Study On The Immunogenicity Of DNA Vaccine And Adeno-vector Vaccine Against H5N1 Influenza A Virus

H5N1 avian influenza A virus has begun to infect humans since 1997 and the cases are accumulating.Recently,limited human-to-human transmission has been reported in small family clusters in Thailand,Indonesia,Pakistan and China.In addition,H5N1 virus continues to mutate and evolve to allow efficient human-to-human transmission.At present,the most effective measure to prevent and control human seasonal or avian influenza is vaccination.However,current licensed vaccine manufacturing procedures for influenza rely heavily on chicken embryo and will not meet the global demand during the period of influenza pandemic. In order to strengthen and expand the vaccine production capacity,methods to enhance the expression of target surface antigen hemagglutinin(HA) of H5N1 virus is essential in the antigen-sparing approach.The aim of the present study is to construct DNA vaccine and adenovirus vector vaccine against H5N1 influenza virus.Expression of HA was improved by codon optimization strategy and the immunogenicity of the vaccines were tested in a mouse model.In this study,a predominant human isolate(A/Anhui/1/2005) in China from clade 2.3.4 was used in the design of vaccine construction.It is recommended by the WHO as the only vaccine candidate for Clade 2.3.4 strain in China and is genetically and antigenically different from the other clades.Current H5N1 vaccine strains are selected from human isolates from clade 1,such as Hong Kong or Vietnam strain. Data in the extent of cross-neutralization between different clades is very limited. In this study,HA DNA sequence of H5N1(A/Anhui/1/2005) influenza virus was optimized in accordance with human codon preference so as to enhance its protein expression in eukaryotic system.Synthetic HA gene product was inserted into the eukaryotic expression vector pDC315.To check and compare the protein expression levels of HA in a eukaryotic system,plasmids containing modified HA (pDC315-Mod.HA) and wild-type HA(pDC315-Wt.HA) were transfected into 293T cells respectively.Secondly,a DNA vaccine for modified HA was constructed using DNA vaccine vector pVR-1012d.Thirdly,AdMax^TM adenovirus packaging system was used to construct two adenovirus vector vaccines,which are Ad-Wt.HA and A.d-Mod.HA.Finally,the different vaccines and immunization strategies of the above DNA and adenovirus vaccines were tested in BALB/c mice for the immunogenicity. Immune responses between vaccinated groups and unvaccinated groups were studied at the level of humoral and cellular immune responses.Antibody levels in the serum samples were determined by standard haemagglutination assay and microneutralisation.Cross-reactivity of the vaccine-induced antibodies against Anhui 01 was tested against eight different H5N1 influenza viruses.Cellular immune responses of the lymphocytes taken from the mouse spleen were determined by IFN-γELISPOT assay using overlapping peptides across the whole H5 HA antigen.The results showed that:1) The expression of codon optimized HA protein in 293 T cells increased significantly,and humoral and cellular immune responses induced by the vaccines were significantly enhanced;2) The adenovirus vector vaccine with codon optimized HA could induce effective neutralizing antibodies against different human H5 isolates in China.3) Two immunizations of single adenovirus vector vaccine could induce strong humoral and cellular immune responses,and are significantly better than two immunizations of individual DNA vaccine;4) DNA Prime-Adenovirus Boost vaccination strategy is the best immunization strategy and is worth further investigation. 5) Two CD8 epitopes of A/Anhui/1/2005(H5N1) HA gene were identified from the lymphocytes of vaccinated BALB/c mouse based on the IFN-γresponse.The sequences HA_212-229:TYISVGTSTLNQRLVPKI and HA_534.543:IYSTVASSLA,and the latter is stronger than the former.