Rational Design And Immune Response Study Of Universal Vaccines Against Influenza B Viruses Based On The Anti-Hemagglutinin Broadly Neutralizing Antibody C12G6

Influenza viruses can cause severe respiratory infections in humans as a serious public health problem.According to the World Health Organization,seasonal influenza viruses,including influenza A viruses and influenza B viruses,cause approximately 35 million severe influenza cases and 290,000-650,000 deaths each year worldwide.However,approximately 25%of all human seasonal influenza infections are related to influenza B viruses.Although the pathogenicity of influnenza B virus is always underestimated,it has been proved actually that influenza B virus infections are as severe as influenza A virus infections.Currently,vaccines are still our best option for preventing influenza virus infections and disease.However,influenza virus vaccines have to be reformulated almost every year against virus antigenic drift,.To solve these problems,a long-lived and broadly protective influenza virus vaccine is an urgently needed.Hemagglutinin(HA)is the most significant glycoprotein in influenza surface and the major target for host immune response.In recent years,broadly neutralizing antibodies against the conserved epitopes of influenza virus HA have been isolated and identified following the relevant technologies development.Such antibodies may greatly reinforce our prophylactic and therapeutic anti-viral drugs against influenza viruses while promoting the foundation for newly vaccine development.Generally,the HA stem region in indeed highly conserved among different strains and subtypes of influenza virus but poorly immunogenic,which can induce broadly antibody response.The majority of the antibody response for influenza virus infection or vaccination is almost directed towards the HA head domain,which is therefore considered immunedominant.The head domain in HA is highly variable and undergoes constant changes which allows the virus to evade the host immune system.So antibodies against this head domain are considered to be strain-specific.Hence,the HA stem region was recently developed as a component in a universal flu vaccine after those finding that broadly neutralizing antibodies(bNAbs)against this region while the universal vaccine based on the epitopes of HA head region has not been made breakthrough.In previous study,an IgG subtype cross-lineage reactive antibody C12G6 was screened and identified,which recognizes a highly conserved epitope around the RBS of HA domain.Interestingly,C12G6 is the neutralizing antibody for influenza virus with most various mechanism at present and provide a novel epitope for further universal vaccine design research.In this study,C12G6 was selected for further rational design of universal influenza B virus vaccine and provide exploring experience for epitope-focused vaccines.At first,we found that C12G6 recognized the RBS domain by engaging interaction with both heavy chain and light chain of C12G6,according to the analysis of the crystal structure of C12G6-HA complex.Notably,C12G6 may bind to the 160 loop at the surface of HA protein and recognize the variable domain so that neutralizing the both lineages of influenza B virus,suggested that the head domain may contain certain conserved and protective epitopes,not only variable and immune-dominant antigenic sites.Then,based on the conformational epitopes recognized by C12G6,we established the series of approaches for rational design of structure-based vaccine and try to focus the immune response to epitope peptide recognized by C12G6,through immunization with C12G6-peptide fused to CRMA carried protein,fusion peptide coupled carriers and HA scaffold,induce non-neutralizing response.However only one of the immunogen with coupled carried showed an excellent in vivo protective efficacies against both lineages for influenza B virus.The molecular mechanism analysis proved that the protective function was based on ADCC response,but the neutralizing antibodies.Finally,to further study the phenomenon which is hard to rebuild the discontinuous and conformational epitope and induce C12G6-like antibodies,we used deep sequencing to study the production and evolution of C12G6 and found it hard to promote the maturation of C12G6-like antibodies.In the meantime,we want to optimize the antibody targeting globular domain in HA for enhancing broadly activities based on structural biology and bioinformatics,explaining and demonstrating that the intact function for broadly neutralizing antibodies could be determined on and reversed by several key residues.The above results showed that there is a broadly neutralizing epitope located on the RBS domain of influenza B recognized by C12G6,but the effective immune responses targeting this discontinuous and conformational epitope may be hard to induced through traditional immunization strategy or immunogen,demonstrating that the advanced design technology for immune-focus by epitope-based vaccine would still be the check point to develop universal influenza vaccine based on broadly neutralizing epitopes.In this study,we established a structural-based platform for rational design of epitope vaccine,providing experience and technology support for further universal vaccine for influenza virus.We also generated a fusion immunogen inducing robust ADCC response and showing an excellent in vivo protective efficacies against both lineages for influenza B virus,which suggested the non-neutralizing antibodies for influenza virus may also play a major role in universal influenza vaccine design.