LcrV Delivered via Type III Secretion System of Live Attenuated Yersinia pseudotuberculosis Enhances Immunogenicity against Pneumonic Plague

ABSTRACT Here, we constructed a Yersinia pseudotuberculosis mutant strain with arabinose-dependent regulated and delayed shutoff of crp expression ( araC P BAD crp ) and replacement of the msbB gene with the Escherichia coli msbB gene to attenuate it. Then, we inserted the asd mutation into this construction to form χ10057 [Δ asd-206 Δ msbB868 ::P msbB msbB (EC) ΔP crp21 ::TT araC P BAD crp ] for use with a balanced-lethal Asd-positive (Asd + ) plasmid to facilitate antigen synthesis. A hybrid protein composed of YopE (amino acids [aa]1 to 138) fused with full-length LcrV (YopE Nt138 -LcrV) was synthesized in χ10057 harboring an Asd + plasmid (pYA5199, yopE Nt138 -lcrV ) and could be secreted through a type III secretion system (T3SS) in vitro and in vivo . Animal studies indicated that mice orally immunized with χ10057(pYA5199) developed titers of IgG response to whole-cell lysates of Y. pestis (YpL) and subunit LcrV similar to those seen with χ10057(pYA3332) (χ10057 plus an empty plasmid). However, only immunization of mice with χ10057(pYA5199) resulted in a significant secretory IgA response to LcrV. χ10057(pYA5199) induced a higher level of protection (80% survival) against intranasal (i.n.) challenge with ∼240 median lethal doses (LD 50 ) (2.4 × 10 4 CFU) of Y. pestis KIM6+(pCD1Ap) than χ10057(pYA3332) (40% survival). Splenocytes from mice vaccinated with χ10057(pYA5199) produced significant levels of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), and interleukin-17 (IL-17) after restimulation with LcrV and YpL antigens. Our results suggest that it is possible to use an attenuated Y. pseudotuberculosis strain delivering the LcrV antigen via the T3SS as a potential vaccine candidate against pneumonic plague.