Evaluation And Optimization Of A Novel HBV Vaccine In An Immunocompetent Mouse Model For HBV Infection

Hepatitis B virus (HBV) infection is a serious public health problem; Over240million people are chronically infected with HBV in the world. Due to introduction of prophylactic vaccines against hepatitis B for mass immunization, the number of new infection cases is continuously decreasing; however,5%to10%of the population does not produce protective antibodies against surface (HBs) antigen, and the prevalence of S variant strains have increased in recent years. Persistent HBV infection and its complications are characterized by impaired HBV-specific immune responses that cannot eliminate the infected hepatocytes, leading to chronic liver cell injury. Therefore, there is a pressing need to develop a therapeutic strategy to prevent, control or cure chronic HBV infection.No appropriate small animal model is available till now because of the strict host restriction of HBV infection. It is imperative to establish a mice model that can simulate HBV infection in human precisely and is convenient for vaccine evaluation. To meet this challenge, we developed a lentiviral-based HBV transducing vector named as pCS-HBV1.3for hydrodynamic injection into immunocompetent mice, in which HBV genes could be continuously expressed and replicated in vivo. To develop a novel HBV vaccine especially therapeutic vaccine, we optimized the immune regimen in mice for various novel HBV vaccine candidates consisting of S-PreSl fusion gene, by combining with recombinant virus vector vaccines or with adjuvant and evaluated antigen-specific immune responses induced in mice; Further, we evaluated the effects of immune protection and immune clearance induced by the novel particle vaccine using the optimal immune regimen in the immunocompetent mouse model for HBV infection.The major results are summarized as below:1. A HBV mouse model could be established by hydrodynamic injection of pCS-HBV1.3into immunocompetent mice. And we found that6-week-old female C57BL/6mice that were injected with5μg pCS-HBV1.3are more suitable for the establishment of HBV persistent infection model.2. We developed three patterns of vaccine candidates in this study, e.g., recombinant vaccinia or adenoviral based HBVvaccines and HBV protein particle vaccine. Among the combinations of vaccination, HBSS1protein prime/RVJSS1boost immunization 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; and also generated more significant level of both CD4+and CD8+T cell responses for Thl cytokines (TNF-a and IFN-γ).In an addition, the immune responses to different adjuvant combinations were assessed in C57BL/6mice by enzyme-linked immunosorbent assay (ELISA), ELISpot and cytokine release assays. Among the combinations tested, a HBV protein particle vaccine with CpG/alum and poly (I:C)/alum priming combinations accelerated specific seroconversion and produced high antibody (anti-PreSl, anti-S antibody) titres with a Thl bias. After boosting with recombinant adenoviral vector vaccine rAdSS1, both groups produced a strong multi-antigen (S and PreS1)-specific cellular immune response. HBSS1immunisation with poly(I:C)/alum priming also generated high-levels CD4+and CD8+T cell responses in terms of Thl cytokines (IFN-γ and IL-2). But CnB/alum priming combinations didn’t showed enhancement of the Ag specific humoral and cellular immune responses.3. Based on the above optimized regimen of HBV vaccine candidates, we evaluated the immune protection in HI mice vaccination with HBV protein particle vaccine HBSS1or HBS both, or combined with different adjuvant, or HBV protein particle vaccine priming and recombinant vaccinia virus boost, or adenovirus vector vaccines boost; The immune responses to different vaccine combinations were assessed in C57BL/6mice by enzyme-linked immunosorbent assay (ELISA), ELISpot, ICS and Luminex. Two weeks after last immunization, all mice were challenged by hydrodynamic injection5μg pCS-HBV1.3to establish HBV infection. The kinetics of serum HBsAg and HBeAg, as well as HBV DNA in serum and liver tissue were analyzed. The results showed that the HBsAg in serum were decreased or cleared; both the HBsAg and HBV DNA in serum were cleared in HBSS1/rAdSS1or HBSS1/RVJSS1combined group. And the levels of serum HBeAg were also reduced. We further found the HBV vaccine induced immune protection responses were not only associated with high titers of S related PreS1-specific antibody response, but also antigen specific cytokines induced by the vaccine.4. We also evaluated the immune clearance effect in the HBV mouse model.6-week-old female C57BL16mice were hydrodynamic injection with5μg pCS-HBV1.3to establish the HBV infection, Five days later, all HI mice were immunized with different HBV vaccine combinations and then the immune responses were also evaluated as described above. And we found that HBSS1combined rAdSS1and RVJSS1immunization could clear the serum HBsAg, reduce the levels of serum HBeAg and HBV DNA. The correlation between the immune response and HBV clearance were analysis. The effect of HBV clearance was closely related with S-or PreSl-specific antibody response as well as Thl cellular immune responses.In summary, we first developed immunocompetent HBV transgenic mice model via hydrodynamic injection with modified lentiviral plasmid; Then we optimized the vaccination strategy of novel HBV vaccine candidates in mice. Finally, we performed the evaluation the immune protection and immune clearance among the HBV mouse model vaccinated with novel HBV vaccine candidates combinations. Our study may provide experimental basis and pave the way for novel HBV vaccine development and applications.