Immune response studies with Wuchereria bancrofti vespid allergen homologue (WbVAH) in human lymphatic filariasis

Abstract

A homologue of Brugia malayi venom allergen (BmVAH) was cloned from the infective stages (L3) of Wuchereria bancrofti. Sequence analysis showed 90% sequence identity between WbVAH and BmVAH. Recombinant WbVAH was then expressed and purified. VAH from other nematode parasites is being evaluated as potential vaccine candidates. Because W. bancrofti infections are more prevalent than B. malayi, it will significantly benefit using W. bancrofti antigens for vaccine development. In this study, we have evaluated the human immune responses to rWbVAH in putatively immune individuals who live in the endemic regions (endemic normal, EN) to determine the vaccine potential of WbVAH. These responses were then compared to those in infected individuals (microfilaraemic, MF and chronic pathology, CP). Results show that EN subjects carry WbVAH-specific IgG1, IgG2, and IgG3 circulating antibodies. It is interesting to note that CP patients also carried antibodies against WbVAH that was mainly of the IgG3 isotype. Peripheral blood mononuclear cells (PBMC) from EN individuals responded strongly to rWbVAH by proliferating and secreting IFN-gamma. PBMC from MF patients also proliferated in response to rWbVAH but secreted mainly IL-10. Thus, there was a clear dichotomy in the cytokine production by infected patients vs individuals who are putatively immune (EN). Although vaccine potential of WbVAH has not been established yet, our findings suggest that WbVAH mediated immune responses in EN individuals is primarily Th1-biased. Further vaccination studies are underway in animal models to determine the role of WbVAH in protective immunity against W. bancrofti and B. malayi infections.

Fig. 1

Expression of WbVAH in E. coli GJ1158 host. a Total protein extracts from WbVAH and control pRSET A were solubilized in 1×SSB, separated on a 12% SDS-PAGE gel, and stained with Coomassie brilliant blue dye; 50 μg of protein was loaded in each of the respective lanes. Lane M molecular weight marker; lane 1 vector-uninduced; lane 2 vector-induced; lane 3 0.3 M NaCl WbVAH-uninduced; lane 4 WbVAH-induced at 3 h; lane 5 purified protein eluted at 200 mM imidazole. b Total protein extracts after inducing the recombinant clone WbVAH with NaCl were separated on 12% SDS-PAGE, transferred to nitrocellulose membrane, and probed with mouse monoclonal anti-Histidine antibody (1:2,000) followed by incubation with goat anti-mouse antibody conjugated with alkaline phosphatase (1:30,000) and developed with NBT and BCIP. Lane P anti-His antibody; lane M marker

Fig. 2

Levels of WbVAH-specific IgG antibodies in the sera of various clinical groups of lymphatic filariasis. Wells were coated with rWbVAH and sera collected from EN, MF, and CP patients were added at 1:100 dilution. a Levels of total IgG reactivity were determined by ELISA. Sera from 10 subjects were tested in each group. Each dot in the scatter plot indicates individual sera. Total IgG reactivity with WbVAH was significantly higher in EN (P<0.05) when compared to CP or MF groups. b Isotype analysis of the WbVAH-specific IgG antibodies in the various clinical groups was determined by isotype-specific ELISA for IgG1, IgG2, IgG3, and IgG4. Levels of IgG1, IgG2, and IgG3 antibodies were significantly higher (P<0.05) in EN individuals. CP individuals had higher levels of IgG3. IgG1, IgG2, and IgG3 antibodies were also present in sera from MF individuals. However, levels were comparatively lower than EN and CP individuals. It is interesting to note that IgG4 levels were consistently low in all individuals

Fig. 3

Lymphoproliferative responses to WbVAH. PBMCs from EN, MF, and CP individuals were cultured at 0.2×10⁶ cells/ml and stimulated with either recombinant WbVAH, BmA or ConA. Cells were incubated for 72 h in 5% CO₂ environment. Lymphocyte proliferation was measured by Cell Titre96^® aqueous non-radio-activecell proliferation assay kit (MTT). Results are expressed as the SI. Vertical bar denotes the geometric mean of all 10 samples from each group. EN endemic normal, CP chronic pathology, MF microfilaraemic

Fig. 4

IFN-γ and IL-10 responses to rWbVAH in human PBMCs. PBMCs collected from EN, MF, and CP individuals were cultured at 4×10⁶ cells/ml and stimulated with rWbVAH (10 μg/ml) for 96 h (a) or 48 h (b) at 37°C. After incubation, culture supernatants were collected and levels of IFN-γ and IL-10 were measured using the ELISA. Cells cultured in media alone were used as controls. Results show that IFN-γ levels (a) were significantly increased (P<0.05) in EN individuals compared to CP or MF, whereas IL-10 levels (b) were significantly elevated in MF individuals compared to the EN and CP groups. Bars represent the geometric mean of 10 samples