Oral administration of genetically modified Bifidobacterium displaying HCV-NS3 multi-epitope fusion protein could induce an HCV-NS3-specific systemic immune response in mice

More than 170 million people worldwide are chronic HCV (Hepatitis C virus) carriers, and about 30% of them will develop progressive liver disease, such as cirrhosis and hepatocellular carcinoma. A combination of pegylated interferon-α with ribavirin, the standard treatment for HCV infection, has been effective in fewer than 50% of patients infected with HCV genotype 1. A strong T cell response against the nonstructural protein 3 (NS3) is important for recovery from acute HCV infection, and an early multi-specific CD4+ helper and CD8+ cytotoxic T cell response is critical for HCV clearance. In the present study, we successfully constructed a genetically modified Bifidobacterium longum (B. longum) displaying recombinant HCV-NS3 peptides containing some CD4 and CD8 epitopes located in the HCV-NS3 region as an oral vaccine against chronic HCV infection. The oral administration of this vaccine could induce NS3-specific immune responses in mice through intestinal mucosal immunity. Our findings suggest that this novel oral vaccine has great potential as a novel oral vaccine against chronic HCV infection.     

Quantification of the number of cytotoxic T cells specific for an immunodominant HCV-specific CTL epitope primed by DNA immunization

Priming of strong cellular immune responses to hepatitis C (HCV) is thought to be important for eradication of infection. Although productive infection of HCV occurs only reproducibly in humans and chimpanzees, definition of HCV-specific T cell epitopes in mice is necessary to screen efficiently HCV vaccine strategies for their ability to prime cellular immune responses. Out of seven strains of mice screened for immunodominant CTL epitopes against HCV-1a Core, E2, NS5a and NS5b, only one epitope (p214K9) in only one mouse strain was identified. Enumeration of p214K9-specific CD8+ cells by flow cytometry revealed that the number of epitope specific CTL primed by 'naked' DNA immunization was lower than that reported during viral infection. The p214K9 epitope described here, combined with analysis of CTL responses by flow cytometry, should be instrumental in ranking various HCV vaccine strategies for their ability to prime CTL responses.     

Characterization of the humoral and cellular immune responses against hepatitis C virus core induced by DNA-based immunization

Hepatitis C Virus (HCV) causes most cases of posttransfusion hepatitis. Chronic HCV infection is highly related to chronic hepatitis, cirrhosis and hepatocellular carcinoma. Current therapies are only minimally effective and no vaccine has been developed. DNA-based immunization could be of prophylactic and therapeutic value for HCV infection. By intramuscular inoculation in BALB/c mice with an HCV recombinant plasmid pCI-HCV-C, we found significant levels of IgM antibody, but no significant IgG rise. After boost the immunized mice with recombinant HCV-core protein (cp1-10; 1-164aa), the anticore IgG, verified by Western-blotting, rose rapidly, which was two weeks earlier than that with control plasmid. Spleen cells from pCI-HCV-C immunized mice gave higher proliferation index (PI) than control (P < 0.05). The PI of cp1-10 boosted mice was even higher. Proliferation blocking assay with mAb proved the responding cell to be of CD4+ CD8- phenotype, supporting specific priming of T helper cells. A 51Cr-releasing CTL assay specific for HCV-core was developed, and a specific CTL response against HCV-core was demonstrated in both pCI-HCV-C immunized mice and mice boosted with cp1-10. Strong cytotoxic activity against peptide-pulsed p815 cells (H-2d), but not EL-4 cells (H-2b), suggested MHC class I restriction of the CTL activity. Blocking of CTL with mAb proved the effector cells to be of CD4- CD8+. Three CTL epitopes in HCV-core protein were demonstrated. We failed to detect CTL when immunized only with core protein. The results suggested that vaccination with HCV-core derived DNA sequences could be an effective method to induce humoral and cellular immune responses to HCV.     

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.     

Induction of CD8+ T-cell responses against subunit antigens by the novel cationic liposomal CAF09 adjuvant

Vaccines inducing cytotoxic T-cell responses are required to achieve protection against cancers and intracellular infections such as HIV and Hepatitis C virus. Induction of CD8+ T cell responses in animal models can be achieved by the use of viral vectors or DNA vaccines but so far without much clinical success. Here we describe the novel CD8+ T-cell inducing adjuvant, cationic adjuvant formulation (CAF) 09, consisting of dimethyldioctadecylammonium (DDA)-liposomes stabilized with monomycoloyl glycerol (MMG)-1 and combined with the TLR3 ligand, Poly(I:C). Different antigens from tuberculosis (TB10.3, H56), HIV (Gag p24), HPV (E7) and the model antigen ovalbumin were formulated with CAF09 and administering these vaccines to mice resulted in a high frequency of antigen-specific CD8+ T cells. CAF09 was superior in its ability to induce antigen-specific CD8+ T cells as compared to other previously described CTL-inducing adjuvants, CAF05 (DDA/trehalose dibehenate (TDB)/Poly(I:C)), Aluminium/monophosphoryl lipid-A (MPL) and Montanide/CpG/IL-2. The optimal effect was obtained when the CAF09-adjuvanted vaccine was administered by the i.p. route, whereas s.c. administration primed limited CD8+ T-cell responses. The CD4+ T cells induced by CAF09 were mainly of an effector-memory-like phenotype and the CD8+ T cells were highly cytotoxic. Finally, in a mouse therapeutic skin tumor model, the HPV-16 E7 antigen formulated in CAF09 significantly reduced the growth of already established subcutaneous E7-expressing TC-1 tumors in 38% of the mice and in a corresponding prophylactic model 100% of the mice were protected. Thus, CAF09 is a potent new adjuvant which is able to induce CD8+ T-cell responses against several antigens and to enhance the protective efficacy of an E7 vaccine both in a therapeutic and in a prophylactic tumor model.     

HCV感染者和HCV/HIV合并感染者免疫调节

目的　探讨我国单纯丙型肝炎病毒(HCV)感染者和HCV/HIV合并感染者免疫应答的相关机制。方法　分离人外周血单个核细胞;流式细胞仪(FACS)检测CD4 + T细胞和CD4 + CD2 5 + T细胞表达水平。结果　CD4 +CD2 5 + T细胞占外周血单个核细胞中CD4 + T细胞比例(%CD4 + CD2 5 + ) ,HCV感染组明显高于健康对照组(19. 2 %>13 8% ,P <0 . 0 5 ) ,HCV/HIV合并感染组明显低于对照组(6. 9% <13. 8% ,P <0 .0 0 1) ,更明显低于HCV感染组(P <0 . 0 0 1)。结论　HCV感染者体内CD4 + CD2 5 + T细胞增殖明显,提示CD4 + CD2 5 + T细胞在HCV慢性感染中发挥免疫调节作用。HCV/HIV合并感染时,CD4 + CD2 5 + T细胞受损,从而影响免疫调节功能。     

A self-adjuvanting lipopeptide-based vaccine candidate for the treatment of hepatitis C virus infection

Effective CD8(+) T cell responses have been induced using totally synthetic self-adjuvanting lipopeptides containing the dipalmitoyl-S-glyceryl cysteine lipid moiety, which is a ligand for Toll-like receptor 2 (TLR2) on dendritic cells (DC). In this study, we evaluated the use of lipopeptide vaccine candidates containing HLA-A2-restricted epitopes for DC-based immunotherapy of HCV infection. Lipopeptides were able to induce specific CD8(+) T cell responses in HLA-A2 transgenic mice and consistently activated human monocyte-derived DC from both healthy individuals and HCV infected patients. Lipopeptide-pulsed human DC were also found to secrete the pro-inflammatory cytokine IL-12p70 and were able to activate antigen-specific IFN-gamma production by autologous CD8(+) T cells obtained from a hepatitis C patient. These results show that DC from HCV patients can be matured and antigen loaded with TLR2-targeting lipopeptides for effective presentation of CD8(+) T cell epitopes; the use of autologous lipopeptide-pulsed DC or direct lipopeptide vaccination may be successful approaches for the priming or boosting of anti-HCV CD8(+) T cell responses to aid in the clearance of the virus in chronically infected individuals.     

DNA vaccine expressing the non-structural proteins of hepatitis C virus diminishes the expression of HCV proteins in a mouse model

Most of the people infected with hepatitis C virus (HCV) develop chronic hepatitis, which in some cases progresses to cirrhosis and ultimately to hepatocellular carcinoma. Although various immunotherapies against the progressive disease status of HCV infection have been studied, a preventive or therapeutic vaccine against this pathogen is still not available. In this study, we constructed a DNA vaccine expressing an HCV structural protein (CN2), non-structural protein (N25) or the empty plasmid DNA as a control and evaluated their efficacy as a candidate HCV vaccine in C57BL/6 and novel genetically modified HCV infection model (HCV-Tg) mice. Strong cellular immune responses to several HCV structural and non-structural proteins, characterized by cytotoxicity and interferon-gamma (IFN-γ) production, were observed in CN2 or N25 DNA vaccine-immunized C57BL/6 mice but not in empty plasmid DNA-administered mice. The therapeutic effects of these DNA vaccines were also examined in HCV-Tg mice that conditionally express HCV proteins in their liver. Though a reduction in cellular immune responses was observed in HCV-Tg mice, there was a significant decrease in the expression of HCV protein in mice administered the N25 DNA vaccine but not in mice administered the empty plasmid DNA. Moreover, both CD8⁺ and CD4⁺ T cells were required for the decrease of HCV protein in the liver. We found that the N25 DNA vaccine improved pathological changes in the liver compared to the empty plasmid DNA. Thus, these DNA vaccines, especially that expressing the non-structural protein gene, may be an alternative approach for treatment of individuals chronically infected with HCV.     

Hepatitis C immunology

This review summarizes what is currently known about the cellular and humoral immune responses to hepatitis C virus. The results of viral protein immunogenicity studies reported so far are analyzed, and points of controversy about the immunology and immunopathology of hepatitis C are discussed. A body of evidence supports the existence in hepatitis C virus-infected subjects of strong humoral and cellular immune responses. HCV Core, NS3 and NS4 proteins are the most immunogenic antigens for B cells and HLA class II-restricted CD4+ T cells. For its part, liver-infiltrating CD8+ CTL recognize epitopes within the Core, E1, E2/NS1 and NS2 proteins in a HLA class I restricted manner. CTL responses to HCV Core, NS3, NS4 and NS5 have been detected in peripheral blood of patients chronically infected with HCV. Although these B and T cell responses may contribute to clear the virus, in most cases they are unable to resolve chronic HCV infections. No protective B or T cell epitope has been found yet. It is becoming clearer that the immune response plays a fundamental role in the pathogenesis of liver disease in hepatitis C patients.     

Enhanced cellular immune responses to SIV Gag by immunization with influenza and vaccinia virus recombinants

An effective vaccination strategy against human immunodeficiency virus type 1 (HIV-1) should include the induction of potent cellular immune responses against conserved HIV-1 antigens. We have generated five replication competent recombinant influenza viruses (rFlu/SIV Gag nos. 1-5) expressing different portions of Gag of simian immunodeficiency virus (SIV). Single intranasal immunizations in mice with each rFlu/SIV Gag viruses resulted in different degrees of protection against a challenge with recombinant vaccinia virus expressing SIV Gag. Immunized BALB/c mice had detectable CD8+ T cell responses specific for Gag peptide 185-199 when mice were vaccinated with rFlu/SIV Gag no. 3 virus, and for Gag peptides 281-295 and 285-299 when vaccinated with rFlu/SIV Gag no. 4 virus. Cellular immune responses against SIV Gag were further enhanced by a booster with a recombinant vaccinia virus expressing SIV Gag in both the spleen and local lymph node tissues, resulting in the induction of robust Gag-specific CD8+ T cell responses at both systemic and mucosal levels. We suggest that a prime-boost immunization regimen using recombinant influenza and vaccinia viruses expressing HIV Gag might represent an effective means to induce potent HIV-specific, protective CD8+ T cell responses.     

Preclinical development of peptide vaccination combined with oncolytic MG1-E6E7 for HPV-associated cancer

Human papilloma virus (HPV)-associated cancer is a significant global health burden and despite the presence of viral transforming antigens within neoplastic cells, therapeutic vaccinations are ineffective for advanced disease. HPV positive TC1 cells are susceptible to viral oncolysis by MG1-E6E7, a custom designed oncolytic Maraba virus. Epitope mapping of mice vaccinated with MG1-E6E7 enabled the rational design of synthetic long peptide (SLP) vaccines against HPV16 and HPV18 antigens. SLPs were able to induce specific CD8+ immune responses and the magnitude of these responses significantly increased when boosted by MG1-E6E7. Logically designed vaccination induced multi-functional CD8+ T cells and provided complete sterilising immunity of mice challenged with TC1 cells. In mice bearing large HPV-positive tumours, SLP vaccination combined with MG1-E6E7 was able to clear tumours in 60% of mice and these mice were completely protected against a long term aggressive re-challenge with the TC1 tumour model. Combining conventional SLPs with the multi-functional oncolytic MG1-E6E7 represents a promising approach against advanced HPV positive neoplasia.     

Involvement of CD8+ T cell-mediated immune responses in LcrV DNA vaccine induced protection against lethal Yersinia pestis challenge

Yersinia pestis (Y. pestis) is the causative pathogen of plague, a highly fatal disease for which an effective vaccine, especially against mucosal transmission, is still not available. Like many bacterial infections, antigen-specific antibody responses have been traditionally considered critical, if not solely responsible, for vaccine-induced protection against Y. pestis. Studies in recent years have suggested the importance of T cell immune responses against Y. pestis infection but information is still limited about the details of Y. pestis antigen-specific T cell immune responses. In current report, studies are conducted to identify the presence of CD8+ T cell epitopes in LcrV protein, the leading antigen of plague vaccine development. Furthermore, depletion of CD8+ T cells in LcrV DNA vaccinated Balb/C mice led to reduced protection against lethal intranasal challenge of Y. pestis. These findings establish that an LcrV DNA vaccine is able to elicit CD8+ T cell immune responses against specific epitopes of this key plague antigen and that a CD8+ T cell immune response is involved in LcrV DNA vaccine-elicited protection. Future studies in plague vaccine development will need to examine if the presence of detectable T cell immune responses, in particular CD8+ T-cell immune responses, will enhance the protection against Y. pestis in higher animal species or humans.     

The immune response of mice and cynomolgus monkeys to macaque mucin 1-mannan

Mice immunised with human epithelial mucin MUC1 coupled to oxidised mannan produce MUC1 specific MHC Class 1 restricted CD8⁺ cytotoxic T cells and are completely protected from the development of MUC1⁺ tumours; such therapy may be applicable to humans. In this light we describe pre-clinical studies in cynomolgus monkeys (Macaca fascicularis), to test the efficacy of mannan-MUC1 in higher primates. Monkey MUC1 genomic clones were isolated from a macaque library, peptides and fusion protein synthesised and mice and monkeys immunised with macaque MUC1-mannan. In mice CTL responses were induced (as has been found with human MUC1 mannan conjugates), but in contrast monkeys produced a humoral response, with no T cell proliferative, cytotoxic responses or CTLp found. In spite of the presence of anti-MUC1 auto-antibodies, there was no toxicity or induction of autoimmunity.     

Vaccination with human papillomavirus-18 E1 protein plus α-galactosyl-ceramide induces CD8+ cytotoxic response and impairs the growth of E1-expressing tumors

CD8+ T cell-mediated immune response plays a major role in the clearance of virus-infected cells, including human papillomavirus (HPV). The effective treatment of women with normal cytology but persistent high risk-HPV infection or with low-grade intraepithelial lesions could take advantage of novel strategies based on vaccination with viral immunological targets with a wide spectrum of cross-protection. The helicase E1, expressed early during viral replication in HPV infection, is among the most conserved papillomavirus proteins, which makes it a good vaccine candidate. In the present study, we examined E1-specific CD8+ T cell and NK immune responses in a mouse model with α-galactosylceramide (α-GalCer) as an adjuvant. We found that mice immunized with E1 combined with α-GalCer elicited an E1-specific CD8+ T and NK cell cytotoxic responses, which correlated with growth inhibition of grafted melanoma B16-F0 cells expressing E1, both in prophylactic and therapeutic protocols.     

Codelivery of PEG-IFN-alpha inhibits HCV DNA vaccine-induced T cell responses but not humoral responses in African green monkeys

Although pegylated interferon alpha (PEG-IFN-alpha) with ribavirin treatment constitutes an effective means of treatment for chronic hepatitis C, novel approaches are needed due to the inefficient effects of the current therapy against chronic infection with genotype 1 virus. In this study, the immunomodulatory effects of PEG-IFN-alpha on multigenic HCV DNA vaccine-induced immunity were investigated in African green monkeys. Multigenic HCV DNA vaccination with and without PEG-IFN-alpha was safe and well tolerated, and induced significant long-term T cell and antibody responses. In addition, the induced immune responses were gradually increased by repeated injection. Interestingly, co-treatment with PEG-IFN-alpha significantly suppressed HCV DNA vaccine-induced T cell responses, but not antibody responses, which demonstrated that IFN-alpha could act as a negative regulator of T cell immune induction. However, the suppression of T cell responses by PEG-IFN-alpha could be overcome by two times more DNA vaccination, which suggests that combined therapy of DNA vaccine with PEG-IFN-alpha might be possible. Our results provide valuable information for the design of an effective therapeutic regimen to treat chronic HCV infection and to understand the immunomodulatory roles of PEG-IFN-alpha in immune induction by DNA vaccination.     

Induction of humoral and cellular responses in cynomolgus monkeys immunised with mannan-human MUC1 conjugates.

Mice immunised with oxidised mannan conjugated to the human mucin 1 (MUC1), produce MHC Class 1 restricted CD8+ cytotoxic T-cells which eradicate MUC1 + tumours, indicating potential for the immunotherapy of MUC1 + cancers in humans. We now describe preclinical studies performed in cynomolgus monkeys immunised with human or murine MUC1 conjugated to oxidised mannan, where immune responses and toxicity were examined. High titred antibodies specific for MUC1 were produced, MUC1 specific CD4+ and CD8+ T-cell proliferative responses and specific cytotoxic precursor cells (CTLp) were found, but not MUC1 specific cytotoxic T-cells (CTL). There was no toxicity and monkeys can be immunised against human MUC1 with mannan-MUC1 conjugates, but a humoral response (Th2 type) predominates. The results contrast with those obtained in mice when a CTL response (Th1 type) predominates.     

Immunogenicity of the E1E2 proteins of hepatitis C virus expressed by recombinant adenoviruses

The E1 and E2 proteins of hepatitis C virus (HCV) are believed to be the viral envelope glycoproteins that are major candidate antigens for HCV vaccine development. We reported previously that the replication-competent recombinant adenovirus encoding core-E1-E2 genes of HCV (Ad/HCV) produces serologically reactive E1 and E2 proteins forming a heterodimer in substantial amounts. Here, we examined immunogenicity of the E1E2 proteins copurified from HeLa cells infected with Ad/HCV virus in mice. Furthermore, we constructed a replication-defective recombinant adenovirus encoding the core-E1-E2 genes of HCV (Ad.CMV.HCV) and examined immunogenicity of the virus in mice. The mice immunized intraperitoneally with the copurified E1E2 proteins induced mainly antibodies to E2, but not to E1 by Western blot analysis. The sera of mice immunized with the E1E2 inhibited the binding of E2 protein to the major extracellular loop of human CD81. E2-specific cytotoxic T cells (CTLs), but not antibodies to the E1E2 antigens were induced in the mice intramuscularly immunized with Ad.CMV.HCV virus. When immunized with both Ad.CMV.HCV virus and the E1E2, mice elicited E2-specific CTLs and antibodies to the E1E2 antigens. The results suggest that immunization of Ad.CMV.HCV virus combined with E2 protein is an effective modality to induce humoral as well as cellular immune response to E2 antigen.     

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.     

Immunotherapy with CTL peptide and VSSP eradicated established human papillomavirus (HPV) type 16 E7-expressing tumors

Peptide-based vaccines aimed at the induction of effective T-cell responses against established tumors have not been successful in clinic and require the use of new adjuvants. One of those is a new adjuvant in which gangliosides are incorporated into the outer membrane protein complex of Neisseria meningitidis to form very small size proteoliposomes (VSSP). In a preclinical model of human papillomavirus HPV16-induced cervical cancer we show that vaccination with HPV 16 E7 derived minimal CTL peptide and VSSP protects mice against tumor challenge, induces regression of established tumors and produces E7-specific CD8⁺ T-cell responses.     

An unbiased peptide-wide discovery approach to select Mycobacterium tuberculosis antigens that target CD8+ T cell response during infection

Accruing data strongly support the possible role of CD8+ T cells in immunity against tuberculosis (TB). Multivalent vaccines against Mycobacterium tuberculosis (Mtb) that incorporate CD8+ T cell antigens with those that elicit CD4+ T cells are therefore highly desirable. To screen for potential CD8+ T cell antigens that are produced by Mtb during infection, we isolated pathogen-derived peptides that bound to MHC Class I molecules expressed in adherent splenocytes obtained from Mtb-infected mice. Mass spectroscopy analysis revealed the following four nonamer peptides that had 100% homology with Mtb proteins: DGYVGAPAH (MT_0401), TTMPLFAD (MT_1164), RSGAATPVR (MT_2160.1) and LAAVVGVVL (MT_0078). The gene MT_0401 codes the protein 5′-phosphoribosylglycinamide transformylase 2 and the other three genes code for hypothetical proteins with unknown function. The NCBI/Blast analysis showed that among the four peptides DGYVGAPAH had the highest maximum alignment score and lowest E value (number of alignments expected by chance). Therefore, we assessed whether MT_0401 expressed in two genetic vaccine formulations was capable of stimulating CD8+ T cell response that is specific to DGYVGAPAH peptide. When mice were immunized with a recombinant plasmid DNA and an E1/E3-deleted Adenovirus 5 expressing MT0401 protein, using both homologous and heterologous prime-boost protocols, they developed strong DGYVGAPAH-specific CD8+ T cell response as well as antibody and CD4+ specific T cell response to the full length MT0401 protein. Equally important was the observation that mice infected with Mtb developed DGYVGAPAH-specific CD8+ T cell responses in both spleen and lungs. These results demonstrate that Mtb antigens that are processed and presented via MHC Class I machinery can be readily identified by the described approach and may be useful candidate antigens to stimulate specific CD8+ T cell responses in vaccine development programs.