Figure 1.
Archaeosome encapsulated Rv3619c induces type I cytokine expression in the immunized mice.
Archaeosome mediated Th1/Th2 bias was ascertained by determining cytokine response in splenocyte culture supernatant belonging to various immunized groups at different time points; (A) IFN-γ, (B) IL-12. To activate splenocytes belonging to group of animals immunized with free Rv3619c, free form of 20 µg Rv3619c was used while lymphocytes isolated from group of animals immunized with physical mixture of sham archaeosome and Rv3619c were activated with physical mixture of 20 µg of free Rv3619c and sham archaeosome (∼1.5 mg archaebacterial lipid) splenocytes isolated from animals immunized with archaeosome entrapped Rv3619c group, were co-cultured with archaeosome entrapped Rv3619c (20 µg antigen). To further confirm the Th1/Th2 polarization upon immunization with archaeosome encapsulated Rv3619c, the sera of immunized animals were analysed for the presence of IgG2a and IgG1 isotype by sandwich ELISA method (C). The data represent mean of three determinants± S.D. and are representative of two different experiments with similar observation. Various immunized groups were compared to determine statistical significance of the data using student's t test analysis with p<0.05 (*), p<0.01(**), P<0.001(***) level of significance. PB (post booster), PC (post challenge), NS (not significant).
Figure 2.
T cell proliferation response in various immunized groups upon stimulation with archaeosome encapsulated Rv3619c.
(A) To determine the effect of amount of Rv3619c on proliferation of T lymphocytes; splenocytes, isolated from various groups of immunized Balb/c mice at two weeks post booster time point, were co-cultured in the presence of increasing amounts (1 to 20 µg) of Rv3619c Ag in flat-bottomed 96 well plates. After 72 h, [3H]-thymidine was added to each well and its incorporation in multiplying cells was measured after 16 h incubation with liquid scintillation spectroscopy. The accumulation of 3H, thymidine was determined in proliferating cells and denoted in term of counting per minute (cpm) values of stimulated cultures to represent Ag specific stimulation. (B) Proliferation of Rv3619c specific T lymphocytes isolated from immunized animals at various time points post booster and also at 4 and 8 weeks post challenge upon stimulation with a fixed amount (20 µg) of free Rv3619c. While (C) represents T cell proliferation in various immunized groups at various time intervals upon activation with corresponding matching formulations of Rv3619c. For stimulation of cells belonging to BCG immunized group, PPD at concentration of 20 µg was used, while maintaining rest of the incubation conditions same to that of archaeosome based cell groups. Similarly, for archaeosome encapsulated Rv3619c group, liposomised form of Rv3619c was used at a concentration of 20 µg (corresponding to ∼1.5 mg Archae lipid) to stimulate splenocytes. Splenocytes belonging to sham group were cultured in the presence of 20 µg of free Rv3619c, while splenocytes isolated from physical mixture group were incubated with empty sham archaeosome (∼1.5 mg lipid) mixed with 20 µg Rv3619c. Data represent the mean of three determinants ± S.D. Figures are representative of four independent experiments. The groups were compared with Student's t test to determine level of significance; p<0.05 (*), p<0.01(**), p<0.001(***).
Figure 3.
Archaeosome encapsulated Rv3619c upregulates costimulatory molecules on antigen presenting cells.
Target cells were isolated following method as described in methodology section on 8th week post challenge with M. tb infection. Co-stimulatory molecules, CD80 (B7-1) and CD86 (B7-2), were determined by staining of target cells with specific antibodies and subsequently analysed by flow cytometry. The histograms correspond to expression of CD80 (A), and CD86 molecules (B) on the surface of target cells. Various immunized groups are BCG (i) Sham (antigen free) archaeosome as control (ii) Free Rv3619c Ag (iii) Sham Archaeosome + free Rv3619c Ag; a physical mixture (iv) Archaeosome entrapped Rv3619c Ag (v) and isotype control (vi). The bar graphs corresponding to CD80 (C), and CD86 (D), depict the mean percent of three determinants (±S.D.). The expression data were analyzed with the Student's t test and are representative of 3 independent experiments. Archaeosome encapsulated Rv3619c Vs BCG p<0.01 (CD80), p<0.05 (CD86); BCG Vs physical mixture p<0.05 (CD80), p = NS (CD86); archaeosome Vs free Rv3619c Ag p<0.05 (CD80, and CD86).
Figure 4.
Augmentation of long-lasting CD4+ T cell (effector/central) memory response upon immunization of mice with archaeosome encapsulated Rv 3619c.
After 8 weeks post challenge, the splenocytes were isolated from various immunized groups and analyzed for the presence of CD44high, CD62Llow/high on gated CD4+ T cells by FACS (A). The gated CD4+ T cells belonging to various immunized groups are denoted in the upper panel. Various experimental groups included in the study are: BCG (a) Sham (antigen free) archaeosome as control (b) Free Rv3619c Ag (c) Sham archaeosome + free Rv3619c Ag, a physical mixture (d) Archaeosome entrapped Rv3619c Ag (e) and isotype control (f). Lower panel indicates the analysed memory markers belonging to the corresponding CD4+ T cells in the upper panel. Bars indicate the total number of (CD4+ CD44high CD62Llow/high) (B). The data were analyzed with the Student's t test and are shown as the means (±S.D.) of 2 independent experiments. Archaeosome Vs BCG p<0.001 CD4+ (CD44high, CD62Llow and CD62Lhigh); physical mixture Vs BCG p = NS CD4+(CD44high, CD62Lhigh), p<0.01 (CD4+CD62Llow); archaeosome Vs free Ag p<0.001 CD4+(CD44high, CD62Lhigh, and CD62Llow).
Figure 5.
Expansion of the long-term CD8+ T cell effector and central memory response after immunization with various forms of Rv3619c on 8 weeks post challenge with M. tb Lymphocytes isolated from spleens of animals belonging to various immunized groups were stained for expression of CD44, CD62L, on CD8+ T cells and analyzed by flow cytometry.
(A) The dot plot graphs in upper panel representing BCG (a), Sham (antigen free) archaeosome as control (b), Free Rv3619c Ag (c), Sham archaeosome + free Rv3619c, a physical mixture (d), Archaeosome entrapped Rv3619c Ag (e), and isotype control (f). The lower panel indicates the population of CD44 and CD62Llow/high on gated CD8+ T cells of corresponding upper panel. Bars indicate the total number of CD8+ T cells expressing CD44highCD62Llow/high (B), The data were analyzed with the Student's t test and are shown as the means (±S.D.) of 2 independent experiments. Archaeosome encapsulated Rv3619c Vs BCG p<0.001 CD8+ (CD44high, CD62Llow and CD62Lhigh); physical mixture of sham archaeosome and free antigen Vs BCG p<0.05 (CD8+CD44high, CD8+CD62Llow), p = NS (CD8+CD62Lhigh); archaeosome encapsulated Rv3619c Vs free Rv3619c Ag p<0.001 CD8+ (CD44high, CD62Llow and CD62Lhigh).
Figure 6.
Archaeosome encapsulated Rv3619c offers protection against M.tb infection in BALB/c mice.
Mycobacterial load, in the lungs (A) and spleens (B) of vaccinated mice, belonging to various groups of animals, was enumerated by plating tissue homogenates of lungs and spleens of vaccinated mice followed by counting the numbers of colony-forming units (CFUs) at 4 and 8 weeks post challenge following method as described in methodology section of the text. The data were expressed as means of three determinants ± S.D. and are representative of 4 independent experiments. Statistical analysis was performed by ANOVA (analysis of variance) using Holm-Sidak method; p<0.01(**), p<0.001(***).
Figure 7.
The histopathological study of lungs obtained from animals belonging to various immunized groups.
At 8 weeks post challenge, the lungs obtained from animals belonging to various immunized groups were fixed in formalin, to prepare them for section cutting. Subsequently, sections were stained with hematoxylin and eosin or Ziehl-Nelson stain to facilitate acid fast staining of bacilli as described in materials and methods section. Various experimental groups included in the study are: BCG (A), Sham control (B), Free Rv3619c (C), physical mixture of sham archaeosome and Rv3619c (D) and archaeosome entrapped Rv3619c (E). The panels on left side represent low power photomicrographs (200×; except D which is 100× of original picture) of sections while right side panel represents the high magnification (1000×) of the inset part of corresponding left panel. Acid fast bacilli can clearly be seen in higher resolution photomicrographs.