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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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Measurement of serum IgG antibody titers in immunized Balb/C mice.[A] Sera collected after first booster (14th day) and second boosters (21st day) from immunized groups (F1, F1+HSP70(II), F1+LcrV, F1+LcrV+HSP70(II)) were measured for F1-specific IgG by indirect ELISA. [B] Determination of LcrV-specific serum IgG antibody titer in the sera from immunized groups (LcrV, LcrV+HSP70(II), F1+LcrV, F1+LcrV+HSP70(II)). Data are represented in mean antibody titers with SD of eight Balb/C mice in each group. The cut-off value for the assays was calculated as the mean OD (+2 SD) from sera of control group assayed at 1∶100 dilution. Serum endpoint IgG titers were calculated as the reciprocal of the highest serum dilution giving an OD more than the cut-off. 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-g002: Measurement of serum IgG antibody titers in immunized Balb/C mice.[A] Sera collected after first booster (14th day) and second boosters (21st day) from immunized groups (F1, F1+HSP70(II), F1+LcrV, F1+LcrV+HSP70(II)) were measured for F1-specific IgG by indirect ELISA. [B] Determination of LcrV-specific serum IgG antibody titer in the sera from immunized groups (LcrV, LcrV+HSP70(II), F1+LcrV, F1+LcrV+HSP70(II)). Data are represented in mean antibody titers with SD of eight Balb/C mice in each group. The cut-off value for the assays was calculated as the mean OD (+2 SD) from sera of control group assayed at 1∶100 dilution. Serum endpoint IgG titers were calculated as the reciprocal of the highest serum dilution giving an OD more than the cut-off. 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: The IgG endpoint titer to F1 was 6.4×104 in sera from F1+LcrV+HSP70(II) group whereas it was 3.2×104 from F1; F1+HSP70(II) and F1+LcrV groups after first booster. The IgG endpoint titer after second booster was 2.56×105 from F1+LcrV+HSP70(II) group and 1.28×105 from F1+LcrV group. However, it was 1.28×105 from F1+HSP70(II) group and only 6.4×104 from F1 group (Figure 2A). HSP70(II) significantly increased the IgG response in the immunized groups i.e., F1+HSP70(II) and F1+LcrV+HSP70(II) in comparison to F1, and F1+LcrV groups respectively.


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 serum IgG antibody titers in immunized Balb/C mice.[A] Sera collected after first booster (14th day) and second boosters (21st day) from immunized groups (F1, F1+HSP70(II), F1+LcrV, F1+LcrV+HSP70(II)) were measured for F1-specific IgG by indirect ELISA. [B] Determination of LcrV-specific serum IgG antibody titer in the sera from immunized groups (LcrV, LcrV+HSP70(II), F1+LcrV, F1+LcrV+HSP70(II)). Data are represented in mean antibody titers with SD of eight Balb/C mice in each group. The cut-off value for the assays was calculated as the mean OD (+2 SD) from sera of control group assayed at 1∶100 dilution. Serum endpoint IgG titers were calculated as the reciprocal of the highest serum dilution giving an OD more than the cut-off. 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

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

pntd-0003322-g002: Measurement of serum IgG antibody titers in immunized Balb/C mice.[A] Sera collected after first booster (14th day) and second boosters (21st day) from immunized groups (F1, F1+HSP70(II), F1+LcrV, F1+LcrV+HSP70(II)) were measured for F1-specific IgG by indirect ELISA. [B] Determination of LcrV-specific serum IgG antibody titer in the sera from immunized groups (LcrV, LcrV+HSP70(II), F1+LcrV, F1+LcrV+HSP70(II)). Data are represented in mean antibody titers with SD of eight Balb/C mice in each group. The cut-off value for the assays was calculated as the mean OD (+2 SD) from sera of control group assayed at 1∶100 dilution. Serum endpoint IgG titers were calculated as the reciprocal of the highest serum dilution giving an OD more than the cut-off. 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: The IgG endpoint titer to F1 was 6.4×104 in sera from F1+LcrV+HSP70(II) group whereas it was 3.2×104 from F1; F1+HSP70(II) and F1+LcrV groups after first booster. The IgG endpoint titer after second booster was 2.56×105 from F1+LcrV+HSP70(II) group and 1.28×105 from F1+LcrV group. However, it was 1.28×105 from F1+HSP70(II) group and only 6.4×104 from F1 group (Figure 2A). HSP70(II) significantly increased the IgG response in the immunized groups i.e., F1+HSP70(II) and F1+LcrV+HSP70(II) in comparison to F1, and F1+LcrV groups respectively.

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