Figure 1.
Structural analysis of BmHsp12.6.
(A) Secondary structure prediction of BmHsp12.6 showing α-helices (H1 and H2), strands by their sheets A, B, (β)- beta turn, (γ)- gamma turn and beta hairpin (⊃). (B) Graphical representation showing N-BmHsp12.6 and BmHsp12.6αc peptide sequences and their functional roles in the BmHsp12.6 protein.
Table 1.
Predicted B-cell, T-cell and CTL epitopes in BmHsp12.6 sequences.
Figure 2.
N-BmHsp12.6 peptide enhances the growth of MC/9 mast cells in vitro. The effects of different peptides of BmHsp12.6 on MC/9 cells were determined by a cell viability assay. 5×103 cells/ml of MC/9 were stimulated with 10 µg/ml of A) N- BmHsp12.6 B) BmHsp12.6 and C) BmHsp12.6αc (10 µg/ml) for 72 h at 37°C. Sera containing antibodies against the respective proteins were added to certain cultures. Sera samples from normal Balb/c mice served as negative controls. Cells stimulated with rhuIL-10 (100 ng/ml) served as positive control. Data presented is representative of one of three similar experiments. * Significant (p<0.005) compared to all the other groups.
Figure 3.
Recombinant BmHsp12.6 prevents thermal aggregation of proteins.
(A) Citrulline synthase (CS) were heat denatured at 45°C in the presence and absence of rBmHsp12.6 at different time intervals (0–40 min). BSA served as control. Thermal aggregation of proteins was determined spectrophotometrically by measuring the light scatter at 300 nm. Results show that ratio of CS: BmHsp12.6 (1∶2) was sufficient to prevent thermal aggregation of CS. Data presented are representative of three similar experiments. (B). BmHsp12.6 can bind to denatured proteins. Binding of rBmHsp12.6 to denatured Citruline synthase (CS) and luciferase (LUC) was determined using an ELISA. CS or luciferase was denatured with 6 M guanidium hydrochloride overnight at 4°C. Wells of 96 wells plate was then coated with the denatured or native CS or LUC and binding of his-tag rBmHsp12.6 or control filarial protein to the coated proteins (CS and LUC) was then analyzed using HRP-labeled penta- his antibodies by ELISA. Results show that BmHsp12.6 preferentially binds to denatured proteins. * Significant (p<0.005) binding of BmHsp12.6 to denatured proteins compared to control and native proteins.
Figure 4.
Anti-rBmHsp12.6 IgG antibody levels in the sera of human.
Levels of total IgG antibodies against (A) rBmHsp12.6 protein, (B) BmHsp12.6αc peptide or (C) N-BmHsp12.6 peptide in the sera of EN, MF, CP and NEN subjects were measured using an indirect ELISA. A total of 20 sera samples were evaluated from EN, MF, and CP and 10 samples from NEN. Each data point represents sera sample from a single individual. Horizontal lines represent geometric mean value. Data is represented as scatter plot where each dot represents absorbance of individual sera.
Figure 5.
Anti-rBmHsp12.6 IgG antibody isotypes in the human sera.
IgG isotype antibodies against (A) rBmHsp12.6 protein, (B) BmHsp12.6αc peptide or (C) N-BmHsp12.6 in the sera of EN, MF, CP and NEN subjects were measured using an indirect ELISA. A total of 20 sera samples were evaluated from EN, MF, and CP from NEN. Each data point represents sera sample from a single individual. Each bar represents the mean ± SD of five patients from each group. * Significant (p<0.05) compared to all the other three groups (CP, MF and NEN).
Table 2.
Antibody-dependant cellular cytotoxicity (ADCC) against B. malayi L3 using human serum.
Figure 6.
Proliferation of human PBMC stimulated with rBmHsp12.6.
PBMC proliferation of (A) Endemic Normal (EN), (B) Microfilaraemic (MF) and (C) Chronic Pathology (CP) groups stimulated with rBmHsp12.6. Concanavalin A (ConA) was used as a positive control. Each data point represents stimulation index (S.I) of individual human sample (n = 10).
Figure 7.
Cytokine levels in human PBMC.
Cytokines (pg/ml) in the culture supernatants of human PBMC were measured using an ELISA. Results show that significant level of IFN-γ is secreted by PBMC of EN individuals in response to rBmHsp12.6. Experiments were repeated two times. Each bar represents mean concentration ± S.D. * Significant (p<0.05) IFN-γ secretions compared to other two groups (CP and MF).
Figure 8.
Mice were immunized with A) BmHsp12.6, B) BmHsp12.6αc or C) N-BmHsp12.6 using homologous DNA vaccine regimen or a heterologous prime boost approach. Approximately, 100 ng of peptides or proteins respectively (100 ng/100 µl/well) were coated on to the wells of an ELISA plate and bound serum IgG was detected using an HRP-labeled anti-mouse IgG secondary antibody. Each data point indicates mean (± S.D) value from five animals.
Figure 9.
Isotype of anti-BmHsp12.6 IgG antibodies in the sera of mice.
Mice were immunized with A) BmHsp12.6, B) BmHsp12.6αc or C) N-BmHsp12.6 using homologous DNA vaccine regimen or a heterologous prime boost approach. Control mice were immunized with vector alone or adjuvant. Isotype specific ELISA was performed as described in the methods section. Bars represent mean O.D ± SD from five mice per group. * Significant (p<0.005) compared to all the other groups.
Table 3.
Killing of B. malayi L3 in rBmHSP12.6 vaccinated mice was evaluated by in vitro (ADCC assay using mouse sera) and in vivo (micropore chamber challenge) assays.
Figure 10.
Splenocytes from vaccinated mice proliferated in response to the antigens.
Single cell suspension of spleen cells (2×105) from vaccinated and control mice were stimulated with respective peptides or protein for 72 hrs at 37°C. Control wells were either stimulated with Con A (positive control) or left unstimulated (negative control). Mice vaccinated with A) BmHsp12.6, B) BmHsp12.6αc or C) N-BmHsp12.6 using homologous DNA vaccine regimen or a heterologous prime boost approach. A non-specific recombinant protein (rSmGBF) was used as negative control. Data is presented as mean stimulation index (S.I.) of five mice ± S.D. * Significant (p<0.005) S.I. value compared to control cells.