Vaccination with live Yersinia pestis primes CD4 and CD8 T cells that synergistically protect against lethal pulmonary Y. pestis infection.

Vaccination with live attenuated Yersinia pestis confers protection against pneumonic plague but is not considered safe for general use. Subunit plague vaccines containing the Y. pestis F1 and LcrV proteins prime robust antibody responses but may not provide sufficient protection. To aid the development of a safe and effective plague vaccine, we are investigating roles for T cells during defense against Y. pestis infection. Here we demonstrate that vaccination of mice with live Y. pestis primes specific CD4 and CD8 T cells that, upon purification and direct transfer to naïve mice, synergistically protect against lethal intranasal Y. pestis challenge. While not preventing extrapulmonary dissemination, the coadministered T cells promote bacterial clearance and reduce bacteremia. These observations strongly suggest that development of pneumonic plague vaccines should strive to prime both CD4 and CD8 T cells. Finally, we demonstrate that vaccination with live Y. pestis primes CD4 and CD8 T cells that respond to Y. pestis strains lacking the capacity to express F1, LcrV, and all pCD1/pPCP-encoded proteins, suggesting that protective T cells likely recognize antigens distinct from those previously defined as targets for humoral immunity.

Anti-LcrV antibody inhibits delivery of Yops by Yersinia pestis KIM5 by directly promoting phagocytosis.

LcrV of Yersinia pestis is a major protective antigen proposed for inclusion in subunit plague vaccines. One way that anti-LcrV antibody is thought to protect is by inhibiting the delivery of toxins called Yops to host cells. The present study characterizes the relation between this inhibition and the phagocytosis of the bacteria. J774A.1 cells were infected with Y. pestis KIM5 in the presence of a protective polyclonal anti-LcrV antibody or a nonprotective polyclonal anti-YopM antibody, and delivery of YopH and YopE into the cytoplasm was assayed by immunoblotting. The ability to inhibit the delivery of these Yops depended upon having antibody bound to the cell surface; blocking conditions that prevented the binding of antibody to Fc receptors prevented the inhibition of Yop delivery. Anti-LcrV antibody also promoted phagocytosis of the yersiniae, whereas F(ab')(2) fragments did not. Further, anti-LcrV antibody could not inhibit the delivery of Yops into cells that were unable to phagocytose due to the presence of cytochalasin D. However, Yops were produced only by extracellular yersiniae. We hypothesize that anti-LcrV antibody does not directly inhibit Yop delivery but instead causes phagocytosis, with consequent inhibition of Yop protein production in the intracellular yersiniae. The prophagocytic effect of anti-LcrV antibody extended to mouse polymorphonuclear neutrophils (PMNs) in vitro, and PMNs were shown to be critical for protection: when PMNs in mice were ablated, the mice lost all ability to be protected by anti-LcrV antibody.

Antibody against V antigen prevents Yop-dependent growth of Yersinia pestis.

The V antigen (LcrV) of the plague bacterium Yersinia pestis is a potent protective antigen that is under development as a vaccine component for humans. LcrV is multifunctional. On the bacterial surface it mediates delivery of a set of toxins called Yops into host cells, and as a released protein it can cause production of the immunosuppressive cytokine interleukin-10 (IL-10) and can inhibit chemotaxis of polymorphonuclear neutrophils. It is not known how these mechanisms of LcrV operate, what their relative importance is, when they function during plague, and which are critical to protection by antibody. This study investigated several of these issues. C57BL/6 mice, mice unable to express IL-10, or mice with the macrophage lineage eliminated were treated with a protective anti-LcrV antibody or a nonprotective antibody against YopM and infected intravenously by Y. pestis KIM5 or a strain that lacked the genes encoding all six effector Yops. Viable bacterial numbers were determined at various times. The data indicated that Yops were necessary for Yersinia growth after the bacteria had seeded liver and spleen. Anti-LcrV antibody prevented this growth, even in IL-10-/- mice, demonstrating that one protective mechanism for anti-LcrV antibody is independent of IL-10. Anti-LcrV antibody had no effect on persistence in organs of Y. pestis lacking effector Yops, even though the yersiniae could strongly express LcrV, suggesting that Yops are necessary for building sufficient bacterial numbers to produce enough LcrV for its immunosuppressive effects. In vitro assays showed that anti-LcrV antibody could partially block delivery of Yops and downstream effects of Yops in infected macrophage-like J774A.1 cells. However, cells of the macrophage lineage were found to be dispensable for protection by anti-LcrV antibody in spleen, although they contributed to protection in liver. Taken together, the data support the hypothesis that one protective effect of the antibody is to block delivery of Yops to host cells and prevent early bacterial growth. The findings also identified the macrophage lineage as one host cell type that mediates protection.