Construction And Immunogenic Study Of Polyvalent Recombinant Vaccinia Virus Vaccine For HPV16, HPV18 And HPV58

Cervical cancer is the second most common cause of cancer deaths in women worldwide. Screening and treatment for cervical cancer result in huge burden in economics in these countries.Persistent infection with high-risk human papillomavirus（HPV） has been identified as a major risk factor for the development of cervical neoplasia and cervical cancer by a wealth of epidemiological,biological,pathological,virological and clinical evidence.Many high-risk HPV type can be detected in the tissue of cervical cancers,including HPV type 16,18,45,31,33,58, 52,35,59,56 and so on.HPV type 16 is the most prevalent HPV types in the world and other types distribution is various in different country.In China,the HPV type 16（61.9%） was the most prevalent in cervical cancers patients,HPV18（7.7%） and HPV58（6.4%） being the second and third respectively.Therefore,it is significant to develop the polyvalent therapeutic vaccine of high-risk HPV including HPV16,HPV18 and HPV58 for treating the serious HPV infection and HPV-associated tumors.E6 and E7 proteins are constitutively expressed in cervical cancer cells, and their continuous expression is necessary for maintenance of the transformation,but they are not present in normal cells.As a consequence,these viral antigens are attractive targets for immunotherapy to treat the cervical cancer and its precursor intraepithelial lesions.Because of safety concerns,E6 and E7 proteins were modified to eliminate their oncogenicity and their antigenicity were preserved as the target antigens for therapy vaccine.Since cell-mediated immunity appears important in controlling HPV infection and disease, the vaccinia virus Tiantan strain was selected as virus vector to construct the polyvalent recombinant vaccinia virus expressing E7E6 fusion protein of HPV type 16,18,58 and bivalent recombinant vaccinia virus expressing E7E6 fusion protein of HPV type 16/18 for treatment of the patients with associated HPV types infection.The aim of the our study was to identify the feasibility of polyvalent recombinant vaccinia virus vaccine by examining their immune responses after separate or combined administration and to provide experimental data for development of polyvalent vaccine to treat cervical cancer and its precursor intraepithelial lesions.The major results are summarized as follow:First,we established a method to detect the humoral immune response and got antibodies to detect the target proteins expression of the recombinant vaccinia virus.The E6 and E7 proteins from three different HPV types were expressed efficiently in prokaryotic system and purified by using affinity chromatography.Then,the ELISA method was established with these materials to estimate the humoral immune response.The purified proteins of HPV type 58 were used to immune animal to prepare polyclonal antibody respectively.As a result,we got six antibodies prepared by our lab or bought from the company to detect the target proteins expressed by the recombinant vaccinaia virus.Second,we established one ELISPOT method to detect the cellular immune response in immunized mice.Three peptides sequence for HPV18E6_41-55,HPV18E6_61-75 and HPV58E6_57-71, which were not reported before,were selected by screening from peptide libraries used ELISPOT（IFN-γ） method.They are suit to the test of the cellular immune induced by the recombinant vaccinia virus in this stuy.Third,the E6 and E7 genes of HPV type 16,18 and 58 were fused in one open reading frame and mutated to inactivate their oncogenic potential without changing their antigenicity. Three recombinant vaccinia viruses（rVVs） were constructed for expressing E7E6 fusion proteins of HPV type 18,58 and bivalent 16/18.The genetic stability of bivalent recombinant vaccinia virus were tested,the results showed that the vector was vaccinia virus Tiantan strain, the exogenous gene sequences were right and stable,and the fusion proteins expression level were stable,the rates of gene deletion were from zero to 2%.Fourth,the immune response of separate or combined administration of the recombinant vaccinia viruses expressing E7E6 fusion protein of HPV type 16,18,58 and bivalent 16/18 were evaluated.The recombinant vaccinia viruses expressing E6E7 fusion protein of HPV type 16 and expressing E7E6 fusion protein of HPV type bivalent 16/18 could elicit cytotoxic T lymphocyte response specific to HPV16 E7 protein in immunized mice.These responses were positively correlated with the delay in time of tumor onset in immunized mice and provided partial protection（30-40%） to immunized mice against challenge with the TC-1 tumor cells.When immunized with the recombinant vaccinia virus expressing E7E6 fusion protein of HPV type 58 in mice,specific cytotoxic T lymphocyte response to HPV58 E6 protein could be elicited and partial immunized mice were protected from RMAHPV58E7 tumor cell challenge.The recombinant vaccinia virus expressing HPV18 E7E6 fusion proteins could elicit cellular immune response specific to HPV18 E6 proteins,but no positive cellular immune responses to HPV18 E7 proteins in immunized mice were observed,which suggested that the mouse animal model be not suitable for examining the cellular immune responses of HPV18E7 proteins.The bivalent recombinant vaccinia virus co-expressing E7E6 fusion proteins of HPV type 16 and 18 could elicit specific cellular immune response to HPV16 E7 protein and HPV18 E6 proteins in immunization mice,which is the same as that of the recombinant vaccinia virus of HPV16 and HPV18 respectively.When immunized with the bivalent recombinant vaccine virus in three rhesus monkeys,specific T cells immune responses were detected.Two showed T cell immune response specific to HPV18 E6 protein or HPV18 E7 protein respectively,one showed specific T cells immune responses to HPV16 E6 protein or HPV16 E7 protein respectively. Among the three monkeys,one showed specific T cells immune responses to all of four proteins.The specific antibodies and corresponding cellular immune response could be elicit in mice when co-immunized with the recombinant vaccinia virus co-expressing E7E6 fusion proteins of HPV type 16 and 18 fusion proteins and the recombinant vaccinia virus expressing E7E6 fusion proteins of HPV type 58,which suggested that co-immunization be not affect the immunogenicity of E7E6 fusion protein between the two recombinant vaccinia virus.In conclusion,our results above suggested that it is feasible to develop the polyvalent recombinant vaccinia virus for vaccine research of HPV,which would provide important evidence for development polyvalent therapeutic vaccine to treat cervical cancer and its precursor intraepithelial lesions caused by many types of HPV infection.