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HSP70 domain II of Mycobacterium tuberculosis modulates immune response and protective potential of F1 and LcrV antigens of Yersinia pestis in a mouse model.

Batra L, Verma SK, Nagar DP, Saxena N, Pathak P, Pant SC, Tuteja U - PLoS Negl Trop Dis (2014)

Bottom Line: A significant difference was noticed in the expression level of IL-2, IFN-γ and TNF-α in splenocytes of immunized animals.We investigated whether the F1, LcrV and HSP70(II) antigens alone or in combination can effectively protect immunized animals from any histopathological changes.A significant difference was observed in the expression of IL-2, IFN-γ, TNF-α, and CD4+/CD8+ T cells secreting IFN-γ in the F1+LcrV+HSP70(II) vaccinated group in comparison to the F1+LcrV vaccinated group.

View Article: PubMed Central - PubMed

Affiliation: Microbiology Division, Defence Research & Development Establishment, Gwalior, India.

ABSTRACT
No ideal vaccine exists to control plague, a deadly dangerous disease caused by Yersinia pestis. In this context, we cloned, expressed and purified recombinant F1, LcrV antigens of Y. pestis and heat shock protein70 (HSP70) domain II of M. tuberculosis in E. coli. To evaluate the protective potential of each purified protein alone or in combination, Balb/C mice were immunized. Humoral and cell mediated immune responses were evaluated. Immunized animals were challenged with 100 LD50 of Y. pestis via intra-peritoneal route. Vaccine candidates i.e., F1 and LcrV generated highly significant titres of anti-F1 and anti-LcrV IgG antibodies. A significant difference was noticed in the expression level of IL-2, IFN-γ and TNF-α in splenocytes of immunized animals. Significantly increased percentages of CD4+ and CD8+ T cells producing IFN-γ in spleen of vaccinated animals were observed in comparison to control group by flow cytometric analysis. We investigated whether the F1, LcrV and HSP70(II) antigens alone or in combination can effectively protect immunized animals from any histopathological changes. Signs of histopathological lesions noticed in lung, liver, kidney and spleen of immunized animals on 3rd day post challenge whereas no lesions in animals that survived to day 20 post-infection were observed. Immunohistochemistry showed bacteria in lung, liver, spleen and kidney on 3rd day post-infection whereas no bacteria was observed on day 20 post-infection in surviving animals in LcrV, LcrV+HSP70(II), F1+LcrV, and F1+LcrV+HSP70(II) vaccinated groups. A significant difference was observed in the expression of IL-2, IFN-γ, TNF-α, and CD4+/CD8+ T cells secreting IFN-γ in the F1+LcrV+HSP70(II) vaccinated group in comparison to the F1+LcrV vaccinated group. Three combinations that included LcrV+HSP70(II), F1+LcrV or F1+LcrV+HSP70(II) provided 100% protection, whereas LcrV alone provided only 75% protection. These findings suggest that HSP70(II) of M. tuberculosis can be a potent immunomodulator for F1 and LcrV containing vaccine candidates against plague.

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Flow cytometric analysis showing the percentage of IFN-γ producing CD4+ T cells in the spleens of immunized Balb/C mice following in vitro stimulation with specific antigens as indicated and anti-CD28 [A].Graphical representation showing the significant difference in the percentage of IFN-γ producing CD4+ T cells in the spleen of different immunized animal groups after stimulation with specific antigens in comparison to the PBS control group. Each bar represents the average of 3 mice/group ± S.D [B]. Analysis was done by one way ANOVA, All Pairwise Multiple Comparison Procedure (Fisher LSD Method). **P<0.01; ***P<0.001; #P<0.001.
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pntd-0003322-g004: Flow cytometric analysis showing the percentage of IFN-γ producing CD4+ T cells in the spleens of immunized Balb/C mice following in vitro stimulation with specific antigens as indicated and anti-CD28 [A].Graphical representation showing the significant difference in the percentage of IFN-γ producing CD4+ T cells in the spleen of different immunized animal groups after stimulation with specific antigens in comparison to the PBS control group. Each bar represents the average of 3 mice/group ± S.D [B]. Analysis was done by one way ANOVA, All Pairwise Multiple Comparison Procedure (Fisher LSD Method). **P<0.01; ***P<0.001; #P<0.001.

Mentions: FACS analysis was performed for CD4+ and CD8+ T cell population producing IFN-γ in the splenocytes of all the immunized animal groups including control group. After stimulating splenocytes with specific antigen/s, an increased percentage of CD4+ T (Figure 4A) and CD8+ T cells expressing IFN-γ (Figure 5A) was observed in all vaccinated groups in comparison to control group. The population count (%) of IFN-γ secreting CD4+ T cells for Control, F1, F1+HSP70(II), LcrV, LcrV+HSP70(II), F1+LcrV, F1+LcrV+HSP70(II) and HSP70(II) groups was 0.46±0.12, 1.75±0.23, 1.16±0.12, 0.925±0.1, 0.98±0.12, 2.48±0.02, 4.43±0.52 and 4.985±0.04 respectively. The population count (%) of IFN-γ secreting CD4+ T cells for Control, F1, F1+HSP70(II), LcrV, LcrV+HSP70(II), F1+LcrV, F1+LcrV+HSP70(II) and HSP70(II) groups was 0.535±0.06, 1.17±0.04, 1.125±0.16, 0.91±0.43, 1.38±0.19, 2.725±0.99, 4.42±0.11 and 1.84±0.14 respectively. As shown by graphical representations, a significant difference (*P<0.05; **P<0.01; ***P<0.001) was noticed in the IFN-γ secreting CD4+ T cells (Figure 4B) and CD8+ T cells (Figure 5B) to all the immunized groups in comparison to control group. We also noticed a remarkable significant difference (#P<0.001) for both CD4+ and CD8+ T cells in F1+LcrV+HSP70(II) group in comparison to F1+LcrV group.


HSP70 domain II of Mycobacterium tuberculosis modulates immune response and protective potential of F1 and LcrV antigens of Yersinia pestis in a mouse model.

Batra L, Verma SK, Nagar DP, Saxena N, Pathak P, Pant SC, Tuteja U - PLoS Negl Trop Dis (2014)

Flow cytometric analysis showing the percentage of IFN-γ producing CD4+ T cells in the spleens of immunized Balb/C mice following in vitro stimulation with specific antigens as indicated and anti-CD28 [A].Graphical representation showing the significant difference in the percentage of IFN-γ producing CD4+ T cells in the spleen of different immunized animal groups after stimulation with specific antigens in comparison to the PBS control group. Each bar represents the average of 3 mice/group ± S.D [B]. Analysis was done by one way ANOVA, All Pairwise Multiple Comparison Procedure (Fisher LSD Method). **P<0.01; ***P<0.001; #P<0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4256173&req=5

pntd-0003322-g004: Flow cytometric analysis showing the percentage of IFN-γ producing CD4+ T cells in the spleens of immunized Balb/C mice following in vitro stimulation with specific antigens as indicated and anti-CD28 [A].Graphical representation showing the significant difference in the percentage of IFN-γ producing CD4+ T cells in the spleen of different immunized animal groups after stimulation with specific antigens in comparison to the PBS control group. Each bar represents the average of 3 mice/group ± S.D [B]. Analysis was done by one way ANOVA, All Pairwise Multiple Comparison Procedure (Fisher LSD Method). **P<0.01; ***P<0.001; #P<0.001.
Mentions: FACS analysis was performed for CD4+ and CD8+ T cell population producing IFN-γ in the splenocytes of all the immunized animal groups including control group. After stimulating splenocytes with specific antigen/s, an increased percentage of CD4+ T (Figure 4A) and CD8+ T cells expressing IFN-γ (Figure 5A) was observed in all vaccinated groups in comparison to control group. The population count (%) of IFN-γ secreting CD4+ T cells for Control, F1, F1+HSP70(II), LcrV, LcrV+HSP70(II), F1+LcrV, F1+LcrV+HSP70(II) and HSP70(II) groups was 0.46±0.12, 1.75±0.23, 1.16±0.12, 0.925±0.1, 0.98±0.12, 2.48±0.02, 4.43±0.52 and 4.985±0.04 respectively. The population count (%) of IFN-γ secreting CD4+ T cells for Control, F1, F1+HSP70(II), LcrV, LcrV+HSP70(II), F1+LcrV, F1+LcrV+HSP70(II) and HSP70(II) groups was 0.535±0.06, 1.17±0.04, 1.125±0.16, 0.91±0.43, 1.38±0.19, 2.725±0.99, 4.42±0.11 and 1.84±0.14 respectively. As shown by graphical representations, a significant difference (*P<0.05; **P<0.01; ***P<0.001) was noticed in the IFN-γ secreting CD4+ T cells (Figure 4B) and CD8+ T cells (Figure 5B) to all the immunized groups in comparison to control group. We also noticed a remarkable significant difference (#P<0.001) for both CD4+ and CD8+ T cells in F1+LcrV+HSP70(II) group in comparison to F1+LcrV group.

Bottom Line: A significant difference was noticed in the expression level of IL-2, IFN-γ and TNF-α in splenocytes of immunized animals.We investigated whether the F1, LcrV and HSP70(II) antigens alone or in combination can effectively protect immunized animals from any histopathological changes.A significant difference was observed in the expression of IL-2, IFN-γ, TNF-α, and CD4+/CD8+ T cells secreting IFN-γ in the F1+LcrV+HSP70(II) vaccinated group in comparison to the F1+LcrV vaccinated group.

View Article: PubMed Central - PubMed

Affiliation: Microbiology Division, Defence Research & Development Establishment, Gwalior, India.

ABSTRACT
No ideal vaccine exists to control plague, a deadly dangerous disease caused by Yersinia pestis. In this context, we cloned, expressed and purified recombinant F1, LcrV antigens of Y. pestis and heat shock protein70 (HSP70) domain II of M. tuberculosis in E. coli. To evaluate the protective potential of each purified protein alone or in combination, Balb/C mice were immunized. Humoral and cell mediated immune responses were evaluated. Immunized animals were challenged with 100 LD50 of Y. pestis via intra-peritoneal route. Vaccine candidates i.e., F1 and LcrV generated highly significant titres of anti-F1 and anti-LcrV IgG antibodies. A significant difference was noticed in the expression level of IL-2, IFN-γ and TNF-α in splenocytes of immunized animals. Significantly increased percentages of CD4+ and CD8+ T cells producing IFN-γ in spleen of vaccinated animals were observed in comparison to control group by flow cytometric analysis. We investigated whether the F1, LcrV and HSP70(II) antigens alone or in combination can effectively protect immunized animals from any histopathological changes. Signs of histopathological lesions noticed in lung, liver, kidney and spleen of immunized animals on 3rd day post challenge whereas no lesions in animals that survived to day 20 post-infection were observed. Immunohistochemistry showed bacteria in lung, liver, spleen and kidney on 3rd day post-infection whereas no bacteria was observed on day 20 post-infection in surviving animals in LcrV, LcrV+HSP70(II), F1+LcrV, and F1+LcrV+HSP70(II) vaccinated groups. A significant difference was observed in the expression of IL-2, IFN-γ, TNF-α, and CD4+/CD8+ T cells secreting IFN-γ in the F1+LcrV+HSP70(II) vaccinated group in comparison to the F1+LcrV vaccinated group. Three combinations that included LcrV+HSP70(II), F1+LcrV or F1+LcrV+HSP70(II) provided 100% protection, whereas LcrV alone provided only 75% protection. These findings suggest that HSP70(II) of M. tuberculosis can be a potent immunomodulator for F1 and LcrV containing vaccine candidates against plague.

Show MeSH
Related in: MedlinePlus