Abstract：Influenza A viruses (IAVs) can infect poultry and many mammals including human (Homo sapiens), causing significant economic losses, and seriously threatening human health. Influenza vaccination is the most effective measure to prevent the occurrence of influenza. However, the effects of different immunization strategies on the immune responses following vaccination remains obscure. In this study, two antigenically distinct H9N2 subtypes of avian influenza viruses, A/chicken/Hubei/YK524/2015 and A/chicken/Shandong/LX830/2014, referred to as YK524 and LX830 were selected, respectively. The effects of homologous immunization and heterologous immunization and virus inactivation on antibody production were investigated. The viruses were purified by sucrose gradient centrifugation. Female BALB/C mice (Mus musculus) of 6~7 week old were immunized with purified Influenza A virus. Haemagglutinin inhibition titer in sera was measured regularly. The results showed that the antibody level produced by live virus antigen was higher than that induced by inactivated virus antigen in mice, with better cross reactivity. It suggested that live viruses were more likely to stimulate the body to produce antibodies with broad cross reactivity. In addition, heterologous immunity stimulated the body to produce more cross-reactive antibodies. Mice with higher antibody titer were selected to prepare monoclonal antibodies against influenza virus using hybridoma technique. Monoclonal antibodies were prepared by fusion of splenocytes and mouse myeloma Sp2/0 cells. Indirect immunofluorescence assay was performed to screen the antibody secreting clones. Following 3 round of sub-cloning, 3 hybridoma cell lines capable of secreting antibodies against influenza virus, namely H9-1, H9-2 and H9-3, respectively, were obtained. All three antibodies showed hemagglutination inhibition and neutralization activity against LX830 and another H9N2 influenza virus Mink/SD/14, although the titer of H9-1 and H9-2 were much higher than H9-3. This results suggested that the antigen recognition epitopes of these three antibodies were located at the haemagglutinin(HA) receptor binding site. All three antibodies could not bind specifically to YK524, none of them displayed hemagglutination inhibitory and neutralization activity towards YK524. We presume that this might be due to the key amino acids difference in the hemagglutinin between LX830 and YK524. This study discussed the way to stimulate the body to produce wider spectrum of antibody response, and would provide experimental basis for the development of influenza vaccine and for the optimal immune strategy design. The monoclonal antibody prepared in this study could be used for further diagnosis of influenza virus.
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