In this study, we demonstrated that the smpB-ssrA mutant of Y. pestis CO92Δpgm was severely attenuated in a mouse model of infection. ΔBA mutant cells were defective in colonization and unable to establish a lethal infection when administered intravenously or intranasally, even at very high doses. Additionally, mice infected with ΔBA cells did not show any signs of infection. Most importantly, our analysis showed that mice immunized via the intranasal route with a low dose of 104 CFU ΔBA bacteria were completely protected against lethal pulmonary Y. pestis challenges. This is a promising finding toward the goal of designing an effective plague vaccine that is highly protective against pneumonic and bubonic plague with minimal side effects.
The SmpB-SsrA system fulfills an important task in maintaining the translational machinery at full capacity by dealing with aberrant mRNAs that stall ribosomes. Although smpB-ssrA
mutations in most bacterial species may not be associated with a growth defect under normal laboratory conditions, it is apparent that mutant cells become compromised in their ability to adapt and survive in hostile environments. For example, it is well documented that smpB-ssrA
mutations render cells more sensitive to translation-specific inhibitors as well as oxidative and nitrosative stress (16
). The versatility to adapt to different environments can be critical for pathogenic bacteria, since they have to deal with a variety of host defense mechanisms. Phagocytosis is one such mechanism, which is essential in guarding against and disposing of facultative intracellular pathogens like Yersinia
mutants of Y. pseudotuberculosis
and Salmonella enterica
serovar Typhimurium were shown to be impaired in intracellular survival and replication within macrophages (6
). Additional functions of the SmpB-SsrA system in bacterial pathogenesis can be illustrated by alterations in the expression of virulence factors. smpB-ssrA
mutants were reported to have deregulated expression of Salmonella ivi
genes and Yersinia yop
). The overall combination of defects suffered by the loss of SmpB-SsrA function contributes to a high level of attenuation. Therefore, such mutant strains should be strongly considered a candidate for live cell vaccines.
A number of studies have utilized recombinant LcrV and the F1 capsular antigen as potential vaccine candidates. Although these subunit vaccines look promising, some shortcomings have been reported (1
). An alternative strategy has been to design vaccines based on live attenuated cells. One theoretical advantage of such a vaccine strain is the potential to stimulate both humoral and cellular immunity while simultaneously priming the host immune system against many antigens (7
). Another advantage of using attenuated live cells is that bacteria can easily be directed to express additional antigens or variants of antigens. Furthermore, any desired modifications to subunit vaccines can be costly, since the procedure will likely require new sets of protein purification and optimization protocols. However, desired modifications could be achieved with minimal cost with live-cell vaccines.
Use of a less-virulent and highly related surrogate pathogen as a vaccine strain has been a known strategy in the field of vaccine research and can historically be exemplified by smallpox vaccines. Similarly, using Y. pseudotuberculosis
as a live plague vaccine has remained an intriguing alternative. Y. pseudotuberculosis
is very closely related to Y. pestis
and possess more than 95% DNA sequence homology. Despite sharing many characteristics, Y. pseudotuberculosis
causes a less dangerous enteric course of illness, which is rarely lethal (12
). Previous studies by Wake et al. (50
) and Simonet et al. (45
) showed that intravenous or subcutaneous administration of Y. pseudotuberculosis
provided 50% protection against bubonic plague in a mouse model. More recently other researchers have shown that oral delivery of a Y. pseudotuberculosis dam
mutant or the naturally attenuated IP32680 strain provides significant protection against bubonic plague (8
). One theoretical advantage of oral delivery of Y. pseudotuberculosis
cells is the possible induction of gut-associated mucosal immunity. Such immunity is usually associated with secretory IgAs and could potentially be protective against infection of other mucosal surfaces, such as lungs. Consistent with these findings, our results indicate that oral immunization of mice with the Y. pseudotuberculosis
ΔBA strain provided ~50% protection against pulmonary Y. pestis
infection (Table ). The aforementioned studies similarly reported that oral immunization with Y. pseudotuberculosis
did not significantly protect mice against pneumonic plague. To explain the lack of protection, several factors have to be considered: Y. pseudotuberculosis
does not possess the F1 capsular protein, an immunodominant antigen. It is possible that oral immunization is unable to trigger the robust IgA and IgG responses that are necessary to protect mice against Y. pestis
. In fact, analysis of serum IgGs from mice immunized with IP2666 ΔBA showed no significant level of antibodies that could cross-react with Y. pestis
(Fig. , lane 5). Therefore, oral vaccines based solely on Y. pseudotuberculosis
may not be effective against pneumonic plague.
EV76 is an attenuated strain of Y. pestis
that lacks the pigmentation locus (Δpgm
) in addition to other genetically undefined modifications. The pigmentation (pgm
) locus is a 102-kb chromosomal DNA region that can be spontaneously lost at low frequencies (11
). The pgm
locus is associated with several virulence factors, such as Ybt and hms
, which are related to iron uptake and biofilm formation, respectively, and ripA
, which is required for survival in activated macrophages (20
). The EV76 strain has been used as a live-cell vaccine, and its effectiveness is strongly supported by field observations (46
). However, the highly reactogenic nature of the EV76 strain is known to cause severe side effects. Also, the possibility of it regaining its virulence highlights the reluctance to use the EV76 strain as a widely accepted vaccine strain. Recent studies identified several promising Y. pestis
mutations that could be protective in animal studies. These mutations include pcm
, and yopH
). In theory, incorporating additional genetically defined mutations into a Y. pestis pgm
-negative strain may eliminate these drawbacks while maintaining the desired efficiency as a live-cell vaccine. Therefore, it is important to identify additional mutations and use the best combination among them to design the safest and the most effective live attenuated plague vaccine strain.
Our studies showed that both intravenous and intranasal delivery of Y. pestis ΔBA cells induced a strong IgG response against several Yersinia proteins (Fig. ), including the F1 antigen. Although passive immunization studies suggest that antibodies against F1 play a major role, innate immunity may also contribute to protection induced by immunization with ΔBA cells. Indeed, mice immunized with the Y. pestis ΔBA mutant provided partial protection (50% protection) against lethal challenges with F1− Y. pestis cells. Consistent with these observations, studies with Y. pseudotuberculosis ΔBA cells, which lack the F1 antigen, showed that immunization of mice with this bacterium provided 50% protection against a lethal challenge with Y. pestis, despite the absence of IgG antibodies that recognize major Y. pestis antigens (Fig. , lane 5). Furthermore, it is possible that any potentially protective IgA response acquired by intranasal immunization may not be utilized in countering a lethal challenge through the intravenous route. Therefore, it is possible that intranasal immunization with the Y. pestis ΔBA strain manifests its full potential against pulmonary Y. pestis infections.
In summary, the smpB-ssrA mutant of Y. pestis is highly attenuated, and this defined mutation could be an excellent candidate for the foundation of a live-cell vaccine.