Sduty On Immune Effect Of Inflenza Virus Matirx Protein2(M2)Vaccine Against H9N2Avian Influenza Virus

In this paper, various vaccines based on matrix protein2from H9N2avian influenza virus were prepared and their immune effects against homologous virus were evaluated. The paper consists of two parts:(1). Construction of M2DNA Vaccines and vaccination effects of the vaccines against lethal homologous virus challenge;(2).Evaluations of M2DNA prime-protein boost vaccination strategy against lethal homologous virus challenge.DNA vaccine can elicit both long-lasting humoral and cell mediated immunity, and can be rapidly constructed and mass-produced.It may be used as an effective weapon to fight against influenza outbreak. In this study, two types of recombinant eukaryotic expression plasmids containing M2segment from avian influenza virus A/Chicken/Jiangsu/7/2002(H9N2) were constructed as DNA vaccines, i.e. pCAGGSP7-M2expressing M2protein alone and pCAGGSP7-GAG-IRES-M2producing virus-like particles (VLPs) containing M2protein. BALB/c mice were immunized five times at a2-week interval with the plasmid DNAs, respectively, at a dose of100ug. Two weeks after the last immunization, mice were challenged with a lethal dose of homologous virus. The effectiveness of the vaccines was evaluated by analyziing M2-specific antibody response, survival rate, body weight loss and lung virus titer in mice compared with those of unimmunized mice. The results showed that, compared with the control, mice in both two immunized groups (i.e. pCAGGSP7-M2and pCAGGSP7-GAG-IRES-M2group) gained partial protection, with the protection rates40%and30%, respectively. Serum anti-M2antibody responses were elicited in the immunized mice, but the antibody level in mice immunized with pCAGGSP7-M2was higher than that in mice immunized with pCAGGSP7-GAG-IRES-M2. It demonstrated that gag-based VLP DNA vaccine could not effectively enhance the immunogenicity and protective efficacy of M2.In our previous study, it showed that intranasal immunization with M2protein adjuvanted with chitosan provided good protection for mice against lethal influenza virus challenge. Considering the unsatisfactory immune effect of M2DNA, we tried to use DNA prime-protein boost immunization strategy. The sM2protein,(i.e. M2protein without transmembrane domains) was expressed in E.coli and purified as previously described. BALB/c mice were grouped and immunized as follows:M2DNA twice (the group named D2), sM2protein twice (P2), M2DNA once+sM2protein once (D1P1), M2DNA once+sM2protein twice (D1P2), and M2DNA twice+sM2protein once (D2P1). The interval was3weeks between two immunizations, Three weeks after the last immunization, the mice were challenged with5LD50homologous virus. The survival rates, weight loss rates, lung virus titers and IgG antibody levels were analyzed. The survival rates in D1P2and P2groups reached100%and90%, respectively, in D1P1and D2P1groups were50%, in D210%, The mice in control all died. D1P2group sufferred less weight loss and recovered faster than P2group. Both D1P2and P2groups have a lower lung virus titer and a significantly higher IgG antibody level than other groups. Above all, immunization with sM2protein twice could provide good protection for mice, and M2DNA prime-protein boost vaccination strategy could provide complete protection against lethal homologous virus challenge and significantly lighten the symptoms of viral infection.