Figure 1.
sIM-1.6.α elicits a long-lasting Th1 immune response.
Mice were vaccinated with sIM-1.6.α and sIM. After 240 days, they were aerosol challenged with M. tb and were sacrificed 35 days later. Lymphocytes Pooled from spleen and lymphnodes of immunized mice were stimulated in vitro with PPD (50 µg/ml) for 48 h. T cell proliferation was monitored by 3H-thymidine incorporation (A); secretion of IFN-γ (B) and IL-2 (C) in the culture SNs by ELISA. Ratio of PPD specific IgG2a/IgG1 antibodies were estimated in serum (1000× dilution) (D); production of IFN-γ (E) and IL-2 (F) was estimated in the culture SNs of lymphocytes isolated from lungs, in vitro stimulated with PPD (50 µg/ml). Data shown are mean ± SEM and representative of two experiments with n = 3 animals per group. ‘*’, ‘**’ and ‘***’ indicate p<0.05, p<0.01 and p<0.001 respectively.
Figure 2.
sIM-1.6.α induces enduring CD4 and CD8 T cells memory response.
Mice were immunized and aerosol challenged as mentioned in Figure 1. Lymphocytes were stimulated in vitro with PPD (50 µg/ml) for 48 h and later stained with fluorochrome conjugated antibodies and analyzed by flowcytometry. A, flow contours depict percentage of gated CD4 T cells expressing IL-7R and CD44 (upper panel); CD62L on CD4 T cells (lower panel). C, gated CD8 T cells expressing IL-7R and CD44 (upper panel) and CD62L on CD8 T cells (lower panel). Bar diagrams show total number of CD4 (B) and CD8 (D) T cells expressing CD44hi, CD62Lhi and CD62Llo. Data shown are mean ± SEM of two independent experiments with n = 3 animals per group. ‘*’ and ‘**’ indicate p<0.05 and p<0.01 respectively.
Figure 3.
sIM-1.6.α provides significantly better protection than BCG.
Mice were immunized and aerosol challenged as mentioned in Figure 1. Mycobacterial load in the lungs was enumerated by CFU plating of diluted lung homogenates. A, data are represented as log10 CFU (n = 6 animals/group) where each triangle indicates single mouse. B, photomicrographs (10×) of formalin fixed lungs sections stained with H & E. ‘+’ indicates active TB lesion with edematous follicular reaction while ‘→’ shows small or large developing follicular granulomas. Data are representative of two independent experiments. ‘**’ and ‘***’indicate p<0.01 and p<0.001 respectively.
Figure 4.
Vaccination with xIM-1.6.α evokes immune response.
Mice were vaccinated with xIM-1.6.α with similar immunization protocol as mentioned in Figure 1. Lymphocytes pooled from spleen and lymphnodes of immunized animals were stimulated in vitro with PPD (50 µg/ml) for 48 h and later T cell proliferation was monitored by 3H-thymidine incorporation (A); secretion of IFN-γ (B) and IL-2 (C) in culture SNs; PPD specific IgG2a/IgG1 antibodies in the serum (1000× dilution) (D) were estimated by ELISA. Production of IFN-γ (E) and IL-2 (F) was estimated in the culture SNs of the lymphocytes isolated from lungs, in vitro stimulated with PPD (50 µg/ml). Data shown as mean ± SEM are representative of two experiments, n = 3 animals per group. ‘*’ and ‘**’ indicate p<0.05 and p<0.01 respectively.
Figure 5.
Vaccination with xIM-1.6.α induces CD4 and CD8 T cells memory response.
Mice were immunized and aerosol challenged as mentioned in Figure 1. Pooled lymphocytes from spleen and lymphnodes of immunized animals were stimulated in vitro with PPD (50 µg/ml) for 48 h and later stained with fluorochrome tagged antibodies and analyzed by flowcytometry. A, flow contours depict percentage of CD4 T cells expressing IL-7R and CD44 (upper panel); CD62L expression on CD4 T cells (lower panel). C, CD8 T cells expressing IL-7R and CD44 (upper panel); CD62L on CD8 T cells (lower panel). Bar diagrams show total number of CD4 (B) and CD8 (D) T cells expressing CD44hi, CD62Lhi and CD62Llo. Data shown are mean ± SEM of two independent experiments with n = 3 animals per group. ‘*’ indicates p<0.05.
Figure 6.
Immunization with xIM-1.6.α provides protection against M. tb.
Mice vaccinated with xIM-1.6.α were rested for 240 days and then aerosol challenged with M. tb. After 35 days, mice were sacrificed and mycobacterial load was enumerated by CFU plating of diluted lung homogenates. A, data are represented as log10 CFU (n = 4–6 animals/group). B, photomicrographs (10×) of H & E stained lungs sections. Control groups of mice were inoculated with uninfected xenogeneic macrophages (xMac). Data are mean ± SEM of two independent experiments, where ‘*’ and ‘**’ indicate p<0.05 and p<0.01 respectively.
Figure 7.
Apoptotic bodies generated by IM transport antigen to bystander dendritic cells.
M. tb infected macrophages were co-cultured with Dendritic cells (DCs) for 48 h. Later, fluorescence microscopy was used to demonstrate generation of apoptotic bodies containing mycobacterial antigens and their uptake by bystander DCs. (A) Left panel, arrows indicate Texas Red labeled M. tb; Middle panel, M. tb infected macrophages (orange, mixed fluorescence) and asterisks depict uninfected macrophages (green); right panel, DIC image of macrophages (images at 100×). (B) Left panel, arrows indicate Texas Red labeled M. tb; middle panel, arrows indicate M. tb within apoptotic bodies (orange, mixed fluorescence) while asterisks indicate apoptotic bodies (green) devoid of mycobacteria; right panel, DIC image of DCs show engulfment of apoptotic bodies containing mycobacterial antigens by DCs (Images at 100×1.6×). Arrow heads indicate association of apoptotic macrophages with DCs. DIC, Differential interference contrast. () Data are representative of three independent experiments.