Yersinia pestis (Yp) - one of the world's most virulent human pathogens - is the gram-negative bacterium that causes pneumonic plague. Virulent, antibiotic-resistant, Yp strains exist and Cold War scientists devised means to effectively aerosolize Yp. Thus, there is grave concern that Yp will be exploited as a bioweapon. To thwart that possibility, it is essential that we develop effective countermeasures. Recent primate studies suggest that the leading vaccine candidate - a recombinant F1-V fusion protein (rF1V) - may not provide sufficient protection. Moreover, resourceful bioweapon engineers could circumvent this vaccine with an F1-negative V-variant strain. While the rF1V vaccine primarily stimulates antibody-mediated humoral immunity, T cell-dependent cellular immunity comprises a second means by which vaccines can prime long-lived protection. However, it is widely accepted that the extreme virulence of Yp results, in large part, from plasmid-encoded factors that dampen inflammation and debilitate phagocytes, thereby compromising cell-mediated defense. Thus, plague vaccine researchers have devoted little attention to cellular immunity. Nevertheless, the Progress Report demonstrates that T cells can protect against pulmonary Yp infection. As such, we propose that next-generation pneumonic plague vaccines should strive to prime both humoral and cellular immunity, as well as incorporate new antigens. In Aim 1 of this application for grant renewal, we will directly measure the extent to which pulmonary Yp infection suppresses na[unreadable]ve and effector/memory T cell responses in vivo and identify mechanisms underlying any suppression that exists. In Aim 2, we will generate T cell clones that confer protection in mice, identify their cognate protein antigens using a series of complementary genetic, molecular and biochemical approaches, and then quantify the utility of these antigens as vaccines. We will also determine which antigens confer synergistic protection when combined with V-specific antibodies. Our findings will allow next-generation F1/V-based vaccines to harness both the neutralizing capacities of antibodies and the antimicrobial capacities of cellular immunity, while simultaneously reducing opportunities for circumvention by bioweapon engineers. Moreover, our studies will also generate powerful new tools for deciphering the fascinating interplay between Yp virulence factors and host defense mechanisms, thereby advancing basic research aimed at exploiting cellular immunity for defense against virulent bacterial pathogens that infect the lung. PUBLIC HEALTH RELEVANCE: To counter the potential use of pneumonic plague as a bioweapon, it is essential that we develop an effective vaccine. Recent animal studies suggest that the current vaccine candidate, which primarily stimulates B cells to make plague-fighting antibodies, may not suffice. This proposal aims to fundamentally improve that vaccine by incorporating the protective capacities of both B and T cells.