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Expression of Trypanosoma brucei gambiense Antigens in Leishmania tarentolae. 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.

SUBMITTER: Rooney B

PROVIDER: S-EPMC4674146 | biostudies-literature | 2015 Dec

REPOSITORIES: biostudies-literature

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

Rooney Barrie B Piening Turid T Büscher Philippe P Rogé Stijn S Smales C Mark CM

PLoS neglected tropical diseases 20151209 12

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 ...[more]

PMID: 26650813

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Project description:BackgroundDiagnosis and treatment are central elements of strategies to control Trypanosoma brucei gambiense human African trypanosomiasis (HAT). Serological screening is a key entry point in diagnostic algorithms. The Card Agglutination Test for Trypanosomiasis (CATT) has been the most widely used screening test for decades, despite a number of practical limitations that were partially addressed by the introduction of rapid diagnostic tests (RDTs). However, current RDTs are manufactured using native antigens, which are challenging to produce.Methodology/principal findingsThe objective of this study was to evaluate the accuracy of a new RDT developed using recombinant antigens (SD BIOLINE HAT 2.0), in comparison with an RDT produced using native antigens (SD BIOLINE HAT) and CATT. A total of 57,632 individuals were screened in the Democratic Republic of the Congo, either passively at 10 health centres, or actively by 5 mobile teams, and 260 HAT cases were confirmed by parasitology. The highest sensitivity was achieved with the SD BIOLINE HAT 2.0 (71.2%), followed by CATT (62.5%) and the SD BIOLINE HAT (59.0%). The most specific test was CATT (99.2%), while the specificity of the SD BIOLINE HAT and SD BIOLINE HAT 2.0 were 98.9% and 98.1%, respectively. Sensitivity of the tests was lower than previously reported, as they identified cases from partially overlapping sub-populations. All three tests were significantly more sensitive in passive than in active screening. Combining two or three tests resulted in a markedly increased sensitivity: When the SD BIOLINE HAT was combined with the SD BIOLINE HAT 2.0, sensitivity reached 98.4% in passive and 83.0% in active screening.Conclusions/significanceThe recombinant antigen-based RDT was more sensitive than, and as specific as, the SD BIOLINE HAT. It was as sensitive as, but slightly less specific than CATT. While the practicality and cost-effectiveness of algorithms including several screening tests would need to be investigated, using two or more tests appears to enhance sensitivity of diagnostic algorithms, although some decrease in specificity is observed as well.

Dataset Information

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

Publications

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

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