22 research outputs found

    Immunization with the <i>FTL_0325</i> mutant induces an early pro-inflammatory cytokine response in lungs.

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    <p>BALB/c mice (n = 4) were infected with 1×10<sup>7</sup> CFU of the indicated mutants and wild type <i>F. tularensis</i> LVS. At the indicated times, the levels of pro-inflammatory cytokines were measured in lung homogenates using a Cytometric Bead Array assay. The data are representative of two independent experiments conducted and were analyzed using ANOVA with Tukey-Kramer Multiple Comparison post-test and <i>P</i> values were recorded. <i>**P</i><0.01; ***<i>P<</i>0.001. Ψ = Mice infected with 1×10<sup>7</sup> CFU of <i>F. tularensis</i> LVS succumbed to infection by day 7 PI and hence were unavailable for comparison.</p

    <i>FTL_0304</i>, <i>FTL_0291</i>, <i>FTL_0325</i> and <i>FTL_0057</i> mutants are highly attenuated for virulence in BALB/c and C57BL/6 mice.

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    a<p>6–8 weeks old mice were infected i.n. with either <i>F. tularensis</i> LVS or the indicated mutants and monitored for mortality for 28 days. Data are shown as number of mice survived/total number of mice infected.</p

    Immunization with the <i>FTL_0325</i> mutant results in a potent memory recall response.

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    <p>Cell culture supernatants collected from BMDMs infected with either the <i>F. tularensis</i> LVS (A–D) or <i>F. tularensis</i> SchuS4 (E and F) and co-cultured with the splenocytes prepared from immunized mice at day 45 post-immunization (A and C) or at day 60 (B–F) were analyzed for IFN-γ (A, B and E) and IL-17a (C,D and F) cytokines by Cytometric Bead Flex sets. The data are representative of two independent experiments and were analyzed using ANOVA with Tukey-Kramer Multiple Comparison post-test and <i>P</i> values recorded. *<i>P<</i>0.05<i>; **P</i><0.01; ***<i>P<</i>0.001.</p

    Vaccine Formulations.

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    <p>Two different vaccine formulations were used. In the first vaccine formulation all three recombinant proteins OmpA, DnaK and Tul4 were conjugated to a single TMV virion (TMV-monoconjugate vaccine). The second vaccine formulation contained each recombinant protein of <i>F</i>. <i>tularensis</i> conjugated individually to TMV and then mixed in equal concentrations to generate a TMV-multiconjugate vaccine.</p

    Immunization with the <i>FTL_0325</i> mutant induces a higher pro-inflammatory cytokine response in the spleen.

    No full text
    <p>BALB/c mice (n = 4) were infected with 1×10<sup>7</sup> CFU of the indicated mutants and wild type <i>F. tularensis</i> LVS. At the indicated times, the levels of pro-inflammatory cytokines were measured in spleen homogenates using a Cytometric Bead Array assay. The data are representative of two independent experiments conducted and were analyzed using ANOVA with Tukey-Kramer Multiple Comparison post-test and <i>P</i> values were recorded. *<i>P<</i>0.05<i>; **P</i><0.01; ***<i>P<</i>0.001. Ψ = Mice infected with 1×10<sup>7</sup> CFU of <i>F. tularensis</i> LVS succumbed to infection by day 7 PI and hence were unavailable for comparison.</p

    Immunization with the <i>FTL_0325</i> mutant induces an early inflammatory response.

    No full text
    <p>BALB/c mice were infected with 1×10<sup>7</sup> CFU of the <i>F. tularensis</i> LVS, <i>FTL_0325</i> or <i>FTL_0304</i> mutant and the lungs were harvested at the indicated times, sectioned and stained with H & E. Representatives of H & E stained lung sections are shown. Infiltration of neutrophils is shown in the inset (Magnification 10×; Inset 100×). Ψ = Mice infected with 1×10<sup>7</sup> CFU of <i>F. tularensis</i> LVS succumbed to infection by day 7 PI and hence were unavailable for comparison. Green arrows indicate the sites of cellular infiltration. Yellow circles in inset show neutrophilic infiltration in the lungs of <i>FTL_0325</i> mutant immunized mice.</p

    The <i>FTL_0325</i> and <i>FTL_0304</i> mutants of <i>F. tularensis</i> LVS exhibit no growth defect under acellular growth conditions.

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    <p>Growth curves for bacteria grown in MHB were generated in a 96-well plate using 200 µl culture volumes while the growth curves for bacteria grown in BHI and CDM were generated using a 25 ml culture volume. Results shown are representative of two independent experiments.</p

    OmpA, DnaK and Tul4 Specific Antibody Responses in Mice Immunized with TMV-Multiconjugate Vaccines using Schedule I and II of Immunization.

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    <p><i>F</i>. <i>tularensis</i> SchuS4 recombinant proteins OmpA, DnaK and Tul4 specific IgG, antibody levels on day 28 in serum samples of C57BL/6 mice immunized with TMV-multiconjugate vaccine using Schedule II were determined by ELISA. The plates were coated with recombinant <i>F</i>. <i>tularensis</i> SchuS4 proteins. Serum samples obtained from naïve mice or those inoculated with TMV alone were used as controls. The data are represented as Mean ±S.D. of absorbance values measured at 450nm. The comparisons are shown with the data obtained from mice immunized with TMV-multiconjugate vaccine using schedule I (shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0130858#pone.0130858.g007" target="_blank">Fig 7</a>). Table shows comparison of antibody titers between groups of mice vaccinated with Schedule I and II vaccination regimens.</p

    Conjugation of DnaK, OmpA and Tul4 Proteins of <i>F</i>. <i>tularensis</i> SchuS4 to TMV.

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    <p>Purified OmpA, DnaK and Tul4 proteins were combined with purified TMV and incubated with EDC and NHS for 0, 30 min, 1, or 2 hours as described in Methods section. Two μg of TMV or recombinant proteins DnaK, OmpA, Tul4 or 4 μg of the TMV-protein mixtures were resolved on an 8–16% SDS-PAGE gel to observe conjugation products indicated by changes in the molecular masses of the starting materials. <b>(A)</b> Conjugation of DnaK, OmpA and Tul4 to a single TMV virion to generate TMV-monoconjugate vaccine. The progress of conjugation process was observed over a period of time: Lane M = Precision Plus Dual Color standard (BioRad) Marker; Lane 1 = TMV-protein mix, 0 min; Lane 2 = TMV-protein mix, 30 min; Lane 3 = TMV-protein mix,1 hour; Lane 4 = TMV-protein mix, 2 hours. <b>(B, C, D)</b> Kinetics of DnaK, OmpA and Tul4 TMV-protein conjugations over a two hour incubation period to generate TMV-protein conjugates. The individual TMV-protein conjugates were then admixed to generate TMV-multiconjugate vaccine. Lane M = Precision Plus Dual Color standard (BioRad) Marker; Lane 1 = TMV; Lane 2 = Recombinant protein; Lane 3 = TMV-protein mix, 0 hour; Lane 4 = TMV-protein mix, 1 hour; Lane 5 = TMV-protein mix, 2 hours. In all cases, 2 hour time points were used for scale-up and vaccine preparation. Solid arrows indicate TMV-protein conjugate(s), dashed arrows indicate free TMV or free proteins.</p
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