Structural And Functional Insights Into Type-specific Neutralization Epitopes Of Human Papillomavirus

Persistent,high-risk human papillomavirus(HPV)infection is the primary cause of cervical cancer,vaginal cancer and penile cancer.Cervical cancer is one of the most common female malignant cancers.More than 500,000 annual new cervical cancer cases worldwide and 100,000 in China are attributed to HPV infection.HPVs are non-enveloped,double-stranded DNA viruses that consist of multiple copies of the major(L1)and minor(L2)capsid proteins.In vitro,each of the 72 pentamers is composed of five copies of the L1 protein,which can self-assemble into an empty T=7 icosahedral shell called a virus-like particle(VLP).Previous studies indicated that neutralizing antibodies elicited by L1-only virus-like particles(VLPs)can block HPV infection by interfering different events during HPV infection process;however,the lack of high-resolution structures has limited our understanding of the mode of virus infection and HPV neutralization mechanism at the molecular level.L1 surface loops of different HPVs exhibit a high degree of variation,and this variability is the main reason why there is limited cross-protection conferred by the current,approved VLP-based vaccines.There are 15 HPV types are associated with the development of cervical cancers and other anogenital cancers.Currently,there are three commercially available,prophylactic HPV vaccines,among which the most widely protective vaccine,Gardasil 9 can provide protection against only 7 high-risk HPV types.Including more HPV types L1 proteins into a vaccine is a big challenge for manufacturing due to the dosage of Gardasil 9 is already up to 270 ?g.Therefore,it is necessary to structurally determine the type-specific epitopes to understand the type specificity for HPV immunology and subsequently help pave a new way to design a wide-spectrum HPV vaccine.To address the above issues,in this study,we two representatively type-specific HPV neutralizing antibodies were selected to analysis their accurate epitope information by determining high resolution immune complex structures.Based on the epitope information,more virus infection assays were conducted with the mutated HPV virus to help us to unravel the process of HPV infection process and type-specificity of HPV.Firstly,two HPV type-specific antibodies HPV58.A12A3 and HPV59.28F10 were selected from the HPV antibody panels in our lab.We measured the binding capabilities,using ELISA assays,of A12A3 and 28F10 IgGs to HPV58 and HPV59 pentamers(p),respectively,and compared this with binding to VLPs.We found that A12A3 IgG did not bind as well to HPV58p as it did to HPV58 VLPs,whereas the binding profiles of 28F10 IgG to the pentamer and VLPs of HPV59 were similar,indicating A12A3 was more sensitive to the minimal conformational changes on epitope between pentamer and VLPs.Using a neutralization assay,we found that the IgGs of A12A3 and 28F10 are more potent at neutralization than their corresponding Fabs in this assay.Electron microscopy observations on VLP:Fab and VLP:IgG of both HPV58:A12A3 and HPV59:28F10 show that both A12A3 and 28F10 IgGs could simultaneously capture two VLPs,indicating the discrepancy of neutralizing activity between Fabs and IgGs may be attributed to the avid binding of the IgGs with its two arms binding to different virus particles,which would produce a much higher apparent affinity than that of the monovalent Fabs.Then,we determined the crystal structures of HPV58p alone,as well as the HPV58p:A12A3 and HPV59p:28F10 immune complexes to resolutions of 2.0 A,3.5 A,and 3.4 A resolution,respectively.From the structural information,we realized that A12A3 and 28F10 could specifically bind to and neutralize HPV58 and HPV59,respectively,through two distinct binding stoichiometries:only one A12A3 Fab is bound to the center of the HPV58 pentamer,whereas five 28F10 Fabs are located around the periphery of the upper rim of the HPV59 pentamer.We show that the epitopes of A12A3 are clustered in the DE loops of two adjacent HPV58 L1 monomers(Q165a,D154b,R161b,Q165b,S168b and N170b),whereas 28F10 recognizes the HPV59 FG loop of a single monomer(M267,G268,Q270,E273,Y276,K278,D281 and R283).We next determined the cryo-EM structures of A12A3 Fab and 28F10 Fab in complex with HPV58 L1 VLPs and HPV59 L1 VLPs at 9.5 A and 8.4 A,respectively.The congruency in the binding modes for both A12A3 and 28F10 to the corresponding pentamer and VLPs was verified by comparing the details in the crystal structures and the capsid immune complexes.Next,to ascertain which residues are critical for antibody recognition,we performed alanine-scanning mutagenesis on pentamers and VLPs.The resultant binding capacities of different variants were evaluated by indirect ELISA and SPR.We found that HPV58-R161,HPV59-E273 and HPV59-R283 play essential roles in the interaction between the antigens and their corresponding antibodies.Finally,to verify the antibody-binding regions associated with the virus infection sites,we generated virus mutants of HPV58 and HPV59 using alanine substitutions for interface amino acids of HPV L1 involved in the interactions with mAbs A12A3 and 28F10,respectively.In HaCaT cells,we found HPV58-D154,S168,N170 and HPV59-M267,Q270,E273,Y276,K278,R283,that play critical roles in virus infection.Moreover,by substituting these strategic epitope residues into other HPV genotypes,we could then redirect the type-specific binding of the antibodies to these genotypes,thus highlighting the importance of these specific residues,HPV58-R161,S168,N308 and HPV59-Q270,E273,D281.Overall,with a combination of various methods of structural biology,biochemistry and celluar biology,we comprehensively characterized the structure and function of type-specific epitopes of HPV,thus help understand the HPV neutralization mechanism at the molecular level.In addition,the extended functional assays based on the identified epitopes also demonstrated the neutralizing epitope of HPV antibodies were involved in the determinants of HPV required for infectivity and type specificity.This knowledge will help better design wide-spectrum HPV vaccine,and potentially be used to drive the development of therapeutics that target neutralization-sensitive sites.