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Sample records for virus particles administered

  1. Chimeric plant virus particles administered nasally or orally induce systemic and mucosal immune responses in mice

    DEFF Research Database (Denmark)

    Brennan, F.R.; Bellaby, T.; Helliwell, S.M.

    1999-01-01

    The humoral immune responses to the D2 peptide of fibronectin-binding protein B (FnBP) of Staphylococcus aureus, expressed on the plant virus cowpea mosaic virus (CPMV), were evaluated after mucosal delivery to mice. Intranasal immunization of these chimeric virus particles (CVPs), either alone o...... demonstrate for the first time that recombinant plant viruses have potential as mucosal vaccines without the requirement for adjuvant and that the nasal route is most effective for the delivery of these nonreplicating particles.......The humoral immune responses to the D2 peptide of fibronectin-binding protein B (FnBP) of Staphylococcus aureus, expressed on the plant virus cowpea mosaic virus (CPMV), were evaluated after mucosal delivery to mice. Intranasal immunization of these chimeric virus particles (CVPs), either alone...... to generate antibody at distant mucosal sites. IgG2a and TgG2b were the dominant IgG subclasses in sera to both CPMV and FnBP, demonstrating a bias in the response toward the T helper 1 type. The sera completely inhibited the binding of human fibronectin to the S. aureus FnBP. Oral immunization of the CVPs...

  2. 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...

  3. 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.

  4. Viruses and viruslike particles of eukaryotic algae.

    OpenAIRE

    Van Etten, J L; Lane, L C; Meints, R H

    1991-01-01

    Until recently there was little interest or information on viruses and viruslike particles of eukaryotic algae. However, this situation is changing. In the past decade many large double-stranded DNA-containing viruses that infect two culturable, unicellular, eukaryotic green algae have been discovered. These viruses can be produced in large quantities, assayed by plaque formation, and analyzed by standard bacteriophage techniques. The viruses are structurally similar to animal iridoviruses, t...

  5. Virus-like particles as nanovaccine candidates

    Science.gov (United States)

    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.

  6. Measurement of intravenously administered γ-Fe2O3 particle amount in mice tissues using vibrating sample magnetometer.

    Science.gov (United States)

    Kishimoto, Mikio; Miyamoto, Ryoichi; Oda, Tatsuya; Ohara, Yusuke; Yanagihara, Hideto; Ohkohchi, Nobuhiro; Kita, Eiji

    2014-12-01

    Dispersions of platelet γ-Fe2O3 particles 30-50nm in size were intravenously administered to mice and the amount of particles accumulated in each tissue was obtained by magnetization measurement using a vibrating sample magnetometer. Background noise was greatly reduced by measuring dried tissues under a magnetic field of 500 Oe so that the effect of diamagnetism was slight. Remarkable particle accumulation was observed in the liver and spleen. Considerable particle accumulation was observed in the lung when a large quantity of γ-Fe2 O3 particles was administered. There was no significant particle accumulation in the kidney and heart.

  7. Characterization of chikungunya virus-like particles.

    Directory of Open Access Journals (Sweden)

    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.

  8. Attenuated Human Parainfluenza Virus Type 1 Expressing Ebola Virus Glycoprotein GP Administered Intranasally Is Immunogenic in African Green Monkeys.

    Science.gov (United States)

    Lingemann, Matthias; Liu, Xueqiao; Surman, Sonja; Liang, Bo; Herbert, Richard; Hackenberg, Ashley D; Buchholz, Ursula J; Collins, Peter L; Munir, Shirin

    2017-05-15

    The recent 2014-2016 Ebola virus (EBOV) outbreak prompted increased efforts to develop vaccines against EBOV disease. We describe the development and preclinical evaluation of an attenuated recombinant human parainfluenza virus type 1 (rHPIV1) expressing the membrane-anchored form of EBOV glycoprotein GP, as an intranasal (i.n.) EBOV vaccine. GP was codon optimized and expressed either as a full-length protein or as an engineered chimeric form in which its transmembrane and cytoplasmic tail (TMCT) domains were replaced with those of the HPIV1 F protein in an effort to enhance packaging into the vector particle and immunogenicity. GP was inserted either preceding the N gene (pre-N) or between the N and P genes (N-P) of rHPIV1 bearing a stabilized attenuating mutation in the P/C gene (CΔ170). The constructs grew to high titers and efficiently and stably expressed GP. Viruses were attenuated, replicating at low titers over several days, in the respiratory tract of African green monkeys (AGMs). Two doses of candidates expressing GP from the pre-N position elicited higher GP neutralizing serum antibody titers than the N-P viruses, and unmodified GP induced higher levels than its TMCT counterpart. Unmodified EBOV GP was packaged into the HPIV1 particle, and the TMCT modification did not increase packaging or immunogenicity but rather reduced the stability of GP expression during in vivo replication. In conclusion, we identified an attenuated and immunogenic i.n. vaccine candidate expressing GP from the pre-N position. It is expected to be well tolerated in humans and is available for clinical evaluation.IMPORTANCE EBOV hemorrhagic fever is one of the most lethal viral infections and lacks a licensed vaccine. Contact of fluids from infected individuals, including droplets or aerosols, with mucosal surfaces is an important route of EBOV spread during a natural outbreak, and aerosols also might be exploited for intentional virus spread. Therefore, vaccines that protect

  9. Immunogenicity of a modified-live virus vaccine against bovine viral diarrhea virus types 1 and 2, infectious bovine rhinotracheitis virus, bovine parainfluenza-3 virus, and bovine respiratory syncytial virus when administered intranasally in young calves.

    Science.gov (United States)

    Xue, Wenzhi; Ellis, John; Mattick, Debra; Smith, Linda; Brady, Ryan; Trigo, Emilio

    2010-05-14

    The immunogenicity of an intranasally-administered modified-live virus (MLV) vaccine in 3-8 day old calves was evaluated against bovine viral diarrhea virus (BVDV) types 1 and 2, infectious bovine rhinotracheitis (IBR) virus, parainfluenza-3 (PI-3) virus and bovine respiratory syncytial virus (BRSV). Calves were intranasally vaccinated with a single dose of a multivalent MLV vaccine and were challenged with one of the respective viruses three to four weeks post-vaccination in five separate studies. There was significant sparing of diseases in calves intranasally vaccinated with the MLV vaccine, as indicated by significantly fewer clinical signs, lower rectal temperatures, reduced viral shedding, greater white blood cell and platelet counts, and less severe pulmonary lesions than control animals. This was the first MLV combination vaccine to demonstrate efficacy against BVDV types 1 and 2, IBR, PI-3 and BRSV in calves 3-8 days of age. Copyright 2010 Elsevier Ltd. All rights reserved.

  10. A dsRNA virus with filamentous viral particles.

    Science.gov (United States)

    Jia, Hengxia; Dong, Kaili; Zhou, Lingling; Wang, Guoping; Hong, Ni; Jiang, Daohong; Xu, Wenxing

    2017-08-01

    Viruses with double-stranded RNA genomes form isometric particles or are capsidless. Here we report a double-stranded RNA virus, Colletotrichum camelliae filamentous virus 1 (CcFV-1) isolated from a fungal pathogen, that forms filamentous particles. CcFV-1 has eight genomic double-stranded RNAs, ranging from 990 to 2444 bp, encoding 10 putative open reading frames, of which open reading frame 1 encodes an RNA-dependent RNA polymerase and open reading frame 4 a capsid protein. When inoculated, the naked CcFV-1 double-stranded RNAs are infectious and induce the accumulation of the filamentous particles in vivo. CcFV-1 is phylogenetically related to Aspergillus fumigatus tetramycovirus-1 and Beauveria bassiana polymycovirus-1, but differs in morphology and in the number of genomic components. CcFV-1 might be an intermediate virus related to truly capsidated viruses, or might represent a distinct encapsidating strategy. In terms of genome and particle architecture, our findings are a significant addition to the knowledge of the virosphere diversity.Viruses with double-stranded RNA (dsRNA) genomes form typically isometric particles or are capsid-less. Here, the authors identify a mycovirus with an eight-segmented dsRNA genome that forms exceptionally long filamentous particles and could represent an evolutionary link between ssRNA and dsRNA viruses.

  11. Evaluation of nanoparticle tracking analysis for total virus particle determination

    Directory of Open Access Journals (Sweden)

    Kramberger Petra

    2012-11-01

    Full Text Available Abstract The NanoSight LM10 with Nanoparticle tracking analysis (NTA software was evaluated for the quantification of latex particles, adenovirus 5, and influenza virus. The inter-day variability was determined by measuring the same sample over several consecutive days and the method’s accuracy was demonstrated by using known concentrations of the subject particles. NTA analysis was also used to quantify chromatographic fractions of adenovirus and influenza virus after purification on a CIM monolithic column. NTA results were compared and evaluated against hemagglutination (HA and end point dilution assay, determining total and infection virus particle number, respectively. The results demonstrated that nanoparticle tracking analysis is a method for fast estimation of virus concentration in different samples. In addition, it can provide a better insight into the sample status, regarding the level of virus aggregation.

  12. Enhanced light microscopy visualization of virus particles from Zika virus to filamentous ebolaviruses.

    Directory of Open Access Journals (Sweden)

    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 infectious ebolavirus particles and virus-like particles. The ability to differentiate and quantify unlabeled virus particles extends the usefulness of traditional light microscopy and can be embodied in a straightforward benchtop approach allowing widespread applications ranging from rapid detection in biological fluids to analysis of virus-like particles for vaccine development and production.

  13. 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,

  14. Intranasal administration of antibody-bound respiratory syncytial virus particles efficiently primes virus-specific immune responses in mice.

    Science.gov (United States)

    Kruijsen, Debby; Einarsdottir, Helga K; Schijf, Marcel A; Coenjaerts, Frank E; van der Schoot, Ellen C; Vidarsson, Gestur; van Bleek, Grada M

    2013-07-01

    Infants are protected from a severe respiratory syncytial virus (RSV) infection in the first months of life by maternal antibodies or by prophylactically administered neutralizing antibodies. Efforts are under way to produce RSV-specific antibodies with increased neutralizing capacity compared to the currently licensed palivizumab. While clearly beneficial during primary infections, preexisting antibodies might affect the onset of adaptive immune responses and the ability to resist subsequent RSV infections. Therefore, we addressed the question of how virus neutralizing antibodies influence the priming of subsequent adaptive immune responses. To test a possible role of the neonatal Fc receptor (FcRn) in this process, we compared the responses in C57BL/6 wild-type (WT) and FcRn(-/-) mice. We observed substantial virus-specific T-cell priming and B-cell responses in mice primed with RSV IgG immune complexes resulting in predominantly Th1-type CD4(+) T-cell and IgG2c antibody responses upon live-virus challenge. RSV-specific CD8(+) T cells were primed as well. Activation of these adaptive immune responses was independent of FcRn. Thus, neutralizing antibodies that localize to the airways and prevent infection-related routes of antigen processing can still facilitate antigen presentation of neutralized virus particles and initiate adaptive immune responses against RSV.

  15. 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

  16. Localization of the nonstructural protein NS1 in bluetongue virus-infected cells and its presence in virus particles.

    Science.gov (United States)

    Eaton, B T; Hyatt, A D; White, J R

    1988-04-01

    Seven monoclonal antibodies to the nonstructural protein NS1 of an Australian isolate of bluetongue virus (BTV) have been used in immunofluoresence and immunogold procedures to locate NS1 in virus-infected cells and cytoskeletons. The antibodies fall into three groups indicating that NS1 contains at least three antigenic sites. One group consists of four antibodies which react solely with cytoskeleton-associated virus-specific tubules. A second group contains one antibody which reacts with cytoskeleton-associated virus particles, released viruses, and purified virus and core particles. Two antibodies constituting a third group react with both tubules and cytoskeleton-associated and released virus particles. NS1 was found in [35S]methionine-labeled, purified virus and core particles. Immunofluorescence tests reveal that those antibodies which react with virus particles also bind to cytoskeleton-associated virus inclusion bodies (VIB). The nature of this association was examined by probing cytoskeletons of BTV-infected cells with antibodies to NS1 and protein A-gold. VIB observed in thin sections were not uniformly labeled. Gold was associated with fibrillar arrays found around virus particles either leaving or in close proximity to the VIB. Fibrillar material was not found in association with all virus particles elsewhere in the cell and this suggests that fibril-virus complexes may be intermediate in virus morphogenesis.

  17. Proteomic analysis of purified Newcastle disease virus particles

    Directory of Open Access Journals (Sweden)

    Ren Xiangpeng

    2012-05-01

    Full Text Available Abstract Background Newcastle disease virus (NDV is an enveloped RNA virus, bearing severe economic losses to the poultry industry worldwide. Previous virion proteomic studies have shown that enveloped viruses carry multiple host cellular proteins both internally and externally during their life cycle. To address whether it also occurred during NDV infection, we performed a comprehensive proteomic analysis of highly purified NDV La Sota strain particles. Results In addition to five viral structural proteins, we detected thirty cellular proteins associated with purified NDV La Sota particles. The identified cellular proteins comprised several functional categories, including cytoskeleton proteins, annexins, molecular chaperones, chromatin modifying proteins, enzymes-binding proteins, calcium-binding proteins and signal transduction-associated proteins. Among these, three host proteins have not been previously reported in virions of other virus families, including two signal transduction-associated proteins (syntenin and Ras small GTPase and one tumor-associated protein (tumor protein D52. The presence of five selected cellular proteins (i.e., β-actin, tubulin, annexin A2, heat shock protein Hsp90 and ezrin associated with the purified NDV particles was validated by Western blot or immunogold labeling assays. Conclusions The current study presented the first standard proteomic profile of NDV. The results demonstrated the incorporation of cellular proteins in NDV particles, which provides valuable information for elucidating viral infection and pathogenesis.

  18. Proteomic analysis of purified Newcastle disease virus particles.

    Science.gov (United States)

    Ren, Xiangpeng; Xue, Chunyi; Kong, Qingming; Zhang, Chengwen; Bi, Yingzuo; Cao, Yongchang

    2012-05-09

    Newcastle disease virus (NDV) is an enveloped RNA virus, bearing severe economic losses to the poultry industry worldwide. Previous virion proteomic studies have shown that enveloped viruses carry multiple host cellular proteins both internally and externally during their life cycle. To address whether it also occurred during NDV infection, we performed a comprehensive proteomic analysis of highly purified NDV La Sota strain particles. In addition to five viral structural proteins, we detected thirty cellular proteins associated with purified NDV La Sota particles. The identified cellular proteins comprised several functional categories, including cytoskeleton proteins, annexins, molecular chaperones, chromatin modifying proteins, enzymes-binding proteins, calcium-binding proteins and signal transduction-associated proteins. Among these, three host proteins have not been previously reported in virions of other virus families, including two signal transduction-associated proteins (syntenin and Ras small GTPase) and one tumor-associated protein (tumor protein D52). The presence of five selected cellular proteins (i.e., β-actin, tubulin, annexin A2, heat shock protein Hsp90 and ezrin) associated with the purified NDV particles was validated by Western blot or immunogold labeling assays. The current study presented the first standard proteomic profile of NDV. The results demonstrated the incorporation of cellular proteins in NDV particles, which provides valuable information for elucidating viral infection and pathogenesis.

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

    NARCIS (Netherlands)

    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

  20. The genetics of virus particle shape in equine influenza A virus.

    Science.gov (United States)

    Elton, Debra; Bruce, Emily A; Bryant, Neil; Wise, Helen M; MacRae, Shona; Rash, Adam; Smith, Nikki; Turnbull, Matthew L; Medcalf, Liz; Daly, Janet M; Digard, Paul

    2013-12-01

    Many human strains of influenza A virus produce highly pleomorphic virus particles that at the extremes can be approximated as either spheres of around 100 nm diameter or filaments of similar cross-section but elongated to lengths of many microns. The role filamentous virions play in the virus life cycle remains enigmatic. Here, we set out to define the morphology and genetics of virus particle shape in equine influenza A virus, using reverse genetics and microscopy of infected cells. The majority of H3N8 strains tested were found to produce filamentous virions, as did the prototype H7N7 A/eq/Prague/56 strain. The exception was the prototype H3N8 isolate, A/eq/Miami/63. Reassortment of equine influenza virus M genes from filamentous and non-filamentous strains into the non-filamentous human virus A/PR/8/34 confirmed that segment 7 is a major determinant of particle shape. Sequence analysis identified three M1 amino acid polymorphisms plausibly associated with determining virion morphology, and the introduction of these changes into viruses confirmed the importance of two: S85N and N231D. However, while either change alone affected filament production, the greatest effect was seen when the polymorphisms were introduced in conjunction. Thus, influenza A viruses from equine hosts also produce filamentous virions, and the major genetic determinants are set by the M1 protein. However, the precise sequence determinants are different to those previously identified in human or porcine viruses. © 2013 Blackwell Publishing Ltd.

  1. Effects of intravenously administered recombinant vesicular stomatitis virus (VSV(deltaM51)) on multifocal and invasive gliomas.

    Science.gov (United States)

    Lun, XueQing; Senger, Donna L; Alain, Tommy; Oprea, Andra; Parato, Kelley; Stojdl, Dave; Lichty, Brian; Power, Anthony; Johnston, Randal N; Hamilton, Mark; Parney, Ian; Bell, John C; Forsyth, Peter A

    2006-11-01

    An ideal virus for the treatment of cancer should have effective delivery into multiple sites within the tumor, evade immune responses, produce rapid viral replication, spread within the tumor, and infect multiple tumors. Vesicular stomatitis virus (VSV) has been shown to be an effective oncolytic virus in a variety of tumor models, and mutations in the matrix (M) protein enhance VSV's effectiveness in animal models. We evaluated the susceptibility of 14 glioma cell lines to infection and killing by mutant strain VSV(deltaM51), which contains a single-amino acid deletion in the M protein. We also examined the activity and safety of this strain against the U87 and U118 experimental models of human malignant glioma in nude mice and analyzed the distribution of the virus in the brains of U87 tumor-bearing mice using fluorescence labeling. Finally, we examined the effect of VSV(deltaM51) on 15 primary human gliomas cultured from surgical specimens. All statistical tests were two-sided. All 14 glioma cell lines were susceptible to VSV(deltaM51) infection and killing. Intratumoral administration of VSV(deltaM51) produced marked regression of malignant gliomas in nude mice. When administered systemically, live VSV(deltaM51) virus, as compared with dead virus, statistically significantly prolonged survival of mice with unilateral U87 tumors (median survival: 113 versus 46 days, P = .0001) and bilateral U87 tumors (median survival: 73 versus 46 days, P = .0025). VSV(deltaM51) infected multifocal gliomas, invasive glioma cells that migrated beyond the main glioma, and all 15 primary human gliomas. There was no evidence of toxicity. Systemically delivered VSV(deltaM51) was an effective and safe oncolytic agent against laboratory models of multifocal and invasive malignant gliomas, the most challenging clinical manifestations of this disease.

  2. 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...

  3. 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.

  4. Progress in Developing Virus-like Particle Influenza Vaccines

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    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

  5. 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

  6. 3D rotating wall vessel and 2D cell culture of four veterinary virus pathogens: A comparison of virus yields, portions of infectious particles and virus growth curves.

    Science.gov (United States)

    Malenovská, Hana

    2016-02-01

    Only very few comparative studies have been performed that evaluate general trends of virus growth under 3D in comparison with 2D cell culture conditions. The aim of this study was to investigate differences when four animal viruses are cultured in 2D and 3D. Suid herpesvirus 1 (SuHV-1), Vesicular stomatitis virus (VSIV), Bovine adenovirus (BAdV) and Bovine parainfluenza 3 virus (BPIV-3) were cultivated in 3D rotating wall vessels (RWVs) and conventional 2D cultures. The production of virus particles, the portion of infectious particles, and the infectious growth curves were compared. For all viruses, the production of virus particles (related to cell density), including the non-infectious ones, was lower in 3D than in 2D culture. The production of only infectious particles was significantly lower in BAdV and BPIV-3 in 3D cultures in relation to cell density. The two cultivation approaches resulted in significantly different virus particle-to-TCID50 ratios in three of the four viruses: lower in SuHV-1 and BPIV-3 and higher in BAdV in 3D culture. The infectious virus growth rates were not significantly different in all viruses. Although 3D RWV culture resulted in lower production of virus particles compared to 2D systems, the portion of infectious particles was higher for some viruses. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. LIPOSOMES AS AN IMMUNOADJUVANT SYSTEM FOR STIMULATION OF MUCOSAL AND SYSTEMIC ANTIBODY-RESPONSES AGAINST INACTIVATED MEASLES-VIRUS ADMINISTERED INTRANASALLY TO MICE

    NARCIS (Netherlands)

    DEHAAN, A; TOMEE, JFC; HUCHSHORN, JP; WILSCHUT, J

    1995-01-01

    This paper reports on the immune-stimulatory activity of liposomes in an inactivated whole measles virus vaccine preparation administered intranasally to mice. Liposomes, simply mixed with inactivated whole measles virus, significantly stimulated the serum IgG response relative to the response to

  8. 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.

  9. Proteomic analysis of purified coronavirus infectious bronchitis virus particles.

    Science.gov (United States)

    Kong, Qingming; Xue, Chunyi; Ren, Xiangpeng; Zhang, Chengwen; Li, Linlin; Shu, Dingming; Bi, Yingzuo; Cao, Yongchang

    2010-06-09

    Infectious bronchitis virus (IBV) is the coronavirus of domestic chickens causing major economic losses to the poultry industry. Because of the complexity of the IBV life cycle and the small number of viral structural proteins, important virus-host relationships likely remain to be discovered. Toward this goal, we performed two-dimensional gel electrophoresis fractionation coupled to mass spectrometry identification approaches to perform a comprehensive proteomic analysis of purified IBV particles. Apart from the virus-encoded structural proteins, we detected 60 host proteins in the purified virions which can be grouped into several functional categories including intracellular trafficking proteins (20%), molecular chaperone (18%), macromolcular biosynthesis proteins (17%), cytoskeletal proteins (15%), signal transport proteins (15%), protein degradation (8%), chromosome associated proteins (2%), ribosomal proteins (2%), and other function proteins (3%). Interestingly, 21 of the total host proteins have not been reported to be present in virions of other virus families, such as major vault protein, TENP protein, ovalbumin, and scavenger receptor protein. Following identification of the host proteins by proteomic methods, the presence of 4 proteins in the purified IBV preparation was verified by western blotting and immunogold labeling detection. The results present the first standard proteomic profile of IBV and may facilitate the understanding of the pathogenic mechanisms.

  10. Proteomic analysis of purified coronavirus infectious bronchitis virus particles

    Directory of Open Access Journals (Sweden)

    Shu Dingming

    2010-06-01

    Full Text Available Abstract Background Infectious bronchitis virus (IBV is the coronavirus of domestic chickens causing major economic losses to the poultry industry. Because of the complexity of the IBV life cycle and the small number of viral structural proteins, important virus-host relationships likely remain to be discovered. Toward this goal, we performed two-dimensional gel electrophoresis fractionation coupled to mass spectrometry identification approaches to perform a comprehensive proteomic analysis of purified IBV particles. Results Apart from the virus-encoded structural proteins, we detected 60 host proteins in the purified virions which can be grouped into several functional categories including intracellular trafficking proteins (20%, molecular chaperone (18%, macromolcular biosynthesis proteins (17%, cytoskeletal proteins (15%, signal transport proteins (15%, protein degradation (8%, chromosome associated proteins (2%, ribosomal proteins (2%, and other function proteins (3%. Interestingly, 21 of the total host proteins have not been reported to be present in virions of other virus families, such as major vault protein, TENP protein, ovalbumin, and scavenger receptor protein. Following identification of the host proteins by proteomic methods, the presence of 4 proteins in the purified IBV preparation was verified by western blotting and immunogold labeling detection. Conclusions The results present the first standard proteomic profile of IBV and may facilitate the understanding of the pathogenic mechanisms.

  11. 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.

  12. 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.

  13. Cytoplasmic Motifs in the Nipah Virus Fusion Protein Modulate Virus Particle Assembly and Egress.

    Science.gov (United States)

    Johnston, Gunner P; Contreras, Erik M; Dabundo, Jeffrey; Henderson, Bryce A; Matz, Keesha M; Ortega, Victoria; Ramirez, Alfredo; Park, Arnold; Aguilar, Hector C

    2017-05-15

    Nipah virus (NiV), a paramyxovirus in the genus Henipavirus, has a mortality rate in humans of approximately 75%. While several studies have begun our understanding of NiV particle formation, the mechanism of this process remains to be fully elucidated. For many paramyxoviruses, M proteins drive viral assembly and egress; however, some paramyxoviral glycoproteins have been reported as important or essential in budding. For NiV the matrix protein (M), the fusion glycoprotein (F) and, to a much lesser extent, the attachment glycoprotein (G) autonomously induce the formation of virus-like particles (VLPs). However, functional interactions between these proteins during assembly and egress remain to be fully understood. Moreover, if the F-driven formation of VLPs occurs through interactions with host cell machinery, the cytoplasmic tail (CT) of F is a likely interactive domain. Therefore, we analyzed NiV F CT deletion and alanine mutants and report that several but not all regions of the F CT are necessary for efficient VLP formation. Two of these regions contain YXXØ or dityrosine motifs previously shown to interact with cellular machinery involved in F endocytosis and transport. Importantly, our results showed that F-driven, M-driven, and M/F-driven viral particle formation enhanced the recruitment of G into VLPs. By identifying key motifs, specific residues, and functional viral protein interactions important for VLP formation, we improve our understanding of the viral assembly/egress process and point to potential interactions with host cell machinery.IMPORTANCE Henipaviruses can cause deadly infections of medical, veterinary, and agricultural importance. With recent discoveries of new henipa-like viruses, understanding the mechanisms by which these viruses reproduce is paramount. We have focused this study on identifying the functional interactions of three Nipah virus proteins during viral assembly and particularly on the role of one of these proteins, the fusion

  14. Possible therapeutic effect of orally administered ribavirin for respiratory syncytial virus-induced acute respiratory distress syndrome in an immunocompetent patient: a case report.

    Science.gov (United States)

    Yoon, Byung Woo; Lee, Seung Hyeun

    2017-12-20

    Human respiratory syncytial virus usually causes self-limiting upper respiratory infection and occasionally causes pneumonia in immunocompromised hosts. Respiratory syncytial virus-induced severe pneumonia or acute respiratory distress syndrome in immunocompetent adults has been rarely described. Unfortunately, optimal treatment has not been established for this potentially fatal condition. We report a case of respiratory syncytial virus-induced acute respiratory distress syndrome occurring in a previously healthy man successfully treated with orally administered ribavirin. An 81-year-old previously healthy Korean man presented with cough, dyspnea, and febrile sensation. He had hypoxemia with diffuse ground glass opacity evident on chest radiography, which progressed and required mechanical ventilation. All microbiological tests were negative except multiplex real-time reverse transcriptase polymerase chain reaction using respiratory specimen, which was positive for human adenovirus. Under the diagnosis of respiratory syncytial virus-induced acute respiratory distress syndrome, orally administered ribavirin was administered and he recuperated completely without complications. This case demonstrates the potential usefulness of orally administered ribavirin as a therapeutic option for severe respiratory syncytial virus infection, at least in an immunocompetent host.

  15. Nasally administered Lactobacillus rhamnosus strains differentially modulate respiratory antiviral immune responses and induce protection against respiratory syncytial virus infection.

    Science.gov (United States)

    Tomosada, Yohsuke; Chiba, Eriko; Zelaya, Hortensia; Takahashi, Takuya; Tsukida, Kohichiro; Kitazawa, Haruki; Alvarez, Susana; Villena, Julio

    2013-08-15

    Some studies have shown that nasally administered immunobiotics had the potential to improve the outcome of influenza virus infection. However, the capacity of immunobiotics to improve protection against respiratory syncytial virus (RSV) infection was not investigated before. The aims of this study were: a) to evaluate whether the nasal administration of Lactobacillus rhamnosus CRL1505 (Lr05) and L. rhamnosus CRL1506 (Lr06) are able to improve respiratory antiviral defenses and beneficially modulate the immune response triggered by TLR3/RIG-I activation; b) to investigate whether viability of Lr05 or Lr06 is indispensable to modulate respiratory immunity and; c) to evaluate the capacity of Lr05 and Lr06 to improve the resistance of infant mice against RSV infection. Nasally administered Lr05 and Lr06 differentially modulated the TLR3/RIG-I-triggered antiviral respiratory immune response. Lr06 administration significantly modulated the production of IFN-α, IFN-β and IL-6 in the response to poly(I:C) challenge, while nasal priming with Lr05 was more effective to improve levels of IFN-γ and IL-10. Both viable Lr05 and Lr06 strains increased the resistance of infant mice to RSV infection while only heat-killed Lr05 showed a protective effect similar to those observed with viable strains. The present work demonstrated that nasal administration of immunobiotics is able to beneficially modulate the immune response triggered by TLR3/RIG-I activation in the respiratory tract and to increase the resistance of mice to the challenge with RSV. Comparative studies using two Lactobacillus rhamnosus strains of the same origin and with similar technological properties showed that each strain has an specific immunoregulatory effect in the respiratory tract and that they differentially modulate the immune response after poly(I:C) or RSV challenges, conferring different degree of protection and using distinct immune mechanisms. We also demonstrated in this work that it is possible

  16. Analysis of Prototype Foamy Virus particle-host cell interaction with autofluorescent retroviral particles

    Directory of Open Access Journals (Sweden)

    Schwille Petra

    2010-05-01

    Full Text Available Abstract Background The foamy virus (FV replication cycle displays several unique features, which set them apart from orthoretroviruses. First, like other B/D type orthoretroviruses, FV capsids preassemble at the centrosome, but more similar to hepadnaviruses, FV budding is strictly dependent on cognate viral glycoprotein coexpression. Second, the unusually broad host range of FV is thought to be due to use of a very common entry receptor present on host cell plasma membranes, because all cell lines tested in vitro so far are permissive. Results In order to take advantage of modern fluorescent microscopy techniques to study FV replication, we have created FV Gag proteins bearing a variety of protein tags and evaluated these for their ability to support various steps of FV replication. Addition of even small N-terminal HA-tags to FV Gag severely impaired FV particle release. For example, release was completely abrogated by an N-terminal autofluorescent protein (AFP fusion, despite apparently normal intracellular capsid assembly. In contrast, C-terminal Gag-tags had only minor effects on particle assembly, egress and particle morphogenesis. The infectivity of C-terminal capsid-tagged FV vector particles was reduced up to 100-fold in comparison to wild type; however, infectivity was rescued by coexpression of wild type Gag and assembly of mixed particles. Specific dose-dependent binding of fluorescent FV particles to target cells was demonstrated in an Env-dependent manner, but not binding to target cell-extracted- or synthetic- lipids. Screening of target cells of various origins resulted in the identification of two cell lines, a human erythroid precursor- and a zebrafish- cell line, resistant to FV Env-mediated FV- and HIV-vector transduction. Conclusions We have established functional, autofluorescent foamy viral particles as a valuable new tool to study FV - host cell interactions using modern fluorescent imaging techniques. Furthermore, we

  17. Analysis of prototype foamy virus particle-host cell interaction with autofluorescent retroviral particles.

    Science.gov (United States)

    Stirnnagel, Kristin; Lüftenegger, Daniel; Stange, Annett; Swiersy, Anka; Müllers, Erik; Reh, Juliane; Stanke, Nicole; Grosse, Arend; Chiantia, Salvatore; Keller, Heiko; Schwille, Petra; Hanenberg, Helmut; Zentgraf, Hanswalter; Lindemann, Dirk

    2010-05-17

    The foamy virus (FV) replication cycle displays several unique features, which set them apart from orthoretroviruses. First, like other B/D type orthoretroviruses, FV capsids preassemble at the centrosome, but more similar to hepadnaviruses, FV budding is strictly dependent on cognate viral glycoprotein coexpression. Second, the unusually broad host range of FV is thought to be due to use of a very common entry receptor present on host cell plasma membranes, because all cell lines tested in vitro so far are permissive. In order to take advantage of modern fluorescent microscopy techniques to study FV replication, we have created FV Gag proteins bearing a variety of protein tags and evaluated these for their ability to support various steps of FV replication. Addition of even small N-terminal HA-tags to FV Gag severely impaired FV particle release. For example, release was completely abrogated by an N-terminal autofluorescent protein (AFP) fusion, despite apparently normal intracellular capsid assembly. In contrast, C-terminal Gag-tags had only minor effects on particle assembly, egress and particle morphogenesis. The infectivity of C-terminal capsid-tagged FV vector particles was reduced up to 100-fold in comparison to wild type; however, infectivity was rescued by coexpression of wild type Gag and assembly of mixed particles. Specific dose-dependent binding of fluorescent FV particles to target cells was demonstrated in an Env-dependent manner, but not binding to target cell-extracted- or synthetic- lipids. Screening of target cells of various origins resulted in the identification of two cell lines, a human erythroid precursor- and a zebrafish- cell line, resistant to FV Env-mediated FV- and HIV-vector transduction. We have established functional, autofluorescent foamy viral particles as a valuable new tool to study FV--host cell interactions using modern fluorescent imaging techniques. Furthermore, we succeeded for the first time in identifying two cell lines

  18. Mediators of protection against lethal systemic vesicular stomatitis virus infection in hamsters: defective interfering particles, polyinosinate-polycytidylate, and interferon

    Energy Technology Data Exchange (ETDEWEB)

    Fultz, P.N.; Shadduck, J.A.; Kang, C.Y.; Streilein, J.W.

    1982-08-01

    Homologous defective interfering (DI) particles protected adult Syrian hamsters against lethal systemic infection with vesicular stomatitis virus (VSV) serotype Indiana. The DI particles had to be biologically active, but did not have to be administered at the same inoculation site as the infectious virus. Serum and tissue levels of VSV postinoculation were significantly lower in DI-protected animals than in unprotected controls, suggesting that true autointerference was occurring. However, some aspects of protection also must be mediated through nonspecific mechanisms, since susceptible hamsters could be protected against VSV Indiana by coinjection with heterologous DI particles prepared from VSV serotype New Jersey or by simultaneous administration of polyinosinic acid-polycytidylic acid. By measuring serum levels of putative hamster interferon (type 1), we found that animals coinjected with VSV and DI particles or polyinosinic acid-polycytidylic acid produced significant levels of interferon. Since similarly high serum levels of interferon were measured in recipients of VSV alone (animals that eventually died from infection), there appeared to be no correlation between protection against lethal disease and induced levels of serum interferon. Instead, serum interferon levels correlated positively with amounts of VSV PFU found in serum and tissues of infected animals, the lowest levels being found in serum of animals protected with homologous DI particles. The data are consistent with the hypothesis that autointerference by DI particles as well as various host defense mechanisms (possibly including induction of interferon) participates in protecting hamsters against lethal VSV infection.

  19. CDC guidance for evaluating health-care personnel for hepatitis B virus protection and for administering postexposure management.

    Science.gov (United States)

    Schillie, Sarah; Murphy, Trudy V; Sawyer, Mark; Ly, Kathleen; Hughes, Elizabeth; Jiles, Ruth; de Perio, Marie A; Reilly, Meredith; Byrd, Kathy; Ward, John W

    2013-12-20

    This report contains CDC guidance that augments the 2011 recommendations of the Advisory Committee on Immunization Practices (ACIP) for evaluating hepatitis B protection among health-care personnel (HCP) and administering post-exposure prophylaxis. Explicit guidance is provided for persons working, training, or volunteering in health-care settings who have documented hepatitis B (HepB) vaccination years before hire or matriculation (e.g., when HepB vaccination was received as part of routine infant [recommended since 1991] or catch-up adolescent [recommended since 1995] vaccination). In the United States, 2,890 cases of acute hepatitis B were reported to CDC in 2011, and an estimated 18,800 new cases of hepatitis B occurred after accounting for underreporting of cases and asymptomatic infection. Although the rate of acute hepatitis B virus (HBV) infections have declined approximately 89% during 1990-2011, from 8.5 to 0.9 cases per 100,000 population in the United States, the risk for occupationally acquired HBV among HCP persists, largely from exposures to patients with chronic HBV infection. ACIP recommends HepB vaccination for unvaccinated or incompletely vaccinated HCP with reasonably anticipated risk for blood or body fluid exposure. ACIP also recommends that vaccinated HCP receive postvaccination serologic testing (antibody to hepatitis B surface antigen [anti-HBs]) 1-2 months after the final dose of vaccine is administered (CDC. Immunization of health-care personnel: recommendations of the Advisory Committee on Immunization Practices [ACIP]. MMWR 2011;60 [No. RR-7]). Increasing numbers of HCP have received routine HepB vaccination either as infants (recommended since 1991) or as catch-up vaccination (recommended since 1995) in adolescence. HepB vaccination results in protective anti-HBs responses among approximately 95% of healthy-term infants. Certain institutions test vaccinated HCP by measuring anti-HBs upon hire or matriculation, even when anti

  20. Tomato spotted wilt virus particle assembly : studying the role of the structural proteins in vivo

    NARCIS (Netherlands)

    Snippe, M.

    2006-01-01

    Members of the Bunyaviridae have spherical, enveloped virus particles that acquire their lipid membrane at the Golgi complex. For the animal-infecting bunyaviruses, virus assembly involves budding of ribonucleoprotein particles (RNPs) into vacuolised lumen of the Golgi complex, after which the

  1. Evaluation of Approaches for Tracking Virus Particles in Fluorescence Microscopy Images

    Science.gov (United States)

    Godinez, W. J.; Lampe, M.; Wörz, S.; Müller, B.; Eils, R.; Rohr, K.

    Tracking virus particles in fluorescence microscopy image sequences enables the characterization of the dynamical behavior of these objects. Several approaches have been developed for the task of virus tracking. However, few studies have quantitatively evaluated the performance of the different approaches. Such a comparison is essential to predict the performance of the approaches under realistic conditions. In this paper, we present a quantitative evaluation of eight approaches for tracking virus particles. We have investigated deterministic and probabilistic approaches. The evaluation is based on nine real microscopy image sequences of virus particles, for which ground truth was obtained by manual tracking.

  2. Treatment of Chronic Hepatitis C Virus Infection With Crushed Ledipasvir/Sofosbuvir Administered via a Percutaneous Endoscopic Gastrostomy Tube.

    Science.gov (United States)

    Jindracek, Lauren; Stark, Jennifer

    2017-01-01

    Ledipasvir/sofosbuvir (Harvoni®) is a fixed-dose tablet indicated for the treatment of chronic hepatitis C virus (HCV) infection. There are currently no data available on the safety and efficacy of crushed ledipasvir/sofosbuvir tablets. This report describes the first documented case of successful treatment of chronic HCV infection in a patient crushing ledipasvir/sofosbuvir for administration via a percutaneous endoscopic gastrostomy (PEG) tube. The patient was treatment experienced and had evidence of compensated cirrhosis. Treatment duration was 24 weeks, and HCV RNA was undetectable 12 weeks after completion of treatment (SVR12) which is the accepted measure of a clinical cure. Issues may arise during or prior to starting HCV treatment that necessitate crushing tablets. Stopping or interrupting HCV treatment could lead to development of resistance or treatment failure. This is the first published case in which crushed ledipasvir/sofosbuvir administered via a PEG tube is documented as a safe and effective option for treatment of chronic HCV infection.

  3. Multiple-Dose Pharmacokinetic Behavior of Elvucitabine, a Nucleoside Reverse Transcriptase Inhibitor, Administered over 21 Days with Lopinavir-Ritonavir in Human Immunodeficiency Virus Type 1-Infected Subjects

    NARCIS (Netherlands)

    Colucci, Philippe; Pottage, John C.; Robison, Heather; Turgeon, Jacques; Schuermann, Dirk; Hoepelman, I. M.; Ducharme, Murray P.

    The purpose of this study was to describe the plasma pharmacokinetics (PK) of elvucitabine at different doses when administered daily or every other day for 21 days with lopinavir-ritonavir ( Kaletra) in human immunodeficiency virus (HIV)-infected subjects. Three different dosing regimens of

  4. 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.

  5. Murine Leukemia Virus (MLV)-based Coronavirus Spike-pseudotyped Particle Production and Infection.

    Science.gov (United States)

    Millet, Jean Kaoru; Whittaker, Gary R

    2016-12-05

    Viral pseudotyped particles (pp) are enveloped virus particles, typically derived from retroviruses or rhabdoviruses, that harbor heterologous envelope glycoproteins on their surface and a genome lacking essential genes. These synthetic viral particles are safer surrogates of native viruses and acquire the tropism and host entry pathway characteristics governed by the heterologous envelope glycoprotein used. They have proven to be very useful tools used in research with many applications, such as enabling the study of entry pathways of enveloped viruses and to generate effective gene-delivery vectors. The basis for their generation lies in the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins of other viruses into a pseudotyped virus particle. These can be engineered to contain reporter genes such as luciferase, enabling quantification of virus entry events upon pseudotyped particle infection with susceptible cells. Here, we detail a protocol enabling generation of MLV-based pseudotyped particles, using the Middle East respiratory syndrome coronavirus (MERS-CoV) spike (S) as an example of a heterologous envelope glycoprotein to be incorporated. We also describe how these particles are used to infect susceptible cells and to perform a quantitative infectivity readout by a luciferase assay.

  6. Influenza Virus Reassortment Is Enhanced by Semi-infectious Particles but Can Be Suppressed by Defective Interfering Particles.

    Science.gov (United States)

    Fonville, Judith M; Marshall, Nicolle; Tao, Hui; Steel, John; Lowen, Anice C

    2015-10-01

    A high particle to infectivity ratio is a feature common to many RNA viruses, with ~90-99% of particles unable to initiate a productive infection under low multiplicity conditions. A recent publication by Brooke et al. revealed that, for influenza A virus (IAV), a proportion of these seemingly non-infectious particles are in fact semi-infectious. Semi-infectious (SI) particles deliver an incomplete set of viral genes to the cell, and therefore cannot support a full cycle of replication unless complemented through co-infection. In addition to SI particles, IAV populations often contain defective-interfering (DI) particles, which actively interfere with production of infectious progeny. With the aim of understanding the significance to viral evolution of these incomplete particles, we tested the hypothesis that SI and DI particles promote diversification through reassortment. Our approach combined computational simulations with experimental determination of infection, co-infection and reassortment levels following co-inoculation of cultured cells with two distinct influenza A/Panama/2007/99 (H3N2)-based viruses. Computational results predicted enhanced reassortment at a given % infection or multiplicity of infection with increasing semi-infectious particle content. Comparison of experimental data to the model indicated that the likelihood that a given segment is missing varies among the segments and that most particles fail to deliver ≥1 segment. To verify the prediction that SI particles augment reassortment, we performed co-infections using viruses exposed to low dose UV. As expected, the introduction of semi-infectious particles with UV-induced lesions enhanced reassortment. In contrast to SI particles, inclusion of DI particles in modeled virus populations could not account for observed reassortment outcomes. DI particles were furthermore found experimentally to suppress detectable reassortment, relative to that seen with standard virus stocks, most likely by

  7. Influenza Virus Reassortment Is Enhanced by Semi-infectious Particles but Can Be Suppressed by Defective Interfering Particles.

    Directory of Open Access Journals (Sweden)

    Judith M Fonville

    2015-10-01

    Full Text Available A high particle to infectivity ratio is a feature common to many RNA viruses, with ~90-99% of particles unable to initiate a productive infection under low multiplicity conditions. A recent publication by Brooke et al. revealed that, for influenza A virus (IAV, a proportion of these seemingly non-infectious particles are in fact semi-infectious. Semi-infectious (SI particles deliver an incomplete set of viral genes to the cell, and therefore cannot support a full cycle of replication unless complemented through co-infection. In addition to SI particles, IAV populations often contain defective-interfering (DI particles, which actively interfere with production of infectious progeny. With the aim of understanding the significance to viral evolution of these incomplete particles, we tested the hypothesis that SI and DI particles promote diversification through reassortment. Our approach combined computational simulations with experimental determination of infection, co-infection and reassortment levels following co-inoculation of cultured cells with two distinct influenza A/Panama/2007/99 (H3N2-based viruses. Computational results predicted enhanced reassortment at a given % infection or multiplicity of infection with increasing semi-infectious particle content. Comparison of experimental data to the model indicated that the likelihood that a given segment is missing varies among the segments and that most particles fail to deliver ≥1 segment. To verify the prediction that SI particles augment reassortment, we performed co-infections using viruses exposed to low dose UV. As expected, the introduction of semi-infectious particles with UV-induced lesions enhanced reassortment. In contrast to SI particles, inclusion of DI particles in modeled virus populations could not account for observed reassortment outcomes. DI particles were furthermore found experimentally to suppress detectable reassortment, relative to that seen with standard virus stocks

  8. Rapid detection of hendra virus using magnetic particles and quantum dots.

    Science.gov (United States)

    Lisi, Fabio; Falcaro, Paolo; Buso, Dario; Hill, Anita J; Barr, Jennifer A; Crameri, Gary; Nguyen, Tich-Lam; Wang, Lin-Fa; Mulvaney, Paul

    2012-09-01

    A proof-of-concept for the development of a fast and portable Hendra virus biosensor is presented. Hendra virus, a deadly emerging pathogen in Australia, can be co-localized, concentrated and revealed using simultaneously magnetic and luminescent functional particles. This method should be applicable for the early detection of any other virus by targeting the specific virus with the corresponding antibody. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Potency of whole virus particle and split virion vaccines using dissolving microneedle against challenges of H1N1 and H5N1 influenza viruses in mice.

    Science.gov (United States)

    Nakatsukasa, Akihiro; Kuruma, Koji; Okamatsu, Masatoshi; Hiono, Takahiro; Suzuki, Mizuho; Matsuno, Keita; Kida, Hiroshi; Oyamada, Takayoshi; Sakoda, Yoshihiro

    2017-05-15

    Transdermal vaccination using a microneedle (MN) confers enhanced immunity compared with subcutaneous (SC) vaccination. Here we developed a novel dissolving MN patch for the influenza vaccine. The potencies of split virion and whole virus particle (WVP) vaccines prepared from A/Puerto Rico/8/1934 (H1N1) and A/duck/Hokkaido/Vac-3/2007 (H5N1), respectively, were evaluated. MN vaccination induced higher neutralizing antibody responses than SC vaccination in mice. Moreover, MN vaccination with a lower dose of antigens conferred protective immunity against lethal challenges of influenza viruses than SC vaccination in mice. These results suggest that the WVP vaccines administered using MN are an effective combination for influenza vaccine to be further validated in humans. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. 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

  11. Anti-tumor effects of inactivated Sendai virus particles with an IL-2 gene on angiosarcoma.

    Science.gov (United States)

    Takehara, Yuki; Satoh, Takahiro; Nishizawa, Aya; Saeki, Kazumi; Nakamura, Masataka; Masuzawa, Mikio; Kaneda, Yasufumi; Katayama, Ichiro; Yokozeki, Hiroo

    2013-10-01

    Cutaneous angiosarcoma is a life-threatening tumor that is resistant to conventional therapies. The therapeutic effects of Sendai virus particles (hemagglutinating virus of Japan envelope: HVJ-E) carrying IL-2 gene (HVJ-E/IL-2) were examined in a mouse model of angiosarcoma. Intra-tumoral injection of HVJ-E/IL-2 effectively inhibited the growth of angiosarcoma cells (ISOS-1) inoculated in mice and improved tumor-free rates. HVJ-E/IL-2 stimulated local accumulation of CD8 (+) T cells and NK cells and reduced regulatory T cells in regional lymph nodes. Notably, the prevalence of myeloid-derived suppressor cells was lower in HVJ-E/IL-2-treated mice than in HVJ-E-treated mice. HVJ-E/IL-2 treatment promoted IFN-γ production from CD8 (+) T cells in response to tumor cells, more significantly than HVJ-E treatment. Greatly improved tumor-free rates were obtained when sunitinib, a tyrosine kinase inhibitor, was administered in combination with HVJ-E/IL-2. Immunogene therapy with HVJ-E/IL-2 with or without sunitinib could be a promising therapeutic option for cutaneous angiosarcoma. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. A humanised murine monoclonal antibody protects mice from Venezuelan equine encephalitis virus, Everglades virus and Mucambo virus when administered up to 48 h after airborne challenge

    Energy Technology Data Exchange (ETDEWEB)

    O' Brien, Lyn M., E-mail: lmobrien@dstl.gov.uk; Goodchild, Sarah A.; Phillpotts, Robert J.; Perkins, Stuart D.

    2012-05-10

    Currently there are no licensed antiviral treatments for the Alphaviruses Venezuelan equine encephalitis virus (VEEV), Everglades virus and Mucambo virus. We previously developed a humanised version of the mouse monoclonal antibody 1A3B-7 (Hu1A3B-7) which exhibited a wide range of reactivity in vitro and was able to protect mice from infection with VEEV. Continued work with the humanised antibody has now demonstrated that it has the potential to be a new human therapeutic. Hu1A3B-7 successfully protected mice from infection with multiple Alphaviruses. The effectiveness of the humanisation process was determined by assessing proliferation responses in human T-cells to peptides derived from the murine and humanised versions of the V{sub H} and V{sub L} domains. This analysis showed that the number of human T-cell epitopes within the humanised antibody had been substantially reduced, indicating that Hu1A3B-7 may have reduced immunogenicity in vivo.

  13. The elemental composition of virus particles: implications for marine biogeochemical cycles.

    Science.gov (United States)

    Jover, Luis F; Effler, T Chad; Buchan, Alison; Wilhelm, Steven W; Weitz, Joshua S

    2014-07-01

    In marine environments, virus-mediated lysis of host cells leads to the release of cellular carbon and nutrients and is hypothesized to be a major driver of carbon recycling on a global scale. However, efforts to characterize the effects of viruses on nutrient cycles have overlooked the geochemical potential of the virus particles themselves, particularly with respect to their phosphorus content. In this Analysis article, we use a biophysical scaling model of intact virus particles that has been validated using sequence and structural information to quantify differences in the elemental stoichiometry of marine viruses compared with their microbial hosts. By extrapolating particle-scale estimates to the ecosystem scale, we propose that, under certain circumstances, marine virus populations could make an important contribution to the reservoir and cycling of oceanic phosphorus.

  14. 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.

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

    Science.gov (United States)

    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.

  16. Experimental investigation of virus and clay particles cotransport in partially saturated columns packed with glass beads.

    Science.gov (United States)

    Syngouna, Vasiliki I; Chrysikopoulos, Constantinos V

    2015-02-15

    Suspended clay particles in groundwater can play a significant role as carriers of viruses, because, depending on the physicochemical conditions, clay particles may facilitate or hinder the mobility of viruses. This experimental study examines the effects of clay colloids on the transport of viruses in variably saturated porous media. All cotransport experiments were conducted in both saturated and partially saturated columns packed with glass beads, using bacteriophages MS2 and ΦX174 as model viruses, and kaolinite (KGa-1b) and montmorillonite (STx-1b) as model clay colloids. The various experimental collision efficiencies were determined using the classical colloid filtration theory. The experimental data indicated that the mass recovery of viruses and clay colloids decreased as the water saturation decreased. Temporal moments of the various breakthrough concentrations collected, suggested that the presence of clays significantly influenced virus transport and irreversible deposition onto glass beads. The mass recovery of both viruses, based on total effluent virus concentrations, was shown to reduce in the presence of suspended clay particles. Furthermore, the transport of suspended virus and clay-virus particles was retarded, compared to the conservative tracer. Under unsaturated conditions both clay particles facilitated the transport of ΦX174, while hindered the transport of MS2. Moreover, the surface properties of viruses, clays and glass beads were employed for the construction of classical DLVO and capillary potential energy profiles, and the results suggested that capillary forces play a significant role on colloid retention. It was estimated that the capillary potential energy of MS2 is lower than that of ΦX174, and the capillary potential energy of KGa-1b is lower than that of STx-1b, assuming that the protrusion distance through the water film is the same for each pair of particles. Moreover, the capillary potential energy is several orders of

  17. Recovery of infective virus particles in ion-exchange and hydrophobic interaction monolith chromatography is influenced by particle charge and total-to-infective particle ratio.

    Science.gov (United States)

    Sviben, Dora; Forcic, Dubravko; Ivancic-Jelecki, Jelena; Halassy, Beata; Brgles, Marija

    2017-06-01

    Viral particles are used in medical applications as vaccines or gene therapy vectors. In order to obtain product of high purity, potency and safety for medical use purification of virus particles is a prerequisite, and chromatography is gaining increased attention to meet this aim. Here, we report on the use of ion-exchange and hydrophobic interaction chromatography on monolithic columns for purification of mumps virus (MuV) and measles virus (MeV). Efficiency of the process was monitored by quantification of infective virus particles (by 50% cell culture infective dose assay) and total virus particles, and monitoring of their size (by Nanoparticle Tracking Analysis). Ion-exchange chromatography was shown to be inefficient for MuV and best results for MeV were obtained on QA column with recovery around 17%. Purification of MuV and MeV by hydrophobic interaction chromatography resulted in recoveries around 60%. Results showed that columns with small channels (d=1.4μm) are not suitable for MuV and MeV, although their size is below 400nm, whereas columns with large channels (6μm) showed to be efficient and recoveries independent on the flow rate up to 10mL/min. Heterogeneity of the virus suspension and its interday variability mostly regarding total-to-infective particle ratio was observed. Interestingly, a trend in recovery depending on the day of the harvest was also observed for both viruses, and it correlated with the total-to-infective particle ratio, indicating influence of the virus sample composition on the chromatography results. Copyright © 2017. Published by Elsevier B.V.

  18. Intranasal administration of antibody-bound respiratory syncytial virus particles efficiently primes virus-specific immune responses in mice

    NARCIS (Netherlands)

    Kruijsen, Debby; Einarsdottir, Helga K.; Schijf, Marcel A.; Coenjaerts, Frank E.; van der Schoot, Ellen C.; Vidarsson, Gestur; van Bleek, Grada M.

    2013-01-01

    Infants are protected from a severe respiratory syncytial virus (RSV) infection in the first months of life by maternal antibodies or by prophylactically administered neutralizing antibodies. Efforts are under way to produce RSV-specific antibodies with increased neutralizing capacity compared to

  19. 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.

  20. 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.

  1. 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...

  2. 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.

  3. 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

  4. 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.

  5. Chimeric hepatitis B virus core particles with parts or copies of the hepatitis C virus core protein.

    OpenAIRE

    Yoshikawa, A.; Tanaka, T.; Hoshi, Y.; Kato, N; Tachibana, K; Iizuka, H; Machida, A; Okamoto, H.; Yamasaki, M; Miyakawa, Y

    1993-01-01

    Either parts or multiple copies of the core gene of hepatitis C virus (HCV) were fused to the 3' terminus of the hepatitis B virus (HBV) core gene with 34 codons removed. As many as four copies of HCV core protein (720 amino acids) were fused to the carboxy terminus of truncated HBV core protein (149 amino acids) without preventing the assembly of HBV core particles. Chimeric core particles were sandwiched between monoclonal antibody to HBV core and that to HCV core, thereby indicating that a...

  6. Lysosomes serve as a platform for hepatitis A virus particle maturation and nonlytic release.

    Science.gov (United States)

    Seggewiß, Nicole; Paulmann, Dajana; Dotzauer, Andreas

    2016-01-01

    Early studies on hepatitis A virus (HAV) in cell culture demonstrated the inclusion of several viral particles in an intracellular lipid-bilayer membrane. However, the origin of these virus-associated membranes and the mechanism for the non-lytic release of HAV into bile are still unknown. Analyzing the association of this virus with cell organelles, we found that newly synthesized HAV particles accumulate in lysosomal organelles and that lysosomal enzymes are involved in the maturation cleavage of the virion. Furthermore, by inhibiting the processes of fusion of lysosomes with the plasma membrane, we found that the nonlytic release of HAV from infected cells occurs via lysosome-related organelles.

  7. Viruses, bacteria and suspended particles in a backwater and main channel site of the Danube (Austria)

    Science.gov (United States)

    Peduzzi, Peter; Luef, Birgit

    2010-01-01

    A short overview of currently available studies on the ecology of viruses in running waters is provided. Additionally, a survey was conducted on the dynamics of both viruses and bacteria in an isolated floodplain segment of the Danube River and in the main channel near Vienna (Austria) during the hydrologically most dynamic phase (spring – summer). The study evaluates the differences between the main channel and the floodplain segment for suspended particle abundance and quality in relation to bacterial and viral parameters; both free-living forms and those attached to particles are examined. The hydrological disconnection of these two contrasting sampling sites influenced particle abundance and quality as well as the distribution of free-living vs. attached bacteria and viruses. The per-cell activity of bacteria attached to particles was significantly higher than that of the free-living fraction, particularly in the isolated water body. The abundance of bacteria and viruses on particles depended on particle quality (size). In the main channel, bacteria were significantly more abundant on surfaces (per mm2) of suspended matter > 5 μm (aggregates with organic constituents) compared to particles 5μm and attached viruses; free-living viruses were less abundant at high > 5μm particle loads. Only in the isolated floodplain section was viral abundance positively influenced by elevated per-cell productivity of potential host bacteria. The results demonstrate that system variability on a relatively small topographical scale (within a river-floodplain system) has consequences for microbial life, including viruses. PMID:21151810

  8. 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.

  9. Dissecting the cell entry pathway of dengue virus by single-particle tracking in living cells.

    Directory of Open Access Journals (Sweden)

    Hilde M van der Schaar

    2008-12-01

    Full Text Available Dengue virus (DENV is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking, and fusion behavior of DENV. Simultaneous tracking of DENV particles and various endocytic markers revealed that DENV enters cells exclusively via clathrin-mediated endocytosis. The virus particles move along the cell surface in a diffusive manner before being captured by a pre-existing clathrin-coated pit. Upon clathrin-mediated entry, DENV particles are transported to Rab5-positive endosomes, which subsequently mature into late endosomes through acquisition of Rab7 and loss of Rab5. Fusion of the viral membrane with the endosomal membrane was primarily detected in late endosomal compartments.

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

    Directory of Open Access Journals (Sweden)

    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.

  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. 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.

  14. 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

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

    Science.gov (United States)

    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.

  16. Size distribution analysis of influenza virus particles using size exclusion chromatography.

    Science.gov (United States)

    Vajda, Judith; Weber, Dennis; Brekel, Dominik; Hundt, Boris; Müller, Egbert

    2016-09-23

    Size exclusion chromatography is a standard method in quality control of biopharmaceutical proteins. In contrast, vaccine analysis is often based on activity assays. The hemagglutination assay is a widely accepted influenza quantification method, providing no insight in the size distribution of virus particles. Capabilities of size exclusion chromatography to complement the hemagglutination assay are investigated. The presented method is comparatively robust regarding different buffer systems, ionic strength and additive concentrations. Addition of 200mM arginine or sodium chloride is necessary to obtain complete virus particle recovery. 0.5 and 1.0M arginine increase the hydrodynamic radius of the whole virus particles by 5nm. Sodium citrate induces virus particle aggregation. Results are confirmed by dynamic light scattering. Retention of a H1N1v strain correlates with DNA contents between 5ng/mL and 670ng/mL. Quantitative elution of the virus preparations is verified on basis of hemagglutination activity. Elution of hemagglutination inducing compounds starts at a flow channel diameter of 7000nm. The universal applicability is demonstrated with three different influenza virus samples, including an industrially produced, pandemic vaccine strain. Size distribution of the pandemic H1N1v 5258, H1N1 PR/8/34, and H3N2 Aichi/2/68 preparations spreads across inter- and intra-particle volume and extends to the secondary interaction dominated range. Thus, virus particle debris seems to induce hemagglutination. Fragments generated by 0.5% Triton™ X-100 treatment increase overall hemagglutination activity. Copyright © 2016 The Author(s). Published by Elsevier B.V. All rights reserved.

  17. VIRUSES

    Indian Academy of Sciences (India)

    and-mouth disease in livestock was an infectious particle smaller than any bacteria. This was the first clue to the nature of viruses, genetic entities that lie somewhere in the gray area between living and non-living states.

  18. 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.

  19. Microstructure of atmospheric particles revealed by TXM and a new mode of influenza virus transmission

    Science.gov (United States)

    Bao, L. M.; Zhang, G. L.; Lei, Q. T.; Li, Y.; Li, X. L.; Hwu, Y. K.; Yi, J. M.

    2015-09-01

    For control of influenza, firstly it is important to find the real virus transmission media. Atmospheric aerosol particles are presumably one of the media. In this study, three typical atmospheric inhaled particles in Shanghai were studied by the synchrotron based transmission X-ray microscopes (TXM). Three dimensional microstructure of the particles reveals that there are many pores contained in, particularly the coal combustion fly particles which may be possible virus carrier. The particles can transport over long distance and cause long-range infections due to its light weight. We suggest a mode which is droplet combining with aerosol mode. By this mode the transmission of global and pandemic influenzas and infection between inland avian far from population and poultry or human living in cities along coast may be explained.

  20. Microstructure of atmospheric particles revealed by TXM and a new mode of influenza virus transmission

    Energy Technology Data Exchange (ETDEWEB)

    Bao, L.M., E-mail: baoliangman@sinap.ac.cn [Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800 (China); Zhang, G.L., E-mail: zhangguilin@sinap.ac.cn [Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800 (China); Lei, Q.T.; Li, Y.; Li, X.L. [Shanghai Institute of Applied Physics, Chinese Academy of Sciences, Shanghai 201800 (China); Hwu, Y.K. [Institute of Physics, Academia Sinica, Taipei 11529, Taiwan (China); Yi, J.M. [Advanced Photon Source, Argonne National Laboratory, Argonne 60439 (United States)

    2015-09-15

    For control of influenza, firstly it is important to find the real virus transmission media. Atmospheric aerosol particles are presumably one of the media. In this study, three typical atmospheric inhaled particles in Shanghai were studied by the synchrotron based transmission X-ray microscopes (TXM). Three dimensional microstructure of the particles reveals that there are many pores contained in, particularly the coal combustion fly particles which may be possible virus carrier. The particles can transport over long distance and cause long-range infections due to its light weight. We suggest a mode which is droplet combining with aerosol mode. By this mode the transmission of global and pandemic influenzas and infection between inland avian far from population and poultry or human living in cities along coast may be explained.

  1. Monitoring virus entry into living cells using DiD-labeled dengue virus particles

    NARCIS (Netherlands)

    Ayala Nunez, Vanesa; Wilschut, Jan; Smit, Jolanda M.

    2011-01-01

    A variety of approaches can be applied to investigate the multiple steps and interactions that occur during virus entry into the host cell. Single-virus tracking is a powerful real-time imaging technique that offers the possibility to monitor virus-cell binding, internalization, intracellular

  2. 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.

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

    Science.gov (United States)

    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.

  4. 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

  5. Viruses, bacteria and suspended particles in a backwater and main channel site of the Danube (Austria)

    OpenAIRE

    Peduzzi, Peter; Luef, Birgit

    2008-01-01

    A short overview of currently available studies on the ecology of viruses in running waters is provided. Additionally, a survey was conducted on the dynamics of both viruses and bacteria in an isolated floodplain segment of the Danube River and in the main channel near Vienna (Austria) during the hydrologically most dynamic phase (spring – summer). The study evaluates the differences between the main channel and the floodplain segment for suspended particle abundance and quality in relation t...

  6. 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.

  7. Measurements of airborne influenza virus in aerosol particles from human coughs.

    Directory of Open Access Journals (Sweden)

    William G Lindsley

    2010-11-01

    Full Text Available Influenza is thought to be communicated from person to person by multiple pathways. However, the relative importance of different routes of influenza transmission is unclear. To better understand the potential for the airborne spread of influenza, we measured the amount and size of aerosol particles containing influenza virus that were produced by coughing. Subjects were recruited from patients presenting at a student health clinic with influenza-like symptoms. Nasopharyngeal swabs were collected from the volunteers and they were asked to cough three times into a spirometer. After each cough, the cough-generated aerosol was collected using a NIOSH two-stage bioaerosol cyclone sampler or an SKC BioSampler. The amount of influenza viral RNA contained in the samplers was analyzed using quantitative real-time reverse-transcription PCR (qPCR targeting the matrix gene M1. For half of the subjects, viral plaque assays were performed on the nasopharyngeal swabs and cough aerosol samples to determine if viable virus was present. Fifty-eight subjects were tested, of whom 47 were positive for influenza virus by qPCR. Influenza viral RNA was detected in coughs from 38 of these subjects (81%. Thirty-five percent of the influenza RNA was contained in particles>4 µm in aerodynamic diameter, while 23% was in particles 1 to 4 µm and 42% in particles<1 µm. Viable influenza virus was detected in the cough aerosols from 2 of 21 subjects with influenza. These results show that coughing by influenza patients emits aerosol particles containing influenza virus and that much of the viral RNA is contained within particles in the respirable size range. The results support the idea that the airborne route may be a pathway for influenza transmission, especially in the immediate vicinity of an influenza patient. Further research is needed on the viability of airborne influenza viruses and the risk of transmission.

  8. High sensitivity detection and sorting of infectious human immunodeficiency virus (HIV-1) particles by flow virometry.

    Science.gov (United States)

    Bonar, Michał M; Tilton, John C

    2017-05-01

    Detection of viruses by flow cytometry is complicated by their small size. Here, we characterized the ability of a standard (FACSAria II) and a sub-micron flow cytometer (A50 Micro) to resolve HIV-1 viruses. The A50 was superior at resolving small particles but did not reliably distinguish HIV-1, extracellular vesicles, and laser noise by light scatter properties alone. However, single fluorescent HIV-1 particles could readily be detected by both cytometers. Fluorescent particles were sorted and retained infectivity, permitting further exploration of the functional consequences of HIV-1 heterogeneity. Finally, flow cytometry had a limit of detection of 80 viruses/ml, nearly equal to PCR assays. These studies demonstrate the power of flow cytometry to detect and sort viral particles and provide a critical toolkit to validate methods to label wild-type HIV-1; quantitatively assess integrity and aggregation of viruses and virus-based therapeutics; and efficiently screen drugs inhibiting viral assembly and release. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. 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.

  10. 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.

  11. Thermal conversion of filamentous potato virus X into spherical particles with different properties from virions.

    Science.gov (United States)

    Nikitin, Nikolai; Ksenofontov, Alexander; Trifonova, Ekaterina; Arkhipenko, Marina; Petrova, Ekaterina; Kondakova, Olga; Kirpichnikov, Mikhail; Atabekov, Joseph; Dobrov, Evgeny; Karpova, Olga

    2016-05-01

    We developed a method for the fast transformation of virions of tobacco mosaic virus (TMV) in so-called spherical particles (SPs) of different sizes. These SPs turned out to be highly useful for the preparation of different kinds of important biotechnological products. In this communication, we report that a representative of the flexuous helical virus group-potato virus X (PVX), produces SPs as well, but these SPs differ from TMV SPs in several important aspects. PVX SPs may be useful biotechnological devices. © 2016 Federation of European Biochemical Societies.

  12. Gamma-irradiated influenza A virus provides adjuvant activity to a co-administered poorly immunogenic SFV vaccine in mice.

    Directory of Open Access Journals (Sweden)

    Rachelle eBabb

    2014-06-01

    Full Text Available Many currently available inactivated vaccines require 'adjuvants' to maximise the protective immune responses generated against the antigens of interest. Recent studies in mice with gamma-irradiated influenza A virus (γ-FLU have shown its superior efficacy compared to other forms of inactivated FLU vaccines and its ability to induce both potent type-I interferon (IFN-I responses and the IFN-I associated partial lymphocyte activation. Commonly, IFN-I responses induced by adjuvants, combined in vaccine preparations, have been shown to effectively enhance the immunogenicity of the antigens of interest. Therefore, we investigated the potential adjuvant activity of γ-FLU and the possible effect on antibody responses against co-administrated antigens, using gamma-irradiated Semliki Forest Virus (γ-SFV as the experimental vaccine in mice. Our data show that co-vaccination with γ-FLU and γ-SFV resulted in enhanced SFV-specific antibody responses in terms of increased titres by 6 fold and greater neutralisation efficacy, when compared to vaccination with γ-SFV alone. This study provides promising evidence related to the possible use of γ-FLU as an adjuvant to poorly immunogenic vaccines without compromising the vaccine efficacy of γ-FLU.

  13. 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...

  14. 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.

  15. 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.

  16. Human immunodeficiency virus type 1 Vpr: oligomerization is an essential feature for its incorporation into virus particles

    Directory of Open Access Journals (Sweden)

    Klein-Seetharaman Judith

    2010-06-01

    Full Text Available Abstract HIV-1 Vpr, a nonstructural viral protein associated with virus particles, has a positive role in the efficient transport of PIC into the nucleus of non-dividing target cells and enhances virus replication in primary T cells. Vpr is a 96 amino acid protein and the structure by NMR shows three helical domains. Vpr has been shown to exist as dimers and higher order oligomers. Considering the multifunctional nature of Vpr, the contribution of distinct helical domains to the dimer/oligomer structure of Vpr and the relevance of this feature to its functions are not clear. To address this, we have utilized molecular modeling approaches to identify putative models of oligomerization. The predicted interface residues were subjected to site-directed mutagenesis and evaluated their role in intermolecular interaction and virion incorporation. The interaction between Vpr molecules was monitored by Bimolecular Fluorescence complementation (BiFC method. The results show that Vpr forms oligomers in live cells and residues in helical domains play critical roles in oligomerization. Interestingly, Vpr molecules defective in oligomerization also fail to incorporate into the virus particles. Based on the data, we suggest that oligomerization of Vpr is essential for virion incorporation property and may also have a role in the events associated with virus infection.

  17. 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.

  18. Mosquito cell-derived West Nile virus replicon particles mimic arbovirus inoculum and have reduced spread in mice.

    Science.gov (United States)

    Boylan, Brendan T; Moreira, Fernando R; Carlson, Tim W; Bernard, Kristen A

    2017-02-01

    Half of the human population is at risk of infection by an arthropod-borne virus. Many of these arboviruses, such as West Nile, dengue, and Zika viruses, infect humans by way of a bite from an infected mosquito. This infectious inoculum is insect cell-derived giving the virus particles distinct qualities not present in secondary infectious virus particles produced by infected vertebrate host cells. The insect cell-derived particles differ in the glycosylation of virus structural proteins and the lipid content of the envelope, as well as their induction of cytokines. Thus, in order to accurately mimic the inoculum delivered by arthropods, arboviruses should be derived from arthropod cells. Previous studies have packaged replicon genome in mammalian cells to produce replicon particles, which undergo only one round of infection, but no studies exist packaging replicon particles in mosquito cells. Here we optimized the packaging of West Nile virus replicon genome in mosquito cells and produced replicon particles at high concentration, allowing us to mimic mosquito cell-derived viral inoculum. These particles were mature with similar genome equivalents-to-infectious units as full-length West Nile virus. We then compared the mosquito cell-derived particles to mammalian cell-derived particles in mice. Both replicon particles infected skin at the inoculation site and the draining lymph node by 3 hours post-inoculation. The mammalian cell-derived replicon particles spread from the site of inoculation to the spleen and contralateral lymph nodes significantly more than the particles derived from mosquito cells. This in vivo difference in spread of West Nile replicons in the inoculum demonstrates the importance of using arthropod cell-derived particles to model early events in arboviral infection and highlights the value of these novel arthropod cell-derived replicon particles for studying the earliest virus-host interactions for arboviruses.

  19. Influenza virus aerosols in human exhaled breath: particle size, culturability, and effect of surgical masks.

    Directory of Open Access Journals (Sweden)

    Donald K Milton

    2013-03-01

    Full Text Available The CDC recommends that healthcare settings provide influenza patients with facemasks as a means of reducing transmission to staff and other patients, and a recent report suggested that surgical masks can capture influenza virus in large droplet spray. However, there is minimal data on influenza virus aerosol shedding, the infectiousness of exhaled aerosols, and none on the impact of facemasks on viral aerosol shedding from patients with seasonal influenza. We collected samples of exhaled particles (one with and one without a facemask in two size fractions ("coarse">5 µm, "fine"≤5 µm from 37 volunteers within 5 days of seasonal influenza onset, measured viral copy number using quantitative RT-PCR, and tested the fine-particle fraction for culturable virus. Fine particles contained 8.8 (95% CI 4.1 to 19 fold more viral copies than did coarse particles. Surgical masks reduced viral copy numbers in the fine fraction by 2.8 fold (95% CI 1.5 to 5.2 and in the coarse fraction by 25 fold (95% CI 3.5 to 180. Overall, masks produced a 3.4 fold (95% CI 1.8 to 6.3 reduction in viral aerosol shedding. Correlations between nasopharyngeal swab and the aerosol fraction copy numbers were weak (r = 0.17, coarse; r = 0.29, fine fraction. Copy numbers in exhaled breath declined rapidly with day after onset of illness. Two subjects with the highest copy numbers gave culture positive fine particle samples. Surgical masks worn by patients reduce aerosols shedding of virus. The abundance of viral copies in fine particle aerosols and evidence for their infectiousness suggests an important role in seasonal influenza transmission. Monitoring exhaled virus aerosols will be important for validation of experimental transmission studies in humans.

  20. Kinetic plots in aqueous size exclusion chromatography of monoclonal antibodies and virus particles.

    Science.gov (United States)

    Vajda, Judith; Conze, Werner; Müller, Egbert

    2015-12-24

    The growing importance of monoclonal antibodies and virus particles has led to a pressure for faster size exclusion chromatography. In recent years, numerous small particle columns for size exclusion chromatography of biologicals have been introduced. Small particles are a strategy to reduce analysis time. In the following study, opportunities of small particles in size exclusion chromatography of large biomolecules are investigated. Poppe plots reveal that the lower particle size limit depends on the size of the sample molecule. Hydrodynamic radii of monoclonal antibody monomer, aggregates and H1N1 as well as the diffusion coefficients were determined. Considering this sample compound dependency, kinetic plots referring to the resolution of a distinct compound pair instead of the plate number of a single analyte are more meaningful. Plate times were found to be equivalent with 4 and 2μm particles for a monoclonal antibody aggregate separation at resolutions smaller than 1.8. Quantification of a H1N1 in clarified cell culture can be accomplished with 17μm and 13μm particles at equal plate times at resolutions smaller than 2.5. Virus polydispersity is likely to be affected by run times of several hours at room temperature and shear forces resulting from particles smaller than 10μm. Comparatively high flow rates should be applied in size exclusion chromatography of the 100nm H1N1 virions. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  1. 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

  2. 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 ...

  3. Defective-interfering particles of the human parvovirus adeno-associated virus. [uv radiation

    Energy Technology Data Exchange (ETDEWEB)

    Laughlin, C.A.; Myers, M.W.; Risin, D.L.; Carter, B.J.

    1979-04-15

    We have previously shown that adeno-associated virus (AAV) grown in KB cells with a helper adenovirus, produced several classes of particles defined by their buoyant density in CsCl. The predominant density classes were referred to as AAV(1.45), AAV(1.41), AAV (1.35), and AAV(1.32), respectively, where the density of the particle was written in the parentheses. The AAV(1.45) and AAV(1.41) particles which contained standard genomes were the only infectious AAV these infectious AAV particles exhibited autointerference. The ligh-density AAV(1.35) and (1.32) particles contained aberrant (deleted and/or snap-back) genomes. We report here experiments which show that the light-density AAV particles were noninfectious but interfered with the replication of AAV(1.41). The interference was intracellular and resulted in inhibition of synthesis of standard (14.5S) AAV genomes. In some cases there was also a concomitant increase in synthesis of aberrant, shorter AAV DNA. The inhibitory activity of the light-density particles was abolished by uv irradiation. These results show that the population of light AAV particles contained DI particles. The observed autointerference of AAV(1.45) or AAV(1.41) virus is postulated to be due to AAV DI particles. Replication of AAV DI genomes appeared to require the presence of replicating, standard AAV genomes. This is interpreted to mean that progeny strand replication of AAV requires an AAV-specified product, presumably the AAV capsid protein. In contrast to standard, infectious AAV, the AAV DI particles alone do not inhibit replication of the helper adenovirus.

  4. Interferon induction by viruses. VIII. Vesicular stomatitis virus: (+-)DI-011 particles induce interferon in the absence of standard virions

    Energy Technology Data Exchange (ETDEWEB)

    Sekellick, M.J.; Marcus, P.I.

    1982-02-01

    Evidence was presented that VSV (+-)DI-011 particles, which contain a genome of covalently linked totally self-complementary RNA, were excellent inducers of interferon (IFN) - by virtue of the dsRNA presumed to form within an infected cell, and that one molecule of that dsRNA per cell sufficed to induce a quantum yield of IFN. While the IFN-inducing capacity of (+-)DI-011 particle preparations has been confirmed, some researchers contend that DIP by themselves cannot induce IFN and that induction requires the presence of coinfecting (contaminating) standard VSV PFP. Consequently, we reexamined this question and now report that under five different conditions where the function of contaminating standard virus is reduced markedly, or eliminated, there was no diminution of the interferon-inducing particle (IFP) activity in preparations of (+-)DI-011 particles. Thus, inactivation of contaminating PFP by uv radiation or heat, the elimination (during induction) of cycling infection through the use of anti-serum (in the case of mouse L cells), and the reduction of PFP by four successive velocity sedimentation-gradient purifications had no adverse affect on the IFN-inducing capacity of DI-011 in either ''aged'' primary chick embryo cells or in mouse L(Y) cells. Furthermore, dilutions of DI-011 stocks which precluded the presence of even a single PFP still induced IFN in ''aged'' chick embryo cells. In concert these data demonstrate convincingly that IFN induction by DI-011 particles does not require coinfection with standard virus. It follows that DI-011 particles are intrinsically capable of inducing IFN.

  5. 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.

  6. Clustering and cellular distribution characteristics of virus particles of Tomato spotted wilt virus and Tomato zonate spot virus in different plant hosts.

    Science.gov (United States)

    Zhang, Zhongkai; Zheng, Kuanyu; Dong, Jiahong; Fang, Qi; Hong, Jian; Wang, Xifeng

    2016-01-19

    Tomato spotted wilt virus (TSWV) and Tomato zonate spot virus (TZSV) are the two dominant species of thrip-transmitted tospoviruses, cause significant losses in crop yield in Yunnan and its neighboring provinces in China. TSWV and TZSV belong to different serogroup of tospoviruses but induce similar symptoms in the same host plant species, which makes diagnostic difficult. We used different electron microscopy preparing methods to investigate clustering and cellular distribution of TSWV and TZSV in the host plant species. Negative staining of samples infected with TSWV and TZSV revealed that particles usually clustered in the vesicles, including single particle (SP), double particles clustering (DPC), triple particles clustering (TPC). In the immunogold labeling negative staining against proteins of TZSV, the antibodies against Gn protein were stained more strongly than the N protein. Ultrathin section and high pressure freeze (HPF)-electron microscopy preparations revealed that TSWV particles were distributed in the cisternae of endoplasmic reticulum (ER), filamentous inclusions (FI) and Golgi bodies in the mesophyll cells. The TSWV particles clustered as multiple particles clustering (MPC) and distributed in globular viroplasm or cisternae of ER in the top leaf cell. TZSV particles were distributed more abundantly in the swollen membrane of ER in the mesophyll cell than those in the phloem parenchyma cells and were not observed in the top leaf cell. However, TZSV virions were mainly present as single particle in the cytoplasm, with few clustering as MPC. In this study, we identified TSWV and TZSV particles had the distinct cellular distribution patterns in the cytoplasm from different tissues and host plants. This is the first report of specific clustering characteristics of tospoviruses particles as well as the cellular distribution of TSWV particles in the FI and globular viroplasm where as TZSV particles inside the membrane of ER. These results indicated that

  7. The pharmacokinetics of daclatasvir and asunaprevir administered in combination in studies in healthy subjects and patients infected with hepatitis C virus.

    Science.gov (United States)

    Eley, Timothy; Sevinsky, Heather; Huang, Shu-Pang; He, Bing; Zhu, Kurt; Kandoussi, Hamza; Gardiner, David; Grasela, Dennis M; Bertz, Richard; Bifano, Marc

    2014-09-01

    The combination of direct-acting antiviral agents in patients with chronic hepatitis C virus (HCV) infection has demonstrated clinical benefit; however, evaluation of potential drug-drug interactions is required prior to therapy. An open-label study assessed the pharmacokinetics and tolerability of the HCV NS5A replication complex inhibitor daclatasvir and the HCV NS3 protease inhibitor asunaprevir when co-administered in healthy subjects. Daclatasvir 60 mg once daily and asunaprevir 600 mg twice daily were dosed for 7 days alone followed by combination dosing for 14 days at 30 mg once daily and 200 mg twice daily, respectively. Further assessments were provided comparing exposures from the current study with those from studies in HCV-infected patients receiving either the same or higher doses of daclatasvir or asunaprevir administered alone or together. Dose-normalized daclatasvir and asunaprevir morning exposures were comparable with control in healthy subjects, with geometric mean area under the concentration-time curve ratios of 1.202 (90 % CI 1.113-1.298) and 0.868 (90 % CI 0.726-1.038), respectively. In HCV patients daclatasvir and asunaprevir exposures were largely comparable, when administered together or alone. Additional data support the conclusion that there is no clinically meaningful interaction between daclatasvir and asunaprevir in either healthy subjects or HCV-infected patients, including those also receiving peginterferon-α/ribavirin, and that the combination of daclatasvir 60 mg once daily and asunaprevir 200 mg twice daily is generally well-tolerated.

  8. From Cells to Virus Particles: Quantitative Methods to Monitor RNA Packaging

    Directory of Open Access Journals (Sweden)

    Mireia Ferrer

    2016-08-01

    Full Text Available In cells, positive strand RNA viruses, such as Retroviridae, must selectively recognize their full-length RNA genome among abundant cellular RNAs to assemble and release particles. How viruses coordinate the intracellular trafficking of both RNA and protein components to the assembly sites of infectious particles at the cell surface remains a long-standing question. The mechanisms ensuring packaging of genomic RNA are essential for viral infectivity. Since RNA packaging impacts on several essential functions of retroviral replication such as RNA dimerization, translation and recombination events, there are many studies that require the determination of RNA packaging efficiency and/or RNA packaging ability. Studies of RNA encapsidation rely upon techniques for the identification and quantification of RNA species packaged by the virus. This review focuses on the different approaches available to monitor RNA packaging: Northern blot analysis, ribonuclease protection assay and quantitative reverse transcriptase-coupled polymerase chain reaction as well as the most recent RNA imaging and sequencing technologies. Advantages, disadvantages and limitations of these approaches will be discussed in order to help the investigator to choose the most appropriate technique. Although the review was written with the prototypic simple murine leukemia virus (MLV and complex human immunodeficiency virus type 1 (HIV-1 in mind, the techniques were described in order to benefit to a larger community.

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

    Science.gov (United States)

    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. A Lagrangian particle model to predict the airborne spread of foot-and-mouth disease virus

    Science.gov (United States)

    Mayer, D.; Reiczigel, J.; Rubel, F.

    Airborne spread of bioaerosols in the boundary layer over a complex terrain is simulated using a Lagrangian particle model, and applied to modelling the airborne spread of foot-and-mouth disease (FMD) virus. Two case studies are made with study domains located in a hilly region in the northwest of the Styrian capital Graz, the second largest town in Austria. Mountainous terrain as well as inhomogeneous and time varying meteorological conditions prevent from application of so far used Gaussian dispersion models, while the proposed model can handle these realistically. In the model, trajectories of several thousands of particles are computed and the distribution of virus concentration near the ground is calculated. This allows to assess risk of infection areas with respect to animal species of interest, such as cattle, swine or sheep. Meteorological input data like wind field and other variables necessary to compute turbulence were taken from the new pre-operational version of the non-hydrostatic numerical weather prediction model LMK ( Lokal-Modell-Kürzestfrist) running at the German weather service DWD ( Deutscher Wetterdienst). The LMK model provides meteorological parameters with a spatial resolution of about 2.8 km. To account for the spatial resolution of 400 m used by the Lagrangian particle model, the initial wind field is interpolated upon the finer grid by a mass consistent interpolation method. Case studies depict a significant influence of local wind systems on the spread of virus. Higher virus concentrations at the upwind side of the hills and marginal concentrations in the lee are well observable, as well as canalization effects by valleys. The study demonstrates that the Lagrangian particle model is an appropriate tool for risk assessment of airborne spread of virus by taking into account the realistic orographic and meteorological conditions.

  11. 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.

  12. Acute infection with venezuelan equine encephalitis virus replicon particles catalyzes a systemic antiviral state and protects from lethal virus challenge.

    Science.gov (United States)

    Konopka, Jennifer L; Thompson, Joseph M; Whitmore, Alan C; Webb, Drue L; Johnston, Robert E

    2009-12-01

    The host innate immune response provides a critical first line of defense against invading pathogens, inducing an antiviral state to impede the spread of infection. While numerous studies have documented antiviral responses within actively infected tissues, few have described the earliest innate response induced systemically by infection. Here, utilizing Venezuelan equine encephalitis virus (VEE) replicon particles (VRP) to limit infection to the initially infected cells in vivo, a rapid activation of the antiviral response was demonstrated not only within the murine draining lymph node, where replication was confined, but also within distal tissues. In the liver and brain, expression of interferon-stimulated genes was detected by 1 to 3 h following VRP footpad inoculation, reaching peak expression of >100-fold over that in mock-infected animals. Moreover, mice receiving a VRP footpad inoculation 6, 12, or 24 h prior to an otherwise lethal VEE footpad challenge were completely protected from death, including a drastic reduction in challenge virus titers. VRP pretreatment also provided protection from intranasal VEE challenge and extended the average survival time following intracranial challenge. Signaling through the interferon receptor was necessary for antiviral gene induction and protection from VEE challenge. However, VRP pretreatment failed to protect mice from a heterologous, lethal challenge with vesicular stomatitis virus, yet conferred protection following challenge with influenza virus. Collectively, these results document a rapid modulation of the host innate response within hours of infection, capable of rapidly alerting the entire animal to pathogen invasion and leading to protection from viral disease.

  13. Influence of solution chemistry on the inactivation of particle-associated viruses by UV irradiation.

    Science.gov (United States)

    Feng, Zhe; Lu, Ruiqing; Yuan, Baoling; Zhou, Zhenming; Wu, Qingqing; Nguyen, Thanh H

    2016-12-01

    MS2 inactivation by UV irradiance was investigated with the focus on how the disinfection efficacy is influenced by bacteriophage MS2 aggregation and adsorption to particles in solutions with different compositions. Kaolinite and Microcystis aeruginosa were used as model inorganic and organic particles, respectively. In the absence of model particles, MS2 aggregates formed in either 1mM NaCl at pH=3 or 50-200mM ionic strength CaCl2 solutions at pH=7 led to a decrease in the MS2 inactivation efficacy because the virions located inside the aggregate were protected from the UV irradiation. In the presence of kaolinite and Microcystis aeruginosa, MS2 adsorbed onto the particles in either 1mM NaCl at pH=3 or 50-200mM CaCl2 solutions at pH=7. In contrast to MS2 aggregates formed without the presence of particles, more MS2 virions adsorbed on these particles were exposed to UV irradiation to allow an increase in MS2 inactivation. In either 1mM NaCl at pH from 4 to 8 or 2-200mM NaCl solutions at pH=7, the absence of MS2 aggregation and adsorption onto the model particles explained why MS2 inactivation was not influenced by pH, ionic strength, and the presence of model particles in these conditions. The influence of virus adsorption and aggregation on the UV disinfection efficiency found in this research suggests the necessity of accounting for particles and cation composition in virus inactivation for drinking water. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. 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

  15. Interferon induction by viruses. XV. Biological characteristics of interferon induction-suppressing particles of vesicular stomatitis virus

    Energy Technology Data Exchange (ETDEWEB)

    Marcus, P.I.; Sekellick, M.J.

    1987-06-01

    A single interferon (IFN) induction-suppressing particle (ISP) of vesicular stomatitis virus (VSV) blocked completely the yield of IFN in a cell otherwise programmed to produce IFN. With mouse L cells as hosts, one lethal hit of UV radiation (D37 = 52.5 ergs/mm2) to the VSV genome sufficed to inactivate ISP activity; however, with ''aged'' primary chick embryo cells as hosts, it took 198 lethal hits (D37 = 10,395 ergs/mm2). ISP expression in chick cells did not require virus replication or amplified RNA synthesis, but did involve functional virion-associated L protein. ISP in chick cells also were capable of inhibiting, in a multiplicity-dependent manner, the plaquing efficiency of two viruses that require cellular polymerase II (pol II) for replication, e.g., pseudorabies and influenza. The refractory state to IFN inducibility that resulted from infection of chick cells with ISP (VSV tsO5 (UV = 100 hits)) was still extant after 6 days. In contrast, the plaquing efficiency of pseudorabies virus returned to control levels by 5 h after ISP infection. Chick cells infected with UV ISP remained viable, served as hosts for the replication of other viruses, and could be subcultured. Models are presented to account for these contrasting effects. The involvement of viral plus-strand leader RNA as an inhibitor of cellular pol II-dependent RNA synthesis, and the multifunctional activities of the virion-associated L protein, are discussed as possible molecules involved in the action of ISP in chick cells.

  16. 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.

  17. 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.

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

    Science.gov (United States)

    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.

  19. Mechanical elasticity as a physical signature of conformational dynamics in a virus particle

    Science.gov (United States)

    Castellanos, Milagros; Pérez, Rebeca; Carrasco, Carolina; Hernando-Pérez, Mercedes; Gómez-Herrero, Julio; de Pablo, Pedro J.; Mateu, Mauricio G.

    2012-01-01

    In this study we test the hypothesis that mechanically elastic regions in a virus particle (or large biomolecular complex) must coincide with conformationally dynamic regions, because both properties are intrinsically correlated. Hypothesis-derived predictions were subjected to verification by using 19 variants of the minute virus of mice capsid. The structural modifications in these variants reduced, preserved, or restored the conformational dynamism of regions surrounding capsid pores that are involved in molecular translocation events required for virus infectivity. The mechanical elasticity of the modified capsids was analyzed by atomic force microscopy, and the results corroborated every prediction tested: Any mutation (or chemical cross-linking) that impaired a conformational rearrangement of the pore regions increased their mechanical stiffness. On the contrary, any mutation that preserved the dynamics of the pore regions also preserved their elasticity. Moreover, any pseudo-reversion that restored the dynamics of the pore regions (lost through previous mutation) also restored their elasticity. Finally, no correlation was observed between dynamics of the pore regions and mechanical elasticity of other capsid regions. This study (i) corroborates the hypothesis that local mechanical elasticity and conformational dynamics in a viral particle are intrinsically correlated; (ii) proposes that determination by atomic force microscopy of local mechanical elasticity, combined with mutational analysis, may be used to identify and study conformationally dynamic regions in virus particles and large biomolecular complexes; (iii) supports a connection between mechanical properties and biological function in a virus; (iv) shows that viral capsids can be greatly stiffened by protein engineering for nanotechnological applications. PMID:22797893

  20. Saliva Proteins of Vector Culicoides Modify Structure and Infectivity of Bluetongue Virus Particles

    Science.gov (United States)

    Darpel, Karin E.; Langner, Kathrin F. A.; Nimtz, Manfred; Anthony, Simon J.; Brownlie, Joe; Takamatsu, Haru-Hisa; Mellor, Philip S.; Mertens, Peter P. C.

    2011-01-01

    Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are related orbiviruses, transmitted between their ruminant hosts primarily by certain haematophagous midge vectors (Culicoides spp.). The larger of the BTV outer-capsid proteins, ‘VP2’, can be cleaved by proteases (including trypsin or chymotrypsin), forming infectious subviral particles (ISVP) which have enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sonorensis). We demonstrate that VP2 present on purified virus particles from 3 different BTV strains can also be cleaved by treatment with saliva from adult Culicoides. The saliva proteins from C. sonorensis (a competent BTV vector), cleaved BTV-VP2 more efficiently than those from C. nubeculosus (a less competent / non-vector species). Electrophoresis and mass spectrometry identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Incubating purified BTV-1 with C. sonorensis saliva proteins also increased their infectivity for KC cells ∼10 fold, while infectivity for BHK cells was reduced by 2–6 fold. Treatment of an ‘eastern’ strain of EHDV-2 with saliva proteins of either C. sonorensis or C. nubeculosus cleaved VP2, but a ‘western’ strain of EHDV-2 remained unmodified. These results indicate that temperature, strain of virus and protein composition of Culicoides saliva (particularly its protease content which is dependent upon vector species), can all play a significant role in the efficiency of VP2 cleavage, influencing virus infectivity. Saliva of several other arthropod species has previously been shown to increase transmission, infectivity and virulence of certain arboviruses, by modulating and/or suppressing the mammalian immune response. The findings presented here, however, demonstrate a novel mechanism by which proteases in Culicoides saliva can also directly modify the orbivirus particle structure, leading to

  1. Saliva proteins of vector Culicoides modify structure and infectivity of bluetongue virus particles.

    Directory of Open Access Journals (Sweden)

    Karin E Darpel

    2011-03-01

    Full Text Available Bluetongue virus (BTV and epizootic haemorrhagic disease virus (EHDV are related orbiviruses, transmitted between their ruminant hosts primarily by certain haematophagous midge vectors (Culicoides spp.. The larger of the BTV outer-capsid proteins, 'VP2', can be cleaved by proteases (including trypsin or chymotrypsin, forming infectious subviral particles (ISVP which have enhanced infectivity for adult Culicoides, or KC cells (a cell-line derived from C. sonorensis. We demonstrate that VP2 present on purified virus particles from 3 different BTV strains can also be cleaved by treatment with saliva from adult Culicoides. The saliva proteins from C. sonorensis (a competent BTV vector, cleaved BTV-VP2 more efficiently than those from C. nubeculosus (a less competent/non-vector species. Electrophoresis and mass spectrometry identified a trypsin-like protease in C. sonorensis saliva, which was significantly reduced or absent from C. nubeculosus saliva. Incubating purified BTV-1 with C. sonorensis saliva proteins also increased their infectivity for KC cells ∼10 fold, while infectivity for BHK cells was reduced by 2-6 fold. Treatment of an 'eastern' strain of EHDV-2 with saliva proteins of either C. sonorensis or C. nubeculosus cleaved VP2, but a 'western' strain of EHDV-2 remained unmodified. These results indicate that temperature, strain of virus and protein composition of Culicoides saliva (particularly its protease content which is dependent upon vector species, can all play a significant role in the efficiency of VP2 cleavage, influencing virus infectivity. Saliva of several other arthropod species has previously been shown to increase transmission, infectivity and virulence of certain arboviruses, by modulating and/or suppressing the mammalian immune response. The findings presented here, however, demonstrate a novel mechanism by which proteases in Culicoides saliva can also directly modify the orbivirus particle structure, leading to

  2. Cloning of an Avian Adeno-Associated Virus (AAAV) and Generation of Recombinant AAAV Particles

    OpenAIRE

    Bossis, Ioannis; Chiorini, John A.

    2003-01-01

    Recent studies have proposed that adeno-associated viruses (AAVs) are not evolutionarily linked to other mammalian autonomous parvoviruses but are more closely linked to the autonomous parvoviruses of birds. To better understand the relationship between primate and avian AAVs (AAAVs), we cloned and sequenced the genome of an AAAV (ATCC VR-865) and generated recombinant AAAV particles. The genome of AAAV is 4,694 nucleotides in length and has organization similar to that of other AAVs. The ent...

  3. A phase I randomized clinical trial of candidate human immunodeficiency virus type 1 vaccine MVA.HIVA administered to Gambian infants.

    Directory of Open Access Journals (Sweden)

    Muhammed O Afolabi

    Full Text Available A vaccine to decrease transmission of human immunodeficiency virus type 1 (HIV-1 during breast-feeding would complement efforts to eliminate infant HIV-1 infection by antiretroviral therapy. Relative to adults, infants have distinct immune development, potentially high-risk of transmission when exposed to HIV-1 and rapid progression to AIDS when infected. To date, there have been only three published HIV-1 vaccine trials in infants.We conducted a randomized phase I clinical trial PedVacc 001 assessing the feasibility, safety and immunogenicity of a single dose of candidate vaccine MVA.HIVA administered intramuscularly to 20-week-old infants born to HIV-1-negative mothers in The Gambia.Infants were followed to 9 months of age with assessment of safety, immunogenicity and interference with Expanded Program on Immunization (EPI vaccines. The trial is the first stage of developing more complex prime-boost vaccination strategies against breast milk transmission of HIV-1.From March to October 2010, 48 infants (24 vaccine and 24 no-treatment were enrolled with 100% retention. The MVA.HIVA vaccine was safe with no difference in adverse events between vaccinees and untreated infants. Two vaccine recipients (9% and no controls had positive ex vivo interferon-γ ELISPOT assay responses. Antibody levels elicited to the EPI vaccines, which included diphtheria, tetanus, whole-cell pertussis, hepatitis B virus, Haemophilus influenzae type b and oral poliovirus, reached protective levels for the vast majority and were similar between the two arms.A single low-dose of MVA.HIVA administered to 20-week-old infants in The Gambia was found to be safe and without interference with the induction of protective antibody levels by EPI vaccines, but did not alone induce sufficient HIV-1-specific responses. These data support the use of MVA carrying other transgenes as a boosting vector within more complex prime-boost vaccine strategies against transmission of HIV-1 and

  4. Immunogenicity of a 2009 pandemic influenza virus A H1N1 vaccine, administered simultaneously with the seasonal influenza vaccine, in children receiving chemotherapy.

    Science.gov (United States)

    Ottóffy, Gábor; Horváth, Petra; Muth, Lajos; Sólyom, Alexander; Garami, Miklós; Kovács, Gábor; Nyári, Tibor; Molnár, Dénes; Pauler, Gábor; Jankovics, István

    2014-06-01

    No examination of simultaneous vaccination against pandemic H1N1 and the seasonal influenza virus strains, in children with cancer receiving chemotherapy, are yet published. We investigated the immunogenicity of a whole-virion, inactivated, adjuvanted pandemic H1N1, and seasonal influenza vaccines administered simultaneously to children with cancer undergoing chemotherapy. We prospectively enrolled 27 pediatric patients receiving therapy for various types of cancer. All received influenza vaccination once in a seasonal risk period. We checked hemaglutination-inhibition (HAI) antibody titers in the sera of patients before, and 21-28 days after vaccination. Seroprotective titer was defined as an antibody titer ≥ 40, and seroresponse as ≥ 4-fold increase in antibody titers after vaccination. The pre- and post-vaccination seroprotective rates were H1N1: 33-48%, H3N2: 56-78%, B: 0-15% for seasonal influenza, and for pandemic H1N1: 15-37%. The seroresponse rates for seasonal influenza H1N1, H3N2, and B were 22%, 37%, and 22%, respectively, and 30% for the pandemic H1N1 vaccine. Whole-virion, inactivated, adjuvanted vaccine for the pandemic H1N1 Influenza A virus and the seasonal influenza vaccines were found safe and partially immunogenic in children with cancer receiving chemotherapy. The only determinants of responsiveness were lymphocyte count and serum immunoglobulin-G. Only influenza B vaccine elicited significant differences in differences in pre- and post-vaccination seroprotective rates. The response to vaccination for pandemic H1N1 is as effective as other vaccines, however administration of a single vaccine during chemotherapy is more comfortable for pediatric cancer patients. © 2014 Wiley Periodicals, Inc.

  5. 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.

  6. Enhancement of blood-brain barrier permeability is required for intravenously administered virus neutralizing antibodies to clear an established rabies virus infection from the brain and prevent the development of rabies in mice.

    Science.gov (United States)

    Huang, Chien-Tsun; Li, Zhenguang; Huang, Ying; Zhang, Guoqing; Zhou, Ming; Chai, Qingqing; Wu, Hua; Fu, Zhen F

    2014-10-01

    Rabies virus (RABV) is a neurotropic virus that causes fatal disease in humans and animals. Currently there is no cure for rabies once clinical signs appear. It is believed that once RABV enters the central nervous system (CNS), virus neutralizing antibodies (VNAs) in the periphery cannot pass through the blood-brain barrier (BBB) and into the CNS. Furthermore, it has been hypothesized that VNAs produced in the CNS by invading B cells, rather than those produced in the periphery and then transported into the CNS, are important in clearing RABV from the CNS. In the present study, mouse serum containing VNA was administered intravenously into mice after infection with wild-type RABV. Our studies demonstrate that exogenous administration of VNAs is crucial in the clearance of RABV from the brain and prevent the development of rabies in both immunocompetent and immunocompromised mice as long as the BBB permeability remains enhanced. This present study therefore provides a foundation for the possibility of developing VNA therapy for clinical rabies in humans. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Foot and mouth disease (FMD) virus: quantification of whole virus particles during the vaccine manufacturing process by size exclusion chromatography.

    Science.gov (United States)

    Spitteler, Marcelo A; Fernández, Ignacio; Schabes, Erika; Krimer, Alejandro; Régulier, Emmanuel G; Guinzburg, Mariela; Smitsaart, Eliana; Levy, M Susana

    2011-09-22

    Foot and mouth disease (FMD) is a highly infectious viral disease that affects cattle, sheep, goats and swine causing severe economic losses worldwide. The efficacy of inactivated vaccines is critically dependent on the integrity of foot and mouth disease virus (FMDV) particles. The recommended method to quantify the active ingredient of vaccines is the 140S quantitative sucrose density gradient analysis. This method has been an immensely valuable tool over the past three decades but it is highly operator dependent and difficult to automate. We developed a method to quantify FMDV particles during the vaccine manufacturing process that is based on separation of components by size-exclusion chromatography and measurement of virus by absorption at 254nm. The method is linear in the 5-70μg/mL range, it is applicable to different FMDV strains, and has a good correlation with the 140S test. The proposed method uses standard chromatographic media and it is amenable to automation. The method has potential as a process analytical technology and for control of final product by manufacturers, international vaccine banks and regulatory agencies. Copyright © 2011 Elsevier Ltd. All rights reserved.

  8. 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.

  9. Actin-myosin network is required for proper assembly of influenza virus particles

    Energy Technology Data Exchange (ETDEWEB)

    Kumakura, Michiko; Kawaguchi, Atsushi, E-mail: ats-kawaguchi@md.tsukuba.ac.jp; Nagata, Kyosuke, E-mail: knagata@md.tsukuba.ac.jp

    2015-02-15

    Actin filaments are known to play a central role in cellular dynamics. After polymerization of actin, various actin-crosslinking proteins including non-muscle myosin II facilitate the formation of spatially organized actin filament networks. The actin-myosin network is highly expanded beneath plasma membrane. The genome of influenza virus (vRNA) replicates in the cell nucleus. Then, newly synthesized vRNAs are nuclear-exported to the cytoplasm as ribonucleoprotein complexes (vRNPs), followed by transport to the beneath plasma membrane where virus particles assemble. Here, we found that, by inhibiting actin-myosin network formation, the virus titer tends to be reduced and HA viral spike protein is aggregated on the plasma membrane. These results indicate that the actin-myosin network plays an important role in the virus formation. - Highlights: • Actin-myosin network is important for the influenza virus production. • HA forms aggregations at the plasma membrane in the presence of blebbistatin. • M1 is recruited to the budding site through the actin-myosin network.

  10. Suramin inhibits Zika virus replication by interfering with virus attachment and release of infectious particles.

    Science.gov (United States)

    Albulescu, Irina C; Kovacikova, Kristina; Tas, Ali; Snijder, Eric J; van Hemert, Martijn J

    2017-07-01

    Zika virus (ZIKV) is a mosquito-borne flavivirus that mostly causes asymptomatic infections or mild disease characterized by low-grade fever, rash, conjunctivitis, and malaise. However, the recent massive ZIKV epidemics in the Americas have also linked ZIKV infection to fetal malformations like microcephaly and Guillain-Barré syndrome in adults, and have uncovered previously unrecognized routes of vertical and sexual transmission. Here we describe inhibition of ZIKV replication by suramin, originally an anti-parasitic drug, which was more recently shown to inhibit multiple viruses. In cell culture-based assays, using reduction of cytopathic effect as read-out, suramin had an EC50 of ∼40 μM and a selectivity index of 48. In single replication cycle experiments, suramin treatment also caused a strong dose-dependent decrease in intracellular ZIKV RNA levels and a >3-log reduction in infectious progeny titers. Time-of-addition experiments revealed that suramin inhibits a very early step of the replication cycle as well as the release of infectious progeny. Only during the first 2 h of infection suramin treatment strongly reduced the fraction of cells that became infected with ZIKV, suggesting the drug affects virus binding/entry. Binding experiments at 4 °C using 35S-labeled ZIKV demonstrated that suramin interferes with attachment to host cells. When suramin treatment was initiated post-entry, viral RNA synthesis was unaffected, while both the release of genomes and the infectivity of ZIKV were reduced. This suggests the compound also affects virion biogenesis, possibly by interfering with glycosylation and the maturation of ZIKV during its traffic through the secretory pathway. The inhibitory effect of suramin on ZIKV attachment and virion biogenesis and its broad-spectrum activity warrant further evaluation of this compound as a potential therapeutic. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. Immunological and biochemical characterization of coxsackie virus A16 viral particles.

    Directory of Open Access Journals (Sweden)

    Pele Chong

    Full Text Available BACKGROUND: Coxsackie virus A16 (CVA16 infections have become a serious public health problem in the Asia-Pacific region. It manifests most often in childhood exanthema, commonly known as hand-foot-and-mouth disease (HFMD. There are currently no vaccine or effective medical treatments available. PRINCIPAL FINDING: In this study, we describe the production, purification and characterization of CVA16 virus produced from Vero cells grown on 5 g/L Cytodex 1 microcarrier beads in a five-liter serum-free bioreactor system. The viral titer was found to be >10(6 the tissue culture's infectious dose (TCID(50 per mL within 7 days post-infection when a multiplicity of infection (MOI of 10(-5 was used for initial infection. Two CVA16 virus fractions were separated and detected when the harvested CVA16 viral concentrate was purified by a sucrose gradient zonal ultracentrifugation. The viral particles detected in the 24-28% sucrose fractions had low viral infectivity and RNA content. The viral particles obtained from 35-38% sucrose fractions were found to have high viral infectivity and RNA content, and composed of four viral proteins (VP1, VP2, VP3 and VP4, as shown by SDS-PAGE analyses. These two virus fractions were formalin-inactivated and only the infectious particle fraction was found to be capable of inducing CVA16-specific neutralizing antibody responses in both mouse and rabbit immunogenicity studies. But these antisera failed to neutralize enterovirus 71. In addition, rabbit antisera did not react with any peptides derived from CVA16 capsid proteins. Mouse antisera recognized a single linear immunodominant epitope of VP3 corresponding to residues 176-190. CONCLUSION: These results provide important information for cell-based CVA16 vaccine development. To eliminate HFMD, a bivalent EV71/CVA16 vaccine formulation is necessary.

  12. 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.

  13. Towards a correlative approach for characterising single virus particles by transmission electron microscopy and nanoscale Raman spectroscopy.

    Science.gov (United States)

    Hermelink, A; Naumann, D; Piesker, J; Lasch, P; Laue, M; Hermann, P

    2017-04-10

    The morphology and structure of biological nanoparticles, such as viruses, can be efficiently analysed by transmission electron microscopy (TEM). To chemically characterise such nanoparticles in heterogeneous samples at the single particle level, we suggest tip-enhanced Raman spectroscopy (TERS) as a correlative method. Here we describe a TERS-compatible staining procedure for TEM which involves sample pre-scanning by TEM imaging, nanoparticle relocalisation by atomic force microscopy (AFM) followed by spectroscopic characterization of the virus nanoparticles using TERS. First successful correlative measurements are demonstrated on tobacco mosaic virus particles deposited on silicon-based TEM sample supports. In addition, the advantages and problems of this methodology are discussed.

  14. Quantitative Evaluation of Protein Heterogeneity within Herpes Simplex Virus 1 Particles.

    Science.gov (United States)

    El Bilali, Nabil; Duron, Johanne; Gingras, Diane; Lippé, Roger

    2017-05-15

    Several virulence genes have been identified thus far in the herpes simplex virus 1 genome. It is also generally accepted that protein heterogeneity among virions further impacts viral fitness. However, linking this variability directly with infectivity has been challenging at the individual viral particle level. To address this issue, we resorted to flow cytometry (flow virometry), a powerful approach we recently employed to analyze individual viral particles, to identify which tegument proteins vary and directly address if such variability is biologically relevant. We found that the stoichiometry of the U L 37, ICP0, and VP11/12 tegument proteins in virions is more stable than the VP16 and VP22 tegument proteins, which varied significantly among viral particles. Most interestingly, viruses sorted for their high VP16 or VP22 content yielded modest but reproducible increases in infectivity compared to their corresponding counterparts containing low VP16 or VP22 content. These findings were corroborated for VP16 in short interfering RNA experiments but proved intriguingly more complex for VP22. An analysis by quantitative Western blotting revealed substantial alterations of virion composition upon manipulation of individual tegument proteins and suggests that VP22 protein levels acted indirectly on viral fitness. These findings reaffirm the interdependence of the virion components and corroborate that viral fitness is influenced not only by the genome of viruses but also by the stoichiometry of proteins within each virion. IMPORTANCE The ability of viruses to spread in animals has been mapped to several viral genes, but other factors are clearly involved, including virion heterogeneity. To directly probe whether the latter influences viral fitness, we analyzed the protein content of individual herpes simplex virus 1 particles using an innovative flow cytometry approach. The data confirm that some viral proteins are incorporated in more controlled amounts, while

  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. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Recombinant rabies virus particles presenting botulinum neurotoxin antigens elicit a protective humoral response in vivo

    Directory of Open Access Journals (Sweden)

    Andrew W Hudacek

    2014-01-01

    Full Text Available Botulinum neurotoxins are one of the most potent toxins found in nature, with broad medical applications from cosmetics to the treatment of various neuropathies. Additionally, these toxins are classified as Category A-Tier 1 agents, with human lethal doses calculated at as little as 90 ng depending upon the route of administration. Of the eight distinct botulinum neurotoxin serotypes, the most common causes of human illness are from serotypes /A, /B, and /E. Protection can be achieved by eliciting antibody responses against the receptor-binding domain of the neurotoxin. Our previous research has shown that recombinant rabies virus–based particles can effectively present heterologous antigens. Here, we describe a novel strategy using recombinant rabies virus particles that elicits a durable humoral immune response against the botulinum neurotoxin receptor binding domains from serotypes /A, /B, and /E. Following intramuscular administration of β-propiolactone-inactivated rabies virus particles, mice elicited specific immune responses against the cognate antigen. Administration of a combination of these vectors also demonstrated antibody responses against all three serotypes based on enzyme-linked immunosorbent assay (ELISA measurements, with minimal decay within the study timeline. Complete protection was achieved against toxin challenge from the serotypes /A and /B and partial protection for /E, indicating that a multivalent approach is feasible.

  1. 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.

  2. 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...

  3. 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

  4. 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.

  5. Single-dose immunization with virus replicon particles confers rapid robust protection against Rift Valley fever virus challenge.

    Science.gov (United States)

    Dodd, Kimberly A; Bird, Brian H; Metcalfe, Maureen G; Nichol, Stuart T; Albariño, César G

    2012-04-01

    Rift Valley fever virus (RVFV) causes outbreaks of severe disease in people and livestock throughout Africa and the Arabian Peninsula. The potential for RVFV introduction outside the area of endemicity highlights the need for fast-acting, safe, and efficacious vaccines. Here, we demonstrate a robust system for the reverse genetics generation of a RVF virus replicon particle (VRP(RVF)) vaccine candidate. Using a mouse model, we show that VRP(RVF) immunization provides the optimal balance of safety and single-dose robust efficacy. VRP(RVF) can actively synthesize viral RNA and proteins but lacks structural glycoprotein genes, preventing spread within immunized individuals and reducing the risk of vaccine-induced pathogenicity. VRP(RVF) proved to be completely safe following intracranial inoculation of suckling mice, a stringent test of vaccine safety. Single-dose subcutaneous immunization with VRP(RVF), although it is highly attenuated, completely protected mice against a virulent RVFV challenge dose which was 100,000-fold greater than the 50% lethal dose (LD(50)). Robust protection from lethal challenge was observed by 24 h postvaccination, with 100% protection induced in as little as 96 h. We show that a single subcutaneous VRP(RVF) immunization initiated a systemic antiviral state followed by an enhanced adaptive response. These data contrast sharply with the much-reduced survivability and immune responses observed among animals immunized with nonreplicating viral particles, indicating that replication, even if confined to the initially infected cells, contributes substantially to protective efficacy at early and late time points postimmunization. These data demonstrate that replicon vaccines successfully bridge the gap between safety and efficacy and provide insights into the kinetics of antiviral protection from RVFV infection.

  6. 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.

  7. 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.

  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. 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.

  10. 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

  11. 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.

  12. Infected dendritic cells are sufficient to mediate the adjuvant activity generated by Venezuelan equine encephalitis virus replicon particles

    OpenAIRE

    Tonkin, Daniel R; Whitmore, Alan; Johnston, Robert E; Barro, Mario

    2012-01-01

    Replicon particles derived from Venezuelan equine encephalitis virus (VEE) are infectious non-propagating particles which act as a safe and potent systemic, mucosal, and cellular adjuvant when delivered with antigen. VEE and VEE replicon particles (VRP) can target multiple cell types including dendritic cells (DCs). The role of these cell types in VRP adjuvant activity has not been previously evaluated, and for these studies we focused on the contribution of DCs to the response to VRP. By ana...

  13. 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...

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

    Directory of Open Access Journals (Sweden)

    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.

  15. Structure of Aichi virus 1 and its empty particle: clues towards kobuvirus genome release mechanism.

    Science.gov (United States)

    Sabin, Charles; Füzik, Tibor; Škubník, Karel; Pálková, Lenka; Lindberg, A Michael; Plevka, Pavel

    2016-09-28

    Aichi virus 1 (AiV-1) is a human pathogen from the Kobuvirus genus of the Picornaviridae family. Worldwide, 80-95% of adults have antibodies against the virus. AiV-1 infections are associated with nausea, gastroenteritis, and fever. Unlike most picornaviruses, kobuvirus capsids are composed of only three types of subunits: VP0, VP1, and VP3. Here we present the structure of the AiV-1 virion determined to a resolution of 2.1 Å using X-ray crystallography. The surface loops puff of VP0 and knob of VP3 in AiV-1 are shorter than those in other picornaviruses. Instead, the 42-residue-long BC-loop of VP0 forms the most prominent surface feature of the AiV-1 virion. We determined the structure of AiV-1 empty particle to a resolution of 4.2 Å using cryo-electron microscopy. The empty capsids are expanded relative to the native virus. The N-terminal arms of capsid proteins VP0, which mediate contacts between the pentamers of capsid protein protomers in the native AiV-1 virion, are disordered in the empty capsid. Nevertheless, the empty particles are stable, at least in vitro, and do not contain pores that might serve as channels for genome release. Therefore, extensive and probably reversible local reorganization of AiV-1 capsid is required for its genome release. Aichi virus 1 (AiV-1) is a human pathogen that can cause diarrhea, abdominal pain, nausea, vomiting, and fever. AiV-1 is identified in environmental screening studies with higher frequency and greater abundance than other human enteric viruses. Accordingly, 80-95% of adults worldwide have suffered from AiV-1 infections. We determined the structure of the AiV-1 virion. Based on the structure, we show that antiviral compounds that were developed against related enteroviruses are unlikely to be effective against AiV-1. The surface of the AiV-1 virion has a unique topology distinct from other related viruses from the Picornaviridae family. We also determined that AiV-1 capsids form compact shells even after genome

  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. Giant Polymersome Protocells Dock with Virus Particle Mimics via Multivalent Glycan-Lectin Interactions

    Science.gov (United States)

    Kubilis, Artur; Abdulkarim, Ali; Eissa, Ahmed M.; Cameron, Neil R.

    2016-08-01

    Despite the low complexity of their components, several simple physical systems, including microspheres, coacervate droplets and phospholipid membrane structures (liposomes), have been suggested as protocell models. These, however, lack key cellular characteristics, such as the ability to replicate or to dock with extracellular species. Here, we report a simple method for the de novo creation of synthetic cell mimics in the form of giant polymeric vesicles (polymersomes), which are capable of behavior approaching that of living cells. These polymersomes form by self-assembly, under electroformation conditions, of amphiphilic, glycosylated block copolymers in aqueous solution. The glycosylated exterior of the resulting polymeric giant unilamellar vesicles (GUVs) allows their selective interaction with carbohydrate-binding receptor-functionalized particles, in a manner reminiscent of the cell-surface docking of virus particles. We believe that this is the first example of a simple protocell model displaying cell-like behavior through a native receptor-ligand interaction.

  18. Giant Polymersome Protocells Dock with Virus Particle Mimics via Multivalent Glycan-Lectin Interactions.

    Science.gov (United States)

    Kubilis, Artur; Abdulkarim, Ali; Eissa, Ahmed M; Cameron, Neil R

    2016-08-31

    Despite the low complexity of their components, several simple physical systems, including microspheres, coacervate droplets and phospholipid membrane structures (liposomes), have been suggested as protocell models. These, however, lack key cellular characteristics, such as the ability to replicate or to dock with extracellular species. Here, we report a simple method for the de novo creation of synthetic cell mimics in the form of giant polymeric vesicles (polymersomes), which are capable of behavior approaching that of living cells. These polymersomes form by self-assembly, under electroformation conditions, of amphiphilic, glycosylated block copolymers in aqueous solution. The glycosylated exterior of the resulting polymeric giant unilamellar vesicles (GUVs) allows their selective interaction with carbohydrate-binding receptor-functionalized particles, in a manner reminiscent of the cell-surface docking of virus particles. We believe that this is the first example of a simple protocell model displaying cell-like behavior through a native receptor-ligand interaction.

  19. Conformations and membrane-driven self-organization of rodlike fd virus particles on freestanding lipid membranes.

    Science.gov (United States)

    Petrova, Anastasiia B; Herold, Christoph; Petrov, Eugene P

    2017-10-11

    Membrane-mediated interactions and aggregation of colloidal particles adsorbed to responsive elastic membranes are challenging problems relevant for understanding the microscopic organization and dynamics of biological membranes. We experimentally study the behavior of rodlike semiflexible fd virus particles electrostatically adsorbed to freestanding cationic lipid membranes and find that their behavior can be controlled by tuning the membrane charge and ionic strength of the surrounding medium. Three distinct interaction regimes of rodlike virus particles with responsive elastic membranes can be observed. (i) A weakly charged freestanding cationic lipid bilayer in a low ionic strength medium represents a gentle quasi-2D substrate preserving the integrity, structure, and mechanical properties of the membrane-bound semiflexible fd virus, which under these conditions is characterized by a monomer length of 884 ± 4 nm and a persistence length of 2.5 ± 0.2 μm, in perfect agreement with its properties in bulk media. (ii) An increase in the membrane charge leads to the membrane-driven collapse of fd virus particles on freestanding lipid bilayers and lipid nanotubes into compact globules. (iii) When the membrane charge is low, and the mutual electrostatic repulsion of membrane-bound virus particles is screened to a considerable degree, membrane-driven self-organization of membrane-bound fd virus particles into long linear tip-to-tip aggregates showing dynamic self-assembly/disassembly and quasi-semiflexible behavior takes place. These observations are in perfect agreement with the results of recent theoretical and simulation studies predicting that membrane-mediated interactions can control the behavior of colloidal particles adsorbed on responsive elastic membranes.

  20. 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.

  1. Plant virus particles carrying tumour antigen activate TLR7 and Induce high levels of protective antibody.

    Directory of Open Access Journals (Sweden)

    Jantipa Jobsri

    Full Text Available 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 natural adjuvant, the viral ssRNA, which acts via TLR7. It induced potent protective anti-Id antibody responses in an in vivo mouse model, superior to the "gold standard" Id vaccine, with prevalence of the IgG2a isotype. Combination with alum further increased antibody levels and maintained the IgG2a bias. Engagement of TLR7 in vivo was followed by secretion of IFN-α by plasmacytoid dendritic cells and by activation of splenic CD11chi conventional dendritic cells. The latter was apparent from up-regulation of co-stimulatory molecules and from secretion of a wide range of inflammatory cytokines and chemokines including the Th1-governing cytokine IL-12, in keeping with the IgG2a antibody isotype distribution. PVP conjugates are a novel cancer vaccine design, offering an attractive molecular form, similar to VLP, and providing T-cell help. In contrast to VLP, they also incorporate a safe "in-built" ssRNA adjuvant.

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

    Science.gov (United States)

    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.

  3. Development of a neutralization assay for Nipah virus using pseudotype particles.

    Science.gov (United States)

    Tamin, Azaibi; Harcourt, Brian H; Lo, Michael K; Roth, James A; Wolf, Mike C; Lee, Benhur; Weingartl, Hana; Audonnet, Jean-Christophe; Bellini, William J; Rota, Paul A

    2009-09-01

    Nipah virus (NiV) and Hendra virus (HeV) are zoonotic paramyxoviruses capable of causing severe disease in humans and animals. These viruses require biosafety level 4 (BSL-4) containment. Like other paramyxoviruses, the plaque reduction neutralization test (PRNT) can be used to detect antibodies to the surface glycoproteins, fusion (F) and attachment (G), and PRNT titers give an indication of protective immunity. Unfortunately, for NiV and HeV, the PRNT must be performed in BSL-4 containment and takes several days to complete. Thus, we have developed a neutralization assay using VSV pseudotype particles expressing the F and G proteins of NiV (pVSV-NiV-F/G) as target antigens. This rapid assay, which can be performed at BSL-2, was evaluated using serum samples from outbreak investigations and more than 300 serum samples from an experimental NiV vaccination study in swine. The results of the neutralization assays with pVSV-NiV-F/G as antigen showed a good correlation with those of standard PRNT. Therefore, this new method has the potential to be a rapid and cost-effective diagnostic method, especially in locations that lack high containment facilities, and will provide a valuable tool for basic research and vaccine development.

  4. African Swine Fever Virus Georgia 2007 with a Deletion of Virulence-Associated Gene 9GL (B119L), when Administered at Low Doses, Leads to Virus Attenuation in Swine and Induces an Effective Protection against Homologous Challenge.

    Science.gov (United States)

    O'Donnell, Vivian; Holinka, Lauren G; Krug, Peter W; Gladue, Douglas P; Carlson, Jolene; Sanford, Brenton; Alfano, Marialexia; Kramer, Edward; Lu, Zhiqiang; Arzt, Jonathan; Reese, Bo; Carrillo, Consuelo; Risatti, Guillermo R; Borca, Manuel V

    2015-08-01

    African swine fever virus (ASFV) is the etiological agent of an often lethal disease of domestic pigs. Disease control strategies have been hampered by the unavailability of vaccines against ASFV. Since its introduction in the Republic of Georgia, a highly virulent virus, ASFV Georgia 2007 (ASFV-G), has caused an epizootic that spread rapidly into Eastern European countries. Currently no vaccines are available or under development to control ASFV-G. In the past, genetically modified ASFVs harboring deletions of virulence-associated genes have proven attenuated in swine, inducing protective immunity against challenge with homologous parental viruses. Deletion of the gene 9GL (open reading frame [ORF] B119L) in highly virulent ASFV Malawi-Lil-20/1 produced an attenuated phenotype even when administered to pigs at 10(6) 50% hemadsorption doses (HAD50). Here we report the construction of a genetically modified ASFV-G strain (ASFV-G-Δ9GLv) harboring a deletion of the 9GL (B119L) gene. Like Malawi-Lil-20/1-Δ9GL, ASFV-G-Δ9GL showed limited replication in primary swine macrophages. However, intramuscular inoculation of swine with 10(4) HAD50 of ASFV-G-Δ9GL produced a virulent phenotype that, unlike Malawi-Lil-20/1-Δ9GL, induced a lethal disease in swine like parental ASFV-G. Interestingly, lower doses (10(2) to 10(3) HAD50) of ASFV-G-Δ9GL did not induce a virulent phenotype in swine and when challenged protected pigs against disease. A dose of 10(2) HAD50 of ASFV-G-Δ9GLv conferred partial protection when pigs were challenged at either 21 or 28 days postinfection (dpi). An ASFV-G-Δ9GL HAD50 of 10(3) conferred partial and complete protection at 21 and 28 dpi, respectively. The information provided here adds to our recent report on the first attempts toward experimental vaccines against ASFV-G. The main problem for controlling ASF is the lack of vaccines. Studies on ASFV virulence lead to the production of genetically modified attenuated viruses that induce protection

  5. 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...

  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. 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.

  8. 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...

  9. 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...

  10. 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

  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. Fatty acid biosynthesis is involved in the production of hepatitis B virus particles

    Energy Technology Data Exchange (ETDEWEB)

    Okamura, Hitomi [Laboratory of Human Tumor Viruses, Institute for Virus Research, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan); Graduate School of Biostudies, Kyoto University, Yoshida-Konoecho, Sakyoku, Kyoto 606-8501 (Japan); Nio, Yasunori, E-mail: yasunori.nio@takeda.com [Takeda Pharmaceutical Company Limited, Pharmaceutical Research Division, 26-1, Muraoka-Higashi 2-Chome, Fujisawa, Kanagawa 251-8555 (Japan); Akahori, Yuichi [Laboratory of Human Tumor Viruses, Institute for Virus Research, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan); Graduate School of Biostudies, Kyoto University, Yoshida-Konoecho, Sakyoku, Kyoto 606-8501 (Japan); Kim, Sulyi [Laboratory of Human Tumor Viruses, Institute for Virus Research, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan); Watashi, Koichi [Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640 (Japan); Department of Applied Biological Science, Tokyo University of Sciences, Noda 278-8510 (Japan); CREST, Japan Science and Technology Agency (JST), Saitama 332-0012 (Japan); Wakita, Takaji [Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640 (Japan); Hijikata, Makoto, E-mail: mhijikat@virus.kyoto-u.ac.jp [Laboratory of Human Tumor Viruses, Institute for Virus Research, Kyoto University, 53 Kawaharacho, Shogoin, Sakyoku, Kyoto 606-8507 (Japan); Graduate School of Biostudies, Kyoto University, Yoshida-Konoecho, Sakyoku, Kyoto 606-8501 (Japan)

    2016-06-17

    Hepatitis B virus (HBV) proliferates in hepatocytes after infection, but the host factors that contribute to the HBV lifecycle are poorly understood at the molecular level. We investigated whether fatty acid biosynthesis (FABS), which was recently reported to contribute to the genomic replication of hepatitis C virus, plays a role in HBV proliferation. We examined the effects of inhibitors of the enzymes in the FABS pathway on the HBV lifecycle by using recombinant HBV-producing cultured cells and found that the extracellular HBV DNA level, reflecting HBV particle production, was decreased by treatment with inhibitors suppressed the synthesis of long-chain saturated fatty acids with little cytotoxicity. The reduced HBV DNA level was reversed when palmitic acid, which is the product of fatty acid synthase (FAS) during FABS, was used simultaneously with the inhibitor. We also observed that the amount of intracellular HBV DNA in the cells was increased by FAS inhibitor treatment, suggesting that FABS is associated with HBV particle production but not its genome replication. This suggests that FABS might be a potent target for anti-HBV drug with a mode of action different from current HBV therapy. -- Highlights: •Inhibitors of ACC1 and FAS but not SCD1 decreased production of extracellular HBV DNA. •Products of FABS, long chain fatty acids, increased production of extracellular HBV DNA. •FAS inhibitor increased intracellular levels of HBV DNA and HBcAg. •FABS was suggested to contribute to HBV particle production without significant relation with secretory pathway of the cells.

  13. 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.

  14. Single-particle detection of virus simulants under microfluidic flow with two-dimensional photonic crystals (Conference Presentation)

    Science.gov (United States)

    Miller, Benjamin L.; Baker, James E.; Sriram, Rashmi

    2017-05-01

    Because of their compatibility with standard CMOS fabrication, small footprint, and exceptional sensitivity, Two-Dimensional Photonic Crystals (2D PhCs) have been posited as attractive components for the development of real-time integrated photonic virus sensors. While detection of single virus-sized particles by 2D PhCs has been demonstrated, specific recognition of a virus simulant under conditions relevant to sensor use (including aqueous solution and microfluidic flow) has remained an unsolved challenge. This talk will describe the design and testing of a W1 waveguide-coupled 2D PhC in the context of addressing that challenge.

  15. Quantitation of influenza virus using field flow fractionation and multi-angle light scattering for quantifying influenza A particles.

    Science.gov (United States)

    Bousse, Tatiana; Shore, David A; Goldsmith, Cynthia S; Hossain, M Jaber; Jang, Yunho; Davis, Charles T; Donis, Ruben O; Stevens, James

    2013-11-01

    Recent advances in instrumentation and data analysis in field flow fractionation and multi-angle light scattering (FFF-MALS) have enabled greater use of this technique to characterize and quantitate viruses. In this study, the FFF-MALS technique was applied to the characterization and quantitation of type A influenza virus particles to assess its usefulness for vaccine preparation. The use of FFF-MALS for quantitation and measurement of control particles provided data accurate to within 5% of known values, reproducible with a coefficient of variation of 1.9%. The methods, sensitivity and limit of detection were established by analyzing different volumes of purified virus, which produced a linear regression with fitting value R2 of 0.99. FFF-MALS was further applied to detect and quantitate influenza virus in the supernatant of infected MDCK cells and allantoic fluids of infected eggs. FFF fractograms of the virus present in these different fluids revealed similar distribution of monomeric and oligomeric virions. However, the monomer fraction of cell grown virus had greater size variety. Notably, β-propialactone (BPL) inactivation of influenza viruses did not influence any of the FFF-MALS measurements. Quantitation analysis by FFF-MALS was compared to infectivity assays and real-time RT-PCR (qRT-PCR) and the limitations of each assay were discussed. Published by Elsevier B.V.

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

    Science.gov (United States)

    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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

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

    Science.gov (United States)

    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.

  6. 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.

  7. 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.

  8. 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.

  9. 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...

  10. Vaccination with recombinant RNA replicon particles protects chickens from H5N1 highly pathogenic avian influenza virus.

    Science.gov (United States)

    Halbherr, Stefan J; Brostoff, Terza; Tippenhauer, Merve; Locher, Samira; Berger Rentsch, Marianne; Zimmer, Gert

    2013-01-01

    Highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV) vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA) was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×10⁸ infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA) by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade). Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry.

  11. Vaccination with recombinant RNA replicon particles protects chickens from H5N1 highly pathogenic avian influenza virus.

    Directory of Open Access Journals (Sweden)

    Stefan J Halbherr

    Full Text Available Highly pathogenic avian influenza viruses (HPAIV of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×10⁸ infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade. Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry.

  12. Preparation of MS2 phage-like particles and their use as potential process control viruses for detection and quantification of enteric RNA viruses in different matrices

    Directory of Open Access Journals (Sweden)

    Pavel Mikel

    2016-12-01

    Full Text Available The detection and quantification of enteric RNA viruses is based on isolation of viral RNA from the sample followed by quantitative reverse transcription polymerase chain reaction (RT-qPCR. To control the whole process of analysis and in order to guarantee the validity and reliability of results, process control viruses (PCV are used. The present article describes the process of preparation and use of such PCV– MS2 phage-like particles (MS2 PLP – in RT-qPCR detection and quantification of enteric RNA viruses. The MS2 PLP were derived from bacteriophage MS2 carrying a unique and specific de novo-constructed RNA target sequence originating from the DNA of two extinct species. The amount of prepared MS2 particles was quantified using four independent methods - UV spectrophotometry, fluorimetry, transmission electron microscopy (TEM and a specifically developed duplex RT-qPCR. To evaluate the usefulness of MS2 PLP in routine diagnostics different matrices known to harbor enteric RNA viruses (swab samples, liver tissue, serum, feces, and vegetables were artificially contaminated with specific amounts of MS2 PLP. The extraction efficiencies were calculated for each individual matrix. The prepared particles fulfill all requirements for PCV – they are very stable, non-infectious, and are genetically distinct from the target RNA viruses. Due to these properties they represent a good morphological and physiochemical model. The use of MS2 PLP as a PCV in detection and quantification of enteric RNA viruses was evaluated in different types of matrices.

  13. Inhibition of full length Hepatitis C Virus particles of 1a genotype through small interference RNA

    Directory of Open Access Journals (Sweden)

    Rehman Sidra

    2011-05-01

    Full Text Available Abstract Background Hepatitis C virus (HCV, a member of the Flaviviridae family of viruses, is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Currently, the only treatment available consists of a combination of Pegylated interferon alpha (INF-α and ribavirin, but only half of the patients treated show a sufficient antiviral response. Thus there is a great need for the development of new treatments for HCV infections. RNA interference (RNAi represents a new promising approach to develop effective antiviral drugs and has been extremely effective against HCV infection. Results This study was design to assess or explore the silencing effect of small interference RNAs (siRNAs against full length HCV particles of genotype 1a. In the present study six 21-bp siRNAs were designed against different regions of HCV structural genes (Core, E1 and E2. Selected siRNAs were labeled as Csi 301, Csi 29, E1si 52, E1si 192, E2si 86 and E2si 493. Our results demonstrated that siRNAs directed against HCV core gene showed 70% reduction in viral titer in HCV infected liver cells. Moreover, siRNAs against E1 and E2 envelop genes showed a dramatic reduction in HCV viral RNA, E2si 86 exhibited 93% inhibition, while E1si 192, E2si 493 and E1si 52 showed 87%, 80%, and 66% inhibition respectively. No significant inhibition was detected in cells transfected with the negative control siRNA. Conclusion Our results suggested that siRNAs targeted against HCV structural genes efficiently silence full length HCV particles and provide an effective therapeutic option against HCV infection.

  14. Plant Virus Particles Carrying Tumour Antigen Activate TLR7 and Induce High Levels of Protective Antibody

    Science.gov (United States)

    Jobsri, Jantipa; Allen, Alex; Rajagopal, Deepa; Shipton, Michael; Kanyuka, Kostya; Lomonossoff, George P.; Ottensmeier, Christian; Diebold, Sandra S.; Stevenson, Freda K.; Savelyeva, Natalia

    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 natural adjuvant, the viral ssRNA, which acts via TLR7. It induced potent protective anti-Id antibody responses in an in vivo mouse model, superior to the “gold standard” Id vaccine, with prevalence of the IgG2a isotype. Combination with alum further increased antibody levels and maintained the IgG2a bias. Engagement of TLR7 in vivo was followed by secretion of IFN-α by plasmacytoid dendritic cells and by activation of splenic CD11chi conventional dendritic cells. The latter was apparent from up-regulation of co-stimulatory molecules and from secretion of a wide range of inflammatory cytokines and chemokines including the Th1-governing cytokine IL-12, in keeping with the IgG2a antibody isotype distribution. PVP conjugates are a novel cancer vaccine design, offering an attractive molecular form, similar to VLP, and providing T-cell help. In contrast to VLP, they also incorporate a safe “in-built” ssRNA adjuvant. PMID:25692288

  15. 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.

  16. Efficient eradication of hormone-resistant human prostate cancers by inactivated Sendai virus particle.

    Science.gov (United States)

    Kawaguchi, Yoshifumi; Miyamoto, Yasuhide; Inoue, Takehiro; Kaneda, Yasufumi

    2009-05-15

    Hormone-refractory prostate cancer is one of the intractable human cancers in the world. Here, we examined the direct tumor-killing activity of inactivated Sendai virus particle [hemagglutinating virus of Japan envelope (HVJ-E)] through induction of Type I interferon (IFN) in the hormone-resistant human prostate cancer cell lines PC3 and DU145. Preferential binding of HVJ-E to PC3 and DU145 over hormone-sensitive prostate cancer cell and normal prostate epithelium was observed, resulting in a number of fused cells. After HVJ-E treatment, a number of IFN-related genes were up-regulated, resulting in Type I IFN production in PC3 cells. Then, retinoic acid-inducible gene-I (RIG-I) helicase which activates Type I IFN expression after Sendai virus infection was up-regulated in cancer cells after HVJ-E treatment. Produced IFN-alpha and -beta enhanced caspase 8 expression via Janus kinases/Signal Transducers and Activators of Transcription pathway, activated caspase 3 and induced apoptosis in cancer cells. When HVJ-E was directly injected into a mass of PC3 tumor cells in SCID (severe combined immunodeficiency) mice, a marked reduction in the bulk of each tumor mass was observed and 85% of the mice became tumor-free. Although co-injection of an anti-asialo GM1 antibody with HVJ-E into each tumor mass slightly attenuated the tumor suppressive activity of HVJ-E, significant suppression of tumor growth was observed even in the presence of anti-asialo GM1 antibody. This suggests that natural killer cell activation made small contribution to tumor regression following HVJ-E treatment in hormone-resistant prostate cancer model in vivo. Thus, HVJ-E effectively targets hormone-resistant prostate cancer by inducing apoptosis in tumor cells, as well as activating anti-tumor immunity. (c) 2009 Wiley-Liss, Inc.

  17. Replicon Particles of Venezuelan Equine Encephalitis Virus as a Reductionist Murine Model for Encephalitis▿

    Science.gov (United States)

    Schäfer, Alexandra; Whitmore, Alan C.; Konopka, Jennifer L.; Johnston, Robert E.

    2009-01-01

    Venezuelan equine encephalitis virus (VEE) replicon particles (VRP) were used to model the initial phase of VEE-induced encephalitis in the mouse brain. VRP can target and infect cells as VEE, but VRP do not propagate beyond the first infected cell due to the absence of the structural genes. Direct intracranial inoculation of VRP into mice induced acute encephalitis with signs similar to the neuronal phase of wild-type VEE infection and other models of virus-induced encephalitis. Using the previously established VRP-mRNP tagging system, a new method to distinguish the host responses in infected cells from those in uninfected bystander cell populations, we detected a robust and rapid innate immune response in the central nervous system (CNS) by infected neurons and uninfected bystander cells. Moreover, this innate immune response in the CNS compromised blood-brain barrier integrity, created an inflammatory response, and directed an adaptive immune response characterized by proliferation and activation of microglia cells and infiltration of inflammatory monocytes, in addition to CD4+ and CD8+ T lymphocytes. Taken together, these data suggest that a naïve CNS has an intrinsic potential to induce an innate immune response that could be crucial to the outcome of the infection by determining the composition and dynamics of the adaptive immune response. Furthermore, these results establish a model for neurotropic virus infection to identify host and viral factors that contribute to invasion of the brain, the mechanism(s) whereby the adaptive immune response can clear the infection, and the role of the host innate response in these processes. PMID:19225006

  18. Venezuelan equine encephalitis replicon particles can induce rapid protection against foot-and-mouth disease virus.

    Science.gov (United States)

    Diaz-San Segundo, Fayna; Dias, Camila C A; Moraes, Mauro P; Weiss, Marcelo; Perez-Martin, Eva; Owens, Gary; Custer, Max; Kamrud, Kurt; de los Santos, Teresa; Grubman, Marvin J

    2013-05-01

    We have previously shown that delivery of the porcine type I interferon gene (poIFN-α/β) with a replication-defective human adenovirus vector (adenovirus 5 [Ad5]) can sterilely protect swine challenged with foot-and-mouth disease virus (FMDV) 1 day later. However, the need of relatively high doses of Ad5 limits the applicability of such a control strategy in the livestock industry. Venezuelan equine encephalitis virus (VEE) empty replicon particles (VRPs) can induce rapid protection of mice against either homologous or, in some cases, heterologous virus challenge. As an alternative approach to induce rapid protection against FMDV, we have examined the ability of VRPs containing either the gene for green fluorescent protein (VRP-GFP) or poIFN-α (VRP-poIFN-α) to block FMDV replication in vitro and in vivo. Pretreatment of swine or bovine cell lines with either VRP significantly inhibited subsequent infection with FMDV as early as 6 h after treatment and for at least 120 h posttreatment. Furthermore, mice pretreated with either 10(7) or 10(8) infectious units of VRP-GFP and challenged with a lethal dose of FMDV 24 h later were protected from death. Protection was induced as early as 6 h after treatment and lasted for at least 48 h and correlated with induction of an antiviral response and production of IFN-α. By 6 h after treatment several genes were upregulated, and the number of genes and the level of induction increased at 24 h. Finally, we demonstrated that the chemokine IP-10, which is induced by IFN-α and VRP-GFP, is directly involved in protection against FMDV.

  19. Replicon particles of Venezuelan equine encephalitis virus as a reductionist murine model for encephalitis.

    Science.gov (United States)

    Schäfer, Alexandra; Whitmore, Alan C; Konopka, Jennifer L; Johnston, Robert E

    2009-05-01

    Venezuelan equine encephalitis virus (VEE) replicon particles (VRP) were used to model the initial phase of VEE-induced encephalitis in the mouse brain. VRP can target and infect cells as VEE, but VRP do not propagate beyond the first infected cell due to the absence of the structural genes. Direct intracranial inoculation of VRP into mice induced acute encephalitis with signs similar to the neuronal phase of wild-type VEE infection and other models of virus-induced encephalitis. Using the previously established VRP-mRNP tagging system, a new method to distinguish the host responses in infected cells from those in uninfected bystander cell populations, we detected a robust and rapid innate immune response in the central nervous system (CNS) by infected neurons and uninfected bystander cells. Moreover, this innate immune response in the CNS compromised blood-brain barrier integrity, created an inflammatory response, and directed an adaptive immune response characterized by proliferation and activation of microglia cells and infiltration of inflammatory monocytes, in addition to CD4(+) and CD8(+) T lymphocytes. Taken together, these data suggest that a naïve CNS has an intrinsic potential to induce an innate immune response that could be crucial to the outcome of the infection by determining the composition and dynamics of the adaptive immune response. Furthermore, these results establish a model for neurotropic virus infection to identify host and viral factors that contribute to invasion of the brain, the mechanism(s) whereby the adaptive immune response can clear the infection, and the role of the host innate response in these processes.

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

    Directory of Open Access Journals (Sweden)

    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.

  1. 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.

  2. 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

  3. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Alix regulates egress of hepatitis B virus naked capsid particles in an ESCRT-independent manner.

    Science.gov (United States)

    Bardens, Andreas; Döring, Tatjana; Stieler, Jens; Prange, Reinhild

    2011-04-01

    Hepatitis B virus (HBV) is an enveloped DNA virus that exploits the endosomal sorting complexes required for transport (ESCRT) pathway for budding. In addition to infectious particles, HBV-replicating cells release non-enveloped (nucleo)capsids, but their functional implication and pathways of release are unclear. Here, we focused on the molecular mechanisms and found that the sole expression of the HBV core protein is sufficient for capsid release. Unexpectedly, released capsids are devoid of a detectable membrane bilayer, implicating a non-vesicular exocytosis process. Unlike virions, naked capsid budding does not require the ESCRT machinery. Rather, we identified Alix, a multifunctional protein with key roles in membrane biology, as a regulator of capsid budding. Ectopic overexpression of Alix enhanced capsid egress, while its depletion inhibited capsid release. Notably, the loss of Alix did not impair HBV production, furthermore indicating that virions and capsids use diverse export routes. By mapping of Alix domains responsible for its capsid release-mediating activity, its Bro1 domain was found to be required and sufficient. Alix binds to core via its Bro1 domain and retained its activity even if its ESCRT-III binding site is disrupted. Together, the boomerang-shaped Bro1 domain of Alix appears to escort capsids without ESCRT. © 2010 Blackwell Publishing Ltd.

  6. 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.

  7. 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

  8. 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

  9. 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...

  10. 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...

  11. The length of vesicular stomatitis virus particles dictates a need for actin assembly during clathrin-dependent endocytosis.

    Directory of Open Access Journals (Sweden)

    David K Cureton

    2010-09-01

    Full Text Available Microbial pathogens exploit the clathrin endocytic machinery to enter host cells. Vesicular stomatitis virus (VSV, an enveloped virus with bullet-shaped virions that measure 70 x 200 nm, enters cells by clathrin-dependent endocytosis. We showed previously that VSV particles exceed the capacity of typical clathrin-coated vesicles and instead enter through endocytic carriers that acquire a partial clathrin coat and require local actin filament assembly to complete vesicle budding and internalization. To understand why the actin system is required for VSV uptake, we compared the internalization mechanisms of VSV and its shorter (75 nm long defective interfering particle, DI-T. By imaging the uptake of individual particles into live cells, we found that, as with parental virions, DI-T enters via the clathrin endocytic pathway. Unlike VSV, DI-T internalization occurs through complete clathrin-coated vesicles and does not require actin polymerization. Since VSV and DI-T particles display similar surface densities of the same attachment glycoprotein, we conclude that the physical properties of the particle dictate whether a virus-containing clathrin pit engages the actin system. We suggest that the elongated shape of a VSV particle prevents full enclosure by the clathrin coat and that stalling of coat assembly triggers recruitment of the actin machinery to finish the internalization process. Since some enveloped viruses have pleomorphic particle shapes and sizes, our work suggests that they may use altered modes of endocytic uptake. More generally, our findings show the importance of cargo geometry for specifying cellular entry modes, even when the receptor recognition properties of a ligand are maintained.

  12. The Length of Vesicular Stomatitis Virus Particles Dictates a Need for Actin Assembly during Clathrin-Dependent Endocytosis

    Science.gov (United States)

    Cureton, David K.; Massol, Ramiro H.; Whelan, Sean P. J.; Kirchhausen, Tomas

    2010-01-01

    Microbial pathogens exploit the clathrin endocytic machinery to enter host cells. Vesicular stomatitis virus (VSV), an enveloped virus with bullet-shaped virions that measure 70×200 nm, enters cells by clathrin-dependent endocytosis. We showed previously that VSV particles exceed the capacity of typical clathrin-coated vesicles and instead enter through endocytic carriers that acquire a partial clathrin coat and require local actin filament assembly to complete vesicle budding and internalization. To understand why the actin system is required for VSV uptake, we compared the internalization mechanisms of VSV and its shorter (75 nm long) defective interfering particle, DI-T. By imaging the uptake of individual particles into live cells, we found that, as with parental virions, DI-T enters via the clathrin endocytic pathway. Unlike VSV, DI-T internalization occurs through complete clathrin-coated vesicles and does not require actin polymerization. Since VSV and DI-T particles display similar surface densities of the same attachment glycoprotein, we conclude that the physical properties of the particle dictate whether a virus-containing clathrin pit engages the actin system. We suggest that the elongated shape of a VSV particle prevents full enclosure by the clathrin coat and that stalling of coat assembly triggers recruitment of the actin machinery to finish the internalization process. Since some enveloped viruses have pleomorphic particle shapes and sizes, our work suggests that they may use altered modes of endocytic uptake. More generally, our findings show the importance of cargo geometry for specifying cellular entry modes, even when the receptor recognition properties of a ligand are maintained. PMID:20941355

  13. 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.

  14. 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.

  15. Ultrastructural Characterization of Turnip Mosaic Virus-Induced Cellular Rearrangements Reveals Membrane-Bound Viral Particles Accumulating in Vacuoles.

    Science.gov (United States)

    Wan, Juan; Basu, Kaustuv; Mui, Jeannie; Vali, Hojatollah; Zheng, Huanquan; Laliberté, Jean-François

    2015-12-01

    Positive-strand RNA [(+) RNA] viruses remodel cellular membranes to facilitate virus replication and assembly. In the case of turnip mosaic virus (TuMV), the viral membrane protein 6K2 plays an essential role in endomembrane alterations. Although 6K2-induced membrane dynamics have been widely studied by confocal microscopy, the ultrastructure of this remodeling has not been extensively examined. In this study, we investigated the formation of TuMV-induced membrane changes by chemical fixation and high-pressure freezing/freeze substitution (HPF/FS) for transmission electron microscopy at different times of infection. We observed the formation of convoluted membranes connected to rough endoplasmic reticulum (rER) early in the infection process, followed by the production of single-membrane vesicle-like (SMVL) structures at the midstage of infection. Both SMVL and double-membrane vesicle-like structures with electron-dense cores, as well as electron-dense bodies, were found late in the infection process. Immunogold labeling results showed that the vesicle-like structures were 6K2 tagged and suggested that only the SMVL structures were viral RNA replication sites. Electron tomography (ET) was used to regenerate a three-dimensional model of these vesicle-like structures, which showed that they were, in fact, tubules. Late in infection, we observed filamentous particle bundles associated with electron-dense bodies, which suggests that these are sites for viral particle assembly. In addition, TuMV particles were observed to accumulate in the central vacuole as membrane-associated linear arrays. Our work thus unravels the sequential appearance of distinct TuMV-induced membrane structures for viral RNA replication, viral particle assembly, and accumulation. Positive-strand RNA viruses remodel cellular membranes for different stages of the infection process, such as protein translation and processing, viral RNA synthesis, particle assembly, and virus transmission. The

  16. Antigen-presenting particle technology using inactivated surface-engineered viruses: induction of immune responses against infectious agents

    Directory of Open Access Journals (Sweden)

    Chang Yung-Nien

    2007-05-01

    Full Text Available Abstract Background Developments in cell-based and gene-based therapies are emerging as highly promising areas to complement pharmaceuticals, but present day approaches are too cumbersome and thereby limit their clinical usefulness. These shortcomings result in procedures that are too complex and too costly for large-scale applications. To overcome these shortcomings, we described a protein delivery system that incorporates over-expressed proteins into viral particles that are non-infectious and stable at room temperature. The system relies on the biological process of viral egress to incorporate cellular surface proteins while exiting their host cells during lytic and non-lytic infections. Results We report here the use of non-infectious surface-engineered virion particles to modulate immunity against three infectious disease agents – human immunodeficiency virus type 1 (HIV-1, herpes simplex virus (HSV, and Influenza. Surface-engineering of particles are accomplished by genetic modification of the host cell surface that produces the egress budding viral particle. Human peripheral blood lymphocytes from healthy donors exposed to CD80/B7.1, CD86/B7.2, and/or antiCD3 single-chain antibody surface-engineered non-infectious HIV-1 and HSV-2 particles stimulate T cell proliferation, whereas particles released from non-modified host cells have no T cell stimulatory activity. In addition to T cell proliferation, HIV-based particles specifically suppress HIV-1 replication (both monocytotropic and lymphocytotropic strains 55 to 96% and HSV-based particles specifically induce cross-reactive HSV-1/HSV-2 anti-herpes virus antibody production. Similar surface engineering of influenza-based particles did not modify the intrinsic ability of influenza particles to stimulate T cell proliferation, but did bestow on the engineered particles the ability to induce cross-strain anti-influenza antibody production. Conclusion We propose that non-infectious viral

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

    Directory of Open Access Journals (Sweden)

    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.

  18. 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.

  19. 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.

  20. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    OpenAIRE

    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 ...

  3. 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...

  4. 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.

  5. 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.

  6. Clustering and cellular distribution characteristics of virus particles of Tomato spotted wilt virus and Tomato zonate spot virus in different plant hosts

    National Research Council Canada - National Science Library

    Zhang, Zhongkai; Zheng, Kuanyu; Dong, Jiahong; Fang, Qi; Hong, Jian; Wang, Xifeng

    2016-01-01

    Tomato spotted wilt virus (TSWV) and Tomato zonate spot virus (TZSV) are the two dominant species of thrip-transmitted tospoviruses, cause significant losses in crop yield in Yunnan and its neighboring provinces in China...

  7. Monitoring processed, mature Human Immunodeficiency Virus type 1 particles immediately following treatment with a protease inhibitor-containing treatment regimen

    Directory of Open Access Journals (Sweden)

    Kuritzkes Daniel R

    2005-04-01

    Full Text Available Abstract Protease inhibitors (PIs block HIV-1 maturation into an infectious virus particle by inhibiting the protease processing of gag and gag-pol precursor proteins. We have used a simple anti-HIV-1 p24 Western blot to monitor the processing of p55gag precursor into the mature p24 capsid immediately following the first dosage of a PI-containing treatment regimen. Evidence of PI activity was observed in plasma virus as early as 72 hours post treatment-initiation and was predictive of plasma viral RNA decrease at 4 weeks.

  8. 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

  9. Safety and tolerability of conserved region vaccines vectored by plasmid DNA, simian adenovirus and modified vaccinia virus ankara administered to human immunodeficiency virus type 1-uninfected adults in a randomized, single-blind phase I trial.

    Directory of Open Access Journals (Sweden)

    Emma-Jo Hayton

    Full Text Available HIV-1 vaccine development has advanced slowly due to viral antigenic diversity, poor immunogenicity and recently, safety concerns associated with human adenovirus serotype-5 vectors. To tackle HIV-1 variation, we designed a unique T-cell immunogen HIVconsv from functionally conserved regions of the HIV-1 proteome, which were presented to the immune system using a heterologous prime-boost combination of plasmid DNA, a non-replicating simian (chimpanzee adenovirus ChAdV-63 and a non-replicating poxvirus, modified vaccinia virus Ankara. A block-randomized, single-blind, placebo-controlled phase I trial HIV-CORE 002 administered for the first time candidate HIV-1- vaccines or placebo to 32 healthy HIV-1/2-uninfected adults in Oxford, UK and elicited high frequencies of HIV-1-specific T cells capable of inhibiting HIV-1 replication in vitro. Here, detail safety and tolerability of these vaccines are reported.Local and systemic reactogenicity data were collected using structured interviews and study-specific diary cards. Data on all other adverse events were collected using open questions. Serum neutralizing antibody titres to ChAdV-63 were determined before and after vaccination.Two volunteers withdrew for vaccine-unrelated reasons. No vaccine-related serious adverse events or reactions occurred during 190 person-months of follow-up. Local and systemic events after vaccination occurred in 27/32 individuals and most were mild (severity grade 1 and predominantly transient (<48 hours. Myalgia and flu-like symptoms were more strongly associated with MVA than ChAdV63 or DNA vectors and more common in vaccine recipients than in placebo. There were no intercurrent HIV-1 infections during follow-up. 2/24 volunteers had low ChAdV-63-neutralizing titres at baseline and 7 increased their titres to over 200 with a median (range of 633 (231-1533 post-vaccination, which is of no safety concern.These data demonstrate safety and good tolerability of the pSG2

  10. Intravenously administered gold nanoparticles pass through the blood-retinal barrier depending on the particle size, and induce no retinal toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jeong Hun; Kim, Jin Hyoung; Yu, Young Suk [Department of Ophthalmology, Seoul National University College of Medicine and Seoul Artificial Eye Center, Clinical Research Institute, Seoul National University Hospital, Seoul 151744 (Korea, Republic of); Kim, Kyu-Won [NeuroVascular Coordination Research Center, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151742 (Korea, Republic of); Kim, Myung Hun, E-mail: hunin315@paran.com, E-mail: ysyu@snu.ac.kr [Department of Chemistry, Yonsei University, 134 Shinchon-dong, Seodaemun-ku, Seoul 120749 (Korea, Republic of)

    2009-12-16

    The retina maintains homeostasis through the blood-retinal barrier (BRB). Although it is ideal to deliver the drug to the retina via systemic administration, it is still challenging due to the BRB strictly regulating permeation from blood to the retina. Herein, we demonstrated that intravenously administered gold nanoparticles could pass through the BRB and are distributed in all retinal layers without cytotoxicity. After intravenous injection of gold nanoparticles into C57BL/6 mice, 100 nm nanoparticles were not detected in the retina whereas 20 nm nanoparticles passed through the BRB and were distributed in all retinal layers. 20 nm nanoparticles in the retina were observed in neurons (75 {+-} 5%), endothelial cells (17 {+-} 6%) and peri-endothelial glial cells (8 {+-} 3%), where nanoparticles were bound on the membrane. In the retina, cells containing nanoparticles did not show any structural abnormality and increase of cell death compared to cells without nanoparticles. Gold nanoparticles never affected the viability of retinal endothelial cells, astrocytes and retinoblastoma cells. Furthermore, gold nanoparticles never led to any change in expression of representative biological molecules including zonula occludens-1 and glut-1 in retinal endothelial cells, neurofilaments in differentiated retinoblastoma cells and glial fibrillary acidic protein in astrocytes. Therefore, our data suggests that small gold nanoparticles (20 nm) could be an alternative for drug delivery across the BRB, which could be safely applied in vivo.

  11. Cloning of an avian adeno-associated virus (AAAV) and generation of recombinant AAAV particles.

    Science.gov (United States)

    Bossis, Ioannis; Chiorini, John A

    2003-06-01

    Recent studies have proposed that adeno-associated viruses (AAVs) are not evolutionarily linked to other mammalian autonomous parvoviruses but are more closely linked to the autonomous parvoviruses of birds. To better understand the relationship between primate and avian AAVs (AAAVs), we cloned and sequenced the genome of an AAAV (ATCC VR-865) and generated recombinant AAAV particles. The genome of AAAV is 4,694 nucleotides in length and has organization similar to that of other AAVs. The entire genome of AAAV displays 56 to 65% identity at the nucleotide level with the other known AAVs. The AAAV genome has inverted terminal repeats of 142 nucleotides, with the first 122 forming the characteristic T-shaped palindromic structure. The putative Rep-binding element consists of a tandem (GAGY)(4) repeat, and the putative terminal resolution site (trs), CCGGT/CG, contains a single nucleotide substitution relative to the AAV(2) trs. The Rep open reading frame of AAAV displays 50 to 54% identity at the amino acid level with the other AAVs, with most of the diversity clustered at the carboxyl and amino termini. Comparison of the capsid proteins of AAAV and the primate dependoviruses indicate that divergent regions are localized to surface-exposed loops. Despite these sequence differences, we were able to produce recombinant AAAV particles carrying a lacZ reporter gene by cotransfection in 293T cells and were able to examine transduction efficiency in both chicken primary cells and several cell lines. Our findings indicate that AAAV is the most divergent AAV described to date but maintains all the characteristics unique to the genera of dependovirus.

  12. 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.

  13. 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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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

  16. 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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    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.

  20. 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. Dissecting the Cell Entry Pathway of Dengue Virus by Single-Particle Tracking in Living Cells

    NARCIS (Netherlands)

    van der Schaar, Hilde M.; Rust, Michael J.; Chen, Chen; van der Ende-Metselaar, Heidi; Wilschut, Jan; Zhuang, Xiaowei; Smit, Jolanda M.

    2008-01-01

    Dengue virus (DENV) is an enveloped RNA virus that causes the most common arthropod-borne infection worldwide. The mechanism by which DENV infects the host cell remains unclear. In this work, we used live-cell imaging and single-virus tracking to investigate the cell entry, endocytic trafficking,

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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

  12. 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.

  13. 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.

  14. 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.

  15. Intragenotypic JFH1 based recombinant hepatitis C virus produces high levels of infectious particles but causes increased cell death

    DEFF Research Database (Denmark)

    Mateu, Guaniri; Donis, Ruben O; Wakita, Takaji

    2008-01-01

    The full-length hepatitis C virus (HCV) JFH1 genome (genotype 2a) produces moderate titers of infectious particles in cell culture but the optimal determinants required for virion production are unclear. It has been shown that intragenotypic recombinants encoding core to NS2 from J6CF in the cont......The full-length hepatitis C virus (HCV) JFH1 genome (genotype 2a) produces moderate titers of infectious particles in cell culture but the optimal determinants required for virion production are unclear. It has been shown that intragenotypic recombinants encoding core to NS2 from J6CF...... into the JFH1 infectious clone. All genomes produced high levels of intracellular HCV RNA and NS3 protein in Huh-7.5 transfected cells. However, JFH1 genomes containing J6 sequences from C to E2 (CE2) or C to p7 (Cp7) secreted up to 100-fold more infectious HCV particles than the parental JFH1 clone....... Subsequent infection of naive Huh-7.5 cells with each of the J6/JFH1 recombinants at a multiplicity of infection of 0.0003 resulted in high viral titers only for CE2 and Cp7 viruses. Comparison of virion production by the Cp7 J6/JFH1 recombinant to previously described J6/JFH1 recombinants showed flexibility...

  16. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  19. 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.

  20. Epitope-tagging approach to determine the stoichiometry of the structural and nonstructural proteins in the virus particles: amount of Vpr in relation to Gag in HIV-1.

    Science.gov (United States)

    Singh, S P; Lai, D; Cartas, M; Serio, D; Murali, R; Kalyanaraman, V S; Srinivasan, A

    2000-03-15

    We used an epitope-tagging approach to determine the ratio of Gag (structural) to Vpr (nonstructural) in the virus particles directed by human immunodeficiency virus type 1. For this purpose, chimeric Gag and Vpr expression plasmids were constructed with the Flag epitope (DYKDDDDK), and the sequences corresponding to the chimeric protein were introduced into human immunodeficiency virus type 1 proviral DNA (NL4-3) to determine the ratio in the virus particles when these proteins are expressed in cis. In addition, NL4-3 DNA was modified to disrupt Vpr synthesis to determine the extent of incorporation of Vpr-FL when it is expressed in trans through a heterologous promoter. The analysis of virus particles generated by transfection of proviral DNA into RD cells indicated that (1) the ratio of Gag to Vpr in virus particles, when Vpr-FL is expressed in cis (in the context of proviral DNA), is in the range of 150-200:1 (14-18 molecules of Vpr per virion) and (2) the expression of Vpr-FL in trans showed efficient incorporation with a Gag to Vpr ratio of 5-7:1 (392-550 molecules of Vpr). These results suggest that the presence of the same epitope on different viral proteins may provide an accurate comparison of these proteins in the virus particles. Copyright 2000 Academic Press.

  1. 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...

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

    Directory of Open Access Journals (Sweden)

    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

  3. 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

  4. Pleiotropic effects of resistance-breaking mutations on particle stability provide insight on life history evolution in a plant RNA virus.

    Science.gov (United States)

    Bera, Sayanta; Moreno-Pérez, Manuel G; García-Figuera, Sara; Pagán, Israel; Fraile, Aurora; Pacios, Luis F; García-Arenal, Fernando

    2017-07-05

    In gene-for-gene host-virus interactions, virus evolution to infect and multiply in previously resistant host genotypes, i.e., resistance-breaking, is a case of host range expansion, predicted to be associated with fitness penalties. Negative effects of resistance-breaking mutations on within-host virus multiplication have been documented for several plant viruses. However, understanding virus evolution requires analyses of potential trade-offs between different fitness components. Here we analyze if coat protein (CP) mutations in Pepper mild mottle virus breaking L -gene resistance in pepper affect particle stability and, thus, survival in the environment. For this purpose, CP mutations determining the overcoming of L 3 and L 4 resistance alleles were introduced in biologically active cDNA clones. The kinetics of the in vitro disassembly of parental and mutants' particles was compared under different conditions. Resistance-breaking mutations variously affected particle stability. Structural analyses identified the number and type of axial and side interactions of adjacent CP subunits in virions, which explained differences in particle stability and contribute to understand tobamovirus disassembly. Resistance-breaking mutations also affected virus multiplication and virulence in the susceptible host, as well as infectivity. The sense and magnitude of the effects of resistance-breaking mutations on particle stability, multiplication, virulence or infectivity depended on the specific mutation, rather than on the ability to overcome the different resistance alleles, and effects on different traits were not correlated. Thus, results do not provide evidence of links or trade-offs between particle stability, i.e., survival, and other components of virus fitness, or virulence. IMPORTANCE The effect of survival on virus evolution remains underexplored, despite that life history trade-offs may constrain virus evolution. We approach this topic by analyzing if breaking of L

  5. An RNA Molecule Derived From Sendai Virus DI Particles Induces Antitumor Immunity and Cancer Cell-selective Apoptosis

    Science.gov (United States)

    Liu, Li-Wen; Nishikawa, Tomoyuki; Kaneda, Yasufumi

    2016-01-01

    Inactivated Sendai virus (hemagglutinating virus of Japan; HVJ) envelope (HVJ-E) induces anticancer immunity and cancer cell-selective apoptosis through the recognition of viral RNA genome fragments by retinoic acid-inducible gene-I (RIG-I). Here, we discovered that the “copy-back” type of defective-interfering (DI) particles that exist in the Cantell strain of HVJ induced the human PC3 prostate cancer cell death more effectively than the Sendai/52 strain or Cantell strain, which contain fewer DI particles. DI particle genomic RNA (~550 bases) activated proapoptotic genes such as Noxa and/or TNF-related apoptosis-inducing ligand (TRAIL) in human prostate cancer cells to induce cancer cell-selective apoptosis. DI particle-derived RNA was synthesized by in vitro transcription (in vitro transcribed (IVT)-B2). IVT-B2 RNA, which has a double-stranded region in its secondary structure, promoted a stronger anticancer effect than IVT-HN RNA, which does not have a double-stranded region in its secondary structure. The intratumoral transfection of IVT-B2 significantly reduced the volume of a human prostate tumor and induced tumor cell apoptosis in the xenograft mouse model. Moreover, the involvement of natural killer (NK) cells in IVT-B2-RNA-induced anticancer effects was also suggested. These findings provide a novel nucleic acid medicine for the treatment of cancer. PMID:26548591

  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. 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.

  8. 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.

  9. 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.

  10. 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.

  11. Purification of nervous necrosis virus (NNV) particles by anion-exchange chromatography.

    Science.gov (United States)

    Gye, Hyun Jung; Nishizawa, Toyohiko

    2016-12-01

    Nervous necrosis virus (NNV) belongs to genus Betanodavirus (family Nodaviridae). It is highly pathogenic to various marine fishes. In the present study, cultured NNV suspension was placed in dialysis tube at molecular weight cut off (MWCO) of 10(6) and dialyzed against Dulbecco's phosphate buffered saline (D-PBS), 15mM Tris-HCl (pH 8.0), or deionized water (DIW) for 14days followed by anion-exchange chromatography. Infectivity titers of NNV suspensions were stable during dialyses. However, the antigenicity of NNV suspension was decreased to 2.5% by D-PBS dialysis, 11.8% by Tris-HCl dialysis, and 56.2% by DIW dialysis. Anion-exchange chromatograms revealed a total of four peaks (P300, P400, P600 and P700) for NNV suspension after D-PBS dialysis. Additional two peaks (P800 and P-OH) were detected in the NNV suspension after Tris-HCl or DIW dialysis. The substance from the P700 peak had the highest NNV-infectivity. Peak P700 commonly shared by the NNV suspensions after dialysis against the three different buffers. After Tris-HCl dialysis, no other protein except NNV coat protein (CP) at Mr 41,000 was detected from P700. However, after D-PBS dialysis, the P700 peak also contained P600 antigens. Therefore, the P700 peak after Tris-HCl dialysis represented the peak of highly purified NNV particles. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. 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.

  13. 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.

    Science.gov (United States)

    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.

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. Venezuelan equine encephalitis virus replicon particle vaccine protects nonhuman primates from intramuscular and aerosol challenge with ebolavirus.

    Science.gov (United States)

    Herbert, Andrew S; Kuehne, Ana I; Barth, James F; Ortiz, Ramon A; Nichols, Donald K; Zak, Samantha E; Stonier, Spencer W; Muhammad, Majidat A; Bakken, Russell R; Prugar, Laura I; Olinger, Gene G; Groebner, Jennifer L; Lee, John S; Pratt, William D; Custer, Max; Kamrud, Kurt I; Smith, Jonathan F; Hart, Mary Kate; Dye, John M

    2013-05-01

    There are no vaccines or therapeutics currently approved for the prevention or treatment of ebolavirus infection. Previously, a replicon vaccine based on Venezuelan equine encephalitis virus (VEEV) demonstrated protective efficacy against Marburg virus in nonhuman primates. Here, we report the protective efficacy of Sudan virus (SUDV)- and Ebola virus (EBOV)-specific VEEV replicon particle (VRP) vaccines in nonhuman primates. VRP vaccines were developed to express the glycoprotein (GP) of either SUDV or EBOV. A single intramuscular vaccination of cynomolgus macaques with VRP expressing SUDV GP provided complete protection against intramuscular challenge with SUDV. Vaccination against SUDV and subsequent survival of SUDV challenge did not fully protect cynomolgus macaques against intramuscular EBOV back-challenge. However, a single simultaneous intramuscular vaccination with VRP expressing SUDV GP combined with VRP expressing EBOV GP did provide complete protection against intramuscular challenge with either SUDV or EBOV in cynomolgus macaques. Finally, intramuscular vaccination with VRP expressing SUDV GP completely protected cynomolgus macaques when challenged with aerosolized SUDV, although complete protection against aerosol challenge required two vaccinations with this vaccine.

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

    Directory of Open Access Journals (Sweden)

    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.

  20. 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.

  1. 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.

  2. Clustering and cellular distribution characteristics of virus particles of Tomato spotted wilt virus and Tomato zonate spot virus in different plant hosts

    OpenAIRE

    ZHANG Zhongkai; Zheng, Kuanyu; Dong, Jiahong; Fang, Qi; Hong, Jian; Wang, Xifeng

    2016-01-01

    Background Tomato spotted wilt virus (TSWV) and Tomato zonate spot virus (TZSV) are the two dominant species of thrip-transmitted tospoviruses, cause significant losses in crop yield in Yunnan and its neighboring provinces in China. TSWV and TZSV belong to different serogroup of tospoviruses but induce similar symptoms in the same host plant species, which makes diagnostic difficult. We used different electron microscopy preparing methods to investigate clustering and cellular distribution of...

  3. Accumulation of defective interfering viral particles in only a few passages in Vero cells attenuates mumps virus neurovirulence.

    Science.gov (United States)

    Šantak, Maja; Markušić, Maja; Balija, Maja Lang; Kopač, Sandra Keć; Jug, Renata; Örvell, Claes; Tomac, Jelena; Forčić, Dubravko

    2015-03-01

    Immunization programs have implemented live attenuated mumps vaccines which reduced mumps incidence ≥97%. Some of the vaccine strains were abandoned due to unwanted side effects and the genetic marker of attenuation has not been identified so far. Our hypothesis was that non-infectious viral particles, in particular defective interfering particles (DIPs), contribute to neuroattenuation. We showed that non-infectious particles of the mumps vaccine L-Zagreb attenuated neurovirulence of wild type mumps virus 9218/Zg98. Then, we attenuated recent wild type mumps virus MuVi/Zagreb.HRV/28.12 in Vero cells through 16 passages but already the fifth passage (p5) showed accumulation of DIPs and attenuated neurovirulence in a newborn rat model when compared to the second passage (p2). Sequence analysis of the p2 and p5 revealed a single mutation in the 5' untranslated region of the HN gene. Analysis of the expression level of the HN protein showed that this mutation does not affect the expression of the protein. We conclude that the passages of MuVi/Zagreb.HRV/28.12 in Vero cells for only three passages accumulated DIPs which attenuate neurovirulence. These findings reveal DIPs as a very promising and general neuroattenuating factor which should be considered in the rational design of the new mumps vaccine. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  4. Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins.

    Directory of Open Access Journals (Sweden)

    Pietro Scaturro

    Full Text Available Non-structural protein 1 (NS1 is one of the most enigmatic proteins of the Dengue virus (DENV, playing distinct functions in immune evasion, pathogenesis and viral replication. The recently reported crystal structure of DENV NS1 revealed its peculiar three-dimensional fold; however, detailed information on NS1 function at different steps of the viral replication cycle is still missing. By using the recently reported crystal structure, as well as amino acid sequence conservation, as a guide for a comprehensive site-directed mutagenesis study, we discovered that in addition to being essential for RNA replication, DENV NS1 is also critically required for the production of infectious virus particles. Taking advantage of a trans-complementation approach based on fully functional epitope-tagged NS1 variants, we identified previously unreported interactions between NS1 and the structural proteins Envelope (E and precursor Membrane (prM. Interestingly, coimmunoprecipitation revealed an additional association with capsid, arguing that NS1 interacts via the structural glycoproteins with DENV particles. Results obtained with mutations residing either in the NS1 Wing domain or in the β-ladder domain suggest that NS1 might have two distinct functions in the assembly of DENV particles. By using a trans-complementation approach with a C-terminally KDEL-tagged ER-resident NS1, we demonstrate that the secretion of NS1 is dispensable for both RNA replication and infectious particle production. In conclusion, our results provide an extensive genetic map of NS1 determinants essential for viral RNA replication and identify a novel role of NS1 in virion production that is mediated via interaction with the structural proteins. These studies extend the list of NS1 functions and argue for a central role in coordinating replication and assembly/release of infectious DENV particles.

  5. Proton-driven assembly of the Rous Sarcoma virus capsid protein results in the formation of icosahedral particles.

    Science.gov (United States)

    Hyun, Jae-Kyung; Radjainia, Mazdak; Kingston, Richard L; Mitra, Alok K

    2010-05-14

    In a mature and infectious retroviral particle, the capsid protein (CA) forms a shell surrounding the genomic RNA and the replicative machinery of the virus. The irregular nature of this capsid shell precludes direct atomic resolution structural analysis. CA hexamers and pentamers are the fundamental building blocks of the capsid, however the pentameric state, in particular, remains poorly characterized. We have developed an efficient in vitro protocol for studying the assembly of Rous sarcoma virus (RSV) CA that involves mild acidification and produces structures modeling the authentic viral capsid. These structures include regular spherical particles with T = 1 icosahedral symmetry, built from CA pentamers alone. These particles were subject to cryoelectron microscopy (cryo-EM) and image processing, and a pseudo-atomic model of the icosahedron was created by docking atomic structures of the constituent CA domains into the cryo-EM-derived three-dimensional density map. The N-terminal domain (NTD) of CA forms pentameric turrets, which decorate the surface of the icosahedron, while the C-terminal domain (CTD) of CA is positioned underneath, linking the pentamers. Biophysical analysis of the icosahedral particle preparation reveals that CA monomers and icosahedra are the only detectable species and that these exist in reversible equilibrium at pH 5. These same acidic conditions are known to promote formation of a RSV CA CTD dimer, present within the icosahedral particle, which facilitates capsid assembly. The results are consistent with a model in which RSV CA assembly is a nucleation-limited process driven by very weak protein-protein interactions.

  6. Proton-driven Assembly of the Rous Sarcoma Virus Capsid Protein Results in the Formation of Icosahedral Particles*

    Science.gov (United States)

    Hyun, Jae-Kyung; Radjainia, Mazdak; Kingston, Richard L.; Mitra, Alok K.

    2010-01-01

    In a mature and infectious retroviral particle, the capsid protein (CA) forms a shell surrounding the genomic RNA and the replicative machinery of the virus. The irregular nature of this capsid shell precludes direct atomic resolution structural analysis. CA hexamers and pentamers are the fundamental building blocks of the capsid, however the pentameric state, in particular, remains poorly characterized. We have developed an efficient in vitro protocol for studying the assembly of Rous sarcoma virus (RSV) CA that involves mild acidification and produces structures modeling the authentic viral capsid. These structures include regular spherical particles with T = 1 icosahedral symmetry, built from CA pentamers alone. These particles were subject to cryoelectron microscopy (cryo-EM) and image processing, and a pseudo-atomic model of the icosahedron was created by docking atomic structures of the constituent CA domains into the cryo-EM-derived three-dimensional density map. The N-terminal domain (NTD) of CA forms pentameric turrets, which decorate the surface of the icosahedron, while the C-terminal domain (CTD) of CA is positioned underneath, linking the pentamers. Biophysical analysis of the icosahedral particle preparation reveals that CA monomers and icosahedra are the only detectable species and that these exist in reversible equilibrium at pH 5. These same acidic conditions are known to promote formation of a RSV CA CTD dimer, present within the icosahedral particle, which facilitates capsid assembly. The results are consistent with a model in which RSV CA assembly is a nucleation-limited process driven by very weak protein-protein interactions. PMID:20228062

  7. 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.

  8. 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.

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

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    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

  10. 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

  11. Nuclear export and import of human hepatitis B virus capsid protein and particles.

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    Hung-Cheng Li

    Full Text Available It remains unclear what determines the subcellular localization of hepatitis B virus (HBV core protein (HBc and particles. To address this fundamental issue, we have identified four distinct HBc localization signals in the arginine rich domain (ARD of HBc, using immunofluorescence confocal microscopy and fractionation/Western blot analysis. ARD consists of four tight clustering arginine-rich subdomains. ARD-I and ARD-III are associated with two co-dependent nuclear localization signals (NLS, while ARD-II and ARD-IV behave like two independent nuclear export signals (NES. This conclusion is based on five independent lines of experimental evidence: i Using an HBV replication system in hepatoma cells, we demonstrated in a double-blind manner that only the HBc of mutant ARD-II+IV, among a total of 15 ARD mutants, can predominantly localize to the nucleus. ii These results were confirmed using a chimera reporter system by placing mutant or wild type HBc trafficking signals in the heterologous context of SV40 large T antigen (LT. iii By a heterokaryon or homokaryon analysis, the fusion protein of SV40 LT-HBc ARD appeared to transport from nuclei of transfected donor cells to nuclei of recipient cells, suggesting the existence of an NES in HBc ARD. This putative NES is leptomycin B resistant. iv We demonstrated by co-immunoprecipitation that HBc ARD can physically interact with a cellular factor TAP/NXF1 (Tip-associated protein/nuclear export factor-1, which is known to be important for nuclear export of mRNA and proteins. Treatment with a TAP-specific siRNA strikingly shifted cytoplasmic HBc to nucleus, and led to a near 7-fold reduction of viral replication, and a near 10-fold reduction in HBsAg secretion. v HBc of mutant ARD-II+IV was accumulated predominantly in the nucleus in a mouse model by hydrodynamic delivery. In addition to the revised map of NLS, our results suggest that HBc could shuttle rapidly between nucleus and cytoplasm via a novel

  12. Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations.

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    Thomas Pietschmann

    2009-06-01

    Full Text Available With the advent of subgenomic hepatitis C virus (HCV replicons, studies of the intracellular steps of the viral replication cycle became possible. These RNAs are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate JFH-1, efficient replication of these HCV RNAs requires replication enhancing mutations (REMs, previously also called cell culture adaptive mutations. These mutations cluster primarily in the central region of non-structural protein 5A (NS5A, but may also reside in the NS3 helicase domain or at a distinct position in NS4B. Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A. However, in spite of efficient replication of HCV genomes containing such mutations, they do not support production of infectious virus particles. By using the genotype 1b isolate Con1, in this study we show that REMs interfere with HCV assembly. Strongest impairment of virus formation was found with REMs located in the NS3 helicase (E1202G and T1280I as well as NS5A (S2204R, whereas a highly adaptive REM in NS4B still allowed virus production although relative levels of core release were also reduced. We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo. Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes.

  13. 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.

  14. 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.

  15. 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

  16. 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...

  17. Nucleolin interacts with the dengue virus capsid protein and plays a role in formation of infectious virus particles.

    Science.gov (United States)

    Balinsky, Corey A; Schmeisser, Hana; Ganesan, Sundar; Singh, Kavita; Pierson, Theodore C; Zoon, Kathryn C

    2013-12-01

    Dengue virus (DENV) is a mosquito-transmitted flavivirus that can cause severe disease in humans and is considered a reemerging pathogen of significant importance to public health. The DENV capsid (C) protein functions as a structural component of the infectious virion; however, it may have additional functions in the virus replicative cycle. Here, we show that the DENV C protein interacts and colocalizes with the multifunctional host protein nucleolin (NCL). Furthermore, we demonstrate that this interaction can be disrupted by the addition of an NCL binding aptamer (AS1411). Knockdown of NCL with small interfering RNA (siRNA) or treatment of cells with AS1411 results in a significant reduction of viral titers after DENV infection. Western blotting and quantitative RT-PCR (qRT-PCR) analysis revealed no differences in viral RNA or protein levels at early time points postinfection, suggesting a role for NCL in viral morphogenesis. We support this hypothesis by showing that treatment with AS1411 alters the migration characteristics of the viral capsid, as visualized by native electrophoresis. Here, we identify a critical interaction between DENV C protein and NCL that represents a potential new target for the development of antiviral therapeutics.

  18. 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

  19. 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.

  20. 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.

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

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    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.

  2. 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.

  3. 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.

  4. 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.

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

    Science.gov (United States)

    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.

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

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    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.

  7. 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.

  8. 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.

  9. 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.

  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. 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.

  12. 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.

  13. 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...

  14. Replication-Deficient Particles: New Insights into the Next Generation of Bluetongue Virus Vaccines.

    Science.gov (United States)

    Celma, Cristina C; Stewart, Meredith; Wernike, Kerstin; Eschbaumer, Michael; Gonzalez-Molleda, Lorenzo; Breard, Emmanuel; Schulz, Claudia; Hoffmann, Bernd; Haegeman, Andy; De Clercq, Kris; Zientara, Stephan; van Rijn, Piet A; Beer, Martin; Roy, Polly

    2017-01-01

    Bluetongue virus (BTV) is endemic in many parts of the world, often causing severe hemorrhagic disease in livestock. To date, at least 27 different serotypes have been recognized. Vaccination against all serotypes is necessary to protect susceptible animals and to prevent onward spread of the virus by insect vectors. In our previous studies, we generated replication-deficient (disabled infectious single-cycle [DISC]) virus strains for a number of serotypes and reported preliminary data on their protective efficacy in animals. In this report, to advance the DISC vaccines to the marketplace, we investigated different parameters of these DISC vaccines. First, we demonstrated the genetic stabilities of these vaccine strains and also the complementing cell line. Subsequently, the optimal storage conditions of vaccines, including additives, temperature, and desiccation, were determined and their protective efficacies in animals confirmed. Furthermore, to test if mixtures of different vaccine strains could be tolerated, we tested cocktails of DISC vaccines in combinations of three or six different serotypes in sheep and cattle, the two natural hosts of BTV. Groups of sheep vaccinated with a cocktail of six different vaccines were completely protected from challenge with individual virulent serotypes, both in early challenge and after 5 months of challenge without any clinical disease. There was no interference in protection between the different vaccines. Protection was also achieved in cattle with a mixture of three vaccine strains, albeit at a lesser level than in sheep. Our data support and validate the suitability of these virus strains as the next-generation vaccines for BTV. Bluetongue (BT) is a debilitating and in many cases lethal disease that affects ruminants of economic importance. Classical vaccines that afford protection against bluetongue virus, the etiological agent, are not free from secondary and undesirable effects. A surge in new approaches to produce

  15. Pattern of shedding of small, round-structured virus particles in stools of patients of outbreaks of food-poisoning from raw oysters.

    Science.gov (United States)

    Haruki, K; Seto, Y; Murakami, T; Kimura, T

    1991-01-01

    The pattern of shedding of the small, round-structured virus (SRSV) particles in the stools of patients who suffered from food-poisoning due to raw oysters was investigated. The duration and concentration of fecal shedding of the SRSV particles were studied by electron microscopic examinations of stool specimens obtained during the course of illness to see a relation of viral shedding to day of illness. It was found that the fecal shedding of the SRSV particles occurred within five days of illness; thereafter, the concentration of the SRSV particles in feces rapidly decreased within a few days during the course of illness.

  16. Characterization of complete particles (VSV-G/SIN-GFP) and empty particles (VSV-G/EMPTY) in human immunodeficiency virus type 1-based lentiviral products for gene therapy: potential applications for improvement of product quality and safety.

    Science.gov (United States)

    Zhao, Yuan; Keating, Kenneth; Dolman, Carl; Thorpe, Robin

    2008-05-01

    Lentiviral vectors persist in the host and are therefore ideally suited for long-term gene therapy. To advance the use of lentiviral vectors in humans, improvement of their production, purification, and characterization has become increasingly important and challenging. In addition to cellular contaminants derived from packaging cells, empty particles without therapeutic function are the major impurities that compromise product safety and efficacy. Removal of empty particles is difficult because of their innate similarity in particle size and protein composition to the complete particles. We propose that comparison of the properties of lentiviral products with those of purposely expressed empty particles may reveal potential differences between empty and complete particles. For this, three forms of recombinant lentiviral samples, that is, recombinant vesicular stomatitis virus glycoprotein (VSV-G) proteins, empty particles (VSV-G/Empty), and complete particles (VSV-G/SIN-GFP) carrying viral RNA, were purified by size-exclusion chromatography (SEC). The SEC-purified samples were further analyzed by immunoblotting with six antibodies to examine viral and cellular proteins associated with the particles. This study has demonstrated, for the first time, important differences between VSV-G/Empty particles and complete VSV-G/SIN-GFP particles. Differences include the processing of Gag protein and the inclusion of cellular proteins in the particles. Our findings support the development of improved production, purification, and characterization methods for lentiviral products.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. 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.

  19. Township Administered Roads

    Data.gov (United States)

    Minnesota Department of Natural Resources — This data set contains roadway centerlines for township administered roads found on the USGS 1:24,000 mapping series. In some areas, these roadways are current...

  20. Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and anti-tumor effects

    Science.gov (United States)

    Osada, Takuya; Berglund, Peter; Morse, Michael A.; Hubby, Bolyn; Lewis, Whitney; Niedzwiecki, Donna; Hobeika, Amy; Burnett, Bruce; Devi, Gayathri R.; Clay, Timothy M.; Smith, Jonathan; Lyerly, H. Kim

    2013-01-01

    We recently demonstrated that Venezuelan equine encephalitis (VEE) virus-based replicon particles (VRP) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP expressing Interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and anti-tumor efficacy. Mice were immunized with VRP encoding the human tumor-associated antigen, carcinoembryonic antigen (CEA) (VRP-CEA(6D)) and VRP-IL-12 was also administered at the same site or at a distant location. CEA-specific T cell and antibody responses were measured. To determine antitumor activity, mice were implanted with MC38-CEA-2 cells and immunized with VRP-CEA with and without VRP-IL-12 and tumor growth and mouse survival were measured. VRP-IL-12 greatly enhanced CEA-specific T cell and antibody responses when combined with VRP-CEA(6D) vaccination. VRP IL-12 was superior to IL-12 protein at enhancing immune responses. Vaccination with VRP-CEA(6D) plus VRP-IL-12 was superior to VRP-CEA(6D) or VRP-IL-12 alone in inducing anti-tumor activity and prolonging survival in tumor-bearing mice. Importantly, local injection of VRP-IL-12 at the VRP-CEA(6D) injection site provided more potent activation of CEA-specific immune responses than VRP-IL-12 injected at a distant site from the VRP-CEA injections. Together, this study shows that VRP-IL-12 enhances vaccination with VRP-CEA(6D) and was more effective at activating CEA-specific T cell responses when locally expressed at the vaccine site. Clinical trials evaluating the adjuvant effect of VRP-IL-12 at enhancing the immunogenicity of cancer vaccines are warranted. PMID:22488274

  1. Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and antitumor effects.

    Science.gov (United States)

    Osada, Takuya; Berglund, Peter; Morse, Michael A; Hubby, Bolyn; Lewis, Whitney; Niedzwiecki, Donna; Yang, Xiao Yi; Hobeika, Amy; Burnett, Bruce; Devi, Gayathri R; Clay, Timothy M; Smith, Jonathan; Kim Lyerly, H

    2012-11-01

    We recently demonstrated that Venezuelan equine encephalitis virus-based replicon particle (VRPs) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP-expressing interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and antitumor efficacy. Mice were immunized with VRP encoding the human tumor-associated antigen, carcinoembryonic antigen (CEA) (VRP-CEA(6D)), and VRP-IL-12 was also administered at the same site or at a distant location. CEA-specific T cell and antibody responses were measured. To determine antitumor activity, mice were implanted with MC38-CEA-2 cells and immunized with VRP-CEA with and without VRP-IL-12, and tumor growth and mouse survival were measured. VRP-IL-12 greatly enhanced CEA-specific T cell and antibody responses when combined with VRP-CEA(6D) vaccination. VRP-IL-12 was superior to IL-12 protein at enhancing immune responses. Vaccination with VRP-CEA(6D) plus VRP-IL-12 was superior to VRP-CEA(6D) or VRP-IL-12 alone in inducing antitumor activity and prolonging survival in tumor-bearing mice. Importantly, local injection of VRP-IL-12 at the VRP-CEA(6D) injection site provided more potent activation of CEA-specific immune responses than that of VRP-IL-12 injected at a distant site from the VRP-CEA injections. Together, this study shows that VRP-IL-12 enhances vaccination with VRP-CEA(6D) and was more effective at activating CEA-specific T cell responses when locally expressed at the vaccine site. Clinical trials evaluating the adjuvant effect of VRP-IL-12 at enhancing the immunogenicity of cancer vaccines are warranted.

  2. 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.

  3. 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.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  6. 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.

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

    Directory of Open Access Journals (Sweden)

    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.

  8. 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.

  9. 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  12. GB Virus Type C Envelope Protein E2 Elicits Antibodies That React with a Cellular Antigen on HIV-1 Particles and Neutralize Diverse HIV-1 Isolates

    Science.gov (United States)

    Mohr, Emma L.; Xiang, Jinhua; McLinden, James H.; Kaufman, Thomas M.; Chang, Qing; Montefiori, David C.; Klinzman, Donna; Stapleton, Jack T.

    2012-01-01

    Broadly neutralizing Abs to HIV-1 are well described; however, identification of Ags that elicit these Abs has proven difficult. Persistent infection with GB virus type C (GBV-C) is associated with prolonged survival in HIV-1–infected individuals, and among those without HIV-1 viremia, the presence of Ab to GBV-C glycoprotein E2 is also associated with survival. GBV-C E2 protein inhibits HIV-1 entry, and an antigenic peptide within E2 interferes with gp41-induced membrane perturbations in vitro, suggesting the possibility of structural mimicry between GBV-C E2 protein and HIV-1 particles. Naturally occurring human and experimentally induced GBV-C E2 Abs were examined for their ability to neutralize infectious HIV-1 particles and HIV-1–enveloped pseudovirus particles. All GBV-C E2 Abs neutralized diverse isolates of HIV-1 with the exception of rabbit anti-peptide Abs raised against a synthetic GBV-C E2 peptide. Rabbit anti–GBV-C E2 Abs neutralized HIV-1–pseudotyped retrovirus particles but not HIV-1–pseudotyped vesicular stomatitis virus particles, and E2 Abs immune-precipitated HIV-1 gag particles containing the vesicular stomatitis virus type G envelope, HIV-1 envelope, GBV-C envelope, or no viral envelope. The Abs did not neutralize or immune-precipitate mumps or yellow fever viruses. Rabbit GBV-C E2 Abs inhibited HIV attachment to cells but did not inhibit entry following attachment. Taken together, these data indicate that the GBV-C E2 protein has a structural motif that elicits Abs that cross-react with a cellular Ag present on retrovirus particles, independent of HIV-1 envelope glycoproteins. The data provide evidence that a heterologous viral protein can induce HIV-1–neutralizing Abs. PMID:20826757

  13. Potentiated virucidal activity of pomegranate rind extract (PRE) and punicalagin against Herpes simplex virus (HSV) when co-administered with zinc (II) ions, and antiviral activity of PRE against HSV and aciclovir-resistant HSV.

    Science.gov (United States)

    Houston, David M J; Bugert, Joachim J; Denyer, Stephen P; Heard, Charles M

    2017-01-01

    There is a clinical need for new therapeutic products against Herpes simplex virus (HSV). The pomegranate, fruit of the tree Punica granatum L, has since ancient times been linked to activity against infection. This work probed the activity of pomegranate rind extract (PRE) and co-administered zinc (II) ions. PRE was used in conjunction with zinc (II) salts to challenge HSV-1 and aciclovir-resistant HSV in terms of virucidal plaque assay reduction and antiviral activities in epithelial Vero host cells. Cytotoxicity was determined by the MTS assay using a commercial kit. Zinc sulphate, zinc citrate, zinc stearate and zinc gluconate demonstrated similar potentiated virucidal activity with PRE against HSV-1 by up to 4-fold. A generally parabolic relationship was observed when HSV-1 was challenged with PRE and varying concentrations of ZnSO4, with a maximum potentiation factor of 5.5. Punicalagin had 8-fold greater virucidal activity than an equivalent mass of PRE. However, antiviral data showed that punicalagin had significantly lower antiviral activity compared to the activity of PRE (EC50 = 0.56 μg mL-1) a value comparable to aciclovir (EC50 = 0.18 μg mL-1); however, PRE also demonstrated potency against aciclovir-resistant HSV (EC50 = 0.02 μg mL-1), whereas aciclovir showed no activity. Antiviral action of PRE was not influenced by ZnSO4. No cytotoxicity was detected with any test solution. The potentiated virucidal activity of PRE by coadministered zinc (II) has potential as a multi-action novel topical therapeutic agent against HSV infections, such as coldsores.

  14. Potentiated virucidal activity of pomegranate rind extract (PRE and punicalagin against Herpes simplex virus (HSV when co-administered with zinc (II ions, and antiviral activity of PRE against HSV and aciclovir-resistant HSV.

    Directory of Open Access Journals (Sweden)

    David M J Houston

    Full Text Available There is a clinical need for new therapeutic products against Herpes simplex virus (HSV. The pomegranate, fruit of the tree Punica granatum L, has since ancient times been linked to activity against infection. This work probed the activity of pomegranate rind extract (PRE and co-administered zinc (II ions.PRE was used in conjunction with zinc (II salts to challenge HSV-1 and aciclovir-resistant HSV in terms of virucidal plaque assay reduction and antiviral activities in epithelial Vero host cells. Cytotoxicity was determined by the MTS assay using a commercial kit.Zinc sulphate, zinc citrate, zinc stearate and zinc gluconate demonstrated similar potentiated virucidal activity with PRE against HSV-1 by up to 4-fold. A generally parabolic relationship was observed when HSV-1 was challenged with PRE and varying concentrations of ZnSO4, with a maximum potentiation factor of 5.5. Punicalagin had 8-fold greater virucidal activity than an equivalent mass of PRE. However, antiviral data showed that punicalagin had significantly lower antiviral activity compared to the activity of PRE (EC50 = 0.56 μg mL-1 a value comparable to aciclovir (EC50 = 0.18 μg mL-1; however, PRE also demonstrated potency against aciclovir-resistant HSV (EC50 = 0.02 μg mL-1, whereas aciclovir showed no activity. Antiviral action of PRE was not influenced by ZnSO4. No cytotoxicity was detected with any test solution.The potentiated virucidal activity of PRE by coadministered zinc (II has potential as a multi-action novel topical therapeutic agent against HSV infections, such as coldsores.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    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.

  17. 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.

  18. Infected dendritic cells are sufficient to mediate the adjuvant activity generated by Venezuelan equine encephalitis virus replicon particles.

    Science.gov (United States)

    Tonkin, Daniel R; Whitmore, Alan; Johnston, Robert E; Barro, Mario

    2012-06-22

    Replicon particles derived from Venezuelan equine encephalitis virus (VEE) are infectious non-propagating particles which act as a safe and potent systemic, mucosal, and cellular adjuvant when delivered with antigen. VEE and VEE replicon particles (VRP) can target multiple cell types including dendritic cells (DCs). The role of these cell types in VRP adjuvant activity has not been previously evaluated, and for these studies we focused on the contribution of DCs to the response to VRP. By analysis of VRP targeting in the draining lymph node, we found that VRP induced rapid recruitment of TNF-secreting monocyte-derived inflammatory dendritic cells. VRP preferentially infected these inflammatory DCs as well as classical DCs and macrophages, with less efficient infection of other cell types. DC depletion suggested that the interaction of VRP with classical DCs was required for recruitment of inflammatory DCs, induction of high levels of many cytokines, and for stable transport of VRP to the draining lymph node. Additionally, in vitro-infected DCs enhanced antigen-specific responses by CD4 and CD8 T cells. By transfer of VRP-infected DCs into mice we showed that these DCs generated an inflammatory state in the draining lymph node similar to that achieved by VRP injection. Most importantly, VRP-infected DCs were sufficient to establish robust adjuvant activity in mice comparable to that produced by VRP injection. These findings indicate that VRP infect, recruit and activate both classical and inflammatory DCs, and those DCs become mediators of the VRP adjuvant activity. Published by Elsevier Ltd.

  19. Processing of the VP1/2A Junction Is Not Necessary for Production of Foot-and-Mouth Disease Virus Empty Capsids and Infectious Viruses: Characterization of “Self-Tagged” Particles

    DEFF Research Database (Denmark)

    Gullberg, Maria; Polacek, Charlotta; Bøtner, Anette

    2013-01-01

    the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction......, resulted in efficient capsid protein production and self-assembly of empty capsid particles. Removal of the 2A peptide from the capsid protein precursor had no effect on capsid protein processing or particle assembly. However, modification of E83K alone abrogated particle assembly with no apparent effect...... on protein processing. Interestingly, the K210E substitution, close to the VP1/2A junction, completely blocked processing by 3Cpro at this cleavage site, but efficient assembly of “self-tagged” empty capsid particles, containing the uncleaved VP1-2A, was observed. These self-tagged particles behaved like...

  20. 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.

    Directory of Open Access Journals (Sweden)

    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

  1. 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

  2. [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.

  3. Isolation of Single-Domain Antibody Fragments That Preferentially Detect Intact (146S Particles of Foot-and-Mouth Disease Virus for Use in Vaccine Quality Control

    Directory of Open Access Journals (Sweden)

    Michiel M. Harmsen

    2017-08-01

    Full Text Available Intact (146S foot-and-mouth disease virus (FMDVs can dissociate into specific (12S viral capsid degradation products. FMD vaccines normally consist of inactivated virions. Vaccine quality is dependent on 146S virus particles rather than 12S particles. We earlier isolated two llama single-domain antibody fragments (VHHs that specifically recognize 146S particles of FMDV strain O1 Manisa and shown their potential use in quality control of FMD vaccines during manufacturing. These 146S-specific VHHs were specific for particular O serotype strains and did not bind strains from other FMDV serotypes. Here, we describe the isolation of 146S-specific VHHs against FMDV SAT2 and Asia 1 strains by phage display selection from llama immune libraries. VHHs that bind both 12S and 146S particles were readily isolated but VHHs that bind specifically to 146S particles could only be isolated by phage display selection using prior depletion for 12S particles. We obtained one 146S-specific VHH—M332F—that binds to strain Asia 1 Shamir and several VHHs that preferentially bind 146S particles of SAT2 strain SAU/2/00, from which we selected VHH M379F for further characterization. Both M332F and M379F did not bind FMDV strains from other serotypes. In a sandwich enzyme-linked immunosorbent assay (ELISA employing unlabeled and biotinylated versions of the same VHH M332F showed high specificity for 146S particles but M379F showed lower 146S-specificity with some cross-reaction with 12S particles. These ELISAs could detect 146S particle concentrations as low as 2.3–4.6 µg/l. They can be used for FMD vaccine quality control and research and development, for example, to identify virion stabilizing excipients.

  4. 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.

  5. 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.

  6. 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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  9. Production and characterization of high-titer serum-free cell culture grown hepatitis C virus particles of genotype 1-6

    DEFF Research Database (Denmark)

    Mathiesen, Christian K; Jensen, Tanja B; Prentoe, Jannick

    2014-01-01

    Recently, cell culture systems producing hepatitis C virus particles (HCVcc) were developed. Establishment of serum-free culture conditions is expected to facilitate development of a whole-virus inactivated HCV vaccine. We describe generation of genotype 1-6 serum-free HCVcc (sf-HCVcc) from Huh7...... systems producing high-titer single-density sf-HCVcc, showing similar biological properties as HCVcc. This methodology has the potential to advance HCV vaccine development and to facilitate biophysical studies of HCV....

  10. 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...

  11. The effect of RNA stiffness on the self-assembly of virus particles

    Science.gov (United States)

    Li, Siyu; Erdemci-Tandogan, Gonca; van der Schoot, Paul; Zandi, Roya

    2018-01-01

    Under many in vitro conditions, some small viruses spontaneously encapsidate a single stranded (ss) RNA into a protein shell called the capsid. While viral RNAs are found to be compact and highly branched because of long distance base-pairing between nucleotides, recent experiments reveal that in a head-to-head competition between an ssRNA with no secondary or higher order structure and a viral RNA, the capsid proteins preferentially encapsulate the linear polymer! In this paper, we study the impact of genome stiffness on the encapsidation free energy of the complex of RNA and capsid proteins. We show that an increase in effective chain stiffness because of base-pairing could be the reason why under certain conditions linear chains have an advantage over branched chains when it comes to encapsidation efficiency. While branching makes the genome more compact, RNA base-pairing increases the effective Kuhn length of the RNA molecule, which could result in an increase of the free energy of RNA confinement, that is, the work required to encapsidate RNA, and thus less efficient packaging.

  12. Humoral Immune Response Induced by PLGA Micro Particle Coupled Newcastle Disease Virus Vaccine in Chickens

    Directory of Open Access Journals (Sweden)

    Sanganagouda K

    2014-02-01

    Full Text Available This experiment was conducted for evaluating the humoral immune responses induced by Poly Lactide-co-Glycolide Acid (PLGA microspheres coupled inactivated Newcastle Disease Virus (NDV vaccine in comparison to an ‘in-house’ prepared inactivated and a live commercial vaccine. PLG microparticles containing inactivated NDV were prepared by a double emulsion technique based on solvent evaporation method. The size of the NDV coupled PLG microparticles was determined by Electron Microscopy. NDV coupled PLG microparticles were spherical having smooth surface, hollow core inside with no pores on the surface. The experiment was conducted in four groups of chickens (n=15. The encapsulation efficiency of NDV coupled PLG microparticles was determined by protein estimation and HA activity in elute. The mean (± SE size of PLG microspheres was found to be 2.409 ± 0.65 µm. The mean percent of encapsulation efficiency of PLG microspheres coupled to NDV was assessed based on the total protein content and HA activity in elute was found to be 8.03 ± 0.50 and 12.5 ± 0.00, respectively. In conclusion, the results of the experiment showed that PLGA coupled NDV vaccine elicited stronger and prolonged humoral immune response in chickens, in comparison to the other tested vaccines, as assessed by haemagglutination inhibition and enzyme linked immuno sorbent asaay titers.

  13. A basic cluster in the N terminus of yellow fever virus NS2A contributes to infectious particle production.

    Science.gov (United States)

    Voßmann, Stephanie; Wieseler, Janett; Kerber, Romy; Kümmerer, Beate Mareike

    2015-05-01

    The flavivirus NS2A protein is involved in the assembly of infectious particles. To further understand its role in this process, a charged-to-alanine scanning analysis was performed on NS2A encoded by an infectious cDNA clone of yellow fever virus (YFV). Fifteen mutants containing single, double, or triple charged-to-alanine changes were tested. Five of them did not produce infectious particles, whereas efficient RNA replication was detectable for two of the five NS2A mutants (R22A-K23A-R24A and R99A-E100A-R101A mutants). Prolonged cultivation of transfected cells resulted in the recovery of pseudorevertants. Besides suppressor mutants in NS2A, a compensating second-site mutation in NS3 (D343G) arose for the NS2A R22A-K23A-R24A mutant. We found this NS3 mutation previously to be suppressive for the NS2Aα cleavage site Q189S mutant, also deficient in virion assembly. In this study, the subsequently suggested interaction between NS2A and NS3 was proven by coimmunoprecipitation analyses. Using selectively permeabilized cells, we could demonstrate that the regions encompassing R22A-K23A-R24A and Q189S in NS2A are localized to the cytoplasm, where NS3 is also known to reside. However, the defect in particle production observed for the NS2A R22A-K23A-R24A and Q189S mutants was not due to a defect in physical interaction between NS2A and NS3, as the NS2A mutations did not interrupt NS3 interaction. In fact, a region just upstream of R22-K23-R24 was mapped to be critical for NS2A-NS3 interaction. Taken together, these data support a complex interplay between YFV NS2A and NS3 in virion assembly and identify a basic cluster in the NS2A N terminus to be critical in this process. Despite an available vaccine, yellow fever remains endemic in tropical areas of South America and Africa. To control the disease, antiviral drugs are required, and an understanding of the determinants of virion assembly is central to their development. In this study, we identified a basic cluster of

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

    Directory of Open Access Journals (Sweden)

    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.

  15. 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.

  16. 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.

  17. 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.

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

    Science.gov (United States)

    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.

  19. 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.

  20. 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.

  1. 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%.

  2. 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.

  3. 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.

  4. Solubilization of Envelopes of HVJ (Sendai virus) with Alkali-Emasol Treatment and Reassembly of Envelope Particles with Removal of the Detergent

    Science.gov (United States)

    Shimizu, Kazufumi; Hosaka, Yashuhiro; Shimizu, Yohko K.

    1972-01-01

    The envelopes of HVJ (Sendai virus) virions were solubilized with alkali-Emasol treatment. The solubilized envelope subunit(s) associated with hemagglutination-inhibiting antibody blocking, neuraminidase, and low hemagglutinating (HA) activities had a sedimentation coefficient of 8.8S. Envelope fragment-like structures were assembled from the solubilized subunits after Emasol was removed by gel filtration. These reassembled envelope particles with HA activity had cell-fusion activity as well as hemolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the reassembled particles revealed that they mainly consisted of two kinds of polypeptides. Images PMID:4337169

  5. Co-delivery of antigen and IL-12 by Venezuelan equine encephalitis virus replicon particles enhances antigen-specific immune responses and anti-tumor effects

    OpenAIRE

    Osada, Takuya; Berglund, Peter; Morse, Michael A.; Hubby, Bolyn; Lewis, Whitney; Niedzwiecki, Donna; Hobeika, Amy; Burnett, Bruce; Devi, Gayathri R.; Clay, Timothy M.; Smith, Jonathan; Lyerly, H. Kim

    2012-01-01

    We recently demonstrated that Venezuelan equine encephalitis (VEE) virus-based replicon particles (VRP) encoding tumor antigens could break tolerance in the immunomodulatory environment of advanced cancer. We hypothesized that local injection of VRP expressing Interleukin-12 (IL-12) at the site of injections of VRP-based cancer vaccines would enhance the tumor-antigen-specific T cell and antibody responses and anti-tumor efficacy. Mice were immunized with VRP encoding the human tumor-associat...

  6. 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.

  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. 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...

  9. Immunogenic and replicative properties of classical swine fever virus replicon particles modified to induce IFN-α/β and carry foreign genes.

    Science.gov (United States)

    Suter, Rolf; Summerfield, Artur; Thomann-Harwood, Lisa J; McCullough, Kenneth C; Tratschin, Jon-Duri; Ruggli, Nicolas

    2011-02-04

    Virus replicon particles (VRP) are genetically engineered infectious virions incapable of generating progeny virus due to partial or complete deletion of at least one structural gene. VRP fulfil the criteria of a safe vaccine and gene delivery system. With VRP derived from classical swine fever virus (CSF-VRP), a single intradermal vaccination protects from disease. Spreading of the challenge virus in the host is however not completely abolished. Parameters that are critical for immunogenicity of CSF-VRP are not well characterized. Considering the importance of type I interferon (IFN-α/β) to immune defence development, we generated IFN-α/β-inducing VRP to determine how this would influence vaccine efficacy. We also evaluated the effect of co-expressing granulocyte macrophage colony-stimulating factor (GM-CSF) in the vaccine context. The VRP were capable of long-term replication in cell culture despite the presence of IFN-α/β. In vivo, RNA replication was essential for the induction of an immune response. IFN-α/β-inducing and GM-CSF-expressing CSF-VRP were similar to unmodified VRP in terms of antibody and peripheral T-cell responses, and in reducing the blood levels of challenge virus RNA. Importantly, the IFN-α/β-inducing VRP did show increased efficacy over the unmodified VRP in terms of B-cell and T-cell responses, when tested with secondary immune responses by in vitro restimulation assay. Copyright © 2010 Elsevier Ltd. All rights reserved.

  10. 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.

  11. 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.

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

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    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

  13. Efficient induction of human T-cell leukemia virus-1-specific CTL by chimeric particle without adjuvant as a prophylactic for adult T-cell leukemia.

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    Kozako, Tomohiro; Fukada, Katsuhiko; Hirata, Shinya; White, Yohann; Harao, Michiko; Nishimura, Yasuharu; Kino, Youichiro; Soeda, Shinji; Shimeno, Hiroshi; Lemonnier, François; Sonoda, Shunro; Arima, Naomichi

    2009-12-01

    Adult T-cell leukemia-lymphoma (ATL) is an aggressive peripheral T-cell neoplasm that develops after long-term infection with the human T-cell leukemia virus-1 (HTLV-1). HTLV-1-specific cytotoxic T lymphocytes (CTLs) play an important role in suppressing proliferation of HTLV-1-infected or transformed T-cells in vitro. Efficient induction of antigen-specific CTLs is important for immunologic suppression of oncogenesis, but has evaded strategies utilizing poorly immunogenic free synthetic peptides. In the present study, we examined the efficient induction of HTLV-1-specific CD8+ T-cell response by an HTLV-1/hepatitis B virus core (HBc) chimeric particle incorporating the HLA-A*0201-restricted HTLV-1 Tax-epitope. The immunization of HLA-A*0201-transgenic mice with the chimeric particle induced antigen-specific gamma-interferon reaction, whereas immunization with epitope peptide only induced no reaction as assessed by enzyme-linked immunospot assay. Immunization with the chimeric particle also induced HTLV-1-specific CD8+ T-cells in spleen and inguinal lymph nodes. Furthermore, upon exposure of dendritic cells from HLA-A*0201-transgenic mice to the chimeric particle, the expression of CD86, HLA-A02, TLR4 and MHC class II was increased. Additionally, our results show that HTLV-1-specific CD8+ T-cells can be induced by peptide with HTLV-1/HBc particle from ATL patient, but not by peptide only and these HTLV-1-specific CD8+ T-cells were able to lyse cells presenting the peptide. These results suggest that HTLV-1/HBc chimeric particle is capable of inducing strong cellular immune responses without adjuvants via effective maturation of dendritic cells and is potentially useful as an effective carrier for therapeutic vaccines in tumors, or in infectious diseases by substituting the epitope peptide.

  14. Mutational analysis of the hepatitis C virus E1 glycoprotein in retroviral pseudoparticles and cell-culture-derived H77/JFH1 chimeric infectious virus particles

    DEFF Research Database (Denmark)

    Russell, R S; Kawaguchi, K; Meunier, J-C

    2009-01-01

    Cell entry by enveloped viruses is mediated by viral glycoproteins, and generally involves a short hydrophobic peptide (fusion peptide) that inserts into the cellular membrane. An internal hydrophobic domain within E1 (aa262-290) of hepatitis C virus (HCV) may function as a fusion peptide....... Retrovirus-based HCV-pseudotyped viruses (HCVpp; genotype 1a) containing Ala or Pro substitutions at conserved amino acid positions within this putative fusion peptide were generated. Mutation of conserved residues significantly reduced efficiency of HCVpp entry into Huh-7 cells. The majority of amino acid...... incorporation into pseudoparticles and normal CD81-binding, and therefore might affect viral fusion. One mutant (S283P) consistently displayed two- to threefold higher infectivity than did wild-type. Three mutations that decreased HCVpp infectivity also reduced levels of HCVcc infectious virus production...

  15. Assembly and characterization of foot-and-mouth disease virus empty capsid particles expressed within mammalian cells

    DEFF Research Database (Denmark)

    Gullberg, Maria; Muszynski, Bartosz; Organtini, Lindsey J.

    2013-01-01

    The foot-and-mouth disease virus (FMDV) structural protein precursor, P1-2A, is cleaved by the virus-encoded 3C protease (3Cpro) into the capsid proteins VP0, VP1 and VP3 (and 2A). In some systems, it is difficult to produce large amounts of these processed capsid proteins since 3Cpro can be toxic...

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

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    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.

  17. 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

  18. 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.

  19. 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.

  20. 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.

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

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    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.

  2. 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...

  3. Immunogenicity of Leishmania-derived hepatitis B small surface antigen particles exposing highly conserved E2 epitope of hepatitis C virus.

    Science.gov (United States)

    Czarnota, Anna; Tyborowska, Jolanta; Peszyńska-Sularz, Grażyna; Gromadzka, Beata; Bieńkowska-Szewczyk, Krystyna; Grzyb, Katarzyna

    2016-04-13

    Hepatitis C virus (HCV) infection is a major health problem worldwide, affecting an estimated 2-3 % of human population. An HCV vaccine, however, remains unavailable. High viral diversity poses a challenge in developing a vaccine capable of eliciting a broad neutralizing antibody response against all HCV genotypes. The small surface antigen (sHBsAg) of hepatitis B virus (HBV) has the ability to form highly immunogenic subviral particles which are currently used as an efficient anti-HBV vaccine. It also represents an attractive antigen carrier for the delivery of foreign sequences. In the present study, we propose a bivalent vaccine candidate based on novel chimeric particles in which highly conserved epitope of HCV E2 glycoprotein (residues 412-425) was inserted into the hydrophilic loop of sHBsAg. The expression of chimeric protein was performed in an unconventional, Leishmania tarentolae expression system resulting in an assembly of particles which retained immunogenicity of both HCV epitope and sHBsAg protein. Direct transmission electron microscopy observation and immunogold staining confirmed the formation of spherical particles approximately 22 nm in diameter, and proper foreign epitope exposition. Furthermore, the sera of mice immunized with chimeric particles proved reactive not only to purified yeast-derived sHBsAg proteins but also HCV E2 412-425 synthetic peptide. Most importantly, they were also able to cross-react with E1E2 complexes from different HCV genotypes. For the first time, we confirmed successful assembly of chimeric sHBsAg virus-like particles (VLPs) in the L. tarentolae expression system which has the potential to produce high-yields of properly N-glycosylated mammalian proteins. We also proved that chimeric Leishmania-derived VLPs are highly immunogenic and able to elicit cross-reactive antibody response against HCV. This approach may prove useful in the development of a bivalent prophylactic vaccine against HBV and HCV and opens up a new

  4. 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

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

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    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

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

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    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

  7. 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

  8. 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

  9. 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

  10. 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.

  11. 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...

  12. 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

  13. [Design and immunogenicity evaluation for the bacteria-like particle vaccine against swine type O foot-and-mouth disease virus].

    Science.gov (United States)

    Hou, Liting; Chen, Jin; Qiao, Xuwen; Yu, Xiaoming; Hou, Jibo; Zheng, Qisheng; Li, Jinnian

    2017-02-25

    Based on gram positive enhancer matrix displaying technology, we designed and evaluated a bacteria-like particle vaccine against swine type O Foot-and-mouth disease virus. Three optimized genes of type O Foot-and-mouth disease virus strain Mya98 were cloned into recombinant prokaryotic expression vector pQZ-PA and renamed as pQZ-BT1B-PA, pQZ-BT2B-PA and pQZ-B (T1BT2) 4B-PA, fused with an anchor protein (PA) binding to Gram-positive enhancer matrix (GEM) particles specifically. The protein expression was identified with SDS-PAGE and Western blotting, and then purified with GEM particles. Five-week old female mice were randomly divided into six groups and all the immunization was developed according to subcutaneous injection. Mice in the first three groups were injected with 50 μg/dose GEM-BT1B, GEM-BT2B and GEM-B (T1BT2) 4B, respectively. Mice in the fourth group were immunized with commercial peptide vaccine as positive control. The fifth group vaccinated with host E. coli transformed with pQZ-PA fulfilled as negative control. Mice in the last group injected with sterile PBS served as blank control. The humoral immunity of recombinant protein vaccine was evaluated with peptide-specific antibody and LPB antibody. The cellular immunity was evaluated with lymphocyte proliferation test and cytokine expression detection. SDS-PAGE and Western blotting showed that the most part of soluble target fusion protein have been purified and displayed on GEM particles. Vaccine GEM-B (T1BT2) 4B stimulated mice produce not only higher level of specific antibody against peptide and Foot-and-mouth disease virus specific liquid phase blocking antibody, but also more vigorous spleen lymph proliferation and higher levels of Th1 type cytokines. To summarize, vaccine of GEM-B (T1BT2) 4B possessed good immunogenicity and opened a new way for further Foot-and-mouth disease virus subunit vaccine design.

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

    OpenAIRE

    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...

  15. 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 ...

  16. Role of Mason-Pfizer Monkey Virus CA-NC Spacer Peptide-Like Domain in Assembly of Immature Particles

    Czech Academy of Sciences Publication Activity Database

    Strohalmová-Böhmová, Karolína; Spiwok, V.; Lepšík, Martin; Hadravová, Romana; Křížová, Ivana; Ulbrich, P.; Pichová, Iva; Bednárová, Lucie; Ruml, T.; Rumlová, Michaela

    2014-01-01

    Roč. 88, č. 24 (2014), s. 14148-14160 ISSN 0022-538X R&D Projects: GA ČR(CZ) GA14-15326S; GA MŠk LO1302; GA ČR GBP208/12/G016 Institutional support: RVO:61388963 Keywords : human immunodeficiency virus * HIV -1 capsid protein * murine leukemia virus Subject RIV: EE - Microbiology, Virology Impact factor: 4.439, year: 2014

  17. Synthesis of empty capsid-like particles of Asia I foot-and-mouth disease virus in insect cells and their immunogenicity in guinea pigs.

    Science.gov (United States)

    Cao, Yimei; Lu, Zengjun; Sun, Jiachuan; Bai, Xingwen; Sun, Pu; Bao, Huifang; Chen, Yingli; Guo, Jianhong; Li, Dong; Liu, Xiangtao; Liu, Zaixin

    2009-05-28

    The assembly of foot-and-mouth disease virus (FMDV) requires the cleavage of the P12A polyprotein into individual structural proteins by protease 3C. In this study, we constructed a recombinant baculovirus that simultaneously expressed the genes for the P12A and 3C proteins of Asia I FMDV from individual promoters. The capsid proteins expressed in High Five insect cells were processed by viral 3C protease, as shown by Western blotting, and were antigenic, as revealed by their reactivity in an indirect sandwich-ELISA, and by immunofluorescent assay. The empty capsid-like particles were similar to authentic 75S empty capsids from FMDV in terms of their shape, size and sedimentation velocity, as demonstrated by sucrose gradient centrifugation. Both empty capsid-like particles and some small-sized particles (about 10nm in diameter) were also observed using immunoelectron microscopy. Furthermore, the empty capsid-like particles or intermediates induced high levels of FMDV-specific antibodies in guinea pigs following immunization, and neutralizing antibodies were induced in the second week after vaccination. These recombinant, non-infectious, FMDV empty capsids are potentially useful for the development of new diagnostic techniques and vaccines.

  18. Computer-assisted 2-D agarose electrophoresis of Haemophilus influenzae type B meningitis vaccines and analysis of polydisperse particle populations in the size range of viruses: a review.

    Science.gov (United States)

    Tietz, Dietmar

    2007-02-01

    When protein-polysaccharide conjugated vaccines were first developed for the immunization of small children against meningitis caused by infection with Haemophilus influenzae type b (Hib), the vaccine preparations varied in immunogenicity. Testing for immunogenicity was time-consuming and alternative analytical procedures for determining vaccine quality were unsatisfactory. For example, due to the very high molecular weight of the vaccine particles, immunogens could only be physically characterized as a fraction in the void volume of Sepharose gel filtration. In search of better analytical methods, a computer-assisted electrophoretic technique for analyzing such vaccines was developed in the period from 1983 to 1995. This new approach made it possible to analyze highly negatively charged particles as large as or larger than intact viruses. 2-D gel patterns were generated that varied depending on the conditions of the particular vaccine preparation and were therefore characteristic of each vaccine sample. Thus, vaccine particle populations with a continuous size variation over a wide range (polydisperse) could be characterized according to size and free mobility (related to particle surface net charge density). These advances are reviewed in this article, since the developed methods are still a promising tool for vaccine quality control and for predicting immunogen effectiveness in the production of vaccines. The technique is potentially beneficial for Hib immunogens and other high-molecular-mass vaccines. Additional biomedical applications for this nondenaturing electrophoretic technique are briefly discussed and detailed information about computational and mathematical procedures and theoretical aspects is provided in the Appendices.

  19. 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...

  20. Radiation and biophysical studies on cells and viruses. Progress report, April 1, 1976--June 30, 1977. [Gamma radiation, alpha particles

    Energy Technology Data Exchange (ETDEWEB)

    Cole, A.

    1977-01-01

    Progress is reported on the following research projects: genetic structure of DNA, chromosomes, and nucleoproteins; particle beam studies of radiosensitive sites; division delay in CHO cells induced by partly penetrating alpha particles; location of cellular sites for mutation induction; sites for radioinduced cell transformation using partly penetrating particle beams; gamma-ray and particle irradiation of nucleoproteins and other model systems; quantitation of surface antigens on normal and neoplastic cells by x-ray fluorescence; hyperthermic effects on cell survival and DNA repair mechanisms; and studies on radioinduced cell transformation. (HLW)

  1. 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.

  2. ORF9p phosphorylation by ORF47p is crucial for the formation and egress of varicella-zoster virus viral particles.

    Science.gov (United States)

    Riva, Laura; Thiry, Marc; Bontems, Sebastien; Joris, Aline; Piette, Jacques; Lebrun, Marielle; Sadzot-Delvaux, Catherine

    2013-03-01

    The role of the tegument during the herpesvirus lytic cycle is still not clearly established, particularly at the late phase of infection, when the newly produced viral particles need to be fully assembled before being released from the infected cell. The varicella-zoster virus (VZV) protein coded by open reading frame (ORF) 9 (ORF9p) is an essential tegument protein, and, even though its mRNA is the most expressed during the productive infection, little is known about its functions. Using a GalK positive/negative selection technique, we modified a bacterial artificial chromosome (BAC) containing the complete VZV genome to create viruses expressing mutant versions of ORF9p. We showed that ORF9p is hyperphosphorylated during the infection, especially through its interaction with the viral Ser/Thr kinase ORF47p; we identified a consensus site within ORF9p recognized by ORF47p and demonstrated its importance for ORF9p phosphorylation. Strikingly, an ultrastructural analysis revealed that the mutation of this consensus site (glutamate 85 to arginine) strongly affects viral assembly and release, reproducing the ORF47 kinase-dead VZV phenotype. It also slightly diminishes the infectivity toward immature dendritic cells. Taken together, our results identify ORF9p as a new viral substrate of ORF47p and suggest a determinant role of this phosphorylation for viral infectivity, especially during the process of viral particle formation and egress.

  3. 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.

  4. 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Directory of Open Access Journals (Sweden)

    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

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

    Science.gov (United States)

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

    Directory of Open Access Journals (Sweden)

    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.

  9. 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.

  10. 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

  11. 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

  12. Hepatitis B virus DNA integration occurs early in the viral life cycle in anin vitroinfection model via NTCP-dependent uptake of enveloped virus particles.

    Science.gov (United States)

    Tu, Thomas; Budzinska, Magdalena A; Vondran, Florian W R; Shackel, Nicholas A; Urban, Stephan

    2018-02-07

    Chronic infection by the Hepatitis B Virus (HBV) is the major contributor to liver disease worldwide. Though HBV replicates via a nuclear episomal DNA (cccDNA), integration of HBV DNA into the host cell genome is regularly observed in the liver of infected patients. While reported as a pro-oncogenic alteration, the mechanism(s) and timing of HBV DNA integration are not well-understood, chiefly due to the lack of in vitro infection models that have detectable integration events. Here, we have established an in vitro system in which integration can be reliably detected following HBV infection. We measured HBV DNA integration using inverse nested PCR in primary human hepatocytes, HepaRG-NTCP, HepG2-NTCP, and Huh7-NTCP cells after HBV infection. Integration was detected in all cell types at a rate of >1 per 10000 cells, with the most consistent detection in Huh7-NTCP cells. Integration rate remained stable between 3 and 9 days post-infection. HBV DNA integration was efficiently blocked by treatment with 200nM of the HBV entry inhibitor Myrcludex B, but not with 10μM Tenofovir, 100U Interferon alpha, or 1μM of the capsid assembly inhibitor GLS4. This suggests integration of HBV DNA occurs immediately after infection of hepatocytes and is likely independent of de novo HBV replication in this model. Site analysis revealed that HBV DNA integrations were distributed over the entire human genome. Further, integrated HBV DNA sequences were consistent with double-stranded linear HBV DNA being the major precursor. Thus, we have established an in vitro system to interrogate the mechanisms of HBV DNA integration. Importance Hepatitis B Virus (HBV) is a common blood-borne pathogen and, following a chronic infection, can cause liver cancer and liver cirrhosis. Integration of HBV DNA into the host genome occurs in all known members of the hepadnaviridae family, despite this form not being necessary for viral replication. HBV DNA integration has been reported to drive liver cancer

  13. 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

  14. Establishment of the black-tailed prairie dog (Cynomys ludovicianus) as a novel animal model for comparing smallpox vaccines administered preexposure in both high- and low-dose monkeypox virus challenges.

    Science.gov (United States)

    Keckler, M S; Carroll, D S; Gallardo-Romero, N F; Lash, R R; Salzer, J S; Weiss, S L; Patel, N; Clemmons, C J; Smith, S K; Hutson, C L; Karem, K L; Damon, I K

    2011-08-01

    The 2003 monkeypox virus (MPXV) outbreak and subsequent laboratory studies demonstrated that the black-tailed prairie dog is susceptible to MPXV infection and that the ensuing rash illness is similar to human systemic orthopoxvirus (OPXV) infection, including a 7- to 9-day incubation period and, likely, in some cases a respiratory route of infection; these features distinguish this model from others. The need for safe and efficacious vaccines for OPVX in areas where it is endemic or epidemic is important to protect an increasingly OPXV-naïve population. In this study, we tested current and investigational smallpox vaccines for safety, induction of anti-OPXV antibodies, and protection against mortality and morbidity in two MPXV challenges. None of the smallpox vaccines caused illness in this model, and all vaccinated animals showed anti-OPXV antibody responses and neutralizing antibody. We tested vaccine efficacy by challenging the animals with 10(5) or 10(6) PFU Congo Basin MPXV 30 days postvaccination and evaluating morbidity and mortality. Our results demonstrated that vaccination with either Dryvax or Acambis2000 protected the animals from death with no rash illness. Vaccination with IMVAMUNE also protected the animals from death, albeit with (modified) rash illness. Based on the results of this study, we believe prairie dogs offer a novel and potentially useful small animal model for the safety and efficacy testing of smallpox vaccines in pre- and postexposure vaccine testing, which is important for public health planning.

  15. 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....

  16. Interaction between the yellow fever virus nonstructural protein NS3 and the host protein Alix contributes to the release of infectious particles.

    Science.gov (United States)

    Carpp, Lindsay N; Galler, Ricardo; Bonaldo, Myrna C

    2011-01-01

    The ESCRT (endosomal sorting complex required for transport) machinery normally executes cargo sorting and internalization during multivesicular body biogenesis, but is also utilized by several enveloped viruses to facilitate their budding from cellular membranes. Although the mechanisms of flavivirus infectious particle assembly and release are poorly understood, the nonstructural protein NS3 has been reported to have an essential role via an undescribed mechanism. Here, we shed light on the role of NS3 by connecting it to the host factor Alix, a protein intimately connected with the ESCRT machinery. We demonstrate that NS3 and Alix interact and show that dominant negative versions of Alix inhibit YFV release. Furthermore, we show that NS3 supplied in trans rescues this effect. We propose that the interaction between NS3 and Alix contributes to YFV release. Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.

  17. Virus Assembly and Maturation

    Science.gov (United States)

    Johnson, John E.

    2004-03-01

    We use two techniques to look at three-dimensional virus structure: electron cryomicroscopy (cryoEM) and X-ray crystallography. Figure 1 is a gallery of virus particles whose structures Timothy Baker, one of my former colleagues at Purdue University, used cryoEM to determine. It illustrates the variety of sizes of icosahedral virus particles. The largest virus particle on this slide is the Herpes simplex virus, around 1200Å in diameter; the smallest we examined was around 250Å in diameter. Viruses bear their genomic information either as positive-sense DNA and RNA, double-strand DNA, double-strand RNA, or negative-strand RNA. Viruses utilize the various structure and function "tactics" seen throughout cell biology to replicate at high levels. Many of the biological principles that we consider general were in fact discovered in the context of viruses ...

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

    Science.gov (United States)

    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.

  19. GB virus C particles inhibit T cell activation via envelope E2 protein-mediated inhibition of T cell receptor signaling

    Science.gov (United States)

    Bhattarai, Nirjal; McLinden, James H.; Xiang, Jinhua; Landay, Alan L.; Chivero, Ernest T.; Stapleton, Jack T.

    2014-01-01

    Viruses enter into complex interactions within human hosts leading to facilitation or suppression of each other's replication. Upon coinfection, GB virus C (GBV-C) suppresses HIV-1 replication in vivo and in vitro, and GBV-C coinfection is associated with prolonged survival in HIV-infected people. GBV-C is a lymphotropic virus capable of persistent infection. GBV-C infection is associated with reduced T cell activation in HIV-infected humans, and immune activation is a critical component of HIV disease pathogenesis. We demonstrate that serum GBV-C particles inhibited activation of primary human T cells. T cell activation inhibition was mediated by the envelope glycoprotein E2, as expression of E2 inhibited T cell receptor (TCR)-mediated activation of tyrosine kinase (Lck). The region on the E2 protein was characterized and revealed a highly conserved peptide motif sufficient to inhibit TCR-mediated signaling. The E2 region contained a predicted Lck substrate site, and substitution of an alanine or histidine for the tyrosine reversed TCR signaling inhibition. GBV-C E2 protein and a synthetic peptide representing the inhibitory amino acid sequence were phosphorylated by Lck in vitro. The synthetic peptide also inhibited TCR-mediated activation of primary human CD4+ and CD8+ T cells. Extracellular microvesicles from GBV-C E2-expressing cells contained E2 protein and inhibited TCR signaling in bystander T cells not expressing E2. Thus, GBV-C reduced global T cell activation via competition between its envelope protein E2 and Lck following TCR engagement. This novel inhibitory mechanism of T cell activation may provide new approaches for HIV and immunoactivation therapy. PMID:23686495

  20. 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.

  1. 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.

  2. 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.

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

    Science.gov (United States)

    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.

  4. 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.

  5. Production and characterization of high-titer serum-free cell culture grown hepatitis C virus particles of genotype 1-6.

    Science.gov (United States)

    Mathiesen, Christian K; Jensen, Tanja B; Prentoe, Jannick; Krarup, Henrik; Nicosia, Alfredo; Law, Mansun; Bukh, Jens; Gottwein, Judith M

    2014-06-01

    Recently, cell culture systems producing hepatitis C virus particles (HCVcc) were developed. Establishment of serum-free culture conditions is expected to facilitate development of a whole-virus inactivated HCV vaccine. We describe generation of genotype 1-6 serum-free HCVcc (sf-HCVcc) from Huh7.5 hepatoma cells cultured in adenovirus expression medium. Compared to HCVcc, sf-HCVcc showed 0.6-2.1 log10 higher infectivity titers (4.7-6.2 log10 Focus Forming Units/mL), possibly due to increased release and specific infectivity of sf-HCVcc. In contrast to HCVcc, sf-HCVcc had a homogeneous single-peak density profile. Entry of sf-HCVcc depended on HCV co-receptors CD81, LDLr, and SR-BI, and clathrin-mediated endocytosis. HCVcc and sf-HCVcc were neutralized similarly by chronic-phase patient sera and by human monoclonal antibodies targeting conformational epitopes. Thus, we developed serum-free culture systems producing high-titer single-density sf-HCVcc, showing similar biological properties as HCVcc. This methodology has the potential to advance HCV vaccine development and to facilitate biophysical studies of HCV. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. 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.

  7. 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.

  8. 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

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

    Science.gov (United States)

    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.

  10. 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.

  11. 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

  12. Structure of the immature HIV-1 capsid in intact virus particles at 8.8 angstrom resolution

    Czech Academy of Sciences Publication Activity Database

    Schur, F. K. M.; Hagen, W. J. H.; Rumlová, Michaela; Ruml, T.; Müller, B.; Kräusslich, H. G.; Briggs, J. A. G.

    2015-01-01

    Roč. 517, č. 7535 (2015), s. 505-508 ISSN 0028-0836 R&D Projects: GA ČR(CZ) GA14-15326S Institutional support: RVO:61388963 Keywords : retrovirus * HIV * M-PMV * capsid protein * CA * assembly * immature particles Subject RIV: CE - Biochemistry Impact factor: 38.138, year: 2015

  13. 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...

  14. Virus Crystallography

    Science.gov (United States)

    Fry, Elizabeth; Logan, Derek; Stuart, David

    Crystallography provides a means of visualizing intact virus particles as well as their isolated constituent proteins and enzymes (1-3) at near-atomic resolution, and is thus an extraordinarily powerful tool in the pursuit of a fuller understanding of the functioning of these simple biological systems. We have already expanded our knowledge of virus evolution, assembly, antigenic variation, and host-cell interactions; further studies will no doubt reveal much more. Although the rewards are enormous, an intact virus structure determination is not a trivial undertaking and entails a significant scaling up in terms of time and resources through all stages of data collection and processing compared to a traditional protein crystallographic structure determination. It is the methodology required for such studies that will be the focus of this chapter. The computational requirements were satisfied in the late 1970s, and when combined with the introduction of phase improvement techniques utilizing the virus symmetry (4,5), the application of crystallography to these massive macromolecular assemblies became feasible. This led to the determination of the first virus structure (the small RNA plant virus, tomato bushy stunt virus), by Harrison and coworkers in 1978 (6). The structures of two other plant viruses followed rapidly (7,8). In the 1980s, a major focus of attention was a family of animal RNA viruses; the Picornaviridae.

  15. 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

  16. 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.

  17. Charge neutralization as the major factor for the assembly of nucleocapsid-like particles from C-terminal truncated hepatitis C virus core protein

    Directory of Open Access Journals (Sweden)

    Theo Luiz Ferraz de Souza

    2016-11-01

    Full Text Available Background Hepatitis C virus (HCV core protein, in addition to its structural role to form the nucleocapsid assembly, plays a critical role in HCV pathogenesis by interfering in several cellular processes, including microRNA and mRNA homeostasis. The C-terminal truncated HCV core protein (C124 is intrinsically unstructured in solution and is able to interact with unspecific nucleic acids, in the micromolar range, and to assemble into nucleocapsid-like particles (NLPs in vitro. The specificity and propensity of C124 to the assembly and its implications on HCV pathogenesis are not well understood. Methods Spectroscopic techniques, transmission electron microscopy and calorimetry were used to better understand the propensity of C124 to fold or to multimerize into NLPs when subjected to different conditions or in the presence of unspecific nucleic acids of equivalent size to cellular microRNAs. Results The structural analysis indicated that C124 has low propensity to self-folding. On the other hand, for the first time, we show that C124, in the absence of nucleic acids, multimerizes into empty NLPs when subjected to a pH close to its isoelectric point (pH ≈ 12, indicating that assembly is mainly driven by charge neutralization. Isothermal calorimetry data showed that the assembly of NLPs promoted by nucleic acids is enthalpy driven. Additionally, data obtained from fluorescence correlation spectroscopy show that C124, in nanomolar range, was able to interact and to sequester a large number of short unspecific nucleic acids into NLPs. Discussion Together, our data showed that the charge neutralization is the major factor for the nucleocapsid-like particles assembly from C-terminal truncated HCV core protein. This finding suggests that HCV core protein may physically interact with unspecific cellular polyanions, which may correspond to microRNAs and mRNAs in a host cell infected by HCV, triggering their confinement into infectious particles.

  18. 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.

  19. 13-Cis retinoic acid can enhance the antitumor activity of non-replicating Sendai virus particle against neuroblastoma.

    Science.gov (United States)

    Nomura, Motonari; Shimbo, Takashi; Miyamoto, Yasuhide; Fukuzawa, Masahiro; Kaneda, Yasufumi

    2013-02-01

    Hemagglutinating virus of Japan-envelope (HVJ-E) is a drug delivery vector based on inactivated Sendai virus. Recently, antitumor activities were found for HVJ-E itself and clinical trials of HVJ-E for some malignant tumors are now ongoing. We investigated the in vitro and in vivo antitumor effects of HVJ-E against neuroblastoma, which is one of the most common malignant solid tumors in childhood. The sensitivity of human neuroblastoma cell lines to HVJ-E correlated with the expression level of gangliosides, Sialylparagloboside (SPG) and GD1a, receptors for HVJ. Among the cell lines, SK-N-SH was the most sensitive to HVJ-E in vitro and total SPG and GD1a expression was the highest. Complete eradication of subcutaneous tumors derived from SK-N-SH cells was achieved by intratumoral injection of HVJ-E in SCID mice and no recurrence was observed for more than 300 days after HVJ-E inoculation. In contrast, NB1 cells expressed the lowest amount of GD1a and SPG and were resistant to HVJ-E in vitro. The expression of GD1a increased by 13-cis retinoic acid (13cRA), which is a therapeutic drug for high risk neuroblastoma, thus leading to an improved sensitivity to HVJ-E in vitro. Only growth inhibition of the subcutaneous tumors derived from NB1 cells was achieved by HVJ-E in the SCID mice, but the combination of 13cRA and HVJ-E could achieve partial eradication of the xenograft and also lead to an improved prognosis. In conclusion, HVJ-E is a promising therapeutic modality for neuroblastoma and 13cRA can be used as an adjuvant to HVJ-E. © 2012 Japanese Cancer Association.

  20. Beyond the God particle

    CERN Document Server

    Lederman, Leon M

    2013-01-01

    On July 4, 2012, the long-sought Higgs Boson--aka "the God Particle"--was discovered at the world's largest particle accelerator, the LHC, in Geneva, Switzerland. On March 14, 2013, physicists at CERN confirmed it. This elusive subatomic particle forms a field that permeates the entire universe, creating the masses of the elementary particles that are the basic building blocks of everything in the known world--from viruses to elephants, from atoms to quasars.

  1. An RNA Vaccine Based on Recombinant Semliki Forest Virus Particles Expressing the Cu,Zn Superoxide Dismutase Protein of Brucella abortus Induces Protective Immunity in BALB/c Mice

    Science.gov (United States)

    Oñate, Angel A.; Donoso, Gabriel; Moraga-Cid, Gustavo; Folch, Hugo; Céspedes, Sandra; Andrews, Edilia

    2005-01-01

    We constructed infectious but replication-deficient Semliki Forest virus (SFV) particles carrying recombinant RNA encoding Brucella abortus Cu,Zn superoxide dismutase (SOD). The recombinant SFV particles (SFV-SOD particles) were then evaluated for their ability to induce a T-cell immune response and to protect BALB/c mice against a challenge with B. abortus 2308. Intraperitoneal injection of mice with recombinant SFV-SOD particles did not lead to the induction of SOD-specific antibodies, at least until week 6 after immunization (the end of the experiment). In vitro stimulation of splenocytes from the vaccinated mice with either recombinant Cu,Zn SOD (rSOD) or crude Brucella protein resulted in a T-cell proliferative response and the induction of gamma interferon secretion but not interleukin-4. In addition, the splenocytes exhibited significant levels of cytotoxic T-lymphocyte activity against Brucella-infected cells. The SFV-SOD particles, but not the control virus particles, induced a significant level of protection in BALB/c mice against challenge with B. abortus virulent strain 2308. These findings indicated that an SFV-based vector carrying the SOD gene has potential for use as a vaccine to induce resistance against B. abortus infections. PMID:15908354<