High-throughput identification of new protective antigens from a Yersinia pestis live vaccine by enzyme-linked immunospot assay.

Yersinia pestis, the plague pathogen, is a facultative intracellular bacterium. Cellular immunity plays important roles in defense against infections. The identification of T-cell targets is critical for the development of effective vaccines against intracellular bacteria; however, the function of cellular immunity in protection from plague was not clearly understood. In this study, 261 genes from Y. pestis were selected on the basis of bioinformatics analysis and previous research results for expression in Escherichia coli BL21(DE3). After purification, 101 proteins were qualified for examination of their abilities to induce the production of gamma interferon in mice immunized with live vaccine EV76 by enzyme-linked immunospot assay. Thirty-four proteins were found to stimulate strong T-cell responses. The protective efficiencies for 24 of them were preliminarily evaluated using a mouse plague model. In addition to LcrV, nine proteins (YPO0606, YPO1914, YPO0612, YPO3119, YPO3047, YPO1377, YPCD1.05c, YPO0420, and YPO3720) may provide partial protection against challenge with a low dose (20 times the 50% lethal dose [20x LD(50)]) of Y. pestis, but only YPO0606 could partially protect mice from infection with Y. pestis at a higher challenge dosage (200x LD(50)). These proteins would be the potential components for Y. pestis vaccine development.