Study On The Expression And Immunogenicity Of HIV-1 Crossing Neutralizing Epitopes Fused With HBV S Protein

Since the discovery of AIDS in 1981, the global spread of HIV has reached pandemic proportions, representing a global developmental and public health threat. Although the propaganda and education, behavior intervention and antiretroviral drugs have led to a remarkable progress in prevention and control of AIDS, the development of a safe, globally effective and affordable HIV vaccine will offer the best hope for the future control of the pandemic. Because of the unique aspects of the virus itself, i.e. high variation, attacking immune system, resulting in persistence infection and inducing immunopathogenesis damage, HIV vaccine research is facing with unprecedented difficulties and challenges. It's the prime approach to develop the cross-protection HIV-1 vaccine, using the antigen inducing different subtype HIV protective immune response, as the main target of vaccine. Finding the antigen is a key and hard job in HIV vaccine study field. In recent 20 years, substantive breakthroughs haven't been achieved on the study. HIV clinical experiment's failure using major neutralizing antigen Env or major cellular immunization antigen Gag illustrated that finding HIV vaccine target antigen was very key and difficult.Different from the study strategy using one or more intact or modified Env as target antigen of HIV vaccine in recent years, our study tried to use broadly neutralizing activity subdominant antigens or internal conserved antigens in the process of natural infection, as the main target of vaccine. As the immunogenicity of these antigen epitopes is very weak, we bring forward such point: It can't be considered as the vaccine antigen, which can't induce strong immune response. The antigen must induce strong immune response, which is the base of successful this virus vaccine. It must be proved whether the assumption is right or not. Central to the strategy is how to prove this assume.The strategy of our investigation is: To enhance immunogenicity of HIV-1 cross neutralizing epitopes, three HIV-1 cross neutralizing epitopes(ELDKWA, NWFDIT, GPGRAFY) or the three-epitope concatemer were fused to 3' end of HBV S gene by PCR cloning technology, respectively. Different immune vectors will be selected to express these antigens separately. Different intensity immune response to these antigens in the mouse model will be explored, using different combinations of antigens and immune vectors, such as single antigen, single antigen in different vectors (prime-boost). Combination antibodies and neutralizing antibodies were evaluated. The major results were summarized as follow:Firstly, Four vaccinia virus (Tiantan strain) recombinants expressing separately the four fusion genes were constructed, named as RVJ1175S-2F5(ELDKWA), RVJ1175S-4E10(NWFDIT), RVJ1175S-44752D(GPGRAFY) and RVJ1175S-multiepitopes (the three-epitope concatemer) respectively. It was confirmed by PCR and sequencing that the fusion genes were inserted into the TK locus of vaccinia virus Tiantan strain correctly. The fusion proteins were expressed efficiently and secreted into supernatant of the infected cells, which was demonstrated by HBsAg ELISA test. Immunofluorescence proved the well expression of the four fusion protein in rVV-infected Hela cells.Secondly, From the supernatants of CEF cells infected by these vaccinia recombinants, four subunit vaccines (PS-2F5, PS-4E10, PS-447-52D and PS- multiepitopes) were prepared after purification. Two typical HBsAg bands of 23kD and 27kD were detected in all the purified samples by SDS-PAGE. These two electrophoresis bands were reacted well with HBsAb and corresponded HIV-1 monoclonal antibodies 2F5,4E10 and 447-52D.Thirdly, Four DNA plasmids expressing separately HIV-1 crossing neutralizing epitopes and HBV S fusion genes were constructed. It was confirmed by restriction digest and sequencing that the fusion genes were correct. ELISA proved the well expression of the four fusion proteins and secreted into supernatant of the transient transfected Hela cells.Fourthly, Construction of HIV-1 pseudotyped virus neutralizing test platformThe lentiviral vector system used in this study consists of the backbone plasmid (pSG3deltaEnv), and the envelop plasmid encoding the HIV-1 subtype B or C Env protein (pcDNA3.1-Env). These plasmids were cotransfected in 293FT cells. Five HIV pseudotyped viruses were successfully packaged, named as SVPB6 (subtype B) , SVPB12 (subtype B) , SVPB13 (subtype B) , SVPB8 (subtype B) , SVPC5 (subtype C) separately. Neutralizing test results showed the five pseudotyped viruses could be neutralized by monoclonal antibodies 2F5, 4E10 and 447-52D.Fifthly, BALB/c mice were immunized with subunit vaccine or recombinant vaccinia vaccine or DNA vaccine separately or combined immunization with different type vaccines. High levels of HBsAb and anti-HIV-1 cross neutralizing epitope combination antibodies in peripheral blood of vaccinia virus and subunit vaccine immunized mice were tested by ELIS A, and all the antibody levels induced by subunit vaccines were higher than that induced by correlated vaccinia recombinants in mice. Neutralizing test results showed that mice serum immunized with three needles protein subunit vaccine (PS-multiepitopes) have weak special and crossing neutralizing activity. The neutralizing antibody levels of mice immunized by three needles DNA vaccine followed by one needle protein (prime-boost) or two needles vaccinia virus vaccine followed by one needle protein (prime-boost) or three needles protein were equivalent. Combined immunization can't enhance crossing neutralizing antibodies level.These above results showed that immunization with HIV crossing neutralizing epitopes fused with HBV S protein many times may induce HIV crossing neutralizing antibodies to a certain extent, but combined immunization with different type vaccines can't enhance HIV crossing neutralizing antibody response. These results hinted that immunization with the same fusion protein more times enhance the immune response of carrier protein only, can't enhance the immune response of HIV weak neutralizing epitope. At the same time, the study on immunization with HIV weak neutralizing epitope and different carriers' fusion protein boosting HIV crossing neutralizing epitope immune response may be valuable. This work undertakes effective exploration on HIV broad-spectrum neutralizing antibody vaccine, also provides valuable experiment data for future study on the virus vaccine.