Detection of antibodies against the African parasite Trypanosoma brucei using synthetic glycosylphosphatidylinositol oligosaccharide fragments

Abstract

Trypanosoma brucei (T. brucei) parasites cause two major infectious diseases in Africa: African trypanosomiasis in humans (HAT) and Nagana in animals. Despite the enormous economic and social impact, vaccines and reliable diagnostic measures are still lacking for these diseases. The main obstacle to developing accurate diagnostic methods and an active vaccine is the parasite’s ability for antigenic variation, impairment of B cell maturation, and loss of B cell memory which collectively prevent the development of a long-lasting, effective immune response. The antigenic variation is sustained by random gene switching, segmental gene conversion, and altered glycosylation states of solvent-exposed regions of the corresponding variant surface glycoproteins (VSGs). These glycoproteins use a glycosylphosphatidylinositol (GPI) anchor for attachment to the membrane. GPIs of T. brucei have specific branched structures that are further heterogeneously galactosylated. Here, we synthesized a glycan fragment library containing T. brucei GPIs’ most prominent structural features and performed an epitope mapping using mice and human sera of infected specimens using glycan microarrays. The studies indicate that in contrast to VSGs, T. brucei GPIs are recognized by infection-induced short-lived Immunoglobulin M (IgM) and long-lasting Immunoglobulin G (IgG), suggesting a specific immune response against GPI structures. These findings enable the development of diagnostic tests based on synthetic antigens for the reliable diagnosis of human African trypanosomiasis and Nagana.

Supplementary Information: The online version contains supplementary material available at 10.1007/s10719-025-10186-x.

Keywords: GPI; Human African Trypanosomiasis; Nagana; VSG; carbohydrate antigens; diagnostics.

Fig. 1

T. brucei GPI derivatives: (a) Representation of the T. brucei GPI core; (b) T. brucei GPI fragments containing mannose with different galactosylation patterns A-E and low variant CTD-peptide of VSG117 (F); (c) Representative Scan of a microarray indicating the positions of structures A–F (left) and Glycan-array printing pattern (right)

Fig. 2

IgM antibodies from mouse sera recognize tetra-galactoside C: left: Box-Plot indicating an increase in GPI fragment recognition after infection; right: ROC curve for the same synthetic structure; ns P > 0.05; * P < 0.05; ** P < 0.01, *** P < 0.001, **** P < 0.0001

Fig. 3

IgM antibodies in human sera from Tbg- or Tbg-infected patients recognize α-galactoside A and tetra-galactoside C: 1st row: Box-Plot and ROC curves indicating an increase in GPI fragment A recognition in specimens with Tbg and a weaker recognition focusing on stage 2 with Tbr infection; 2nd row: GPI fragment C recognition in specimens with Tbg and a weaker recognition focusing on stage 2 with Tbr infection; nsP > 0.05; * P < 0.05; ** P < 0.01, *** P < 0.001, **** P < 0.0001

Fig. 4

IgG antibodies in human sera from Tbg-infected patients and endemic controls interact with tetra-galactoside C: Box-Plot and ROC curves show an increase in GPI fragment C recognition in specimens with Tbg phase 2 infection; ns P > 0.05; * P < 0.05; ** P < 0.01, *** P < 0.001, **** P < 0.0001