Acknowledgements This study received financial support from the Flanders Fund for Scientific Research (FWO; Krediet aan Navorsers 1516907N), the Institute of Tropical Medicine (RATT project) and from the EC Health Cooperation Work Programme, FP7 NIDIAG project (Grant Agreement 260260, website: www.nidiag.org)

Conclusions •  rRoTat 1.2, rLiTat 1.3 and rLiTat 1.5 are expressed in Pichia pastoris GlycoSwitch™ M5 with yields up to 20 mg per liter cell culture

•  ELISA confirms the diagnostic potential of rRoTat 1.2, rLiTat 1.3 and rLiTat 1.5 VSG

•  Recombinant VSGs produced in P. pastoris can replace native antigens for serodiagnosis

•  Lateral flow tests are applicable on any host species, have a high sensitivity and specificity, and are individual, thermostable tests with a rapid readout

1Institute of Tropical Medicine, Department of Biomedical Sciences, Nationalestraat 155, 2000 Antwerp, Belgium 2University of Antwerp, Laboratory for Molecular Plant Physiology and Biotechnology, Department of Biology, Groenenborgerlaan 171, 2020 Antwerp, Belgium 3Coris BioConcept, Science Park Crealys, Rue Jean Sonet 4A, 5032 Gembloux, Belgium

Materials & Methods A. Cloning of VSGs and transfection into the Pichia pastoris M5-strain •  PCR amplification of the N-terminal part of VSGs from cDNA •  Plasmid linearisation and electroporation into the Pichia pastoris

GlycoSwitch™ M5 strain •  Integration into yeast genomic DNA by homologous recombination under

control of the methanol-inducible AOX1 promotor

B. Secretion of recombinant VSGs in Ultra Yield flasks with AirOtop seals •  Production of biomass in BMGY at 29 °C and 200 rpm •  Induction in BMMY at 22 °C and 250 rpm •  Collection of supernatant with the secreted recombinant

after 3-4 days induction

C. Protein purification •  His tag affinity purification of the recombinants from the supernatant •  Desalting of the purified recombinants •  Protein concentration measurement

D. ELISA on animal and human sera •  Surra: sera from 25 experimentally infected goats and from 93

naturally infected and 92 non-infected dromedary camels •  HAT: sera from 88 HAT patients, 66 endemic controls and 8 non-

endemic controls E. Development of diagnostic tests for surra and HAT •  Incorporation of the recombinants in a lateral flow format

Introduction Surra is an infectious disease caused by Trypanosoma (T.) evansi. It affects camels, horses, buffaloes and cattle in Africa, the Middle East, Asia and Latin America. The parasite is transmitted by bloodsucking flies such as Tabanidae and Stomoxys species. Trypanosoma brucei (T.b.) gambiense and T.b. rhodesiense cause human African trypanosomiasis (HAT) and are transmitted by tsetse flies. Serodiagnosis of both diseases is based on detection of antibodies against predominantly expressed variant surface glycoproteins (VSGs) that are prepared by massive trypanosome cultures in lab rodents. We aimed to develop lateral flow diagnostic tests based on recombinant fragments of the VSGs RoTat 1.2, LiTat 1.3 and LiTat 1.5, expressed in the methylotrophic yeast Pichia pastoris.

Results

Surra HAT

Expression of rRoTat 1.2 by Pichia pastoris A.   Coomassie stained gel; B. WB (anti-His tag Ab); C. WB (anti-RoTat 1.2 antiserum)

lane 1: 20x concentrated supernatant of non-transfected Pichia pastoris M5 lane 2: 20x concentrated supernatant of transfected Pichia pastoris M5 (46 h induction)

lane 3: His tag purified recombinant RoTat 1.2 lane 4: 20x concentrated flow-through

Expression and purification of rRoTat 1.2

A B C

97 kDa 66 kDa

45 kDa

30 kDa

20.1 kDa

14.4 kDa

75 kDa

25 kDa

37 kDa

50 kDa

Expression and purification of rLiTat 1.3 & rLiTat 1.5

A B A

97 kDa 66 kDa

45 kDa

30 kDa

20.1 kDa

14.4 kDa

75 kDa

25 kDa

37 kDa

50 kDa

B

1 2 3 4 2 3 4 2 3 4

Expression of rLiTat 1.3 (left) and rLiTat 1.5 (right) by Pichia pastoris A.   Coomassie stained gel; B. WB (anti-Strep tag Ab)

lane 1: 20x concentrated supernatant of non-transfected Pichia pastoris M5 lane 2: 20x concentrated supernatant of transfected Pichia pastoris M5 (69 h induction)

lane 3: His tag purified recombinant LiTat 1.3 or LiTat 1.5 lane 4: 20x concentrated flow-through

1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

Diagnostic potential of rRoTat 1.2 in ELISA

0,0

0,5

1,0

1,5

2,0

2,5

3,0

3,5

Native RoTat 1.2 Recombinant RoTat 1.2

OD

at

41

4n

m

Antigen

Negative camel sera (average)

Positive camel sera (average)

Development of lateral flow tests

Surra Sero K-SeT and rHAT Sero K-SeT

Diagnostic potential of rLiTat 1.3 & 1.5 in ELISA

ROC-curves of recombinant and native LiTat 1.3 and LiTat 1.5 tested in ELISA on 162 human serum samples

A: area under the curve