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

Large extracellular loop of tetraspanin as a potential vaccine candidate for filariasis.

Lymphatic filariasis affects nearly 120 million people worldwide and mass preventive chemotherapy is currently used as a strategy to control this infection. This has substantially reduced the incidence of the infection in several parts of the world. However, a prophylactic vaccine would be more effective in preventing future infections and will supplement the mass chemotherapy efforts. Unfortunately, there is no licensed vaccine available currently to prevent this infection. Molecules expressed on the surface of the parasite are potential candidates for vaccine development as they are exposed to the host immune system. In this study we show that the large extracellular loop of tetraspanin (TSP LEL), a protein expressed on the cuticle of Brugia malayi and Wuchereria bancrofti is a potential vaccine candidate. Our results showed that BmTSP LEL is expressed on the surface of B. malayi infective third stage larvae (L3) and sera from human subjects who are putatively immune to lymphatic filariasis carry high titer of IgG1 and IgG3 antibodies against BmTSP LEL and WbTSP LEL. We also showed that these antibodies in the sera of human subjects can participate in the killing of B. malayi L3 in an antibody dependent cell-mediated cytotoxicity mechanism. Vaccination trials in mice showed that close to 64% protection were achieved against challenge infections with B. malayi L3. Immunized animals showed high titer of anti-WbTSP LEL IgG1, IgG2a and IgG2b antibodies in the sera and IFN-γ secreting cells in the spleen. Onchocerca volvulus another filarial parasite also expresses TSP LEL. Cross-reactivity studies showed that IgG1 antibody in the sera of endemic normal subjects, recognize OvTSP LEL. Similarly, anti-OvTSP LEL antibodies in the sera of subjects who are immune to O. volvulus were also shown to cross-react with rWbTSP LEL and rBmTSP LEL. These findings thus suggested that rTSP LEL can be developed as a potential vaccine candidate against multiple filarial infections

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

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

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

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

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