Identification of Peptide Mimotopes of Trypanosoma brucei gambiense Variant Surface Glycoproteins

The control of human African trypanosomiasis or sleeping sickness, a deadly disease in sub-Saharan Africa, mainly depends on a correct diagnosis and treatment. The aim of our study was to identify mimotopic peptides (mimotopes) that may replace the native proteins in antibody detection tests for sleeping sickness and hereby improve the diagnostic sensitivity and specificity. We selected peptide expressing phages from the PhD.-12 and PhD.-C7C phage display libraries with mouse monoclonal antibodies specific to variant surface glycoprotein (VSG) LiTat 1.3 or LiTat 1.5 of Trypanosoma brucei gambiense. The peptide coding genes of the selected phages were sequenced and the corresponding peptides were synthesised. Several of the synthetic peptides were confirmed as mimotopes for VSG LiTat 1.3 or LiTat 1.5 since they were able to inhibit the binding of their homologous monoclonal to the corresponding VSG. These peptides were biotinylated and their diagnostic potential was assessed with human sera. We successfully demonstrated that human sleeping sickness sera recognise some of the mimotopes of VSG LiTat 1.3 and LiTat 1.5, indicating the diagnostic potential of such peptides

Amino acid sequences of peptides obtained by phage display with anti- VSG LiTat 1.5 mAb H12H3.

<p>The first sequence displayed is amino acid 265 to 288 of the protein sequence of VSG LiTat 1.5. Homologous amino acids are indicated in grey and amino acids that are identical to those of the VSG protein sequence are in bold and grey. All peptide sequences include the GGGS-spacer (connecting phage protein pIII and the random peptide in the original library) at the C-terminal. When expressed on the phage, the first cysteine of the C7C-peptides is preceded by alanine. AAA: eluted via antigen competition. PPP: eluted via acidification. Freq: number of times this sequence was found amongst seventy-nine selected phage clones. Name: name of the synthesised peptide.*: peptide selected for biotinylation. NW: not withheld. % identity: percentage identity of the peptide sequence with a stretch of 14 AA (AA 268 to 281) within the protein sequence of VSG LiTat 1.5. % RA mAb-VSG: percentage remaining of the mAb binding to its corresponding native VSG after inhibition by the synthetic peptide at 67 µg/ml.</p

Average percent remaining activity of the mAb binding to the biotinylated synthetic peptides.

<p>The ability of human serum antibodies to bind to the mimotopic peptides selected with anti-VSG monoclonal antibodies was assessed by means of an inhibition ELISA. *: <i>p</i><0.05 and **: <i>p</i><0.01. Compared to the ten HAT negative sera, the nine HAT positive sera significantly inhibited the binding of anti-LiTat 1.5 mAb H12H3 to peptide 21, 22, 23, 28, C59 and C60 and the binding of anti-LiTat 1.3 mAb H13F7 to peptides 25, 60 and 61.</p