Prediction And Identification Of CTL Epitoper Of Nucleoprotein From An H5 Avian Influenza Virus In Chickens

Avian influenza virus(AIV) is a causative agent of highly contagious viral disease in chickens. Previous studies showed that T cell-mediated immune responses play a critical role in defense against influenza virus infection. The nucleoprotein (NP) encoded by the fifth fragment of AIV is a conserved inner protein, and it contains many T cell epitopes. Those epitopes can induce Cytotoxic T lymphocyte(CTL) response against the infection of different subtypes of influenza viruses. Some T cell epitopes from various proteins of influenza virus have been identified in human and mouse. However, little is known about which in chicken partially for lacking of the structure of the chicken major histocompatibility complex (MHC).In this study, we have developed three-dimensional structures ofα1 andα2 domains of chicken MHC classΙmolecules from B4, B12, B15 and B19 haplotypes, those models provide clear insight into the structural characters of their peptide binding grooves. The surface of peptide binding grooves in those four structures are high lipothilic; The B pockets of the four structures are more flexible than the F pockets; The electrostatic potential of the peptide binding groove of B4 haplotype is highly positive charged,B15 and B19 haplotypes are negatively charged, while that of B12 is neutral. Based on structural properties, we screened antigen peptides derived from an AIV NP according to peptide-binding motifs and molecular docking, all together 25 peptides (10 for B4, 6 for B12, 2 for B15 and 7 for B19 haplotype) were predicted with potentials to bind to those MHC class I molecules. The top 9 peptides with high binding energy were selected to verify their activity.In order to induce CD8~+ CTL specific to AIV NP in SPF chicken, NP gene, which was optimized with chicken biased codons, was sub-cloned into the eukaryotic expression vector pCAGGS. The recombinant plasmid pCAGGS-NP was constructed and the expression of NP was confirmed by IFA and western blot in transfected 293T cells. SPF chickens were immunized with 100μg pCAGG-NP and boosted with the same dose three weeks later. A dramatically increase NP antibody was detected by ELISA after being boosted. The spleens were isolated from vaccinated chickens for the preparation of lymphocytes. The peptide-stimulated lymphocytes were used to detect the secretions of chicken IFN-γand proliferation of CD8~+ T cell by ELISA kit and flow cytometry analysis respectively. The results showed that the increase of chicken IFN-γsecretions and proliferation of CD8~+ T lymphocytes by 13.8%, 11.9 % were detected in cells stimulated with peptides NP89-97 and NP198-206. The results indicate that peptides NP89-97 and NP198-206 are NP T cell epitopes in chicken. The homology modeling structures of chicken MHC class I molecules and the identified epitopes will extend our understanding of the mechanisms of immune response to AIV in chickens.