Expression And Immunogenicity Study Of Human Enterovirus71and Epstein-Barr Virus Vaccine Candidate Antigens In Pichia Pastoris

The methylotrophic yeast Pichia pastoris is one of the most widely used systems for heterologous protein expression. P. pastoris offers several advantageous features including high production levels, easy manipulation, stable expression and low cost. In addition, P. pastoris is capable of performing post-translational modifications, which makes this system very suitable for high-level production of eukaryotic proteins. In this thesis, we employed P. pastoris system to produce candidate antigens for human enterovirus71(EV71) and Epstein-Barr virus (EBV) vaccines, and further evaluated the immunogenicity of these antigens.EV71is one of the major causative agents of hand, foot and mouth disease (HFMD) and is associated with serious neurological diseases or even death in children. EV71has strong infectivity and can cause high morbidity, thus EV71has led to multiple epidemic outbreaks of HFMD. However, there are currently no effective antiviral drugs or preventive vaccines against EV71infection. Previous studies demonstrated that the capsid protein VP1of EV71exhibits strong immunogenicity. Recombinant VP1could induce neutralizing antibodies and protect newborn mice against lethal EV71infection. Therefore, VP1is a promising candidate antigen of an EV71vaccine.EBV is a ubiquitous y-herpesvirus that latently infects human B cells and can establish chronic infection in more than95%of the world population. EBV is the causative agent of infectious mononucleosis and is closely associated with both lymphoid and epithelial tumors. The carcinogenicity of EBV determines the urgency and necessity to develop effective vaccines against EBV. Currently therapeutic EBV vaccine development has focused on nuclear antigen1(EBNA1), while for prophylactic vaccines, viral envelope glycoprotein (gp350) is the principal focus of research attention. EBNA1is the only viral protein expressed in all EBV-associated malignancies and plays important roles in EBV latency. Previous studies showed that EBNA1-specific T cells could effectively kill EBV-positive tumor cells. Thus, EBNA1is thought to be a promising antigen for immunotherapy of EBV-associated malignancies. Gp350plays an essential role during EBV infection. The binding of gp350by complement receptor type2(CR2) on the surface of B lymphocytes mediates EBV entry and infection. Increasing evidence indicates that gp350is the predominant target for neutralizing antibodies, and is a principal candidate antigen for a prophylactic EBV vaccine. In this thesis, recombinant VP1, EBNA1and gp350were successfully expressed in P. pastoris. Our innovative results were presented as follows:1. The eukaryotic expression vectors comprising the codon-optimized VP1gene insert were constructed and transformed into P. pastoris cells for the induction of protein expression. The recombinant VP1was expressed and secreted into the culture medium. Ni-NTA affinity chromatography was then used to obtain the purified VP1protein. Immunogenicity and antiviral efficacy of the recombinant VP1were assessed in mouse models. The results showed that the recombinant VP1could efficiently induce VP1-specific antibodies in BALB/c mice, which were able to neutralize EV71viruses in an in vitro neutralization assay. Passive protection of neonatal mice confirmed the prophylactic efficacy of VP1vaccines. Furthermore, VP1was able to induce lymphoproliferative and Thl-type responses. In conclusion, the P. pastoris-expressed VP1protein retained good immunogenicity, and could induce antiviral antibody responses and activate the immune system, therefore the recombinant VP1was a potential subunit vaccine against EV71.2. Bioinformatics analysis showed that the C-terminal domain of EBNA1contains the major immunogenic epitopes, therefore, the truncated form of EBNA1protein (E1ΔGA, codons390-641) was produced and used as an immunogen for therapeutic EBV vaccine. After optimizing E1ΔGA sequence, we successfully constructed the eukaryotic recombinant vectors expressing codon-optimized E1ΔGA, and then transformed the recombinant vectors into P. pastoris to induce the expression of recombinant protein. SDS-PAGE/Western blot analysis showed that the recombinant E1ΔGA was successfully expressed in P. pastoris system. The secreted E1ΔGA was easily purified from culture supernatants by using Ni-NTA affinity chromatography. The purified proteins were then used as antigens to immunize BALB/c mice for the production of polyclonal antibodies. Western blot analysis showed that the polyclonal antibodies could specifically recognize the EBNA1protein in B95-8cell lysates. The recombinant E1ΔGA could stimulate the proliferation of T lymphocytes, and induce Thl-type immune responses. Moreover, increased numbers of CD4+T and CD8+T cells were observed in spleen and peripheral blood of immunized mice. These findings showed that the P. pastoris-expressed recombinant E1ΔGA retained strong immunogenicity and might be a promising candidate against EBV-associated malignancies. 3. Epitope analysis indicates that the N-terminal domain of gp350protein contains critical neutralizing epitopes and has the potential to be developed into an EBV vaccine. In this study, the N-terminal domain of gp350(codons1-443) was successfully expressed in P. pastoris through codon optimization, and the recombinant protein could be efficiently purified by one-step affinity chromatography. This study laid the foundation for large-scale production and research into antiviral activity of the recombinant gp350.In this thesis, we successfully expressed EV71and EBV vaccine candidate antigens in P. pastoris. Our data demonstrated that the recombinant VP1and E1ΔGA could induce specific antibodies and activate the immune system. Furthermore, these candidate antigens were able to stimulate the secretion of cytokines and the proliferation of lymphocytes. These results indicated that the yeast-expressed antigens displayed strong immunogenicity and had the potential to be developed into antiviral vaccines.