Luminescent Multiplex Viability Assay for T.b. gambiense

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1 Luminescent Multiplex Viability Assay for T.b. gambiense Van Reet N., Pyana P., Rogé S., Claes F. and Büscher, P. Institute of Tropical Medicine Antwerp 32nd ISCTRC conference 8th 12th September 2013

2 Introduction New drugs for HAT ideally should be safe active against both subspecies active against both disease stages oral and single administration Novel trypanotoxic compounds are discovered using whole cell in vitro high-throughput screenings (HTS) HTS are almost exclusively performed with one particular non human pathogenic strain: T. brucei brucei 427

3 Introduction II More than 95 % of all sleeping sickness in patients is caused by Trypanosoma brucei gambiense Genetically and phenotypically different from T.b. brucei / T.b. rhodesiense Differences in sensitivity to drugs: pentamidine and eflornithine Why no HTS screening with T.b. gambiense? biosafety concerns difficult in vitro and in vivo isolation current viability assays are not efficient due to metabolism (resazurin) or not tested (ATP luminescence measurement)

4 Objectives I To improve cell viability measurement for T.b. gambiense by making use of 2 independent luminescent assays within the same experiment To compare this Luminescent Multiplex Viability Assay with the standard fluorimetric resazurin assay

5 Objectives II 1. Measure Renilla luciferase reporter gene activity with EnduRen (Promega) 2. Combine with a sensitive assay based on ATP measurement with a recombinant firefly luciferase : CellTiter-Glo (Promega)

6 Isolation of strain T.b. gambiense strain from Mbuyi-Mayi in RDC isolated from a sleeping sickness patient in 2006 with known cured outcome after 10 days melarsoprol treatment MHOM/CD/INRB/2006/23A or alias 348 BT THARSAT study (Mumba et al, JID 2010) In vivo isolation in G. surdaster (Pyana P et al 2011 PloS NTD) In vitro isolation in methylcellulose containing HMI9 and human serum (Van Reet et al 2011 Exp Para)

7 Renilla luciferase Strain adapted to HMI9 culture medium without methylcellulose Transfected by electroporation with a Renilla luciferase reporter gene (RLUC) in a promotorless vector for constitutive expression Selection of most luminescent population based on hygromycin resistance RLUC parasites can be used for in vivo bioluminescence imaging (Claes et al & Giroud et al PLOS NTD 2009)

8 LMVA: very easy assay 1. Renilla luciferase 2. ATP measurement Reagents that are fit for use in HTS on 96-well plates (or higher) Both assays are performed within the same experiment on the same dataset = multiplex Multiplex assays may decrease false positive hits in HTS

9 LMVA: detection limits The most luminescent population had a detection limit of 2 x 10 4 cells for the EnduRen assay 5 x 10 3 cells for the CellTiter-Glo assay Both assays of the LMVA give linear results up to 10 6 cells ml -1

10 LMVA: growth profile Both assays of the LMVA can be used to monitor growth Population doubling time is similar as deterimined with microscopy Correlation with log phase trypanosome growth, but not in stationary phase

11 LMVA: compared to resazurin No difference in phenotype due to RLuc integration Both assays of the LMVA gave similar results in IC 50 Similar IC 50 as found at STPHI with othe strain from Mbuji Mayi, used for fexinidazol study Measurement with resazurin may yield higher IC 50 values The IC 50 value for pentamidine is very high ~ resistance?

12 Conclusions LMVA Sensitive, fast and two assays simultaneously Expensive, but fully adapted to HTS In vivo bioluminescence imaging is possible with RLUC strain 348 BT Pentamidine resistant strain! What is going on with T.b. gambiense in Mbuyi Mayi?

13 Acknowledgements Van Reet Nick ITM Unit of Parasite Diagnostics University of Antwerp Bio-Imaging Lab FWO project # Lucicor