Application Of Luciferase Reporter Influenza Virus Carrying Chimeric Hemagglutinin In The Screening Of Broad-spectrum Neutralizing Influenza Antibody

The outbreak of seasonal flu and pandemic bring a huge threat to human health.Influenza virus belongs to the genus orthomyxoviruses and its genome consists of series of negative strand RNA segments.Influenza A viruses infection is the most extensive and most harmful among all three types of influenza viruses.Influenza virus surface hemagglutinin molecule is three HA monomer polymerization trimer.It is divided into two part,the globular head and rod-like stem are connected by a disulfide bond consisting of a conserved 52-277 cys.The head domain contains a sialic acid receptor binding site that mediates the binding of the virus to the host cell.The HA head domain of different subtypes is highly variable in amino acid and the major target of the traditional neutralizing antibody prevents the virus from binding to the host cell.The stem domain is highly conserved and can be used as a binding target for broad-spectrum neutralizing antibodies.However,due to the exposed surface antigen epitope rarely,these antibodies to get more difficult.The HA stem domain is relatively conserved in all HA subtypes and can be used as a binding target for broad-spectrum neutralizing antibodies.At present,the screening of broad-spectrum neutralizing antibody is a hotspot in the research of influenza antibody.However,traditional hemagglutination inhibition and micro neutralization experiments are difficult to quickly screen for antibodies that detect a conserved region for HA stems.The use of reverse genetic technology to produce chimeric HA influenza virus,will be able to quickly screen for the conservative stem of the broad-spectrum neutralizing antibodies.Previously,we have rescued a recombinant influenza virus carrying Gaussia.luciferase reporter gene,which we named IAV-luc.IAV-luc is replication-competent both in vitro and in vivo.It can cause the onset and death of mice.In this study,we try to rescue new type of luciferase reporter influenza viruses carrying chimeric globular head and stem domain form different subtype of HA genes in the background of IAV-luc.Furthermore,we aim to develop high-throughput method for screening the broad-spectrum neutralizing antibodies which target the conserved stem domain by these chimeric HA luciferase reporter influenza viruses with high sensitivity and feasibility.We generated luciferase reporter influenza viruses carrying chimeric hemagglutinins from different subtypes of head and stem combinations.These chimeric recombinant reporter viruses named H1-PR8/H1-CA09-luci,H1-PR8/H5-HK-luci,H9-HK/H1-PR8-luci,H1-CA09/H1-PR8-luci.Chimeric HA reporter viruses were pre-incubation with the tested monoclonal antibodies.After incubation,the mixture was inoculated into the cell plates.At 24 hours post infection,the substrate was added into the cell culture medium and then cell plates were analyzed by fluorescent illuminitor or IVIS imaging system.As shown in the 96-well plate,the chimeric luciferase reporter viruses could be inhibited by stem-specific broad neutralizing antibody and shown no fluorescent signals.On the other hand,fluorescence signal could be detected.The neutralizing antibody(4E6CR9114?C179?8852)was used to react with three luciferase reporter influenza viruses of HK97-H5-luci,PR8-NA-luci,H1-PR8/H5-HK-luci.Among them,4E6 only reacts with H5-HK97-luci,and CR9114,C179,8852 reacts with all fluorescent viruses of H5-HK97-luci,H1-PR8-luci,H1-PR8/H5-HK-luci.The above experiment can effectively distinguish the antibody recognition domain.In summary,this experiment for the first time rescued the chimeric HA influenza virus carrying Gussia.luciferase reporter gene.The use of this virus can establish a highly efficient broad-spectrum influenza neutralizing antibody screening method.This method overcomes the shortcomings of the traditional HA stem antibody detection method,which is time consuming and low sensitivity.At the same time,this method provides a viable solution for broad-spectrum anti-influenza virus neutralizing antibody screening and new ideas for the prevention and epidemiological investigation of influenza virus.