Characterization of cytotoxic T-lymphocyte epitopes and immune responses to SARS coronavirus spike DNA vaccine expressing the RGD-integrin-binding motif.

Integrins are critical for initiating T-cell activation events. The integrin-binding motif Arg-Gly-Asp (RGD) was incorporated into the pcDNA 3.1 mammalian expression vector expressing the codon-optimized extracellular domain of SARS coronavirus (SARS-CoV) spike protein, and tested by immunizing C57BL/6 mice. Significant cell-mediated immune responses were characterized by cytotoxic T-lymphocyte (51)Cr release assay and interferon-gamma secretion ELISPOT assay against RMA-S target cells presenting predicted MHC class I H2-Kb epitopes, including those spanning residues 884-891 and 1116-1123 within the S2 subunit of SARS-CoV spike protein. DNA vaccines incorporating the Spike-RGD/His motif or the Spike-His construct generated robust cell-mediated immune responses. Moreover, the Spike-His DNA vaccine construct generated a significant antibody response. Immunization with these DNA vaccine constructs elicited significant cellular and humoral immune responses. Additional T-cell epitopes within the SARS-CoV spike protein that may contribute to cell-mediated immunity in vivo were also identified.