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

Abstract

ABSTRACTHere, we constructed aYersinia pseudotuberculosismutant strain with arabinose-dependent regulated and delayed shutoff ofcrpexpression (araCPBADcrp) and replacement of themsbBgene with theEscherichia colimsbBgene to attenuate it. Then, we inserted theasdmutation into this construction to form χ10057 [Δasd-206ΔmsbB868::PmsbBmsbB(EC)ΔPcrp21::TTaraCPBADcrp] 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 (YopENt138-LcrV) was synthesized in χ10057 harboring an Asd+plasmid (pYA5199,yopENt138-lcrV) and could be secreted through a type III secretion system (T3SS)in vitroandin vivo. Animal studies indicated that mice orally immunized with χ10057(pYA5199) developed titers of IgG response to whole-cell lysates ofY. 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 (LD50) (2.4 × 104CFU) ofY. pestisKIM6+(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 attenuatedY. pseudotuberculosisstrain delivering the LcrV antigen via the T3SS as a potential vaccine candidate against pneumonic plague.