Identification and evaluation of protective ability of a T cell epitope targeting nucleoprotein of H5N1 influenza virus

Abstract

The outbreak of human influenza caused by highly pathogenic influenza A (H5N1) virus has emphasized the urgent need of developing new vaccines.[1, 2] Although the vaccines targeting at hemagglutinin (HA) have shown efficient immunogenicity, the antigenic variations of HA may lead to resistance against such vaccines. While previous studies have shown that the nucleoprotein (NP), one of the most conserved proteins of influenza virus, is a significant target for diverse protection.[3, 4] In our previous study, we identified a novel HLA-DR1 restricted T cell epitope, named NP-7. The objective of this study is to investigate whether NP-7 may increase protection against H5N1 virus infection. DNA vaccine is an effective means of eliciting both humeral and cellular immunity. We constructed a DNA vaccine, pVAX-tNP, by inserting the full length of NP gene of A/Viet Nam/1194/2004 (H5N1) into plasmid vector pVAX-1. To verify the expression of NP, pVAX-tNP was transfected into 293FT cells in six-well plates using Lipofectamine™ 2000. The cells were harvested at 72 hours post-transfection, lysed and analyzed by Western blotting. Protective ability of NP-7 was studied on HLA-A2.1HLA-DR1transgenic mice (SURE/L1). Mice were primed with the DNA vaccine then boosted with twice or once of NP-7 peptide. Ten days after the last boosting, mice were challenged with lethal dose of H5N1 influenza virus in biosafety level 3 laboratories. The survival rate of each group was calculated as percentage. The splenocytes of mice were collected for ELISpot to evaluate T cell responses. In Western Blotting analysis, NP expression was probed by a rabbit anti-NP polyclonal Ab. The results of ELISPot showed that the IFNgamma secreting T cells in mice group which were boosted twice with NP-7 peptide is much more than other groups. Two weeks after challenge, 57% of mice in mice group which were boosted twice with NP-7 peptide survived while 14% survived in blank vector and adjuvant control group. These results indicated that NP-7 can provide partial protection against challenge of H5N1 influenza virus, which may benefit future vaccine design.