Dual-Function Antibodies to Yersinia pestis LcrV Required for Pulmonary Clearance of Plague

Yersinia pestis causes pneumonic plague, a necrotic pneumonia that rapidly progresses to death without early treatment. Antibodies to the protective antigen LcrV are thought to neutralize its essential function in the type III secretion system (TTSS) and by themselves are capable of inducing immunity to plague in mouse models. To develop multivalent LcrV antibodies as a therapeutic treatment option, we screened for monoclonal antibodies (MAbs) to LcrV that could prevent its function in the TTSS. Although we were able to identify single and combination MAbs that provided the high-level inhibition of the TTSS, these did not promote phagocytosis in vitro and were only weakly protective in a mouse pneumonic plague model. Only one MAb, BA5, was able to protect mice from pneumonic plague. In vitro, MAb BA5 blocked the TTSS with efficiency equal to or even less than that of other MAbs as single agents or as combinations, but its activity led to increased phagocytic uptake. Polyclonal anti-LcrV was superior to BA5 in promoting phagocytosis and also was more efficient in protecting mice from pneumonic plague. Taken together, the data support a hypothesis whereby the pulmonary clearance of Y. pestis by antibodies requires both the neutralization of the TTSS and the simultaneous stimulation of innate signaling pathways used by phagocytic cells to destroy pathogens.Yersinia pestis, the etiologic agent of bubonic, pneumonic, and septicemic plague, has been responsible for more human death than any other bacterial pathogen (42). Fortunately, naturally occurring cases of plague in humans now are uncommon, largely due to advances in basic sanitation and public awareness of infectious disease (32). Nevertheless, the disease remains endemic in many areas of the world, and periodic human bubonic and, to a lesser extent, pneumonic plague cases appear each year. Yersinia pestis is believed to have evolved recently from Yersinia pseudotuberculosis, acquiring flea transmission and respiratory invasion properties through mobile genetic elements (1, 9). The flea transmission cycle provides an opportunity for further evolution, because the bacteria reside in the nonsterile environment of the flea gut, where the formation of a biofilm provides an opportunity for horizontal gene exchange with other microbes (30). Multidrug-resistant Y. pestis isolates have been recovered from human plague patients, suggesting that the bacteria do indeed continue to evolve mechanisms of survival in the mammalian host (22, 25, 54). For these reasons, as well as for its potential use as a biological weapon, Y. pestis continues to be a significant public health concern and is a priority pathogen for the development of new vaccines and alternative therapeutics (32, 43).There currently are no plague vaccines that are licensed for human use in the United States. The licensing of current candidates is likely to fall under the U.S. Food and Drug Administration''s Animal Rule for the demonstration of efficacy and potency due to a lack of naturally occurring human plague cases (19). Thus, efficacy trials and the evaluation of vaccine potency in humans will be dependent on our ability to understand the molecular mechanism of protection. Current subunit vaccine candidates are formulated from two protective antigens, Fraction 1 (F1) and LcrV, which are undergoing extensive testing to satisfy the Animal Rule requirements (2, 5, 13, 26, 55, 57-59). Both antigens elicit a neutralizing antibody response that can be translated to passive antibody or even gene therapies (2, 4, 13, 28, 37, 48). These protective antibodies act directly on the bacteria and alter its interactions with innate immune cells such that the host clears the infection. T-cell responses also are believed to play an important role in host defense against Yersinia pestis (40, 41).CaF1, or F1, is an abundant cell surface antigen of the type I pilin family that forms a capsule-like structure on Y. pestis at 37°C (8). Although F1 appears to be antiphagocytic, it is not essential for virulence and thus would not contribute to immunity against Y. pestis mutant caF1 (18, 21). In contrast, LcrV is essential for all forms of plague due to its role in the type III secretion system (TTSS) (12, 45, 47). LcrV is positioned on the surface of bacteria at 37°C, where it mediates the translocation of anti-host factors, collectively known as Yersinia outer proteins (Yops), whose antiphagocytic, cytolytic, and proapoptotic activities allow Yersinia to avoid being killed by the host''s immune system (38, 46). Polyclonal antibodies to recombinant LcrV (α-LcrV) can bind to this needle tip and lead to the inhibition of the TTSS and the phagocytosis of the bacteria (14, 24, 53). However, it remains controversial whether the direct inhibition of the TTSS by α-LcrV leads to phagocytosis or if the direct promotion of phagocytosis leads to the inhibition of the TTSS because it cannot function intracellularly (59, 60). Three monoclonal antibodies (MAbs) have been independently cloned that can protect mice from bubonic and pneumonic plague (2, 27, 48). Although it is unclear whether each of these targets the same epitope, deletion studies of LcrV antigen suggest multiple protective epitopes exist (13, 39, 44, 51).We were interested in developing antibody therapeutics and maximizing the potency of anti-LcrV therapy. In this work, we investigated the mechanism of protection from pneumonic plague to determine if the multivalent occupancy of antibody to LcrV improved protection. We found that antibodies that promoted phagocytosis directly were more potent at neutralizing pneumonic plague, although the inhibition of the TTSS alone led to partial protection. Only a single LcrV epitope led to antibodies that by themselves promoted uptake, while the multivalent occupation of antigen with MAbs did not increase either phagocytosis or protection. These data provide new insight into the mechanism of LcrV and support the use of assays that measure the phagocytic uptake of Y. pestis as correlates of immunity for the evaluation of plague vaccines.