Design And Evaluation Of Epitope Polypeptide Vaccine Based On L1 Capsid Protein Of HPV6 And 11

Human papillomaviruses (HPV) are double strand DNA viruses of approximately 8 kb. Among all types of HPV, type 6, 11, 16 and 18 infect basal and suprabasal layers of genital stratified epithelium and are the major cause of condyloma acuminata, which is known as the most frequent sexually transmitted diseases (STD) in the world. The immunobiology of HPV infections further warrants the development of HPV vaccines. Though the strict species specificity of HPV that precludes the development of simple animal models, some results from studies of papillomavirus infection in animals, including rabbit, bovine and canine model, have supported the concept that vaccines can prevent or hasten clearance of HPV infection. Vaccines derived from structural proteins (L1, L2 and their subfragments) and non-structural protein (E1, E2, E6 and E7) of HPV have been successfully produced in bacteria system, generally as proteins fused to bacterial proteins.In animal papillomaviruses models, vaccination against L1 and/or L2 viral capsid proteins can provide the animals an efficient protection from infection, and the mechanism of protection inculed the production of virus type-specific neutralizing antibodies. Vaccination against non-structural E1, E2, E6 or E7 viral proteins do not prevent infection, unless administered altogether with the structure proteins. Prophylactic vaccines based on the use of virus-like particles (VLPs) obtained from auto-assembly of L1 or L1 and L2 proteins produced by recombinant DNA technology are under phase I/II clinical trials for HPV6/11 associated with condylomas. Second generation vaccines are chimeric proteins in which the structural proteins (L1 or L2) fused to a non-structural protein (E6, E7 or E2) or VLPs. These second generation vaccines have been verified to be able to induce both humoral and cellular immunity. Vaccine valency (number of genotypes), route of administration (humoral versus local immunity) and so on are under optimization. Therapeutic vaccines based on recombinant HPV E6 and/or E7 vaccinia virus, L2-E7 fusion proteins or E7 peptides corresponding to cytotoxic T cell epitopes are currently tested (phase I/II trials) in patients with cervical carcinomas of advanced clinical stages or high- grade intraepithelial lesions. In this research, basing on the features of L1 capsid proteins of HPV6 or 11, we utilized the Goldkey and PC/gene software to seek for B-cell epitopes from L1 protein and evaluated the B-cell dominant epitopes using antigen index (AI). Four B-cell dominant epitopes (named I, II, III, and IV respectively) were predicted and synthesized. The secondary structure and the B cell epitope motif were analysised and investgated. The percentage of the Turn(T), Extended(E), Helical(H) and Coil structure(C)in L1 protein was 27.54%, 32.34%, 20.76% and 19.36% respectively. The B cell epitope motives of the L1 protein of the human papilloma virus-11 were located at the aa120-140, 345-360, 420-440 and 480-501. The average antigenic index of the motif 426-439aa and 487-501aa was 0.0508 and 0.0578 respectively. To compare the immunogenicity of the four epitope polypeptide vaccines on the basis of the different assembly peptide, different amount of the four epitopes (i.e.I, II, III and IV) were emulsified with Complete Freund's Adjuvant (CFA) and inoculated i.p.into Balb/c mice on week 0, 2 and 4 respectively. When the inocaluations were completed, the stereotype of HPV from patien's wart were evaluated with PCR, antibody titer of the sera from animals immunized with the same or different antigen dose and the antibody cross-reactivities with HPV from different patien's wart were deterimined. Then the production of cytokines (IFN-γand IL-4) and stimulation proliferation of lymphocyte from spleens of immunized mcie were investigated. The results showed that the antibody titer and stimulation proliferation of spleen lymphocyte from mice inoculated with the same antigen dose (80 μg per mouse) are Ⅳ>Ⅰ>Ⅲ>Ⅱ at weeks 2, 4 and 6. The minus amount of antigen that could induce th...