Immunoinformatics Design and Assessment of a Multiepitope Antigen (OvMCBL02) for Onchocerciasis Diagnosis and Monitoring

Abstract

Onchocerciasis is a Neglected Tropical Disease that has a significant socioeconomic impact, especially in Sub-Saharan Africa. Numerous reports indicate that the Expanded Special Project for the Elimination of Neglected Tropical Diseases needs novel diagnostic tools before achieving its goal of successful elimination of onchocerciasis in Africa. The current diagnostic tests are either invasive, insensitive, or not applicable in the field and about 25% of persons infected cannot mount immune responses against the single antigen used in the only approved Ov-16 serological test. In the quest to identify novel biomarkers that can be used to certify that a patient is free from the disease, evaluate the progress of elimination programmes, and conduct post elimination surveillances, mass spectrometric analysis of Onchocerca volvulus crude extract revealed that 1392 proteins are expressed in the adult and microfilariae stages of the parasite. Computational analysis predicted six of the proteins as O. volvulus potential diagnostic targets. Linear B-epitopes were predicted from the six proteins and used to construct a multiepitope antigen (OvMCBL02). Serological analysis revealed that the OvMCBL02 test significantly differentiated between serum samples of onchocerciasis patients from the Kombone Health Area in the South West Region of Cameroon (n = 63) and control serum samples from Rwanda (n = 29) and Europe (n = 26) as well as between serum samples from the onchocerciasis hyperendemic region of Kombone Health Area (n = 63) and the hypoendemic region of Bandjoun Health District (n = 54). Interestingly, the test did not cross-react with serum samples from patients suffering from related nematode infections, thereby suggesting that further characterization of the OvMCBL02 multiepitope antigen will render it an additional member of the diagnostic toolbox for the elimination of onchocerciasis.

Keywords: IgG; OvMCBL02; diagnosis; multiepitope antigen; onchocerciasis.

Figure 1

Schematic representation of OvMCBL02 multiepitope antigen with methionine coupled at the N-terminus and 6X His tag to its C-terminus. The various linear B-epitopes (LBE) are represented with different colors.

Figure 2

Secondary structure of OvMCBL02 multiepitope antigen predicted using RaptorX server with alpha-helix (8.9%), beta strands (13.1%), and coils (78.0%).

Figure 3

Humoral immune responses to OvMCBL02 multiepitope antigen by ELISA. OVS = O. volvulus serum (n = 63), HES = Hypo-endemic serum (n = 29), ECS = European control serum (n = 26), ITS = Ivermectin treated serum (n = 54). Kruskal Wallis test followed by Dunn’s test was used for multiple comparisons among the groups.

Figure 4

Evaluation of the humoral immune responses of related nematode sera to the OvMCBL02 multiepitope antigen. O. volvulus sera (OVS, n = 63), Wuchereria bancrofti sera (WBS, n = 6), Mansonella perstans sera (MPS, n = 6), Brugia malayi sera (BMS, n = 3) and Ascaris lumbricoides sera (ALS, n = 6). Kruskal–Wallis test followed by Dunn’s test were used for multiple comparisons among the groups.

Figure 5

Measurement of IgG subclasses and total IgG response to OvMCBL02 multiepitope antigen using sera pools from infected (OVS, n = pool of 10 infected serum samples) and non-infected individuals (ESC, n = pool of 10 control serum samples). Microtiter plates were coated with purified antigen for 2 h and blocking was performed overnight. Plates were incubated with serum pools from either OVS or ECS at different dilutions (1:250 to 1:32,000) followed by incubation with (A) anti-human IgG1 (Fc-specific) antibody (B), mouse monoclonal anti-Human IgG2, (C) mouse monoclonal anti-Human IgG3, (D) mouse monoclonal anti-Human IgG (HRP), or (E) goat anti-human IgG peroxidase conjugate as the secondary antibody. TMB was used for revelation and optical densities read at 450 nm. OD values were plotted against the different serum types.