Splenocytes from vaccinated mice proliferated in response to the antigens.

<p>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 <b>A</b>) BmHsp12.6, <b>B</b>) BmHsp12.6αc or <b>C</b>) 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.</p

MC/9 proliferation assay.

<p>N-BmHsp12.6 peptide enhances the growth of MC/9 mast cells <i>in vitro</i>. 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 <b>A</b>) N- BmHsp12.6 <b>B</b>) BmHsp12.6 and <b>C</b>) 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.</p

Cytokine levels in human PBMC.

<p>Cytokines (pg/ml) in the culture supernatants of human PBMC were measured using an ELISA. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034077#s3" target="_blank">Results</a> 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).</p

Anti-rBmHsp12.6 IgG antibody levels in the sera of human.

<p>Levels of total IgG antibodies against (<b>A</b>) rBmHsp12.6 protein, (<b>B</b>) BmHsp12.6αc peptide or (<b>C</b>) 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.</p

Killing of <i>B. malayi</i> L3 in rBmHSP12.6 vaccinated mice was evaluated by <i>in vitro</i> (ADCC assay using mouse sera) and <i>in vivo</i> (micropore chamber challenge) assays.

a<p>ADCC assay was performed by incubating 50 µl of pooled mice sera (n = 5) samples with 2×10⁵ normal peritoneal exudates cells and 10 <i>B. malayi</i> L3 at 37°C for 48 hrs. Values represent mean ± SD of three wells.</p>b<p><i>In vivo</i> micropore chamber assay was performed by surgically implanting 20 <i>B. malayi</i> L3 into the peritoneal cavity of each mouse. 48 hrs after implantation, chambers were removed and larval viability and death determined. Values are mean ± SD. N = 5. Data presented is from one of two similar experiments showing comparable results.</p>*<p>Significant larval death (P<0.01) compared to other mice groups.</p

Isotype of anti-BmHsp12.6 IgG antibodies in the sera of mice.

<p>Mice were immunized with <b>A</b>) BmHsp12.6, <b>B</b>) BmHsp12.6αc or <b>C</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034077#s2" target="_blank">methods</a> section. Bars represent mean O.D ± SD from five mice per group. * Significant (p<0.005) compared to all the other groups.</p

Structural analysis of BmHsp12.6.

<p>(<b>A</b>) Secondary structure prediction of BmHsp12.6 showing α-helices (H1 and H2), strands by their sheets A, B, (<b>β</b>)- beta turn, (<b>γ</b>)- gamma turn and beta hairpin (<b>⊃</b>). (<b>B</b>) Graphical representation showing N-BmHsp12.6 and BmHsp12.6αc peptide sequences and their functional roles in the BmHsp12.6 protein.</p