14 research outputs found

    Expression and Purification of Recombinant DnaK, OmpA and Tul4 Proteins of <i>F</i>. <i>tularensis</i> SchuS4.

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    <p>Purification of recombinant OmpA, DnaK and Tul4 proteins of <i>F</i>. <i>tularensis</i> SchuS4 proteins was confirmed by SDS-PAGE and western blot analysis using anti-His antibodies.</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

    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

    OmpA, DnaK and Tul4 Specific Antibody Responses in Mice Immunized with TMV-Monoconjugate and TMV-Multiconjugate Vaccines using Schedule I 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-monoconjugate and TMV-multiconjugate vaccine using Schedule I were determined by ELISA. 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. Table shows comparison of antibody titers between groups of mice vaccinated with these vaccine formulations.</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

    Protective Efficacy of TMV-Conjugate Vaccine.

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    <p><b>(A)</b> C57BL/6 mice (N = 8 per group) immunized with TMV-monoconjugate vaccine; <b>(C)</b> with TMV-multiconjugate vaccine (schedule I) or <b>(E)</b> with TMV-multiconjugate vaccine (Schedule II) were challenged i.n. with 10xLD<sub>100</sub> of <i>F</i>. <i>tularensis</i> LVS on day 28 post-immunization. Mice vaccinated with TMV alone were used as controls. Challenged mice were observed for morbidity and mortality for a period of 21 days post-challenge. The survival results are expressed as Kaplan-Meier survival curves and statistical analysis was performed using Log-rank test. <b>(B, D and F)</b> Body weight of the challenged mice at the indicate time points. The data are represented as Mean ± S.D.</p

    The <i>emrA1</i> mutant vaccinated mice induce sustained production of pro-inflammatory cytokines and a potent antibody response following lethal <i>Ft</i> LVS challenge.

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    <p>C57BL/6 mice immunized i.n. with 1×10<sup>6</sup> CFU of the <i>emrA1</i> mutant were challenged i.n. with 1×10<sup>7</sup> CFU of wild type <i>Ft</i> LVS 42 days post-immunization. <b>(A-D)</b> On days 5, 7 and 14 post-challenge, mice (n = 3 per group/time point) were euthanized and their excised lungs were homogenized. Clear lung homogenates were used for quantification of indicated pro-inflammatory cytokines using flow cytometric analysis. The data are represented as Mean ± S.D. <b>(E)</b> On day 21 post-challenge, mice (n = 3 per group) were anesthetized and bled retroorbitally to obtain serum. <i>Ft</i> specific total IgG, IgG2a, IgG2b, IgG1 and IgA levels in serum samples were determined by ELISA. The data are represented as Mean ± S.D. of absorbance values measured at 450 nm. Red arrows indicate antibody titers. ND = Not detected.</p

    Immunization with the <i>emrA1</i> mutant results in minimal weight loss, rapid bacterial clearance, and histopathological lesions in lung, liver and spleen.

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    <p>C57BL/6 mice were immunized i.n. with 1×10<sup>6</sup> CFU of the <i>emrA1</i> mutant. Mice infected with equal numbers of wild type <i>Ft</i> LVS were used as controls. <b>(A)</b> The immunized mice were weighed at the indicated times post-immunization to track the progress of infection. <b>(B)</b> On days 1, 5, 7, 14 and 21 post-immunization, mice (n = 4 per group/time point) were euthanized and bacterial burdens were quantified in their lung, liver and spleen. Bacterial counts in organs are expressed as Log<sub>10</sub>CFU/mL. The <i>P</i> values were determined using one way ANOVA. *<i>P<0</i>.<i>05; **P<0</i>.<i>01; ***P<0</i>.<i>001</i>. <b>(C)</b> Excised lungs, livers and spleens were preserved in 10% formalin, paraffin embedded, sliced into 5 μM thin sections and stained with Hematoxylene & Eosin. Stained sections were observed for histopathological lesions under a light microscope (Magnification 100×). # = <i>Ft</i> LVS infected mice succumbed to infection.</p

    Mice immunized with the <i>emrA1</i> mutant are protected against 1000LD100–10,000LD100 challenge dose of <i>Ft</i> LVS.

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    <p>C57BL/6 mice (n = 5–10 per group) were immunized i.n. with 1×10<sup>6</sup> CFU of the <i>emrA1</i> mutant. <b>(A, B)</b> On day 42 of the primary immunization mice were challenged i.n. with 1×10<sup>7</sup> CFU of wild type <i>Ft</i> LVS. Age matched unvaccinated mice challenged with a similar dose of <i>Ft</i> LVS were kept as controls. (<b>C, D)</b> On day 42 of the primary immunization mice were challenged i.n. with 1×10<sup>8</sup> CFU of wild type <i>Ft</i> LVS. Age matched unvaccinated mice challenged with a similar dose of <i>Ft</i> LVS were kept as controls. (<b>E, F)</b> On day 75 of the primary immunization mice were challenged i.n. with 1×10<sup>7</sup> CFU of wild type <i>Ft</i> LVS. Age matched unvaccinated mice challenged with a similar dose of <i>Ft</i> LVS were kept as controls. The Challenged mice were observed for morbidity and mortality for a period of 21 days post-challenge (<b>A, C, E</b>). The mice were weighed at the indicated times post-challenge to monitor the progression of infection (<b>B, D, F</b>). The survival results are expressed as Kaplan-Meier survival curves and P values were determined by Log-rank test. Body weights of mice are expressed percent body weights.</p

    Immunization with <i>emrA1</i> mutant using a prime-boost vaccination regimen improves the extent of protection against <i>Ft</i> SchuS4 challenge.

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    <p><b>(A)</b> C57BL/6 mice (n = 10 per group) were immunized i.n. with 1×10<sup>6</sup> CFU of the <i>emrA1</i> mutant and boosted i.d. on day 21 with a similar dose. On day 42 of the primary immunization mice were challenged i.n. with 17 CFU of <i>Ft</i> SchuS4. Age matched unvaccinated mice challenged with a similar dose of <i>Ft</i> SchuS4 served as controls. <b>(B)</b> C57BL/6 mice (n = 10 per group) were immunized i.d. with 1×10<sup>6</sup> CFU of the <i>emrA1</i> mutant and boosted i.n. on day 21 with a similar dose. On day 42 of the primary immunization mice were challenged i.n. with 17 CFU of <i>Ft</i> SchuS4. Age matched unvaccinated mice challenged with a similar dose of <i>Ft</i> SchuS4 served as controls. The survival results are expressed as Kaplan-Meier survival curves and P values were determined by Log-rank test.</p
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