Preliminary Study Of The Influenza H5N1Virus Vaccine Based On Baculovirus Surface-display System

Since1997, H5N1avian influenza A virus has begun to infect human and caused them dead. With the unprecedented globe spread and continuing evolution of H5N1HPAIV (Highly pathogenic avian influenza virus), the pandemic potential of this virus still exist. Undoubtedly, Vaccines offer the most viable and effective measure for pandemic preparedness. However, egg-based productions of H5N1influenza vaccine unlikely to be able to satisfy the globe demand due to its limited production capacity within a short period of time at the emergency of pandemic.Thus, alternative, egg-independent vaccine manufacturing strategies should be exploited to supplement the traditional egg derived influenza vaccine manufacturing.For the purpose of obtaining a new influenza vaccine with high immunogenicity, broad protective effieney and rapid manufacturing system, this study explores preliminary study on the influenza H5N1virus vaccine using the technology of baculovirus surface-display system. Main contents and research method are as following:(1) Insert HA and NA sequence of H5N1strain(A/Hubei/1/2010) into modified expression vector pFast BacTM Dual to construct recombinant plasmid pFast-HA-NA, then transfect Sf9cell using Bac-to-Bac expression system, package the recombinant baculovirus named Bac-HA-NA which displaying HA and NA protein in its surface.(2) Test the two different immunization strategies of the above vaccine in BALB/c mice. Immunized Bac-HA-NA100μl/mouse by intramuscular injection (HA titer of log28) and vaccine Bac-HA-NA25μl/mouse by intranasal (HA titer of log210). The vaccinations were boosted with the same strategy and dose after28days. HA specific serum IgG antibodies were quantified by indirect ELISA. Cellular immune responses of the lymphocytes, taken from the mouse spleen, were determined by IFN-γ-ELISPOT assay using purified HA protein.(3) Functional cross-protective efficacy of the vaccine was assessed by host challenge against A/PR/8/34H1N1strains.The results showed that:1) HA and NA protein can be expressed simultaneously on the surface of recombinant baculovirus and sustained their authentic cleavage, hemagglutination activity, neuraminidase activity and antigenicity respectivly.2) The HA specific IgG titer could reach1:40000by intramuscular injection. While intranasal vaccination strategies couldn’t induce any HA specific IgG antibody.3) Both of the two vaccination strategies could induce effective cellular immune response. Intramuscular injection could induce479SFCs/million splenocytes, while intranasal vaccination induced325SFCs/million splenocytes.4) To against A/PR8/34(H1N1), the cross-protective effect of this vaccine can reach25%by intramuscular route, while there is no cross-protective effect by intranasal immune.This baculovirus surface-displayed vaccine is more efficacious than inactivated H5N1influenza vaccine when administered by intramusculal route and has no biosafety concerns associated with isolation, purification and production of the latter vaccine.