Antigenic variation in malaria involves a highly structured

Antigenic variation in malaria involves a highly structured switching pattern Mario Recker Department of Zoology, University of Oxford

Mathematical approach to (understand) malaria “the mathematical method of treatment is really nothing but the application of careful reasoning to the problems at issue. ” Sir Ronald Ross 20. Aug. 1897 Ross R, 1911. The Prevention of Malaria. London: John Murray. ? ? ? Macdonald G, 1957. The Epidemiology and Control of Malaria from Mc. Kenzie & Samba, AJTMH 2004

Most targets of protective immunity polymorphic surface proteins Development of immunity / effective vaccines hindered by extensive antigenic diversity: - mutation / recombination (genotypic change) - antigenic variation (no genotypic change) circumsporozoite protein (CSP) merozoite surface proteins (MSP) diversity variant surface antigens (VSA) www. fda. gov/Cb. ER/blood/malaria 071206 sk 5. gif Major multigene families: o rif o stevor o Pfmc-2 TM > 150 copies per genome 30 copies per genome 13 copies per genome o var 60 copies per genome Scherf et al. , Annu Rev Microbiol 2008

Sequence diversity of var genes is immense! adapted from Gardner, M. et al. , 2002, Kyes, S. et al. , 2002 cumulative diversity of DBLa seqnuences from Barry et al, PLo. S Pathog. 2007 pairwise sharing among DBLa seqnuences

Antigenic variation in P. falciparum Pf. EMP 1 (P. falciparum Erythrocyte Membrane Protein 1) IE binding to endothelium • embedded on surface of red cell • causes severe disease through adherence to host cell receptors • important immune target IE binding to erythrocytes IE binding to dendritic cell EM by D. Ferguson, Oxford Univ.

Infected blood cells sequester in tissue capillaries EM by D. Ferguson, Oxford Univ.

(Molecular) Requirement for antigenic variation - every var gene recognised as part of a family - mechanism to limit expression to a single copy - activation coinciding with silencing of previously active gene - cellular memory to avoid ‘early’ repertoire exhaustion Result: Result Scherf et al. , Annu Rev Microbiol 2008 Pf. Sir 2: P. falciparum silent information regulator TPE: telomere position effect succeeding waves of parasitaemia dominated by a single variant of Pf. EMP 1 what orchestrates expression at population level?

What orchestrates sequential dominance? - use mathematical models to create and test hypotheses For example: • differences in growth rates or probabilities in switch rates (e. g. Kosinski, 1980) • differential susceptibilities assigned to variants expressing two surface proteins (e. g. Agur et al. , 1989) • modifications of switch rates by ‘natural selection’ (Frank, 1999) • immunological interaction, e. g. cross-immunity (e. g. Recker et al, 2004)

Increases in levels of antibodies to VSA expressed by heterologous isolates are transient and limited. 60 50 55 50 30 20 10 0 60 58 50 40 30 20 10 0 time after infection 18 Agglutinating antibody titer Percentage of infected red cells positive 40

Model assumption: each variant comprises a unique major epitope which elicits variant specific, long-lived immune response a number of minor epitopes which elicits transient, cross-reactive immune response

The model dynamics of variant i: intrinsic growth rate clearance by cross specific response -reactive response dynamics of specific response zi: immune response proportional to antigen decay rate m’>>m dynamics of transient, cross-reactive response, wi: transient immune response proportional to antigen variants with shared epitopes

Mathematical model without switching Recker et al, Nature 2004 Model suggested that parasite-host relationship has evolved to favour some short-lived immune responses that allow the parasite to persist and the host to survive

In vitro switching dynamics Horrocks et al. (PNAS, 2004) showed • on and off rates for a given variant are • on and off rates vary dramatically among different variants Abundance dissimilar var 1 var 2 var 3 time - rates appear to be intrinsic property of a particular gene - → could introduce a hierarchy of expression whereby stable variants are more prominently expressed, at least during the early phases of infection?

To investigate the nature of var gene switching, generate transcription profiles for the entire repertoire in clonal parasite populations and measure the change in that profile over time stable dominance of initial variant 1 st generation 2 nd generation initial variant replaced

change over generations determined by:

- use mathematical model to determine most likely switching pathway - off rate switch bias on rate use iterative approach to find ‘best-fit’ switch matrix and off-rate vector

Switch matrix: Switch sequence: 1→ 2 → 4 → 3 →

Clone 3 D 7_AS 2

Clone It_B 2 B

even for a stable clone…

Clone B 12 Data provided by Dzwikowski, Frank & Deitsch Clone B 10

To test the validity of this prediction, examine the var transcript distribution in Clone 2 every few generations

Evolutionary conflict: protection of repertoire vs. protection against immune attack repertoire protection: immune evasion:

Evolutionary conflict: protection of repertoire vs. protection against immune attack Assume var gene repertoire as a network where - nodes = variants - edges = switch / transition probabilities Task: optimise network over two traits - pathlength (= repertoire protection) - robustness (= adaptability to selection pressure)

Clone 3 D 7_AS 2

Clone It_B 2 B

Investigate effects of hierarchical switching for in vivo dynamics

naïve host highly structured switching results in (significantly? ) increased length of infection.

naïve host highly structured switching results in (significantly? ) increased length of infection. semi-immune host sms and lattice-type pathways far more flexible in overcoming pressure from pre-existing immune responses to help set up chronic infections.

Antigenic relationship between variants minor epitope 1 a b c d e minor epitope 2 u v x y z Switch sequence: (au) → (bu, av) → (cx) → (dx, cy) →…

Summary • for pathogens with a limited antigenic pool, such as P. falciparum, tight control over variant expression is essential • tightly ordered gene activation requires every subsequent variant to be able to evade current immune responses and therefore may be compromised by previous infections • highly structured switching in P. falciparum has evolved as an evolutionary compromise between the protection of its limited antigenic repertoire and the flexibility to fully utilise this repertoire when needed

Acknowledgements University of Oxford Department of Zoology • Sunetra Gupta • Caroline Buckee • Robert Noble • Chris Newbold • Andrew Serazin • Sue Kyes • Zóe Christodoulou • Robert Pinches • Sam Kinyanjui • Pete Bull • Kevin Marsh