Immunogenicity of a Salmonella typhimurium aroA aroD vaccine expressing a nontoxic domain of Clostridium difficile toxin A.
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The C-terminal repeat domain of Clostridium difficile toxin A harbors toxin-neutralizing epitopes and is considered to be a candidate component of a vaccine against C. difficile-associated disease (CDAD). Fourteen of the 38 C-terminal toxin A repeats (14CDTA) were cloned into pTECH-1 in frame with the immunogenic fragment C of tetanus toxin (TETC) to generate plasmid p56TETC. Expression of the TETC-14CDTA fusion protein was driven from the anaerobically inducible nirB promoter within attenuated Salmonella typhimurium BRD509 (aroA aroD). The TETC-14CDTA fusion protein was purified and shown to bind to known toxin A receptors found on the surface of rabbit erythrocytes. Intranasal (i.n.) and intragastric (i.g.) immunization with 10(7) and 10(10) CFU, respectively, of BRD509(p56TETC) generated significant (P < 0.05) anti-toxin A serum responses after a single dose. Antibody titers were elevated following a boosting dose with either live vaccine or a subcutaneous injection of 0.5 microgram of purified 14CDTA protein. Importantly, serum from mice immunized with BRD509(p56TETC) neutralized toxin A cytotoxicity. Both i.n. and i.g. immunizations also generated toxin A-specific immunoglobulin A on the pulmonary and intestinal mucosa, respectively. Intranasal vaccination induced consistently higher serum and mucosal anti-toxin A antibody responses. Significant anti-tetanus toxoid serum and mucosal antibodies were also generated by both immunization routes. The availability of live attenuated Salmonella typhi for human use may allow the development of a multivalent mucosal vaccine against CDAD, tetanus, and typhoid. (+info)
Acute systemic inflammation impairs endothelium-dependent dilatation in humans.
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BACKGROUND: We tested the hypothesis that endothelial dysfunction underlies the association between an acute inflammatory episode and the transiently increased risk of a cardiovascular event by examining the effects of an experimental inflammatory stimulus on endothelium-dependent vasodilation. METHODS AND RESULTS: Salmonella typhi vaccine was used to generate a systemic inflammatory response in healthy volunteers. In 12 subjects, dilatation of the brachial artery to flow and to sublingual nitroglycerin (NTG) was recorded (conduit vessel response), and in 6 subjects, venous occlusion plethysmography was used to measure forearm blood flow during intrabrachial infusion of the endothelium-dependent dilators acetylcholine (ACh) and bradykinin (BK) and the endothelium-independent dilators NTG and verapamil (resistance vessel response). Responses were assessed 16 hours before and 8 and 32 hours after vaccination. Vaccination resulted in elevations in white cell count and serum levels of interleukin-6 and interleukin-1 receptor antagonist. Eight hours after vaccination, resistance vessel responses to BK (P:=0.0099) and ACh (P:=0.0414) were markedly attenuated, and brachial artery flow-mediated dilatation was depressed. Resistance vessel responses to verapamil and NTG were unchanged, as was the conduit vessel response to NTG. Thirty-two hours after vaccination, resistance vessel responses to BK and ACh had returned to normal. CONCLUSIONS: S typhi vaccine generates a mild inflammatory reaction associated with temporary but profound dysfunction of the arterial endothelium in both resistance and conduit vessels to both physical and pharmacological dilator stimuli. This finding might explain the association between infection and inflammation and the enhanced risk of an acute cardiovascular event. (+info)
Protection against murine listeriosis by oral vaccination with recombinant Salmonella expressing hybrid Yersinia type III proteins.
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In the present study, we have investigated the possibility to engage the Yersinia outer protein E (YopE) as a carrier molecule for heterologous Ag delivery by the type III secretion system of Salmonella typhimurium. Defined secretion and translocation domains of YopE were fused to the immunodominant T cell Ags listeriolysin O and p60 of Listeria monocytogenes. In vitro experiments showed that S. typhimurium allows secretion and translocation of large hybrid YopE proteins in a type III-dependent fashion. Translocation and cytosolic delivery of these chimeric proteins into host cells, but not secretion into endosomal compartments, led to efficient MHC class I-restricted Ag presentation of listerial nonamer peptides. Mice orally vaccinated with a single dose of attenuated S. typhimurium expressing translocated hybrid YopE proteins revealed high numbers of IFN-gamma-producing cells reactive with listeriolysin O 91-99 or p60 217-225, respectively. This CD8 T cell response protected mice against a challenge with L. monocytogenes. In conclusion, these findings suggest that YopE is a versatile carrier molecule for type III-mediated foreign Ag delivery by Salmonella vaccine strains. (+info)
Impaired mucosal immunity in L-selectin-deficient mice orally immunized with a Salmonella vaccine vector.
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Lymphocyte trafficking in the gastrointestinal tract is primarily mediated by interactions with the mucosal addressin cell adhesion molecule 1 and its lymphocyte ligand, alpha(4)beta(7), and partly by L-selectin (L-Sel) interactions with peripheral node addressin coexpressed on some mucosal addressin cell adhesion molecule 1. We inquired whether intestinal responses in mice lacking L-Sel would be enhanced. L-Sel-deficient (L-Sel(-/-)) mice were orally immunized with either Salmonella vaccine vector or Salmonella vector-expressing colonization factor Ag I (CFA/I) from enterotoxigenic Escherichia coli. In L-Sel(-/-) mice, mucosal IgA anti-CFA/I fimbrial responses were greatly reduced, and systemic IgG2a anti-CFA/I fimbrial responses were 26-fold greater compared with C57BL/6 (L-Sel(+/+)) mice. L-Sel(-/-) Peyer's patch (PP) CD4(+) Th cells revealed IFN-gamma-dominated responses and an unprecedented absence of IL-4, whereas the expected mixed Th cell phenotype developed in L-Sel(+/+) mice. PP CD4(+) Th cell anti-Salmonella responses were nearly nonexistent in L-Sel(-/-) mice immunized with either Salmonella vaccine. Splenic CD4(+) Th cell anti-Salmonella responses were reduced but did show cytokine production in Ag restimulation assays. Increased colonization of PP and spleen was noted only with the Salmonella vector in L-Sel(-/-) mice, resulting in increased splenomegaly, suggesting that the Salmonella-CFA/I vaccine was not as infectious or that the presence of the fimbriae improved clearance, possibly because of reduced neutrophil recruitment. However, sufficient anti-Salmonella immunity was induced, because Salmonella vector-immunized L-Sel(-/-) mice showed complete protection against wild-type Salmonella challenge, unlike L-Sel(+/+) mice. This evidence shows that L-Sel is important for development of mucosal immunity, and absence of L-Sel is protective against salmonellosis. (+info)
Construction, genotypic and phenotypic characterization, and immunogenicity of attenuated DeltaguaBA Salmonella enterica serovar Typhi strain CVD 915.
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A promising live attenuated typhoid vaccine candidate strain for mucosal immunization was developed by introducing a deletion in the guaBA locus of pathogenic Salmonella enterica serovar Typhi strain Ty2. The resultant DeltaguaBA mutant, serovar Typhi CVD 915, has a gene encoding resistance to arsenite replacing the deleted sequence within guaBA, thereby providing a marker to readily identify the vaccine strain. CVD 915 was compared in in vitro and in vivo assays with wild-type strain Ty2, licensed live oral typhoid vaccine strain Ty21a, or attenuated serovar Typhi vaccine strain CVD 908-htrA (harboring mutations in aroC, aroD, and htrA). CVD 915 was less invasive than CVD 908-htrA in tissue culture and was more crippled in its ability to proliferate after invasion. In mice inoculated intraperitoneally with serovar Typhi and hog gastric mucin (to estimate the relative degree of attenuation), the 50% lethal dose of CVD 915 (7.7 x 10(7) CFU) was significantly higher than that of wild-type Ty2 (1.4 x 10(2) CFU) and was only slightly lower than that of Ty21a (1.9 x 10(8) CFU). Strong serum O and H antibody responses were recorded in mice inoculated intranasally with CVD 915, which were higher than those elicited by Ty21a and similar to those stimulated by CVD 908-htrA. CVD 915 also elicited potent proliferative responses in splenocytes from immunized mice stimulated with serovar Typhi antigens. Used as a live vector, CVD 915(pTETlpp) elicited high titers of serum immunoglobulin G anti-fragment C. These encouraging preclinical data pave the way for phase 1 clinical trials with CVD 915. (+info)
Host response to various treatments to reduce Salmonella infections in swine.
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Host response was evaluated following the administration of various treatments, such as probiotics, prebiotics, and vaccination, to reduce Salmonella in swine. Response to the treatments were studied by the evaluation of phagocytosis rates by flow cytometry, by studying the activation of whole-blood phagocytes by bioluminescence, the production of IgA against S. Typhimurium, and by histopathology. Significant differences were observed in the activation of whole-blood phagocytes in all groups of treated pigs (P = 0.0001). In SC54 vaccinated pigs, a significant reduction of Salmonella in the ileum was observed (P < 0.05) and the production of IgA against S. Typhimurium was higher in this group in comparison to uninfected control pigs (P = 0.0007). Furthermore, significant histopathological (P < 0.05) changes were observed in SC54 vaccinated pigs. Villus height and mucus and goblet cells density in the small intestine were reduced in vaccinated pigs in comparison to infected control pigs. Taken together, these findings suggest that SC54 vaccine can stimulate local immunity and reduce the presence of Salmonella in the ileum in swine. Use of SC54 vaccine should thus be considered in further field experiments. (+info)
Salmonella DNA adenine methylase mutants confer cross-protective immunity.
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Salmonella isolates that lack or overproduce DNA adenine methylase (Dam) elicited a cross-protective immune response to different Salmonella serovars. The protection afforded by the Salmonella enterica serovar Typhimurium Dam vaccine was greater than that elicited in mice that survived a virulent infection. S. enterica serovar Typhimurium Dam mutant strains exhibited enhanced sensitivity to mediators of innate immunity such as antimicrobial peptides, bile salts, and hydrogen peroxide. Also, S. enterica serovar Typhimurium Dam(-) vaccines were not immunosuppressive; unlike wild-type vaccines, they failed to induce increased nitric oxide levels and permitted a subsequent robust humoral response to diptheria toxoid antigen in infected mice. Dam mutant strains exhibited a low-grade persistence which, coupled with the nonimmunosuppression and the ectopic protein expression caused by altered levels of Dam, may provide an expanded source of potential antigens in vaccinated hosts. (+info)
Regulated antigen expression in live recombinant Salmonella enterica serovar Typhimurium strongly affects colonization capabilities and specific CD4(+)-T-cell responses.
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Regulated antigen expression can influence the immunogenicity of live recombinant Salmonella vaccines, but a rational optimization has remained difficult since important aspects of this effect are incompletely understood. Here, attenuated Salmonella enterica serovar Typhimurium SL3261 strains expressing the model antigen GFP_OVA were used to quantify in vivo antigen levels by flow cytometry and to simultaneously follow the crucial early steps of antigen-specific T-cell responses in mice that are transgenic for a T-cell receptor recognizing ovalbumin. Among seven tested promoters, P(pagC) has the highest activity in murine tissues combined with low in vitro expression, whereas P(tac) has a comparable in vivo and a very high in vitro activity. Both SL3261 (pP(pagC)GFP_OVA) and SL3261 (pP(tac)GFP_OVA) cells can induce potent ovalbumin-specific cellular immune responses following oral administration, but doses almost 1,000-fold lower are sufficient for the in vivo-inducible construct SL3261 (pP(pagC)GFP_OVA) compared to SL3261 (pP(tac)GFP_OVA). This efficacy difference is largely explained by impaired early colonization capabilities of SL3261 (pP(tac)GFP_OVA) cells. Based on the findings of this study, appropriate in vivo expression levels for any given antigen can be rationally selected from the increasing set of promoters with defined properties. This will allow the improvement of recombinant Salmonella vaccines against a wide range of pathogens. (+info)