Factors associated with typical enteropathogenic Escherichia coli infection among children <5 years old with moderate-to-severe diarrhoea in rural western Kenya, 2008-2012.

Typical enteropathogenic Escherichia coli (tEPEC) infection is a major cause of diarrhoea and contributor to mortality in children <5 years old in developing countries. Data were analysed from the Global Enteric Multicenter Study examining children <5 years old seeking care for moderate-to-severe diarrhoea (MSD) in Kenya. Stool specimens were tested for enteric pathogens, including by multiplex polymerase chain reaction for gene targets of tEPEC. Demographic, clinical and anthropometric data were collected at enrolment and ~60-days later; multivariable logistic regressions were constructed. Of 1778 MSD cases enrolled from 2008 to 2012, 135 (7.6%) children tested positive for tEPEC. In a case-to-case comparison among MSD cases, tEPEC was independently associated with presentation at enrolment with a loss of skin turgor (adjusted odds ratio (aOR) 2.08, 95% confidence interval (CI) 1.37-3.17), and convulsions (aOR 2.83, 95% CI 1.12-7.14). At follow-up, infants with tEPEC compared to those without were associated with being underweight (OR 2.2, 95% CI 1.3-3.6) and wasted (OR 2.5, 95% CI 1.3-4.6). Among MSD cases, tEPEC was associated with mortality (aOR 2.85, 95% CI 1.47-5.55). This study suggests that tEPEC contributes to morbidity and mortality in children. Interventions aimed at defining and reducing the burden of tEPEC and its sequelae should be urgently investigated, prioritised and implemented.

Diarrhoea, enteric pathogen detection and nutritional indicators among controls in the Global Enteric Multicenter Study, Kenya site: an opportunity to understand reference populations in case-control studies of diarrhoea.

Given the challenges in accurately identifying unexposed controls in case-control studies of diarrhoea, we examined diarrhoea incidence, subclinical enteric infections and growth stunting within a reference population in the Global Enteric Multicenter Study, Kenya site. Within 'control' children (0-59 months old without diarrhoea in the 7 days before enrolment, n = 2384), we examined surveys at enrolment and 60-day follow-up, stool at enrolment and a 14-day post-enrolment memory aid for diarrhoea incidence. At enrolment, 19% of controls had ⩾1 enteric pathogen associated with moderate-to-severe diarrhoea ('MSD pathogens') in stool; following enrolment, many reported diarrhoea (27% in 7 days, 39% in 14 days). Controls with and without reported diarrhoea had similar carriage of MSD pathogens at enrolment; however, controls reporting diarrhoea were more likely to report visiting a health facility for diarrhoea (27% vs. 7%) or fever (23% vs. 16%) at follow-up than controls without diarrhoea. Odds of stunting differed by both MSD and 'any' (including non-MSD pathogens) enteric pathogen carriage, but not diarrhoea, suggesting control classification may warrant modification when assessing long-term outcomes. High diarrhoea incidence following enrolment and prevalent carriage of enteric pathogens have implications for sequelae associated with subclinical enteric infections and for design and interpretation of case-control studies examining diarrhoea.

Overexpression of O-polysaccharide chain length regulators in Gram-negative bacteria using the Wzx-/Wzy-dependent pathway enhances production of defined modal length O-polysaccharide polymers for use as haptens in glycoconjugate vaccines.

AIMS: O-polysaccharide (OPS) molecules are protective antigens for several bacterial pathogens, and have broad utility as components of glycoconjugate vaccines. Variability in the OPS chain length is one obstacle towards further development of these vaccines. Introduction of sizing steps during purification of OPS molecules of suboptimal or of mixed lengths introduces additional costs and complexity while decreasing the final yield. The overall goal of this study was to demonstrate the utility of engineering Gram-negative bacteria to produce homogenous O-polysaccharide populations that can be used as the basis of carbohydrate vaccines by overexpressing O-polysaccharide chain length regulators of the Wzx-/Wzy-dependent pathway. METHOD AND RESULTS: The O-polysaccharide chain length regulators wzzB and fepE from Salmonella Typhimurium I77 and wzz2 from Pseudomonas aeruginosa PAO1 were cloned and expressed in the homologous organism or in other Gram-negative bacteria. Overexpression of these Wzz proteins in the homologous organism significantly increased the proportion of long or very long chain O-polysaccharides. The same observation was made when wzzB was overexpressed in Salmonella Paratyphi A and Shigella flexneri, and wzz2 was overexpressed in two other strains of P. aeruginosa. CONCLUSIONS: Overexpression of Wzz proteins in Gram-negative bacteria using the Wzx/Wzy-dependant pathway for lipopolysaccharide synthesis provides a genetic method to increase the production of an O-polysaccharide population of a defined size. SIGNIFICANCE AND IMPACT OF THE STUDY: The methods presented herein represent a cost-effective and improved strategy for isolating preferred OPS vaccine haptens, and could facilitate the further use of O-polysaccharides in glycoconjugate vaccine development.

Adaptation of red blood cell lysis represents a fundamental breakthrough that improves the sensitivity of Salmonella detection in blood.

AIMS: Isolation of Salmonella Typhi from blood culture is the standard diagnostic for confirming typhoid fever but it is unavailable in many developing countries. We previously described a Microwave Accelerated Metal Enhanced Fluorescence (MAMEF)-based assay to detect Salmonella in medium. Attempts to detect Salmonella in blood were unsuccessful, presumably due to the interference of erythrocytes. The objective of this study was to evaluate various blood treatment methods that could be used prior to PCR, real-time PCR or MAMEF to increase sensitivity of detection of Salmonella. METHODS AND RESULTS: We tested ammonium chloride and erythrocyte lysis buffer, water, Lymphocyte Separation Medium, BD Vacutainer(®) CPT(™) Tubes and dextran. Erythrocyte lysis buffer was the best isolation method as it is fast, inexpensive and works with either fresh or stored blood. The sensitivity of PCR- and real-time PCR detection of Salmonella in spiked blood was improved when whole blood was first lysed using erythrocyte lysis buffer prior to DNA extraction. Removal of erythrocytes and clotting factors also enabled reproducible lysis of Salmonella and fragmentation of DNA, which are necessary for MAMEF sensing. CONCLUSIONS: Use of the erythrocyte lysis procedure prior to DNA extraction has enabled improved sensitivity of Salmonella detection by PCR and real-time PCR and has allowed lysis and fragmentation of Salmonella using microwave radiation (for future detection by MAMEF). SIGNIFICANCE AND IMPACT OF THE STUDY: Adaptation of the blood lysis method represents a fundamental breakthrough that improves the sensitivity of DNA-based detection of Salmonella in blood.

Antigen-specific IgA B memory cell responses to Shigella antigens elicited in volunteers immunized with live attenuated Shigella flexneri 2a oral vaccine candidates.

We studied the induction of antigen-specific IgA memory B cells (B(M)) in volunteers who received live attenuated Shigella flexneri 2a vaccines. Subjects ingested a single oral dose of 10(7), 10(8) or 10(9) CFU of S. flexneri 2a with deletions in guaBA (CVD 1204) or in guaBA, set and sen (CVD 1208). Antigen-specific serum and stool antibody responses to LPS and Ipa B were measured on days 0, 7, 14, 28 and 42. IgA B(M) cells specific to LPS, Ipa B and total IgA were assessed on days 0 and 28. We show the induction of significant LPS-specific IgA B(M) cells in anti-LPS IgA seroresponders. Positive correlations were found between anti-LPS IgA B(M) cells and anti-LPS IgA in serum and stool; IgA B(M) cell responses to IpaB were also observed. These B(M) cell responses are likely play an important role in modulating the magnitude and longevity of the humoral response.

Immunogenicity in humans of a recombinant bacterial antigen delivered in a transgenic potato.

Compared with vaccine delivery by injection, oral vaccines offer the hope of more convenient immunization strategies and a more practical means of implementing universal vaccination programs throughout the world. Oral vaccines act by stimulating the immune system at effector sites (lymphoid tissue) located in the gut. Genetic engineering has been used with variable success to design living and non-living systems as a means to deliver antigens to these sites and to stimulate a desired immune response. More recently, plant biotechnology techniques have been used to create plants which contain a gene derived from a human pathogen; the resultant plant tissues will accumulate an antigenic protein encoded by the foreign DNA. In pre-clinical trials, we found that antigenic proteins produced in transgenic plants retained immunogenic properties when purified; if injected into mice the antigen caused production of protein-specific antibodies. Moreover, in some experiments, if the plant tissues were simply fed to mice, a mucosal immune response occurred. The present study was conducted as a proof of principle to determine if humans would also develop a serum and/or mucosal immune response to an antigen delivered in an uncooked foodstuff.

Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans.

Isogenic mutant strains of V. cholerae O1 lacking elements of a genetic regulon controlled by toxR and implicated in virulence were tested in volunteers. A deletion mutation in ctxA, the gene encoding the A subunit of cholera toxin, markedly attenuated disease symptoms without affecting intestinal colonization. Deletion of toxR, the gene encoding the cholera toxin-positive regulatory protein resulted in a diminution in colonizing capacity. A deletion mutation in tcpA, encoding the major subunit of the toxin coregulated pilus (regulated by toxR), abolished the colonizing capacity of this strain. These results show for the first time the role of a specific pilus structure in colonization of the human intestine by V. cholerae O1 and exemplify the significance of a genetic regulon in pathogenesis.

CfaE tip mutations in enterotoxigenic Escherichia coli CFA/I fimbriae define critical human intestinal binding sites.

Enterotoxigenic Escherichia coli (ETEC) use colonization factors to attach to the human intestinal mucosa, followed by enterotoxin expression that induces net secretion and diarrhoeal illness. ETEC strain H10407 expresses CFA/I fimbriae, which are composed of multiple CfaB structural subunits and a CfaE tip subunit. Currently, the contribution of these individual fimbrial subunits in intestinal binding remains incompletely defined. To identify the role of CfaE in attachment in the native ETEC background, an R181A single-amino-acid substitution was introduced by recombination into the H10407 genome. The substitution of R181A eliminated haemagglutination and binding of intestinal mucosa biopsies in in vitro organ culture assays, without loss of CFA/I fimbriae expression. Wild-type in trans plasmid-expressed cfaE restored the binding phenotype. In contrast, in trans expression of cfaE containing amino acid 181 substitutions with similar amino acids, lysine, methionine and glutamine did not restore the binding phenotype, indicating that the loss of the binding phenotype was due to localized areas of epitope disruption. R181 appears to have an irreplaceable role in the formation of a receptor-binding feature on CFA/I fimbriae. The results specifically indicate that the CfaE tip protein is a required binding factor in CFA/I-mediated ETEC colonization, making it a potentially important vaccine antigen.

Kinetics of maternally-derived serogroup A, C, Y and W-specific meningococcal immunoglobulin G in Malian women and infants.

A prospective, randomised, controlled observer-blind trial measuring the efficacy and immunogenicity of trivalent influenza vaccine (TIV) and the immunogenicity of quadrivalent meningococcal conjugate vaccine (MCV) in pregnant women and their infants up to 6 months of age was conducted in Mali. Here we reported the immunogenicity of MCV, which was used as a comparator vaccine to TIV, in this population. Third-trimester pregnant Malian women were randomized to receive TIV or MCV. Blood samples were collected from women prior to vaccination, 28 days post-vaccination, at delivery and 3 and 6 months post-delivery and from infants at birth and 3 and 6 months of age. Meningococcal-specific serogroup (Men) A, C, Y and W-specific antibodies were measured by enzyme linked immunosorbent assay in a randomly selected subset of 50 mother-infant pairs where the mother had received MCV. At birth, 94.0% (47/50) of infants had MenA specific IgG levels ≥ 2 µg/mL decreasing to 72.9% and 30.4% at 3 and 6 months of age. For MenC, 81.3% (39/48) of infants had MenC specific IgG levels ≥ 2 µg/mL at birth decreasing to 29.4% and 17.8% at 3 and 6 months of age. For MenY, 89.6% (43/48) of infants had MenY specific IgG levels ≥ 2 µg/mL at birth decreasing to 64.6% and 62.5% at 3 and 6 months of age. For MenW, 89.6% (43/48) of infants had MenW specific IgG levels ≥ 2 µg/ml at birth decreasing to 62.5% and 41.7% at 3 and 6 months of age. Maternal immunization with MCV conveyed protective levels of IgG at birth through to 3 months of age in the majority of infants.

Acute bacterial meningitis among children, in Manhiça, a rural area in Southern Mozambique.

INTRODUCTION: Acute bacterial meningitis (ABM) is one of the most severe diseases in Sub-Saharan Africa. Although data for the continent is very limited, more than one million cases are estimated per year, with mortality and life-long sequelae occurring in 50% of these cases. METHODS: As part of the clinical management of children admitted to the Manhiça District Hospital, information on cases of ABM was recorded. We analysed data from June 1998 to November 2003. RESULTS: During the study period, 475 cerebrospinal-fluid (CSF) samples were collected from 20,173 children <15 years of age admitted to hospital. Culture results confirmed 71 (15%) cases of ABM. The most prevalent bacterial aetiologies were Streptotoccus pneumoniae (pneumococcus, n=31), Haemophilus influenzae (n=13) and Neisseria meningitis (n=8). Other important bacteria were Streptococcus sp. (n=7), Salmonella sp. (n=4) and Staphylococcus aureus (n=3). Crude incidence rates of ABM and pneumococcal meningitis were 20/100,000 and 10/100,000 children-year-at-risk, respectively. Incidences were more than three times higher in the <1 year age group. Overall case fatality rate was 36%, and was highest for H. influenzae and pneumococcal meningitis (55% and 45%, respectively, p=0.044). Pneumococcal susceptibility was 81% for oxacillin and 93% for chloramphenicol. For H. influenzae isolates, susceptibility was 54% for ampicillin and 62% for chloramphenicol. CONCLUSIONS: S. pneumoniae and H. influenzae are the main aetiologies responsible for the high burden of morbidity and mortality associated with ABM in rural Mozambique. These findings are important to evaluate treatment guidelines and potential impact of control measures.

Enteroaggregative Escherichia coli elaborate a heat-stable enterotoxin demonstrable in an in vitro rabbit intestinal model.

Enteroaggregative Escherichia coli (EAggEC) have been associated with persistent diarrhea in young children, but little is known about its pathogenesis. We assayed for enterotoxic activity in culture filtrates (CF) of EAggEC strains in Ussing chambers mounted with rabbit ileal mucosa. CF from strain 17-2, a prototype Chilean EAggEC strain, caused a greater rise in potential difference and short circuit current (SCC) than that seen in HB101 control, and this effect was abolished by protease pretreatment and partially stable after heat treatment. Ultrafiltration of 17-2 CF preparations localized the active moiety to the 2-5 kD Mr size range. CF from HB101 transformed with the 17-2 plasmid showed Ussing chamber activity. less than 10-kD CF fractions from five of six other EAggEC strains screened in Ussing chambers gave SCC responses of similar magnitude to 17-2. The 17-2 CF activity was not neutralized after pretreatment with polyclonal anti-STa antibody. Additionally, all of the seven EAggEC strains studied were nonreactive by heat-stable enterotoxin variant STa ELISA, were negative in the suckling mouse assay, and failed to hybridize with heat-stable enterotoxin variant STh and STp DNA probes. In summary, our data indicate that 17-2 produces a low molecular weight, partially heat-stable, protease-sensitive enterotoxin which appears to be plasmid associated, and genetically and immunologically distinct from E. coli STa. Preliminary screening suggests that this tox+ phenotype may be common among EAggEC.

Pathogenesis of shigella diarrhea. Serum anticytotoxin antibody response produced by toxigenic and nontoxigenic Shigella dysenteriae 1.

The serum antitoxin response to the cytotoxin contained in preparations of Shigella dysenteriae 1 (Shiga's bacillus) exotoxin was studied in natural and experimental infections of man. Natural infection resulted in the rapid appearance of toxin-neutralizing antibody, which disappeared some time between 9 and 18 mo after infection. Experimental infection of human volunteers provided the opportunity to study immunoglobulin class of the antibody in sera obtained serially from 7 to 50 days after infection. Neutralizing antibody was present only in the IgM fraction isolated by sucrose density gradient ultracentrifugation. This was confirmed by the use of solid-phase immunoaffinity chromatography. Even though the time-course and immunoglobulin class of the antitoxin antibody response was similar to that previously observed for anti-O polysaccharide antibody, the biologically active cytotoxin was shown to be highly susceptible to destruction by proteolytic enzymes. Sera from subjects infected with a virulent invasive chlorate-resistant Shiga mutant thought to be "nontoxigenic" also contained antibody which was similarly restricted to the IgM fraction. Biologically active cytotoxin was recovered when this mutant organism was grown in liquid media with controlled ion concentration. The mutant cytotoxin was heat labile, neutralized by antiwild-type cytotoxin antibody, and was separable by isoelectric focusing into two fractions with pI 7.2 and 6.1 like the wild-type toxin. These studies show that cytotoxin antigen is produced during in vivo infection with Shiga bacilli, resulting in a serum antitoxin antibody response. Without explanation is the restriction of the antibody to the IgM class and lack of evidence for an IgG antibody to the protein cytotoxin. Finally, mutant strain 725, previously designated "nontoxigenic," was shown to produce biologically active cytotoxin in vitro and, in experimentally infected volunteers, to result in a serum IgM antibody similar to that observed during infection with the wild-type strain.

Shigella enterotoxin 1: an enterotoxin of Shigella flexneri 2a active in rabbit small intestine in vivo and in vitro.

Culture filtrates of Shigella flexneri 2a strain M4243 grown in iron-depleted medium, caused significant fluid accumulation in rabbit ileal loops. Also, when tested in Ussing chambers, a greater rise in potential difference and short circuit current was seen with such filtrates compared with the medium control. Analogous filtrates from two M4243 derivatives lacking the 140-MD invasiveness plasmid (either M4243avir or BS103) retained 60-65% of the wild-type enterotoxic activity. Ultrafiltration and gel exclusion size fractionation of M4243 filtrate revealed that the activity was approximately 60 kD. SDS-PAGE performed on this fraction showed 18 bands, 5 of which reacted with human convalescent sera. Genes encoding this enterotoxin, named ShET1 for Shigella enterotoxin 1, were cloned from the S. flexneri 2a chromosome, and two separate open reading frames of 534 and 186 bp were sequenced. These observations suggest that S. flexneri 2a elaborates two distinct enterotoxins: ShET1, encoded by genes located on the chromosome, and ShET2, encoded by a gene on the 140-MD invasiveness plasmid. ShET1, which is composed of two distinct subunits and is elaborated in vivo, where it elicits an immune response, may be important in the pathogenesis of diarrheal illness due to S. flexneri 2a.

Evaluation in volunteers of a candidate live oral attenuated Salmonella typhi vector vaccine.

Candidate vector vaccine strain CVD 906 (aroC- and aroD- derivative of virulent Salmonella typhi strain ISP1820) was evaluated in phase 1 clinical trials. The first nine volunteers ingested a single dose of 5 x 10(7) CVD 906 bacilli. At this dose CVD 906 stimulates remarkable systemic and mucosal immune responses, inasmuch as 89% of volunteers developed marked serum antibody levels to S. typhi antigens and high numbers of antigen-specific gut-derived antibody-secreting cells. Four (44%) volunteers developed asymptomatic vaccinemia 4-10 d after immunization and all volunteers excreted CVD 906 on at least one occasion. However, two volunteers developed febrile adverse reactions, one on the day of vaccination and the other on day 4. Of 11 volunteers who ingested a single dose of 5 x 10(3) CVD 906 bacilli, none displayed side effects but 27% developed significant serum responses to S. typhi LPS. In vitro, CVD 906 replicates for only nine generations in pooled human serum, indicating that CVD 906 growth is limited in this physiologically relevant medium. In phorbol myristate acetate-induced U937 human macrophage-like cells, CVD 906 replicates intracellularly to a lesser extent than parent strain ISP1820. Although, strain CVD 906 is attenuated and highly immunogenic, the occasional febrile reactions at high doses indicate that further attenuation of this strain is necessary.

Serum cytokine profiles in experimental human malaria. Relationship to protection and disease course after challenge.

Serum cytokine profiles were evaluated in immunized and nonimmunized human volunteers after challenge with infectious Plasmodium falciparum sporozoites. Three volunteers had been immunized with x-irradiated sporozoites and were fully protected from infection. Four nonimmune volunteers all developed symptomatic infection at which time they were treated. Sera from all volunteers were collected at approximately 20 time points during the 28-d challenge period; levels of IL-1 alpha, IL-1 beta, IL-2, IFN-gamma, tumor necrosis factor-alpha, IL-4, IL-6, granulocyte macrophage-colony-stimulating factor, and soluble CD4, CD8, and IL-2 receptor (sCD4, sCD8, and sIL-2R, respectively) were determined by ELISA. C-reactive protein (CRP) was assayed by radial immunodiffusion. Parasitemic subjects developed increases in CRP and IFN-gamma, with less marked increases in sIL-2R and sCD8; the other cytokines tested did not change. CRP increases were abrupt and occurred at the onset of fever (day 14 after challenge). IFN-gamma increases were also abrupt, preceding those of fever and CRP by one day. Increases in sIL-2R and sCD8 were more gradual. Increases in fever, CRP, IFN-gamma, and sCD8 were concordant in each volunteer. Early IL-6 increases were noted in the protected vaccinees. Thus, after challenge with virulent P. falciparum, unique systemic cytokine profiles were detectable both in immunized, nonparasitemic volunteers and in unvaccinated, parasitemic subjects. The contrasting cytokine profiles in the two groups may relate to mechanisms of protection and immunopathology in experimental human malaria.

Safety, infectivity, immunogenicity, and in vivo stability of two attenuated auxotrophic mutant strains of Salmonella typhi, 541Ty and 543Ty, as live oral vaccines in humans.

Two Salmonella typhi mutants, 541Ty (Vi+) and 543Ty (Vi-), auxotrophic for p-aminobenzoate and adenine, were evaluated as live oral vaccines. 33 volunteers ingested single doses of 10(8), 10(9), or 10(10) vaccine organisms, while four others received two 2 X 10(9) organism doses 4 d apart. No adverse reactions were observed. Vaccine was recovered from coprocultures of 29 of 37 vaccinees (78%) and from duodenal string cultures of two; repeated blood cultures were negative. The humoral antibody response to S. typhi O, H, Vi, and lysate antigens in serum and intestinal fluid was meager. In contrast, all vaccinees manifested cell-mediated immune responses. After vaccination, 69% of vaccinees overall and 89% of recipients of doses greater than or equal to 10(9) responded to S. typhi particulate or purified O polysaccharide antigens in lymphocyte replication studies but not to antigens of other Salmonella or Escherichia coli. All individuals, postvaccination, demonstrated a significant plasma-dependent mononuclear cell inhibition of wild S. typhi.

Role of the eaeA gene in experimental enteropathogenic Escherichia coli infection.

Enteropathogenic Escherichia coli (EPEC) infections are a leading cause of infant diarrhea in developing countries. Recently eaeA, a gene necessary for the characteristic intimate attachment of EPEC to epithelial cells in tissue culture, was described. We conducted a randomized, double-blind study to determine the role of the eaeA gene in human EPEC infection. 11 adult volunteers ingested 2 x 10(10) colony-forming units of O127:H6 EPEC strain E2348/69, and an equal number received the same dose of an isogenic eaeA deletion mutant constructed from E2348/69. Volunteers were monitored for the development of diarrhea, fever, and systemic and gastrointestinal complaints. Diarrhea developed in all 11 volunteers who received E2348/69 and in 4 of 11 who received the mutant (P = 0.002). Fever was more common in recipients of the wild-type strain (P = 0.024). Stool volumes were lower in recipients of the mutant. All volunteers seroconverted to E2348/69 LPS, but the geometric mean peak titers of serum IgG and IgA in recipients of the mutant were lower than those of recipients of the wild-type strain. IgA against LPS was detected in the jejunal fluid of six of six recipients of E2348/69 and 5/6 recipients of the mutant. This study unambiguously assigns a role for eaeA as an EPEC virulence gene, but the residual diarrhea seen in recipients of the mutant indicates that other factors are involved.

Can a 'flawless' live vector vaccine strain be engineered?

The efficiency of any live bacterial vector vaccine hinges on its ability to present sufficient foreign antigen to the human immune system to initiate the desired protective immune response(s). However, synthesis of sufficient levels of heterologous antigen can result in an increase in metabolic burden with an accompanying decrease in the fitness of the live vector, which can ultimately lower desired immune responses to both live vector and heterologous antigen. Here, we explore the underlying mechanisms of metabolic load and propose ways of minimizing such burdens to enhance the fitness and immunogenicity of Salmonella-based live vector vaccines.

Immunization with Ty21a live oral typhoid vaccine elicits crossreactive multifunctional CD8+ T-cell responses against Salmonella enterica serovar Typhi, S. Paratyphi A, and S. Paratyphi B in humans.

Previously we have extensively characterized Salmonella enterica serovar Typhi (S. Typhi)-specific cell-mediated immune (CMI) responses in volunteers orally immunized with the licensed Ty21a typhoid vaccine. In this study we measured Salmonella-specific multifunctional (MF) CD8+ T-cell responses to further investigate whether Ty21a elicits crossreactive CMI against S. Paratyphi A and S. Paratyphi B that also cause enteric fever. Ty21a-elicited crossreactive CMI responses against all three Salmonella serotypes were predominantly observed in CD8+ T effector/memory (T(EM)) and, to a lesser extent, in CD8+CD45RA+ T(EM) (T(EMRA)) subsets. These CD8+ T-cell responses were largely mediated by MF cells coproducing interferon-γ and macrophage inflammatory protein-1ß and expressing CD107a with or without tumor necrosis factor-α. Significant proportions of Salmonella-specific MF cells expressed the gut-homing molecule integrin α4ß7. In most subjects, similar MF responses were observed to S. Typhi and S. Paratyphi B, but not to S. Paratyphi A. These results suggest that Ty21a elicits MF CMI responses against Salmonella that could be critical in clearing the infection. Moreover, because S. Paratyphi A is a major public concern and Ty21a was shown in field studies not to afford cross-protection to S. Paratyphi A, these results will be important in developing a S. Typhi/S. Paratyphi A bivalent vaccine against enteric fevers.

Host-Salmonella interaction: human trials.

Human clinical trials, including experimental challenges of volunteers with pathogenic Salmonella enterica serovar Typhi, small phase I and II trials that monitor the immune responses to vaccines, and large-scale controlled field trials that assess vaccine efficacy under conditions of natural challenge, have helped elucidate the interactions between Salmonella typhi and human hosts.