Construction And Immunoefficiency Of DNA Vaccines Against H1N1 Subtype Of Influenza Virus

Influenza is the first disease under worldwide intensified surveillance disease and it is an ancient, contagious and acute respiratory disease. Influenza outbreaks are responsible for tremendous morbidity, mortality and economic loss. Pandemic influenza threats human health, even the steadiness of society. The prominent characteristic of influenza virus is continuously antigen change and thus bypassing the host's acquired immunity to influenza .Vaccination is still the most effective method of prophylaxis.Recently, DNA vaccines become appreciated because they are able to induce cellular and humoral response to the encoded antigens and can be used repeatedly due to lack of immune response to the vector itself.The viral HA is the main determinant of virulence of the virus and plays an important role in the viral life cycle. The NA cleaves sialic acid and plays important roles in viral entry and release. NP segment and the RNA form a ribonucleotide NP(RNP) complex.HA, NA and NP gene of Influenza virus subtype A/PR/8/34 were amplified from cDNA by PCR and were inserted into the eukaryotic expression vector pVAX1 to construct recombinant eukaryotic expression plasmids. Then we amplified the gene of fusion HA1-NA1 protein by over-lapping extension PCR to construct the bivalent DNA vaccine. Immunochemistry method and western blot analysis indicated that influenza virus HA, NA, NP and HA1-NA1 were expressed in COS-7 cells 48 h after being transfected of recombinant plasmids.When BALB/c mice were immunized with 100μg recombinant plasmids intramuscularly, specific antibody were detectable in sera of mice by the first immunization. The specific IgG levels were significantly increased following 3 times of enhanced immunization. Furthermore immunoglobulin G isotype analysis showed IgG2a level were higher than IgG1, and the value of CD4⁺/CD8⁺T was lower than that of the control mice. All the above indicted that 4 DNA vaccines can induce Th1 type response. Meanwhile the immunogenicity of bivalent HA1-NA1 DNA vaccine was not enhanced but equal to NA DNA vaccine? HA.NA and NP DNA vaccines were able to induce effective immuno-protection against intranasal challenge of the same subtype virus with 40LD₅₀.Recombinant asd-pVAX1/HA and asd-pVAX1/NA were transformed into an attenuated S . typhimurium strain X4550, namely X4550(asd-pVAX1/HA) and X4550 (asd-pVAX1/NA), and were administered into BALB/c mice orally. They could induce mucosal and humoral immunoresponses. The plasmids were steadily maintained by a host-plasmid balanced lethal system based asd gene. Both X4550 (asd-pVAX1/HA) and X4550 (asd-pVAX1/NA) induced HA-specific and NA-specific serum IgG, IgA and SIgA antibodies. They were capable of inducing effective immuno-protection against challenge of 4OLD₅₀ influenza virus. These results demonstrated that two Salmonella-based delivering systems can release harboured DNA vaccines in vivo, and elicit protective immune responses.