Vigorous hepatitis C virus-specific CD4+ and CD8+ T cell responses induced by protein immunization in the presence of Montanide ISA720 plus synthetic oligodeoxynucleotides containing immunostimulatory cytosine-guanine dinucleotide motifs.
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BACKGROUND: A prophylactic vaccine for hepatitis C virus (HCV) requires generation of strong humoral as well as CD4(+) and CD8(+) T cell responses. METHODS: The immunomodulatory effects of the combination of 2 adjuvants, synthetic oligodeoxynucleotides containing immunostimulatory cytosine-guanine dinucleotide motifs emulsified with Montanide ISA720 (M-ISA720/CpG), were investigated using the murine model. RESULTS: Administration of recombinant HCV (rHCV) nonstructural (NS) 3 and NS5B proteins plus M-ISA720/CpG (hereafter, "M-ISA720/CpG/rHCV protein") induced high anti-NS3 and anti-NS5B immunoglobulin (Ig) G titers, with the IgG2a isotype being predominant. NS3- and NS5B-specific interferon (IFN)- gamma - and interleukin-2-producing CD4(+) T cell responses, as assessed by enzyme-linked immunospot assay, were significantly more vigorous in mice immunized with M-ISA720/CpG/rHCV protein than in control mice immunized without adjuvant. NS3- and NS5B-specific IFN- gamma -producing CD8(+) T cell percentages, as measured by direct ex vivo intracellular cytokine staining assay, were, respectively, a mean+/-SD of 0.14% +/- 0.04% and 0.15% +/- 0.05% in mice immunized with M-ISA720/CpG/rHCV protein. Furthermore, boosting with recombinant NS3 expression plasmid DNA after priming with M-ISA720/CpG-adjuvanted rNS3 strikingly enhanced both CD4(+) and CD8(+) T cell responses. CONCLUSION: Immunization with M-ISA720/CpG/rHCV protein is capable of inducing potent humoral as well as HCV-specific T helper type 1-biased CD4(+) and CD8(+) T cell responses. A DNA boost after a protein prime--a reversal of the conventional approach--may provide an alternative path to the development of an effective HCV vaccine. (+info)
Hepatitis E virus chimeric DNA vaccine elicits immunologic response in mice.
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AIM: To construct the plasmid pcHEV23 containing fragments of HEV ORF2 and ORF3 chimeric gene and to assess its ability to elicit specific immunologic response in mice. METHODS: The gene encoding the structural protein of HEV ORF2 fragment and full-length ORF3 was amplified by PCR. The PCR products were cloned into an eucaryotic expression plasmid pcDNA3. The resulting plasmid pcHEV23 was used as a DNA vaccine to inoculate BALB/c mice intramuscularly thrice at a dose of 100 or 200 microg. Mice injected with empty pcDNA3 DNA or saline served as control and then specific immune responses in the mice were detected. RESULTS: After 2-3 times of inoculation, all mice injected with pcHEV23 had anti-HEV IgG seroconversion and specific T lymphocyte proliferation. The lymphocyte stimulation index in the group immunized with pcHEV23 (3.1+/-0.49) was higher than that in the control group (0.787+/-0.12, P<0.01). None in the control group had a detectable level of anti-HEV IgG. CONCLUSION: DNA vaccine containing HEV ORF2 and ORF3 chimeric gene can successfully induce specific humoral and cellular immune response in mice. (+info)
A novel adenovirus type 6 (Ad6)-based hepatitis C virus vector that overcomes preexisting anti-ad5 immunity and induces potent and broad cellular immune responses in rhesus macaques.
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Success in resolving hepatitis C virus (HCV) infection has been correlated to vigorous, multispecific, and sustained CD8(+) T-cell response in humans and chimpanzees. The efficacy of inducing T-cell-mediated immunity by recombinant serotype 5 adenovirus vector has been proven in many animal models of infectious diseases, but its immunogenicity can be negatively influenced by preexisting immunity against the vector itself. To evaluate the less prevalent adenovirus serotype 6 (Ad6) as an alternative vector for and HCV vaccine development, we have generated serotype 5 and 6 adenoviral vectors directing expression of the nonstructural region of HCV (MRKAd5-NSmut and MRKAd6-NSmut). Immunogenicity studies in mice showed that the two vectors induced comparable T-cell responses but that only MRKAd6-NSmut was not suppressed in the presence of anti-Ad5 immunity. In contrast, preexisting anti-Ad5 immunity dramatically blunted the immunogenicity of the serotype 5-based HCV vector. Furthermore, MRKAd6-NSmut showed equivalent potency, breadth, and longevity of HCV-specific T-cell responses in rhesus macaques as the corresponding Ad5-based vector over a wide range of doses and was capable of boosting DNA-primed animals even if administered at low doses. These data support the use of the MRKAd6-NSmut for anti-HCV immunotherapy and, more generally, for the Ad6 serotype as a better genetic vaccine vehicle than Ad5. (+info)
Vaccination of ducks with a whole-cell vaccine expressing duck hepatitis B virus core antigen elicits antiviral immune responses that enable rapid resolution of de novo infection.
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As a first step in developing immuno-therapeutic vaccines for patients with chronic hepatitis B virus infection, we examined the ability of a whole-cell vaccine, expressing the duck hepatitis B virus (DHBV) core antigen (DHBcAg), to target infected cells leading to the resolution of de novo DHBV infections. Three separate experiments were performed. In each experiment, ducks were vaccinated at 7 and 14 days of age with primary duck embryonic fibroblasts (PDEF) that had been transfected 48 h earlier with plasmid DNA expressing DHBcAg with and without the addition of anti-DHBcAg (anti-DHBc) antibodies. Control ducks were injected with either 0.7% NaCl or non-transfected PDEF. The ducks were then challenged at 18 days of age by intravenous inoculation with DHBV (5 x 10(8) viral genome equivalents). Liver biopsies obtained on day 4 post-challenge demonstrated that vaccination did not prevent infection of the liver as similar numbers of infected hepatocytes were detected in all vaccinated and control ducks. However, analysis of liver tissue obtained 9 or more days post-challenge revealed that 9 out of 11 of the PDEF-DHBcAg vaccinated ducks and 8 out of 11 ducks vaccinated with PDEF-DHBcAg plus anti-DHBc antibodies had rapidly resolved the DHBV infection with clearance of infected cells. In contrast, 10 out of 11 of the control unvaccinated ducks developed chronic DHBV infection. In conclusion, vaccination of ducks with a whole-cell PDEF vaccine expressing DHBcAg elicited immune responses that induced a rapid resolution of DHBV infection. The results establish that chronic infection can be prevented via the vaccine-mediated induction of a core-antigen-specific immune response. (+info)
Putative neutralization epitopes and broad cross-genotype neutralization of Hepatitis E virus confirmed by a quantitative cell-culture assay.
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Monolayers of Hep G2/C3A cells were inoculated with genotype 1 Hepatitis E virus (HEV) mixed with either anti-HEV or an appropriate control. After 5 or 6 days, cell monolayers were stained with anti-HEV and infected cells were identified by immunofluorescence microscopy and counted. Anti-HEV from vaccinated or infected rhesus monkeys neutralized the virus, as did mAbs that recognized epitopes on the C terminus of a recombinant vaccine protein. Antibodies were broadly cross-reactive, since convalescent serum from animals infected with any one of the four mammalian genotypes all neutralized the genotype 1 virus. (+info)
A truncated hepatitis E virus ORF2 protein expressed in tobacco plastids is immunogenic in mice.
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AIM: To cost-effectively express the 23-ku pE2, the most promising subunit vaccine encoded by the E2 fragment comprising of the 3'-portion of hepatitis E virus (HEV) open reading frame 2 (ORF2) in plastids of tobacco (Nicotiana tabacum cv. SR1), to investigate the transgene expression and pE2 accumulation in plastids, and to evaluate the antigenic effect of the plastid-derived pE2 in mice. METHODS: Plastid-targeting vector pRB94-E2 containing the E2 fragment driven by rice psbA promoter was constructed. Upon delivery into tobacco plastids, this construct could initiate homologous recombination in psaB-trnfM and trnG-psbC fragments in plastid genome, and result in transgene inserted between the two fragments. The pRB94-E2 was delivered with a biolistic particle bombardment method, and the plastid-transformed plants were obtained following the regeneration of the bombarded leaf tissues on a spectinomycin-supplemented medium. Transplastomic status of the regenerated plants was confirmed by PCR and Southern blot analysis, transgene expression was investigated by Northern blot analysis, and accumulation of pE2 was measured by ELISA. Furthermore, protein extracts were used to immunize mice, and the presence of the pE2-reactive antibodies in serum samples of the immunized mice was studied by ELISA. RESULTS: Transplastomic lines confirmed by PCR and Southern blot analysis could actively transcribe the E2 mRNA. The pE2 polypeptide was accumulated to a level as high as 13.27 microg/g fresh leaves. The pE2 could stimulate the immunized mice to generate pE2-specific antibodies. CONCLUSION: HEV-E2 fragment can be inserted into the plastid genome and the recombinant pE2 antigen derived is antigenic in mice. Hence, plastids may be a novel source for cost-effective production of HEV vaccines. (+info)
Improving birth dose coverage of hepatitis B vaccine.
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Administration of a birth dose of hepatitis B vaccine (HepB vaccine) to neonates is recommended to prevent mother-to-infant transmission and chronic infection with the hepatitis B virus (HBV). Although manufacturers recommend HepB vaccine distribution and storage at 2-8 degrees C, recognition of the heat stability of hepatitis B surface antigen stimulated research into its use after storage at, or exposure to, ambient or high temperatures. Storage of HepB vaccine at ambient temperatures would enable birth dosing for neonates delivered at home in remote areas or at health posts lacking refrigeration. This article reviews the current evidence on the thermostability of HepB vaccine when stored outside the cold chain (OCC). The reports reviewed show that the vaccines studied were safe and effective whether stored cold or OCC. Field and laboratory data also verifies the retained potency of the vaccine after exposure to heat. The attachment of a highly stable variety of a vaccine vial monitor (measuring cumulative exposure to heat) on many HepB vaccines strongly supports policies allowing their storage OCC, when this will benefit birth dose coverage. We recommend that this strategy be introduced to improve birth dose coverage, especially in rural and remote areas. Concurrent monitoring and evaluation should be undertaken to affirm the safe implementation of this strategy, and assess its cost, feasibility and effect on reducing HBV infection rates. Meanwhile, release of manufacturer data verifying the potency of currently available HepB vaccines after exposure to heat will increase confidence in the use of vaccine vial monitors as a managerial tool during storage of HepB vaccine OCC. (+info)
A recombinant vaccinia virus expressing hepatitis A virus structural polypeptides: characterization and demonstration of protective immunogenicity.
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A recombinant vaccinia virus containing most of the P1 region of hepatitis A virus (HAV) was constructed. Cell lysates of cultures infected with the virus contained HAV proteins detectable by radioimmunoassay. Western blot analysis revealed the presence of a single protein of Mr 60K to 62K, bearing epitopes from structural polypeptides VP4, -3 and -2, and the N terminus of VP1. The size of the protein suggests that at least some of the vaccinia virus thymidine kinase is also expressed. Inoculation of tamarin monkeys with the recombinant virus resulted in the development of a specific anti-HAV immune response which was protective against challenge with a virulent strain of HAV. Recombinant viruses expressing the above region of HAV or the proteins expressed by such viruses may be useful in the development of a vaccine suitable for use in man. (+info)