Yersinia pestis CO92 mutant deleted for the genes encoding Braun lipoprotein and plasminogen activator protease: Characterization of a potential live-attenuated vaccine candidate
Van lier, Christina J
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There is no FDA-approved vaccine against Yersinia pestis, the causative agent of bubonic and pneumonic plague. Since both humoral- and cell- mediated immunity are essential in providing protection to the host against plague, we developed a live-attenuated vaccine strain by deleting Braun lipoprotein (lpp) and plasminogen-activating protease (pla) genes from Y. pestis CO92. The lpp pla double isogenic mutant was highly attenuated in evoking both bubonic and pneumonic plague in a mouse model. Further, animals immunized with the mutant by either the intranasal or the subcutaneous routes were significantly protected from developing subsequent pneumonic plague. In mice, the mutant poorly disseminated to peripheral organs and the production of pro-inflammatory cytokines concurrently decreased. Histopathologically, reduced damage to the lungs and liver of mice infected with the lpp pla double mutant was observed when compared to the WT CO92-challenged animals. The lpp pla mutant-immunized mice elicited a humoral immune response to the WT bacterium, as well as to CO92-specific antigens. Moreover, T cells from the mutant-immunized animals exhibited significantly higher proliferative responses when stimulated ex vivo with the heat-killed WT CO92 antigens, compared to responses in mice immunized with the same sub-lethal dose of WT CO92. Likewise, T cells from the mutant-immunized mice produced more IFN-γ and interleukin-4. These animals had an increasing number of TNF-α-producing CD4+ and CD8+ T cells compared to WT CO92-infected mice. These data emphasized the role of TNF-α and IFN-γ in protecting mice against pneumonic plague. Overall, our in vivo studies provided evidence that deletion of lpp and pla genes acted synergistically in protecting animals against pneumonic plague, and we have demonstrated an immunological basis for this protection. We further characterized the ∆lpp ∆pla double mutant in two murine macrophage cell lines as well as in primary human monocyte-derived macrophages to gauge its potential as a live-attenuated vaccine candidate. We first demonstrated that the ∆pla single and the ∆lpp ∆pla double mutant were unable to survive efficiently in murine and human macrophages, unlike WT CO92. We observed that the levels of Pla and its associated protease activity were not affected in the ∆lpp single mutant, and, likewise, deletion of the pla gene from WT CO92 did not alter Lpp levels. Further, our study revealed that both Lpp and Pla contributed to the intracellular survival of WT CO92 via different mechanisms. Importantly, the ability of the ∆lpp ∆pla double mutant to be phagocytized by macrophages, to stimulate production of tumor necrosis factor-α and interleukin-6, and to activate the nitric oxide killing pathways of the host cells remained unaltered when compared to the WT CO92-infected macrophages. Finally, macrophages infected with either the WT CO92 or the ∆lpp ∆pla double mutant were equally efficient in their uptake of zymosan particles as determined by flow cytometric analysis. Overall, our data indicated that although the ∆lpp ∆pla double mutant of Y. pestis CO92 was highly attenuated, it retained the ability to elicit innate and subsequent acquired immune responses in the host similar to that of WT CO92, which are highly desirable in a live-attenuated vaccine candidate.