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Pneumonic plague is a severe, rapidly progressing disease for which there is no effective vaccine. Since the efficacy of new vaccines cannot be tested in humans, it is essential to develop in vitro surrogate assays that are valid predictors of immunity. The F1 capsule antigen stimulates a protective immune response to most strains of Yersinia pestis. However, strains of Y. pestis that are F1؊ but still virulent have been isolated, and an in vitro assay, the results which can predict protection against both F1؉ and F1 ؊ strains, is needed. The virulence antigen (V) is an essential virulence factor of Y. pestis and stimulates protective antibodies. We investigated potential correlates of plague immunity that are based on anti-V antibodymediated neutralization of Yersinia-induced macrophage cytotoxicity. The neutralizing activity of sera from mice vaccinated with an F1-V fusion candidate vaccine was determined. The decrease in the level of the apoptosis-specific enzyme caspase-3 significantly predicted survival in one-and two-dose vaccination experiments. Sera from F1-V-vaccinated nonhuman primates were evaluated with macrophage assays based on caspase-3 and on other markers manifested at the different stages in cell death. Using murineand human-derived macrophages in microscopic and fluorescence-activated-cell-sorting-based live/dead staining assays of terminal necrosis, we demonstrated a strong association between in vitro neutralization of macrophage cytotoxicity induced by serum-treated Yersinia and in vivo protection against lethal infection. These results provide a strong base for the development of reliable in vitro correlate bioassays that are predictive of protective immunity to plague.Pneumonic plague is a severe and rapidly progressing disease for which no fully effective vaccine exists. There is no licensed vaccine available that elicits complete immunity in animal models to pneumonic plague. Although the previously licensed vaccine for plague protected experimental animals against parenteral challenge, it was ineffective against pneumonic challenge (19, 27; M. L. Pitt, unpublished data). Furthermore, the former vaccine, which consisted of killed whole cells of virulent Yersinia pestis (27), did not protect against virulent nonencapsulated (F1 Ϫ ) strains (19) since the vaccine did not contain immunogenic quantities of the virulence antigen (V) or other potentially protective immunogens (8,9,21). It was previously demonstrated that a combination of both F1 capsular protein antigen and V effectively protects against both encapsulated (F1 ϩ ) and nonencapsulated (F1 Ϫ ) strains of Y. pestis (3). New candidate plague vaccines containing a single recombinant F1-V fusion protein or a combination of these two proteins have been developed (19, 49a).The protective efficacy against lethal challenge of these new candidate plague vaccines cannot be ethically tested in humans. Thus, it is essential that an in vitro surrogate marker that can reliably predict the level of protective immunity in sera from vaccinated i...

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