A high-throughput assay for the identification of malarial transmission-blocking drugs and vaccines

https://doi.org/10.1016/j.ijpara.2012.08.009Get rights and content

Abstract

Following the cessation of the global malaria eradication initiative in the 1970s, the prime objective of malarial intervention has been to reduce morbidity and mortality. This motivated the development of high throughput assays to determine the impact of interventions on asexual bloodstage parasites. In response to the new eradication agenda, interrupting parasite transmission from the human to the mosquito has been recognised as an important and additional target for intervention. Current assays for Plasmodium mosquito stage development are very low throughput and resource intensive, and are therefore inappropriate for high throughput screening. Using an ookinete-specific GFP reporter strain of the rodent parasite Plasmodium berghei, it has been possible to develop and validate a high biological complexity, high throughput bioassay that can rapidly, reproducibly and accurately evaluate the effect of transmission-blocking drugs or vaccines on the ability of host-derived gametocytes to undergo the essential onward steps of gamete formation, fertilisation and ookinete maturation. This assay may greatly accelerate the development of malaria transmission-blocking interventions.

Highlights

► A novel Plasmodium transmission-stage screening assay is reported. ► The assay used in this study is high throughput, easy to perform and reliable. ► The assay is able to identify both transmission-blocking drugs and antibodies. ► The utility of the assay was demonstrated by screening 40 known and marketed antimalarial drugs. ► The assay will be of use in identifying new transmission-blocking interventions.

Introduction

The recent appreciation that decreasing malaria prevalence locally or globally requires strategies to reduce malaria transmission through the mosquito (Feacham and The Malaria Elimination Group, 2009, Alonso et al., 2011), has prompted a renewed search for transmission blocking drugs and vaccines, alongside other novel interventions. Transmission of the parasite from the human to the mosquito is exclusively mediated by a small fraction (∼0.2–1%) (Sinden, 1983) of bloodstage parasites: the terminally-, and sexually-differentiated gametocytes. Commitment of Plasmodium falciparum to sexual development in the blood of the host begins one asexual cycle before gametocytes are morphologically distinguishable, with all of the merozoites from one asexual schizont forming either asexual or sexual parasites (Bruce et al., 1990). Following red blood cell (RBC) invasion, sexually committed merozoites take ∼10 days to differentiate into mature gametocytes that are infectious to the mosquito (Garnham, 1966). For the first 6 days the young gametocyte grows rapidly, followed by a period of cell cycle arrest (G0) from day 6 to 10 during which the mature gametocyte progressively becomes insensitive to the majority of blood schizonticides (Smalley, 1977, Butcher, 1997). These mature gametocytes remain quiescent but infectious for as long as 22 days (Sinden and Smalley, 1979). In all other human-infective Plasmodium spp. and Plasmodium berghei, gametocyte maturation takes only 26–29 h and is almost concurrent with asexual parasitemia. Unlike P. falciparum, the mature gametocytes of these species remain infective for only a further 24 h (Sinden et al., 1996).

Female mosquitoes ingest mature male and female gametocytes from the peripheral blood of the host. In the gut of the mosquito, the parasite undergoes a period of rapid and explosive development. The resultant drop in temperature, together with the presence of mosquito-derived xanthurenic acid induces gamete formation and egress (Billker et al., 1998). In just 20 min, each male gametocyte produces eight sperm-like male gametes by a process termed ‘exflagellation’ (Toyé et al., 1977). Fertilisation ensues within the bloodmeal, with gamete recognition being dependent on Pfs48/45-P230 expression (van Dijk et al., 2001) and membrane fusion mediated by the conserved protein, HAP2 (Liu et al., 2008). Early zygote development requires de-repression of mRNAs held in stable complexes with the protein DOZI in the female gametocyte (Mair et al., 2006), leading to new rounds of AP2-O mediated transcription of ‘late’ genes and subsequent differentiation into a motile ookinete ∼22 h after ingestion (Sinden et al., 1985). Maturation of the ookinete requires the assembly of a large population of secretory organelles, the micronemes, within which is found the product of a ‘late’ gene, the circumsporozoite TRAP-related protein (CTRP), a protein essential for ookinete gliding motility (Dessens et al., 1999, Yuda et al., 1999, Ramakrishnan et al., 2011). The mature ookinete penetrates the mosquito midgut wall and on reaching the basal lamina transforms into an oocyst. Eight to 21 days later the oocyst produces thousands of sporozoites that migrate through the haemolymph to the salivary glands, which they invade and where they remain ready to infect another mammalian host when a subsequent bloodmeal is taken (Sinden, 1997).

Due to the comparative ease of bulk culture, high throughput (HTP) drug assays have been extensively performed against the asexual form of the parasite, screening millions of compounds for activity (Plouffe et al., 2008, Gamo et al., 2010, Guiguemde et al., 2010) and identifying exciting new lead compounds (Rottmann et al., 2010, Yeung et al., 2010). Recently, due to the renewed focus on parasite transmission, potential gametocyte drug screening assays have been reported in the literature. These broadly fall into four categories: those that assay gametocytogenesis (Dixon et al., 2009, Buchholz et al., 2011); those that focus on gametocyte development (Adjalley et al., 2011); those that assay for lethal activity against the mature gametocyte, either with an ATP production readout (Peatey et al., 2011, Tanaka and Williamson, 2011), or stage-specific GFP expression (Adjalley et al., 2011); and finally those that assay the onward development of mature gametocytes (Delves et al., 2012). Given that early gametocytes are susceptible to schizonticides (Kumar and Zheng, 1990, Chutmongkonkul et al., 1992, Smalley, 1977) and patients often present to the clinic already possessing infectious mature gametocytes (Giao et al., 2005, Tangpukdee et al., 2008), assays focussing on gametocyte induction and early development may be prone to rediscovery of previously identified schizonticidal compounds that will not necessarily affect the schizonticide-insensitive mature cell and so not impact transmission. Assays against the mature gametocyte using a cell viability readout will only identify compounds that are gametocytocidal (=’immediate death’), but will overlook those which do not kill the gametocyte directly, but fatally impair onward development in the mosquito (=’delayed death’). Consequently, the multifaceted remodelling that the gametocyte undergoes during the first 22 h after uptake in the mosquito provides an underexploited resource for the discovery of new transmission blocking interventions. Additionally, this transformation occurs in an environment almost entirely composed of host blood and so provides a convenient conduit for sustained exposure to interventions. For decades, the standard membrane feeding assay (SMFA) has been the “gold-standard” assay for evaluation of any transmission-blocking intervention (van der Kolk et al., 2005, Delves et al., 2012). It involves feeding drug- or antibody-treated gametocyte-infected blood to mosquitoes in artificial membrane feeders and then observing midgut oocyst infections approximately 7–10 days later. Whilst a powerful assay with high biological complexity, it is very low throughput and expensive, requiring the rearing of mosquito colonies, the training of skilled researchers, and laborious data collection and analysis. In contrast, P. berghei gametocyte-to-ookinete development can be reproduced successfully in vitro with high efficiency (Yoeli and Upmanis, 1968) and in bulk (Lal et al., 2009), and encapsulates all of the diverse and essential parasite biological processes occurring over the first 22 h in the mosquito, offering a prolonged drug/antibody exposure time. To date, quantification of ookinete production has been achieved by microscopic counting of Giemsa-stained ookinete slides (Ramjanee et al., 2007) or of cells stained with antibodies to ookinete-expressed proteins such as P28 (Blagborough and Sinden, 2009). The throughput achieved is consequently low, and accuracy varies between observers (Supplementary Fig. S1). This report outlines and validates a novel fluorescent assay measuring, in cultured P. berghei ookinetes, the intensity of GFP expressed under the control of the CTRP promoter (CTRPp.GFP) (Vlachou et al., 2004). Herein, it is demonstrated that this method can measure rapidly, accurately and reproducibly, the ability of drugs and antibodies to inhibit essential and complex events in the biology of gametocyte-ookinete differentiation at high throughput. This will enable future screening and identification of novel antimalarial drugs that have the potential to target a key bottleneck in Plasmodium development (Sinden, 2010), and facilitate the evaluation of new antigens as candidates in transmission-blocking vaccines.

Section snippets

Materials and methods

All work involving laboratory animals was performed in accordance with the EU regulations ‘EU Directive 86/609/EEC’ and within the regulations of the United Kingdom Animals (Scientific Procedures) Act 1986.

GFP expression by CTRPp.GFP ookinetes

At 22 h in culture, CTRPp.GFP parasites were observed to express GFP only in morphologically mature ookinetes as observed by fluorescence microscopy (Fig. 1A). Transformation from gametocytes into ookinetes and hence GFP expression is completely inhibited in the presence of the protein synthesis inhibitor cycloheximide (CH) (Fig. 1B; Supplementary Fig. S2). To establish whether the gross GFP fluorescence of the parasite line CTRPp.GFP (Vlachou et al., 2004) would reliably monitor ookinete

Discussion

Current practice, even in regions with the most effective malaria diagnosis and P. falciparum/Plasmodium vivax treatment programmes, has the consequence that committed mature gametocytes are likely to be present in the blood before the patient is treated (Giao et al., 2005, Tangpukdee et al., 2008), therefore treatments targeting the asexual bloodstage parasites alone are unlikely to break the cycle of Plasmodium transmission. Consequently, any programme of malaria elimination or eradication

Acknowledgements

The CTRPp.GFP parasite line was a gift from Dr D. Vlachou at Imperial College, London, UK. This study was supported by grants to RES and MJD from the Medicines for Malaria Venture (MMV), Switzerland.

References (55)

  • T. Tanaka et al.

    A malaria gametocytocidal assay using oxidoreduction indicator, alamarBlue

    Mol. Biochem. Parasitol.

    (2011)
  • R. Tewari et al.

    The systematic functional analysis of Plasmodium protein kinases identifies essential regulators of mosquito transmission

    Cell Host Microbe

    (2010)
  • M. van Dijk et al.

    A central role for P48/45 in malaria parasite male gamete fertility

    Cell

    (2001)
  • M. Yoeli et al.

    Plasmodium berghei ookinete formation in vitro

    Exp. Parasitol.

    (1968)
  • S. Adjalley et al.

    Quantitative assessment of Plasmodium falciparum sexual development reveals potent transmission-blocking activity by methylene blue

    Proc. Natl. Acad. Sci.

    (2011)
  • P. Alonso et al.

    A research agenda to underpin malaria eradication

    PLoS Med.

    (2011)
  • O. Billker et al.

    Identification of xanthurenic acid as the putative inducer of malaria development in the mosquito

    Nature

    (1998)
  • V. Bounkeua et al.

    In Vitro Generation of Plasmodium falciparum Ookinetes

    Am. J. Trop. Med. Hyg.

    (2010)
  • M. Bruce et al.

    Commitment of the malaria parasite Plasmodium falciparum to sexual and asexual development

    Parasitology

    (1990)
  • K. Buchholz et al.

    A high-throughput screen targeting malaria transmission stages opens new avenues for drug development

    J. Infect. Dis.

    (2011)
  • M. Chutmongkonkul et al.

    A new model for testing gametocytocidal effects of some antimalarial drugs on Plasmodium falciparum in vitro

    Ann. Trop. Med. Parasitol.

    (1992)
  • R. Coleman et al.

    Gametocytocidal and sporontocidal activity of antimalarials against Plasmodium berghei ANKA in ICR Mice and Anopheles stephensi mosquitoes

    Am. J. Trop. Med. Hyg.

    (1992)
  • M. Delves et al.

    The activities of current antimalarial drugs on the life cycle stages of Plasmodium: a comparative study with human and rodent parasites

    PLOS Med.

    (2012)
  • J. Dessens et al.

    CTRP is essential for mosquito infection by malaria ookinetes

    EMBO J.

    (1999)
  • Feacham, R., and The Malaria Elimination Group, 2009. A Guide on Malaria Elimination for Policy Makers. The Global...
  • R. Fowler et al.

    Inhibitory activity of the anti-malarial atovaquone (566C80) against ookinetes, oocysts, and sporozoites of Plasmodium berghei

    J. Parasitol.

    (1995)
  • F. Gamo et al.

    Thousands of chemical starting points for antimalarial lead identification

    Nature

    (2010)
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