7 research outputs found

    The B subunits of cholera and Escherichia coli heat-labile toxins enhance the immune responses in mice orally immunised with a recombinant live P-fimbrial vaccine for avian pathogenic E. coli

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    This study aimed to investigate the adjuvant effect of recombinant attenuated Salmonella expressing cholera toxin B subunit (CTB) and Escherichia coli heat-labile enterotoxin B subunit (LTB) for the P-fimbriae subunit-based vaccine of avian pathogenic E. coli (APEC) in a murine model. The PapA-specific sIgA and IgG responses were significantly enhanced after immunisation with the Salmonella-PapA vaccine in the presence of CTB or LTB. The group immunised with the Salmonella-LTB strain promoted Th1-type immunity, whereas that immunised with the Salmonella-CTB strain produced Th2-type immunity. We concluded that both Salmonella-CTB and -LTB strains can enhance the immune response to PapA, and that the LTB strain may be a more effective adjuvant for APEC vaccination, which requires higher Th1-type immunity for protection. Thus, our findings provide evidence that immunisation with an adjuvant, LTB, is one of the strategies of developing effective vaccines against P-fimbriated APEC

    Vector-primed mice display hypo-responsiveness to foreign antigen presented by recombinant Salmonella regardless of the route of delivery

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    C1 - Journal Articles RefereedOur previous studies have shown that mice which have been orally primed with an attenuated Salmonella vector [S. enterica serovar Stanley] are hypo-responsive to foreign antigens later delivered orally by the same vector strain, responding with significantly impaired serum and intestinal antibody responses compared with those seen in unprimed controls. Initial vector priming of the gut-associated lymphoid tissue (GALT) is likely to result in impaired persistence of recombinant Salmonella later administered orally. Delivery of recombinant bacteria by the intra-peritoneal or intra-nasal route, to avoid exposure to a primed GALT, did not allow vector-primed recipients to mount normal antibody responses to the foreign pilus protein K88. The negative impact of vector priming could be largely overcome, however, if mice were exposed to the foreign protein just prior to priming with the vector strain. Using this strategy, vector-primed mice displayed normal gut IgA and intermediate serum IgG responses to K88 following oral administration of recombinant Salmonella. Our findings are compatible with the concept of epitopic suppression, in which failure to respond to the foreign vaccine antigen reflects the clonal dominance of B cells specific for epitopes associated with the vector strain

    Molecular and genetic characterization of the capsule biosynthesis locus of Streptococcus pneumoniae type 23F

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    The authors have previously reported the nucleotide sequence of the 5' and 3' portions of the Streptococcus pneumoniae type 23F capsular polysaccharide biosynthesis locus (cps23f) (from dexB to cps23fB and from cps23fL to aliA). These regions of cps23f were very similar to the sequence reported for cps19f, the capsule locus of S. pneumoniae type 19F. However, Southern hybridization analysis indicated that no other genes closely related to cps19f are present in the cps23f locus. In this study long-range PCR was used to amplify and clone the section of the S. pneumoniae type 23F capsule locus between cps23fB and cps23fL. This region is 13 kb in size and contains 12 new ORFs, designated cps23fC-E, I, J, and T-Z. Functions are proposed for all of the protein products, including functional homologues of Cps19fC-E, Cps19fI and Cps19fJ. A biosynthetic pathway for type 23F capsular polysaccharide is also proposed

    Reactive oxygen species are the major antibacterials against Salmonella Typhimurium purine auxotrophs in the phagosome of RAW 264.7 cells

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    Intramacrophage survival appears to be a pathogenic trait common to Salmonellae and definition of the metabolic requirements of Salmonella within macrophages might provide opportunities for novel therapeutic interventions. We show that loss of PurG function in Salmonella enterica serovar Typhimurium SL1344 leads to death of the bacterium in RAW264.7 cells, which was due to unavailability of purine nucleotides but not thiamine in the phagosome of RAW264.7 cells. Phagosomal escape of purG mutant restored growth, suggesting that the phagosomal environment, but not the cytosol, is toxic to Salmonella purine auxotrophs. NADPH oxidase inhibition restored the growth of purG mutant in RAW264.7 cells, implying that the Salmonella-containing vacuole acquires reactive oxygen species (ROS) that are lethal to purine auxotrophs. Under purine limiting conditions, purG mutant was unable to repair the damage caused by hydrogen peroxide or UV irradiation, suggesting that ROS-mediated DNA damage may have been responsible for the attenuated phenotype of purG mutant in RAW264.7 cells and in mice

    Overexpression and topology of the Shigella flexneri O-antigen polymerase (Rfc/Wzy).

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    Lipopolysaccharides (LPS), particularly the O-antigen component, are one of many virulence determinants necessary for Shigella flexneri pathogenesis. O-antigen biosynthesis is determined mostly by genes located in the rfb region of the chromosome. The rfc/wzy gene encodes the O-antigen polymerase, an integral membrane protein, which polymerizes the O-antigen repeat units of the LPS. The wild-type rfc/wzy gene has no detectable ribosome-binding site (RBS) and four rare codons in the translation initiation region (TIR). Site-directed mutagenesis of the rare codons at positions 4, 9 and 23 to those corresponding to more abundant tRNAs and introduction of a RBS allowed detection of the rfc/wzy gene product via a T7 promoter/polymerase expression assay. Complementation studies using the rfc/wzy constructs allowed visualization of a novel LPS with unregulated O-antigen chain length distribution, and a modal chain length could be restored by supplying the gene for the O-antigen chain length regulator (Rol/Wzz) on a low-copy-number plasmid. This suggests that the O-antigen chain length distribution is determined by both Rfc/Wzy and Rol/Wzz proteins. The effect on translation of mutating the rare codons was determined using an Rfc::PhoA fusion protein as a reporter. Alkaline phosphatase enzyme assays showed an approximately twofold increase in expression when three of the rare codons were mutated. Analysis of the Rfc/Wzy amino acid sequence using TM-PREDICT indicated that Rfc/Wzy had 10-13 transmembrane segments. The computer prediction models were tested by genetically fusing C-terminal deletions of Rfc/Wzy to alkaline phosphatase and beta-galactosidase. Rfc::PhoA fusion proteins near the amino-terminal end were detected by Coomassie blue staining and Western blotting using anti-PhoA serum. The enzyme activities of cells with the rfc/wzy fusions and the location of the fusions in rfc/wzy indicated that Rfc/Wzy has 12 transmembrane segments with two large periplasmic domains, and that the amino- and carboxy-termini are located on the cytoplasmic face of the membrane

    Evaluation of Mucosal and Systemic Immune Responses Elicited by GPI-0100-Adjuvanted Influenza Vaccine Delivered by Different Immunization Strategies

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    <p>Vaccines for protection against respiratory infections should optimally induce a mucosal immune response in the respiratory tract in addition to a systemic immune response. However, current parenteral immunization modalities generally fail to induce mucosal immunity, while mucosal vaccine delivery often results in poor systemic immunity. In order to find an immunization strategy which satisfies the need for induction of both mucosal and systemic immunity, we compared local and systemic immune responses elicited by two mucosal immunizations, given either by the intranasal (IN) or the intrapulmonary (IPL) route, with responses elicited by a mucosal prime followed by a systemic boost immunization. The study was conducted in BALB/c mice and the vaccine formulation was an influenza subunit vaccine supplemented with GPI-0100, a saponin-derived adjuvant. While optimal mucosal antibody titers were obtained after two intrapulmonary vaccinations, optimal systemic antibody responses were achieved by intranasal prime followed by intramuscular boost. The latter strategy also resulted in the best T cell response, yet, it was ineffective in inducing nose or lung IgA. Successful induction of secretory IgA, IgG and T cell responses was only achieved with prime-boost strategies involving intrapulmonary immunization and was optimal when both immunizations were given via the intrapulmonary route. Our results underline that immunization via the lungs is particularly effective for priming as well as boosting of local and systemic immune responses.</p>
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