Studies Of DNA Vaccines Against H5N1 Avian Influenza Virus And A Molecular Adjuvant

The influenza A viruses are enveloped, segmented, single-stranded negative sense RNA members of the Orthomyxoviridae family. Eight gene segments in the influenza A viruses encode 11 viral proteins which represent surface and internal proteins:hemagglutinin (HA), neuraminidase (NA), the polymerase proteins (PB1, PB2, PA, PB1-F2), nucleocapsid proteins (NP), matrix proteins (M1, M2), and nonstructural proteins (NS1, NS2). HA and NA, spikes anchored in the virus membrane in a ratio of about 8:1, are the most important antigens since they are capable of stimulating protective immune response against infection. NP is one of the major components of the functional ribonucleoprotein complex (RNP). NP is a highly conserved protein, and induces cross-protective immunity against different influenza A virus strains and subtypes.Cornfirmed human cases of H5N1 avian influenza virus were first reported in 1997 in Hong Kong. The H5N1 outbreaked in poultry since late 2003 and affected more than 10 Asian countries, and viruses have been isolated from wild birds and poultry in Asia, Europe, and Africa. The continued circulation of H5N1 viruses in birds provides ample opportunity for them to infect humans. From December 2003 to March 2010, confirmed human H5N1 cases reported to WHO were 442, leading to 262 deaths with a fatality rate of approximately 60%. Therefore, there is an urgent need to develop effective vaccines against the H5N1 viruses.In the present study, we focused on the DNA vaccines against H5N1 avian influenza virus and a molecular adjuvant. A H5N1 influenza virus A/chicken/Hubei/489/2004 (A/Hubei/489) was used in the following research.First, NP gene was recovered from the H5N1 influenza virus and cloned into prokaryotic expression vector pET-28a to generate pET-△NP, which was transformed into E. coli BL21codon plus(DE3)-RIL. Expression of the protein was induced by IPTG and the recombinant NP protein was purified and analyzed by SDS-PAGE and Western blot. Rabbit polyclonal antibodies against NP were prepared by immunizing with the purified NP. ELISA analysis showed that the antibody titer was as high as 105. Western blot and indirect immunofluorescence analysis showed that the antibodies could specifically react with the NP protein expressed in prokaryotes or eukaryotes.Second, a fusion NP with the tissue plasminogen activator secretory signal sequence (tPAs) was amplified and cloned into pVAX1 vector to generate ptPAs/NP. A plasmid encoding the wildtype full-length NP, pflNP, was also constructed for comparison. Expression and subcellular localization of the fusion NP protein were analyzed. In a mouse model, the humoral and cellular immune responses induced by ptPAs/NP were studied. The protective efficacy of ptPAs/NP DNA vaccine against the homologous or heterologous H5N1 influenza virus infection was investigated. The results demonstrated that compared with pflNP, the NP-based DNA vaccine with the fusion tPAs (ptPAs/NP) showed enhanced specific humoral and cellular immune responses. The protective immunity against heterologous and homologous H5N1 influenza virus challenge was significantly improved.Third, eukaryotic expression plasmids pVAX1-TRIF and pVAX1-VISA were constructed as molecular adjuvants. The IFN-βand NF-κB promoter activities stimulated by the plasmids pVAX1-TRIF and pVAX1-VISA were detected using the luciferase reporter gene assay system. Data indicated that both pVAXl-TRIF and pVAX1-VISA could induce IFN-βand NF-κB transcription in cells. BALB/c mice were co-immunized with the plasmid pHA/NP147-155 (constructed by ourself) and pVAX1-TRIF or pVAX1-VISA. Our results demonstrated that both pVAX1-TRIF and pVAX1-VISA could induce enhanced immunity of DNA vaccine against heterologous and homologous H5N1 influenza virus challenge in mice, including enhanced cellular immune response, higher survival rate and decreased virus titer of the lungs. The results indicate the cellular signaling molecule VISA potentiates the efficacy of a DNA vaccine and could be used as a potential molecular adjuvant of DNA vaccines.