12 research outputs found

    Preclinical testing of a vaccine candidate against tularemia.

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    Tularemia is caused by a gram-negative, intracellular bacterial pathogen, Francisella tularensis (Ft). The history weaponization of Ft in the past has elevated concerns that it could be used as a bioweapon or an agent of bioterrorism. Since the discovery of Ft, three broad approaches adopted for tularemia vaccine development have included inactivated, live attenuated, or subunit vaccines. Shortcomings in each of these approaches have hampered the development of a suitable vaccine for prevention of tularemia. Recently, we reported an oxidant sensitive mutant of Ft LVS in putative EmrA1 (FTL_0687) secretion protein. The emrA1 mutant is highly sensitive to oxidants, attenuated for intramacrophage growth and virulence in mice. We reported that EmrA1 contributes to oxidant resistance by affecting the secretion of antioxidant enzymes SodB and KatG. This study investigated the vaccine potential of the emrA1 mutant in prevention of respiratory tularemia caused by Ft LVS and the virulent SchuS4 strain in C57BL/6 mice. We report that emrA1 mutant is safe and can be used at an intranasal (i. n.) immunization dose as high as 1x106 CFU without causing any adverse effects in immunized mice. The emrA1 mutant is cleared by vaccinated mice by day 14-21 post-immunization, induces minimal histopathological lesions in lungs, liver and spleen and a strong humoral immune response. The emrA1 mutant vaccinated mice are protected against 1000-10,000LD100 doses of i.n. Ft LVS challenge. Such a high degree of protection has not been reported earlier against respiratory challenge with Ft LVS using a single immunization dose with an attenuated mutant generated on Ft LVS background. The emrA1 mutant also provides partial protection against i.n. challenge with virulent Ft SchuS4 strain in vaccinated C57BL/6 mice. Collectively, our results further support the notion that antioxidants of Ft may serve as potential targets for development of effective vaccines for prevention of tularemia

    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√ó10<sup>6</sup> CFU of the <i>emrA1</i> mutant were challenged i.n. with 1√ó10<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 ¬Ī 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 ¬Ī 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√ó10<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 őľM thin sections and stained with Hematoxylene & Eosin. Stained sections were observed for histopathological lesions under a light microscope (Magnification 100√ó). # = <i>Ft</i> LVS infected mice succumbed to infection.</p

    Immunization with <i>emrA1</i>-mAb complexes 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) immunized i.n. with 1√ó10<sup>6</sup> CFU of the <i>emrA1</i> mutant-mAb immune complexes. On day 42 of the primary immunization mice were challenged i.n. with 32 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. <b>(B)</b> The mice were weighed at the indicated times post-challenge to monitor the progression of infection. <b>(C)</b> The indicated <i>Ft</i> specific antibodies were determined in serum from immunized mice on day 14 post-immunization. The results are expressed as antibody titers.</p

    The <i>emrA1</i> mutant vaccinated mice clear bacteria rapidly and exhibit minimal histopathological lesions in lung, liver and spleen following lethal <i>Ft</i> LVS challenge.

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    <p>C57BL/6 mice immunized i.n. with 1√ó10<sup>6</sup> CFU of the <i>emrA1</i> mutant or the unvaccinated control mice were challenged i.n. with 1√ó10<sup>7</sup> CFU of <i>Ft</i> LVS 42 days post-immunization. <b>(A)</b> On days 5, 7, and 14 post-challenge, mice (n = 3 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>01;</i> ***<i>P<0</i>.<i>001</i>. <b>(B)</b> Lungs, livers and spleens collected at the indicated times post-challenge were preserved in 10% formalin, embedded in paraffin blocks, sliced into 5 őľM thin sections and stained with Hematoxylene & Eosin. Stained sections were observed for histopathological lesions under a light microscope (Magnification 100√ó). # = Unvaccinated mice succumbed to infection.</p

    Immunization with <i>emrA1</i>-mAb complexes using a prime-boost vaccination regimen or a low dose immunization 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.d. with 1√ó10<sup>6</sup> CFU of the <i>emrA1</i> mutant-mAb immune complex 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. <b>(B)</b> C57BL/6 mice (n = 10 per group) immunized i.n. with 1√ó10<sup>6</sup> CFU of the <i>emrA1</i>mutant-mAb immune complex 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>(C)</b> C57BL/6 mice (n = 10 per group) immunized i.n. with 1√ó10<sup>3</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>(D)</b> C57BL/6 mice (n = 10 per group) immunized i.d. with 1√ó10<sup>3</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

    Mice immunized with the <i>emrA1</i> mutant induce regulated production of pro-inflammatory cytokines and a potent antibody response.

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    <p>C57BL/6 mice were immunized i.n. with <i>1</i>√ó10<sup>6</sup> CFU of the <i>emrA1</i> mutant or <i>Ft</i> LVS. On days 1, 5, 7 and 14 post-immunization, mice (n = 4 per group/time point) were euthanized and their excised lungs and spleens were homogenized. Clear lung <b>(A-D)</b> and spleen <b>(E-H)</b> homogenates were used for quantification of indicated pro-inflammatory cytokines using flow cytometric analysis. The data are represented as Mean ¬Ī S.D. The <i>P</i> values were determined using one way ANOVA. *<i>P<0</i>.<i>05; **P<0</i>.<i>01</i>. <b>(I)</b> On day 42 post-immunization, mice (n = 3 per group/ time point) 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 ¬Ī S.D. of absorbance values measured at 450 nm. Red arrows indicate antibody titers. # = <i>Ft</i> LVS immunized mice succumbed to infection; ND = Not detected.</p

    Mice immunized with the <i>emrA1</i> mutant are protected against 1000LD100‚Äď10,000LD100 challenge dose of <i>Ft</i> LVS.

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    <p>C57BL/6 mice (n = 5‚Äď10 per group) were immunized i.n. with 1√ó10<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√ó10<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√ó10<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√ó10<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√ó10<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√ó10<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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