Washington Global Health Alliance Discovery Series Robert Sinden, PhD July 28, 2008 'Understanding Malaria Development in the Mosquito, and its Pivotal Role in the Formulation of Effective Control Strategies'

1. Understanding malaria development in the mosquito, and its pivotal role in the formulation of effective control strategies. R.E.Sinden DSc, FMedSci. The Malaria Research Centre Imperial College London

3. Genomics / Immune mechanisms Structure / Function analysis Modelling Population & Evolutionary Biology Transgenics / Drive systems Sinden / Billker Kafatos / Christophides / Vlachou Crisanti / Catterucia / Burt Koella Basanez / Ghani GM refractory mosquitoes Transmission blocking vaccines & drugs Indoor antimalarial smart chemicals

6. Parasite and/or vector? Vector

9. Plasmodium is a ‘local’ parasite distributed by mosquitoes

10. Malaria-Vector Control Works! Environmental House position House screens --Bednets Breeding site-- elimination DDT ( 19 50 s -60 s ) Synthetic Pyrethroid s/ ITN : Permethrin, Deltamethrin ( since 19 80 s ) Biological Control Bacillus thuringiensis israelensis (Bti) ( since 1980s ) Insecticidal

11. Reducing malaria profile (2001-2006) Source WHO Reports from countries Francis Omaswa, ITN’s; Drugs; Societal change

12. Why attacking (malaria in) the mosquito is an essential component of eradication Ro- the basic reproductive rate, is defined as the number of secondary cases derived from one individual. For malaria this can be as high as 200, but must be <1 to reduce the number of persons infected. ma 2 bp n r(-log e p) R o =

13. ma 2 bp n r(-log e p) R o = Mosquito:man ratio Mosquito biting rate Proportion of mosquitoes that are infectious Mosquito daily survival rate The daily proportion of infected people who become non-infectious to the mosquito Days taken for parasite in mosquito to become infective What factors determine the value of Ro?

14. ma 2 bp n r(-log e p) R o = Environment mgmt Insecticides Larvicides SIT/RIDL Biological control GM-sex ratio Housing Clothing Repellents Deterrents ITN GM-Olfaction mutants GM-Biological clocks GM-Parasite/vector interactions GM-Vector immunity Sporontocidal drugs ‘ Terminator’ genes ITN/IRS Gametocytocidal drugs Transmission-blocking vaccines and drugs L iver/ABS drugs/vaccines… only if they reduce infectivity to vector

15. Parasite and/or vector? Parasite…..How?

16. Biology of Plasmodium development in the mosquito 8-20 day 1 hour 12 hour 24-36 hour 15 min Gametocytes  Gametes  Zygote  Ookinete  Oocyst  Sporozoite

18. Population bottlenecks With thanks to Georgos Christophides

21. Host cell lysis Mitosis Axoneme Polymerisation Differential gene expression DNA synthesis Chromatin condensation, Axoneme motility Cytokinesis Toye: Kawamoto: Ogwang: Janse Billker et al ., Cell, 2004 Tewari et al ., Mol. Mic., 2005 Azadirachtin Aphidicolin Colchicine Vinblastine Actinomycin D CycloheximidePuromycin Emetine Cytochalsasin B TMB8 W-7

22. Male gamete formation Targets: P48/45, P230

23. Target parasite energy metabolism in gamete? Arthur Talman D-Glucose Export? N HT A

24. Ookinete Invasion of PTM and Midgut Cells Vlachou et al, Midgut R.Moon Bloodmeal PTM Midgut

25. Target parasite energy metabolism in ookinete? Vertebrate Invertebrate Oxidative phosphorylation Mitochondrial transport TCA cycle Glycolysis Atovaquone very active vs ookinete and oocyst Glycolysis and conversion of pyruvate to L-lactate TCA cycle Oxidative Phosphorylation Other mitochondrial proteins ABS Gct Okn

27. Impact of anti-gamete (P230) antibody on microgametes Control Anti-230 SEM Ferritin -TEM

29. Expression of FusM fragments in E.coli N C TM 1 828 680 708 33 N C MBP - 40 kDa TVMV TEV His 6 FusM polypeptide 37 kDa 25 kDa 1 2 3 4 5

30. Anti-HAP2 rabbit sera inhibits P.berghei ookinete development in vitro Currently evaluating transmission-blocking ability of sera in vivo using SMFA. Affinity purification on rProtein 37 kDa 25 kDa 1 2

31. Ookinete Secretome Mass culture ookinetes (5L) Purify Sonicate Fractionate MudPIT Fraction 6/7= 1.17M sucrose SOAP Kalpana Lal CTRP Fraction 6

32. Defining the ookinete/mosquito interactome 184 6 518 Includes : hypotheticals with protease or carbohydrate binding domains and botulinum neurotoxin-related homologies (regulated pathway) Includes : known vaccine candidates P25, P28 (default pathway) WARP and 3 hypotheticals ‘ Surface’ Proteome Bioinformatics Micronemes 4 3 90 26 POSH Etramp Pepsinogen

33. Proteomics: Understanding the Ookinete….Surface (Vaccine discovery) 1985 2005 2007 R. Sinden et al. -of micronemal origin

34. Surface/secreted molecules with known roles in gamete-ookinete-oocyst development P230 P48/45 HAP2* P47 P25 P28 Chitinase WARP MAOP CTRP PPLP5* Fertilization Ookinete ‘ Epithelial’ invasion

35. Standard Membrane Feeding Assay for transmission-blocking vaccines. Blood containing gametocytes + test antiserum Artificial or Natural Membrane Jacket for warm water Infected mosquito gut 6 days post feed Cultured P.f gametocytes + transmission blocking antibodies give reduced numbers of oocysts per mosquito and fewer infected mosquitoes ( An. stephensi and An. gambiae )

36. Human trials of Pv25 vaccine If 80% of the gametocyte carriers immunised vaccination would result in 33% reduction in transmission in the field Malkin et al; Vaccine, 2005 Medley et al; Parasitology, 1993 Antibody Activity (ELISA units) Percent blockade of oocyst intensity

37. Modelling transmission Sporozoites in bite Emma Dawes in Sinden et al PLoS Pathogens 2007 Hyperbolic No. of Macrogametocytes No. of Ookinetes Sigmoid No. of Ookinetes No. of Oocysts Sigmoid ? Hyperbolic

39. 1. Modelling:- Implications A vaccine that kills 90% of the parasites will have markedly different impacts upon transmission ( i.e rendering each mosquito incapable of transmitting malaria) dependent upon the initial parasite burden Sinden et al PLoS Pathogens 2007 Percent reduction in infected mosquitoes Initial parasite number in mosquito Low transmission High transmission

40. Intensity/Prevalence relationship in P. berghei infections of the mosquito Medley et al 1993

41. Number of P. falciparum oocysts per positive mosquito in Simbock, Cameroon Frequency Number of Oocysts 30% of infected mosquito population have just one oocyst Mean infection intensity 2.2 oocysts Annan at al. 2005

42. Malaria Research Centre Current Research Collaborations Malaria Research Centre Institute for Systems Biology FILM Imaging Centre Scripps Research Institute (Proteomics) Metabolomics Research Biomalpar NoE Sanger Institute IRDC/IRSS Burkina Faso Malaria Research Centre Centre for Bioinformatics

44. Gene expression during ookinete development DNA replication Meiosis Gametocyte activation 0 1h 24h Fertilisation 3h 8h Midgut Invasion 10d www.lumc.nl www.lumc.nl What essential function do aspartyl protease and the LAPs fulfil so early in development, and yet the phenotype is visible so late? Maternal transcription, gametic translation e.g p28, p25 Maternal transcription, ??????? translation e.g AP essential function phenotype Pre-zygotic maternal t/t/function e.g LAPs Zygotic (m/f) gene activation e.g. CTRP

45. Gene disruption (double crossover) studies were carried out in the rodent malaria model P.berghei. Plasmodial (and C. reinhardtii) HAP2 KO male gametes exflagellate normally, retain the ability to form tight, pre-fusion membrane attachments with female macrogametes, but are unable to fertilise normally, blocking the transmission of the parasite in the mosquito. IFAs using tagged transgenic parasites, and genetic crosses using male (CDPK4 KO) and female (NEK4 KO) deficient strains of P.berghei demonstrate that HAP2 functions is located (and essential) on the male gametocyte/gamete:

46. Binding of Pv25 to Mab Fragment Heavy chain Light chain GPI- anchor Mike Sternberg

47. Sequence variation between P25 orthologues Face ‘A’ Face ‘B’

48. In Silico modelling of ookinete surface molecules and their interactions M.Sternberg

49. Transmission-Blocking Epitope Distribution on Generic P25/28 GPI- anchor 1 2 3