A novel sORF gene mutant strain of Yersinia pestis vaccine EV76 offers enhanced safety and improved protection against plague

We recently identified two virulence-associated small open reading frames (sORF) of Yersinia pestis, named yp1 and yp2, and null mutants of each individual genes were highly attenuated in virulence. Plague vaccine strain EV76 is known for strong reactogenicity, making it not suitable for use in humans. To improve the immune safety of EV76, three mutant strains of EV76, Delta yp1, Delta yp2, and Delta yp1&yp2 were constructed and their virulence attenuation, immunogenicity, and protective efficacy in mice were evaluated. All mutant strains were attenuated by the subcutaneous (s.c.) route and exhibited more rapid clearance in tissues than the parental strain EV76. Under iron overload conditions, only the mice infected with EV76 Delta yp1 survived, accompanied by less draining lymph nodes damage than those infected by EV76. Analysis of cytokines secreted by splenocytes of immunized mice found that EV76 Delta yp2 induced higher secretion of multiple cytokines including TNF-alpha, IL-2, and IL-12p70 than EV76. On day 42, EV76 Delta yp2 or EV76 Delta yp1&yp2 immunized mice exhibited similar protective efficacy as EV76 when exposed to Y. pestis 201, both via s.c. or intranasal (i.n.) routes of administration. Moreover, when exposed to 200-400 LD50 Y. pestis strain 201 Delta caf1 (non-encapsulated Y. pestis), EV76 Delta yp2 or EV76 Delta yp1&yp2 are able to afford about 50% protection to i.n. challenges, significantly better than the protection afforded by EV76. On 120 day, mice immunized with EV76 Delta yp2 or EV76 Delta yp1&yp2 cleared the i.n. challenge of Y. pestis 201-lux as quickly as those immunized with EV76, demonstrating 90-100% protection. Our results demonstrated that deletion of the yp2 gene is an effective strategy to attenuate virulence of Y. pestis EV76 while improving immunogenicity. Furthermore, EV76 Delta yp2 is a promising candidate for conferring protection against the pneumonic and bubonic forms of plague.