Biochemical Characterization and Evaluation of a Brugia malayi Small Heat Shock Protein as a Vaccine against Lymphatic Filariasis

Filarial nematodes enjoy one of the longest life spans of any human pathogen due to effective immune evasion strategies developed by the parasite. Among the various immune evasion strategies exhibited by the parasite, Interleukin 10 (IL-10) productions and IL-10 mediated immune suppression has significant negative impact on the host immune system. Recently, we identified a small heat shock protein expressed by Brugia malayi (BmHsp12.6) that can bind to soluble human IL-10 receptor alpha (IL-10R) and activate IL-10 mediated effects in cell lines. In this study we show that the IL-10R binding region of BmHsp12.6 is localized to its N-terminal region. This region has significant sequence similarity to the receptor binding region of human IL-10. In vitro studies confirm that the N-terminal region of BmHsp12.6 (N-BmHsp12.6) has IL-10 like activity and the region containing the alpha crystalline domain and C-terminus of BmHsp12.6 (BmHsp12.6αc) has no IL-10 like activity. However, BmHsp12.6αc contains B cell, T cell and CTL epitopes. Members of the sHSP families are excellent vaccine candidates. Evaluation of sera samples from putatively immune endemic normal (EN) subjects showed IgG1 and IgG3 antibodies against BmHsp12.6αc and these antibodies were involved in the ADCC mediated protection. Subsequent vaccination trials with BmHsp12.6αc in a mouse model using a heterologous prime boost approach showed that 83% protection can be achieved against B. malayi L3 challenge. Results presented in this study thus show that the N-BmHsp12.6 subunit of BmHsp12.6 has immunoregulatory function, whereas, the BmHsp12.6αc subunit of BmHsp12.6 has significant vaccine potential

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

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

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

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

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

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