Evaluation of Wuchereria bancrofti GST as a vaccine candidate for lymphatic filariasis

Abstract

Background: Lymphatic filarial parasites survive within the lymphatic vessels for years despite the complex immune environment surrounding them. Parasites possibly accomplish this by adopting various immunomodulatory strategies, which include release of glutathione-S-transferases (GSTs) that counteract the oxidative free radicals produced by the host. Since GSTs produced by parasites appear to be critical for the survival of parasites in the host, several studies evaluated the potential of parasite GSTs as vaccine candidates especially against schistosomiasis, fascioliasis and Seteria cervi. However, vaccine potential of GSTs of lymphatic filarial parasites has not been evaluated before.

Methods/principal findings: In the present study, the GST gene was cloned from the third stage larval (L3) cDNA libraries of Wuchereria bancrofti, and recombinant GST (WbGST) was expressed and purified. Serum samples from individuals living in an endemic area were analyzed for their reactivity with rWbGST. These findings showed that sera from endemic normal individuals (EN) carry significant levels of anti-WbGST IgG antibodies compared to subjects who are microfilaraemic (Mf) or show symptoms of clinical pathology (CP). Isotype analysis of the anti-WbGST IgG antibodies showed a predominance of IgG1 and IgG3 antibodies in EN individuals. Subsequent functional analysis of the rWbGST showed that the rWbGST protein retained the enzymatic activity of GST and the antibodies in EN sera could inhibit this enzymatic activity. Similar results were obtained when anti-rWbGST antibodies raised in mice were used in the neutralization assay. Brugia malayi GST and WbGST show significant sequence similarity. Therefore, to evaluate the vaccine potential of rWbGST, we used B. malayi L3 as challenge parasites. Vaccine potential of rWbGST was initially evaluated by confirming the role of human and mice WbGST antibodies in an antibody dependent cellular cytotoxicity (ADCC) assay. Subsequent vaccination studies in a jird model showed that approximately 61% protection could be achieved against a B. malayi L3 challenge infection in jirds immunized with rWbGST.

Conclusions: Results of this study show that rWbGST is a potential vaccine candidate against lymphatic filariasis. Nearly 61% protection can be achieved against a B. malayi challenge infection in a jird model. The study also showed that the WbGST protein retained the enzymatic activity of GST and this enzymatic activity appears to be critical for the survival of the parasite in the host.

Figure 1. Multiple sequence alignment of WbGST.

Multiple sequence alignments (CLUSTAL) of the amino acid sequences of GST family of proteins from W. bancrofti (WbGST; accession no. AY195867), Brugia malayi (BmGST, accession no. Y12788), Onchocerca volvulus (OvGST, accession no. L28771) and Dirofilaria immitis (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.

Figure 2. Expression and purification of rWbGST.

Cultures of E. coli 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.

Figure 3. Human immune responses to WbGST.

A. Immunoreactivity of rWbGST with sera from different clinical groups of bancroftian filariasis. Approximately 1 µg of rWbGST was resolved on 15% SDS-PAGE, transferred to nitrocellulose membrane and blots were probed with pooled sera from MF, CP, EN or NEN individuals. Results showed strong immunoreactivity with pooled EN sera followed by CP and MF but no reactivity was detected with control NEN sera. Data is representative of one of three experiments using the same sera samples. B. Humoral immune response to rWbGST in human subjects. Total IgG levels against rWbGST in various clinical groups were evaluated by ELISA. Each data point represents single individual absorbance from the four different groups. Horizontal lines represent geometric mean value of EN (43), Mf (45), CP (45) and NEN (39) samples respectively. C. WbGST-specific IgG subclasses in the sera from different clinical groups. Isotype of anti-WbGST IgG antibodies in the sera from various groups of human subjects (EN, MF, CP, TPE and NEN) were evaluated using an isotype-specific ELISA. Data presented is mean±SD value from EN (43), Mf (45), CP (45) and NEN (39). * Significant (p<0.005) compared to all the other three groups (CP, MF and NEN). The statistical significance was calculated by Kruskal–Wallis test.

Figure 4. Subclass specific anti-WbGST IgG antibodies in the sera of mice immunized with rWbGST protein.

Subclass analysis was performed using a mouse antibody isotyping ELISA kit. The bars represent the mean O.D.±SD at 405 nm of five mice per group.

Figure 5. Stage-specific expression of filarial GST.

WbGST gene was PCR amplified form W.bancrofti stage-specific cDNA libraries using WbGST gene specific forward and reverse primers. Results show that the WbGST gene is predominantly expressed in the L3 stage of the W.bancrofti and is barely detectable in adult female and microfilarial stages. Lane 1: 100 bp ladder; 2: W. bancrofti L3; 3: Adult Female; and 4: Mf.

Figure 6. Anti-WbGST antibodies can neutralize the enzymatic activity of rWbGST.

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.

Figure 7. Photomicrograph of L3 recovered from cultures after ADCC assay.

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.