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Enteric fever remains a major public health problem in South and Southeast Asia. The recent roll-out of the typhoid conjugate vaccine protecting against S. Typhi exhibits great promise for disease reduction in high burden areas. However, some endemic regions remain vulnerable to S. Paratyphi A due to a lack of licensed vaccines and inadequate WASH. Several developmental S. Paratyphi A vaccines exploit O-antigen as the target antigen. It has been hypothesised that O-antigen is under selective and environmental pressure, with mutations in O-antigen biosynthesis genes being reported, but their phenotypic effects are unknown. Here, we aimed to evaluate O-antigen variation in S. Paratyphi A originating from Nepal, and the potential effect of this variation on antibody binding. O-antigen variation was determined by measuring LPS laddering shift following electrophoresis; this analysis was complemented with genomic characterisation of the O-antigen region. We found structural O-antigen variation in <10 % of S. Paratyphi A organisms, but a direct underlying genetic cause could not be identified. High-content imaging was performed to determine antibody binding by commercial O2 monoclonal (mAb) and polyclonal antibodies, as well as polyclonal sera from convalescent patients naturally infected with S. Paratyphi A. Commercial mAbs detected only a fraction of an apparently "clonal" bacterial population, suggesting phase variation and nonuniform O-antigen composition. Notably, and despite visible subpopulation clusters, O-antigen structural changes did not appear to affect the binding ability of polyclonal human antibody considerably, which led to no obvious differences in the functionality of antibodies targeting organisms with different O-antigen conformations. Although these results need to be confirmed in organisms from alternative endemic areas, they are encouraging the use of O-antigen as the target antigen in S. Paratyphi A vaccines., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.)

A safety enhanced Salmonella Gallinarum (SG) ghost was constructed using an antibiotic resistance gene free plasmid and evaluated its potential as fowl typhoid (FT) vaccine candidate. The antibiotic resistance free pYA3342 plasmid possesses aspartate semialdehyde dehydrogenase gene which is complimentary to the deletion of the chromosomal asd gene in the bacterial host. This plasmid was incorporated with a ghost cassette containing the bacteriophage PhiX174 lysis gene E, designated as pJHL101. The plasmid pJHL101 was transformed into a two virulence genes-deleted SG. The SG ghosts with tunnel formation and loss of cytoplasmic contents were observed by scanning electron microscopy and transmission electron microscopy. The cell viability of the culture solution was decreased to 0% at 24h after the induction of gene E expression by an increase in temperature from 37°C to 42°C. The safety and protective efficacy of the SG ghost vaccine was further examined in chickens which were divided into three groups: group A (non-immunized control), group B (orally immunized), and group C (intramuscularly immunized). The birds were immunized at 7d of age. No clinical symptoms associated with FT such as anorexia, depression and greenish diarrhea were observed in the immunized chickens. Upon challenge with a virulent SG strain at 3 week post-immunization, the chickens immunized with the SG ghost via various routes were efficiently protected, as shown by significantly lower mortality and post-mortem lesions in comparison with control group. In addition, all the immunized chickens showed significantly higher antibody responses accompanied by a potent antigen-specific lymphocyte proliferative response along with significantly increased numbers of CD4⁺ and CD8⁺ T lymphocytes. Overall, our results provide a promising approach of generating SG ghosts using the antibiotic resistance free plasmid in order to prepare a non-living bacterial vaccine candidate which could be environmentally safe yet efficient to prevent FT in chickens., (Copyright © 2013 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.)

Live attenuated Salmonellae may overcome limitations with conventional methods of DNA immunisation. This study examined the impact of plasmid stability on oral DNA delivery by the attenuated Salmonella enterica serovar Typhimurium vaccine strain BRD509. A DNA vaccine cassette comprising the C fragment of tetanus toxin under control of the cytomegalovirus (CMV) promoter was ligated into plasmid pcDNA3, pUC18, pBBR122, pACYC184, pRSF1010/CAT, pBR322 and pAT153. In vitro and in vivo stability studies revealed that, with the exception of pcDNA3 and pUC18, the plasmids were retained by BRD509. However, pAT153 was the only plasmid to induce a tetanus toxoid-specific antibody response following oral delivery. Plasmid copy number was found to impact on plasmid stability and the induction of antigen-specific humoral responses.

CVD 909 is a novel live attenuated S. Typhi oral vaccine candidate derived from strain CVD 908-htrA which constitutively expresses Vi. Herein we investigated whether the genetic manipulations involved in modifying CVD 908-htrA altered its ability to induce potent T-cell immune responses (CMI) after a single dose (five subjects) and, in a separate trial, whether a second dose (eight subjects) further enhanced its immunogenicity. In these clinical trials we observed that CVD 909 immunization elicits a wide array of CMI, including cytotoxic T cells (CTL), IFN-gamma, TNF-alpha and IL-10 (but not IL-2, IL-4 or IL-5) production, and proliferation to S. Typhi antigens. However, the administration of a second dose did not result in increases in CMI. These results suggest that the genetic manipulations to constitutively express Vi did not adversely affect the ability of CVD 909 to elicit a wide array of CMI responses. These observations add impetus for the continuing evaluation of CVD 909 as a typhoid vaccine candidate.

Cytokine and inflammatory response against virulent LT2 strain and its attenuated aroA deletion mutant of Salmonella enterica serovar Typhimurium were compared in gnotobiotic pigs. Contrary to the parental strain, the auxotrofic mutant did not induce IL-1beta, IL-18, TNF-alpha, and IFN-gamma in the ileum and plasma 24h after the infection, did not cause pathological changes in ileal epithelium and mesenteric lymph nodes or immunoreactivity of gp91 phox and peroxynitrite and was not immunostained for GroEL stress protein. The absence of induction of proinflammatory cytokines may be a reason why aroA mutant was unable to elicit any inflammatory response and protect pigs against challenge with virulent LT2 strain administered 24h later.

Live bacterial vaccines represent a highly valid preventive strategy in the fight against infectious disease. However, the road from research to market is peppered with hurdles, one of which is the requirement for high biosafety characteristics, which the candidate vaccine has to display. In Europe, the European Agency for the evaluation of medicinal products (EMEA) is the relevant authority regulating the licensure of genetically engineered vaccines. For this purpose, the agency may rely on several directives and guidelines defined in the past 15 years. As for live vaccines containing genetically modified organisms (GMOs) susceptible to be released into the environment, Directive 2001/18/EC determines the framework and principles of an environmental risk assessment (ERA) process, the results of which constitute an important section of the vaccine registration package submitted to registration authorities. In this article, we address the implications of current European regulations for the approval of live oral bacterial vaccines with emphasis on the assessment of potential risks associated with environmental release. Biosafety aspects of already registered and some promising live bacterial vaccine strains will be briefly discussed.

Attenuated strains of Salmonella enterica serovar Typhimurium are used as carriers of heterologous antigens as candidate oral vaccines and, more recently, as carriers of DNA vaccines. In this study, recombinant Salmonella strains that were altered in their ability to colonise murine tissues in vivo when compared to parent strains were not, however, equally altered in their ability to invade murine cells in vitro. These results suggest that in vitro invasion studies may not be a representative model for colonisation of tissues in vivo, and that in vitro studies should ideally be used in conjunction with in vivo studies for the assessment of potential Salmonella vaccines.

Gnotobiotic pigs were inoculated orally with wild-type or defined aroA or cya crp vaccine strains of Salmonella enterica serovars Typhimurium and S. Enteritidis. The parental strains were virulent, inducing severe diarrhoea, dehydration and systemic disease. The cya crp mutant was also highly virulent whereas the aroA strains were attenuated.

A 13 amino acid peptide corresponding to a potent BALB/c mouse T cell epitope of hen's egg lysozyme (HEL) was substituted singly at five sites in the d flagellin of Salmonella muenchen. The resulting chimeric proteins were unable to expand T cells capable of being stimulated by the HEL epitope and induced T cell populations which either failed to respond or responded at a low level to a normally highly stimulatory flagellin T cell epitope that was present in all chimeras. The results suggested that substitution of a T cell epitope in flagellin may alter the processing of the resulting immunogen.

Temperature-sensitive (ts) mutants of Salmonella typhi were isolated following treatment with nitrosoguanidine, and characterized with respect to cut-off temperature, ts phenotype and reversion frequency. Linkage of the ts mutations to selectable chromosomal markers was established by generalized transduction with bacteriophage phage Vi I, and the appropriate antibiotic resistances were transduced into the ts mutants. Multiple mutant S. typhi were then constructed by combining three independent ts mutations in one strain, utilizing linkage of three of the mutations to erythromycin-, streptomycin- and methylglyoxal-resistance. Several recombinants are genetically stable, with calculated reversion rates of less than 10(-22), and induce both protection from intraperitoneal challenge with the virulent parental wild-type S. typhi in mice and the formation of antibodies to the somatic O-9 and O-12, the flagellar H and the capsular Vi antigens.

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.

The construction of a Salmonella typhi Ty2 strain harbouring defined deletions in both the aroA and aroC genes is described. These deletions have been fully defined at the molecular level by DNA sequencing and have been introduced in such a way that no foreign DNA remains in the S. typhi genome. This strain is attenuated in mice when given by the intraperitoneal route suspended in hog gastric mucin and is attenuated to a similar level to strains harbouring deletions in aroA or aroC alone indicating that both lesions are capable of attenuating independently. We have used this defined S. typhi aroA aroC strain to express stably a non-toxic 50 kDa fragment of tetanus toxin (fragment C) from a gene incorporated into the chromosome. This strain has the advantage of harbouring no antibiotic-resistance markers and we consider it to be a candidate bivalent oral typhoid-tetanus vaccine.