The Research Of Human Papillomavirus Types Of 39/68/70 Cross-genotype Vaccine

Sustained infection with high-risk Human Papillomavirus(HPV)can cause of mucosal tissue epithelial malignant lesions,which is the main cause of cervical cancer in women.According to epidemiological statistics,cervical cancer has become the second leading cause of cancer in women.Cervical cancer was showing an increasing trend year by year in C.hina and even in the world.The capsid proteins of HPV include major(LI)and minor(L2)capsid protein.L1 protein can be expressed in vitro and assembled into virus-like particles(VLPs)in a certain environment,and its surface retains the structure similar to native HPV The current market-available prophylactic HPV vaccines were developed based on the form of VLPs,which could induce neutralizing antibodies and block HPV infection efficiently.Because of the strict type specificity of HPV,it is difficult to generated high titer cross-neutralizing antibody.Currently,Merck's Gardasil 9 could provide protection against infection from only seven high-risk HPV types,which are responsible for about 90%of cervical cancers.But there are more than fifteen HPV types are associated with the development of cervical cancers.In order to prevent more types of HPV infection,additional types of VLPs need to be introduced on the basis of existing vaccines.However,this will lead to a surge in vaccine production costs,a more complicated production process and vaccine safety hazards.Therefore,the use of fewer types of VLPs to prevent more types of HPV infection("one against many")is the concern of the next generation of HPV prophylactic vaccine research.According to the problems,this study analyzed the genetic relationship of HPV L1 protein,and established chimeric HPV VLPs by epitope grafting,and carried out comprehensive qualitative identification and immunological evaluation,screening of candidate vaccine molecules that can induce high degrees of cross-neutralization antibody,exploring feasibility of chimeric VLPs vaccines to achieve "one against many".According to the carcinogenicity classification of IARC,HPV39,HPV68 and HPV70 belong to class 1,class 2 A and class 2B respectively.Epidemiological statistics showed that 3%of cervical cancer were caused by HPV39,HPV68 and HPV70.In china,the infection rate of HPV39 and HPV68 was 3-5%,and that of HPV70 was about 1%.Therefore,in order to expand the scope of vaccine protection,this study designed candidate molecules of cross-vaccine based on HPV39,HPV68 and HPV70,which might differ slightly in conformation.Firstly,three types of VLPs and pseudovirus of HPV39,HPV68 and HPV70 were created and the physical and chemical properties and immunogenicity of HPV39,HPV68 and HPV70 VLPs were identified.The result showed that the morphology of VLPs is regular,and the icosahedral structure is T=7;Immunogenicity analysis showed that all three types of VLPs could elicit high titers neutralizing antibodies.Further,HPV39/68 and HPV68/39 chimeric molecules were created and purified using HPV39 and HPV68 VLPs,which are closely related to HPV39,HPV68 and HPV70,and determined the physical and chemical properties and immunogenicity.The pseudo-virion-based cell neutralization assay showed that the H3 9-68FG VLPs and H68-39BC VLPs elicited the neutralizing antibodies against HPV39 and HPV68.Then H39-68FG VLPs and H68-39BC VLPs were used as backbone to create chimeric molecules of triple-type HPV39/68/70,the same method was used to analyze the physicochemical properties and immunogenicity,and ED50 values was used to screen out H39-68FG-70DE VLPs and H39-68FG-70HI VLPs,which could elicit high titer cross-neutralizing antibodies.And that thermal stability was good and can be further evaluated as candidate molecules in subsequent vaccine development.Secondly,monoclonal antibodies(mAbs)screened by HPV68 and HPV70 VLPs were used for epitope analysis of H39-68FG-70DE VLPs and H39-68FG-70HI VLPs.HPV68 and HPV70 specific mAbs were determined by ELISA assay with H39-68FG-70DE VLPs and H39-68FG-70HI VLPs,among which HPV68 mAb 9F4 and HPV70 mAb 12B11 had higher binding activity with H39-68FG-70HI VLPs,which was equivalent to the result of wild-type VLPs.This indicates that homologous substitution of surface loop regions could graft the epitopes of HPV68 and HPV70 into HPV39 L1 VLPs successfully,and subsequently further analyze the VLPs-Fab complexes structure of H39-68FG-70HI VLPs and 9F4 Fab and 12B11 Fab,and indicate its epitope information.In conclusion,this study designed HPV39/68/70 triple-type chimeric particles by swapping epitopes of HPV L1 and expressed in E.coli successfully,these chimeric molecules were similar to wild types,and determined the pseudo-virion-based cell neutralization assay to screen candidate vaccine molecules with cross-protection,achieved fewer types of VLPs to prevent more types of HPV infection,laying the foundation for the next generation of HPV prophylactic vaccine development.