Delivery of <i>Yersinia pestis</i> antigens via <i>Escherichia coli</i> outer membrane vesicles offered improved protection against plague

Outer membrane vesicles (OMVs) from Gram-negative bacteria can be used as a vaccine platform to deliver heterologous antigens. Here, the major protective antigens of <i>Yersinia pestis,</i> F1 and LcrV, were fused either with the leader sequence or the transmembrane domain of the outer membrane protein A (OmpA), resulting in chimeric proteins OmpA-ls-F1V and OmpA_46-159-F1V, respectively. We show that OmpA-ls-F1V and OmpA_46-159-F1V can be successfully delivered into the lumen and membrane of the OMVs of <i>Escherichia coli,</i> respectively. Mutation of <i>ompA</i> but not <i>tolR</i> in <i>E. coli</i> enhanced the delivery efficiency of OmpA-ls-F1V into OMVs. The OmpA-ls-F1V protein comprises up to 20% of the total protein in OMVs derived from the <i>ompA</i> mutant (OMV_dA-ALS-F1V), a proportion significantly higher than the 1% observed for OmpA_46-159-F1V in OMVs produced by an <i>ompA</i> mutant that expresses OmpA46-159-F1V, referred to as OMV_dA-LATM5-F1V. Intramuscular (<i>i.m</i>.) immunization of mice with OMV_dA-ALS-F1V induced significantly higher levels of serum anti-LcrV and anti-F1 IgG, and provided higher efficacy in protection against subcutaneous (<i>s.c.</i>) <i>Y. pestis</i> infection compared to OMV_dA-LATM5-F1V and the purified recombinant F1V (rF1V) protein adsorbed to aluminum hydroxide. The three-dose <i>i.m</i>. immunization with OMV_dA-ALS-F1V, administered at 14-day intervals, provides complete protection to mice against <i>s.c.</i> infection with 130 LD₅₀ of <i>Y. pestis</i> 201 and conferred 80% against intranasal (<i>i.n</i>.) challenge with 11.4 LD₅₀ of <i>Y. pestis</i> 201. Taken together, our findings indicate that the engineered OMVs containing F1V fused with the leader sequence of OmpA provide significantly higher protection than rF1V against both <i>s.c</i>. and <i>i.n</i>. infection of <i>Y. pestis</i> and more balanced Th1/Th2 responses.IMPORTANCEThe two major protective antigens of <i>Y. pestis</i>, LcrV and F1, have demonstrated the ability to elicit systemic and local mucosal immune responses as subunit vaccines. However, these vaccines have failed to provide adequate protection against pneumonic plague in African green monkeys. Here, <i>Y. pestis</i> F1 and LcrV antigens were successfully incorporated into the lumen and the surface of the outer membrane vesicles (OMVs) of <i>E. coli</i> by fusion either with the leader sequence or the transmembrane domain of OmpA. We compared the humoral immune response elicited by these OMV formulations and their protective efficacy in mice against <i>Y. pestis</i>. Our results demonstrate that the plague OMV vaccine candidates can induce robust protective immunity against both <i>s.c</i>. and <i>i.n. Y. pestis</i> infections, surpassing the effectiveness of rF1V. In addition, immunization with OMVs generated a relatively balanced Th1/Th2 immune response compared to rF1V immunization. These findings underscore the potential of OMVs-based plague vaccines for further development.