Microbiology & Immunology - Research Publications
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ItemA Randomised Trial Evaluating the Safety and Immunogenicity of the Novel Single Oral Dose Typhoid Vaccine M01ZH09 in Healthy Vietnamese Children(PUBLIC LIBRARY SCIENCE, 2010-07-26)BACKGROUND: The emergence of drug resistant typhoid fever is a major public health problem, especially in Asia. An oral single dose typhoid vaccine would have major advantages. M01ZH09 is a live oral single dose candidate typhoid vaccine containing Salmonella enterica serovar Typhi (Ty2 aroC(-)ssaV(-)) ZH9 with two independently attenuating deletions. Studies in healthy adults demonstrated immunogenicity and an acceptable safety profile. OBJECTIVES: We conducted a randomised placebo controlled, single-blind trial to evaluate the safety and immunogenicity of M01ZH09 in healthy Vietnamese children aged 5 to 14 years. METHODS: Subjects were randomly assigned to receive either a nominal dose of 5x10(9) CFU of M01ZH09 or placebo and were followed up for 28 days. The primary safety outcome was the proportion of subjects with any adverse event attributed to M01ZH09. The primary immunogenicity endpoint was the proportion of subjects who showed a positive immune response to M01ZH09 in the Salmonella Typhi lipopolysaccharide (LPS) specific serum IgA and IgG ELISA. PRINCIPAL FINDINGS: One hundred and fifty-one children were enrolled, 101 subjects received M01ZH09 and 50 subjects received placebo. An intention to treat analysis was conducted. There were no serious adverse events and no bacteraemias. In the M01ZH09 group, 26 (26%; 95% CI, 18-5%) of 101 subjects experienced adverse events compared to 11 (22%; 95% CI, 12-36%) of 50 subjects in the placebo group (odds ratio (OR) [95%CI] = 1.23 [0.550-2.747]; p = 0.691). Faecal shedding of S. Typhi (Ty2 aroC(-)ssaV(-)) ZH9 was detected in 51 (51%; 95% CI, 41-61%) of 100 M01ZH09 subjects. No shedding was detected beyond day 3. A positive immune response, defined as 70% increase (1.7 fold change) in LPS specific serum IgG (day 14 or 28) and/or 50% increase (1.5 fold change) in LPS specific serum IgA (day 7 or 14) from baseline was detected in 98 (97%; 95% CI, 92-99%) of 101 M01ZH09 recipients and 8 (16%; 95% CI, 7-29%) of 50 placebo recipients. Twenty-eight (100%; 95% CI, 88-100%) of 28 vaccine recipients who were evaluated in the LPS specific IgA ELISPOT assay showed a positive response compared to none of the 14 placebo recipients tested. CONCLUSIONS: This was the first phase II trial of a novel oral candidate typhoid vaccine in children in an endemic country. M01ZH09 had an appropriate safety profile and was immunogenic in children. TRIAL REGISTRATION: Controlled-trials.com ISRCTN91111837.
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ItemModulation of dendritic cell endocytosis and antigen processing pathways by Escherichia coli heat-labile enterotoxin and mutant derivatives(ELSEVIER SCI LTD, 2003-03-28)Escherichia coli heat-labile enterotoxin (LT) is known to be a potent adjuvant of both the mucosal and systemic immune systems but the mechanism of action leading to adjuvant activity remains incompletely understood. This study investigates the action of LT and LT mutants with impaired enzymatic activity, on the function of dendritic cells. Wild-type LT and LTR72, which retains some ADP ribosyltransferase activity, induced a selective increase in cell surface expression of B7.1, and a selective decrease of CD40 expression on mouse bone marrow derived dendritic cells. LTK63 and LT-B had no obvious effect on the expression of these antigens on similar dendritic cells. LT-treated dendritic cells also showed a profoundly impaired ability to present protein antigen (ovalbumin) to cognate T cells, although this effect was not observed with non-toxic LT mutants. LT and LTR72-treated cells showed a slower rate of receptor-mediated endocytosis as measured by flow cytometric analysis of uptake of fluorescently labelled dextran. Furthermore, confocal microscopy showed changes in the intracellular distribution of endocytosed molecules, and of the class II containing acidic antigen processing compartments. This response of dendritic cells to toxin is likely to play an important role in determining the adjuvant activity of these molecules.
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ItemIdentification of a novel type IV pilus gene cluster required for gastrointestinal colonization of Citrobacter rodentium(WILEY, 2003-05)Citrobacter rodentium is used as an in vivo model system for clinically significant enteric pathogens such as enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). These pathogens all colonize the lumen side of the host gastrointestinal tract via attaching and effacing (A/E) lesion formation. In order to identify genes required for the colonization of A/E-forming pathogens, a library of signature-tagged transposon mutants of C. rodentium was constructed and screened in mice. Of the 576 mutants tested, 14 were attenuated in their ability to colonize the descending colon. Of these, eight mapped to the locus of enterocyte effacement (LEE), which is required for the formation of A/E lesions, underlying the importance of this mechanism for pathogenesis. Another mutant, P5H2, was found to have a transposon insertion in an open reading frame that has strong similarity to type IV pilus nucleotide-binding proteins. The region flanking the transposon insertion was sequenced, identifying a cluster of 12 genes that encode the first described pilus of C. rodentium (named colonization factor Citrobacter, CFC). The proteins encoded by cfc genes have identity to proteins of the type IV COF pilus of enterotoxigenic E. coli (ETEC), the toxin co-regulated pilus of Vibrio cholerae and the bundle-forming pilus of EPEC. A non-polar mutation in cfcI, complementation of this strain with wild-type cfcI and complementation of strain P5H2 with wild-type cfcH confirmed that these genes are required for colonization of the gastrointestinal tract by C. rodentium. Thus, CFC provides a convenient model to study type IV pilus-mediated pathogen-host interactions under physiological conditions in the natural colonic environment.
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ItemIntracellular adhesion molecule 1 plays a key role in acquired immunity to Salmonellosis(AMER SOC MICROBIOLOGY, 2003-10)This study investigated the role of intracellular adhesion molecule 1 (ICAM-1) during Salmonella enterica serovar Typhimurium infection of mice. We show that ICAM-1 is expressed in and around granulomas on day 4 of infection in wild-type mice. However, when naive ICAM-1(-/-) mice were challenged with a sublethal dose of serovar Typhimurium, there were no detectable differences in systemic bacterial burden over the first 9 days of infection compared to wild-type control mice. When mice were immunized with the S. enterica serovar Typhimurium vaccine strain SL2361 and then challenged with the virulent S. enterica serovar Typhimurium strain C5, 100% of the ICAM-1(-/-) mice succumbed to infection, compared to 30% of wild-type mice. T-cell responses, as measured by activation via interleukin-2 production, as well as antibody responses were comparable in the ICAM-1(-/-) and wild-type mice. Following challenge, counts in organs were significantly higher in the ICAM-1(-/-) mice, and histological examination of organs showed pathological differences. Strain SL3261-immunized wild-type mice had cellular infiltrate and normal granuloma formation in the liver and spleen on days 5 and 10 after challenge with strain C5. ICAM-1(-/-) mice had a similar infiltrate on day 5, whereas on day 10 the infiltrate was more widespread and there were fewer macrophages associated with the granulomas. High circulating levels of tumor necrosis factor alpha and gamma interferon, as well as a high burden of strain C5 in the blood, accompanied the differences in histopathology. In this study we show that ICAM-1 plays a critical role during rechallenge of immunized mice with virulent S. enterica serovar Typhimurium.
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ItemSalmonella enterica serovar Typhimurium interaction with dendritic cells:: impact of the sifA gene(BLACKWELL PUBLISHING LTD, 2004-11)Salmonella enterica serovar Typhimurium (S. Typhimurium) and several mutant derivatives were able to enter efficiently murine bone marrow-derived dendritic cells using mechanisms predominantly independent of the Salmonella pathogenicity island 1 type III secretion system. The levels of intracellular bacteria did not increase significantly over many hours after invasion. Using fluid endocytic tracers and other markers, S. Typhimurium-containing vacuoles (SCVs) were physically distinguishable from early endocytic compartments. Fifty to eighty per cent of SCVs harbouring wild-type S. Typhimurium or aroA, invH and ssaV mutant derivatives were associated with late endosome markers. In contrast, S. Typhimurium sifA was shown to escape the SCVs into the cytosol of infected dendritic cells. S. Typhimurium aroC sifA was more efficient than S. Typhimurium aroC in delivering a eukaryotic promoter-driven green fluorescent protein reporter gene for expression in dendritic cells. In contrast, S. Typhimurium aroC sifA did not detectably increase the efficiency of MHC class I presentation of the model antigen ovalbumin to T cells compared to a similar aroC derivative. Mice infected with the S. Typhimurium aroC sifA expressing ovalbumin did not develop detectably enhanced levels of cytotoxic T cell or interferon-gamma production compared to S. Typhimurium aroC derivatives.
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ItemUnderstanding mucosal responsiveness: lessons from enteric bacterial pathogens(ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD, 2001-06)Mucosal immune responses must discriminate between commensal flora within the lumen and potential pathogens. These responses are highly adapted to induce protection without excessive inflammation. The balances that regulate mucosal immune and inflammatory responses have to be understood if effective mucosal immunity is to be induced through local immunization. This review will summarize some of the lessons learnt from studies of antigens derived from enteric bacterial pathogens and discuss how the gastrointestinal epithelia can 'fight back' when it encounters pathogens.
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ItemIntimin-specific immune responses prevent bacterial colonization by the attaching-effacing pathogen Citrobacter rodentium(AMER SOC MICROBIOLOGY, 2001-09)The formation of attaching and effacing (A/E) lesions on gut enterocytes is central to the pathogenesis of enterohemorrhagic (EHEC) Escherichia coli, enteropathogenic E. coli (EPEC), and the rodent pathogen Citrobacter rodentium. Genes encoding A/E lesion formation map to a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Here we show that the LEE-encoded proteins EspA, EspB, Tir, and intimin are the targets of long-lived humoral immune responses in C. rodentium-infected mice. Mice infected with C. rodentium developed robust acquired immunity and were resistant to reinfection with wild-type C. rodentium or a C. rodentium derivative, DBS255(pCVD438), which expressed intimin derived from EPEC strain E2348/69. The receptor-binding domain of intimin polypeptides is located within the carboxy-terminal 280 amino acids (Int280). Mucosal and systemic vaccination regimens using enterotoxin-based adjuvants were employed to elicit immune responses to recombinant Int280alpha from EPEC strain E2348/69. Mice vaccinated subcutaneously with Int280alpha, in the absence of adjuvant, were significantly more resistant to oral challenge with DBS255(pCVD438) but not with wild-type C. rodentium. This type-specific immunity could not be overcome by employing an exposed, highly conserved domain of intimin (Int388-667) as a vaccine. These results show that anti-intimin immune responses can modulate the outcome of a C. rodentium infection and support the use of intimin as a component of a type-specific EPEC or EHEC vaccine.
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ItemSite-directed mutagenesis of intimin α modulates intimin-mediated tissue tropism and host specificity(WILEY, 2001-04)The hallmark of enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherchia coli adhesion to host cells is intimate attachment leading to the formation of distinctive 'attaching and effacing' lesions. This event is mediated, in part, by binding of the bacterial adhesion molecule intimin to a second bacterial protein, Tir, delivered by a type III secretion system into the host cell plasma membrane. The receptor-binding activity of intimin is localized to the C-terminal 280 amino acids (Int280) and at least five distinct intimin types (alpha, beta, gamma, delta and epsilon) have been identified thus far. In addition to binding to Tir, intimin can also bind to a component encoded by the host. The consequence of latter intimin-binding activity may determine tissue tropism and host specificity. In this study we selected three amino acids in intimin, which are implicated in Tir binding, for site-directed mutagenesis. We used the yeast two-hybrid system and gel overlays to study intimin-Tir protein interaction. In addition, the biological consequences of the mutagenesis was tested using a number of infection models (cultured epithelial cells, human intestinal explants and a mouse model). We report that while an I237/897A substitution (positions numbered according to Int280alpha/whole intimin alpha) in intimin alpha did not have any affect on its biological activity, a T255/914A substitution attenuated intimin activity in vivo. In contrast, the mutation V252/911A affected tissue targeting in the human intestinal explant model and attenuated the biological activity of intimin in the mouse model. This study provides the first clues of the molecular basis of how intimin mediates tissue tropism and host specificity.
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ItemImpaired resistance and enhanced pathology during infection with a noninvasive, attaching-effacing enteric bacterial pathogen, Citrobacter rodentium, in mice lacking IL-12 or IFN-γ(AMER ASSOC IMMUNOLOGISTS, 2002-02-15)Mice infected with Citrobacter rodentium represent an excellent model in which to examine immune defenses against an attaching-effacing enteric bacterial pathogen. Colonic tissue from mice infected with C. rodentium harbors increased transcripts for IL-12 and IFN-gamma and displays mucosal pathology compared with uninfected controls. In this study, the role of IL-12 and IFN-gamma in host defense and mucosal injury during C. rodentium infection was examined using gene knockout mice. IL-12p40(-/-) and IFN-gamma(-/-) mice were significantly more susceptible to mucosal and gut-derived systemic C. rodentium infection. In particular, a proportion of IL-12p40(-/-) mice died during infection. Analysis of the gut mucosa of IL-12p40(-/-) mice revealed an influx of CD4(+) T cells and a local IFN-gamma response. Infected IL-12p40(-/-) and IFN-gamma(-/-) mice also mounted anti-Citrobacter serum and gut-associated IgA responses and strongly expressed inducible NO synthase (iNOS) in mucosal tissue, despite diminished serum nitrite/nitrate levels. However, iNOS does not detectably contribute to host defense against C. rodentium, as iNOS(-/-) mice were not more susceptible to infection. However, C57BL/6 mice infected with C. rodentium up-regulated expression of the mouse beta-defensin (mBD)-1 and mBD-3 in colonic tissue. In contrast, expression of mBD-3, but not mBD-1, was significantly attenuated during infection of IL-12- and IFN-gamma-deficient mice, suggesting mBD-3 may contribute to host defense. These studies are among the first to examine mechanisms of host resistance to an attaching-effacing pathogen and show an important role for IL-12 and IFN-gamma in limiting bacterial infection of the colonic epithelium.
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ItemMutagenesis of conserved tryptophan residues within the receptor-binding domain of intimin: influence on binding activity and virulence(MICROBIOLOGY SOC, 2002-03)Intimate bacterial adhesion to intestinal epithelium is a pathogenic mechanism shared by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli and Citrobacter rodentium. The proteins directly involved in this process are the outer-membrane adhesion molecule intimin and the translocated intimin receptor, Tir. The receptor-binding activity of intimin resides within the carboxy terminus 280 aa (Int280) of the polypeptide. Four tryptophan residues, W117/776, W136/795, W222/881 and W240/899, are conserved within different Int280 molecules that otherwise show considerable sequence variation. In this study the influence of site-directed mutagenesis of each of the four tryptophan residues on intimin-Tir interactions and on intimin-mediated intimate attachment was determined. The mutant intimins were also studied using a variety of in vitro and in vivo infection models. The results show that all the substitutions modulated intimin activity, although some mutations had more profound effects than others.