Cellular immune responses play a key role in the control of viral infection. The nucleocapsid (N) protein of infectious bronchitis virus (IBV) is a major immunogenic protein that can induce protective immunity. To screen for potential T-cell epitopes on IBV N protein, forty overlapping peptides covering the entirety of the N protein were designed and synthesized. Four T-cell epitope peptides were identified by IFN-γ ELISpot, intracellular cytokine staining, and CFSE lymphocyte proliferation assays; among them, three peptides (N
211–230
, N
271–290
, and N
381–400
) were CTL epitopes, and one peptide (N
261–280
) was a dual-specific T-cell epitope, which can be recognized by both CD8
+
and CD4
+
T cells. Multi-epitope gene transcription cassettes comprising four neutralizing epitope domains and four T-cell epitope peptides were synthesized and inserted into the genome of Newcastle disease virus strain La Sota between the P and M genes. Recombinant IBV multi-epitope vaccine candidate rLa Sota/SBNT was generated via reverse genetics, and its immune protection efficacy was evaluated in specific-pathogen-free chickens. Our results show that rLa Sota/SBNT induced IBV-specific neutralizing antibody and T-cell responses and provided significant protection against homologous and heterologous IBV challenge. Thus, the T-cell epitope peptides identified in this study could be good candidates for IBV vaccine development, and recombinant Newcastle disease virus expressing IBV multi-epitope genes represents a safe and effective vaccine candidate for controlling infectious bronchitis.
IMPORTANCE
T-cell-mediated immune responses are critical for the elimination of IBV-infected cells. To screen conserved T-cell epitopes in the IBV N protein, forty overlapping peptides covering the entirety of the N protein were designed and synthesized. By combining IFN-γ ELISpot, intracellular cytokine staining, and CFSE lymphocyte proliferation assays, we identified three CTL epitopes and one dual-specific T-cell epitope. The value of T-cell epitope peptides identified in the N protein was further verified by the design of an IBV multi-epitope vaccine. Results show that IBV multi-epitope vaccine candidate rLa Sota/SBNT provided cross protection against challenges with a QX-like or a TW-like IBV strain. So T-cell-mediated immune responses play an important role in the control of viral infection and conserved T-cell epitopes serve as promising candidates for use in multi-epitope vaccine construction. Our results provide a new perspective for the development of a safer and more effective IBV vaccine.
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