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Persistence and activation of malaria hypnozoites in long-term primary hepatocyte cultures

Nature Medicine volume 20, pages 307–312 (2014)Cite this article

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Abstract

Malaria relapses, resulting from the activation of quiescent hepatic hypnozoites of Plasmodium vivax and Plasmodium ovale, hinder global efforts to control and eliminate malaria. As primaquine, the only drug capable of eliminating hypnozoites, is unsuitable for mass administration, an alternative drug is needed urgently. Currently, analyses of hypnozoites, including screening of compounds that would eliminate them, can only be made using common macaque models, principally Macaca rhesus and Macaca fascicularis, experimentally infected with the relapsing Plasmodium cynomolgi. Here, we present a protocol for long-term in vitro cultivation of P. cynomolgi–infected M. fascicularis primary hepatocytes during which hypnozoites persist and activate to resume normal development. In a proof-of-concept experiment, we obtained evidence that exposure to an inhibitor of histone modification enzymes implicated in epigenetic control of gene expression induces an accelerated rate of hypnozoite activation. The protocol presented may further enable investigations of hypnozoite biology and the search for compounds that kill hypnozoites or disrupt their quiescence.

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Figure 1: Persistence of P. cynomolgi hypnozoites.
Figure 2: Activation of hypnozoites over time.
Figure 3: Induction of hypnozoite activation by a histone methyltransferase inhibitor.
Figure 4: Schematic diagram of the long-term primary hepatocyte cultures.

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Acknowledgements

This work was supported in part by a translational research grant (WT078285) from the Wellcome Trust, a grant from the Medicines for Malaria Venture to the Novartis Institute for Tropical Diseases (Singapore), the Biomedical Primate Research Centre (the Netherlands), the Université Pierre et Marie Curie–Paris 6 (France), a Seventh Framework Programme EU European Research Council Executive Agency Advanced Grant (Plasmoescape 250320) to A.S. and a Laboratoires d'Excellence (LABEX) grant (ANR-11-LABX0024) from the Agence Nationale de la Recherche (France). We are grateful to T.A. Moss (Imperial College London) for the chemical synthesis of TM2-115. HepaRG cells were provided by C. Guguen-Guillouzo (Biopredic International). pTRIP-CMV-GFP ΔU3 vector was provided by P. Ravassard (le Centre de Recherche de l'Institut du Cerveau et de la Moelle Epinière). We thank the staff of the Flow Cytometry Core CyPS at the Université Pierre et Marie Curie (Pitié-Salpêtrière Hospital, Paris, France) for their help and assistance. Finally, we thank A.C. Grüner for her excellent critical comments on the manuscript.

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Author notes

  1. Laurent Dembélé and Jean-François Franetich: These authors contributed equally to this work.

Authors and Affiliations

  1. Sorbonne Universités, Université Pierre et Marie Curie Paris 6, UMR S945, Paris, France

    Laurent Dembélé, Jean-François Franetich, Audrey Lorthiois, Audrey Gego, Georges Snounou & Dominique Mazier

  2. Institut National de la Santé et de la Recherche Médicale, U945, Paris, France

    Laurent Dembélé, Jean-François Franetich, Audrey Lorthiois, Audrey Gego, Georges Snounou & Dominique Mazier

  3. Department of Parasitology, Biomedical Primate Research Centre, Rijswijk, The Netherlands

    Anne-Marie Zeeman & Clemens H M Kocken

  4. Division of Immuno-Virology, Institute of Emerging Diseases and Innovative Therapies, Commissariat à l'Energie Atomique et aux Energies Alternatives, Fontenay-aux-Roses, France

    Roger Le Grand & Nathalie Dereuddre-Bosquet

  5. Université Paris-Sud XI, UMRE01, Orsay, France

    Roger Le Grand & Nathalie Dereuddre-Bosquet

  6. Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

    Geert-Jan van Gemert & Robert Sauerwein

  7. Service de Chirurgie Digestive, Hépato-Bilio-Pancréatique et Transplantation Hépatique, Hôpital Pitié-Salpêtrière, Paris, France

    Jean-Christophe Vaillant & Laurent Hannoun

  8. Department of Chemistry, Imperial College London, South Kensington Campus, London, UK

    Matthew J Fuchter

  9. Novartis Institute for Tropical Diseases, Singapore

    Thierry T Diagana

  10. Unité de Biologie des Interactions Hôte-Parasite, Institut Pasteur, Paris, France

    Nicholas A Malmquist & Artur Scherf

  11. Centre National de la Recherche Scientifique, Unité de Recherche Associée 2581, Paris, France

    Nicholas A Malmquist & Artur Scherf

  12. Assistance Publique–Hôpitaux de Paris, Groupe Hospitalier Pitié-Salpêtrière, Service Parasitologie-Mycologie, Paris, France

    Dominique Mazier

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Contributions

D.M., G.S. and A.S. designed the study. L.D., J.-F.F., A.L. and A.G. designed and performed the experiments. A.-M.Z., C.H.M.K., R.L.G., N.D.-B., G.-J.v.G., R.S., J.-C.V., L.H., M.J.F., T.T.D. and N.A.M. contributed essential materials. G.S. and D.M. wrote the paper.

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Correspondence to Georges Snounou.

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T.T.D. is a Novartis employee and owns Novartis shares.

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Dembélé, L., Franetich, JF., Lorthiois, A. et al. Persistence and activation of malaria hypnozoites in long-term primary hepatocyte cultures. Nat Med 20, 307–312 (2014). https://doi.org/10.1038/nm.3461

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