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

Salmonella enterica serovar Typhimurium strain lacking pmrG-HM-D provides excellent protection against salmonellosis in murine typhoid model

The superiority of live attenuated vaccines in systemic salmonellosis has been proven over killed and subunit vaccines, because of its ability to induce protective cell mediated immunity by CD8+ T cells. A live attenuated Salmonella enterica serovar Typhimurium vaccine has been developed by systematic site directed deletion of the pmrG-HM-D chromosomal genomic loci. This gene confers involved in antimicrobial peptide resistance and is involved in LPS modification, both of which are the major immune evasive mechanisms in Salmonella. The efficacy of the newly developed strain in inducing protection against mortality after challenge with the virulent wild type Salmonella typhimurium 12023 was evaluated in mice model of typhoid fever. Animals were immunized and then boosted on days 7 and 14. Following challenge with virulent S. typhimurium 12023, organ burden and mortality of vaccinated mice were less compared to non-immunized controls. The vaccine strain also induced elevated CD8+ T cells in the vaccinated mice. This multiple mutant vaccine candidate appears to be safe for use in pregnant mice and provides a model for the development of live vaccine candidates against naturally occurring salmonellosis and typhoid fever

Typhoid fever & vaccine development: a partially answered question

Typhoid fever is a systemic disease caused by the human specific Gram-negative pathogen Salmonella enterica serovar Typhi (S Typhi). The extra-intestinal infections caused by Salmonella are very fatal. The incidence of typhoid fever remains very high in impoverished areas and the emergence of multidrug resistance has made the situation worse. To combat and to reduce the morbidity and mortality caused by typhoid fever, many preventive measures and strategies have been employed, the most important being vaccination. In recent years, many Salmonella vaccines have been developed including live attenuated as well as DNA vaccines and their clinical trials have shown encouraging results. But with the increasing antibiotic resistance, the development of potent vaccine candidate for typhoid fever is a need of the hour. This review discusses the latest trends in the typhoid vaccine development and the clinical trials which are underway

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

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

Th1 cytokines were significantly increased in unvaccinated mice as compared to the vaccinated pregnant mice.

<p>There is an increase in TNFα (2-fold) in amniotic fluid of unvaccinated mice (A). The serum INFγ levels also increased (B). The IL-12 level in serum as well as in amniotic fluid was higher in unvaccinated group (C). * p<0.05, *** 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

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

Transmission profile of bacteria during pregnancy in control and vaccinated mice.

<p>Undelivered pups were dissected aseptically from WT and DV-STM-07 strain infected mice and homogenized after three days of infection. Homogenate was plated on LB agar plate and analyzed for CFU growth. There was no trans-placental transfer of <i>Salmonella</i> both in case of vaccine and WT infected mice. But WT mothers were positive for <i>Salmonella</i> in blood culture and vaginal swab culture (2 mouse) Experiment was done thrice to reproduce the results. (+; Positive, ND; Not determined).</p