Clonal analysis of Salmonella-specific effector T cells reveals serovar-specific and cross-reactive T cell responses.

To tackle the complexity of cross-reactive and pathogen-specific T cell responses against related Salmonella serovars, we used mass cytometry, unbiased single-cell cloning, live fluorescence barcoding, and T cell-receptor sequencing to reconstruct the Salmonella-specific repertoire of circulating effector CD4+ T cells, isolated from volunteers challenged with Salmonella enterica serovar Typhi (S. Typhi) or Salmonella Paratyphi A (S. Paratyphi). We describe the expansion of cross-reactive responses against distantly related Salmonella serovars and of clonotypes recognizing immunodominant antigens uniquely expressed by S. Typhi or S. Paratyphi A. In addition, single-amino acid variations in two immunodominant proteins, CdtB and PhoN, lead to the accumulation of T cells that do not cross-react against the different serovars, thus demonstrating how minor sequence variations in a complex microorganism shape the pathogen-specific T cell repertoire. Our results identify immune-dominant, serovar-specific, and cross-reactive T cell antigens, which should aid in the design of T cell-vaccination strategies against Salmonella.

Very long O-antigen chains of Salmonella Paratyphi A inhibit inflammasome activation and pyroptotic cell death.

Salmonella Paratyphi A (SPtA) remains one of the leading causes of enteric (typhoid) fever. Yet, despite the recent increased rate of isolation from patients in Asia, our understanding of its pathogenesis is incomplete. Here we investigated inflammasome activation in human macrophages infected with SPtA. We found that SPtA induces GSDMD-mediated pyroptosis via activation of caspase-1, caspase-4 and caspase-8. Although we observed no cell death in the absence of a functional Salmonella pathogenicity island-1 (SPI-1) injectisome, HilA-mediated overexpression of the SPI-1 regulon enhances pyroptosis. SPtA expresses FepE, an LPS O-antigen length regulator, which induces the production of very long O-antigen chains. Using a ΔfepE mutant we established that the very long O-antigen chains interfere with bacterial interactions with epithelial cells and impair inflammasome-mediated macrophage cell death. Salmonella Typhimurium (STm) serovar has a lower FepE expression than SPtA, and triggers higher pyroptosis, conversely, increasing FepE expression in STm reduced pyroptosis. These results suggest that differential expression of FepE results in serovar-specific inflammasome modulation, which mirrors the pro- and anti-inflammatory strategies employed by STm and SPtA, respectively. Our studies point towards distinct mechanisms of virulence of SPtA, whereby it attenuates inflammasome-mediated detection through the elaboration of very long LPS O-polysaccharides.

Multi-epitope DnaK peptide vaccine accords protection against lethal S. typhimurium challenge: Elicits both cell mediated immunity and long-lasting serum-neutralizing antibody titers.

Typhoid vaccine development has been impeded by inability of currently available vaccines to induce cellular immunity along with neutralizing antibodies against all serovars of S. Typhi and S. Paratyphi. Unfortunately, antibiotic treatment has shown to be an ineffective therapy due to development of resistance against multiple antibiotics. In the present study, we have explored the immunogenicity and protective efficacy of in-silico designed multi-epitope DnaK peptides as candidate vaccine molecules against Salmonella. Immunization studies in mouse typhoid model revealed three of these peptides (DP1, DP5 and DP7) are highly efficacious, stimulating both humoral and cell mediated immunity along with long lasting antibody memory response. There was significant increase in antibody titers (IgG, IgG1, IgG2a, IgA and IgM), lymphocyte proliferative responses and cytokine levels. Immunized groups showed marked reduction in organ bacterial load, fecal shedding and pronounced protection (upto 80%) as compared to unimmunized controls after challenge with S. typhimurium. Our results demonstrate the huge potential of DnaK peptide vaccine candidates (DP1, DP5 and DP7) to accord protective immunity with significant increase in survivability against Salmonella infection in mice, thus commending these molecules as promising agents to tackle typhoid.

Syntheses of Salmonella Paratyphi†A Associated Oligosaccharide Antigens and Development towards Anti-Paratyphoid Fever Vaccines.

With the emergence of multidrug resistant Salmonella strains, the development of anti-Salmonella vaccines is an important task. Currently there are no approved vaccines against Salmonella Paratyphi A, the leading cause of paratyphoid fever. To fill this gap, oligosaccharides corresponding to the O-polysaccharide repeating units from the surface of Salmonella Paratyphi A have been synthesized through convergent stereoselective glycosylations. The synthetic glycan antigen was conjugated with a powerful immunogenic carrier system, the bacteriophage Qß. The resulting construct was able to elicit strong and long-lasting anti-glycan IgG antibody responses, which were highly selective toward Salmonella Paratyphi A associated glycans. The availability of well-defined glycan antigen enabled the determination that one repeating unit of the polysaccharide is sufficient to induce protective antibodies, and the paratose residue and/or the O-acetyl modifications on the backbone are important for recognition by antibodies elicited by a Qß-tetrasaccharide conjugate. Immune sera provided excellent protection to mice from lethal challenge with Salmonella Paratyphi A, highlighting the potential of the synthetic glycan-based vaccine.

Cell mediated immune responses elicited in volunteers following immunization with candidate live oral Salmonella enterica serovar Paratyphi A attenuated vaccine strain CVD 1902.

The incidence of Salmonella enterica serovar Paratyphi A (PA) infection is on the rise and no licensed vaccines are available. We evaluated cell mediated immune (CMI) responses elicited in volunteers following immunization with a single dose (109 or 1010 cfu) of a novel attenuated live oral PA-vaccine strain (CVD 1902). Results showed increases in PA-lipopolysaccharide-specific IgG- and/or IgA B-memory cells and production of IFN-γ, TNF-α, IL-10, IL-23 and RANTES following stimulation with PA-antigens by peripheral blood mononuclear cells obtained 28 days post immunization. Flow cytometry assays revealed that vaccine elicited PA-specific CD8+ and/or CD4+ T effector/memory cells were predominantly multifunctional concomitantly expressing CD107a and/or producing IFN-γ, TNF-α and/or IL-2. Similar proportions of these MF cells expressed, or not, the gut homing marker integrin α4ß7. The results suggest that immunization with CVD 1902 elicits CMI responses against PA supporting its further evaluation as a potential vaccine candidate against paratyphoid A fever.

Characterization and protective efficacy of a Salmonella pathogenicity island 2 (SPI2) mutant of Salmonella Paratyphi A.

Paratyphoid fever caused by Salmonella Paratyphi A is a serious public health problem in many countries. In order to and develop a live attenuated candidate vaccine of Salmonella Paratyphi A, a Salmonella pathogenicity island 2 (SPI2, approximate 40 kb) deletion mutant of Salmonella Paratyphi A was constructed by lambda Red recombination, then the biological characteristics and protective ability of the Salmonella Paratyphi A SPI2 mutant were evaluated. Our results showed that the growth and biochemical properties of the SPI2 mutant were consistent with that of its parent strain, and the mutant was stable with the loss of SPI2. The mice lethal test showed that the virulence of the SPI2 mutant was significantly decreased, it can colonize and persistent more than 14 days in the liver and spleen of mice. Vaccination with the SPI2 mutant in mice revealed no significant effect on body weight and clinical symptoms compared to control animals, and specific humoral and cellular immune responses were also significantly induced. Immunization of mice offered efficient protection against Salmonella Paratyphi A strain challenge at 14 days post vaccination based on mortality and clinical symptoms relative to control group. Overall, these findings suggested that SPI2 plays an important role in pathogenicity of Salmonella Paratyphi A, and the SPI2 mutant showed its potential to develop a live attenuated vaccine candidate.

Cross-reactive multifunctional CD4+ T cell responses against Salmonella enterica serovars Typhi, Paratyphi A and Paratyphi B in humans following immunization with live oral typhoid vaccine Ty21a.

The live oral typhoid vaccine Ty21a elicits predominantly CD8+, as well as CD4+ T cells mediated immune responses. Clinical field studies showed that Ty21a is moderately effective against S. Typhi and S. Paratyphi B, but not S. Paratyphi A infections. In this study we describe the in depth characterization of S. Typhi, S. Paratyphi A and S. Paratyphi B cross-reactive CD4+ T cell responses elicited following immunization with Ty21a. PBMC samples were collected from 16 healthy volunteers before and 42/84days after Ty21a immunization and stimulated ex-vivo with Salmonella-infected targets. Multiparametric flow cytometry was used to detect the vaccine elicited Salmonella-specific responses in T effector/memory (TEM) and CD45RA+ T effector/memory (TEMRA) CD4+ cell subsets, by measuring CD4+ multifunctional (MF) cells that concomitantly produced IFN-γ, TNF-α, IL-2, MIP-1ß, IL-17A and/or expressed CD107a. Post-vaccination increases in S. Typhi-specific MF cells were observed in CD4+ TEM and TEMRA subsets which predominantly produced IFN-γ and/or TNF-α, while IL-2 was produced by a smaller cell subset. A small proportion of those MF cells also produced MIP-1ß, IL-17A and expressed CD107a (a marker associated with cytotoxicity). Approximately one third of these specific MF cells have the potential to migrate to the gut mucosa, as evidenced by co-expression of the gut-homing molecule integrin α4ß7. In contrast to our previous observations with CD8+ T cells, MF CD4+ T cell responses to the different Salmonella serovars evaluated were similar in magnitude and characteristics. We conclude that although induction of cross-reactive CD4+ MF effector T cells suggest a possible role in Salmonella-immunity, these responses are unlikely to provide an immunological basis for the observed efficacy of Ty21a against S. Typhi and S. Paratyphi B, but not to S. Paratyphi A.

Typhoid fever.

Control of typhoid fever relies on clinical information, diagnosis, and an understanding for the epidemiology of the disease. Despite the breadth of work done so far, much is not known about the biology of this human-adapted bacterial pathogen and the complexity of the disease in endemic areas, especially those in Africa. The main barriers to control are vaccines that are not immunogenic in very young children and the development of multidrug resistance, which threatens efficacy of antimicrobial chemotherapy. Clinicians, microbiologists, and epidemiologists worldwide need to be familiar with shifting trends in enteric fever. This knowledge is crucial, both to control the disease and to manage cases. Additionally, salmonella serovars that cause human infection can change over time and location. In areas of Asia, multidrug-resistant Salmonella enterica serovar Typhi (S Typhi) has been the main cause of enteric fever, but now S Typhi is being displaced by infections with drug-resistant S enterica serovar Paratyphi A. New conjugate vaccines are imminent and new treatments have been promised, but the engagement of local medical and public health institutions in endemic areas is needed to allow surveillance and to implement control measures.

Safety and immunogenicity of an attenuated Salmonella enterica serovar Paratyphi A vaccine candidate.

Enteric fever caused by Salmonella enterica serovar Paratyphi A has progressively increased in recent years and became a global health issue. Currently licensed typhoid vaccines do not confer adequate cross-immunoprotection against S. Paratyphi A infection. Therefore, vaccines specifically against enteric fever caused by S. Paratyphi A are urgently needed. In the present study, an attenuated vaccine strain was constructed from S. Paratyphi A CMCC50093 by the deletions of aroC and yncD. The obtained strain SPADD01 showed reduced survival within THP-1 cells and less bacterial burden in spleens and livers of infected mice compared with the wild-type strain. The 50% lethal doses of SPADD01 and the wild-type strain were assessed using a murine infection model. The virulence of SPADD01 is approximately 40,000-fold less than that of the wild-type strain. In addition, SPADD01 showed an excellent immunogenicity in mouse model. Single intranasal inoculation elicited striking humoral and mucosal immune responses in mice and yielded effective protection against lethal challenge of the wild-type strain. A high level of cross-reactive humoral immune response against LPS of Salmonella enterica serovar Typhi was also detected in immunized mice. However, SPADD01 vaccination only conferred a low level of cross-protection against S. Typhi. Our data suggest that SPADD01 is a promising vaccine candidate against S. Paratyphi A infection and deserves further evaluation in clinical trial. To date, no study has demonstrated a good cross-protection between serovars of S. Typhi and S. Paratyphi A, suggesting that the dominant protective antigens of both serovars are likely different and need to be defined in future study.

Construction of an attenuated Salmonella enterica serovar Paratyphi A vaccine strain harboring defined mutations in htrA and yncD.

The global epidemic features of enteric fever have changed greatly in recent years. The incidence of enteric fever caused by Salmonella enterica serovar Paratyphi A has progressively increased. In some areas of Asia, infections with S. Paratyphi A have exceeded those with S. Typhi, resulting in S. Paratyphi A becoming the main causative agent of enteric fever. However, two currently licensed typhoid vaccines do not confer adequate cross-protection against S. Paratyphi A infection. Therefore, development of specific vaccines against enteric fever caused by S. Paratyphi A is urgently needed. In the present study, an attenuated strain was constructed by double deletion of the htrA and yncD genes in a wild-type strain of S. Paratyphi A and its safety and immunogenicity assessed. In a mouse model, the 50% lethal dose of the double deletion mutant and the wild-type strain were 3.0 × 10(8) CFU and 1.9 × 10(3) CFU, respectively, suggesting that the double deletion resulted in remarkably decreased bacterial virulence. Bacterial colonization of the double deletion mutant in the livers and spleens of infected mice was strikingly less than that of the wild-type strain. A single nasal administration of the attenuated vaccine candidate elicited high concentrations of anti-LPS and anti-flagellin IgG in a mouse model and protected immunized mice against lethal challenge with the wild-type strain. Thus, our findings suggest that the attenuated vaccine strain is a promising candidate worthy of further evaluation both as a human enteric fever vaccine and as a vaccine delivery vector for heterologous antigens.

Cross-reactive immune response induced by the Vi capsular polysaccharide typhoid vaccine against Salmonella Paratyphi strains.

There are no vaccines in clinical use against paratyphoid fever, caused by Salmonella Paratyphi A and B or, rarely, C. Oral Salmonella Typhi Ty21a typhoid vaccine elicits a significant cross-reactive immune response against S. Paratyphi A and B, and some reports suggest cross-protective efficacy against the disease. These findings are ascribed to the O-12 antigen shared between the strains. The Vi capsular polysaccharide vaccine has been shown to elicit antibodies reactive with O-9,12. Twenty-five volunteers immunized with the parenteral Vi vaccine (Typherix(®) ) were explored for plasmablasts cross-reactive with paratyphoid strains; the responses were compared to those in 25 age- and gender-matched volunteers immunized with Ty21a (Vivotif(®) ). Before vaccination, 48/50 vaccinees had no plasmablasts reactive with the antigens. Seven days after vaccination, 15/25 and 22/25 Vi- and Ty21a-vaccinated volunteers had circulating plasmablasts producing antibodies cross-reactive with S. Paratyphi A, 18/25 and 23/25 with S. Paratyphi B and 16/25 and 9/25 with Paratyphi C, respectively. Compared to the Ty21a group, the Vi group showed significantly lower responses to S. Paratyphi A and B and higher to S. Paratyphi C. To conclude, the Vi vaccine elicited a cross-reactive plasmablast response to S. Paratyphi C (Vi antigen in common) and less marked responses to S. Paratyphi A and B than the Ty21a preparation. S. Paratyphi A and B both being Vi-negative, the result can be explained by trace amounts of bacterial cell wall O-12 antigen in the Vi preparation, despite purification. The clinical significance of this finding remains to be determined.

A novel tool for capture and detection of typhoid fever using Ag-labeled nanocomposites.

This work investigated a simple and versatile modification to a solid substrate to develop antibody recognition using nanoparticles. The new immobilized metal ion affinity adsorbent containing nanoparticles and hydrophilic resins is proposed here to improve the binding of antigen on its surface. The light-scattering properties of submicroscopic metal particles ranging from 100 to 120 nm in diameter were confirmed by scanning electron microscopy. We found that synthesized nanoparticles have an inherent enzyme mimetic activity similar to that found in natural peroxidases. The synthesized nanoparticles were coated with Salmonella typhi and Salmonella paratyphi antigens and were allowed to react with Salmonella-infected serum. Positive reactions were detected visually with the naked eye. The color changes of substrate (TMB) were observed even in 1:800 dilutions. This report helps in developing a specific immunoassay using nano-conjugated antigen for the rapid detection of S. typhi and S. paratyphi antibodies in infected serum.

[Distribution of Salmonella paratyphi A pagC gene and immunoprotective effect of its recombinant expressed products].

OBJECTIVE: To determine the distribution and sequence conservation of pagC gene in Salmonella paratyphi A isolates, and the immunogenicity and immunoprotection of its recombinant expression products (rPagC). METHODS: The distribution of pagC gene in Salmonella paratyphi A isolates and its sequence conservation were examined by PCR and sequencing. A prokaryotic expression system of pagC gene was constructed and the expressed rPagC was extracted by Ni-NTA affinity chromatography. SDS-PAGE and Bio-Rad Gel Image Analyzer were applied to examine the expression and yield of rPagC. The antigenicity and immunoreactivity of rPagC were detected by immunodiffusion test, ELISA and Western Blot assay. The immunoprotective effect of rPagC against infection of Salmonella paratyphi A in mice was determined, while the agglutinative effect of sera from rPagC-immunized mice was measured by micro-Widal's test. RESULTS: All the Salmonella paratyphi A isolates tested had the pagC gene, the similarity of nucleotide and amino acid sequences was 99.1 %-100 % and 98.4 %-100 %, respectively. The constructed prokaryotic expression system expressed rPagC with high efficiency. The rPagC immunized rabbit produced a high level antibody and it also combined with antiserum against whole cell of S. paratyphi A to generate a positive Western hybridization signal. ELISA results indicated that 97.1 % (66/68) paratyphoid patients infected with Salmonella paratyphi A were positive for rPagC antibody in their serum specimens. When mice were immunized with 100 µg or 200 µg rPagC, the immunoprotective rates were 73.3 % (11/15) or 86.7 % (13/15), respectively. The sera from rPagC-immunized mice offered 1:10-1:40 agglutination titers with the H antigens of Salmonella paratyphi A and Salmonella typhi. CONCLUSION: PagC gene has an extensive distribution in Salmonella paratyphi A isolates. rPagC can be used as the candidate antigen in genetic engineering vaccine due to its fine immunogenicity and powerful immunoprotective effect.

Vaccines for typhoid fever and other salmonelloses.

PURPOSE OF REVIEW: This review summarizes the recent advances in vaccination against Salmonella enterica serovar Typhi and highlights the data supporting the development of next generation vaccines to address paratyphoid fever and invasive nontyphoidal Salmonella (iNTS) disease. RECENT FINDINGS: There has been increasing awareness of the disease burden caused by S. Typhi particularly in Africa and greater recognition of S. Paratyphi A's contribution to enteric fever episodes throughout Asia. Groups have been working to improve the existing typhoid vaccines and provide comprehensive data on the feasibility of their implementation in endemic settings. These data have resulted in modifications to the recommendations for typhoid vaccination in traveller markets and endemic settings, and has also led to the development of S. Paratyphi A vaccine components that can be combined with existing typhoid vaccines to generate bivalent formulations against enteric fever. The epidemiology of iNTS serovars as cause of appreciable morbidity and mortality in Africa, and the need for vaccines, has also become more widely appreciated. SUMMARY: Current typhoid vaccines, although moderately effective for short periods of time, cannot be used in all age groups and only target one of the clinically relevant Salmonella serovars. Greater effort must be placed on the development and implementation of improved vaccines for the disease burden resulting from Typhi, Paratyphi A or iNTS infections.

O-Antigen Extraction, Purification, and Chemical Conjugation to a Carrier Protein.

A variety of bacterial infections have been tackled by glycoconjugates over the recent years, and more vaccines are either under development at preclinical level or in clinical trials. So far, licensed glycoconjugate vaccines have made use of capsular polysaccharides or derived fragments. Today, many glycoconjugates are making use of other classes of sugars, in particular, the O-antigen portion of lipopolysaccharide molecules. Here, we report a simplified method for O-antigen extraction and purification that avoids the step of lipopolysaccharide isolation. Also, a selective chemistry for terminal linkage of O-antigen chains to a carrier protein is described, together with analytical methods for intermediates and final conjugate characterization.

Roles of Salmonella typhi and Salmonella paratyphi in Gallbladder Cancer Development.

BACKGROUND: Typhoid (Salmonella typhi and paratyphi) carriers and gall bladder cancer (GBC) are endemic in northern India. Results of previous studies about association of typhoid carriers with GBC are inconsistent. We studied antibodies against Salmonella typhi and paratyphi in serum samples of patients with GBC. METHODS: We performed modified Widal test for antibodies against Salmonella typhi (Vi and O) and Salmonella paratyphi (AO and BO) antigens in patients with GBC (n=100), xanthogranulomatous cholecystitis (XGC, n=24), chronic cholecystitis (CC, n=200) and healthy controls (HC, n=200). RESULTS: Serum antibodies against Salmonella were more frequently positive in GBC (22%) and XGC (29%), particularly in males in age ≥50 years (GBC: 47% and XGC: 50%) vs. HC (0) (p <0.01). Vi antibody was more common in GBC (13%, OR:9.8) and XGC (8%, OR:5.9) than HC (2%). O antibody was more common in GBC (8%, OR: 8.6) and XGC (8%, OR: 9.0) than HC (1%). O antibody was also more common in males with GBC (12%) than CC (1%) and HC (1%) (P=0.02 and p <0.001, respectively). AO (6%) and BO (4%) antibodies were detected in GBC, particularly in males, than HC (0), (p <0.01). Salmonella antibodies were more frequent in GBC with GS than those without GS (50% vs. 20%, OR=3.94, P=0.01). CONCLUSIONS: Salmonella carrier state was more common in GBC and XGC, particularly in elderly males than HC. The Vi antibody was more common in GBC and XGC than HC. Salmonella infection was more common in GBC with GS than those without GS.

Characterization and protective efficacy of a sptP mutant of Salmonella Paratyphi A.

BACKGROUND: Salmonella Paratyphi A causes paratyphoid A, a severe systemic disease of people and remains a major public health problem in many parts of the world. In the interest of researching the roles of sptP on Salmonella Paratyphi A and developing a live-attenuated vaccine candidate, an sptP mutant of Salmonella Paratyphi A SPA017 (SPA017ΔsptP) was constructed, and then its characterization, immunogenicity, and protective ability were evaluated. RESULTS: The deletion of sptP had no effect on growth and biochemical properties. Adhesion and invasion assays showed that the lack of sptP did not affect the adhesion of Salmonella Paratyphi A, but the invasive ability of the mutant strain was significantly decreased, the half-lethal dose (LD50 ) of the mutant strain was 1.43 × 104 times of the parent strain in intraperitoneally injected mice. Single intraperitoneal vaccination with SPA017ΔsptP (1 × 105 CFU) in mice did not affect the body weight or elicit clinical symptoms relative to the control group, SPA017ΔsptP bacteria were isolated from livers and spleens of vaccinated mice at 14 days postvaccination. Notably, specific humoral and cellular immune responses were significantly induced. The protective assessment showed that the mutant strain could provide high-level protection against subsequent challenge with the wild-type SPA017 strain. CONCLUSIONS: These results demonstrated that SptP plays an essential role in the pathogenicity of Salmonella Paratyphi A, and Salmonella Paratyphi A lacking sptP is immunogenic and protective in mice.

Homologous and heterologous re-challenge with Salmonella Typhi and Salmonella Paratyphi A in a randomised controlled human infection model.

Enteric fever is a systemic infection caused by Salmonella Typhi or Paratyphi A. In many endemic areas, these serovars co-circulate and can cause multiple infection-episodes in childhood. Prior exposure is thought to confer partial, but incomplete, protection against subsequent attacks of enteric fever. Empirical data to support this hypothesis are limited, and there are few studies describing the occurrence of heterologous-protection between these closely related serovars. We performed a challenge-re-challenge study using a controlled human infection model (CHIM) to investigate the extent of infection-derived immunity to Salmonella Typhi or Paratyphi A infection. We recruited healthy volunteers into two groups: naïve volunteers with no prior exposure to Salmonella Typhi/Paratyphi A and volunteers previously-exposed to Salmonella Typhi or Paratyphi A in earlier CHIM studies. Within each group, participants were randomised 1:1 to oral challenge with either Salmonella Typhi (104 CFU) or Paratyphi A (103 CFU). The primary objective was to compare the attack rate between naïve and previously challenged individuals, defined as the proportion of participants per group meeting the diagnostic criteria of temperature of ≥38°C persisting for ≥12 hours and/or S. Typhi/Paratyphi bacteraemia up to day 14 post challenge. The attack-rate in participants who underwent homologous re-challenge with Salmonella Typhi was reduced compared with challenged naïve controls, although this reduction was not statistically significant (12/27[44%] vs. 12/19[63%]; Relative risk 0.70; 95% CI 0.41-1.21; p = 0.24). Homologous re-challenge with Salmonella Paratyphi A also resulted in a lower attack-rate than was seen in challenged naïve controls (3/12[25%] vs. 10/18[56%]; RR0.45; 95% CI 0.16-1.30; p = 0.14). Evidence of protection was supported by a post hoc analysis in which previous exposure was associated with an approximately 36% and 57% reduced risk of typhoid or paratyphoid disease respectively on re-challenge. Individuals who did not develop enteric fever on primary exposure were significantly more likely to be protected on re-challenge, compared with individuals who developed disease on primary exposure. Heterologous re-challenge with Salmonella Typhi or Salmonella Paratyphi A was not associated with a reduced attack rate following challenge. Within the context of the model, prior exposure was not associated with reduced disease severity, altered microbiological profile or boosting of humoral immune responses. We conclude that prior Salmonella Typhi and Paratyphi A exposure may confer partial but incomplete protection against subsequent infection, but with a comparable clinical and microbiological phenotype. There is no demonstrable cross-protection between these serovars, consistent with the co-circulation of Salmonella Typhi and Paratyphi A. Collectively, these data are consistent with surveillance and modelling studies that indicate multiple infections can occur in high transmission settings, supporting the need for vaccines to reduce the burden of disease in childhood and achieve disease control. Trial registration NCT02192008; clinicaltrials.gov.

Crosstalk between leukocytes triggers differential immune responses against Salmonella enterica serovars Typhi and Paratyphi.

Enteric fevers, caused by the Salmonella enterica serovars Typhi (ST), Paratyphi A (PA) and Paratyphi B (PB), are life-threatening illnesses exhibiting very similar clinical symptoms but with distinct epidemiologies, geographical distributions and susceptibilities to antimicrobial treatment. Nevertheless, the mechanisms by which the host recognizes pathogens with high levels of homology, such as these bacterial serovars, remain poorly understood. Using a three-dimensional organotypic model of the human intestinal mucosa and PA, PB, and ST, we observed significant differences in the secretion patterns of pro-inflammatory cytokines and chemokines elicited by these serovars. These cytokines/chemokines were likely to be co-regulated and influenced the function of epithelial cells, such as the production of IL-8. We also found differing levels of polymorphonuclear leukocyte (PMN) migration among various infection conditions that either included or excluded lymphocytes and macrophages (Mϕ), strongly suggesting feedback mechanisms among these cells. Blocking experiments showed that IL-1ß, IL-6, IL-8, TNF-α and CCL3 cytokines were involved in the differential regulation of migration patterns. We conclude that the crosstalk among the lymphocytes, Mϕ, PMN and epithelial cells is cytokine/chemokine-dependent and bacterial-serotype specific, and plays a pivotal role in orchestrating the functional efficiency of the innate cells and migratory characteristics of the leukocytes.

Clinical Evaluation of a Multiplex PCR for the Detection of Salmonella enterica Serovars Typhi and Paratyphi A from Blood Specimens in a High-Endemic Setting.

Enteric fever is a major public health concern in endemic areas, particularly in infrastructure-limited countries where Salmonella Paratyphi A has emerged in increasing proportion of cases. We aimed to evaluate a method to detect Salmonella Typhi (S. Typhi) and Salmonella Paratyphi A (S. Paratyphi A) in febrile patients in Bangladesh. We conducted a prospective study enrolling patients with fever > 38°C admitted to two large urban hospitals and two outpatient clinics located in Dhaka, Bangladesh. We developed and evaluated a method combining short culture with a new molecular assay to simultaneously detect and differentiate S. Typhi and S. Paratyphi A from other Salmonella directly from 2 to 4 mL of whole blood in febrile patients (n = 680). A total of 680 cases were enrolled from the four participating sites. An increase in the detection rate (+38.8%) in S. Typhi and S. Paratyphi A was observed with a multiplex polymerase chain reaction (PCR) assay, and absence of non-typhoidal Salmonella detection was reported. All 45 healthy controls were culture and PCR negative, generating an estimated 92.9% of specificity on clinical samples. When clinical performance was assessed in the absence of blood volume prioritization for testing, a latent class model estimates clinical performance ≥ 95% in sensitivity and specificity with likelihood ratio (LR) LR+ > 10 and LR- < 0.1 for the multiplex PCR assay. The alternative method to blood culture we developed may be useful alone or in combination with culture or serological tests for epidemiological studies in high disease burden settings and should be considered as secondary endpoint test for future vaccine trials.