Efficacy Evaluation Of DNA Vaccines Co-expressing Consensus HA And NA Protein Of H5N1Subtype Avian Influenza Virus

Avian influenza is a kind of avian infectious disease caused by type A influenza virus. Highlypathogenic H5N1Avian Influenza threatens poultry industry most seriously for its high morbidity andmortality in chickens. DNA vaccine, known as the third generation vaccine, has many distinct features.It generates all three arms of adaptive immunity: antibodies responses, helper T cells and CTLs. Mostimportantly, it can provide excellent cross protection. DNA vaccine is regarded as the best vaccineagainst influenza virus, which is undergoing remarkable antigenic shift and antigenic drift.In order to develop a universe vaccine, which could provide broad protection against divergentH5N1influenza virus strains, hundreds of sequences of HA and NA gene of AIV strains isolated inChina in recent years were analyzed and two consensus sequences were obtained and named as Cons-CHA and Cons-CNA. Three DNA vaccines pCons-CHA, pCons-CNA and pCons-CHANA wereconstructed. In vitro expression was identified in293T cells by IFA assay and western blot assay. Theresults indicated that the recombinant consensus HA protein and consensus NA protein were effectivelyexpressed in293T cells. The expressed proteins had very good cross-reactions with different avianinfluenza positive serum.In order to evaluate the protective efficacy of those DNA vaccines, groups of SPF chickens wereimmunized with30g pCons-CHA, pCons-CHA and pCons-CNA combined vaccine and pCons-CHANA, respectively, and boosters were conducted with same dose after three weeks. Challenge wasperformed with lethal dose of HPAIV DK/GD/322/2010（Clade2.3.2.c）, DK/HuB/S1513/2010（Clade2.3.2.b）, CK/SD/A-10/2011（Clade7.2） and DK/FJ/31/2007（Clade2.3.4.a）, respectively two weeks afterthe booster. Sera samples were collected weekly to detect the HI antibodies. Results demonstrate thatpCons-CHA could provide100%,100%,90%and100%death protection against the challenge ofDK/GD/322/2010,DK/HuB/S1513/2010, CK/SD/A-10/2011, DK/FJ/31/2007and the correspondingaverage HI antibodies were4.2log₂,5.1log₂,4.6log₂and1.9log₂, respectively. The pCons-CHA andpCons-CNA combined vaccine could provide100%,100%,100%and90%death protection against thechallenge of DK/GD/322/2010,K/HuB/S1513/2010, CK/SD/A-10/2011and, DK/FJ/31/2007, and theaverage HI antibodies were5.1log₂，4.1log₂,4.6log₂and5.1log₂, respectively. The vaccine pCons-CHANA could provide completely protection （no disease signs, no virus shedding, and no deaths）against the challenge of DK/FJ/31/2007and CK/SD/A-10/2011.While it provided death protectionagainst the challenge of DK/GD/322/2010and DK/HuB/S1513/2010strains. The average HI antibodieswere6.3log₂,4.6log₂,5.4log₂,and5.3log₂, respectively.In summary, the vaccine strain pCons-CHANA has the potential to be a universal vaccinecandidate for against H5N1viruses of different antigenicity from different clades.