Evaluation of Wuchereria bancrofti GST as a Vaccine Candidate for Lymphatic Filariasis

Lymphatic parasites survive for years in a complex immune environment by adopting various strategies of immune modulation, which includes counteracting the oxidative free radical damage caused by the host. We now know that the filarial parasites secrete antioxidant enzymes. Among these, the glutathione-S-transferases (GSTs) have the potent ability to effectively neutralize cytotoxic products arising from reactive oxygen species (ROS) that attack cell membranes. Thus, GSTs have the potential to protect the parasite against host oxidative stress. GSTs of several helminthes, including schistosomes, fasciola and the filarial parasite Seteria cervi, are also involved in inducing protective immunity in the host. The schistosome 28 kDa GST has been successfully developed into a vaccine and is currently in Phase II clinical trials. Thus, GST appears to be a potential target for vaccine development. Therefore, in the present study, we cloned W. bancrofti GST, and expressed and purified the recombinant protein. Immunization and challenge experiments showed that 61% of protection could be achieved against B. malayi infections in a jird model. In vitro studies confirm that the anti-WbGST antibodies participate in the killing of B. malayi L3 through an ADCC mechanism and enzymatic activity of WbGST appears to be critical for this larvicidal function

Targeting folate metabolism for therapeutic option: A bioinformatics approach

762-766Lymphatic filariasis, commonly called elephantiasis, poses a burden of estimated level of 5.09 million disability adjusted life year. Limitations of its sole drug, diethylcarbamazine (DEC) drive exploration of effective filarial target. A few plant extracts having polyphenolic ingredients and some synthetic compounds possess potential dihydrofolate reductase (DHFR) inhibitory effect. Here, we postulated a plausible link between folates and polyphenolics based on their common precursor in shikimate metabolism. Considering its implication in structural resemblance based antagonism, we have attempted to validate parasitic DHFR protein as a target. The bioinformatics approach, in the absence of crystal structure of the proposed target, used to authenticate and for virtual docking with suitable tested compounds, showed remarkably lower thermodynamic parameters as opposed to the positive control. A comparative docking analysis between human and Brugia malayi DHFR also showed effective binding parameters with lower inhibition constants of these ligands with parasitic target, but not with human counterpart highlighting safety and efficacy. This study suggests that DHFR could be a valid drug target for lymphatic filariasis, and further reveal that bioinformatics may be an effective tool in reverse pharmacological approach for drug design

<i style="mso-bidi-font-style:normal"><span style="font-size:11.0pt;font-family:"Times New Roman";mso-fareast-font-family: "Times New Roman";mso-bidi-font-family:Mangal;mso-ansi-language:EN-GB; mso-fareast-language:EN-US;mso-bidi-language:HI" lang="EN-GB">Brugia malayi</span></i><span style="font-size:11.0pt;font-family:"Times New Roman";mso-fareast-font-family: "Times New Roman";mso-bidi-font-family:Mangal;mso-ansi-language:EN-GB; mso-fareast-language:EN-US;mso-bidi-language:HI" lang="EN-GB"> abundant larval transcript 2 protein treatment attenuates experimentally-induced colitis in mice</span>

732-739Helminths are known to modulate host’s immunity by suppressing host protective pro-inflammatory responses. Such immunomodulatory effects have been experimentally shown to have therapeutic implications in immune mediated disorders. In the present study, we have explored a filarial protein i.e. Brugia malayi recombinant abundant larval transcript 2 (rBmALT2) for its therapeutic effect in dextran sodium sulfate (DSS) induced colitis in mouse model. The immunomodulatory activity of rBmALT-2 was initially confirmed by demonstrating that it suppressed the lipopolysaccharide (LPS) induced nitric oxide synthesis and down-regulated the expression of pro-inflammatory cytokines in vitro by peritoneal exudate cells of mice. Treatment with rBmALT2 reduced severity of colitis associated with significant reduction in weight loss, disease activity, colon damage, mucosal edema and histopathological score including myeloperoxidase activity in colon tissues. rBmALT2 was comparatively more effective in attenuation of colitis when used in the preventive mode than when used for curative purpose. The therapeutic effect of rBmALT2 was found to be associated with downregulation of IFN-γ, IL-6, IL-17 and upregulation of IL-10 cytokines. These results provide strong experimental evidence that BmALT2 could be a potential alternative therapeutic agent in colitis

Multiple sequence alignment of WbGST.

<p>Multiple sequence alignments (CLUSTAL) of the amino acid sequences of GST family of proteins from <i>W. bancrofti</i> (WbGST; accession no. AY195867), <i>Brugia malayi</i> (BmGST, accession no. Y12788), <i>Onchocerca volvulus</i> (OvGST, accession no. L28771) and <i>Dirofilaria immitis</i> (DiGST, accession no. P46426). The amino acid positions are numbered above the amino acid sequences. Multiple alignment results show that GSTs from filarial parasites are highly identical to each other. (*) red color denotes identical amino acids, (:) green color denotes strongly similar amino acids and (.) blue color denotes weakly similar amino acids.</p

Photomicrograph of L3 recovered from cultures after ADCC assay.

<p>A. L3s incubated in NEN sera and PBMCs. There are no cells adhered to the larva and the larva was active. B. L3s incubated in EN sera and PBMCs. Note the cluster of cells adhered throughout the surface of larvae, but more specifically to the anterior and posterior end of the larva.</p

Expression and purification of rWbGST.

<p>Cultures of <i>E. coli</i> containing pRSETA and rWbGST expression construct were induced with 1 mM IPTG. Following induction, rWbGST was purified from the cultures using a cobalt metal affinity chromatography column. Approximately 1 µg of the purified protein was then separated in a 15% SDS-PAGE and stained with coomassie brilliant blue R250. Lane 1- pRSET-A uninduced, Lane 2- pRSET-A induced, Lane 3- rWbGST uninduced, Lane 4- rWbGST induced, Lane 5- Purified rWbGST and Lane M is protein molecular weight marker.</p

Anti-WbGST antibodies can neutralize the enzymatic activity of rWbGST.

<p>rWbGST was added to various test sera (pooled EN sera, sera from mouse immunized with rWbGST, EN and mouse sera depleted of anti WbGST antibodies) and incubated for 1 hr at 37°C followed by another incubation at 4°C for 4 h. Following incubation, GST enzyme activity was determined spectrophotometrically at 340 nm by measuring enzymatic conjugation of glutathione with 1-chloro-2, 4-dinitrobenzene (CDNB). Individual bar represents mean±SD of GST activity (µM/min/mg). Samples were read in triplicate wells. Data is representative of one of three similar experiments. Pre-immune sera from mice and sera from NEN individuals were used as control samples. * Significant (p<0.005) compared to pre-immune or NEN sera samples. ** significant (p<0.01) compared to non-depleted immune sera or EN sera samples.</p

The larval specific lymphatic filarial ALT-2: Induction of protection using protein or DNA vaccination

Genes from the infective stage of lymphatic filarial parasites expressed at the time of host invasion have been identified as potential vaccine candidates. By screening an L3 cDNA library with sera from uninfected longstanding residents of an area endemic for onchocerciasis, so-called “endemic normals” (EN), we have cloned and characterized one such gene termed the abundant larval transcript two (ALT-2). The stage specificity of ALT-2 gene transcription and protein synthesis was confirmed by PCR using genespecific primers, and by western blot analysis of protein extracts from various stages of the parasite life cycle using specific antisera. Significant differences in antibody response to the recombinant ALT-2 were observed in endemic populations with differing clinical manifestations of lymphatic filariasis with an antibody response present in sera from 18 of 25 (72%) EN subjects compared to 9 of 25 (36%) with subclinical microfilaracmia (MF) and 14 of 25 (52%) of those with chronic lymphatic obstruction (CP) (P=0.01 for comparison of EN to CP or to MF). This differential responsiveness suggests that the protective immunity postulated to account for their uninfected status might be associated with a response to this protein. When the utility of ALT-2 as a vaccine candidate was tested in a murine model using either recombinant protein or a DNA vaccine construct, statistically significant protection was observed when compared to a control filarial gene product expressed across all stages of the parasite lifecycle (SXP-1; P=0.02 for protein and P=0.01 for the DNA vaccine) or compared to adjuvant alone. This level of protection indicates that this vaccine is a promising candidate for further development

Evaluation of preventive effect of <em>Brugia malayi</em> recombinant cystatin on mBSA-induced experimental arthritis

655-660Epidemiological and experimental studies have demonstrated the therapeutic efficacy of the helminths derived immunomodulatory molecules. In this study, we investigated the preventive effect of Brugia malayi recombinant cystatin (rBmCys) in methylated bovine serum albumin (mBSA)-induced arthritis. Mastomys coucha rats were treated with 4 doses of rBmCys (intraperitoneal) in alum adjuvant (25 µg/dose/200 µL) in intervals of 15 days. Control rats received alum only. mBSA-arthritis induction was done 10 days after the last dose of rBmCys/alum. Rats were sacrificed when all the rats in mBSA group developed arthritis. Administration of rBmCys significantly (P=0.0005) protected rats from arthritis by reducing paw swelling and arthritic index. In rBmCys treated rats, histopathology of hind paw joints showed decreased synovitis, bone erosion, fibrosis and influx of inflammatory cells. This protective effect was found to be associated with significantly (P BmCys can benefit in rheumatoid arthritis prevention

Results of antibody-dependent cellular cytotoxicity (ADCC) assay against heterologous <i>B. malayi</i> L3.

<p>In these assays, L3s were incubated with pooled sera from EN individuals and 1×10⁵ PBMCs. One group of L3s were treated with 14 µM of 1-chloro-2, 4-dinitrobenzene for 2 hrs then used in the ADCC assay.</p>*<p>Statistically significant (p<0.001) compared to alum controls.</p>**<p>Statistically significant (p<0.001) compared to non-depleted sera.</p