Identification of sVSG117 as an immunodiagnostic antigen and evaluation of a dual-antigen lateral flow test for the diagnosis of human african trypanosomiasis

The diagnosis of human African trypanosomiasis (HAT) caused by Trypanosoma brucei gambiense relies mainly on the Card Agglutination Test for Trypanosomiasis (CATT). There is no immunodiagnostic for HAT caused by T. b. rhodesiense. Our principle aim was to develop a prototype lateral flow test that might be an improvement on CATT.Pools of infection and control sera were screened against four different soluble form variant surface glycoproteins (sVSGs) by ELISA and one, sVSG117, showed particularly strong immunoreactivity to pooled infection sera. Using individual sera, sVSG117 was shown to be able to discriminate between T. b. gambiense infection and control sera by both ELISA and lateral flow test. The sVSG117 antigen was subsequently used with a previously described recombinant diagnostic antigen, rISG65, to create a dual-antigen lateral flow test prototype. The latter was used blind in a virtual field trial of 431 randomized infection and control sera from the WHO HAT Specimen Biobank.In the virtual field trial, using two positive antigen bands as the criterion for infection, the sVSG117 and rISG65 dual-antigen lateral flow test prototype showed a sensitivity of 97.3% (95% CI: 93.3 to 99.2) and a specificity of 83.3% (95% CI: 76.4 to 88.9) for the detection of T. b. gambiense infections. The device was not as good for detecting T. b. rhodesiense infections using two positive antigen bands as the criterion for infection, with a sensitivity of 58.9% (95% CI: 44.9 to 71.9) and specificity of 97.3% (95% CI: 90.7 to 99.7). However, using one or both positive antigen band(s) as the criterion for T. b. rhodesiense infection improved the sensitivity to 83.9% (95% CI: 71.7 to 92.4) with a specificity of 85.3% (95% CI: 75.3 to 92.4). These results encourage further development of the dual-antigen device for clinical use

Modeling of the N-Glycosylated Transferrin Receptor Suggests How Transferrin Binding Can Occur within the Surface Coat of Trypanosoma brucei

The transferrin receptor of bloodstream form Trypanosoma brucei is a heterodimer encoded by expression site associated genes 6 and 7. This low-abundance glycoprotein with a single glycosylphosphatidylinositol membrane anchor and eight potential N-glycosylation sites is located in the flagellar pocket. The receptor is essential for the parasite, providing its only source of iron by scavenging host transferrin from the bloodstream. Here, we demonstrate that both receptor subunits contain endoglycosidase H-sensitive and endoglycosidase H-resistant N-glycans. Lectin blotting of the purified receptor and structural analysis of the released N-glycans revealed oligomannose and paucimannose structures but, contrary to previous suggestions, no poly-N-acetyllactosamine structures were found. Overlay experiments suggest that the receptor can bind to other trypanosome glycoproteins, which may explain this discrepancy. Nevertheless, these data suggest that a current model, in which poly-N-acetyllactosamine glycans are directly involved in receptor-mediated endocytosis in bloodstream form Trypanosoma brucei, should be revised. Sequential endoglycosidase H and peptide-N-glycosidase F treatment, followed by tryptic peptide analysis, allowed the mapping of oligomannose and paucimannose structures to four of the receptor N-glycosylation sites. These results are discussed with respect to the current model for protein N-glycosylation in the parasite. Finally, the glycosylation data allowed the creation of a molecular model for the parasite transferrin receptor. This model, when placed in the context of a model for the dense variant surface glycoprotein coat in which it is embedded, suggests that receptor N-glycosylation may play an important role in providing sufficient space for the approach and binding of transferrin to the receptor, without significantly disrupting the continuity of the protective variant surface glycoprotein coat