Rational Design Of A Multi-Type Human Papillomavirus Vaccine Based On The Structural Details Of A Cross-Neutralization Epitope

Human papillomavirus(HPV)persistent infection can lead to proliferative lesions of the epithelial tissue and a variety of benign and malignant tumors.More than 200 genotypes of HPV have been identified,of which at least 25 high-risk types are highly correlated with cervical cancer.Cervical cancer is the third most malignant tumor of women in the world.About 569,000 annual new cervical cancer cases and 311,000 deaths worldwild are attributed to HPV,causing a serious threat to women’s health.Vaccination with prophylactic HPV vaccine is an effective means of preventing cervical cancer.There are currently bivalent,tetravalent and nine-valent vaccines on the market.HPV L1 protein can self-assemble in vitro to form virus-like particle(viruslike particle,VLP),which has strong immunogenicity and is the main immunogen form of the commercial HPV vaccines.Due to the type-restricted immune response of HPV VLP,it is difficult to induce cross-neutralizing antibodies.For this reason,HPV vaccines use a mixture of multiple VLPs to prevent different types.The HPV ninevalent vaccine contains 7 high-risk types that can prevent about 90%of cervical cancer,but it cannot effectively protect about 10%of cervical cancer and related lesions caused by 18 high-risk non-vaccine types.The expensive price of the nine-valent vaccine also limits its widespread vaccination in less developed regions.In order to achieve the widely protection of vaccines,the types of VLPs have to be continuously increased,resulting in excessively high doses of immunogens,high costs,more complex production process and potential safety issues.Therefore,it is of great significance to design multi-type HPV vaccines.Previous work of our research group has contructed a triple-type HPV chimeric VLP through loop swapping,which can induce cross neutralization titers against three types.Although this method can provide three types protection through single chimeric VLP,at least 7 types of VLPs are still needed to protect more than 20 high-risk HPVs.In addition,triple-type chimeric VLP can not generate cross-protection between distantly related types.Whether accurate design can achieve more types of cross-protection through a single immunogen is a key scientific question for the next generation of vaccines against multi-genotype pathogen including HPV.In recent years,with the development of structural biology,conserved epitopes on target proteins of highly variable pathogens such as influenza virus and Human immunodeficiency virus(HIV)have been mapped by resolving the structure of antigenantibody immune complex,which makes it possible to guide the design of crossprotective vaccines based on cross-neutralizing antibodies.However,due to the lack of high-resolution structural information of HPV and cross-neutralizing antibody immune complexes,it is difficult to deeply understand the spatial structure of cross-neutralizing epitopes and antibody functional mechanism,which greatly limits the research of multitype HPV vaccines.In response to the above scientific problems and challenges,this study starts with cross-neutralizing antibodies against high-risk types,resolves the high-resolution structure of virus and antibody immune complexes,reveals the spatial distribution characteristics of cross-neutralizing epitopes,and clarifies the functional mechanism of cross-neutralization.We attempt to complete the reconstruction of cross-neutralization epitope on wild-type VLP,try to construct cross-type immunogen,and lay a theoretical foundation for the design of HPV multi-type vaccines.First,this study established a panel of cross-neutralizing antibodies against highrisk HPV types for the first time.A comprehensive characterization revealed two different neutralization modes:antibodies 4H4,6C8,6H4 and 12B9 can prevent the PsV from binding the extracellular matrix and cell surface;antibody 13A10 allows viral attachment but prohibits PsV from entering the cell.13A10 is the most optimal antibody in the panel and have potent neutralizing efficiency against four high-risk types HPV 16,-31,-33 and 58.We further determined the affinity of 13A10 and prepared Fab to identify the functional difference between the full-length antibody and Fab.The results showed that full-length antibody has stronger abilities of cross-neutralization and crosslinking.Second,this study elucidated the structure basis of cross-neutralization with HPV antibody.Pufrifed HPV pseudovirus(PsV)was obtained by WAVE bioreactor and ultracentrifugation and reacted with 13A10 Fab for the preparation of immune complexes.The cryo-EM structures of the four immune complexes HPV 16:13A10 Fab,HPV31:13A10 Fab,HPV33:13A10 Fab and HPV58:13A10 Fab were resolved by single particle reconstruction,with resolutions of 4.19 A,13.76 A,6.40 A and 4.92 A,respectively.The structure of HPV16:13A10 Fab and HPV58:13A10 Fab was optimized by sub-particle reconstruction and corresponding atomic models was built to find that each pentamer bound five Fabs.13A10 can simultaneously bind the DE loop,FG loop and HI loop from three adjacent L1 monomers.13A10 recognizes eight epitope amino acids(S280,G281,N285,S349,E352,K356,T358,and N359)of HPV16 L1 protein,and recognizes ten epitope amino acids(Q139,P140,S142,S280,G281,T349,E351,K355,D357 and N358)of HPV58 L1 protein.The five key sites(S,G,E,K,and N)are highly sequence conserved and structurally consistent on HPV16 and 58,which is the structural basis for cross-neutralization.In addition,the potential model of 13 A10 bivalent binding was analyzed,explaining the reason why the neutralization ability of full-length antibody is stronger than that of Fab.Further,this study systematically characterized the key amino acids of crossneutralization epitope.Alanine scanning was used to anlyze the functions of the crossneutralizing epitope sites by mutant VLPs and PsVs of HPV16,-31,-33,and 58.The four completely conserved sites(S280,G281,K356,and N359)among four types are not only important in antibody binding,but also play a critical role in PsV infection.The neutralization mechanism of 13A10 is revealed:antibody may recognize some specific receptor binding sites on HPV L1 protein,which interferes with the continuous interaction between HPV capsid with the receptor after viral primary attachment;antibody can also lock the HPV capsid and inhibit the conformational changes,which is required for cell-entry.Finally,this study is the first to design the HPV multi-types immunogen based on cross-neutralizing epitopes.Through the reconstruction of cross-neutralization epitopes,not only is it possible to break through the type-specificity among the closely related types,but also to establish cross-immunogenicity between the distantly related types.The single-type VLP can induce significant cross-neutralizing antibody titers by changing at least five key sites.The principles of epitope reconstruction were summarized and a multi-type HPV vaccine candidate HPV35FG+/HI+VLP was designed to obtain cross-immunogenicity against HPV31,-33,-35,-52 and 58.In summary,based on methods of structural vaccinology,we studied the recognition epitopes of high-risk HPV cross-neutralizing antibodies,and revealed the molecular mechanism and structural basis of HPV cross-neutralization induced by antibodies.According to the accurate structural and functional atlas of crossneutralization epitopes,we complete the design of a multi-type HPV vaccine candidate with cross-immunogenicity of five types.Our work provides a theoretical basis to develop more efficient and available multi-type HPV vaccines and other universal vaccine for highly variable viral pathogens.