Antoine Claessens 24 October 2019

1. Le silence assourdissant du portage asymptomatique du Plasmodium falciparum A misleading title because:(a) a major part of this lecture is about antigenic variation (b) I will speak in English ;-) Antoine Claessens 24 October 2019 antoineclaessens@gmail.com

2. Outline Part 1: Brief introduction to malaria Part 2: Introduction to var genes and antigenic variation Part 3: On the origin of var gene sequence diversity Part 4: Asymptomatic infections and field sample collection Part 5: Brainstorming and debate: how would you save the world from malaria? Feel free to interrupt, ask questions, comments, at any time! And tell me to slow down if I speak too fast

3. PhD: Microarray transcriptome analysis of P. falciparum binding to human brain endothelial cells PostDoc: Mutation rate and genomics of P. falciparum Malaria clinical trial La Pitié-Salpêtrière Paris

4. U. Montpellier Chargé de Recherche INSERM Research Fellowshipat the London School of Hygiene & Tropical Medicine and MRC-Gambia Plasmodium falciparum asymptomatic infections (Genomics) MRC-Gambia,The Gambia

5. MALARIAAn introduction Acknowledgements: Some slides from http://courses.bio.unc.edu

6. infectious diseases remain big killers Leading causes of death in Sub-Saharan Africa, South Asia, and Southeast Asia for persons age 0-44 (World Health Organization)

7. Number 3: Malaria Leading causes of death in Sub-Saharan Africa, South Asia, and Southeast Asia for persons age 0-44 (World Health Organization)

8. 2 billion people are at risk of malaria

10. What Is malaria? • A mosquito-borne infectious disease caused by Protozoan parasites of the genus Plasmodium • It’s not a bacteria or a virus! Mammals Plasmodium plants • Plasmodium species able to infect humans: • P. falciparum • P. vivax • P. ovale(2 subspecies) • P. malariae • P. knowlesi

11. What Is malaria? • Transmitted only by Anopheles Mosquitoes (>60 species!)

12. Plasmodium life cycle

13. All age groups are at risk of developing malaria However, in high endemicity areas, only children develop severe symptoms Kinyanjui 2012

14. Malaria pathogenesis and the crucial role of var genes

15. Malaria Ring-stage Plasmodium infected red blood cell(0 to 24h post invasion) The spleen removes old red blood cells P. falciparum infected red blood cells Rigid Sequestration P. vivax infected red blood cells Deformable No or little sequestration

16. P. falciparum Infected red blood cells at pigmented trophozoite stages cytoadhere to microvessels(sequestration) • P. falciparum Infected red blood cells at pigmented trophozoite stages cytoadhere to microvessels (sequestration)

17. Why botherabout var genes? The Var protein, or PfEMP1 (P. falciparum Erythrocyte Membrane Protein 1), locates at the surface of the iRBC and mediates cytoadherence to endothelial cells

18. var genes encode Erythrocyte Membrane Protein (PfEMP1) • Encoded by ~50 var genes per P. falciparum genome. • Located in the subtelomeres and in clusters in the core genome. • All var genes share a few conserved motifs but are mostly extremely variable. • Each clone, or strain, has got a different set of 50 var genes. • LOADS of strains in the wild. • Thus, the total number of var genes in the world = LOADS x 50 • However, each parasite expresses only 1 var at a time.

19. var genes encode Erythrocyte Membrane Protein (PfEMP1) • In a culture flask, in the absence of any selective pressure, virtually all of the 50 var genes are expressed.

20. Var genes are classified into 3 major groups: A, B and C according to their upstream region. upsA upsB upsC var A var B var C var genes encode Erythrocyte Membrane Protein (PfEMP1) • In a culture flask, in the absence of any selective pressure, virtually all of the 50 var genes are expressed. • The var gene subgrouping has been linked with PfEMP1 functions: • For example, only group B and C PfEMP1s are able to bind to receptor CD36. • Group A var genes expression is associated with Cerebral Malaria (Avril2012, Claessens2012, Lavstsen2012, PNAS)

21. Group A EPCR Group B/C PfEMP1 Group A PfEMP1

22. P. falciparum’s stroke of genius: Extreme polymorphism, yet functional var sequences How is the var gene sequence diversity generated? Conserved region Conserved region One var gene = 4 to 7 DBL and CIDR domains

23. Outline Part 1: Brief introduction to malaria Part 2: Introduction to var genes and antigenic variation Part 3: On the origin of var gene sequence diversity Part 4: Asymptomatic infections and field sample collection Part 5: Brainstorming and debate: how would you save the world from malaria? Feel free to interrupt, ask questions, comments, at any time! And tell me to slow down if I speak too fast

24. Mitotic recombination of var genes: The most polymorphic gene family in P. falciparum just got more diverse GOAL: To identify new mutations (SNP, indels…) in cultures of P. falciparum

25. Whole Genome Sequencing (Illumina) Sequencing raw data = Millions of ‘reads’, each of them about 100bp long Bioinformatic challenge: to reconstruct a 23Mbp-long genome from pieces that are 100bp long Luckily there is a “reference genome” (3D7 strain) available. => All reads are ‘mapped’ against the reference genome

26. After each round of mitosis, P. falciparum, like any living organism, will accumulate mutations The parasite population accumulates mutations. However, each mutation occurs randomly in a single genome. Overall, the genome of the population is still “wild-type”. (assuming no selection!!) one individual mutation one individual parasite genome DNA extraction Sequencing DNA extraction Sequencing DNA extraction Sequencing T T G A

27. Building up a clone tree to record mutation accumulation in vitro Whole genome sequencing Cloning Parent Progeny, clone 2 Progeny,clone 1 Whole genome sequencing Growth

28. Days in culture (‘A branch’) * * * * Clone tree with P. falciparum strain Dd2 0 20 60 40 80 160 180 100 140 120 200 • Clone trees generated with 6 strains, including 2 field isolates. • 350 subclones were whole genome sequenced at >20X coverage 200 0 20 60 40 80 160 180 100 140 120 * * * * Days in culture (‘B branch’)

29. Single Nucleotide Polymorphisms (SNPs) are scattered throughout the genome • P. falciparum genome: • 14 chromosomes • 23Mb • 80% AT • Haploid Core genome Subtelomere / internal var region SNP in 3D7 SNP in Dd2 SNP in W2 SNP in HB3

30. SNPs are scattered throughout the genome • Structural Variants: • Deletion • Duplication • Translocation Core genome Subtelomere / internal var region SNP in 3D7 SNP in Dd2 SNP in W2 SNP in HB3 Non-genic interchromosomal structural variant Non-genic intrachromosomal structural variant Var gene interchromosomal structural variant Var gene intrachromosomal structural variant But Structural Variants are found in var regions (subtelomeres and internal)

31. Recombinations generate new “chimeric” var sequences Parental clone Progeny subclone Chromosome 5 Chromosome 5 2 Var10 exon 1 Var5 exon 1 Var5 exon 1 2 2 2 Chromosome 10 Chromosome 10 Var10-5 X X X

32. Real-time evolution Days in culture (‘A branch’) * * * * 0 20 60 40 80 160 180 100 140 120 200 Var34-45-34 present Var34-45-34 absent Var34-45-34 extra recombinations Var34 deleted 200 0 20 60 40 80 160 180 100 140 120 * * * * Days in culture (‘B branch’)

33. Patterns of var gene recombination Pair of recombining var genes, here with 3 recombination events

34. Order within chaos: The var gene structure is always conserved var34 DBLα0.1 CIDRα3.1 CIDRα8 DBLα16 DBL δ1 var45 DBL α0.1 CIDRα3.1 CIDRα4 DBLδ1 DBLα 0.1 DBLδ 1 CIDRα8 DBLα 0.1 DBLδ 1 DBLα0.1 CIDRα3.1 CIDRα3.1 CIDRα4 DBLα16 DBLδ1

35. Order within chaos: Recombination occurs at loci with elevated homology between recombining var genes Recombinationbreakpoints

36. Lessons from the clone trees • Ectopic recombination occurs specifically in var regions. • Recombination creates new chimeric var sequences. Rate: Every 48h, 1 out of 500 parasites will generate a new chimeric var gene. • The resulting chimeric var sequence is always in frame. • The structure of the gene is preserved. • Recombination does not occur randomly but within regions of higher homology. • All observed recombinations were in group B and C var, never group A. SNP mutation ratevar recombination rate Art-S Art-R

37. P. falciparum life cycle Hundreds of mitoses! A single meiotic event Bousema &Drakeley, Clin. Microbiol. Rev., 2011 • The origin of var gene sequence polymorphism • New var gene sequences are mainly generated by the numerous rounds of mitosis. • Meiosis has a crucial role in shuffling var genes from the 2 parents.

38. Per 48-hour life cycle inside a single infected individual… An infected human with clinical malaria carries ~ 1010 parasites Mutation rate ~ 3.2 * 10-10 SNPs / life cycle / nucleotide, in all tested strains. = Every nucleotide in the genome is substituted for alternatives several times Exon 1var recombination rate ~ 2 * 10-3 recombining var pairs / life cycle = millions of chimeric var genes generated • All above predictions are based on in vitro results... What remains to be tested: • Do chimeric var genes occur in vivo? • If so, are they expressed? • If so, are they ‘new antigens’ ? • More generally, does antigenic variation explain chronic infections?

39. Var genes mediate immune evasion by regularly switching the antigen present at the surface of the infected red blood cell 1. This hypothesis has not been tested in humans yet! 2. Mathematical models predict that, for a single infection, the parasite would exhaust all of its var gene repertoire after a few weeks. Hypothesis to be tested: what if the parasite was generating novel var gene sequences during the course of an infection? To test this hypothesis, we need blood samples from chronically infected individuals…

40. Outline Part 1: Brief introduction to malaria Part 2: Introduction to var genes and antigenic variation Part 3: On the origin of var gene sequence diversity Part 4: Asymptomatic infections and field sample collection Part 5: Brainstorming and debate: how would you save the world from malaria? Feel free to interrupt, ask questions, comments, at any time! And tell me to slow down if I speak too fast

41. In Africa, very strong correlation between humidity and malaria cases ARIDITY MALARIA ENDEMICITY

42. RAIN MALARIA CASES

43. How does Plasmodium falciparum survive during the dry season?

45. Unresolved questions: • Why do P. falciparum infections lead to clinical cases in the wet season but not in the dry season? • Is the parasite able to regulate its virulence?

47. Longitudinal study in a Gambian village

48. Longitudinal study in a Gambian village