A Safe Vaccine (DV-STM-07) against Salmonella Infection Prevents Abortion and Confers Protective Immunity to the Pregnant and New Born Mice

Pregnancy is a transient immuno-compromised condition which has evolved to avoid the immune rejection of the fetus by the maternal immune system. The altered immune response of the pregnant female leads to increased susceptibility to invading pathogens, resulting in abortion and congenital defects of the fetus and a subnormal response to vaccination. Active vaccination during pregnancy may lead to abortion induced by heightened cell mediated immune response. In this study, we have administered the highly attenuated vaccine strain ΔpmrG-HM-D (DV-STM-07) in female mice before the onset of pregnancy and followed the immune reaction against challenge with virulent S. Typhimurium in pregnant mice. Here we demonstrate that DV-STM-07 vaccine gives protection against Salmonella in pregnant mice and also prevents Salmonella induced abortion. This protection is conferred by directing the immune response towards Th2 activation and Th1 suppression. The low Th1 response prevents abortion. The use of live attenuated vaccine just before pregnancy carries the risk of transmission to the fetus. We have shown that this vaccine is safe as the vaccine strain is quickly eliminated from the mother and is not transmitted to the fetus. This vaccine also confers immunity to the new born mice of vaccinated mothers. Since there is no evidence of the vaccine candidate reaching the new born mice, we hypothesize that it may be due to trans-colostral transfer of protective anti-Salmonella antibodies. These results suggest that our vaccine DV-STM-07 can be very useful in preventing abortion in the pregnant individuals and confer immunity to the new born. Since there are no such vaccine candidates which can be given to the new born and to the pregnant women, this vaccine holds a very bright future to combat Salmonella induced pregnancy loss

Differentially Evolved Genes of Salmonella Pathogenicity Islands: Insights into the Mechanism of Host Specificity in Salmonella

BACKGROUND: The species Salmonella enterica (S. enterica) includes many serovars that cause disease in avian and mammalian hosts. These serovars differ greatly in their host range and their degree of host adaptation. The host specificity of S. enterica serovars appears to be a complex phenomenon governed by multiple factors acting at different stages of the infection process, which makes identification of the cause/s of host specificity solely by experimental methods difficult. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we have employed a molecular evolution and phylogenetics based approach to identify genes that might play important roles in conferring host specificity to different serovars of S. enterica. These genes are 'differentially evolved' in different S. enterica serovars. This list of 'differentially evolved' genes includes genes that encode translocon proteins (SipD, SseC and SseD) of both Salmonella pathogenicity islands 1 and 2 encoded type three secretion systems, sptP, which encodes an effector protein that inhibits the mitogen-activated protein kinase pathway of the host cell, and genes which encode effector proteins (SseF and SifA) that are important in placing the Salmonella-containing vacuole in a juxtanuclear position. CONCLUSIONS/SIGNIFICANCE: Analysis of known functions of these 'differentially evolved genes' indicates that the products of these genes directly interact with the host cell and manipulate its functions and thereby confer host specificity, at least in part, to different serovars of S. enterica that are considered in this study

Efficacy of DV-STM-07 vaccine in pregnant mice.

<p>Pregnant BALB/c mice were infected with WT and Vaccine strain DV-STM-07 (10⁷ cfu) and were observed for abortion and the offspring outcome. With WT infection, abortion and the death of pups after delivery was more than DV-STM-07 infected mice. The data shows the representative of three independent experiments performed. Fisher's exact test was used to analyze the data. <i>p</i>-values are provided for statistically significant data.</p

Th1 cytokine levels are lower in the splenocytes of vaccinated mothers.

<p>Isolated splenocytes (details in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009139#s4" target="_blank">material and methods</a>) were cultured for 48 hrs and 72 hrs. Level of TNFα (A) and INFγ (B) was significantly reduced in primed splenocytes as compared to the control followed by the infection. The level of IFNγ was significantly more in serum of single dose infection with WT <i>Salmonella</i> not in control and primed (C). Data shows the representative of three independent experiments performed. * p<0.05, ** p<0.001 *** p<0.0001 (Student “t” test).</p

Pregnancy outcome in pre-pregnancy vaccinated mice without WT challenge.

<p>Cohorts of 4 mice were given 3 doses of 10⁴ CFU/mouse of DV-STM-07 at an interval of 7 days. The control mice received PBS. One week after the last dose the mice were mated and observed for the outcome of pregnancy. Experiment was done thrice to reproduce the results.</p

Th2 cytokine levels were higher in vaccinated than unvaccinated pregnant mice.

<p>Vaccinated and unvaccinated mice were sacrificed and checked for the cytokine profile. IL-4 was significantly more in vaccinated mice as compared to the unvaccinated mice (A) Levels of Serum IL-6 was enhanced in vaccinated mice but reverse was observed in case of the amniotic fluid of vaccinated and unvaccinated mice (B). Data shows the representative of three independent experiments performed. * p<0.05, ** p<0.001(Student “t” test).</p

Pre-pregnancy vaccination of mice reduces bacterial burden in pups.

<p>Non- pregnant nice were given 3 doses of 10⁴ cfu of vaccine strain at an interval of one week. A week after the last vaccination the mice were mated. The pups of vaccinated and unvaccinated mice were challenged with 10⁷ cfu of WT bacteria and on the 5th day of post infection of pups (4 weeks), organ CFU of Spleen and Liver (A) and PP and MLN (B) was recorded. Data shows the representative of three independent experiments performed. * p<0.05, ** p<0.001 (Mann-Whitney test).</p

TolA mediates the differential detergent resistance pattern between the Salmonella enterica subsp enterica serovars Typhi and Typhimurium

The tol-pal genes are essential for maintaining the outer membrane integrity and detergent resistance in various Gram-negative bacteria, including Salmonella. The role of TolA has been well established for the bile resistance of Salmonella enterica subsp. enterica serovar Typhimurium. We compared the bile resistance pattern between the S. enterica serovars Typhi and Typhimurium and observed that Typhi is more resistant to bile-mediated damage. A closer look revealed a significant difference in the TolA sequence between the two serovars which contributes to the differential detergent resistance. The tolA knockout of both the serovars behaves completely differently in terms of membrane organization and morphology. The role of the Pal proteins and difference in LPS organization between the two serovars were verified and were found to have no direct connection with the altered bile resistance. In normal Luria broth (LB), S. Typhi Delta tolA is filamentous while S. Typhimurium Delta tolA grows as single cells, similar to the wildtype. In low osmolarity LB, however, S. Typhimurium Delta tolA started chaining and S. Typhi Delta tolA showed no growth. Further investigation revealed that the chaining phenomenon observed was the result of failure of the outer membrane to separate in the dividing cells. Taken together, the results substantiate the evolution of a shorter TolA in S. Typhi to counteract high bile concentrations, at the cost of lower osmotic tolerance

List of primers used for semi-quantitative RT-PCR of cryptdine levels.

<p>List of primers used for semi-quantitative RT-PCR of cryptdine levels.</p