Delivery of Yersinia pestis antigens via Escherichia coli 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 Yersinia pestis, 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 Escherichia coli, respectively. Mutation of ompA but not tolR in E. coli 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 ompA mutant (OMVdA-ALS-F1V), a proportion significantly higher than the 1% observed for OmpA(46-159)-F1V in OMVs produced by an ompA mutant that expresses OmpA46-159-F1V, referred to as OMVdA-LATM5-F1V. Intramuscular (i.m.) immunization of mice with OMVdA-ALS-F1V induced significantly higher levels of serum anti-LcrV and anti-F1 IgG, and provided higher efficacy in protection against subcutaneous (s.c.) Y. pestis infection compared to OMVdA-LATM5-F1V and the purified recombinant F1V (rF1V) protein adsorbed to aluminum hydroxide. The three-dose i.m. immunization with OMVdA-ALS-F1V, administered at 14-day intervals, provides complete protection to mice against s.c. infection with 130 LD50 of Y. pestis 201 and conferred 80% against intranasal (i.n.) challenge with 11.4 LD50 of Y. pestis 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 s.c. and i.n. infection of Y. pestis and more balanced Th1/Th2 responses.