Prediction and identification of novel IBV S1 protein derived CTL epitopes in chicken

Infectious bronchitis virus (IBV) is a major pathogen common in the poultry industry. Broad cytotoxic T lymphocyte (CTL) response against IBV is one of the crucial factors that help to control viral replication. Spike glycoproteins on the surface of the IBV virion harbor major T cell epitopes. In this study, based on the peptide-binding motifs of chicken MHC I molecules for the BF2*4, BF2*12, BF2*15, and BF2*19 haplotypes, potential CTL epitopes were predicted using S1 proteins from different IBV strains. Twenty-one peptides were predicted to be potential CTL epitopes; they were manually synthesized and the CTL responses to them tested in vitro. Spleen lymphocytes were collected from specific-pathogen free (SPF) chicken that had been immunized with the S1 protein expression plasmid, pV-S1, and were stimulated by the synthesized peptides. IFN-γ secretion and CD8(+) T cell proliferation in chickens were tested by ELISpot array and flow cytometry, respectively. Four epitopes (P8SRIQTATDP, P9SRNATGSQP, P18GAYAVVNV, and P19SRIQTATQP) were identified to stimulate CD8(+) T cell proliferation and IFN-γ secretion, indicating their efficacy as CTL epitopes in chicken. Poly-CTL-epitope DNA vaccine (pV-S1T) was constructed by inserting nucleotide sequences encoding the P8, P9, P18, and P19 CTL epitopes into the pVAX1 vector. Chickens were vaccinated with either pV-S1, pV-S1T, or pVAX1 and the protection efficacy was analyzed, revealing that ninety percent of chickens immunized with pV-S1T were protected after challenge with 10(6) ELD50 of IBV, demonstrating that these novel CTL epitopes were effective against IBV challenge. This study provides a new method to screen virus CTL epitopes in chicken and to develop poly-CTL-epitope DNA vaccines.