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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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a. Schematic diagram of three recombinant vaccine candidates; F1, LcrV and HSP70(II) showing the histidine tag and orientation of the open reading frame.b. 16% SDS-PAGE analysis of F1 protein expression [A]. 12% SDS–PAGE analysis of LcrV [B] and of HSP70(II) domain II of M. tuberculosis protein expression in E. coli [C]. The panels depict protein molecular mass marker (lane M), and Coomassie-stained polypeptide profiles of E. coli lysates un-induced (lane U) and induced with IPTG (lane I). The arrows at the right of the panels indicate the position of expressed recombinant proteins. c. SDS-PAGE analysis of purified F1 [A], LcrV [B] and HSP70(II) domain II of M. tuberculosis [C] metal affinity chromatography using Ni-NTA column. Each purified protein (3 µg/well) was analysed on SDS-PAGE. d. The humoral and cell mediated immune responses, protective potential and histopathological examinations of F1 and LcrV from Y. pestis with or without HSP70(II) of M. tuberculosis were evaluated in a mouse model. [A] Balb/C mice (8/group) were immunized with plague vaccine candidates with HSP70(II) as an immunomodulator in formulation aluminium hydroxide gel. [B] Schematic representation of immunization schedule following challenge experiments.
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pntd-0003322-g001: a. Schematic diagram of three recombinant vaccine candidates; F1, LcrV and HSP70(II) showing the histidine tag and orientation of the open reading frame.b. 16% SDS-PAGE analysis of F1 protein expression [A]. 12% SDS–PAGE analysis of LcrV [B] and of HSP70(II) domain II of M. tuberculosis protein expression in E. coli [C]. The panels depict protein molecular mass marker (lane M), and Coomassie-stained polypeptide profiles of E. coli lysates un-induced (lane U) and induced with IPTG (lane I). The arrows at the right of the panels indicate the position of expressed recombinant proteins. c. SDS-PAGE analysis of purified F1 [A], LcrV [B] and HSP70(II) domain II of M. tuberculosis [C] metal affinity chromatography using Ni-NTA column. Each purified protein (3 µg/well) was analysed on SDS-PAGE. d. The humoral and cell mediated immune responses, protective potential and histopathological examinations of F1 and LcrV from Y. pestis with or without HSP70(II) of M. tuberculosis were evaluated in a mouse model. [A] Balb/C mice (8/group) were immunized with plague vaccine candidates with HSP70(II) as an immunomodulator in formulation aluminium hydroxide gel. [B] Schematic representation of immunization schedule following challenge experiments.

Mentions: Immunogenicity of recombinant proteins alone or in combination and protection of immunized mice against virulent Y. pestis (S1 strain) was evaluated in 6–8 week old female Balb/C mice. The animals were taken in three batches and divided into 8 groups/batch (8 mice/group) i.e., Control group; HSP70(II) group; F1 group; LcrV group; F1+HSP70(II) group; LcrV+HSP70(II) group; F1+LcrV group and F1+LcrV+HSP70(II) group (Figure 1d [A]). The animals of batch-I were used for evaluation of IgG antibody response and protection studies against Y. pestis challenge; batch-II for evaluation of cell mediated immune response (cytokine profiling and the estimation of CD4+ and CD8+ T cells) and batch-III for histopathological/immunohistochemical studies. All the animal groups were immunized subcutaneously with 10 µg/mouse of each purified corresponding antigen/s as designated by their group name in formulation with aluminium hydroxide gel (0.35% in sterile phosphate buffer saline, PBS). The animals of control group were injected with PBS only. The prime dose was given on day 0 followed by two boosters on day 14 and 21. Blood was collected after first and second booster from each group on day 0, 21 and 28, sera were separated for IgG antibody response (Figure 1d [B]).


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)

a. Schematic diagram of three recombinant vaccine candidates; F1, LcrV and HSP70(II) showing the histidine tag and orientation of the open reading frame.b. 16% SDS-PAGE analysis of F1 protein expression [A]. 12% SDS–PAGE analysis of LcrV [B] and of HSP70(II) domain II of M. tuberculosis protein expression in E. coli [C]. The panels depict protein molecular mass marker (lane M), and Coomassie-stained polypeptide profiles of E. coli lysates un-induced (lane U) and induced with IPTG (lane I). The arrows at the right of the panels indicate the position of expressed recombinant proteins. c. SDS-PAGE analysis of purified F1 [A], LcrV [B] and HSP70(II) domain II of M. tuberculosis [C] metal affinity chromatography using Ni-NTA column. Each purified protein (3 µg/well) was analysed on SDS-PAGE. d. The humoral and cell mediated immune responses, protective potential and histopathological examinations of F1 and LcrV from Y. pestis with or without HSP70(II) of M. tuberculosis were evaluated in a mouse model. [A] Balb/C mice (8/group) were immunized with plague vaccine candidates with HSP70(II) as an immunomodulator in formulation aluminium hydroxide gel. [B] Schematic representation of immunization schedule following challenge experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pntd-0003322-g001: a. Schematic diagram of three recombinant vaccine candidates; F1, LcrV and HSP70(II) showing the histidine tag and orientation of the open reading frame.b. 16% SDS-PAGE analysis of F1 protein expression [A]. 12% SDS–PAGE analysis of LcrV [B] and of HSP70(II) domain II of M. tuberculosis protein expression in E. coli [C]. The panels depict protein molecular mass marker (lane M), and Coomassie-stained polypeptide profiles of E. coli lysates un-induced (lane U) and induced with IPTG (lane I). The arrows at the right of the panels indicate the position of expressed recombinant proteins. c. SDS-PAGE analysis of purified F1 [A], LcrV [B] and HSP70(II) domain II of M. tuberculosis [C] metal affinity chromatography using Ni-NTA column. Each purified protein (3 µg/well) was analysed on SDS-PAGE. d. The humoral and cell mediated immune responses, protective potential and histopathological examinations of F1 and LcrV from Y. pestis with or without HSP70(II) of M. tuberculosis were evaluated in a mouse model. [A] Balb/C mice (8/group) were immunized with plague vaccine candidates with HSP70(II) as an immunomodulator in formulation aluminium hydroxide gel. [B] Schematic representation of immunization schedule following challenge experiments.
Mentions: Immunogenicity of recombinant proteins alone or in combination and protection of immunized mice against virulent Y. pestis (S1 strain) was evaluated in 6–8 week old female Balb/C mice. The animals were taken in three batches and divided into 8 groups/batch (8 mice/group) i.e., Control group; HSP70(II) group; F1 group; LcrV group; F1+HSP70(II) group; LcrV+HSP70(II) group; F1+LcrV group and F1+LcrV+HSP70(II) group (Figure 1d [A]). The animals of batch-I were used for evaluation of IgG antibody response and protection studies against Y. pestis challenge; batch-II for evaluation of cell mediated immune response (cytokine profiling and the estimation of CD4+ and CD8+ T cells) and batch-III for histopathological/immunohistochemical studies. All the animal groups were immunized subcutaneously with 10 µg/mouse of each purified corresponding antigen/s as designated by their group name in formulation with aluminium hydroxide gel (0.35% in sterile phosphate buffer saline, PBS). The animals of control group were injected with PBS only. The prime dose was given on day 0 followed by two boosters on day 14 and 21. Blood was collected after first and second booster from each group on day 0, 21 and 28, sera were separated for IgG antibody response (Figure 1d [B]).

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