Development And Immunological Evaluation In Mice Of Several Novel Vaccine Candidates Against Hepatitis C Virus Infection

As one of the acute and chronic hepatitis pathogens, Hepatitis C virus (HCV) was identified in 1989 mainly transmitted via transfusion or needle-share. Among all HCV infected individuals, 80% of them remain chronically infected,10%-20% of them develop cirrhosis, and more than 20% of them acquire liver cancer over years. According to statutory infectious disease surveillance report announced by the Chinese Center for Disease Control and Prevention, HCV infected more than 150,000 in china in 2010, with an increase by 15.49% compared with the number in 2009. HCV infection and transmission have been a global public health problem, and in theory, HCV vaccine is the most cost-effective strategy for hepatitis C control and prevention. HCV is a positive-sense single-stranded RNA virus, encoding a polypeptide about 3011aa which is cleaved into 10 proteins by host and viral protease, including structural proteins (C/E1/E2/p7) and non-structural proteins (NS2/NS3/NS4A/NS4B/NS5A/NS5B). There is a large HCV genome heterogeneity, at least 7 major genotypes and about 70 subtypes. The major epidemic subtypes in China are subtypes of lb and 2a, and other subtypes were also reported. The presence of quasispecies is one of the mechanisms leading host immune evasion and chronic HCV infection. This is a serious issue impact on the HCV vaccine development. The lack of an efficient in vitro replication and infection model system has long hampered the characterization of neutralizing antibodies and functional studies of viral variants escaping B and T cell responses. Studies suggest that except for broad spectrum neutralizing antibodies, the broad spectrum cellular immunity is also critical to improve the efficiency of HCV vaccine. Employing broad spectrum of cross reactive antigens, combined immunization with different antigens and a prime-boost regimen will become important strategies for HCV vaccine development.There are many similar characteristics between HCV and HIV infection (persistence, highly variable genome and immune evasion mechanisms, the lack of cross neutralizing antibody, etc.). HCV vaccine development has experienced many challenges. Strategies been explored so far include:peptide vaccines, recombinant subunit vaccines, DNA vaccines, recombinant viral vector vaccines (mainly include recombinant adenovirus and recombinant vaccinia virus), VLP and a variety of combination with different vaccines, but none of them (especially prophylactic vaccine) showed an ideal immunity and applied to a large scale population. For chimpanzees is the only HCV infectious animal model, several experiments based on chimpanzees in recent years showed that:combined immunization of recombinant adenovirus and DNA vaccine carrying non-structural proteins or the VLP vaccine formed by structural protein (C/E1/E2) with adjuvant achieved a certain degree of protection which is promising for HCV vaccine development. A Meta-Analysis of the protective effectiveness about more than ten HCV experimental vaccines in chimpanzees indicated that structural proteins are able to induce T cell response which mediated the virus clearance.According to the needs and status of HCV vaccine development and the latest progress of DNA vaccine, viral vector vaccines, integration-defective lentiviral particle vaccines and combined immunization, this investigation selected HCV structure protein (core, E1, E2)and non-structure protein NS3 as target antigen, and used varied forms of vaccines:DNA vaccines, viral vector vaccines, lentiviral particle vaccines, systematically analyzed the characteristics of the different vaccine immune response, assessed the immune effectiveness via alternative heterogeneous challenge model (replicating vaccinia virus rTTV-JFH 1(3011) expressing the full ORF of subtype 2a), in order to obtain antigen components and immunization programs of HCV vaccines with strong immune response and broad spectrum protective effect.To achieve the purposes above, this paper conducted the following investigations:1. Preparation and identification of DNA vaccines, viral vector vaccines, integration-deficient pseudotyped particles vaccines.2. Analysis of immune responses and protective effect of different vaccines. The main results are as follows:1. Development and identification of HCV DNA vaccines, viral vector vaccines, integration-deficient pseudotyped lentiviral particles vaccines(IDLVpp).a. Various DNA vaccines were constructed. Conventional DNA vaccine vector pVRC and replicating DNA vaccine vector pSC or pSCK were employed. Based on previous work, DC targeting NS3 (DEC-NS3 fusion protein) or E2 (DEC-E2 fusion protein) was choose as target antigen, pVRC-DEC-NS3, pVRC-DEC-E2 and pSC-DEC-NS3 were constructed. At the same time, with the deletion of hypervariable region HVR1 and codon optimization, CE1E2Y and H155 (fusion CE1E2 with NS3) were synthesized, and cloned into replicating DNA vaccine vector pSCK respectively, constructed two novel HCV DNA vaccines(pSCK-CE1E2Y and pSCK-H155). Endonucleases digestion and DNA sequencing confirmed the structure; Western blot confirmed the expression of the target antigens. These DNA vaccines were extracted for further immunization.b. Development of the recombinant adenovirus expressing HCV structural gene (C/E1/E2) or expressing partial of NS3 and core (C44P) fusion gene (rAd5-CE1E2 and rAd5-C44P). Development of the replicating and non-replicating recombinant vaccinia TianTan strain viruses expressing NS3 or CE1E2NS3(H155), and replicating recombinant vaccinia TianTan strain virus expressing full-ORF of HCV 2a subtype JFH1 strain. Western blot and indirect immunofluorescence assay(IFA) identified that the target proteins are correctly expressed in host cell infected with recombinant virus vaccines.c. Modification of the three plasmid-base lentiviral system and development of HCV IDLVpp. Constructed integration-defective packaging plasmid of lentiviral system, and confirmed the integration-defective properties in vitro. The reporter gene (GFP) in transfer plasmid was replaced by the HCV non-structural gene NS3. HCV envelope plasmid with different subtypes (from three representative subtypes 1a, 1b,2a) combined with the above two modified lentiviral plasmids, HCV integration-defective lentiviral particle (IDLVpp) vaccines carrying HCV NS3 were packaged. The HCV IDLVpp were identified by Western blot, and the particle structure was observed by electron microscope. HCV integration-defective lentiviral particles(HCV IDLVpp) were titrated according to p24 quantification2. Characteristics of of immune responses and protective effect of different HCV vaccine candidates in micea. In order to analyze the immunological responses of HCV DNA vaccines, two DNA vaccines of HCV based on NS3 and CE1E2, were selected.1) The characteristics of immunological responses of NS3-based DNA vaccines, non-replicating DNA vaccine pVRC-DEC-NS3 and replicating DNA vaccine pSC-DEC-NS3, were compared. The results showed that NS3 antibody induced by pVRC-DEC-NS3 (3000 above) and IFN-γ(above 200 SFC) are higher than pSC-DEC-NS3.2) CE1E2-based DNA vaccines(pSCK-CE1E2Y and pSCK-H155)with replicating DNA vector backbone. Our data showed that antigen specific humoral and cellular immune response can detected by ELISA and ELISPOT after twice immunization with HCV DNA vaccine pSCK-CE1E2Y and pSCK-H155. Compared with twice immunization, the third immunization boosted the antibody level of E1, E2, core and NS3, but the cellular immune response did not increase. The E1, E2 antibody level was lower, and cross-neutralizing antibody was not detected. Experiments of alternative heterogeneous challenge model showed that no cross-protection was observed in mice with above HCV DNA vaccine immunization alone.b. In order to analyze the immune response of HCV recombinant adenovirus vaccine candidates with different inoculation routes and immunization program, mice were immunized with rAd5-C44P and rAd5-CE1E2 respectively. The results showed that intramuscular (i.m.)or intradermal (i.d.) injection of rAd5-CE1E2 vaccines were able to induce robust cellular response specific to HCV E1 and E2 antigen(by ELISPOT), mainly CD8+ CTL response (by ICS). Both of the cellular responses induced by rAd5-CE1E2 intranasal(i.n.) immunized alone or intranasal prime plus intramuscular boost were lower than other groups, but the anti-core specific antibody induced by intranasal prime intramuscular boost was significantly higher than other groups. Experiments of alternative heterogeneous challenge model showed HCV recombinant adenovirus vaccines with different immunization programs have protective effect, but the immune responses induced by recombinant adenovirus vaccine expressing different antigens were varied. Twice immunized with rAd5-C44P did not increase the level of single-dose response, and the cellular immune response decreased, while the specific cellular immune response to core, E1, E2 were strengthen by rAd5-CE1E2 boost, and could last for long time.c. In order to analyze the immune response of HCV IDLVpp, three HCV IDLVpp with different subtype (1a, 1b,2a) were packaged. Comparison among sequential immunization, mixed immunization and immunization with the same subtype showed that HCV IDLVpp alone could induce humoral immune response including the IgG antibody to E1, E2, NS3 and cross-neutralizing antibodies to subtype 5a HCV pp, which would be gradually increased along with times of HCV IDLVpp immunization. Antibody to NS3 and cross-neutralizing antibody to subtype 5a HCV pp induced by sequential immunization with different subtype or mixed immunization were higher than the immunization with same subtype. The addition of adjuvant (Al(OH)3+CpG) had no benefit effect on the immunity of HCV IDLVpp. We also compared the immune response characteristics of HCV IDLVpp involved combined immunization. DNA (pVRC-DEC-E2) primed twice and HCV IDLVpp boost, significantly increased the humoral immunity to E2 antigen induced by DNA vaccine alone. rAd5-CElE2 prime and HCV IDLVpp boost regimen can induce cellular and humoral immune responses against multiple target antigens (core, E1, E2, NS3) which would be gradually increased along with times of HCV IDLVpp immunization. We also compared the regimens between rAd5-CE1E2 prime plus HCV IDLVpp boost and that HCV IDLVpp prime plus rAd5-CE1E2 boost. The results showed that in view of the immune response (antibody level against E2 and SFC against El), the former is stronger than the latter.d. In order to analyze the immune response of combined immunization with DNA vaccines and recombinant viral vector vaccines, several vaccine programs were selected:including:1) DNA vaccines prime and recombinant adenovirus vaccines boost, based on NS3 or CE1E2; 2) DNA vaccines prime and recombinant vaccinia Tiantan vaccines (replicating or non-replicating) boost. The specific binding antibodies were detected by ELISA, antigen-specific IFN-g secretion detected by ELISPOT, various cytokines (IL-2, IFN-g, IL-4, TNF-a) in CD4+ or CD8+ T cells were detected by ICS, protective efficacy was evaluated by alternative heterogeneous challenge model or in vivo CTL. Systematic analysis of immunological responses and protective efficacy for combined immunization programs were performed. These results showed that the combined immunization with DNA vaccines and recombinant adenovirus vector vaccines can induce higher humoral and cellular immune response and have cross-protection against heterogeneous vaccinia. The cross-protection produced by replicating plasmid (pSC-DEC-NS3) priming group was better than conventional DNA vaccines (pVRC-DEC-NS3) priming group. Although the antigen specific binding antibody induced by pSC-DEC-NS3 was lower obviously than the latter, the pattern of intracellular cytokine secretion was varied. Antibody level and cellular response (results from ELISPOT and ICS) induced by DNA vaccine (pSCK-CE1E2Y) twice plus rAd5-CE1E2 boost regimen was different from the group that immunized DNA vaccine three times. IFN-g secretion against core and E1, TNF-αsecretion of CD4- T cells, IFN-g secretion of CD8+ T cells, IFN-g and IL-2 secretion of E2 specific CD4+ T cells, IFN-g and TNF-a secretion of CD8+ T cells induced by pSCK-CE1E2Y prime twice plus rAd5-CE1E2 boost were higher than the group immunized DNA vaccine three times. Combined immunization with DNA vaccines and recombinant vaccinia vaccines can also induce higher humoral and cellular immune response, and higher killing rate of in vivo CTL. For pSC-DEC-NS3 priming plus HCV recombinant vaccinia TianTan strain vaccine boosting, both target specific antibody level (ELISA, IgG) and NS3 specific cellular immunity (ELISPOT, IFN-g) were induced. The results of ICS indicated that a variety of cytokines secretion (IL-2, IFN-g, IL-4, TNF-a) of CD4+ and CD8 +T cells were induced against NS3-1 and NS3-2 peptide pools, in all, the immunity of replicating TianTan vaccinia (rTTV) vaccines boosting group were higher than non-replicating TianTan vaccinia (rTTVACK) vaccines boosting group. The killing rate of CTL activated by NS3 in rTTV boosting group was 16%, while rTTVACK boosting group were 10%. For pSCK-H155 priming, recombinant vaccinia vectors boosting could increase the cellular immunity but little impact on the antibody to E1 and E2. In the two groups, little cross-neutralization antibody were detected, but the antibody to core and NS3 was increased by 4 times (rTTVACK boosting) to 32 times (rTTV boosting). The rTTV△CK boosting group mainly produced E1 and E2 specific IFN-g (CD8 +T cells), NS3 specific TNF-a and IL-2 (CD4+ and CD8+T cells). In vivo CTL studies showed that the killing rate of CTL activated by E1 was 99.7%(rTTV boosting) and 99.76%(rTTVACK boosting).In summary, we developed a variety of novel HCV vaccine candidates based on structural gene(core, E1, E2)or non-structural gene NS3, systematically analyzed the immune response characteristics of these vaccines in mouse model, and established comprehensive methods for evaluation of HCV immunology. These vaccines immunized alone or in combination can induce antigen specific humoral and/or cellular immune response. The immune responses and immune protection were varied with different inoculation routes and components. In combined immunization programs, recombinant viral vector vaccines can induce potent cellular immune responses and cross-protective immunity. HCV integration-defective lentiviral particle vaccines (HCV IDLVpp) alone or in combination (DNA or recombinant adenovirus) application can induce potent humoral immune responses (including cross-neutralizing antibodies). Many innovative results reported for the first time in this thesis provide a scientific and experimental basis for novel effective HCV vaccines development and applications of combination and optimization with different vector vaccines.