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Discovery of novel IgM binding PfEMP1 variants in Plasmodium falciparum line NF54

Discovery of novel IgM binding PfEMP1 variants in Plasmodium falciparum line NF54. Anine E Jeppesen PhD-student Centre for Medical Parasitology, University of Copenhagen and Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark. PfEMP1 variants.

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Discovery of novel IgM binding PfEMP1 variants in Plasmodium falciparum line NF54

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  1. Centre for Medical Parasitology Discovery of novel IgM binding PfEMP1 variants in Plasmodium falciparum line NF54 Anine E Jeppesen PhD-student Centre for Medical Parasitology, University of Copenhagen and Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark

  2. Centre for Medical Parasitology PfEMP1 variants • Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) • Mediate adhesion to host receptors • Build up of DBL and CIDR domains

  3. Centre for Medical Parasitology PfEMP1 variants • PfEMP1 proteins are encoded by var genes • 60 different copies pr. genome • Variation between genomes • huge PfEMP1 variant repertoire • Mutually exclusive expression • Epigenetic memory

  4. Centre for Medical Parasitology PfEMP1 and non-immune IgM binding • PfEMP1 variants binding non-immune IgM binding: • VAR2CSA variants (Creasey et al. 2003. Infection and Immunity, Rasti et al. 2006. PNAS) • Some rosetting variants (Rowe et al. 2002, Am. J. Trop. Med. Hyg.)

  5. Centre for Medical Parasitology PfEMP1 and non-immune IgM binding • Role of non-immune IgM binding • Immune evasion by masking IgGepitopes (VAR2CSA) (Barfod et al. 2011, PNAS) • Role in rosette formation (Clough et al. 1998, Exp. Parasit.) • Questions: • Other roles of non-immune IgM binding? • Other non-immune IgM binding variants?

  6. Centre for Medical Parasitology Selecting IE for non-immune IgM binding PfEMP1 variants • Problems: • In vitro cultures tend to express the same PfEMP1 variants and have a low switching rate towards other variants • Solution: • Using the var transcription memory reset parasite G6 of the NF54 line. IgM

  7. Centre for Medical Parasitology The NF54 G6 parasite line • NF54 (3D7) line transfected with the VBH plasmid (Dzikowski et al. 2007. EMBO reports) • VBH resets the epigenetic memory of what var genes was being transcribed (Dzikowski and Deitsch. 2008. J. Mol. Biol.) Dzikowski et al. 2007 EMBO reports •  Heterogenous population expressing a vast variety of vargenes Fastman et al. 2012. PLoS One

  8. Centre for Medical Parasitology Method IgM Test for IE by Ethidium Bromide staining in flow cytometry Fastman et al. 2012. PLoS One Transfer to 25 ml flask culture, test for non-immune IgM binding and var transcription

  9. Centre for Medical Parasitology Results – IgM binding in flow cytometry EthidiumBromide IgM Positive (5 clones) Mix (13 clones) Negative (1 clone)

  10. Centre for Medical Parasitology Results - var gene transcription PFL0030c (VAR2CSA) PFL0020w (/MAL6P1.4) (2 clones) (4 clones) PF07_0139 MAL6P1.316 (3 clones) (2 clones)

  11. Centre for Medical Parasitology MAL6P1.4 binds to non-immune IgM antibodies • 3D7 line selected by MAL6P1.4 specific antiserum • Not rosetting

  12. Centre for Medical Parasitology MAL6P1.4 binds to IgM at the C-terminal end

  13. Centre for Medical Parasitology PFL0020w binds to non-immune IgM antibodies • NF54 line transcribing PFL0020w

  14. Centre for Medical Parasitology PFL0020w binds IgM at the C-terminal end

  15. Centre for Medical Parasitology MAL6P1.316 binds to non-immune IgM antibodies • 3D7 line selected by MAL6P1.316 specific antiserum 10 nMIgM Control

  16. Centre for Medical Parasitology Discussion • 5 predicted non-immune IgM binding variants in NF54 • MAL6P1.4, PFL0020w and MAL6P1.316 bind non-immune IgM • Indication of binding via the C-terminal end HB3VAR06 VAR2CSA MAL6P1.4 PFL0020w PF07_0139 MAL6P1.316 • Ongoing work: Characterization of remaining variants, • Determination of binding site • What role does non-immune IgM binding play?

  17. Centre for Medical Parasitology Thank you for your attention! Liz Stevenson, CMP Ron Dzikowski Hebrew University, Jerusalem Louise Turner, CMP Supervisors: A special thank to: Christian Wang Michele Kreutzer Erik Laursen Lisbeth Stolpe Financial support: University of Copenhagen EviMalaR The Danish Research Council Rigshospitalet The Novo foundation The Lundbeck foundation Lea Barfod, CMP Lars Hviid, CMP

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