Induction of hepatitis C virus-specific cytotoxic T lymphocytes in mice by immunization with dendritic cells transduced with replication-defective recombinant adenovirus

We studied the potential of dendritic cells (DCs) in priming hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTLs) in mice. Recombinant adenovirus expressing HCV core (Adex1SR3ST) was employed to express core in DCs. Core-specific CTLs are effectively elicited by injecting Adex1SR3ST-transduced DCs, whereas injection of Adex1SR3ST does not result in effective priming. Further, Adex1SR3ST-transduced DCs more efficiently prime core-specific CTLs than Adex1SR3ST-transduced macrophages, or DCs treated with an anthrax toxin fusion protein reported previously. Upon challenge with recombinant HCV-core-expressing vaccinia virus, vaccinia titers are significantly reduced in mice immunized with Adex1SR3ST-transduced DCs. Thus, adenovirus-transduced DCs may be a promising candidate for a CTL-based vaccine against HCV.     

Deletion of N-glycosylation sites of hepatitis C virus envelope protein E1 enhances specific cellular and humoral immune responses

N-linked glycosylations of viral proteins have been implicated in immunogenicity. In this study, the effects of the N-linked glycosylation of the hepatitis C virus (HCV) E1 protein, a naturally poor immunogen, on the induction of specific immune response were examined. We constructed the plasmids containing the genes encoding both wild type and mutant E1 proteins in which N-linked glycosylation sites are mutated individually or in combination by site-directed mutagenesis. The immunogenicity of wild type E1 and six mutated E1 proteins was analyzed in BALB/C mice using a DNA-based vaccination approach. We found that E1-M2 mutant (at site of N209SS) significantly enhanced E1-specific CD8(+)T cells cytotoxic T lymphocytes (CTL) activities, expression of IFN-gamma producing T cells, and suppression of tumor growth. While E1-M4 mutant (at site of N305CS) induced the highest specific antibody response among all groups. Moreover, E1 wild-type vaccinated mice developed a mixture of IgG1 and Ig2a, but E1-M2 mutant induced only IgG2a isotype, and E1-M4 mutant dominantly developed IgG1 isotype. Our data showed that N-linked glycosylation can limit both cellular and antibody response to the HCV E1 protein and deletion of the N-glycosylation sites at N209SS and N305CS of hepatitis C virus envelope protein E1 provided potential applications for the development of DNA vaccine with enhanced immunogenicity.     

Evaluation of adenovirus 19a as a novel vector for mucosal vaccination against influenza A viruses

Since preexisting immunity and enhanced infection rates in a clinical trial of an HIV vaccine have raised some concerns on adenovirus (Ad) serotype 5-based vaccines, we evaluated the subgroup D adenovirus serotype Ad19a for its suitability as novel viral vector vaccine against mucosal infections. In BALB/c mice, we compared the immunogenicity and efficacy of E1/E3-deleted Ad19a vectors encoding the influenza A virus (IAV)-derived antigens hemagglutinin (HA) and nucleoprotein (NP) to the most commonly used Ad5 vectors. The adenoviral vectors were applied intranasally and induced detectable antigen-specific T cell responses in the lung and in the spleen as well as robust antibody responses. A prior DNA immunization significantly improved the immunogenicity of both vectors and resulted in full protection against a lethal infection with a heterologous H3N2 virus. Nevertheless, the Ad5-based vectors were slightly superior in reducing viral replication in the lung which corresponded to higher NP-specific T cell responses measured in the lungs.     

DNA immunization of mice and macaques with plasmids encoding hepatitis C virus envelope E2 protein expressed intracellularly and on the cell surface

We analyzed the humoral immune response elicited by hepatitis C virus (HCV) E2 protein expressed in vivo after injection of plasmid DNA into mice and rhesus macaques. Three plasmids were used for immunization: a plasmid containing the entire sequence of the E2 and p7 genes (pE2); a plasmid encoding a truncated form of the E2 protein targeted to the cell surface (pE2surf); a control plasmid (pDisplay) lacking an HCV insert. Each plasmid was injected intramuscularly into 5 mice and intraepidermally (via gene gun) into 5 mice. Immunization was repeated three times at three week intervals. Five macaques were injected intramuscularly (two with pE2, two with pE2surf and one with pDisplay) and immunization was repeated after 8 weeks. All mice immunized via gene gun with pE2 or pE2surf developed anti-E2. The animals immunized with pE2surf developed an earlier and stronger humoral immune response than those immunized with pE2. Only 2 of the mice injected by the intramuscular route, both immunized with pE2surf, developed detectable anti-E2. One of the two macaques immunized with pE2 and both macaques immunized with pE2surf developed anti-E2; the humoral immune response was much stronger in the animals immunized with pE2surf. Our results suggest that presentation of HCV E2 on the cell surface may increase its immunogenicity while preserving its ability to react with antibodies generated during a natural infection.     

丙型肝炎病毒E2蛋白DNA疫苗诱导小鼠产生中和抗体的研究

目的探讨丙型肝炎病毒(HCV)包膜E2蛋白DNA疫苗诱导小鼠产生中和抗体的可行性。方法构建截除疏水性羧基末端的HCV包膜蛋白表达质粒pCI-1b661以及同时截除疏水性羧基末端和高变区1(HVR1)的表达质粒pCI-1b661Δ,转染293T细胞,以Western blot和ELISA检测细胞内和培养上清中的HCVE2蛋白,将两种表达质粒及空载体分别肌注免疫BALB/c小鼠,以ELISA检测小鼠血清中的HVR1抗体,以HCV假病毒颗粒(HCVpp)分析小鼠血清的中和活性。结果 2种表达质粒均能表达分泌性截短型E2蛋白。pCI-1b661免疫的8只小鼠血清中均可检测到HVR1抗体,而pCI-1b661Δ免疫血清中未检测到HVR1抗体。pCI-1b661和pCI-1b661Δ免疫血清对HCVpp的中和率分别为(78.5±13.8)%和(38.7±6.5)%,差异有显著性(P<0.01)。pCI-1b661免疫组小鼠血清的中和率与HVR1抗体水平呈正相关(r=0.967,P<0.01)。结论表达截短型E2蛋白的DNA疫苗能诱导产生HCV中和抗体,其主要成员为HVR1抗体。     

Complete protection of mice against a lethal dose challenge of western equine encephalitis virus after immunization with an adenovirus-vectored vaccine

Western equine encephalitis virus (WEEV) is an important pathogen for both humans and equines. The virus is also listed as a bioterrorism agent due to its ability for aerosol transmission with high mortality. No commercial vaccines or antiviral drugs are available for the prevention and treatment of WEEV infection in humans. In this paper, we constructed a recombinant WEEV vaccine, designated as Ad5-WEEV, using a replication defective, human adenovirus serotype 5 (HAd5) as a delivery vector. Ad5-WEEV contains the E3-E2-6K-E1 structural protein gene of the 71V-1658 strain of WEEV and the E1 and E2 proteins were synthesized in cells inoculated with Ad5-WEEV. After intramuscular immunization of mice with two doses of Ad5-WEEV, neutralizing antibodies against WEEV were generated and the mice were completely protected from a lethal dose challenge of 71V-1658. In addition, we showed that passive transfer of serum from the Ad5-WEEV-immunized mice could partially control WEEV infection. These results demonstrate that HAd5 vectors are promising for WEEV vaccine development.     

Engineering of N-glycosylation of hepatitis C virus envelope protein E2 enhances T cell responses for DNA immunization

N-linked oligosaccharides are known to be important in modulating immunogenicity of some viral proteins. However, the roles of N-glycans of hepatitis C virus (HCV) E2 envelope glycoprotein in specific cellular immune responses remain elusive. Previous studies showed that the epitopes aa481-500 and aa551-570 of E2 might be important for immunoreactivity and that the binding site of E2 for hCD81 is located at aa480-493 and aa544-551 within the E2 protein. Here, we made plasmids containing genes encoding either wild type or mutant E2 proteins in which N-glycosylation sites (N560NT and N576ST) close to these important regions were mutated separately or in combination. The immunogenicities of wild type E2 and three mutated E2 proteins were analyzed in BALB/c mice using DNA vaccination. The E2-M2 mutant (at N576ST) significantly enhanced (compared to control) E2-specific CTL activity (P<0.05), expression of IFN-gamma (P<0.05) and suppression of tumor growth (P<0.05). Also, high IgG2a/IgG1 ratios were elicited in a Th1-type response. These results indicate that engineering of the N-glycosylation site N576ST of HCV E2 protein enhances specific cellular immune responses, providing insights into the development of E2-based DNA vaccines with enhanced immunogenicity.     

Envelope protein E1 as vaccine target for western equine encephalitis virus

Western equine encephalitis virus (WEEV) is a mosquito-borne RNA virus which causes lethal infection in humans and equines. There are no commercial vaccines or anti-WEEV drugs available for humans. We used replication-defective, human adenovirus serotype-5 (HAd5) as a delivery vector for developing WEEV vaccine. Our previous study found delivery of both E1 and E2 envelope proteins of WEEV by HAd5 vector offers complete protection against lethal challenge of WEEV. In this paper, we constructed a HAd5-vectored E1 vaccine, Ad5-E1. Mice given single-dose vaccination of Ad5-E1 were completely protected against both homologous and heterologous WEEV strains. The protection was rapid, which was achieved as early as day 7 after vaccination. In addition, Ad5-E1 induced a strong WEEV-specific T cell response. Our data suggest E1 is a potential target for developing single-dose, fast-acting, HAd5-vectored vaccine for WEEV.     

Novel Adenovirus type 5 vaccine platform induces cellular immunity against HIV-1 Gag,Pol, Nef despite the presence of Ad5 immunity

Recombinant Adenovirus serotype 5 (Ad5) vectors have been used as vaccine platforms in numerous animal and human clinical studies. The immune response induced by Ad5 vaccines can be mitigated due to pre-existing Ad5 immunity. We previously reported the use of a novel Ad5 platform to induce cellular immune responses (CMI) against HIV-1 Gag in Ad5 hyper immune mice. Here, the effectiveness of the Ad5 [E1-, E2b-] vaccine platform was evaluated using a triad mixture of HIV-1 Gag, Pol, and Nef as antigenic transgenes. Broad CMI was induced following vaccination with the HIV-1 expressing vectors in Ad5 naïve and Ad5 immunized mice. A mixture of the three vaccines induced CMI against each transgene product even in the presence of hyper Ad5 immunity. These studies revealed that CMI responses to immunization with Ad5 [E1-, E2b-]-gag, Ad5 [E1-, E2b-]-pol or Ad5 [E1-, E2b-]-nef vectors were transgene specific and did not induce CMI responses against irrelevant antigens such as carcinoembryonic antigen (CEA), herpes simplex virus glycoprotein B (HSV), cytomegalovirus (CMV) or influenza virus antigens. We are evaluating this recombinant triad viral vector as an HIV-1 vaccine in a non-human primate model and the data indicate that the vaccine is worthy of clinical evaluation.     

DNA-based vaccination against hepatitis C virus (HCV): effect of expressing different forms of HCV E2 protein and use of CpG-optimized vectors in mice

DNA-based immunization may be of prophylactic and therapeutic value for hepatitis C virus (HCV) infection. In efforts to improve the immunogenicity of a plasmid expressing the second envelope protein (E2) of HCV, we evaluated in mice the role of the antigen localization and demonstrated that membrane-bound and secreted forms induced higher titers of E2-specific antibodies, as well as earlier and higher seroconversion rates, than the intracellular form, but all three forms induced strong CTL. We also investigated whether E2-specific antibody responses could be enhanced by CpG optimization of the plasmid backbone and showed that removal of neutralizing CpG dinucleotides did not have a significant effect but addition of 64 immunostimulatory CpG motifs significantly enhanced anti-E2 titers. These results may have implications for the design and development of HCV DNA vaccines.     

Comparison of protective efficacies of plasmid DNAs encoding Japanese encephalitis virus proteins that induce neutralizing antibody or cytotoxic T lymphocytes in mice

Mice immunized with a plasmid DNA encoding the premembrane (prM) and envelope (E) proteins of Japanese encephalitis (JE) virus (designated pcJEME) produce neutralizing antibodies and are protected from JE. To determine the role of the immune response to other viral proteins in protection, we constructed plasmid DNAs encoding other JE virus proteins and made a direct comparison among these plasmids using a mouse model. Cytotoxic T lymphocytes (CTLs) were induced by plasmids encoding capsid (C) or nonstructural proteins, NS1, NS2A, NS2B, NS3 or NS5. However, these plasmids provided only a partial protection against intraperitoneal challenge with a lethal dose of JE virus, whereas mice immunized with pcJEME were fully protected. In mice inoculated with CTL-inducing plasmids, high virus titers were detected in plasma immediately (1h) following challenge and in brain on day 4 post-challenge, but no virus infectivity was detected in plasma and brain of pcJEME-immunized mice during the 5 days following challenge. These results indicate that protection provided by the prM/E-encoding DNA consists of neutralizing antibody that prevents virus dissemination from the peripheral site to the brain, and that this antibody-mediated mechanism of protection is more efficient than the immunity induced by plasmids that generate CTL responses capable of killing JE virus-infected cells.     

Comparative immunogenicity analysis of modified vaccinia Ankara vectors expressing native or modified forms of hepatitis C virus E1 and E2 glycoproteins

We have evaluated in C57/Bl6 and HLA-A2.1 transgenic mice the immunogenicity of three MVA vectors expressing either native HCV E1E2 polyprotein, truncated and secreted E1 (E'1(311)) and E2 (E'2(661)) proteins, or a chimeric E1E2 heterodimer presented at the plasma membrane. Immunization induced mainly a Th1 response in HLA-A2.1 transgenic mice while a Th2-type response was detected in C57/Bl6 mice. Comparison of the three vectors shows an increase in the humoral response when antigens are secreted or membrane bound, and slightly in the cellular response when antigens are exposed on the cell surface.     

Neutralizing antibodies and broad,functional T cell immune response following immunization with hepatitis C virus proteins-based vaccine formulation

HCV is a worldwide health problem despite the recent advances in the development of more effective therapies. No preventive vaccine is available against this pathogen. However, non-sterilizing immunity has been demonstrated and supports the potential success of HCV vaccines. Induction of cross-neutralizing antibodies and T cell responses targeting several conserved epitopes, have been related to hepatitis C virus (HCV) clearance. Therefore, in this work, the immunogenicity of a preparation (MixprotHC) based on protein variants of HCV Core, E1, E2 and NS3 was evaluated in mice and monkeys. IgG from MixprotHC immunized mice and monkeys neutralized the infectivity of heterologous HCVcc. Moreover, strong CD4+ and CD8+ T cells proliferative and IFN-γ secretion responses were elicited against HCV proteins. Remarkably, immunization with MixprotHC induced control of viremia in a surrogate challenge model in mice. These results suggest that MixprotHC might constitute an effective immunogen against HCV in humans with potential for reducing the likelihood of immune escape and viral persistence.     

Control of SIV infection and subsequent induction of pandemic H1N1 immunity in rhesus macaques using an Ad5 [E1-, E2b-] vector platform

Anti-vector immunity mitigates immune responses induced by recombinant adenovirus vector vaccines, limiting their prime-boost capabilities. We have developed a novel gene delivery and expression platform (Ad5 [E1-, E2b-]) that induces immune responses despite pre-existing and/or developed concomitant Ad5 immunity. In the present study, we evaluated if this new Ad5 platform could overcome the adverse condition of pre-existing Ad5 immunity to induce effective immune responses in prime-boost immunization regimens against two different infectious diseases in the same animal. Ad5 immune rhesus macaques (RM) were immunized multiple times with the Ad5 [E1-, E2b-] platform expressing antigens from simian immunodeficiency virus (SIV). Immunized RM developed cell-mediated immunity against SIV antigens Gag, Pol, Nef and Env as well as antibody against Env. Vaccinated and vector control RMs were challenged intra-rectally with homologous SIVmac239. During a 7-week follow-up, there was perturbation of SIV load in some immunized RM. At 7 weeks post-challenge, eight immunized animals (53%) did not have detectable SIV, compared to two RM controls (13%) (P < 0.02; log-rank Mantel–Cox test). There was no correlation of protective MHC contributing to infection control. The RM without detectable circulating SIV, now hyper immune to Ad5, were then vaccinated with the same Ad5 [E1-, E2b-] platform expressing H1N1 influenza hemagglutinin (HA). Thirty days post Ad5 [E1-, E2b-]-HA vaccination, significant levels of influenza neutralizing antibody were induced in all animals that increased after an Ad5 [E1-, E2b-]-HA homologous boost. These data demonstrate the versatility of this new vector platform to immunize against two separate disease targets in the same animal despite the presence of immunity against the delivery platform, permitting homologous repeat immunizations with an Ad5 gene delivery platform.     

Oral immunization with attenuated Salmonella carrying a co-expression plasmid encoding the core and E2 proteins of hepatitis C virus capable of inducing cellular immune responses and neutralizing antibodies in mice

Hepatitis C virus (HCV) core protein has long been considered an attractive candidate for inclusion in a protective vaccine. However, this protein may hamper the development of systemic immune responses because of its immune suppressive properties. We previously reported that immune responses to HCV core protein could be efficiently induced by attenuated Salmonella carrying the HCV core protein, but not the HCV core DNA vaccine. To optimize the combination of the core protein and envelope protein 2 (E2) into a vaccine formulation to induce cellular immune responses and neutralizing antibodies, we constructed a plasmid containing two expression cassettes. One expression cassette was included to regulate the expression of HCV core protein by an inducible in vivo-activated Salmonella promoter, the other was included to regulate the expression of HCV E2 protein by the cytomegalovirus enhancer/promoter. Oral immunization of BALB/c mice with the attenuated Salmonella strain SL7207 carrying this plasmid efficiently induced HCV core and E2-specific cellular immune responses and antibodies. IgG purified from immunized mice could neutralize the infectivity of HCV pseudoparticles (HCVpp) of both the autologous Con 1 isolate and the heterologous H77 isolate, and cell culture produced HCV (HCVcc) of Con1-JFH1 chimera. These results indicated that this vaccine strategy can effectively deliver core and E2 protein to the immune system and provide a promising approach for the development of prophylactic and therapeutic vaccines against HCV infection.     

Efficient induction of T helper 1 CD4+ T-cell responses to hepatitis C virus core and E2 by a DNA prime-adenovirus boost

Hepatitis C virus (HCV) is an important causative agent of liver disease, but currently there is no available prophylactic vaccine against HCV infection. Here, we investigated the HCV E2- and core-specific T-cell responses induced by DNA (D) and/or recombinant adenovirus (A) vaccines. In single (D versus A) or double immunizations (D-D versus A-A), the recombinant adenovirus vaccines induced higher levels of IFN-gamma secreting T-cell response and cytotoxic T lymphocytes (CTL) response than the DNA vaccines. However, a heterologous (D-A) regimen elicited the highest level of T helper 1 (Th1) CD4(+) T-cell responses. Furthermore, three E2-specific CTL epitopes were mapped using a peptide pool spanning the E2 protein sequence (a.a. 384-713) in BALB/c mice, and one of these (E2 405-414: SGPSQKIQLV) was shown to be immunodominant. Interestingly, no significant differences were found in the repertoire of E2-specific T-cell responses or in the immunodominance hierarchy of the three epitopes induced by D-D, D-A, A-A, and A-D, indicating that the breadth and hierarchy of T-cell responses is independent of these different vaccination regimens. In conclusion, the heterologous DNA prime-recombinant adenovirus boost regimen described offers an efficient promising strategy for the development of an effective T-cell-based HCV vaccine.     

Immunogenic properties of RSV-B1 fusion (F) protein gene-encoding recombinant adenoviruses

Two recombinant adenoviruses designated rAd-F0ΔTM and rAd-F0 carrying the transmembrane truncated and full length of the F gene of the RSV-B1 strain, respectively, were engineered. Comparative immunogenicity studies in BALB/c mice showed that each vector was capable of inducing RSV-B1-specific antibodies that cross-reacted with the RSV-long and RSV-A2 viruses. The anti-RSV-B1 antibodies were neutralizing, and exhibited strong cross-neutralizing activity against the RSV-long and RSV-A2 isolates as well. Analysis of the cellular responses revealed that animals immunized with rAd-F0ΔTM and rAd-F0 elicited CD4⁺ T-cell responses of the Th1 and Th2 phenotypes, as well as F protein-specific CTLs. Production of Th2 cytokines (IL-4, IL-5 and IL-13) by splenocytes of the rAd-F0ΔTM and rAd-F0 immunized mice was markedly lower than those released by animals administered with heat-inactivated RSV-B1 (HIRSV-B1). Comparison of the overall humoral and cellular responses suggests that rAd-F0ΔTM is significantly more immunogenic than rAd-F0. The anti-viral immunity generated by both recombinant adenovirus vectors has conferred protection against live RSV-B1 challenge as judged by the lower viral load recovered in the lungs, a faster rate of recovery of body weight loss, and a lower count of eosinophils as compared to eosinophilia in mice immunized with HIRSV-B1. Results from these studies suggest that rAd-F0ΔTM or rAd-F0 possess immunogenic properties that meet the requirements expected from potential RSV vaccine candidates.     

Expression, processing, and immunogenicity of the structural proteins of Venezuelan equine encephalitis virus from recombinant baculovirus vectors

Recombinant baculoviruses expressing the structural proteins of Venezuelan equine encephalitis virus (VEE) have been constructed and the intracellular processing, antigenicity, and immunogenicity of the expression products have been assessed. Baculoviruses expressing the entire structural protein region (C-E3-E2-6K-E1), or the complete glycoprotein region (E3-E2-6K-E1), generated products in Sf9 cells that were accurately and completely processed, and resulted in mature proteins that were antigenically and electrophoretically indistinguishable from authentic viral proteins. These products were highly immunogenic in BALB/c mice, induced efficient VEE neutralizing responses, and protected these animals against challenge with virulent VEE. Expression of individual glycoprotein regions (E3-E2 and 6K-E1) generated products that were accurately but incompletely processed, and induced non-neutralizing antibodies that reacted more efficiently with denatured than native VEE proteins. Nonetheless, immunization with the 6K-E1 expression product provided complete protection against VEE challenge.     

Neutralization effects of antibody elicited by chimeric HBV S antigen viral-like particles presenting HCV neutralization epitopes

Hepatitis C virus (HCV) infection is a major public health problem despite effectual direct-acting antivirals (DAAs) therapy. Development of a prophylactic vaccine is essential to block spread of HCV infection. The HBV small surface antigen (HBsAg-S) can self-assemble into virus-like particles (VLPs), has higher immunogenicity and is used as a vaccine against HBV infections. Chimeric HBsAg-S proteins with foreign epitopes allow VLP formation and induce the specific humoral and cellular immune responses against the foreign proteins. In this study, we investigated the immune responses induced by chimeric VLPs with HCV neutralizing epitopes and HBV S antigen in mice. The chimeric HCV-HBV VLPs expressing neutralizing epitopes were prepared and purified. BALB/c mice were immunized with purified chimeric VLPs and the serum neutralizing antibodies were analyzed. We found that these chimeric VLPs induced neutralizing antibodies against HCV in mice. Additionally, the murine serum neutralized infections with HCV pseudoparticles and cell-cultured viruses derived from different heterologous 1a, 1b and 2a genotypes. We also found that immunization with chimeric VLPs induced anti-HBsAg antibodies. This study provides a novel strategy for development of a HCV prophylactic neutralizing epitope vaccine and a HCV-HBV bivalent prophylactic vaccine.     

Enhancement of CD4 and CD8 immunity by anti-CD137 (4-1BB) monoclonal antibodies during hepatitis C vaccination with recombinant adenovirus

The induction of protective or therapeutic cellular immunity against hepatitis C virus (HCV) is a difficult goal. In a previous work we showed that immunization with a recombinant adenovirus encoding HCV-NS3 (RAdNS3) could partially protect mice from challenge with a vaccinia virus encoding HCV antigens. We sought to investigate whether systemic administration of an immunostimulatory monoclonal antibody directed against the lymphocyte surface molecule CD137 could enhance the immunity elicited by RAdNS3. It was found that treatment with anti-CD137 mAb after the administration of a suboptimal dose of RAdNS3 enhanced cytotoxic and T helper cell responses against HCV NS3. Importantly, the ability of RAdNS3 to induce protective immunity against challenge with a recombinant vaccinia virus expressing HCV proteins was markedly augmented. Thus, combination of immunostimulatory anti-CD137 mAb with recombinant adenoviruses expressing HCV proteins might be useful in strategies of immunization against HCV.