Limits...
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.

Show MeSH

Related in: MedlinePlus

Measurement of cytokines expressed by splenocytes of immunized mice groups.Cytokines expressed by splenocytes collected from mice immunized with F1, F1+HSP70(II), LcrV, LcrV+HSP70(II), F1+LcrV+HSP70(II) and HSP70(II) including control group were measured. Concentrations of cytokines detected in splenocytes supernatant after 48 h of stimulation with specific antigens (5 µg/ml) are shown. Graphs showed concentrations of (A) IL-2, (B) IFN-γ, (C) TNF-α in picograms per millilitre (pg/ml). Each bar represents the average of 8 mice/group ± S.D and is representative of three independent experiments. Analysis was done by one way ANOVA, All Pairwise Multiple Comparison Procedure (Fisher LSD Method). *P<0.05; **P<0.01; ***P<0.001; #P<0.001.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4256173&req=5

pntd-0003322-g003: Measurement of cytokines expressed by splenocytes of immunized mice groups.Cytokines expressed by splenocytes collected from mice immunized with F1, F1+HSP70(II), LcrV, LcrV+HSP70(II), F1+LcrV+HSP70(II) and HSP70(II) including control group were measured. Concentrations of cytokines detected in splenocytes supernatant after 48 h of stimulation with specific antigens (5 µg/ml) are shown. Graphs showed concentrations of (A) IL-2, (B) IFN-γ, (C) TNF-α in picograms per millilitre (pg/ml). Each bar represents the average of 8 mice/group ± S.D and is representative of three independent experiments. Analysis was done by one way ANOVA, All Pairwise Multiple Comparison Procedure (Fisher LSD Method). *P<0.05; **P<0.01; ***P<0.001; #P<0.001.

Mentions: Cytokine profiles of all immunized animal groups were determined by estimating the levels of IL-2, IL-4, IL-10, IFN-γ and TNF-α in supernatants of splenocytes stimulated with specific antigen/s. Significantly high (***p<0.001) expression levels of IL-2 (Figure 3A), and TNF-α (Figure 3C) were noticed in all the immunized animal groups in comparison control group. In case of IFN-γ (Figure 3B), a significant difference (*P<0.05; ***P<0.001) was noticed to all the immunized groups with respect to control except F1 group. No significant difference was noticed in the expression levels of IL-4 and IL-10 (Figure S2). Splenocytes from all groups responded to ConA non-specifically. The significant difference was observed in the expression level of IFN-γ (#P<0.001) in F1+LcrV+HSP70(II); LcrV+HSP70(II) and F1+HSP70(II) groups in comparison to F1+LcrV; LcrV and F1 groups respectively. The significant difference was observed in the expression level of IL-2 (#P<0.001) in F1+LcrV+HSP70(II) and LcrV+HSP70(II) groups in comparison to F1+LcrV; LcrV groups respectively. In the case of TNF-α, a significant difference (#P<0.001) was observed in F1+LcrV+HSP70(II) group in comparison to F1+LcrV group. The expression level of cytokines (IL-2, IL-4, IL-10, IFN-γ and TNF-α) in animal groups have been shown in Table 2.


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)

Measurement of cytokines expressed by splenocytes of immunized mice groups.Cytokines expressed by splenocytes collected from mice immunized with F1, F1+HSP70(II), LcrV, LcrV+HSP70(II), F1+LcrV+HSP70(II) and HSP70(II) including control group were measured. Concentrations of cytokines detected in splenocytes supernatant after 48 h of stimulation with specific antigens (5 µg/ml) are shown. Graphs showed concentrations of (A) IL-2, (B) IFN-γ, (C) TNF-α in picograms per millilitre (pg/ml). Each bar represents the average of 8 mice/group ± S.D and is representative of three independent experiments. Analysis was done by one way ANOVA, All Pairwise Multiple Comparison Procedure (Fisher LSD Method). *P<0.05; **P<0.01; ***P<0.001; #P<0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003322-g003: Measurement of cytokines expressed by splenocytes of immunized mice groups.Cytokines expressed by splenocytes collected from mice immunized with F1, F1+HSP70(II), LcrV, LcrV+HSP70(II), F1+LcrV+HSP70(II) and HSP70(II) including control group were measured. Concentrations of cytokines detected in splenocytes supernatant after 48 h of stimulation with specific antigens (5 µg/ml) are shown. Graphs showed concentrations of (A) IL-2, (B) IFN-γ, (C) TNF-α in picograms per millilitre (pg/ml). Each bar represents the average of 8 mice/group ± S.D and is representative of three independent experiments. Analysis was done by one way ANOVA, All Pairwise Multiple Comparison Procedure (Fisher LSD Method). *P<0.05; **P<0.01; ***P<0.001; #P<0.001.
Mentions: Cytokine profiles of all immunized animal groups were determined by estimating the levels of IL-2, IL-4, IL-10, IFN-γ and TNF-α in supernatants of splenocytes stimulated with specific antigen/s. Significantly high (***p<0.001) expression levels of IL-2 (Figure 3A), and TNF-α (Figure 3C) were noticed in all the immunized animal groups in comparison control group. In case of IFN-γ (Figure 3B), a significant difference (*P<0.05; ***P<0.001) was noticed to all the immunized groups with respect to control except F1 group. No significant difference was noticed in the expression levels of IL-4 and IL-10 (Figure S2). Splenocytes from all groups responded to ConA non-specifically. The significant difference was observed in the expression level of IFN-γ (#P<0.001) in F1+LcrV+HSP70(II); LcrV+HSP70(II) and F1+HSP70(II) groups in comparison to F1+LcrV; LcrV and F1 groups respectively. The significant difference was observed in the expression level of IL-2 (#P<0.001) in F1+LcrV+HSP70(II) and LcrV+HSP70(II) groups in comparison to F1+LcrV; LcrV groups respectively. In the case of TNF-α, a significant difference (#P<0.001) was observed in F1+LcrV+HSP70(II) group in comparison to F1+LcrV group. The expression level of cytokines (IL-2, IL-4, IL-10, IFN-γ and TNF-α) in animal groups have been shown in Table 2.

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