Immunogenicity Of The Novel Preparations Of Hepatitis B Vaccines

[Objective]:The two novel formulations were explored to solve the problems existed in the current vaccine in the prevention and control of HBV infection, such as low and non-responder, ineffectively provoking immune cells, unable to break immune tolerance, the side effects from aluminum hydroxide adjuvant. The formulations included chimeric Hepatitis B virus (HBV) core virus-like particles carrying immunodominant epitopes of HBV on the surface and biological adjuvants-HBsAg covalently conjugated vaccine preparation.[Methods]:In the section I, the immunodominant epitopes of HBV (HBsAg "a" epitope aa119-152; HBsAg "a" epitope aa139-148; preSl aa21-47; preSl aa37-45) were inserted into major immunodominant region (MIR) or carboxyl terminus (following aa144) of the full-length (aa1-183) or truncated (aa1-144) HBc to construct a series of chimeric HBc virus-like particles with these epitopes on the surface whose immunogenicity were evaluated by animal experiments. Experimental techniques mainly included codon optimization, gene cloning, ordinary and recombinant PCR technique, prokaryotic expression, protein purification, protein identification, ELISPOT, flow cytometry and so on.In the section Ⅱ, by borrowing from the successful experience of polysaccharide-protein D (PD) of Haemophilus influenzae conjugated vaccine, the non-acylated region of PD (named H17) was amplified from the genome of Haemophilus influenzae by nested PCR, inserted into pET43.1a expression vector, and expressed in E.coli BL21(DE3). H17was purified by ammonium sulfate precipitation and ion exchange chromatography to a high purity and high concentration. And using glutaraldehyde two-step cross-linking procedure, H17was covalently conjugated to HBsAg which was the main ingredient of Hepatitis B vaccine to prepare experimental conjugated vaccine formulations. The immunogenicity of the conjugated vaccine formulation was evaluated by animal experiments.[Results]:In the section I, a series of chimeric HBc virus-like particles (H2, H3, H8, H9, H10, HI1, S47, H20, H21) was successfully constructed and expressed in prokaryotic expression system. By ammonium sulfate precipitation, ion exchange chromatography, protein concentration, protein refolding, size-exclusion chromatography (SEC) and ultracentrifugation technique, H2, H3, H8, H9, H11, S47of high homogeneity were obtained with the immunodominant epitopes of HBV on the surface of chimeric HBc virus-like particles. Finally, the immunogenicity of H8, H11were evaluated by animal experiments. The antibodies against HBsAg and preSl were induced in the Balb/c mice immunized with H8; The antibody level of anti-preSl was relatively lower than anti-HBs (p<0.05), while both of them reached the peaks at Day42-49after immunization; ELISPOT showed that H8could stimulate the proliferation of IFN-y-secreting cells of splenocytes of the immunized mice, indicating that Thl type cellular immune response was provoked effectively; Flow cytometry showed that compare to the PBS control group, the proportion of CD4+T cells and CD8+T cells in the splenocytes increased significantly (p<0.05), while CD4+T/CD8+T cell ratios decreased, suggesting that H8could stimulate specific CD8+T cell differentiation. H11could induce anti-HBs in mice with the peak at Day49-56; ELISPOT revealed that H11could also stimulate the proliferation of IFN-y-secreting cells of splenocytes of the immunized mice, indicating that Thl type cellular immune response was provoked effectively; Flow cytometry showed that compare to the PBS control group, the proportion of CD4+T cells and CD8+T cells in the splenocytes increased significantly (p<0.05), while CD4+T/CD8+T cell ratios decreased, suggesting that H11could also stimulate specific CD8+T cells differentiation.In the section II, non-acylated17protein was obtained with a high purity and high concentration by gene cloning and purification techniques such as ammonium sulfate precipitation, protein refolding and ion exchange chromatography. The antigenicity of H17was preliminarily confirmed by Western Blotting. By glutaraldehyde two-step cross-linking, H17was conjugated covalently to HBsAg to prepare experimental conjugated vaccine formulations whose antigenicity titer was determined to1:1600by double-antibody sandwich ELISA which included antibodies specific to H17and antibodies specific to HBsAg. Animal experiments confirmed that conjugated vaccine formulations could induce antibodies against HBsAg with the peak of antibody levels at Day35-42which was held in the whole tracking period for15weeks in mice. The antibody titer was higher than HBsAg-alone and PBS group (p<0.05), while it was lower than current hepatitis B vaccine group (p<0.05). However, after first-dose vaccination, conjugated vaccine formulation was higher than any other groups in antibody level (p<0.05). ELISPOT showed that there was not a statistical difference in the level of proliferation of IL-4-secreting cells in splenocytes between the conjugated vaccine formulation and hepatitis B vaccine (p>0.05), but both of which were higher than HBsAg-alone and PBS group (p<0.05), indicating that conjugated vaccine and hepatitis B vaccine formulation were stronger than the other two groups in stimulating Th2type cellular immune response. However, considering the proliferation of IFN-y-secreting cells, the conjugated vaccine formulation was superior to any other groups (p<0.05), demonstrating its superiority in the stimulation of Th1cellular immune response. Flow cytometry showed that compare to the PBS control group, the proportion of CD4+T cells and CD8+T cells in the splenocytes increased significantly (p<0.05), while CD4+T/CD8+T cell ratios decreased obviously, suggesting that the conjugated vaccine formulation could stimulate the differentiation of specific CD8+T cells.[Conclusion]:In the section I, The chimeric HBc VLPs (H8and H11) displaying the immunodominant epitopes of HBV on their surface assembled with the monomers which arranged the epitopes from HBc and HBsAg "a" determinants and/or preSl neutralizing epitopes on the identical protein molecule (essential to stimulate the body to induce a comprehensive HBV immune response) were successfully constructed. The VLPs effectively challenged the mice to produce antibodies against specific epitopes and provoked Thl type cellular immune responses in mice, and the proportion and the number of CD8+T cells in splenocytes also increased obviously, which fully demonstrated their potential as a novel vaccine preparation.In the section Ⅱ, the conjugated vaccine formulation covalently coupled non-acylated protein D of Haemophilus influenza that possessed strong T cell epitopes and a vaccine-vector potential to HBsAg-the main ingredient of Hepatitis B vaccine, would change the ways of presenting antigen possibly, break immune tolerance and eliminate the issues of low non-response and others. Animal experiments confirmed conjugated vaccine formulation fully induced the antibodies against HBsAg in mice. Although the final antibody levels was below that of Hepatitis B vaccine, a strong Thl cellular response was induced (Comparable in Th2response between the two) and the number and the ratio of CD8+T cell in splenocytes increased significantly.