Background At present, screening of the population at risk for gambiense human African trypanosomiasis (HAT) is based on detection of antibodies against native variant surface glycoproteins (VSGs) of Trypanosoma brucei ( T.b. ) gambiense . Drawbacks of these native VSGs include culture of infective T.b. gambiense trypanosomes in laboratory rodents, necessary for production, and the exposure of non-specific epitopes that may cause cross-reactions. We therefore aimed at identifying peptides that mimic epitopes, hence called “mimotopes,” specific to T.b. gambiense VSGs and that may replace the native proteins in antibody detection tests. Methodology/Principal Findings A Ph.D.-12 peptide phage display library was screened with polyclonal antibodies from patient sera, previously affinity purified on VSG LiTat 1.3 or LiTat 1.5. The peptide sequences were derived from the DNA sequence of the selected phages and synthesised as biotinylated peptides. Respectively, eighteen and twenty different mimotopes were identified for VSG LiTat 1.3 and LiTat 1.5, of which six and five were retained for assessment of their diagnostic performance. Based on alignment of the peptide sequences on the original protein sequence of VSG LiTat 1.3 and 1.5, three additional peptides were synthesised. We evaluated the diagnostic performance of the synthetic peptides in indirect ELISA with 102 sera from HAT patients and 102 endemic negative controls. All mimotopes had areas under the curve (AUCs) of ≥0.85, indicating their diagnostic potential. One peptide corresponding to the VSG LiTat 1.3 protein sequence also had an AUC of ≥0.85, while the peptide based on the sequence of VSG LiTat 1.5 had an AUC of only 0.79. Conclusions/Significance We delivered the proof of principle that mimotopes for T.b. gambiense VSGs, with diagnostic potential, can be selected by phage display using polyclonal human antibodies.

BackgroundScreening tests for gambiense sleeping sickness, such as the CATT/T. b. gambiense and a recently developed lateral flow tests, are hitherto based on native variant surface glycoproteins (VSGs), namely LiTat 1.3 and LiTat 1.5, purified from highly virulent trypanosome strains grown in rodents.Methodology/Principal FindingsWe have expressed SUMO (small ubiquitin-like modifier) fusion proteins of the immunogenic N-terminal part of these antigens in the yeast Pichia pastoris. The secreted recombinant proteins were affinity purified with yields up to 10 mg per liter cell culture.Conclusions/SignificanceThe diagnostic potential of each separate antigen and a mixture of both antigens was confirmed in ELISA on sera from 88 HAT patients and 74 endemic non-HAT controls. Replacement of native antigens in the screening tests for sleeping sickness by recombinant proteins will eliminate both the infection risk for the laboratory staff during antigen production and the need for laboratory animals. Upscaling production of recombinant antigens, e.g. in biofermentors, is straightforward thus leading to improved standardisation of antigen production and reduced production costs, which on their turn will increase the availability and affordability of the diagnostic tests needed for the elimination of gambiense HAT.

Objective To evaluate the diagnostic accuracy of a peptide corresponding to the variant surface glycoprotein (VSG) LiTat 1.5 amino acid sequence 268–281, and identified through alignment of monoclonal antibody selected mimotopes, for diagnosis of Trypanosoma brucei gambiense sleeping sickness. Methods A synthetic biotinylated peptide (peptide 1.5/268–281), native VSG LiTat 1.3 and VSG LiTat 1.5 were tested in an indirect ELISA with 102 sera from HAT patients and 102 endemic HAT negative controls. Results The area under the curve (AUC) of peptide 1.5/268–281 was 0.954 (95% confidence interval 0.918– 0.980), indicating diagnostic potential. The areas under the curve of VSG LiTat 1.3 and LiTat 1.5 were 1.000 (0.982–1.000) and 0.997 (0.973–1.000), respectively, and significantly higher than the AUC of peptide 1.5/268–281. On a model of VSG LiTat 1.5, peptide 1.5/268–281 was mapped near the top of the VSG. Conclusions A biotinylated peptide corresponding to AA 268–281 of VSG LiTat 1.5 may replace the native VSG in serodiagnostic tests, but the diagnostic accuracy is lower than for the full length native VSG LiTat 1.3 and VSG LiTat 1.5.

ABSTRACT. At present, all available diagnostic antibody detection tests for Trypanosoma brucei gambiense human African trypanosomiasis are based on predominant variant surface glycoproteins (VSGs), such as VSG LiTat 1.5. During investigations aiming at replacement of the native VSGs by recombinant proteins or synthetic peptides, the sequence of VSG LiTat 1.5 was derived from cDNA and direct N-terminal amino acid sequencing. Characterization of the VSG based on cysteine distribution in the amino acid sequence revealed an unusual cysteine pattern identical to that of VSG Kinu 1 of T. b. brucei. Even though both VSGs lack the third of four conserved cysteines typical for type A N-terminal domains, they can be classified as type A.

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