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.

Fig 1. SDS-PAGE analysis of the expression of rISG65, rLiTat1.3 and rLiTat1.5 by Leishmania tarentolae after purification following a single round of metal affinity chromatography.

Coomassie stained 10% SDS-PAGE; Precision Plus Protein Unstained Standards, BioRad (M). Lane 1: rISG65, Lane 2: rLiTat1.3, Lane 3: rLiTat1.5. A = 1 μg and B = 10 μg protein.

Fig 2. 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.

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.

Fig 3. Analysis of the deglycosylation of rISG65 using PNGase F (1.5 U) (Sigma-Aldrich, P7367) for two hours at 37°C.

Coomassie blue stained pre-cast 4–12% BisTris gradient SDS-PAGE gel (Novex) using the MOPS running system; Precision Plus Protein Unstained Standards, BioRad (M). Lane 1: rISG65 (1 ug) before treatment with PNGase F, Lane 2: rISG65 (1 ug) after treatment with PNGase F.

Fig 4. Receiver operator characteristic (ROC) curves and area under the curve (AUC) constructed from ELISA results obtained by testing sera from 172 g-HAT patients, 119 non-g-HAT controls and 50 non-r-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).

Fig 5. Receiver operator characteristic (ROC) curves and area under the curve (AUC) constructed from ELISA results obtained by testing sera from 78 r-HAT patients, 50 non-r-HAT controls and 50 non-g-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).