Research On Evaluation Technique Of Hepatitis B Vaccines

Major histocompatibility (MHC) class I tetramers are used in the quantitative analysis of epitope peptide-specific CD8+ T-cells. An MHC class I tetramer was composed of 4 MHC class I complexes and a fluorescently labeled streptavidin (SA) molecule. Each MHC class I complex consists of an MHC heavy chain, aβ2-microglobulin (β2m) molecule and a synthetic epitope peptide. In most previous studies, an MHC class I complex was formed in the refolding buffer with an expressed MHC heavy chain molecule andβ2m, respectively. This procedure inevitably resulted in the disadvantages of forming unwanted multimers and self-refolding products, and the purification of each kind of monomer was time-consuming. In the present study, the genes of a human/murine chimeric MHC heavy chain (HLA-A2α1, HLA-A2α2 and MHC-H2Dα3) andβ2m were tandem-cloned into plasmid pET17b and expressed as a fusion protein. Such chimeric MHC class I tetramers could monitor HLA/A2 restrictive cytotoxic T lymphocytes (CTLs) in immunized HLA/A*0201 transgenic mice.A lack of relevant animal models has hampered preclinical screening and critical evaluation of immunogenicity of human Hepatitis B virus (HBV) vaccines in vivo. In this study, HBV/HLA-A2.1 double transgenic mice (dTg) were generated for the first time, confirmed by the integration and expression of HBV and HLA genes. HBV/HLA-A2.1 dTg not only display the tolerance to HBV antigens (Ags), but also have the ability to process and present HLA-A2 restricted antigenic epitopes. We established the utility 4of this model by demonstrating that HLA-A2 restricted HBc18-27 or HBs260-269 epitope peptide vaccine induced effective HLA-A2 restricted HBc18-27 or HBs260-269-specific CTL responses. This finding was supported by the evidence of CTL, ELISPOT and tetramer staining. Importantly, T cell tolerance against HBV Ags in HBV/HLA-A2.1 dTg was broken by the HBc18-27 or HBs260-269 peptide vaccine. In conclusion, HBV/HLA-A2.1 dTg were demonstrated to be good candidates for in vivo immunogenicity testing of various human HBV based vaccines.In summary, the results indicated that such chimeric MHC class I tetramers showed a sensitive binding activity in monitoring HLA/A2 restrictive cytotoxic T lymphocytes (CTLs) in immunized HLA/A*0201 transgenic mice. In addition, HBV/HLA-A2.1 dTg showed specific CTL and humoral responses in several parameters: antibody production, IFN-γsecretion, specific cell lysis, proportions of specific CD8+ T cells and depressed the level of antigen. HBV/HLA-A2.1 dTg represented a most relevant in vivo animal model to study human immune responses towards human HBV-based vaccines and could be used for in vivo preclinical evaluations of the vaccines for immunogenicity and therapeutic efficacy.