The Research Of Protection Of Synthetic HA2Peptide And Recombinant M2e Protein Of Avian Influenza Virus

Avian Influenza is a poultry disease syndrome caused by Influenza A virus. AI is ahighly contagious disease which spreads rapidly. AIV causes huge damage tohuman-beings’ life and production is one of the most important infectious disease. Notonly poultry, but also human-beings could be infected with it. According to differenthemagglutinin and neuraminidase, AIV is divided into17HA subtypes and9NA subtypes.High variability of HA and NA and low cross-protection among different subtypes of AIVboth limit the control of AIV and the vaccine development.HA, which induces neutralizing antibody and has multiple T/B cell epitopes is one of theprotective antigen of influenza virus. HA2locates in the C-terminal of HA had highlyconserved fusion sequence. So universal vaccine based on the conserved sequence of HA2is always a hot spot. Matrix protein2(M2) composed of97amino acids is atransmembrane protein of influenza virus. The sequence of M2is highly conserved,especially the extracellular domain composed of23amino acids located in the N-terminalof M2. Despite the little difference of M2e during four worldwide flu pandemic, it maydevelop into a candidate antigen of universal vaccine with cross-protection. In order to finda broad-spectrum influenza vaccine, the study synthesizes a peptide based on the HA2according to the HA of Chinese H5/H9subtype influenza viruses. Another way, we expressrecombinant protein based on the highly conserved sequence of M2e. The immuneprotection of HA2synthetic peptide and M2e recombinant protein are compared andevaluated by mice challenged detection, which paves a way for the further study of thebroad-spectrum influenza virus vaccine. The contents of the paper as follows:1Study of immune protection of HA2synthetic peptide of avian influenza virusThe peptide was synthesized based on the conserved regions of HA2and KLH wascoupled to its C-terminus by comparing the HA gene of Chinese H5/H9subtype influenzaviruses. Mice were intramuscularly immunized with LAH-KLH、LAH-KLH+CpG、KLH、PBS at0week and3weeks. Sera taken from mice at two weeks after boost immunizationwere to test the LAH-specific IgG and IgG1/IgG2a/IgG2b by ELISA. At the same time,splenocytes and lung cells were taken to detect the amounts of LAH-specific T/B cell byELISPOT. Then the immune protection of HA2synthetic peptide was evaluated by micechallenged detection、viral titers and histopathological changes in lung after infection.Results of ELISA showed only LAH-KLH+CpG-immunized group had tested high levels with extremely significant difference (p﹤0.01) of LAH-specific IgG compared with thecontrol group. Antibody sub-type results indicated LAH-KLH+CpG-immunized groupinduced secretion both of IgG1/IgG2a/IgG2b, partial to IgG1. ELISPOT resultsdemonstrated the numbers of spot formed in every1×106spleen lymphocytes secretingIFN-γ of the LAH-KLH+CpG-immunized group were up to80, which had extremelysignificantly difference (p﹤0.01) compared with control group. In all, it indicated CpGpromoted HA2synthetic peptide induced both TH1and TH2immune response. The resultof animal experiment suggested HA2synthetic peptide couldn’t protect mice from beenattacked by CXG11(H5N1) avian influenza virus, it only can lower viral titers andpathological damage in lung.2Study of immune protection of M2e recombinant protein of avian influenza virusBased on the sequences of the M2gene of Chinese H5/H9subtype influenza virusesreported in GenBank, four copies of M2extracellular domain composed of23amino acidsof H5M2e and H9M2e were synthesized and linked with three T/B cell epitopes located ininfluenza virus HA245-260respectively, tGCN4was added at their C-terminus. Then theywere cloned into pGEX-6p-1vector with Bam HI and Xho I. SDS-PAGE and Western-blotresults showed the recombinant proteins-SH5M2e and SH9M2e were successfullyexpressed and their immunogenicity was observed on BALB/c mice by an intramuscularinjection with Freund’s adjuvant. Sera taken from mice at two weeks after boostimmunization were to test the antibody and its subtype levels by ELISA. At the same time,splenocytes were taken to detect the amounts of specific T-cell by ELISPOT. Immuneprotection of recombinant proteins (SH5M2e/SH9M2e) were analysed by mice challengeddetection、viral titers and histopathological changes in lung after infection. Resultsdemonstrated the recombinant proteins (SH5M2e/SH9M2e) could induce high level ofM2e-specific IgG. Antibody sub-type results indicated recombinant proteins(SH5M2e/SH9M2e) induced secretion both of IgG1/IgG2a/IgG2b, partial to IgG1. Andonly SH9M2e can induce HA-specific IgG and IgG1. The numbers of spot formed in every1×106spleen lymphocytes of the immunized groups were up to150after being stimulatedby M2e peptide and the numbers of spot formed in every1×106spleen lymphocytes of theimmunized groups were up to130after being stimulated by HA peptide. Above all,recombinant proteins (SH5M2e/SH9M2e) with good immunogenicity could induce bothTH1and TH2immune response. Results of animal experiment demonstrated recombinantproteins (SH5M2e/SH9M2e) could partially protect mice from being attacked byCXG11(H5N1) avian influenza virus. And viral titers and histopathological changes also confirmed it.In all, the synthetic HA2peptide didn’t protected mice from been attacked byCXG11(H5N1) avian influenza virus. But the highly efficient recombinant protein withmultiple copies of M2e strongly paved a way for the further study of the broad-spectruminfluenza virus subunit vaccine.