Prediction and validation of the structural features of Ov58GPCR, an immunogenic determinant of Onchocerca volvulus

Abstract

Onchocerciasis is a severely debilitating yet neglected tropical disease (NTD) that creates social stigma, generates and perpetuates poverty, and leads ultimately in some cases to irreversible unilateral or bilateral blindness if untreated. Consequently, the disease is a major impediment to socioeconomic development. Many control programs have been launched for the disease with moderate successes achieved. This mitigated hit is partially due to the lingering need for reliable, non-invasive and easily applicable tools for mapping endemic regions and post-elimination surveillance. In this work, bioinformatics analyses combined with immunological assays were applied in a bid to develop potential tools for diagnosis and assessing the success of drug treatment programs. We report that (i) the O. volvulus antigen, Ov58GPCR is a G-protein coupled receptor (GPCR) conserved in related nematodes, (ii) synthetic peptides predicted to be in the extracellular domain (ECD) of Ov58GPCR are indeed immunogenic epitopes in actively-infected individuals, (iii) synthetic peptide cocktails discriminate between actively-infected individuals, treated individuals and healthy African controls, (iv) polyclonal antibodies against one of the peptides or against the bacterially-expressed ECD reacted specifically with the native antigen of O. volvulus total and surface extracts, (v) Ov58GPCR is transcribed in both larvae and adult parasite stages, (vi) IgG and IgE responses to the recombinant ECD decline with ivermectin treatment. All these findings suggest that the extracellular domain and synthetic peptides of Ov58GPCR, as well as the specific immune response generated could be harnessed in the context of disease diagnosis and surveillance.

Fig 1. Ov58GPCR is a 7-transmembrane protein conserved in related nematodes.

(A) Pictorial presentation of Ov58GPCR transmembrane topology showing the long extracellular domain and the 7 transmembrane helix regions (numbered 1 to 7) as generated by MEMSATSVM and TOPCONS and illustrated by PROTTER (Red residues indicate the signal peptide). (B) Multiple sequence alignment of Ov58GPCR with homologues in some key nematodes revealing the respective identity percentages: O. volvulus (OVOC5284), O. flexuosa (A0A183HRS3), 76.0%; O. ochengi (A0A182E0H8), 97.2%; Wuchereria bancrofti (A0A1I8EAM4), 82.3%; Brugia malayi (A0A0K0JJD3) 81.5%; Brugia pahangi (A0A0N4TKQ9) 81.5%; Brugia timori (A0A0R3QZE7) 81.3%;, Ascaris suum (U1MP61) 53.8%; A. lumbicoides (A0A0M3HY48) 53.8%; Loa loa (A0A1S0TX81) 63.5% and Caenorhabditis elegans (Q22162) 44.2%; data were retrieved from WormBase and aligned using Multalin. Consensus levels were set at the default parameters of the program: red color indicates a high consensus (90%), blue color a low consensus (50%) and black color stands for neutral. (C) The above related orthologues were used to depict the phylogenetic relationship between Ov58GPCR in O. volvulus and its homologues in other parasites. The evolutionary history was inferred by using the Maximum Likelihood method based on the JTT matrix-based model to estimate the distances. and phylogeny was tested using the Bootstrap method. The tree was drawn to scale and the Bootstrap values are indicated on the tree.

Fig 2. Ov58GPCR harbours immunogenic epitopes in its extracellular domain.

(A) Serum samples collected from all participants (OVS, ITS and HES) were investigated for the presence of epitope-specific IgG against 4 linear B-epitopes of Ov58GPCR (P1, P2, P3 and P4 (a shorter version of P1) predicted using bioinformatics tools (see Table 1) and selected for synthesis. (B) ELISA plates were coated overnight with the indicated synthetic peptide. IgG responses to each peptide were assessed for all individual samples diluted 1:800 from infected persons (OVS) initially to evaluate the immunoreactivity of each epitope. (C-F) Thereafter, the ability of each of the peptides to distinguish between infected individuals and controls was tested using individual serum samples from Onchocerca volvulus-infected individuals (OVS) and controls (ITS & HES). Following ANOVA analysis, p-value less than 0.05 was considered statistically significant. OVS: Onchocerca volvulus serum or Human onchocerciasis serum (serum from actively infected people), ITS: Ivermectin Treatment Serum (serum from an onchocerciasis near-elimination setting), HES: serum from an onchocerciasis-hypoendemic setting). The confidence intervals are indicated.

Fig 3. Increased antigen-specific antibody responses to Ov58GPCR peptide cocktails are associated with onchocerciasis infection.

ELISA plates were coated with the different indicated peptide combinations (A-I). The combination P1-P4 was not considered as the two sequences overlap. IgG responses to the peptide cocktails were measured for all individual serum samples diluted 1:800 for infected persons and the controls. OVS: Human onchocerciasis serum, ITS (Ivermectin treatment serum, HES:hypo-endemic African serum). Following ANOVA analysis, p-values less than 0.05 were considered statistically significant. The confidence intervals are indicated.

Fig 4. The recombinant extra-cellular domain of Ov58GPCR (Ov58GPCR-ECD) can be found in monomeric and dimeric forms.

(A) The gene fragment coding for the extra-cellular domain of Ov58GPCR (green portion), without the predicted signal peptide (red portion) was cloned in pET30a+ and the construct was expressed in E. coli cells. (B) and (C) The protein, initially purified on a Ni⁺⁺-IMAC column (B) and further purified by size exclusion chromatography (C) was resolved on a 12% SDS-PAGE gel and stained with Coomassie blue,. (D) Anti-6xHis monoclonal antibodies and (E) anti-Ov58GPCR-ECD antibodies detected two forms of the protein on nitrocellulose membranes at molecular weights of approximately 23 kDa and 46 kDa matching the expected monomeric and dimeric forms respectively. Green and black arrow heads indicate the positions of the putative monomeric and dimeric forms of Ov58GPCR-ECD respectively. Arrows indicate contaminants. M: protein ladder, P: pellet, F: flow-through, W: wash, E: Eluate, E(x): Eluate Fraction, PI: pre-immune serum, α-HIS: anti-His and α-ECD: anti-ECD.

Fig 5. Exposure of host to parasite transmission leads to increased Ov58GPCR-ECD-specific IgG but not IgE response following treatment.

ELISA plates were coated with purified recombinant protein at 4 °C. Humoral responses were measured in all individual serum samples diluted 1:1000 for (A) IgG and 1:30 for (B) IgE (after IgG depletion) in all infected persons and controls. OVS = Human Onchocerciasis Serum (58); ENS = Endemic Non symptomatic Serum (24); ITS = Ivermectin treatment serum (68); LLS = Human Loiasis Serum (50); HES = Hypo-endemic African Serum (50), and ECS = European Serum Control (3). Following ANOVA analysis, p-values less than 0.05 were considered statistically significant. The confidence intervals are indicated. The number of samples selected for each group is indicated into brackets.

Fig 6. Increased ivermectin treatment length is associated with decline in IgG subclasses and total IgE responses to Ov58GPCR-ECD.

ELISA plates were coated with purified recombinant protein and incubated overnight at 4°C. Total IgG responses were measured in all individual serum samples of the ITS group diluted 1:1000 for IgG (A) and 1:30 for IgE (B) and for IgG subclasses (Figs 6C-6F) after grouping sera according to years of ivermectin treatment. Following ANOVA analysis, p-values less than 0.05 were considered statistically significant. (0: never taken ivernectin, 1–6: taken ivermectin from 1 to 6 years, 7–12: taken ivermectin for between 7 and 12 years, >13: taken ivermectin for 13 years and above).

Fig 7. Ov58GPCR is transcriptionally and translationally activated in larva and adult parasite stages.

(A) Two sets of primers targeting specific regions of Ov58GPCR across adjacent introns were used for stage-specific detection of Ov58GPCR in both genomic DNA and total cDNA preparations. (B) cDNA and genomic DNA of O. volvulus Mf, L2, L3, adult male (AM) and adult female (AF) parasitic stages were prepared and analysed. The first primer pair F2-R3 produced amplicons of 390 bp (from genomic DNA) and 111 bp (from cDNA) as predicted. The second primer set, F8-R9 specifically amplified a 338 bp product from genomic DNA and a 187 bp product from cDNA as predicted. The amplicons were resolved, ethidium bromide-stained and UV-visualized on agarose gels. (C) SDS-PAGE analysis and Coomassie staining of worm total protein extracts. (D-E) Western blot analysis of Ov58GPCR (green arrow) expression in O. volvulus extracts. (F) Western blot monitoring of Ov58GPCR (green arrow) in cuticular protein extract. mf: microfilaria extract, AM: adult male extract, AF: adult female extract, L2 and L3: parasite larval stages, CE: cuticular surface extract, TE: total extract.