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Salmonella is one of the key global causes of food and water borne enteric infections, responsible for significant morbidity and mortality worldwide especially in developing countries. Currently available vaccines against typhoid are moderately effective with several side effects and not efficacious against all Salmonella serovars. Due to limitations of these vaccines and emerging threats of multidrug resistance, developing an effective vaccine against these infections has increasingly become a priority. Heat shock proteins (Hsps), being evolutionarily conserved, represent dominant antigens in the host immune response. In continuation of our earlier studies on the development of S. Typhi DnaK and GroEL vaccine candidates, highly efficacious against Salmonella and multiple pathogens, in the present study, we have designed multi-epitope vaccine candidates common to multiple serovars of Salmonella using bioinformatics approach. Implementing various immunoinformatics tools such as IEDB, EpiJen, BCPRED, ElliPro and VaxiJen, led to the identification of many immunogenic B and T cell epitopes. The 3-D structure model of DnaK was generated to predict conformational B-cell epitopes using ElliPro server. Most promising T cell epitopes (29 CTLs, 18 T-helper cells) were selected based on their binding efficiency with commonly occurring MHC alleles. Finally we narrowed down to 5 protective antigenic peptides (PAPs), comprising highly conserved, antigenic and immunogenic B /T cell epitopes, least homologous with human host. These PAPs were predicted to be non-allergenic by allergenicity prediction tools (SORTALLER and AllerHunter). Hence, these immunogenic epitopes can be used for prophylactic or therapeutic usages specifically to defeat antibiotic-resistant Salmonella. These antigens have been reported for the first time and their conserved nature endow them as potential future vaccine candidates against other multiple pathogens as well., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Live, attenuated, orally-administered Salmonella strains are excellent vectors for vaccine antigens and are attractive as vaccines based on previous use of S. Typhimurium in animals. A Phase I dose escalation trial was conducted to evaluate the safety and immunogenicity of three newly constructed recombinant attenuated Salmonella enterica serovar Typhi vaccine (RASV) vectors synthesizing Streptococcus pneumoniae surface protein A (PspA)., Methods: The 3 S. Typhi strains used as vectors to deliver PspA were S. Typhi ISP1820; S. Typhi Ty2 RpoS(-); and S. Typhi Ty2 RpoS(+). Sixty healthy adults (median age 25.2 years) were enrolled into 4 Arms (total 15 subjects per Arm); within each Arm, subjects were randomized 1:1:1 into 3 Groups of 5. All subjects in the same Group received the same vaccine vector, and all subjects in the same Arm received the same titer of vaccine (10(7), 10(8), 10(9) or 10(10)CFU). Adverse events, safety, shedding, and IgG and IgA titers against Salmonella outer membrane proteins (OMPs), lipopolysaccharide (LPS) and PspA were evaluated., Results: In the highest dose group, no subject experienced severe reactions or serious adverse events. Most adverse events were mild; one subject had a positive blood culture. No subject shed vaccine in stool. No statistically significant differences for post vaccination ELISA or ELISPOT results between Groups were detected. However, a limited number of ≥ 4 fold increases from baseline for IgA anti-OMPs, IgA and IgG anti-LPS, and IgA anti-PspA occurred for a few individuals as measured by ELISA, and IgA anti-OMPs as measured by ELISPOT assay., Conclusions: All three S. Typhi vectored pneumococcal vaccines were safe and well-tolerated. Immunogenicity was limited possibly due to pre-existing high antibody titers prior to vaccination. Increases in IgA were most often observed., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The attenuation and immunoenhancing effects of rpoS and phoP Salmonella enterica serovar strain Typhi (Salmonella typhi) mutants have not been compared. Here, three S. typhi deletion mutants (phoP, rpoS, and rpoS-phoP double mutant) are constructed and these mutants are characterized with respect to invasiveness, virulence, and protective immune response compared with wild-type Ty2. It was found that phoP and phoP-rpoS deletion mutants are less invasive to HT-29 cells than the wild-type Ty2 and the rpoS single-deleted strain. The LD(50) of immunized mice was higher for phoP than for rpoS mutants, and the highest for the phoP-rpoS double mutant. In addition, all S. typhi mutants showed an increase in the specific serum IgG levels and T-cell-mediated immunity, and showed equal protection abilities against a wild-type Ty2 challenge after two rounds of immunization in BALB/c mice. It is concluded that phoP genes appear to play a more important role than rpoS genes in both cellular invasion and virulence of S. typhi, but not in immunogenicity in mice. Furthermore, the data indicate that the phoP-rpoS double mutant may show promise as a candidate for an attenuated typhoid vaccine.

Salmonella typhimurium SL3261 (aroA mutant) expressing a recombinant Mycoplasma hyopneumoniae antigen was used to orally immunize swine against porcine enzootic pneumonia. This construct, designated S. typhimurium aro A SL3261 (pKF1), expressed a recombinant protein containing the carboxy-terminal 11 kDa of a 42 kDa M. hyopneumoniae NrdF ribonucleotide reductase R2 subunit protein. Here we demonstrate that this antigen is present in all seven geographically diverse strains of M. hyopneumoniae tested, and is recognized by the swine immune system after experimental infection with the virulent M. hyopneumoniae Beaufort strain. The immune response of swine orally immunized twice with S. typhimurium SL3261 (pKF1) on day 0 and day 14 was evaluated. Oral immunization with S. typhimurium SL3261 (pKF1) primed the immune system to elicit a significant (P<0.05) secretory IgA response against the 15 kDa NrdF antigen in the respiratory tract of swine, post-challenge, compared to control groups. Blood lymphocytes from swine immunized with S. typhimurium SL3261 (pKF1) proliferated significantly (P<0.05) following stimulation with M. hyopneumoniae whole-cell extracts compared to control groups 14 days post-vaccination. Following challenge with virulent M. hyopneumoniae, swine immunized with S. typhimurium SL3261 (pKF1) showed higher average daily weight gains and reduced lung pathology compared to control groups., (Copyright 2001 Crown Copyright.)

Salmonella requires its alternative sigma factor sigma S (RpoS) for virulence in mice. rpoS mutants can be frequently isolated from highly passaged Salmonella laboratory strains. In particular, the live typhoid oral vaccine Salmonella typhi Ty21a and its parental strain Ty2, a 'wild-type' strain widely used for vaccine development, are rpoS mutants. Here, we show that the nucleotide sequence of the rpoS mutant allele of Ty2 is identical to that of the rpoS mutant allele of Ty21a. This demonstrates that the rpoS mutation arose in Ty2 before the isolation of Ty21a in 1975, an observation that may have implications for vaccine research.

The rpoS (katF) gene, which encodes a RNA polymerase sigma factor (sigma s), regulates the virulence of Salmonella typhimurium in mice. In the present study, we show that rpoS mutants can be frequently found among laboratory strains of Salmonella. In addition, a rpoS mutation was identified in the S. typhi live oral vaccine Ty21a. Introduction of a wild-type rpoS gene in Ty21a allowed the bacteria to survive better under starvation conditions and increased their resistance to other stresses. These results contribute to a better understanding of the genetic background of the live typhoid oral vaccine Ty21a and suggest that the rpoS mutation may contribute to the safety of this strain in humans.

The PhoP virulence regulon is essential to Salmonella typhimurium mouse typhoid fever pathogenesis and survival within macrophages. This virulence regulon is composed of the PhoP (transcriptional regulator) and PhoQ (environmental sensor) proteins and the genetic loci they positively (pags for PhoP activated genes) and negatively (prgs for PhoP repressed genes) regulate. Three regulated loci pagC, pagD, and prgH, when singly mutated, affect the virulence of S. typhimurium for mice. Strains with phoP locus mutations are effective as live vaccines in mice, and strains with a constitutive regulatory mutation, a point mutation in PhoQ, can protect mice against typhoid fever when only very few organisms are administered. The addition of various PhoP regulon mutations further attenuates aroA mutants of S. typhimurium, suggesting that these mutations would be useful in further attenuating vaccine strains with metabolic pathway mutations. The phoP, phoQ, pagC, and pagD genes are highly conserved between S. typhimurium and S. typhi and may be valuable as mutations in live vaccines for human typhoid fever. A plasmid suicide vector that allows deletion of the pagC gene and stable insertion of heterologous antigen genes within the deleted pagC locus has been constructed and used successfully in S. typhimurium and S. typhi.