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

Expression of Trypanosoma brucei gambiense Antigens in Leishmania tarentolae. Potential for Use in Rapid Serodiagnostic Tests (RDTs)

The development of rapid serodiagnostic tests for sleeping sickness and other diseases caused by kinetoplastids relies on the affordable production of parasite-specific recombinant antigens. Here, we describe the production of recombinant antigens from Trypanosoma brucei gambiense (T.b. gambiense) in the related species Leishmania tarentolae (L. tarentolae), and compare their diagnostic sensitivity and specificity to native antigens currently used in diagnostic kits against a panel of human sera. A number of T.b. gambiense protein antigen candidates were chosen for recombinant expression in L. tarentolae based on current diagnostics in field use and recent findings on immunodiagnostic antigens found by proteomic profiling. In particular, the extracellular domains of invariant surface glycoprotein 65 (ISG65), variant surface glycoproteins VSG LiTat 1.3 and VSG LiTat 1.5 were fused with C-terminal histidine tags and expressed as soluble proteins in the medium of cultured, recombinant L. tarentolae. Using affinity chromatography, on average 10 mg/L of recombinant protein was purified from cultures and subsequently tested against a panel of sera from sleeping sickness patients from controls, i.e. persons without sleeping sickness living in HAT endemic countries. The evaluation on sera from 172 T.b. gambiense human African trypanosomiasis (HAT) patients and from 119 controls showed very high diagnostic potential of the two recombinant VSG and the rISG65 fragments with areas under the curve between 0.97 and 0.98 compared to 0.98 and 0.99 with native VSG LiTat 1.3 and VSG LiTat 1.5 (statistically not different). Evaluation on sera from 78 T.b. rhodesiense HAT patients and from 100 controls showed an acceptable diagnostic potential of rISG65 with an area under the curve of 0.83. These results indicate that a combination of these recombinant antigens has the potential to be used in next generation rapid serodiagnostic tests. In addition, the L. tarentolae expression system enables simple, cheap and efficient production of recombinant kinetoplatid proteins for use in diagnostic, vaccine and drug discovery research that does not rely on animal use to generate materials

Potential for Use in Rapid Serodiagnostic Tests (RDTs)

Abstract The development of rapid serodiagnostic tests for sleeping sickness and other diseases caused by kinetoplastids relies on the affordable production of parasite-specific recombinant antigens. Here, we describe the production of recombinant antigens from Trypanosoma brucei gambiense (T.b. gambiense) in the related species Leishmania tarentolae (L. tarentolae), and compare their diagnostic sensitivity and specificity to native antigens currently used in diagnostic kits against a panel of human sera. A number of T.b. gambiense protein antigen candidates were chosen for recombinant expression in L. tarentolae based on current diagnostics in field use and recent findings on immunodiagnostic antigens found by proteomic profiling. In particular, the extracellular domains of invariant surface glycoprotein 65 (ISG65), variant surface glycoproteins VSG LiTat 1.3 and VSG LiTat 1.5 were fused with C-terminal histidine tags and expressed as soluble proteins in the medium of cultured, recombinant L. tarentolae. Using affinity chromatography, on average 10 mg/L of recombinant protein was purified from cultures and subsequently tested against a panel of sera from sleeping sickness patients from controls, i.e. persons without sleeping sickness living in HAT endemic countries. The evaluation on sera from 172 T.b. gambiense human African trypanosomiasis (HAT) patients and from 119 controls showed very high diagnostic potential of the two recombinant VSG and the rISG65 fragments with areas under the curve between 0.97 and 0.98 compared to 0.98 and 0.99 with native VSG LiTat 1.3 and VSG LiTat 1.5 (statistically not different). Evaluation on sera from 78 T.b. rhodesiense HAT patients and from 100 controls showed an acceptable diagnostic potential of rISG65 with an area under the curve of 0.83. These results indicate that a combination of these recombinant antigens has the potential to be used in next generation rapid serodiagnostic tests. In addition, the L. tarentolae expression system enables simple, cheap and efficient production of recombinant kinetoplatid proteins for use in diagnostic, vaccine and drug discovery research that does not rely on animal use to generate materials. Funding: This work was funded via a Biotechnology and Biological Sciences Research Council (BBSRC, UK) Flexible Interchange Programme (FLIP) award (grant number BB/L026279/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Author Summary The development of rapid serodiagnostic tests for African sleeping sickness and other diseases caused by kinetoplastids relies in part on the affordable production of parasite-specific recombinant antigens. The majority of cases of sleeping sickness are caused by the parasite Trypanosoma brucei gambiense (T.b. gambiense) which is transmitted when bitten by an infected tsetse fly. Existing tests rely on the utilisation of extracts from the parasite or use antigens raised in animal models. In this study we have shown that using a cell culture system devised from a parasite similar to T.b. gambiense recombinant antigens can be produced that are as effective in rapid diagnostic tests as the native antigens purified from T.b. gambiense parasites grown in laboratory rodents. We compared the diagnostic sensitivity and specificity of the antigens we produced recombinantly to native antigens currently used in diagnostic kits against a panel of human sera. The evaluation on sera from 172 T.b. gambiense patients and from 119 controls without sleeping sickness showed very high diagnostic potential of two recombinant antigens where the response was not significantly different to that from the native antigens. These results indicate that a combination of these recombinant antigens has the potential to be used in next generation rapid serodiagnostic tests

Ribosomal DNA analysis of tsetse and non-tsetse transmitted Ethiopian Trypanosoma vivax strains in view of improved molecular diagnosis

Animal trypanosomosis caused by Trypanosoma vivax (T. vivax) is a devastating disease causing serious economic losses. Most molecular diagnostics for T. vivax infection target the ribosomal DNA locus (rDNA) but are challenged by the heterogeneity among T. vivax strains. In this study, we investigated the rDNA heterogeneity of Ethiopian T. vivax strains in relation to their presence in tsetse-infested and tsetse-free areas and its effect on molecular diagnosis. We sequenced the rDNA loci of six Ethiopian (three from tsetse-infested and three from tsetse-free areas) and one Nigerian T. vivax strain. We analysed the obtained sequences in silico for primer-mismatches of some commonly used diagnostic PCR assays and for GC content. With these data, we selected some rDNA diagnostic PCR assays for evaluation of their diagnostic accuracy. Furthermore we constructed two phylogenetic networks based on sequences within the smaller subunit (SSU) of 18S and within the 5.8S and internal transcribed spacer 2 (ITS2) to assess the relatedness of Ethiopian T. vivax strains to strains from other African countries and from South America. In silico analysis of the rDNA sequence showed important mismatches of some published diagnostic PCR primers and high GC content of T. vivax rDNA. The evaluation of selected diagnostic PCR assays with specimens from cattle under natural T. vivax challenge showed that this high GC content interferes with the diagnostic accuracy of PCR, especially in cases of mixed infections with T. congolense. Adding betain to the PCR reaction mixture can enhance the amplification of T. vivax rDNA but decreases the sensitivity for T. congolense and Trypanozoon. The networks illustrated that Ethiopian T. vivax strains are considerably heterogeneous and two strains (one from tsetse-infested and one from tsetse-free area) are more related to the West African and South American strains than to the East African strains. The rDNA locus sequence of six Ethiopian T. vivax strains showed important differences and higher GC content compared to other animal trypanosomes but could not be related to their origin from tsetse-infested or tsetse-free area. The high GC content of T. vivax DNA renders accurate diagnosis of all pathogenic animal trypanosomes with one single PCR problematic.publisher: Elsevier articletitle: Ribosomal DNA analysis of tsetse and non-tsetse transmitted Ethiopian Trypanosoma vivax strains in view of improved molecular diagnosis journaltitle: Veterinary Parasitology articlelink: http://dx.doi.org/10.1016/j.vetpar.2016.02.013 content_type: article copyright: Copyright © 2016 The Authors. Published by Elsevier B.V.status: publishe

Ribosomal DNA analysis of tsetse and non-tsetse transmitted Ethiopian Trypanosoma vivax strains in view of improved molecular diagnosis

AbstractAnimal trypanosomosis caused by Trypanosoma vivax (T. vivax) is a devastating disease causing serious economic losses. Most molecular diagnostics for T. vivax infection target the ribosomal DNA locus (rDNA) but are challenged by the heterogeneity among T. vivax strains. In this study, we investigated the rDNA heterogeneity of Ethiopian T. vivax strains in relation to their presence in tsetse-infested and tsetse-free areas and its effect on molecular diagnosis.We sequenced the rDNA loci of six Ethiopian (three from tsetse-infested and three from tsetse-free areas) and one Nigerian T. vivax strain. We analysed the obtained sequences in silico for primer-mismatches of some commonly used diagnostic PCR assays and for GC content. With these data, we selected some rDNA diagnostic PCR assays for evaluation of their diagnostic accuracy. Furthermore we constructed two phylogenetic networks based on sequences within the smaller subunit (SSU) of 18S and within the 5.8S and internal transcribed spacer 2 (ITS2) to assess the relatedness of Ethiopian T. vivax strains to strains from other African countries and from South America.In silico analysis of the rDNA sequence showed important mismatches of some published diagnostic PCR primers and high GC content of T. vivax rDNA. The evaluation of selected diagnostic PCR assays with specimens from cattle under natural T. vivax challenge showed that this high GC content interferes with the diagnostic accuracy of PCR, especially in cases of mixed infections with T. congolense. Adding betain to the PCR reaction mixture can enhance the amplification of T. vivax rDNA but decreases the sensitivity for T. congolense and Trypanozoon. The networks illustrated that Ethiopian T. vivax strains are considerably heterogeneous and two strains (one from tsetse-infested and one from tsetse-free area) are more related to the West African and South American strains than to the East African strains.The rDNA locus sequence of six Ethiopian T. vivax strains showed important differences and higher GC content compared to other animal trypanosomes but could not be related to their origin from tsetse-infested or tsetse-free area. The high GC content of T. vivax DNA renders accurate diagnosis of all pathogenic animal trypanosomes with one single PCR problematic

Pairwise difference and <i>p</i> value of the Chi square between the areas under the curve recorded for the different antigens when tested with sera from 172 <i>T</i>.<i>b</i>. <i>g-</i>HAT patients, 119 non-<i>g-</i>HAT controls and 50 non-<i>r-</i>HAT controls.

<p>* = significantly different</p

Gel filtration of purified rISG65 on a Superdex 200 10/300 GL column (GE Healthcare Lifesciences) in sodium phosphate buffer, pH7.5, O.5 M NaCl.

<p>A. Molecular weight markers (Sigma-Aldrich MWGF100) 1 = thyroglobulin 669 kDa, 2 = apoferritin 443 kDa, 3 = β-amylase 200 kDa, 4 = alcohol dehydrogenase 150 kDa, 5 = bovine serum albumin 66 kDa, 6 = carbonic anhydrase 29 kDa. B. Elution profile of rISG following one round of metal affinity chromatography purification.</p

Area under the curve (AUC) and its 95% confidence interval (CI), Youden index, percent sensitivity (Se %), percent specificity (Sp %) and their respective 95% CI recorded for the different antigens when tested with sera from 172 <i>g-</i>HAT patients, 119 non-<i>g-</i> HAT controls and 50 non-<i>r-</i>HAT controls.

<p>Area under the curve (AUC) and its 95% confidence interval (CI), Youden index, percent sensitivity (Se %), percent specificity (Sp %) and their respective 95% CI recorded for the different antigens when tested with sera from 172 <i>g-</i>HAT patients, 119 non-<i>g-</i> HAT controls and 50 non-<i>r-</i>HAT controls.</p

Receiver operator characteristic (ROC) curves and area under the curve (AUC) constructed from ELISA results obtained by testing sera from 172 <i>g-</i>HAT patients, 119 non-<i>g-</i>HAT controls and 50 non-<i>r-</i>HAT controls with nLiTat 1.3 (2 μg/ml), rLiTat 1.3 (4 μg/ml), nLiTat 1.5 (2 μg/ml), rLiTat 1.5 (4 μg/ml) and rISG65 (4 μg/ml).

<p>Receiver operator characteristic (ROC) curves and area under the curve (AUC) constructed from ELISA results obtained by testing sera from 172 <i>g-</i>HAT patients, 119 non-<i>g-</i>HAT controls and 50 non-<i>r-</i>HAT controls with nLiTat 1.3 (2 μg/ml), rLiTat 1.3 (4 μg/ml), nLiTat 1.5 (2 μg/ml), rLiTat 1.5 (4 μg/ml) and rISG65 (4 μg/ml).</p