Characterization And Functional Validation Of Influenza Virus Antibody CR9114 Mutants

Objective:Influenza is an epidemic infectious disease caused by influenza virus infection.Seasonal influenza causes large-scale worldwide spread every year,causing great harm to the economy and health.Every year,the World Health Organization constantly monitors influenza virus strains and transmission patterns in response to the next influenza pandemic.Prophylactic vaccination is an important prevention and treatment strategy to deal with the infection and transmission of influenza virus,but the diversity and high frequency of variation of influenza vaccine bring great challenges to the research and development of influenza vaccine.the drug resistance of influenza virus also greatly hinders the prevention and treatment of influenza virus infection.In recent years,therapeutic influenza monoclonal antibody is a new strategy to deal with influenza virus infection,in which general broad-spectrum influenza monoclonal antibody is an important drug for the treatment of influenza infection.At present,most of the antibodies produced by influenza vaccine are aimed at the head of influenza hemagglutinin,and influenza hemagglutinin,as the main glycoprotein coated on the surface of influenza virus,is the main medium for influenza virus to invade the body.the stem area of influenza hemagglutinin is also the main site of antibody recognition.Monoclonal antibody CR9114 is a universal influenza antibody.At present,it has been proved that it can recognize many different subtypes of influenza virus and has a certain neutralizing effect.However,the antibody mainly targets influenza virus strain H1N1,and its neutralization effect on other types of virus strains is relatively weak.In this study,we will first explore the basic characteristics of wild-type CR9114 antibodies,further modify the amino acid sites of influenza antibody CR9114,and evaluate the binding ability,affinity characteristics,recognition epitope information and neutralization activity of mutated CR9114 antibodies in vitro,in order to confirm the effect of these amino acid sites on the basic characteristics of influenza antibodies.This study provides a new idea for the follow-up screening and evaluation of therapeutic antibodies against highly pathogenic influenza viruses.Methods:Firstly,wild type CR9114 antibody and different mutant influenza monoclonal antibodies,including heavy chain single mutation antibody(H-A+L),light chain single mutation antibody(H+L-A)and heavy chain light chain double mutation antibody(H-A+L-A),were expressed and purified.In the second step,the RNA of influenza virus strain was obtained by reverse transcription to obtain cDNA,and the antigenic target fragments of different influenza virus strains were amplified by specific primer PCR:H1N1-HA1,H1N1-HA2,H7N9-HA1,H7N9-HA2.The antigenic expression vectors of pmCherry-H1N1-HA 1,pmCherry-H1N1-HA2,pmCherry-H7N9-HA1 and pmCherry-H7N9-HA2,influenza viruses were constructed.In the third step,the affinity and neutralization activity of wild-type and mutant antibodies with different influenza virus antigens were verified by enzyme-linked immunosorbent assay,energy resonance transfer,co-immunoprecipitation,micro-neutralization test and so on.Results:Both mutant and wild type influenza antibodies could recognize influenza virus antigens including H1N1-HA,H5N1-HA and H7N9murha,and could neutralize H1N1 and H7N9 strains.The results suggest that the change of amino acid site has a certain effect on the expression,affinity and neutralization effect of influenza antibody.Among them,the mutation of single amino acid site of light chain or heavy chain can have a certain effect on the affinity of CR9114 antibody,and the affinity of double mutation antibody of light and heavy chain decreases obviously.The results of neutralization experiment showed that the neutralization effect of light chain single mutation antibody was better than that of the other two mutants in vitro.In summary,the single mutation modification of the light chain of CR9114 can enhance the broad spectrum and neutralization effect of the antibody to a certain extent.