Preparation, Optimization And Immunogenicity Of Virus-like Particle Vaccine Of 2009 H1N1 Influenza

Influenza caused by influenza virus is an acute respiratory infectious disease,and spread fast, highly infectious.The number of seasonal influenza-associated deaths per year in the world has ranged from 250,000 to 500,000.The pandemic 2009 H1N1 influenza had posed a serious threat to public health security, then the influenza evolved into seasonal influenza,and epidemics occur yearly.The most efficiency strategy to reduce the impact of influenza infections is vaccination. Therefore, the development of a more safe, affordable, and potent influenza vaccine is one of the main concerns of influenza research.In a number of vaccine research programs,VLP mimic the morphology of the natural virus, and lead to highly immunogenic. There was no genetic material in VLP, which non-replicative and non-infective.Another strategy is to modify the VLP with the immunostimulatory molecule adjuvant to construct a chimeric influenza virus-like particle.Therefore, virus-like particles (VLP) based on A/Changchun/01/2009 (H1N1) and comprising hemagglutinin (HA), neuraminidase (NA), and matrix (M1) were produced using a baculovirus expression system.Then we generated the chimeric VLPs composed of membrane-anchored forms of the Cholera toxin B subunit (CTB) or Ricin toxicity B subunit (RTB) or staphylococcal enterotoxins A (SEA),and evaluated the immune responses induced by these VLPs in mice.1. Construction and identification of 2009 H1N1 influenza virus-like particle and chimeric influenza virus-like particle of CTB-VLPs^ RTB-VLPs.VLP based on A/Changchun/01/2009 (H1N1) and comprising hemagglutinin (HA), neuraminidase (NA), and matrix (M1)were produced using a baculovirus expression system, then we generated the chimeric VLPs composed of membrane-anchored forms of the Cholera toxin B subunit (CTB) or Ricin toxicity B subunit (RTB).By western blot and Immunofluorescence assay, all proteins expressed correctly; by electron microscopy, influenza VLPs and chimeric influenza VLPs were round, and with diameter were approximately 100 nm, obvious surface spikes were observed, and with functional activity. The incorporation of CTB and RTB did not alter the morphology of influenza VLPs.2. Immunogenicity study of 2009 H1N1 influenza virus-like particle and chimeric influenza virus-like particle of CTB-VLPs、RTB-VLPs.The immunogenicity of influenza VLPs、CTB-VLPs and RTB-VLPs were evaluated by intranasal vaccination in mouse.Influenza VLPs、CTB-VLPs and RTB-VLPs were immunogenic, but compared with influenza VLPs, both CTB-VLPs and RTB-VLPs induce significantly enhanced humoral and cellular immune responses, showed 5 ～7-fold higher virus-specific IgG titers.Immunization with influenza VLPs only provide a partial protection for FM1-6 virus (80% survival), however, CTB-VLPs and RTB-VLPs provided complete protection against lethal challenge with UI182 virus and FM1-6 virus.3. Construction and identification of chimeric influenza virus-like particle of SEA-VLPs.We constructed and rescued recombinant baculoviruses (rBVs) of rpFastBac1-HA、 rpFastBac1-NA、rpFastBacl-M1 and rpFastBacl-SEA that express HA、NA and M1 of A/Changchun/01/2009 (H1N1) and SEA, then co-infected Sf9 cells. By western blot and Immunofluorescence assay, all proteins expressed correctly. By electron microscopy, the chimeric influenza VLPs has the typical characteristics of influenza virus, and with functional activity.We assembled the chimeric influenza virus-like particle of CTB-VLPs successfully.4. Immunogenicity study of chimeric influenza virus-like particle of SEA-VLPs.Mice were immunized by intramuscular,then challenge at days 7 post-vaccination or 10 post-vaccination to evaluate the protective effect. Compared with influenza VLPs, SEA-VLPs induce significantly enhanced humoral and cellular immune responses. Reduce the vaccine immune response time and shorten the immune blank period.The results indicate that chimeric of molecular adjuvants improved the immunogenicity of VLPs effectively. CTB and RTB enhanced humoral and cellular immune responses effectively, and stimulated the production of mucosal immune response, improved the protection efficiency of vaccine. SEA increased the rate of immune response significantly,and shorten the immune blank period. This study provides technical support for the development of new influenza vaccine.