The Development And Antiviral Mechanism Study Of Broadly Neutralizing Antibodies Against The Receptor Binding Site Of Influenza B Virus

Annual seasonal influenza epidemics caused by influenza virus types A and B give rise to considerable mortality and morbidity in humans globally.Influenza B can dominate certain influenza seasons and accounts for about 25%of all influenza cases,the disease burden caused by influenza B should not be overlooked.The efficacy of current influenza antiviral drugs and vaccines is limited due to the evolution and diversity of influenza viras,there is an urgent medical requirement to discover more high-efficacy antiviral drugs and vaccines against influenza infection.Hemagglutinin(HA)is the major influenza viral surface glycoprotein,which include many functional epitopes such as the receptor binding site(RBS)domain,and can induce various protective antibodies.Hence HA is a crucial target protein for the development of novel influenza vaccines and antiviral drugs.In recent years,the development and epitope identification of influenza HA broadly neutralizing antibodies(bnAbs)is creating new hope for the development of novel influenza vaccines and antiviral drugs.Studies have confirmed the presence of broadly neutralizing epitope on the RBS domain of influenza B virus,however,current influenza vaccines are unable to induce cross-lineage neutralizing antibodies against influenza B.Therefore,some scientific problems are definitely worth exploring:i.whether the broadly neutralizing epitopes on RBS domain of influenza B exist widely,ii:how to induce the cross-lineage bnAbs efficiently,iii:what kinds of antiviral mechanisms do the bnAbs use.In this study,we tried many immunization strategies to induce cross-lineage bnAbs against RBS domain of influenza B,and we generated an IgG subtype cross-lineage reactive antibody C12G6 by using live virus sequential intranasal immunization strategy.C12G6 recognizes a highly conserved epitope around the RBS domain of influenza B,and is the first antibody which shows HI activity against Victoria and Yamagata lineages of influenza B.In addition,C12G6 showed higher antiviral activity in vitro when compared to other previously reported influenza B bnAbs.C12G6 revealed more potent protection against influenza B viruses in ferrets and mice when made a comparison with those of other previously reported influenza B HA bnAbs and antiviral drug oseltamivir and revealed additive antiviral effect with oseltamivir,and encouragingly,protective efficacy was still observed even when C12G6 treatment was delayed.C12G6 neutralized influenza B viruses via various mechanisms,including blocking viral infection,virion release and membrane fusion,and triggering complement-dependent cytotoxicity(CDC)and antibody-dependent cell-mediated cytotoxicity(ADCC)antiviral responses.The multiple mechanisms possessed by C12G6 explained the reason why C12G6 inhibited influenza B viruses so potently in vitro and vivo.Notably,C12G6 is the most multimechanistic bnAb against influenza virus developed at present.Influenza B virus is constantly mutating,although C12G6 conferred potent antiviral activity aganst influenz B viruses,the ecsape mutant strains could emerge at any time,the formulation of therapeutic antibody cocktails contained different kinds of broadly neutralizing antibodies could dramatically lowered the risk of drug resistance.In order to generate various kinds of bnAbs against influenza B,we found that,for the intranasal live virus immunization strategy,the early mice immune antisera containing large proportions of IgM antibodies revealed broader antiviral activities when compared to those of the late immune antisera containing large proportions of IgG antibodies,suggested that some IgM subtype antibodies might possess broader anvirial activities to the IgG subtype antibodies.According to this result,we generated several high-efficacy bnAbs against RBS of influenza B,the best antibody C7G6-IgM was able to inhibit the viral entry of all the tested strains,demonstrated stronger potency and broader breadth of antiviral activity in vitro than all the other bnAbs in this study and reported previously and conferred potent broad protection against distinct lineages of influenza B viruses in mice and ferrets.The above results showed that there were certain broadly neutralizing epitopes existed on the RBS domain of influenza B,immune responses against such epitope could be induced through live virus sequential intranasal immunization strategy but not routine immunization strategies.This finding can provide the significant scientific evidence for the development of universal influenza vaccines.In this study,we generated several bnAbs against the RBS domain of influenza B,demonstrated that such kind of bnAbs could neutralize influenza viruses through multiple mechanisms.This finding can also provide the significant scientific evidence for the development of novel anti-influenza drugs.We also demonstrated that the IgM subtype antibodies which recognized certain epitopes on the RBS were able to inhibit the viral infections efficiently,suggested that IgM subtype antibodies could play an important role in prevention and control of highly variable pathogens.