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WGHA Discovery Series: Robert Sinden
 

WGHA Discovery Series: Robert Sinden

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Washington Global Health Alliance Discovery Series...

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'

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WGHA Discovery Series: Robert Sinden WGHA Discovery Series: Robert Sinden Presentation Transcript

  • 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
    • To pursue world leading research and training programmes on malaria
    • To strengthen international collaborations (Africa, India, USA & Europe)
    • To understand the biology of the malaria parasite, its vector, and their interactions
    • To pioneer methods to render endemic populations of mosquitoes incapable of malaria transmission
    IC Malaria Research Centre A Developing Cluster of 11 PI’s/50 scientists OBJECTIVES
  • 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
  • The dual drivers for intervention: treatment and control
    • There is a clear and urgent need to save the life of each infected individual, but unless we stem the input of new cases such campaigns are unsustainable.
    • Elimination/eradication, as an objective, refocuses our attention on the critical need to break the parasite transmission cycle (Ro-><1).
  • Malaria: a diverse/dynamic target for intervention
    • 5 species of parasite. P.falciparum P. vivax P. malariae P. ovale P.knowlesi
    • Transmitted by ~51 /~470 mosquito species
  • Parasite and/or vector? Vector
  • With such vector diversity, why target malaria in the mosquito?
    • Experience says vector control can reduce transmission.
    • Average infection 5 parasites (vs 10 9 -10 12 in human).
    • Extracellular for 24hrs vs. 15 sec in man. (window-of-opportunity)
    • The target of T-B vaccines are remarkably in variant.
  • Hotspots of Malaria Transmission
        • Malaria transmission is local and focal with estimation that typically 20% of the hosts is responsible for 80% of the disease transmission
    S. Brooker, et al., Trop. Med. Int. Health 9(7), 757 (2004); J. Gaudart, et al., BMC. Public Health 6, 286 (2006); Y. Ye, et al., Malar. J. 6, 46 (2007) ; D. L. Smith, et al., Nature 438(7067), 492 (2005); M. E. Woolhouse, et al., Proc. Natl. Acad. Sci. U. S. A 94(1), 338 (1997).
  • Plasmodium is a ‘local’ parasite distributed by mosquitoes
  • 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
  • Reducing malaria profile (2001-2006) Source WHO Reports from countries Francis Omaswa, ITN’s; Drugs; Societal change
  • 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 =
  • 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?
  • 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
  • Parasite and/or vector? Parasite…..How?
  • 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
  • Why target the parasite in the mosquito?
    • Past experience says vector control can be very effective.
    • Average infection 1-5 parasites (vs 10 9 -10 12 in human).
    • Extracellular for 24hrs vs. 15 sec in man. (Window-of-opportunity)
    • The target of T-B vaccines are remarkably In variant.
  • Population bottlenecks With thanks to Georgos Christophides
  • Gametocytes and transmission-blocking drugs
    • Mature gametocytes are arrested in G 0 of the cell cycle, and are insusceptible to many antimetabolites.
    • Some schizonticidal drugs increase the unit-infectivty (chloroquine), others reportedly increase the proportion of gametocytes (Fansidar).
    • Artemesinin has gametocytocidal activity
    • We have neglected to prioritise the search for drugs that kill gametocytes , or those vulnerable stages in early development in the mosquito (gametes; ookinete).
    • Drugs to target signalling pathways
    • Provide insights into life cycle regulation
    • Identify essential genes for prioritisation as drug targets
    Billker et al ., CamK-like CDPK4 MAP-2 Nek-4 Nek-2 CDPK3 CDPK6 PbPK7 PKG PDE GC
  • 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
  • Male gamete formation Targets: P48/45, P230
  • Target parasite energy metabolism in gamete? Arthur Talman D-Glucose Export? N HT A
  • Ookinete Invasion of PTM and Midgut Cells Vlachou et al, Midgut R.Moon Bloodmeal PTM Midgut
  • 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
  • The role of transmission-blocking vaccines
    • Past experience says vector control can be very effective.
    • Average infection 5 parasites (vs 10 9 -10 12 in human).
    • Extracellular for 24hrs vs. 15 sec in man. (Window-of-opportunity)
    • The target of T-B vaccines are remarkably In variant.
  • Impact of anti-gamete (P230) antibody on microgametes Control Anti-230 SEM Ferritin -TEM
  • HAP2/GSC-1 – a potential transmission blocking target?
    • 85.68 kDa (828 aa) protein, located on male gametocytes/gametes in Plasmodium spp.
    • Originally identified in screens for zygote formation mutants in the green alga Chlamydomonas reinhardtii (Bill Snell; University of Texas Southwestern Medical Center)
    Activated malarial male gametocyte with HA tagged HAP2 Male malarial microgamete with HA tagged HAP2 Liu et al; Genes & Dev. 2008
  • 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
  • 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
  • Ookinete Secretome Mass culture ookinetes (5L) Purify Sonicate Fractionate MudPIT Fraction 6/7= 1.17M sucrose SOAP Kalpana Lal CTRP Fraction 6
  • 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
  • Proteomics: Understanding the Ookinete….Surface (Vaccine discovery) 1985 2005 2007 R. Sinden et al. -of micronemal origin
  • 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
  • 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 )
  • 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
  • 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
    • Measuring intensity and prevalence of infection in vectors is essential to understand the population dynamics of Plasmodium within the mosquito
    • Assessing the potential impact of control measures
    Implications
      • If linear: Intervention efforts result in a proportional effect
      • If negative density dependence:
      • Areas with high parasite intensities sustained intervention required before significant impact.
    0 20 40 60 80 100 0 20 40 60 80 100 Parasite Density Measure of Transmission 50% 50% 50% 10%
  • 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
  • Intensity/Prevalence relationship in P. berghei infections of the mosquito Medley et al 1993
  • 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
  • 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
  • Participating groups
    • Genetic Manipulation
    • University of Leiden (Waters)
    • P. falciparum
    • Cornell University (Templeton)
    • Wurzburg (Pradel)
    • Proteomics
    • US Navy (Carucci)
    • Scripps Institute (Florens/Yates)
    • TIGR (Carlton)
    • Sanger Centre (Hall/Berriman)
    • Imperial College
    • Parasite Cell Biology Group
    • Ecker ; K. Lal ; J.Mendoza; D.Raine ;
    • C. Ramakrishnan; R.Stanway; H.Trueman ;
    • M. Tufet; J.Robertson; K.Baker
    Infectious Disease Epidemiology M-G Basanez, E.Dawes
  • 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
  • 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:
  • Binding of Pv25 to Mab Fragment Heavy chain Light chain GPI- anchor Mike Sternberg
  • Sequence variation between P25 orthologues Face ‘A’ Face ‘B’
  • In Silico modelling of ookinete surface molecules and their interactions M.Sternberg
  • Transmission-Blocking Epitope Distribution on Generic P25/28 GPI- anchor 1 2 3