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Sample records for chimeric protein vaccine

  1. Chimeric Pestivirus Experimental Vaccines.

    Science.gov (United States)

    Reimann, Ilona; Blome, Sandra; Beer, Martin

    2016-01-01

    Chimeric pestiviruses have shown great potential as marker vaccine candidates against pestiviral infections. Exemplarily, we describe here the construction and testing of the most promising classical swine fever vaccine candidate "CP7_E2alf" in detail. The description is focused on classical cloning technologies in combination with reverse genetics. PMID:26458840

  2. EspA-Intimin chimeric protein, a candidate vaccine against Escherichia coli O157:H7.

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    Hamid Sedighian Rad

    2013-09-01

    Full Text Available Enterohemorrhagic Escherichia coli (EHEC O157:H7 is an important enteric pathogen in human causing bloody or nonbloody diarrhea, which may be complicated by hemolytic uremic syndrome (HUS. Cattle are an important reservoir of EHEC. This research aims at vaccination with a divalent chimer protein composed of EspA120 and Intimin 282 and its preventive effect of EHEC O157 colonization in mice rectal epithelium.A divalent recombinant EspA-Intimin (EI protein containing EspA120 and Intimin280 attached with a linker was amplified from a trivalent construct and cloned in pET-28a (+ vector. The immunization was conducted in mice after expression and purification of the recombinant EI (rEI.Mice subcutaneously immunized with rEI, elicited significant rEI specific serum IgG antibodies and showed significantly decreased E.coli O157:H7 shedding compared to the control group.The chimeric recombinant protein induced strong humoral response as well as protection against oral challenges with live E.coli O157:H7.

  3. Vaccination of dogs with six different candidate leishmaniasis vaccines composed of a chimerical recombinant protein containing ribosomal and histone protein epitopes in combination with different adjuvants.

    Science.gov (United States)

    Poot, J; Janssen, L H M; van Kasteren-Westerneng, T J; van der Heijden-Liefkens, K H A; Schijns, V E J C; Heckeroth, A

    2009-07-16

    Chimerical protein "Q", composed of antigenic ribosomal and histone sequences, in combination with live BCG is a promising canine leishmaniasis vaccine candidate; one of the few vaccine candidates that have been tested successfully in dogs. Unfortunately, live BCG is not an appropriate adjuvant for commercial application due to safety problems in dogs. In order to find a safe adjuvant with similar efficacy to live BCG, muramyl dipeptide, aluminium hydroxide, Matrix C and killed Propionibacterium acnes in combination with either E. coli- or baculovirus-produced recombinant JPCM5_Q protein were tested. Groups of five or seven dogs were vaccinated with six different adjuvant-antigen combinations and challenged with a high dose intravenous injection of Leishmania infantum JPC strain promastigotes. All candidate vaccines proved to be safe, and both humoral and cellular responses to the recombinant proteins were detected at the end of the prime-boost vaccination scheme. However, clinical and parasitological data obtained during the 10 month follow-up period indicated that protection was not induced by either of the six candidate vaccines. Although no direct evidence was obtained, our data suggest that live BCG may have a significant protective effect against challenge with L. infantum in dogs. PMID:19500553

  4. Development of a chimeric Plasmodium berghei strain expressing the repeat region of the P. vivax circumsporozoite protein for in vivo evaluation of vaccine efficacy.

    Science.gov (United States)

    Espinosa, Diego A; Yadava, Anjali; Angov, Evelina; Maurizio, Paul L; Ockenhouse, Christian F; Zavala, Fidel

    2013-08-01

    The development of vaccine candidates against Plasmodium vivax-the most geographically widespread human malaria species-is challenged by technical difficulties, such as the lack of in vitro culture systems and availability of animal models. Chimeric rodent Plasmodium parasites are safe and useful tools for the preclinical evaluation of new vaccine formulations. We report the successful development and characterization of chimeric Plasmodium berghei parasites bearing the type I repeat region of P. vivax circumsporozoite protein (CSP). The P. berghei-P. vivax chimeric strain develops normally in mosquitoes and produces highly infectious sporozoites that produce patent infection in mice that are exposed to the bites of as few as 3 P. berghei-P. vivax-infected mosquitoes. Using this transgenic parasite, we demonstrate that monoclonal and polyclonal antibodies against P. vivax CSP strongly inhibit parasite infection and thus support the notion that these antibodies play an important role in protective immunity. The chimeric parasites we developed represent a robust model for evaluating protective immune responses against P. vivax vaccines based on CSP. PMID:23716612

  5. Solution structure of a Plasmodium falciparum AMA-1/MSP 1 chimeric protein vaccine candidate (PfCP-2.9 for malaria

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    Jin Changwen

    2010-03-01

    Full Text Available Abstract Background The Plasmodium falciparum chimeric protein PfCP-2.9 is a promising asexual-stage malaria vaccine evaluated in clinical trials. This chimeric protein consists of two cysteine-rich domains: domain III of the apical membrane antigen 1 (AMA-1 [III] and the C-terminal region of the merozoite surface protein 1 (MSP1-19. It has been reported that the fusion of these two antigens enhanced their immunogenicity and antibody-mediated inhibition of parasite growth in vitro. Methods The 15N-labeled and 13C/15N-labeled PfCP-2.9 was produced in Pichia pastoris for nuclear magnetic resonance (NMR structure analysis. The chemical shift assignments of PfCP-2.9 were compared with those previously reported for the individual domains (i.e., PfAMA-1(III or PfMSP 1-19. The two-dimensional spectra and transverse relaxation rates (R2 of the PfMSP1-19 alone were compared with that of the PfCP-2.9. Results Confident backbone assignments were obtained for 122 out of 241 residues of PfCP-2.9. The assigned residues in PfCP-2.9 were very similar to those previously reported for the individual domains. The conformation of the PfMSP1-19 in different constructs is essentially the same. Comparison of transverse relaxation rates (R2 strongly suggests no weak interaction between the domains. Conclusions These data indicate that the fusion of AMA-1(III and MSP1-19 as chimeric protein did not change their structures, supporting the use of the chimeric protein as a potential malaria vaccine.

  6. Safety and immunogenicity of a malaria vaccine, Plasmodium falciparum AMA-1/MSP-1 chimeric protein formulated in montanide ISA 720 in healthy adults.

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    Jinhong Hu

    Full Text Available BACKGROUND: The P. falciparum chimeric protein 2.9 (PfCP-2.9 consisting of the sequences of MSP1-19 and AMA-1 (III is a malaria vaccine candidate that was found to induce inhibitory antibodies in rabbits and monkeys. This was a phase I randomized, single-blind, placebo-controlled, dose-escalation study to evaluate the safety and immunogenicity of the PfCP-2.9 formulated with a novel adjuvant Montanide ISA720. Fifty-two subjects were randomly assigned to 4 dose groups of 10 participants, each receiving the test vaccine of 20, 50, 100, or 200 microg respectively, and 1 placebo group of 12 participants receiving the adjuvant only. METHODS AND FINDINGS: The vaccine formulation was shown to be safe and well-tolerated, and none of the participants withdrew. The total incidence of local adverse events (AEs was 75%, distributed among 58% of the placebo group and 80% of those vaccinated. Among the vaccinated, 65% had events that were mild and 15% experienced moderate AEs. Almost all systemic adverse reactions observed in this study were graded as mild and required no therapy. The participants receiving the test vaccine developed detectable antibody responses which were boosted by the repeated vaccinations. Sixty percent of the vaccinated participants had high ELISA titers (>1:10,000 of antigen-specific antibodies which could also recognize native parasite proteins in an immunofluorescence assay (IFA. CONCLUSION: This study is the first clinical trial for this candidate and builds on previous investigations supporting PfCP-2.9/ISA720 as a promising blood-stage malaria vaccine. Results demonstrate safety, tolerability (particularly at the lower doses tested and immunogenicity of the formulation. Further clinical development is ongoing to explore optimizing the dose and schedule of the formulation to decrease reactogenicity without compromising immunogenicity. TRIAL REGISTRATION: Chinese State Food and Drug Administration (SFDA 2002SL0046; Controlled

  7. Exploiting the yeast L-A viral capsid for the in vivo assembly of chimeric VLPs as platform in vaccine development and foreign protein expression.

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    Frank Powilleit

    Full Text Available A novel expression system based on engineered variants of the yeast (Saccharomyces cerevisiae dsRNA virus L-A was developed allowing the in vivo assembly of chimeric virus-like particles (VLPs as a unique platform for a wide range of applications. We show that polypeptides fused to the viral capsid protein Gag self-assemble into isometric VLP chimeras carrying their cargo inside the capsid, thereby not only effectively preventing proteolytic degradation in the host cell cytosol, but also allowing the expression of a per se cytotoxic protein. Carboxyterminal extension of Gag by T cell epitopes from human cytomegalovirus pp65 resulted in the formation of hybrid VLPs that strongly activated antigen-specific CD8(+ memory T cells ex vivo. Besides being a carrier for polypeptides inducing antigen-specific immune responses in vivo, VLP chimeras were also shown to be effective in the expression and purification of (i a heterologous model protein (GFP, (ii a per se toxic protein (K28 alpha-subunit, and (iii a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A. Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production.

  8. An E2-Substituted Chimeric Pestivirus With DIVA Vaccine Properties

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; Nielsen, Jens;

    An advantage of the use of chimeric pestiviruses as modified live vaccines against classical swine fever (CSF) resides in their capacity to be manipulated to achieve the characteristics desired for safe and efficacious DIVA vaccines. We have recently generated a new chimeric virus, Riems26_E2gif...

  9. Immunogenicity of a virosomally-formulated Plasmodium falciparum GLURP-MSP3 chimeric protein-based malaria vaccine candidate in comparison to adjuvanted formulations

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    Tamborrini Marco

    2011-12-01

    Full Text Available Abstract Background In clinical trials, immunopotentiating reconstituted influenza virosomes (IRIVs have shown great potential as a versatile antigen delivery platform for synthetic peptides derived from Plasmodium falciparum antigens. This study describes the immunogenicity of a virosomally-formulated recombinant fusion protein comprising domains of the two malaria vaccine candidate antigens MSP3 and GLURP. Methods The highly purified recombinant protein GMZ2 was coupled to phosphatidylethanolamine and the conjugates incorporated into the membrane of IRIVs. The immunogenicity of this adjuvant-free virosomal formulation was compared to GMZ2 formulated with the adjuvants Montanide ISA 720 and Alum in three mouse strains with different genetic backgrounds. Results Intramuscular injections of all three candidate vaccine formulations induced GMZ2-specific antibody responses in all mice tested. In general, the humoral immune response in outbred NMRI mice was stronger than that in inbred BALB/c and C57BL/6 mice. ELISA with the recombinant antigens demonstrated immunodominance of the GLURP component over the MSP3 component. However, compared to the Al(OH3-adjuvanted formulation the two other formulations elicited in NMRI mice a larger proportion of anti-MSP3 antibodies. Analyses of the induced GMZ2-specific IgG subclass profiles showed for all three formulations a predominance of the IgG1 isotype. Immune sera against all three formulations exhibited cross-reactivity with in vitro cultivated blood-stage parasites. Immunofluorescence and immunoblot competition experiments showed that both components of the hybrid protein induced IgG cross-reactive with the corresponding native proteins. Conclusion A virosomal formulation of the chimeric protein GMZ2 induced P. falciparum blood stage parasite cross-reactive IgG responses specific for both MSP3 and GLURP. GMZ2 thus represents a candidate component suitable for inclusion into a multi-valent virosomal

  10. Virulence, immunogenicity and vaccine properties of a novel chimeric pestivirus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; Reimann, Ilona;

    2007-01-01

    avirulent and neither chimeric virus nor viral RNA was detected in serum after vaccination. The safety of the vaccine was tested by horizontal transmission to sentinel pigs, which remained uninfected. The vaccine efficacy was examined by challenge infection with classical swine fever virus (CSFV) Eystrup...

  11. Protective and immunological behavior of chimeric yellow fever dengue vaccine.

    Science.gov (United States)

    Halstead, Scott B; Russell, Philip K

    2016-03-29

    Clinical observations from the third year of the Sanofi Pasteur chimeric yellow fever dengue tetravalent vaccine (CYD) trials document both protection and vaccination-enhanced dengue disease among vaccine recipients. Children who were 5 years-old or younger when vaccinated experienced a DENV disease resulting in hospitalization at 5 times the rate of controls. On closer inspection, hospitalized cases among vaccinated seropositives, those at highest risk to hospitalized disease accompanying a dengue virus (DENV) infection, were greatly reduced by vaccination. But, seronegative individuals of all ages after being vaccinated were only modestly protected from mild to moderate disease throughout the entire observation period despite developing neutralizing antibodies at high rates. Applying a simple epidemiological model to the data, vaccinated seronegative individuals of all ages were at increased risk of developing hospitalized disease during a subsequent wild type DENV infection. The etiology of disease in placebo and vaccinated children resulting in hospitalization during a DENV infection, while clinically similar are of different origin. The implications of the observed mixture of DENV protection and enhanced disease in CYD vaccinees are discussed. PMID:26873054

  12. DIVA vaccine properties of the live chimeric pestivirus strain CP7_E2gif

    DEFF Research Database (Denmark)

    von Rosen, Tanya; Rangelova, Desislava Yordanova; Nielsen, Jens;

    2014-01-01

    Live modified vaccines to protect against classical swine fever virus (CSFV), based on chimeric pestiviruses, have been developed to enable serological Differentiation of Infected from Vaccinated Animals (DIVA). In this context, the chimeric virus CP7_E2gif vaccine candidate is unique as it does...

  13. Immunogenicity of a virosomally-formulated Plasmodium falciparum GLURP-MSP3 chimeric protein-based malaria vaccine candidate in comparison to adjuvanted formulations

    DEFF Research Database (Denmark)

    Tamborrini, Marco; Stoffel, Sabine A; Westerfeld, Nicole; Amacker, Mario; Theisen, Michael; Zurbriggen, Rinaldo; Pluschke, Gerd

    2011-01-01

    In clinical trials, immunopotentiating reconstituted influenza virosomes (IRIVs) have shown great potential as a versatile antigen delivery platform for synthetic peptides derived from Plasmodium falciparum antigens. This study describes the immunogenicity of a virosomally-formulated recombinant ...... fusion protein comprising domains of the two malaria vaccine candidate antigens MSP3 and GLURP....

  14. Antigenic properties of a transport-competent influenza HA/HIV Env chimeric protein

    International Nuclear Information System (INIS)

    The transmembrane subunit (gp41) of the HIV Env glycoprotein contains conserved neutralizing epitopes which are not well-exposed in wild-type HIV Env proteins. To enhance the exposure of these epitopes, a chimeric protein, HA/gp41, in which the gp41 of HIV-1 89.6 envelope protein was fused to the C-terminus of the HA1 subunit of the influenza HA protein, was constructed. Characterization of protein expression showed that the HA/gp41 chimeric proteins were expressed on cell surfaces and formed trimeric oligomers, as found in the HIV Env as well as influenza HA proteins. In addition, the HA/gp41 chimeric protein expressed on the cell surface can also be cleaved into 2 subunits by trypsin treatment, similar to the influenza HA. Moreover, the HA/gp41 chimeric protein was found to maintain a pre-fusion conformation. Interestingly, the HA/gp41 chimeric proteins on cell surfaces exhibited increased reactivity to monoclonal antibodies against the HIV Env gp41 subunit compared with the HIV-1 envelope protein, including the two broadly neutralizing monoclonal antibodies 2F5 and 4E10. Immunization of mice with a DNA vaccine expressing the HA/gp41 chimeric protein induced antibodies against the HIV gp41 protein and these antibodies exhibit neutralizing activity against infection by an HIV SF162 pseudovirus. These results demonstrate that the construction of such chimeric proteins can provide enhanced exposure of conserved epitopes in the HIV Env gp41 and may represent a novel vaccine design strategy for inducing broadly neutralizing antibodies against HIV

  15. CHIMERIC WEST NILE/DENGUE VIRUS VACCINE CANDIDATE: PRECLINICAL EVALUATION IN MICE, GEESE, AND MONKEYS FOR SAFETY AND IMMUNOGENICITY

    Science.gov (United States)

    A live attenuated virus vaccine is being developed to protect against West Nile virus (WN) disease in humans. Previously, it was found that chimeric West Nile/dengue viruses (WN/DEN4 and WN/DEN4-delta-30) bearing the membrane precursor and envelope protein genes of WN on a backbone of dengue type 4 ...

  16. Murine immune responses to a Plasmodium vivax-derived chimeric recombinant protein expressed in Brassica napus

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    Chung Nam-Jun

    2011-04-01

    Full Text Available Abstract Background To develop a plant-based vaccine against Plasmodium vivax, two P. vivax candidate proteins were chosen. First, the merozoite surface protein-1 (MSP-1, a major asexual blood stage antigen that is currently considered a strong vaccine candidate. Second, the circumsporozoite protein (CSP, a component of sporozoites that contains a B-cell epitope. Methods A synthetic chimeric recombinant 516 bp gene encoding containing PvMSP-1, a Pro-Gly linker motif, and PvCSP was synthesized; the gene, named MLC, encoded a total of 172 amino acids. The recombinant gene was modified with regard to codon usage to optimize gene expression in Brassica napus. The Ti plasmid inducible gene transfer system was used for MLC chimeric recombinant gene expression in B. napus. Gene expression was confirmed by polymerase chain reaction (PCR, beta-glucuronidase reporter gene (GUS assay, and Western blot. Results The MLC chimeric recombinant protein expressed in B. napus had a molecular weight of approximately 25 kDa. It exhibited a clinical sensitivity of 84.21% (n = 38 and a clinical specificity of 100% (n = 24 as assessed by enzyme-linked immunosorbent assay (ELISA. Oral immunization of BALB/c mice with MLC chimeric recombinant protein successfully induced antigen-specific IgG1 production. Additionally, the Th1-related cytokines IL-12 (p40, TNF, and IFN-γ were significantly increased in the spleens of the BALB/c mice. Conclusions The chimeric MLC recombinant protein produced in B. napus has potential as both as an antigen for diagnosis and as a valuable vaccine candidate for oral immunization against vivax malaria.

  17. Enhanced protective efficacy of a chimeric form of the schistosomiasis vaccine antigen Sm-TSP-2.

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    Mark S Pearson

    Full Text Available The large extracellular loop of the Schistosoma mansoni tetraspanin, Sm-TSP-2, when fused to a thioredoxin partner and formulated with Freund's adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. Moreover, Sm-TSP-2 is uniquely recognised by IgG(1 and IgG(3 from putatively resistant individuals resident in S. mansoni endemic areas in Brazil. In the present study, we expressed Sm-TSP-2 at high yield and in soluble form in E. coli without the need for a solubility enhancing fusion partner. We also expressed in E. coli a chimera called Sm-TSP-2/5B, which consisted of Sm-TSP-2 fused to the immunogenic 5B region of the hookworm aspartic protease and vaccine antigen, Na-APR-1. Sm-TSP-2 formulated with alum/CpG showed significant reductions in adult worm and liver egg burdens in two separate murine schistosomiasis challenge studies. Sm-TSP-2/5B afforded significantly greater protection than Sm-TSP-2 alone when both antigens were formulated with alum/CpG. The enhanced protection obtained with the chimeric fusion protein was associated with increased production of anti-Sm-TSP-2 antibodies and IL-4, IL-10 and IFN-γ from spleen cells of vaccinated animals. Sera from 666 individuals from Brazil who were infected with S. mansoni were screened for potentially deleterious IgE responses to Sm-TSP-2. Anti-Sm-TSP-2 IgE to this protein was not detected (also shown previously for Na-APR-1, suggesting that the chimeric antigen Sm-TSP-2/5B could be used to safely and effectively vaccinate people in areas where schistosomes and hookworms are endemic.

  18. Enhanced protective efficacy of a chimeric form of the schistosomiasis vaccine antigen Sm-TSP-2.

    Science.gov (United States)

    Pearson, Mark S; Pickering, Darren A; McSorley, Henry J; Bethony, Jeffrey M; Tribolet, Leon; Dougall, Annette M; Hotez, Peter J; Loukas, Alex

    2012-01-01

    The large extracellular loop of the Schistosoma mansoni tetraspanin, Sm-TSP-2, when fused to a thioredoxin partner and formulated with Freund's adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. Moreover, Sm-TSP-2 is uniquely recognised by IgG(1) and IgG(3) from putatively resistant individuals resident in S. mansoni endemic areas in Brazil. In the present study, we expressed Sm-TSP-2 at high yield and in soluble form in E. coli without the need for a solubility enhancing fusion partner. We also expressed in E. coli a chimera called Sm-TSP-2/5B, which consisted of Sm-TSP-2 fused to the immunogenic 5B region of the hookworm aspartic protease and vaccine antigen, Na-APR-1. Sm-TSP-2 formulated with alum/CpG showed significant reductions in adult worm and liver egg burdens in two separate murine schistosomiasis challenge studies. Sm-TSP-2/5B afforded significantly greater protection than Sm-TSP-2 alone when both antigens were formulated with alum/CpG. The enhanced protection obtained with the chimeric fusion protein was associated with increased production of anti-Sm-TSP-2 antibodies and IL-4, IL-10 and IFN-γ from spleen cells of vaccinated animals. Sera from 666 individuals from Brazil who were infected with S. mansoni were screened for potentially deleterious IgE responses to Sm-TSP-2. Anti-Sm-TSP-2 IgE to this protein was not detected (also shown previously for Na-APR-1), suggesting that the chimeric antigen Sm-TSP-2/5B could be used to safely and effectively vaccinate people in areas where schistosomes and hookworms are endemic. PMID:22428079

  19. Chimeric hepatitis B virus (HBV)/hepatitis C virus (HCV) subviral envelope particles induce efficient anti-HCV antibody production in animals pre-immunized with HBV vaccine.

    Science.gov (United States)

    Beaumont, Elodie; Roingeard, Philippe

    2015-02-18

    The development of an effective, affordable prophylactic vaccine against hepatitis C virus (HCV) remains a medical priority. The recently described chimeric HBV-HCV subviral envelope particles could potentially be used for this purpose, as they could be produced by industrial procedures adapted from those established for the hepatitis B virus (HBV) vaccine. We show here, in an animal model, that pre-existing immunity acquired through HBV vaccination does not influence the immunogenicity of the HCV E2 protein presented by these chimeric particles. Thus, these chimeric HBV-HCV subviral envelope particles could potentially be used as a booster in individuals previously vaccinated against HBV, to induce protective immunity to HCV. PMID:25596457

  20. Chimeric Epitope Vaccine from Multistage Antigens for Lymphatic Filariasis.

    Science.gov (United States)

    Anugraha, G; Madhumathi, J; Prince, P R; Prita, P J Jeya; Khatri, V K; Amdare, N P; Reddy, M V R; Kaliraj, P

    2015-10-01

    Lymphatic filariasis, a mosquito-borne parasitic disease, affects more than 120 million people worldwide. Vaccination for filariasis by targeting different stages of the parasite will be a boon to the existing MDA efforts of WHO which required repeated administration of the drug to reduce the infection level and sustained transmission. Onset of a filaria-specific immune response achieved through antigen vaccines can act synergistically with these drugs to enhance the parasite killing. Multi-epitope vaccine approach has been proved to be successful against several parasitic diseases as it overcomes the limitations associated with the whole antigen vaccines. Earlier results from our group suggested the protective efficacy of multi-epitope vaccine comprising two immunodominant epitopes from Brugia malayi antioxidant thioredoxin (TRX), several epitopes from transglutaminase (TGA) and abundant larval transcript-2 (ALT-2). In this study, the prophylactic efficacy of the filarial epitope protein (FEP), a chimera of selective epitopes identified from our earlier study, was tested in a murine model (jird) of filariasis with L3 larvae. FEP conferred a significantly (P < 0.0001) high protection (69.5%) over the control in jirds. We also observed that the multi-epitope recombinant construct (FEP) induces multiple types of protective immune responses, thus ensuring the successful elimination of the parasite; this poses FEP as a potential vaccine candidate. PMID:26179420

  1. Construction and analysis of variants of a polyvalent Lyme disease vaccine: approaches for improving the immune response to chimeric vaccinogens

    OpenAIRE

    Earnhart, Christopher G.; Marconi, Richard T.

    2007-01-01

    There is currently no Lyme disease vaccine commercially available for use in humans. Outer surface protein C (OspC) of the Borrelia has been widely investigated as a potential vaccinogen. At least 38 OspC types have been defined. While the antibody response to OspC is protective, the range of protection is narrow due to the localization of protective epitopes within OspC type-specific domains. To develop a broadly protective vaccine, we previously constructed a tetravalent chimeric vaccinogen...

  2. In Silico Design of a Chimeric Protein Containing Antigenic Fragments of Helicobacter pylori; A Bioinformatic Approach

    Science.gov (United States)

    Mohammad, Nazanin; Karsabet, Mehrnaz Taghipour; Amani, Jafar; Ardjmand, Abolfazl; Zadeh, Mohsen Razavi; Gholi, Mohammad Khalifeh; Saffari, Mahmood; Ghasemi, Amir

    2016-01-01

    Helicobacter pylori is a global health problem which has encouraged scientists to find new ways to diagnose, immunize and eradicate the H. pylori infection. In silico studies are a promising approach to design new chimeric antigen having the immunogenic potential of several antigens. In order to obtain such benefit in H. pylori vaccine study, a chimeric gene containing four fragments of FliD sequence (1-600 bp), UreB (327-334 bp),VacA (744-805 bp) and CagL(51-100 bp) which have a high density of B- and T-cell epitopes was designed. The secondary and tertiary structures of the chimeric protein and other properties such as stability, solubility and antigenicity were analyzed. The in silico results showed that after optimizing for the purpose of expression in Escherichia coli BL21, the solubility and antigenicity of the construct fragments were highly retained. Most regions of the chimeric protein were found to have a high antigenic propensity and surface accessibility. These results would be useful in animal model application and accounted for the development of an epitope-based vaccine against the H. pylori. PMID:27335622

  3. Custom-engineered chimeric foot-and-mouth disease vaccine elicits protective immune responses in pigs

    Science.gov (United States)

    Chimeric foot-and-mouth disease viruses (FMDV) of which the antigenic properties can be readily manipulated is a potentially powerful approach in the control of foot-and-mouth disease (FMD) in sub-Saharan Africa. FMD vaccine application is complicated by the extensive variability of the South Africa...

  4. Chimeric DNA Vaccines against ErbB2+ Carcinomas: From Mice to Humans

    International Nuclear Information System (INIS)

    DNA vaccination exploits a relatively simple and flexible technique to generate an immune response against microbial and tumor-associated antigens (TAAs). Its effectiveness is enhanced by the application of an electrical shock in the area of plasmid injection (electroporation). In our studies we exploited a sophisticated electroporation device approved for clinical use (Cliniporator, IGEA, Carpi, Italy). As the target antigen is an additional factor that dramatically modulates the efficacy of a vaccine, we selected ErbB2 receptor as a target since it is an ideal oncoantigen. It is overexpressed on the cell membrane by several carcinomas for which it plays an essential role in driving their progression. Most oncoantigens are self-tolerated molecules. To circumvent immune tolerance we generated two plasmids (RHuT and HuRT) coding for chimeric rat/human ErbB2 proteins. Their immunogenicity was compared in wild type mice naturally tolerant for mouse ErbB2, and in transgenic mice that are also tolerant for rat or human ErbB2. In several of these mice, RHuT and HuRT elicited a stronger anti-tumor response than plasmids coding for fully human or fully rat ErbB2. The ability of heterologous moiety to blunt immune tolerance could be exploited to elicit a significant immune response in patients. A clinical trial to delay the recurrence of ErbB2+ carcinomas of the oral cavity, oropharynx and hypopharynx is awaiting the approval of the Italian authorities

  5. Chimeric DNA Vaccines against ErbB2{sup +} Carcinomas: From Mice to Humans

    Energy Technology Data Exchange (ETDEWEB)

    Quaglino, Elena; Riccardo, Federica; Macagno, Marco; Bandini, Silvio; Cojoca, Rodica; Ercole, Elisabetta [Molecular Biotechnology Center, Department of Clinical and Biological Sciences, University of Turin, 10126 Turin (Italy); Amici, Augusto [Department of Molecular Cellular and Animal Biology, University of Camerino, 62032 Camerino (Italy); Cavallo, Federica, E-mail: federica.cavallo@unito.it [2 Department of Molecular Cellular and Animal Biology, University of Camerino, 62032 Camerino (Italy)

    2011-08-10

    DNA vaccination exploits a relatively simple and flexible technique to generate an immune response against microbial and tumor-associated antigens (TAAs). Its effectiveness is enhanced by the application of an electrical shock in the area of plasmid injection (electroporation). In our studies we exploited a sophisticated electroporation device approved for clinical use (Cliniporator, IGEA, Carpi, Italy). As the target antigen is an additional factor that dramatically modulates the efficacy of a vaccine, we selected ErbB2 receptor as a target since it is an ideal oncoantigen. It is overexpressed on the cell membrane by several carcinomas for which it plays an essential role in driving their progression. Most oncoantigens are self-tolerated molecules. To circumvent immune tolerance we generated two plasmids (RHuT and HuRT) coding for chimeric rat/human ErbB2 proteins. Their immunogenicity was compared in wild type mice naturally tolerant for mouse ErbB2, and in transgenic mice that are also tolerant for rat or human ErbB2. In several of these mice, RHuT and HuRT elicited a stronger anti-tumor response than plasmids coding for fully human or fully rat ErbB2. The ability of heterologous moiety to blunt immune tolerance could be exploited to elicit a significant immune response in patients. A clinical trial to delay the recurrence of ErbB2{sup +} carcinomas of the oral cavity, oropharynx and hypopharynx is awaiting the approval of the Italian authorities.

  6. Structural Characterization by NMR of a Double Phosphorylated Chimeric Peptide Vaccine for Treatment of Alzheimer’s Disease

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    Stefan Berger

    2013-04-01

    Full Text Available Rational design of peptide vaccines becomes important for the treatment of some diseases such as Alzheimer’s disease (AD and related disorders. In this study, as part of a larger effort to explore correlations of structure and activity, we attempt to characterize the doubly phosphorylated chimeric peptide vaccine targeting a hyperphosphorylated epitope of the Tau protein. The 28-mer linear chimeric peptide consists of the double phosphorylated B cell epitope Tau229-237[pThr231/pSer235] and the immunomodulatory T cell epitope Ag85B241-255 originating from the well-known antigen Ag85B of the Mycobacterium tuberculosis, linked by a four amino acid sequence -GPSL-. NMR chemical shift analysis of our construct demonstrated that the synthesized peptide is essentially unfolded with a tendency to form a β-turn due to the linker. In conclusion, the -GPSL- unit presumably connects the two parts of the vaccine without transferring any structural information from one part to the other. Therefore, the double phosphorylated epitope of the Tau peptide is flexible and accessible.

  7. Molecularly engineered live-attenuated chimeric West Nile/dengue virus vaccines protect rhesus monkeys from West Nile virus

    International Nuclear Information System (INIS)

    Two molecularly engineered, live-attenuated West Nile virus (WN) vaccine candidates were highly attenuated and protective in rhesus monkeys. The vaccine candidates are chimeric viruses (designated WN/DEN4) bearing the membrane precursor and envelope protein genes of WN on a backbone of dengue 4 virus (DEN4) with or without a deletion of 30 nucleotides (Δ30) in the 3' noncoding region of DEN4. Viremia in WN/DEN4- infected monkeys was reduced 100-fold compared to that in WN- or DEN4-infected monkeys. WN/DEN4-3'Δ30 did not cause detectable viremia, indicating that it is even more attenuated for monkeys. These findings indicate that chimerization itself and the presence of the Δ30 mutation independently contribute to the attenuation phenotype for nonhuman primates. Despite their high level of attenuation in monkeys, the chimeras induced a moderate-to-high titer of neutralizing antibodies and prevented viremia in monkeys challenged with WN. The more attenuated vaccine candidate, WN/DEN4-3'Δ30, will be evaluated first in our initial clinical studies

  8. Preclinical and Clinical Development of a YFV 17 D-Based Chimeric Vaccine against West Nile Virus

    Directory of Open Access Journals (Sweden)

    Gustavo H. Dayan

    2013-12-01

    Full Text Available Substantial success has been achieved in the development and implementation of West Nile (WN vaccines for horses; however, no human WN vaccines are approved. This review focuses on the construction, pre-clinical and clinical characterization of ChimeriVax-WN02 for humans, a live chimeric vaccine composed of a yellow fever (YF 17D virus in which the prM-E envelope protein genes are replaced with the corresponding genes of the WN NY99 virus. Pre-clinical studies demonstrated that ChimeriVax-WN02 was significantly less neurovirulent than YF 17D in mice and rhesus and cynomolgus monkeys. The vaccine elicited neutralizing antibody titers after inoculation in hamsters and monkeys and protected immunized animals from lethal challenge including intracerebral inoculation of high dose of WN NY99 virus. Safety, viremia and immunogenicity of ChimeriVax-WN02 were assessed in one phase I study and in two phase II clinical trials. No safety signals were detected in the three clinical trials with no remarkable differences in incidence of adverse events (AEs between vaccine and placebo recipients. Viremia was transient and the mean viremia levels were low. The vaccine elicited strong and durable neutralizing antibody and cytotoxic T cell responses. WN epidemiology impedes a classical licensure pathway; therefore, innovative licensure strategies should be explored.

  9. Chimeric Proteins to Detect DNA Damage and Mismatches

    Energy Technology Data Exchange (ETDEWEB)

    McCutchen-Maloney, S; Malfatti, M; Robbins, K M

    2002-01-14

    The goal of this project was to develop chimeric proteins composed of a DNA mismatch or damage binding protein and a nuclease, as well as methods to detect DNA mismatches and damage. We accomplished this through protein engineering based on using polymerase chain reactions (PCRs) to create chimeras with novel functions for damage and mismatch detection. This project addressed fundamental questions relating to disease susceptibility and radiation-induced damage in cells. It also supported and enhanced LLNL's competency in the emerging field of proteomics. In nature, DNA is constantly being subjected to damaging agents such as exposure to ultraviolet (UV) radiation and various environmental and dietary carcinogens. If DNA damage is not repaired however, mutations in DNA result that can eventually manifest in cancer and other diseases. In addition to damage-induced DNA mutations, single nucleotide polymorphisms (SNPs), which are variations in the genetic sequence between individuals, may predispose some to disease. As a result of the Human Genome Project, the integrity of a person's DNA can now be monitored. Therefore, methods to detect DNA damage, mutations, and SNPs are useful not only in basic research but also in the health and biotechnology industries. Current methods of detection often use radioactive labeling and rely on expensive instrumentation that is not readily available in many research settings. Our methods to detect DNA damage and mismatches employ simple gel electrophoresis and flow cytometry, thereby alleviating the need for radioactive labeling and expensive equipment. In FY2001, we explored SNP detection by developing methods based on the ability of the chimeric proteins to detect mismatches. Using multiplex assays with flow cytometry and fluorescent beads to which the DNA substrates where attached, we showed that several of the chimeras possess greater affinity for damaged and mismatched DNA than for native DNA. This affinity was

  10. Cross-presentation of HCMV chimeric protein enables generation and measurement of polyclonal T cells.

    Science.gov (United States)

    Nguyen, Thi H O; Sullivan, Lucy C; Kotsimbos, Tom C; Schwarer, Anthony P; Mifsud, Nicole A

    2010-08-01

    CD8(+) T cell immunity has a critical function in controlling human cytomegalovirus (HCMV) infection. In immunocompromized individuals, HCMV reactivation or disease can lead to increased morbidity and mortality, particularly in transplant recipients. In this setting, adoptive transfer of HCMV-specific CD8(+) T cells is a promising vaccine strategy to restore viral immunity, with most clinical approaches focussing on the use of peptides for the generation of single epitope-specific CD8(+) T cells. We show that using an IE1-pp65 chimeric protein as the antigen source promotes effective cross-presentation, by monocyte-derived dendritic cells (MoDCs), to generate polyclonal CD8(+) T cell epitopes. By exploring human leukocyte antigen (HLA)-restricted immunodominance hierarchies both within and across two immunodominant proteins, we show that HLA-B7 epitopes elicit higher CD8(+) T cell responses compared with HLA-A1, -A2 or -B8. This study provides important evidence highlighting both the efficacy of the IE1-pp65 chimeric protein and the importance of immunodominance in designing future therapeutic vaccines. PMID:20195281

  11. Incorporation of chimeric HIV-SIV-Env and modified HIV-Env proteins into HIV pseudovirions

    International Nuclear Information System (INIS)

    Low level incorporation of the viral glycoprotein (Env) into human immunodeficiency virus (HIV) particles is a major drawback for vaccine strategies against HIV/AIDS in which HIV particles are used as immunogen. Within this study, we have examined two strategies aimed at achieving higher levels of Env incorporation into non-infectious pseudovirions (PVs). First, we have generated chimeric HIV/SIV Env proteins containing the truncated C-terminal tail region of simian immunodeficiency virus (SIV)mac239-Env767stop, which mediates strongly increased incorporation of SIV-Env into SIV particles. In a second strategy, we have employed a truncated HIV-Env protein (Env-Tr752N750K) which we have previously demonstrated to be incorporated into HIV virions, generated in infected T-cells, to a higher level than that of Wt-HIV-Env. Although the chimeric HIV/SIV Env proteins were expressed at the cell surface and induced increased levels of cell-cell fusion in comparison to Wt-HIV-Env, they did not exhibit increased incorporation into either HIV-PVs or SIV-PVs. Only Env-Tr752N750K exhibited significantly higher (threefold) levels of incorporation into HIV-PVs, an improvement, which, although not dramatic, is worthwhile for the large-scale preparation of non-infectious PVs for vaccine studies aimed at inducing Env humoral responses

  12. A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen

    OpenAIRE

    Vijayan, Aneesh; García-Arriaza, Juan; C. Raman, Suresh; Conesa, José Javier; Chichón, Francisco Javier; Santiago, César; Sorzano, Carlos Óscar S.; Carrascosa, José L.; Esteban, Mariano

    2015-01-01

    In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size excl...

  13. Construction and analysis of variants of a polyvalent Lyme disease vaccine: approaches for improving the immune response to chimeric vaccinogens.

    Science.gov (United States)

    Earnhart, Christopher G; Marconi, Richard T

    2007-04-30

    There is currently no Lyme disease vaccine commercially available for use in humans. Outer surface protein C (OspC) of the Borrelia has been widely investigated as a potential vaccinogen. At least 38 OspC types have been defined. While the antibody response to OspC is protective, the range of protection is narrow due to the localization of protective epitopes within OspC type-specific domains. To develop a broadly protective vaccine, we previously constructed a tetravalent chimeric vaccinogen containing epitopes from OspC types A, B, K, and D. While this construct elicited bactericidal antibody against strains bearing each of the four OspC types, its solubility was low, and decreasing IgG titer to epitopes near the C-terminus of the construct was observed. In this report, construct solubility and immunogenicity were increased by dialysis against an Arg/Glu buffer. We also demonstrate the immunogenicity of the construct in alum. To further optimize epitope-specific immune responses, several constructs were generated with differing epitope organization or with putative C-terminal protective motifs. Analyses of murine antibody titers and isotype profiles induced by these constructs revealed that while the C-terminal tags did not enhance antibody titer, specific epitope reorganization and reiteration did. These analyses provide important information that can be exploited in the development of chimeric vaccinogens in general. PMID:17239505

  14. Chimeric Foot-and-Mouth Disease Viruses: Evaluation of Their Efficacy as Potential Marker Vaccines in Cattle

    Science.gov (United States)

    Previous work in swine has demonstrated that full protection against Foot-and-Mouth Disease (FMD) can be achieved following vaccination with chimeric Foot-and-Mouth Disease Virus (FMDV) vaccines, whereby the VP1 G-H loop has been substituted with a non-homologous alternative. If proven to be effect...

  15. PRELIMINARY STUDY OF A NOVEL HUMAN PAPILLOMAVIRUS TYPE 16 L1/E6-E7 CHIMERIC RECOMBINANT DNA VACCINE

    Institute of Scientific and Technical Information of China (English)

    郑瑾; 马军; 张福萍; 杨筱凤; 董小平; 司履生; 王一理

    2004-01-01

    Objective Preparations of HPV16 L1/E6 and L1/E7 prophylactic and therapeutic DNA vaccines. Methods The nucleotides within HPV16 E6 and E7 genes, which are responsible for viral transforming activity, were mutated by mage primer site-directed mutagenesis method. The correctly mutated E6 and E7 fragments were separately cloned into an eukaryotic expression vector pVAX1, together with HPV16 L1 gene, generating chimeric recombinants plasmids 1MpVAX1-L1E6, 2MpVAX1-L1E6, 1MpVAX1-L1E7, 2MpVAX1-L1E7 and 3MpVAX1-L1E7. CHO cells were transiently transfected with the individual DNA vaccines by calcium phosphate method. Target protein expressions in the extracts of the transfected cell lines were measured by ELISA and immunohistochemistry, with HPV16 L1 and E6 specific monoclonal antibodies. Results ELISA assays showed the P/N ratios in the cell extracts transfected with L1E6 and L1E7 plasmids were more than 2.1. Immunohistochemistry revealed brownish precipitant signal in cytoplasm and nuclei of the transfected cells. Conclusion Successful constructions of prophylactic and therapeutic DNA vaccine plasmids lay solid foundation for future animal experiment and clinical trial.

  16. Chimeras taking shape: potential functions of proteins encoded by chimeric RNA transcripts.

    Science.gov (United States)

    Frenkel-Morgenstern, Milana; Lacroix, Vincent; Ezkurdia, Iakes; Levin, Yishai; Gabashvili, Alexandra; Prilusky, Jaime; Del Pozo, Angela; Tress, Michael; Johnson, Rory; Guigo, Roderic; Valencia, Alfonso

    2012-07-01

    Chimeric RNAs comprise exons from two or more different genes and have the potential to encode novel proteins that alter cellular phenotypes. To date, numerous putative chimeric transcripts have been identified among the ESTs isolated from several organisms and using high throughput RNA sequencing. The few corresponding protein products that have been characterized mostly result from chromosomal translocations and are associated with cancer. Here, we systematically establish that some of the putative chimeric transcripts are genuinely expressed in human cells. Using high throughput RNA sequencing, mass spectrometry experimental data, and functional annotation, we studied 7424 putative human chimeric RNAs. We confirmed the expression of 175 chimeric RNAs in 16 human tissues, with an abundance varying from 0.06 to 17 RPKM (Reads Per Kilobase per Million mapped reads). We show that these chimeric RNAs are significantly more tissue-specific than non-chimeric transcripts. Moreover, we present evidence that chimeras tend to incorporate highly expressed genes. Despite the low expression level of most chimeric RNAs, we show that 12 novel chimeras are translated into proteins detectable in multiple shotgun mass spectrometry experiments. Furthermore, we confirm the expression of three novel chimeric proteins using targeted mass spectrometry. Finally, based on our functional annotation of exon organization and preserved domains, we discuss the potential features of chimeric proteins with illustrative examples and suggest that chimeras significantly exploit signal peptides and transmembrane domains, which can alter the cellular localization of cognate proteins. Taken together, these findings establish that some chimeric RNAs are translated into potentially functional proteins in humans. PMID:22588898

  17. A recombinant, chimeric tetravalent dengue vaccine candidate based on a dengue virus serotype 2 backbone.

    Science.gov (United States)

    Osorio, Jorge E; Wallace, Derek; Stinchcomb, Dan T

    2016-04-01

    Dengue fever is caused by infection with one of four dengue virus (DENV) serotypes (DENV-1-4), necessitating tetravalent dengue vaccines that can induce protection against all four DENV. Takeda's live attenuated tetravalent dengue vaccine candidate (TDV) comprises an attenuated DENV-2 strain plus chimeric viruses containing the prM and E genes of DENV-1, -3 and -4 cloned into the attenuated DENV-2 'backbone'. In Phase 1 and 2 studies, TDV was well tolerated by children and adults aged 1.5-45 years, irrespective of prior dengue exposure; mild injection-site symptoms were the most common adverse events. TDV induced neutralizing antibody responses and seroconversion to all four DENV as well as cross-reactive T cell-mediated responses that may be necessary for broad protection against dengue fever. PMID:26635182

  18. Immunogenicity of IMS 1113 plus soluble subunit and chimeric proteins containing Mycoplasma hyopneumoniae P97 C-terminal repeat regions.

    Science.gov (United States)

    Barate, Abhijit K; Cho, Youngjae; Truong, Quang Lam; Hahn, Tae-Wook

    2014-03-01

    The surface adhesin P97 mediates the adherence of Mycoplasma hyopneumoniae to swine cilia. Two reiterated repeats R1 and R2 are located at the C-terminus of P97. The purpose of this study was to evaluate the immunogenicity of Montanide adjuvant IMS 1113 plus soluble subunit proteins rR1, rR1R2 and their chimeric forms coupled with B subunit of the heat-labile enterotoxin of Escherichia coli (LTB). Each recombinant protein in this study was capable of eliciting anti-R1 specific humoral antibodies (IgG), mucosal antibodies (IgG and IgA) and IFN-γ production. The chimeric protein rLTBR1R2 elicited the quickest humoral antibody response among the recombinant proteins. Serum and bronchoalveolar lavage analysis revealed that each recombinant protein was capable of inducing both Th1 and Th2 responses. Importantly, all of the proteins induced an anti-R1-specific Th2-biased response in both humoral and mucosal compartments, similar to the response observed in a natural infection or vaccination process. These observations indicate that rR1, rR1R2, rLTBR1 and rLTBR1R2 with IMS 1113 might represent a promising subunit vaccine strategy against porcine enzootic pneumonia in pigs. PMID:24461070

  19. Ag85A/ESAT-6 chimeric DNA vaccine induces an adverse response in tuberculosis-infected mice

    Science.gov (United States)

    Liang, Yan; Bai, Xuejuang; Zhang, Junxian; Song, Jingying; Yang, Yourong; Yu, Qi; Li, Ning; Wu, Xueqiong

    2016-01-01

    The Mycobacterium tuberculosis (M. tb) antigens encoded by the 6 kDa early secretory antigenic target (esat-6) and antigen 85A (ag85a) genes are known to exert protective effects against tuberculosis in animal models. In addition, these antigens represent vaccine components that were tested in early human clinical trials. In the present study, a chimeric DNA vaccine was constructed that contained two copies of the esat-6 gene inserted into the ag85a gene from M. tb. BALB/c mice were treated with this chimeric vaccine following infection with either M. tb H37Rv or a clinical multi drug resistant tuberculosis isolate. Treatment of both groups of mice with the chimeric vaccine resulted in accelerated mortality. These findings are in contrast with previous results, which indicated that DNA vaccines expressing the individual antigens were either beneficial or at least not harmful. The results of the present study suggested that the ESAT-6 antigen is not suitable for inclusion in therapeutic vaccines. PMID:27279275

  20. Immunogenicity of a chimeric peptide corresponding to T helper and B cell epitopes of the Chlamydia trachomatis major outer membrane protein

    OpenAIRE

    1992-01-01

    The immunogenicity of a chimeric T/B cell peptide corresponding to antigenically characterized epitopes of the Chlamydia trachomatis major outer membrane protein (MOMP) was studied in mice to further define its potential use in the development of a subunit vaccine in preventing blinding trachoma in humans. The chimeric peptide, designated A8-VDI, corresponds to a conserved MOMP T helper (Th) cell epitope(s) (A8, residues 106-130) and serovar A VDI (residues 66-80), which contains the serovar-...

  1. Safety and immunogenicity of a live attenuated Japanese encephalitis chimeric virus vaccine (IMOJEV®) in children.

    Science.gov (United States)

    Chokephaibulkit, K; Houillon, G; Feroldi, E; Bouckenooghe, A

    2016-02-01

    JE-CV (IMOJEV®, Sanofi Pasteur, France) is a live attenuated virus vaccine constructed by inserting coding sequences of the prM and E structural proteins of the Japanese encephalitis SA14-14-2 virus into the genome of yellow fever 17D virus. Primary immunization with JE-CV requires a single dose of the vaccine. This article reviews clinical trials of JE-CV in children aged up to 6 years conducted in countries across South-East Asia. Strong and persistent antibody responses were observed after single primary and booster doses, with 97% of children seroprotected up to five years after booster vaccination. Models of long-term antibody persistence predict a median duration of protection of approximately 30 years after a booster dose. The safety and reactogenicity profiles of JE-CV primary and booster doses are comparable to other widely used childhood vaccines. PMID:26588242

  2. Chimeric spider silk proteins mediated by intein result in artificial hybrid silks.

    Science.gov (United States)

    Lin, Senzhu; Chen, Gefei; Liu, Xiangqin; Meng, Qing

    2016-07-01

    Hybrid silks hold a great potential as specific biomaterials due to its controlled mechanical properties. To produce fibers with tunable properties, here we firstly made chimeric proteins in vitro, called W2C4CT and W2C8CT, with ligation of MaSp repetitive modules (C) with AcSp modules (W) by intein trans splicing technology from smaller precursors without final yield reduction. Intein mediated chimeric proteins form fibers at a low concentration of 0.4 mg/mL in 50 mM K3 PO4 pH 7.5 just drawn by hand. Hybrid fibers show smoother surface, and also have stronger chemical resistance as compared with fibers from W2CT (W fibers) and mixture of W2CT/C8CT (MHF8 fibers). Fibers from chimeric protein W2C4CT (HFH4) have improved mechanical properties than W fibers; however, with more C modules W2C8CT fibers (HFH8) properties decreased, indicates the length proportion of various modules is very important and should be optimized for fibers with specific properties. Generally, hybrid silks generated via chimeric proteins, which can be simplified by intein trans splicing, has greater potential to produce fibers with tunable properties. Our research shows that intein mediated directional protein ligation is a novel way to make large chimeric spider silk proteins and hybrid silks. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 385-392, 2016. PMID:26948769

  3. Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein

    KAUST Repository

    Mahfouz, Magdy M.

    2011-12-14

    Transcriptional activator-like effectors (TALEs) are proteins secreted by Xanthomonas bacteria when they infect plants. TALEs contain a modular DNA binding domain that can be easily engineered to bind any sequence of interest, and have been used to provide user-selected DNA-binding modules to generate chimeric nucleases and transcriptional activators in mammalian cells and plants. Here we report the use of TALEs to generate chimeric sequence-specific transcriptional repressors. The dHax3 TALE was used as a scaffold to provide a DNA-binding module fused to the EAR-repression domain (SRDX) to generate a chimeric repressor that targets the RD29A promoter. The dHax3. SRDX protein efficiently repressed the transcription of the RD29A

  4. Preparation and Characterization of a Novel Chimeric Protein VEGI-CTT in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Jiping Cai

    2008-08-01

    Full Text Available Vascular endothelial cell growth inhibitor (VEGI is a recently identified antiangiogenic cytokine that belongs to the TNF superfamily, and could effectively inhibit endothelial cell proliferation and angiogenesis. Synthetic peptide CTT (CTTHWGFTLC has been found to suppress invasion and migration of both tumor and endothelial cells by potent and selective inhibition of MMP-2 and MMP-9. To prepare chimeric protein VEGI-CTT for more potent antitumor therapy, the recombinant expression vector pET-VEGI-CTT was constructed. This fusion protein was expressed in inclusion bodies in E. coli BL21 (DE3, and was refolded and purified by immobilized metal affinity chromatography using His-tag. Purified VEGI-CTT protein was characterized by proliferation assays of the endothelial cells and casein degradation assay in vitro. The results demonstrated that chimeric protein VEGI-CTT had a potent activity of antiangiogenesis through inhibiting the proliferation of endothelial cells, and could effectively reduce the activity of MMP-2 and MMP-9. The preliminarily in vivo study demonstrated that chimeric protein VEGI-CTT had more potent antitumor activity than VEGI and/or CTT peptide against CA46 human lymphoma xenografts in nude mice. Thus, these facts that are derived from the present study suggest that the chimeric protein VEGI-CTT may be used for tumor therapy in the future.

  5. Chimeric elk/mouse prion proteins in transgenic mice

    OpenAIRE

    Tamguney, G; Giles, K; Oehler, A.; Johnson, NL; DeArmond, SJ; Prusiner, SB

    2013-01-01

    Chronic wasting disease (CWD) of deer and elk is a highly communicable neurodegenerative disorder caused by prions. Investigations of CWD are hampered by slow bioassays in transgenic (Tg) mice. Towards the development of Tg mice that will be more susceptible to CWD prions, we created a series of chimeric elk/mouse transgenes that encode the N terminus of elk PrP (ElkPrP) up to residue Y168 and the C terminus of mouse PrP (MoPrP) beyond residue 169 (mouse numbering), designated Elk3M(SNIVVK). ...

  6. Thermostability of multidomain proteins: chimeric mesophilic/thermophilic elongation factors EF-Tu

    Czech Academy of Sciences Publication Activity Database

    Šanderová, Hana; Maloň, Petr; Hůlková, Marta; Jonák, Jiří

    Varšava : FEBS, 2004, s. 7. [FEBS Forum for Young Scientists. Varšava (PL), 24.06.2004-26.06.2004] R&D Projects: GA ČR GA303/02/0689 Institutional research plan: CEZ:AV0Z5052915 Keywords : thermostability * EF-Tu * chimeric protein Subject RIV: EB - Genetics ; Molecular Biology

  7. Thermostability of multidomain proteins: chimeric mesophilic/thermophilic elongation factors EF-Tu

    Czech Academy of Sciences Publication Activity Database

    Šanderová, Hana; Maloň, Petr; Hůlková, Marta; Jonák, Jiří

    Oxford : Blackwell Publishing, 2004, s. 219. [Meeting of the Federation of the European Biochemical Societies /29./. Varšava (PL), 26.06.2004-01.07.2004] R&D Projects: GA ČR GA303/02/0689 Institutional research plan: CEZ:AV0Z5052915 Keywords : thermostability * EF-Tu * chimeric protein Subject RIV: EB - Genetics ; Molecular Biology

  8. Exploiting chimeric human antibodies to characterize a protective epitope of Neisseria adhesin A, one of the Bexsero vaccine components.

    Science.gov (United States)

    Bertoldi, Isabella; Faleri, Agnese; Galli, Barbara; Lo Surdo, Paola; Liguori, Alessia; Norais, Nathalie; Santini, Laura; Masignani, Vega; Pizza, Mariagrazia; Giuliani, Marzia Monica

    2016-01-01

    Neisseria adhesin A (NadA) is one of the antigens of Bexsero, the recently licensed multicomponent vaccine against serogroup B Neisseria meningitidis (MenB). NadA belongs to the class of oligomeric coiled-coil adhesins and is able to mediate adhesion and invasion of human epithelial cells. As a vaccine antigen, NadA has been shown to induce high levels of bactericidal antibodies; however, the domains important for protective response are still unknown. In order to further investigate its immunogenic properties, we have characterized the murine IgG1 mAb (6E3) that was able to recognize the 2 main antigenic variants of NadA on the surface of MenB strains. The epitope targeted by mAb 6E3 was mapped by hydrogen-deuterium exchange mass spectrometry and shown to be located on the coiled-coil stalk region of NadA (aa 206-249). Although no serum bactericidal activity was observed for murine IgG1 mAb 6E3, functional activity was restored when using chimeric antibodies in which the variable regions of the murine mAb 6E3 were fused to human IgG3 constant regions, thus confirming the protective nature of the mAb 6E3 epitope. The use of chimeric antibody molecules will enable future investigations of complement-mediated antibody functionality independently of the Fc-mediated differences in complement activation. PMID:26304221

  9. A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27 Protein as an HIV Immunogen.

    Directory of Open Access Journals (Sweden)

    Aneesh Vijayan

    Full Text Available In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K by fusing gp120 from clade B with the vaccinia virus (VACV 14K oligomeric protein (derived from A27L gene. Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size exclusion chromatography, electron microscopy and binding to anti-Env antibodies. These approaches indicate that gp120-14K protein is oligomeric and reacts with a wide spectrum of HIV-1 neutralizing antibodies. Furthermore, in human monocyte-derived dendritic cells (moDCs, gp120-14K protein upregulates the levels of several proinflammatory cytokines and chemokines associated with Th1 innate immune responses (IL-1β, IFN-γ, IL-6, IL-8, IL-12, RANTES. Moreover, we showed in a murine model, that a heterologous prime/boost immunization protocol consisting of a DNA prime with a plasmid expressing gp120-14K protein followed by a boost with MVA-B [a recombinant modified vaccinia virus Ankara (MVA expressing HIV-1 gp120, Gag, Pol and Nef antigens from clade B], generates stronger, more polyfunctional, and greater effector memory HIV-1-specific CD4+ and CD8+ T-cell immune responses, than immunization with DNA-gp120/MVA-B. The DNA/MVA protocol was superior to immunization with the combination of protein/MVA and the latter was superior to a prime/boost of MVA/MVA or protein/protein. In addition, these immunization protocols enhanced antibody responses against gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune response. These results demonstrate that fusing HIV-1 gp120 with VACV 14K forms an oligomeric protein which is highly antigenic as it activates a Th1 innate immune response in human moDCs, and in vaccinated mice triggers polyfunctional HIV-1

  10. A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen

    Science.gov (United States)

    Vijayan, Aneesh; García-Arriaza, Juan; C. Raman, Suresh; Conesa, José Javier; Chichón, Francisco Javier; Santiago, César; Sorzano, Carlos Óscar S.; Carrascosa, José L.; Esteban, Mariano

    2015-01-01

    In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size exclusion chromatography, electron microscopy and binding to anti-Env antibodies. These approaches indicate that gp120-14K protein is oligomeric and reacts with a wide spectrum of HIV-1 neutralizing antibodies. Furthermore, in human monocyte-derived dendritic cells (moDCs), gp120-14K protein upregulates the levels of several proinflammatory cytokines and chemokines associated with Th1 innate immune responses (IL-1β, IFN-γ, IL-6, IL-8, IL-12, RANTES). Moreover, we showed in a murine model, that a heterologous prime/boost immunization protocol consisting of a DNA prime with a plasmid expressing gp120-14K protein followed by a boost with MVA-B [a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120, Gag, Pol and Nef antigens from clade B], generates stronger, more polyfunctional, and greater effector memory HIV-1-specific CD4+ and CD8+ T-cell immune responses, than immunization with DNA-gp120/MVA-B. The DNA/MVA protocol was superior to immunization with the combination of protein/MVA and the latter was superior to a prime/boost of MVA/MVA or protein/protein. In addition, these immunization protocols enhanced antibody responses against gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune response. These results demonstrate that fusing HIV-1 gp120 with VACV 14K forms an oligomeric protein which is highly antigenic as it activates a Th1 innate immune response in human moDCs, and in vaccinated mice triggers polyfunctional HIV-1-specific adaptive

  11. Protective Efficacy of a Single Immunization of a Chimeric Adenovirus Vector-Based Vaccine against Simian Immunodeficiency Virus Challenge in Rhesus Monkeys▿

    OpenAIRE

    Barouch, Dan H.; Liu, Jinyan; Lynch, Diana M; O'Brien, Kara L.; La Porte, Annalena; Simmons, Nathaniel L.; Riggs, Ambryice M.; Clark, Sarah; Abbink, Peter; Montefiori, David C.; Landucci, Gary; Forthal, Donald N.; Self, Steven G.; Carville, Angela; Mansfield, Keith

    2009-01-01

    Rare serotype and chimeric recombinant adenovirus (rAd) vectors that evade anti-Ad5 immunity are currently being evaluated as potential vaccine vectors for human immunodeficiency virus type 1 and other pathogens. We have recently reported that a heterologous rAd prime-boost regimen expressing simian immunodeficiency virus (SIV) Gag afforded durable partial immune control of an SIV challenge in rhesus monkeys. However, single-shot immunization may ultimately be preferable for global vaccine de...

  12. The chimeric VirA-tar receptor protein is locked into a highly responsive state.

    OpenAIRE

    Turk, S C; van Lange, R P; Sonneveld, E; Hooykaas, P J

    1993-01-01

    The wild-type VirA protein is known to be responsive not only to phenolic compounds but also to sugars via the ChvE protein (G. A. Cangelosi, R. G. Ankenbauer, and E. W. Nester, Proc. Natl. Acad. Sci. USA 87:6708-6712, 1990, and N. Shimoda, A. Toyoda-Yamamoto, J. Nagamine, S. Usami, M. Katayama, Y. Sakagami, and Y. Machida, Proc. Natl. Acad. Sci. USA 87:6684-6688, 1990). It is shown here that the mutant VirA(Ser-44, Arg-45) protein and the chimeric VirA-Tar protein are no longer responsive to...

  13. SAT Type Foot-and-Mouth Disease (FMD) Chimeric Vaccine Elicits Protection in Pigs

    Science.gov (United States)

    The recent development of infectious cDNA clone technology for foot-and-mouth disease (FMD), Southern African Territories (SAT) viruses has provided a valuable tool for genetic and biological characterization of field and laboratory strains. Recombinant chimeric viruses, containing the capsid-coding...

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

    Directory of Open Access Journals (Sweden)

    Bettina Huber

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

  15. Chimeric avian paramyxovirus-based vector immunization against highly pathogenic avian influenza followed by conventional Newcastle disease vaccination eliminates lack of protection from virulent ND virus

    Directory of Open Access Journals (Sweden)

    C. Steglich

    2014-01-01

    Full Text Available Recently, we described a chimeric, hemagglutinin of highly pathogenic avian influenza virus (HPAIV H5 expressing Newcastle disease virus (NDV-based vector vaccine (chNDVFHNPMV8H5 in which NDV envelope glycoproteins were replaced by those of avian paramyxovirus-8 (APMV-8. This chimeric vaccine induced solid protection against lethal HPAIV H5N1 even in chickens with maternal antibodies against NDV (MDA+. However, due to the absence of the major NDV immunogens it failed to induce protection against Newcastle disease (ND. Here, we report on protection of MDA+ chickens against HPAI H5N1 and ND, by vaccination with chNDVFHNPMV8H5 either on day 1 or day seven after hatch, and subsequent immunization with live attenuated NDV seven days later. Vaccination was well tolerated and three weeks after immunization, challenge infections with highly pathogenic NDV as well as HPAIV H5N1 were carried out. All animals remained healthy without exhibiting any clinical signs, whereas non-vaccinated animals showed morbidity and mortality. Therefore, vaccination with chNDVFHNPMV8H5 can be followed by NDV vaccination to protect chickens from HPAIV as well as NDV, indicating that the antibody response against chNDVFHNPMV8H5 does not interfere with live ND vaccination.

  16. Boosting BCG-primed mice with chimeric DNA vaccine HG856A induces potent multifunctional T cell responses and enhanced protection against Mycobacterium tuberculosis.

    Science.gov (United States)

    Ji, Ping; Hu, Zhi-Dong; Kang, Han; Yuan, Qin; Ma, Hui; Wen, Han-Li; Wu, Juan; Li, Zhong-Ming; Lowrie, Douglas B; Fan, Xiao-Yong

    2016-02-01

    The tuberculosis pandemic continues to rampage despite widespread use of the current Bacillus Calmette-Guerin (BCG) vaccine. Because DNA vaccines can elicit effective antigen-specific immune responses, including potent T cell-mediated immunity, they are promising vehicles for antigen delivery. In a prime-boost approach, they can supplement the inadequate anti-TB immunological memory induced by BCG. Based on this, a chimeric DNA vaccine HG856A encoding Mycobacterium tuberculosis (M. tuberculosis) immunodominant antigen Ag85A plus two copies of ESAT-6 was constructed. Potent humoral immune responses, as well as therapeutic effects induced by this DNA vaccine, were observed previously in M. tuberculosis-infected mice. In this study, we further evaluated the antigen-specific T cell immune responses and showed that repeated immunization with HG856A gave modest protection against M. tuberculosis challenge infection and significantly boosted the immune protection primed by BCG vaccination. Enhanced protection was accompanied by increased multifunctional Th1 CD4(+) T cell responses, most notably by an elevated frequency of M. tuberculosis antigen-specific IL-2-producing CD4(+) T cells post-vaccination. These data confirm the potential of chimeric DNA vaccine HG856A as an anti-TB vaccine candidate. PMID:26111521

  17. Construction and characterization of chimeric BHIV (BIV/HIV-1) viruses carrying the bovine immunodeficiency virus gag gene

    Institute of Scientific and Technical Information of China (English)

    Yi-Xin Zhu; Chang Liu; Xin-Lei Liu; Wen-Tao Qiao; Qi-Min Chen; Yi Zeng; Yun-Qi Geng

    2005-01-01

    AIM: To explore the possibility of the replacement of the gag gene between human immunodeficiency virus and bovine immunodeficiency virus, to achieve chimeric virions,and thereby gain a new kind of AIDS vaccine based on BHIV chimeric viruses.METHODS: A series of chimeric BHIV proviral DNAs differing in the replacement regions in gag gene were constructed, and then were transfected into 293T cells. The expression of chimeric viral genes was detected at the RNA and protein level. The supematant of 293T cell was ultra centrifuged to detect the probable chimeric virion. Once the chimeric virion was detected, its biological activities were also assayed by infecting HIV-sensitive MT4 cells.RESULTS: Four chimeric BHIV proviral DNAs were constructed. Genes in chimeric viruses expressed correctly in transfected 293T cells. All four constructs assembled chimeric virions with different degrees of efficiency. These virions had complete structures common to retroviruses and packaged genomic RNAs, but the cleavages of the precursor Gag proteins were abnormal to some extent. Three of these virions tested could attach and enter into MT4 cells, and one of them could complete the course of reverse transcription. Yet none of them could replicate in MT4 cells.CONCLUSION: The replacement of partial gag gene of HIV with BIV gaggene is feasible. Genes in chimeric BHIVs are accurately expressed, and virions are assembled. These chimeric BHIVs (proviral DNA together with virus particles) have the potential to become a new kind of HIV/AIDS vaccine.

  18. Construction and evaluation of a chimeric protein made from Fasciola hepatica leucine aminopeptidase and cathepsin L1.

    Science.gov (United States)

    Hernández-Guzmán, K; Sahagún-Ruiz, A; Vallecillo, A J; Cruz-Mendoza, I; Quiroz-Romero, H

    2016-01-01

    Leucine aminopeptidase (LAP) and cathepsin L1 (CL1) are important enzymes for the pathogenesis and physiology of Fasciola hepatica. These enzymes were analysed in silico to design a chimeric protein containing the most antigenic sequences of LAP (GenBank; AAV59016.1; amino acids 192-281) and CL1 (GenBank CAC12806.1; amino acids 173-309). The cloned 681-bp chimeric fragment (rFhLAP-CL1) contains 270 bp from LAP and 411 bp from CL1, comprising three epitopes, DGRVVHLKY (amino acids 54-62) from LAP, VTGYYTVHSGSEVELKNLV (amino acids 119-137) and YQSQTCLPF (amino acids 161-169) from CL1. The ~25 kDa rFhLAP-CL1 chimeric protein was expressed from the pET15b plasmid in the Rosetta (DE3) Escherichia coli strain. The chimeric protein rFhLAP-CL1, which showed antigenic and immunogenic properties, was recognized in Western blot assays using F. hepatica-positive bovine sera, and induced strong, specific antibody responses following immunization in rabbits. The newly generated chimeric protein may be used as a diagnostic tool for detection of antibodies against F. hepatica in bovine sera and as an immunogen to induce protection against bovine fasciolosis. PMID:25274570

  19. Crystal Structures of Yeast-Produced Enterovirus 71 and Enterovirus 71/Coxsackievirus A16 Chimeric Virus-Like Particles Provide the Structural Basis for Novel Vaccine Design against Hand-Foot-and-Mouth Disease

    Science.gov (United States)

    Lyu, Ke; He, Ya-Ling; Li, Hao-Yang

    2015-01-01

    ABSTRACT Human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the two major causative agents for hand-foot-and-mouth disease (HFMD). Previously, we demonstrated that a virus-like particle (VLP) for EV71 produced from Saccharomyces cerevisiae is a potential vaccine candidate against EV71 infection, and an EV71/CVA16 chimeric VLP can elicit protective immune responses against both virus infections. Here, we presented the crystal structures of both VLPs, showing that both the linear and conformational neutralization epitopes identified in EV71 are mostly preserved on both VLPs. The replacement of only 4 residues in the VP1 GH loop converted strongly negatively charged surface patches formed by portions of the SP70 epitope in EV71 VLP into a relatively neutral surface in the chimeric VLP, which likely accounted for the additional neutralization capability of the chimeric VLP against CVA16 infection. Such local variations in the amino acid sequences and the surface charge potential are also present in different types of polioviruses. In comparison to EV71 VLP, the chimeric VLP exhibits structural changes at the local site of amino acid replacement and the surface loops of all capsid proteins. This is consistent with the observation that the VP1 GH loop located near the pseudo-3-fold junction is involved in extensive interactions with other capsid regions. Furthermore, portions of VP0 and VP1 in EV71 VLP are at least transiently exposed, revealing the structural flexibility of the VLP. Together, our structural analysis provided insights into the structural basis of enterovirus neutralization and novel vaccine design against HFMD and other enterovirus-associated diseases. IMPORTANCE Our previous studies demonstrated that the enterovirus 71 (EV71) virus-like particle (VLP) produced from yeast is a vaccine candidate against EV71 infection and that a chimeric EV71/coxsackievirus A16 (CVA16) VLP with the replacement of 4 amino acids in the VP1 GH loop can confer

  20. Eliciting neutralizing antibodies against the membrane proximal external region of HIV-1 Env by chimeric live attenuated influenza A virus vaccines.

    Science.gov (United States)

    Zang, Yang; Du, Dongchuan; Li, Na; Su, Weiheng; Liu, Xintao; Zhang, Yan; Nie, Jianhui; Wang, Youchun; Kong, Wei; Jiang, Chunlai

    2015-07-31

    Despite significant efforts directed toward research on HIV-1 vaccines, a truly effective immunogen has not been achieved. However, the broadly neutralizing antibodies (BnAbs) 2F5 and 4E10, targeting the highly conserved membrane proximal external region (MPER) of HIV-1, are two promising tools for vaccine development. Here we engrafted the MPER into the linker domain between the trimeric core structure and the transmembrane domain of influenza A virus HA2 to investigate the potential of such chimeric viruses to elicit HIV-1 neutralizing antibodies. In the context of proliferating attenuated influenza A viruses, these HIV-1 neutralizing antibody epitopes could be continuously expressed and mimicked their native conformation to induce humoral immune responses. While MPER-specific antibodies could be detected in serum of guinea pigs vaccinated with the chimeric viruses, they exhibited only weakly neutralizing activities. These antisera from vaccinated animals neutralized viruses of clades B and BC (tier 1), but not of clades AE (tier 1) and C (tier 2). These results suggest that influenza A virus can be used as a vehicle for displaying MPER and inducing BnAbs, but it provides limited protection against HIV-1 infection. In the future development of HIV-1 vaccines by rational design, a more effective live virus vector or multiple antigens should be chosen to facilitate the process of neutralizing antibody maturation. PMID:26126669

  1. 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. PMID:27154395

  2. Generation and characterization of potential dengue vaccine candidates based on domain III of the envelope protein and the capsid protein of the four serotypes of dengue virus.

    Science.gov (United States)

    Suzarte, Edith; Marcos, Ernesto; Gil, Lázaro; Valdés, Iris; Lazo, Laura; Ramos, Yassel; Pérez, Yusleidi; Falcón, Viviana; Romero, Yaremis; Guzmán, María G; González, Sirenia; Kourí, Juan; Guillén, Gerardo; Hermida, Lisset

    2014-07-01

    Dengue is currently one of the most important arthropod-borne diseases, causing up to 25,000 deaths annually. There is currently no vaccine to prevent dengue virus infection, which needs a tetravalent vaccine approach. In this work, we describe the cloning and expression in Escherichia coli of envelope domain III-capsid chimeric proteins (DIIIC) of the four dengue serotypes as a tetravalent dengue vaccine candidate that is potentially able to generate humoral and cellular immunity. The recombinant proteins were purified to more than 85 % purity and were recognized by anti-dengue mouse and human sera. Mass spectrometry analysis verified the identity of the proteins and the correct formation of the intracatenary disulfide bond in the domain III region. The chimeric DIIIC proteins were also serotype-specific, and in the presence of oligonucleotides, they formed aggregates that were visible by electron microscopy. These results support the future use of DIIIC recombinant chimeric proteins in preclinical studies in mice for assessing their immunogenicity and efficacy. PMID:24420159

  3. Genetic Stability of a Genetically-Engineered Chimeric Porcine Circovirus (PCV) Vaccine, PCV1-2

    OpenAIRE

    Gillespie, Jennifer Ann

    2009-01-01

    Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus associated disease (PCVAD), an economically important swine disease that causes wasting in pigs 5-18 weeks of age. There exist two different types of porcine circoviruses: porcine circovirus type 1 (PCV1) was discovered as a contaminant of porcine kidney (PK-15) cells and was determined to be nonpathogenic in swine; whereas porcine circovirus type 2 (PCV2) is pathogenic. A recently released vaccine for PCVA...

  4. Enhanced Protective Efficacy of a Chimeric Form of the Schistosomiasis Vaccine Antigen Sm-TSP-2

    OpenAIRE

    Pearson, Mark S.; Pickering, Darren A.; McSorley, Henry J.; Bethony, Jeffrey M; Tribolet, Leon; Dougall, Annette M.; Hotez, Peter J.; Loukas, Alex

    2012-01-01

    The large extracellular loop of the Schistosoma mansoni tetraspanin, Sm-TSP-2, when fused to a thioredoxin partner and formulated with Freund's adjuvants, has been shown to be an efficacious vaccine against murine schistosomiasis. Moreover, Sm-TSP-2 is uniquely recognised by IgG1 and IgG3 from putatively resistant individuals resident in S. mansoni endemic areas in Brazil. In the present study, we expressed Sm-TSP-2 at high yield and in soluble form in E. coli without the need for a solubilit...

  5. A novel recombinant 6Aβ15-THc-C chimeric vaccine (rCV02) mitigates Alzheimer’s disease-like pathology, cognitive decline and synaptic loss in aged 3 × Tg-AD mice

    Science.gov (United States)

    Yu, Yun-Zhou; Liu, Si; Wang, Hai-Chao; Shi, Dan-Yang; Xu, Qing; Zhou, Xiao-Wei; Sun, Zhi-Wei; Huang, Pei-Tang

    2016-01-01

    Alzheimer’s disease (AD) is a neurodegenerative disorder that impairs memory and cognition. Targeting amyloid-β (Aβ) may be currently the most promising immunotherapeutic strategy for AD. In this study, a recombinant chimeric 6Aβ15-THc-C immunogen was formulated with alum adjuvant as a novel Aβ B-cell epitope candidate vaccine (rCV02) for AD. We examined its efficacy in preventing the cognitive deficit and synaptic impairment in 3 × Tg-AD mice. Using a toxin-derived carrier protein, the rCV02 vaccine elicited robust Aβ-specific antibodies that markedly reduced AD-like pathology and improved behavioral performance in 3 × Tg-AD mice. Along with the behavioral improvement in aged 3 × Tg-AD mice, rCV02 significantly decreased calpain activation concurrent with reduced soluble Aβ or oligomeric forms of Aβ, probably by preventing dynamin 1 and PSD-95 degradation. Our data support the hypothesis that reducing Aβ levels in rCV02-immunized AD mice increases the levels of presynaptic dynamin 1 and postsynaptic PSD-95 allowing functional recovery of cognition. In conclusion, this novel and highly immunogenic rCV02 shows promise as a new candidate prophylactic vaccine for AD and may be useful for generating rapid and strong Aβ-specific antibodies in AD patients with pre-existing memory Th cells generated after immunization with conventional tetanus toxoid vaccine. PMID:27255752

  6. A novel recombinant 6Aβ15-THc-C chimeric vaccine (rCV02) mitigates Alzheimer's disease-like pathology, cognitive decline and synaptic loss in aged 3 × Tg-AD mice.

    Science.gov (United States)

    Yu, Yun-Zhou; Liu, Si; Wang, Hai-Chao; Shi, Dan-Yang; Xu, Qing; Zhou, Xiao-Wei; Sun, Zhi-Wei; Huang, Pei-Tang

    2016-01-01

    Alzheimer's disease (AD) is a neurodegenerative disorder that impairs memory and cognition. Targeting amyloid-β (Aβ) may be currently the most promising immunotherapeutic strategy for AD. In this study, a recombinant chimeric 6Aβ15-THc-C immunogen was formulated with alum adjuvant as a novel Aβ B-cell epitope candidate vaccine (rCV02) for AD. We examined its efficacy in preventing the cognitive deficit and synaptic impairment in 3 × Tg-AD mice. Using a toxin-derived carrier protein, the rCV02 vaccine elicited robust Aβ-specific antibodies that markedly reduced AD-like pathology and improved behavioral performance in 3 × Tg-AD mice. Along with the behavioral improvement in aged 3 × Tg-AD mice, rCV02 significantly decreased calpain activation concurrent with reduced soluble Aβ or oligomeric forms of Aβ, probably by preventing dynamin 1 and PSD-95 degradation. Our data support the hypothesis that reducing Aβ levels in rCV02-immunized AD mice increases the levels of presynaptic dynamin 1 and postsynaptic PSD-95 allowing functional recovery of cognition. In conclusion, this novel and highly immunogenic rCV02 shows promise as a new candidate prophylactic vaccine for AD and may be useful for generating rapid and strong Aβ-specific antibodies in AD patients with pre-existing memory Th cells generated after immunization with conventional tetanus toxoid vaccine. PMID:27255752

  7. A chimeric protein of aluminum-activated malate transporter generated from wheat and Arabidopsis shows enhanced response to trivalent cations.

    Science.gov (United States)

    Sasaki, Takayuki; Tsuchiya, Yoshiyuki; Ariyoshi, Michiyo; Ryan, Peter R; Yamamoto, Yoko

    2016-07-01

    TaALMT1 from wheat (Triticum aestivum) and AtALMT1 from Arabidopsis thaliana encode aluminum (Al)-activated malate transporters, which confer acid-soil tolerance by releasing malate from roots. Chimeric proteins from TaALMT1 and AtALMT1 (Ta::At, At::Ta) were previously analyzed in Xenopus laevis oocytes. Those studies showed that Al could activate malate efflux from the Ta::At chimera but not from At::Ta. Here, functions of TaALMT1, AtALMT1 and the chimeric protein Ta::At were compared in cultured tobacco BY-2 cells. We focused on the sensitivity and specificity of their activation by trivalent cations. The activation of malate efflux by Al was at least two-fold greater in the chimera than the native proteins. All proteins were also activated by lanthanides (erbium, ytterbium, gadolinium, and lanthanum), but the chimera again released more malate than TaALMT1 or AtALMT1. In Xenopus oocytes, Al, ytterbium, and erbium activated inward currents from the native TaALMT1 and the chimeric protein, but gadolinium only activated currents from the chimera. Lanthanum inhibited currents from both proteins. These results demonstrated that function of the chimera protein was altered compared to the native proteins and was more responsive to a range of trivalent cations when expressed in plant cells. PMID:27039280

  8. Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

    Science.gov (United States)

    Liu, Z.; Xia, M.; Poovaiah, B. W.

    1998-01-01

    cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.

  9. Inducible Expression of Chimeric EWS/ETS Proteins Confers Ewing's Family Tumor-Like Phenotypes to Human Mesenchymal Progenitor Cells

    OpenAIRE

    Miyagawa, Yoshitaka; Okita, Hajime; Nakaijima, Hideki; Horiuchi, Yasuomi; Sato, Ban; TAGUCHI, Tomoko; Toyoda, Masashi; Katagiri, Yohko U; Fujimoto, Junichiro; Hata, Jun-Ichi; Umezawa, Akihiro; Kiyokawa, Nobutaka

    2008-01-01

    Ewing's family tumor (EFT) is a rare pediatric tumor of unclear origin that occurs in bone and soft tissue. Specific chromosomal translocations found in EFT cause EWS to fuse to a subset of ets transcription factor genes (ETS), generating chimeric EWS/ETS proteins. These proteins are believed to play a crucial role in the onset and progression of EFT. However, the mechanisms responsible for the EWS/ETS-mediated onset remain unclear. Here we report the establishment of a tetracycline-controlle...

  10. Construction, Expression and Characterization of a Chimeric Protein Targeting Carcinoembryonic Antigen in Lung Cancer

    Institute of Scientific and Technical Information of China (English)

    LI Yang; HUA Shu-cheng; MA Cheng-yuan; YU Zhen-xiang; XU Li-jun; LI Dan; SUN Li-li; LI Xiao; PENG Li-ping

    2011-01-01

    The carcinoembryonic antigen(CEA) is an oncofetal glycoprotein known as an important clinical tumor marker and is overexpressed in several types of tumors, including colorectal and lung carcinomas. We constructed a chimeric protein that exhibits both specific binding and immune stimulating activities, by fusing staphylococcal enterotoxin A(SEA) to the C-terminus of an anti-CEA single-chain disulfide-stabilized Fv(scdsFv) antibody (single-chain-C-terminus/SEA, SC-C/SEA). The SC-C/SEA protein was expressed in Escherichia coli(E. coli), refolded, and purified on an immobilized Ni2+ affinity chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot analysis reveal that the target protein was expressed sufficiently. We used immunofluorescence assays to demonstrate that SC-C/SEA could bind specifically to human lung carcinoma cells(A549), but almost human uterine cervix cells(HeLa). We also used the L-lactate dehydrogenase(LDH) release assay to show that SC-C/SEA elicits a strong A549 tumor-specific cytotoxic T lymphocyte(CTL) response in vitro. The results suggest that SC-C/SEA shows specific activity against CEA-positive cells and has potential application in CEA-targeted cancer immunotherapy.

  11. Improved humoral and cellular immune response against the gp120 V3 loop of HIV-1 following genetic immunization with a chimeric DNA vaccine encoding the V3 inserted into the hepatites B surface antigen

    DEFF Research Database (Denmark)

    Fomsgaard, A.; Nielsen, H.V.; Bryder, K.;

    1998-01-01

    The gp120-derived V3 loop of HIV-1 is involved in co-receptor interaction, it guides cell tropism, and contains an epitope for antibody neutralization. Thus, HIV-1 V3 is an attractive vaccine candidate. The V3 of the MN strain (MN V3) contains both B- and T-cell epitopes, including a known mouse H......-2d-restricted cytotoxic T lymphocyte (CTL) epitope. In an attempt to improve the immunogenicity of V3 in DNA vaccines, a plasmid expressing MN V3 as a fusion protein with the highly immunogenic middle (pre-S2+S) surface antigen of hepatitis B virus (HBsAg) was constructed. Epidermal inoculation by...... response and a uniform strong anti-HBs CTL response already 1 week p.i. in all mice. DNA vaccination with the chimeric MN V2/HBsAg plasmid elicited humoral responses against both viruses within 3-6 weeks which peaked at 6-12 weeks and remained stable for at least 25 weeks. In addition, specific CTL...

  12. Improved humoral and cellular immune responses against the gp120 V3 loop of HIV-1 following genetic immunization with a chimeric DNA vaccine encoding the V3 inserted into the hepatitis B surface antigen

    DEFF Research Database (Denmark)

    Fomsgaard, A; Nielsen, H V; Bryder, K;

    1998-01-01

    The gp120-derived V3 loop of HIV-1 is involved in co-receptor interaction, it guides cell tropism, and contains an epitope for antibody neutralization. Thus, HIV-1 V3 is an attractive vaccine candidate. The V3 of the MN strain (MN V3) contains both B- and T-cell epitopes, including a known mouse H......-2d-restricted cytotoxic T lymphocyte (CTL) epitope. In an attempt to improve the immunogenicity of V3 in DNA vaccines, a plasmid expressing MN V3 as a fusion protein with the highly immunogenic middle (pre-S2 + S) surface antigen of hepatitis B virus (HBsAg) was constructed. Epidermal inoculation by...... response and a uniform strong anti-HBs CTL response already 1 week p.i. in all mice. DNA vaccination with the chimeric MN V3/HBsAg plasmid elicited humoral responses against both viruses within 3-6 weeks which peaked at 6-12 weeks and remained stable for at least 25 weeks. In addition, specific CTL...

  13. Characterization of Hepatitis C Virus Recombinants with Chimeric E1/E2 Envelope Proteins and Identification of Single Amino Acids in the E2 Stem Region Important for Entry

    OpenAIRE

    Carlsen, Thomas H. R.; Scheel, Troels K. H.; Ramirez, Santseharay; Foung, Steven K. H.; Bukh, Jens

    2013-01-01

    The hepatitis C virus (HCV) envelope proteins E1 and E2 play a key role in host cell entry and represent important targets for vaccine and drug development. Here, we characterized HCV recombinants with chimeric E1/E2 complexes in vitro. Using genotype 1a/2a JFH1-based recombinants expressing 1a core-NS2, we exchanged E2 with functional isolate sequences of genotypes 1a (alternative isolate), 1b, and 2a. While the 1a-E2 exchange did not impact virus viability, the 2a-E2 recombinant was nonviab...

  14. Reduction of porcine circovirus type 2 (PCV2 viremia by a reformulated inactivated chimeric PCV1-2 vaccine-induced humoral and cellular immunity after experimental PCV2 challenge

    Directory of Open Access Journals (Sweden)

    Seo Hwi

    2012-10-01

    Full Text Available Abstract Background The objective of the present study was to elucidate the humoral and cellular immune response mechanisms by which a reformulated inactivated chimeric PCV1-2 vaccine reduces the PCV2 viremia. Forty PCV2 seronegative 3-week-old pigs were randomly divided into the following four groups: vaccinated challenged (T01, vaccinated non-challenged (T02, non-vaccinated challenged (T03, and non-vaccinated non-challenged (T04 animals. The pigs in groups T01 and T02 were immunized with a reformulated inactivated chimeric PCV1-2 vaccine (Fostera™ PCV; Pfizer Animal Health administered as a 2.0 ml dose at 21 days of age. At 35 days of age (0 days post-challenge, the pigs in groups T01 and T03 were inoculated intranasally with 2 ml each of PCV2b. Results A reduction of PCV2 viremia coincided with the appearance of both PCV2-specific neutralizing antibodies (NA and interferon-γ-secreting cells (IFN-γ-SCs in the vaccinated animals. However, the presence of anti-PCV2 IgG antibodies did not correlate with the reduction of PCV2 viremia. Lymphocyte subset analysis indicated that the numbers of CD3+ and CD4+ cells increased in vaccinated animals but the numbers of CD4+ cells decreased transiently in non-vaccinated animals. The observation of a delayed type hypersensitivity response in only the vaccinated animals also supports a CD4+ cell-associated protective cellular immune response induced by the reformulated inactivated chimeric PCV1-2 vaccine. Conclusions The induction of PCV2-specific NA and IFN-γ-SCs, and CD4+ cells by the reformulated inactivated chimeric PCV1-2 vaccine is the important protective immune response leading to reduction of the PCV2 viremia and control of the PCV2 infection. To our knowledge this is the first demonstration of protective humoral and cellular immunity induced by the reformulated inactivated chimeric PCV1-2 vaccine and its effect on reduction of PCV2 viremia by vaccination.

  15. In vitro and in vivo characterization of chimeric duck Tembusu virus based on Japanese encephalitis live vaccine strain SA14-14-2.

    Science.gov (United States)

    Wang, Hong-Jiang; Liu, Long; Li, Xiao-Feng; Ye, Qing; Deng, Yong-Qiang; Qin, E-De; Qin, Cheng-Feng

    2016-07-01

    Duck Tembusu virus (DTMUV), a newly identified flavivirus, has rapidly spread to China, Malaysia and Thailand. The potential threats to public health have been well-highlighted; however its virulence and pathogenesis remain largely unknown. Here, by using reverse genetics, a recombinant chimeric DTMUV based on Japanese encephalitis live vaccine strain SA14-14-2 was obtained by substituting the corresponding prM and E genes (named ChinDTMUV). In vitro characterization demonstrated that ChinDTMUV replicated efficiently in mammalian cells with small-plaque phenotype in comparison with its parental viruses. Mouse tests showed ChinDTMUV exhibited avirulent phenotype in terms of neuroinvasiveness, while it retained neurovirulence from its parental virus DTMUV. Furthermore, immunization with ChinDTMUV was evidenced to elicit robust IgG and neutralizing antibody responses in mice. Overall, we successfully developed a viable chimeric DTMUV, and these results provide a useful platform for further investigation of the pathogenesis of DTMUV and development of a live attenuated DTMUV vaccine candidate. PMID:27100268

  16. Local and systemic immune responses induced by a recombinant chimeric protein containing Mycoplasma hyopneumoniae antigens fused to the B subunit of Escherichia coli heat-labile enterotoxin LTB.

    Science.gov (United States)

    Marchioro, Silvana Beutinger; Fisch, Andressa; Gomes, Charles K; Jorge, Sérgio; Galli, Vanessa; Haesebrouck, Freddy; Maes, Dominiek; Dellagostin, Odir; Conceição, Fabricio R

    2014-09-17

    A multi-antigen chimera composed of three antigens of Mycoplasma hyopneumoniae (R1, P42, and NrdF) and the mucosal adjuvant Escherichia coli heat-labile enterotoxin B subunit (LTB) was constructed, and its antigenic and immunogenic properties were evaluated in mice and pigs. In addition, we compared the effect of the fusion and co-administration of these proteins in mice. Antibodies against each subunit recognized the chimeric protein. Intranasal and intramuscular immunization of mice with the chimeric protein significantly increased IgG and IgA levels in the serum and tracheobronchial lavages, respectively, against some of the antigens present in the chimeric. Swine immunized with the chimeric protein developed an immune response against all M. hyopneumoniae antigens present in the fusion with a statistically significant difference (Phyopneumoniae infection. PMID:25091529

  17. ReX: A suite of computational tools for the design, visualization, and analysis of chimeric protein libraries.

    Science.gov (United States)

    Huang, Weiliang; Johnston, Wayne A; Boden, Mikael; Gillam, Elizabeth M J

    2016-02-01

    Directed evolution has greatly facilitated protein engineering and provided new insights into protein structure-function relationships. DNA shuffling using restriction enzymes is a particularly simple and cost-effective means of recombinatorial evolution that is well within the capability of most molecular biologists, but tools for the design and analysis of such experiments are limited. Here we introduce a suite of freely available online tools to make the construction and analysis of chimeric libraries readily accessible to the novice. REcut (http://qpmf.rx.umaryland.edu/REcut.html) facilitates the choice of DNA fragmentation strategy, while Xover (http://qpmf.rx.umaryland.edu/Xover.html) analyzes chimeric mutants to reveal recombination patterns and extract quantitative data. PMID:26842355

  18. A Chimeric Protein That Functions as both an Anthrax Dual-Target Antitoxin and a Trivalent Vaccine▿

    OpenAIRE

    Wu, Gaobing; Hong, Yuzhi; Guo, Aizhen; Feng, Chunfang; Cao, Sha; Zhang, Cheng-Cai; Shi, Ruiping; Tan, Yadi; Liu, Ziduo

    2010-01-01

    Effective measures for the prophylaxis and treatment of anthrax are still required for counteracting the threat posed by inhalation anthrax. In this study, we first demonstrated that the chimeric protein LFn-PA, created by fusing the protective antigen (PA)-binding domain of lethal factor (LFn) to PA, retained the functions of the respective molecules. On the basis of this observation, we attempted to develop an antitoxin that targets the binding of lethal factor (LF) and/or edema factor (EF)...

  19. A chimeric measles virus with a lentiviral envelope replicates exclusively in CD4+/CCR5+ cells

    International Nuclear Information System (INIS)

    We generated a replicating chimeric measles virus in which the hemagglutinin and fusion surface glycoproteins were replaced with the gp160 envelope glycoprotein of simian immunodeficiency virus (SIVmac239). Based on a previously cloned live-attenuated Schwarz vaccine strain of measles virus (MV), this chimera was rescued at high titers using reverse genetics in CD4+ target cells. Cytopathic effect consisted in the presence of large cell aggregates evolving to form syncytia, as observed during SIV infection. The morphology of the chimeric virus was identical to that of the parent MV particles. The presence of SIV gp160 as the only envelope protein on chimeric particles surface altered the cell tropism of the new virus from CD46+ to CD4+ cells. Used as an HIV candidate vaccine, this MV/SIVenv chimeric virus would mimic transient HIV-like infection, benefiting both from HIV-like tropism and the capacity of MV to replicate in dendritic cells, macrophages and lymphocytes.

  20. Biochemical characterization and evaluation of cytotoxicity of antistaphylococcal chimeric protein P128

    Directory of Open Access Journals (Sweden)

    George Shilpa E

    2012-06-01

    Full Text Available Abstract Background Antibiotic resistant S. aureus infection is a global threat. Newer approaches are required to control this organism in the current scenario. Cell wall degrading enzymes have been proposed as antibacterial agents for human therapy. P128 is a novel antistaphylococcal chimeric protein under development against S. aureus for human use which derives its bacterial cell wall degrading catalytic endopeptidase domain from ORF56, the Phage K tail-structure associated enzyme. Lead therapeutic entities have to be extensively characterized before they are assessed in animals for preclinical safety and toxicity. P128 is effective against antibiotic resistant strains as well as against a panel of isolates of global significance. Its efficacy against S. aureus in vivo has been established in our lab. Against this background, this study describes the characterization of this protein for its biochemical properties and other attributes. Results We evaluated the requirement or effect of divalent cations and the metal ion chelator, EDTA upon biological activity of P128. As the protein is intended for therapeutic use, we tested its activity in presence of body fluids and antibodies specific to P128. For the same reason, we used standard human cell lines to evaluate cytotoxic effects, if any. The divalent cations, calcium and magnesium at upto 25 mM and Zinc upto 2.5 mM neither inhibited nor enhanced P128 activity. Incubation of this protein with EDTA, human serum, plasma and blood also did not alter the antibacterial properties of the molecule. No inhibitory effect was observed in presence of hyper-immune sera raised against the protein. Finally, P128 did not show any cytotoxic effect on HEp2 and Vero cells at the highest concentration (5 mg/mL tested. Conclusions The results presented here throw light on several properties of protein P128. Taken together, these substantiate the potential of P128 for therapeutic use against S. aureus

  1. Calcium-stimulated autophosphorylation site of plant chimeric calcium/calmodulin-dependent protein kinase

    Science.gov (United States)

    Sathyanarayanan, P. V.; Siems, W. F.; Jones, J. P.; Poovaiah, B. W.

    2001-01-01

    The existence of two molecular switches regulating plant chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK), namely the C-terminal visinin-like domain acting as Ca(2+)-sensitive molecular switch and calmodulin binding domain acting as Ca(2+)-stimulated autophosphorylation-sensitive molecular switch, has been described (Sathyanarayanan, P. V., Cremo, C. R., and Poovaiah, B. W. (2000) J. Biol. Chem. 275, 30417-30422). Here we report the identification of Ca(2+)-stimulated autophosphorylation site of CCaMK by matrix-assisted laser desorption ionization time of flight-mass spectrometry. Thr(267) was confirmed as the Ca(2+)-stimulated autophosphorylation site by post-source decay experiments and by site-directed mutagenesis. The purified T267A mutant form of CCaMK did not show Ca(2+)-stimulated autophosphorylation, autophosphorylation-dependent variable calmodulin affinity, or Ca(2+)/calmodulin stimulation of kinase activity. Sequence comparison of CCaMK from monocotyledonous plant (lily) and dicotyledonous plant (tobacco) suggests that the autophosphorylation site is conserved. This is the first identification of a phosphorylation site specifically responding to activation by second messenger system (Ca(2+) messenger system) in plants. Homology modeling of the kinase and calmodulin binding domain of CCaMK with the crystal structure of calcium/calmodulin-dependent protein kinase 1 suggests that the Ca(2+)-stimulated autophosphorylation site is located on the surface of the kinase and far from the catalytic site. Analysis of Ca(2+)-stimulated autophosphorylation with increasing concentration of CCaMK indicates the possibility that the Ca(2+)-stimulated phosphorylation occurs by an intermolecular mechanism.

  2. Developmental regulation of the gene for chimeric calcium/calmodulin-dependent protein kinase in anthers

    Science.gov (United States)

    Poovaiah, B. W.; Xia, M.; Liu, Z.; Wang, W.; Yang, T.; Sathyanarayanan, P. V.; Franceschi, V. R.

    1999-01-01

    Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) was cloned from developing anthers of lily (Lilium longiflorum Thumb. cv. Nellie White) and tobacco (Nicotiana tabacum L. cv. Xanthi). Previous biochemical characterization and structure/function studies had revealed that CCaMK has dual modes of regulation by Ca(2+) and Ca(2+)/calmodulin. The unique structural features of CCaMK include a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain. The existence of these three features in a single polypeptide distinguishes it from other kinases. Western analysis revealed that CCaMK is expressed in a stage-specific manner in developing anthers. Expression of CCaMK was first detected in pollen mother cells and continued to increase, reaching a peak around the tetrad stage of meiosis. Following microsporogenesis, CCaMK expression rapidly decreased and at later stages of microspore development, no expression was detected. A tobacco genomic clone of CCaMK was isolated and transgenic tobacco plants were produced carrying the CCaMK promoter fused to the beta-glucuronidase reporter gene. Both CCaMK mRNA and protein were detected in the pollen sac and their localizations were restricted to the pollen mother cells and tapetal cells. Consistent results showing a stage-specific expression pattern were obtained by beta-glucuronidase analysis, in-situ hybridization and immunolocalization. The stage- and tissue-specific appearance of CCaMK in anthers suggests that it could play a role in sensing transient changes in free Ca(2+) concentration in target cells, thereby controlling developmental events in the anther.

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

    Directory of Open Access Journals (Sweden)

    Alma Gedvilaite

    2015-07-01

    Full Text Available 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. Evaluation of cellular responses for a chimeric HBsAg-HCV core DNA vaccine in BALB/c mice

    Directory of Open Access Journals (Sweden)

    Maryam Yazdanian

    2015-01-01

    Conclusion: Fusion of HBsAg to HCVcp in the context of a DNA vaccine modality could augment Th1-oriented cellular and CTL responses toward a protective epitope, comparable to that of HCVcp (subunit HCV vaccine immunization.

  5. Protein carriers of conjugate vaccines: characteristics, development, and clinical trials.

    Science.gov (United States)

    Pichichero, Michael E

    2013-12-01

    The immunogenicity of polysaccharides as human vaccines was enhanced by coupling to protein carriers. Conjugation transformed the T cell-independent polysaccharide vaccines of the past to T cell-dependent antigenic vaccines that were much more immunogenic and launched a renaissance in vaccinology. This review discusses the conjugate vaccines for prevention of infections caused by Hemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis. Specifically, the characteristics of the proteins used in the construction of the vaccines including CRM, tetanus toxoid, diphtheria toxoid, Neisseria meningitidis outer membrane complex, and Hemophilus influenzae protein D are discussed. The studies that established differences among and key features of conjugate vaccines including immunologic memory induction, reduction of nasopharyngeal colonization and herd immunity, and antibody avidity and avidity maturation are presented. Studies of dose, schedule, response to boosters, of single protein carriers with single and multiple polysaccharides, of multiple protein carriers with multiple polysaccharides and conjugate vaccines administered concurrently with other vaccines are discussed along with undesirable consequences of conjugate vaccines. The clear benefits of conjugate vaccines in improving the protective responses of the immature immune systems of young infants and the senescent immune systems of the elderly have been made clear and opened the way to development of additional vaccines using this technology for future vaccine products. PMID:23955057

  6. RNA-guided Transcriptional Regulation in Plants via dCas9 Chimeric Proteins

    KAUST Repository

    Baazim, Hatoon

    2014-05-01

    Developing targeted genome regulation approaches holds much promise for accelerating trait discovery and development in agricultural biotechnology. Clustered Regularly Interspaced Palindromic Repeats (CRISPRs)/CRISPR associated (Cas) system provides bacteria and archaea with an adaptive molecular immunity mechanism against invading nucleic acids through phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing purposes across a variety of cell types and organisms. Recently, the catalytically inactive Cas9 (dCas9) protein combined with guide RNAs (gRNAs) were used as a DNA-targeting platform to modulate the expression patterns in bacterial, yeast and human cells. Here, we employed this DNA-targeting system for targeted transcriptional regulation in planta by developing chimeric dCas9-based activators and repressors. For example, we fused to the C-terminus of dCas9 with the activation domains of EDLL and TAL effectors, respectively, to generate transcriptional activators, and the SRDX repression domain to generate transcriptional repressor. Our data demonstrate that the dCas9:EDLL and dCas9:TAD activators, guided by gRNAs complementary to promoter elements, induce strong transcriptional activation on episomal targets in plant cells. Moreover, our data suggest that the dCas9:SRDX repressor and the dCas9:EDLL and dCas9:TAD activators are capable of markedly repressing or activating, respectively, the transcription of an endogenous genomic target. Our data indicate that the CRISPR/dCas9:TFs DNA targeting system can be used in plants as a functional genomic tool and for biotechnological applications.

  7. Autophosphorylation-dependent inactivation of plant chimeric calcium/calmodulin-dependent protein kinase

    Science.gov (United States)

    Sathyanarayanan, P. V.; Poovaiah, B. W.

    2002-01-01

    Chimeric calcium/calmodulin dependent protein kinase (CCaMK) is characterized by the presence of a visinin-like Ca(2+)-binding domain unlike other known calmodulin- dependent kinases. Ca(2+)-Binding to the visinin-like domain leads to autophosphorylation and changes in the affinity for calmodulin [Sathyanarayanan P.V., Cremo C.R. & Poovaiah B.W. (2000) J. Biol. Chem. 275, 30417-30422]. Here, we report that the Ca(2+)-stimulated autophosphorylation of CCaMK results in time-dependent loss of enzyme activity. This time-dependent loss of activity or self-inactivation due to autophosphorylation is also dependent on reaction pH and ATP concentration. Inactivation of the enzyme resulted in the formation of a sedimentable enzyme due to self-association. Specifically, autophosphorylation in the presence of 200 microm ATP at pH 7.5 resulted in the formation of a sedimentable enzyme with a 33% loss in enzyme activity. Under similar conditions at pH 6.5, the enzyme lost 67% of its activity and at pH 8.5, 84% enzyme activity was lost. Furthermore, autophosphorylation at either acidic or alkaline reaction pH lead to the formation of a sedimentable enzyme. Transmission electron microscopic studies on autophosphorylated kinase revealed particles that clustered into branched complexes. The autophosphorylation of wild-type kinase in the presence of AMP-PNP (an unhydrolyzable ATP analog) or the autophosphorylation-site mutant, T267A, did not show formation of branched complexes under the electron microscope. Autophosphorylation- dependent self-inactivation may be a mechanism of modulating the signal transduction pathway mediated by CCaMK.

  8. Chimeric human parainfluenza virus bearing the Ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against Ebola virus challenge

    International Nuclear Information System (INIS)

    We generated a new live-attenuated vaccine against Ebola virus (EBOV) based on a chimeric virus HPIV3/ΔF-HN/EboGP that contains the EBOV glycoprotein (GP) as the sole transmembrane envelope protein combined with the internal proteins of human parainfluenza virus type 3 (HPIV3). Electron microscopy analysis of the virus particles showed that they have an envelope and surface spikes resembling those of EBOV and a particle size and shape resembling those of HPIV3. When HPIV3/ΔF-HN/EboGP was inoculated via apical surface of an in vitro model of human ciliated airway epithelium, the virus was released from the apical surface; when applied to basolateral surface, the virus infected basolateral cells but did not spread through the tissue. Following intranasal (IN) inoculation of guinea pigs, scattered infected cells were detected in the lungs by immunohistochemistry, but infectious HPIV3/ΔF-HN/EboGP could not be recovered from the lungs, blood, or other tissues. Despite the attenuation, the virus was highly immunogenic, and a single IN dose completely protected the animals against a highly lethal intraperitoneal challenge of guinea pig-adapted EBOV

  9. The neurovirulence and neuroinvasiveness of chimeric tick-borne encephalitis/dengue virus can be attenuated by introducing defined mutations into the envelope and NS5 protein genes and the 3' non-coding region of the genome

    International Nuclear Information System (INIS)

    Tick-borne encephalitis (TBE) is a severe disease affecting thousands of people throughout Eurasia. Despite the use of formalin-inactivated vaccines in endemic areas, an increasing incidence of TBE emphasizes the need for an alternative vaccine that will induce a more durable immunity against TBE virus (TBEV). The chimeric attenuated virus vaccine candidate containing the structural protein genes of TBEV on a dengue virus genetic background (TBEV/DEN4) retains a high level of neurovirulence in both mice and monkeys. Therefore, attenuating mutations were introduced into the envelope (E315) and NS5 (NS5654,655) proteins, and into the 3' non-coding region (Δ30) of TBEV/DEN4. The variant that contained all three mutations (vΔ30/E315/NS5654,655) was significantly attenuated for neuroinvasiveness and neurovirulence and displayed a reduced level of replication and virus-induced histopathology in the brains of mice. The high level of safety in the central nervous system indicates that vΔ30/E315/NS5654,655 should be further evaluated as a TBEV vaccine.

  10. The rapid generation of chimerical genes expanding protein diversity in zebrafish

    Directory of Open Access Journals (Sweden)

    Zou Ming

    2010-11-01

    Full Text Available Abstract Background Variation of gene number among species indicates that there is a general process of new gene origination. One of the major mechanism providing raw materials for the origin of new genes is gene duplication. Retroposition, as a special type of gene duplication- the RNA-based duplication, has been found to play an important role in new gene evolution in mammals and plants, but little is known about the process in the teleostei genome. Results Here we screened the zebrafish genome for identification of retrocopies and new chimerical retrogenes and investigated their origination and evolution. We identified 652 retrocopies, of which 440 are intact retrogenes and 212 are pseudogenes. Retrocopies have long been considered evolutionary dead ends without functional significance due to the presumption that retrocopies lack the regulatory element needed for expression. However, 437 transcribed retrocopies were identified from all of the retrocopies. This discovery combined with the substitution analysis suggested that the majority of all retrocopies are subject to negative selection, indicating that most of the retrocopies may be functional retrogenes. Moreover, we found that 95 chimerical retrogenes had recruited new sequences from neighboring genomic regions that formed de novo splice sites, thus generating new intron-containing chimeric genes. Based on our analysis of 38 pairs of orthologs between Cyprinus carpio and Danio rerio, we found that the synonymous substitution rate of zebrafish genes is 4.13×10-9 substitution per silent site per year. We also found 10 chimerical retrogenes that were created in the last 10 million years, which is 7.14 times the rate of 0.14 chimerical retrogenes per million years in the primate lineage toward human and 6.25 times the rate of 0.16 chimerical genes per million years in Drosophila. This is among the most rapid rates of generation of chimerical genes, just next to the rice. Conclusion There is

  11. A HIV-Tat/C4-binding protein chimera encoded by a DNA vaccine is highly immunogenic and contains acute EcoHIV infection in mice.

    Science.gov (United States)

    Tomusange, Khamis; Wijesundara, Danushka; Gummow, Jason; Garrod, Tamsin; Li, Yanrui; Gray, Lachlan; Churchill, Melissa; Grubor-Bauk, Branka; Gowans, Eric J

    2016-01-01

    DNA vaccines are cost-effective to manufacture on a global scale and Tat-based DNA vaccines have yielded protective outcomes in preclinical and clinical models of human immunodeficiency virus (HIV), highlighting the potential of such vaccines. However, Tat-based DNA vaccines have been poorly immunogenic, and despite the administration of multiple doses and/or the addition of adjuvants, these vaccines are not in general use. In this study, we improved Tat immunogenicity by fusing it with the oligomerisation domain of a chimeric C4-binding protein (C4b-p), termed IMX313, resulting in Tat heptamerisation and linked Tat to the leader sequence of tissue plasminogen activator (TPA) to ensure that the bulk of heptamerised Tat is secreted. Mice vaccinated with secreted Tat fused to IMX313 (pVAX-sTat-IMX313) developed higher titres of Tat-specific serum IgG, mucosal sIgA and cell-mediated immune (CMI) responses, and showed superior control of EcoHIV infection, a surrogate murine HIV challenge model, compared with animals vaccinated with other test vaccines. Given the crucial contribution of Tat to HIV-1 pathogenesis and the precedent of Tat-based DNA vaccines in conferring some level of protection in animal models, we believe that the virologic control demonstrated with this novel multimerised Tat vaccine highlights the promise of this vaccine candidate for humans. PMID:27358023

  12. Abbreviated incubation times for human prions in mice expressing a chimeric mouse–human prion protein transgene

    OpenAIRE

    Korth, Carsten; Kaneko, Kiyotoshi; Groth, Darlene; Heye, Norbert; Telling, Glenn; Mastrianni, James; Parchi, Piero; Gambetti, Pierluigi; Will, Robert; Ironside, James; Heinrich, Cornelia; Tremblay, Patrick; Stephen J DeArmond; Prusiner, Stanley B.

    2003-01-01

    Transgenic (Tg) mouse lines that express chimeric mouse–human prion protein (PrP), designated MHu2M, are susceptible to prions from patients with sporadic Creutzfeldt–Jakob disease (sCJD). With the aim of decreasing the incubation time to fewer than 200 days, we constructed transgenes in which one or more of the nine human residues in MHu2M were changed to mouse. The construct with murine residues at positions 165 and 167 was expressed in Tg(MHu2M,M165V,E167Q) mice and resulted in shortening ...

  13. The role of bone marrow-derived cells in bone fracture repair in a green fluorescent protein chimeric mouse model

    International Nuclear Information System (INIS)

    We investigated the role of bone marrow cells in bone fracture repair using green fluorescent protein (GFP) chimeric model mice. First, the chimeric model mice were created: bone marrow cells from GFP-transgenic C57BL/6 mice were injected into the tail veins of recipient wild-type C57BL/6 mice that had been irradiated with a lethal dose of 10 Gy from a cesium source. Next, bone fracture models were created from these mice: closed transverse fractures of the left femur were produced using a specially designed device. One, three, and five weeks later, fracture lesions were extirpated for histological and immunohistochemical analyses. In the specimens collected 3 and 5 weeks after operation, we confirmed calluses showing intramembranous ossification peripheral to the fracture site. The calluses consisted of GFP- and osteocalcin-positive cells at the same site, although the femur consisted of only osteocalcin-positive cells. We suggest that bone marrow cells migrated outside of the bone marrow and differentiated into osteoblasts to make up the calluses

  14. Iron carrier proteins facilitate engraftment of allogeneic bone marrow and enduring hemopoietic chimerism in the lethally irradiated host

    International Nuclear Information System (INIS)

    Cell-free supernatants of rabbit bone marrow were fractionated, separated, and purified by Ultrogel and Superose chromatography. A single fraction promoted engraftment of allogeneic bone marrow and enduring hemopoietic chimerism across the H-2 barrier in lethally irradiated mice. This 'bio-active' fraction, analyzed by reducing SDS-PAGE electrophoresis, and transblotted on PVDF membrane, and purified by reverse-phase HPLC and SDS-PAGE electrophoresis yielded a main prealbumin band that when examined for primary structure by Edman degradation, proved to be rabbit transferrin. This was also attested by highly specific precipitation of the prealbumin band with polyclonal antibodies to rabbit transferrin. Iron-saturated human transferrin, lactotransferrin, and egg transferrin (conalbumin) were assayed in irradiated C57BL/6 mice infused with bone marrow from histoincompatible BALB/c donors. Mice treated with iron-loaded transferrins survive and develop enduring allogeneic chimerism with no discernible signs of graft-versus-host disease. Iron carrier proteins thus provide an unique means of achieving successful engraftment of allogeneic bone marrow in immunologically hostile murine H-2 combinations

  15. A Live-Attenuated Chimeric Porcine Circovirus Type 2 (PCV2) Vaccine Is Transmitted to Contact Pigs but Is Not Upregulated by Concurrent Infection with Porcine Parvovirus (PPV) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Is Efficacious in a PCV2b-PRRSV-PPV Challenge Model▿

    OpenAIRE

    Opriessnig, T.; Shen, H. G.; Pal, N; Ramamoorthy, S.; Huang, Y. W.; Lager, K M; Beach, N. M.; Halbur, P G; Meng, X. J.

    2011-01-01

    The live chimeric porcine circovirus type 2 (PCV2) vaccine with the capsid gene of the emerging subtype 2b cloned in the genomic backbone of the nonpathogenic PCV1 is attenuated in vivo and induces protective immunity against PCV2. To further determine the safety and efficacy of this experimental vaccine, we tested for evidence of pig-to-pig transmission by commingling nonvaccinated and vaccinated pigs, determined potential upregulation by simultaneous vaccination and infection with porcine p...

  16. A live-attenuated chimeric porcine circovirus type 2 (PCV2) vaccine is transmitted to contact pigs but is not upregulated by concurrent infection with porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus (PRRSV) and is efficacious in a PCV2b-PRRSV-PPV challenge model

    OpenAIRE

    Opriessnig, T.; Shen, H. G.; Pal, N; Ramamoorthy, S.; Huang, Y. W.; Lager, K M; Beach, N. M.; Halbur, P G; Meng, X. J.

    2011-01-01

    The live chimeric porcine circovirus type 2 (PCV2) vaccine with the capsid gene of the emerging subtype 2b cloned in the genomic backbone of the nonpathogenic PCV1 is attenuated in vivo and induces protective immunity against PCV2. To further determine the safety and efficacy of this experimental vaccine, we tested for evidence of pig-to-pig transmission by commingling nonvaccinated and vaccinated pigs, determined potential upregulation by simultaneous vaccination and infection with porcine p...

  17. Improving bacterial cellulose for blood vessel replacement: functionalization with a chimeric protein containing a cellulose-binding module and an adhesion peptide

    OpenAIRE

    Andrade, Fábia K.; Costa, Raquel; Domingues, Lucília; Soares, Raquel; Gama, F. M.

    2010-01-01

    Chimeric proteins containing a cellulose-binding module (CBM) and an adhesion peptide (RGD or GRGDY) were produced and used to improve the adhesion of human microvascular endothelial cells (HMEC) to bacterial cellulose (BC). The effect of these proteins on the HMEC–BC interaction was studied. The results obtained demonstrated that recombinant proteins containing adhesion sequences were able to significantly increase the attachment of HMEC to BC surfaces, especially the RGD sequenc...

  18. Recent advances in recombinant protein-based malaria vaccines.

    Science.gov (United States)

    Draper, Simon J; Angov, Evelina; Horii, Toshihiro; Miller, Louis H; Srinivasan, Prakash; Theisen, Michael; Biswas, Sumi

    2015-12-22

    Plasmodium parasites are the causative agent of human malaria, and the development of a highly effective vaccine against infection, disease and transmission remains a key priority. It is widely established that multiple stages of the parasite's complex lifecycle within the human host and mosquito vector are susceptible to vaccine-induced antibodies. The mainstay approach to antibody induction by subunit vaccination has been the delivery of protein antigen formulated in adjuvant. Extensive efforts have been made in this endeavor with respect to malaria vaccine development, especially with regard to target antigen discovery, protein expression platforms, adjuvant testing, and development of soluble and virus-like particle (VLP) delivery platforms. The breadth of approaches to protein-based vaccines is continuing to expand as innovative new concepts in next-generation subunit design are explored, with the prospects for the development of a highly effective multi-component/multi-stage/multi-antigen formulation seeming ever more likely. This review will focus on recent progress in protein vaccine design, development and/or clinical testing for a number of leading malaria antigens from the sporozoite-, merozoite- and sexual-stages of the parasite's lifecycle-including PfCelTOS, PfMSP1, PfAMA1, PfRH5, PfSERA5, PfGLURP, PfMSP3, Pfs48/45 and Pfs25. Future prospects and challenges for the development, production, human delivery and assessment of protein-based malaria vaccines are discussed. PMID:26458807

  19. Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen.

    Science.gov (United States)

    Alves, Rúbens Prince Dos Santos; Pereira, Lennon Ramos; Fabris, Denicar Lina Nascimento; Salvador, Felipe Scassi; Santos, Robert Andreata; Zanotto, Paolo Marinho de Andrade; Romano, Camila Malta; Amorim, Jaime Henrique; Ferreira, Luís Carlos de Souza

    2016-06-01

    Dengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed in Escherichia coli at large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses. PMID:27030586

  20. Nanoscale orientation and lateral organization of chimeric metal-binding green fluorescent protein on lipid membrane determined by epifluorescence and atomic force microscopy

    International Nuclear Information System (INIS)

    Epifluorescence microscopy as well as atomic force microscopy was successfully applied to explore the orientation and lateral organization of a group of chimeric green fluorescent proteins (GFPs) on lipid membrane. Incorporation of the chimeric GFP carrying Cd-binding region (His6CdBP4GFP) to the fluid phase of DPPC monolayer resulted in a strong fluorescence intensity at the air-water interface. Meanwhile, non-specific adsorption of the GFP having hexahistidine (His6GFP) led to the perturbation of the protein structure in which very low fluorescence was observed. Specific binding of both of the chimeric GFPs to immobilized zinc ions underneath the metal-chelating lipid membrane was revealed. This specific binding could be reversibly controlled by addition of metal ions or metal chelator. Binding of the chimeric GFPs to the metal-chelating lipid membrane was proven to be the end-on orientation while the side-on adsorption was contrarily noted in the absence of metal ions. Increase of lateral mobility owing to the fluidization effect on the chelating lipid membrane subsequently facilitated crystal formation. All these findings have opened up a potential approach for a specific orientation of immobilization of protein at the membrane interface. This could have accounted for a better opportunity of sensor development

  1. Ectopic bone formation cannot occur by hydroxyapatite/β-tricalcium phosphate bioceramics in green fluorescent protein chimeric mice

    International Nuclear Information System (INIS)

    Highlights: ► Firstly, chimeric mouse model could be established successfully by bone marrow transplantation after irradiation. ► Secondly, bone induction can occur in wild-type mice 90 days after implantation, but not occur in chimeric mice. ► Thirdly, destruction of immune function will block osteoinduction by calcium phosphate ceramics. - Abstract: Many studies have shown that calcium phosphate ceramics (CP) have osteoconductive and osteoinductive properties; however, the exact mechanism of bone induction has not yet been reported. This study was performed to investigate if destroying immunological function will influence osteogenesis, to explain the mechanism which is unclear. In this study, twenty C57BL/6 mice were divided into two groups (n = 10), in group 1, a hydroxyapatite/β-tricalcium phosphate (HA/β-TCP) ceramic was implanted into both the left and right leg muscles of each mouse; in group 2, ten mice experienced lethal irradiation, then were injected bone marrow (BM) cells from green fluorescent protein (GFP) transgenic mice by tail veil, after bone marrow transplantation (BMT), heart, liver, spleen, lung, kidney, and muscle were harvested for biological analysis, after the GFP chimera model was established successfully, the same HA/β-TCP ceramic was implanted into both leg muscles of each mouse immediately after irradiation. 45 and 90 days after implantation, the ceramics of the two groups were harvested to perform with hematoxylin and eosin (HE) and immunohistochemistry (IHC) staining; the results showed that there was no bone formation in group 2, while new bone tissues were detected in group 1. Our findings suggest that the BM cell from GFP transgenic mice is a good biomarker and it could set a good platform for chimera model; it also shows that BM cell is one of cell resources of bone induction, and destruction of immune function will impede osteoinduction by CP. Overall, our results may shed light on clear mechanism study of bone

  2. Ectopic bone formation cannot occur by hydroxyapatite/{beta}-tricalcium phosphate bioceramics in green fluorescent protein chimeric mice

    Energy Technology Data Exchange (ETDEWEB)

    Cheng Lijia [Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu (China); Duan Xin [Department of Orthopaedics, Chengdu Second People' s Hospital, Chengdu (China); Department of Orthopaedics, West China Hospital, Sichuan University, Chengdu (China); Xiang Zhou [Department of Orthopaedics, West China Hospital, Sichuan University, Chengdu (China); Shi Yujun; Lu Xiaofeng; Ye Feng [Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu (China); Bu Hong, E-mail: hongbu@scu.edu.cn [Key Laboratory of Transplant Engineering and Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu (China); Department of Pathology, West China Hospital, Sichuan University, Chengdu (China)

    2012-12-01

    Highlights: Black-Right-Pointing-Pointer Firstly, chimeric mouse model could be established successfully by bone marrow transplantation after irradiation. Black-Right-Pointing-Pointer Secondly, bone induction can occur in wild-type mice 90 days after implantation, but not occur in chimeric mice. Black-Right-Pointing-Pointer Thirdly, destruction of immune function will block osteoinduction by calcium phosphate ceramics. - Abstract: Many studies have shown that calcium phosphate ceramics (CP) have osteoconductive and osteoinductive properties; however, the exact mechanism of bone induction has not yet been reported. This study was performed to investigate if destroying immunological function will influence osteogenesis, to explain the mechanism which is unclear. In this study, twenty C57BL/6 mice were divided into two groups (n = 10), in group 1, a hydroxyapatite/{beta}-tricalcium phosphate (HA/{beta}-TCP) ceramic was implanted into both the left and right leg muscles of each mouse; in group 2, ten mice experienced lethal irradiation, then were injected bone marrow (BM) cells from green fluorescent protein (GFP) transgenic mice by tail veil, after bone marrow transplantation (BMT), heart, liver, spleen, lung, kidney, and muscle were harvested for biological analysis, after the GFP chimera model was established successfully, the same HA/{beta}-TCP ceramic was implanted into both leg muscles of each mouse immediately after irradiation. 45 and 90 days after implantation, the ceramics of the two groups were harvested to perform with hematoxylin and eosin (HE) and immunohistochemistry (IHC) staining; the results showed that there was no bone formation in group 2, while new bone tissues were detected in group 1. Our findings suggest that the BM cell from GFP transgenic mice is a good biomarker and it could set a good platform for chimera model; it also shows that BM cell is one of cell resources of bone induction, and destruction of immune function will impede

  3. Latent Membrane Protein 1 as a molecular adjuvant for single-cycle lentiviral vaccines

    Directory of Open Access Journals (Sweden)

    Rahmberg Andrew R

    2011-05-01

    Full Text Available Abstract Background Molecular adjuvants are a promising method to enhance virus-specific immune responses and protect against HIV-1 infection. Immune activation by ligands for receptors such as CD40 can induce dendritic cell activation and maturation. Here we explore the incorporation of two CD40 mimics, Epstein Barr Virus gene LMP1 or an LMP1-CD40 chimera, into a strain of SIV that was engineered to be limited to a single cycle of infection. Results Full length LMP1 or the chimeric protein LMP1-CD40 was cloned into the nef-locus of single-cycle SIV. Human and Macaque monocyte derived macrophages and DC were infected with these viruses. Infected cells were analyzed for activation surface markers by flow cytometry. Cells were also analyzed for secretion of pro-inflammatory cytokines IL-1β, IL-6, IL-8, IL-12p70 and TNF by cytometric bead array. Conclusions Overall, single-cycle SIV expressing LMP1 and LMP1-CD40 produced a broad and potent TH1-biased immune response in human as well as rhesus macaque macrophages and DC when compared with control virus. Single-cycle SIV-LMP1 also enhanced antigen presentation by lentiviral vector vaccines, suggesting that LMP1-mediated immune activation may enhance lentiviral vector vaccines against HIV-1.

  4. Chimeric Plant Calcium/Calmodulin-Dependent Protein Kinase Gene with a Neural Visinin-Like Calcium-Binding Domain

    Science.gov (United States)

    Patil, Shameekumar; Takezawa, D.; Poovaiah, B. W.

    1995-01-01

    Calcium, a universal second messenger, regulates diverse cellular processes in eukaryotes. Ca-2(+) and Ca-2(+)/calmodulin-regulated protein phosphorylation play a pivotal role in amplifying and diversifying the action of Ca-2(+)- mediated signals. A chimeric Ca-2(+)/calmodulin-dependent protein kinase (CCaMK) gene with a visinin-like Ca-2(+)- binding domain was cloned and characterized from lily. The cDNA clone contains an open reading frame coding for a protein of 520 amino acids. The predicted structure of CCaMK contains a catalytic domain followed by two regulatory domains, a calmodulin-binding domain and a visinin-like Ca-2(+)-binding domain. The amino-terminal region of CCaMK contains all 11 conserved subdomains characteristic of serine/threonine protein kinases. The calmodulin-binding region of CCaMK has high homology (79%) to alpha subunit of mammalian Ca-2(+)/calmodulin-dependent protein kinase. The calmodulin-binding region is fused to a neural visinin-like domain that contains three Ca-2(+)-binding EF-hand motifs and a biotin-binding site. The Escherichia coli-expressed protein (approx. 56 kDa) binds calmodulin in a Ca-2(+)-dependent manner. Furthermore, Ca-45-binding assays revealed that CCaMK directly binds Ca-2(+). The CCaMK gene is preferentially expressed in developing anthers. Southern blot analysis revealed that CCaMK is encoded by a single gene. The structural features of the gene suggest that it has multiple regulatory controls and could play a unique role in Ca-2(+) signaling in plants.

  5. Multivalent Fusion Protein Vaccine for Lymphatiac filariasis

    OpenAIRE

    Dakshinamoorthy, Gajalakshmi; Samykutty, Abhilash Kumble; Munirathinam, Gnanasekar; Reddy, Maryada Venkatarami; Kalyanasundaram, Ramaswamy

    2012-01-01

    Lymphatic filariasis affects approximately 3% of the whole world population. Mass drug administration is currently the major control strategy to eradicate this infection from endemic regions by year 2020. Combination drug treatments are highly efficient in controlling the infection. However, there are no effective vaccines available for human or animal lymphatic filariasis despite the identification of several subunit vaccines. Lymphatic filariasis parasites are multicellular organisms and po...

  6. Generation and evaluation of a chimeric classical swine fever virus expressing a visible marker gene.

    Science.gov (United States)

    Li, Yongfeng; Wang, Xiao; Sun, Yuan; Li, Lian-Feng; Zhang, Lingkai; Li, Su; Luo, Yuzi; Qiu, Hua-Ji

    2016-03-01

    Classical swine fever virus (CSFV) is a noncytopathogenic virus, and the incorporation of an enhanced green fluorescent protein (EGFP) tag into the viral genome provides a means of direct monitoring of viral infection without immunostaining. It is well established that the 3' untranslated region (3'-UTR) of the CSFV plays an important role in viral RNA replication. Although CSFV carrying a reporter gene and chimeric CSFV have been generated and evaluated, a chimeric CSFV with a visible marker has not yet been reported. Here, we generated and evaluated a chimeric virus containing the EGFP tag and the 3'-UTR from vaccine strain HCLV (C-strain) in the genetic background of the highly virulent CSFV Shimen strain. The chimeric marker CSFV was fluorescent and had an approximately 100-fold lower viral titer, lower replication level of viral genome, and weaker fluorescence intensity than the recombinant CSFV with only the EGFP tag or the parental virus. Furthermore, the marker chimera was avirulent and displayed no viremia in inoculated pigs, which were completely protected from lethal CSFV challenge as early as 15 days post-inoculation. The chimeric marker virus was visible in vitro and attenuated in vitro and in vivo, which suggests that CSFV can be engineered to produce attenuated variants with a visible marker to facilitate in vitro studies of CSFV infection and replication and to develop of novel vaccines against CSF. PMID:26614259

  7. A chimeric affinity tag for efficient expression and chromatographic purification of heterologous proteins from plants

    OpenAIRE

    Frank eSainsbury; Philippe V. Jutras; Juan eVorster; Marie-Claire eGoulet; Dominique eMichaud

    2016-01-01

    The use of plants as expression hosts for recombinant proteins is an increasingly attractive option for the production of complex and challenging biopharmaceuticals. Tools are needed at present to marry recent developments in high-yielding gene vectors for heterologous expression with routine protein purification techniques. In this study we designed the Cysta-tag, a new purification tag for immobilized metal affinity chromatography (IMAC) of plant-made proteins based on the protein-stabilizi...

  8. Preclinical studies on new proteins as carrier for glycoconjugate vaccines.

    Science.gov (United States)

    Tontini, M; Romano, M R; Proietti, D; Balducci, E; Micoli, F; Balocchi, C; Santini, L; Masignani, V; Berti, F; Costantino, P

    2016-07-29

    Glycoconjugate vaccines are made of carbohydrate antigens covalently bound to a carrier protein to enhance their immunogenicity. Among the different carrier proteins tested in preclinical and clinical studies, five have been used so far for licensed vaccines: Diphtheria and Tetanus toxoids, the non-toxic mutant of diphtheria toxin CRM197, the outer membrane protein complex of Neisseria meningitidis serogroup B and the Protein D derived from non-typeable Haemophilus influenzae. Availability of novel carriers might help to overcome immune interference in multi-valent vaccines containing several polysaccharide-conjugate antigens, and also to develop vaccines which target both protein as well saccharide epitopes of the same pathogen. Accordingly we have conducted a study to identify new potential carrier proteins. Twenty-eight proteins, derived from different bacteria, were conjugated to the model polysaccharide Laminarin and tested in mice for their ability in inducing antibodies against the carbohydrate antigen and eight of them were subsequently tested as carrier for serogroup meningococcal C oligosaccharides. Four out of these eight were able to elicit in mice satisfactory anti meningococcal serogroup C titers. Based on immunological evaluation, the Streptococcus pneumoniae protein spr96/2021 was successfully evaluated as carrier for serogroups A, C, W, Y and X meningococcal capsular saccharides. PMID:27317455

  9. Influenza A virus hemagglutinin protein subunit vaccine elicits vaccine-associated enhanced respiratory disease in pigs.

    Science.gov (United States)

    Rajão, Daniela S; Loving, Crystal L; Gauger, Phillip C; Kitikoon, Pravina; Vincent, Amy L

    2014-09-01

    Vaccine-associated enhanced respiratory disease (VAERD) can occur when pigs are challenged with heterologous virus in the presence of non-neutralizing but cross-reactive antibodies elicited by whole inactivated virus (WIV) vaccine. The aim of this study was to compare the effects of heterologous δ1-H1N2 influenza A virus (IAV) challenge of pigs after vaccination with 2009 pandemic H1N1 virus (H1N1pdm09) recombinant hemagglutinin (HA) subunit vaccine (HA-SV) or temperature-sensitive live attenuated influenza virus (LAIV) vaccine, and to assess the role of immunity to HA in the development of VAERD. Both HA-SV and LAIV vaccines induced high neutralizing antibodies to virus with homologous HA (H1N1pdm09), but not heterologous challenge virus (δ1-H1N2). LAIV partially protected pigs, resulting in reduced virus shedding and faster viral clearance, as no virus was detected in the lungs by 5 days post infection (dpi). HA-SV vaccinated pigs developed more severe lung and tracheal lesions consistent with VAERD following challenge. These results demonstrate that the immune response against the HA protein alone is sufficient to cause VAERD following heterologous challenge. PMID:25077416

  10. Analysis of serpin inhibitory function by mutagenesis of ovalbumin and generation of chimeric ovalbumin/PAI-2 fusion proteins.

    Science.gov (United States)

    McCarthy, B J; Worrall, D M

    1997-04-01

    Ovalbumin is a non-inhibitory serpin which lacks the ability to undergo the S --> R transition or conformational change. Amino acid residues in the hinge region (P11 to P14) of ovalbumin and other non-inhibitory serpins differ from the concensus sequence of this region of inhibitory serpins, and have been proposed to be responsible for lack of inhibitory properties, particularly the P14 charged residue. Site directed mutagenesis using PCR overlap extension was performed on these residues in ovalbumin to create a mutant with three amino acid changes, R340T, V342A and V343A. However analysis of the mutant recombinant ovalbumin with the consensus residues failed to show inhibitory activity or decreased stability, indicating that the hinge region alone is not responsible for lack of inhibition. A series of three fusion proteins were then constructed by replacing varying C-terminal regions of ovalbumin with the corresponding region of the inhibitory ov-serpin PAI-2 in order to further analyse serpin inhibitory function. Fusion proteins F1 and F2 contained approximately 16% and 35% PAI-2, respectively. This resulted in the replacing of structural features such as the reactive site loop, hinge region and beta sheet strands 5A and 6A. However both fusion proteins showed no inhibitory activity with the PAI-2 target protease urokinase (uPA) and no decrease in stability as analysed by transverse urea gradient (TUG) gels. The third chimeric fusion protein constructed (F3) contained 64% PAI-2 and did demonstrate inhibition of uPA, SDS-PAGE stable complex formation with uPA and increased instability on TUG gels. Structural differences between the inactive F2 and active F3 include the replacement of helix F and beta sheet strand 3A of ovalbumin with those of PAI-2, suggesting that these features may have a key role in serpin beta-sheet opening and inhibitory function. PMID:9126838

  11. Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

    Science.gov (United States)

    Wang, W.; Poovaiah, B. W.

    1999-01-01

    A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

  12. Protein L: a novel reagent for the detection of Chimeric Antigen Receptor (CAR expression by flow cytometry

    Directory of Open Access Journals (Sweden)

    Zheng Zhili

    2012-02-01

    Full Text Available Abstract Background There has been significant progress in the last two decades on the design of chimeric antigen receptors (CAR for adoptive immunotherapy targeting tumor-associated antigens. Structurally CARs consist of a single chain antibody fragment directed against a tumor-associated antigen fused to an extracellular spacer and transmembrane domain followed by T cell cytoplasmic signaling moieties. Currently several clinical trials are underway using gene modified peripheral blood lymphocytes (PBL with CARs directed against a variety of tumor associated antigens. Despite the improvements in the design of CARs and expansion of the number of target antigens, there is no universal flow cytometric method available to detect the expression of CARs on the surface of transduced lymphocytes. Methods Currently anti-fragment antigen binding (Fab conjugates are most widely used to determine the expression of CARs on gene-modified lymphocytes by flow cytometry. The limitations of these reagents are that many of them are not commercially available, generally they are polyclonal antibodies and often the results are inconsistent. In an effort to develop a simple universal flow cytometric method to detect the expression of CARs, we employed protein L to determine the expression of CARs on transduced lymphocytes. Protein L is an immunoglobulin (Ig-binding protein that binds to the variable light chains (kappa chain of Ig without interfering with antigen binding site. Protein L binds to most classes of Ig and also binds to single-chain antibody fragments (scFv and Fab fragments. Results We used CARs derived from both human and murine antibodies to validate this novel protein L based flow cytometric method and the results correlated well with other established methods. Activated human PBLs were transduced with retroviral vectors expressing two human antibody based CARs (anti-EGFRvIII, and anti-VEGFR2, two murine antibody derived CARs (anti-CSPG4, and anti

  13. The expression and genetic immunization of chimeric fragment of Hantaan virus M and S segments

    International Nuclear Information System (INIS)

    Hemorrhagic fever with renal syndrome (HFRS), which is characterized by severe symptoms and high mortality, is caused by hantavirus. There are still no effective prophylactic vaccines directed to HFRS until now. In this research, we fused expressed G2 fragment of M segment and 0.7 kb fragment of S segment. We expect it could be a candidate vaccine. Chimeric gene G2S0.7 was first expressed in prokaryotic expression system pGEX-4T. After inducing expressed fusion proteins, GST-G2S0.7 was induced and its molecular weight was about 100 kDa. Meanwhile, the fusion protein kept the activity of its parental proteins. Further, BALB/c mice were vaccinated by the chimeric gene. ELISA, cell microculture neutralization test in vitro were used to detect the humoral immune response in immunized BALB/c mice. Lymphocyte proliferation assay was used to detect the cellular immune response. The results showed that the chimeric gene could simultaneously evoke specific antibody against nucleocapsid protein (NP) and glycoprotein (GP). And the immunized mice of every group elicited neutralizing antibodies with different titers. But the titers were low. Lymphocyte proliferation assay results showed that the stimulation indexes of splenocytes of chimeric gene to NP and GP were significantly higher than that of control. It suggested that the chimeric gene of Hantaan virus containing G2 fragment of M segment and 0.7 kb fragment of S segment could directly elicit specific anti-Hantaan virus humoral and cellular immune response in BALB/c mice

  14. Use of hepadnavirus core proteins as vaccine platforms

    OpenAIRE

    Whitacre, David C.; Lee, Byung O.; Milich, David R.

    2009-01-01

    The first virus-like particle to be tested for use as a vaccine carrier was based on the hepatitis B virus nucleocapsid protein. This viral subunit, while not infectious on its own, is a 36-nm particle that is highly immunogenic during a natural infection. The self-assembly and high degree of immunogenicity is maintained when expressed as a recombinant protein and, moreover, can confer a high degree of immunogenicity on foreign antigens linked to the particle, either chemically or genetically...

  15. Interaction analysis of chimeric metal-binding green fluorescent protein and artificial solid-supported lipid membrane by quartz crystal microbalance and atomic force microscopy

    International Nuclear Information System (INIS)

    Non-specific adsorption and specific interaction between a chimeric green fluorescent protein (GFP) carrying metal-binding region and the immobilized zinc ions on artificial solid-supported lipid membranes was investigated using the quartz crystal microbalance technique and the atomic force microscopy (AFM). Supported lipid bilayer, composed of octanethiol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-[N- (5-amino-1-carboxypentyl iminodiacetic acid)succinyl] (NTA-DOGS)-Zn2+, was formed on the gold electrode of quartz resonator (5 MHz). Binding of the chimeric GFP to zinc ions resulted in a rapid decrease of resonance frequency. Reversibility of the process was demonstrated via the removal of metal ions by EDTA. Nanoscale structural orientation of the chimeric GFP on the membrane was imaged by AFM. Association constant of the specific binding to metal ions was 2- to 3-fold higher than that of the non-specific adsorption, which was caused by the fluidization effect of the metal-chelating lipid molecules as well as the steric hindrance effect. This infers a possibility for a further development of biofunctionalized membrane. However, maximization is needed in order to attain closer advancement to a membrane-based sensor device

  16. Efficient, trans-complementing packaging systems for chimeric, pseudoinfectious dengue 2/yellow fever viruses

    International Nuclear Information System (INIS)

    In our previous studies, we have stated to build a new strategy for developing defective, pseudoinfectious flaviviruses (PIVs) and applying them as a new type of vaccine candidates. PIVs combined the efficiency of live vaccines with the safety of inactivated or subunit vaccines. The results of the present work demonstrate further development of chimeric PIVs encoding dengue virus 2 (DEN2V) glycoproteins and yellow fever virus (YFV)-derived replicative machinery as potential vaccine candidates. The newly designed PIVs have synergistically functioning mutations in the prM and NS2A proteins, which abolish processing of the latter proteins and make the defective viruses capable of producing either only noninfectious, immature and/or subviral DEN2V particles. The PIV genomes can be packaged to high titers into infectious virions in vitro using the NS1-deficient YFV helper RNAs, and both PIVs and helpers can then be passaged as two-component genome viruses at an escalating scale.

  17. Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

    Science.gov (United States)

    Wu, Jinxia; Hu, Zhangli; Wang, Chaogang; Li, Shuangfei; Lei, Anping

    2008-08-01

    To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein (GFP) was expressed in C. reinhardtii under the control of promoters: RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae: RBCS2 mediated strain Tran-I and HSP70A-RBCS2 mediated strain Tran-II. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-II was at least double of that in Tran-I. In addition, a threefold increase of GFP in Tran-II was induced by heat shock at 40°C. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.

  18. Efficient expression of green fluorescent protein (GFP) mediated by a chimeric promoter in Chlamydomonas reinhardtii

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein (GFP) was expressed in C. Reinhardtii under the control of promoters: RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae: RBCS2 mediated strain Tran-I and HSP70A-RBCS2 mediated strain Tran-II. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-II was at least double of that in Tran-I. In addition, a threefold increase of GFP in Tran-II was induced by heat shock at 40°C. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. Reinhardtii.

  19. Construction and characterization of chimeric BHIV (BIV/HIV-1) viruses carrying the bovine immunodeficiency virus gag gene

    OpenAIRE

    Zhu, Yi-Xin; Liu, Chang; Liu, Xin-Lei; Qiao, Wen-Tao; Chen, Qi-Min; Zeng, Yi; Geng, Yun-Qi

    2005-01-01

    AIM: To explore the possibility of the replacement of the gag gene between human immunodeficiency virus and bovine immunodeficiency virus, to achieve chimeric virions, and thereby gain a new kind of AIDS vaccine based on BHIV chimeric viruses.

  20. Evaluation of the protective immunity of a novel subunit fusion vaccine in a murine model of systemic MRSA infection.

    Directory of Open Access Journals (Sweden)

    Qian-Fei Zuo

    Full Text Available Staphylococcus aureus is a common commensal organism in humans and a major cause of bacteremia and hospital acquired infection. Because of the spread of strains resistant to antibiotics, these infections are becoming more difficult to treat. Therefore, exploration of anti-staphylococcal vaccines is currently a high priority. Iron surface determinant B (IsdB is an iron-regulated cell wall-anchored surface protein of S. aureus. Alpha-toxin (Hla is a secreted cytolytic pore-forming toxin. Previous studies reported that immunization with IsdB or Hla protected animals against S. aureus infection. To develop a broadly protective vaccine, we constructed chimeric vaccines based on IsdB and Hla. Immunization with the chimeric bivalent vaccine induced strong antibody and T cell responses. When the protective efficacy of the chimeric bivalent vaccine was compared to that of individual proteins in a murine model of systemic S. aureus infection, the bivalent vaccine showed a stronger protective immune response than the individual proteins (IsdB or Hla. Based on the results presented here, the chimeric bivalent vaccine affords higher levels of protection against S. aureus and has potential as a more effective candidate vaccine.

  1. An Eimeria vaccine candidate based on Eimeria tenella immune mapped protein 1 and the TLR-5 agonist Salmonella typhimurium FliC flagellin

    Energy Technology Data Exchange (ETDEWEB)

    Yin, Guangwen; Qin, Mei [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Liu, Xianyong [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Key Laboratory of Zoonosis, China Ministry of Agriculture and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Suo, Jingxia; Tang, Xinming; Tao, Geru [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Han, Qian [Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061 (United States); Suo, Xun [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Key Laboratory of Zoonosis, China Ministry of Agriculture and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Wu, Wenxue, E-mail: labboard@126.com [National Animal Protozoa Laboratory and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China); Key Laboratory of Zoonosis, China Ministry of Agriculture and College of Veterinary Medicine, China Agricultural University, Beijing 100193 (China)

    2013-10-25

    Highlights: •We found a new protective protein – (IMPI) in Eimeria tenella. •EtIMP1-flagellin fusion protein is an effective immunogen against Eimeria infection. •Flagellin can be as an apicomplexan parasite vaccine adjuvant in chickens. -- Abstract: Immune mapped protein-1 (IMP1) is a new protective protein in apicomplexan parasites, and exits in Eimeria tenella. But its structure and immunogenicity in E. tenella are still unknown. In this study, IMPI in E. tenella was predicted to be a membrane protein. To evaluate immunogenicity of IMPI in E. tenella, a chimeric subunit vaccine consisting of E. tenella IMP1 (EtIMP1) and a molecular adjuvant (a truncated flagellin, FliC) was constructed and over-expressed in Escherichia coli and its efficacy against E. tenella infection was evaluated. Three-week-old AA broiler chickens were vaccinated with the recombinant EtIMP1-truncated FliC without adjuvant or EtIMP1 with Freund’s Complete Adjuvant. Immunization of chickens with the recombinant EtIMP1-truncated FliC fusion protein resulted in stronger cellular immune responses than immunization with only recombinant EtIMP1 with adjuvant. The clinical effect of the EtIMP1-truncated FliC without adjuvant was also greater than that of the EtIMP1 with adjuvant, which was evidenced by the differences between the two groups in body weight gain, oocyst output and caecal lesions of E. tenella-challenged chickens. The results suggested that the EtIMP1-flagellin fusion protein can be used as an effective immunogen in the development of subunit vaccines against Eimeria infection. This is the first demonstration of antigen-specific protective immunity against avian coccidiosis using a recombinant flagellin as an apicomplexan parasite vaccine adjuvant in chickens.

  2. An Eimeria vaccine candidate based on Eimeria tenella immune mapped protein 1 and the TLR-5 agonist Salmonella typhimurium FliC flagellin

    International Nuclear Information System (INIS)

    Highlights: •We found a new protective protein – (IMPI) in Eimeria tenella. •EtIMP1-flagellin fusion protein is an effective immunogen against Eimeria infection. •Flagellin can be as an apicomplexan parasite vaccine adjuvant in chickens. -- Abstract: Immune mapped protein-1 (IMP1) is a new protective protein in apicomplexan parasites, and exits in Eimeria tenella. But its structure and immunogenicity in E. tenella are still unknown. In this study, IMPI in E. tenella was predicted to be a membrane protein. To evaluate immunogenicity of IMPI in E. tenella, a chimeric subunit vaccine consisting of E. tenella IMP1 (EtIMP1) and a molecular adjuvant (a truncated flagellin, FliC) was constructed and over-expressed in Escherichia coli and its efficacy against E. tenella infection was evaluated. Three-week-old AA broiler chickens were vaccinated with the recombinant EtIMP1-truncated FliC without adjuvant or EtIMP1 with Freund’s Complete Adjuvant. Immunization of chickens with the recombinant EtIMP1-truncated FliC fusion protein resulted in stronger cellular immune responses than immunization with only recombinant EtIMP1 with adjuvant. The clinical effect of the EtIMP1-truncated FliC without adjuvant was also greater than that of the EtIMP1 with adjuvant, which was evidenced by the differences between the two groups in body weight gain, oocyst output and caecal lesions of E. tenella-challenged chickens. The results suggested that the EtIMP1-flagellin fusion protein can be used as an effective immunogen in the development of subunit vaccines against Eimeria infection. This is the first demonstration of antigen-specific protective immunity against avian coccidiosis using a recombinant flagellin as an apicomplexan parasite vaccine adjuvant in chickens

  3. Chimeric classical swine fever (CSF)-Japanese encephalitis (JE) viral particles as a non-transmissible bivalent marker vaccine candidate against CSF and JE infections

    Science.gov (United States)

    A trans-complemented CSF- JE chimeric viral replicon was constructed using an infectious cDNA clone of the CSF virus (CSFV) Alfort/187 strain. The E2 gene of CSFV Alfort/187 strain was deleted and the resultant plasmid pA187delE2 was inserted by a fragment containing the region coding for a truncate...

  4. Development of antifertility vaccine using sperm specific proteins

    Directory of Open Access Journals (Sweden)

    A H Bandivdekar

    2014-01-01

    Full Text Available Sperm proteins are known to be associated with normal fertilization as auto- or iso-antibodies to these proteins may cause infertility. Therefore, sperm proteins have been considered to be the potential candidate for the development of antifertility vaccine. Some of the sperm proteins proved to be promising antigens for contraceptive vaccine includes lactate dehydrogenase (LDH-C4, protein hyaluronidase (PH-20, and Eppin. Immunization with LDH-C4 reduced fertility in female baboons but not in female cynomolgus macaques. Active immunization with PH-20 resulted in 100 per cent inhibition of fertility in male guinea pigs but it induced autoimmune orchitis. Immunization with Eppin elicited high antibody titres in 78 per cent of immunized monkeys and induced infertility but the immunopathological effect of immunization was not examined. Human sperm antigen (80kDa HSA is a sperm specific, highly immunogenic and conserved sperm protein. Active immunization with 80kDa HSA induced immunological infertility in male and female rats. Partial N-terminal amino acid sequence of 80kDa HSA (Peptide NT and its peptides (Peptides 1, 2, 3 and 4 obtained by enzymatic digestion did not show homology with any of the known proteins in gene bank. Peptides NT, 1, 2 and 4 were found to mimic immunobiological activity of native protein. Passive administration of antibodies to peptides NT, 1, 2 and 4 induced infertility in male and female rats and peptide 1 was found to be most effective in suppressing fertility. Active immunization with keyhole limpet haemocynin (KLH conjugated synthetic peptide 1 impaired fertility in all the male rabbits and six of the seven male marmosets. The fertility was restored following decline in antibody titre. All these findings on 80kDA HAS suggest that the synthetic Peptide-1 of 80kDa HSA is the promising candidate for development of male contraceptive vaccine.

  5. Development of antifertility vaccine using sperm specific proteins.

    Science.gov (United States)

    Bandivdekar, A H

    2014-11-01

    Sperm proteins are known to be associated with normal fertilization as auto- or iso-antibodies to these proteins may cause infertility. Therefore, sperm proteins have been considered to be the potential candidate for the development of antifertility vaccine. Some of the sperm proteins proved to be promising antigens for contraceptive vaccine includes lactate dehydrogenase (LDH-C4), protein hyaluronidase (PH-20), and Eppin. Immunization with LDH-C4 reduced fertility in female baboons but not in female cynomolgus macaques. Active immunization with PH-20 resulted in 100 per cent inhibition of fertility in male guinea pigs but it induced autoimmune orchitis. Immunization with Eppin elicited high antibody titres in 78 per cent of immunized monkeys and induced infertility but the immunopathological effect of immunization was not examined. Human sperm antigen (80 kDa HSA) is a sperm specific, highly immunogenic and conserved sperm protein. Active immunization with 80 kDa HSA induced immunological infertility in male and female rats. Partial N-terminal amino acid sequence of 80 kDa HSA (Peptide NT) and its peptides (Peptides 1, 2, 3 and 4) obtained by enzymatic digestion did not show homology with any of the known proteins in gene bank. Peptides NT, 1, 2 and 4 were found to mimic immunobiological activity of native protein. Passive administration of antibodies to peptides NT, 1, 2 and 4 induced infertility in male and female rats and peptide 1 was found to be most effective in suppressing fertility. Active immunization with keyhole limpet haemocynin (KLH) conjugated synthetic peptide 1 impaired fertility in all the male rabbits and six of the seven male marmosets. The fertility was restored following decline in antibody titre. All these findings on 80 kDA HAS suggest that the synthetic Peptide-1 of 80 kDa HSA is the promising candidate for development of male contraceptive vaccine. PMID:25673547

  6. Effective polymer adjuvants for sustained delivery of protein subunit vaccines.

    Science.gov (United States)

    Adams, Justin R; Haughney, Shannon L; Mallapragada, Surya K

    2015-03-01

    We have synthesized thermogelling cationic amphiphilic pentablock copolymers that have the potential to act as injectable vaccine carriers and adjuvants that can simultaneously provide sustained delivery and enhance the immunogenicity of released antigen. While these pentablock copolymers have shown efficacy in DNA delivery in past studies, the ability to deliver both DNA and protein for subunit vaccines using the same polymeric carrier can provide greater flexibility and efficacy. We demonstrate the ability of these pentablock copolymers, and the parent triblock Pluronic copolymers to slowly release structurally intact and antigenically stable protein antigens in vitro, create an antigen depot through long-term injection-site persistence and enhance the in vivo immune response to these antigens. We show release of the model protein antigen ovalbumin in vitro from the thermogelling block copolymers with the primary, secondary and tertiary structures of the released protein unchanged compared to the native protein, and its antigenicity preserved upon release. The block copolymers form a gel at physiological temperatures that serves as an antigenic depot and persists in vivo at the site of injection for over 50days. The pentablock copolymers show a significant fivefold enhancement in the immune response compared to soluble protein alone, even 6weeks after the administration, based on measurement of antibody titers. These results demonstrate the potential of these block copolymers hydrogels to persist for several weeks and sustain the release of antigen with minimal effects on protein stability and antigenicity; and their ability to be used simultaneously as a sustained delivery device as well as a subunit vaccine adjuvant platform. PMID:25484331

  7. Peptide/protein vaccine delivery system based on PLGA particles.

    Science.gov (United States)

    Allahyari, Mojgan; Mohit, Elham

    2016-03-01

    Due to the excellent safety profile of poly (D,L-lactide-co-glycolide) (PLGA) particles in human, and their biodegradability, many studies have focused on the application of PLGA particles as a controlled-release vaccine delivery system. Antigenic proteins/peptides can be encapsulated into or adsorbed to the surface of PLGA particles. The gradual release of loaded antigens from PLGA particles is necessary for the induction of efficient immunity. Various factors can influence protein release rates from PLGA particles, which can be defined intrinsic features of the polymer, particle characteristics as well as protein and environmental related factors. The use of PLGA particles encapsulating antigens of different diseases such as hepatitis B, tuberculosis, chlamydia, malaria, leishmania, toxoplasma and allergy antigens will be described herein. The co-delivery of antigens and immunostimulants (IS) with PLGA particles can prevent the systemic adverse effects of immunopotentiators and activate both dendritic cells (DCs) and natural killer (NKs) cells, consequently enhancing the therapeutic efficacy of antigen-loaded PLGA particles. We will review co-delivery of different TLR ligands with antigens in various models, highlighting the specific strengths and weaknesses of the system. Strategies to enhance the immunotherapeutic effect of DC-based vaccine using PLGA particles can be designed to target DCs by functionalized PLGA particle encapsulating siRNAs of suppressive gene, and disease specific antigens. Finally, specific examples of cellular targeting where decorating the surface of PLGA particles target orally administrated vaccine to M-cells will be highlighted. PMID:26513024

  8. Protein L: a novel reagent for the detection of Chimeric Antigen Receptor (CAR) expression by flow cytometry

    OpenAIRE

    Zheng Zhili; Chinnasamy Nachimuthu; Morgan Richard A

    2012-01-01

    Abstract Background There has been significant progress in the last two decades on the design of chimeric antigen receptors (CAR) for adoptive immunotherapy targeting tumor-associated antigens. Structurally CARs consist of a single chain antibody fragment directed against a tumor-associated antigen fused to an extracellular spacer and transmembrane domain followed by T cell cytoplasmic signaling moieties. Currently several clinical trials are underway using gene modified peripheral blood lymp...

  9. Lipidation of intact proteins produces highly immunogenic vaccine candidates.

    Science.gov (United States)

    Zeng, Weiguang; Eriksson, Emily M; Lew, Andrew; Jackson, David C

    2011-01-01

    In this study we investigate the feasibility of generating self-adjuvanting vaccines capable of inducing high titre antibody responses following the covalent attachment of the TLR2 agonist Pam(2)Cys to intact proteins. Three Pam(2)Cys-based lipid moieties were prepared which contain a solubilising spacer composed of either lysine residues or polyethyleneglycol. A model protein, hen egg white lysozyme (HEL), was lipidated individually with each of these lipid modules and the immunogenicity of the lipidated species studied in mice by measuring antibody responses. We found that lipidated HEL elicited antibodies which is much stronger than the responses obtained when the HEL was administered in Freund's adjuvant or in Alum. Little or no antibody was elicited by the lipidated HEL in CD4 T cell-deficient mice indicating that the antibody response is T cell dependent. Furthermore, the lipidated protein elicited similar antibody responses in two different strains of mice indicating that sufficient helper T cell epitopes are available to enable antibody production across the histocompatability barrier. In a similar way, lipidated bovine insulin was found to be highly immunogenic in mice despite the largely conserved sequences of bovine and murine insulin. The results provide evidence that lipidation of proteins provides a simple and safe method for the manufacture of soluble self-adjuvanting protein-based vaccines. PMID:21056473

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

    OpenAIRE

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

    2015-01-01

    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 R...

  11. 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. PMID:26790940

  12. Unexpected fold in the circumsporozoite protein target of malaria vaccines

    Energy Technology Data Exchange (ETDEWEB)

    Doud, Michael B.; Koksal, Adem C.; Mi, Li-Zhi; Song, Gaojie; Lu, Chafen; Springer, Timothy A. (Harvard-Med)

    2012-10-09

    Circumsporozoite (CS) protein is the major surface component of Plasmodium falciparum sporozoites and is essential for host cell invasion. A vaccine containing tandem repeats, region III, and thrombospondin type-I repeat (TSR) of CS is efficacious in phase III trials but gives only a 35% reduction in severe malaria in the first year postimmunization. We solved crystal structures showing that region III and TSR fold into a single unit, an '{alpha}TSR' domain. The {alpha}TSR domain possesses a hydrophobic pocket and core, missing in TSR domains. CS binds heparin, but {alpha}TSR does not. Interestingly, polymorphic T-cell epitopes map to specialized {alpha}TSR regions. The N and C termini are unexpectedly close, providing clues for sporozoite sheath organization. Elucidation of a unique structure of a domain within CS enables rational design of next-generation subunit vaccines and functional and medicinal chemical investigation of the conserved hydrophobic pocket.

  13. Cloning, expression, and purification of a highly immunogenic recombinant gonadotropin-releasing hormone (GnRH) chimeric peptide.

    Science.gov (United States)

    Xu, Jinshu; Zhu, Zheng; Duan, Peng; Li, Wenjia; Zhang, Yin; Wu, Jie; Hu, Zhuoyi; Roque, Rouel S; Liu, Jingjing

    2006-12-01

    To design an anti-gonadotropin-releasing hormone (GnRH) vaccine capable of eliciting strong immunogenicity, a gene fragment encoding a chimeric peptide was constructed using polymerase chain reaction and ligated into a novel expression vector for recombinant expression in a T7 RNA polymerase-based expression system. The chimeric peptide called GnRH3-hinge-MVP contained three linear repeats of GnRH (GnRH3), a fragment of the human IgG1 hinge region, and a T-cell epitope of measles virus protein (MVP). The expression plasmid contained the GnRH3-hinge-MVP construct ligated to its fusion partner (AnsB-C) via an unique acid labile Asp-Pro linker. The recombinant fusion protein was expressed in an inclusion body in Escherichia coli under IPTG or lactose induction and the target peptide was easily purified using washing of urea and ethanol precipitation. The target chimeric peptide was isolated from the fusion partner following acid hydrolysis and purified using DEAE-Sephacel chromatography. The purified GnRH3-hinge-MVP was determined to be highly homogeneous by IEF analysis and the N-terminal sequencing. Further, immunization of female mice with the recombinant chimeric peptide resulted in generation of high-titer antibodies specific for GnRH. The results showed that GnRH3-hinge-MVP could be considered as a candidate anti-GnRH vaccine. PMID:17064933

  14. Enhancement of protein vaccine potency by in vivo electroporation mediated intramuscular injection

    OpenAIRE

    Kang, Tae Heung; Monie, Archana; Wu, Lee S.-F.; Pang, Xiaowu; Hung, Chien-Fu; Wu, T.-C.

    2010-01-01

    Protein-based vaccines have emerged as a potentially promising approach for the generation of antigen-specific immune responses. However, due to their low immunogenicity, there is a need for innovative approaches to enhance protein-based vaccine potency. One approach to enhance protein-based vaccine potency is the employment of toll-like receptor ligands, such as CpG oligonucleotides, to activate the antigen-specific T cell immune responses. Another approach involves employing a method capabl...

  15. Vaccines and immunization strategies for dengue prevention.

    Science.gov (United States)

    Liu, Yang; Liu, Jianying; Cheng, Gong

    2016-01-01

    Dengue is currently the most significant arboviral disease afflicting tropical and sub-tropical countries worldwide. Dengue vaccines, such as the multivalent attenuated, chimeric, DNA and inactivated vaccines, have been developed to prevent dengue infection in humans, and they function predominantly by stimulating immune responses against the dengue virus (DENV) envelope (E) and nonstructural-1 proteins (NS1). Of these vaccines, a live attenuated chimeric tetravalent DENV vaccine developed by Sanofi Pasteur has been licensed in several countries. However, this vaccine renders only partial protection against the DENV2 infection and is associated with an unexplained increased incidence of hospitalization for severe dengue disease among children younger than nine years old. In addition to the virus-based vaccines, several mosquito-based dengue immunization strategies have been developed to interrupt the vector competence and effectively reduce the number of infected mosquito vectors, thus controlling the transmission of DENV in nature. Here we summarize the recent progress in the development of dengue vaccines and novel immunization strategies and propose some prospective vaccine strategies for disease prevention in the future. PMID:27436365

  16. Vaccinations

    Science.gov (United States)

    ... vaccinated? For many years, a set of annual vaccinations was considered normal and necessary for dogs and ... to protect for a full year. Consequently, one vaccination schedule will not work well for all pets. ...

  17. Protein conjugate polysaccharide vaccines: Challenges in development and global implementation

    Directory of Open Access Journals (Sweden)

    Manisha Nair

    2012-01-01

    Replacement by nonvaccine serotypes;capsule switching;time duration of the antibody protective effect following vaccination;costs of the vaccines, programme costs, lack of knowledge of the disease burden, and targeting population groups for vaccination.

  18. A DNA vaccine encoding a chimeric allergen derived from major group 1 allergens of dust mite can be used for specific immunotherapy

    OpenAIRE

    Sun, Tong; Yin, Kang; Wu, Lu-Yi; Jin, Wen-Jie; Li, Yang; Sheng, Bin; Jiang, Yu-Xin

    2014-01-01

    Immunization with DNA-based constructs has been shown to be against the antigen and the response is skewed in such a way as to ameliorate the symptoms of allergic disease. This approach is particularly useful in the treatment of allergic inflammatory diseases, such as asthma. The major group 1 allergen from house dust mites is one of the triggers of allergic asthma. This study explores whether a chimeric gene R8, derived from the major group 1 allergen of house dust mite species (Dermatophago...

  19. A Review of Pneumococcal Vaccines: Current Polysaccharide Vaccine Recommendations and Future Protein Antigens

    OpenAIRE

    Daniels, Calvin C.; Rogers, P. David; Shelton, Chasity M.

    2016-01-01

    This review describes development of currently available pneumococcal vaccines, provides summary tables of current pneumococcal vaccine recommendations in children and adults, and describes new potential vaccine antigens in the pipeline. Streptococcus pneumoniae, the bacteria responsible for pneumonia, otitis media, meningitis and bacteremia, remains a cause of morbidity and mortality in both children and adults. Introductions of unconjugated and conjugated pneumococcal polysaccharide vaccine...

  20. Immunization with Human Papillomavirus 16 L1+E2 Chimeric Capsomers Elicits Cellular Immune Response and Antitumor Activity in a Mouse Model.

    Science.gov (United States)

    López-Toledo, Gabriela; Schädlich, Lysann; Alonso-Castro, Ángel Josabad; Monroy-García, Alberto; García-Rocha, Rosario; Guido, Miriam C; Gissmann, Lutz; García-Carrancá, Alejandro

    2016-06-01

    Development of cervical cancer is associated with persistent infections by high-risk human papillomavirus (HPV). Although current HPV L1-based prophylactic vaccines prevent infection, they do not help to eliminate prevalent infections or lesions. Our aims were (i) to generate a vaccine combining prophylactic and therapeutic properties by producing chimeric capsomers after fusion of the L1 protein to different fragments of E2 from HPV 16, and (ii) to evaluate their capacity to generate an antitumoral cellular response, while conserving L1 neutralizing epitopes. Chimeric proteins were produced in Escherichia coli and purified by glutathione S-transferase (GST)-affinity chromatography. Their structure was characterized using size exclusion chromatography, sucrose gradient centrifugation, electron microscopy, and anti-L1 enzyme-linked immunosorbent assay. All chimeric proteins form capsomers and heterogeneous aggregates. One, containing part of the carboxy-terminal domain of E2 and its hinge region (L1Δ+E2H/NC, aa 206-307), conserved the neutralizing epitope H16.V5. We then evaluated the capacity of this chimeric protein to induce a cytotoxic T-cell response against HPV 16 E2. In (51)Cr release cytotoxicity assays, splenocytes from C57BL/6 immunized mice recognized and lysed TC-1/E2 cells, which express and present endogenously processed E2 peptides. Moreover, this E2-specific cytotoxic response inhibited the growth of tumors of TC-1/E2 cells in mice. Finally, we identified an epitope (aa 292-301) of E2 involved in this cytotoxic response. We conclude that the L1Δ+E2H/NC chimeric protein produced in bacteria can be an effective and economically interesting candidate for a combined prophylactic and therapeutic vaccine that could help eliminating HPV16-positive low-grade cervical lesions and persistent viral infections, thus preventing the development of lesions and, at the same time, the establishment of new infections. PMID:27058179

  1. Immunogenicity of next-generation HPV vaccines in non-human primates: Measles-vectored HPV vaccine versus Pichia pastoris recombinant protein vaccine.

    Science.gov (United States)

    Gupta, Gaurav; Giannino, Viviana; Rishi, Narayan; Glueck, Reinhard

    2016-09-01

    Human papillomavirus (HPV) infection is the most common sexually transmitted disease worldwide. HPVs are oncogenic small double-stranded DNA viruses that are the primary causal agent of cervical cancer and other types of cancers, including in the anus, oropharynx, vagina, vulva, and penis. Prophylactic vaccination against HPV is an attractive strategy for preventing cervical cancer and some other types of cancers. However, there are few safe and effective vaccines against HPV infections. Current first-generation commercial HPV vaccines are expensive to produce and deliver. The goal of this study was to develop an alternate potent HPV recombinant L1-based vaccines by producing HPV virus-like particles into a vaccine that is currently used worldwide. Live attenuated measles virus (MV) vaccines have a well-established safety and efficacy record, and recombinant MV (rMV) produced by reverse genetics may be useful for generating candidate HPV vaccines to meet the needs of the developing world. We studied in non-human primate rMV-vectored HPV vaccine in parallel with a classical alum adjuvant recombinant HPV16L1 and 18L1 protein vaccine produced in Pichia pastoris. A combined prime-boost approach using both vaccines was evaluated, as well as immune interference due to pre-existing immunity against the MV. The humoral immune response induced by the MV, Pichia-expressed vaccine, and their combination as priming and boosting approaches was found to elicit HPV16L1 and 18L1 specific total IgG and neutralizing antibody titres. Pre-existing antibodies against measles did not prevent the immune response against HPV16L1 and 18L1. PMID:27523740

  2. Use of a Chimeric Hsp70 to Enhance the Quality of Recombinant Plasmodium falciparum S-Adenosylmethionine Decarboxylase Protein Produced in Escherichia coli.

    Directory of Open Access Journals (Sweden)

    Xolani Henry Makhoba

    Full Text Available S-adenosylmethionine decarboxylase (PfAdoMetDC from Plasmodium falciparum is a prospective antimalarial drug target. The production of recombinant PfAdoMetDC for biochemical validation as a drug target is important. The production of PfAdoMetDC in Escherichia coli has been reported to result in unsatisfactory yields and poor quality product. The co-expression of recombinant proteins with molecular chaperones has been proposed as one way to improve the production of the former in E. coli. E. coli heat shock proteins DnaK, GroEL-GroES and DnaJ have previously been used to enhance production of some recombinant proteins. However, the outcomes were inconsistent. An Hsp70 chimeric protein, KPf, which is made up of the ATPase domain of E. coli DnaK and the substrate binding domain of P. falciparum Hsp70 (PfHsp70 has been previously shown to exhibit chaperone function when it was expressed in E. coli cells whose resident Hsp70 (DnaK function was impaired. We proposed that because of its domain constitution, KPf would most likely be recognised by E. coli Hsp70 co-chaperones. Furthermore, because it possesses a substrate binding domain of plasmodial origin, KPf would be primed to recognise recombinant PfAdoMetDC expressed in E. coli. First, using site-directed mutagenesis, followed by complementation assays, we established that KPf with a mutation in the hydrophobic residue located in its substrate binding cavity was functionally compromised. We further co-expressed PfAdoMetDC with KPf, PfHsp70 and DnaK in E. coli cells either in the absence or presence of over-expressed GroEL-GroES chaperonin. The folded and functional status of the produced PfAdoMetDC was assessed using limited proteolysis and enzyme assays. PfAdoMetDC co-expressed with KPf and PfHsp70 exhibited improved activity compared to protein co-expressed with over-expressed DnaK. Our findings suggest that chimeric KPf may be an ideal Hsp70 co-expression partner for the production of recombinant

  3. A Library of Plasmodium vivax Recombinant Merozoite Proteins Reveals New Vaccine Candidates and Protein-Protein Interactions.

    Directory of Open Access Journals (Sweden)

    Jessica B Hostetler

    2015-12-01

    Full Text Available A vaccine targeting Plasmodium vivax will be an essential component of any comprehensive malaria elimination program, but major gaps in our understanding of P. vivax biology, including the protein-protein interactions that mediate merozoite invasion of reticulocytes, hinder the search for candidate antigens. Only one ligand-receptor interaction has been identified, that between P. vivax Duffy Binding Protein (PvDBP and the erythrocyte Duffy Antigen Receptor for Chemokines (DARC, and strain-specific immune responses to PvDBP make it a complex vaccine target. To broaden the repertoire of potential P. vivax merozoite-stage vaccine targets, we exploited a recent breakthrough in expressing full-length ectodomains of Plasmodium proteins in a functionally-active form in mammalian cells and initiated a large-scale study of P. vivax merozoite proteins that are potentially involved in reticulocyte binding and invasion.We selected 39 P. vivax proteins that are predicted to localize to the merozoite surface or invasive secretory organelles, some of which show homology to P. falciparum vaccine candidates. Of these, we were able to express 37 full-length protein ectodomains in a mammalian expression system, which has been previously used to express P. falciparum invasion ligands such as PfRH5. To establish whether the expressed proteins were correctly folded, we assessed whether they were recognized by antibodies from Cambodian patients with acute vivax malaria. IgG from these samples showed at least a two-fold change in reactivity over naïve controls in 27 of 34 antigens tested, and the majority showed heat-labile IgG immunoreactivity, suggesting the presence of conformation-sensitive epitopes and native tertiary protein structures. Using a method specifically designed to detect low-affinity, extracellular protein-protein interactions, we confirmed a predicted interaction between P. vivax 6-cysteine proteins P12 and P41, further suggesting that the proteins

  4. Vaccination of Cattle with a CpG Oligodeoxynucleotide-Formulated Mycobacterial Protein Vaccine and Mycobacterium bovis BCG Induces Levels of Protection against Bovine Tuberculosis Superior to Those Induced by Vaccination with BCG Alone

    OpenAIRE

    Wedlock, D. Neil; Denis, Michel; Skinner, Margot A.; Koach, Jessica; de Lisle, Geoffrey W.; Vordermeier, H. Martin; Hewinson, R Glyn; van Drunen Littel-van den Hurk, Sylvia; Babiuk, Lorne A.; Hecker, Rolf; Buddle, Bryce M.

    2005-01-01

    The development of a subunit protein vaccine for bovine tuberculosis which could be used either in combination with Mycobacterium bovis BCG (to improve the efficacy of that vaccine) or alone would offer significant advantages over currently available strategies. A study was conducted with cattle to determine the protective efficacy of a strategy based on concurrent immunization with an M. bovis culture filtrate (CFP) vaccine and BCG compared to vaccination with either vaccine alone. One group...

  5. Enhancement of mucosal immune responses by chimeric influenza HA/SHIV virus-like particles

    International Nuclear Information System (INIS)

    To enhance mucosal immune responses using simian/human immunodeficiency virus-like particles (SHIV VLPs), we have produced novel phenotypically mixed chimeric influenza HA/SHIV VLPs and used them to immunize C57BL/6J mice intranasally. Antibody and cytotoxic T-cell (CTL) responses as well as cytokine production in both systemic and mucosal sites were compared after immunization with SHIV VLPs or chimeric HA/SHIV VLPs. By using enzyme-linked immunosorbent assay (ELISA), the levels of serum IgG and mucosal IgA to the HIV envelope protein (Env) were found to be highest in the group immunized with chimeric HA/SHIV VLPs. Furthermore, the highest titer of serum neutralizing antibody against HIV Env was found with the group immunized with chimeric HA/SHIV VLPs. Analysis of the IgG1/IgG2a ratio indicated that a TH1-oriented immune response resulted from these VLP immunizations. HA/SHIV VLP-immunized mice also showed significantly higher CTL responses than those observed in SHIV VLP-immunized mice. Moreover, a MHC class I restricted T-cell activation ELISPOT assay showed a mixed type of TH1/TH2 cytokines in the HA/SHIV VLP-immunized mice, indicating that the chimeric VLPs can enhance both humoral and cellular immune responses to the HIV Env protein at multiple mucosal and systemic sites. The results indicate that incorporation of influenza HA into heterotypic VLPs may be highly effective for targeting vaccines to mucosal surfaces

  6. HCV prototype vaccine based on hepatitis B core virus-like particles

    OpenAIRE

    Marija Mihailova

    2008-01-01

    HCV prototype vaccine based on hepatitis B core virus-like particles Abstract In the current study the C-terminally truncated HBc expression vectors were used for exposure of different hepatitis C virus (HCV) protein (core, E2, and NS3) fragments. All created chimeric constructs directed high level of recombinant protein synthesis in E.coli. However, not all chimeric proteins were able to self-assemble into virus-like particles (VLPs). HBcCterm/HVR1tetramer VLPs turned ou...

  7. Shape of Key Malaria Protein Could Help Improve Vaccine Efficacy

    Science.gov (United States)

    ... children. The development of a safe and effective malaria vaccine is critical to control malaria globally, especially given ... in July 2015 and is the first licensed malaria vaccine. In clinical trials, it was shown to protect ...

  8. Effects of RING-SH2Grb², a chimeric protein containing the E3 ligase domain of Cbl, on the EGFR pathway.

    Science.gov (United States)

    Lee, Wei-Hao; Wang, Pei-Yu; Lin, Yu-Hung; Chou, He-Yen; Lee, Yen-Hsien; Lee, Chien-Kuo; Pai, Li-Mei

    2014-12-31

    The E3 ubiquitin-protein ligase Casitas B-lineage lymphoma protein (Cbl) negatively regulates epidermal growth factor receptor (EGFR) signaling pathway in many organisms, and has crucial roles in cell growth, development and human pathologies, including lung cancers. RING-SH2Grb² a chimeric protein of 215 amino acids containing the RING domain of Cbl that provides E3 ligase activity, and the SH2 domain of Grb2 that serves as an adaptor for EGFR. In this study, we demonstrated that RING-SH2Grb² could promote the ubiquitinylation and degradation of EGFR in a human non-small cell lung carcinoma cell line H1299. Moreover, we discovered that the RING-SH2Grb² chimera promoted the internalization of ligand-bound EGFR, inhibited the growth of H1299 cells, and significantly suppressed tumor growth in a xenograft mouse model. In summary, our results revealed a potential new cancer therapeutic approach for non-small cell lung cancer. PMID:25575524

  9. COMPARED OF IMMUNOGENESITY AND PROTEIN COMPOSITION OF EXPERIMENTAL AND OFFICINAL VYROSOMAL INFLUENZA VIRUS VACCINE

    Directory of Open Access Journals (Sweden)

    Volyanskiy A.Yu

    2014-10-01

    Full Text Available Introduction Due to the high mutagenicity that distinguishes influenza A virus, there are viruses of this type of new antigenic properties, the prevention of which the immune system has no immunological memory. It is therefore important to find new ways to create and study patterns of immunological impact of influenza vaccines. The success of liposomal vaccines and the growing interest in their improvement can be attributed to a number of key advantages over other systems of delivery of antigens and enhance the immune response. The main advantage of liposomes is their safety and good tolerance, as demonstrated by the use of approved based on liposomes of anticancer and anti-infective drugs. Material and methods As part of the search for new ways to create, study functional characteristics and patterns of immunological impact of influenza vaccine immunogenicity was investigated newly formed liposomal vaccine "Lipos 2" compared to the trivalent vaccine officinal virosomal vaccine Infleksal and analyzed their protein composition using bioanalyzer «Agilent 2100." Immunogenicity of vaccines studied by hemagglutination inhibition test (HIT with a specific antigen (influenza virus. The composition of proteins containing vaccine was studied using bioanalyzer «Agilent-2100» («Agilent Technologies», USA using the method of SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis. Analysis on «Agilent-2100" conducted by using chips to analyze 12 samples simultaneously studied vaccines. The protein composition of the vaccine was determined by elektrophoregram receiving data on molecular weight protein fragments, their concentration and percentage of study medication. Also, determine the average number of proteins in vaccine strain. Results and discussion Considerable experimental vaccine immunogenicity, which caused a persistent increase in the level of specific antibodies to all three viral antigens in most experimental animals compared

  10. Plant chimeric Ca2+/Calmodulin-dependent protein kinase. Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

    Science.gov (United States)

    Sathyanarayanan, P. V.; Cremo, C. R.; Poovaiah, B. W.

    2000-01-01

    Chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca(2+)-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known calmodulin-dependent kinases. Biological functions of the plant visinin-like proteins or visinin-like domains in plant proteins are not well known. Using EF-hand deletions in the visinin-like domain, we found that the visinin-like domain regulated Ca(2+)-stimulated autophosphorylation of CCaMK. To investigate the effects of Ca(2+)-stimulated autophosphorylation on the interaction with calmodulin, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated calmodulin. Binding was 8-fold tighter after Ca(2+)-stimulated autophosphorylation. This shift in affinity did not occur in CCaMK deletion mutants lacking Ca(2+)-stimulated autophosphorylation. A variable calmodulin affinity regulated by Ca(2+)-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and calmodulin-dependent protein kinases. Our experiments demonstrate the existence of two functional molecular switches in a protein kinase regulating the kinase activity, namely a visinin-like domain acting as a Ca(2+)-triggered switch and a CaM-binding domain acting as an autophosphorylation-triggered molecular switch.

  11. Immunization against Rumen Methanogenesis by Vaccination with a New Recombinant Protein.

    Science.gov (United States)

    Zhang, Litai; Huang, Xiaofeng; Xue, Bai; Peng, Quanhui; Wang, Zhisheng; Yan, Tianhai; Wang, Lizhi

    2015-01-01

    Vaccination through recombinant proteins against rumen methanogenesis provides a mitigation approach to reduce enteric methane (CH4) emissions in ruminants. The objective of present study was to evaluate the in vivo efficacy of a new vaccine candidate protein (EhaF) on methanogenesis and microbial population in the rumen of goats. We amplified the gene mru 1407 encoding protein EhaF using fresh rumen fluid samples of mature goats and successfully expressed recombinant protein (EhaF) in Escherichia coli Rosetta. This product was evaluated using 12 mature goats with half for control and other half injected with 400ug/goat the purified recombinant protein in day 1 and two subsequent booster immunizations in day 35 and 49. All measurements were undertaken from 63 to 68 days after the initial vaccination, with CH4 emissions determined using respiration calorimeter chambers. The results showed that the vaccination caused intensive immune responses in serum and saliva, although it had no significant effect on total enteric CH4 emissions and methanogen population in the rumen, when compared with the control goats. However, the vaccination altered the composition of rumen bacteria, especially the abundance of main phylum Firmicutes and genus Prevotella. The results indicate that protein EhaF might not be an effective vaccine to reduce enteric CH4 emissions but our vaccine have potential to influence the rumen ecosystem of goats. PMID:26445479

  12. Electroejaculation of chimeric rats

    OpenAIRE

    McCoy, Marina R.; Montonye, Daniel; Bryda, Elizabeth C.

    2013-01-01

    With the advent of genetic engineering of rodents came the need to assess fertility and germline competency, especially in chimeric rodents generated using embryonic stem cells. Traditional methods rely on natural mating and progeny testing, which is time- and cost-intensive. Electroejaculation is a faster method of collecting sperm for genetic analysis and offers the additional benefit of using fewer animals. This column describes a refined electroejaculation technique for chimeric rats usin...

  13. An avirulent chimeric Pestivirus with altered cell tropism protects pigs against lethal infection with classical swine fever virus

    International Nuclear Information System (INIS)

    A chimeric Pestivirus was constructed using an infectious cDNA clone of bovine viral diarrhea virus (BVDV) [J. Virol. 70 (1996) 8606]. After deletion of the envelope protein E2-encoding region, the respective sequence of classical swine fever virus (CSFV) strain Alfort 187 was inserted in-frame resulting in plasmid pA/CP7E2alf. After transfection of in vitro-transcribed CP7E2alf RNA, autonomous replication of chimeric RNA in bovine and porcine cell cultures was observed. Efficient growth of chimeric CP7E2alf virus, however, could only be demonstrated on porcine cells, and in contrast to the parental BVDV strain CP7, CP7E2alf only inefficiently infected and propagated in bovine cells. The virulence, immunogenicity, and 'marker vaccine' properties of the generated chimeric CP7E2alf virus were determined in an animal experiment using 27 pigs. After intramuscular inoculation of 1 x 107 TCID50, CP7E2alf proved to be completely avirulent, and neither viremia nor virus transmission to contact animals was observed; however, CSFV-specific neutralizing antibodies were detected from day 11 after inoculation. In addition, sera from all animals reacted positive in an E2-specific CSFV-antibody ELISA, but were negative for CSFV-ERNS-specific antibodies as determined with a CSFV marker ELISA. After challenge infection with highly virulent CSFV strain Eystrup, pigs immunized with CP7E2alf were fully protected against clinical signs of CSFV infection, viremia, and shedding of challenge virus, and almost all animals scored positive in a CSFV marker ELISA. From our results, we conclude that chimeric CP7E2alf may not only serve as a tool for a better understanding of Pestivirus attachment, entry, and assembly, but also represents an innocuous and efficacious modified live CSFV 'marker vaccine'

  14. The yellow fever 17D vaccine virus as a vector for the expression of foreign proteins: development of new live flavivirus vaccines

    Directory of Open Access Journals (Sweden)

    Myrna C Bonaldo

    2000-01-01

    Full Text Available The Flaviviridae is a family of about 70 mostly arthropod-borne viruses many of which are major public health problems with members being present in most continents. Among the most important are yellow fever (YF, dengue with its four serotypes and Japanese encephalitis virus. A live attenuated virus is used as a cost effective, safe and efficacious vaccine against YF but no other live flavivirus vaccines have been licensed. The rise of recombinant DNA technology and its application to study flavivirus genome structure and expression has opened new possibilities for flavivirus vaccine development. One new approach is the use of cDNAs encopassing the whole viral genome to generate infectious RNA after in vitro transcription. This methodology allows the genetic mapping of specific viral functions and the design of viral mutants with considerable potential as new live attenuated viruses. The use of infectious cDNA as a carrier for heterologous antigens is gaining importance as chimeric viruses are shown to be viable, immunogenic and less virulent as compared to the parental viruses. The use of DNA to overcome mutation rates intrinsic of RNA virus populations in conjunction with vaccine production in cell culture should improve the reliability and lower the cost for production of live attenuated vaccines. The YF virus despite a long period ignored by researchers probably due to the effectiveness of the vaccine has made a come back, both in nature as human populations grow and reach endemic areas as well as in the laboratory being a suitable model to understand the biology of flaviviruses in general and providing new alternatives for vaccine development through the use of the 17D vaccine strain.

  15. Construction of an oral recombinant DNA vaccine from H pylori neutrophil activating protein and its immunogenicity

    OpenAIRE

    Sun, Bo; Li, Zhao-Shen; Tu, Zhen-Xing; Xu, Guo-Ming; Du, Yi-qi

    2006-01-01

    AIM: To construct a live attenuated Salmonella typhimurium (S. typhimurium) strain harboring the H pylori neutrophil activating protein (HP-NAP) gene as an oral recombinant DNA vaccine, and to evaluate its immunogenicity.

  16. Influenza A virus hemagglutinin protein subunit vaccine elicits vaccine-associated enhanced respiratory disease

    Science.gov (United States)

    Vaccine-associated enhanced respiratory disease (VAERD) can occur when pigs are challenged with heterologous virus in the presence of non-neutralizing but cross-reactive antibodies elicited by whole inactivated virus (WIV) vaccine. The aim of this study was to compare the effects of heterologous del...

  17. Vaccine testing of a recombinant activation-associated secreted protein (ASP1) from Ostertagia ostertagi

    OpenAIRE

    Geldhof, Peter; Meyvis, Yves; Vercruysse, Jozef; Claerebout, Edwin

    2008-01-01

    Previous vaccination trials against the economically important cattle parasite Ostertagia ostertagi have indicated the protective capacity of activation-associated secreted proteins (ASPs). The further development of these antigens into a commercial vaccine will require their recombinant expression. The aim of the current study was to clone and express Oo-asp1 in a baculovirus expression system and to evaluate the protective capacity of the recombinant protein against an O. ostertagi challeng...

  18. Serum antibody immunoreactivity to equine zona protein after SpayVac vaccination.

    Science.gov (United States)

    Mask, Tracy A; Schoenecker, Kathryn A; Kane, Albert J; Ransom, Jason I; Bruemmer, Jason E

    2015-07-15

    Immunocontraception with porcine ZP (pZP) can be an effective means of fertility control in feral horses. Previous studies suggest that antibodies produced after pZP vaccination may both inhibit fertilization and cause follicular dysgenesis. Zonastat-H, PZP-22, and SpayVac are three pZP vaccines proposed for use in horses. Although all these vaccines contain the pZP antigen, variations in antigen preparation and vaccine formulation lead to differences in antigenic properties among them. Likewise, despite numerous efficacy and safety studies of Zonastat-H and PZP-22, the contraceptive mechanisms of SpayVac remain unclear. The preparation of pZP for SpayVac is thought to include more nonzona proteins, making it less pure than the other two vaccines. This may result in increased antigenicity of the vaccine. We therefore investigated the immunoreactivity of serum antibodies from SpayVac-vaccinated mares to equine zona protein. Western blot analyses revealed an immunoreactivity of these antibodies to protein isolated from mature equine oocytes, ZP, follicular tissues, and ovarian tissues. Immunohistochemical analyses were used to locate the binding of serum antibodies to the ZP of immature oocytes in ovarian stromal tissue. We also found serum antibodies from SpayVac-treated mares to be predominantly specific for zona protein 3. Collectively, our results suggest a model where serum antibodies produced in response to SpayVac vaccination are immunoreactive to equine zona protein in vitro. Our study lends insight into the contraceptive mechanisms underlying the infertility observed after SpayVac vaccination. PMID:25922172

  19. Novel recombinant chimeric virus-like particle is immunogenic and protective against both enterovirus 71 and coxsackievirus A16 in mice.

    Science.gov (United States)

    Zhao, Hui; Li, Hao-Yang; Han, Jian-Feng; Deng, Yong-Qiang; Zhu, Shun-Ya; Li, Xiao-Feng; Yang, Hui-Qin; Li, Yue-Xiang; Zhang, Yu; Qin, E-De; Chen, Rong; Qin, Cheng-Feng

    2015-01-01

    Hand-foot-and-mouth disease (HFMD) has been recognized as an important global public health issue, which is predominantly caused by enterovirus 71 (EV-A71) and coxsackievirus A16 (CVA16). There is no available vaccine against HFMD. An ideal HFMD vaccine should be bivalent against both EV-A71 and CVA16. Here, a novel strategy to produce bivalent HFMD vaccine based on chimeric EV-A71 virus-like particles (ChiEV-A71 VLPs) was proposed and illustrated. The neutralizing epitope SP70 within the capsid protein VP1 of EV-A71 was replaced with that of CVA16 in ChiEV-A71 VLPs. Structural modeling revealed that the replaced CVA16-SP70 epitope is well exposed on the surface of ChiEV-A71 VLPs. These VLPs produced in Saccharomyces cerevisiae exhibited similarity in both protein composition and morphology as naive EV-A71 VLPs. Immunization with ChiEV-A71 VLPs in mice elicited robust Th1/Th2 dependent immune responses against EV-A71 and CVA16. Furthermore, passive immunization with anti-ChiEV-A71 VLPs sera conferred full protection against lethal challenge of both EV-A71 and CVA16 infection in neonatal mice. These results suggested that this chimeric vaccine, ChiEV-A71 might have the potential to be further developed as a bivalent HFMD vaccine in the near future. Such chimeric enterovirus VLPs provide an alternative platform for bivalent HFMD vaccine development. PMID:25597595

  20. DNA prime-protein boost based vaccination with a conserved region of leptospiral immunoglobulin-like A and B proteins enhances protection against leptospirosis.

    Science.gov (United States)

    Forster, Karine M; Hartwig, Daiane D; Oliveira, Thaís L; Bacelo, Kátia L; Schuch, Rodrigo; Amaral, Marta G; Dellagostin, Odir A

    2015-12-01

    Leptospirosis is a zoonotic disease caused by pathogenic spirochetes of the Leptospira genus. Vaccination with bacterins has severe limitations. Here, we evaluated the N-terminal region of the leptospiral immunoglobulin-like B protein (LigBrep) as a vaccine candidate against leptospirosis using immunisation strategies based on DNA prime-protein boost, DNA vaccine, and subunit vaccine. Upon challenge with a virulent strain ofLeptospira interrogans, the prime-boost and DNA vaccine approaches induced significant protection in hamsters, as well as a specific IgG antibody response and sterilising immunity. Although vaccination with recombinant fragment of LigBrep also produced a strong antibody response, it was not immunoprotective. These results highlight the potential of LigBrep as a candidate antigen for an effective vaccine against leptospirosis and emphasise the use of the DNA prime-protein boost as an important strategy for vaccine development. PMID:26676320

  1. 人乳头瘤病毒11型L1/E7嵌合DNA疫苗的构建及其诱导的免疫效应%Construction and immunogenicity evaluation of chimerical DNA vaccine of human papillomavirus type 11

    Institute of Scientific and Technical Information of China (English)

    黄朝晖; 李莉华; 郭子健; 刘志辉; 任金冬; 宋明旭; 周希科; 王飞; 毕志刚

    2009-01-01

    目的 构建人乳头瘤病毒11型L1/E7嵌合DNA疫苗pcDNA3 L1-E7并研究其在小鼠中诱导的免疫效应.方法 用分子克隆技术构建重组真核表达质粒peDNA3 L1-E7.重组质粒DNA股四头肌注射免疫小鼠,用ELISA方法 检测L1、E7特异性抗体和脾细胞分泌的IL-2、γ-INF;MTT法检测脾淋巴细胞增殖反应.结果 成功构建pcDNA3 L1-E7.免疫小鼠后,重组质粒可诱导机体产生特异性脾淋巴细胞增殖及IL-2、γ-INF分泌增加,并诱导机体产生HPV 11-E7 IgG和HPV 11-L1 IgG抗体.结论 嵌合DNA疫苗pcDNA3 L1-E7能诱导小鼠产生特异性的细胞免疫和体液免疫反应.%Objective To construct chimerical DNA vaccine plasmid of human papiUomavirus type 11 (HPV11) L1-E7, and to evaluate its immunogenicity. Methods Molecular cloning techniques were used to construct recombinant plasmid PeDNA3 L1-E7 as a DNA vaccine. BALB/c mice were vaccinated with DNA recombinants through muscle injection. IL-2 and γ-INF secreted by immunized spleens lymphocyte and HPV 11 LI or E7 specific antibodies were assayed by ELISA method. Spleens lymphocyte proliferation was measured by MTT assay. Results The chimerical DNA plasmid of pcDNA3 LI-E7 was constructed correctly. Specific anti-HPV11 E7 and L1 antibodies, specific lymphocyte proliferation and secretions of IL-2 and γ-INF were detected in vaccinated mice. Conclusion Specific immune response, including cellular immunity and humoral immunity, could been detected in mice vaccinated with chimerical DNA vaccine of pcDNA3 L1-E7.

  2. Electroejaculation of chimeric rats.

    Science.gov (United States)

    McCoy, Marina R; Montonye, Daniel; Bryda, Elizabeth C

    2013-06-01

    With the advent of genetic engineering of rodents came the need to assess fertility and germline competency, especially in chimeric rodents generated using embryonic stem cells. Traditional methods rely on natural mating and progeny testing, which is time- and cost-intensive. Electroejaculation is a faster method of collecting sperm for genetic analysis and offers the additional benefit of using fewer animals. This column describes a refined electroejaculation technique for chimeric rats using light gas anesthesia and a custom-made platform for sperm collection. PMID:23689457

  3. Chimeric HIV-1 envelope glycoproteins with potent intrinsic granulocyte-macrophage colony-stimulating factor (GM-CSF activity.

    Directory of Open Access Journals (Sweden)

    Gözde Isik

    Full Text Available HIV-1 acquisition can be prevented by broadly neutralizing antibodies (BrNAbs that target the envelope glycoprotein complex (Env. An ideal vaccine should therefore be able to induce BrNAbs that can provide immunity over a prolonged period of time, but the low intrinsic immunogenicity of HIV-1 Env makes the elicitation of such BrNAbs challenging. Co-stimulatory molecules can increase the immunogenicity of Env and we have engineered a soluble chimeric Env trimer with an embedded granulocyte-macrophage colony-stimulating factor (GM-CSF domain. This chimeric molecule induced enhanced B and helper T cell responses in mice compared to Env without GM-CSF. We studied whether we could optimize the activity of the embedded GM-CSF as well as the antigenic structure of the Env component of the chimeric molecule. We assessed the effect of truncating GM-CSF, removing glycosylation-sites in GM-CSF, and adjusting the linker length between GM-CSF and Env. One of our designed Env(GM-CSF chimeras improved GM-CSF-dependent cell proliferation by 6-fold, reaching the same activity as soluble recombinant GM-CSF. In addition, we incorporated GM-CSF into a cleavable Env trimer and found that insertion of GM-CSF did not compromise Env cleavage, while Env cleavage did not compromise GM-CSF activity. Importantly, these optimized Env(GM-CSF proteins were able to differentiate human monocytes into cells with a macrophage-like phenotype. Chimeric Env(GM-CSF should be useful for improving humoral immunity against HIV-1 and these studies should inform the design of other chimeric proteins.

  4. PRODUCTION IN PICHIA PASTORIS AND CHARACTERIZATION OF GENETIC ENGINEERED CHIMERIC HBV/HEV VIRUS-LIKE PARTICLES

    Institute of Scientific and Technical Information of China (English)

    Hong-zhao Li; Hong-ying Gang; Qiang-ming Sun; Xiao Liu; Yan-bing Ma; Mao-sheng Sun; Chang-bai Dai

    2004-01-01

    Objective To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV)on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg).Methods The gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg.The resulted fusion gene was then integrated through transformation into the genome of Pichiapastoris under the control of a methanol-induced alcohol oxidase 1 (A OX 1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualization.Results The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichiapastoris with an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological, physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immunogenicity on surface-displayed HEnAg.Conclusion The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.

  5. Application of a Chimeric Protein Construct having Enterotoxin B and Toxic Shock Syndrome Toxin Domains of S. aureus in Immunodiagnostics

    OpenAIRE

    Shylaja, R.; Thakasi, Devi Kalyan Kumar; H.S. Murali; Reddy, K. Prakash Narayana; Batra, H. V.

    2012-01-01

    Staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 are the super antigens responsible for diseases such as staphylococcal food poisoning and toxic shock syndrome. At low serum concentrations, SEB can trigger toxic shock, profound hypotension and multi organ failure and hence is recognized as biowarfare molecule. In this study, a multidomain fusion protein (r-TE) was generated with specificity for SEB and toxic shock syndrome toxin (Tsst-1). The fusion gene comprising the cons...

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

    International Nuclear Information System (INIS)

    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⁎02:01-restricted, cytotoxic T lymphocyte (CTL) epitope derived from influenza A virus. HLA-A⁎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

  7. 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

    2016-01-01

    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. PMID:27170066

  8. 以轮状病毒VP6为载体的RV/HBV嵌合蛋白的构建及抗原性研究%Construction of a rotavirus/hepatitis B virus plasmid and the immunogenic analysis of the expressed chimeric proteins

    Institute of Scientific and Technical Information of China (English)

    袁静; 吴绪伟; 郑红梅; 曹向红; 潘小霞; 滕玉梅; 李琦涵; 陈元鼎

    2012-01-01

    Objective To construct a pETP6v carrier vector that expressed rotavirus (RV) VP6 protein and insert a gene fragment encoding the epitope of hepatitis B virus (HBV) S protein that expressed a RV/HBV chimeric protein, and to study the immunogenicity of this chimeric protein and assess its potential as a combined vaccine.Methods Using gene cloning and recombinant techniques,the gene of rotavirus VP6,the group (subgroup) of antigenic protein of the virus,was inserted into plasmid pETL to construct a carrier vector named pETP6v; then,a DNA fragment from HBV S gene that encodes the highly conserved epitope consisting of 21 amino acids (TTPAQGNSMFPSCCCSKPTDG) was inserted into the carrier vector pETP6v.The expression of this rotavirus VP6/HBV S epitope chimeric protein was analyzed in E.coli and further evaluated by ELISA and Western blot.Results A RV/HBV expression plasmid (pETP6/Hs) that carries the HBV S determinant was constructed and the RV/HBV chimeric protein expressed at very high levels in E.coli (about 36.2% of the total bacterial proteins) and the purity of the recombinant protein reached above 90%.The purified chemeric protein could react with anti-HBs serum from HBV-infected individuals and serum from guinea pigs immunized with rotavirus VP6 protein by ELISA and/or Western blot analysis.Conclusions The recombinant expression plasmid pETP6/Hs can be constructed to express chimeric proteins such as RV/HBV with high immunogenicity and reactivity and may be used in the development of RV/HBV and RV/other virus combined vaccines.%目的 构建轮状病毒(rotavirus,RV) VP6载体及携带乙型肝炎病毒(hepatitis B virus,HBV)外源抗原表位的嵌合质粒,进一步探索研发携带HBV抗原表位的重组嵌合疫苗的技术.方法 运用基因克隆和重组技术,构建轮状病毒VP6载体,同时将HBV编码中和抗原表位21个氨基酸的基因序列构建到VP6载体上相应酶切位点,在大肠杆菌中表达嵌合蛋白,并进行相关

  9. A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles.

    Science.gov (United States)

    Muthamilselvan, Thangarasu; Lee, Chin-Wei; Cho, Yu-Hsin; Wu, Feng-Chao; Hu, Chung-Chi; Liang, Yu-Chuan; Lin, Na-Sheng; Hsu, Yau-Heiu

    2016-01-01

    We describe a novel strategy to produce vaccine antigens using a plant cell-suspension culture system in lieu of the conventional bacterial or animal cell-culture systems. We generated transgenic cell-suspension cultures from Nicotiana benthamiana leaves carrying wild-type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot-and-mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co-expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large-scale production of immunopeptide vaccines in a cost-effective manner using a plant cell-suspension culture system. PMID:25879277

  10. Matrix protein 2 vaccination and protection against influenza viruses, including subtype H5N1.

    Science.gov (United States)

    Tompkins, Stephen Mark; Zhao, Zi-Shan; Lo, Chia-Yun; Misplon, Julia A; Liu, Teresa; Ye, Zhiping; Hogan, Robert J; Wu, Zhengqi; Benton, Kimberly A; Tumpey, Terrence M; Epstein, Suzanne L

    2007-03-01

    Changes in influenza viruses require regular reformulation of strain-specific influenza vaccines. Vaccines based on conserved antigens provide broader protection. Influenza matrix protein 2 (M2) is highly conserved across influenza A subtypes. To evaluate its efficacy as a vaccine candidate, we vaccinated mice with M2 peptide of a widely shared consensus sequence. This vaccination induced antibodies that cross-reacted with divergent M2 peptide from an H5N1 subtype. A DNA vaccine expressing full-length consensus-sequence M2 (M2-DNA) induced M2-specific antibody responses and protected against challenge with lethal influenza. Mice primed with M2-DNA and then boosted with recombinant adenovirus expressing M2 (M2-Ad) had enhanced antibody responses that crossreacted with human and avian M2 sequences and produced T-cell responses. This M2 prime-boost vaccination conferred broad protection against challenge with lethal influenza A, including an H5N1 strain. Vaccination with M2, with key sequences represented, may provide broad protection against influenza A. PMID:17552096

  11. The use of green fluorescent protein as a marker for Brucella vaccines.

    Science.gov (United States)

    Chacón-Díaz, Carlos; Muñoz-Rodríguez, Melissa; Barquero-Calvo, Elías; Guzmán-Verri, Caterina; Chaves-Olarte, Esteban; Grilló, María Jesús; Moreno, Edgardo

    2011-01-10

    Brucellosis is an important malady of productive and wildlife animals and a worldwide zoonosis. The use of effective vaccines and the corresponding diagnostic tests that allow differentiating infected from vaccinated animals are essential tools to control the disease. For this, a prototype of Brucella abortus S19 vaccine expressing green fluorescent protein (S19-GFP) was constructed. The S19-GFP was readily identified under ultraviolet light by macroscopic and microscopic examination and maintained all the biochemical characteristics of the parental S19 vaccine. S19-GFP replicated ex vivo and in vivo, and protected mice against challenge with virulent B. abortus to the same extent as the isogenic S19. An immunoenzymatic assay designed to measure anti-GFP antibodies allowed the discrimination between mice vaccinated with S19-GFP and those immunized with S19. Both vaccines raised antibodies against lipopolysaccharide molecule to similar levels. This experimental model constitutes a "proof of concept" for the use of Brucella-GFP vaccines and associated diagnostic tests to distinguish vaccinated from naturally Brucella infected animals. PMID:21056079

  12. Protective value of immune responses developed in goats vaccinated with insoluble proteins from Sarcoptes Scabiei

    Directory of Open Access Journals (Sweden)

    Simson Tarigan

    2005-06-01

    Full Text Available Vaccines developed from certain membrane proteins lining the lumen of arthropod’s gut have been demonstrated effective in the control of some arthropod ectoparasites. A similar approach could also be applied to Sarcoptes scabiei since this parasite also ingests its host immunoglobulins. To evaluate immune protection of the membrane proteins, insoluble mite proteins were fractionated by successive treatment in the solutions of 1.14 M NaCl, 2% SB 3-14 Zwitterion detergent, 6 M urea, 6 M guanidine-HCl and 5% SDS. Five groups of goats (6 or 7 goats per group were immunised respectively with the protein fractions. Vaccination was performed 6 times, each with a dosage of 250 μg proteins, and 3 week intervals between vaccination. Group 6 (7 goats received PBS and adjuvant only, and served as an unvaccinated control. One week after the last vaccination, all goats were challenged with 2000 live mites on the auricles. The development of lesions were examined at 1 day, 2 days, and then every week from week 1 to 8. All animals were bled and weighed every week, and at the end of the experiment, skin scrapings were collected to determine the mite burden. Antibody responses induced by vaccination and challenge were examined by ELISA and Western blotting. This experiment showed that vaccination with the insoluble-protein fractions resulted in the development of high level of specific antibodies but the responses did not have any protective value. The severity of lesions and mite burden in the vaccinated animals were not different from those in the unvaccinated control.

  13. Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice.

    Directory of Open Access Journals (Sweden)

    Camila Figueiredo Pinzan

    Full Text Available Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6 or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6 to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection.

  14. Protective Efficacy of Baculovirus Dual Expression System Vaccine Expressing Plasmodium falciparum Circumsporozoite Protein

    OpenAIRE

    Iyori, Mitsuhiro; Nakaya, Hiroki; Inagaki, Katsuya; Pichyangkul, Sathit; Yamamoto, Daisuke S.; Kawasaki, Masanori; Kwak, Kyungtak; Mizukoshi, Masami; Goto, Yoshihiro; Matsuoka, Hiroyuki; Matsumoto, Makoto; Yoshida, Shigeto

    2013-01-01

    We have previously developed a new malaria vaccine delivery system based on the baculovirus dual expression system (BDES). In this system, expression of malaria antigens is driven by a dual promoter consisting of the baculovirus-derived polyhedrin and mammal-derived cytomegalovirus promoters. To test this system for its potential as a vaccine against human malaria parasites, we investigated immune responses against the newly developed BDES-based Plasmodium falciparum circumsporozoite protein ...

  15. Redirecting Therapeutic T Cells against Myelin-Specific T Lymphocytes Using a Humanized Myelin Basic Protein-HLA-DR2-{zeta} Chimeric Receptor

    DEFF Research Database (Denmark)

    Moisini, Ioana; Nguyen, Phuong; Fugger, Lars;

    2008-01-01

    Therapies that Ag-specifically target pathologic T lymphocytes responsible for multiple sclerosis (MS) and other autoimmune diseases would be expected to have improved therapeutic indices compared with Ag-nonspecific therapies. We have developed a cellular immunotherapy that uses chimeric receptors...... humanized mouse model system. Finally, the chimeric receptor-modified CTL ameliorated or blocked experimental allergic encephalomyelitis (EAE) disease mediated by MBP(84-102)/DR2-specific T lymphocytes. These results provide support for the further development of redirected therapeutic T cells able to...

  16. Identification of the immunogenic spore and vegetative proteins of Bacillus anthracis vaccine strain A16R.

    Directory of Open Access Journals (Sweden)

    Xiankai Liu

    Full Text Available Immunoproteomics was used to screen the immunogenic spore and vegetative proteins of Bacillus anthracis vaccine strain A16R. The spore and vegetative proteins were separated by 2D gel electrophoresis and transferred to polyvinylidene difluoride membranes, and then western blotting was performed with rabbit immune serum against B.anthracis live spores. Immunogenic spots were cut and digested by trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was performed to identify the proteins. As a result, 11 and 45 immunogenic proteins were identified in the spores and vegetative cells, respectively; 26 of which have not been reported previously. To verify their immunogenicity, 12 of the identified proteins were selected to be expressed, and the immune sera from the mice vaccinated by the 12 expressed proteins, except BA0887, had a specific western blot band with the A16R whole cellular lytic proteins. Some of these immunogenic proteins might be used as novel vaccine candidates themselves or for enhancing the protective efficacy of a protective-antigen-based vaccine.

  17. Design and Construction of Chimeric VP8-S2 Antigen for Bovine Rotavirus and Bovine Coronavirus

    Science.gov (United States)

    Nasiri, Khadijeh; Nassiri, Mohammadreza; Tahmoorespur, Mojtaba; Haghparast, Alireza; Zibaee, Saeed

    2016-01-01

    Purpose: Bovine Rotavirus and Bovine Coronavirus are the most important causes of diarrhea in newborn calves and in some other species such as pigs and sheep. Rotavirus VP8 subunit is the major determinant of the viral infectivity and neutralization. Spike glycoprotein of coronavirus is responsible for induction of neutralizing antibody response. Methods: In the present study, several prediction programs were used to predict B and T-cells epitopes, secondary and tertiary structures, antigenicity ability and enzymatic degradation sites. Finally, a chimeric antigen was designed using computational techniques. The chimeric VP8-S2 antigen was constructed. It was cloned and sub-cloned into pGH and pET32a(+) expression vector. The recombinant pET32a(+)-VP8-S2 vector was transferred into E.oli BL21CodonPlus (DE3) as expression host. The recombinant VP8-S2 protein was purified by Ni-NTA chromatography column. Results: The results of colony PCR, enzyme digestion and sequencing showed that the VP8-S2 chimeric antigen has been successfully cloned and sub-cloned into pGH and pET32a(+).The results showed that E.coli was able to express VP8-S2 protein appropriately. This protein was expressed by induction of IPTG at concentration of 1mM and it was confirmed by Ni–NTA column, dot-blotting analysis and SDS-PAGE electrophoresis. Conclusion: The results of this study showed that E.coli can be used as an appropriate host to produce the recombinant VP8-S2 protein. This recombinant protein may be suitable to investigate to produce immunoglobulin, recombinant vaccine and diagnostic kit in future studies after it passes biological activity tests in vivo in animal model and or other suitable procedure. PMID:27123423

  18. Protection against bovine tuberculosis induced by oral vaccination of cattle with Mycobacterium bovis BCG is not enhanced by co-administration of mycobacterial protein vaccines.

    Science.gov (United States)

    Wedlock, D Neil; Aldwell, Frank E; Vordermeier, H Martin; Hewinson, R Glyn; Buddle, Bryce M

    2011-12-15

    Mycobacterium bovis bacille Calmette-Guérin (BCG) delivered to calves by the oral route in a formulated lipid matrix has been previously shown to induce protection against bovine tuberculosis. A study was conducted in cattle to determine if a combination of a low dose of oral BCG and a protein vaccine could induce protective immunity to tuberculosis while not sensitising animals to tuberculin. Groups of calves (10 per group) were vaccinated by administering 2 × 10(7)colony forming units (CFU) of BCG orally or a combination of 2 × 10(7)CFU oral BCG and a protein vaccine comprised of M. bovis culture filtrate proteins (CFP) formulated with the adjuvants Chitin and Gel 01 and delivered by the intranasal route, or CFP formulated with Emulsigen and the TLR2 agonist Pam(3)CSK(4) and administered by the subcutaneous (s.c.) route. Two further groups were vaccinated with the CFP/Chitin/Gel 01 or CFP/Emulsigen/Pam(3)CSK(4) vaccines alone. Positive control groups were given 10(8)CFU oral BCG or 10(6)CFU s.c. BCG while a negative control group was non-vaccinated. All animals were challenged with M. bovis 15 weeks after vaccination and euthanized and necropsied at 16 weeks following challenge. Groups of cattle vaccinated with s.c. BCG, 10(8)CFU or 2 × 10(7)CFU oral BCG showed significant reductions in seven, three and four pathological or microbiological disease parameters, respectively, compared to the results for the non-vaccinated group. There was no evidence of protection in calves vaccinated with the combination of oral BCG and CFP/Emulsigen/Pam(3)CSK(4) or oral BCG and CFP/Chitin/Gel 01 or vaccinated with the protein vaccines alone. Positive responses in the comparative cervical skin test at 12 weeks after vaccination were only observed in animals vaccinated with s.c. BCG, 10(8)CFU oral BCG or a combination of 2 × 10(7)CFU oral BCG and CFP/Chitin/Gel 01. In conclusion, co-administration of a protein vaccine, administered by either systemic or mucosal routes with oral

  19. Glycan masking of Plasmodium vivax Duffy Binding Protein for probing protein binding function and vaccine development.

    Directory of Open Access Journals (Sweden)

    Sowmya Sampath

    Full Text Available Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of Plasmodium vivax Duffy Binding Protein (PvDBP and the Duffy Antigen Receptor for Chemokines (DARC and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development.

  20. Establishment and characterization of a chimeric infectious cDNA clone of classical swine fever virus.

    Science.gov (United States)

    Zhao, T S; Xia, Y H

    2016-01-01

    Classical swine fever virus (CSFV) causes a highly contagious disease among swine that has an important economic impact worldwide. There are two important CSFV strains in China, Shimen and hog cholera lapinized virus (HCLV). Shimen strain is highly virulent while HCLV, also referred to as C-strain, is a live attenuated vaccine strain considered to be one of the most effective and safest live vaccines. In this study, a chimeric infectious cDNA clone of CSFV named pT7SM-c was engineered by replacing the Erns genomic region of an infectious clone of CSFV Shimen strain, pT7SM, with the same region obtained from HCLV. RNA transcripts of pT7SM-c containing an engineered EcoRI site that served as a genetic marker were directly infectious in PK15 cells. The rescued virus vT7SM-c showed similar growth kinetics and cytopathic effect with the parental virus vT7SM in the cells. The chimeric infectious cDNA clone can be used as a practical tool for further studying of the virulence, protein function and pathogenesis of CSFV through genetic manipulation. PMID:27265471

  1. Recommendation for use of the newly introduced pneumococcal protein conjugate vaccines in Korea

    Directory of Open Access Journals (Sweden)

    Eun Hwa Choi

    2011-04-01

    Full Text Available Streptococcus pneumoniae remains a leading cause of invasive infections including bacteremia and meningitis, as well as mucosal infections such as otitis media and pneumonia among children and adults. The 7-valent pneumococcal conjugate vaccine (PCV7 was licensed for use among infants and young children in many countries including Korea. The routine use of PCV7 has resulted in a decreased incidence of invasive pneumococcal disease (IPD by the vaccine serotypes among the vaccinees and substantial declines in IPD among unvaccinated populations such as older children and adults as well. In addition, there are increasing evidences to suggest that routine immunization with PCV7 is changing the epidemiology of pneumococcal diseases such as serotype distribution of IPD, nasopharyngeal colonization, and antibiotic resistance patterns. In contrast, there is an increase in the number of IPDs caused by nonvaccine serotypes, though it is much smaller than overall declines of vaccine serotype diseases. Several vaccines containing additional serotypes have been developed and tested clinically in order to expand the range of serotypes of Streptococcus pneumoniae. Recently two new pneumococcal protein conjugate vaccines, 10-valent pneumococcal conjugate vaccine (PCV10 and 13-valent pneumococcal conjugate vaccine (PCV13, have been approved for use in several countries including Korea. This report summarizes the recommendations approved by the Committee on Infectious Diseases, the Korean Pediatric Society.

  2. Recent advances in recombinant protein-based malaria vaccines

    DEFF Research Database (Denmark)

    Draper, Simon J; Angov, Evelina; Horii, Toshihiro;

    2015-01-01

    Plasmodium parasites are the causative agent of human malaria, and the development of a highly effective vaccine against infection, disease and transmission remains a key priority. It is widely established that multiple stages of the parasite's complex lifecycle within the human host and mosquito...

  3. Mosaic protein and nucleic acid vaccines against hepatitis C virus

    Energy Technology Data Exchange (ETDEWEB)

    Yusim, Karina; Korber, Bette T. M.; Kuiken, Carla L.; Fischer, William M.

    2013-06-11

    The invention relates to immunogenic compositions useful as HCV vaccines. Provided are HCV mosaic polypeptide and nucleic acid compositions which provide higher levels of T-cell epitope coverage while minimizing the occurrence of unnatural and rare epitopes compared to natural HCV polypeptides and consensus HCV sequences.

  4. Elucidating the mechanisms of protein antigen adsorption to the CAF/NAF liposomal vaccine adjuvant systems

    DEFF Research Database (Denmark)

    Hamborg, Mette; Rose, Fabrice; Jorgensen, Lene;

    2014-01-01

    The reverse vaccinology approach has recently resulted in the identification of promising protein antigens, which in combination with appropriate adjuvants can stimulate customized, protective immune responses. Although antigen adsorption to adjuvants influences vaccine efficacy and safety, little...... attractive interaction with the zwitterionic liposomes. In contrast, the net cationic lysozyme showed very little interaction with either types of liposome. Adsorption of α-lactalbumin altered its tertiary structure, affected lipid membrane packing below and above the phase transition temperature, and...... way vaccine antigens are presented to antigen-presenting cells, and may play an important role for the efficacy of the vaccine-induced immune response. These studies thus exemplify the importance of characterizing the molecular interactions between the vaccine antigen and adjuvant along with...

  5. Correlates of Protection for M Protein-Based Vaccines against Group A Streptococcus

    Directory of Open Access Journals (Sweden)

    Shu Ki Tsoi

    2015-01-01

    Full Text Available Group A streptococcus (GAS is known to cause a broad spectrum of illness, from pharyngitis and impetigo, to autoimmune sequelae such as rheumatic heart disease, and invasive diseases. It is a significant cause of infectious disease morbidity and mortality worldwide, but no efficacious vaccine is currently available. Progress in GAS vaccine development has been hindered by a number of obstacles, including a lack of standardization in immunoassays and the need to define human correlates of protection. In this review, we have examined the current immunoassays used in both GAS and other organisms, and explored the various challenges in their implementation in order to propose potential future directions to identify a correlate of protection and facilitate the development of M protein-based vaccines, which are currently the main GAS vaccine candidates.

  6. Polymorphism in liver-stage malaria vaccine candidate proteins: immune evasion and implications for vaccine design.

    Science.gov (United States)

    Flanagan, Katie L; Wilson, Kirsty L; Plebanski, Magdalena

    2016-03-01

    The pre-erythrocytic stage of infection by malaria parasites represents a key target for vaccines that aim to eradicate malaria. Two important broad immune evasion strategies that can interfere with vaccine efficacy include the induction of dendritic cell (DC) dysfunction and regulatory T cells (Tregs) by blood-stage malaria parasites, leading to inefficient priming of T cells targeting liver-stage infections. The parasite also uses 'surgical strike' strategies, whereby polymorphism in pre-erythrocytic antigens can interfere with host immunity. Specifically, we review how even single amino acid changes in T cell epitopes can lead to loss of binding to major histocompatibility complex (MHC), lack of cross-reactivity, or antagonism and immune interference, where simultaneous or sequential stimulation with related variants of the same T cell epitope can cause T cell anergy or the conversion of effector to immunosuppressive T cell phenotypes. PMID:26610026

  7. Th1-directing adjuvants increase the immunogenicity of oligosaccharide-protein conjugate vaccines related to Streptococcus pneumoniae type 3

    NARCIS (Netherlands)

    Lefeber, DJ; Benaissa-Trouw, B; Vliegenthart, JFG; Kamerling, JP; Jansen, WTM; Kraaijeveld, K; Snippe, H

    2003-01-01

    Oligosaccharide (OS)-protein conjugates are promising candidate vaccines against encapsulated bacteria, such as Haemophilus influenzae, Neisseria meningitidis, and Streptococcus pneumoniae. Although the effects of several variables such as OS chain length and protein carrier have been studied, littl

  8. Candidate mosaic proteins for a pan-filoviral cytotoxic T-Cell lymphocyte vaccine

    Energy Technology Data Exchange (ETDEWEB)

    Fenimore, Paul W [Los Alamos National Laboratory; Fischer, William M [Los Alamos National Laboratory; Kuiken, Carla [Los Alamos National Laboratory; Foley, Brian T [Los Alamos National Laboratory; Thurmond, J R [Los Alamos National Laboratory; Yusim, K [Los Alamos National Laboratory; Korber, B T [Los Alamos National Laboratory

    2008-01-01

    The extremely high fatality rates of many filovirus (FILV) strains the recurrent but rarely identified origin of human epidemics, the only partly identified viral reservoirs and the continuing non-human primate epizootics in Africa make a broadly-protective filovirus vaccine highly desirable. Cytotoxic T-cells (CTL) have been shown to be protective in mice, guinea pigs and non-human primates. In murine models the cytotoxic T-cell epitopes that are protective against Ebola virus have been mapped and in non-human primates CTL-mediated protection between viral strains (John Dye: specify) has been demonstrated using two filoviral proteins, nucleoprotein (NP) and glycoprotein (GP). These immunological results suggest that the CTL avenue of immunity deserves consideration for a vaccine. The poorly-understood viral reservoirs means that it is difficult to predict what strains are likely to cause epidemics. Thus, there is a premium on developing a pan-filoviral vaccine. The genetic diversity of FILV is large, roughly the same scale as human immunodeficiency virus (HIV). This presents a serious challenge for the vaccine designer because a traditional vaccine aspiring to pan-filoviral coverage is likely to require the inclusion of many antigenic reagents. A recent method for optimizing cytotoxic T-cell lymphocyte epitope coverage with mosaic antigens was successful in improving potential CTL epitope coverage against HIV and may be useful in the context of very different viruses, such as the filoviruses discussed here. Mosaic proteins are recombinants composed of fragments of wild-type proteins joined at locations resulting in exclusively natural k-mers, 9 {le} k {le} 15, and having approximately the same length as the wild-type proteins. The use of mosaic antigens is motivated by three conjectures: (1) optimizing a mosaic protein to maximize coverage of k-mers found in a set of reference proteins will give better odds of including broadly-protective CTL epitopes in a vaccine

  9. Vaccine delivery system for tuberculosis based on nano-sized hepatitis B virus core protein particles

    Directory of Open Access Journals (Sweden)

    Dhanasooraj D

    2013-02-01

    Full Text Available Dhananjayan Dhanasooraj, R Ajay Kumar, Sathish MundayoorMycobacterium Research Group, Rajiv Gandhi Centre for Biotechnology, Kerala, IndiaAbstract: Nano-sized hepatitis B virus core virus-like particles (HBc-VLP are suitable for uptake by antigen-presenting cells. Mycobacterium tuberculosis antigen culture filtrate protein 10 (CFP-10 is an important vaccine candidate against tuberculosis. The purified antigen shows low immune response without adjuvant and tends to have low protective efficacy. The present study is based on the assumption that expression of these proteins on HBc nanoparticles would provide higher protection when compared to the native antigen alone. The cfp-10 gene was expressed as a fusion on the major immunodominant region of HBc-VLP, and the immune response in Balb/c mice was studied and compared to pure proteins, a mixture of antigens, and fusion protein-VLP, all without using any adjuvant. The humoral, cytokine, and splenocyte cell proliferation responses suggested that the HBc-VLP bearing CFP-10 generated an antigen-specific immune response in a Th1-dependent manner. By virtue of its self-adjuvant nature and ability to form nano-sized particles, HBc-VLPs are an excellent vaccine delivery system for use with subunit protein antigens identified in the course of recent vaccine research.Keywords: Mycobacterium tuberculosis, VLP, hepatitis B virus core particle, CFP-10, self-adjuvant, vaccine delivery

  10. Use of procalcitonin and C-reactive protein to evaluate vaccine efficacy against pneumonia.

    Directory of Open Access Journals (Sweden)

    Shabir A Madhi

    2005-02-01

    Full Text Available BACKGROUND: Pneumonia remains the leading cause of death in young children. The poor specificity of chest radiographs (CXRs to diagnose pneumococcal pneumonia may underestimate the efficacy of pneumococcal conjugate vaccine in preventing pneumococcal pneumonia. METHODS AND FINDINGS: The efficacy of nine-valent pneumococcal conjugate vaccine among children not infected with HIV (21%; 95% confidence interval, 1%-37% increased when CXR-confirmed pneumonia was associated with serum C-reactive protein of 120 mg/l (12 mg/dl or more and procalcitonin of 5.0 ng/ml or more (64%; 95% confidence interval, 23%-83%. Similar results were observed in children infected with HIV. CONCLUSION: C-reactive protein and procalcitonin improve the specificity of CXR to diagnose pneumococcal pneumonia and may be useful for the future evaluation of the effectiveness of pneumococcal conjugate vaccine in preventing pneumococcal pneumonia.

  11. Characterization of the enterovirus 71 VP1 protein as a vaccine candidate.

    Science.gov (United States)

    Zhou, Shi-Li; Ying, Xiao-Ling; Han, Xue; Sun, Xian-Xun; Jin, Qi; Yang, Fan

    2015-02-01

    Enterovirus 71 (EV71) is an important agent responsible for hand-foot-and-mouth disease (HFMD), which can cause severe neurological complications and death in children. However, there is no specific treatment for EV71 infection, and a safe and effective vaccine is needed urgently. In this study, an effective and economical method for the production of EV71-VP1 protein was developed, and the VP1 protein was evaluated in humoral and cellular immune responses as an EV71 vaccine. The results revealed that the VP1 protein induced high titers of cross-neutralizing antibodies for different EV71 subtypes, and elicited significant splenocyte proliferation. The high levels of IFN-r and IL-10 showed the VP1 protein induced a mixed Th1 and Th2 immune response. Vaccinated female mice could confer protection in their neonatal offspring. Compared with the inactivated EV71, the VP1 protein elicited similar humoral and cellular responses, but the engineered protein is safer, less expensive and can be produced more efficiently. Therefore, EV71-VP1 protein can induce effective immunologic protection against EV71 and is an ideal candidate against EV71 infection. PMID:25043151

  12. Lesser protein degradation machinery correlates with higher BM86 tick vaccine efficacy in Rhipicephalus annulatus when compared to Rhipicephalus microplus.

    Science.gov (United States)

    Popara, Marina; Villar, Margarita; Mateos-Hernández, Lourdes; de Mera, Isabel G Fernández; Marina, Anabel; del Valle, Mercedes; Almazán, Consuelo; Domingos, Ana; de la Fuente, José

    2013-10-01

    Infestations with cattle ticks, Rhipicephalus (Boophilus) microplus and Rhipicephalus annulatus, economically impact cattle production in tropical and subtropical regions of the world. Vaccines containing the recombinant R. microplus BM86 gut antigen were developed and commercialized to induce an immunological protection in cattle against tick infestations. These vaccines demonstrated that tick control by vaccination is cost-effective, reduces environmental contamination and prevents the selection of drug resistant ticks that result from repeated acaricide applications. The protection elicited by BM86-containing vaccines against tick infestations is mediated by a collaborative action between the complement system and IgG antibodies. The efficacy of the vaccination with BM86 and other tick antigens is always higher for R. annulatus than against R. microplus, suggesting that tick genetic and/or physiological factors may affect tick vaccine efficacy. These factors may be related to BM86 protein levels or tick physiological processes such as feeding and protein degradation that could result in more efficient antibody-antigen interactions and vaccine efficacy. To test this hypothesis, we compared the proteome in R. annulatus and R. microplus female ticks after feeding on BM86-vaccinated and control cattle. The results showed that cattle proteins were under represented in R. annulatus when compared to R. microplus, suggesting that R. annulatus ticks ingested less blood, a difference that increased when feeding on vaccinated cattle, probably reflecting the effect of antibody-BM86 interactions on this process. The results also showed that tick protein degradation machinery was under represented in R. annulatus when compared to R. microplus. BM86 mRNA and protein levels were similar in both tick species, suggesting that lesser protease activity in R. annulatus results in more efficient antibody-antigen interactions and higher vaccine efficacy. These results have important

  13. Elicitation of strong immune responses by a DNA vaccine expressing a secreted form of hepatitis C virus envelope protein E2 in murine and porcine animal models

    DEFF Research Database (Denmark)

    Li, Yiping; Kang, H.N.; Babiuk, L.A.;

    2006-01-01

    . RESULTS: Intradermal injection of E2 DNA vaccine induced strong Th1-like immune responses in mice. In piglets, E2 DNA vaccine elicited moderate and more balanced immune responses. A DNA vaccine prime and protein boost vaccination, strategy induced significantly higher E2-specific antibody levels and...

  14. Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development.

    Science.gov (United States)

    Connolly, Joseph P; Comerci, Diego; Alefantis, Timothy G; Walz, Alexander; Quan, Marian; Chafin, Ryan; Grewal, Paul; Mujer, Cesar V; Ugalde, Rodolfo A; DelVecchio, Vito G

    2006-07-01

    Brucella abortus is the etiologic agent of bovine brucellosis and causes a chronic disease in humans known as undulant fever. In livestock the disease is characterized by abortion and sterility. Live, attenuated vaccines such as S19 and RB51 have been used to control the spread of the disease in animals; however, they are considered unsafe for human use and they induce abortion in pregnant cattle. For the development of a safer and equally efficacious vaccine, immunoproteomics was utilized to identify novel candidate proteins from B. abortus cell envelope (CE). A total of 163 proteins were identified using 2-DE with MALDI-TOF MS and LC-MS/MS. Some of the major protein components include outer-membrane protein (OMP) 25, OMP31, Omp2b porin, and 60 kDa chaperonin GroEL. 2-DE Western blot analyses probed with antiserum from bovine and a human patient infected with Brucella identified several new immunogenic proteins such as fumarate reductase flavoprotein subunit, F0F1-type ATP synthase alpha subunit, and cysteine synthase A. The elucidation of the immunome of B. abortus CE identified a number of candidate proteins for developing vaccines against Brucella infection in bovine and humans. PMID:16739129

  15. CD46 Measles Virus Receptor Polymorphisms Influence Receptor Protein Expression and Primary Measles Vaccine Responses in Naive Australian Children

    OpenAIRE

    Clifford, Holly D.; Hayden, Catherine M.; Khoo, Siew-Kim; Zhang, Guicheng; Le Souëf, Peter N.; Richmond, Peter

    2012-01-01

    Despite the availability of measles vaccines, infants continue to die from measles. Measles vaccine responses vary between individuals, and poor immunogenicity is likely to preclude protection against measles. CD46 is a ubiquitously expressed specific receptor for vaccine strains of measles virus. CD46 polymorphisms have not been functionally investigated but may affect CD46 protein expression, which in turn may mediate primary measles antibody responses in infants. In a cohort of children ag...

  16. Immune reactivity of sera obtained from brucellosis patients and vaccinated-rabbits to a fusion protein from Brucella melitensis

    Directory of Open Access Journals (Sweden)

    Jafar Amani

    2015-04-01

    Full Text Available Objective(s: Brucella spp. are facultative intracellular pathogens which can stay alive and multiply in professional and nonprofessional phagocytes. Immunity against Brucella melitensis involves antigen-specific CD4+ and CD8+ T-cells activation and humoral immune responses. Due to negative aspects of live attenuated vaccines, much attention has been focused on finding Brucella-protective antigens to introduce them as potential subunit vaccine candidates. Materials and Methods: A chimeric gene encoding trigger factor (TF, Omp3148-74 and BP2687-111 fragments (TOB from B. melitensis was successfully cloned, expressed in Escherichia coliBL21-DE3 and purified by Ni-NTA agarose column. Antibodies to recombinant TOB (rTOB have been investigated in Brucella-infected human sera and a pool serum prepared from B. melitensis-vaccinated rabbits. Results: Our results showed that the immunized rabbit pool serum strongly reacted with rTOB. In addition, antibodies against rTOB were detectable in 76.5% of sera obtained from infected patients. Conclusion: These findings suggest that rTOB may provide a potential immunogenic candidate which could be considered in future vaccine studies.

  17. Immunogenicity and protective efficacy of Semliki forest virus replicon-based DNA vaccines encoding goatpox virus structural proteins

    International Nuclear Information System (INIS)

    Goatpox, caused by goatpox virus (GTPV), is an acute feverish and contagious disease in goats often associated with high morbidity and high mortality. To resolve potential safety risks and vaccination side effects of existing live attenuated goatpox vaccine (AV41), two Semliki forest virus (SFV) replicon-based bicistronic expression DNA vaccines (pCSm-AAL and pCSm-BAA) which encode GTPV structural proteins corresponding to the Vaccinia virus proteins A27, L1, A33, and B5, respectively, were constructed. Then, theirs ability to induce humoral and cellular response in mice and goats, and protect goats against virulent virus challenge were evaluated. The results showed that, vaccination with pCSm-AAL and pCSm-BAA in combination could elicit strong humoral and cellular responses in mice and goats, provide partial protection against viral challenge in goats, and reduce disease symptoms. Additionally, priming vaccination with the above-mentioned DNA vaccines could significantly reduce the goats' side reactions from boosting vaccinations with current live vaccine (AV41), which include skin lesions at the inoculation site and fevers. Data obtained in this study could not only facilitate improvement of the current goatpox vaccination strategy, but also provide valuable guidance to suitable candidates for evaluation and development of orthopoxvirus vaccines.

  18. Elicitation of strong immune responses by a DNA vaccine expressing a secreted form of hepatitis C virus envelope protein E2 in murine and porcine animal models

    Institute of Scientific and Technical Information of China (English)

    Yi-Ping Li; Hye Na Kang; Lorne A Babiuk; Qiang Liu

    2006-01-01

    AIM: To characterize the immunogenicity of a hepatitis C virus (HCV) E2 DNA vaccine alone or with a protein vaccine boost in murine and porcine animal models.METHODS: A DNA vaccine expressing a secreted form of HCV E2 protein was constructed and used to vaccinate mice and piglets with or without boosting with a recombinant E2 protein vaccine formulated with CpG ODN and 10% Emulsigen. The immunogenicity of HCV E2 vaccines was analyzed by ELISA for antibody responses, MTT assay for lymphocyte proliferation,ELISPOT for the number of interferon-γ secreting cells,and cytotoxic T lymphocyte assays.RESULTS: Intradermal injection of E2 DNA vaccine induced strong Th1-like immune responses in mice. In piglets, E2 DNA vaccine elicited moderate and more balanced immune responses. A DNA vaccine prime and protein boost vaccination strategy induced significantly higher E2-specific antibody levels and shifted the immune response towards Th2-like ones in piglets.CONCLUSION: A DNA vaccine expressing a secreted form of HCV E2 protein elicited E2-specific immune responses in mice and piglets. Recombinant E2 protein vaccination following DNA immunization significantly increased the antibody response in piglets. These HCV E2 vaccines may represent promising hepatitis C vaccine candidates for further investigations.

  19. Outer Membrane Protein Complex of Meningococcus Enhances the Antipolysaccharide Antibody Response to Pneumococcal Polysaccharide–CRM197 Conjugate Vaccine

    OpenAIRE

    Lai, Zengzu; Schreiber, John R.

    2011-01-01

    Bacterial polysaccharides (PS) are T cell-independent antigens that do not induce immunologic memory and are poor immunogens in infants. Conjugate vaccines in which the PS is covalently linked to a carrier protein have enhanced immunogenicity that resembles that of T cell-dependent antigens. The Haemophilus influenzae type b (Hib) conjugate vaccine, which uses the outer membrane protein complex (OMPC) from meningococcus as a carrier protein, elicits protective levels of anti-capsular PS antib...

  20. Human Enterovirus 71 Protein Displayed on the Surface of Saccharomyces cerevisiae as an Oral Vaccine.

    Science.gov (United States)

    Zhang, Congdang; Wang, Yi; Ma, Shuzhi; Li, Leike; Chen, Liyun; Yan, Huimin; Peng, Tao

    2016-06-01

    Human enterovirus 71 (EV-A71), a major agent of hand, foot, and mouth disease, has become an important public health issue in recent years. No effective antiviral or vaccines against EV-A71 infection are currently available. EV-A71 infection intrudes bodies through the gastric mucosal surface and it is necessary to enhance mucosal immune response to protect children from these pathogens. Recently, the majority of EV-A71 vaccine candidates have been developed for parenteral immunization. However, parenteral vaccine candidates often induce poor mucosal responses. On the other hand, oral vaccines could induce effective mucosal and systemic immunity, and could be easily and safely administered. Thus, proper oral vaccines have attached more interest compared with parenteral vaccine. In this study, the major immunogenic capsid protein of EV-A71 was displayed on the surface of Saccharomyces cerevisiae. Oral immunization of mice with surface-displayed VP1 S. cerevisiae induced systemic humoral and mucosal immune responses, including virus-neutralizing titers, VP1-specific antibody, and the induction of Th1 immune responses in the spleen. Furthermore, oral immunization of mother mice with surface-displayed VP1 S. cerevisiae conferred protection to neonatal mice against the lethal EV-A71 infection. Furthermore, we observed that multiple boost immunization as well as higher immunization dosage could induce higher EV-A71-specific immune response. Our results demonstrated that surface-displayed VP1 S. cerevisiae could be used as potential oral vaccine against EV-A71 infection. PMID:27259043

  1. The Pneumovirinae fusion (F) protein: A common target for vaccines and antivirals.

    Science.gov (United States)

    Melero, José A; Mas, Vicente

    2015-11-01

    The Pneumovirinae fusion (F) protein mediates fusion of the virus and cell membrane, an essential step for entry of the viral genome in the cell cytoplasm and initiation of a new infectious cycle. Accordingly, potent inhibitors of virus infectivity have been found among antibodies and chemical compounds that target the Pneumovirinae F protein. Recent developments in structure-based vaccines have led to a deeper understanding of F protein antigenicity, unveiling new conformations and epitopes which should assist in development of efficacious vaccines. Similarly, structure-based studies of potent antiviral inhibitors have provided information about their mode of action and mechanisms of resistance. The advantages and disadvantages of the different options to battle against important pathogens, such as human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are summarized and critically discussed in this review. PMID:25738581

  2. Immune Reactivity of Brucella Melitensis–Vaccinated Rabbit Serum with Recombinant Omp31 and Dnak Proteins

    Directory of Open Access Journals (Sweden)

    Mahmood Jeddi-Tehrani

    2013-03-01

    Full Text Available Background and objectives: Brucella melitensis infection is still a major health problem for human and cattle in developing countries and the Middle East.Materials and Methods: In this study, in order to screen immunogenic candidate antigens for the development of a Brucella subunit vaccine, a cytoplasmic protein (DnaK and an outer membrane protein (Omp31 of B. melitensis were cloned, expressed in E.coli BL21 and then purified using Ni-NTA agarose. Immunized serum was prepared from a rabbit inoculated with attenuated B. melitensis.Results and Conclusion: It was proved that immunized serum contains antibodies against recombinant Omp31 (rOmp31 and DnaK (rDnaK by Western blot and ELISA assays. The results may suggest the importance of these proteins as subunit vaccines against B. melitensis as well as targets for immunotherapy.

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

    Directory of Open Access Journals (Sweden)

    Huawei Zhang

    2014-12-01

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

  4. Virus-like particles of chimeric recombinant porcine circovirus type 2 as antigen vehicle carrying foreign epitopes.

    Science.gov (United States)

    Zhang, Huawei; Qian, Ping; Liu, Lifeng; Qian, Suhong; Chen, Huanchun; Li, Xiangmin

    2014-12-01

    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. PMID:25490764

  5. Sulfate-binding protein, CysP, is a candidate vaccine antigen of Moraxella catarrhalis.

    Science.gov (United States)

    Murphy, Timothy F; Kirkham, Charmaine; Johnson, Antoinette; Brauer, Aimee L; Koszelak-Rosenblum, Mary; Malkowski, Michael G

    2016-07-19

    Moraxella catarrhalis causes otitis media in children and respiratory tract infections in adults with chronic obstructive pulmonary disease (COPD). A vaccine to prevent M. catarrhalis infections would have an enormous impact globally in preventing morbidity caused by M. catarrhalis in these populations. Using a genome mining approach we have identified a sulfate binding protein, CysP, of an ATP binding cassette (ABC) transporter system as a novel candidate vaccine antigen. CysP expresses epitopes on the bacterial surface and is highly conserved among strains. Immunization with CysP induces potentially protective immune responses in a murine pulmonary clearance model. In view of these features that indicate CysP is a promising vaccine antigen, we conducted further studies to elucidate its function. These studies demonstrated that CysP binds sulfate and thiosulfate ions, plays a nutritional role for the organism and functions in intracellular survival of M. catarrhalis in human respiratory epithelial cells. The observations that CysP has features of a vaccine antigen and also plays an important role in growth and survival of the organism indicate that CysP is an excellent candidate vaccine antigen to prevent M. catarrhalis otitis media and infections in adults with COPD. PMID:27265455

  6. Differentiation of infected and vaccinated animals (DIVA) using the NS1 protein of avian influenza virus in chickens

    Science.gov (United States)

    The use of avian influenza (AI) vaccination in poultry would have greater world-wide acceptance if a reliable test that clearly discriminates naturally infected from vaccinated only animals (DIVA) was available. Because the non-structural protein (NS1) is expressed in infected cells, and is not pac...

  7. Proteins mediating the Neospora caninum-host cell interaction as targets for vaccination.

    Science.gov (United States)

    Hemphill, Andrew; Debache, Karim; Monney, Thierry; Schorer, Michelle; Guionaud, Christophe; Alaeddine, Ferial; Mueller, Norbert; Mueller, Joachim

    2013-01-01

    Neospora caninum is an apicomplexan parasite that is capable of infecting, a wide range of tissues. The fact that Neospora represents an important abortion-causing parasite in cattle has transformed neosporosis research from an earlier, rather esoteric field, to a significant research topic, and considerable investments have been made in the last years to develop an efficacious vaccine or other means of intervention that would prevent infection and abortion due to N. caninum infection in cattle. Antigenic molecules associated with proteins involved in adhesion/invasion or other parasite-host-cell interaction processes can confer protection against Neospora caninum infection, and such proteins represent valuable targets for the development of a vaccine to limit economical losses due to neosporosis. Although not ideal, small laboratory animal models that mimic cerebral infection, acute disease and fetal loss upon infection during pregnancy have been used for the assessment of vaccine candidates, in parallel with studies on experimental infections in cattle. Herein, we review and critically assess these vaccination approaches and discuss potential options for improvements. PMID:23276967

  8. 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.; Jones, T.D.; Kamstrup, Søren; Dalsgaard, Kristian; Flock, J.I.; Hamilton, W.D.O.

    1999-01-01

    of CVPs 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......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 or....... These studies 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....

  9. Worldwide genetic variability of the Duffy binding protein: insights into Plasmodium vivax vaccine development.

    Directory of Open Access Journals (Sweden)

    Taís Nóbrega de Sousa

    Full Text Available The dependence of Plasmodium vivax on invasion mediated by Duffy binding protein (DBP makes this protein a prime candidate for development of a vaccine. However, the development of a DBP-based vaccine might be hampered by the high variability of the protein ligand (DBP(II, known to bias the immune response toward a specific DBP variant. Here, the hypothesis being investigated is that the analysis of the worldwide DBP(II sequences will allow us to determine the minimum number of haplotypes (MNH to be included in a DBP-based vaccine of broad coverage. For that, all DBP(II sequences available were compiled and MNH was based on the most frequent nonsynonymous single nucleotide polymorphisms, the majority mapped on B and T cell epitopes. A preliminary analysis of DBP(II genetic diversity from eight malaria-endemic countries estimated that a number between two to six DBP haplotypes (17 in total would target at least 50% of parasite population circulating in each endemic region. Aiming to avoid region-specific haplotypes, we next analyzed the MNH that broadly cover worldwide parasite population. The results demonstrated that seven haplotypes would be required to cover around 60% of DBP(II sequences available. Trying to validate these selected haplotypes per country, we found that five out of the eight countries will be covered by the MNH (67% of parasite populations, range 48-84%. In addition, to identify related subgroups of DBP(II sequences we used a Bayesian clustering algorithm. The algorithm grouped all DBP(II sequences in six populations that were independent of geographic origin, with ancestral populations present in different proportions in each country. In conclusion, in this first attempt to undertake a global analysis about DBP(II variability, the results suggest that the development of DBP-based vaccine should consider multi-haplotype strategies; otherwise a putative P. vivax vaccine may not target some parasite populations.

  10. Construction of an oral recombinant DNA vaccine from H pylori neutrophil activating protein and its immunogenicity

    Institute of Scientific and Technical Information of China (English)

    Bo Sun; Zhao-Shen Li; Zhen-Xing Tu; Guo-Ming Xu; Yi-Qi Du

    2006-01-01

    AIM: To construct a live attenuated Salmonella typhimurium (S.typhimurium) strain harboring the H pylori neutrophil activating protein (HP-NAP) gene as an oral recombinant DNA vaccine, and to evaluate its immunogenicity.METHODS: By genetic engineering methods, the genomic DNA of H pylori was extracted as a template. The total length of the HP-NAP gene was amplified by polymerase chain reaction (PCR) and cloned into pBT vector for sequencing and BLAST analysis, then subcloned into a eukaryotic expression vector pIRES followed by PCR identification and restriction enzyme digestion. The identified recombinant plasmid pIRES-NAP was transfected into COS-7 cells for target fusion protein expression, and its antigenicity was detected by Western blotting. Then the recombinant plasmid was transformed into a live attenuated S. typhimurium strain SL7207 as an oral vaccine strain, and its immunogenicity was evaluated with animal experiments.RESULTS: A 435 bp product was cloned using high homology with HP-NAP gene in GenBank (more than 98%). With identification by PCR and restriction enzyme digestion, a recompinant eukaryotic expression plasmid pIRES-NAP containing the HP-NAP gene of H pylori was successfully constructed. The expressed target protein had a specific reaction with H pylor(i) whole cell antibody and showed a single strip result detected by Western blotting. Oral immunization of mice with recombinant DNA vaccine strain SL7207 (pIRES-NAP) also induced a specific immune response.CONCLUSION: The successful construction of HP-NAP oral DNA vaccine with good immunogenicity may help to further investigate its immunoprotection effects and develop vaccine against H pylori infection.

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

    Directory of Open Access Journals (Sweden)

    Weiss-Steider Benny

    2009-01-01

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

  12. Radioiodination of the protein complex of the VA-MENGOC-BC vaccine

    International Nuclear Information System (INIS)

    In this work was made the labelling of the protein complex of the vaccine VA-MEMGOC-BC with I-125 in order to study its immunological responses. These proteins were in both forms: dissolved and conjugated with polisacarids of the C-group. There were used three methods of iodination: chloramine-T iodogen and lactoperoxidase. Was found out that dissolved proteins can be iodinated using these methods with 0,1 mCi of I-125, and the obtained specific activities were similar

  13. Helminth Protein Vaccine Induced Follicular T Helper Cell for Enhancement of Humoral Immunity against Schistosoma japonicum

    Directory of Open Access Journals (Sweden)

    Jingyao Zhang

    2013-01-01

    Full Text Available Protein vaccines combined with adjuvants have been widely used to induce immune responses, especially the humoral immune response, against molecular targets including parasites. Follicular T helper (Tfh cells are the specialized providers of B-cell help, however, the induction of Tfh cells in protein vaccination has been rarely studied. Here, we report that the Schistosoma japonicum recombinant protein (SjGST-32 combined with tacrolimus (FK506 augmented the induction of Tfh cells, which expressed the canonical markers CXCR5, BCL6, and IL-21, and enhanced the humoral immune responses in BALB/c mice. Furthermore, the expression of IL-21R on germinal center (GC B cells and memory B cells increased in immunized mice, which indicated that IL-21 from the induced Tfh cells interacted with IL-21R for activation of B cells and maintenance of long-lived humoral immunity. Our results suggest that helminth protein vaccine combined with FK506 induces Tfh cell for stimulating humoral immune responses and inducing long-lived humoral immunity.

  14. Live virus vaccines based on a yellow fever vaccine backbone: Standardized template with key considerations for a risk/benefit assessment

    OpenAIRE

    Monath, Thomas P.; Seligman, Stephen J.; Robertson, James S; Guy, Bruno; Hayes, Edward B.; Condit, Richard C.; Excler, Jean Louis; Mac, Lisa Marie; Carbery, Baevin; Chen, Robert T.

    2014-01-01

    The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety of live, recombinant viral vaccines incorporating genes from heterologous viruses inserted into the backbone of another virus (so-called "chimeric virus vaccines"). Many viral vector vaccines are in advanced clinical trials. The first such vaccine to be approved for mark...

  15. Expression of Plasmodium falciparum Circumsporozoite Proteins in Escherichia coli for Potential Use in a Human Malaria Vaccine

    Science.gov (United States)

    Young, James F.; Hockmeyer, Wayne T.; Gross, Mitchell; Ripley Ballou, W.; Wirtz, Robert A.; Trosper, James H.; Beaudoin, Richard L.; Hollingdale, Michael R.; Miller, Louis H.; Diggs, Carter L.; Rosenberg, Martin

    1985-05-01

    The circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum may be the most promising target for the development of a malaria vaccine. In this study, proteins composed of 16, 32, or 48 tandem copies of a tetrapeptide repeating sequence found in the CS protein were efficiently expressed in the bacterium Escherichia coli. When injected into mice, these recombinant products resulted in the production of high titers of antibodies that reacted with the authentic CS protein on live sporozoites and blocked sporozoite invasion of human hepatoma cells in vitro. These CS protein derivatives are therefore candidates for a human malaria vaccine.

  16. Enhanced Protection against Bovine Tuberculosis after Coadministration of Mycobacterium bovis BCG with a Mycobacterial Protein Vaccine-Adjuvant Combination but Not after Coadministration of Adjuvant Alone▿

    OpenAIRE

    Wedlock, D. Neil; Denis, Michel; Painter, Gavin F; Ainge, Gary D.; Vordermeier, H. Martin; Hewinson, R Glyn; Buddle, Bryce M.

    2008-01-01

    Current efforts are aimed at optimizing the protective efficacy of Mycobacterium bovis BCG by the use of vaccine combinations. We have recently demonstrated that the protection afforded by BCG alone is enhanced by vaccinating cattle with a combination of vaccines comprising BCG and a protein tuberculosis vaccine, namely, culture filtrate proteins (CFPs) from M. bovis plus an adjuvant. In the current study, three different adjuvant systems were compared. The CFP was formulated with a depot adj...

  17. Immunogenicity of a Haemophilus influenzae polysaccharide-Neisseria meningitidis outer membrane protein complex conjugate vaccine.

    Science.gov (United States)

    Donnelly, J J; Deck, R R; Liu, M A

    1990-11-01

    Polysaccharide-protein conjugate vaccines made with different carriers vary in their ability to elicit antipolysaccharide IgG antibody responses in young infants and an adult mouse model, suggesting that the carrier proteins used in the conjugate vaccines differ in their ability to act as carriers, or that additional mechanisms of immunogenicity play a role. A conjugate vaccine of Haemophilus influenzae PRP coupled to the outer membrane protein complex (OMPC) of Neisseria meningitidis serogroup B is immunogenic in children as young as 2 mo of age and is immunogenic in infant rhesus monkeys, an animal model for infant humans. In the present study, PRP-OMPC was found to induce efficient IgM to IgG switching of anti-PRP serum antibody in adult mice, whereas PRP conjugated to two other protein carriers did not. Thus the PRP-OMPC conjugate was examined in order to determine why PRP coupled to OMPC was so immunogenic, even more immunogenic than conjugates made with other carrier proteins. The OMPC carrier differs from the other protein carriers in that the proteins are present in a liposomal form containing lipids (including LPS) derived from the outer membrane of N. meningitidis. We studied the OMPC to see whether the different components or the nature of the OMPC carrier could contribute to its enhanced immunogenicity. Specifically we evaluated the OMPC for both classic Th cell carrier activity and adjuvanticity, and the LPS component of OMPC for systemic polyclonal B cell activation. Carrier recognition of the OMPC moiety of PRP-OMPC was demonstrated. In addition the PRP-OMPC conjugate vaccine was observed to have adjuvant properties for both T cell-dependent and T cell-independent Ag in the absence of LPS-induced systemic polyclonal B cell activation. These observations suggest that in addition to functioning as a classic protein carrier whereby the proteins in OMPC provide Th cell epitopes, the OMPC also has adjuvant activity that distinguishes it from other protein

  18. Bacterial Protein Characterization of Streptococcus agalactiae by SDS-page Method for Subclinical Mastitis Irradiated Vaccine Materials in Dairy Cattle

    OpenAIRE

    B.J. Tuasikal; I.W.T. Wibawan2; F.H. Pasaribu2; S. Estuningsih2

    2012-01-01

    A study have been conducted to isolate and characterize bacterial protein S. agalactiae, which is antigenic and can be used to test immunogenicity of vaccine in order to manufacture irradiated mastitis (inflammation of the udder) vaccine in ruminant. The study aims to determine the Molecular Weight (MW) bacterial protein S. agalactiae irradiation, which can be used to test the nature of its antigenic caharacteristic. The character of S. agalactiae antigenic stimulates antibody induction of th...

  19. Merozoite Surface Antigen 2 Proteins of Babesia bovis Vaccine Breakthrough Isolates Contain a Unique Hypervariable Region Composed of Degenerate Repeats

    OpenAIRE

    Berens, Shawn J.; Brayton, Kelly A.; Molloy, John B.; Bock, Russell E.; Lew, Ala E.; McElwain, Terry F.

    2005-01-01

    The merozoite surface antigen 2 (MSA-2) proteins of Babesia bovis are members of the variable merozoite surface antigen (VMSA) family that have been implicated in erythrocyte invasion and are important targets for antibody-mediated blocking of invasion. Extensive sequence variation in another VMSA member, MSA-1, has been shown in all vaccine breakthrough isolates. To test the hypothesis that the msa-2 genes of vaccine breakthrough isolates would also encode a diverse set of proteins, the comp...

  20. Viral-mimicking protein nanoparticle vaccine for eliciting anti-tumor responses

    OpenAIRE

    Molino, NM; Neek, M; Tucker, JA; Nelson, EL; Wang, S-W

    2016-01-01

    The immune system is a powerful resource for the eradication of cancer, but to overcome the low immunogenicity of tumor cells, a sufficiently strong CD8(+) T cell-mediated adaptive immune response is required. Nanoparticulate biomaterials represent a potentially effective delivery system for cancer vaccines, as they can be designed to mimic viruses, which are potent inducers of cellular immunity. We have been exploring the non-viral pyruvate dehydrogenase E2 protein nanoparticle as a biomimet...

  1. Characterization of hepatitis C virus recombinants with chimeric E1/E2 envelope proteins and identification of single amino acids in the E2 stem region important for entry

    DEFF Research Database (Denmark)

    Carlsen, Thomas H R; Scheel, Troels K H; Ramirez, Santseharay;

    2013-01-01

    . For recovered 1b-E2 recombinants, single E2 stem region amino acid changes were identified at residues 706, 707, and 710. In reverse genetic studies, these mutations increased infectivity titers by ~100-fold, apparently without influencing particle stability or cell binding although introducing slight...... decrease in particle density. In addition, the 1b-E2 exchange led to a decrease in secreted core protein of 25 to 50%, which was further reduced by the E2 stem region mutations. These findings indicated that compensatory mutations permitted robust infectious virus production, without increasing assembly....../release. Studies of E1/E2 heterodimerization showed no differences in intracellular E1/E2 interaction for chimeric constructs with or without E2 stem region mutations. Interestingly, the E2 stem region mutations allowed efficient entry, which was verified in 1a-E1/1b-E2 HCV pseudoparticle assays. A CD81 inhibition...

  2. DNA vaccines

    OpenAIRE

    Coban, Cevayir; Kobiyama, Kouji; Jounai, Nao; Tozuka, Miyuki; Ishii, Ken J.

    2013-01-01

    Since the introduction of DNA vaccines two decades ago, this attractive strategy has been hampered by its low immunogenicity in humans. Studies conducted to improve the immunogenicity of DNA vaccines have shown that understanding the mechanism of action of DNA vaccines might be the key to successfully improving their immunogenicity. Our current understanding is that DNA vaccines induce innate and adaptive immune responses in two ways: (1) encoded protein (or polypeptide) antigen(s) by the DNA...

  3. Merozoite surface proteins in red blood cell invasion, immunity and vaccines against malaria.

    Science.gov (United States)

    Beeson, James G; Drew, Damien R; Boyle, Michelle J; Feng, Gaoqian; Fowkes, Freya J I; Richards, Jack S

    2016-05-01

    Malaria accounts for an enormous burden of disease globally, with Plasmodium falciparum accounting for the majority of malaria, and P. vivax being a second important cause, especially in Asia, the Americas and the Pacific. During infection with Plasmodium spp., the merozoite form of the parasite invades red blood cells and replicates inside them. It is during the blood-stage of infection that malaria disease occurs and, therefore, understanding merozoite invasion, host immune responses to merozoite surface antigens, and targeting merozoite surface proteins and invasion ligands by novel vaccines and therapeutics have been important areas of research. Merozoite invasion involves multiple interactions and events, and substantial processing of merozoite surface proteins occurs before, during and after invasion. The merozoite surface is highly complex, presenting a multitude of antigens to the immune system. This complexity has proved challenging to our efforts to understand merozoite invasion and malaria immunity, and to developing merozoite antigens as malaria vaccines. In recent years, there has been major progress in this field, and several merozoite surface proteins show strong potential as malaria vaccines. Our current knowledge on this topic is reviewed, highlighting recent advances and research priorities. PMID:26833236

  4. Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae

    DEFF Research Database (Denmark)

    Stentebjerg-Olesen, Bodil; Pallesen, Lars; Jensen, Lars Bogø;

    1997-01-01

    inserted. Several of the chosen positions seemed amenable even for large foreign inserts; the chimeric proteins were exposed on the bacterial surface and the cholera toxin epitope was authentically displayed, i.e. it was recognized on bacteria by specific antiserum. Display of chimeric fimbriae was tested...

  5. Authentic display of a cholera toxin epitope by chimeric type 1 fimbriae

    DEFF Research Database (Denmark)

    Stentebjerg-Olesen, Bodil; Pallesen, Lars; Jensen, Lars Bogø; Christiansen, Gunnar; Klemm, Per

    inserted. Several of the chosen positions seemed amenable even for large foreign inserts; the chimeric proteins were exposed on the bacterial surface and the cholera toxin epitope was authentically displayed, i.e. it was recognized on bacteria by specific antiserum. Display of chimeric fimbriae was tested...

  6. Antitumor immunity induced by DNA vaccine encoding alpha-fetoprotein/heat shock protein 70

    Institute of Scientific and Technical Information of China (English)

    Xiao-Ping Wang; Guo-Zhen Liu; Ai-Li Song; Hai-Yan Li; Yu Liu

    2004-01-01

    AIM: To construct a DNA vaccine encoding human alphafetoprotein (hAFP)/heat shock protein 70 (HSP70), and to study its ability to induce specific CTL response and its protective effect against AFP-expressing tumor.METHODS: A DNA vaccine was constructed by combining hAFP gene with HSP70 gene. SP2/0 cells were stably transfected with pBBS212-hAFP and pBBS212-hAFP/HSP70eukaryotic expression vectors. Mice were primed and boosted with DNA vaccine hAFP/HSP70 by intramuscular injection, whereas plasmid with hAFP or HSP70 was used as controls. ELISPOT and ELISA were used to detect IFN-γ-producing splenocytes and the level of serum anti-AFP antibody from immunized mice respectively. In vivo tumor challenge was measured to assess the immune effect of the DNA vaccine.RESULTS: By DNA vaccine immunization, the results of ELISPOT and ELISA showed that the number of IFN-γ-producing splenocytes and the level of serum anti-AFP antibody were significantly higher in rhAFP/HSP70 group than in hAFP and empty plasmid groups (95.50±10.90IFN-γ spots/106 cells vs 23.60±11.80 IFN-γ spots/106 cells,7.17±4.24 IFN-γ spots/106 cells, P<0.01; 126.50±8.22 μg/mL vs 51.72±3.40 μg/mL, 5.83±3.79 μg/mL, P<0.01). The tumor volume in rhAFP/HSP70 group was significantly smaller than that in pBBS212-hAFP and empty plasmid groups (37.41±7.34 mm3 vs381.13±15.48 mm3, 817.51±16.25 mm3,P<0.01).CONCLUSION: Sequential immunization with a recombinant DNA vaccine encoding AFP and heat shock protein70 could generate effective AFP-specific T cell responses and induce definite antitumor effects on AFP-producing tumors, which may be suitable for some clinical testing as a vaccine for HCC.

  7. Antibody recognition of porcine circovirus type 2 capsid protein epitopes after vaccination, infection, and disease.

    Science.gov (United States)

    Trible, Benjamin R; Kerrigan, Maureen; Crossland, Nicholas; Potter, Megan; Faaberg, Kay; Hesse, Richard; Rowland, Raymond R R

    2011-05-01

    Open reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) codes for the 233-amino-acid capsid protein (CP). Baculovirus-based vaccines that express only ORF2 are protective against clinical disease following experimental challenge or natural infection. The goal of this study was to identify regions in CP preferentially recognized by sera from experimentally infected and vaccinated pigs and to compare these responses to those of pigs diagnosed with porcine circovirus-associated disease (PCVAD), including porcine multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). The approach was to react porcine sera with CP polypeptide fragments followed by finer mapping studies using overlapping oligopeptides that covered amino acids 141 to 200. The results showed that vaccinated pigs preferentially recognized only the largest polypeptide fragment, CP(43-233). A subset of experimentally infected pigs and pigs with PDNS showed strong reactivity against a CP oligopeptide, 169-STIDYFQPNNKR-180. Alanine scanning identified Y-173, F-174, Q-175, and K-179 as important for antibody recognition. The results from this study support the notion of PCV2 modulation of immunity, including antibody responses that may represent a precursor for disease. The recognition of CP(169-180) and other polypeptides provides opportunities to devise diagnostic tests for monitoring the immunological effectiveness of vaccination. PMID:21430122

  8. Elicitation of strong immune responses by a DNA vaccine expressing a secreted form of hepatitis C virus envelope protein E2 in murine and porcine animal models

    DEFF Research Database (Denmark)

    Li, Yiping; Kang, H.N.; Babiuk, L.A.; Liu, Q.

    2006-01-01

    AIM: To characterize the immunogenicity of a hepatitis C virus (HCV) E2 DNA vaccine alone or with a protein vaccine boost in murine and porcine animal models. METHODS: A DNA vaccine expressing a secreted form of HCV E2 protein was constructed and used to vaccinate mice and piglets with or without...... piglets. These HCV E2 vaccines may represent promising hepatitis C vaccine candidates for further investigations....... boosting with a recombinant E2 protein vaccine formulated with CpG ODN and 10% Emulsigen. The immunogenicity of HCV E2 vaccines was analyzed by ELISA for antibody responses, MTT assay for lymphocyte proliferation, ELISPOT for the number of interferon-gamma secreting cells, and cytotoxic T lymphocyte assays...

  9. Characterization of the neutralization determinants of equine arteritis virus using recombinant chimeric viruses and site-specific mutagenesis of an infectious cDNA clone

    International Nuclear Information System (INIS)

    We have used an infectious cDNA clone of equine arteritis virus (EAV) and reverse genetics technology to further characterize the neutralization determinants in the GP5 envelope glycoprotein of the virus. We generated a panel of 20 recombinant viruses, including 10 chimeric viruses that each contained the ORF5 (which encodes GP5) of different laboratory, field, and vaccine strains of EAV, a chimeric virus containing the N-terminal ectodomain of GP5 of a European strain of porcine reproductive and respiratory syndrome virus, and 9 mutant viruses with site-specific substitutions in their GP5 proteins. The neutralization phenotype of each recombinant chimeric/mutant strain of EAV was determined with EAV-specific monoclonal antibodies and EAV strain-specific polyclonal equine antisera and compared to that of their parental viruses from which the substituted ORF5 was derived. The data unequivocally confirm that the GP5 ectodomain contains critical determinants of EAV neutralization. Furthermore, individual neutralization sites are conformationally interactive, and the interaction of GP5 with the unglycosylated membrane protein M is likely critical to expression of individual epitopes in neutralizing conformation. Substitution of individual amino acids within the GP5 ectodomain usually resulted in differences in neutralization phenotype of the recombinant viruses, analogous to differences in the neutralization phenotype of field strains of EAV and variants generated during persistent infection of EAV carrier stallions

  10. Vector Development for the Expression of Foreign Proteins in the Vaccine Strain Brucella abortus S19

    OpenAIRE

    Diego J Comerci; Pollevick, Guido D.; Vigliocco, Ana M.; Frasch, Alberto C. C.; Ugalde, Rodolfo A.

    1998-01-01

    A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a goo...

  11. Protein energy malnutrition during vaccination has limited influence on vaccine efficacy but abolishes immunity if administered during Mycobacterium tuberculosis infection

    DEFF Research Database (Denmark)

    Hoang, Truc; Agger, Else Marie; Cassidy, Joseph P;

    2015-01-01

    Protein energy malnutrition (PEM) increases susceptibility to infectious diseases, including tuberculosis (TB), but it is not clear how PEM influences vaccine-promoted immunity to TB. We demonstrate that PEM during low-level steady-state TB infection in a mouse model results in rapid relapse of M...

  12. Immunogenicity of a 2-dose priming and booster vaccination with the 10-valent pneumococcal nontypeable Haemophilus influenzae protein D conjugate vaccine

    DEFF Research Database (Denmark)

    Silfverdal, Sven Arne; Høgh, Birthe; Bergsaker, Marianne Riise;

    2009-01-01

    BACKGROUND: The immunogenicity of the 10-valent pneumococcal nontypeable Haemophilus influenzae protein D-conjugate vaccine (PHiD-CV) was determined following a simplified 2-dose priming and the more commonly employed 3-dose priming both followed by a booster dose. METHODS: A total of 351 healthy...

  13. Quantitative Determination of Lethal Toxin Proteins in Culture Supernatant of Human Live Anthrax Vaccine Bacillus anthracis A16R.

    Science.gov (United States)

    Zai, Xiaodong; Zhang, Jun; Liu, Ju; Liu, Jie; Li, Liangliang; Yin, Ying; Fu, Ling; Xu, Junjie; Chen, Wei

    2016-03-01

    Bacillus anthracis (B. anthracis) is the etiological agent of anthrax affecting both humans and animals. Anthrax toxin (AT) plays a major role in pathogenesis. It includes lethal toxin (LT) and edema toxin (ET), which are formed by the combination of protective antigen (PA) and lethal factor (LF) or edema factor (EF), respectively. The currently used human anthrax vaccine in China utilizes live-attenuated B. anthracis spores (A16R; pXO1+, pXO2-) that produce anthrax toxin but cannot produce the capsule. Anthrax toxins, especially LT, have key effects on both the immunogenicity and toxicity of human anthrax vaccines. Thus, determining quantities and biological activities of LT proteins expressed by the A16R strain is meaningful. Here, we explored LT expression patterns of the A16R strain in culture conditions using another vaccine strain Sterne as a control. We developed a sandwich ELISA and cytotoxicity-based method for quantitative detection of PA and LF. Expression and degradation of LT proteins were observed in culture supernatants over time. Additionally, LT proteins expressed by the A16R and Sterne strains were found to be monomeric and showed cytotoxic activity, which may be the main reason for side effects of live anthrax vaccines. Our work facilitates the characterization of anthrax vaccines components and establishment of a quality control standard for vaccine production which may ultimately help to ensure the efficacy and safety of the human anthrax vaccine A16R. PMID:26927174

  14. Transmission-blocking activity of antibodies to Plasmodium falciparum GLURP.10C chimeric protein formulated in different adjuvants

    DEFF Research Database (Denmark)

    Roeffen, Will; Theisen, Michael; van de Vegte-Bolmer, Marga;

    2015-01-01

    BACKGROUND: Plasmodium falciparum is transmitted from person to person by Anopheles mosquitoes after completing its sexual reproductive cycle within the infected mosquito. An efficacious vaccine holds the potential to interrupt development of the parasite in the mosquito leading to control and...

  15. Development and Characterization of Protective Haemophilus parasuis Subunit Vaccines Based on Native Proteins with Affinity to Porcine Transferrin and Comparison with Other Subunit and Commercial Vaccines

    OpenAIRE

    Frandoloso, Rafael; Martínez, Sonia; Rodríguez-Ferri, Elías F.; García-Iglesias, María José; Pérez-Martínez, Claudia; Martínez-Fernández, Beatriz; Gutiérrez-Martín, César B.

    2010-01-01

    Haemophilus parasuis is the agent responsible for causing Glässer's disease, which is characterized by fibrinous polyserositis, polyarthritis, and meningitis in pigs. In this study, we have characterized native outer membrane proteins with affinity to porcine transferrin (NPAPT) from H. parasuis serovar 5, Nagasaki strain. This pool of proteins was used as antigen to developed two vaccine formulations: one was adjuvanted with a mineral oil (Montanide IMS 2215 VG PR), while the other was poten...

  16. Live virus vaccines based on a yellow fever vaccine backbone: standardized template with key considerations for a risk/benefit assessment.

    Science.gov (United States)

    Monath, Thomas P; Seligman, Stephen J; Robertson, James S; Guy, Bruno; Hayes, Edward B; Condit, Richard C; Excler, Jean Louis; Mac, Lisa Marie; Carbery, Baevin; Chen, Robert T

    2015-01-01

    The Brighton Collaboration Viral Vector Vaccines Safety Working Group (V3SWG) was formed to evaluate the safety of live, recombinant viral vaccines incorporating genes from heterologous viruses inserted into the backbone of another virus (so-called "chimeric virus vaccines"). Many viral vector vaccines are in advanced clinical trials. The first such vaccine to be approved for marketing (to date in Australia, Thailand, Malaysia, and the Philippines) is a vaccine against the flavivirus, Japanese encephalitis (JE), which employs a licensed vaccine (yellow fever 17D) as a vector. In this vaccine, two envelope proteins (prM-E) of YF 17D virus were exchanged for the corresponding genes of JE virus, with additional attenuating mutations incorporated into the JE gene inserts. Similar vaccines have been constructed by inserting prM-E genes of dengue and West Nile into YF 17D virus and are in late stage clinical studies. The dengue vaccine is, however, more complex in that it requires a mixture of four live vectors each expressing one of the four dengue serotypes. This vaccine has been evaluated in multiple clinical trials. No significant safety concerns have been found. The Phase 3 trials met their endpoints in terms of overall reduction of confirmed dengue fever, and, most importantly a significant reduction in severe dengue and hospitalization due to dengue. However, based on results that have been published so far, efficacy in preventing serotype 2 infection is less than that for the other three serotypes. In the development of these chimeric vaccines, an important series of comparative studies of safety and efficacy were made using the parental YF 17D vaccine virus as a benchmark. In this paper, we use a standardized template describing the key characteristics of the novel flavivirus vaccine vectors, in comparison to the parental YF 17D vaccine. The template facilitates scientific discourse among key stakeholders by increasing the transparency and comparability of

  17. Obtaining classical swine fever virus E2 recombinant protein and DNA-vaccine on the basis of one subunit

    International Nuclear Information System (INIS)

    Three forms of E2 recombinant protein were expressed in E. coli. Swine sera obtained against different forms of the recombinant protein were cross-studied with indirect ELISA. Using individual proteins as an antigen, only 15% of sera against other forms of protein reacted positively, while 100% of heterologous sera showed positive reaction with fused protein. Challenge experiments showed the existence of protective action only from the individual protein. Specificity and activity of sera obtained from the animals after control challenge was confirmed in a blocking variant of ELISA. Genetic construction used a eukaryotic vector that contained the E2 protein gene. Immunization of mice with the resulting DNA induced synthesis of specific antibodies, the titre of which increased considerably after additional single immunization with the E2 recombinant protein, expressed in E. coli. This demonstrated the effectiveness of animal priming by DNA vaccine, and the possibility of using the E2 recombinant protein in E. coli for booster vaccination. (author)

  18. 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.

  19. Chimeric hepatitis B virus core particles as probes for studying peptide-integrin interactions.

    OpenAIRE

    Chambers, M A; Dougan, G; Newman, J.; Brown, F.; Crowther, J.; Mould, A P; Humphries, M J; Francis, M. J.; Clarke, B.; Brown, A L; Rowlands, D.

    1996-01-01

    An RGD-containing epitope from the foot-and-mouth disease virus (FMDV) VP1 protein was inserted into the e1 loop of the hepatitis B virus core (HBc) protein. This chimeric protein was expressed at high levels in Escherichia coli and spontaneously assembled into virus-like particles which could be readily purified. These fusion particles elicited high levels of both enzyme-linked immunosorbent assay- and FMDV-neutralizing antibodies in guinea pigs. The chimeric particles bound specifically to ...

  20. Outer Membrane Proteins of Brucella abortus Vaccinal and Field Strains and their Immune Response in Buffaloes

    Directory of Open Access Journals (Sweden)

    Rukhshanda Munir*, M. Afzal1, M. Hussain2, S. M. S. Naqvi3 and A. Khanum3

    2010-04-01

    Full Text Available Outer membrane proteins (OMPs of three strains of B. abortus i.e. S19, RB51 and a local field isolate of biotype 1 were isolated through disrupting cells to generate membranes by centrifugation and sodium lauryl sarcosinate solubilisation of inner membrane proteins. Distinct OMP profiles of each strain were seen on SDS-PAGE. SDS-PAGE analysis of S19 and field isolate revealed eight protein bands in each strain. The OMPs of S19 had molecular masses 89.0, 73.0, 53.7, 49.0, 38.0, 27.0, 22.3, and 17.7 kDa, while field isolate had OMPs of 151.3, 89.0, 75.8, 67.6, 37.0, 27.0, 24.0 and 19.0 kDa. B. abortus RB51 yielded 11 OMP bands ranging from 12.5 to 107.1 kDa, with 34.2, 15.8 and 12.5 kDa as additional OMPs. Western immunoblot analysis using antisera raised against all three strains in buffaloes indicated an almost similar pattern of immuno-reactive OMPs in S19 and field strain. Two OMPs of molecular weight 37-38 and 19 kDa were immuno-reactive in all strains in buffaloes. There is possibility of use of these OMPs in a recombinant vaccine for B. abortus. A distinct protein of molecular weight of 151.3 kDa was identified in field strain but not in both vaccine strains of B. abortus. Use of this OMP in a diagnostic assay may differentiate between vaccinated and infected animals.

  1. Complexes of trophoblastic peptides and heat shock protein 70 as a novel contraceptive vaccine in a mouse model.

    Science.gov (United States)

    Han, Mei; Yao, Yuan; Zeng, Wangjiang; Wang, Yanfang; Feng, Lin; Zhao, Jie

    2016-04-01

    The concept of contraceptive vaccines has interested reproductive biologists and immunologists for nearly 2 decades, but no approach has been approved. In this study, a new immunocontraceptive vaccine that targets placental trophoblasts was expored. We demonstrated that after in-vitro binding with heat shock protein 70, trophoblast-derived peptides can activate T cells both in vitro and in vivo. The activated T cells have a Th1 bias and specifically cause cytolysis of trophoblasts, leading to the termination of pregnancy. Such activated T cells seem to have an effect on early gestation, rather than influencing preimplantation. We did not observe side-effects of this vaccine in mice. In conclusion, a novel contraceptive strategy is described that uses heat shock protein 70-trophoblastic peptide complexes to generate a specific T-cell immune response against placental trophoblasts. This type of vaccine targeting the post-implantation phase does not generate a permanent effect but possibly raises an ethical issue. PMID:26847794

  2. Efficient amplification of chimeric adenovirus 5/40S vectors carrying the short fiber protein of Ad40 in suspension cell cultures.

    Directory of Open Access Journals (Sweden)

    Marta Miralles

    Full Text Available The human adenovirus 40 (Ad40 is a promising tool for gene therapy of intestinal diseases. Since the production of Ad40 in vitro is extremely inefficient, chimeric Adenovirus 5/40S vectors carrying the Ad40 short fiber on the Ad5 capsid have been developed. However, Ad5/40S productivity is low. We hypothesized that low productivity was a result of inefficient viral entry into producer cells during amplification. To this end, we have developed a production strategy based on using 211B cells (expressing Ad5 fiber during amplification steps, while Ad5/40S infectivity is further improved by adding polybrene during infections. In addition, the optimal harvesting time was determined by evaluating the Ad5/40S viral cycle. The developed production strategy significantly reduces the number of amplification cycles and duration of the process. Finally, to further facilitate Ad5/40S production, 211B cells were adapted to suspension thus allowing to easily upscale the production process in bioreactors.

  3. Induction of indoleamine 2, 3-dioxygenase in human dendritic cells by a cholera toxin B subunit-proinsulin vaccine.

    Directory of Open Access Journals (Sweden)

    Jacques C Mbongue

    Full Text Available Dendritic cells (DC interact with naïve T cells to regulate the delicate balance between immunity and tolerance required to maintain immunological homeostasis. In this study, immature human dendritic cells (iDC were inoculated with a chimeric fusion protein vaccine containing the pancreatic β-cell auto-antigen proinsulin linked to a mucosal adjuvant the cholera toxin B subunit (CTB-INS. Proteomic analysis of vaccine inoculated DCs revealed strong up-regulation of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1. Increased biosynthesis of the immunosuppressive enzyme was detected in DCs inoculated with the CTB-INS fusion protein but not in DCs inoculated with proinsulin, CTB, or an unlinked combination of the two proteins. Immunoblot and PCR analyses of vaccine treated DCs detected IDO1mRNA by 3 hours and IDO1 protein synthesis by 6 hours after vaccine inoculation. Determination of IDO1 activity in vaccinated DCs by measurement of tryptophan degradation products (kynurenines showed increased tryptophan cleavage into N-formyl kynurenine. Vaccination did not interfere with monocytes differentiation into DC, suggesting the vaccine can function safely in the human immune system. Treatment of vaccinated DCs with pharmacological NF-κB inhibitors ACHP or DHMEQ significantly inhibited IDO1 biosynthesis, suggesting a role for NF-κB signaling in vaccine up-regulation of dendritic cell IDO1. Heat map analysis of the proteomic data revealed an overall down-regulation of vaccinated DC functions, suggesting vaccine suppression of DC maturation. Together, our experimental data indicate that CTB-INS vaccine induction of IDO1 biosynthesis in human DCs may result in the inhibition of DC maturation generating a durable state of immunological tolerance. Understanding how CTB-INS modulates IDO1 activity in human DCs will facilitate vaccine efficacy and safety, moving this immunosuppressive strategy closer to clinical applications for prevention

  4. A Novel Immunogenic Spore Coat-Associated Protein in Bacillus Anthracis: Characterization via Proteomics Approaches and a Vector-Based Vaccine System

    OpenAIRE

    Liu, Yu-Tsueng; Lin, Shwu-Bin; Huang, Cheng-Po; Huang, Chun-Ming

    2007-01-01

    New generation anthrax vaccines have been actively explored with the aim of enhancing efficacies and decreasing undesirable side effects that could be caused by licensed vaccines. Targeting novel antigens and/or eliminating the requirements for multiple needle injections and adjuvants are major objectives in the development of new anthrax vaccines. Using proteomics approaches, we identified a spore coat-associated protein (SCAP) in Bacillus anthracis. An E. coli vector-based vaccine system wa...

  5. Efficacy of bacterin-, outer membrane protein- and fimbriae extract-based vaccines for the control of Salmonella Enteritidis experimental infection in chickens

    OpenAIRE

    2013-01-01

    The efficacy of three vaccines was evaluated in chickens for the control of experimental infection with Salmonella Enteritidis (SE) phage type 4. The vaccines were produced with bacterin, outer membrane proteins (OMP) and fimbriae crude extract (FE). The chickens were vaccinated intramuscularly with two doses of each vaccine at 12 and 15 weeks of age. The chickens were then orally challenged with 10(9) CFU/chicken Salmonella Enteritidis phage type 4 at 18 weeks of age. Fecal swabs were perfor...

  6. Identification of a Protein Subset of the Anthrax Spore Immunome in Humans Immunized with the Anthrax Vaccine Adsorbed Preparation

    OpenAIRE

    Kudva, Indira T.; Griffin, Robert W.; Garren, Jeonifer M.; Calderwood, Stephen B.; John, Manohar

    2005-01-01

    We identified spore targets of Anthrax Vaccine Adsorbed (AVA)-induced immunity in humans by screening recombinant clones of a previously generated, limited genomic Bacillus anthracis Sterne (pXO1+, pXO2−) expression library of putative spore surface (spore-associated [SA]) proteins with pooled sera from human adults immunized with AVA (immune sera), the anthrax vaccine currently approved for use by humans in the United States. We identified 69 clones that reacted specifically with pooled immu...

  7. Myeloma cell line–derived, pooled heat shock proteins as a universal vaccine for immunotherapy of multiple myeloma

    OpenAIRE

    Qian, Jianfei; Hong, Sungyoul; Wang, Siqing; Zhang, Liang; Sun, Luhong; Wang, Michael; Yang, Jing; Kwak, Larry W.; Hou, Jian; Yi, Qing

    2009-01-01

    Tumor cell–derived heat shock proteins are used as vaccines for immunotherapy of cancer patients. However, current approaches require the generation of custom-made products and are clinically ineffective. To improve the applicability of heat shock protein–based immunotherapy in cancers and to enhance clinical efficacy, we explored combinational treatments in a myeloma setting using pooled heterogeneous or allogeneic myeloma cell line–derived glycoprotein 96 (gp96) as universal vaccines, and c...

  8. Heat shock proteins and cancer vaccines: developments in the past decade and chaperoning in the decade to come

    OpenAIRE

    Murshid, Ayesha; Gong, Jianlin; Stevenson, Mary Ann; Calderwood, Stuart K.

    2011-01-01

    Molecular chaperone–peptide complexes extracted from tumors (heat shock protein [HSP] vaccines) have been intensively studied in the preceding two decades, proving to be safe and effective in treating a number of malignant diseases. They offer personalized therapy and target a cross-section of antigens expressed in patients' tumors. Future advances may rely on understanding the molecular underpinnings of this approach to immunotherapy. One property common to HSP vaccines is the ability to sti...

  9. Antibody Recognition of Porcine Circovirus Type 2 Capsid Protein Epitopes after Vaccination, Infection, and Disease▿†

    OpenAIRE

    Trible, Benjamin R.; Kerrigan, Maureen; Crossland, Nicholas; Potter, Megan; Faaberg, Kay; Hesse, Richard; Rowland, Raymond R. R.

    2011-01-01

    Open reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) codes for the 233-amino-acid capsid protein (CP). Baculovirus-based vaccines that express only ORF2 are protective against clinical disease following experimental challenge or natural infection. The goal of this study was to identify regions in CP preferentially recognized by sera from experimentally infected and vaccinated pigs and to compare these responses to those of pigs diagnosed with porcine circovirus-associated disease (...

  10. Nanogel antigenic protein-delivery system for adjuvant-free intranasal vaccines.

    Science.gov (United States)

    Nochi, Tomonori; Yuki, Yoshikazu; Takahashi, Haruko; Sawada, Shin-ichi; Mejima, Mio; Kohda, Tomoko; Harada, Norihiro; Kong, Il Gyu; Sato, Ayuko; Kataoka, Nobuhiro; Tokuhara, Daisuke; Kurokawa, Shiho; Takahashi, Yuko; Tsukada, Hideo; Kozaki, Shunji; Akiyoshi, Kazunari; Kiyono, Hiroshi

    2010-07-01

    Nanotechnology is an innovative method of freely controlling nanometre-sized materials. Recent outbreaks of mucosal infectious diseases have increased the demands for development of mucosal vaccines because they induce both systemic and mucosal antigen-specific immune responses. Here we developed an intranasal vaccine-delivery system with a nanometre-sized hydrogel ('nanogel') consisting of a cationic type of cholesteryl-group-bearing pullulan (cCHP). A non-toxic subunit fragment of Clostridium botulinum type-A neurotoxin BoHc/A administered intranasally with cCHP nanogel (cCHP-BoHc/A) continuously adhered to the nasal epithelium and was effectively taken up by mucosal dendritic cells after its release from the cCHP nanogel. Vigorous botulinum-neurotoxin-A-neutralizing serum IgG and secretory IgA antibody responses were induced without co-administration of mucosal adjuvant. Importantly, intranasally administered cCHP-BoHc/A did not accumulate in the olfactory bulbs or brain. Moreover, intranasally immunized tetanus toxoid with cCHP nanogel induced strong tetanus-toxoid-specific systemic and mucosal immune responses. These results indicate that cCHP nanogel can be used as a universal protein-based antigen-delivery vehicle for adjuvant-free intranasal vaccination. PMID:20562880

  11. A novel method for synthetic vaccine construction based on protein assembly

    OpenAIRE

    Zhida Liu; Hang Zhou; Wenjun Wang; Wenjie Tan; Yang-Xin Fu; Mingzhao Zhu

    2014-01-01

    In the history of vaccine development, the synthetic vaccine is a milestone that is in stark contrast with traditional vaccines based on live-attenuated or inactivated microorganisms. Synthetic vaccines not only are safer than attenuated or inactivated microorganisms but also provide the opportunity for vaccine design for specific purposes. The first generation of synthetic vaccines has been largely based on DNA recombination technology and genetic manipulation. This de novo generation is occ...

  12. Use of non-structural proteins of FMDV to differentiate vaccinated and infected animals in Argentina

    International Nuclear Information System (INIS)

    In this paper, we present the results of the use of serological tests to detect antibodies to non-structural proteins (NSP) in cattle and pigs from different epidemiological situations in Argentina. The work was made under the sponsoring of the IAEA/FAO from 2000 to 2004, as part of the Coordinated Research Project. Serological tests are used at the National Reference Laboratory (DILAB) for sero-surveillance; monitoring; control of animal movement and as diagnostic tool in case of outbreaks. The control of vaccine purity in terms of NSP contents was examined using various assays. (author)

  13. Chimeric enzymes with improved cellulase activities

    Science.gov (United States)

    Xu, Qi; Baker, John O; Himmel, Michael E

    2015-03-31

    Nucleic acid molecules encoding chimeric cellulase polypeptides that exhibit improved cellulase activities are disclosed herein. The chimeric cellulase polypeptides encoded by these nucleic acids and methods to produce the cellulases are also described, along with methods of using chimeric cellulases for the conversion of cellulose to sugars such as glucose.

  14. Status of vaccine research and development of vaccines for HIV-1.

    Science.gov (United States)

    Safrit, Jeffrey T; Fast, Patricia E; Gieber, Lisa; Kuipers, Hester; Dean, Hansi J; Koff, Wayne C

    2016-06-01

    Human immunodeficiency virus (HIV) is the cause of one of the most lethal pandemics in human history, although in recent years access to highly effective anti-retroviral therapy has provided new hope worldwide. Transmission of HIV by sexual contact, childbirth and injection drug use has been reduced, but 2 million are newly infected each year, and much of the transmission is from people who do not know their status. In addition to known methods, a preventive vaccine is needed to end the pandemic. The extraordinary mutability and genetic diversity of HIV is an enormous challenge, but vaccines are being designed for broad coverage. Computer-aided design of mosaic immunogens, incorporating many epitopes from the entire genome or from conserved regions aim to induce CD8+ T cells to kill virus-infected cells or inhibit virus replication, while trimeric envelope proteins or synthetic mimics aim to induce broadly reactive neutralizing antibodies similar to those cloned from some infected patients. Induction of more potent and durable responses may require new adjuvants or replicating chimeric vectors chimeras that bear HIV genes. Passive or genetic delivery of broadly neutralizing antibodies may provide broad protection and/or lead to insights for vaccine designers. Proof-of-concept trials in non-human primates and in one human efficacy trial have provided scientific clues for a vaccine that could provide broad and durable protection against HIV. The use of vaccines to destroy HIV reservoirs as part of therapy or cure is now also being explored. PMID:26993335

  15. Active immunotherapy of allergic asthma with a recombinant human interleukin-5 protein as vaccine in a murine model

    Institute of Scientific and Technical Information of China (English)

    TAN Guang-hong; WANG Cai-chun; HUANG Feng-ying; WANG Hua; HUANG Yong-hao; LIN Ying-ying

    2007-01-01

    Background Eosinophils are highly related to allergic asthma inflammation. Interleukin (IL)-5 is the major chemokine of eosinophils, inhibition of the activity of IL-5 thus seems to be a potential approach to asthma therapy. The current study was performed to determine whether a recombinant human IL-5 protein as a xenogeneic vaccine has the capability of inducing anti-asthma activities.Methods Recombinant human IL-5 was used as a protein vaccine. Mouse asthma model was established to observe the anti-asthma activities. Lung histology was observed; eosinophils in blood and bronchoalveolar lavage were stained and counted. Airway hyperresponsiveness was determined by whole body plethysmograph. Antibody characters and cytokines were detected with enzyme linked immunosorbent assay (ELISA) and Western blot assay.Results Vaccination with recombinant human IL-5 protein as vaccine significantly reduced airway inflammation and airway hyperresponsiveness, and shifted the cytokine production from Th2 (IL-4) to Th1 (INF-γ) in mice allergic-asthma model. Immunization with recombinant human IL-5 protein vaccine bypassed the immunological tolerance and induced production of polyclonal antibodies that were cross-reactive with murine IL-5.Conclusions Active immunization with xenogeneic homologous IL-5 may be a possible therapeutic approach to the treatment of asthma and potentially of other eosinophilic disorders.

  16. Immunity Elicited by an Experimental Vaccine Based on Recombinant Flagellin-Porcine Circovirus Type 2 Cap Fusion Protein in Piglets

    Science.gov (United States)

    Wang, Jing; Wei, Li; Quan, Rong; Yang, Jiayu; Yan, Xu; Li, Zixuan; She, Ruiping; Hu, Fengjiao; Liu, Jue

    2016-01-01

    In a recent study, we reported that a recombinant protein from fusion expression of flagellin to porcine circovirus type 2 (PCV2) Cap induced robust humoral and cell-mediated immunity that afforded full protection for PCV2 infection using BALB/c mice. Here, we further evaluated the immunogenicity and protection of the recombinant protein using specific pathogen free (SPF) pigs. Twenty-five 3-week-old piglets without passively acquired immunity were divided into 5 groups. All piglets except negative controls were challenged with a virulent PCV2 at 21 days after booster vaccination and necropsied at 21 days post-challenge. Vaccination of piglets with the recombinant protein without adjuvant induced strong humoral and cellular immune responses as observed by high levels of PCV2-specific IgG antibodies and neutralizing antibodies, as well as frequencies of PCV2-specific IFN-γ-secreting cells that conferred good protection against PCV2 challenge, with significant reduced PCV2 viremia, mild lesions, low PCV2 antigen-positive cells, as well as improved body weight gain, comparable to piglets vaccinated with a commercial PCV2 subunit vaccine. These results further demonstrated that the recombinant flagellin-Cap fusion protein is capable of inducing solid protective humoral and cellular immunity when administered to pigs, thereby becoming an effective PCV2 vaccine candidate for control of PCV2 infection. PMID:26848967

  17. Chimeric newcastle disease virus protects chickens against avian influenza in the presence of maternally derived NDV immunity.

    Directory of Open Access Journals (Sweden)

    Constanze Steglich

    Full Text Available Newcastle disease virus (NDV, an avian paramyxovirus type 1, is a promising vector for expression of heterologous proteins from a variety of unrelated viruses including highly pathogenic avian influenza virus (HPAIV. However, pre-existing NDV antibodies may impair vector virus replication, resulting in an inefficient immune response against the foreign antigen. A chimeric NDV-based vector with functional surface glycoproteins unrelated to NDV could overcome this problem. Therefore, an NDV vector was constructed which carries the fusion (F and hemagglutinin-neuraminidase (HN proteins of avian paramyxovirus type 8 (APMV-8 instead of the corresponding NDV proteins in an NDV backbone derived from the lentogenic NDV Clone 30 and a gene expressing HPAIV H5 inserted between the F and HN genes. After successful virus rescue by reverse genetics, the resulting chNDVFHN PMV8H5 was characterized in vitro and in vivo. Expression and virion incorporation of the heterologous proteins was verified by Western blot and electron microscopy. Replication of the newly generated recombinant virus was comparable to parental NDV in embryonated chicken eggs. Immunization with chNDVFHN PMV8H5 stimulated full protection against lethal HPAIV infection in chickens without as well as with maternally derived NDV antibodies. Thus, tailored NDV vector vaccines can be provided for use in the presence or absence of routine NDV vaccination.

  18. Sublingual vaccination with sonicated Salmonella proteins and mucosal adjuvant induces mucosal and systemic immunity and protects mice from lethal enteritis.

    Science.gov (United States)

    Huang, Ching-Feng; Wu, Tzee-Chung; Wu, Chia-Chao; Lee, Chin-Cheng; Lo, Wen-Tsung; Hwang, Kwei-Shuai; Hsu, Mu-Ling; Peng, Ho-Jen

    2011-07-01

    Salmonella enteritidis is one of the most common pathogens of enteritis. Most experimental vaccines against Salmonella infection have been applied through injections. This is a new trial to explore the effect of sublingual administration of Salmonella vaccines on systemic and mucosal immunity. Adult BALB/c mice were sublingually vaccinated with sonicated Salmonella proteins (SSP) alone, or plus adjuvant CpG DNA (CpG) or cholera toxin (CT). They were boosted 2 weeks later. Saliva specific secretory IgA (SIgA) antibody responses were significantly stimulated in the mice vaccinated with SSP only or together with CpG or CT. Whereas the mice sublingually vaccinated with SSP and CpG had higher spleen cell IFN-γ production and serum specific IgG2a antibody responses, those receiving SSP and CT showed enhanced spleen cell IL-4, IL-5 and IL-6 production, and serum specific IgG1 antibody responses. After oral challenge with live S. enteritidis, the same strain of the source of SSP, immune protection in those sublingually vaccinated with SSP and CpG or CT was found to prevent intestinal necrosis and to render a higher survival rate. In conclusion, sublingual vaccination together with mucosal adjuvant CpG or CT is a simple but effective way against enteric bacterial pathogens. PMID:21635554

  19. A Plasmodium Promiscuous T Cell Epitope Delivered within the Ad5 Hexon Protein Enhances the Protective Efficacy of a Protein Based Malaria Vaccine.

    Science.gov (United States)

    Fonseca, Jairo Andres; Cabrera-Mora, Monica; Kashentseva, Elena A; Villegas, John Paul; Fernandez, Alejandra; Van Pelt, Amelia; Dmitriev, Igor P; Curiel, David T; Moreno, Alberto

    2016-01-01

    A malaria vaccine is a public health priority. In order to produce an effective vaccine, a multistage approach targeting both the blood and the liver stage infection is desirable. The vaccine candidates also need to induce balanced immune responses including antibodies, CD4+ and CD8+ T cells. Protein-based subunit vaccines like RTS,S are able to induce strong antibody response but poor cellular reactivity. Adenoviral vectors have been effective inducing protective CD8+ T cell responses in several models including malaria; nonetheless this vaccine platform exhibits a limited induction of humoral immune responses. Two approaches have been used to improve the humoral immunogenicity of recombinant adenovirus vectors, the use of heterologous prime-boost regimens with recombinant proteins or the genetic modification of the hypervariable regions (HVR) of the capsid protein hexon to express B cell epitopes of interest. In this study, we describe the development of capsid modified Ad5 vectors that express a promiscuous Plasmodium yoelii T helper epitope denominated PyT53 within the hexon HVR2 region. Several regimens were tested in mice to determine the relevance of the hexon modification in enhancing protective immune responses induced by the previously described protein-based multi-stage experimental vaccine PyCMP. A heterologous prime-boost immunization regime that combines a hexon modified vector with transgenic expression of PyCMP followed by protein immunizations resulted in the induction of robust antibody and cellular immune responses in comparison to a similar regimen that includes a vector with unmodified hexon. These differences in immunogenicity translated into a better protective efficacy against both the hepatic and red blood cell stages of P. yoelii. To our knowledge, this is the first time that a hexon modification is used to deliver a promiscuous T cell epitope. Our data support the use of such modification to enhance the immunogenicity and protective

  20. DEVELOPMENT OF RECOMBINANT VACCINE AGAINST A(H1N1) 2009 INFLUENZA BASED ON VIRUS-LIKE NANOPARTICLES CARRYING THE EXTRACELLULAR DOMAIN OF M2 PROTEIN

    OpenAIRE

    Kotlyarov, R.; Kuprianov, V.; Migunov, A.; Stepanova, L.; Tsybalova, L.; Kiselev, O.; Ravin, N.; Skryabin, K.

    2010-01-01

    The conventional vaccines currently being used to deal with influenza are based on a virus obtained in chicken embryos or its components. The high variability of the major immunogenic surface proteins – hemagglutinin and neuraminidase–require the development of strain–specific vaccines that match the antigenic specificity of a newly emerging virus. Recombinant vaccines based on single viral proteins that could be easily produced in standard expression systems are attractive alternatives to tr...

  1. EXPERIMENTAL MEASLES VACCINES: A RESEARCH TOOL IN VACCINATION EVENTS

    OpenAIRE

    V. A. Liashenko

    2014-01-01

    Abstract. The review article considers different variants of measles vaccine that may be classified into two groups, i.e., vaccines that do not contain viable measles virus, and attenuated measles vaccines which could be employed in unusual manner.The first group includes DNA-vaccines, recombinant vaccine strains encoding synthesis of measles hemagglutinin and fusion protein, as well as peptide vaccines containing molecular fragments of these proteins. The mentioned variants of vaccines were ...

  2. Vaccination with pcDNA3-15/60 Naked DNA Encoding the Surface Protein of Sporozoites in Cryptosporidium parvum

    Institute of Scientific and Technical Information of China (English)

    HE Hong-xuan; ZHANG Xi-chen; YIN Ji-gang; LI Jian-hua; YANG Ju

    2004-01-01

    The CP15/60 gene encoding the CP15/60 surface protein of sporozoites in Cryptosporidium parvum was obtained by PCR so as to research the nucleic vaccine against C.parvum. The eukaryotic expressing vector pcDNA3-15/60 was constructed by inserting CP15/60 gene into pcDNA3 (+) in Xho Ⅰ and EcoR Ⅰ. A vaccination protocol was the adult pregnant goats inoculated intranasally with the pcDNA3-15/60 plasmid and their offspring were infected with C.parvum oocysts. The results showed that the pcDNA3-15/60 plasmid can induce the immune response of goats and the vaccinated goats can transfer the immunity to offspring conferring protection against C.parvum infection. These suggested that the recombinant plasmid could be a DNA vaccine candidate.

  3. Nucleic Acid Vaccines

    Institute of Scientific and Technical Information of China (English)

    LU Shan

    2004-01-01

    @@ Anew method of immunization was discovered in the early 1990s. Several research groups independently demonstrated that direct inoculation of DNA plasmids coding for a specific protein antigen could elicit immune responses against that antigen[1-4].Since in theory the mRNA molecules also have the potential to be translated into the protein antigen, this vaccination approach was officially named by WHO as the nucleic acid vaccination even though the term DNA vaccine has been used more commonly in the literature. This novel approach is considered the fourth generation of vaccines after live attenuated vaccines, killed or inactivated vaccines and recombinant protein based subunit vaccines.

  4. Proteomic Analysis of Mecistocirrus digitatus and Haemonchus contortus Intestinal Protein Extracts and Subsequent Efficacy Testing in a Vaccine Trial

    OpenAIRE

    Dicker, Alison J; Inglis, Neil F.; Manson, Erin D. T.; Subhadra, Subhra; Illangopathy, Manikkavasagan; Muthusamy, Raman; Knox, David P

    2014-01-01

    Background Gastrointestinal nematode infections, such as Haemonchus contortus and Mecistocirrus digitatus, are ranked in the top twenty diseases affecting small-holder farmers' livestock, yet research into M. digitatus, which infects cattle and buffalo in Asia is limited. Intestine-derived native protein vaccines are effective against Haemonchus, yet the protective efficacy of intestine-derived M. digitatus proteins has yet to be determined. Methodology/Principal Findings A simplified protein...

  5. Limited variation in vaccine candidate Plasmodium falciparum Merozoite Surface Protein-6 over multiple transmission seasons

    Directory of Open Access Journals (Sweden)

    Branch OraLee H

    2010-05-01

    Full Text Available Abstract Background Plasmodium falciparum Merozoite Surface Protein-6 (PfMSP6 is a component of the complex proteinacious coat that surrounds P. falciparum merozoites. This location, and the presence of anti-PfMSP6 antibodies in P. falciparum-exposed individuals, makes PfMSP6 a potential blood stage vaccine target. However, genetic diversity has proven to be a major hurdle for vaccines targeting other blood stage P. falciparum antigens, and few endemic field studies assessing PfMSP6 gene diversity have been conducted. This study follows PfMSP6 diversity in the Peruvian Amazon from 2003 to 2006 and is the first longitudinal assessment of PfMSP6 sequence dynamics. Methods Parasite DNA was extracted from 506 distinct P. falciparum infections spanning the transmission seasons from 2003 to 2006 as part of the Malaria Immunology and Genetics in the Amazon (MIGIA cohort study near Iquitos, Peru. PfMSP6 was amplified from each sample using a nested PCR protocol, genotyped for allele class by agarose gel electrophoresis, and sequenced to detect diversity. Allele frequencies were analysed using JMP v.8.0.1.0 and correlated with clinical and epidemiological data collected as part of the MIGIA project. Results Both PfMSP6 allele classes, K1-like and 3D7-like, were detected at the study site, confirming that both are globally distributed. Allele frequencies varied significantly between transmission seasons, with 3D7-class alleles dominating and K1-class alleles nearly disappearing in 2005 and 2006. There was a significant association between allele class and village location (p-value = 0.0008, but no statistically significant association between allele class and age, sex, or symptom status. No intra-allele class sequence diversity was detected. Conclusions Both PfMSP6 allele classes are globally distributed, and this study shows that allele frequencies can fluctuate significantly between communities separated by only a few kilometres, and over time in the

  6. The Effects of Salt on the Physicochemical Properties and Immunogenicity of Protein Based Vaccine Formulated in Cationic Liposome

    OpenAIRE

    Yan, Weili; Huang, Leaf

    2008-01-01

    Recently, we have developed a simple and potent therapeutic cancer vaccine consisting of a cationic lipid and a peptide antigen. In this report, we expanded the utility of this formulation to a protein based vaccine. First, we formulated the human papillomavirus (HPV) 16 E7 protein (E7) in different doses of DOTAP liposome. The results showed that these formulations failed to regress an established tumor. However, when sodium chloride (30 mM) was added to the DOTAP (100 nmol) / E7 (20 μg) for...

  7. Immunogenicity of a plasmid DNA vaccine encoding 42kDa fragment of Plasmodium vivax merozoite surface protein-1.

    Science.gov (United States)

    Sheikh, Inayat Hussain; Kaushal, Deep C; Chandra, Deepak; Kaushal, Nuzhat A

    2016-10-01

    Plasmodium vivax is the second major human malaria parasite that inflicts debilitating morbidity and consequent economic impact in South-East Asian countries. The relapsing nature of P. vivax along with the emergence of drug-resistant P. vivax strains has emphasized the urgent need for a vaccine. However, the development of an effective vivax vaccine is seriously hampered due to the diversity and variation in parasite antigens and non-availability of suitable animal models. DNA based vaccines represent an alternative approach in inducing immunity to multiple targets from different stages of malaria parasite. DNA prime-boosting strategies induce both antibody mediated and cell-mediated immune responses that are the major mechanisms of protection against malaria parasites. We have earlier studied the immunogenicity and protective efficacy of the soluble and refolded forms of recombinant 42kDa fragment of Plasmodium vivax merozoite surface protein-1 (PvMSP-142) using P. cynomolgi rhesus monkey model. In the present study, we have constructed a recombinant DNA vaccine encoding 42kDa fragment of P. vivax MSP-1 and studied the immunogenicity of PvMSP-142 DNA vaccine construct in mice. The 42kDa gene fragment of PvMSP-1 was PCR amplified using gene specific primers and subcloned into pcDNA 3.1 (+) eukaryotic expression vector. In vitro expression of PvMSP-142 plasmid construct was checked by transfection in COS-1 cell line. Indirect immunofluorescence of transfected COS-1 cells probed with monoclonal antibodies against PvMSP-142 exhibited positive fluorescence. Immunization of BALB/c mice with PvMSP-142-pcDNA vaccine construct revealed the immunogenicity of recombinant vaccine plasmid that can be enhanced by prime boosting with recombinant protein corresponding to the DNA vaccine as evidenced by significant elevation of antibody and the cytokines responses. PMID:27311385

  8. The effects of salt on the physicochemical properties and immunogenicity of protein based vaccine formulated in cationic liposome.

    Science.gov (United States)

    Yan, Weili; Huang, Leaf

    2009-02-23

    Recently, we have developed a simple and potent therapeutic cancer vaccine consisting of a cationic lipid and a peptide antigen. In this report, we expanded the utility of this formulation to protein based vaccines. First, we formulated the human papillomavirus (HPV) 16 E7 protein (E7) in different doses of DOTAP liposome. The results showed that these formulations failed to regress an established tumor. However, when sodium chloride (30 mM) was added to the DOTAP (100 nmol)/E7 (20 microg) formulation, anti-tumor activity was generated in the immunized mice. Correlatively, 30 mM NaCl in the DOTAP/E7 protein formulation increased the particle size from approximately 350 to 550 nm, decreased the protein loading capacity (from 95 to 90%), and finally increased the zeta potential (from 29 to 38 mV). Next, a model protein antigen ovalbumin (OVA) was formulated in different doses of DOTAP liposomes. Similarly, the results showed that 20 microg OVA formulated in 200 nmol DOTAP with 30 mM NaCl had the best OVA-specific antibody response, including both IgG(1) and IgG(2a), suggesting both Th1 and Th2 immune responses were generated by this formulation. In conclusion, we have expanded the application of cationic DOTAP liposome formulation to protein based vaccines and also identified that small amounts of salt could change the physicochemical properties of the vaccine formulation and enhance the activity of the DOTAP/protein based vaccine. The enhancement of immune responses by salt is possibly due to its interference of the electrostatic interaction between the cationic lipid and the protein antigen to facilitate the antigen release from the carrier and at the same time activate the antigen presenting cells. PMID:18992312

  9. Evaluation of a multi-epitope subunit vaccine against avian leukosis virus subgroup J in chickens.

    Science.gov (United States)

    Xu, Qingqing; Ma, Xingjiang; Wang, Fangkun; Li, Hongmei; Zhao, Xiaomin

    2015-12-01

    The intricate sequence and antigenic variability of avian leukosis virus subgroup J (ALV-J) have led to unprecedented difficulties in the development of vaccines. Much experimental evidence demonstrates that ALV-J mutants have caused immune evasion and pose a challenge for traditional efforts to develop effective vaccines. To investigate the potential of a multi-epitope vaccination strategy to prevent chickens against ALV-J infections, a recombinant chimeric multi-epitope protein X (rCMEPX) containing both immunodominant B and T epitope concentrated domains selected from the major structural protein of ALV-J using bioinformatics approach was expressed in Escherichia coli Rosetta (DE3). Its immunogenicity and protective efficacy was studied in chickens. The results showed that rCMEPX could elicit neutralizing antibodies and cellular responses, and antibodies induced by rCMEPX could specifically recognize host cell naturally expressed ALV-J proteins, which indicated that the rCMEPX is a good immunogen. Challenge experiments showed 80% chickens that received rCMEPX were well protected against ALV-J challenge. This is the first report of a chimeric multi-epitope protein as a potential immunogen against ALV-J. PMID:26196055

  10. The DNA Binding Domain of a Papillomavirus E2 Protein Programs a Chimeric Nuclease To Cleave Integrated Human Papillomavirus DNA in HeLa Cervical Carcinoma Cells▿

    OpenAIRE

    Horner, Stacy M.; DiMaio, Daniel

    2007-01-01

    Viral DNA binding proteins that direct nucleases or other protein domains to viral DNA in lytically or latently infected cells may provide a novel approach to modulate viral gene expression or replication. Cervical carcinogenesis is initiated by high-risk human papillomavirus (HPV) infection, and viral DNA persists in the cancer cells. To test whether a DNA binding domain of a papillomavirus protein can direct a nuclease domain to cleave HPV DNA in cervical cancer cells, we fused the DNA bind...

  11. A Two-Component DNA-Prime/Protein-Boost Vaccination Strategy for Eliciting Long-Term, Protective T Cell Immunity against Trypanosoma cruzi

    OpenAIRE

    Shivali Gupta; Garg, Nisha J.

    2015-01-01

    In this study, we evaluated the long-term efficacy of a two-component subunit vaccine against Trypanosoma cruzi infection. C57BL/6 mice were immunized with TcG2/TcG4 vaccine delivered by a DNA-prime/Protein-boost (D/P) approach and challenged with T. cruzi at 120 or 180 days post-vaccination (dpv). We examined whether vaccine-primed T cell immunity was capable of rapid expansion and intercepting the infecting T. cruzi. Our data showed that D/P vaccine elicited CD4+ (30-38%) and CD8+ (22-42%) ...

  12. Luteinizing hormone-releasing hormone fusion protein vaccines block estrous cycle activity in beef heifers.

    Science.gov (United States)

    Stevens, J D; Sosa, J M; deAvila, D M; Oatley, J M; Bertrand, K P; Gaskins, C T; Reeves, J J

    2005-01-01

    Two LHRH fusion proteins, thioredoxin and ovalbumin, each containing seven LHRH inserts were tested for their ability to inhibit estrous cycle activity. The objective was to evaluate immune and biological responses from alternating the two fusion proteins in an immunization schedule. One hundred ten heifers were divided equally into 11 groups. Two control groups consisted of either spayed or intact, untreated heifers. Heifers in the other nine groups were immunized on wk 0, 4, and 9. Treatments were immunizations of the same protein throughout or alternating the proteins in different booster sequences. Blood was collected weekly for 22 wk, and serum was assayed for concentrations of progesterone and titers of anti-LHRH. At slaughter, reproductive tracts were removed from each heifer and weighed. Heifers with >or=1 ng/mL of progesterone were considered to have a functional corpus luteum and thus to have estrous cycle activity. All LHRH-immunized groups of heifers had a smaller (P spayed heifers during wk 9 to 22. Anti-LHRH did not differ among immunized groups during wk 1 to 9. Starting at wk 10 and continuing through the conclusion of the study, there was an overall difference among treatment groups for anti-LHRH (P spaying in suppression of estrous cycle activity, but alternating the two proteins in an immunization schedule did not enhance the immunological or biological effectiveness of the vaccine. PMID:15583055

  13. Exploiting the Campylobacter jejuni protein glycosylation system for glycoengineering vaccines and diagnostic tools directed against brucellosis

    Directory of Open Access Journals (Sweden)

    Iwashkiw Jeremy A

    2012-01-01

    Full Text Available Abstract Background Immune responses directed towards surface polysaccharides conjugated to proteins are effective in preventing colonization and infection of bacterial pathogens. Presently, the production of these conjugate vaccines requires intricate synthetic chemistry for obtaining, activating, and attaching the polysaccharides to protein carriers. Glycoproteins generated by engineering bacterial glycosylation machineries have been proposed to be a viable alternative to traditional conjugation methods. Results In this work we expressed the C. jejuni oligosaccharyltansferase (OTase PglB, responsible for N-linked protein glycosylation together with a suitable acceptor protein (AcrA in Yersinia enterocolitica O9 cells. MS analysis of the acceptor protein demonstrated the transfer of a polymer of N-formylperosamine to AcrA in vivo. Because Y. enterocolitica O9 and Brucella abortus share an identical O polysaccharide structure, we explored the application of the resulting glycoprotein in vaccinology and diagnostics of brucellosis, one of the most common zoonotic diseases with over half a million new cases annually. Injection of the glycoprotein into mice generated an IgG response that recognized the O antigen of Brucella, although this response was not protective against a challenge with a virulent B. abortus strain. The recombinant glycoprotein coated onto magnetic beads was efficient in differentiating between naïve and infected bovine sera. Conclusion Bacterial engineered glycoproteins show promising applications for the development on an array of diagnostics and immunoprotective opportunities in the future.

  14. Potential of Translationally Controlled Tumor Protein-Derived Protein Transduction Domains as Antigen Carriers for Nasal Vaccine Delivery.

    Science.gov (United States)

    Bae, Hae-Duck; Lee, Joohyun; Jin, Xing-Hai; Lee, Kyunglim

    2016-09-01

    Nasal vaccination offers a promising alternative to intramuscular (i.m.) vaccination because it can induce both mucosal and systemic immunity. However, its major drawback is poor absorption of large antigens in the nasal epithelium. Protein transduction domains (PTDs), also called cell-penetrating peptides, have been proposed as vehicles for nasal delivery of therapeutic peptides and proteins. Here, we evaluated the potential of a mutant PTD derived from translationally controlled tumor protein (designated TCTP-PTD 13) as an antigen carrier for nasal vaccines. We first compared the l- and d-forms of TCTP-PTD 13 isomers (l- or d-TCTP-PTD 13) as antigen carriers. Studies in mice demonstrated that nasally administered mixtures of the model antigen ovalbumin (OVA) and d-TCTP-PTD 13 induced higher plasma IgG titers and secretory IgA levels in nasal washes than nasally administered OVA alone, OVA/l-TCTP-PTD 13, or i.m.-injected OVA. Plasma IgG subclass responses (IgG1 and IgG2a) of mice nasally administered OVA/d-TCTP-PTD 13 showed that the predominant IgG subclass was IgG1, indicating a Th2-biased immune response. We also used synthetic CpG oligonucleotides (CpG) as a Th1 immune response-inducing adjuvant. Nasally administered CpG plus OVA/d-TCTP-PTD 13 was superior in eliciting systemic and mucosal immune responses compared to those induced by nasally administered OVA/d-TCTP-PTD 13. Furthermore, the OVA/CpG/d-TCTP-PTD 13 combination skewed IgG1 and IgG2a profiles of humoral immune responses toward a Th1 profile. These findings suggest that TCTP-derived PTD is a suitable vehicle to efficiently carry antigens and to induce more powerful antigen-specific immune responses and a more balanced Th1/Th2 response when combined with a DNA adjuvant. PMID:27454469

  15. Bm86 antigen induces a protective immune response against Boophilus microplus following DNA and protein vaccination in sheep.

    Science.gov (United States)

    De Rose, R; McKenna, R V; Cobon, G; Tennent, J; Zakrzewski, H; Gale, K; Wood, P R; Scheerlinck, J P; Willadsen, P

    1999-11-30

    Vaccination of sheep with a plasmid bearing the full length gene for the tick antigen Bm86 either alone or co-administered with plasmid carrying the ovine genes for the cytokines, granulocyte and macrophage colony stimulating factor (GM-CSF) or interleukin (IL)-1beta induced a relatively low level of protection against subsequent tick infestation. This tick damage reached statistical significance only for the groups which were vaccinated with plasmid encoding for Bm86, co-administered with plasmid encoding for ovine GM-CSF. Antibody titres measured against Bm86 were also low in all groups injected with the Bm86 DNA vaccine. Antibody production and anti-tick effect were significantly less than that achieved by two vaccinations with recombinant Bm86 protein. In all cases only a low level of antigen-specific stimulation of peripheral blood lymphocytes was recorded, as measured either by the incorporation of tritiated thymidine or the release of IFN-gamma. Injection of DNA encoding for Bm86, either alone or with co-administered cytokine genes, did however prime for a strong subsequent antibody response following a single injection of recombinant Bm86 protein in adjuvant. Antibody production nevertheless appeared to be slightly less effective than following two vaccinations with recombinant protein. The persistence of antibody following vaccination was the same regardless of the method of primary sensitization. In all cases the half-life of the antibody response was approximately 40-50 days indicating that, in contrast to results reported in mice, DNA vaccination in sheep did not result in sustained antibody production. PMID:10587297

  16. Enhanced protective immunity of the chimeric vector-based vaccine rAdV-SFV-E2 against classical swine fever in pigs by a Salmonella bacterial ghost adjuvant.

    Science.gov (United States)

    Xia, Shui-Li; Lei, Jian-Lin; Du, Mingliang; Wang, Yimin; Cong, Xin; Xiang, Guang-Tao; Li, Lian-Feng; Yu, Shenye; Du, Enqi; Liu, Siguo; Sun, Yuan; Qiu, Hua-Ji

    2016-01-01

    Classical swine fever (CSF) is a highly contagious swine disease caused by classical swine fever virus (CSFV). Previously, we demonstrated that rAdV-SFV-E2, an adenovirus-delivered, Semliki Forest virus replicon-vectored marker vaccine against CSF, is able to protect pigs against lethal CSFV challenge. From an economical point of view, it will be beneficial to reduce the minimum effective dose of the vaccine. This study was designed to test the adjuvant effects of Salmonella enteritidis-derived bacterial ghosts (BG) to enhance the protective immunity of rAdV-SFV-E2 in pigs. Groups of 5-week-old pigs (n = 4) were immunized intramuscularly twice with 10(5) median tissue culture infective doses (TCID50) rAdV-SFV-E2 combined with 10(10) colony forming units (CFU) BG, 10(6) or 10(5) TCID50 rAdV-SFV-E2 alone or 10(10) CFU BG alone at an interval of 3 weeks, and challenged with the highly virulent CSFV Shimen strain at 1 week post-booster immunization. The results show that the pigs inoculated with 10(5) TCID50 rAdV-SFV-E2 plus BG or 10(6) TCID50 rAdV-SFV-E2 alone were completely protected from lethal CSFV challenge, in contrast with the pigs vaccinated with 10(5) TCID50 rAdV-SFV-E2 or BG alone, which displayed partial or no protection following virulent challenge. The data indicate that BG are a promising adjuvant to enhance the efficacy of rAdV-SFV-E2 and possibly other vaccines. PMID:27301745

  17. Designing an efficient multi-epitope peptide vaccine against Vibrio cholerae via combined immunoinformatics and protein interaction based approaches.

    Science.gov (United States)

    Nezafat, Navid; Karimi, Zeinab; Eslami, Mahboobeh; Mohkam, Milad; Zandian, Sanam; Ghasemi, Younes

    2016-06-01

    Cholera continues to be a major global health concern. Among different Vibrio cholerae strains, only O1 and O139 cause acute diarrheal diseases that are related to epidemic and pandemic outbreaks. The currently available cholera vaccines are mainly lived and attenuated vaccines consisting of V. cholerae virulence factors such as toxin-coregulated pili (TCP), outer membrane proteins (Omps), and nontoxic cholera toxin B subunit (CTB). Nowadays, there is a great interest in designing an efficient epitope vaccine against cholera. Epitope vaccines consisting of immunodominant epitopes and adjuvant molecules enhance the possibility of inciting potent protective immunity. In this study, V. cholerae protective antigens (OmpW, OmpU, TcpA and TcpF) and the CTB, which is broadly used as an immunostimulatory adjuvant, were analyzed using different bioinformatics and immunoinformatics tools. The common regions between promiscuous epitopes, binding to various HLA-II supertype alleles, and B-cell epitopes were defined based upon the aforementioned protective antigens. The ultimately selected epitopes and CTB adjuvant were fused together using proper GPGPG linkers to enhance vaccine immunogenicity. A three-dimensional model of the thus constructed vaccine was generated using I-TASSER. The model was structurally validated using the ProSA-web error-detection software and the Ramachandran plot. The validation results indicated that the initial 3D model needed refinement. Subsequently, a high-quality model obtained after various refinement cycles was used for defining conformational B-cell epitopes. Several linear and conformational B-cell epitopes were determined within the epitope vaccine, suggesting likely antibody triggering features of our designed vaccine. Next, molecular docking was performed between the 3D vaccine model and the tertiary structure of the toll like receptor 2 (TLR2). To gain further insight into the interaction between vaccine and TLR2, molecular dynamics

  18. A novel subunit vaccine co-expressing GM-CSF and PCV2b Cap protein enhances protective immunity against porcine circovirus type 2 in piglets.

    Science.gov (United States)

    Zhang, Huawei; Qian, Ping; Peng, Bo; Shi, Lin; Chen, Huanchun; Li, Xiangmin

    2015-05-15

    Porcine circovirus type 2 (PCV2) causes porcine circovirus-associated disease. Capsid (Cap) protein of PCV2 is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. GM-CSF is an immune adjuvant that enhances responses to vaccines. In this study, recombinant baculoviruses Ac-Cap and Ac-Cap-GM-CSF expressing the Cap protein alone and co-expressing the Cap protein and porcine GM-CSF, respectively, were constructed successfully. The target proteins were analyzed by western blotting and IFA. Further, these proteins were confirmed by electron microscopy, which showed that Cap proteins could self-assemble into virus-like particles having diameters of 17-25nm. Animal experiments showed that pigs immunized with Cap-GM-CSF subunit vaccine showed significantly higher levels of PCV2-specific antibodies and neutralizing antibodies than pigs immunized with the Cap subunit vaccine and a commercial vaccine (Ingelvac CircoFLEX; PPigs receiving the Cap-GM-CSF subunit vaccine showed significantly higher average daily weight gain after wild-type PCV2 challenge than pigs receiving the other three vaccines (Ppigs. Thus, the novel Cap-GM-CSF subunit vaccine has the potential to be used as an effective and safe vaccine candidate against PCV2 infection. PMID:25863115

  19. Use of non-structural proteins to FMDV to assess antibodies in vaccinated and infected cattle in Peru

    International Nuclear Information System (INIS)

    The identification of animals that have been infected with FMDV is very important for the control measures of the disease, because the animals frequently become carriers and can be the cause of new outbreaks. In countries were vaccination is applied it is recognised that there is the necessity to use tests that can differentiate the antibodies resulted from the vaccine and the antibodies from infection. In recent years tests have been developed that identify antibodies against virus-specific proteins present only in infected animals, being the non-structural protein 2C and non-structural 3ABC polyprotein the most studied. The work made under the CRP can be divided into three phases. (1) Examination of a set of tests provided up to 2002; (2) Examination of tests provided from 2002 to present and; (3) Studies on post vaccinated animal to examine whether antibodies to NSP are produced. (author)

  20. A Novel Adeno-Associated Virus-Based Genetic Vaccine Encoding the Hepatitis C Virus NS3/4 Protein Exhibits Immunogenic Properties in Mice Superior to Those of an NS3-Protein-Based Vaccine.

    Directory of Open Access Journals (Sweden)

    Fengqin Zhu

    Full Text Available More than 170 million individuals worldwide are infected with hepatitis C virus (HCV, and up to an estimated 30% of chronically infected individuals will go on to develop progressive liver disease. Despite the recent advances in antiviral treatment of HCV infection, it remains a major public health problem. Thus, development of an effective vaccine is urgently required. In this study, we constructed novel adeno-associated virus (AAV vectors expressing the full-length NS3 or NS3/4 protein of HCV genotype 1b. The expression of the NS3 or NS3/4 protein in HepG2 cells was confirmed by western blotting. C57BL/6 mice were intramuscularly immunised with a single injection of AAV vectors, and the resultant immune response was investigated. The AAV2/rh32.33.NS3/4 vaccine induced stronger humoral and cellular responses than did the AAV2/rh32.33.NS3 vaccine. Our results demonstrate that AAV-based vaccines exhibit considerable potential for the development of an effective anti-HCV vaccine.

  1. Subunit Protein Vaccine Delivery System for Tuberculosis Based on Hepatitis B Virus Core VLP (HBc-VLP) Particles.

    Science.gov (United States)

    Dhanasooraj, Dhananjayan; Kumar, R Ajay; Mundayoor, Sathish

    2016-01-01

    Despite the development of modern medicine, tuberculosis (TB), caused by the pathogenic bacterium, Mycobacterium tuberculosis (Mtb), remains one of the deadliest diseases. This bacterium can lay dormant in individuals and get activated when immunity goes down and has also shown considerable prowess in mutating into drug resistant forms. The global emergence of such drug resistant Mtb and the lack of efficacy of Bacille Calmette Guérin (BCG), the only vaccine available so far, have resulted in a situation which cries out for a safe and effective tuberculosis vaccine.Number of different strategies has been used for developing new anti-TB vaccines and several protective antigens have been identified so far. One strategy, the use of protein subunits, has the potential to develop into a powerful tuberculosis vaccine, not only because of its efficacy and safety, but also because they are economical. The proper delivery of protein subunit vaccines with adjuvants or novel delivery systems is necessary for inducing protective immune responses. The available adjuvants or delivery systems are inadequate for generating such a response. In the present method, we have constructed a vaccine delivery system for tuberculosis based on Virus-Like Particles (VLPs). Hepatitis B Virus core antigen gene was recombinantly modified using Overlap Extension PCR (OEPCR). The final construct was designed to express HBc-VLP carrying external antigen (fusion VLP). Mycobacterium tuberculosis antigen CFP-10 was used for the construction of fusion VLP. The recombinant gene for the construct was cloned into a pET expression system and transformed into E. coli BL21(DE3) and induced with IPTG to express the protein. The fusion protein was purified using the Histidine tag and allowed to form VLPs. The preformed VLPs were purified by sucrose density gradient centrifugation. The VLPs were characterized using Transmission Electron Microscopy (TEM). PMID:27076312

  2. Immunity conferred by an experimental vaccine based on the recombinant PCV2 Cap protein expressed in Trichoplusia ni-larvae.

    Science.gov (United States)

    Pérez-Martín, Eva; Gómez-Sebastián, Silvia; Argilaguet, Jordi M; Sibila, Marina; Fort, María; Nofrarías, Miquel; Kurtz, Sherry; Escribano, José M; Segalés, Joaquim; Rodríguez, Fernando

    2010-03-01

    Porcine circovirus type 2 (PCV2) vaccination has been recently included as a measure to control postweaning multisystemic wasting syndrome (PMWS) in the field. Aiming to obtain a more affordable vaccine to be extensively implemented in the field, a highly efficient non-fermentative expression platform based on Trichoplusia ni (T. ni) larvae was used to produce a baculovirus-derived recombinant PCV2 Cap protein (rCap) for vaccine purposes. Vaccination of pigs with rCap induced solid protection against PCV2 experimental infection, inhibiting both the viremia and the viral shedding very efficiently. The protection afforded by the rCap vaccine strongly correlated with the induction of specific humoral immune responses, even in the presence of PCV2-specific maternal immunity, although cellular responses also seemed to play a partial role. In summary, we have shown that rCap expressed in T. ni larvae could be a cost-effective PCV2 vaccine candidate to be tested under field conditions. PMID:20056179

  3. Vaccination against Very Virulent Infectious Bursal Disease Virus Using Recombinant T4 Bacteriophage Displaying Viral Protein VP2

    Institute of Scientific and Technical Information of China (English)

    Yong-Chang CAO; Quan-Cheng SHI; Jing-Yun MA; Qing-Mei XIE; Ying-Zuo BI

    2005-01-01

    In order to develop a desirable inexpensive, effective and safe vaccine against the very virulent infectious bursal disease virus (vvIBDV), we tried to take advantage of the emerging T4 bacteriophage surface protein display system. The major immunogen protein VP2 from the vvIBDV strain HK46 was fused to the nonessential T4 phage surface capsid protein, a small outer capsid (SOC) protein, resulting in the 49 kDa SOC-VP2 fusion protein, which was verified by sodium dodecylsulfate polyacrylamide gel electrophoresis and Western blot. Immunoelectromicroscopy showed that the recombinant VP2 protein was successfully displayed on the surface of the T4 phage. The recombinant VP2 protein is antigenic and showed reactivities to various monoclonal antibodies (mAbs) against IBDV, whereas the wild-type phage T4 could not react to any mAb. In addition, the recombinant VP2 protein is immunogenic and elicited specific antibodies in immunized specific pathogen free (SPF) chickens. More significantly, immunization of SPF chickens with the recombinant T4-VP2 phage protected them from infection by the vvIBDV strain HK46. When challenged with the vvIBDV strain HK46 at a dose of 100 of 50% lethal dose (LD50) per chicken 4 weeks after the booster was given, the group vaccinated with the T4-VP2 recombinant phage showed no clinical signs of disease or death, wh ereas the unvaccinated group and the group vaccinated with the wild-type T4phage exhibited 100% clinical signs of disease and bursal damages, and 30%-40% mortality. Collectively,the data herein showed that the T4-displayed VP2 protein might be an inexpensive, effective and safe vaccine candidate against vvIBDV.

  4. Cationic lipid-formulated DNA vaccine against hepatitis B virus: immunogenicity of MIDGE-Th1 vectors encoding small and large surface antigen in comparison to a licensed protein vaccine.

    Directory of Open Access Journals (Sweden)

    Anne Endmann

    Full Text Available Currently marketed vaccines against hepatitis B virus (HBV based on the small (S hepatitis B surface antigen (HBsAg fail to induce a protective immune response in about 10% of vaccinees. DNA vaccination and the inclusion of PreS1 and PreS2 domains of HBsAg have been reported to represent feasible strategies to improve the efficacy of HBV vaccines. Here, we evaluated the immunogenicity of SAINT-18-formulated MIDGE-Th1 vectors encoding the S or the large (L protein of HBsAg in mice and pigs. In both animal models, vectors encoding the secretion-competent S protein induced stronger humoral responses than vectors encoding the L protein, which was shown to be retained mainly intracellularly despite the presence of a heterologous secretion signal. In pigs, SAINT-18-formulated MIDGE-Th1 vectors encoding the S protein elicited an immune response of the same magnitude as the licensed protein vaccine Engerix-B, with S protein-specific antibody levels significantly higher than those considered protective in humans, and lasting for at least six months after the third immunization. Thus, our results provide not only the proof of concept for the SAINT-18-formulated MIDGE-Th1 vector approach but also confirm that with a cationic-lipid formulation, a DNA vaccine at a relatively low dose can elicit an immune response similar to a human dose of an aluminum hydroxide-adjuvanted protein vaccine in large animals.

  5. Protection of Mice from Fatal Measles Encephalitis by Vaccination with Vaccinia Virus Recombinants Encoding Either the Hemagglutinin or the Fusion Protein

    Science.gov (United States)

    Drillien, Robert; Spehner, Daniele; Kirn, Andre; Giraudon, Pascale; Buckland, Robin; Wild, Fabian; Lecocq, Jean-Pierre

    1988-02-01

    Vaccinia virus recombinants encoding the hemagglutinin or fusion protein of measles virus have been constructed. Infection of cell cultures with the recombinants led to the synthesis of authentic measles proteins as judged by their electrophoretic mobility, recognition by antibodies, glycosylation, proteolytic cleavage, and presentation on the cell surface. Mice vaccinated with a single dose of the recombinant encoding the hemagglutinin protein developed antibodies capable of both inhibiting hemagglutination activity and neutralizing measles virus, whereas animals vaccinated with the recombinant encoding the fusion protein developed measles neutralizing antibodies. Mice vaccinated with either of the recombinants resisted a normally lethal intracerebral inoculation of a cell-associated measles virus subacute sclerosing panencephalitis strain.

  6. Strategic evaluation of vaccine candidate antigens for the prevention of Visceral Leishmaniasis.

    Science.gov (United States)

    Duthie, Malcolm S; Favila, Michelle; Hofmeyer, Kimberley A; Tutterrow, Yeung L; Reed, Steven J; Laurance, John D; Picone, Alessandro; Guderian, Jeffrey; Bailor, H Remy; Vallur, Aarthy C; Liang, Hong; Mohamath, Raodoh; Vergara, Julie; Howard, Randall F; Coler, Rhea N; Reed, Steven G

    2016-05-27

    Infection with Leishmania parasites results in a range of clinical manifestations and outcomes, the most severe of which is visceral leishmaniasis (VL). Vaccination will likely provide the most effective long-term control strategy, as the large number of vectors and potential infectious reservoirs renders sustained interruption of Leishmania parasite transmission extremely difficult. Selection of the best vaccine is complicated because, although several vaccine antigen candidates have been proposed, they have emerged following production in different platforms. To consolidate the information that has been generated into a single vaccine platform, we expressed seven candidates as recombinant proteins in E. coli. After verifying that each recombinant protein could be recognized by VL patients, we evaluated their protective efficacy against experimental L. donovani infection of mice. Administration in formulation with the Th1-potentiating adjuvant GLA-SE indicated that each antigen could elicit antigen-specific Th1 responses that were protective. Considering the ability to reduce parasite burden along with additional factors such as sequence identity across Leishmania species, we then generated a chimeric fusion protein comprising a combination of the 8E, p21 and SMT proteins. This E. coli -expressed fusion protein was also demonstrated to protect against L. donovani infection. These data indicate a novel recombinant vaccine antigen with the potential for use in VL control programs. PMID:27142329

  7. In vitro assembly properties of mutant and chimeric intermediate filament proteins: insight into the function of sequences in the rod and end domains of IF

    International Nuclear Information System (INIS)

    The factors and mechanisms regulating assembly of intermediate filament (IF) proteins to produce filaments with their characteristic 10 nm diameter are not fully understood. All IF proteins contain a central rod domain flanked by variable head and tail domains. To elucidate the role that different domains of IF proteins play in filament assembly, we used negative staining and electron microscopy (EM) to study the in vitro assembly properties of purified bacterially expressed IF proteins, in which specific domains of the proteins were either mutated or swapped between a cytoplasmic (mouse neurofilament-light (NF-L) subunit) and nuclear intermediate filament protein (human lamin A). Our results indicate that filament formation is profoundly influenced by the composition of the assembly buffer. Wild type (wt) mouse NF-L formed 10 nm filaments in assembly buffer containing 175 mM NaCl, whereas a mutant deleted of 18 NH2-terminal amino acids failed to assemble under similar conditions. Instead, the mutant assembled efficiently in buffers containing CaCl2 ≥ 6 mM forming filaments that were 10 times longer than those formed by wt NF-L, although their diameter was significantly smaller (6-7 nm). These results suggest that the 18 NH2-terminal sequence of NF-L might serve two functions, to inhibit filament elongation and to promote lateral association of NF-L subunits. We also demonstrate that lengthening of the NF-L rod domain, by inserting a 42 aa sequence unique to nuclear IF proteins, does not compromise filament assembly in any noticeable way. Our results suggests that the known inability of nuclear lamin proteins to assemble into 10 nm filaments in vitro cannot derive solely from their longer rod domain. Finally, we demonstrate that the head domain of lamin A can substitute for that of NF-L in filament assembly, whereas substitution of both the head and tail domains of lamins for those of NF-L compromises assembly. Therefore, the effect of lamin A 'tail' domain alone

  8. Identification of potential new protein vaccine candidates through pan-surfomic analysis of pneumococcal clinical isolates from adults.

    Directory of Open Access Journals (Sweden)

    Alfonso Olaya-Abril

    Full Text Available Purified polysaccharide and conjugate vaccines are widely used for preventing infections in adults and in children against the Gram-positive bacterium Streptococcus pneumoniae, a pathogen responsible for high morbidity and mortality rates, especially in developing countries. However, these polysaccharide-based vaccines have some important limitations, such as being serotype-dependent, being subjected to losing efficacy because of serotype replacement and high manufacturing complexity and cost. It is expected that protein-based vaccines will overcome these issues by conferring a broad coverage independent of serotype and lowering production costs. In this study, we have applied the "shaving" proteomic approach, consisting of the LC/MS/MS analysis of peptides generated by protease treatment of live cells, to a collection of 16 pneumococcal clinical isolates from adults, representing the most prevalent strains circulating in Spain during the last years. The set of unique proteins identified in all the isolates, called "pan-surfome", consisted of 254 proteins, which included most of the protective protein antigens reported so far. In search of new candidates with vaccine potential, we identified 32 that were present in at least 50% of the clinical isolates analyzed. We selected four of them (Spr0012, Spr0328, Spr0561 and SP670_2141, whose protection capacity has not yet been tested, for assaying immunogenicity in human sera. All of them induced the production of IgM antibodies in infected patients, thus indicating that they could enter the pipeline for vaccine studies. The pan-surfomic approach shows its utility in the discovery of new proteins that can elicit protection against infectious microorganisms.

  9. Layer-By-Layer Nanoparticle Vaccines Carrying the G Protein CX3C Motif Protect against RSV Infection and Disease

    Directory of Open Access Journals (Sweden)

    Patricia A. Jorquera

    2015-10-01

    Full Text Available Respiratory syncytial virus (RSV is the single most important cause of serious lower respiratory tract infections in young children; however no effective treatment or vaccine is currently available. Previous studies have shown that therapeutic treatment with a monoclonal antibody (clone 131-2G specific to the RSV G glycoprotein CX3C motif, mediates virus clearance and decreases leukocyte trafficking to the lungs of RSV-infected mice. In this study, we show that vaccination with layer-by-layer nanoparticles (LbL-NP carrying the G protein CX3C motif induces blocking antibodies that prevent the interaction of the RSV G protein with the fractalkine receptor (CX3CR1 and protect mice against RSV replication and disease pathogenesis. Peptides with mutations in the CX3C motif induced antibodies with diminished capacity to block G protein-CX3CR1 binding. Passive transfer of these anti-G protein antibodies to mice infected with RSV improved virus clearance and decreased immune cell trafficking to the lungs. These data suggest that vaccination with LbL-NP loaded with the CX3C motif of the RSV G protein can prevent manifestations of RSV disease by preventing the interaction between the G protein and CX3CR1 and recruitment of immune cells to the airways.

  10. Effect of Bacterial Flora on Postimmunization Gastritis following Oral Vaccination of Mice with Helicobacter pylori Heat Shock Protein 60

    OpenAIRE

    Yamaguchi, Hiroyuki; Osaki, Takako; Taguchi, Haruhiko; Sato, Noriko; Toyoda, Atushi; Takahashi, Motomichi; Kai, Masanori; Nakata, Noboru; Komatsu, Akio; Atomi, Yutaka; Kamiya, Shigeru

    2003-01-01

    In order to assess the efficacy of oral Helicobacter pylori heat shock protein 60 (HSP60) as a vaccine, protection against H. pylori infection in specific-pathogen-free (SPF) C57BL/6 and germfree (GF) IQI mice was examined. Prophylactic oral vaccination of these two strains of mice with either H. pylori HSP60 or Escherichia coli GroEL inhibited H. pylori colonization by 90 to 95% at 3 weeks postinfection (p.i.). However, these mice were only partially protected because bacterial loads increas...

  11. A computational method for identification of vaccine targets from protein regions of conserved human leukocyte antigen binding

    DEFF Research Database (Denmark)

    Olsen, Lars Rønn; Simon, Christian; Kudahl, Ulrich J.;

    2015-01-01

    variable regions, where all variants bind HLA. These regions, although variable, can thus be considered stable in terms of HLA binding and represent valuable vaccine targets. Results: We applied this method to predict CD8+ T-cell targets in influenza A H7N9 hemagglutinin and significantly increased the...... number of potential vaccine targets compared to the number of targets discovered using the traditional approach where low-frequency peptides are excluded. Conclusions: We developed a webserver with an intuitive visualization scheme for summarizing the T cell-based antigenic potential of any given protein...

  12. Bioinformatics Study on Zaire Ebolavirus (EBOV Protein for Better Understanding the Vaccine Development

    Directory of Open Access Journals (Sweden)

    D.S. Mundaganur

    2014-12-01

    Full Text Available Nine, Ebola viruse EBOV (Zaire ebolavirus, proteins are extracted from the NCBI repository and their study was carried out. The physicchemical properties and evolutionary link with other such viruses by homology modeling were carried out. All the proteins show rich in leucine domain an ideal requirement for fast attachment of the virus to the receptor molecule on the host cell surface. The prediction of trans-membrane sequence for the entire glycoprotein component reveals the ability of the virus to enter the host with ease. The lack of adequate homology model for the viral proteins indicates its novel origin and lack of well traceable evolutionary link. We studied the homology based model by using various available tools and find similar approach in all, hence finally concentrated only on one method. The model predicted shows well acceptable region on Ramchandran plot. This discrepancy is only due to the fact that we validate the model to Ramchandran plot and the model predicted were not under the well acceptable ‘e’ value range i.e. >1. Therefore we suggests that vaccine production against this deadly virus should be concentrated on the structure and functions of glycoprotein like low quality secreted glycoprotein (NP_066248, low quality spike glycoprotein(NP_0662460, small secreted glycoprotein (NP_066247 and RNA dependent RNA polymerase (NP_066251.

  13. Merozoite Surface Antigen 2 Proteins of Babesia bovis Vaccine Breakthrough Isolates Contain a Unique Hypervariable Region Composed of Degenerate Repeats

    Science.gov (United States)

    Berens, Shawn J.; Brayton, Kelly A.; Molloy, John B.; Bock, Russell E.; Lew, Ala E.; McElwain, Terry F.

    2005-01-01

    The merozoite surface antigen 2 (MSA-2) proteins of Babesia bovis are members of the variable merozoite surface antigen (VMSA) family that have been implicated in erythrocyte invasion and are important targets for antibody-mediated blocking of invasion. Extensive sequence variation in another VMSA member, MSA-1, has been shown in all vaccine breakthrough isolates. To test the hypothesis that the msa-2 genes of vaccine breakthrough isolates would also encode a diverse set of proteins, the complete msa-2 locus was characterized from 12 Australian B. bovis strains and isolates, including two vaccine strains and eight vaccine breakthrough isolates, and compared to the loci in previously and newly characterized American strains. In contrast to American strains, the msa-2 loci of all Australian strains and isolates examined contain, in addition to msa-2c, only a solitary gene (designated msa-2a/b) closely related to American strain msa-2a and msa-2b. Nevertheless, the proteins encoded by these genes are quite diverse both between and within geographic regions and harbor evidence of genetic exchange among other VMSA family members, including msa-1. Moreover, all but one of the Australian breakthrough isolate MSA-2a/b proteins is markedly different from the vaccine strain from which immune escape occurred, consistent with their role in strain-specific protective immunity. The densest distribution of polymorphisms occurs in a hypervariable region (HVR) within the carboxy third of the molecule that is highly proline rich. Variation in length and content of the HVR is primarily attributable to differences in the order and number of degenerate nucleotide repeats encoding three motifs of unknown function. PMID:16239512

  14. Metabolizable protein supply modulated the acute-phase response following vaccination of beef steers.

    Science.gov (United States)

    Moriel, P; Arthington, J D

    2013-12-01

    Our objective was to evaluate the effects of MP supply, through RUP supplementation, on the acute-phase response of beef steers following vaccination. On d 0, Brangus-crossbred steers (n = 24; 173 ± 31 kg; 175 ± 16 d of age) were randomly assigned to receive 1 of 3 isocaloric diets formulated to provide 85, 100, and 115% of the daily MP requirements of a beef steer gaining 0.66 kg of BW daily. Diets were limit-fed at 1.8% of BW (DM basis) and individually provided to steers once daily (0800 h) from d 0 to 29. Steers were weighed on d 0 and 29, following a 12-h period of feed and water withdrawal. On d 7, steers were vaccinated against Mannheimia haemolytica (OneShot, Pfizer), and blood samples were collected on d 0, 7, 8, 10, 14, 21, and 30. Plasma metabolites were analyzed as repeated measures using the MIXED procedure of SAS. Final BW and ADG were similar (P ≥ 0.50) among treatments (mean = 184 ± 9 kg and 0.5 ± 0.08 kg/d, respectively). Effects of time were detected (P < 0.01) for plasma concentrations of all acute-phase proteins, which peaked between d 7 to 14, returning to baseline concentrations by d 29. Treatment effects were not detected (P ≥ 0.19) for plasma concentrations of acid-soluble protein, albumin, fibrinogen, IGF-1 and serum amyloid-A. Plasma concentrations of total protein (TP) and plasma urea nitrogen (PUN) increased (P ≤ 0.05) with increasing supply of MP (87.1, 89.6, and 90.1 ± 1.09 mg TP/mL and 6.1, 8.3, and 10.3 ± 0.41 mg PUN/dL for 85, 100, and 115% MP steers, respectively). From d 10 to 29, steers provided 115% MP had less (P < 0.001) plasma concentrations of ceruloplasmin than steers fed 85 and 100% MP, which had similar plasma ceruloplasmin concentrations. On d 14, plasma concentrations of haptoglobin were greatest (P ≤ 0.06) for steers fed 115% MP, intermediate for 100% MP, and least for 85% MP (0.98, 0.71 and 0.44 ± 0.099 mg/mL, respectively). On d 10, plasma concentrations of creatinine were greater (P = 0.01) for steers

  15. Immunogenicity of a novel tetravalent vaccine formulation with four recombinant lipidated dengue envelope protein domain IIIs in mice

    Science.gov (United States)

    Chiang, Chen-Yi; Pan, Chien-Hsiung; Chen, Mei-Yu; Hsieh, Chun-Hsiang; Tsai, Jy-Ping; Liu, Hsueh-Hung; Liu, Shih-Jen; Chong, Pele; Leng, Chih-Hsiang; Chen, Hsin-Wei

    2016-01-01

    We developed a novel platform to express high levels of recombinant lipoproteins with intrinsic adjuvant properties. Based on this technology, our group developed recombinant lipidated dengue envelope protein domain IIIs as vaccine candidates against dengue virus. This work aims to evaluate the immune responses in mice to the tetravalent formulation. We demonstrate that 4 serotypes of recombinant lipidated dengue envelope protein domain III induced both humoral and cellular immunity against all 4 serotypes of dengue virus on the mixture that formed the tetravalent formulation. Importantly, the immune responses induced by the tetravalent formulation in the absence of the exogenous adjuvant were functional in clearing the 4 serotypes of dengue virus in vivo. We affirm that the tetravalent formulation of recombinant lipidated dengue envelope protein domain III is a potential vaccine candidate against dengue virus and suggest further detailed studies of this formulation in nonhuman primates. PMID:27470096

  16. Immunogenicity of a novel tetravalent vaccine formulation with four recombinant lipidated dengue envelope protein domain IIIs in mice.

    Science.gov (United States)

    Chiang, Chen-Yi; Pan, Chien-Hsiung; Chen, Mei-Yu; Hsieh, Chun-Hsiang; Tsai, Jy-Ping; Liu, Hsueh-Hung; Liu, Shih-Jen; Chong, Pele; Leng, Chih-Hsiang; Chen, Hsin-Wei

    2016-01-01

    We developed a novel platform to express high levels of recombinant lipoproteins with intrinsic adjuvant properties. Based on this technology, our group developed recombinant lipidated dengue envelope protein domain IIIs as vaccine candidates against dengue virus. This work aims to evaluate the immune responses in mice to the tetravalent formulation. We demonstrate that 4 serotypes of recombinant lipidated dengue envelope protein domain III induced both humoral and cellular immunity against all 4 serotypes of dengue virus on the mixture that formed the tetravalent formulation. Importantly, the immune responses induced by the tetravalent formulation in the absence of the exogenous adjuvant were functional in clearing the 4 serotypes of dengue virus in vivo. We affirm that the tetravalent formulation of recombinant lipidated dengue envelope protein domain III is a potential vaccine candidate against dengue virus and suggest further detailed studies of this formulation in nonhuman primates. PMID:27470096

  17. Genetic screen of a library of chimeric poxviruses identifies an ankyrin repeat protein involved in resistance to the avian type I interferon response.

    Science.gov (United States)

    Buttigieg, Karen; Laidlaw, Stephen M; Ross, Craig; Davies, Marc; Goodbourn, Stephen; Skinner, Michael A

    2013-05-01

    Viruses must be able to resist host innate responses, especially the type I interferon (IFN) response. They do so by preventing the induction or activity of IFN and/or by resisting the antiviral effectors that it induces. Poxviruses are no exception, with many mechanisms identified whereby mammalian poxviruses, notably, vaccinia virus (VACV), but also cowpox and myxoma viruses, are able to evade host IFN responses. Similar mechanisms have not been described for avian poxviruses (avipoxviruses). Restricted for permissive replication to avian hosts, they have received less attention; moreover, the avian host responses are less well characterized. We show that the prototypic avipoxvirus, fowlpox virus (FWPV), is highly resistant to the antiviral effects of avian IFN. A gain-of-function genetic screen identified fpv014 to contribute to increased resistance to exogenous recombinant chicken alpha IFN (ChIFN1). fpv014 is a member of the large family of poxvirus (especially avipoxvirus) genes that encode proteins containing N-terminal ankyrin repeats (ANKs) and C-terminal F-box-like motifs. By binding the Skp1/cullin-1 complex, the F box in such proteins appears to target ligands bound by the ANKs for ubiquitination. Mass spectrometry and immunoblotting demonstrated that tandem affinity-purified, tagged fpv014 was complexed with chicken cullin-1 and Skp1. Prior infection with an fpv014-knockout mutant of FWPV still blocked transfected poly(I·C)-mediated induction of the beta IFN (ChIFN2) promoter as effectively as parental FWPV, but the mutant was more sensitive to exogenous ChIFN1. Therefore, unlike the related protein fpv012, fpv014 does not contribute to the FWPV block to induction of ChIFN2 but does confer resistance to an established antiviral state. PMID:23427151

  18. A novel vaccine against Streptococcus equi ssp. zooepidemicus infections: the recombinant swinepox virus expressing M-like protein.

    Science.gov (United States)

    Lin, Hui-xing; Huang, Dong-yan; Wang, Ye; Lu, Cheng-ping; Fan, Hong-jie

    2011-09-16

    To develop a safer, more immunogenic and efficacious vaccine against Streptococcus equi ssp. zooepidemicus (SEZ) infections, the gene of M-like protein (SzP) was placed under the strong vaccinia virus promoter P28 and then inserted into swinepox virus (SPV) genome. The recombinant swinepox virus (rSPV-szp) was isolated in a non-selective medium by the co-expression of Escherichia coli LacZ gene and verified by PCR, western blotting and immunofluorescence assays. To evaluate the immunogenicity of this rSPV-szp, ICR mice were immunized with the rSPV-szp, inactivated SEZ vaccine (positive control), wild type SPV (negative control), or PBS (challenge control). All mice were intraperitoneally challenged with 5 LD(50) of homogenous ATCC 35246 strain 14 days post-vaccination. The results showed that at least 70% mice in rSPV-szp-vaccinated group were protected against homogenous ATCC 35246 challenge, the survival rate was significantly higher compared with mice in the negative control group and the challenge control group (Panalysis showed a marked increased in levels of IL-4 and IFN-γ mRNA in immunized mice. The results suggested that the recombinant rSPV-szp provided mice with significant protection from the SEZ infections. It is a promising candidate for the vaccine development against SEZ infections. PMID:21807055

  19. 恶性疟原虫多表位蛋白疫苗M.RCAg-1和M.RCAg-3佐剂的配伍%Effects and immune mechanism of different adjuvants on polyepitope chimeric vaccine M.RCAg-1 & M.RCAg-3

    Institute of Scientific and Technical Information of China (English)

    马瑞森; 蔺亚晖; 张文文; 麻明; 王恒

    2011-01-01

    Objective To investigate the formulations of Plasmodium falciparum polyepitope chimeric vaccine M.RCAg-1 & M.RCAg-3 with different adjuvants in order to increase their immunogenicities and to find out their basic immune mechanisms.Methods The polyepitope chimeric vaccines formulated with different adjuvants (AD,ADL, CpG, CpG&Al) were subcutaneously injected into mice for three times.The immunization was performed on week 0, 2, 4.The concentration of anti-M.RCAg-1 and anti-M.RCAg-3 IgG, the IgG isotype and the recognition level with M.RCAg-1/M.RCAg-3 single epitopes were detected.The recognition of antibodies in serum was tested with indirect immunofluorescence assay (IFA).Results ADL and AD can enhance the immunogenieity of M.RCAg-1 and M.RCAg-3 respectively.Compared with another groups, ADL and AD formulated with M.RCAg-1 and M.RCAg-3 respectively can greatly enhance their antibodies recognition with natural parasites.Their antibodies can recognize the single epitopes generally.Conclusion ADL and AD can greatly increase the antibody level against M.RCAg-1 and M.RCAg-3 and their recognition with natural parasites.In conclusion, ADL and AD, as potential adjuvants, pave the way for the clinical use of M.RCAg-1 and M.RCAg-3 respectively.%目的 评价恶性疟原虫多表位蛋白疫苗M.RCAg-1和M.RCAg-3与不同佐剂最优配伍方式并进行初步的免疫机制研究.方法 将多表位疫苗表达纯化后混合4种佐剂(AD、ADL、CpG及CpG&A1)经皮下注射BALB/c小鼠,免疫3次,每次间隔2周.测定血清中抗M.RcAg-1和M.RCAg-3抗体滴度、IgG分型及血清抗体对抗原单表位识别.间接免疫荧光检测血清对天然疟原虫的识别.结果 佐剂ADL和AD分别能显著增强M.RCAg-1(P<0.05)和M.RCAg-3(P<0.05)免疫反应.而佐剂CpG和CpG&A1增强抗原免疫反应的能力相对较弱.与M.RCAg-1、M.RCAg-3和CpG、CpG&A1配伍的组相比,AD和ADL佐剂组的抗体水平和间接免疫荧光反应也都显著增强(P<0.05,P<0.05),抗

  20. Recombinant methionine aminopeptidase protein of Babesia microti: immunobiochemical characterization as a vaccine candidate against human babesiosis.

    Science.gov (United States)

    Munkhjargal, Tserendorj; Yokoyama, Naoaki; Igarashi, Ikuo

    2016-09-01

    Human babesiosis is the most important zoonotic protozoan infection in the world. This is the first report of the cloning, expression, purification, and immunobiochemical characterization of a methionine aminopeptidase 1 (MetAP1) protein from Babesia microti (B. microti). The gene encodes a MetAP1 protein of B. microti (BmMetAP1) of approximately 66.8 kDa that includes glutathione S-transferase (GST) tag and shows MetAP activity. BmMetAP1 was detected in a lysate of B. microti and further localized in cytoplasm of the B. microti merozoite. rBmMetAP1 was found to be immunogenic, eliciting a high antibody titer in mice. Moreover, rBmMetAP1 stimulated the production of IFN-γ and IL-12 but not IL-4. Finally, rBmMetAP1 was able to provide considerable protection to mice against a B. microti challenge infection based on a reduction in peak parasitemia levels and earlier clearance of the parasite as compared with control mice. Taken together, these results suggest that rBmMetAP1 confers significant protection against experimental B. microti infection and might be considered a potential vaccine target against human babesiosis. PMID:27306898

  1. Exploration of genetically encoded voltage indicators based on a chimeric voltage sensing domain

    Directory of Open Access Journals (Sweden)

    Yukiko Mishina

    2014-09-01

    We recently introduced a new VSFP design in which the voltage-sensing domain (VSD is sandwiched between a FRET pair of fluorescent proteins (termed VSFP-Butterflies and also demonstrated a series of chimeric VSD in which portions of the VSD of Ciona intestinalis voltage-sensitive phosphatase (Ci-VSP are substituted by homologous portions of a voltage-gated potassium channel subunit. These chimeric VSD had faster sensing kinetics than that of the native Ci-VSD. Here, we describe a new set of VSFPs that combine chimeric VSD with the Butterfly structure. We show that these chimeric VSFP-Butterflies can report membrane voltage oscillations of up to 200 Hz in cultured cells and report sensory evoked cortical population responses in living mice. This class of GEVIs may be suitable for imaging of brain rhythms in behaving mammalians.

  2. Immunogenicity Analysis of a Novel Subunit Vaccine Candidate Molecule-Recombinant L7/L12 Ribosomal Protein of Brucella suis.

    Science.gov (United States)

    Du, Zhi-Qiang; Li, Xin; Wang, Jian-Ying

    2016-08-01

    Brucella was an intracellular parasite, which could infect special livestock and humans. After infected by Brucella, livestock's reproductive system could be affected and destroyed resulting in huge economic losses. More seriously, it could be contagious from livestock to humans. So far, there is no available vaccine which is safe enough for humans. On this point, subunit vaccine has become the new breakthrough of conquering brucellosis. In this study, Brucella rL7/L12-BLS fusion protein was used as an antigen to immunize rabbits to detect the immunogenicity. The results of antibody level testing assay of rabbit antiserum indicated rL7/L12-BLS fusion protein could elicit rabbits to produce high-level IgG. And gamma interferon (IFN-γ) concentrations in rabbit antiserum were obviously up-regulated in both the rL7/L12 group and rL7/L12-BLS group. Besides, the results of quantitative real-time PCR (qRT-PCR) showed the IFN-γ gene's expression levels of both the rL7/L12 group and rL7/L12-BLS group were obviously up-regulated. All these results suggested Brucella L7/L12 protein was an ideal subunit vaccine candidate and possessed good immunogenicity. And Brucella lumazine synthase (BLS) molecule was a favorable transport vector for antigenic protein. PMID:27075455

  3. Bacterial Protein Characterization of Streptococcus agalactiae by SDS-page Method for Subclinical Mastitis Irradiated Vaccine Materials in Dairy Cattle

    Directory of Open Access Journals (Sweden)

    B.J. Tuasikal

    2012-08-01

    Full Text Available A study have been conducted to isolate and characterize bacterial protein S. agalactiae, which is antigenic and can be used to test immunogenicity of vaccine in order to manufacture irradiated mastitis (inflammation of the udder vaccine in ruminant. The study aims to determine the Molecular Weight (MW bacterial protein S. agalactiae irradiation, which can be used to test the nature of its antigenic caharacteristic. The character of S. agalactiae antigenic stimulates antibody induction of the immune system, in which case is the body's defense system against mastitis disease in cattle. In this study, irradiation of gamma ray is used to attenuate the pathogenicity of bacteria by reducing S. agalactiae antigenic caharacteristic. Previous research, in irradiation dose orientation before antigenic protein isolation of S. agalactiae, indicated that irradiation lethal dose to 50% (LD50 is 17 Gy. The characterization of S. agalactiae bacteria isolate using SDS-page method results in no significance different between irradiated and non-irradiated group, which indicated by MW range 75 – 100 kDa base on marker standard which used, or 99 kDa by the linier equation of Y = 11,60 – 0.05X (where Y = bands distance; X = MW standard protein; r2 = 0.99. In conclusion, 17 Gy irradiation dose does not impair antigenic property of S. agalactiae and therefore, can be applied to produce base material of irradiated vaccine for mastitis

  4. Bacterial Protein Characterization of Streptococcus agalactiae by SDS-page Method for Subclinical Mastitis Irradiated Vaccine Materials in Dairy Cattle

    International Nuclear Information System (INIS)

    A study have been conducted to isolate and characterize bacterial protein S. agalactiae, which is antigenic and can be used to test immunogenicity of vaccine in order to manufacture irradiated mastitis (inflammation of the udder) vaccine in ruminant. The study aims to determine the Molecular Weight (MW) bacterial protein S. agalactiae irradiation, which can be used to test the nature of its antigenic caharacteristic. The character of S. agalactiae antigenic stimulates antibody induction of the immune system, in which case is the body's defense system against mastitis disease in cattle. In this study, irradiation of gamma ray is used to attenuate the pathogenicity of bacteria by reducing S. agalactiae antigenic characteristic. Previous research, in irradiation dose orientation before antigenic protein isolation of S. agalactiae, indicated that irradiation lethal dose to 50% (LD50) is 17 Gy. The characterization of S. agalactiae bacteria isolate using SDS-page method results in no significance different between irradiated and non-irradiated group, which indicated by MW range 75 - 100 kDa base on marker standard which used, or 99 kDa by the linier equation of Y = 11,60 - 0.05X (where Y = bands distance; X = MW standard protein); r2 = 0.99. In conclusion, 17 Gy irradiation dose does not impair antigenic property of S. agalactiae and therefore, can be applied to produce base material of irradiated vaccine for mastitis. (author)

  5. Protective antibody and CD8+ T-cell responses to the Plasmodium falciparum circumsporozoite protein induced by a nanoparticle vaccine.

    Directory of Open Access Journals (Sweden)

    Stephen A Kaba

    Full Text Available BACKGROUND: The worldwide burden of malaria remains a major public health problem due, in part, to the lack of an effective vaccine against the Plasmodium falciparum parasite. An effective vaccine will most likely require the induction of antigen specific CD8(+ and CD4(+ T-cells as well as long-lasting antibody responses all working in concert to eliminate the infection. We report here the effective modification of a self-assembling protein nanoparticle (SAPN vaccine previously proven effective in control of a P. berghei infection in a rodent model to now present B- and T-cell epitopes of the human malaria parasite P. falciparum in a platform capable of being used in human subjects. METHODOLOGY/PRINCIPAL FINDINGS: To establish the basis for a SAPN-based vaccine, B- and CD8(+ T-cell epitopes from the P. falciparum circumsporozoite protein (PfCSP and the universal CD4 T-helper epitope PADRE were engineered into a versatile small protein (∼125 amino acids that self-assembles into a spherical nanoparticle repetitively displaying the selected epitopes. P. falciparum epitope specific immune responses were evaluated in mice using a transgenic P. berghei malaria parasite of mice expressing the human malaria full-length P. falciparum circumsporozoite protein (Tg-Pb/PfCSP. We show that SAPN constructs, delivered in saline, can induce high-titer, long-lasting (1 year protective antibody and poly-functional (IFNγ(+, IL-2(+ long-lived central memory CD8(+ T-cells. Furthermore, we demonstrated that these Ab or CD8(+ T-cells can independently provide sterile protection against a lethal challenge of the transgenic parasites. CONCLUSION: The SAPN construct induces long-lasting antibody and cellular immune responses to epitope specific sequences of the P. falciparum circumsporozoite protein (PfCSP and prevents infection in mice by a transgenic P. berghei parasite displaying the full length PfCSP.

  6. A full-length Plasmodium falciparum recombinant circumsporozoite protein expressed by Pseudomonas fluorescens platform as a malaria vaccine candidate.

    Directory of Open Access Journals (Sweden)

    Amy R Noe

    Full Text Available The circumsporozoite protein (CSP of Plasmodium falciparum is a major surface protein, which forms a dense coat on the sporozoite's surface. Preclinical research on CSP and clinical evaluation of a CSP fragment-based RTS, S/AS01 vaccine have demonstrated a modest degree of protection against P. falciparum, mediated in part by humoral immunity and in part by cell-mediated immunity. Given the partial protective efficacy of the RTS, S/AS01 vaccine in a recent Phase 3 trial, further improvement of CSP-based vaccines is crucial. In this report, we describe the preclinical development of a full-length, recombinant CSP (rCSP-based vaccine candidate against P. falciparum malaria suitable for current Good Manufacturing Practice (cGMP production. Utilizing a novel high-throughput Pseudomonas fluorescens expression platform, we demonstrated greater efficacy of full-length rCSP as compared to N-terminally truncated versions, rapidly down-selected a promising lead vaccine candidate, and developed a high-yield purification process to express immunologically active, intact antigen for clinical trial material production. The rCSP, when formulated with various adjuvants, induced antigen-specific antibody responses as measured by enzyme-linked immunosorbent assay (ELISA and immunofluorescence assay (IFA, as well as CD4+ T-cell responses as determined by ELISpot. The adjuvanted rCSP vaccine conferred protection in mice when challenged with transgenic P. berghei sporozoites containing the P. falciparum repeat region of CSP. Furthermore, heterologous prime/boost regimens with adjuvanted rCSP and an adenovirus type 35-vectored CSP (Ad35CS showed modest improvements in eliciting CSP-specific T-cell responses and anti-malarial protection, depending on the order of vaccine delivery. Collectively, these data support the importance of further clinical development of adjuvanted rCSP, either as a stand-alone product or as one of the components in a heterologous prime

  7. Efficacy of bacterin-, outer membrane protein- and fimbriae extract-based vaccines for the control of Salmonella Enteritidis experimental infection in chickens

    Directory of Open Access Journals (Sweden)

    Márcia C. Menão

    2013-03-01

    Full Text Available The efficacy of three vaccines was evaluated in chickens for the control of experimental infection with Salmonella Enteritidis (SE phage type 4. The vaccines were produced with bacterin, outer membrane proteins (OMP and fimbriae crude extract (FE. The chickens were vaccinated intramuscularly with two doses of each vaccine at 12 and 15 weeks of age. The chickens were then orally challenged with 10(9 CFU/chicken Salmonella Enteritidis phage type 4 at 18 weeks of age. Fecal swabs were performed for the recovery of shedding SE, and SE was recovered from the liver and spleen. Additionally, antibody titers were measured in the serum by micro-agglutination test. The results indicated that the vaccine produced with bacterin yielded better results and resulted in reduction of fecal shedding and organ invasion by SE after oral challenge, although no vaccine was 100% effective for the control of SE experimental infection.

  8. Is there a potential role for protein-conjugate pneumococcal vaccine in older adults?

    OpenAIRE

    Ridda, Iman; Musher, Daniel M.

    2012-01-01

    Longstanding controversy over the efficacy of 23-valent pneumococcal polysaccharide vaccine (PPV23) led to a recommendation by the Joint Committee on Vaccination and Immunisation (JCVI) of the United Kingdom in March 2011, to discontinue routine use of PPV23 in older adults.1 Following careful review of the evidence and feedback from stakeholders, the JCVI decided to retain the original policy of uniform vaccination of adults >65 years of age, while keeping the subject under continued review....

  9. Tissue factor expression in ovarian cancer: implications for immunotherapy with hI-con1, a factor VII-IgGF(c) chimeric protein targeting tissue factor.

    Science.gov (United States)

    Cocco, Emiliano; Varughese, Joyce; Buza, Natalia; Bellone, Stefania; Lin, Ken-Yu; Bellone, Marta; Todeschini, Paola; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E; Rutherford, Thomas J; Carrara, Luisa; Tassi, Renata; Pecorelli, Sergio; Lockwood, Charles J; Santin, Alessandro D

    2011-10-01

    We evaluated the expression of tissue factor (TF) in ovarian cancer (EOC) and the potential of hI-con1, an antibody-like molecule targeting TF, as a novel form of therapy against chemotherapy-resistant ovarian disease. We studied the expression of TF in 88 EOC by immunohistochemistry (IHC) and real-time-PCR (qRT-PCR) and the levels of membrane-bound-complement-regulatory-proteins CD46, CD55 and CD59 in primary EOC cell lines by flow-cytometry. Sensitivity to hI-con1-dependent-cell-mediated-cytotoxicity (IDCC), complement-dependent-cell-cytotoxicity and inhibition of IDCC by γ-immunoglobulin were evaluated in 5-h (51)chromium-release-assays. Cytoplasmic and/or membrane TF expression was observed in 24 out of 25 (96%) of the EOC samples tested by IHC, but not in normal ovarian-tissue. EOC with clear cell histology significantly overexpress TF when compared to serous, endometrioid, or undifferentiated tumors by qRT-PCR. With a single exception, all primary EOC that overexpressed TF demonstrated high levels of CD46, CD55 and CD59 and regardless of their histology or resistance to chemotherapy, were highly sensitive to IDCC. The effect of complement and physiologic doses of γ-immunoglobulin on IDCC in ovarian cancer cell lines overexpressing TF was tumor specific and related to the overexpression of CD59 on tumor cells. Small-interfering-RNA-mediated knockdown of CD59 expression in ovarian tumors significantly increased hI-con1-mediated cytotoxic activity in vitro. Finally, low doses of interleukin-2 further increased the cytotoxic effect induced by hI-con1 (P < 0.01). hI-con1 molecule induces strong cytotoxicity against primary chemotherapy-resistant ovarian cancer cell lines overexpressing TF and may represent a novel therapeutic agent for the treatment of ovarian tumors refractory to standard treatment modalities. PMID:21725665

  10. Tissue factor expression in ovarian cancer: implications for immunotherapy with hI-con1, a factor VII-IgGFc chimeric protein targeting tissue factor

    Science.gov (United States)

    Cocco, Emiliano; Varughese, Joyce; Buza, Natalia; Bellone, Stefania; Lin, Ken-Yu; Bellone, Marta; Todeschini, Paola; Silasi, Dan-Arin; Azodi, Masoud; Schwartz, Peter E.; Rutherford, Thomas J.; Carrara, Luisa; Tassi, Renata; Pecorelli, Sergio; Lockwood, Charles J.

    2013-01-01

    We evaluated the expression of tissue factor (TF) in ovarian cancer (EOC) and the potential of hI-con1, an antibody-like molecule targeting TF, as a novel form of therapy against chemotherapy-resistant ovarian disease. We studied the expression of TF in 88 EOC by immunohistochemistry (IHC) and real-time-PCR (qRT-PCR) and the levels of membrane-bound-complement-regulatory-proteins CD46, CD55 and CD59 in primary EOC cell lines by flow-cytometry. Sensitivity to hI-con1-dependent-cell-mediated-cytotoxicity (IDCC), complement-dependent-cell-cytotoxicity and inhibition of IDCC by γ-immunoglobulin were evaluated in 5-h 51chromium-release-assays. Cytoplasmic and/or membrane TF expression was observed in 24 out of 25 (96%) of the EOC samples tested by IHC, but not in normal ovarian-tissue. EOC with clear cell histology significantly overexpress TF when compared to serous, endometrioid, or undifferentiated tumors by qRT-PCR. With a single exception, all primary EOC that overexpressed TF demonstrated high levels of CD46, CD55 and CD59 and regardless of their histology or resistance to chemotherapy, were highly sensitive to IDCC. The effect of complement and physiologic doses of γ-immunoglobulin on IDCC in ovarian cancer cell lines overexpressing TF was tumor specific and related to the overexpression of CD59 on tumor cells. Small-interfering-RNA-mediated knockdown of CD59 expression in ovarian tumors significantly increased hI-con1-mediated cytotoxic activity in vitro. Finally, low doses of interleukin-2 further increased the cytotoxic effect induced by hI-con1 (P < 0.01). hI-con1 molecule induces strong cytotoxicity against primary chemotherapy-resistant ovarian cancer cell lines overexpressing TF and may represent a novel therapeutic agent for the treatment of ovarian tumors refractory to standard treatment modalities. PMID:21725665

  11. Chimeric viruses containing the N-terminal ectodomains of GP5 and M proteins of porcine reproductive and respiratory syndrome do not change the cellular tropism of equine arteritis virus

    Science.gov (United States)

    Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) are members of family Arteriviridae; they share many biological properties but differ significantly in cellular tropism. Using an infectious cDNA clone of EAV, we engineered a panel of six chimeric viruses b...

  12. Delineation of structural domains involved in the subtype specificity of tachykinin receptors through chimeric formation of substance P/substance K receptors.

    OpenAIRE

    Y. Yokota; Akazawa, C; Ohkubo, H; Nakanishi, S.

    1992-01-01

    The mammalian tachykinin receptors belong to the family of G protein-coupled receptors and consist of the substance P, substance K and neuromedin K receptors (SPR, SKR and NKR). We constructed 14 chimeric receptors in which seven transmembrane segments were sequentially exchanged between the rat SPR and SKR and examined the subtype specificity of the chimeric receptors by radioligand binding and inositol phosphate measurements after transfection into COS cells. All chimeric receptors showed m...

  13. Adsorption of recombinant poxvirus L1-protein to aluminum hydroxide/CpG vaccine adjuvants enhances immune responses and protection of mice from vaccinia virus challenge

    Science.gov (United States)

    Xiao, Yuhong; Zeng, Yuhong; Alexander, Edward; Mehta, Shyam; Joshi, Sangeeta B.; Buchman, George W.; Volkin, David B.; Middaugh, C. Russell; Isaacs, Stuart N.

    2012-01-01

    The stockpiling of live vaccinia virus vaccines has enhanced biopreparedness against the intentional or accidental release of smallpox. Ongoing research on future generation smallpox vaccines is providing key insights into protective immune responses as well as important information about subunit vaccine design strategies. For protein-based recombinant subunit vaccines, the formulation and stability of candidate antigens with different adjuvants are important factors to consider for vaccine design. In this work, a non-tagged secreted L1-protein, a target antigen on mature virus, was expressed using recombinant baculovirus technology and purified. To identify optimal formulation conditions for L1, a series of biophysical studies was performed over a range of pH and temperature conditions. The overall physical stability profile was summarized in an empirical phase diagram. Another critical question to address for development of an adjuvanted-vaccine was if immunogenicity and protection could be affected by the interactions and binding of L1 to aluminum salts (Alhydrogel) with and without a second adjuvant, CpG. We thus designed a series of vaccine formulations with different binding interactions between the L1 and the two adjuvants, and then performed a series of vaccination-challenge experiments in mice including measurement of antibody responses and post-challenge weight-loss and survival. We found that better humoral responses and protection were conferred with vaccine formulations when the L1-protein was adsorbed to Alhydrogel. These data demonstrate that designing vaccine formulation conditions to maximize antigen-adjuvant interactions is a key factor in smallpox subunit vaccine immunogenicity and protection. PMID:23153450

  14. Immunization with chlamydial plasmid protein pORF5 DNA vaccine induces protective immunity against genital chlamydial infection in mice

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    To validate the immune protective efficacy of pORF5 DNA vaccine and to analyze potential mechanisms related to this protection. In this study, pORF5 DNA vaccine was constructed and evaluated for its protective immunity in a mouse model of genital chlamydial infection. Groups of BALB/c mice were immunized intranasally with pORF5 DNA vaccine. Humoral and cell mediated immune responses were evaluated. The clearance ability of chlamydial challenge from the genital tract and the chlamy- dia-induced upper genital tract gross pathology and histopathological characterization were also de- tected. The results showed that the total and the IgG2a anti-pORF5 antibody levels in serum were sig- nificantly elevated after pcDNA3.1-pORF5 vaccination, as were the total antibody and IgA levels in vaginal fluids. pcDNA3.1-pORF5 induced a significantly high level of Th1 response as measured by robust gamma interferon (IFN-γ). Minimal IL-4 was produced by immune T cells in response to the re-stimulation with pORF5 protein or the inactive elementary body in vitro. pcDNA3.1-pORF5-vacci- nated mice displayed significantly reduced bacterial shedding upon a chlamydial challenge and an accelerated resolution of infection. 100% of pcDNA3.1-pORF5 vaccinated mice successfully resolved the infection by day 24. pcDNA3.1-pORF5-immunized mice also exhibited protection against patho- logical consequences of chlamydial infection. The stimulated index was significantly higher than that of mice immunized with pcDNA3.1 and PBS (P<0.05). Together, these results demonstrated that immu- nization with pORF5 DNA vaccine is a promising approach for eliciting a protective immunity against a genital chlamydial challenge.

  15. n Silico Analysis of Envelope Dengue Virus-2 and Envelope Dengue Virus-3 Protein as the Backbone of Dengue Virus Tetravalent Vaccine by Using Homology Modeling Method

    Directory of Open Access Journals (Sweden)

    Rizky I. Taufik

    2009-01-01

    Full Text Available Problem statement: Dengue fever, which was caused by Dengue virus infection, had became a major public health problem in the tropic and subtropical countries. Dengue virus (DENV had four serotypes (DENV-1, DENV-2, DENV-3 and DENV-4, based on their immunogenic in the human body. Preventive measure will be necessary to decrease the prevalence of dengue fever, by developing modern vaccine. Approach: This research was focused on in silico study of dengue virus vaccines, by using envelope (E protein of DENV-2 and DENV-3 as their backbones. T cell epitope prediction was determined by using MULTIPRED server and B cell epitope prediction was determined by using Conformational Epitope Prediction server (CEP. Homology modeling study of E DENV-3 protein as the vaccine backbone had produced six dengue vaccine peptides (HMM Vaccine 1-6. Moreover, homology modeling study of E DENV-2 protein as vaccine backbone had produced six dengue vaccine peptides (ANN vaccine 1-6. Results: The BLAST analysis of HMM and ANN vaccines had produced 93% and 91% identity, respectively. The Ramachandran Plot of both vaccines had shown less than 15% non glycine residue in the disallowed region, therefore it showed the solid stability of the proteins. The VAST analysis of E DENV-3 backbone vaccines had determined, that HMM4 and HMM6 had the highest structure similarity with native E DENV-3. HMM4 and HMM6 had the highest VAST score of 64.5. Moreover, the VAST analysis of E DENV-2 backbone vaccines had determined, that ANN1, ANN3, ANN4, ANN5 and ANN6 had the highest structure similarity with native E DENV-2. ANN1, ANN3, ANN4, ANN5 and ANN6 have the highest VAST score of 64.7. Conclusion/Recommendation: It could be inferred from this research that HMM4; HMM6; ANN1; ANN3; ANN4; ANN5; and ANN6 were the best in silico vaccine design, based on their similarity with native E DENV Proteins. This research could be applied for the wet

  16. Vaccination with recombinant Boophilus annulatus Bm86 ortholog protein, Ba86, protects cattle against B. annulatus and B. microplus infestations

    OpenAIRE

    Jongejan Frans; Naranjo Victoria; Almazán Consuelo; Canales Mario; de la Fuente José

    2009-01-01

    Abstract Background The cattle ticks, Boophilus spp., affect cattle production in tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The recombinant B. microplus Bm86 protective antigen has been shown to protect cattle against tick infestations. Recently, the gene coding for B. annulatus Bm86 ortholog, Ba86, was cloned and the recombinant protein was secreted and purified from the yeast Pichia past...

  17. Cell-penetrating peptides mediated protein cross-membrane delivery and its use in bacterial vector vaccine.

    Science.gov (United States)

    Ma, Jimei; Xu, Jinmei; Guan, Lingyu; Hu, Tianjian; Liu, Qin; Xiao, Jingfan; Zhang, Yuanxing

    2014-07-01

    It is an attractive strategy to develop a recombinant bacterial vector vaccine by expressing exogenous protective antigen to induce the immune response, and the main concern is how to enhance the cellular internalization of antigen produced by bacterial vector. Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides which facilitate cellular uptake of various molecular cargoes and therefore have great potentials in vector vaccine design. In this work, eleven different CPPs were fused to the C-terminus of EGFP respectively, and the resultant EGFP-CPP fusion proteins were expressed and purified to assay their cross-membrane transport in macrophage J774 A.1 cells. Among the tested CPPs, TAT showed an excellent capability to deliver the cargo protein EGFP into cytoplasm. In order to establish an efficient antigen delivery system in Escherichia coli, the EGFP-TAT synthesis circuit was combined with an in vivo inducible lysis circuit PviuA-E in E. coli to form an integrated antigen delivery system, the resultant E. coli was proved to be able to lyse upon the induction of a mimic in vivo signal and thus release intracellular EGFP-TAT intensively, which were assumed to undergo a more efficient intracellular delivery by CPP to evoke protective immune responses. Based on the established antigen delivery system, the protective antigen gene flgD from an invasive intracellular fish pathogen Edwardsiella tarda EIB202, was applied to establish an E. coli recombinant vector vaccine. This E. coli vector vaccine presented superior immune protection (RPS = 63%) under the challenge with E. tarda EIB202, suggesting that the novel antigen delivery system had great potential in bacterial vector vaccine applications. PMID:24746937

  18. Comparison of spleen cell proliferation in response to Brucella abortus 2308 lipopolysaccharide or proteins in mice vaccinated with strain 19 or RB51.

    OpenAIRE

    Stevens, M G; S. C. Olsen; Pugh, G W

    1995-01-01

    Mice vaccinated with Brucella abortus 19 (S19) or RB51 (SRB51) had spleen cells which proliferated in response to proteins of 32, 27, 18, and < 18 kDa but not in response to proteins of 106, 80, and 49 kDa from B. abortus 2308 (S2308) following vaccination and challenge infection with S2308. Spleen cells from mice vaccinated with S19 but not with SRB51 had increased proliferation in response to S2308 lipopolysaccharide (LPS) following challenge infection with S2308. We previously reported tha...

  19. Differential induction of type I interferons in macaques by wild-type measles virus alone or with the hemagglutinin protein of the Edmonston vaccine strain.

    Science.gov (United States)

    Van Nguyen, Nguyen; Kato, Sei-Ich; Nagata, Kyosuke; Takeuchi, Kaoru

    2016-07-01

    Measles vaccines are highly effective and safe; however, the mechanism(s) underlying their attenuation has not been well understood. In this study, type I IFNs (IFN-α and IFN-β) induction in macaques infected with measles virus (MV) strains was examined. Type I IFNs were not induced in macaques infected with wild-type MV. However, IFN-α was sharply induced in most macaques infected with recombinant wild-type MV bearing the hemagglutinin (H) protein of the Edmonston vaccine strain. These results indicate that the H protein of MV vaccine strains may have a role in MV attenuation. PMID:27278100

  20. Prime-Boost Vaccination Using Chemokine-Fused gp120 DNA and HIV Envelope Peptides Activates Both Immediate and Long-Term Memory Cellular Responses in Rhesus Macaques

    Directory of Open Access Journals (Sweden)

    Hong Qin

    2010-01-01

    Full Text Available HIV vaccine candidates with improved immunogenicity and induction of mucosal T-cell immunity are needed. A prime-boost strategy using a novel HIV glycoprotein 120 DNA vaccine was employed to immunize rhesus macaques. The DNA vaccine encoded a chimeric gp120 protein in fusion with monocyte chemoattractant protein-3, which was hypothesized to improve the ability of antigen-presenting cells to capture viral antigen through chemokine receptor-mediated endocytosis. DNA vaccination induced virus-reactive T cells in peripheral blood, detectable by T cell proliferation, INFγ ELISPOT and sustained IL-6 production, without humoral responses. With a peptide-cocktail vaccine containing a set of conserved polypeptides of HIV-1 envelope protein, given by nasogastric administration, primed T-cell immunity was significantly boosted. Surprisingly, long-term and peptide-specific mucosal memory T-cell immunity was detected in both vaccinated macaques after one year. Therefore, data from this investigation offer proof-of-principle for potential effectiveness of the prime-boost strategy with a chemokine-fused gp120 DNA and warrant further testing in the nonhuman primate models for developing as a potential HIV vaccine candidate in humans.

  1. A new Leishmania-specific hypothetical protein, LiHyT, used as a vaccine antigen against visceral leishmaniasis.

    Science.gov (United States)

    Martins, Vívian Tamietti; Lage, Daniela Pagliara; Duarte, Mariana Costa; Costa, Lourena Emanuele; Garde, Esther; Rodrigues, Marcella Rezende; Chávez-Fumagalli, Miguel Angel; Menezes-Souza, Daniel; Roatt, Bruno Mendes; Tavares, Carlos Alberto Pereira; Soto, Manuel; Coelho, Eduardo Antonio Ferraz

    2016-02-01

    The present study aimed to evaluate a new Leishmania-specific hypothetical protein, LiHyT, as a vaccine candidate against VL. The immunogenicity of the recombinant protein (rLiHyT) plus saponin was evaluated in BALB/c mice. In the results, it is shown that rLiHyT plus saponin vaccinated mice produced high levels of IFN-γ, IL-12, and GM-CSF after in vitro stimulation of spleen cells using both rLiHyT and Leishmania infantum SLA. The protective efficacy was evaluated after subcutaneous challenge with stationary promastigotes of L. infantum. Immunized and infected mice, when compared to the controls, showed significant reductions in the number of parasites in the liver, spleen, bone marrow, and in the paws' draining lymph nodes. Protection was associated with an IL-12-dependent production of IFN-γ, mainly by CD4(+) T cells, with a minor contribution of CD8(+) T cells. In these mice, a decrease in the parasite-mediated IL-4 and IL-10 responses, as well as a predominance of LiHyT- and parasite-specific IgG2a isotype antibodies, were also observed. The present study showed that a new Leishmania-specific protein, when combined with a Th1-type adjuvant, presents potential to be used as a vaccine against VL. PMID:26593442

  2. Vaccination of Gerbils with Bm-103 and Bm-RAL-2 Concurrently or as a Fusion Protein Confers Consistent and Improved Protection against Brugia malayi Infection.

    Directory of Open Access Journals (Sweden)

    Sridhar Arumugam

    2016-04-01

    Full Text Available The Brugia malayi Bm-103 and Bm-RAL-2 proteins are orthologous to Onchocerca volvulus Ov-103 and Ov-RAL-2, and which were selected as the best candidates for the development of an O. volvulus vaccine. The B. malayi gerbil model was used to confirm the efficacy of these Ov vaccine candidates on adult worms and to determine whether their combination is more efficacious.Vaccine efficacy of recombinant Bm-103 and Bm-RAL-2 administered individually, concurrently or as a fusion protein were tested in gerbils using alum as adjuvant. Vaccination with Bm-103 resulted in worm reductions of 39%, 34% and 22% on 42, 120 and 150 days post infection (dpi, respectively, and vaccination with Bm-RAL-2 resulted in worm reductions of 42%, 22% and 46% on 42, 120 and 150 dpi, respectively. Vaccination with a fusion protein comprised of Bm-103 and Bm-RAL-2 resulted in improved efficacy with significant reduction of worm burden of 51% and 49% at 90 dpi, as did the concurrent vaccination with Bm-103 and Bm-RAL-2, with worm reduction of 61% and 56% at 90 dpi. Vaccination with Bm-103 and Bm-RAL-2 as a fusion protein or concurrently not only induced a significant worm reduction of 61% and 42%, respectively, at 150 dpi, but also significantly reduced the fecundity of female worms as determined by embryograms. Elevated levels of antigen-specific IgG were observed in all vaccinated gerbils. Serum from gerbils vaccinated with Bm-103 and Bm-RAL-2 individually, concurrently or as a fusion protein killed third stage larvae in vitro when combined with peritoneal exudate cells.Although vaccination with Bm-103 and Bm-RAL-2 individually conferred protection against B. malayi infection in gerbils, a more consistent and enhanced protection was induced by vaccination with Bm-103 and Bm-RAL-2 fusion protein and when they were used concurrently. Further characterization and optimization of these filarial vaccines are warranted.

  3. Design of a heterosubtypic epitope-based peptide vaccine fused with hemokinin-1 against influenza viruses

    Institute of Scientific and Technical Information of China (English)

    Shahla; Shahsavandi; Mohammad; Majid; Ebrahimi; Kaveh; Sadeghi; Homayoon; Mahravani

    2015-01-01

    Influenza viruses continue to emerge and re-emerge, posing new threats for public health. Control and treatment of influenza depends mainly on vaccination and chemoprophylaxis with approved antiviral drugs. Identification of specific epitopes derived from influenza viruses has significantly advanced the development of epitope-based vaccines. Here, we explore the idea of using HLA binding data to design an epitope-based vaccine that can elicit heterosubtypic T-cell responses against circulating H7N9, H5N1, and H9N2 subtypes. The hemokinin-1(HK-1) peptide sequence was used to induce immune responses against the influenza viruses. Five conserved high score cytotoxic T lymphocyte(CTL) epitopes restricted to HLA-A*0201-binding peptides within the hemagglutinin(HA) protein of the viruses were chosen, and two HA CTL/HK-1 chimera protein models designed. Using in silico analysis, which involves interferon epitope scanning, protein structure prediction, antigenic epitope determination, and model quality evaluation, chimeric proteins were designed. The applicability of one of these proteins as a heterosubtypic epitopebased vaccine candidate was analyzed.

  4. Non-carrier nanoparticles adjuvant modular protein vaccine in a particle-dependent manner.

    Science.gov (United States)

    Seth, Arjun; Ritchie, Fiona K; Wibowo, Nani; Lua, Linda H L; Middelberg, Anton P J

    2015-01-01

    Nanoparticles are increasingly used to adjuvant vaccine formulations due to their biocompatibility, ease of manufacture and the opportunity to tailor their size, shape, and physicochemical properties. The efficacy of similarly-sized silica (Si-OH), poly (D,L-lactic-co-glycolic acid) (PLGA) and poly caprolactone (PCL) nanoparticles (nps) to adjuvant recombinant capsomere presenting antigenic M2e modular peptide from Influenza A virus (CapM2e) was investigated in vivo. Formulation of CapM2e with Si-OH or PLGA nps significantly boosted the immunogenicity of modular capsomeres, even though CapM2e was not actively attached to the nanoparticles prior to injection (i.e., formulation was by simple mixing). In contrast, PCL nps showed no significant adjuvant effect using this simple-mixing approach. The immune response induced by CapM2e alone or formulated with nps was antibody-biased with very high antigen-specific antibody titer and less than 20 cells per million splenocytes secreting interferon gamma. Modification of silica nanoparticle surface properties through amine functionalization and pegylation did not lead to significant changes in immune response. This study confirms that simple mixing-based formulation can lead to effective adjuvanting of antigenic protein, though with antibody titer dependent on nanoparticle physicochemical properties. PMID:25756283

  5. Differentiation of infected and vaccinated animals (DIVA) using the NS1 protein of avian influenza virus

    Science.gov (United States)

    Vaccination against avian influenza (AI) virus, a powerful tool for control of the disease, may result in issues related to surveillance programs and international trade of poultry and poultry products. The use of AI vaccination in poultry would have greater world-wide acceptance if a reliable test...

  6. The use of non-structural proteins of FMDV to differentiate between vaccinated and infected animals in Thailand

    International Nuclear Information System (INIS)

    The detection of antibody to non-structural proteins (NSP) of foot and mouth disease virus (FMDV) using various types of NSP kits produced from World Reference Laboratory (WRL), United Biomedical Inc.(UBI), Bommeli/Intervet, CEDI and other research institutes, has been studied. The Liquid Phase blocking ELISA (LPBE) for measurement of antibody to FMDV type O, A and Asia1 was used in parallel with the NSP tests to study the specificity and sensitivity of each NSP test kit. Three thousand nine hundred and twenty two serum samples from cattle, buffaloes and pigs were grouped according to known history comprising: non vaccinated, single vaccinated, multiple vaccinated and field infected animals. The NSP tests all gave showed high specificity in non vaccinated cattle of 100%, 100%, 99% and 100% respectively. In pigs both the WRL and UBI kits gave 100%. In the single vaccination group, the specificities were WRL 99.5% and UBI = 98.7%. The NSP test in multiple vaccinated cattle and buffaloes gave specificities of WRL 97.73%, and UBI = 100%. In the FMD field outbreak area, clinically distinguishable infected cattle gave sensitivity results by NSP tests of WRL, 98.3%; UBI, 88.9%; Bommeli/Intervet, 84.6%; CEDI, 90.1%; Inoue/Japan, 93.8%; USDA, 88.3%; and Pen Side/Korea, 84.6%. This study indicates that the NSP kits from WRL, UBI, Bommeli/ Intervet, and CEDI had a high specificity for cattle and pigs from 99-100% and a high sensitivity from 84.6-93.8% in cattle and 73.33-80% in pigs. In addition, the NSP tests have been applied to FMD sero surveillance in animals being quarantined at the international quarantine station in a multilateral Malaysia Thailand Myanmar (MTM) project and also used in sero-monitoring of field animals in the Thailand national plan for the FMD vaccination campaign programme in the country. Hence, the use of NSP test to differentiate between vaccinated and infected animals would be used as a standard diagnostic test for the control and eradication of

  7. Antitumor activity of mixed heat shock protein/peptide vaccine and cyclophosphamide plus interleukin-12 in mice sarcoma

    Directory of Open Access Journals (Sweden)

    Sui Xiang

    2011-02-01

    Full Text Available Abstract Background The immune factors heat shock protein (HSP/peptides (HSP/Ps can induce both adaptive and innate immune responses. Treatment with HSP/Ps in cancer cell-bearing mice and cancer patients revealed antitumor immune activity. We aimed to develop immunotherapy strategies by vaccination with a mixture of HSP/Ps (mHSP/Ps, HSP60, HSP70, Gp96 and HSP110 enhanced with cyclophosphamide (CY and interleukin-12 (IL-12. Methods We extracted mHSP/Ps from the mouse sarcoma cell line S180 using chromatography. The identity of proteins in this mHSP/Ps was assayed using SDS-PAGE and Western blot analysis with antibodies specific to various HSPs. BALB/C mice bearing S180 cells were vaccinated with mHSP/Ps ×3, then were injected intraperitoneally with low-dose CY and subcutaneously with IL-12, 100 μg/day, ×5. After vaccination, T lymphocytes in the peripheral blood were analyzed using FACScan and Cytotoxicity (CTL was analyzed using lactate dehydrogenase assay. ELISPOT assay was used to evaluate interferon γ (IFN-γ, and immune cell infiltration in tumors was examined in the sections of tumor specimen. Results In mice vaccinated with enhanced vaccine (mHSP/Ps and CY plus IL-12, 80% showed tumor regression and long-term survival, and tumor growth inhibition rate was 82.3% (30 days, all controls died within 40 days. After vaccination, lymphocytes and polymorphonuclear leukocytes infiltrated into the tumors of treated animals, but no leukocytes infiltrated into the tumors of control mice. The proportions of natural killer cells, CD8+, and interferon-γ-secreting cells were all increased in the immune group, and tumor-specific cytotoxic T lymphocyte activity was increased. Conclusions In this mice tumor model, vaccination with mHSP/Ps combined with low-dose CY plus IL-12 induced an immunologic response and a marked antitumor response to autologous tumors. The regimen may be a promising therapeutic agent against tumors.

  8. Antibody Recognition of Porcine Circovirus Type 2 Capsid Protein Epitopes after Vaccination, Infection, and Disease▿†

    Science.gov (United States)

    Trible, Benjamin R.; Kerrigan, Maureen; Crossland, Nicholas; Potter, Megan; Faaberg, Kay; Hesse, Richard; Rowland, Raymond R. R.

    2011-01-01

    Open reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) codes for the 233-amino-acid capsid protein (CP). Baculovirus-based vaccines that express only ORF2 are protective against clinical disease following experimental challenge or natural infection. The goal of this study was to identify regions in CP preferentially recognized by sera from experimentally infected and vaccinated pigs and to compare these responses to those of pigs diagnosed with porcine circovirus-associated disease (PCVAD), including porcine multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). The approach was to react porcine sera with CP polypeptide fragments followed by finer mapping studies using overlapping oligopeptides that covered amino acids 141 to 200. The results showed that vaccinated pigs preferentially recognized only the largest polypeptide fragment, CP(43-233). A subset of experimentally infected pigs and pigs with PDNS showed strong reactivity against a CP oligopeptide, 169-STIDYFQPNNKR-180. Alanine scanning identified Y-173, F-174, Q-175, and K-179 as important for antibody recognition. The results from this study support the notion of PCV2 modulation of immunity, including antibody responses that may represent a precursor for disease. The recognition of CP(169-180) and other polypeptides provides opportunities to devise diagnostic tests for monitoring the immunological effectiveness of vaccination. PMID:21430122

  9. Poliovirus Vaccines

    OpenAIRE

    Isik Yalcin

    2008-01-01

    The two types of poliovirus vaccines are inactivated vaccine, given parenterally, and live virus vaccine, given orally. Oral poliovirus is the vaccine of choice for global eradication. Either inactivated vaccine or oral vaccine may be given concurrently with other routinely recommended childhood vaccines. No serious adverse events have been associated with the vaccine. Oral poliovirus vaccine can cause vaccine associated paralytic poliomyelitis.

  10. Recombinant infectious bronchitis virus (IBV) H120 vaccine strain expressing the hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) protects chickens against IBV and NDV challenge.

    Science.gov (United States)

    Yang, Xin; Zhou, Yingshun; Li, Jianan; Fu, Li; Ji, Gaosheng; Zeng, Fanya; Zhou, Long; Gao, Wenqian; Wang, Hongning

    2016-05-01

    Infectious bronchitis (IB) and Newcastle disease (ND) are common viral diseases of chickens, which are caused by infectious bronchitis virus (IBV) and Newcastle disease virus (NDV), respectively. Vaccination with live attenuated strains of IBV-H120 and NDV-LaSota are important for the control of IB and ND. However, conventional live attenuated vaccines are expensive and result in the inability to differentiate between infected and vaccinated chickens. Therefore, there is an urgent need to develop new efficacious vaccines. In this study, using a previously established reverse genetics system, we generated a recombinant IBV virus based on the IBV H120 vaccine strain expressing the haemagglutinin-neuraminidase (HN) protein of NDV. The recombinant virus, R-H120-HN/5a, exhibited growth dynamics, pathogenicity and viral titers that were similar to those of the parental IBV H120, but it had acquired hemagglutination activity from NDV. Vaccination of SPF chickens with the R-H120-HN/5a virus induced a humoral response at a level comparable to that of the LaSota/H120 commercial bivalent vaccine and provided significant protection against challenge with virulent IBV and NDV. In summary, the results of this study indicate that the IBV H120 strain could serve as an effective tool for designing vaccines against IB and other infectious diseases, and the generation of IBV R-H120-HN/5a provides a solid foundation for the development of an effective bivalent vaccine against IBV and NDV. PMID:26873815

  11. The Complexity of a Dengue Vaccine : A Review of the Human Antibody Response

    NARCIS (Netherlands)

    Flipse, Jacky; Smit, Jolanda M.

    2015-01-01

    Dengue is the most prevalent mosquito-borne viral disease worldwide. Yet, there are no vaccines or specific antivirals available to prevent or treat the disease. Several dengue vaccines are currently in clinical or preclinical stages. The most advanced vaccine is the chimeric tetravalent CYD-TDV vac

  12. A recombinant West Nile virus envelope protein vaccine candidate produced in Spodoptera frugiperda expresSF+ cells

    OpenAIRE

    Bonafé, Nathalie; Rininger, Joseph A.; Chubet, Richard G.; Foellmer, Harald G.; Fader, Stacey; Anderson, John F.; Bushmich, Sandra L.; Anthony, Karen; Ledizet, Michel; Fikrig, Erol; Koski, Raymond A.; Kaplan, Paul

    2008-01-01

    In this study, a recombinant truncated West Nile virus envelope protein antigen (rWNV-E) was produced in serum-free cultures of the expresSF+ insect cell line via baculovirus infection. This production system was selected based on its use in the production of candidate human and animal vaccine antigens. A defined fermentation and purification process for the rWNV-E antigen was established to control for purity and immunogenicity of each protein batch. The material formulated with aluminum hyd...

  13. Studies on recombinant glucokinase (r-glk) protein of Brucella abortus as a candidate vaccine molecule for brucellosis.

    Science.gov (United States)

    Vrushabhendrappa; Singh, Amit Kumar; Balakrishna, Konduru; Sripathy, Murali Harishchandra; Batra, Harsh Vardhan

    2014-09-29

    Brucellosis is one of the most prevalent zoonotic diseases of worldwide distribution caused by the infection of genus Brucella. Live attenuated vaccines such as B. abortus S19, B. abortus RB51 and B. melitensis Rev1 are found most effective against brucellosis infection in animals, contriving a number of serious side effects and having chances to revert back into their active pathogenic form. In order to engineer a safe and effective vaccine candidate to be used in both animals and human, a recombinant subunit vaccine molecule comprising the truncated region of glucokinase (r-glk) gene from B. abortus S19 was cloned and expressed in Escherichia coli BL21DE3 host. Female BALB/c mice immunized with purified recombinant protein developed specific antibody titer of 1:64,000. The predominant IgG2a and IgG2b isotypes signified development of Th1 directed immune responses. In vitro cell cytotoxicity assay using anti-r-glk antibodies incubated with HeLa cells showed 81.20% and 78.5% cell viability against lethal challenge of B. abortus 544 and B. melitensis 16M, respectively. The lymphocyte proliferative assay indicated a higher splenic lymphocyte responses at 25μg/ml concentration of protein which implies the elevated development of memory immune responses. In contrast to control, the immunized group of mice intra-peritoneal (I.P.) challenged with B. abortus 544 were significantly protected with no signs of necrosis and vacuolization in their liver and spleen tissue. The elevated B-cell response associated with Th1 adopted immunity, significant in vitro cell viability as well as protection afforded in experimental animals after challenge, supplemented with histopathological analysis are suggestive of r-glk protein as a prospective candidate vaccine molecule against brucellosis. PMID:25131740

  14. Mosaic origins of a complex chimeric mitochondrial gene in Silene vulgaris.

    Directory of Open Access Journals (Sweden)

    Helena Storchova

    Full Text Available Chimeric genes are significant sources of evolutionary innovation that are normally created when portions of two or more protein coding regions fuse to form a new open reading frame. In plant mitochondria astonishingly high numbers of different novel chimeric genes have been reported, where they are generated through processes of rearrangement and recombination. Nonetheless, because most studies do not find or report nucleotide variation within the same chimeric gene, evolution after the origination of these chimeric genes remains unstudied. Here we identify two alleles of a complex chimera in Silene vulgaris that are divergent in nucleotide sequence, genomic position relative to other mitochondrial genes, and expression patterns. Structural patterns suggest a history partially influenced by gene conversion between the chimeric gene and functional copies of subunit 1 of the mitochondrial ATP synthase gene (atp1. We identified small repeat structures within the chimeras that are likely recombination sites allowing generation of the chimera. These results establish the potential for chimeric gene divergence in different plant mitochondrial lineages within the same species. This result contrasts with the absence of diversity within mitochondrial chimeras found in crop species.

  15. HBV和HIV的复合型DNA免疫可诱发BALB/c小鼠的细胞免疫应答%Vaccination of BALB/c Mice with Chimeric HBV and HIV DNA can Induce Cellular Immune Responses

    Institute of Scientific and Technical Information of China (English)

    和绍清; 董承红; 孙明; 李琦涵

    2001-01-01

    本研究是通过细胞免疫检测来研究含有两种不同病毒基因的复合型DNA免疫的效果,同时和单一病毒基因的DNA免疫作比较。在本实验中,将HBV的S基因和(或)HⅣ-1的gp120基因插入到真核表达载体pcDNA3中,得到能表达HBsAg和gp120融合蛋白的重组体pcDNA-S-120,和能表达HBsAg的重组体pcDNA-S。在实验中,将重组质粒DNA直接注射到BALB/c小鼠股四头肌内,按每只小鼠100μg/100μl的剂量经3次免疫后,在T细胞增殖实验中,用HBsAg蛋白刺激体外培养的被免疫小鼠T细胞后,出现了明显的T细胞增殖。采用”cr释放法检测特异性CTL杀伤作用时,发现致敏的CTL对HIV gp120多肽孵育后的靶细胞P815有明显的杀伤作用。%Recent studies support the notion that vaccination with plasmid DNA encording genes for viral or bacterial antigens can elicite both humoral and cellular immune responses in rodents and non-human primates, but their major advantage at immunologic level are their capacity to induce antigen-specific CD8 + T cell responses. In the present study,BALB/c mice were immunilized intramuscularly with recombinant plasmid pcDNA-S-120 in which the S gene of HBV and gp120 gene of HIV-1 were cloned into pcDNA3 in fusion form, and also with pcDNA-S in which the S gene of HBV had been cloned into pcDNA3. Mice were immunilized 3 times with 100 μg/100 μi per mouse. In the T cell proliferation assays, it was showed that T cells proliferated in vitro obviously after stimulation with pcDNA-S and pcDNA-S-120. The sensitized CTL could specifically kill the target cell P815 which were pulsed by 10-AA peptide of HIV. It concludes that cellular immune responses could be elicited in the experimental animals after direct injections of recombinatant plasmid DNA in which the S gene and gp120 gene were cloned in fusion form.

  16. Dengue vaccine: hypotheses to understand CYD-TDV-induced protection.

    Science.gov (United States)

    Guy, Bruno; Jackson, Nicholas

    2016-01-01

    Dengue virus (DENV) is a human pathogen with a large impact on public health. Although no vaccine against DENV is currently licensed, a recombinant vaccine - chimeric yellow fever virus-DENV tetravalent dengue vaccine (CYD-TDV) - has shown efficacy against symptomatic dengue disease in two recent Phase III clinical trials. Safety observations were also recently reported for these trials. In this Opinion article, we review the data from recent vaccine clinical trials and discuss the putative mechanisms behind the observed efficacy of the vaccine against different forms of the disease, focusing on the interactions between the infecting virus, pre-existing host immunity and vaccine-induced immune responses. PMID:26639777

  17. Optimization of ammonium sulfate concentration for purification of colorectal cancer vaccine candidate recombinant protein GA733-FcK isolated from plants

    OpenAIRE

    Se-Ra ePark; Chae-Yeon eLim; Deuk-Su eKim; Kisung eKo

    2015-01-01

    A protein purification procedure is required to obtain high-value recombinant injectable vaccine proteins produced in plants as a bioreactor. However, existing purification procedures for plant-derived recombinant proteins are often not optimized and are inefficient, with low recovery rates. In our previous study, we used 25-30% ammonium sulfate to precipitate total soluble proteins (TSPs) in purification process for recombinant proteins from plant leaf biomass which has not been optimized. T...

  18. Progress in Chimeric Vector and Chimeric Gene Based Cardiovascular Gene Therapy

    Institute of Scientific and Technical Information of China (English)

    HU Chun-Song; YOON Young-sup; ISNER Jeffrey M.; LOSORDO Douglas W.

    2003-01-01

    Gene therapy for cardiovascular diseases has developed from preliminary animal experiments to clinical trials. However, vectors and target genes used currently in gene therapy are mainly focused on viral, nonviral vector and single target gene or monogene. Each vector system has a series of advantages and limitations. Chimeric vectors which combine the advantages of viral and nonviral vector,chimeric target genes which combine two or more target genes and novel gene delivery modes are being developed. In this article, we summarized the progress in chimeric vectors and chimeric genes based cardiovascular gene therapy, which including proliferative or occlusive vascular diseases such as atheroslerosis and restenosis, hypertonic vascular disease such as hypertension and cardiac diseases such as myocardium ischemia, dilated cardiomyopathy and heart failure, even heart transplantation. The development of chimeric vector, chimeric gene and their cardiovascular gene therapy is promising.

  19. A Comparison of Red Fluorescent Proteins to Model DNA Vaccine Expression by Whole Animal In Vivo Imaging.

    Directory of Open Access Journals (Sweden)

    Ekaterina Kinnear

    Full Text Available DNA vaccines can be manufactured cheaply, easily and rapidly and have performed well in pre-clinical animal studies. However, clinical trials have so far been disappointing, failing to evoke a strong immune response, possibly due to poor antigen expression. To improve antigen expression, improved technology to monitor DNA vaccine transfection efficiency is required. In the current study, we compared plasmid encoded tdTomato, mCherry, Katushka, tdKatushka2 and luciferase as reporter proteins for whole animal in vivo imaging. The intramuscular, subcutaneous and tattooing routes were compared and electroporation was used to enhance expression. We observed that overall, fluorescent proteins were not a good tool to assess expression from DNA plasmids, with a highly heterogeneous response between animals. Of the proteins used, intramuscular delivery of DNA encoding either tdTomato or luciferase gave the clearest signal, with some Katushka and tdKatushka2 signal observed. Subcutaneous delivery was weakly visible and nothing was observed following DNA tattooing. DNA encoding haemagglutinin was used to determine whether immune responses mirrored visible expression levels. A protective immune response against H1N1 influenza was induced by all routes, even after a single dose of DNA, though qualitative differences were observed, with tattooing leading to high antibody responses and subcutaneous DNA leading to high CD8 responses. We conclude that of the reporter proteins used, expression from DNA plasmids can best be assessed using tdTomato or luciferase. But, the disconnect between visible expression level and immunogenicity suggests that in vivo whole animal imaging of fluorescent proteins has limited utility for predicting DNA vaccine efficacy.

  20. Vaccine Safety

    Science.gov (United States)

    ... the safety of Tdap, Meningococcal, and HPV vaccines Human Papillomavirus (HPV) Vaccine is Very Safe Read about the safety of ... Hepatitis A Vaccine Safety Hepatitis B Vaccine Safety Human Papillomavirus (HPV) Vaccine Safety FAQs about HPV Safety Influenza (Flu) Vaccine ...

  1. A New Approach for Designing a Potentially Vaccine Candidate against Urinary Tract Infection by Using Protein Display on Lactobacillus

    Directory of Open Access Journals (Sweden)

    Gholamreza Goudarzi

    2015-10-01

    Full Text Available Background: The prevalence of Urinary Tract Infection (UTI is really high in the world. Escherichia coli is a major agent of UTI. One of the strategies for decreasing UTI infections is vaccine development. As the attachment is a really important stage in colonization and infection, at- tachment inhibition has an applied strategy.  FimH protein is a major factor during bacterial colonization in urinary tract and could be used as a vaccine. Thus, it was considered in this research as a candidate anti- gen.Methods: The sequences of fimH and acmA genes were used for de- signing a synthetic gene. It was cloned to pET23a expression vector and transformed  to E. coli (DE3 Origami.  To confirm the expression  of recombinant  protein,  SDS-PAGE  and western  blotting  methods  were used.  Subsequently,  recombinant  protein  was  purified.  On  the  other hand, Lactobacillus reuteri was cultured and mixed with FimH / AcmA recombinant  protein. The rate of protein localization  on lactobacillus surface was assessed using ELISA method.Results: It was showed that the recombinant protein was expressed inE. coli (DE3 Origami and purified by affinity chromatography. More- over, this protein could be localized on lactobacillus surface by 5 days. Conclusion:  In current study,  a fusion recombinant  protein was pre- pared and displayed on L. reuteri surface. This strain could be used for animal  experiment  as  a  competitor  against  Uropathogenic   E.  coli (UPEC. Using manipulated probiotics strains instead of antibiotic ther- apy could decrease the antibiotic consumption  and reduce multi-drug resistant strains.

  2. A new strategy based on SmRho protein loaded chitosan nanoparticles as a candidate oral vaccine against schistosomiasis.

    Directory of Open Access Journals (Sweden)

    Carolina R Oliveira

    Full Text Available BACKGROUND: Schistosomiasis is one of the most important neglected tropical diseases and an effective control is unlikely in the absence of improved sanitation and vaccination. A new approach of oral vaccination with alginate coated chitosan nanoparticles appears interesting because their great stability and the ease of target accessibility, besides of chitosan and alginate immunostimulatory properties. Here we propose a candidate vaccine based on the combination of chitosan-based nanoparticles containing the antigen SmRho and coated with sodium alginate. METHODS AND FINDINGS: Our results showed an efficient performance of protein loading of nanoparticles before and after coating with alginate. Characterization of the resulting nanoparticles reported a size around 430 nm and a negative zeta potential. In vitro release studies of protein showed great stability of coated nanoparticles in simulated gastric fluid (SGF and simulated intestinal fluid (SIF. Further in vivo studies was performed with different formulations of chitosan nanoparticles and it showed that oral immunization was not able to induce high levels of antibodies, otherwise intramuscular immunization induced high levels of both subtypes IgG1 and IgG2a SmRho specific antibodies. Mice immunized with nanoparticles associated to CpG showed significant modulation of granuloma reaction. Mice from all groups immunized orally with nanoparticles presented significant levels of protection against infection challenge with S. mansoni worms, suggesting an important role of chitosan in inducing a protective immune response. Finally, mice immunized with nanoparticles associated with the antigen SmRho plus CpG had 38% of the granuloma area reduced and also presented 48% of protection against of S. mansoni infection. CONCLUSIONS: Taken together, this results support this new strategy as an efficient delivery system and a potential vaccine against schistosomiasis.

  3. In silico analysis and recombinant expression of BamA protein as a universal vaccine against Escherichia coli in mice.

    Science.gov (United States)

    Guan, Qingfeng; Wang, Xiao; Wang, Xiumin; Teng, Da; Wang, Jianhua

    2016-06-01

    Colibacillosis, caused by pathogenic Escherichia coli, is a common disease in animals and human worldwide with extensive losses in breeding industry and with millions of people death annually. There is thus an urgent need for the development of universal vaccines against colibacillosis. In this study, the BamA protein was analyzed in silico for sequence homology, physicochemical properties, allergenic prediction, and epitopes prediction. The BamA protein (containing 286 amino acids) clusters in E. coli were retrieved in UniProtKB database, in which 81.7 % sequences were identical (Uniref entry A7ZHR7), and sequences with 94.82 % identity were above 93.4 %. Moreover, BamA was highly conserved among Salmonella and Shigella and has no allergenicity to mice and human. The epitopes of BamA were located principally in periplasm and extracellular domain. Surf_Ag_VNR domain (at position 448-810 aa) of BamA was expressed, purified, and then used for immunization of mice. Titers of the rBamA sera were 1:736,000 and 1:152,000 against rBamA and E. coli and over 1:27,000 against Salmonella and Shigella. Opsonophagocytosis result revealed that the rBamA sera strengthened the phagocytic activity of neutrophils against E. coli. The survival rate of mice vaccinated with rBamA and PBS was 80 and 20 %, respectively. These data indicated that BamA could serve as a promising universal vaccine candidate for the development of a protective subunit vaccine against bacterial infection. Thus, the above protocol would provide more feasible technical clues and choices for available control of pathogenic E. coli, Salmonella, and Shigella. PMID:27020285

  4. Polysaccharide-Based Vaccines

    Science.gov (United States)

    Santana, Violeta Fernández; Balbin, Yury Valdés; Calderón, Janoi Chang; Icart, Luis Peña; Verez-Bencomo, Vicente

    Capsular polysaccharides (CPS) and lipopolysaccharides from bacteria are employed for the production of vaccines against human diseases. Initial development of CPS as a vaccine was followed by the development and introduction of conjugate polysaccharide-protein vaccines. The principles leading to both developments are reviewed.

  5. Recommendation for use of the newly introduced pneumococcal protein conjugate vaccines in Korea

    OpenAIRE

    Eun Hwa Choi; Kyung Hyo Kim; Yae Jean Kim; Jong Hyun Kim; Su Eun Park; Hoan Jong Lee; Byung Wook Eun; Dae Sun Jo; Kyong Min Choi; Young Jin Hong

    2011-01-01

    Streptococcus pneumoniae remains a leading cause of invasive infections including bacteremia and meningitis, as well as mucosal infections such as otitis media and pneumonia among children and adults. The 7-valent pneumococcal conjugate vaccine (PCV7) was licensed for use among infants and young children in many countries including Korea. The routine use of PCV7 has resulted in a decreased incidence of invasive pneumococcal disease (IPD) by the vaccine serotypes among the vaccinees and substa...

  6. Evaluation of the non-toxic mutant of the diphtheria toxin K51E/E148K as carrier protein for meningococcal vaccines.

    Science.gov (United States)

    Pecetta, S; Vijayakrishnan, B; Romano, M R; Proietti, D; Surdo, P Lo; Balocchi, C; Mori, E; Davis, B G; Berti, F

    2016-03-01

    Diphtheria toxin mutant CRM197 is a common carrier protein for glycoconjugate vaccines, which has been proven an effective protein vector for, among others, meningococcal carbohydrates. The wide-range use of this protein in massive vaccine production requires constant increase of production yields and adaptability to an ever-growing market. Here we compare CRM197 with the alternative diphtheria non-toxic variant DT-K51E/E148K, an inactive mutant that can be produced in the periplasm of Escherichia coli. Biophysical characterization of DT-K51E/E148K suggested high similarity with CRM197, with main differences in their alpha-helical content, and a suitable purity for conjugation and vaccine preparation. Meningococcal serogroup A (MenA) glycoconjugates were synthesized using CRM197 and DT-K51E/E148K as carrier proteins, obtaining the same conjugation yields and comparable biophysical profiles. Mice were then immunized with these CRM197 and DT-K51E/E148K conjugates, and essentially identical immunogenic and protective effects were observed. Overall, our data indicate that DT-K51E/E148K is a readily produced protein that now allows the added flexibility of E. coli production in vaccine development and that can be effectively used as protein carrier for a meningococcal conjugate vaccine. PMID:26845738

  7. Ns1 is a key protein in the vaccine composition to protect Ifnar(-/- mice against infection with multiple serotypes of African horse sickness virus.

    Directory of Open Access Journals (Sweden)

    Francisco de la Poza

    Full Text Available African horse sickness virus (AHSV belongs to the genus Orbivirus. We have now engineered naked DNAs and recombinant modified vaccinia virus Ankara (rMVA expressing VP2 and NS1 proteins from AHSV-4. IFNAR((-/- mice inoculated with DNA/rMVA-VP2,-NS1 from AHSV-4 in an heterologous prime-boost vaccination strategy generated significant levels of neutralizing antibodies specific of AHSV-4. In addition, vaccination stimulated specific T cell responses against the virus. The vaccine elicited partial protection against an homologous AHSV-4 infection and induced cross-protection against the heterologous AHSV-9. Similarly, IFNAR((-/- mice vaccinated with an homologous prime-boost strategy with rMVA-VP2-NS1 from AHSV-4 developed neutralizing antibodies and protective immunity against AHSV-4. Furthermore, the levels of immunity were very high since none of vaccinated animals presented viraemia when they were challenged against the homologous AHSV-4 and very low levels when they were challenged against the heterologous virus AHSV-9. These data suggest that the immunization with rMVA/rMVA was more efficient in protection against a virulent challenge with AHSV-4 and both strategies, DNA/rMVA and rMVA/rMVA, protected against the infection with AHSV-9. The inclusion of the protein NS1 in the vaccine formulations targeting AHSV generates promising multiserotype vaccines.

  8. A rapid immunization strategy with a live attenuated tetravalent dengue vaccine elicits protective neutralizing antibody responses in non-human primates

    Directory of Open Access Journals (Sweden)

    Yuping eAmbuel

    2014-06-01

    Full Text Available Dengue viruses (DENVs cause approximately 390 million cases of DENV infections annually and over 3 billion people worldwide are at risk of infection. No dengue vaccine is currently available nor is there an antiviral therapy for DENV infections. We have developed a tetravalent live-attenuated DENV vaccine (TDV that consists of a molecularly characterized attenuated DENV-2 strain (TDV-2 and three chimeric viruses containing the pre-membrane and envelope genes of DENV-1, -3 and -4 expressed in the context of the TDV-2 genome. To impact dengue vaccine delivery in endemic areas and immunize travelers, a simple and rapid immunization strategy (RIS is preferred. We investigated RIS consisting of two full vaccine doses being administered subcutaneously or intradermally on the initial vaccination visit (day 0 at two different anatomical locations with a needle-free disposable syringe jet injection (DSJI delivery devices (PharmaJet in non-human primates (NHP. This vaccination strategy resulted in efficient priming and induction of neutralizing antibody responses to all four DENV serotypes comparable to those elicited by the traditional prime and boost (two months later vaccination schedule. In addition, the vaccine induced CD4+ and CD8+ T cells producing IFN-γ, IL-2, and TNF-α, and targeting the DENV-2 NS1, NS3 and NS5 proteins. Moreover, vaccine-specific T cells were cross-reactive with the non-structural NS3 and NS5 proteins of DENV-4. When animals were challenged with DENV-2 they were protected with no detectable viremia, and exhibited sterilizing immunity (no increase of neutralizing titers post- challenge. RIS could decrease vaccination visits and provide quick immune response to all four DENV serotypes. This strategy could increase vaccination compliance and would be especially advantageous for travelers into endemic areas.

  9. 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.

  10. 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

    2015-03-01

    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. PMID:25768031

  11. Combining Viral Vectored and Protein-in-adjuvant Vaccines Against the Blood-stage Malaria Antigen AMA1: Report on a Phase 1a Clinical Trial

    OpenAIRE

    Susanne H. Hodgson; Choudhary, Prateek; Elias, Sean C; Milne, Kathryn H; Thomas W Rampling; Biswas, Sumi; Ian D Poulton; Miura, Kazutoyo; Douglas, Alexander D.; Alanine, Daniel GW; Illingworth, Joseph J.; de Cassan, Simone C.; ZHU, DAMING; Nicosia, Alfredo; Long, Carole A.

    2014-01-01

    The development of effective vaccines against difficult disease targets will require the identification of new subunit vaccination strategies that can induce and maintain effective immune responses in humans. Here we report on a phase 1a clinical trial using the AMA1 antigen from the blood-stage Plasmodium falciparum malaria parasite delivered either as recombinant protein formulated with Alhydrogel adjuvant with and without CPG 7909, or using recombinant vectored vaccines—chimpanzee adenovir...

  12. Proteomics Reveals that Proteins Expressed During the Early Stage of Bacillus anthracis Infection Are Potential Targets for the Development of Vaccines and Drugs

    Institute of Scientific and Technical Information of China (English)

    Chun-Ming Huang; Craig A. Elmets; De-chu C. Tang; Fuming Li; Nabiha Yusuf

    2004-01-01

    In this review, we advance a new concept in developing vaccines and/or drugs to target specific proteins expressed during the early stage of Bacillus anthracis (an thrax) infection and address existing challenges to this concept. Three proteins (immune inhibitor A, GPR-like spore protease, and alanine racemase) initially identified by proteomics in our laboratory were found to have differential expres sions during anthrax spore germination and early outgrowth. Other studies of different bacillus strains indicate that these three proteins are involved in either germination or cytotoxicity of spores, suggesting that they may serve as potential targets for the design of anti-anthrax vaccines and drugs.

  13. Bioinformatics and protein modelling of the GS element of Mycobacteriumavium subsp. paratuberculosis (MAP) and GS-encoded proteins as drugtargets and vaccine components

    Institute of Scientific and Technical Information of China (English)

    Joe Sheridan; Tim Bull; Nazira Sumar; Jun Cheng; John Hermon-Taylor

    2000-01-01

    AIM To determine the function and cellular localization of GS-encoded proteins and to assess their potentialas drug targets and vaccine components.METHODS Bioinformatics software was used to predict the function of GS-encoded proteins and theirlocation within MAP. Protein modelling software was used to build protein structures.RESULTS The gene gsa is a truncated glycosyl transferase and probably non-functional. gsbA and gsbBproduce GDP-fucose which is methylated by gsc and acetylated by mpa. gsd is a fucosyl transferase whichattaches fucose to subterminal rhamnose on cell surface glycopeptidolipid. gsa, gsbA and gsbB and gsc arelocated within the cytoplasm. mpa is embedded in the plasma membrane with 10 transmembrane regions anda conspicuous extracellular loop. gsd is lipid-linked and predicted to localize to the microbial cell surface.CONCLUSION GS encodes the biosynthetic machinery to give MAP a surface coat of methylated andacetylated fucose which may contribute to its protease-resistant nature and ability to minimize immunerecognition. The gsbA/gsbB operon and gsd are promising drug targets and gsd is a good candidatecomponent of a new class of anti-MAP vaccines.

  14. Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP fused antigens: a potential tool to develop DNA vaccines against flaviviruses

    Directory of Open Access Journals (Sweden)

    Rafael Dhalia

    2009-12-01

    Full Text Available Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the developent of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP. The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.A vacinação é a estratégia mais prática e o melhor custo-benefício para prevenir a maioria das infecções dos flavivirus, para os quais existe vacina disponível. Entretanto, as vacinas baseadas em vírus atenuados podem potencialmente promover efeitos colaterais e, mais raramente, reações fatais. Diante deste cenário, o desenvolvimento de estratégias alternativas de vacinação, como vacinas baseadas em DNA codificando seqüências específicas dos flavivirus, está sendo considerado

  15. A novel dual promoter DNA vaccine induces CD8+ response against Toxoplasma gondii sporozoite specific surface protein “SporoSAG” through non-apoptotic cells

    Directory of Open Access Journals (Sweden)

    Sultan Gülçe İz

    2014-01-01

    The results of this study reveal the ability of SporoSAG protein to induce CD8+ T lymphocyte response for the first time. Overall, SporoSAG protein can be included to multivalant vaccine formulations in future studies to increase the protection in infections acquired through T. gondii oocysts.

  16. A New Gene Family (ariel) Encodes Asparagine-Rich Entamoeba histolytica Antigens, Which Resemble the Amebic Vaccine Candidate Serine-Rich E. histolytica Protein

    OpenAIRE

    Mai, Zhiming; Samuelson, John

    1998-01-01

    A family of genes, called ariel, are named for and encode asparagine-rich Entamoeba histolytica antigens containing 2 to 16 octapeptide repeats. Ariel proteins, which are constitutively expressed by trophozoites, belong to a large antigen family that includes the serine-rich E. histolytica protein (SREHP), an amebic vaccine candidate.

  17. Construction, purification, and characterization of a chimeric TH1 antagonist

    Directory of Open Access Journals (Sweden)

    Javier-González Luís

    2006-05-01

    Full Text Available Abstract Background TH1 immune response antagonism is a desirable approach to mitigate some autoimmune and inflammatory reactions during the course of several diseases where IL-2 and IFN-γ are two central players. Therefore, the neutralization of both cytokines could provide beneficial effects in patients suffering from autoimmune or inflammatory illnesses. Results A chimeric antagonist that can antagonize the action of TH1 immunity mediators, IFN-γ and IL-2, was designed, engineered, expressed in E. coli, purified and evaluated for its in vitro biological activities. The TH1 antagonist molecule consists of the extracellular region for the human IFNγ receptor chain 1 fused by a four-aminoacid linker peptide to human 60 N-terminal aminoacid residues of IL-2. The corresponding gene fragments were isolated by RT-PCR and cloned in the pTPV-1 vector. E. coli (W3110 strain was transformed with this vector. The chimeric protein was expressed at high level as inclusion bodies. The protein was partially purified by pelleting and washing. It was then solubilized with strong denaturant and finally refolded by gel filtration. In vitro biological activity of chimera was demonstrated by inhibition of IFN-γ-dependent HLA-DR expression in Colo 205 cells, inhibition of IFN-γ antiproliferative effect on HEp-2 cells, and by a bidirectional effect in assays for IL-2 T-cell dependent proliferation: agonism in the absence versus inhibition in the presence of IL-2. Conclusion TH1 antagonist is a chimeric protein that inhibits the in vitro biological activities of human IFN-γ, and is a partial agonist/antagonist of human IL-2. With these attributes, the chimera has the potential to offer a new opportunity for the treatment of autoimmune and inflammatory diseases.

  18. Combining viral vectored and protein-in-adjuvant vaccines against the blood-stage malaria antigen AMA1: report on a phase 1a clinical trial.

    Science.gov (United States)

    Hodgson, Susanne H; Choudhary, Prateek; Elias, Sean C; Milne, Kathryn H; Rampling, Thomas W; Biswas, Sumi; Poulton, Ian D; Miura, Kazutoyo; Douglas, Alexander D; Alanine, Daniel Gw; Illingworth, Joseph J; de Cassan, Simone C; Zhu, Daming; Nicosia, Alfredo; Long, Carole A; Moyle, Sarah; Berrie, Eleanor; Lawrie, Alison M; Wu, Yimin; Ellis, Ruth D; Hill, Adrian V S; Draper, Simon J

    2014-12-01

    The development of effective vaccines against difficult disease targets will require the identification of new subunit vaccination strategies that can induce and maintain effective immune responses in humans. Here we report on a phase 1a clinical trial using the AMA1 antigen from the blood-stage Plasmodium falciparum malaria parasite delivered either as recombinant protein formulated with Alhydrogel adjuvant with and without CPG 7909, or using recombinant vectored vaccines--chimpanzee adenovirus ChAd63 and the orthopoxvirus MVA. A variety of promising "mixed-modality" regimens were tested. All volunteers were primed with ChAd63, and then subsequently boosted with MVA and/or protein-in-adjuvant using either an 8- or 16-week prime-boost interval. We report on the safety of these regimens, as well as the T cell, B cell, and serum antibody responses. Notably, IgG antibody responses primed by ChAd63 were comparably boosted by AMA1 protein vaccine, irrespective of whether CPG 7909 was included in the Alhydrogel adjuvant. The ability to improve the potency of a relatively weak aluminium-based adjuvant in humans, by previously priming with an adenoviral vaccine vector encoding the same antigen, thus offers a novel vaccination strategy for difficult or neglected disease targets when access to more potent adjuvants is not possible. PMID:25156127

  19. Targeting hepatitis B virus antigens to dendritic cells by heat shock protein to improve DNA vaccine potency

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To investigate a novel DNA vaccination based upon expression of the HBV e antigen fused to a heat shock protein (HSP) as a strategy to enhance DNA vaccine potency.METHODS: A pCMV-HBeAg-HSP DNA vaccine and a control DNA vaccine were generated. Mice were immunized with these different construct. Immune responses were measured 2 wk after a second immunization by a T cell response assay, CTL cytotoxicity assay, and an antibody assay in C57BL/6 and BALB/c mice. CT26-HBeAg tumor cell challenge test in vivo was performed in BALB/c mice to monitor anti-tumor immune responses.RESULTS: In the mice immunized with pCMV-HBe-HSP DNA, superior CTL activity to target HBV-positive target cells was observed in comparison with mice immunized with pCMV-HBeAg (44% ± 5% vs 30% ± 6% in E: T > 50:1, P < 0.05). ELISPOT assays showed a stronger T-cell response from mice immunized with pCMV-HBe-HSP than that from pCMV-HBeAg immunized animals when stimulated either with MHC class Ⅰ or class Ⅱ epitopes derived from HBeAg (74% ± 9% vs 31% ± 6%, P < 0.01). ELISA assays revealed an enhanced HBeAg antibody response from mice immunized with pCMV-HBe-HSP than from those immunized with pCMV-HBeAg. The lowest tumor incidence and the slowest tumor growth were observed in mice immunized with pCMV-HBe-HSP when challenged with CT26-HBeAg.CONCLUSION: The results of this study demonstrate a broad enhancement of antigen-specific CD4+ helper,CD8+ cytotoxic T-cell, and B-cell responses by a novel DNA vaccination strategy. They also proved a stronger antigen-specific immune memory, which may be superior to currently described HBV DNA vaccination strategies for the treatment of chronic HBV infection.

  20. Use of recombinant capsid proteins in the development of a vaccine against the foot-and-mouth disease virus

    Directory of Open Access Journals (Sweden)

    Belsham GJ

    2015-02-01

    Full Text Available Graham J Belsham, Anette Bøtner National Veterinary Institute, Technical University of Denmark, Kalvehave, Denmark Abstract: Foot-and-mouth disease remains one of the world's most economically important diseases of livestock. It is caused by foot-and-mouth disease virus, a member of the picornavirus family. The virus replicates very rapidly and can be efficiently transmitted between hosts by a variety of routes. The disease has been effectively controlled in some parts of the world but remains endemic in many others, thus there is a constant risk of introduction of the disease into areas that are normally free of foot-and-mouth disease with potentially huge economic consequences. To reduce the need for large-scale culling of infected, and potentially infected, animals there has been significant effort to develop new vaccines against this disease which avoid some, or all, of the deficiencies of current vaccines. A major focus has been on the use of systems that express the structural proteins of the virus that self-assemble to generate “empty capsid” particles which share many features with the intact virus but lack the ribonucleic acid genome and are therefore non-infectious. Such particles can be “designed” to improve their stability or modify their antigenicity and can be produced without “high containment” facilities. The development and use of such improved vaccines should assist in the global efforts to control this important disease. Keywords: picornavirus, diagnostic assays, virus structure, infection, immune responses

  1. Immunogenicity of DNA- and recombinant protein-based Alzheimer disease epitope vaccines.

    Science.gov (United States)

    Davtyan, Hayk; Bacon, Andrew; Petrushina, Irina; Zagorski, Karen; Cribbs, David H; Ghochikyan, Anahit; Agadjanyan, Michael G

    2014-01-01

    Alzheimer disease (AD) process involves the accumulation of amyloid plaques and tau tangles in the brain, nevertheless the attempts at targeting the main culprits, neurotoxic β-amyloid (Aβ) peptides, have thus far proven unsuccessful for improving cognitive function. Important lessons about anti-Aβ immunotherapeutic strategies were learned from the first active vaccination clinical trials. AD progression could be safely prevented or delayed if the vaccine (1) induces high titers of antibodies specific to toxic forms of Aβ; (2) does not activate the harmful autoreactive T cells that may induce inflammation; (3) is initiated before or at least at the early stages of the accumulation of toxic forms of Aβ. Data from the recent passive vaccination trials with bapineuzumab and solanezumab also indicated that anti-Aβ immunotherapy might be effective in reduction of the AD pathology and even improvement of cognitive and/or functional performance in patients when administered early in the course of the disease. For the prevention of AD the active immunization strategy may be more desirable than passive immunotherapy protocol and it can offer the potential for sustainable clinical and commercial advantages. Here we discuss the active vaccine approaches, which are still in preclinical development and vaccines that are already in clinical trials. PMID:24525778

  2. Generation and Characterization of a Human/Mouse Chimeric GD2-Mimicking Anti-Idiotype Antibody Ganglidiximab for Active Immunotherapy against Neuroblastoma.

    Directory of Open Access Journals (Sweden)

    Christin Eger

    Full Text Available Vaccination with proteins mimicking GD2 that is highly expressed on neuroblastoma (NB cells is a promising strategy in treatment of NB, a pediatric malignancy with poor prognosis. We previously showed efficacy of ganglidiomab in vivo, a murine anti-idiotype (anti-Id IgG1. In order to tailor immune responses to variable regions, we generated a new human/mouse chimeric anti-Id antibody (Ab ganglidiximab by replacing murine constant fragments with corresponding human IgG1 regions. DNA sequences encoding for variable regions of heavy (VH and light chains (VL were synthesized by RT-PCR from total RNA of ganglidiomab-producing hybridoma cells and further ligated into mammalian expression plasmids with coding sequences for constant regions of human IgG1 heavy and light chains, respectively. We established a stable production cell line using Chinese hamster ovarian (CHO cells co-transfected with two expression plasmids driving the expression of either ganglidiximab heavy or light chain. After purification from supernatants, anti-idiotypic characteristics of ganglidiximab were demonstrated. Binding of ganglidiximab to anti-GD2 Abs of the 14.18 family as well as to NK-92tr cells expressing a GD2-specific chimeric antigen receptor (scFv(ch14.18-zeta was shown using standard ELISA and flow cytometry analysis, respectively. Ganglidiximab binding affinities to anti-GD2 Abs were further determined by surface plasmon resonance technique. Moreover, binding of anti-GD2 Abs to the nominal antigen GD2 as well as GD2-specific Ab-mediated cytotoxicity (ADCC, CDC was competitively inhibited by ganglidiximab. Finally, ganglidiximab was successfully used as a protein vaccine in vivo to induce a GD2-specific humoral immune response. In summary, we report generation and characterization of a new human/mouse chimeric anti-Id Ab ganglidiximab for active immunotherapy against NB. This Ab may be useful to tailor immune responses to the paratope regions mimicking GD2

  3. Streptococcus iniae M-like protein contributes to virulence in fish and is a target for live attenuated vaccine development.

    Directory of Open Access Journals (Sweden)

    Jeffrey B Locke

    Full Text Available BACKGROUND: Streptococcus iniae is a significant pathogen in finfish aquaculture, though knowledge of virulence determinants is lacking. Through pyrosequencing of the S. iniae genome we have identified two gene homologues to classical surface-anchored streptococcal virulence factors: M-like protein (simA and C5a peptidase (scpI. METHODOLOGY/PRINCIPAL FINDINGS: S. iniae possesses a Mga-like locus containing simA and a divergently transcribed putative mga-like regulatory gene, mgx. In contrast to the Mga locus of group A Streptococcus (GAS, S. pyogenes, scpI is located distally in the chromosome. Comparative sequence analysis of the Mgx locus revealed only one significant variant, a strain with an insertion frameshift mutation in simA and a deletion mutation in a region downstream of mgx, generating an ORF which may encode a second putative mga-like gene, mgx2. Allelic exchange mutagenesis of simA and scpI was employed to investigate the potential role of these genes in S. iniae virulence. Our hybrid striped bass (HSB and zebrafish models of infection revealed that M-like protein contributes significantly to S. iniae pathogenesis whereas C5a peptidase-like protein does not. Further, in vitro cell-based analyses indicate that SiMA, like other M family proteins, contributes to cellular adherence and invasion and provides resistance to phagocytic killing. Attenuation in our virulence models was also observed in the S. iniae isolate possessing a natural simA mutation. Vaccination of HSB with the Delta simA mutant provided 100% protection against subsequent challenge with a lethal dose of wild-type (WT S. iniae after 1,400 degree days, and shows promise as a target for live attenuated vaccine development. CONCLUSIONS/SIGNIFICANCE: Analysis of M-like protein and C5a peptidase through allelic replacement revealed that M-like protein plays a significant role in S. iniae virulence, and the Mga-like locus, which may regulate expression of this gene, has an

  4. Expression of immunogenic epitopes of hepatitis B surface antigen with hybrid flagellin proteins by a vaccine strain of Salmonella.

    OpenAIRE

    Wu, J Y; Newton, S; Judd, A; Stocker, B; Robinson, W S

    1989-01-01

    A nonvirulent Salmonella dublin flagellin-negative, aromatic-dependent live vaccine strain has been used to express hepatitis B virus surface antigen epitopes in an immunogenic form. The envelope proteins of the virion are encoded by the S gene, which contains the pre-S1, pre-S2, and S coding regions. Synthetic oligonucleotides corresponding to amino acid residues S-(122-137) and pre-S2-(120-145) were inserted in-frame into the hypervariable region of a cloned Salmonella flagellin gene, and t...

  5. The fimbrial protein FlfA from Gallibacterium anatis is a virulence factor and vaccine candidate

    DEFF Research Database (Denmark)

    Bager, Ragnhild Jørgensen; Nesta, Barbara; Pors, Susanne Elisabeth;

    2013-01-01

    antigenic diversity hampers disease prevention by classical vaccines. Thus, insight into its pathogenesis and knowledge about important virulence factors is urgently required. A key event during the colonization and invasion of mucosal surfaces is adherence, and recently, at least three F17-like fimbrial...... gene clusters were identified in the genomes of several G. anatis strains. The objective of this study was to characterize the putative F17-like fimbrial subunit protein FlfA from G. anatis 12656-12 and determine its importance for virulence. In vitro expression and surface exposure of FlfA was...

  6. Expression of the classical swine fever E2 recombinant protein and examination of DNA-vaccine based on one subunit

    International Nuclear Information System (INIS)

    Full text: The aim of the work was to study the possibility of using the Classical Swine Fever Virus (CSFV) E2 (gp51-C55) recombinant protein expressed in E.coli in an immunologically active form and the creation of a DNA-vaccine model based on the gene. For minimizing the refolding problems of the recombinant protein one of two subunits of CSFV E2protein was chosen for production. This subunit has the domains A and D with the conservative epitopes some of which, in the domain A, induce synthesis of virus neutralizing antibodies. Viral RNA was isolated from the CSFV virulent strain Shi-Min, which was produced in a continuous swine kidney PK-15 cell culture (passage 37) Oligonucleotide primers were designed based on the Brescia (GeneBank, M31768) strain sequence Sn 2647-C2668 and Asn 3492-C3471. For additional RT-PCR controls the obtained fragment (846 n. p.) from isolates from CSF outbreak in Ukraine in 1994. It was shown that only the samples from the acute form of disease, wherever the amplicon sizes corresponded to the Shi-Min fragment, readily hybridized under this in stringency conditions. The subsequent sequence analysis of the fragment and the phylogenetic analyses with the use of sequences of various origin strains and isolates, placed the virus in group 1, subgroup 1.1, according to the classification suggested by D. Paton in 1995. For protein synthesis 3 constructions for plasmids expressing with the insert of CSFV E2 gene were created: CSFV-pET24a (+) -C as the individual form of the protein with the molecular mass (m.m.) 34 kD; CSFV-pGEX-2T - as fused with glutathion-S-tsansphesase, m.m. 59 kD and CSFV-pLY -C as fused with one subunit of γ-galactisidase, m.m. 107 kD. All 3 variants of the protein were synthesized as inclusion bodies with an expression level of 15-C20%. After optimizing the purification and refolding conditions, antigenic properties of the proteins were characterized in an indirect ELISA and immunoperoxidase test with homologous and

  7. Construction, Expression and Identification of a Recombinant BCG Vaccine Encoding Human Mycobacterium Tuberculosis Heat Shock Protein 65

    Institute of Scientific and Technical Information of China (English)

    戴五星; 梁靓; 高红; 黄海浪; 陈智浩; 程继忠; 皇甫永穆

    2004-01-01

    Heat shock protein 65 (HSP65) is one of the most important protective immunogens against the tuberculosis infection. The signal sequence of antigen 85B and the whole HSP65 DNA sequence of human Mycobacterium tuberculosis (M. tuberculosis) were amplified from BCG genome and plasmid pCMV-MTHSP65 respectively by polymerase chain reactions (PCR). These two sequences were cloned into the plasmid pBCG-2100 under the control of the promoter of heat shock protein 70 (HSP70) from human M. tuberculosis, yielding the prokaryotic shuttle expression plasmid pBCG-SP-HSP65. Results of restriction endonuclease analysis, PCR detection and DNA sequencing analysis showed that the two cloned DNA sequences were consistent with those previously reported, and the direction of their inserting into the recombinant was correct and the reading frame had been maintained. The recombinants were electroporated into BCG to construct the recombinant BCG vaccine and induced by heating. The induced expression detected by SDS-PAGE showed that the content of 65 kD protein expressed in recombinant BCG was 35.69 % in total bacterial protein and 74.09 % in the cell lysate supernatants, suggesting that the recombinant HSP65 gene could express in BCG with high efficiency and the expressed proteins were mainly soluble. Western-blot showed that the secretive recombinant proteins could specifically combine with antibody against M.tuberculosis HSP65, indicating that the recombinant proteins possess the biological activity of HSP65.

  8. Optimisation of prime-boost immunization in mice using novel protein-based and recombinant vaccinia (Tiantan-based HBV vaccine.

    Directory of Open Access Journals (Sweden)

    Hong Chen

    Full Text Available BACKGROUND: A therapeutic vaccine for chronic hepatitis B virus (HBV infection that enhances virus-specific cellular immune responses is urgently needed. The "prime-boost" regimen is a widely used vaccine strategy against many persistence infections. However, few reports have addressed this strategy applying for HBV therapeutic vaccine development. METHODOLOGY/PRINCIPAL FINDINGS: To develop an effective HBV therapeutic vaccine, we constructed a recombinant vaccinia virus (Tiantan containing the S+PreS1 fusion antigen (RVJSS1 combined with the HBV particle-like subunit vaccine HBVSS1 to explore the most effective prime-boost regimen against HBV. The immune responses to different prime-boost regimens were assessed in C57BL/C mice by ELISA, ELISpot assay and Intracellular cytokine staining analysis. Among the combinations tested, an HBV protein particle vaccine priming and recombinant vaccinia virus boosting strategy accelerated specific seroconversion and produced high antibody (anti-PreS1, anti-S antibody titres as well as the strongest multi-antigen (PreS1, and S-specific cellular immune response. HBSS1 protein prime/RVJSS1 boost immunization was also generated more significant level of both CD4+ and CD8+ T cell responses for Th1 cytokines (TNF-α and IFN-γ. CONCLUSIONS: The HBSS1 protein-vaccine prime plus RVJSS1 vector boost elicits specific antibody as well as CD4 and CD8 cells secreting Th1-like cytokines, and these immune responses may be important parameters for the future HBV therapeutic vaccines.

  9. Examination on the protein profiles of salivary glands of P. berghei infected anopheles Sp. post gamma irradiation using SDS-PAGE technique for developing malaria vaccine

    International Nuclear Information System (INIS)

    Sporozoite is a step of malaria parasitic live cycle that is most invasive and appropriate vaccine candidate. Result of experiments showed that malaria vaccine created by attenuating Plasmodium sp sporozoites with gamma rays was proven more effective. Study on the effects of irradiation to the profiles of protein in vaccine development is also important. The aim of this research was to examine the protein profile of salivary glands in sporozoite infected Anopheles sp post gamma irradiation using Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) technique. Examination covered the infection of Anopheles sp with Plasmodium sp, maintenance of infected mosquitoes for 14-16 days to obtain sporozoites, in vivo - in vitro irradiation of mosquitoes, preparation of salivary glands, electrophoresis on 10% SDS-PAGE, and Commassie blue staining. Results showed a different protein profile of infected and non infected salivary glands of Anopheles sp. There was additional protein band numbers at higher dose of irradiation (200 Gy) from sporozoite protein of P. berghei (MW 62 kDa). However, no difference of the profiles of circumsporozoite protein (CSP) observed among gamma irradiation doses of 150, 175 and 200 Gy. These results provide basic information that would lead to further study on the role of sporozoite proteins in malaria vaccine development. (author)

  10. Granulocyte-macrophage colony-stimulating factor DNA prime-protein boost strategy to enhance efficacy of a recombinant pertussis DNA vaccine

    Institute of Scientific and Technical Information of China (English)

    Qing-tian LI; Yong-zhang ZHU; Jia-you CHU; Ke DONG; Ping HE; Chun-yan FENG; Bao-yu HU; Shu-min ZHANG; Xiao-kui GUO

    2006-01-01

    Aim: To investigate a new strategy to enhance the efficacy of a recombinant pertussis DNA vaccine. The strategy is co-injection with cytokine plasmids as prime, and boosted with purified homologous proteins. Method: A recombinant pertussis DNA vaccine containing the pertussis toxin subunit 1 (PTS1), fragments of the filamentous hemagglutinin (FHA) gene and pertactin (PRN) gene encoding filamentous hemagglutinin and pertactin were constructed. Balb/c mice were immunized with several DNA vaccines and antigen-specific antibodies anti-PTSl, anti-PRN, anti-FHA, cytokines interleukin (IL)-10, IL-4, IFN-γ, TNF-oc, and spleno-cyte-proliferation assay were used to describe immune responses. Results: The recombinant DNA vaccine could elicit similar immune responses in mice as that of separate plasmids encoding the 3 fragments, respectively. Mice immunized with DNA and boosted with the corresponding protein elicited more antibodies than those that received DNA as boost. In particular, when the mice were co-immunized with murine granulocyte-macrophage colony-stimulating factor plasmids and boosted with proteins, all 4 cytokines and the 3 antigen-specific antibodies were significantly increased compared to the pVAXl group. Anti-PTSl, anti-FHA, IL-4 and TNF-α elicited in the colony stimulating factor (CSF) prime-protein boost group showed significant increase compared to all the other groups. Conclusion: This prime and boost strategy has proven to be very useful in improving the immunogenicity of DNA vaccines against pertussis.

  11. Novel vector vaccine against Brucella abortus based on influenza A viruses expressing Brucella L7/L12 or Omp16 proteins: evaluation of protection in pregnant heifers.

    Science.gov (United States)

    Tabynov, Kaissar; Yespembetov, Bolat; Sansyzbay, Abylai

    2014-10-14

    The present study provides the first information about the protection of a novel influenza viral vector vaccine expressing the Brucella proteins ribosomal L7/L12 or Omp16 containing the adjuvant Montanide Gel01 in pregnant heifers. Immunization of pregnant heifers was conducted via the conjunctival (n=10) or subcutaneous (n=10) route using cross prime and booster vaccination schedules at an interval of 28 days. The vector vaccine was evaluated in comparison with positive control groups vaccinated with Brucella abortus S19 (n=10) or B. abortus RB51 (n=10) and a negative (PBS+Montanide Gel01; n=10) control group. Via both the conjunctival or subcutaneous route, evaluation of protectiveness against abortion, effectiveness of vaccination and index of infection (in heifers and their fetuses or calves) demonstrated the vector vaccine provided good protection against B. abortus 544 infection compared to the negative control group (PBS+Montanide Gel01) and comparable protection to commercial vaccines B. abortus S19 or B. abortus RB51. PMID:25218295

  12. Co-expression of Interleukin-15 Enhances the Protective Immune Responses Induced by Immunization with a Murine Malaria MVA-Based Vaccine Encoding the Circumsporozoite Protein.

    Directory of Open Access Journals (Sweden)

    Marcela Parra

    Full Text Available Malaria remains a major global public health problem with an estimated 200 million cases detected in 2012. Although the most advanced candidate malaria vaccine (RTS,S has shown promise in clinical trials, its modest efficacy and durability have created uncertainty about the impact of RTS,S immunization (when used alone on global malaria transmission. Here we describe the development and characterization of a novel modified vaccinia virus Ankara (MVA-based malaria vaccine which co-expresses the Plasmodium yoelii circumsporozoite protein (CSP and IL-15. Vaccination/challenge studies showed that C57BL/6 mice immunized with the MVA-CSP/IL15 vaccine were protected significantly better against a P. yoelii 17XNL sporozoite challenge than either mice immunized with an MVA vaccine expressing only CSP or naïve controls. Importantly, the levels of total anti-CSP IgG were elevated about 100-fold for the MVA-CSP/IL15 immunized group compared to mice immunized with the MVA-CSP construct that does not express IL-15. Among the IgG subtypes, the IL-15 expressing MVA-CSP vaccine induced levels of IgG1 (8 fold and IgG2b (80 fold higher than the MVA-CSP construct. The significantly enhanced humoral responses and protection detected after immunization with the MVA-CSP/IL15 vaccine suggest that this IL-15 expressing MVA construct could be considered in the development of future malaria immunization strategies.

  13. Co-expression of Interleukin-15 Enhances the Protective Immune Responses Induced by Immunization with a Murine Malaria MVA-Based Vaccine Encoding the Circumsporozoite Protein.

    Science.gov (United States)

    Parra, Marcela; Liu, Xia; Derrick, Steven C; Yang, Amy; Molina-Cruz, Alvaro; Barillas-Mury, Carolina; Zheng, Hong; Thao Pham, Phuong; Sedegah, Martha; Belmonte, Arnel; Litilit, Dianne D; Waldmann, Thomas A; Kumar, Sanjai; Morris, Sheldon L; Perera, Liyanage P

    2015-01-01

    Malaria remains a major global public health problem with an estimated 200 million cases detected in 2012. Although the most advanced candidate malaria vaccine (RTS,S) has shown promise in clinical trials, its modest efficacy and durability have created uncertainty about the impact of RTS,S immunization (when used alone) on global malaria transmission. Here we describe the development and characterization of a novel modified vaccinia virus Ankara (MVA)-based malaria vaccine which co-expresses the Plasmodium yoelii circumsporozoite protein (CSP) and IL-15. Vaccination/challenge studies showed that C57BL/6 mice immunized with the MVA-CSP/IL15 vaccine were protected significantly better against a P. yoelii 17XNL sporozoite challenge than either mice immunized with an MVA vaccine expressing only CSP or naïve controls. Importantly, the levels of total anti-CSP IgG were elevated about 100-fold for the MVA-CSP/IL15 immunized group compared to mice immunized with the MVA-CSP construct that does not express IL-15. Among the IgG subtypes, the IL-15 expressing MVA-CSP vaccine induced levels of IgG1 (8 fold) and IgG2b (80 fold) higher than the MVA-CSP construct. The significantly enhanced humoral responses and protection detected after immunization with the MVA-CSP/IL15 vaccine suggest that this IL-15 expressing MVA construct could be considered in the development of future malaria immunization strategies. PMID:26505634

  14. Co-expression of Interleukin-15 Enhances the Protective Immune Responses Induced by Immunization with a Murine Malaria MVA-Based Vaccine Encoding the Circumsporozoite Protein

    Science.gov (United States)

    Parra, Marcela; Liu, Xia; Derrick, Steven C.; Yang, Amy; Molina-Cruz, Alvaro; Barillas-Mury, Carolina; Zheng, Hong; Thao Pham, Phuong; Sedegah, Martha; Belmonte, Arnel; Litilit, Dianne D.; Waldmann, Thomas A.; Kumar, Sanjai; Morris, Sheldon L.; Perera, Liyanage P.

    2015-01-01

    Malaria remains a major global public health problem with an estimated 200 million cases detected in 2012. Although the most advanced candidate malaria vaccine (RTS,S) has shown promise in clinical trials, its modest efficacy and durability have created uncertainty about the impact of RTS,S immunization (when used alone) on global malaria transmission. Here we describe the development and characterization of a novel modified vaccinia virus Ankara (MVA)–based malaria vaccine which co-expresses the Plasmodium yoelii circumsporozoite protein (CSP) and IL-15. Vaccination/challenge studies showed that C57BL/6 mice immunized with the MVA-CSP/IL15 vaccine were protected significantly better against a P. yoelii 17XNL sporozoite challenge than either mice immunized with an MVA vaccine expressing only CSP or naïve controls. Importantly, the levels of total anti-CSP IgG were elevated about 100-fold for the MVA-CSP/IL15 immunized group compared to mice immunized with the MVA-CSP construct that does not express IL-15. Among the IgG subtypes, the IL-15 expressing MVA-CSP vaccine induced levels of IgG1 (8 fold) and IgG2b (80 fold) higher than the MVA-CSP construct. The significantly enhanced humoral responses and protection detected after immunization with the MVA-CSP/IL15 vaccine suggest that this IL-15 expressing MVA construct could be considered in the development of future malaria immunization strategies. PMID:26505634

  15. Tomato bushy stunt virus (TBSV), a versatile platform for polyvalent display of antigenic epitopes and vaccine design

    International Nuclear Information System (INIS)

    Viruses-like particles (VLPs) are frequently being used as platforms for polyvalent display of foreign epitopes of interest on their capsid surface to improve their presentation enhancing the antigenicity and host immune response. In the present study, we used the VLPs of Tomato bushy stunt virus (TBSV), an icosahedral plant virus, as a platform to display 180 copies of 16 amino acid epitopes of ricin toxin fused to the C-terminal end of a modified TBSV capsid protein (NΔ52). Expression of the chimeric recombinant protein in insect cells resulted in spontaneous assembly of VLPs displaying the ricin epitope. Cryo-electron microscopy and image reconstruction of the chimeric VLPs at 22 A resolution revealed the locations and orientation of the ricin epitope exposed on the TBSV capsid surface. Furthermore, injection of chimeric VLPs into mice generated antisera that detected the native ricin toxin. The ease of fusing of short peptides of 15-20 residues and their ability to form two kinds (T = 1, T = 3) of bio-nanoparticles that result in the display of 60 or 180 copies of less constrained and highly exposed antigenic epitopes makes TBSV an attractive and versatile display platform for vaccine design.

  16. DNA vaccines encoding the envelope protein of West Nile virus lineages 1 or 2 administered intramuscularly, via electroporation and with recombinant virus protein induce partial protection in large falcons (Falco spp.).

    Science.gov (United States)

    Fischer, Dominik; Angenvoort, Joke; Ziegler, Ute; Fast, Christine; Maier, Kristina; Chabierski, Stefan; Eiden, Martin; Ulbert, Sebastian; Groschup, Martin H; Lierz, Michael

    2015-01-01

    As West Nile virus (WNV) can cause lethal diseases in raptors, a vaccination prophylaxis of free-living and captive populations is desirable. In the absence of vaccines approved for birds, equine vaccines have been used in falcons, but full protection against WNV infection was not achieved. Therefore, two DNA vaccines encoding the ectodomain of the envelope protein of WNV lineages 1 and 2, respectively, were evaluated in 28 large falcons. Four different vaccination protocols were used, including electroporation and booster-injections of recombinant WNV domain III protein, before challenge with the live WNV lineage 1 strain NY99. Drug safety, plasmid shedding and antibody production were monitored during the vaccination period. Serological, virological, histological, immunohistochemical and molecular biological investigations were performed during the challenge trials. Antibody response following vaccination was low overall and lasted for a maximum of three weeks. Plasmid shedding was not detected at any time. Viremia, mortality and levels, but not duration, of oral virus shedding were reduced in all of the groups during the challenge trial compared to the non-vaccinated control group. Likewise, clinical scoring, levels of cloacal virus shedding and viral load in organs were significantly reduced in three vaccination groups. Histopathological findings associated with WNV infections (meningo-encephalitis, myocarditis, and arteritis) were present in all groups, but immunohistochemical detection of the viral antigen was reduced. In conclusion, the vaccines can be used safely in falcons to reduce mortality and clinical signs and to lower the risk of virus transmission due to decreased levels of virus shedding and viremia, but full protection was not achieved in all groups. PMID:26282836

  17. Immunobiology of Influenza Vaccines

    OpenAIRE

    Gomez Lorenzo, Margarita M.; Fenton, Matthew J.

    2013-01-01

    Vaccination is the primary strategy for prevention and control of influenza. The surface hemagglutinin (HA) protein of the influenza virus contains two structural elements (head and stalk) that differ in their potential utility as vaccine targets. The head of the HA protein is the primary target of antibodies that confer protective immunity to influenza viruses. The underlying health status, age, and gene polymorphisms of vaccine recipients and, just as importantly, the extent of the antigeni...

  18. Influenza Vaccines

    OpenAIRE

    Ellebedy, A. H.; Webby, R J

    2009-01-01

    Influenza A viruses pose a substantial threat to the human population whether by purposeful manipulation and release or by the natural process of interspecies transmissions from animal reservoirs. The challenge with preparing for these events with vaccination strategies is that the best forms of protective immunity target the most variably of the viral proteins, hemagglutinin. Add to this even just the natural extent of variation in this protein and the challenges to vaccinologists become gre...

  19. Blood chimerism in a dizygotic dichorionic pregnancy.

    Science.gov (United States)

    Jang, Ja-Hyun; Jung, Haiyoung; Kim, Jong-Hwa; Park, Won-Soon; Kim, Sun-Hee

    2010-10-01

    Blood chimerism in twins is known to occur through the transfer of hematopoietic stem cells between the fetuses via a common placenta. We present a case of blood chimerism in a dizygotic dichorionic twin pregnancy. The female twin was delivered at 34 weeks of gestation, and the male twin was stillborn. Pathologic examination confirmed dichorionic diamniotic placentas. The karyotype of the female child was obtained using peripheral blood sample, and it revealed a mixture of 46,XX and 46,XY cells (chi 46,XY[13]/46,XX[7]). FISH analysis performed on the buccal cells by using CEP X/Y probe (Abbott Molecular Inc., USA) revealed 100% XX signals (nuc ish Xcen(DXZ1x2)[500]). Gross examination of the external genitalia and abdominal ultrasonography revealed no definitive abnormal findings in relation to sex differentiation. When XX/XY chimerism is present in blood lymphocytes, careful examination of external genitalia and reproductive organs and further studies are required to detect chimerism in non-hematopoetic tissues. This is a rare case of blood chimerism in dichorionic placentas, in contrast to those in monochorionic placentas. PMID:20890086

  20. Immune Responses in Pigs Induced by Recombinant DNA Vaccine Co-Expressing Swine IL-18 and Membrane Protein of Porcine Reproductive and Respiratory Syndrome Virus

    Directory of Open Access Journals (Sweden)

    Zhuang Ding

    2012-05-01

    Full Text Available In this study, two DNA vaccines, which express the membrane (M protein of porcine respiratory and reproductive syndrome virus (PRRSV (pEGFP-M and co-express both M and swine IL-18 (pEGFP-IL18-M, were constructed and their abilities to induce humoral and cellular responses in piglets were comparatively evaluated. Experimental results showed that both recombinant DNA vaccines could not elicit neutralizing antibodies in the immunized piglets. However, both DNA vaccines elicited Th1-biased cellular immune responses. Notably, pigs immunized with the plasmid pEGFP-IL18-M developed significantly higher levels of IFN-γ and IL-2 production response and stronger specific T-lymphocyte proliferation response than the pigs inoculated with the plasmids pEGFP-M and pEGFP-IL18 (P < 0.05. These results illustrated that co-expression of M and IL-18 proteins could significantly improve the potency of DNA vaccination on the activation of vaccine-induced virus-specific cell-mediated immune responses in pigs, which may be used as a strategy to develop a new generation of vaccines against highly pathogenic PRRSV.

  1. Nanogel-based pneumococcal surface protein A nasal vaccine induces microRNA-associated Th17 cell responses with neutralizing antibodies against Streptococcus pneumoniae in macaques.

    Science.gov (United States)

    Fukuyama, Y; Yuki, Y; Katakai, Y; Harada, N; Takahashi, H; Takeda, S; Mejima, M; Joo, S; Kurokawa, S; Sawada, S; Shibata, H; Park, E J; Fujihashi, K; Briles, D E; Yasutomi, Y; Tsukada, H; Akiyoshi, K; Kiyono, H

    2015-09-01

    We previously established a nanosized nasal vaccine delivery system by using a cationic cholesteryl group-bearing pullulan nanogel (cCHP nanogel), which is a universal protein-based antigen-delivery vehicle for adjuvant-free nasal vaccination. In the present study, we examined the central nervous system safety and efficacy of nasal vaccination with our developed cCHP nanogel containing pneumococcal surface protein A (PspA-nanogel) against pneumococcal infection in nonhuman primates. When [(18)F]-labeled PspA-nanogel was nasally administered to a rhesus macaque (Macaca mulatta), longer-term retention of PspA was noted in the nasal cavity when compared with administration of PspA alone. Of importance, no deposition of [(18)F]-PspA was seen in the olfactory bulbs or brain. Nasal PspA-nanogel vaccination effectively induced PspA-specific serum IgG with protective activity and mucosal secretory IgA (SIgA) Ab responses in cynomolgus macaques (Macaca fascicularis). Nasal PspA-nanogel-induced immune responses were mediated through T-helper (Th) 2 and Th17 cytokine responses concomitantly with marked increases in the levels of miR-181a and miR-326 in the serum and respiratory tract tissues, respectively, of the macaques. These results demonstrate that nasal PspA-nanogel vaccination is a safe and effective strategy for the development of a nasal vaccine for the prevention of pneumonia in humans. PMID:25669148

  2. Vaccination with F1-V fusion protein protects black-footed ferrets (Mustela nigripes) against plague upon oral challenge with Yersinia pestis

    Science.gov (United States)

    Rocke, T.E.; Smith, S.; Marinari, P.; Kreeger, J.; Enama, J.T.; Powell, B.S.

    2008-01-01

    Previous studies have established that vaccination of black-footed ferrets (Mustela nigripes) with F1-V fusion protein by subcutaneous (SC) injection protects the animals against plague upon injection of the bacterium Yersinia pestis. This study demonstrates that the F1-V antigen can also protect ferrets against plague contracted via ingestion of a Y. pestis-infected mouse, a probable route for natural infection. Eight black-footed ferret kits were vaccinated with F1-V protein by SC injection at approximately 60 days-of-age. A booster vaccination was administered 3 mo later via SC injection. Four additional ferret kits received placebos. The animals were challenged 6 wk after the boost by feeding each one a Y. pestis-infected mouse. All eight vaccinates survived challenge, while the four controls succumbed to plague within 3 days after exposure. To determine the duration of antibody postvaccination, 18 additional black-footed ferret kits were vaccinated and boosted with F1-V by SC injection at 60 and 120 days-of-age. High titers to both F1 and V (mean reciprocal titers of 18,552 and 99,862, respectively) were found in all vaccinates up to 2 yr postvaccination, whereas seven control animals remained antibody negative throughout the same time period. ?? Wildlife Disease Association 2008.

  3. Molecular chimerization of Pasteurella haemolytica leukotoxin to interleukin-2: effects on cytokine and antigen function.

    OpenAIRE

    Hughes, H P; Campos, M.; Potter, A A; Babiuk, L. A.

    1992-01-01

    A chimeric recombinant protein composed of the lktA gene product from Pasteurella haemolytica fused to bovine interleukin-2 (IL-2) was made. The LKT-IL-2 chimera was compared with recombinant bovine IL-2 with regard to the ability to induce proliferative responses and LAK cell activity in bovine peripheral blood mononuclear cells in vitro. In both instances, chimerization had no effect on IL-2 activity. Similarly, the LKT component was unaffected in its ability to induce an effective immune r...

  4. Ligand-mediated negative regulation of a chimeric transmembrane receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Desai, D M; Sap, J; Schlessinger, J;

    1993-01-01

    inactivate the EGFR-CD45 chimera in a manner that is dependent on dimerization of the chimeric protein. Inactivation of EGFR-CD45 chimera function results in the loss of TCR signaling, indicating that CD45 function is continuously required for TCR-mediated proximal signaling events. These results suggest...

  5. Construction of exogenous multiple epitopes of helper T lymphocytes and DNA immunization of its chimeric plasmid with HBV pre-S2/S gene

    Institute of Scientific and Technical Information of China (English)

    Wen-Jun Gao; Xiao-Mou Peng; Dong-Ying Xie; Qi-Feng Xie; Zhi-Liang Gao; Ji-Lu Yao

    2004-01-01

    AIM: To design and construct an exogenous multiple epitope of helper T lymphocytes (HTL), and to evaluate its effect on anti-HBs response through DNA immunization.METHODS: Artificial HTL epitope, PADRE and four other HTL epitopes from different proteins were linked together using splicing by overlap extension to generate exogenous multiple epitopes of HTL, MTE5. pcMTE5 and pcHB weregenerated by cloning MTE5 and fragments of HBV pre-S2/S gene into mammalian expression plasmid pcDNA3. Four chimeric plasmids were constructed by cloning MTE5 into the region of pre-S2 gene (Bam HI), 5′ terminal of S gene (HincⅡ, Xba Ⅰ) and 3′ terminal of S gene (Acc Ⅰ) of pcHB respectively. BALB/c mice were used in DNA immunization of the recombinant plasmids. Anti-HBs was detected using Abbott IMx AUSAB test kits.RESULTS: The sequences of MTE5 and the 6 constructs of recombinant plasmids were confirmed to be correct by DNA sequencing. The anti-HBs response of the coinoculation of pcHB and pcMTE5 was much higher than that of the inoculation of pcHB only (136.7±69.1 mIU/mL vs 27.6±17.3 mIU/mL, P<0.01, t = -6.56). Among the 4 chimeric plasmids, only the plasmid in which MTE5 was inserted into the pre-S2 region had good anti-HBs response (57.54±7.68 mIU/mL), and had no significant difference compared with those of pcHB and the co-inoculation of pcHB and pcMTE5.CONCLUSION: Exogenous multiple epitopes of HTL had immune enhancement when they were co-inoculated with pre-S2/S gene or inoculated in the chimeric form at a proper site of pre-S2/S gene of HBV. It might suggest that it was possible to improve hepatitis B vaccine using exogenous multiple epitopes of HTL. The antibody responses were very low using DNA immunization in the study. Thus, the immune enhancement effect of exogenous multiple epitopes of HTL has to be confirmed and the effect on overcoming the drawback of the polymorphism of HLA Ⅱ antigens should also be evaluated after these chimeric plasmids are expressed

  6. Evaluation of recombinant P23 protein as a vaccine for passive immunization of newborn calves against Cryptosporidium parvum.

    Science.gov (United States)

    Askari, N; Shayan, P; Mokhber-Dezfouli, M R; Ebrahimzadeh, E; Lotfollahzadeh, S; Rostami, A; Amininia, N; Ragh, M J

    2016-05-01

    Cryptosporidiosis is a zoonotic protozoan disease that affects the gastrointestinal tract of animals and humans. Diarrhoea as the most important indication of the infection leads to high economic losses in livestock industries and is a life threatening infection in immunocompromised individuals. In the absence of the effective drugs, vaccine has an effective role in the prevention of infection. For this purpose we developed a vaccine utilizing recombinant P23 protein and immunized pregnant cows four times from 70 days to parturition every 2 weeks. After parturition, each calf received his dam colostrum and challenged with 1 × 10(7) Cryptosporidium parvum oocysts at 12 h of age. Results showed that in contrast with the control group, the antibody titre in the sera and first milking colostra of the immunized cows significantly increased and calves fed hyperimmune colostrum did not show cryptosporidiosis signs. Moreover, enriched colostrum not only reduced significantly the amount of oocyst excretion but also delayed its onset. Our study showed that recombinant P23 protein could be used for passive immunization of newborn calves against Cryptosporidium parvum. PMID:27012710

  7. Three-Dimensional Visualizations in Teaching Genomics and Bioinformatics: Mutations in HIV Envelope Proteins and Their Consequences for Vaccine Design

    Directory of Open Access Journals (Sweden)

    Kathy Takayama

    2009-11-01

    Full Text Available This project addresses the need to provide a visual context to teach the practical applications of genome sequencing and bioinformatics. Present-day research relies on indirect visualization techniques (e.g., fluorescence-labeling of DNA in sequencing reactions and sophisticated computer analysis. Such methods are impractical and prohibitively expensive for laboratory classes. More importantly, there is a need for curriculum resources that visually demonstrate the application of genome sequence information rather than the DNA sequencing methodology itself. This project is a computer-based lesson plan that engages students in collaborative, problem-based learning. The specific example focuses on approaches to Human Immunodeficiency Virus-1 (HIV-1 vaccine design based on HIV-1 genome sequences using a case study. Students performed comparative alignments of variant HIV-1 sequences available from a public database. Students then examined the consequences of HIV-1 mutations by applying the alignments to three-dimensional images of the HIV-1 envelope protein structure, thus visualizing the implications for applications such as vaccine design. The lesson enhances problem solving through the application of one type of information (genomic or protein sequence into concrete visual conceptualizations. Assessment of student comprehension and problem-solving ability revealed marked improvement after the computer tutorial. Furthermore, contextual presentation of these concepts within a case study resulted in student responses that demonstrated higher levels of cognitive ability than was expected by the instructor.

  8. Crystallization and X-ray study of the artificial TBI protein, an experimental multiple-epitope vaccine against type 1 human immunodeficiency virus

    International Nuclear Information System (INIS)

    The artificial protein TBI constructed from four T-cell epitopes and five neutralizing B-cell epitopes is an experimental new-generation multiple-epitope vaccine against human immunodeficiency virus (HIV-1). The TBI protein was expressed in Escherichia coli cells, isolated, and purified to the homogeneous state. The growth conditions for protein crystal were found, and the crystals thus grown were studied by the method of X-ray diffraction analysis

  9. Sequence Variation and Immunologic Cross-Reactivity among Babesia bovis Merozoite Surface Antigen 1 Proteins from Vaccine Strains and Vaccine Breakthrough Isolates

    Science.gov (United States)

    LeRoith, Tanya; Brayton, Kelly A.; Molloy, John B.; Bock, Russell E.; Hines, Stephen A.; Lew, Ala E.; McElwain, Terry F.

    2005-01-01

    The Babesia bovis merozoite surface antigen 1 (MSA-1) is an immunodominant membrane glycoprotein that is the target of invasion-blocking antibodies. While antigenic variation has been demonstrated in MSA-1 among strains from distinct geographical areas, the extent of sequence variation within a region where it is endemic and the effect of variation on immunologic cross-reactivity have not been assessed. In this study, sequencing of MSA-1 from two Australian B. bovis vaccine strains and 14 breakthrough isolates from vaccinated animals demonstrated low sequence identity in the extracellular region of the molecule, ranging from 19.8 to 46.7% between the T vaccine strain and eight T vaccine breakthrough isolates, and from 18.7 to 99% between the K vaccine strain and six K vaccine breakthrough isolates. Although MSA-1 amino acid sequence varied substantially among strains, overall predicted regions of hydrophilicity and hydrophobicity in the extracellular domain were conserved in all strains examined, suggesting a conserved functional role for MSA-1 despite sequence polymorphism. Importantly, the antigenic variation created by sequence differences resulted in a lack of immunologic cross-reactivity among outbreak strains using sera from animals infected with the B. bovis vaccine strains. Additionally, sera from cattle hyperinfected with the Mexico strain of B. bovis and shown to be clinically immune did not cross-react with MSA-1 from any other isolate tested. The results indicate that isolates of B. bovis capable of evading vaccine-induced immunity contain an msa-1 gene that is significantly different from the msa-1 of the vaccine strain, and that the difference can result in a complete lack of cross-reactivity between MSA-1 from vaccine and breakthrough strains in immunized animals. PMID:16113254

  10. A suicidal DNA vaccine expressing the fusion protein of peste des petits ruminants virus induces both humoral and cell-mediated immune responses in mice.

    Science.gov (United States)

    Wang, Yong; Yue, Xiaolin; Jin, Hongyan; Liu, Guangqing; Pan, Ling; Wang, Guijun; Guo, Hao; Li, Gang; Li, Yongdong

    2015-12-01

    Peste des petits ruminants (PPR), a highly contagious disease induced by PPR virus (PPRV), affects sheep and goats. PPRV fusion (F) protein is important for the induction of immune responses against PPRV. We constructed a Semliki Forest virus (SFV) replicon-vectored DNA vaccine ("suicidal DNA vaccine") and evaluated its immunogenicity in BALB/c mice. The F gene of PPRV was cloned and inserted into the SFV replicon-based vector pSCA1. The antigenicity of the resultant plasmid pSCA1/F was identified by indirect immunofluorescence and western blotting. BALB/c mice were then intramuscularly injected with pSCA1/F three times at 14-d intervals. Specific antibodies and virus-neutralizing antibodies against PPRV were quantified by indirect ELISA and microneutralization tests, respectively. Cell-mediated immune responses were examined by cytokine and lymphocyte proliferation assays. The pSCA1/F expressed F protein in vitro and induced specific and neutralizing antibody production, and lymphocyte proliferation in mice. Mice vaccinated with pSCA1/F had increased IL-2 and IL-10 levels after 24-h post first immunization. IFN-γ and TNF-α levels increased from that time point and gradually decreased thereafter. Thus, the Semliki Forest virus replicon-vectored DNA vaccine expressing the F protein of PPRV induced both humoral and cell-mediated immune responses in mice. This could be considered as a novel strategy for vaccine development against PPR. PMID:26343487

  11. Immune responses of a chimaeric protein vaccine containing Mycoplasma hyopneumoniae antigens and LTB against experimental M. hyopneumoniae infection in pigs.

    Science.gov (United States)

    Marchioro, Silvana B; Sácristan, Rubén Del Pozo; Michiels, Annelies; Haesebrouck, Freddy; Conceição, Fabricio R; Dellagostin, Odir A; Maes, Dominiek

    2014-08-01

    A recombinant chimaeric protein containing three Mycoplasma hyopneumoniae antigens (C-terminal portion of P97, heat shock protein P42, and NrdF) fused to an adjuvant, the B subunit of heat-labile enterotoxin of Escherichia coli (LTB), was used to immunize pigs against enzootic pneumonia. The systemic and local immune responses, as well as the efficacy of the chimaeric protein in inducing protection against experimental M. hyopneumoniae infection were evaluated. In total, 60 male piglets, purchased from a M. hyopneumoniae-free herd, at 4 weeks of age were randomly allocated to six different experimental groups of 10 animals each: recombinant chimaeric protein by intramuscular (IM) (1) or intranasal (IN) (2) administration, commercial bacterin by IM administration (3), and the adjuvant LTB by IM (4, control group A) or IN (5, control group B) administration. All groups were immunized at 24 and 38 days of age and challenged at 52 days of age. The sixth group that was not challenged was used as the negative control (IN [n=5] or IM [n=5] administration of the LTB adjuvant). Compared with the non-challenged group, administration of the chimaeric protein induced significant (Phyopneumoniae infection in pigs. This lack of effectiveness points towards the need for further studies to improve the efficacy of this subunit-based vaccine approach. PMID:24909331

  12. Role of C-terminal domain and transmembrane helices 5 and 6 in function and quaternary structure of major intrinsic proteins: analysis of aquaporin/glycerol facilitator chimeric proteins.

    Science.gov (United States)

    Duchesne, Laurence; Pellerin, Isabelle; Delamarche, Christian; Deschamps, Stephane; Lagree, Valerie; Froger, Alexandrine; Bonnec, Georgette; Thomas, Daniel; Hubert, Jean-Francois

    2002-06-01

    We previously observed that aquaporins and glycerol facilitators exhibit different oligomeric states when studied by sedimentation on density gradients following nondenaturing detergent solubilization. To determine the domains of major intrinsic protein (MIP) family proteins involved in oligomerization, we constructed protein chimeras corresponding to the aquaporin AQPcic substituted in the loop E (including the proximal part of transmembrane domain (TM) 5) and/or the C-terminal part (including the distal part of TM 6) by the equivalent domain of the glycerol channel aquaglyceroporin (GlpF) (chimeras called AGA, AAG, and AGG). The analogous chimeras of GlpF were also constructed (chimeras GAG, GGA, and GAA). cRNA corresponding to all constructs were injected into Xenopus oocytes. AQPcic, GlpF, AAG, AGG, and GAG were targeted to plasma membranes. Water or glycerol membrane permeability measurements demonstrated that only the AAG chimera exhibited a channel function corresponding to water transport. Analysis of all proteins expressed either in oocytes or in yeast by velocity sedimentation on sucrose gradients following solubilization by 2% n-octyl glucoside indicated that only AQPcic and AAG exist in tetrameric forms. GlpF, GAG, and GAA sediment in a monomeric form, whereas GGA and AGG were found mono/dimeric. These data bring new evidence that, within the MIP family, aquaporins and GlpFs behave differently toward nondenaturing detergents. We demonstrate that the C-terminal part of AQPcic, including the distal half of TM 6, can be substituted by the equivalent domain of GlpF (AAG chimera) without modifying the transport specificity. Our results also suggest that interactions of TM 5 of one monomer with TM 1 of the adjacent monomer are crucial for aquaporin tetramer stability. PMID:11927589

  13. Safety and immunogenicity of GMZ2 - a MSP3-GLURP fusion protein malaria vaccine candidate

    DEFF Research Database (Denmark)

    Esen, Meral; Kremsner, Peter G; Schleucher, Regina; Gässler, Michael; Imoukhuede, Egeruan Babatunde; Imbault, Nathalie; Leroy, Odile; Jepsen, Søren; Knudsen, Birgitte Walther; Schumm, Michael; Knobloch, Jürgen; Theisen, Michael; Mordmüller, Benjamin

    2009-01-01

    -immune individuals. Ten, 30 and 100 microg of GMZ2 were well tolerated in 30 healthy malaria-naïve German volunteers when given three times in monthly intervals. Antigen-specific antibodies as well as memory B-cells were induced and detectable throughout the one year follow-up of the study. We conclude that GMZ2 is......Malaria is a major public health problem in Sub-Saharan Africa. In highly endemic regions infants, children and pregnant women are mostly affected. An effective malaria vaccine would complement existing malaria control strategies because it can be integrated in existing immunization programs easily....... Here we present the results of the first phase Ia clinical trial of GMZ2 adjuvanted in aluminium hydroxide. GMZ2 is a malaria vaccine candidate, designed upon the rationale to induce immune responses against asexual blood stages of Plasmodium falciparum similar to those encountered in semi...

  14. Vector prime/protein boost vaccine that overcomes defects acquired during aging and cancer

    DEFF Research Database (Denmark)

    Tang, Y.; Akbulut, H.; Maynard, J.;

    2006-01-01

    following the Ad-sig-TAA/ecdCD40L vector, the levels of the TAA-specific CD8 T cells and Abs increase dramatically over that seen with vector alone, in young (2-mo-old) as well as old (18-mo-old) mice. The Abs induced against hMUC-1 react with human breast cancer. This vaccine also induces a 4-fold...

  15. Molecular evolution of hypoallergenic hybrid proteins for vaccination against grass pollen allergy.

    Science.gov (United States)

    Linhart, Birgit; Focke-Tejkl, Margarete; Weber, Milena; Narayanan, Meena; Neubauer, Angela; Mayrhofer, Hannes; Blatt, Katharina; Lupinek, Christian; Valent, Peter; Valenta, Rudolf

    2015-04-15

    More than 10% of the population in Europe and North America suffer from IgE-associated allergy to grass pollen. In this article, we describe the development of a vaccine for grass pollen allergen-specific immunotherapy based on two recombinant hypoallergenic mosaic molecules, designated P and Q, which were constructed out of elements derived from the four major timothy grass pollen allergens: Phl p 1, Phl p 2, Phl p 5, and Phl p 6. Seventeen recombinant mosaic molecules were expressed and purified in Escherichia coli using synthetic genes, characterized regarding biochemical properties, structural fold, and IgE reactivity. We found that depending on the arrangement of allergen fragments, mosaic molecules with strongly varying IgE reactivity were obtained. Based on an extensive screening with sera and basophils from allergic patients, two hypoallergenic mosaic molecules, P and Q, incorporating the primary sequence elements of the four grass pollen allergens were identified. As shown by lymphoproliferation experiments, they contained allergen-specific T cell epitopes required for tolerance induction, and upon immunization of animals induced higher allergen-specific IgG Abs than the wild-type allergens and a registered monophosphoryl lipid A-adjuvanted vaccine based on natural grass pollen allergen extract. Moreover, IgG Abs induced by immunization with P and Q inhibited the binding of patients' IgE to natural allergens from five grasses better than IgG induced with the wild-type allergens or an extract-based vaccine. Our results suggest that vaccines based on the hypoallergenic grass pollen mosaics can be used for immunotherapy of grass pollen allergy. PMID:25786690

  16. Vaccination of cats with an attenuated recombinant myxoma virus expressing feline calicivirus capsid protein.

    Science.gov (United States)

    McCabe, Victoria J; Tarpey, Ian; Spibey, Norman

    2002-06-01

    Myxoma virus, a member of the Poxviridae family (genus Leporipoxvirus) is the agent responsible for myxomatosis in the European rabbit. Recombinant myxoma viruses expressing the capsid gene of an F9 strain of feline calicivirus (FCV) were constructed from an apathogenic, laboratory attenuated, isolate of myxoma virus. The FCV capsid genes were recombined into the myxoma growth factor (MGF) locus of the myxoma genome and expressed from synthetic poxvirus promoters. Myxoma virus is unable to replicate productively in feline cells in vitro, however, cells infected with recombinant viruses do express the heterologous antigens from both late and early/late synthetic promoters. Cats immunised with myxoma-FCV recombinant virus generated high levels of serum neutralising antibody and were protected from disease on subsequent challenge with virulent FCV. Furthermore, there was no evidence of transmission of myxoma-FCV recombinant virus from vaccinated to non-vaccinated cats. These results demonstrate the potential of myxoma virus as a safe vaccine vector for use in non-lepori species and in particular the cat. PMID:12057600

  17. Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP) fused antigens: a potential tool to develop DNA vaccines against flaviviruses

    OpenAIRE

    Rafael Dhalia; Milton Maciel Jr.; Fábia S.P. Cruz; Isabelle F.T. Viana; Mariana L. Palma; Thomas August; Ernesto T.A. Marques Jr.

    2009-01-01

    Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the developent of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmi...

  18. Vaccination with recombinant Boophilus annulatus Bm86 ortholog protein, Ba86, protects cattle against B. annulatus and B. microplus infestations

    Directory of Open Access Journals (Sweden)

    Jongejan Frans

    2009-03-01

    Full Text Available Abstract Background The cattle ticks, Boophilus spp., affect cattle production in tropical and subtropical regions of the world. Tick vaccines constitute a cost-effective and environmentally friendly alternative to tick control. The recombinant B. microplus Bm86 protective antigen has been shown to protect cattle against tick infestations. Recently, the gene coding for B. annulatus Bm86 ortholog, Ba86, was cloned and the recombinant protein was secreted and purified from the yeast Pichia pastoris. Results Recombinant Ba86 (Israel strain was used to immunize cattle to test its efficacy for the control of B. annulatus (Mercedes, Texas, USA strain and B. microplus (Susceptible, Mexico strain infestations. Bm86 (Gavac and Mozambique strain and adjuvant/saline were used as positive and negative controls, respectively. Vaccination with Ba86 reduced tick infestations (71% and 40%, weight (8% and 15%, oviposition (22% and 5% and egg fertility (25% and 50% for B. annulatus and B. microplus, respectively. The efficacy of both Ba86 and Bm86 was higher for B. annulatus than for B. microplus. The efficacy of Ba86 was higher for B. annulatus (83.0% than for B. microplus (71.5%. The efficacy of Bm86 (Gavac; 85.2% but not Bm86 (Mozambique strain; 70.4% was higher than that of Ba86 (71.5% on B. microplus. However, the efficacy of Bm86 (both Gavac and Mozambique strain; 99.6% was higher than that of Ba86 (83.0% on B. annulatus. Conclusion These experiments showed the efficacy of recombinant Ba86 for the control of B. annulatus and B. microplus infestations in cattle and suggested that physiological differences between B. microplus and B. annulatus and those encoded in the sequence of Bm86 orthologs may be responsible for the differences in susceptibility of these tick species to Bm86 vaccines.

  19. A mouse model based on replication-competent Tiantan vaccinia expressing luciferase/HIV-1 Gag fusion protein for the evaluation of protective efficacy of HIV vaccine

    Institute of Scientific and Technical Information of China (English)

    HUANG Yang; QIU Chao; LIU Lian-xing; FENG Yan-meng; ZHU Ting; XU Jian-qing

    2009-01-01

    Background Developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1) remains a grand challenge after more than two decades of intensive effort. It is partially due to the lack of suitable animal models for screening and prioritizing vaccine candidates. In this study, we aim to develop a mice model to test HIV-1 vaccine efficacy. Methods We constructed a recombinant vaccinia expressing firefly luciferase and HIV-1 Gag fusion protein based on Tiantan strain, an attenuated but replication-competent poxvirus (rTTV-lucgag). By quantifying the luciferase activity as its read out, we defined the biodistribution of Tiantan strain poxvirus in mice inoculated intraperitoneally and attempted to apply this model to evaluate the HIV-1 vaccine efficacy. Results Our data demonstrated that the rTTV-lucgag was able to express high level of luciferase (≤106 relative luciferase units (RLU)/mg protein) and HIV-1 Gag (>3 folds increase comparing to the control). After intraperitoneal inoculation, this virus had dominant replication in the ovary, uterus, and cervix of mice and the luciferase activities in those organs are significantly correlated with viral titers (r2=0.71, P <0.01). Pre-immunization with an HIV gag DNA vaccine reduced the luciferase activity in ovary from (6006+3141) RLU/mg protein in control group to (1538±463) RLU/mg protein in vaccine group (P=0.1969). Conclusions The luciferase activity in ovary could represent viral replication in vivo;, this rTTV-lucgag/mice model may be suitable to assess the protective efficacy of cytotoxic T-cell responses to HIV Gag with less tedious work and high through-put.

  20. Comparative efficacy of hemagglutinin, nucleoprotein, and matrix 2 protein gene-based vaccination against H5N1 influenza in mouse and ferret.

    Science.gov (United States)

    Rao, Srinivas S; Kong, Wing-Pui; Wei, Chih-Jen; Van Hoeven, Neal; Gorres, J Patrick; Nason, Martha; Andersen, Hanne; Tumpey, Terrence M; Nabel, Gary J

    2010-01-01

    Efforts to develop a broadly protective vaccine against the highly pathogenic avian influenza A (HPAI) H5N1 virus have focused on highly conserved influenza gene products. The viral nucleoprotein (NP) and ion channel matrix protein (M2) are highly conserved among different strains and various influenza A subtypes. Here, we investigate the relative efficacy of NP and M2 compared to HA in protecting against HPAI H5N1 virus. In mice, previous studies have shown that vaccination with NP and M2 in recombinant DNA and/or adenovirus vectors or with adjuvants confers protection against lethal challenge in the absence of HA. However, we find that the protective efficacy of NP and M2 diminishes as the virulence and dose of the challenge virus are increased. To explore this question in a model relevant to human disease, ferrets were immunized with DNA/rAd5 vaccines encoding NP, M2, HA, NP+M2 or HA+NP+M2. Only HA or HA+NP+M2 vaccination conferred protection against a stringent virus challenge. Therefore, while gene-based vaccination with NP and M2 may provide moderate levels of protection against low challenge doses, it is insufficient to confer protective immunity against high challenge doses of H5N1 in ferrets. These immunogens may require combinatorial vaccination with HA, which confers protection even against very high doses of lethal viral challenge. PMID:20352112

  1. Design and expression of fusion protein consists of HBsAg and Polyepitope of HCV as an HCV potential vaccine

    Science.gov (United States)

    Gholizadeh, Monireh; Khanahmad, Hossein; Memarnejadian, Arash; Aghasadeghi, Mohammad Reza; Roohvand, Farzin; Sadat, Seyed Mehdi; Cohan, Reza Ahangari; Nazemi, Ali; Motevalli, Fatemeh; Asgary, Vahid; Arezumand, Roghaye

    2015-01-01

    Background: Hepatitis C virus (HCV) infection is a serious public health threat worldwide. Cellular immune responses, especially cytotoxic T-lymphocytes (CTLs), play a critical role in immune response toward the HCV clearance. Since polytope vaccines have the ability to stimulate the cellular immunity, a recombinant fusion protein was developed in this study. Materials and Methods: The designed fusion protein is composed of hepatitis B surface antigen (HBsAg), as an immunocarrier, fused to an HCV polytope sequence. The polytope containing five immunogenic epitopes of HCV was designed to induce specific CTL responses. The construct was cloned into the pET-28a, and its expression was investigated in BL21 (DE3), BL21 pLysS, BL21 pLysE, and BL21 AI Escherichia coli strains using 12% gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Finally, the identity of expressed fusion protein was confirmed by Western blotting using anti-His monoclonal antibody and affinity chromatography was applied to purify the expressed protein. Results: The accuracy of the construct was confirmed by restriction map analysis and sequencing. The transformation of the construct into the BL21 (DE3), pLysS, and pLysE E. coli strains did not lead to any expression. The fusion protein was found to be toxic for E. coli DE3. By applying two steps inhibition, the fusion protein was successfully expressed in BL21 (AI) E. coli strain. Conclusion: The HBsAg-polytope fusion protein expressed in this study can be further evaluated for its immunogenicity in animal models. PMID:26682209

  2. Expression and intercellular trafficking of the VP22 protein of CVI988/Rispens vaccine strain of Marek's disease virus

    Institute of Scientific and Technical Information of China (English)

    CHEN HongJun; SONG CuiPing; QIN AiJian; ZHANG ChenFei

    2007-01-01

    The viral protein 22 (VP22) in the tegument of Marek's disease virus serotype 1 (MDV-1) plays an important role in cell-to-cell spread and viral propagation. Antiserum against the carboxyl terminus of VP22 was prepared by immunizing mice with recombinant VP22 expressed in E. coli, and used to investigate its expression in chicken embryo fibroblast (CEF) cells infected with different MDV-1 strains. At an infection dose of PFU=50, intercellular trafficking of the VP22 into the nuclei of the surrounding receipt cells was detected as early as 3 hours post infection. By 6 hours after infection (before viral plague formation), the protein was detected in the whole nuclei of the recipient cells with no difference among MDV-1 strains CVI988/Rispens, GA and RB1B. Intra-nuclear accumulation of the VP22 protein was further increased when the viral plagues started to form. These results indicate that, albeit the existence of the 201TKSERT206 deletion, the VP22 of the CVI988/Rispens vaccine strain has also intercellular-trafficking function, which might serve as a potential alternative delivering protein instead of virulent strains VP22.

  3. Gene variability and degree of expression of vaccine candidate factor H binding protein in clinical isolates of Neisseria meningitidis.

    Science.gov (United States)

    Kelly, Anne; Jacobsson, Susanne; Hussain, Shahida; Olcén, Per; Mölling, Paula

    2013-01-01

    The factor H binding protein (fHbp) is currently being evaluated in clinical trials as a vaccine candidate for a meningococcal group B vaccine. We have previously described the prevalence and sequence variation of fHbp (Jacobsson et al., 2009) and here we investigate the expression of the antigen. The present study includes isolates from carriers (n = 62) and patients with invasive Neisseria meningitidis infections (n = 146), of which 62 had a fatal outcome. Among the invasive isolates from patients with fatal and non-fatal infections fHbp allele 1 was most common (42% and 29% respectively), but it was only identified in 3% of the carrier isolates, where allele 16 was most frequent (13%). The Fluorescence-activated cell sorting analysis identified fHbp expression in all except seven isolates and further analysis by Western blot showed that five of these seven samples were indeed negative using a polyclonal anti-fHbp serum. The negative isolates belonged to serogroup B fHbp allele 24, Y allele 104, and W-135 allele 16 (all invasive). Two were non-serogroupable carrier isolates (allele 21 and 101). An interesting finding is that isolates from invasive infections with fatal outcome had lower expression of fHbp or lower affinity for the fHbp antibody compared to isolates from non-fatal invasive infections and carriers. PMID:23030708

  4. Protective vaccination against experimental canine visceral leishmaniasis using a combination of DNA and protein immunization with cysteine proteinases type I and II of L. infantum.

    Science.gov (United States)

    Rafati, Sima; Nakhaee, Alireza; Taheri, Tahere; Taslimi, Yasaman; Darabi, Haideh; Eravani, Davood; Sanos, Stephanie; Kaye, Paul; Taghikhani, Mohammad; Jamshidi, Shahram; Rad, Mohammad Ali

    2005-05-25

    Leishmania infantum is known to be associated with visceral leishmaniasis in Iran and canids are natural reservoirs. Control of disease in dogs appears to be one of the most effective approaches for interrupting the domestic cycle of the disease. In search for successful vaccine strategies, we evaluated the cysteine proteinases (CPs) type I and II using a heterologous prime-boost regime for vaccination against experimental visceral leishmaniasis in dogs. Following vaccination and challenge, dogs were followed for 12 months. Ten dogs vaccinated by prime/boost with DNA/recombinant CPs (in combination with CpG ODN and Montanide 720) remained free of infection in their bone morrow. In contrast, three out of four dogs in the control groups had infection in their bone marrow. The peripheral lymphocytes from protected animals had generally higher proliferation responses to F/T antigen, recombinant CPA (rCPA) and recombinant CPB (rCPB) than controls. During post-challenge period, the difference in stimulation index is significant (pdogs had elevated IFN-gamma mRNA in peripheral blood mononuclear cells (PBMC), whereas there was a consistent increase in the level of IL-10 in the control groups and some vaccinated dogs. The level of total IgG and IgG2, but not IgG1, to rCPA and rCPB was significantly higher in the vaccinated group (pdog, all dogs in the vaccinated group in comparison to control dogs had strong DTH responses. We propose that the combination of DNA and recombinant protein vaccination using CPs could be instrumental to control (VL) in dogs. PMID:15882533

  5. Chimerism in health, transplantation and autoimmunity

    NARCIS (Netherlands)

    Koopmans, Marije; Kremer Hovinga, Idske Cornelia Lydia

    2009-01-01

    The term “chimerism” originates from Greek mythology and refers to the creature Chimaera, whose body was in front a lion, the back a serpent and the midsection a goat. In medicine, the term chimerism refers to an individual, organ or part consisting of tissues of diverse genetic constitution. Pregna

  6. Recombinant trimeric HA protein immunogenicity of H5N1 avian influenza viruses and their combined use with inactivated or adenovirus vaccines.

    Directory of Open Access Journals (Sweden)

    Shih-Chang Lin

    Full Text Available BACKGROUND: The highly pathogenic avian influenza (HPAI H5N1 virus continues to cause disease in poultry and humans. The hemagglutinin (HA envelope protein is the primary target for subunit vaccine development. METHODOLOGY/PRINCIPAL FINDINGS: We used baculovirus-insect cell expression to obtain trimeric recombinant HA (rHA proteins from two HPAI H5N1 viruses. We investigated trimeric rHA protein immunogenicity in mice via immunizations, and found that the highest levels of neutralizing antibodies resulted from coupling with a PELC/CpG adjuvant. We also found that the combined use of trimeric rHA proteins with (a an inactivated H5N1 vaccine virus, or (b a recombinant adenovirus encoding full-length HA sequences for prime-boost immunization, further improved antibody responses against homologous and heterologous H5N1 virus strains. Data from cross-clade prime-boost immunization regimens indicate that sequential immunization with different clade HA antigens increased antibody responses in terms of total IgG level and neutralizing antibody titers. CONCLUSION/SIGNIFICANCE: Our findings suggest that the use of trimeric rHA in prime-boost vaccine regimens represents an alternative strategy for recombinant H5N1 vaccine development.

  7. Studies on Nanoparticle Based Avian Influenza Vaccines to Present Immunogenic Epitopes of the Virus with Concentration on Ectodomain of Matrix 2 (M2e) Protein

    Science.gov (United States)

    Babapoor Dighaleh, Sankhiros

    2011-12-01

    Avian influenza is an infectious disease of avian species caused by type A influenza viruses with a significant economic impact on the poultry industry. Vaccination is the main prevention strategy in many countries worldwide. However, available vaccines elicit antibodies against two major surface protein of the virus hemagglutinin (HA) and neuraminidase (NA), where they constantly change by point mutations. Influenza viruses can also easily undergo gene reassortment. Therefore, to protect chickens against new strain of avian influenza virus, as well as control and prevent virus spread among farms, new vaccines needed to be designed which is a tedious, time consuming and expensive. Recently, conserved regions of the influenza genome have been evaluated as possible universal vaccines to eliminate constant vaccine updates based on circulating virus. In this study, peptide nanotechnology was used to generate vaccine nanoparticles that carry the highly conserved external domain of matrix 2 protein (M2e). These nanoparticles presented M2e in monomeric or tetrameric forms, designated as PSC-M2e-CH and BNSC-M2eN-CH. respectively. First, to demonstrate immunogenicity of these nanoparticles, we measured anti-M2e antibody in chickens, particularly when a high dose was applied. Prior to vaccination-challenge study, the challenge dose were determined by oculonasal inoculation of 10 6 EID50 or 107.7 EID50 of low pathogenicity AI virus HSN2 followed by measuring cloacal and tracheal virus shedding. A biphasic virus shedding pattern was observed with two peaks of virus shedding at days 4 and 8 for both tracheal and cloacal swabs. The chickens infected with 107.7 EID50 had significant virus shedding as compared with 106 EID50. Based on results of mentioned studies, a vaccination-challenge study was conducted by using 75mug of each vaccine construct per inoculation (with and without adjuvant) and higher dose of virus for challenge. BN5C-M2e-CH with adjuvant significantly reduced the

  8. 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. PMID:27123586

  9. Molecular Evolution of Hypoallergenic Hybrid Proteins for Vaccination against Grass Pollen Allergy

    OpenAIRE

    Linhart, Birgit; Focke-Tejkl, Margarete; Weber, Milena; Narayanan, Meena; Neubauer, Angela; Mayrhofer, Hannes; Blatt, Katharina; Lupinek, Christian; Valent, Peter; Valenta, Rudolf

    2015-01-01

    More than 10% of the population in Europe and North America suffer from IgE-associated allergy to grass pollen. In this article, we describe the development of a vaccine for grass pollen allergen-specific immunotherapy based on two recombinant hypoallergenic mosaic molecules, designated P and Q, which were constructed out of elements derived from the four major timothy grass pollen allergens: Phl p 1, Phl p 2, Phl p 5, and Phl p 6. Seventeen recombinant mosaic molecules were expressed and pur...

  10. Origination of an X-linked testes chimeric gene by illegitimate recombination in Drosophila.

    Directory of Open Access Journals (Sweden)

    2006-05-01

    Full Text Available The formation of chimeric gene structures provides important routes by which novel proteins and functions are introduced into genomes. Signatures of these events have been identified in organisms from wide phylogenic distributions. However, the ability to characterize the early phases of these evolutionary processes has been difficult due to the ancient age of the genes or to the limitations of strictly computational approaches. While examples involving retrotransposition exist, our understanding of chimeric genes originating via illegitimate recombination is limited to speculations based on ancient genes or transfection experiments. Here we report a case of a young chimeric gene that has originated by illegitimate recombination in Drosophila. This gene was created within the last 2-3 million years, prior to the speciation of Drosophila simulans, Drosophila sechellia, and Drosophila mauritiana. The duplication, which involved the Bällchen gene on Chromosome 3R, was partial, removing substantial 3' coding sequence. Subsequent to the duplication onto the X chromosome, intergenic sequence was recruited into the protein-coding region creating a chimeric peptide with approximately 33 new amino acid residues. In addition, a novel intron-containing 5' UTR and novel 3' UTR evolved. We further found that this new X-linked gene has evolved testes-specific expression. Following speciation of the D. simulans complex, this novel gene evolved lineage-specifically with evidence for positive selection acting along the D. simulans branch.

  11. Roles of adjuvant and route of vaccination in antibody response and protection engendered by a synthetic matrix protein 2-based influenza A virus vaccine in the mouse

    Directory of Open Access Journals (Sweden)

    Cudic Mare

    2007-10-01

    Full Text Available Abstract Background The M2 ectodomain (M2e of influenza A virus (IAV strains that have circulated in humans during the past 90 years shows remarkably little structural diversity. Since M2e-specific antibodies (Abs are capable of restricting IAV replication in vivo but are present only at minimal concentration in human sera, efforts are being made to develop a M2e-specific vaccine. We are exploring a synthetic multiple antigenic peptide (MAP vaccine and here report on the role of adjuvants (cholera toxin and immunostimulatory oligodeoxynucleotide and route of immunization on Ab response and strength of protection. Results Independent of adjuvants and immunization route, on average 87% of the M2e-MAP-induced Abs were specific for M2e peptide and a variable fraction of these M2e(pep-specific Abs (average 15% cross-reacted with presumably native M2e expressed by M2-transfected cells. The titer of these cross-reactive M2e(pep-nat-specific Abs in sera of parenterally immunized mice displayed a sigmoidal relation to level of protection, with EC50 of ~20 μg Ab/ml serum, though experiments with passive M2e(pep-nat Abs indicated that serum Abs did not fully account for protection in parenterally vaccinated mice, particularly in upper airways. Intranasal vaccination engendered stronger protection and a higher proportion of G2a Abs than parenteral vaccination, and the strength of protection failed to correlate with M2e(pep-nat-specific serum Ab titers, suggesting a role of airway-associated immunity in protection of intranasally vaccinated mice. Intranasal administration of M2e-MAP without adjuvant engendered no response but coadministration with infectious IAV slightly enhanced the M2e(pep-nat Ab response and protection compared to vaccination with IAV or adjuvanted M2e-MAP alone. Conclusion M2e-MAP is an effective immunogen as ~15% of the total M2e-MAP-induced Ab response is of desired specificity. While M2e(pep-nat-specific serum Abs have an important

  12. Effect of natural and chimeric haemagglutinin genes on influenza A virus replication in baby hamster kidney cells.

    Science.gov (United States)

    van Wielink, R; Harmsen, M M; Martens, D E; de Leeuw, O S; Peeters, B P H; Wijffels, R H; Moormann, R J M

    2012-12-31

    Baby hamster kidney (BHK21) cells are used to produce vaccines against various viral veterinary diseases, including rabies and foot-and-mouth-disease. Although particular influenza virus strains replicate efficiently in BHK21 cells the general use of these cells for influenza vaccine production is prohibited by the poor replication of most strains, including model strain A/PR/8/34 [H1N1] (PR8). We now show that in contrast to PR8, the related strain A/WSN/33 [H1N1] (WSN) replicates efficiently in BHK21 cells. This difference is determined by the haemagglutinin (HA) protein since reciprocal reassortant viruses with swapped HAs behave similarly with respect to growth on BHK21 cells as the parental virus from which their HA gene is derived. The ability or inability of six other influenza virus strains to grow on BHK21 cells appears to be similarly dependent on the nature of the HA gene since reassortant PR8 viruses containing the HA of these strains grow to similar titres as the parental virus from which the HA gene was derived. However, the growth to low titres of a seventh influenza strain was not due to the nature of the HA gene since a reassortant PR8 virus containing this HA grew efficiently on BHK21 cells. Taken together, these results suggest that the HA gene often primarily determines influenza replication efficiency on BHK21 cells but that in some strains other genes are also involved. High virus titres could be obtained with reassortant PR8 strains that contained a chimeric HA consisting of the HA1 domain of PR8 and the HA2 domain of WSN. HA1 contains most antigenic sites and is therefore important for vaccine efficacy. This method of producing the HA1 domain as fusion to a heterologous HA2 domain could possibly also be used for the production of HA1 domains of other viruses to enable the use of BHK21 cells as a generic platform for veterinary influenza vaccine production. PMID:23079079

  13. Amino acid sequence diversity of the major human papillomavirus capsid protein: Implications for current and next generation vaccines

    OpenAIRE

    Ahmed, Amina I.; Bissett, Sara L; Beddows, Simon

    2013-01-01

    Despite the fidelity of host cell polymerases, the human papillomavirus (HPV) displays a degree of genomic polymorphism resulting in distinct genotypes and intra-type variants. The current HPV vaccines target the most prevalent genotypes associated with cervical cancer (HPV16/18) and genital warts (HPV6/11). Although these vaccines confer some measure of cross-protection, a multivalent HPV vaccine is in the pipeline that aims to broaden vaccine protection against other cervical cancer-associa...

  14. The Immunodominance Change and Protection of CD4+ T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice.

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    Hsin-Wei Chen

    Full Text Available Dengue is the leading cause of mosquito-borne viral infections and no vaccine is available now. Envelope protein domain III (ED3 is the major target for the binding of dengue virus neutralizing antibodies; however, the ED3-specifc T-cell response is less well understood. To investigate the T-cell responses to four serotypes of dengue virus (DENV-1 to 4, we immunized mice using either a tetravalent ED3-based DNA or protein vaccine, or combined both as a DNA prime-protein boost strategy (prime-boost. A significant serotype-dependent IFN-γ or IL-4 response was observed in mice immunized with either the DNA or protein vaccine. The IFN-γ response was dominant to DENV-1 to 3, whereas the IL-4 response was dominant to DENV-4. Although the similar IgG titers for the four serotypes were observed in mice immunized with the tetravalent vaccines, the neutralizing antibody titers varied and followed the order of 2 = 3>1>4. Interestingly, the lower IFN-γ response to DENV-4 is attributable to the immunodominance change between two CD4+ T-cell epitopes; one T-cell epitope located at E349-363 of DENV-1 to 3 was more immunogenic than the DENV-4 epitope E313-327. Despite DENV-4 specific IFN-γ responses were suppressed by immunodominance change, either DENV-4-specific IFN-γ or neutralizing antibody responses were still recalled after DENV-4 challenge and contributed to virus clearance. Immunization with the prime-boost elicited both IFN-γ and neutralizing antibody responses and provided better protection than either DNA or protein immunization. Our findings shed light on how ED3-based tetravalent dengue vaccines sharpen host CD4 T-cell responses and contribute to protection against dengue virus.

  15. HPV vaccine

    Science.gov (United States)

    Vaccine - HPV; Immunization - HPV; Gardasil; Cervarix; HPV2; HPV4; Vaccine to prevent cervical cancer ... Girls ages 11 and 12 should receive the HPV vaccine series: The vaccine is given in three shots ...

  16. Immune response to influenza A virus hemagglutinin protein is sufficient to induce vaccine-associated enhanced respiratory disease

    Science.gov (United States)

    The antigenic diversity of influenza A virus (IAV) circulating in pigs continues to complicate control of swine influenza through the use of vaccination in the United States. The antibody response elicited by whole inactivated virus (WIV) vaccines can lead to vaccine-associated enhanced respiratory ...

  17. Production of full-length soluble Plasmodium falciparum RH5 protein vaccine using a Drosophila melanogaster Schneider 2 stable cell line system

    Science.gov (United States)

    Hjerrild, Kathryn A.; Jin, Jing; Wright, Katherine E.; Brown, Rebecca E.; Marshall, Jennifer M.; Labbé, Geneviève M.; Silk, Sarah E.; Cherry, Catherine J.; Clemmensen, Stine B.; Jørgensen, Thomas; Illingworth, Joseph J.; Alanine, Daniel G. W.; Milne, Kathryn H.; Ashfield, Rebecca; de Jongh, Willem A.; Douglas, Alexander D.; Higgins, Matthew K.; Draper, Simon J.

    2016-01-01

    The Plasmodium falciparum reticulocyte-binding protein homolog 5 (PfRH5) has recently emerged as a leading candidate antigen against the blood-stage human malaria parasite. However it has proved challenging to identify a heterologous expression platform that can produce a soluble protein-based vaccine in a manner compliant with current Good Manufacturing Practice (cGMP). Here we report the production of full-length PfRH5 protein using a cGMP-compliant platform called ExpreS2, based on a Drosophila melanogaster Schneider 2 (S2) stable cell line system. Five sequence variants of PfRH5 were expressed that differed in terms of mutagenesis strategies to remove potential N-linked glycans. All variants bound the PfRH5 receptor basigin and were recognized by a panel of monoclonal antibodies. Analysis following immunization of rabbits identified quantitative and qualitative differences in terms of the functional IgG antibody response against the P. falciparum parasite. The antibodies induced by one protein variant were shown to be qualitatively similar to responses induced by other vaccine platforms. This work identifies Drosophila S2 cells as a clinically-relevant platform suited for the production of ‘difficult-to-make’ proteins from Plasmodium parasites, and identifies a PfRH5 sequence variant that can be used for clinical production of a non-glycosylated, soluble full-length protein vaccine immunogen. PMID:27457156

  18. Plant-based vaccines: novel and low-cost possible route for Mediterranean innovative vaccination strategies.

    Science.gov (United States)

    Aboul-Ata, Aboul-Ata E; Vitti, Antonella; Nuzzaci, Maria; El-Attar, Ahmad K; Piazzolla, Giuseppina; Tortorella, Cosimo; Harandi, Ali M; Olson, Olof; Wright, Sandra A; Piazzolla, Pasquale

    2014-01-01

    A plant bioreactor has enormous capability as a system that supports many biological activities, that is, production of plant bodies, virus-like particles (VLPs), and vaccines. Foreign gene expression is an efficient mechanism for getting protein vaccines against different human viral and nonviral diseases. Plants make it easy to deal with safe, inexpensive, and provide trouble-free storage. The broad spectrum of safe gene promoters is being used to avoid risk assessments. Engineered virus-based vectors have no side effect. The process can be manipulated as follows: (a) retrieve and select gene encoding, use an antigenic protein from GenBank and/or from a viral-genome sequence, (b) design and construct hybrid-virus vectors (viral vector with a gene of interest) eventually flanked by plant-specific genetic regulatory elements for constitutive expression for obtaining chimeric virus, (c) gene transformation and/or transfection, for transient expression, into a plant-host model, that is, tobacco, to get protocols processed positively, and then moving into edible host plants, (d) confirmation of protein expression by bioassay, PCR-associated tests (RT-PCR), Northern and Western blotting analysis, and serological assay (ELISA), (e) expression for adjuvant recombinant protein seeking better antigenicity, (f) extraction and purification of expressed protein for identification and dosing, (g) antigenicity capability evaluated using parental or oral delivery in animal models (mice and/or rabbit immunization), and (h) growing of construct-treated edible crops in protective green houses. Some successful cases of heterologous gene-expressed protein, as edible vaccine, are being discussed, that is, hepatitis C virus (HCV). R9 mimotope, also named hypervariable region 1 (HVR1), was derived from the HVR1 of HCV. It was used as a potential neutralizing epitope of HCV. The mimotope was expressed using cucumber mosaic virus coat protein (CP), alfalfa mosaic virus CP P3/RNA3, and

  19. Evaluation of Indirect Enzyme-Linked Immunosorbent Assays and IgG Avidity Assays Using a Protein A-Peroxidase Conjugate for Serological Distinction between Brucella abortus S19-Vaccinated and -Infected Cows ▿

    OpenAIRE

    Pajuaba, Ana C. A. M.; Deise A O Silva; Mineo, José R.

    2010-01-01

    This study aimed to evaluate the use of protein A-peroxidase (horseradish peroxidase [HRPO]) in indirect enzyme-linked immunosorbent assays (iELISAs) and IgG avidity assays for serological distinction between Brucella abortus S19-vaccinated and -infected cows. Four groups were analyzed: GI, 41 nonvaccinated seropositive cows; GII, 79 S19-vaccinated heifers analyzed at 3 months postvaccination; GIII, 105 S19-vaccinated cows analyzed after 24 months of age; and GIV, 278 nonvaccinated seronegati...

  20. Induction of Broad and Potent Anti-Human Immunodeficiency Virus Immune Responses in Rhesus Macaques by Priming with a DNA Vaccine and Boosting with Protein-Adsorbed Polylactide Coglycolide Microparticles

    OpenAIRE

    Otten, Gillis; Schaefer, Mary; Greer, Catherine; Calderon-Cacia, Maria; Coit, Doris; Kazzaz, Jina; Medina-Selby, Angelica; Selby, Mark; Singh, Manmohan; Ugozzoli, Mildred; zur Megede, Jan; Barnett, Susan W.; O'Hagan, Derek; Donnelly, John; Ulmer, Jeffrey

    2003-01-01

    Several vaccine technologies were evaluated for their abilities to induce anti-human immunodeficiency virus Gag immune responses in rhesus macaques. While no vaccine alone was able to induce broad and strong immune responses, these were achieved by priming with Gag DNA and boosting with Gag protein adsorbed to polylactide coglycolide microparticles. This regimen elicited strong antibodies, helper T cells, and cytotoxic T lymphocytes and thus holds promise as an effective vaccination scheme.

  1. Co-transformation of canola by chimeric chitinase and tlp genes towards improving resistance to Sclerotinia sclerotiorum.

    Science.gov (United States)

    Aghazadeh, Rustam; Zamani, Mohammadreza; Motallebi, Mostafa; Moradyar, Mehdi; Moghadassi Jahromi, Zahra

    2016-09-01

    Canola (Brassica napus) plants were co-transformed with two pathogenesis-related protein genes expressing a Trichoderma atroviride chitinase with a chitin-binding domain (chimeric chitinase) and a thaumatin-like protein (tlp) from Oryza sativa conferring resistance to phytopatogenic fungi by Agrobacterium-mediated transformation. The putative transgenic plants were confirmed by PCR. After measuring the specific activity of the chimeric chitinase and glucanase activity for tlp genes, transgenic plants with high specific activity were selected for southern blot analysis to confirm the copy number of the genes. In vitro assays, the antifungal activity of crude extracted protein against Sclerotinia sclerotiorum showed that the inhibition percentage in double transgenic plants was between 55 and 62, whereas the inhibition percentage in single-gene transformants (chimeric chitinase) ranged from 35 to 45 percent. Importantly, in greenhouse conditions, the double transgenic plants showed significant resistance than the single-gene transformant and wild type plants. The results in T2 generation using the intact leaf inoculation method showed that the average lesion diameters were 10, 14.7 and 29 mm for the double transformant, single-gene transformant and non-transgenic plants, respectively. Combined expression of chimeric chitinase and tlp in transgenic plants showed significantly enhanced resistance against S. sclerotiorum than the one that express single-gene transformant plants. These results suggest that the co-expression of chimeric chitinase and tlp can confer enhanced disease resistance in canola plant. PMID:27430511

  2. Efficacy of marker vaccine candidate CP7 E2alf in piglets with maternally derived C-strain antibodies

    DEFF Research Database (Denmark)

    Rangelova, Desislava Yordanova; Nielsen, Jens; Strandbygaard, Bertel;

    2012-01-01

    Marker vaccines offer the possibility to differentiate classical swine fever (CSF) infected from CSF vaccinated animals based on serology and their implementation will ensure free trade with pigs. Therefore, new generations of promising marker vaccines have been developed, among them the chimeric...... intramuscularly with C-strain vaccine 4 weeks before farrowing. Thus, these piglets were vaccinated intramuscularly with CP7_E2alf at the age of 5 or 8 weeks. Subsequently, the piglets and their mock-vaccinated littermate controls were challenged 2 weeks post vaccination with highly virulent Classical swine fever...

  3. Novel recombinant chimeric virus-like particle is immunogenic and protective against both enterovirus 71 and coxsackievirus A16 in mice

    OpenAIRE

    Hui Zhao; Hao-Yang Li; Jian-Feng Han; Yong-Qiang Deng; Shun-Ya Zhu; Xiao-Feng Li; Hui-Qin Yang; Yue-Xiang Li; Yu Zhang; E-De Qin; Rong Chen; Cheng-Feng Qin

    2015-01-01

    Hand-foot-and-mouth disease (HFMD) has been recognized as an important global public health issue, which is predominantly caused by enterovirus 71 (EV-A71) and coxsackievirus A16 (CVA16). There is no available vaccine against HFMD. An ideal HFMD vaccine should be bivalent against both EV-A71 and CVA16. Here, a novel strategy to produce bivalent HFMD vaccine based on chimeric EV-A71 virus-like particles (ChiEV-A71 VLPs) was proposed and illustrated. The neutralizing epitope SP70 within the cap...

  4. In silico analysis of chimeric espA, eae and tir fragments of Escherichia coli O157:H7 for oral immunogenic applications

    Directory of Open Access Journals (Sweden)

    Rafati Sima

    2009-12-01

    Full Text Available Abstract Background In silico techniques are highly suited for both the discovery of new and development of existing vaccines. Enterohemorrhagic Escherichia coli O157:H7 (EHEC exhibits a pattern of localized adherence to host cells, with the formation of microcolonies, and induces a specific histopathological lesion (attaching/effacing. The genes encoding the products responsible for this phenotype are clustered on a 35-kb pathogenicity island. Among these proteins, Intimin, Tir, and EspA, which are expressed by attaching-effacing genes, are responsible for the attachment to epithelial cell that leads to lesions. Results We designed synthetic genes encoding the carboxy-terminal fragment of Intimin, the middle region of Tir and the carboxy-terminal part of EspA. These multi genes were synthesized with codon optimization for a plant host and were fused together by the application of four repeats of five hydrophobic amino acids as linkers. The structure of the synthetic construct gene, its mRNA and deduced protein and their stabilities were analyzed by bioinformatic software. Furthermore, the immunogenicity of this multimeric recombinant protein consisting of three different domains was predicted. Conclusion a structural model for a chimeric gene from LEE antigenic determinants of EHEC is presented. It may define accessibility, solubility and immunogenecity.

  5. Protecting capacity against malaria of chemically defined tetramer forms based on the Plasmodium falciparum apical sushi protein as potential vaccine components.

    Science.gov (United States)

    Vanegas, Magnolia; Bermúdez, Adriana; Guerrero, Yuly Andrea; Cortes-Vecino, Jesús Alfredo; Curtidor, Hernando; Patarroyo, Manuel Elkin; Lozano, José Manuel

    2014-08-15

    Developing novel generations of subunit-based antimalarial vaccines in the form of chemically-defined macromolecule systems for multiple antigen presentation represents a classical problem in the field of vaccine development. Many efforts involving synthesis strategies leading to macromolecule constructs have been based on dendrimer-like systems, the condensation of large building blocks and conventional asymmetric double dimer constructs, all based on lysine cores. This work describes novel symmetric double dimer and condensed linear constructs for presenting selected peptide multi-copies from the apical sushi protein expressed in Plasmodium falciparum. These molecules have been proved to be safe and innocuous, highly antigenic and have shown strong protective efficacy in rodents challenged with two Plasmodium species. Insights into systematic design, synthesis and characterisation have led to such novel antigen systems being used as potential platforms for developing new anti-malarial vaccine candidates. PMID:25063026

  6. Enhanced mucosal immune responses induced by a combined candidate mucosal vaccine based on Hepatitis A virus and Hepatitis E virus structural proteins linked to tuftsin.

    Directory of Open Access Journals (Sweden)

    Yan Gao

    Full Text Available Hepatitis A virus (HAV and Hepatitis E virus (HEV are the most common causes of infectious hepatitis. These viruses are spread largely by the fecal-oral route and lead to clinically important disease in developing countries. To evaluate the potential of targeting hepatitis A and E infection simultaneously, a combined mucosal candidate vaccine was developed with the partial open reading frame 2 (ORF2 sequence (aa 368-607 of HEV (HE-ORF2 and partial virus protein 1 (VP1 sequence (aa 1-198 of HAV (HA-VP1, which included the viral neutralization epitopes. Tuftsin is an immunostimulatory peptide which can enhance the immunogenicity of a protein by targeting it to macrophages and dendritic cells. Here, we developed a novel combined protein vaccine by conjugating tuftsin to HE-ORF2 and HA-VP1 and used synthetic CpG oligodeoxynucleotides (ODNs as the adjuvant. Subsequent experiments in BALB/c mice demonstrated that tuftsin enhanced the serum-specific IgG and IgA antibodies against HEV and HAV at the intestinal, vaginal and pulmonary interface when delivered intranasally. Moreover, mice from the intranasally immunized tuftsin group (HE-ORF2-tuftsin + HA-VP1-tuftsin + CpG showed higher levels of IFN-γ-secreting splenocytes (Th1 response and ratio of CD4+/CD8+ T cells than those of the no-tuftsin group (HE-ORF2 + HA-VP1 + CpG. Thus, the tuftsin group generated stronger humoral and cellular immune responses compared with the no-tuftsin group. Moreover, enhanced responses to the combined protein vaccine were obtained by intranasal immunization compared with intramuscular injection. By integrating HE-ORF2, HA-VP1 and tuftsin in a vaccine, this study validated an important concept for further development of a combined mucosal vaccine against hepatitis A and E infection.

  7. Clinical and immunological evaluation of anti-apoptosis protein, survivin-derived peptide vaccine in phase I clinical study for patients with advanced or recurrent breast cancer

    Directory of Open Access Journals (Sweden)

    Asanuma Hiroko

    2008-05-01

    effectively than vaccination with the peptide alone, although neither vaccination could induce efficient clinical responses. Considering the above, the addition of another effectual adjuvant such as a cytokine, heat shock protein, etc. to the vaccination with survivin-2B peptide mixed with IFA might induce improved immunological and clinical responses.

  8. Incorporation of membrane-bound, mammalian-derived immunomodulatory proteins into influenza whole virus vaccines boosts immunogenicity and protection against lethal challenge

    Directory of Open Access Journals (Sweden)

    Roberts Paul C

    2009-04-01

    Full Text Available Abstract Background Influenza epidemics continue to cause morbidity and mortality within the human population despite widespread vaccination efforts. This, along with the ominous threat of an avian influenza pandemic (H5N1, demonstrates the need for a much improved, more sophisticated influenza vaccine. We have developed an in vitro model system for producing a membrane-bound Cytokine-bearing Influenza Vaccine (CYT-IVAC. Numerous cytokines are involved in directing both innate and adaptive immunity and it is our goal to utilize the properties of individual cytokines and other immunomodulatory proteins to create a more immunogenic vaccine. Results We have evaluated the immunogenicity of inactivated cytokine-bearing influenza vaccines using a mouse model of lethal influenza virus challenge. CYT-IVACs were produced by stably transfecting MDCK cell lines with mouse-derived cytokines (GM-CSF, IL-2 and IL-4 fused to the membrane-anchoring domain of the viral hemagglutinin. Influenza virus replication in these cell lines resulted in the uptake of the bioactive membrane-bound cytokines during virus budding and release. In vivo efficacy studies revealed that a single low dose of IL-2 or IL-4-bearing CYT-IVAC is superior at providing protection against lethal influenza challenge in a mouse model and provides a more balanced Th1/Th2 humoral immune response, similar to live virus infections. Conclusion We have validated the protective efficacy of CYT-IVACs in a mammalian model of influenza virus infection. This technology has broad applications in current influenza virus vaccine development and may prove particularly useful in boosting immune responses in the elderly, where current vaccines are minimally effective.

  9. Baculovirus as a PRRSV and PCV2 bivalent vaccine vector: baculovirus virions displaying simultaneously GP5 glycoprotein of PRRSV and capsid protein of PCV2.

    Science.gov (United States)

    Xu, Xin-Gang; Wang, Zhi-Sheng; Zhang, Qi; Li, Zhao-Cai; Ding, Li; Li, Wei; Wu, Hung-Yi; Chang, Ching-Dong; Lee, Long-Huw; Tong, De-Wen; Liu, Hung-Jen

    2012-02-01

    The GP5 glycoprotein of PRRSV is the main target for inducing neutralizing antibodies and protective immunity in the natural host. The capsid (Cap) protein is the major immunogenic protein and associated with the production of PCV2-specific neutralizing antibodies. In the present study, one genetic recombinant baculovirus BacSC-Dual-GP5-Cap was constructed. This virus displays simultaneously histidine-tagged GP5 and Cap proteins with the baculovirus glycoprotein gp64 TM and CTD on the virion surface as well as the surface of the virus-infected cells. After infection, the GP5 and Cap proteins were expressed and anchored simultaneously on the plasma membrane of Sf-9 cells, as revealed by Western blot and confocal microscopy. This report demonstrated first that both GP5 and Cap proteins were displayed successfully on the viral surface, revealed by immunogold electron microscopy. Vaccination of swine with recombinant baculovirus BacSC-Dual-GP5-Cap elicited significantly higher GP5 and Cap ELISA antibody titers in swine than the control groups. Virus neutralization test also showed that serum from the BacSC-Dual-GP5-Cap treated swine had significant levels of virus neutralization titers. Lymphocyte proliferation responses could be induced in swine immunized with BacSC-Dual-GP5-Cap than the control groups. These findings demonstrate that the BacSC-Dual-GP5-Cap bivalent subunit vaccine can be a potential vaccine against PRRSV and PCV2 infections. PMID:22172969

  10. Comparison of the immunogenicity and protection against bovine tuberculosis following immunization by BCG-priming and boosting with adenovirus or protein based vaccines.

    Science.gov (United States)

    Dean, G; Whelan, A; Clifford, D; Salguero, F J; Xing, Z; Gilbert, S; McShane, H; Hewinson, R G; Vordermeier, M; Villarreal-Ramos, B

    2014-03-01

    There is a requirement for vaccines or vaccination strategies that confer better protection against TB than the current live attenuated Mycobacterium bovis Bacillus Calmette-Guerin (BCG) vaccine for use in cattle. Boosting with recombinant viral vectors expressing mycobacterial proteins, such as Ag85A, has shown a degree of promise as a strategy for improving on the protection afforded by BCG. Experiments in small animal models have indicated that broadening the immune response to include mycobacterial antigens other than Ag85A, such as Rv0288, induced by boosting with Ad5 constructs has a direct effect on the protection afforded against TB. Here, we compared the immunogenicity and protection against challenge with M. bovis afforded by boosting BCG-vaccinated cattle with a human type 5 (Ad5)-based vaccine expressing the mycobacterial antigens Ag85A (Ad5-85A); or Ag85A, Rv0251, Rv0287 and Rv0288 (Ad5-TBF); or with protein TBF emulsified in adjuvant (Adj-TBF). Boosting with TBF broaden the immune response. The kinetics of Ad5-TBF and Adj-TBF were shown to be different, with effector T cell responses from the latter developing more slowly but being more durable than those induced by Ad5-TBF. No increase in protection compared to BCG alone was afforded by Ad5-TBF or Adj-TBF by gross pathology or bacteriology. Using histopathology, as a novel parameter of protection, we show that boosting BCG vaccinated cattle with Ad5-85A induced significantly better protection than BCG alone. PMID:24269321

  11. Secretion of dengue virus envelope protein ectodomain from mammalian cells is dependent on domain II serotype and affects the immune response upon DNA vaccination.

    Science.gov (United States)

    Slon Campos, J L; Poggianella, M; Marchese, S; Bestagno, M; Burrone, O R

    2015-11-01

    Dengue virus (DENV) is currently among the most important human pathogens and affects millions of people throughout the tropical and subtropical regions of the world. Although it has been a World Health Organization priority for several years, there is still no efficient vaccine available to prevent infection. The envelope glycoprotein (E), exposed on the surface on infective viral particles, is the main target of neutralizing antibodies. For this reason it has been used as the antigen of choice for vaccine development efforts. Here we show a detailed analysis of factors involved in the expression, secretion and folding of E ectodomain from all four DENV serotypes in mammalian cells, and how this affects their ability to induce neutralizing antibody responses in DNA-vaccinated mice. Proper folding of E domain II (DII) is essential for efficient E ectodomain secretion, with DIII playing a significant role in stabilizing soluble dimers. We also show that the level of protein secreted from transfected cells determines the strength and efficiency of antibody responses in the context of DNA vaccination and should be considered a pivotal feature for the development of E-based DNA vaccines against DENV. PMID:26358704

  12. Effect of Plasmodium yoelii YM Infection on Vaccination with 19 kDa Carboxylterminus of the Merozoite Surface Protein 1 (MSP1 19)

    Institute of Scientific and Technical Information of China (English)

    徐沪济; JiraprapaWIPASA; 刘雪琴; AnthonySTOWERS; 杨晓平; MichaelFGOOD

    2004-01-01

    We have previously demonstrated the ability of malaria parasites to interfere with specific immune responses. CD4 T cells specific to parasite antigens, but not CD4 T cells specific to an irrelevant antigen, ovalbumin (OVA), are deleted via apoptosis during malaria infection. It is of interest, therefore, to investigate the immune responses that developed following vaccination with the 19 kDa carboxylterminus of the merozoite surface protein 1 (MSP1 19) in mice that had previously experienced malaria infection. In this study, pre-exposure of mice to Plasmodium yoelii elicited native anti-MSP1 19 antibody responses, which could be boosted by vaccination with recombinant MSP1 19 ,Likewise, infection of MSP1 19-primed mice with Plasmodium yoelii (P. yoelii) led to an increase of anti-MSP1 19 antibodies. MSP1 19 vaccination of malaria preexposed mice or immunization by infection/cure of MSP1 19-primed mice enabled the mice to survive challenge infection, with the former group having slightly lower parasitaemia. The data suggest that exposure to malaria infection primes a natural immune response which can be boosted by vaccination. This information is relevant to the development of a vaccine for use in individuals living in malaria-endemic areas.

  13. High-Level Systemic Expression of Conserved Influenza Epitope in Plants on the Surface of Rod-Shaped Chimeric Particles

    Directory of Open Access Journals (Sweden)

    Natalia V. Petukhova

    2014-04-01

    Full Text Available Recombinant viruses based on the cDNA copy of the tobacco mosaic virus (TMV genome carrying different versions of the conserved M2e epitope from influenza virus A cloned into the coat protein (CP gene were obtained and partially characterized by our group previously; cysteines in the human consensus M2e sequence were changed to serine residues. This work intends to show some biological properties of these viruses following plant infections. Agroinfiltration experiments on Nicotiana benthamiana confirmed the efficient systemic expression of M2e peptides, and two point amino acid substitutions in recombinant CPs significantly influenced the symptoms and development of viral infections. Joint expression of RNA interference suppressor protein p19 from tomato bushy stunt virus (TBSV did not affect the accumulation of CP-M2e-ser recombinant protein in non-inoculated leaves. RT-PCR analysis of RNA isolated from either infected leaves or purified TMV-M2e particles proved the genetic stability of TMV‑based viral vectors. Immunoelectron microscopy of crude plant extracts demonstrated that foreign epitopes are located on the surface of chimeric virions. The rod‑shaped geometry of plant-produced M2e epitopes is different from the icosahedral or helical filamentous arrangement of M2e antigens on the carrier virus-like particles (VLP described earlier. Thereby, we created a simple and efficient system that employs agrobacteria and plant viral vectors in order to produce a candidate broad-spectrum flu vaccine.

  14. Phase 1/2a Trial of Plasmodium vivax Malaria Vaccine Candidate VMP001/AS01B in Malaria-Naive Adults: Safety, Immunogenicity, and Efficacy.

    Directory of Open Access Journals (Sweden)

    Jason W Bennett

    2016-02-01

    Full Text Available A vaccine to prevent infection and disease caused by Plasmodium vivax is needed both to reduce the morbidity caused by this parasite and as a key component in efforts to eradicate malaria worldwide. Vivax malaria protein 1 (VMP001, a novel chimeric protein that incorporates the amino- and carboxy- terminal regions of the circumsporozoite protein (CSP and a truncated repeat region that contains repeat sequences from both the VK210 (type 1 and the VK247 (type 2 parasites, was developed as a vaccine candidate for global use.We conducted a first-in-human Phase 1 dose escalation vaccine study with controlled human malaria infection (CHMI of VMP001 formulated in the GSK Adjuvant System AS01B. A total of 30 volunteers divided into 3 groups (10 per group were given 3 intramuscular injections of 15 μg, 30 μg, or 60 μg respectively of VMP001, all formulated in 500 μL of AS01B at each immunization. All vaccinated volunteers participated in a P. vivax CHMI 14 days following the third immunization. Six non-vaccinated subjects served as infectivity controls.The vaccine was shown to be well tolerated and immunogenic. All volunteers generated robust humoral and cellular immune responses to the vaccine antigen. Vaccination did not induce sterile protection; however, a small but significant delay in time to parasitemia was seen in 59% of vaccinated subjects compared to the control group. An association was identified between levels of anti-type 1 repeat antibodies and prepatent period.This trial was the first to assess the efficacy of a P. vivax CSP vaccine candidate by CHMI. The association of type 1 repeat-specific antibody responses with delay in the prepatency period suggests that augmenting the immune responses to this domain may improve strain-specific vaccine efficacy. The availability of a P. vivax CHMI model will accelerate the process of P. vivax vaccine development, allowing better selection of candidate vaccines for advancement to field trials.

  15. Use of a Th1 Stimulator Adjuvant for Vaccination against Neospora caninum Infection in the Pregnant Mouse Model

    Directory of Open Access Journals (Sweden)

    Denis Grandgirard

    2013-03-01

    Full Text Available Vertical transmission from an infected cow to its fetus accounts for the vast majority of new Neospora caninum infections in cattle. A vaccine composed of a chimeric antigen named recNcMIC3-1-R, based on predicted immunogenic domains of the two microneme proteins NcMIC1 and NcMIC3, the rhoptry protein NcROP2, and emulsified in saponin adjuvants, significantly reduced the cerebral infection in non-pregnant BALB/c mice. Protection was associated with a mixed Th1/Th2-type cytokine response. However, the same vaccine formulation elicited a Th2-type immune response in pregnant mice and did not prevent vertical transmission or disease, neither in dams nor in offspring mice. In this study, an alternative vaccine formulation containing recNcMIC3-1-R emulsified in Freund’s incomplete adjuvant, a stimulator of the cellular immunity, was investigated. No protection against vertical transmission and cerebral infection in the pregnant mice and a very limited protective effect in the non-pregnant mice were observed. The vaccine induced a Th1-type immune response characterized by high IgG2a titres and strong IFN-γ expression, which appeared detrimental to pregnancy.

  16. Performance Assessment of Four Chimeric Trypanosoma cruzi Antigens Based on Antigen-Antibody Detection for Diagnosis of Chronic Chagas Disease.

    Science.gov (United States)

    Santos, Fred Luciano Neves; Celedon, Paola Alejandra Fiorani; Zanchin, Nilson Ivo Tonin; Brasil, Tatiana de Arruda Campos; Foti, Leonardo; Souza, Wayner Vieira de; Silva, Edmilson Domingos; Gomes, Yara de Miranda; Krieger, Marco Aurélio

    2016-01-01

    The performance of serologic tests in chronic Chagas disease diagnosis largely depends on the type and quality of the antigen preparations that are used for detection of anti-Trypanosoma cruzi antibodies. Whole-cell T. cruzi extracts or recombinant proteins have shown variation in the performance and cross-reactivity. Synthetic chimeric proteins comprising fragments of repetitive amino acids of several different proteins have been shown to improve assay performances to detect Chagasic infections. Here, we describe the production of four chimeric T. cruzi proteins and the assessment of their performance for diagnostic purposes. Circular Dichroism spectra indicated the absence of well-defined secondary structures, while polydispersity evaluated by Dynamic Light Scattering revealed only minor aggregates in 50 mM carbonate-bicarbonate (pH 9.6), demonstrating that it is an appropriate buffering system for sensitizing microplates. Serum samples from T. cruzi-infected and non-infected individuals were used to assess the performance of these antigens for detecting antibodies against T. cruzi, using both enzyme-linked immunosorbent assay and a liquid bead array platform. Performance parameters (AUC, sensitivity, specificity, accuracy and J index) showed high diagnostic accuracy for all chimeric proteins for detection of specific anti-T. cruzi antibodies and differentiated seropositive individuals from those who were seronegative. Our data suggest that these four chimeric proteins are eligible for phase II studies. PMID:27517281

  17. Evidence for transcript networks composed of chimeric RNAs in human cells.

    Science.gov (United States)

    Djebali, Sarah; Lagarde, Julien; Kapranov, Philipp; Lacroix, Vincent; Borel, Christelle; Mudge, Jonathan M; Howald, Cédric; Foissac, Sylvain; Ucla, Catherine; Chrast, Jacqueline; Ribeca, Paolo; Martin, David; Murray, Ryan R; Yang, Xinping; Ghamsari, Lila; Lin, Chenwei; Bell, Ian; Dumais, Erica; Drenkow, Jorg; Tress, Michael L; Gelpí, Josep Lluís; Orozco, Modesto; Valencia, Alfonso; van Berkum, Nynke L; Lajoie, Bryan R; Vidal, Marc; Stamatoyannopoulos, John; Batut, Philippe; Dobin, Alex; Harrow, Jennifer; Hubbard, Tim; Dekker, Job; Frankish, Adam; Salehi-Ashtiani, Kourosh; Reymond, Alexandre; Antonarakis, Stylianos E; Guigó, Roderic; Gingeras, Thomas R

    2012-01-01

    The classic organization of a gene structure has followed the Jacob and Monod bacterial gene model proposed more than 50 years ago. Since then, empirical determinations of the complexity of the transcriptomes found in yeast to human has blurred the definition and physical boundaries of genes. Using multiple analysis approaches we have characterized individual gene boundaries mapping on human chromosomes 21 and 22. Analyses of the locations of the 5' and 3' transcriptional termini of 492 protein coding genes revealed that for 85% of these genes the boundaries extend beyond the current annotated termini, most often connecting with exons of transcripts from other well annotated genes. The biological and evolutionary importance of these chimeric transcripts is underscored by (1) the non-random interconnections of genes involved, (2) the greater phylogenetic depth of the genes involved in many chimeric interactions, (3) the coordination of the expression of connected genes and (4) the close in vivo and three dimensional proximity of the genomic regions being transcribed and contributing to parts of the chimeric RNAs. The non-random nature of the connection of the genes involved suggest that chimeric transcripts should not be studied in isolation, but together, as an RNA network. PMID:22238572

  18. Loss of sialic acid binding domain redirects protein σ1 to enhance M cell-directed vaccination.

    Directory of Open Access Journals (Sweden)

    Dagmara Zlotkowska

    Full Text Available Ovalbumin (OVA genetically fused to protein sigma 1 (pσ1 results in tolerance to both OVA and pσ1. Pσ1 binds in a multi-step fashion, involving both protein- and carbohydrate-based receptors. To assess the relative pσ1 components responsible for inducing tolerance and the importance of its sialic binding domain (SABD for immunization, modified OVA-pσ1, termed OVA-pσ1(short, was deleted of its SABD, but with its M cell targeting moiety intact, and was found to be immunostimulatory and enhanced CD4(+ and CD8(+ T cell proliferation. When used to nasally immunize mice given with and without cholera toxin (CT adjuvant, elevated SIgA and serum IgG responses were induced, and OVA-pσ1(s was more efficient for immunization than native OVA+CT. The immune antibodies (Abs were derived from elevated Ab-forming cells in the upper respiratory tissues and submaxillary glands and were supported by mixed Th cell responses. Thus, these studies show that pσ1(s can be fused to vaccines to effectively elicit improved SIgA responses.

  19. Selection of glutamate-rich protein long synthetic peptides for vaccine development: antigenicity and relationship with clinical protection and immunogenicity.

    Science.gov (United States)

    Theisen, M; Dodoo, D; Toure-Balde, A; Soe, S; Corradin, G; Koram, K K; Kurtzhals, J A; Hviid, L; Theander, T; Akanmori, B; Ndiaye, M; Druilhe, P

    2001-09-01

    Antibodies against three long synthetic peptides (LSPs) derived from the glutamate-rich protein (GLURP) of Plasmodium falciparum were analyzed in three cohorts from Liberia, Ghana, and Senegal. Two overlapping LSPs, LR67 and LR68, are derived from the relatively conserved N-terminal nonrepeat region (R0), and the third, LR70, is derived from the R2 repeat region. A high prevalence of antibody responses to each LSP was observed in all three areas of endemic infection. Levels of cytophilic immunoglobulin G (IgG) antibodies against both GLURP regions were significantly correlated with protection from clinical P. falciparum malaria. Protected children from the Ghana cohort possessed predominantly IgG1 antibodies against the nonrepeat epitope and IgG3 antibodies against the repeat epitope. T-cell proliferation responses, studied in the cohort from Senegal, revealed that T-helper-cell epitopes were confined to the nonrepeat region. When used as immunogens, the LR67 and LR68 peptides elicited strong IgG responses in outbred mice and LR67 also induced antibodies in mice of different H-2 haplotypes, confirming the presence of T-helper-cell epitopes in these constructs. Mouse antipeptide antisera recognized parasite proteins as determined by immunofluorescence and immunoblotting. This indicates that synthetic peptides derived from relatively conserved epitopes of GLURP might serve as useful immunogens for vaccination against P. falciparum malaria. PMID:11500389

  20. Oral Vaccination with the Porcine Rotavirus VP4 Outer Capsid Protein Expressed by Lactococcus lactis Induces Specific Antibody Production

    Directory of Open Access Journals (Sweden)

    Yi-jing Li

    2010-01-01

    Full Text Available The objective of this study to design a delivery system resistant to the gastrointestinal environment for oral vaccine against porcine rotavirus. Lactococcus lactis NZ9000 was transformed with segments of vP4 of the porcine rotavirus inserted into the pNZ8112 surface-expression vector, and a recombinant L. lactis expressing VP4 protein was constructed. An approximately 27 kDa VP4 protein was confirmed by SDS-PAGE , Western blot and immunostaining analysis. BALB/c mice were immunized orally with VP4-expression recombinant L. lactis and cellular, mucosal and systemic humoral immune responses were examined. Specific anti-VP4 secretory IgA and IgG were found in feces, ophthalmic and vaginal washes and in serum. The induced antibodies demonstrated neutralizing effects on porcine rotavirus infection on MA104 cells. Our findings suggest that oral immunization with VP4-expressing L. lactis induced both specific local and systemic humoral and cellular immune responses in mice.

  1. Expression of rabies glycoprotein and ricin toxin B chain (RGP-RTB) fusion protein in tomato hairy roots: a step towards oral vaccination for rabies.

    Science.gov (United States)

    Singh, Ankit; Srivastava, Subhi; Chouksey, Ankita; Panwar, Bhupendra Singh; Verma, Praveen C; Roy, Sribash; Singh, Pradhyumna K; Saxena, Gauri; Tuli, Rakesh

    2015-04-01

    Transgenic hairy roots of Solanum lycopersicum were engineered to express a recombinant protein containing a fusion of rabies glycoprotein and ricin toxin B chain (rgp-rtxB) antigen under the control of constitutive CaMV35S promoter. Asialofetuin-mediated direct ELISA of transgenic hairy root extracts was performed using polyclonal anti-rabies antibodies (Ab1) and epitope-specific peptidal anti-RGP (Ab2) antibodies which confirmed the expression of functionally viable RGP-RTB fusion protein. Direct ELISA based on asialofetuin-binding activity was used to screen crude protein extracts from five transgenic hairy root lines. Expressions of RGP-RTB fusion protein in different tomato hairy root lines varied between 1.4 and 8 µg in per gram of tissue. Immunoblotting assay of RGP-RTB fusion protein from these lines showed a protein band on monomeric size of ~84 kDa after denaturation. Tomato hairy root line H03 showed highest level of RGP-RTB protein expression (1.14 %) and was used further in bench-top bioreactor for the optimization of scale-up process to produce large quantity of recombinant protein. Partially purified RGP-RTB fusion protein was able to induce the immune response in BALB/c mice after intra-mucosal immunization. In the present investigation, we have not only successfully scaled up the hairy root culture but also established the utility of this system to produce vaccine antigen which subsequently will reduce the total production cost for implementing rabies vaccination programs in developing nations. This study in a way aims to provide consolidated base for low-cost preparation of improved oral vaccine against rabies. PMID:25519901

  2. Enhanced Th1-biased immune efficacy of porcine circovirus type 2 Cap-protein-based subunit vaccine when coadministered with recombinant porcine IL-2 or GM-CSF in mice.

    Science.gov (United States)

    Wang, Yiping; Lu, Yuehua; Liu, Dan; Wei, Yanwu; Guo, Longjun; Wu, Hongli; Huang, Liping; Liu, Jianbo; Liu, Changming

    2015-02-01

    Porcine circovirus type 2 (PCV2) capsid (Cap) protein is the primary protective antigen responsible for inducing PCV2-specific protective immunity, so it is a desirable target for the development of recombinant subunit vaccines to prevent PCV2-associated diseases. Interleukin 2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF), used as immune adjuvants, have been shown to enhance the immunogenicity of certain antigens or vaccines in various experimental models. In this study, five different subunit vaccines (the PCV2-Cap, Cap-PoIL-2, PCV2-Cap + PoIL-2, Cap-PoGM-CSF, and PCV2-Cap + PoGM-CSF vaccines) were prepared based on baculovirus-expressed recombinant proteins. The immunogenicity of these vaccines was evaluated to identify the immunoenhancement by PoIL-2 and PoGM-CSF of the Cap-protein-based PCV2 subunit vaccine in mice. The PCV2-Cap + PoIL-2, Cap-PoGM-CSF, PCV2-Cap + PoGM-CSF, and PCV2-Cap vaccines induced significantly higher levels of PCV2-specific antibodies than the Cap-PoIL-2 vaccine, whereas there was no apparent difference between these four vaccines. Our results indicate that neither PoIL-2 nor PoGM-CSF had effect on the enhancement of the humoral immunity induced by the PCV2-Cap vaccine. Furthermore, the PCV2-Cap + PoIL-2, Cap-PoGM-CSF, and PCV2-Cap + PoGM-CSF vaccines elicited stronger lymphocyte proliferative responses and greater IL-2 and interferon gamma (IFN-γ) secretion. This suggests that PoIL-2 and PoGM-CSF substantially augmented the Th1-biased immune response to the PCV2-Cap vaccine. Following challenge, the viral loads in the lungs of the PCV2-Cap + PoIL-2-, Cap-PoGM-CSF-, and PCV2-Cap + PoGM-CSF-treated groups were dramatically lower than those in the Cap-PoIL-2- and PCV2-Cap-treated groups, indicating that the three vaccines induced stronger protective effects against challenge. These findings show that PoIL-2 and PoGM-CSF essentially enhanced the Th1-biased protective efficacy of the

  3. Control of tick infestations in cattle vaccinated with bacterial membranes containing surface-exposed tick protective antigens.

    Science.gov (United States)

    Almazán, Consuelo; Moreno-Cantú, Orlando; Moreno-Cid, Juan A; Galindo, Ruth C; Canales, Mario; Villar, Margarita; de la Fuente, José

    2012-01-01

    Vaccines containing the Rhipicephalus (Boophilus) microplus BM86 and BM95 antigens protect cattle against tick infestations. Tick subolesin (SUB), elongation factor 1a (EF1a) and ubiquitin (UBQ) are new candidate protective antigens for the control of cattle tick infestations. Previous studies showed that R. microplus BM95 immunogenic peptides fused to the Anaplasma marginale major surface protein (MSP) 1a N-terminal region (BM95-MSP1a) for presentation on the Escherichia coli membrane were protective against R. microplus infestations in rabbits. In this study, we extended these results by expressing SUB-MSP1a, EF1a-MSP1a and UBQ-MSP1a fusion proteins on the E. coli membrane using this system and demonstrating that bacterial membranes containing the chimeric proteins BM95-MSP1a and SUB-MSP1a were protective (>60% vaccine efficacy) against experimental R. microplus and Rhipicephalus annulatus infestations in cattle. This system provides a novel, simple and cost-effective approach for the production of tick protective antigens by surface display of antigenic protein chimera on the E. coli membrane and demonstrates the possibility of using recombinant bacterial membrane fractions in vaccine preparations to protect cattle against tick infestations. PMID:22085549

  4. A Synthetic Virus-Like Particle Streptococcal Vaccine Candidate Using B-Cell Epitopes from the Proline-Rich Region of Pneumococcal Surface Protein A

    Directory of Open Access Journals (Sweden)

    Marco Tamborrini

    2015-10-01

    Full Text Available Alternatives to the well-established capsular polysaccharide-based vaccines against Streptococcus pneumoniae that circumvent limitations arising from limited serotype coverage and the emergence of resistance due to capsule switching (serotype replacement are being widely pursued. Much attention is now focused on the development of recombinant subunit vaccines based on highly conserved pneumococcal surface proteins and virulence factors. A further step might involve focusing the host humoral immune response onto protective protein epitopes using as immunogens structurally optimized epitope mimetics. One approach to deliver such epitope mimetics to the immune system is through the use of synthetic virus-like particles (SVLPs. SVLPs are made from synthetic coiled-coil lipopeptides that are designed to spontaneously self-assemble into 20–30 nm diameter nanoparticles in aqueous buffer. Multivalent display of epitope mimetics on the surface of SVLPs generates highly immunogenic nanoparticles that elicit strong epitope-specific humoral immune responses without the need for external adjuvants. Here, we set out to demonstrate that this approach can yield vaccine candidates able to elicit a protective immune response, using epitopes derived from the proline-rich region of pneumococcal surface protein A (PspA. These streptococcal SVLP-based vaccine candidates are shown to elicit strong humoral immune responses in mice. Following active immunization and challenge with lethal doses of streptococcus, SVLP-based immunogens are able to elicit significant protection in mice. Furthermore, a mimetic-specific monoclonal antibody is shown to mediate partial protection upon passive immunization. The results show that SVLPs combined with synthetic epitope mimetics may have potential for the development of an effective vaccine against Streptococcus pneumoniae.

  5. Outer Membrane Proteins of Brucella abortus Vaccinal and Field Strains and their Immune Response in Buffaloes

    OpenAIRE

    Rukhshanda Munir*, M. Afzal1, M. Hussain2, S. M. S. Naqvi3 and A. Khanum3

    2010-01-01

    Outer membrane proteins (OMPs) of three strains of B. abortus i.e. S19, RB51 and a local field isolate of biotype 1 were isolated through disrupting cells to generate membranes by centrifugation and sodium lauryl sarcosinate solubilisation of inner membrane proteins. Distinct OMP profiles of each strain were seen on SDS-PAGE. SDS-PAGE analysis of S19 and field isolate revealed eight protein bands in each strain. The OMPs of S19 had molecular masses 89.0, 73.0, 53.7, 49.0, 38.0, 27.0, 22.3, a...

  6. Current status and future prospects of yellow fever vaccines.

    Science.gov (United States)

    Beck, Andrew S; Barrett, Alan D T

    2015-11-01

    Yellow fever 17D vaccine is one of the oldest live-attenuated vaccines in current use that is recognized historically for its immunogenic and safe properties. These unique properties of 17D are presently exploited in rationally designed recombinant vaccines targeting not only flaviviral antigens but also other pathogens of public health concern. Several candidate vaccines based on 17D have advanced to human trials, and a chimeric recombinant Japanese encephalitis vaccine utilizing the 17D backbone has been licensed. The mechanism(s) of attenuation for 17D are poorly understood; however, recent insights from large in silico studies have indicated particular host genetic determinants contributing to the immune response to the vaccine, which presumably influences the considerable durability of protection, now in many cases considered to be lifelong. The very rare occurrence of severe adverse events for 17D is discussed, including a recent fatal case of vaccine-associated viscerotropic disease. PMID:26366673

  7. Vaccines against leptospirosis.

    Science.gov (United States)

    Adler, Ben

    2015-01-01

    Vaccines against leptospirosis followed within a year of the first isolation of Leptospira, with the first use of a killed whole cell bacterin vaccine in guinea pigs published in 1916. Since then, bacterin vaccines have been used in humans, cattle, swine, and dogs and remain the only vaccines licensed at the present time. The immunity elicited is restricted to serovars with related lipopolysaccharide (LPS) antigen. Likewise, vaccines based on LPS antigens have clearly demonstrated protection in animal models, which is also at best serogroup specific. The advent of leptospiral genome sequences has allowed a reverse vaccinology approach for vaccine development. However, the use of inadequate challenge doses and inappropriate statistical analysis invalidates many of the claims of protection with recombinant proteins. PMID:25388138

  8. Structural characterization of low molecular weight polysaccharide from Astragalus membranaceus and its immunologic enhancement in recombinant protein vaccine against systemic candidiasis.

    Science.gov (United States)

    Yang, Fan; Xiao, Chunyu; Qu, Jing; Wang, Guiyun

    2016-07-10

    Structure and immunologic enhancement of low molecular weight polysaccharide (LMW-ASP) isolated from the root of Astragalus membranaceus (Fisch) Bge. Were detected in recombinant protein vaccine. Structure analysis of LMW-ASP revealed that LMW-ASP (Mw=5.6kDa) was an acid heteropolysaccharide, which consisted of Glc, Gal, Ara, Xyl and GalA in ratio of 10.0:1.3:1.7:1.0:0.9. Recombinant protein (rP-HSP90C) contained epitope C (LKVIRK) from heat shock protein 90 (HSP90) of Candida albicans was used as a vaccine. The results indicated that LMW-ASP significantly promoted specific antibody titers IgG, IgG1, IgG2b, and IL-2, IL-4, IL-10, IL-12 in sera of mice immunized with rP-HSP90C (p<0.05). It was also found LMW-ASP improved DTH response in HSP90C-injceted mice. More importantly, the mice immunized with rP-HSP90C/LMW-ASP had fewer CFU (colony forming unites) in the kidneys compared to the mice immunized with rP-HSP90C (p<0.05). Therefore, LMW-ASP could be exploited into the novel adjuvant to enhance the efficacy of recombinant protein vaccine. PMID:27106150

  9. FACIAL EXPRESSION RECOGNITION WITH THE USE OF CHIMERIC FACE TECHNIQUE

    OpenAIRE

    Menshikova, Galina

    2010-01-01

    The aim of this study was to investigate holistic / feature processing for encoding face expressions employing the chimeric face technique. In the course of our experiment we tested the recognition accuracy of universal and chimeric countenance. As the study has revealed there was a considerable difference between distributions of subject responses depending on the localization of expression features (top / bottom parts of the face). For chimeric face identification accuracy substantially dec...

  10. Vaccination with the Defined Chlamydial Secreted Protein CPAF Induces Robust Protection Against Female Infertility Following Repeated Genital Chlamydial Challenge

    OpenAIRE

    Murthy, Ashlesh K.; Li, Weidang; Guentzel, M. Neal; Zhong, Guangming; Arulanandam, Bernard P.

    2011-01-01

    We previously have shown the efficacy of recombinant (r) chlamydial protease-like activity factor (CPAF) vaccination against hydrosalpinx development following primary genital chlamydial challenge. In this study, we evaluated further the protection induced by rCPAF vaccination against infertility. Following primary challenge, fertility levels were not significantly different between the mock- and CPAF-vaccinated and Chlamydia alone challenged mice. However, following secondary genital chlamyd...

  11. A New Strategy Based on Smrho Protein Loaded Chitosan Nanoparticles as a Candidate Oral Vaccine against Schistosomiasis

    OpenAIRE

    Oliveira, Carolina R.; Rezende, Cíntia M. F.; Silva, Marina R.; Ana Paula Pêgo; Olga Borges; Alfredo M. Goes

    2012-01-01

    BACKGROUND: Schistosomiasis is one of the most important neglected tropical diseases and an effective control is unlikely in the absence of improved sanitation and vaccination. A new approach of oral vaccination with alginate coated chitosan nanoparticles appears interesting because their great stability and the ease of target accessibility, besides of chitosan and alginate immunostimulatory properties. Here we propose a candidate vaccine based on the combination of chitosan-based nanoparticl...

  12. Immunogenic multistage recombinant protein vaccine confers partial protection against experimental toxoplasmosis mimicking natural infection in murine model

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    Yaprak Gedik

    2016-01-01

    To generate a protective vaccine against toxoplasmosis, multistage vaccines and usage of challenging models mimicking natural route of infection are critical cornerstones. In this study, we generated a BAG1 and GRA1 multistage vaccine that induced strong immune response in which the protection was not at anticipated level. In addition, the murine model was orally challenged with tissue cysts to mimic natural route of infection.

  13. Vesicular Stomatitis Virus glycoprotein G carrying a tandem dimer of Foot and Mouth Disease Virus antigenic site A can be used as DNA and peptide vaccine for cattle.

    Science.gov (United States)

    Capozzo, Alejandra V; Wilda, Maximiliano; Bucafusco, Danilo; de los Ángeles Lavoria, María; Franco-Mahecha, Olga L; Mansilla, Florencia C; Pérez-Filgueira, Daniel M; Grigera, Pablo R

    2011-11-01

    Effective Foot and Mouth Disease Virus (FMDV) peptide vaccines for cattle have two major constraints: resemblance of one or more of the multiple conformations of the major VP1 antigenic sites to induce neutralizing antibodies, and stimulation of T cells despite the variable bovine-MHC polymorphism. To overcome these limitations, a chimeric antigen was developed, using Vesicular Stomatitis Virus glycoprotein (VSV-G) as carrier protein of an in tandem-dimer of FMDV antigenic site A (ASA), the major epitope on the VP1 capsid protein (aa 139-149, FMDV-C3 serotype). The G-ASA construct was expressed in the Baculovirus system to produce a recombinant protein (DEL BAC) (cloned in pCDNA 3.1 plasmid) (Invitrogen Corporation, Carlsbad, CA) and was also prepared as a DNA vaccine (pC DEL). Calves vaccinated with both immunogens elicited antibodies that recognized the ASA in whole virion and were able to neutralize FMDV infectivity in vitro. After two vaccine doses, DEL BAC induced serum neutralizing titers compatible with an "expected percentage of protection" above 90%. Plasmid pC DEL stimulated FMDV specific humoral responses earlier than DEL BAC, though IgG1 to IgG2 ratios were lower than those induced by both DEL BAC and inactivated FMDV-C3 after the second dose. DEL BAC induced FMDV-specific secretion of IFN-γ in peripheral blood mononuclear cells of outbred cattle immunized with commercial FMDV vaccine, suggesting its capacity to recall anamnestic responses mediated by functional T cell epitopes. The results show that exposing FMDV-VP1 major neutralizing antigenic site in the context of N-terminal sequences of the VSV G protein can overcome the immunological limitations of FMDV-VP1 peptides as effective protein and DNA vaccines for cattle. PMID:21889542

  14. Ingredients of Vaccines

    Science.gov (United States)

    ... is packaged. Monosodium glutamate ( MSG ) and 2-phenoxy-ethanol which are used as stabilizers in a few ... mercury, aluminum, formaldehyde, human serum albumin, antibiotics, and yeast proteins in vaccines have not been found to ...

  15. Mutations in GTP binding protein Obg of Mycoplasma synoviae vaccine strain MS-H: implications in temperature-sensitivity phenotype.

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    Muhammad A Shahid

    Full Text Available Mycoplasma synoviae strain MS-H, developed by chemical mutagenesis of the Australian field strain 86079/7NS, is a live temperature-sensitive (ts (+ vaccine used for control of M. synoviae infection in poultry worldwide. Genetic basis of temperature sensitivity and attenuation of MS-H has not been revealed thus far. Comparison of the complete genome sequence of MS-H, its parent strain 86079/7NS and two non-temperature sensitive (ts (- reisolates of MS-H revealed a mutation in a highly conserved domain of GTP binding protein Obg of MS-H, with reversion in ts (- MS-H reisolates. Nucleotide change from G to A at position 369 of the obg gene resulted in an alteration of glycine to arginine at position 123 in Obg fold. Further analysis of the complete obg gene sequence in several MS-H reisolates revealed that a Gly123Arg substitution was associated with alteration in temperature sensitivity phenotype of MS-H. A second mutation, C to T at position 629, in obg gene was found in some of the MS-H reisolates and appeared to suppress the effects of the Gly123Arg substitution. In silico analysis of point mutations revealed that Gly123Arg has highly destabilizing effect on the MS-H Obg structure that can potentially abolish its biological functions in vivo especially at non-permissive temperature. Findings of this study implicate Obg alteration (Gly123Arg as one of the possible causes of MS-H attenuation/temperature sensitivity and warrant further investigations into exploring the role of Obg-like proteins, an evolutionarily conserved protein from human to bacteria, in the biology of mycoplasmas.

  16. Improving the MVA vaccine potential by deleting the viral gene coding for the IL-18 binding protein.

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    Juliana Falivene

    Full Text Available BACKGROUND: Modified Vaccinia Ankara (MVA is an attenuated strain of Vaccinia virus (VACV currently employed in many clinical trials against HIV/AIDS and other diseases. MVA still retains genes involved in host immune response evasion, enabling its optimization by removing some of them. The aim of this study was to evaluate cellular immune responses (CIR induced by an IL-18 binding protein gene (C12L deleted vector (MVAΔC12L. METHODOLOGY/PRINCIPAL FINDINGS: BALB/c and C57BL/6 mice were immunized with different doses of MVAΔC12L or MVA wild type (MVAwt, then CIR to VACV epitopes in immunogenic proteins were evaluated in spleen and draining lymph nodes at acute and memory phases (7 and 40 days post-immunization respectively. Compared with parental MVAwt, MVAΔC12L immunization induced a significant increase of two to three-fold in CD8(+ and CD4(+ T-cell responses to different VACV epitopes, with increased percentage of anti-VACV cytotoxic CD8(+ T-cells (CD107a/b(+ during the acute phase of the response. Importantly, the immunogenicity enhancement was also observed after MVAΔC12L inoculation with different viral doses and by distinct routes (systemic and mucosal. Potentiation of MVA's CIR was also observed during the memory phase, in correlation with a higher protection against an intranasal challenge with VACV WR. Of note, we could also show a significant increase in the CIR against HIV antigens such as Env, Gag, Pol and Nef from different subtypes expressed from two recombinants of MVAΔC12L during heterologous DNA prime/MVA boost vaccination regimens. CONCLUSIONS/SIGNIFICANCE: This study demonstrates the relevance of IL-18 bp contribution in the immune response evasion during MVA infection. Our findings clearly show that the deletion of the viral IL-18 bp gene is an effective approach to increase MVA vaccine efficacy, as immunogenicity improvements were observed against vector antigens and more importantly to HIV antigens.

  17. Vaccine process technology.

    Science.gov (United States)

    Josefsberg, Jessica O; Buckland, Barry

    2012-06-01

    The evolution of vaccines (e.g., live attenuated, recombinant) and vaccine production methods (e.g., in ovo, cell culture) are intimately tied to each other. As vaccine technology has advanced, the methods to produce the vaccine have advanced and new vaccine opportunities have been created. These technologies will continue to evolve as we strive for safer and more immunogenic vaccines and as our understanding of biology improves. The evolution of vaccine process technology has occurred in parallel to the remarkable growth in the development of therapeutic proteins as products; therefore, recent vaccine innovations can leverage the progress made in the broader biotechnology industry. Numerous important legacy vaccines are still in use today despite their traditional manufacturing processes, with further development focusing on improving stability (e.g., novel excipients) and updating formulation (e.g., combination vaccines) and delivery methods (e.g., skin patches). Modern vaccine development is currently exploiting a wide array of novel technologies to create safer and more efficacious vaccines including: viral vectors produced in animal cells, virus-like particles produced in yeast or insect cells, polysaccharide conjugation to carrier proteins, DNA plasmids produced in E. coli, and therapeutic cancer vaccines created by in vitro activation of patient leukocytes. Purification advances (e.g., membrane adsorption, precipitation) are increasing efficiency, while innovative analytical methods (e.g., microsphere-based multiplex assays, RNA microarrays) are improving process understanding. Novel adjuvants such as monophosphoryl lipid A, which acts on antigen presenting cell toll-like receptors, are expanding the previously conservative list of widely accepted vaccine adjuvants. As in other areas of biotechnology, process characterization by sophisticated analysis is critical not only to improve yields, but also to determine the final product quality. From a regulatory

  18. A high-throughput method for quantifying alleles and haplotypes of the malaria vaccine candidate Plasmodium falciparum merozoite surface protein-1 19 kDa

    Directory of Open Access Journals (Sweden)

    Thera Mahamadou A

    2006-04-01

    Full Text Available Abstract Background Malaria vaccine efficacy may be compromised if the frequency of non-target alleles increases following vaccination with a genetically polymorphic target. Methods are needed to monitor genetic diversity in polymorphic vaccine antigens, but determining which genetic variants of such antigens are present in infected individuals is complicated by the frequent occurrence of mixed infections. Methods Pyrosequencing was used to determine allele frequencies at each of six single nucleotide polymorphisms in the Plasmodium falciparum blood-stage vaccine antigen merozoite surface protein 1 19 kDa (MSP-119 in field samples from a vaccine-testing site in Mali. Mixtures of MSP-119 clones were created to validate a haplotype-estimating algorithm that uses maximum likelihood methods to determine the most probable combination of haplotypes given the allele frequencies for an infection and the haplotypes known to be circulating in the population. Results Fourteen unique MSP-119 haplotypes were identified among 351 genotyped infections. After adjustment to a standard curve, Pyrosequencing provided accurate and precise estimates of allele frequencies in mixed infections. The haplotype-estimating algorithm provided accurate estimates of haplotypes in mixed infections containing up to three haplotypes. Based on the MSP-119 locus, approximately 90% of the 351 infections contained two or fewer haplotypes. Conclusion Pyrosequencing in conjunction with a haplotype-estimating algorithm provides accurate estimates of haplotypes present in infections with up to 3 haplotypes, and can be used to monitor genetic diversity in parasite populations prior to and following introduction of MSP-1-based malaria vaccines.

  19. Phase I clinical study of anti-apoptosis protein, survivin-derived peptide vaccine therapy for patients with advanced or recurrent colorectal cancer

    Directory of Open Access Journals (Sweden)

    Minamida Hidetoshi

    2004-06-01

    Full Text Available Abstract Survivin is a member of the inhibitor of apoptosis protein (IAP family containing a single baculovirus IAP repeat domain. It is expressed during fetal development but becomes undetectable in terminally differentiated normal adult tissues. We previously reported that survivin and its splicing variant survivin-2B was expressed abundantly in various types of tumor tissues as well as tumor cell lines and was suitable as a target antigen for active-specific anti-cancer immunization. Subsequently, we identified an HLA-A24-restricted antigenic peptide, survivin-2B80-88 (AYACNTSTL recognized by CD8+ cytotoxic T lymphocytes (CTLs. We, therefore, started a phase I clinical study assessing the efficacy of survivin-2B peptide vaccination in patients with advanced or recurrent colorectal cancer expressing survivin. Vaccinations with survivin-2B peptide were given subcutaneously six times at 14-day intervals. Of 15 patients who finished receiving the vaccination schedule, three suffered slight toxicities, including anemia (grade 2, general malaise (grade 1, and fever (grade 1. No severe adverse events were observed in any patient. In 6 patients, tumor marker levels (CEA and CA19-9 decreased transiently during the period of vaccination. Slight reduction of the tumor volume was observed in one patient, which was considered a minor responder. No changes were noted in three patients while the remaining eleven patients experienced tumor progression. Analysis of peripheral blood lymphocytes of one patient using HLA-A24/peptide tetramers revealed an increase in peptide-specific CTL frequency from 0.09% to 0.35% of CD8+ T cells after 4 vaccinations. This phase I clinical study indicates that survivin-2B peptide-based vaccination is safe and should be further considered for potential immune and clinical efficacy in HLA-A24-expression patients with colorectal cancer.

  20. Vaccination of Goats with 31 kDa and 32 kDa Schistosoma japonicum Antigens by DNA Priming and Protein Boosting

    Institute of Scientific and Technical Information of China (English)

    Lianfei Tang; Zhijun Zhou; Yuxiao Chen; Yonghui Luo; Linqian Wang; Liyu Chen; Fushen Huang; Xianfang Zeng; Xinyuan Yi

    2007-01-01

    Two Schistosoma japonicum vaccine candidate antigens Sj 31 and Sj 32, which have shown particular promise to induce protective immunity in mice, were used to immunize goats by using a DNA priming-protein boosting strategy in present work. DNA vaccine formulations of the two antigens (VRSj31 and VRSj32) were produced and injected intramuscularly twice at a 2-week interval and then recombinant proteins (rSj31 and rSj32) together with Freund Complete Adjuvant (FCA) were used to boost the goats. The experiment was repeated in different batche cercariae. A strong anamnestic antibody response was induced after boost. A significant reduction of liver egg counts and miracidial hatching was showed in both experiments. Significant protections against challenge infection were elicited with 31.6% of percentage reduction for worm recovery in the second experiment and 20.9% in the first experiment, respectively.

  1. Humoral immune response against lipopolysaccharide and cytoplasmic proteins of Brucella abortus in cattle vaccinated with B. abortus S19 or experimentally infected with Yersinia enterocolitica serotype 0:9.

    OpenAIRE

    Baldi, P C; Giambartolomei, G H; Goldbaum, F A; Abdón, L F; C.A. Velikovsky; Kittelberger, R; Fossati, C A

    1996-01-01

    The humoral immune responses against three different antigens of Brucella abortus were monitored by enzyme-linked immunosorbent assay in cattle vaccinated with B. abortus S19 or experimentally infected with Yersinia enterocolitica serotype 0:9. Immunoglobulin G (IgG) and IgM responses against (i) B. abortus lipopolysaccharide (LPS), (ii) total cytoplasmic proteins depleted of LPS (LPS-free CYT), and (iii) B. abortus 18-kDa cytoplasmic protein were measured. Vaccinated animals and Yersinia-inf...

  2. Chimeric viruses blur the borders between the major groups of eukaryotic single-stranded DNA viruses

    OpenAIRE

    Roux, Simon; Enault, Francois; Bronner, Gisèle; Vaulot, Daniel; Forterre, Patrick; Krupovic, Mart

    2013-01-01

    Metagenomic studies have uncovered an astonishing diversity of ssDNA viruses encoding replication proteins (Reps) related to those of eukaryotic Circoviridae, Geminiviridae or Nanoviridae; however, exact evolutionary relationships among these viruses remain obscure. Recently, a unique chimeric virus (CHIV) genome, which has apparently emerged via recombination between ssRNA and ssDNA viruses, has been discovered. Here we report on the assembly of 13 new CHIV genomes recovered f...

  3. Oxidative stress and the mechanical properties of naturally occurring chimeric collagen-containing fibers.

    OpenAIRE

    Sun, C.; E. Vaccaro; Waite, J. H.

    2001-01-01

    The byssal threads of marine mussels are a fiber-reinforced composite material. Fibers are continuous, separated by matrix, and consist of chimeric collagens that encompass within the same primary protein structure domains corresponding to collagen, polyhistidine, and either elastin or dragline spider silk. The elastic modulus (stiffness) of the proximal portion of byssal threads was measured by cyclic stress-strain analysis at 50% extension. Before measurement, the threads were conditioned b...

  4. Design and Development of Therapies using Chimeric Antigen Receptor-Expressing T cells

    OpenAIRE

    Dotti, Gianpietro; Gottschalk, Stephen; Savoldo, Barbara; Brenner, Malcolm K

    2014-01-01

    Investigators developed chimeric antigen receptors (CARs) for expression on T cells more than 25 years ago. When the CAR is derived from an antibody, the resultant cell should combine the desirable targeting features of an antibody (e.g. lack of requirement for major histocompatibility complex recognition, ability to recognize non-protein antigens) with the persistence, trafficking and effector functions of a T-cell. This article describes how the past two decades have seen a crescendo of res...

  5. Evaluation of recombinant Leishmania poly-protein plus GLA-SE vaccines against sand fly-transmitted Leishmania major in C57Bl/6 mice1

    OpenAIRE

    Peters, Nathan C.; Bertholet, Sylvie; Lawyer, Phillip G; Charmoy, Melanie; Romano, Audrey; Ribeiro-Gomes, Flavia L.; Stamper, Lisa W.; Sacks, David L.

    2012-01-01

    Numerous experimental Leishmania vaccines have been developed to prevent the visceral and cutaneous forms of Leishmaniasis, which occur after exposure to the bite of an infected sand fly, yet only one is under evaluation in humans. KSAC and L110f, recombinant Leishmania poly-proteins delivered in a stable emulsion (SE) with the TLR 4 agonists monophosphoryl lipid A (MPL) or glucopyranosyl lipid A (GLA) have shown protection in animal models. KSAC+GLA-SE protected against cutaneous disease fol...

  6. The Ag85B protein of the BCG vaccine facilitates macrophage uptake but is dispensable for protection against aerosol Mycobacterium tuberculosis infection.

    Science.gov (United States)

    Prendergast, Kelly A; Counoupas, Claudio; Leotta, Lisa; Eto, Carolina; Bitter, Wilbert; Winter, Nathalie; Triccas, James A

    2016-05-17

    Defining the function and protective capacity of mycobacterial antigens is crucial for progression of tuberculosis (TB) vaccine candidates to clinical trials. The Ag85B protein is expressed by all pathogenic mycobacteria and is a component of multiple TB vaccines under evaluation in humans. In this report we examined the role of the BCG Ag85B protein in host cell interaction and vaccine-induced protection against virulent Mycobacterium tuberculosis infection. Ag85B was required for macrophage infection in vitro, as BCG deficient in Ag85B expression (BCG:(Δ85B)) was less able to infect RAW 264.7 macrophages compared to parental BCG, while an Ag85B-overexpressing BCG strain (BCG:(oex85B)) demonstrated improved uptake. A similar pattern was observed in vivo after intradermal delivery to mice, with significantly less BCG:(Δ85B) present in CD64(hi)CD11b(hi) macrophages compared to BCG or BCG:(oex85B). After vaccination of mice with BCG:(Δ85B) or parental BCG and subsequent aerosol M. tuberculosis challenge, similar numbers of activated CD4(+) and CD8(+) T cells were detected in the lungs of infected mice for both groups, suggesting the reduced macrophage uptake observed by BCG:(Δ85B) did not alter host immunity. Further, vaccination with both BCG:(Δ85B) and parental BCG resulted in a comparable reduction in pulmonary M. tuberculosis load. These data reveal an unappreciated role for Ag85B in the interaction of mycobacteria with host cells and indicates that single protective antigens are dispensable for protective immunity induced by BCG. PMID:27060378

  7. The role of Montanide ISA 70 as an adjuvant in immune responses against Leishmania major induced by thiol-specific antioxidant-based protein vaccine.

    Science.gov (United States)

    Khabazzadeh Tehrani, Narges; Mahdavi, Mehdi; Maleki, Fatemeh; Zarrati, Somayeh; Tabatabaie, Fatemeh

    2016-09-01

    Leishmaniasis is a parasitic disease caused by several species of the genus Leishmania. Montanide ISA 70 is an adjuvant composed of a natural metabolizable oil and a very refined emulsifier from the manide monooleate family. The TSA (thiol-specific antioxidant) is a important antigen of Leishmania major. The purpose of this work was protein-vaccine efficacy as an protection and excellent candidate in the presence Montanide. The expression of recombinant protein was confirmed with SDS (sodium dodecyl sulfate) page and Western bloting. 48 BALB/c mice were divided into four groups (TSA/Freund,TSA/Alum + BCG, TSA/Montanide and PBS groups) and immunized with 20 μg of vaccine subcutaneously three times intervals on days 0, 14 and 28. The mice were challenged with parasite 21 days after final immunization. The lymphocyte proliferation was evaluated with Brdu method. Cytokines and also total antibody and subclasses were evaluated with ELISA method. The vaccine formulated with the recombinant TSA protein with Montanide induced lymphocytes proliferation cytokines and total antibody and subclasses as compared with the control group. PMID:27605780

  8. Evaluation of novel Streptococcus pyogenes vaccine candidates incorporating multiple conserved sequences from the C-repeat region of the M-protein.

    Science.gov (United States)

    Bauer, Michelle J; Georgousakis, Melina M; Vu, Therese; Henningham, Anna; Hofmann, Andreas; Rettel, Mandy; Hafner, Louise M; Sriprakash, Kadaba S; McMillan, David J

    2012-03-01

    A major challenge for Streptococcus pyogenes vaccine development is the identification of epitopes that confer protection from infection by multiple S. pyogenes M-types. Here we have identified and characterised the distribution of common variant sequences from individual repeat units of the C-repeat region (CRR) of M-proteins representing 77 different M-types. Three polyvalent fusion vaccine candidates (SV1, SV2 and SV3) incorporating the most common variants were subsequently expressed and purified, and demonstrated to be alpha-helical by Circular Dichroism (CD), a secondary conformational characteristic of the CRR in the M-protein. Antibodies raised against each of these constructs recognise M-proteins that vary in their CRR, and bind to the surface of multiple S. pyogenes isolates. Antibodies raised against SV1, containing five variant sequences, also kill heterologous S. pyogenes isolates in in vitro bactericidal assays. Further structural characterisation of this construct demonstrated the conformation of SV1 was stable at different pHs, and thermal unfolding of SV1 is a reversible process. Our findings demonstrate that linkage of multiple variant sequences into a single recombinant construct overcomes the need to embed the variant sequences in foreign helix promoting flanking sequences for conformational stability, and demonstrates the viability of the polyvalent candidates as global S. pyogenes vaccine candidates. PMID:22265945

  9. Immunogenicity when utilizing adenovirus serotype 4 and 5 vaccines expressing circumsporozoite protein in naïve and Adenovirus (Ad5 immune mice

    Directory of Open Access Journals (Sweden)

    Schuldt Nathaniel J

    2012-06-01

    Full Text Available Abstract Background Induction of potent long lasting effector T cell responses against liver stage malaria antigens strongly correlates with protection from malaria. While Adenovirus serotype 5 (Ad5 based malaria vaccine platforms have the ability to induce potent effector T cell responses against transgenes, high rates of pre-existing Ad5 immunity in malaria endemic regions has prompted study of alternative Ad serotype based malaria vaccines as replacements for Ad5 based malaria vaccines. The research described in this article examines the utility of alternative serotype adenovirus serotype 4 (Ad4 expressing a sporozoite surface protein (circumsporozoite protein (CSP (Ad4-CSP to induce immune responses against CSP. The immunogenicity of Ad4-CSP was also tested in homologous and heterologous prime boost vaccinations in both Ad5 naïve and Ad5 immune backgrounds as compared to use of Ad5-CSP. Results In Ad5 naïve animals, use of Ad4-CSP priming vaccinations followed by boosting with Ad5-CSP (Ad4-CSP/Ad5-CSP maximally increased the numbers of CSP specific cytokine secreting cytotoxic T cells relative to repeated use of Ad5-CSP. The Ad4-CSP/Ad5-CSP regimen also induced equivalent levels of CSP specific cell killing as did homologous prime-boost vaccinations with Ad5-CSP, despite stimulating lower numbers of CSP specific cytotoxic T cells. Priming with Ad4-CSP followed by a homologous boost resulted in significantly less CSP specific humoral responses than any other vaccination regimen tested in Ad naïve animals. In Ad5 immune animals, addition of Ad4-CSP in homologous or heterologous prime boost resulted in inductions of higher CSP specific responses than animals repeatedly vaccinated with Ad5-CSP alone. However, the observed responses were well below those observed in similarly treated Ad naïve mice. Conclusions While the Ad4-CSP/Ad5-CSP and Ad5-CSP/Ad5-CSP vaccination regimens resulted in equivalent CSP specific killing in Ad naïve animals

  10. Response to Hepatocarcinoma Hca-F of Mice Immunized with Heat Shock Protein 70 from Elemene Combo Tumor Cell Vaccine

    Institute of Scientific and Technical Information of China (English)

    Lianying Guo; Guangxia Shi; Zhihong Gao; Jie Shen; Rong Xing; Zhenchao Qian

    2006-01-01

    To analyze immune response to murine hepatocarcinoma Hca-F of mice immunized with heat shock protein 70(HSP70) derived from elemene combo tumor cell vaccine (EC-TCV) of Hca-F, HSP70 was isolated from EC-TCV by ADP affinity chromatography. Mice were immunized with HSP70 intraperitoneally three times and spleen cells were sampled. For cells, their proliferation and cytotoxicity against Hca-F were measured with MTT assay and their phenotypes were analyzed with flow cytometry. Spleen cells of immunized mice with HSP70 exhibited more potent cytotoxicity against Hca-F and proliferation than that of normal control mice, but less potent than that of mice immunized with EC-TCV. Among three groups, the percent of γδ T lymphocytes in the mice immunized with HSP70 (35.5%) was the highest compared with 6.25% in normal mice, and 28.4% in the mice immunized with EC-TCV. Immunization of HSP70 derived from EC-TCV could elicit potent immune response to Hca-F. HSP70 is one of elements inducing anti-tumor immune responses against Hca-F. Cellular & Molecular Immunology. 2006;3(4):291-295.

  11. An immunotherapeutic treatment against flea allergy dermatitis in cats by co-immunization of DNA and protein vaccines.

    Science.gov (United States)

    Jin, Jin; Ding, Zheng; Meng, Fengxia; Liu, Qiyong; Ng, Terry; Hu, Yanxin; Zhao, Gan; Zhai, Bing; Chu, Hsien-Jue; Wang, Bin

    2010-02-23

    Flea allergy dermatitis (FAD) is considered a harmful and persistent allergic disease in cats, dogs and humans. Effective and safe antigen-specific treatments are lacking. Previously we reported that the simultaneous co-immunization with a DNA vaccine and its cognate coded protein antigen could induce antigen-specific iTreg cells (inducible Treg cells); demonstrating its potential to protect animals from FAD in a murine model. Its clinical efficacy however, remains to be demonstrated. In this report, we clinically tested this protocol to treat established FAD in cats following flea infestations. We present data showing a profound therapeutic improvement of dermatitis in these FAD cats following two co-immunizations, not only in relieving clinical symptoms, but also the amelioration of the allergic responses, including antigen-induced wheal formation, elevated T cell proliferation, infiltration of lymphocytes and migration of mast cells to the sites. This study demonstrates that a co-immunization approach as described can be used to treat flea-induced allergic disease in animals, thus implicating its potential for a practical clinical application. PMID:20188255

  12. Adjuvant effect of docetaxel on HPV16 L2E6E7 fusion protein vaccine in a mouse model.

    Science.gov (United States)

    Su, Xiaoyan; Xu, Wei; Guan, Ran; Wang, Yunhao; Wu, Jie; Zhai, Lijuan; Chen, Gang; Hu, Songhua

    2016-09-01

    We previously demonstrated that the antineoplastic agent docetaxel enhanced the immune response to an influenza vaccine. This study evaluated the adjuvant effect of docetaxel (DOC) on the therapeutic efficacy of HPV16 L2E6E7 fusion protein (HPV-LFP) in mice inoculated with TC-1 cells. The results demonstrated that docetaxel significantly enhanced the therapeutic effect of HPV-LFP on TC-1 cell-induced tumors in mice. The injection of HPV-LFP in combination with docetaxel in TC-1 tumor-bearing mice significantly reduced tumor volume and weight, and a greater percent survival was detected than mice treated with HPV-LFP alone. The inhibition of tumors was associated with significantly increased serum antigen-specific IgG and isotypes, activated CTLs, increased IFN-γ-secreting T cells, and decreased Treg cells and IL-10-secreting cells in spleen. In addition, down-regulation of IL-10, VEGF and STAT3, up-regulation of IFN-γ and decreased Treg cells in the tumor microenvironment may also important contributing factors to the antitumor effect. It may be valuable to use a DOC-containing water to dilute HPV-LFP powder before injection in patients because of its excellent adjuvant effect on HPV-LFP and solubility in water. PMID:27233002

  13. Development of marker vaccines for rinderpest virus using reverse genetics technology

    International Nuclear Information System (INIS)

    animals depended on its mode of expression. The form of GFP that was only expressed in the cytoplasm failed to elicit an immune response in any of the vaccinated animals, despite very high expression levels of the protein during in vitro experiments. Animals vaccinated with a virus expressing a secreted form of GFP varied in their responses to the marker protein with only 50% showing strong anti-GFP antibody levels. Only the membrane-anchored form of the protein gave a strong antibody response in all vaccinated animals and thus was a suitable marker protein. In addition to positive markers, a 'negatively' marked vaccine, i.e. one lacking a RPV-specific antigenic component, was also produced. To achieve this a chimeric virus in which the nucleocapsid protein (N) gene of RPV was replaced by that from the related morbillivirus Peste des petits ruminants virus (PPRV), was produced (rRPV-PPRN) and tested in cattle. Use of this recombinant virus vaccine enables vaccinated animals to be distinguished from those that have been naturally infected. This distinction can be made using two currently available ELISAs that detect antibodies specific to either the N or haemagglutinin (H) proteins of these two viruses. Vaccinated animals become positive in the PPRV N-specific ELISA and negative in the RPV N-specific assay. The opposite is true for the H proteinspecific ELISAs. Vaccinated animals which subsequently become infected with wild type virus will become double positive for the N protein antibodies of both viruses. In a further development we have introduced a positive marker gene into this virus genome, namely that encoding the membrane-anchored GFP protein. This vaccine (rRPV-PPRN-ancGFP), in addition to being distinguishable using the above mentioned tests, produces antibodies against the marker protein in vaccinated animals which are absent in naturally infected and recovered animals. Unfortunately this 'positive-negative' marker vaccine, unlike the other marker vaccines

  14. Superior Immune Response to Protein-Conjugate versus Free Pneumococcal Polysaccharide Vaccine in Chronic Obstructive Pulmonary Disease

    OpenAIRE

    Dransfield, Mark T.; Nahm, Moon H.; Han, MeiLan K.; Harnden, Sarah; Criner, Gerard J.; Fernando J Martinez; Scanlon, Paul D.; Woodruff, Prescott G.; Washko, George R.; Connett, John E.; Anthonisen, Nicholas R.; Bailey, William C.

    2009-01-01

    Rationale: Debate exists about the immunogenicity and protective efficacy of antibodies produced by the 23-valent pneumococcal polysaccharide vaccine (PPSV23) in chronic obstructive pulmonary disease (COPD). The 7-valent diphtheria-conjugated pneumococcal polysaccharide vaccine (PCV7) induces a more robust immune response than PPSV23 in healthy elderly adults.

  15. Enhancement of DNA vaccine potency through linkage of antigen to filamentous bacteriophage coat protein III domain I

    DEFF Research Database (Denmark)

    Cuesta, Àngel M; Suárez, Eduardo; Larsen, Martin; Jensen, Kim Bak; Sanz, Laura; Compte, Marta; Kristensen, Peter; Álvarez-Vallina, Luis

    2006-01-01

    Although DNA-based cancer vaccines have been successfully tested in mouse models, a major drawback of cancer vaccination still remains, namely that tumour antigens are weak and fail to generate a vigorous immune response in tumour-bearing patients. Genetic technology offers strategies for promoti...

  16. A commercial PCV2a-based vaccine is effective in protection from experimental challenge of PCV2 mutant with two amino acids elongation in capsid protein.

    Science.gov (United States)

    Guo, Long-Jun; Fu, Yu-Jie; Huang, Li-Ping; Wang, Yi-Ping; Wei, Yan-Wu; Wu, Hong-Li; Liu, Chang-Ming

    2015-07-17

    Current commercial PCV2 vaccines are almost based on PCV2a and have been shown to be effective in reducing PCV2a and PCV2b viremia and PCV2-associated lesions and diseases. The recent emergence of novel mutant PCV2 (mPCV2) strains and linkage of mPCV2 with cases of porcine circovirus associated disease (PCVAD) in pig herds have raised concerns over emergence of vaccine-escape mutants and reduced efficacy of PCV2a-based vaccines. The aim of this study was to determine the ability of a commercial PCV2a-based vaccine developed by our laboratory to protect conventional pigs against experimental challenge with mPCV2 at 9 weeks of age. Twenty 4-week-old pigs free of PCV2 infection were randomly divided into four treatment groups with 5 pigs each. Two groups were unvaccinated as positive and negative controls. Another two groups were vaccinated with the commercial PCV2a-based vaccine (PCV2-LG strain, China) at 4 weeks of age and identical booster immunization was conducted 3 weeks post primary immunization. At 9 weeks of age, all pigs except the negative control were challenged with a mutant PCV2b/YJ (mPCV2b/YJ) with two amino acids elongation in capsid protein. The experiment was terminated 28 days after challenge. Under the conditions of this study, vaccinated pigs were protected against PCV2 viremia and lesions whereas unvaccinated pigs were not. Moreover, mPCV2b/YJ infection was demonstrated in positive control and almost all had macroscopic or microscopic lesions consistent with PCVAD while negative control did not develop PCVAD. This study indicates that mPCV2b/YJ infection alone can trigger PCVAD development and that the commercial vaccine (PCV2-LG) is still effective in protecting conventional pigs against the emerging mPCV2b/YJ strain in China. PMID:26051516

  17. Combination of pneumococcal surface protein A (PspA with whole cell pertussis vaccine increases protection against pneumococcal challenge in mice.

    Directory of Open Access Journals (Sweden)

    Maria Leonor S Oliveira

    Full Text Available Streptococcus pneumoniae is the leading cause of respiratory acute infections around the world. In Latin America, approximately 20,000 children under 5 years of age die of pneumococcal diseases annually. Pneumococcal surface protein A (PspA is among the best-characterized pneumococcal antigens that confer protection in animal models of pneumococcal infections and, as such, is a good alternative for the currently available conjugated vaccines. Efficient immune responses directed to PspA in animal models have already been described. Nevertheless, few low cost adjuvants for a subunit pneumococcal vaccine have been proposed to date. Here, we have tested the adjuvant properties of the whole cell Bordetella pertussis vaccine (wP that is currently part of the DTP (diphtheria-tetanus-pertussis vaccine administrated to children in several countries, as an adjuvant to PspA. Nasal immunization of BALB/c mice with a combination of PspA5 and wP or wP(low--a new generation vaccine that contains low levels of B. pertussis LPS--conferred protection against a respiratory lethal challenge with S. pneumoniae. Both PspA5-wP and PspA5-wP(low vaccines induced high levels of systemic and mucosal antibodies against PspA5, with similar profile, indicating no essential requirement for B. pertussis LPS in the adjuvant properties of wP. Accordingly, nasal immunization of C3H/HeJ mice with PspA5-wP conferred protection against the pneumococcal challenge, thus ruling out a role for TLR4 responses in the adjuvant activity and the protection mechanisms triggered by the vaccines. The high levels of anti-PspA5 antibodies correlated with increased cross-reactivity against PspAs from different clades and also reflected in cross-protection. In addition, passive immunization experiments indicated that antibodies played an important role in protection in this model. Finally, subcutaneous immunization with a combination of PspA5 with DTP(low protected mice against challenge with two

  18. HIV-1 Env DNA vaccine plus protein boost delivered by EP expands B- and T-cell responses and neutralizing phenotype in vivo.

    Directory of Open Access Journals (Sweden)

    Kar Muthumani

    Full Text Available An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs, and the elicitation of antibody-dependent cellular cytotoxicity (ADCC. Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP. However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted.

  19. Pneumococcal vaccine.

    OpenAIRE

    1999-01-01

    Streptococcus pneumoniae is a frequent cause of pneumonia and meningitis. This article looks at the pneumococcal vaccine, its uses, efficacy, and adverse effects and how vaccination may be improved. We also look at the role of the new conjugate vaccines.

  20. Polio Vaccination

    Science.gov (United States)

    ... to its advantages over IPV in providing intestinal immunity and providing secondary spread of the vaccine to unprotected contacts. Who needs this vaccine and when? Side Effects Excerpt from Vaccine Information Statement A Polio-Free ...

  1. Smallpox Vaccination

    Science.gov (United States)

    ... Newsletters Events Also Known As Smallpox = Vaccinia Smallpox Vaccination Recommend on Facebook Tweet Share Compartir The smallpox ... like many other vaccines. For that reason, the vaccination site must be cared for carefully to prevent ...

  2. Protective efficacy of a Pasteurella haemolytica Serotype A2 outer membrane protein polysaccharide (OMP-PS) complex vaccine in sheep

    International Nuclear Information System (INIS)

    The aim is to study the immunogenicity of the crude and purified OMP-PS complex in conventional sheep and to determine the possible contribution of this candidate antigen to Pasteurella vaccines. This allows improvements of the existing vaccines for an effective control of pneumonic pasteurellosis in small ruminants. The trial has indicated that the purified preparation of OMP-PS complex vaccine was most successful in enhancing the serological response. The complex was immunogenic in adult sheet, with induction of humoral anticapsular antibody in the IHA test and with OMP in immunoblotting. The sera from adult sheep immunized with OMP-PS passively protected mice against A2 challenge. The study has demonstrated that immunity and protection in sheep are not confined to vaccination with live organisms, and that the OMP-PS can act as very effective vaccines in mice and sheep

  3. Two potential recombinant rabies vaccines expressing canine parvovirus virion protein 2 induce immunogenicity to canine parvovirus and rabies virus.

    Science.gov (United States)

    Luo, Jun; Shi, Hehe; Tan, Yeping; Niu, Xuefeng; Long, Teng; Zhao, Jing; Tian, Qin; Wang, Yifei; Chen, Hao; Guo, Xiaofeng

    2016-08-17

    Both rabies virus (RABV) and canine parvovirus (CPV) cause lethal diseases in dogs. In this study, both high egg passage Flury (HEP-Flury) strains of RABV and recombinant RABV carrying double RABV glycoprotein (G) gene were used to express the CPV virion protein 2 (VP2) gene, and were designated rHEP-VP2 and, rHEP-dG-VP2 respectively. The two recombinant RABVs maintained optimal virus titration according to their viral growth kinetics assay compared with the parental strain HEP-Flury. Western blotting indicated that G protein and VP2 were expressed in vitro. The expression of VP2 in Crandell feline kidney cells post-infection by rHEP-VP2 and rHEP-dG-VP2 was confirmed by indirect immunofluorescence assay with antibody against VP2. Immunogenicity of recombinant rabies viruses was tested in Kunming mice. Both rHEP-VP2 and rHEP-dG-VP2 induced high levels of rabies antibody compared with HEP-Flury. Mice immunized with rHEP-VP2 and rHEP-dG-VP2 both had a high level of antibodies against VP2, which can protect against CPV infection. A challenge experiment indicated that more than 80% mice immunized with recombinant RABVs survived after infection of challenge virus standard 24 (CVS-24). Together, this study showed that recombinant RABVs expressing VP2 induced protective immune responses to RABV and CPV. Therefore, rHEP-VP2 and rHEP-dG-VP2 might be potential combined vaccines for RABV and CPV. PMID:27449079

  4. Optimized Blanching Reduces the Host Cell Protein Content and Substantially Enhances the Recovery and Stability of Two Plant-Derived Malaria Vaccine Candidates.

    Science.gov (United States)

    Menzel, Stephan; Holland, Tanja; Boes, Alexander; Spiegel, Holger; Bolzenius, Johanna; Fischer, Rainer; Buyel, Johannes F

    2016-01-01

    Plants provide an advantageous expression platform for biopharmaceutical proteins because of their low pathogen burden and potential for inexpensive, large-scale production. However, the purification of target proteins can be challenging due to issues with extraction, the removal of host cell proteins (HCPs), and low expression levels. The heat treatment of crude extracts can reduce the quantity of HCPs by precipitation thus increasing the purity of the target protein and streamlining downstream purification. In the overall context of downstream process (DSP) development for plant-derived malaria vaccine candidates, we applied a design-of-experiments approach to enhance HCP precipitation from Nicotiana benthamiana extracts generated after transient expression, using temperatures in the 20-80°C range, pH values of 3.0-8.0 and incubation times of 0-60 min. We also investigated the recovery of two protein-based malaria vaccine candidates under these conditions and determined their stability in the heat-treated extract while it was maintained at room temperature for 24 h. The heat precipitation of HCPs was also carried out by blanching intact plants in water or buffer prior to extraction in a blender. Our data show that all the heat precipitation methods reduced the amount of HCP in the crude plant extracts by more than 80%, simplifying the subsequent DSP steps. Furthermore, when the heat treatment was performed at 80°C rather than 65°C, both malaria vaccine candidates were more stable after extraction and the recovery of both proteins increased by more than 30%. PMID:26925077

  5. Genetic diversity of anaplasma species major surface proteins and implications for anaplasmosis serodiagnosis and vaccine development

    OpenAIRE

    de la Fuente, J.; Lew, A.; Lutz, H.; Meli, M. L.; Hofmann-Lehmann, R.; Shkap, V; Molad, T; Mangold, A J; Almazán, C; Naranjo, V.; Gortázar, C.; Torina, A; Caracappa, S.; García-Pérez, A. L.; Barral, M.

    2005-01-01

    The genus Anaplasma (Rickettsiales: Anaplasmataceae) includes several pathogens of veterinary and human medical importance. An understanding of the diversity of Anaplasma major surface proteins (MSPs), including those MSPs that modulate infection, development of persistent infections, and transmission of pathogens by ticks, is derived in part, by characterization and phylogenetic analyses of geographic strains. Information concerning the genetic diversity of Anaplasma spp. MSPs will likely in...

  6. Outer membrane protein (OMP) based vaccine for Neisseria meningitidis serogroup B.

    Science.gov (United States)

    Pillai, Subramonia; Howell, Alan; Alexander, Kristin; Bentley, B Erin; Jiang, Han-Qian; Ambrose, Karita; Zhu, Duzhang; Zlotnick, Gary

    2005-03-18

    A family of outer membrane lipoproteins of Neisseria meningitidis, LP2086, has been shown to induce serum bactericidal activity against a broad variety of meningococcal strains. Two sub-families of serologically distinct LP2086 proteins (A and B) have been identified. In the present study, we have shown that polyclonal anti-serum against rLP2086 is protective in vivo in an infant rat passive-protection model. Additionally, the LP2086 protein is displayed on the surface of 91% meningococcal strains as measured in a whole cell ELISA using polyclonal anti-sera raised against these proteins. We also demonstrate based on the reactivity of anti-rLP2086 antibody with recombinantly expressed C- and N-terminal fragments of rLP2086 in a Western blot assay that the C-terminal fragment of LP2086 dictates sub-family specificity and the N-terminal fragment determines the family specificity. A formulation containing family A and B of LP2086 potentially would provide broad protection against a majority of Neisseria meningitidis strains. PMID:15755596

  7. Immobilization antigen vaccine adjuvanted by parasitic heat shock protein 70C confers high protection in fish against cryptocaryonosis.

    Science.gov (United States)

    Josepriya, T A; Chien, Kuo-Hsuan; Lin, Hsin-Yun; Huang, Han-Ning; Wu, Chang-Jer; Song, Yen-Ling

    2015-08-01

    The immobilization antigen (iAg) has been demonstrated as a protective immunogen against Cryptocaryon irritans infection. In this study, C-terminal domain of heat shock protein 70 cloned from C. irritans (Hsp70C) was tested for its immuno-stimulatory effects. The iAg and Hsp70C cDNAs were constructed independently in secretory forms and were encapsulated in chitosan nanoparticles. In the first immunization trial, grouper fingerlings orally intubated with iAg and iAg:Hsp70C presented 96% and 100% relative percent survival (RPS), respectively, after a lethal challenge. In the second trial, both iAg and iAg:Hsp70C groups showed 100% RPS and the skin trophont burden was significantly lowered. The iAg:Hsp70C still provides a significantly high protection of 51% RPS at 49 days post immunization, when an even more serious lethal infection occurs. RT-qPCR results showed that Hsp70C could up-regulate the expression of i) T cell markers: Cluster of Differentiation 8 alpha (CD8α) and CD4, ii) cytokine genes: Interferon gamma (IFNγ), Tumor Necrosis Factor alpha (TNFα) and Interleukin 12 p40 (IL-12/P40), iii) antibody genes: Immunoglobulin M heavy chain (IgMH) and IgTH, and iv) major histocompatibility complex (MHC-I & MHC-II), in the spleen of iAg:Hsp70C group. Furthermore, significantly high levels of iAg-specific IgM was detected in skin mucus which efficiently immobilized live theronts in iAg- and iAg:Hsp70C-immunized fish at 5 weeks post immunization. Hsp70C significantly increased the number of nonspecific CD8(+) skin leucocytes which exerted cytotoxicity against theronts, although cytotoxic activity showed no difference among the various groups. Because of this complementary cooperation of cellular and humoral immune responses, Hsp70C enhances the efficacy of iAg vaccine and constrains C. irritans infection. In view of the severe loss caused by cryptocaryonosis, application of this parasitic vaccine in farmed and ornamental fish, is worthy to be considered. PMID

  8. Expression and immunoactivity of chimeric particulate antigens of receptor binding site-core antigen of hepatitis B virus

    Institute of Scientific and Technical Information of China (English)

    Hai-Jie Yang; Ning-Shao Xia; Min Chen; Tong Cheng; Shui-Zhen He; Shao-Wei Li; Bao-Quan Guan; Zi-Heng Zhu; Ying Gu; Jun Zhang

    2005-01-01

    AIM: To improve the immunogenicity of receptor binding site of hepatitis B virus (HBV) on preS1 antigen using HBV core antigen as an immuno-carrier.METHODS: One to 6 tandem copies of HBV preS1 (21-47)fragment were inserted into HBcAg at the sites of aa 78 and 82, and expressed in E. coli. ELISA, Western blot and animal immunization were used to analyze the antigenicity and immmunogenicity of purified particulate antigens. The ability to capture HBV by antibodies elicited by chimeric partides was detected with immuno-capture PCR.RESULTS: Recombinant antigens CⅠ, CⅡ, CⅢ carrying 1-3 copies of HBV preS1 (21-47) individually could form viruslike particles (VLPs), similar to HBcAg in morphology. But recombinant antigens carrying 4-6 copies of HBV preS1 (21-47) were poorly expressed in E.coli. Chimeric antigens were lacking of immunoreactivity with anti-HBc monoclonal antibodies (McAbs), but still reserved good immunoreactivity with anti-HBe McAbs. CⅠ, CⅡ, CⅢ could strongly react with anti-preS1 McAb, suggesting that preS1 (21-47) fragment was well exposed on the surface of chimeric VLPs. Three chimeric VLP antigens (CⅠ, CⅡ and CⅢ) could stimulate mice to produce high-level antibody responses, and their immunogenicity was stronger than non-particulate antigen 21-47*6, containing 6 copies of preS1 (21-47). Mouse antibodies to CⅠ, CⅡ and CⅢ were able to capture HBV virions in immuno-capture PCR assay in vitro.CONCLUSION: Chimeric particulate antigens of receptor binding site-core antigen of HBV can elicit strong antibody responses to preS1. They have a potential to be developed into prophylactic or therapeutic vaccines against HBV infection.

  9. Vaccine Hesitancy.

    Science.gov (United States)

    Jacobson, Robert M; St Sauver, Jennifer L; Finney Rutten, Lila J

    2015-11-01

    Vaccine refusal received a lot of press with the 2015 Disneyland measles outbreak, but vaccine refusal is only a fraction of a much larger problem of vaccine delay and hesitancy. Opposition to vaccination dates back to the 1800 s, Edward Jenner, and the first vaccine ever. It has never gone away despite the public's growing scientific sophistication. A variety of factors contribute to modern vaccine hesitancy, including the layperson's heuristic thinking when it comes to balancing risks and benefits as well as a number of other features of vaccination, including falling victim to its own success. Vaccine hesitancy is pervasive, affecting a quarter to a third of US parents. Clinicians report that they routinely receive requests to delay vaccines and that they routinely acquiesce. Vaccine rates vary by state and locale and by specific vaccine, and vaccine hesitancy results in personal risk and in the failure to achieve or sustain herd immunity to protect others who have contraindications to the vaccine or fail to generate immunity to the vaccine. Clinicians should adopt a variety of practices to combat vaccine hesitancy, including a variety of population health management approaches that go beyond the usual call to educate patients, clinicians, and the public. Strategies include using every visit to vaccinate, the creation of standing orders or nursing protocols to provide vaccination without clinical encounters, and adopting the practice of stating clear recommendations. Up-to-date, trusted resources exist to support clinicians' efforts in adopting these approaches to reduce vaccine hesitancy and its impact. PMID:26541249

  10. Lassa fever vaccine.

    Science.gov (United States)

    Fisher-Hoch, Susan P; McCormick, Joseph B

    2004-04-01

    Lassa fever remains a serious challenge to public health in West Africa threatening both local residents in rural areas and those who serve them, particularly medical care providers. Given the ecology of the rodent host and conditions in the endemic area, a vaccine is mandatory for control. The challenge is to overcome the scientific, political and economic obstacles to producing a human use vaccine candidate. There are some scientific issues to resolve. It is known that the G-protein confers protection but we do not know its duration. If the N-protein is also included there may be a better duration of protection but it is unclear whether the N-protein as a vaccine may possibly enhance the infection. The original vaccinia vector must be replaced by new vectors, chimeras or by delivering DNA in some format. A live vaccine is attractive because it can confer protection in a single shot. A killed vaccine is more stable, particularly for distribution in the tropics but usually requires repeated shots. For practical reasons a live vaccine format should probably be pursued, which could then be combined with a yellow fever vaccine, using the same cold chains, since this disease occupies the same endemic areas in West Africa. Lassa vaccine initiatives have suffered from a lack of funding in the past but bioterrorism has brought new resources to Lassa virus science. Adequate funding and applications of new vaccine technologies give hope that we may soon see a vaccine in clinical trials. However, the difficulty of conducting trials in endemic areas and lack of political stability remain serious problems. PMID:15056044

  11. Absolute quantification of dengue virus serotype 4 chimera vaccine candidate in Vero cell culture by targeted mass spectrometry.

    Science.gov (United States)

    Rougemont, Blandine; Simon, Romain; Carrière, Romain; Biarc, Jordane; Fonbonne, Catherine; Salvador, Arnaud; Huillet, Céline; Berard, Yves; Adam, Olivier; Manin, Catherine; Lemoine, Jérôme

    2015-10-01

    Infection by dengue flavivirus is transmitted by mosquitoes and affects tens to hundreds of millions people around the world each year. Four serotypes have been described, all of which cause similar disease. Currently, there no approved vaccines or specific therapeutics for dengue, although several vaccine prototypes are in different stages of clinical development. Among them, a chimeric vaccine, built from the replication machinery of the yellow fever 17D virus, has shown promising results in phase III trials. Accurate quantitation of expressed viral particles in alive attenuated viral antigen vaccine is essential and determination of infectious titer is usually the method of choice. The current paper describes an alternative or orthogonal strategy, namely, a multiplexed and absolute assay of four proteins of the chimera yellow fever/dengue serotype 4 virus using targeted MS in SRM mode. Over 1 month, variability of the assay using a partially purified Vero cell extract was between 8 and 17%, and accuracy was between 80 and 120%. In addition, the assay was linear between 6.25 and 200 nmol/L and could therefore be used in the near future to quantify dengue virus type 4 during production and purification from Vero cells. PMID:26205729

  12. Fiber knob domain lacking the shaft sequence but fused to a coiled coil is a candidate subun