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Veerle Lejon - One of the best experts on this subject based on the ideXlab platform.
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Identification of mimotopes with diagnostic potential for trypanosoma brucei gambiense Variant Surface Glycoproteins using human antibody fractions
PLoS Neglected Tropical Diseases, 2012Co-Authors: Liesbeth Van Nieuwenhove, Fatima Balharbi, Tessa Dieltjens, Michael Humbert, Yves Guisez, Philippe Buscher, Veerle LejonAbstract: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.\n\nMETHODOLOGY/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.\n\nCONCLUSIONS/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.
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identification of peptide mimotopes of trypanosoma brucei gambiense Variant Surface Glycoproteins
PLOS Neglected Tropical Diseases, 2011Co-Authors: Liesbeth Van Nieuwenhove, Fatima Balharbi, Tessa Dieltjens, Yves Guisez, Philippe Buscher, Stijn Roge, Thierry Laurent, Veerle LejonAbstract:BACKGROUND: The current antibody detection tests for the diagnosis of gambiense human African trypanosomiasis (HAT) are based on native Variant Surface Glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. These native VSGs are difficult to produce, and contain non-specific epitopes that may cause cross-reactions. We aimed to identify mimotopic peptides for epitopes of T.b. gambiense VSGs that, when produced synthetically, can replace the native proteins in antibody detection tests. METHODOLOGY/PRINCIPAL FINDINGS: PhD.-12 and PhD.-C7C phage display peptide libraries were screened with mouse monoclonal antibodies against the predominant VSGs LiTat 1.3 and LiTat 1.5 of T.b. gambiense. Thirty seven different peptide sequences corresponding to a linear LiTat 1.5 VSG epitope and 17 sequences corresponding to a discontinuous LiTat 1.3 VSG epitope were identified. Seventeen of 22 synthetic peptides inhibited the binding of their homologous monoclonal to VSG LiTat 1.5 or LiTat 1.3. Binding of these monoclonal antibodies to respectively six and three synthetic mimotopic peptides of LiTat 1.5 and LiTat 1.3 was significantly inhibited by HAT sera (p<0.05). CONCLUSIONS/SIGNIFICANCE: We successfully identified peptides that mimic epitopes on the native trypanosomal VSGs LiTat 1.5 and LiTat 1.3. These mimotopes might have potential for the diagnosis of human African trypanosomiasis but require further evaluation and testing with a large panel of HAT positive and negative sera.
John E Donelson - One of the best experts on this subject based on the ideXlab platform.
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Analysis of expressed sequence tags from the four main developmental stages of Trypanosoma congolense
2015Co-Authors: Jared R. Helm, Christiane Hertz-fowler, Martin Aslett, Matthew Berriman, Michael A. Quail, Marcelo B. Soares, Maria F. Bonaldo, Tatsuya Sakurai, Noboru Inoue, John E DonelsonAbstract:Trypanosoma congolense is one of the most economically important pathogens of livestock in Africa. Culture-derived parasites of each of the three main insect stages of the T. congolense life cycle, i.e., the procyclic, epimastigote and metacyclic stages, and bloodstream stage parasites isolated from infected mice, were used to construct stage-specific cDNA libraries and expressed sequence tags (ESTs or cDNA clones) in each library were sequenced. Thirteen EST clusters encoding different Variant Surface Glycoproteins (VSGs) were detected in the metacyclic library and twenty-six VSG EST clusters were found in the bloodstream library, six of which are shared by the metacyclic library. Rare VSG ESTs are present in the epimastigote library, and none were detected in the procyclic library. ESTs encoding enzymes that catalyze oxidative phosphorylation and amino acid metabolism are about twice as abundant in the procyclic and epimastigote stages as in the metacyclic and bloodstream stages. In contrast, ESTs encoding enzymes involved in glycolysis, the citric acid cycle and nucleotide metabolism are about the same in all four developmental stages. Cysteine proteases, kinases and phosphatases are the most abundant enzyme groups represented by the ESTs. All four libraries contain T. congolense-specific expressed sequences not present in the T. brucei and T
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analysis of a donor gene region for a Variant Surface glycoprotein and its expression site in african trypanosomes
Nucleic Acids Research, 2001Co-Authors: Douglas J Lacount, Najib M Elsayed, Samir Kaul, David Wanless, Michael C R Turner, John E DonelsonAbstract:African trypanosomes evade the immune response of their mammalian hosts by sequentially expressing genes for different Variant Surface Glycoproteins (VSGs) from telomere-linked VSG expression sites. In the Trypanosoma brucei clone whose genome is being sequenced (GUTat 10.1), we show that the expressed VSG (VSG 10.1) is duplicated from a silent donor VSG located at another telomere-linked site. We have determined two 130 kb sequences representing the VSG 10.1 donor and expression sites. The telomere-linked donor VSG 10.1 resembles metacyclic VSG expression sites, and is preceded by a cluster of 35 or more tandem housekeeping genes, all of which are transcribed away from the telomere. The 45 kb telomere-linked VSG 10.1 expression site contains a promoter followed by seven expression site-associated genes (ESAGs), three pseudo ESAGs, two pseudo VSGs and VSG 10.1. The 80 kb preceding the expression site has few, if any, functional ORFs, but contains 50 bp repeats, INGI retrotransposon-like elements, and novel 4–12 kb repeats found near other telomeres. This analysis provides the first look over a 130 kb range of a telomere-linked donor VSG and its corresponding telomere-linked VSG expression site and forms the basis for studies on antigenic variation in the context of a completely sequenced genome.
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a monocistronic transcript for a trypanosome Variant Surface glycoprotein
Molecular and Cellular Biology, 1994Co-Authors: C M Alarcon, Hyeung Jin Son, T Hall, John E DonelsonAbstract:Many protein-encoding genes of African trypanosomes are transcribed as large polycistronic pre-mRNAs that are processed into individual mRNAs containing a 5' spliced leader and 3' poly(A). The 45- to 60-kb pre-mRNAs encoding some Variant Surface Glycoproteins (VSGs) contain as many as eight unrelated coding regions. Here we identify the promoter for a metacyclic VSG gene that is expressed without duplication in a bloodstream trypanosome clone. This 70-bp promoter is located 2 kb upstream of the telomere-linked VSG gene and directs the synthesis of a monocistronic VSG pre-mRNA lacking the 5' spliced leader. Its sequence only slightly resembles those of other known trypanosome promoters, but it does cross-hybridize with several related sequences elsewhere in the genome. These results suggest that a new class of trypanosome promoters has been found, whose function is to initiate monocistronic transcription of those VSG genes normally expressed during the metacyclic stage.
Liesbeth Van Nieuwenhove - One of the best experts on this subject based on the ideXlab platform.
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recombinant antigens expressed in pichia pastoris for the diagnosis of sleeping sickness caused by trypanosoma brucei gambiense
PLOS Neglected Tropical Diseases, 2014Co-Authors: Stijn Roge, Liesbeth Van Nieuwenhove, Magali Meul, Annick Heykers, Annette Brouwer De Koning, Nicolas Bebronne, Yves GuisezAbstract:BACKGROUND Screening 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 FINDINGS We 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/SIGNIFICANCE The 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.
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Recombinant antigens expressed in Pichia pastoris for the diagnosis of sleeping sickness caused by Trypanosoma brucei gambiense.
Public Library of Science (PLoS), 2014Co-Authors: Stijn Roge, Liesbeth Van Nieuwenhove, Yves Guisez, Magali Meul, Annick Heykers, Annette Brouwer De Koning, Nicolas Bebronne, Philippe BuscherAbstract:Screening 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.We 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.The 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
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Identification of mimotopes with diagnostic potential for trypanosoma brucei gambiense Variant Surface Glycoproteins using human antibody fractions
PLoS Neglected Tropical Diseases, 2012Co-Authors: Liesbeth Van Nieuwenhove, Fatima Balharbi, Tessa Dieltjens, Michael Humbert, Yves Guisez, Philippe Buscher, Veerle LejonAbstract: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.\n\nMETHODOLOGY/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.\n\nCONCLUSIONS/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.
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identification of peptide mimotopes of trypanosoma brucei gambiense Variant Surface Glycoproteins
PLOS Neglected Tropical Diseases, 2011Co-Authors: Liesbeth Van Nieuwenhove, Fatima Balharbi, Tessa Dieltjens, Yves Guisez, Philippe Buscher, Stijn Roge, Thierry Laurent, Veerle LejonAbstract:BACKGROUND: The current antibody detection tests for the diagnosis of gambiense human African trypanosomiasis (HAT) are based on native Variant Surface Glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. These native VSGs are difficult to produce, and contain non-specific epitopes that may cause cross-reactions. We aimed to identify mimotopic peptides for epitopes of T.b. gambiense VSGs that, when produced synthetically, can replace the native proteins in antibody detection tests. METHODOLOGY/PRINCIPAL FINDINGS: PhD.-12 and PhD.-C7C phage display peptide libraries were screened with mouse monoclonal antibodies against the predominant VSGs LiTat 1.3 and LiTat 1.5 of T.b. gambiense. Thirty seven different peptide sequences corresponding to a linear LiTat 1.5 VSG epitope and 17 sequences corresponding to a discontinuous LiTat 1.3 VSG epitope were identified. Seventeen of 22 synthetic peptides inhibited the binding of their homologous monoclonal to VSG LiTat 1.5 or LiTat 1.3. Binding of these monoclonal antibodies to respectively six and three synthetic mimotopic peptides of LiTat 1.5 and LiTat 1.3 was significantly inhibited by HAT sera (p<0.05). CONCLUSIONS/SIGNIFICANCE: We successfully identified peptides that mimic epitopes on the native trypanosomal VSGs LiTat 1.5 and LiTat 1.3. These mimotopes might have potential for the diagnosis of human African trypanosomiasis but require further evaluation and testing with a large panel of HAT positive and negative sera.
Yves Guisez - One of the best experts on this subject based on the ideXlab platform.
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recombinant antigens expressed in pichia pastoris for the diagnosis of sleeping sickness caused by trypanosoma brucei gambiense
PLOS Neglected Tropical Diseases, 2014Co-Authors: Stijn Roge, Liesbeth Van Nieuwenhove, Magali Meul, Annick Heykers, Annette Brouwer De Koning, Nicolas Bebronne, Yves GuisezAbstract:BACKGROUND Screening 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 FINDINGS We 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/SIGNIFICANCE The 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.
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Recombinant antigens expressed in Pichia pastoris for the diagnosis of sleeping sickness caused by Trypanosoma brucei gambiense.
Public Library of Science (PLoS), 2014Co-Authors: Stijn Roge, Liesbeth Van Nieuwenhove, Yves Guisez, Magali Meul, Annick Heykers, Annette Brouwer De Koning, Nicolas Bebronne, Philippe BuscherAbstract:Screening 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.We 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.The 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
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Identification of mimotopes with diagnostic potential for trypanosoma brucei gambiense Variant Surface Glycoproteins using human antibody fractions
PLoS Neglected Tropical Diseases, 2012Co-Authors: Liesbeth Van Nieuwenhove, Fatima Balharbi, Tessa Dieltjens, Michael Humbert, Yves Guisez, Philippe Buscher, Veerle LejonAbstract: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.\n\nMETHODOLOGY/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.\n\nCONCLUSIONS/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.
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identification of peptide mimotopes of trypanosoma brucei gambiense Variant Surface Glycoproteins
PLOS Neglected Tropical Diseases, 2011Co-Authors: Liesbeth Van Nieuwenhove, Fatima Balharbi, Tessa Dieltjens, Yves Guisez, Philippe Buscher, Stijn Roge, Thierry Laurent, Veerle LejonAbstract:BACKGROUND: The current antibody detection tests for the diagnosis of gambiense human African trypanosomiasis (HAT) are based on native Variant Surface Glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. These native VSGs are difficult to produce, and contain non-specific epitopes that may cause cross-reactions. We aimed to identify mimotopic peptides for epitopes of T.b. gambiense VSGs that, when produced synthetically, can replace the native proteins in antibody detection tests. METHODOLOGY/PRINCIPAL FINDINGS: PhD.-12 and PhD.-C7C phage display peptide libraries were screened with mouse monoclonal antibodies against the predominant VSGs LiTat 1.3 and LiTat 1.5 of T.b. gambiense. Thirty seven different peptide sequences corresponding to a linear LiTat 1.5 VSG epitope and 17 sequences corresponding to a discontinuous LiTat 1.3 VSG epitope were identified. Seventeen of 22 synthetic peptides inhibited the binding of their homologous monoclonal to VSG LiTat 1.5 or LiTat 1.3. Binding of these monoclonal antibodies to respectively six and three synthetic mimotopic peptides of LiTat 1.5 and LiTat 1.3 was significantly inhibited by HAT sera (p<0.05). CONCLUSIONS/SIGNIFICANCE: We successfully identified peptides that mimic epitopes on the native trypanosomal VSGs LiTat 1.5 and LiTat 1.3. These mimotopes might have potential for the diagnosis of human African trypanosomiasis but require further evaluation and testing with a large panel of HAT positive and negative sera.
Don C Wiley - One of the best experts on this subject based on the ideXlab platform.
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a structural motif in the Variant Surface Glycoproteins of trypanosoma brucei
Nature, 1993Co-Authors: M Blum, James Down, Anne Gurnett, Mark Carrington, Mervyn J Turner, Don C WileyAbstract:The variable domain of the trypanosome Variant Surface glycoprotein (VSG) ILTat 1.24 has been shown by X-ray crystallography to resemble closely the structures of VSG MITat 1.2, despite their low sequence similarity. Specific structural features of these VSGs, including substitution of carbohydrate for an alpha-helix, can be found in other VSG sequences. Thus antigenic variation in trypanosomes is accomplished by sequence variation, not gross structural alteration; the extensive sequence differences among VSGs may be required for another reason, such as the avoidance of recognition by helper T cells. Additionally, VSG sequences are found to define families, within a VSG superfamily, which have evolved in the trypanosome genome.
