar properties to rotavirus. In vitro experiments showed that VLP were able to enter into MA104 cells and deliver the reporter protein. Intragastric administration of fluorescent VLP in healthy and TNBS-treated mice resulted in the detection of GFP and viral proteins in intestinal samples. Our results demonstrate an efficient entry of non-replicative rotavirus VLP into the epithelial cell line MA104 and provide the first in vivo evidence of the potential of these nanoparticles as a promising safe candidate for drug delivery to intestinal cells.

  8. An interferon-alpha-induced tethering mechanism inhibits HIV-1 and Ebola virus particle release but is counteracted by the HIV-1 Vpu protein.

    Science.gov (United States)

    Neil, Stuart J D; Sandrin, Virginie; Sundquist, Wesley I; Bieniasz, Paul D

    2007-09-13

    Type 1 interferon (IFN) inhibits the release of HIV-1 virus particles via poorly defined mechanisms. Here, we show that IFNalpha induces retention of viral particles on the surface of fibroblasts, T cells, or primary lymphocytes infected with HIV-1 lacking the Vpu protein. Retained particles are tethered to cell surfaces, can be endocytosed, appear fully assembled, exhibit mature morphology, and can be detached by protease. Strikingly, expression of the HIV-1 Vpu protein attenuates the ability of human cells to adhere to, and thereby retain, nascent HIV-1 particles upon IFNalpha treatment. Vpu also counteracts the IFNalpha-induced retention of virus-like particles assembled from the Ebola virus matrix protein. Furthermore, levels of IFNalpha that suppress replication of Vpu-defective HIV-1 have little effect on wild-type HIV-1. Thus, we propose that HIV-1 expresses Vpu to counteract an IFNalpha-induced, general host defense that inhibits dissemination of enveloped virions from the surface of infected cells.

  9. The induction of CXCR4 expression in human osteoblast-like cells (MG63) by CoCr particles is regulated by the PLC-DAG-PKC pathway.

    Science.gov (United States)

    Drynda, Andreas; Ren, Qiang; Buchhorn, Gottfried H; Lohmann, Christoph H

    2017-11-01

    Osteolysis which leads to aseptic loosening of implants is a fundamental problem in joint replacement surgery (arthroplasty) and the leading cause for implant failure and revision surgery. Metal (CoCr) particles separated from implants by wear cause osteolysis and the failure of orthopedic implants, but the molecular mechanism is not clear. The chemokine receptor CXCR4 has been shown to play a pivotal role in periprosthetic osteolysis. The aim of this study was to determine which signal transduction pathway (PLC-DAG-PKC or MAPK/ERK) induces CXCR4 expression in osteoblast-like cells (MG63) cells. MG63 and Jurkat cells were stimulated with different amounts of particles (10 7 , 10 6 , and 10 5 ) for different time periods (30 min to 24 h), in the presence and absence of specific inhibitors (chelerythrine for the PLC-DAG-PKC pathway and PD98059 for the MAPK/ERK pathway). The expression of CXCR4-specific mRNA was determined by real-time polymerase chain reaction (PCR), and the PKC activity was measured by Western Blot using an antibody specific for PKC-related phosphorylation. Real-time PCR data showed that CXCR4 mRNA expression in MG63 cells induced by CoCr particles was significantly diminished by the PKC-specific inhibitor chelerythrine. This effect was not observed with the MAPK/ERK inhibitor PD98059. The involvement of PKC was also confirmed by an intensified phosphorylation pattern after stimulation with CoCr particles. In Jurkat cells, none of the inhibitors exhibited any effect. The induction of CXCR4-specific mRNA expression in MG63 cells after stimulation with CoCr particles is regulated by the PLC-DAG-PKC pathway and not by the MAPK/ERK pathway. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2326-2332, 2017. © 2016 Wiley Periodicals, Inc.

  10. Changes of gene expression in developing mouse brain after exposures to x-rays, in comparison with exposures to accelerated heavy ion particles

    Energy Technology Data Exchange (ETDEWEB)

    Yaoi, Takeshi; Fushiki, Shinji [Kyoto Prefectural Univ. of Medicine, Dept. of Pathology and Applied Neurobiology, Kyoto (Japan); Nojima, Kumie [National Institute of Radiological Sciences, International Space Radiation Lab., Anagawa, Chiba (Japan)

    2003-07-01

    Prenatal exposure to ionizing radiation of low doses in rodents impedes neuronal migration during the period of cortical histogenesis, and results in disorganized cortical architecture in mature brain. On the contrary, exposure to heavy ion beams during fetal period mainly affects cell survival, viz., induction of apoptosis. However, the molecular mechanisms underlying to produce such difference in the effects between exposure to heavy particles and exposure to X-rays remain unknown. We have attempted to elucidate whether the changes of gene expression after exposure to heavy ions differ from those after X-irradiation in fetal brains. We thus applied two molecular biological techniques, i.e., the Restriction Landmark cDNA Scanning (RLCS) method and the suppression subtractive PCR method. Approximately 13,000 cDNA species were scanned and it turned out that more than twenty genes among the genes scanned were differentially expressed between X-irradiated embryos and non-irradiated ones. One of the genes showing up-regulation is Rab6A that is known to be associated with vesicle transport from trans-Golgi network. In addition, expression of some genes encoding RAB6A-interacting proteins was up-regulated. When expression of these genes was compared between animals after heavy-ion irradiation and those after X-irradiation, the changing pattern was different. Taking our previous observation that prenatal exposure to carbon particles induces apoptotic cell death in developing cerebral cortex into consideration, the difference in gene expression herein reported may contribute to better understand the difference in effects between exposures to heavy-ion particles and to X-rays. In conclusion, we identified Rab6A and its interacting proteins as candidates for the migration-associated genes, whose expression in fetal brain is up-regulated by carbon beam irradiation. (author)

  11. Low-dose gamma-rays and simulated solar particle event protons modify splenocyte gene and cytokine expression patterns.

    Science.gov (United States)

    Rizvi, Asma; Pecaut, Michael J; Gridley, Daila S

    2011-01-01

    The goal was to investigate the T helper (Th) response in splenocytes of mice exposed to low-dose/low-dose-rate (LDR) γ-rays, simulated solar particle event protons (sSPE), or combination of both. C57BL/6 mice were exposed to LDR γ-radiation ((57)Co) to a total dose of 0.05 Gray (Gy) at 0.024 cGy/h, either with or without subsequent exposure to 2 Gy sSPE protons. Expression of genes related to Th cells was evaluated immediately after exposure (day 0). On day 21, intra- and extracellular cytokine production was assessed after activation with anti-CD3 monoclonal antibodies (mAb) or phorbol 12-myristate 13-acetate/ionophore (PMA/I). Five genes were significantly modulated on day 0 in one or more of the irradiated groups compared to controls (p < 0.05): Ccl11, Ccr5, Cd80, Inha, and Il9. On day 21, numbers of cells positive for interferon-γ were high in the LDR + sSPE group versus 0 Gy and LDR γ-rays (p < 0.05), but there was no difference in IL-2 and TNF-α. Levels of secreted cytokines after anti-CD3 mAb activation were high for 5 (MIP-1α, GM-CSF, IFN-γ, TNF-α, IL-13) and low for 2 (IL-7, IL-9) in all irradiated groups. Priming with LDR photons had a significant effect on IFN-γ and IL-17 compared to sSPE protons alone; IL-2 was low only in the LDR + sSPE group. The cytokine patterns after anti-PMA/I activation were different compared to anti-CD3 mAb and with fewer differences among groups. The data show that total-body exposure to space-relevant radiation has profound effects on Th cell status and that priming with LDR γ-rays can in some cases modulate the response to sSPE.

  12. One-prime multi-boost strategy immunization with recombinant DNA, adenovirus, and MVA vector vaccines expressing HPV16 L1 induces potent, sustained, and specific immune response in mice.

    Science.gov (United States)

    Li, Li-Li; Wang, He-Rong; Zhou, Zhi-Yi; Luo, Jing; Xiao, Xiang-Qian; Wang, Xiao-Li; Li, Jin-Tao; Zhou, Yu-Bai; Zeng, Yi

    2016-04-01

    Human papillomavirus (HPV) is associated with various human diseases, including cancer, and developing vaccines is a cost-efficient strategy to prevent HPV-related disease. The major capsid protein L1, which an increasing number of studies have confirmed is typically expressed early in infection, is a promising antigen for such a vaccine, although the E6 and E7 proteins have been characterized more extensively. Thus, the L1 gene from HPV16 was inserted into a recombinant vector, AdHu5, and MVA viral vectors, and administered by prime-boost immunization. Virus-like particles were used as control antigens. Our results indicate that prime-boost immunization with heterologous vaccines induced robust and sustained cellular and humoral response specific to HPV16 L1. In particular, sera obtained from mice immunized with DNA + DNA + Ad + MVA had excellent antitumor activity in vivo. However, the data also confirm that virus-like particles can only elicit low levels cellular immunity and not be long-lasting, and are therefore unsuitable for treatment of existing HPV infections. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Multigene expression of protein complexes by iterative modification of genomic Bacmid DNA

    Directory of Open Access Journals (Sweden)

    Celma Cristina C

    2009-09-01

    Full Text Available Abstract Background Many cellular multi-protein complexes are naturally present in cells at low abundance. Baculovirus expression offers one approach to produce milligram quantities of correctly folded and processed eukaryotic protein complexes. However, current strategies suffer from the need to produce large transfer vectors, and the use of repeated promoter sequences in baculovirus, which itself produces proteins that promote homologous recombination. One possible solution to these problems is to construct baculovirus genomes that express each protein in a complex from a separate locus within the viral DNA. However current methods for selecting such recombinant genomes are too inefficient to routinely modify the virus in this way. Results This paper reports a method which combines the lambda red and bacteriophage P1 Cre-recombinase systems to efficiently generate baculoviruses in which protein complexes are expressed from multiple, single-locus insertions of foreign genes. This method is based on an 88 fold improvement in the selection of recombinant viruses generated by red recombination techniques through use of a bipartite selection cassette. Using this system, seven new genetic loci were identified in the AcMNPV genome suitable for the high level expression of recombinant proteins. These loci were used to allow the recovery two recombinant virus-like particles with potential biotechnological applications (influenza A virus HA/M1 particles and bluetongue virus VP2/VP3/VP5/VP7 particles and the mammalian chaperone and cancer drug target CCT (16 subunits formed from 8 proteins. Conclusion 1. Use of bipartite selections can significantly improve selection of modified bacterial artificial chromosomes carrying baculovirus DNA. Furthermore this approach is sufficiently robust to allow routine modification of the virus genome. 2. In addition to the commonly used p10 and polyhedrin loci, the ctx, egt, 39k, orf51, gp37, iap2 and odv-e56 loci in Ac

  14. Molecular and process design for rotavirus-like particle production in Saccharomyces cerevisiae.

    Science.gov (United States)

    Rodríguez-Limas, William A; Tyo, Keith E J; Nielsen, Jens; Ramírez, Octavio T; Palomares, Laura A

    2011-05-14

    Virus-like particles (VLP) have an increasing range of applications including vaccination, drug delivery, diagnostics, gene therapy and nanotechnology. These developments require large quantities of particles that need to be obtained in efficient and economic processes. Production of VLP in yeast is attractive, as it is a low-cost protein producer able to assemble viral structural proteins into VLP. However, to date only single-layered VLP with simple architecture have been produced in this system. In this work, the first steps required for the production of rotavirus-like particles (RLP) in S. cerevisiae were implemented and improved, in order to obtain the recombinant protein concentrations required for VLP assembly. The genes of the rotavirus structural proteins VP2, VP6 and VP7 were cloned in four Saccharomyces cerevisiae strains using different plasmid and promoter combinations to express one or three proteins in the same cell. Performance of the best constructs was evaluated in batch and fed-batch cultures using a complete synthetic media supplemented with leucine, glutamate and succinate. The strain used had an important effect on recombinant protein concentration, while the type of plasmid, centromeric (YCp) or episomal (YEp), did not affect protein yields. Fed-batch culture of the PD.U-267 strain resulted in the highest concentration of rotavirus proteins. Volumetric and specific productivities increased 28.5- and 11-fold, respectively, in comparison with batch cultures. Expression of the three rotavirus proteins was confirmed by immunoblotting and RLP were detected using transmission electron microscopy. We present for the first time the use of yeast as a platform to express multilayered rotavirus-like particles. The present study shows that the combined use of molecular and bioprocess tools allowed the production of triple-layered rotavirus RLP. Production of VLP with complex architecture in yeasts could lead to the development of new vaccine candidates

  15. Exploiting plant virus-derived components to achieve in planta expression and for templates for synthetic biology applications.

    Science.gov (United States)

    Saunders, Keith; Lomonossoff, George P

    2013-10-01

    This review discusses the varying roles that have been played by many plant-viral regulatory sequences and proteins in the creation of plant-based expression systems and virus particles for use in nanotechnology. Essentially, there are two ways of expressing an exogenous protein: the creation of transgenic plants possessing a stably integrated gene construction, or the transient expression of the desired gene following the infiltration of the gene construct. Both depend on disarmed strains of Agrobacterium tumefaciens to deliver the created gene construction into cell nuclei, usually through the deployment of virus-derived components. The importance of efficient mRNA translation in the latter process is highlighted. Plant viruses replicate to sustain an infection to promote their survival. The major product of this, the virus particle, is finding increasing roles in the emerging field of bionanotechnology. One of the major products of plant-viral expression is the virus-like particle (VLP). These are increasingly playing a role in vaccine development. Similarly, many VLPs are suitable for the investigation of the many facets of the emerging field of synthetic biology, which encompasses the design and construction of new biological functions and systems not found in nature. Genetic and chemical modifications to plant-generated VLPs serve as ideal starter templates for many downstream synthetic biology applications. © 2013 JIC. New Phytologist © 2013 New Phytologist Trust.

  16. A Simple Add-and-Display Method for Immobilisation of Cancer Drug on His-tagged Virus-like Nanoparticles for Controlled Drug Delivery.

    Science.gov (United States)

    Biabanikhankahdani, Roya; Bayat, Saadi; Ho, Kok Lian; Alitheen, Noorjahan Banu Mohamed; Tan, Wen Siang

    2017-07-13

    pH-responsive virus-like nanoparticles (VLNPs) hold promising potential as drug delivery systems for cancer therapy. In the present study, hepatitis B virus (HBV) VLNPs harbouring His-tags were used to display doxorubicin (DOX) via nitrilotriacetic acid (NTA) conjugation. The His-tags served as pH-responsive nanojoints which released DOX from VLNPs in a controlled manner. The His-tagged VLNPs conjugated non-covalently with NTA-DOX, and cross-linked with folic acid (FA) were able to specifically target and deliver the DOX into ovarian cancer cells via folate receptor (FR)-mediated endocytosis. The cytotoxicity and cellular uptake results revealed that the His-tagged VLNPs significantly increased the accumulation of DOX in the ovarian cancer cells and enhanced the uptake of DOX, which improved anti-tumour effects. This study demonstrated that NTA-DOX can be easily displayed on His-tagged VLNPs by a simple Add-and-Display step with high coupling efficiency and the drug was only released at low pH in a controlled manner. This approach facilitates specific attachment of any drug molecule on His-tagged VLNPs at the very mild conditions without changing the biological structure and native conformation of the VLNPs.

  17. Deep sequencing reveals the complete genome and evidence for transcriptional activity of the first virus-like sequences identified in Aristotelia chilensis (Maqui Berry).

    Science.gov (United States)

    Villacreses, Javier; Rojas-Herrera, Marcelo; Sánchez, Carolina; Hewstone, Nicole; Undurraga, Soledad F; Alzate, Juan F; Manque, Patricio; Maracaja-Coutinho, Vinicius; Polanco, Victor

    2015-04-03

    Here, we report the genome sequence and evidence for transcriptional activity of a virus-like element in the native Chilean berry tree Aristotelia chilensis. We propose to name the endogenous sequence as Aristotelia chilensis Virus 1 (AcV1). High-throughput sequencing of the genome of this tree uncovered an endogenous viral element, with a size of 7122 bp, corresponding to the complete genome of AcV1. Its sequence contains three open reading frames (ORFs): ORFs 1 and 2 shares 66%-73% amino acid similarity with members of the Caulimoviridae virus family, especially the Petunia vein clearing virus (PVCV), Petuvirus genus. ORF1 encodes a movement protein (MP); ORF2 a Reverse Transcriptase (RT) and a Ribonuclease H (RNase H) domain; and ORF3 showed no amino acid sequence similarity with any other known virus proteins. Analogous to other known endogenous pararetrovirus sequences (EPRVs), AcV1 is integrated in the genome of Maqui Berry and showed low viral transcriptional activity, which was detected by deep sequencing technology (DNA and RNA-seq). Phylogenetic analysis of AcV1 and other pararetroviruses revealed a closer resemblance with Petuvirus. Overall, our data suggests that AcV1 could be a new member of Caulimoviridae family, genus Petuvirus, and the first evidence of this kind of virus in a fruit plant.

  18. Plant Virus Particles Carrying Tumour Antigen Activate TLR7 and Induce High Levels of Protective Antibody

    OpenAIRE

    Jantipa Jobsri; Alex Allen; Deepa Rajagopal; Michael Shipton; Kostya Kanyuka; Lomonossoff, George P.; Christian Ottensmeier; Diebold, Sandra S.; Stevenson, Freda K.; Natalia Savelyeva

    2015-01-01

    Induction of potent antibody is the goal of many vaccines targeted against infections or cancer. Modern vaccine designs that use virus-like particles (VLP) have shown efficacy for prophylactic vaccination against virus-associated cancer in the clinic. Here we used plant viral particles (PVP), which are structurally analogous to VLP, coupled to a weak idiotypic (Id) tumour antigen, as a conjugate vaccine to induce antibody against a murine B-cell malignancy. The Id-PVP vaccine incorporates a n...

  19. Differential gene expression in human fibroblasts after alpha-particle emitter (211)At compared with (60)Co irradiation

    DEFF Research Database (Denmark)

    Danielsson, Anna; Claesson, Kristina; Parris, Toshima Z

    2013-01-01

    trastuzumab monoclonal antibody (0.25, 0.5, and 1 Gy) and (60)Co (1, 2, and 3 Gy). Results: We report gene expression profiles that distinguish the effect different radiation qualities and absorbed doses have on cellular functions in human fibroblasts. In addition, we identified commonly expressed transcripts...

  20. Simulating biodegradation under mixing-limited conditions using Michaelis-Menten (Monod) kinetic expressions in a particle tracking model

    Science.gov (United States)

    Ding, Dong; Benson, David A.

    2015-02-01

    Recent studies have demonstrated that effective field-scale bioremediation reactions rates are significantly lower than batch- or lab-scale rates, when the same law of mass action is used to represent the reaction at both scales. The mismatch is usually attributed to poor mixing of reactants brought about by heterogeneity. A recent method, based on a purely Lagrangian particle tracking (PT) theoretical development, successfully reproduces the effects of mixing-limited bimolecular reaction (A + B → C) from two benchmark experiments. In this numerical method, the reactants are represented by particles, and the small-scale physics are directly translated into a combination of two probabilities that govern whether: (1) reactant particles are collocated during a short time interval, and (2) two collocated particles favorably transform into a reaction. The latter is due to thermodynamics and is independent of scale of mixing. The former directly accounts for the degree of mixing in any system. We extend the application of the PT method to biodegradation, which is commonly characterized by more complex Michaelis-Menten (Monod) chemical kinetics. The advantage of the PT method is that it explains the variation of reaction rate based on mixing-controlled particle collisions instead of using empirical parameters. The PT method not only matches the Michaelis-Menten (Monod) equation under ideal conditions, but also captures the characteristics of non-ideal conditions such as imperfect mixing, disequilibrium, and limited availability of the active sites. We show these using hypothetical systems and also successfully apply the method to a column study of carbon tetrachloride biodegradation.

  1. Particle bombardment-mediated transient expression to identify localization signals in plant disease resistance proteins and target sites for the proteolytic activity of pathogen effectors.

    Science.gov (United States)

    Takemoto, Daigo; Jones, David A

    2014-01-01

    Plant pathogens, including fungi, oomycetes, bacteria, aphids, and nematodes, produce a variety of effector proteins to counter plant disease resistance mechanisms. After delivery into the cytosol of the plant cell, effectors may target proteins localized to different compartments within the plant cell. Plants, in turn, have evolved disease resistance (R) proteins to recognize the action of effectors. Elucidation of the subcellular localization of pathogen effectors, the plant proteins they target, and plant disease resistance proteins is essential to fully understand their interactions during pathogen challenge. In recent years, expression of fluorescent protein fusions has been widely used to determine the subcellular localization of plant proteins and pathogen effectors. Use of fluorescent proteins enables researchers to monitor the dynamic behavior of proteins in living cells. Among various methods available for the introduction of genes into plant cells, particle bombardment-mediated transient expression is the most rapid method suitable for both the identification of localization signals in proteins of interest and their dissection via amino acid substitutions generated using site-directed mutagenesis. This chapter describes a rapid procedure for particle bombardment-mediated transient expression in leaf epidermal cells. This method is also applicable to detection of pathogen effector protease activities directed against target proteins in the plant cell and analysis of protease recognition sites within these target proteins.

  2. Negative effects of ultrafine particle exposure during forced exercise on the expression of Brain-Derived Neurotrophic Factor in the hippocampus of rats.

    Science.gov (United States)

    Bos, I; De Boever, P; Int Panis, L; Sarre, S; Meeusen, R

    2012-10-25

    Exercise improves cognitive function, and Brain-Derived Neurotrophic Factor (BDNF) plays a key role in this process. We recently reported that particulate matter (PM) exposure negatively contributed to the exercise-induced increase in human serum BDNF concentration. Furthermore, PM exposure is associated with neuroinflammation and cognitive decline. The aim of this study was to investigate the effect of exposure to ultrafine particles (UFP) during a single bout of forced exercise on the expression of inflammatory (IL1α, IL1β, TNF, IL6, NOS2, NOS3) and oxidative stress (NFE2L2)-related genes, as well as BDNF in the brain of rats. Four groups (n=6/group) of Wistar rats were exposed for 90 min to one of the following exposure regimes: UFP+exercise, UFP+rest, ambient air+exercise, ambient air+rest (control). Hippocampus, olfactory bulb and prefrontal cortex were collected 24h after exposure. Gene expression changes were analyzed with real-time PCR. In the condition ambient air+exercise, hippocampal expression of BDNF and NFE2L2 was up-regulated, while the expression of IL1α and NOS3 in the prefrontal cortex and IL1α in the olfactory bulb was down-regulated compared to the control. In contrast, gene expression in the condition UFP+exercise did not differ from the control. In the condition UFP+rest, hippocampal expression of NFE2L2 was down-regulated and there was a trend toward down-regulation of BDNF expression compared to the control. This study shows a negative effect of UFP exposure on the exercise-induced up-regulation of BDNF gene expression in the hippocampus of rats. Copyright © 2012 IBRO. Published by Elsevier Ltd. All rights reserved.

  3. Expression

    Directory of Open Access Journals (Sweden)

    Wang-Xia Wang

    2014-02-01

    Full Text Available The miR-15/107 family comprises a group of 10 paralogous microRNAs (miRNAs, sharing a 5′ AGCAGC sequence. These miRNAs have overlapping targets. In order to characterize the expression of miR-15/107 family miRNAs, we employed customized TaqMan Low-Density micro-fluid PCR-array to investigate the expression of miR-15/107 family members, and other selected miRNAs, in 11 human tissues obtained at autopsy including the cerebral cortex, frontal cortex, primary visual cortex, thalamus, heart, lung, liver, kidney, spleen, stomach and skeletal muscle. miR-103, miR-195 and miR-497 were expressed at similar levels across various tissues, whereas miR-107 is enriched in brain samples. We also examined the expression patterns of evolutionarily conserved miR-15/107 miRNAs in three distinct primary rat brain cell preparations (enriched for cortical neurons, astrocytes and microglia, respectively. In primary cultures of rat brain cells, several members of the miR-15/107 family are enriched in neurons compared to other cell types in the central nervous system (CNS. In addition to mature miRNAs, we also examined the expression of precursors (pri-miRNAs. Our data suggested a generally poor correlation between the expression of mature miRNAs and their precursors. In summary, we provide a detailed study of the tissue and cell type-specific expression profile of this highly expressed and phylogenetically conserved family of miRNA genes.

  4. Evaluation of Gaussia luciferase and foot-and-mouth disease virus 2A translational interrupter chimeras as polycistronic reporters for transgene expression.

    Science.gov (United States)

    Puckette, Michael; Burrage, Thomas; Neilan, John G; Rasmussen, Max

    2017-06-12

    The Gaussia princeps luciferase is used as a stand-alone reporter of transgene expression for in vitro and in vivo expression systems due to the rapid and easy monitoring of luciferase activity. We sought to simultaneously quantitate production of other recombinant proteins by transcriptionally linking the Gaussia princeps luciferase gene to other genes of interest through the foot-and-mouth disease virus 2A translational interrupter sequence. We produced six plasmids, each encoding a single open reading frame, with the foot-and-mouth disease virus 2A sequence placed either N-terminal or C-terminal to the Gaussia princeps luciferase gene. Two plasmids included novel Gaussia princeps luciferase variants with the position 1 methionine deleted. Placing a foot-and-mouth disease virus 2A translational interrupter sequence on either the N- or C-terminus of the Gaussia princeps luciferase gene did not prevent the secretion or luminescence of resulting chimeric luciferase proteins. We also measured the ability of another polycistronic plasmid vector with a 2A-luciferase sequence placed downstream of the foot-and-mouth disease virus P1 and 3C protease genes to produce of foot-and-mouth disease virus-like particles and luciferase activity from transfected cells. Incorporation of the 2A-luciferase sequence into a transgene encoding foot-and-mouth disease virus structural proteins retained luciferase activity and the ability to form virus-like particles. We demonstrated a mechanism for the near real-time, sequential, non-destructive quantitative monitoring of transcriptionally-linked recombinant proteins and a valuable method for monitoring transgene expression in recombinant vaccine constructs.

  5. Motorcycle exhaust particles up-regulate expression of vascular adhesion molecule-1 and intercellular adhesion molecule-1 in human umbilical vein endothelial cells.

    Science.gov (United States)

    Lee, Chen-Chen; Huang, Shih-Hsuan; Yang, Ya-Ting; Cheng, Yu-Wen; Li, Ching-Hao; Kang, Jaw-Jou

    2012-06-01

    Epidemiological studies have shown that there is a strong correlation between atherosclerosis and ambient air pollution. In this study, we found that motorcycle exhaust particles (MEP) induced adhesion between cells of the human monocytic leukemia cell line (THP-1) and human umbilical vein endothelial cells (HUVECs) in a time-and dose-dependent manner. In addition, MEP treatment induced both mRNA and protein expression of vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) in HUVECs. The IκB degradation and p65 nuclear translocation was found in MEP-treated HUVECs, suggested the involvement of nuclear factor-κB (NF-κB). MEP-induced VCAM-1 and ICAM-1 protein expression was inhibited by NF-κB inhibitor BAY 11-7085. Oxidative stress was also involved in the signaling of VCAM-1 and ICAM-1 expression. MEP treatment caused hydrogen peroxide and superoxide formation. Pretreatment with α-tocopherol could inhibit MEP-induced reactive oxygen intermediates generation and suppressed MEP-induced IκB degradation and adhesion molecules expression. Furthermore, the carbon black (CB) nanoparticles with different diameters could induce VCAM-1 and ICAM-1 protein expression; however, polycyclic aromatic hydrocarbons (PAHs) only increased the expression of ICAM-1 but not that of VCAM-1 in HUVECs. In this study, we found that MEPs could induce ICAM-1 and VCAM-1 expression through oxidative stress and NF-κB activation in HUVECs. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Norovirus-like VP1 particles exhibit isolate dependent stability profiles

    Science.gov (United States)

    Pogan, Ronja; Schneider, Carola; Reimer, Rudolph; Hansman, Grant; Uetrecht, Charlotte

    2018-02-01

    Noroviruses are the main cause of viral gastroenteritis with new variants emerging frequently. There are three norovirus genogroups infecting humans. These genogroups are divided based on the sequence of their major capsid protein, which is able to form virus-like particles (VLPs) when expressed recombinantly. VLPs of the prototypical GI.1 Norwalk virus are known to disassemble into specific capsid protein oligomers upon alkaline treatment. Here, native mass spectrometry and electron microscopy on variants of GI.1 and of GII.17 were performed, revealing differences in terms of stability between these groups. Beyond that, these experiments indicate differences even between variants within a genotype. The capsid stability was monitored in different ammonium acetate solutions varying both in ionic strength and pH. The investigated GI.1 West Chester isolate showed comparable disassembly profiles to the previously studied GI.1 Norwalk virus isolate. However, differences were observed with the West Chester being more sensitive to alkaline pH. In stark contrast to that, capsids of the variant belonging to the currently prevalent genogroup GII were stable in all tested conditions. Both variants formed smaller capsid particles already at neutral pH. Certain amino acid substitutions in the S domain of West Chester relative to the Norwalk virus potentially result in the formation of these T  =  1 capsids.

  7. Physico-chemical requirements and kinetics of membrane fusion of flavivirus-like particles.

    Science.gov (United States)

    Espósito, Danillo L A; Nguyen, Jennifer B; DeWitt, David C; Rhoades, Elizabeth; Modis, Yorgo

    2015-07-01

    Flaviviruses deliver their RNA genome into the host-cell cytoplasm by fusing their lipid envelope with a cellular membrane. Expression of the flavivirus pre-membrane and envelope glycoprotein genes in the absence of other viral genes results in the spontaneous assembly and secretion of virus-like particles (VLPs) with membrane fusion activity. Here, we examined the physico-chemical requirements for membrane fusion of VLPs from West Nile and Japanese encephalitis viruses. In a bulk fusion assay, optimal hemifusion (or lipid mixing) efficiencies were observed at 37 °C. Fusion efficiency increased with decreasing pH; half-maximal hemifusion was attained at pH 5.6. The anionic lipids bis(monoacylglycero)phosphate and phosphatidylinositol-3-phosphate, when present in the target membrane, significantly enhanced fusion efficiency, consistent with the emerging model that flaviviruses fuse with intermediate-to-late endosomal compartments, where these lipids are most abundant. In a single-particle fusion assay, VLPs catalysed membrane hemifusion, tracked as lipid mixing with the cellular membrane, on a timescale of 7-20 s after acidification. Lipid mixing kinetics suggest that hemifusion is a kinetically complex, multistep process.

  8. Structure of birnavirus-like particles determined by combined electron cryomicroscopy and X-ray crystallography.

    Science.gov (United States)

    Pous, Joan; Chevalier, Christophe; Ouldali, Malika; Navaza, Jorge; Delmas, Bernard; Lepault, Jean

    2005-08-01

    Birnaviruses possess a capsid with a single protein layer in contrast to most double-stranded RNA viruses infecting multicellular eukaryotes. Using freeze-drying and heavy metal shadowing, the capsids of two birnaviruses, infectious bursal disease virus (IBDV) and infectious pancreatic necrosis virus, as well as of an IBDV virus-like particle (VLP) are shown to follow the same T=13 laevo icosahedral geometry. The structure of the VLP was determined at a resolution of approximately 15 A (1.5 nm) by a combination of electron cryomicroscopy and a recently developed three-dimensional reconstruction method, where the scattering density is expressed in terms of symmetry-adapted functions. This reconstruction methodology is well adapted to the icosahedral symmetry of viruses and only requires a small number of images to analyse. The atomic model of the external capsid protein, VP2, recently determined by X-ray crystallography, fits well into the VLP reconstruction and occupies all the electron densities present in the map. Thus, similarly to the IBDV virion, only VP2 forms the icosahedral layer of the VLP. The other components of both VLP and IBDV particles that play a crucial role in the capsid assembly, VP1, VP3 and the peptides arising from the processing of pVP2, do not follow the icosahedral symmetry, allowing them to be involved in other processes such as RNA packaging.

  9. Analysis of Venezuelan equine encephalitis replicon particles packaged in different coats.

    Science.gov (United States)

    Kamrud, Kurt I; Alterson, Kim D; Andrews, Chasity; Copp, Laura O; Lewis, Whitney C; Hubby, Bolyn; Patel, Deepa; Rayner, Jonathan O; Talarico, Todd; Smith, Jonathan F

    2008-07-16

    The Venezuelan equine encephalitis (VEE) virus replicon system was used to produce virus-like replicon particles (VRP) packaged with a number of different VEE-derived glycoprotein (GP) coats. The GP coat is believed to be responsible for the cellular tropism noted for VRP and it is possible that different VEE GP coats may have different affinities for cells. We examined VRP packaged in four different VEE GP coats for their ability to infect cells in vitro and to induce both humoral and cellular immune responses in vivo. The VRP preparations were characterized to determine both infectious units (IU) and genome equivalents (GE) prior to in vivo analysis. VRP packaged with different VEE GP coats demonstrated widely varying GE/IU ratios based on Vero cell infectivity. BALB/c mice were immunized with the different VRP based on equal GE titers and the humoral and cellular responses to the expressed HIV gag gene measured. The magnitude of the immune responses measured in mice revealed small but significant differences between different GP coats when immunization was based on GE titers. We suggest that care should be taken when alternative coat proteins are used to package vector-based systems as the titers determined by cell culture infection may not represent accurate particle numbers and in turn may not accurately represent actual in vivo dose.

  10. Analysis of Venezuelan equine encephalitis replicon particles packaged in different coats.

    Directory of Open Access Journals (Sweden)

    Kurt I Kamrud

    2008-07-01

    Full Text Available The Venezuelan equine encephalitis (VEE virus replicon system was used to produce virus-like replicon particles (VRP packaged with a number of different VEE-derived glycoprotein (GP coats. The GP coat is believed to be responsible for the cellular tropism noted for VRP and it is possible that different VEE GP coats may have different affinities for cells. We examined VRP packaged in four different VEE GP coats for their ability to infect cells in vitro and to induce both humoral and cellular immune responses in vivo.The VRP preparations were characterized to determine both infectious units (IU and genome equivalents (GE prior to in vivo analysis. VRP packaged with different VEE GP coats demonstrated widely varying GE/IU ratios based on Vero cell infectivity. BALB/c mice were immunized with the different VRP based on equal GE titers and the humoral and cellular responses to the expressed HIV gag gene measured. The magnitude of the immune responses measured in mice revealed small but significant differences between different GP coats when immunization was based on GE titers.We suggest that care should be taken when alternative coat proteins are used to package vector-based systems as the titers determined by cell culture infection may not represent accurate particle numbers and in turn may not accurately represent actual in vivo dose.

  11. An analytical expression for the electric field and particle tracing in modelling of Be erosion experiments at the JET ITER-like Wall

    Energy Technology Data Exchange (ETDEWEB)

    Borodkina, I. [National Research Nuclear University MEPHI, Moscow (Russian Federation); Forschungszentrum Juelich GmbH, Juelich (Germany); EUROfusion Consortium, JET, Culham Science Centre, Abingdon (United Kingdom); Borodin, D.; Kirschner, A. [Forschungszentrum Juelich GmbH, Juelich (Germany); EUROfusion Consortium, JET, Culham Science Centre, Abingdon (United Kingdom); Tsvetkov, I.V.; Kurnaev, V.A. [National Research Nuclear University MEPHI, Moscow (Russian Federation); EUROfusion Consortium, JET, Culham Science Centre, Abingdon (United Kingdom); Komm, M.; Dejarnac, R. [Institute of Plasma Physics, Academy of Sciences of the Czech Republic, Association EURATOM/IPP.CR, Prague (Czech Republic); EUROfusion Consortium, JET, Culham Science Centre, Abingdon (United Kingdom); Collaboration: JET contributors

    2016-08-15

    A new analytical approximation for the electric potential profile in the presence of an oblique magnetic field and the analytical solution for the particle motion just before the impact with a plasma-facing surface are presented. These approximations are in good agreement with fluid solutions and the corresponding PIC simulations. These expressions were applied to provide effective physical erosion yields for Be, which have in a second step been used in ERO code simulations of spectroscopy at Be limiters of the JET ITER-like wall. These new analytical expressions lead to an increase of the effective physical sputtering yields of Be by deuteron impact up to 30% in comparison with earlier pure numerical simulations. (copyright 2016 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  12. R-SNARE ykt6 resides in membrane-associated protease-resistant protein particles and modulates cell cycle progression when over-expressed.

    Science.gov (United States)

    Thayanidhi, Nandhakumar; Liang, Yingjian; Hasegawa, Haruki; Nycz, Deborah C; Oorschot, Viola; Klumperman, Judith; Hay, Jesse C

    2012-07-01

    The arginine-type soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE) ykt6 possesses several atypical properties including selective high expression in neurons, a lipidated C-terminus, localization to punctae that do not correspond with known endomembrane markers, a potent ability to protect the secretory pathway from alpha-synuclein over-expression and specific up-regulation in tumors. We have followed up on several of these features that together suggest nontraditional SNARE structures and functions. A significant portion of ykt6 in PC12 cells was found in a protease-resistant state suggestive of a large complex or aggregate. Other endoplasmic reticulum/Golgi SNAREs were not protease resistant, demonstrating that SNARE complexes per se did not cause protease resistance. Mutagenesis indicated that lipidation of the ykt6 C-terminus was also not involved, implicating its longin domain in particle formation. Immunogold electron microscopy revealed ykt6 labeling of ∼100 nm electron densities associated with diverse membranes. Density gradient analysis of the protease-resistant structures confirmed their tight association with membranes. Since excess ykt6 has been correlated with tumorigenesis, we tested whether ykt6 over-expression in normal rat kidney cells that normally express little ykt6 affected the cell cycle. Ykt6 over-expression was found to result in altered cell division cycles as evidenced by significantly smaller cells, a higher mitotic index and increased DNA synthesis. Mutagenesis studies dis-correlated SNARE function with the cell cycle effects; instead, the cell cycle effects correlated better with ykt6 properties related to the longin domain or particle formation. The ykt6 particles/aggregates may represent ykt6 engaged in a non-SNARE function(s) or else nonfunctional, stored and/or excess ykt6. Whether the particulate ykt6 structures represent a means of buffering the apparent proliferative activity or are in fact

  13. A vaccinia virus recombinant transcribing an alphavirus replicon and expressing alphavirus structural proteins leads to packaging of alphavirus infectious single cycle particles.

    Directory of Open Access Journals (Sweden)

    Juana M Sánchez-Puig

    Full Text Available Poxviruses and Alphaviruses constitute two promising viral vectors that have been used extensively as expression systems, or as vehicles for vaccine purposes. Poxviruses, like vaccinia virus (VV are well-established vaccine vectors having large insertion capacity, excellent stability, and ease of administration. In turn, replicons derived from Alphaviruses like Semliki Forest virus (SFV are potent protein expression and immunization vectors but stocks are difficult to produce and maintain. In an attempt to demonstrate the use of a Poxvirus as a means for the delivery of small vaccine vectors, we have constructed and characterized VV/SFV hybrid vectors. A SFV replicon cDNA was inserted in the VV genome and placed under the control of a VV early promoter. The replicon, transcribed from the VV genome as an early transcript, was functional, and thus capable of initiating its own replication and transcription. Further, we constructed a VV recombinant additionally expressing the SFV structural proteins under the control of a vaccinia synthetic early/late promoter. Infection with this recombinant produced concurrent transcription of the replicon and expression of SFV structural proteins, and led to the generation of replicon-containing SFV particles that were released to the medium and were able to infect additional cells. This combined VV/SFV system in a single virus allows the use of VV as a SFV delivery vehicle in vivo. The combination of two vectors, and the possibility of generating in vivo single-cycle, replicon containing alphavirus particles, may open new strategies in vaccine development or in the design of oncolytic viruses.

  14. High throughput screening of particle conditioning operations: II. Evaluation of scale-up heuristics with prokaryotically expressed polysaccharide vaccines.

    Science.gov (United States)

    Noyes, Aaron; Huffman, Ben; Berrill, Alex; Merchant, Nick; Godavarti, Ranga; Titchener-Hooker, Nigel; Coffman, Jonathan; Sunasara, Khurram; Mukhopadhyay, Tarit

    2015-08-01

    Multivalent polysaccharide conjugate vaccines are typically comprised of several different polysaccharides produced with distinct and complex production processes. Particle conditioning steps, such as precipitation and flocculation, may be used to aid the recovery and purification of such microbial vaccine products. An ultra scale-down approach to purify vaccine polysaccharides at the micro-scale would greatly enhance productivity, robustness, and speed the development of novel conjugate vaccines. In part one of this series, we described a modular and high throughput approach to develop particle conditioning processes (HTPC) for biologicals that combines flocculation, solids removal, and streamlined analytics. In this second part of the series, we applied HTPC to industrially relevant feedstreams comprised of capsular polysaccharides (CPS) from several bacterial species. The scalability of HTPC was evaluated between 0.8 mL and 13 L scales, with several different scaling methodologies examined. Clarification, polysaccharide yield, impurity clearance, and product quality achieved with HTPC were reproducible and comparable with larger scales. Particle sizing was the response with greatest sensitivity to differences in processing scale and enabled the identification of useful scaling rules. Scaling with constant impeller tip speed or power per volume in the impeller swept zone offered the most accurate scale up, with evidence that time integration of these values provided the optimal basis for scaling. The capability to develop a process at the micro-scale combined with evidence-based scaling metrics provide a significant advance for purification process development of vaccine processes. The USD system offers similar opportunities for HTPC of proteins and other complex biological molecules. © 2015 Wiley Periodicals, Inc.

  15. Virus replicon particles expressing porcine reproductive and respiratory syndrome virus proteins elicit immune priming but do not confer protection from viremia in pigs.

    Science.gov (United States)

    Eck, Melanie; Durán, Margarita García; Ricklin, Meret E; Locher, Samira; Sarraseca, Javier; Rodríguez, María José; McCullough, Kenneth C; Summerfield, Artur; Zimmer, Gert; Ruggli, Nicolas

    2016-02-19

    Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of one of the most devastating and economically significant viral disease of pigs worldwide. The vaccines currently available on the market elicit only limited protection. Recombinant vesicular stomatitis virus (VSV) replicon particles (VRP) have been used successfully to induce protection against influenza A virus (IAV) in chickens and bluetongue virus in sheep. In this study, VSV VRP expressing the PRRSV envelope proteins GP5, M, GP4, GP3, GP2 and the nucleocapsid protein N, individually or in combination, were generated and evaluated as a potential vector vaccine against PRRSV infection. High level expression of the recombinant PRRSV proteins was demonstrated in cell culture. However, none of the PRRSV antigens expressed from VRP, with the exception of the N protein, did induce any detectable antibody response in pigs before challenge infection with PRRSV. After challenge however, the antibody responses against GP5, GP4 and GP3 appeared in average 2 weeks earlier than in pigs vaccinated with the empty control VRP. No reduction of viremia was observed in the vaccinated group compared with the control group. When pigs were co-vaccinated with VRP expressing IAV antigens and VRP expressing PRRSV glycoproteins, only antibody responses to the IAV antigens were detectable. These data show that the VSV replicon vector can induce immune responses to heterologous proteins in pigs, but that the PRRSV envelope proteins expressed from VSV VRP are poorly immunogenic. Nevertheless, they prime the immune system for significantly earlier B-cell responses following PRRSV challenge infection.

  16. Persistent Expression Changes of Fibrosis-Related Genes in the Lung Tissues of Rats Exposed to Lunar Dust Particles

    Science.gov (United States)

    Zhang, Ye; Lam, Chiu-Wing; Scully, Robert R.; Yeshitla, Samrawit A.; Wu, Honglu; Meyers, Valerie; James, John T.

    2014-01-01

    The Moon's surface is covered by a layer of fine, potential reactive dust. Lunar dust contain about 1-2% of very fine respirable dust (less than 3 micrometers). The habitable area of any lunar landing vehicle and outpost would inevitably be contaminated with lunar dust that could pose a health risk. The purpose of the study is to evaluate the toxicity of Apollo moon dust in rodents to assess the health risk of dust exposures to humans. One of the particular interests in the study is to evaluate dust-induced changes of the expression of fibrosis-related genes, and to identify specific signaling pathways involved in lunar dustinduced toxicity. F344 rats were exposed for 4 weeks (6h/d; 5d/wk) in nose-only inhalation chambers to concentrations of 0 (control air), 2.1, 6.8, 21, and 61 milligrams per cubic meters of lunar dust. Five rats per group were euthanized at 1 day, 1 week, 1 month, and 3 months after the last inhalation exposure. The bronchoalveolar lavage fluid (BALF) was collected by lavaging with phosphate-buffered saline (PBS). A zymosan-induced luminolbased chemiluminescence assay was used to assess the activity of BAL cells. The lavaged lung tissue was snap frozen in LN2 and total RNA was isolated using the Qigen RNeasy kit. The expression of 84 fibrosisrelated genes were analyzed using the RT2 Profiler PCR Array technique. The expression of 18 genes of interest were further measured using real-time PCR technique in all the samples. 10 out of 18 genes of interest showed persistently significant expression changes in the local lung tissue exposed to lunar dust, indicating a prolonged proinflammatory response. The expressions of several of these genes were dose- and time-dependent and were significantly correlated with other pathological parameters. The potential signaling pathways and upstream regulators were further analyzed using IPA pathway analysis tool based on the gene expression data. The data presented in this study, for the first time, explore the

  17. Characterization of the ectodomain of the envelope protein of dengue virus type 4: expression, membrane association, secretion and particle formation in the absence of precursor membrane protein.

    Directory of Open Access Journals (Sweden)

    Szu-Chia Hsieh

    Full Text Available The envelope (E of dengue virus (DENV is the major target of neutralizing antibodies and vaccine development. After biosynthesis E protein forms a heterodimer with precursor membrane (prM protein. Recent reports of infection enhancement by anti-prM monoclonal antibodies (mAbs suggest anti-prM responses could be potentially harmful. Previously, we studied a series of C-terminal truncation constructs expressing DENV type 4 prM/E or E proteins and found the ectodomain of E protein alone could be recognized by all 12 mAbs tested, suggesting E protein ectodomain as a potential subunit immunogen without inducing anti-prM response. The characteristics of DENV E protein ectodomain in the absence of prM protein remains largely unknown.In this study, we investigated the expression, membrane association, glycosylation pattern, secretion and particle formation of E protein ectodomain of DENV4 in the presence or absence of prM protein. E protein ectodomain associated with membrane in or beyond trans-Golgi and contained primarily complex glycans, whereas full-length E protein associated with ER membrane and contained high mannose glycans. In the absence of prM protein, E protein ectodomain can secrete as well as form particles of approximately 49 nm in diameter, as revealed by sucrose gradient ultracentrifugation with or without detergent and electron microscopy. Mutational analysis revealed that the secretion of E protein ectodomain was affected by N-linked glycosylation and could be restored by treatment with ammonia chloride.Considering the enhancement of DENV infectivity by anti-prM antibodies, our findings provide new insights into the expression and secretion of E protein ectodomain in the absence of prM protein and contribute to future subunit vaccine design.

  18. Persistent Expression Changes of Fibrosis Related Genes in the Lung Tissues of Rats Exposed to Lunar Dust Particles

    Science.gov (United States)

    Zhang, Ye; Lam, Chiu-Wing; Scully, Robert R.; Theriot, Corey; Zalesak, Selina; Yeshitla, Samrawit; Williams, Kyle; Wu, Honglu; James, John T.

    2014-01-01

    The Moon's surface is covered by a layer of reactive dust, containing 1-2% of respirable fine dust (expression changes in the lung tissue. The expression of several of these genes were dose- and time- dependent, and were significantly correlated with other pathological. Our previous data showed that no pathological changes were detected in low dose groups. However, several genes, primarily produced by lung epithelial, were significantly altered persistently in response to low-dose dust exposure. The data presented in this study, for the first time, explores the molecular mechanisms of lunar dust induced toxicity, contributing not only the risk assessment for future space exploration, but also understandings of the dust-induced toxicity to humans on earth.

  19. Up-regulation of thromboxane A2 receptor expression by lipid soluble smoking particles through post-transcriptional mechanisms

    DEFF Research Database (Denmark)

    Zhang, Wei; Zhang, Yaping; Edvinsson, Lars

    2008-01-01

    Atherosclerosis is a key factor in vascular disease, and cigarette smoking is a well-known risk factor that may induce an inflammatory response and enhance plaque formation in arteries. Thromboxane (Tx) is one key inflammatory mediator involved in the pathogenesis of cardiovascular disease. The p...... are responsible for the up-regulation of TP receptor by DSP, in which enhanced translation is the major cause of the elevated protein expression and the enhanced contraction.......Atherosclerosis is a key factor in vascular disease, and cigarette smoking is a well-known risk factor that may induce an inflammatory response and enhance plaque formation in arteries. Thromboxane (Tx) is one key inflammatory mediator involved in the pathogenesis of cardiovascular disease....../ml for 24h) resulted in markedly elevated contractile responses to the Tx analog U46619, compared with the control DMSO. There was no increase in TP receptor mRNA expression, while the protein expression was significantly enhanced. This up-regulation was not affected by a general transcriptional inhibitor...

  20. Detection and localisation of picorna-like virus particles in tissues of Varroa destructor, an ectoparasite of the honey bee, Apis mellifera

    NARCIS (Netherlands)

    Zhang, Q.; Ongus, J.R.; Boot, W.J.; Calis, J.; Bonmatin, J.M.; Bengsch, E.; Peters, D.

    2007-01-01

    Virus-like particles, 27 nm in diameter, were observed in extracts of individual Varroa destructor mites and in sections of mite tissue. Application of a purification procedure resulted in virus preparations that were used to prepare an antiserum to detect the virus in individual mites.

  1. Particle Pollution

    Science.gov (United States)

    ... Your Health Particle Pollution Public Health Issues Particle Pollution Recommend on Facebook Tweet Share Compartir Particle pollution — ... see them in the air. Where does particle pollution come from? Particle pollution can come from two ...

  2. PEI-engineered respirable particles delivering a decoy oligonucleotide to NF-κB: inhibiting MUC2 expression in LPS-stimulated airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Francesca Ungaro

    Full Text Available A specific and promising approach to limit inflammation and mucin iperproduction in chronic lung diseases relies on specific inhibition of nuclear Factor-κB (NF-κB by a decoy oligonucleotide (dec-ODN. To fulfill the requirements dictated by translation of dec-ODN therapy in humans, inhalable dry powders were designed on a rational basis to provide drug protection, sustained release and to optimize pharmacological response. To this end, large porous particles (LPP for dec-ODN delivery made of a sustained release biomaterial (poly(lactic-co-glycolic acid, PLGA and an "adjuvant" hydrophilic polymer (polyethylenimine, PEI were developed and their effects on LPS-stimulated human airway epithelial cells evaluated. The composite PLGA/PEI particles containing dec-ODN (i.e., LPP(PEI were successfully engineered for widespread deposition in the lung and prolonged release of intact dec-ODN in vitro. LPP(PEI caused a prolonged inhibition of IL-8 and MUC2 expression in CF human bronchial epithelial cells and human epithelial pulmonary NCI-H292 cells, respectively, as compared to naked dec-ODN. Nonetheless, as compared to previously developed LPP, the presence of PEI was essential to construct a dec-ODN delivery system able to act in mucoepidermoid lung epithelial cells. In perspective, engineering LPP with PEI may become a key factor for tuning carrier properties, controlling lung inflammation and mucin production which, in turn, can foster in vivo translation of dec-ODN therapy.

  3. [Optimized expression of the L1 protein of human papillomavirus in Hansenula polymorpha].

    Science.gov (United States)

    Li, Weiwei; He, Xiuping; Guo, Xuena; Zhang, Zhenying; Zhang, Borun

    2009-10-01

    The heterologously expressed L1 protein of human papilomavirus 16 can assembly into virus-like particles (VLPs), which has been used as prophylactic vaccine for cervical carcinoma. To express L1 protein in Hansenula polymorpha, we analyzed the codon usage of the native gene of L1 protein and redesigned the encoding sequence according to the codon bias of H. polymorpha. We used assembly PCR to synthesize the native gene HPV16L1-N and the codon optimized gene HPV16L1. The synthesized genes were cloned into pMOXZa-A vector to generate plasmids pMOXZ-HPV16N and pMOXZ-HPV16. The expression cassettes MOXp-HPV16L1(N)-AOXTT were cloned into YEp352 vector and transferred into H. polymorpha. After methanol inducement, the expression of L1 protein in H. polymorpha was detected from the codon optimized gene HPV16L1 rather than the native gene HPVI6L1-N. The parameters for induced cultivation for strain HP-U-16L with HPV16L1 were investigated in shaking flask cultures. After induced cultivation in YPM (pH 7.0) medium supplemented with methanol to a final concentration of 1.0% every 12 h at 37 degrees C for 72 h, the recombinant produced 78.6 mg/L of L1 protein. This work offers the possibility for the production of prophylactic vaccine for cervical carcinoma by H. polymorpha.

  4. A novel recombinant pseudorabies virus expressing parvovirus VP2 gene: Immunogenicity and protective efficacy in swine.

    Science.gov (United States)

    Chen, Yang; Guo, Wanzhu; Xu, Zhiwen; Yan, Qigui; Luo, Yan; Shi, Qian; Chen, Dishi; Zhu, Ling; Wang, Xiaoyu

    2011-06-16

    Porcine parvovirus (PPV) VP2 gene has been successfully expressed in many expression systems resulting in self-assembly of virus-like particles (VLPs) with similar morphology to the native capsid. Here, a pseudorabies virus (PRV) system was adopted to express the PPV VP2 gene. A recombinant PRV SA215/VP2 was obtained by homologous recombination between the vector PRV viral DNA and a transfer plasmid. Then recombinant virus was purified with plaque purification, and its identity confirmed by PCR amplification, Western blot and indirect immunofluorescence (IFA) analyses. Electronic microscopy of PRV SA215/VP2 confirmed self-assembly of both pseudorabies virus and VLPs from VP2 protein. Immunization of piglets with recombinant virus elicited PRV-specific and PPV-specific humoral immune responses and provided complete protection against a lethal dose of PRV challenges. Gilts immunized with recombinant viruses induced PPV-specific antibodies, and significantly reduced the mortality rate of (1 of 28) following virulent PPV challenge compared with the control (7 of 31). Furthermore, PPV virus DNA was not detected in the fetuses of recombinant virus immunized gilts. In this study, a recombinant PRV SA215/VP2 virus expressing PPV VP2 protein was constructed using PRV SA215 vector. The safety, immunogenicity, and protective efficacy of the recombinant virus were demonstrated in piglets and primiparous gilts. This recombinant PRV SA215/VP2 represents a suitable candidate for the development of a bivalent vaccine against both PRV and PPV infection.

  5. Diesel exhaust particles increase IL-1β-induced human β-defensin expression via NF-κB-mediated pathway in human lung epithelial cells

    Directory of Open Access Journals (Sweden)

    Lee Chun

    2006-05-01

    Full Text Available Abstract Background Human β-defensin (hBD-2, antimicrobial peptide primarily induced in epithelial cells, is a key factor in the innate immune response of the respiratory tract. Several studies showed increased defensin levels in both inflammatory lung diseases, such as cystic fibrosis, diffuse panbronchiolitis, idiopathic pulmonary fibrosis and acute respiratory distress syndrome, and infectious diseases. Recently, epidemiologic studies have demonstrated acute and serious adverse effects of particulate air pollution on respiratory health, especially in people with pre-existing inflammatory lung disease. To elucidate the effect of diesel exhaust particles (DEP on pulmonary innate immune response, we investigated the hBD-2 and interleukin-8 (IL-8 expression to DEP exposure in interleukin-1 beta (IL-1β-stimulated A549 cells. Results IL-1β markedly up-regulated the hBD-2 promoter activity, and the subsequent DEP exposure increased dose-dependently the expression of hBD-2 and inflammatory cytokine IL-8 at the transcriptional level. In addition, DEP further induced the NF-κB activation in IL-1β-stimulated A549 cells more rapidly than in unstimulated control cells, which was showed by nuclear translocation of p65 NF-κB and degradation of IκB-α. The experiment using two NF-κB inhibitors, PDTC and MG132, confirmed that this increase of hBD-2 expression following DEP exposure was regulated through NF-κB-mediated pathway. Conclusion These results demonstrated that DEP exposure increases the expression of antimicrobial peptide and inflammatory cytokine at the transcriptional level in IL-1β-primed A549 epithelial cells and suggested that the increase is mediated at least partially through NF-κB activation. Therefore, DEP exposure may contribute to enhance the airway-responsiveness especially on the patients suffering from chronic respiratory disease.

  6. Transient expression of Human papillomavirus type 16 L1 protein in Nicotiana benthamiana using an infectious tobamovirus vector.

    Science.gov (United States)

    Varsani, Arvind; Williamson, Anna-Lise; Stewart, Debbie; Rybicki, Edward P

    2006-09-01

    A Tobacco mosaic virus (TMV)-derived vector was used to express a native Human papillomavirus type 16 (HPV-16) L1 gene in Nicotiana benthamiana by means of infectious in vitro RNA transcripts inoculated onto N. benthamiana plants. HPV-16 L1 protein expression was quantitated by enzyme-linked immunosorbent assays (ELISA) after concentration of the plant extract. We estimated that the L1 product yield was 20-37 microg/kg of fresh leaf material. The L1 protein in the concentrated extract was antigenically characterised using the neutralising and conformation-specific Mabs H16:V5 and H16:E70, which bound to the plant-produced protein. Particles observed by transmission electron microscopy were mainly capsomers but virus-like particles (VLPs) similar to those produced in other systems were also present. Immunisation of rabbits with the concentrated plant extract induced a weak immune response. This is the first report of the successful expression of an HPV L1 gene in plants using a plant virus vector.

  7. Quantum Dots Encapsulated with Canine Parvovirus-Like Particles Improving the Cellular Targeted Labeling.

    Directory of Open Access Journals (Sweden)

    Dan Yan

    Full Text Available Quantum dots (QDs have a promising prospect in live-cell imaging and sensing because of unique fluorescence features. QDs aroused significant interest in the bio-imaging field through integrating the fluorescence properties of QDs and the delivery function of biomaterial. The natural tropism of Canine Parvovirus (CPV to the transferrin receptor can target specific cells to increase the targeting ability of QDs in cell imaging. CPV virus-like particles (VLPs from the expression of the CPV-VP2 capsid protein in a prokaryotic expression system were examined to encapsulate the QDs and deliver to cells with an expressed transferrin receptor. CPV-VLPs were used to encapsulate QDs that were modified using 3-mercaptopropionic acid. Gel electrophoresis, fluorescence spectrum, particle size, and transmission electron microscopy verified the conformation of a complex, in which QDs were encapsulated in CPV-VLPs (CPV-VLPs-QDs. When incubated with different cell lines, CPV-VLPs-QDs significantly reduced the cytotoxicity of QDs and selectively labeled the cells with high-level transferrin receptors. Cell-targeted labeling was achieved by utilizing the specific binding between the CPV capsid protein VP2 of VLPs and cellular receptors. CPV-VLPs-QDs, which can mimic the native CPV infection, can recognize and attach to the transferrin receptors on cellular membrane. Therefore, CPV-VLPs can be used as carriers to facilitate the targeted delivery of encapsulated nanomaterials into cells via receptor-mediated pathways. This study confirmed that CPV-VLPs can significantly promote the biocompatibility of nanomaterials and could expand the application of CPV-VLPs in biological medicine.

  8. Canine parvovirus-like particles, a novel nanomaterial for tumor targeting

    Directory of Open Access Journals (Sweden)

    Destito Giuseppe

    2006-02-01

    Full Text Available Abstract Specific targeting of tumor cells is an important goal for the design of nanotherapeutics for the treatment of cancer. Recently, viruses have been explored as nano-containers for specific targeting applications, however these systems typically require modification of the virus surface using chemical or genetic means to achieve tumor-specific delivery. Interestingly, there exists a subset of viruses with natural affinity for receptors on tumor cells that could be exploited for nanotechnology applications. For example, the canine parvovirus (CPV utilizes transferrin receptors (TfRs for binding and cell entry into canine as well as human cells. TfRs are over-expressed by a variety of tumor cells and are widely being investigated for tumor-targeted drug delivery. We explored whether the natural tropism of CPV to TfRs could be harnessed for targeting tumor cells. Towards this goal, CPV virus-like particles (VLPs produced by expression of the CPV-VP2 capsid protein in a baculovirus expression system were examined for attachment of small molecules and delivery to tumor cells. Structural modeling suggested that six lysines per VP2 subunit are presumably addressable for bioconjugation on the CPV capsid exterior. Between 45 and 100 of the possible 360 lysines/particle could be routinely derivatized with dye molecules depending on the conjugation conditions. Dye conjugation also demonstrated that the CPV-VLPs could withstand conditions for chemical modification on lysines. Attachment of fluorescent dyes neither impaired binding to the TfRs nor affected internalization of the 26 nm-sized VLPs into several human tumor cell lines. CPV-VLPs therefore exhibit highly favorable characteristics for development as a novel nanomaterial for tumor targeting.

  9. Preparation by alkaline treatment and detailed characterisation of empty hepatitis B virus core particles for vaccine and gene therapy applications

    Science.gov (United States)

    Strods, Arnis; Ose, Velta; Bogans, Janis; Cielens, Indulis; Kalnins, Gints; Radovica, Ilze; Kazaks, Andris; Pumpens, Paul; Renhofa, Regina

    2015-06-01

    Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications.

  10. Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5 ' untranslated regions are efficiently translated in cells by a cap-dependent mechanism

    DEFF Research Database (Denmark)

    Belsham, Graham; Nielsen, Inge; Normann, Preben

    2008-01-01

    secondary structure and multiple upstream AUG codons. These features can be expected to inhibit cap-dependent initiation of translation. However, we have now shown that certain mutant hepatitis C virus-like picornavirus IRES elements (from porcine teschovirus-1 and avian encephalomyelitis virus), which...... cleavage of eIF4G) and is also inhibited by hippuristanol, a specific inhibitor of eIF4A function, in contrast to their parental wild-type IRES elements. These results provide a possible basis for the evolution of viral IRES elements within the context of functional mRNAs that are translated by a cap...

  11. Vaccine efficacy against malaria by the combination of porcine parvovirus-like particles and vaccinia virus vectors expressing CS of Plasmodium.

    Directory of Open Access Journals (Sweden)

    Dolores Rodríguez

    Full Text Available With the aim to develop an efficient and cost-effective approach to control malaria, we have generated porcine parvovirus-like particles (PPV-VLPs carrying the CD8(+ T cell epitope (SYVPSAEQI of the circumsporozoite (CS protein from Plasmodium yoelii fused to the PPV VP2 capsid protein (PPV-PYCS, and tested in prime/boost protocols with poxvirus vectors for efficacy in a rodent malaria model. As a proof-of concept, we have characterized the anti-CS CD8(+ T cell response elicited by these hybrid PPV-VLPs in BALB/c mice after immunizations with the protein PPV-PYCS administered alone or in combination with recombinant vaccinia virus (VACV vectors from the Western Reserve (WR and modified virus Ankara (MVA strains expressing the entire P. yoelii CS protein. The results of different immunization protocols showed that the combination of PPV-PYCS prime/poxvirus boost was highly immunogenic, inducing specific CD8+ T cell responses to CS resulting in 95% reduction in liver stage parasites two days following sporozoite challenge. In contrast, neither the administration of PPV-PYCS alone nor the immunization with the vectors given in the order poxvirus/VLPs was as effective. The immune profile induced by VLPs/MVA boost was associated with polyfunctional and effector memory CD8+ T cell responses. These findings highlight the use of recombinant parvovirus PPV-PYCS particles as priming agents and poxvirus vectors, like MVA, as booster to enhance specific CD8+ T cell responses to Plasmodium antigens and to control infection. These observations are relevant in the design of T cell-inducing vaccines against malaria.

  12. Construction and characterisation of a recombinant fowlpox virus that expresses the human papilloma virus L1 protein

    Directory of Open Access Journals (Sweden)

    Zanotto Carlo

    2011-11-01

    Full Text Available Abstract Background Human papilloma virus (HPV-16 is the most prevalent high-risk mucosal genotype. Virus-like-particle (VLP-based immunogens developed recently have proven to be successful as prophylactic HPV vaccines, but are still too expensive for developing countries. Although vaccinia viruses expressing the HPV-16 L1 protein (HPV-L1 have been studied, fowlpox-based recombinants represent efficient and safer vectors for immunocompromised hosts due to their ability to elicit a complete immune response and their natural host-range restriction to avian species. Methods A new fowlpox virus recombinant encoding HPV-L1 (FPL1 was engineered and evaluated for the correct expression of HPV-L1 in vitro, using RT-PCR, immunoprecipitation, Western blotting, electron microscopy, immunofluorescence, and real-time PCR assays. Results The FPL1 recombinant correctly expresses HPV-L1 in mammalian cells, which are non-permissive for the replication of this vector. Conclusion This FPL1 recombinant represents an appropriate immunogen for expression of HPV-L1 in human cells. The final aim is to develop a safe, immunogenic, and less expensive prophylactic vaccine against HPV.

  13. Expression of the bovine papillomavirus type 1, 2 and 4 L1 genes in the yeast Pichia pastoris.

    Science.gov (United States)

    Jesus, A L S; Mariz, F C; Souza, H M; Cordeiro, M N; Coimbra, E C; Leitão, M C G; Nascimento, L M; Stocco, R C; Beçak, W; Freitas, A C

    2012-08-16

    Papillomaviruses are known to cause benign or malignant lesions in various animals. In cattle, bovine papillomavirus (BPV) is the etiologic agent of papillomatosis and neoplasia of the upper gastrointestinal tract and urinary bladder. Currently, there are no standard diagnostic tests or prophylactic vaccines. Protection against papillomavirus infection is conferred by neutralizing antibodies directed towards the major structural protein L1. These antibodies can be efficiently induced by immunization with virus-like particles that are formed spontaneously after L1 gene expression in recombinant systems. The yeast Pichia pastoris is known to provide an efficient system for expression of proteins due to reduced cost and high levels of protein production. We evaluated P. pastoris for expression of the L1 gene from BPV1, BPV2 and BPV4. After methanol induction, the recombinants were able to produce L1 proteins of the three different BPV types. To increase heterologous L1 protein levels, a codon optimization strategy was used for production under bioreactor conditions. The BPV1 L1 protein was identified by monoclonal antibody anti-6xHis. This is the first report of BPV L1 expression in yeast.

  14. Partial Protection against Porcine Influenza A Virus by a Hemagglutinin-Expressing Virus Replicon Particle Vaccine in the Absence of Neutralizing Antibodies.

    Science.gov (United States)

    Ricklin, Meret E; Vielle, Nathalie J; Python, Sylvie; Brechbühl, Daniel; Zumkehr, Beatrice; Posthaus, Horst; Zimmer, Gert; Summerfield, Artur

    2016-01-01

    This work was initiated by previous reports demonstrating that mismatched influenza A virus (IAV) vaccines can induce enhanced disease, probably mediated by antibodies. Our aim was, therefore, to investigate if a vaccine inducing opsonizing but not neutralizing antibodies against the hemagglutinin (HA) of a selected heterologous challenge virus would enhance disease or induce protective immune responses in the pig model. To this end, we immunized pigs with either whole inactivated virus (WIV)-vaccine or HA-expressing virus replicon particles (VRP) vaccine based on recombinant vesicular stomatitis virus (VSV). Both types of vaccines induced virus neutralizing and opsonizing antibodies against homologous virus as shown by a highly sensitive plasmacytoid dendritic cell-based opsonization assay. Opsonizing antibodies showed a broader reactivity against heterologous IAV compared with neutralizing antibodies. Pigs immunized with HA-recombinant VRP vaccine were partially protected from infection with a mismatched IAV, which was not neutralized but opsonized by the immune sera. The VRP vaccine reduced lung lesions, lung inflammatory cytokine responses, serum IFN-α responses, and viral loads in the airways. Only the VRP vaccine was able to prime IAV-specific IFNγ/TNFα dual secreting CD4(+) T cells detectable in the peripheral blood. In summary, this work demonstrates that with the virus pair selected, a WIV vaccine inducing opsonizing antibodies against HA which lack neutralizing activity, is neither protective nor does it induce enhanced disease in pigs. In contrast, VRP-expressing HA is efficacious vaccines in swine as they induced both potent antibodies and T-cell immunity resulting in a broader protective value.

  15. Modifications in Dynamic Contrast-Enhanced Magnetic Resonance Imaging Parameters After α-Particle-Emitting {sup 227}Th-trastuzumab Therapy of HER2-Expressing Ovarian Cancer Xenografts

    Energy Technology Data Exchange (ETDEWEB)

    Heyerdahl, Helen, E-mail: Helen.Heyerdahl@rr-research.no [Department of Radiation Biology, Institute for Cancer Research, Oslo University Hospital - The Norwegian Radium Hospital, Oslo (Norway); Røe, Kathrine [Department of Oncology, Division of Medicine, Akershus University Hospital, Lørenskog (Norway); Brevik, Ellen Mengshoel [Department of Research and Development, Algeta ASA, Oslo (Norway); Dahle, Jostein [Nordic Nanovector AS, Oslo (Norway)

    2013-09-01

    Purpose: The purpose of this study was to investigate the effect of α-particle-emitting {sup 227}Th-trastuzumab radioimmunotherapy on tumor vasculature to increase the knowledge about the mechanisms of action of {sup 227}Th-trastuzumab. Methods and Materials: Human HER2-expressing SKOV-3 ovarian cancer xenografts were grown bilaterally in athymic nude mice. Mice with tumor volumes 253 ± 36 mm{sup 3} (mean ± SEM) were treated with a single injection of either {sup 227}Th-trastuzumab at a dose of 1000 kBq/kg body weight (treated group, n=14 tumors) or 0.9% NaCl (control group, n=10 tumors). Dynamic T1-weighted contrast-enhanced magnetic resonance imaging (DCEMRI) was used to study the effect of {sup 227}Th-trastuzumab on tumor vasculature. DCEMRI was performed before treatment and 1, 2, and 3 weeks after therapy. Tumor contrast-enhancement curves were extracted voxel by voxel and fitted to the Brix pharmacokinetic model. Pharmacokinetic parameters for the tumors that underwent radioimmunotherapy were compared with the corresponding parameters of control tumors. Results: Significant increases of k{sub ep}, the rate constant of diffusion from the extravascular extracellular space to the plasma (P<.05), and k{sub el,} the rate of clearance of contrast agent from the plasma (P<.01), were seen in the radioimmunotherapy group 2 and 3 weeks after injection, compared with the control group. The product of k{sub ep} and the amplitude parameter A, associated with increased vessel permeability and perfusion, was also significantly increased in the radioimmunotherapy group 2 and 3 weeks after injection (P<.01). Conclusions: Pharmacokinetic modeling of MRI contrast-enhancement curves evidenced significant alterations in parameters associated with increased tumor vessel permeability and tumor perfusion after {sup 227}Th-trastuzumab treatment of HER2-expressing ovarian cancer xenografts.

  16. Exploiting the yeast L-A viral capsid for the in vivo assembly of chimeric VLPs as platform in vaccine development and foreign protein expression.

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    Frank Powilleit

    Full Text Available A novel expression system based on engineered variants of the yeast (Saccharomyces cerevisiae dsRNA virus L-A was developed allowing the in vivo assembly of chimeric virus-like particles (VLPs as a unique platform for a wide range of applications. We show that polypeptides fused to the viral capsid protein Gag self-assemble into isometric VLP chimeras carrying their cargo inside the capsid, thereby not only effectively preventing proteolytic degradation in the host cell cytosol, but also allowing the expression of a per se cytotoxic protein. Carboxyterminal extension of Gag by T cell epitopes from human cytomegalovirus pp65 resulted in the formation of hybrid VLPs that strongly activated antigen-specific CD8(+ memory T cells ex vivo. Besides being a carrier for polypeptides inducing antigen-specific immune responses in vivo, VLP chimeras were also shown to be effective in the expression and purification of (i a heterologous model protein (GFP, (ii a per se toxic protein (K28 alpha-subunit, and (iii a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A. Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production.

  17. Binding to histo-blood group antigen-expressing bacteria protects human norovirus from acute heat stress

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    Dan eLi

    2015-07-01

    Full Text Available This study aims to investigate if histo-blood group antigen (HBGA expressing bacteria have any protective role on human norovirus (NoV from acute heat stress. Eleven bacterial strains were included, belonging to Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, Clostridium difficile, Bifidobacterium adolescentis, and Bifidobacterium longum. HBGA expression of the bacteria as well as binding of human NoV virus-like particles (VLPs, GI.1 and GII.4 strains to the bacteria were detected by flow cytometry. NoV VLPs pre-incubated with HBGA expressing or non-HBGA expressing bacteria were heated and detected by both direct ELISA and porcine gastric mucin-binding assay. The NoV-binding abilities of the bacteria correlated well with their HBGA expression profiles. Two HBGA expressing E.coli (LMG8223 and LFMFP861, both GI.1 and GII.4 binders and one non-HBGA expressing E.coli (ATCC8739, neither GI.1 nor GII.4 binder were selected for the heat treatment test with NoV VLPs. Compared with the same cell numbers of non-HBGA expressing E.coli, the presence of HBGA-expressing E.coli could always maintain higher antigen integrity, as well as mucin-binding ability of NoV VLPs of both GI.1 and GII.4 after heat-treatment at 90°C for 2 min. These results indicate that HBGA-expressing bacteria may protect NoVs during the food processing treatments, thereby facilitating their transmission.

  18. Transient gene expression optimization and expression vector comparison to improve HIV-1 VLP production in HEK293 cell lines.

    Science.gov (United States)

    Fuenmayor, Javier; Cervera, Laura; Gutiérrez-Granados, Sonia; Gòdia, Francesc

    2017-11-04

    Transient gene expression (TGE) has been used at small and medium scale for the production of biologicals in sufficient quantities to perform pre-clinical and characterization studies. Polyethyleneimine (PEI)-mediated transfection offers a low toxicity and non-expensive method for cell transfection. DNA and PEI concentration for transient gene expression has been extensively optimized in order to increase product titers. However, the possibility to extrapolate the optimal concentrations found for a specific bioprocess when expression vectors or cell lines need to be changed has not been investigated.In this work, the combination of three different HEK293 cell lines with three different vectors was studied for the production of HIV-1 virus-like particles (VLPs). The concentration of DNA and PEI was optimized for the nine combinations. The obtained results were very similar in all cases (DNA = 2.34 ± 0.18 μg/mL and PEI = 5.81 ± 0.18 μg/mL), revealing that transfection efficiency is not dependent on the cell line or vector type, but on DNA and PEI quantities. Furthermore, two of the cell lines tested stably expressed a protein able to recognize specific origins of replication: HEK293T/SV40 and HEK293E/oriP. Origins of replication were included in the vector sequences in order to test their capacity to increase production titers. HEK293T/SV40 resulted in a decrease of cell density and productivity of 2.3-fold compared to a control plasmid. On the other hand, HEK293E/OriP platform enabled a threefold improvement in HIV-1 VLP production keeping the same cell densities and viabilities compared to a control plasmid.

  19. Molecular and process design for rotavirus-like particle production in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Palomares Laura A

    2011-05-01

    Full Text Available Abstract Background Virus-like particles (VLP have an increasing range of applications including vaccination, drug delivery, diagnostics, gene therapy and nanotechnology. These developments require large quantities of particles that need to be obtained in efficient and economic processes. Production of VLP in yeast is attractive, as it is a low-cost protein producer able to assemble viral structural proteins into VLP. However, to date only single-layered VLP with simple architecture have been produced in this system. In this work, the first steps required for the production of rotavirus-like particles (RLP in S. cerevisiae were implemented and improved, in order to obtain the recombinant protein concentrations required for VLP assembly. Results The genes of the rotavirus structural proteins VP2, VP6 and VP7 were cloned in four Saccharomyces cerevisiae strains using different plasmid and promoter combinations to express one or three proteins in the same cell. Performance of the best constructs was evaluated in batch and fed-batch cultures using a complete synthetic media supplemented with leucine, glutamate and succinate. The strain used had an important effect on recombinant protein concentration, while the type of plasmid, centromeric (YCp or episomal (YEp, did not affect protein yields. Fed-batch culture of the PD.U-267 strain resulted in the highest concentration of rotavirus proteins. Volumetric and specific productivities increased 28.5- and 11-fold, respectively, in comparison with batch cultures. Expression of the three rotavirus proteins was confirmed by immunoblotting and RLP were detected using transmission electron microscopy. Conclusions We present for the first time the use of yeast as a platform to express multilayered rotavirus-like particles. The present study shows that the combined use of molecular and bioprocess tools allowed the production of triple-layered rotavirus RLP. Production of VLP with complex architecture in yeasts

  20. Polygamous particles

    OpenAIRE

    Wu, Kun-Ta; Feng, Lang; Sha, Ruojie; Dreyfus, Rémi; Grosberg, Alexander Y.; Seeman, Nadrian C.; Chaikin, Paul M.

    2012-01-01

    DNA is increasingly used as an important tool in programming the self-assembly of micrometer- and nanometer-scale particles. This is largely due to the highly specific thermoreversible interaction of cDNA strands, which, when placed on different particles, have been used to bind precise pairs in aggregates and crystals. However, DNA functionalized particles will only reach their true potential for particle assembly when each particle can address and bind to many different kinds of particles. ...

  1. Bacterially expressed human papillomavirus type 6 and 11 bivalent vaccine: Characterization, antigenicity and immunogenicity.

    Science.gov (United States)

    Pan, Huirong; Li, Zhihai; Wang, Jin; Song, Shuo; Wang, Daning; Wei, Minxi; Gu, Ying; Zhang, Jun; Li, Shaowei; Xia, Ningshao

    2017-05-31

    Human papillomavirus (HPV)-6 and HPV11 are the major etiological causes of condylomata acuminate. HPV neutralization by vaccine-elicited neutralizing antibodies can block viral infection and prevent subsequent disease. Currently, two commercially available HPV vaccines cover these two genotypes, expressed by Saccharomyces cerevisiae. Here we describe another HPV6/11 bivalent vaccine candidate derived from Escherichia coli. The soluble expression of N-terminally truncated L1 proteins was optimized to generate HPV6- and HPV11 L1-only virus-like particles (VLPs) as a scalable process. In a pilot scale, we used various biochemical, biophysical and immunochemical approaches to comprehensively characterize the scale and lot consistency of the vaccine candidate at 30L and 100L. Cryo-EM structure analysis showed that these VLPs form a T=7 icosahedral lattice, imitating the L1 capsid of the authentic HPV virion. This HPV6/11 bivalent vaccine confers a neutralization titer and antibody production profile in monkey that is comparable with the quadrivalent vaccine, Gardasil. This study demonstrates the robustness and scalability of a potential HPV6/11 bivalent vaccine using a prokaryotic system for vaccine production. Copyright © 2017. Published by Elsevier Ltd.

  2. Hsp70 expression and free radical release after exposure to non-thermal radio-frequency electromagnetic fields and ultrafine particles in human Mono Mac 6 cells.

    Science.gov (United States)

    Simkó, M; Hartwig, C; Lantow, M; Lupke, M; Mattsson, M-O; Rahman, Q; Rollwitz, J

    2006-02-08

    The contemporary urban environment has become increasingly complex in its composition, leading to discussions regarding possible novel health effects. Two factors that recently have received considerable attention are ultrafine particles (UFP; spectrum. Several studies have shown biological effects of both these exposures in various cell systems. Here we investigate if exposure to UFP (12-14 nm, 100 microg/ml) and RF-electromagnetic fields (EMF; 2 W/kg specific absorption rate (SAR); continuous wave (CW) or modulated (217Hz or GSM-nonDTX)), alone or in combination influences levels of the superoxide radical anion or the stress protein heat-shock protein (Hsp70) in the human monocyte cell line Mono Mac 6. Heat treatment (42-43 degrees C, 1h) was used as positive control for both stress reaction and for heat development in the RF exposure setup. Our results clearly show that Mono Mac 6 cells are capable to internalise UFP, and that this phagocytic activity is connected to an increased release of free radicals. This increase (40-45% above negative control) is stronger than the effect of heat treatment. On the other hand, none of the employed RF exposures showed any effects on free radical levels. Co-exposure of RF and UFP did not potentiate the UFP effect either. Our investigations showed a significantly increased Hsp70 expression level by heat treatment in a time-dependent manner, whereas UFP, RF, or UFP+RF were without any effect. Therefore, we conclude that in the investigated Mono Mac 6 cells, RF exposure alone or in combination with UFP cannot influence stress-related responses.

  3. Modeling rotavirus-like particles production in a baculovirus expression vector system: Infection kinetics, baculovirus DNA replication, mRNA synthesis and protein production.

    Science.gov (United States)

    Roldão, António; Vieira, Helena L A; Charpilienne, Annie; Poncet, Didier; Roy, Polly; Carrondo, Manuel J T; Alves, Paula M; Oliveira, R

    2007-03-10

    Rotavirus is the most common cause of severe diarrhoea in children worldwide, responsible for more than half a million deaths in children per year. Rotavirus-like particles (Rota VLPs) are excellent vaccine candidates against rotavirus infection, since they are non-infectious, highly immunogenic, amenable to large-scale production and safer to produce than those based on attenuated viruses. This work focuses on the analysis and modeling of the major events taking place inside Spodoptera frugiperda (Sf-9) cells infected by recombinant baculovirus that may be critical for the expression of rotavirus viral proteins (VPs). For model validation, experiments were performed adopting either a co-infection strategy, using three monocistronic recombinant baculovirus each one coding for viral proteins VP(2), VP(6) and VP(7), or single-infection strategies using a multigene baculovirus coding for the three proteins of interest. A characteristic viral DNA (vDNA) replication rate of 0.19+/-0.01 h(-1) was obtained irrespective of the monocistronic or multigene vector employed, and synthesis of progeny virus was found to be negligible in comparison to intracellular vDNA concentrations. The timeframe for vDNA, mRNA and VP synthesis tends to decrease with increasing multiplicity of infection (MOI) due to the metabolic burden effect. The protein synthesis rates could be ranked according to the gene size in the multigene experiments but not in the co-infection experiments. The model exhibits acceptable prediction power of the dynamics of intracellular vDNA replication, mRNA synthesis and VP production for the three proteins involved. This model is intended to be the basis for future Rota VLPs process optimisation and also a means to evaluating different baculovirus constructs for Rota VLPs production.

  4. Engineering and expression of a human rotavirus candidate vaccine in Nicotiana benthamiana.

    Science.gov (United States)

    Pêra, Francisco F P G; Mutepfa, David L R; Khan, Ayesha M; Els, Johann H; Mbewana, Sandiswa; van Dijk, Alberdina A A; Rybicki, Edward P; Hitzeroth, Inga I

    2015-12-02

    Human rotaviruses are the main cause of severe gastroenteritis in children and are responsible for over 500 000 deaths annually. There are two live rotavirus vaccines currently available, one based on human rotavirus serotype G1P[8], and the other a G1-G4 P[8] pentavalent vaccine. However, the recent emergence of the G9 and other novel rotavirus serotypes in Africa and Asia has prompted fears that current vaccines might not be fully effective against these new varieties. We report an effort to develop an affordable candidate rotavirus vaccine against the new emerging G9P[6] (RVA/Human-wt/ZAF/GR10924/1999/G9P[6]) strain. The vaccine is based on virus-like particles which are both highly immunogenic and safe. The vaccine candidate was produced in Nicotiana benthamiana by transient expression, as plants allow rapid production of antigens at lower costs, without the risk of contamination by animal pathogens. Western blot analysis of plant extracts confirmed the successful expression of two rotavirus capsid proteins, VP2 and VP6. These proteins assembled into VLPs resembling native rotavirus particles when analysed by transmission electron microscopy (TEM). Expression of the rotavirus glycoprotein VP7 and the spike protein VP4 was also tried. However, VP7 expression caused plant wilting during the course of the time trial and expression could never be detected for either protein. We therefore created three fusion proteins adding the antigenic part of VP4 (VP8*) to VP6 in an attempt to produce more appropriately immunogenic particles. Fusion protein expression in tobacco plants was detected by western blot using anti-VP6 and anti-VP4 antibodies, but no regular particles were observed by TEM, even when co-expressed with VP2. Our results suggest that the rotavirus proteins produced in N. benthamiana are candidates for a subunit vaccine specifically for the G9P[6] rotavirus strain. This could be more effective in developing countries, thereby possibly providing a higher

  5. Safety and persistent immunogenicity of a quadrivalent human papillomavirus types 6, 11, 16, 18 L1 virus-like particle vaccine in preadolescents and adolescents: a randomized controlled trial.

    Science.gov (United States)

    Reisinger, Keith S; Block, Stan L; Lazcano-Ponce, Eduardo; Samakoses, Rudiwilai; Esser, Mark T; Erick, Joanne; Puchalski, Derek; Giacoletti, Katherine E D; Sings, Heather L; Lukac, Suzanne; Alvarez, Frances B; Barr, Eliav

    2007-03-01

    Administration of a quadrivalent HPV-6/ 1/16/18 vaccine to 16- to 26-year-old women was highly effective in preventing HPV-6/ 1/16/18-related cervical/vulvar/vaginal precancerous lesions and genital warts. As the risk of acquiring HPV significantly rises after sexual debut, HPV vaccines should have the greatest benefit in sexually naive adolescents. We evaluated the tolerability and immunogenicity of quadrivalent vaccine in males and females 9 to 15 years of age through 18 months postenrollment. In this randomized, double-blind trial, 1781 sexually naive children were assigned (2:1) to quadrivalent HPV-6/11/16/18 vaccine or saline placebo administered at day 1 and months 2 and 6. Serum neutralizing anti-HPV-6/11/16/18 responses were summarized as geometric mean titers (GMTs) and seroconversion rates. Primary analyses were done per-protocol (subjects received 3 doses, had no major protocol violations and were HPV type-specific seronegative at day 1). Adverse experiences were collected by diary card. At month 7, seroconversion rates were > or =99.5% for the 4 vaccine-HPV-types. GMTs and seroconversion rates in boys were noninferior to those in girls (P or =91.5% of vaccine recipients were seropositive, regardless of gender. A higher proportion of vaccine recipients (75.3%) than placebo recipients (50.0%) reported one or more injection-site adverse experiences following any vaccination. Rates of fever were similar between vaccination groups. No serious vaccine-related adverse experiences were reported. In 9- to 15-year-old adolescents, the quadrivalent vaccine was generally well tolerated and induced persistent anti-HPV serologic responses in the majority of subjects for at least 12 months following completion of a three-dose regimen. The vaccine durability supports universal HPV vaccination programs in adolescents to reduce the burden of clinical HPV disease, particularly cervical cancer and precancers.

  6. Self-Assembly of the Recombinant Capsid Protein of a Swine Norovirus into Virus-Like Particles and Evaluation of Monoclonal Antibodies Cross-Reactive with a Human Strain from Genogroup II

    NARCIS (Netherlands)

    Almanza, H.; Cubillos, C.; Angulo, I.; Mateos, F.; Castön, J.R.; Poel, van der W.H.M.; Vinje, J.; Bárcena, J.; Mena, I.

    2008-01-01

    Noroviruses (NoVs) are responsible for the majority of gastroenteritis outbreaks in humans. Recently, NoV strains which are genetically closely related to human genogroup II (GII) NoVs have been detected in fecal specimens from swine. These findings have raised concern about the possible role of

  7. Phase III, randomized controlled trial in girls 9-15 years old to evaluate lot consistency of a novel nine-valent human papillomavirus L1 virus-like particle vaccine.

    Science.gov (United States)

    Luxembourg, Alain; Moreira, Edson D; Samakoses, Rudiwilai; Kim, Kyung-Hyo; Sun, Xiao; Maansson, Roger; Moeller, Erin; Christiano, Susan; Chen, Joshua

    2015-01-01

    A 9-valent human papillomavirus (6/11/16/18/31/33/45/52/58) VLP (9vHPV) vaccine has recently been proven highly efficacious in preventing disease associated with vaccine HPV types in a pivotal Phase III study. The demonstration of lot-to-lot consistency to confirm the reliability of the manufacturing process is a regulatory requirement for vaccine licensure in the United States. A randomized trial was conducted to demonstrate that three lots of 9vHPV vaccine elicit equivalent antibody response for all 9 vaccine types. The study required thorough planning because it required success on 27 separate statistical comparisons. An innovative statistical approach was used taking into account between-lot variance for more conservative power calculations. The study demonstrated equivalence of three lots of 9vHPV vaccine for all 9 vaccine types.

  8. Transient expression of Human papillomavirus type 16 L2 epitope fused to N- and C-terminus of coat protein of Potato virus X in plants.

    Science.gov (United States)

    Cerovska, Noemi; Hoffmeisterova, Hana; Moravec, Tomas; Plchova, Helena; Folwarczna, Jitka; Synkova, Helena; Ryslava, Helena; Ludvikova, Viera; Smahel, Michal

    2012-03-01

    Transient expression of foreign genes based on plant viral vectors is a suitable system for the production of relevant immunogens that can be used for the development of a new generation of vaccines against a variety of infectious diseases. In the present study the epitope derived from HPV-16 L2 minor capsid protein (amino acids 108-120) was expressed from Potato virus X (PVX)-based vector pGR106 as N- or C-terminal fusion with the PVX coat protein (PVX CP) in transgenic Nicotiana benthamiana plants. The fusion protein L2 108-120-PVX CP was successfully expressed in plants at a level of 170 mg/kg of fresh leaf tissue. The C-terminal fusion protein PVX CP- L2 108-120 was expressed using mutated vector sequence to avoid homologous recombination at a level of 8 mg/kg of fresh leaf tissue. Immunogenicity of L2 108-120-PVX CP virus-like particles was tested after immunization of mice by subcutaneous injection or tattoo administration. In animal sera the antibodies against the PVX CP and the L2 108-120 epitope were found after both methods of vaccine delivery.

  9. Treatment of norovirus particles with citrate.

    Science.gov (United States)

    Koromyslova, Anna D; White, Peter A; Hansman, Grant S

    2015-11-01

    Human norovirus is a dominant cause of acute gastroenteritis around the world. Several norovirus disinfectants label citric acid as an active ingredient. In this study, we showed that norovirus virus-like particles (VLPs) treated with citrate buffer caused the particles to alter their morphology, including increased diameters associated with a new ring-like structure. We also found that epitopes on the protruding (P) domain on these particles were more readily accessible to antibodies after the citrate treatment. These results suggested that citrate had a direct effect on the norovirus particles. Using X-ray crystallography, we showed that the P domain bound citrate from lemon juice and a disinfectant containing citric acid. Importantly, citrate binds at the histo-blood group antigen binding pocket, which are attachment factors for norovirus infections. Taken together, these new findings suggested that it might be possible to treat/reduce norovirus infections with citrate, although further studies are needed. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Enhanced nigrostriatal neuron-specific, long-term expression by using neural-specific promoters in combination with targeted gene transfer by modified helper virus-free HSV-1 vector particles

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    Kong Lingxin

    2008-04-01

    Full Text Available Abstract Background Direct gene transfer into neurons has potential for developing gene therapy treatments for specific neurological conditions, and for elucidating neuronal physiology. Due to the complex cellular composition of specific brain areas, neuronal type-specific recombinant gene expression is required for many potential applications of neuronal gene transfer. One approach is to target gene transfer to a specific type of neuron. We developed modified Herpes Simplex Virus (HSV-1 particles that contain chimeric glycoprotein C (gC – glial cell line-derived neurotrophic factor (GDNF or brain-derived neurotrophic factor (BDNF proteins. HSV-1 vector particles containing either gC – GDNF or gC – BDNF target gene transfer to nigrostriatal neurons, which contain specific receptors for GDNF or BDNF. A second approach to achieve neuronal type-specific expression is to use a cell type-specific promoter, and we have used the tyrosine hydroxylase (TH promoter to restrict expression to catecholaminergic neurons or a modified neurofilament heavy gene promoter to restrict expression to neurons, and both of these promoters support long-term expression from HSV-1 vectors. To both improve nigrostriatal-neuron specific expression, and to establish that targeted gene transfer can be followed by long-term expression, we performed targeted gene transfer with vectors that support long-term, neuronal-specific expression. Results Helper virus-free HSV-1 vector packaging was performed using either gC – GDNF or gC – BDNF and vectors that contain either the TH promoter or the modified neurofilament heavy gene promoter. Vector stocks were injected into the midbrain proximal to the substantia nigra, and the rats were sacrificed at either 4 days or 1 month after gene transfer. Immunofluorescent costaining was performed to detect both recombinant gene products and nigrostriatal neurons. The combination of targeted gene transfer with neuronal

  11. Enhanced light microscopy visualization of virus particles from Zika virus to filamentous ebolaviruses.

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    George G Daaboul

    Full Text Available Light microscopy is a powerful tool in the detection and analysis of parasites, fungi, and prokaryotes, but has been challenging to use for the detection of individual virus particles. Unlabeled virus particles are too small to be visualized using standard visible light microscopy. Characterization of virus particles is typically performed using higher resolution approaches such as electron microscopy or atomic force microscopy. These approaches require purification of virions away from their normal millieu, requiring significant levels of expertise, and can only enumerate small numbers of particles per field of view. Here, we utilize a visible light imaging approach called Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS that allows automated counting and sizing of thousands of individual virions. Virions are captured directly from complex solutions onto a silicon chip and then detected using a reflectance interference imaging modality. We show that the use of different imaging wavelengths allows the visualization of a multitude of virus particles. Using Violet/UV illumination, the SP-IRIS technique is able to detect individual flavivirus particles (~40 nm, while green light illumination is capable of identifying and discriminating between vesicular stomatitis virus and vaccinia virus (~360 nm. Strikingly, the technology allows the clear identification of filamentous infe