The Development Of Potent Neutralizing Antibodies Targeting EBV GB And Exploratory Study Of Epitope-based Vaccine

Epstetin-Barr virus(EBV)is a ubiquitous pathogen and infects 95%of adults worldwide.EBV is also the first identified human oncogenic virus.About 200,000 new cancer cases and 140,000 deaths are related with EBV infection each year worldwide.Diseases including tumors caused by EBV infection cause tremendous health and economic burdens.Vaccines are the most efficient weapon in fighting against viral infection.However,there is no available licensed EBV vaccines,suggesting that more suitable candidates for EBV vaccines are needed.gB is the fusion protein of EBV and the key component in the viral infection process.gB is also the most conserved herpesvirus envelope glycoprotein in sequence and structure.Several neutralizing antibodies(nAb)and prophylactic vaccines based on HCMV gB have been advanced to the clinical stage,suggesting that gB is an important candidate for the development of prophylactic vaccine against herpesvirus.However,there are few studies about nAbs and vaccines based on EBV gB.Therefore,it is of great significance to screen more efficient nAbs against gB,analyze the structure of the immune complexes and elucidate the mechanism of antibody neutralization,which is important for in-depth study of the functional mechanism of gB and revealing the potent neutralizing epitopes on gB.It also contributes to the development of EBV prophylactic vaccines and targeted drugs.In this study,we had isolated two potent gB-specific nAbs,3A3 and 3A5,using the rabbit monoclonal antibody platform.3 A3 and 3 A5 are the first reported gB-specific nAbs that potently neutralize EBV infection of B cells and epithelial cells in vitro.We further systematically evaluated the binding ability,neutralizing activity in vitro and protection efficiency in a humanized mouse model of 3A3 and 3A5.We successfully determined the Cryo-electron microscope(Cryo-EM)structure of the ternary immune complexes composed of two antibodies and gB.We analyzed the potent neutralizing epitopes on gB for the first time and carried out preliminary research including the design and evaluation of epitope vaccine based on the structural information.First,we evaluated the binding and neutralizing activities of antibodies in vitro and in vivo systematically.We found that 3A3 and 3A5 had higher affinities than AMMO5 and 3A3 and 3A5 also bound to full-length gB anchored on the cell surfaces more efficient than AMM05.In the in vitro neutralization assay,3 A3 and 3A5 were capable of completely blocking epithelial and B cell infection of EBV,while AMM05 failed to inhibit virus infection of B cells even at the highest concentration.3A3 and 3A5 exhibited synergistic neutralization effects in both in vitro infection models.We further evaluated the protection effect of the antibodies in a humanized mouse model.The survival rate of mice was 100%for 3 A3 and 3A5 groups and 66.67%for AMMO5 group against lethal EBV challenge.3A3 and 3A5 can inhibit virus replication and EBV-induced lymphoma formation effectively in humanized mice.Next,we determined the Cryo-EM structure of the immune complexes of gB and nAbs for the first time and elucidated the structural basis of the antibodies’ efficient neutralizing activities.We had determined the Cryo-EM structures of the ternary immune complex with a resolution of 3.9 A and found that 3A3 targeted the D-II on the stem region and 3A5 targeted the D-IV on the head region of gB.3A3 mainly recognized aB of gB and 3A5 mainly recognized the loops connecting β27 and β28,P28 and P29,and P33 of gB.Furthermore,we clarified the molecular mechanism of efficient neutralization activity of antibodies.We determined the key residues recognized by 3A3(H352,E356 and D360)and 3A5(K540,T567,H610 and T613)using alanine-scanning mutagenesis.The mutations of key residues recognized by 3A5 impaired the efficiency of gBmediated membrane fusion,indicating that the epitope recognized by 3A5 is involved in the process of membrane fusion.3A5 may inhibit gB activation and subsequent membrane fusion by blocking the binding site.3A3 inhibited gB binding to the cell surface and its epitope overlapped with NRP1 binding site on gB.The introduction of mutations recognized by 3A5 to gB reduced the binding efficiency of gB with NRP1,suggesting that 3 A3 neutralized EBV infection by inhibiting the binding of gB to NRP1.Finally,we carried out the design,construction and evaluation of epitope vaccine based on the epitope of 3A3.The results showed that the epitope of 3A3 was displayed successfully based on the HBc149 particle and exhibited good antigenicity by retaining the binding activity to 3A3.Immunized serum induced by epitope vaccine was capable of neutralizing EBV infection in both in vitro models.In summary,we evaluated the potent gB-specific nAbs for the first time and found that 3A3 and 3A5 had excellent antiviral effects in vitro and in vivo,suggesting that 3A3 and 3A5 would be potential candidates for the prevention and treatment of EBV infection.We used structural information and biochemical results to elucidate the epitope and molecular mechanism of neutralization.Furthermore,we found that the epitope recognized by 3A3 could induce neutralizing antibodies in vivo.By exploring and studying the key neutralizing epitopes recognized by gB-specific nAbs,it is of great scientific significance and clinical application value to guide the design of prophylactic vaccines and targeted drugs for herpesviruses including EBV.