Epitope Mapping Of Human Papillomavirous Type-hybrid Virus Like Particle By Subtractive Immunization Strategy

The persistent infection of HPV usually results in cervical cancer. Approximately560,000new cancer cases with200,000fatal cases worldwide and100,000new cervical cancer cases with30,000deaths occured in China are attributed to HPV infection annually. Cervical cancer ranks second in the most common malignant tumors threatening women’s health. It is known that there are at least15types of high-risk HPV, therefore, vaccines which can against15types of HPV are needed to reduce the cervical cancer incidence. There are two vaccines available in the market, only cover two of the the high-risk HPV, HPV16and18, account for approximately60%to70%of cervical cancer. Therefore, the development of vaccines which can prevent more HPVs has the great significance and huge challenges.In this study, HPV16L1proteins with C175mutants and HPV52proteins with C428mutants couldn’t assemble into VLPs were prepared. But two mutants’ proteins can co-assemble to homogeneous hybrid VLPs with the mixing proportion of1:1. The hybrid VLPs can induce the same of IgG mAbs titer against other types. The hybrid VLPs and wild type HPV VLPs have different spatial structure on account of only one disulfide bond between two pentamers. The hybrid VLPs contains more rare epitopes between two adjacent pentamers. New epitopes of hybrid VLPs study has important implications by the method of screening specific antibodies.Routine immunization cannot prepare specific antibody against hybrid VLPs. In this study, cyclophosphamide subtractive immunization was uesd to prepare hybrid VLPs monoclonal antibodies. Through a series of research, immunization programs were established:20μg antigen dose without the use of adjuvants, intraperitoneal injection,100mg/kg cyclophosphamide and immunosuppression could be sustained for4weeks.By screening antibodies, we found two specific antibodies against hybrid VLPs, and four antibodies titer10times higher against wild tyle VLPs. To investigate the hybrid VLP’s specific epitopes, moleculer homology modeling and moleculer docking were uesd to predict epitope recognized by2B3. The results show that the interaction region of2B3and hybrid VLP is located between two pentamers, the key amino acids are Ser170and Lys439in HPV16pentamer,Asn240、Ser255、Glu378、Lys382and Glu469in HPV52pentamer.All these works in this study will contribute to the research of HPV L1molecular evolution, specific neutralizing epitope of HPV, Assembly mechanism of Type-hybrid VLP and pave the way for further design of new generation of HPV vaccine.