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

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.

Figure 1. 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.

Figure 2. Measurement of serum IgG antibody titers in immunized Balb/C mice.

[A] Sera collected after first booster (14^th day) and second boosters (21^st 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.

Figure 3. 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.

Figure 4. 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.

Figure 5. Flow cytometric analysis showing the percentage of IFN-γ producing CD8+ 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 CD8+ 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<0.05; **P<0.01; ***P<0.001; ^#P<0.001.

Figure 6. Determination of survival of Balb/C mice infected with Y. pestis.

The immunized and PBS control groups (8 mice/group) were challenged with 100 LD₅₀ of Y. pestis (S1 strain). The protective efficacy of vaccine candidate alone or in mixture of antigens was determined by Kaplan Meier's method to compare percentage survivals (****P<0.0001, ***P<0.001).

Figure 7. Histopathology of the organs collected from the immunized group animals on 3^rd and 20^th day post infection with Y. pestis and the naive control animals that were neither immunized nor challenged with Y. pestis.

Tissue sections were stained with hematoxylin and eosin for pathological examination. Tissue section collected from naive control and immunized animals on 3^rd day post infection i.e., Naive control (A); PBS control (B); HSP70(II) (C); F1 (D); F1+HSP70(II) (E); LcrV (F); LcrV+HSP70(II) (G); F1+LcrV (H); F1+LcrV+HSP70(II) (I). Tissue sections were collected from the survived animal groups on 20^th day post infection i.e., LcrV (J); LcrV+HSP70(II) (K); F1+LcrV (L); F1+LcrV+HSP70(II) (M). Photomicrograph represents the histopathology of Lung[a]: the arrows in the panel B indicate the infiltration of neutrophils. Photomicrograph of spleen [b]: in the panel B, reduced density of white pulp follicle and congestion in the red pulp, lymphoid follicle depletion shown by arrow and the presence of megakaryocytes shown by bold arrow. Photomicrograph of kidney [c]: the granular degeneration of parenchyma was observed in the panel B (bold arrows) and swelling in renal tubules (arrows). Photomicrograph of liver [d]: in the panel B, the hepatocytes degeneration was observed as indicated by arrow.

Figure 8. Immunohistochemistry (IHC) staining for localization of Y. pestis in the organs collected from immunized group animals on 3^rd and 20^th day post infection with Y. pestis and the naive control animals that were neither immunized nor challenged.

The F1 antigen of Y. pestis was identified with anti-mouse FITC conjugated secondary antibody in the tissue sections collected from immunized animal groups on 3^rd day post infection including naive control i.e., Naive control (A); PBS control (B); HSP70(II) (C); F1 (D); F1+HSP70(II) (E); LcrV (F); LcrV+HSP70(II) (G); F1+LcrV (H); F1+LcrV+HSP70(II) (I). Tissue sections were collected from the survived animal groups on 20^th day post infection i.e., LcrV (J); LcrV+HSP70(II) (K); F1+LcrV (L); F1+LcrV+HSP70(II) (M). Fluorescent images representing the localization of Y. pestis in tissue sections of Lung [a]; Spleen [b]; Kidney [c]; and Liver [d].