TSP LEL specific IgG antibodies in the sera of mice.

<p>Mice were immunized four times at two weeks interval using 15 µg of r<i>Wb</i>TSP LEL combined with alum adjuvant. <b>A</b>) <b>Titer of anti-TSP LEL IgG antibodies</b>. Approximately, 100 ng of recombinant proteins (100 ng /100µl /well) were coated onto 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. <b>B</b>). <b>Isotype of anti-TSP LEL IgG antibodies in the sera of mice</b>. Isotypes specific ELISA was performed as described in the methods section. Bars represent mean ±SD from five mice per group. ** Significant (P<0.001) and * (P<0.05) compared to control groups (Student’s t-test).</p

Sera from individuals who are putatively immune against <i>O. volvulus</i> infections (PI) has antibodies that cross react with r<i>Wb</i>TSP LEL.

<p>Isotype of IgG antibody <b>A</b>) IgG1 and <b>B</b>) IgG3 reactivity was measured in the sera of putatively immune individuals (PI) and <i>O. volvulus</i> infected individuals (INF) against r<i>Wb</i>TSP LEL. Each data point represents sera sample from a single individual (n=20). Horizontal lines represent median value. Data is represented as scatter plot where each dot represents absorbance of individual sera. PI sera showing significant (P<0.001) cross reactivity of IgG1 antibodies compared to INF individuals. The OD of NEN (NYC individuals) was always below OD 0.1. Significant (P<0.001) levels of isotype antibodies in PI compared to INF group (Student’s t-test). </p

TSP LEL specific IgG isotype of antibodies in the sera of human.

<p>Isotype of IgG antibodies A) IgG1, B) IgG2, C) IgG3 and D) IgG4 against r<i>Wb</i>TSP LEL were measured in the sera of putatively immune individuals (n=10) using an indirect ELISA. 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. Significant *(P<0.05) levels of isotype antibodies in EN individuals compared to other groups (One way ANOVA along with Tukey-Kramer post statistics test was used).</p

Characterizations of TSP LEL.

<p>A. <i>Bm</i>TSP LEL is expressed on the cuticle of <i>B. malayi</i> L3. Presence of <i>Bm</i>TSP LEL on the surface of <i>B. malayi</i> L3 was demonstrated by staining L3 with mouse anti-<i>Bm</i>TSP LEL antibodies followed by FITC conjugated anti-mouse IgG (1,2). Green fluorescence denotes regions where antibody was bound. No fluorescence found when L3 was stained with control negative sera (3). Scale bar is 11µm. B. Multiple sequence alignment of Tetraspanin was performed using clustalW online tools. Sequence alignment showed that LEL domain of <i>Wb</i>TSP is 100% similar to <i>Bm</i>TSP and 97% similar to <i>Ov</i>TSP. Arrow showing only two amino acids (C₃₅-Y₃₅ /D₄₈ -G₄₈) of <i>Ov</i>TSP LEL was different from <i>Bm</i>TSP LEL and/or <i>Wb</i>TSP LEL. C. Immunoblot was performed by probing anti-His tag, anti-<i>Bm</i>TSPLEL or anti-<i>Wb</i>TSPLEL antibodies. Both anti-<i>Wb</i>TSPLEL and anti-<i>Bm</i>TSP LEL antibodies cross reacted with r<i>Ov</i>TSP LEL proteins.</p

Recombinant BmHsp12.6 prevents thermal aggregation of proteins.

<p>(<b>A</b>) 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. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034077#s3" target="_blank">Results</a> 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>B</b>). <b>BmHsp12.6 can bind to denatured proteins</b>. 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. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034077#s3" target="_blank">Results</a> 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.</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

Predicted B-cell, T-cell and CTL epitopes in BmHsp12.6 sequences.

a<p>Immune Epitope Database and Analysis Resource (IEDB) was used for the prediction.</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

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