Isolation And Characterization Of Full Human Neutralizing Antibodies Against Respiratory Syncytial Virus

Respiratory syncytial virus（RSV）is the most common cause of acute respiratory infections in infants and young children,and is also a disease burden in the elderly or those individuals with chronic or immunocompromised conditions.The rates of hospitalization and mortality caused by RSV and influenza infection were similar in the elderly population.Currently,there is no available effective vaccine for RSV on the market,and there is also a lack of RSV-specific antiviral drugs to treat RSV infection.Humanized palivizumab is the only anti-infective monoclonal antibody approved for RSV prevention in high-risk children.However,palivizumab does not effectively treat RSV infection and has been demonstrated to select escape mutants in vitro and in vivo.Due to the limited efficacy of palivizumab and the lack of effective vaccines,the development of new fully human neutralizing antibody alternative drugs is urgently needed.RSV belongs to the Paramyxoviridae family,which has a negative-sense single stranded RNA genome.Fusion protein（F,fusion protein）and attachment protein（G）are the major glycoproteins on the surface of RSV virus,which play an important role in the process of virus entry into cells.The F protein and the G protein are important antigens that could induce a protective immune response.They are the main targets of neutralizing antibodies（NAbs）and important targets for vaccine design and small molecular inhibitors.Firstly,although neutralizing antibodies play an important protective role in the prevention of RSV infection,our understanding of human antibody responses during the natural infection of RSV is limited,especially for antibodies against G protein.In order to better understand the antibody immune response against F and G proteins during RSV natural infection and to develop new antibody molecules as potential drugs for passive immunization,we initially characterized human serum response from 31healthy adults,including neutralizing antibody titers,binding activity to the prefusion F protein（pre-F）,G protein and the central conserved domain（CCD）of G protein.By analysis of the correlation using statistical methods,it is revealed that the titer of serum neutralizing antibodies is not directly related to the F protein binding titer,and may be more correlated with neutralizing antibodies targeting certain antigenic sites.Furthermore,we demonstrate that antibodies directed to CCD domain are ubiquitous in serum.Secondly,we isolated 30 antibodies against pre-F and G proteins from peripheral blood memory B cells of one of the volunteers with the highest titer of neutralizing antibodies using single-cell PCR.We determined the binding activity,microneutralizing activity and epitope mapping of F protein specific antibodies.Our results show that neutralizing antibodies against F protein target two main epitopes:antigenic siteφand antigenic site III-based quaternary epitope,suggesting the serum neutralizing activity of volunteers may be related to the presence of antibodies specific for these two antigenic sites.Thirdly,in order to understand how the neutralizing antibody evolved and whether it has some similar genetic features to recognize certain neutralizing sites,we analyzed inferred germline,the frequency of somatic hypermutation and the length of the CDR3 of the neutralizing antibodies.The results suggest that B cells tend to target a more conserved sensitive antigenic site III on the RSV F protein using a combination of IGHV3-21*01 and IGLV1-40*1,which may possess some common molecular structural features to recognize a susceptive epitope.Furthermore,we confirmed that the 3F4 targeting this epitope cross-binds the F protein of respiratory syncytial virus（RSV）and human metapneumovirus（hMPV）.Finally,we compared the protective activities of 4E6,4F1 and palivizumab in vitro and in vivo.The results of microneutralization experiments showed that the neutralizing activity of 4E6 and 4F1 antibodies against RSV A and B subtype was greater than that of palivizumab.Notably,the IC₅₀ of 4F1 is nearly 80-100 times lower than palivizumab.We tested in vivo efficacy of 4E6 and 4F1 in prophylactic settings,4E6 and 4F1 were more potent than palivizumab in reducing HRSV lung titres.The evaluation of lung histopathology showed that compared with palivizumab,4F1 from the high and low dose groups can significantly inhibit the infiltration of monocytes into the bronchi,pulmonary interstitial and alveolar spaces during acute infection.Overall,4F1 has great potential for the development of drugs for the prevention of RSV infection.In addition,we demonstrated that the combination of 4F1 and 4E6 targeting two different epitopes has a synergistic effect on antiviral effects,and based on this result,we built bispecific antibodies and test the binding activity and neutralizing activity.It has been shown that bispecific antibodies exhibit improved binding and neutralization potency compared to their parental NAbs and cocktails.In addition,bispecific antibody possess more broadly antiviral activity compared with the parental NAbs.Taken together,this study provides strong support for the important antigenic sites that are needed to be retained based on pre-F protein vaccine candidates.Meanwhile the newly discovered antibody molecule is a potential drug for RSV antibody based immunotherapy.Furthermore,we report for the first time that a combination of antibodies targeting two different epitopes of F protein has synergistic effects against RSV infection.The bispecific antibody we designed provides a new idea for passive immunotherapy to prevent RSV severe infection.In addition,by analyzing the sera of dozens of healthy volunteers,we found that antibodies against the central conserved domain of G protein are ubiquitous in healthy adults,and we have also isolated antibodies that target this site.The function and mechanism of antibodies targeting CCD in anti-RSV infection deserves further investigation.