| Highly Pathogenic Avian Influenza virus(HP-AIV) seriously endanger the development of poultry industry because of its high morbidity and mortality in birds, and seriously threat to human life and health because of it was break through species barriers and infect human being directly. The best way to against AIV was vaccinal vaccinate. The new avian influenza vaccine was need because the traditionary vaccine several serious disagvantages including limited egg supply in case of a pandemic and the need for high-growth reassortants for new antigenic variants. Furthermore, contamination with egg-derived protein can cause allergic reactions to vaccines.It’s an important strategy for the new avian influenza vaccine development because AI-VLPs were safe, efficient and of good immunogenicity. However, there are still many difficulties, such as the complex ingredient of AI-VLPs and the protein purification limitation. It has been confirmed that influenza VLPs vaccine could elicit varying degrees of protective immunity in mice, ferrets and other animal models, but rarely reported in poultry.In this study, hemagglutinin (HA) alone or in combination with matrix1(M1) were expressed in Bac-to-Bac eukaryotic expression system and named with rBV-HA or rBV-HA-M1. The bioactivities of VLPs were identified by immunohistochemistry and hemagglutination test. The SDS-PAGE and western blot analysis indicated that the HA and HA-M1were efficiently expressed, but the VLPs were observed under transmission electron microscopy in Sf9cells, which was infected only with the recombinant baculovirus coexpressing HA and M1of H5subtype AIV. The immunohistochemistry and hemagglutination test demonstrated that both the HA and HA-M1expressed in the cells, and the HA titers reached1:512and1:1024, respectively.BALB/c mice and SPF chickens were vaccinated with both rHA and VLPs vaccine. The mice and chickens were vaccinated with Sf9cells as a negative control, and immunized with inactivated vaccines as a positive control. The serum antibody levels were tested by hemagglutination inhibition test (HI) and neutralization test. The mice were challenged with A/Chicken/Anhui/2009(H5N1) virus and the chickens were challenged with A/Goose/QFY/2004(H5N1) after vaccination. It was shown that VLPs vaccine or rHA vaccine as well as inactivated vaccine inducted protective immune responses in mice and chickens, and were protected from morbidity and mortality resulting from lethal influenza infections. The results of Immunization showed that the immune effect achieved the best state following the boost vaccination in BALB/c mice, and VLPs vaccine induced the highest antibody levels which could reach1:718, the rHA vaccine group and the inactivated vaccine could reach1:285and1:201, respectively. The statistically analysis showed that significant difference among rHA vaccine group, VLPs vaccine and inactivated vaccine were insignificant (P>0.05), and difference between VLPs vaccine and inactivated vaccine group were obvious (P<0.01), the difference between three immune groups and control group were obvious (P<0.01). It was demonstrated that the inactivated vaccine induced the highest antibody levels in SPF chickens models which achieved1:1024, the VLPs vaccine and the rHA vaccine were reached1:331or1:181, respectively. The statistical analysis showed that the inactivated vaccine group was extremely significant difference with VLPs vaccine group or the rHA group(P<0.01), but the difference between VLPs vaccine group and the rHA vaccine group was insignificant (P>0.05), the difference between three immune groups and control group were obvious (P<0.01). Neutralizing antibody titers were changed with HI antibody, a good neutralizing antibody could be induced in BALB/c mice and SPF chickens after vaccinated with VLPs vaccine two weeks, and the antibody levels were higher in mice. Weight change of the immuned BALB/c mice were insignificant after infected with AIV, the control group was reduced with23%on the average, and it demonstrated that thesr vaccine could protected mice from AIV infection. There were no death in immuned SPF chickens, however, all of the control group were died, the statistical analysis showed that significant difference between immuned group and control group was obvious(P<0.01), the VLPs vaccine and rHA vaccine could provide protection against the AIV infections in SPF chickens as well as inactivated vaccine.These results confirmed the influenza VLPs vaccine was able to elicit specific immune response, and a strong antibody reponse was generated following the boost injection in immuned BALB/c mice and SPF chickens. These results also indicated that VLPs vaccine can induce high titers of neutralizing antibody and HI antibody to provide sufficient protection against the H5-subtype influenza virus infection in BALB/c mice and chickens. The VLPs produced in Sf9cells facilitated for further study and development of the novel avian influenza vaccines. Influenza VLPs vaccine have been paid significant attention for their potential application in development of effective and safe vaccine against seasonal as well as pandemic influenza viruses. |
