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Journal articleChallenger J, Bruxvoort K, Ghani AC, et al., 2017,
Assessing the impact of imperfect adherence to artemether-lumefantrine on malaria treatment outcomes using within-host modelling
, Nature Communications, Vol: 8, ISSN: 2041-1723Artemether-lumefantrine (AL) is the most widely-recommended treatment for uncomplicated Plasmodium falciparum malaria worldwide. Its safety and efficacy have been extensively demonstrated in clinical trials; however, its performance in routine health care settings, where adherence to drug treatment is unsupervised and therefore may be suboptimal, is less well characterised. Here we develop a within-host modelling framework for estimating the effects of sub-optimal adherence to AL treatment on clinical outcomes in malaria patients. Our model incorporates data on the human immune response to the parasite, and AL’s pharmacokinetic and pharmacodynamic properties. Utilising individual-level data of adherence to AL in 482 Tanzanian patients as input for our model predicted higher rates of treatment failure than were obtained when adherence was optimal (9% compared to 4%). Our model estimates that the impact of imperfect adherence was worst in children, highlighting the importance of advice to caregivers.
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Journal articleHammond AM, Kyrou K, Bruttini M, et al., 2017,
The creation and selection of mutations resistant to a gene drive over multiple generations in the malaria mosquito
, PLoS Genetics, Vol: 13, ISSN: 1553-7390Gene drives have enormous potential for the control of insect populations of medical and agricultural relevance. By preferentially biasing their own inheritance, gene drives can rapidly introduce genetic traits even if these confer a negative fitness effect on the population. We have recently developed gene drives based on CRISPR nuclease constructs that are designed to disrupt key genes essential for female fertility in the malaria mosquito. The construct copies itself and the associated genetic disruption from one homologous chromosome to another during gamete formation, a process called homing that ensures the majority of offspring inherit the drive. Such drives have the potential to cause long-lasting, sustainable population suppression, though they are also expected to impose a large selection pressure for resistance in the mosquito. One of these population suppression gene drives showed rapid invasion of a caged population over 4 generations, establishing proof of principle for this technology. In order to assess the potential for the emergence of resistance to the gene drive in this population we allowed it to run for 25 generations and monitored the frequency of the gene drive over time. Following the initial increase of the gene drive we observed a gradual decrease in its frequency that was accompanied by the spread of small, nuclease-induced mutations at the target gene that are resistant to further cleavage and restore its functionality. Such mutations showed rates of increase consistent with positive selection in the face of the gene drive. Our findings represent the first documented example of selection for resistance to a synthetic gene drive and lead to important design recommendations and considerations in order to mitigate for resistance in future gene drive applications.
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Journal articleWatson O, Slater HC, Verity R, et al., 2017,
Modelling the drivers of the spread of Plasmodium falciparum hrp2 gene deletions in sub-Saharan Africa
, eLife, Vol: 6, ISSN: 2050-084XRapid diagnostic tests (RDTs) have transformed malaria diagnosis. The most prevalent P. falciparum RDTs detect histidine-rich protein 2 (PfHRP2). However, pfhrp2 gene deletions yielding false-negative RDTs, first reported in South America in 2010, have been confirmed in Africa and Asia. We developed a mathematical model to explore the potential for RDT-led diagnosis to drive selection of pfhrp2-deleted parasites. Low malaria prevalence and high frequencies of people seeking treatment resulted in the greatest selection pressure. Calibrating our model against confirmed pfhrp2-deletions in the Democratic Republic of Congo, we estimate a starting frequency of 6% pfhrp2-deletion prior to RDT introduction. Furthermore, the patterns observed necessitate a degree of selection driven by the introduction of PfHRP2-based RDT-guided treatment. Combining this with parasite prevalence and treatment coverage estimates, we map the model-predicted spread of pfhrp2-deletion, and identify the geographic regions in which surveillance for pfhrp2-deletion should be prioritised.
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Journal articleWerther R, Hallinan JP, Lambert AR, et al., 2017,
Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity
, Nucleic Acids Research, Vol: 45, Pages: 8621-8634, ISSN: 0305-1048The retargeting of protein–DNA specificity, outsideof extremely modular DNA binding proteins suchas TAL effectors, has generally proved to be quitechallenging. Here, we describe structural analysesof five different extensively retargeted variants of asingle homing endonuclease, that have been shownto function efficiently in ex vivo and in vivo applications.The redesigned proteins harbor mutationsat up to 53 residues (18%) of their amino acid sequence,primarily distributed across the DNA bindingsurface, making them among the most signifi-cantly reengineered ligand-binding proteins to date.Specificity is derived from the combined contributionsof DNA-contacting residues and of neighboringresidues that influence local structural organization.Changes in specificity are facilitated by theability of all those residues to readily exchange bothform and function. The fidelity of recognition is notprecisely correlated with the fraction or total numberof residues in the protein–DNA interface that areactually involved in DNA contacts, including directionalhydrogen bonds. The plasticity of the DNArecognitionsurface of this protein, which allows substantialretargeting of recognition specificity withoutrequiring significant alteration of the surroundingprotein architecture, reflects the ability of the correspondinggenetic elements to maintain mobility andpersistence in the face of genetic drift within potentialhost target sites.
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Journal articleBretscher MT, Griffin JT, Ghani AC, et al., 2017,
Modelling the benefits of long-acting or transmission-blocking drugs for reducing Plasmodium falciparum transmission by case management or by mass treatment
, MALARIA JOURNAL, Vol: 16, ISSN: 1475-2875BackgroundAnti-malarial drugs are an important tool for malaria control and elimination. Alongside their direct benefit in the treatment of disease, drug use has a community-level effect, clearing the reservoir of infection and reducing onward transmission of the parasite. Different compounds potentially have different impacts on transmission—with some providing periods of prolonged chemoprophylaxis whilst others have greater transmission-blocking potential. The aim was to quantify the relative benefit of such properties for transmission reduction to inform target product profiles in the drug development process and choice of first-line anti-malarial treatment in different endemic settings.MethodsA mathematical model of Plasmodium falciparum epidemiology was used to estimate the transmission reduction that can be achieved by using drugs of varying chemoprophylactic (protection for 3, 30 or 60 days) or transmission-blocking activity (blocking 79, 92 or 100% of total onward transmission). Simulations were conducted at low, medium or high transmission intensity (slide-prevalence in 2–10 year olds being 1, 10 or 40%, respectively), with drugs administered either via case management or mass drug administration (MDA).ResultsTransmission reductions depend strongly on deployment strategy, treatment coverage and endemicity level. Transmission-blocking was most effective at low endemicity, whereas chemoprophylaxis was most useful at high endemicity levels. Increasing the duration of protection as much as possible was beneficial. Increasing transmission-blocking activity from the level of ACT to a 100% transmission-blocking drug (close to the effect estimated for ACT combined with primaquine) produced moderate impact but was not as effective as increasing the duration of protection in medium-to-high transmission settings (slide prevalence 10–40%). Combining both good transmission-blocking activity (e.g. as achieved by ACT or ACT + primaquine) and a long durat
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Journal articleOkell L, Griffin JT, Roper C, 2017,
Mapping sulphadoxine-pyrimethamine-resistant Plasmodium falciparum malaria in infected humans and in parasite populations in Africa
, Scientific Reports, Vol: 7, ISSN: 2045-2322Intermittent preventive treatment (IPT) with sulphadoxine-pyrimethamine in vulnerable populations reduces malaria morbidity in Africa, but resistance mutations in the parasite dhps gene (combined with dhfr mutations) threaten its efficacy. We update a systematic review to map the prevalence of K540E and A581G mutations in 294 surveys of infected humans across Africa from 2004-present. Interpreting these data is complicated by multiclonal infections in humans, especially in high transmission areas. We extend statistical methods to estimate the frequency, i.e. the proportion of resistant clones in the parasite population at each location, and so standardise for varying transmission levels. Both K540E and A581G mutations increased in prevalence and frequency in 60% of areas after 2008, highlighting the need for ongoing surveillance. Resistance measures within countries were similar within 300 km, suggesting an appropriate spatial scale for surveillance. Spread of the mutations tended to accelerate once their prevalence exceeded 10% (prior to fixation). Frequencies of resistance in parasite populations are the same or lower than prevalence in humans, so more areas would be classified as likely to benefit from IPT if similar frequency thresholds were applied. We propose that the use of resistance frequencies as well as prevalence measures for policy decisions should be evaluated.
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Journal articlePatouillard E, Ghani ACH, Bhatt S, et al., 2017,
Global investment targets for malaria control and elimination between 2016 and 2030
, BMJ Global Health, Vol: 2, ISSN: 2059-7908Background Access to malaria control interventions falls short of universal health coverage. The Global Technical Strategy for malaria targets at least 90% reduction in case incidence and mortality rates, and elimination in 35 countries by 2030. The potential to reach these targets will be determined in part by investments in malaria. This study estimates the financing required for malaria control and elimination over the 2016–2030 period.Methods A mathematical transmission model was used to explore the impact of increasing intervention coverage on burden and costs. The cost analysis took a public provider perspective covering all 97 malaria endemic countries and territories in 2015. All control interventions currently recommended by the WHO were considered. Cost data were sourced from procurement databases, the peer-reviewed literature, national malaria strategic plans, the WHO-CHOICE project and key informant interviews.Results Annual investments of $6.4 billion (95% uncertainty interval (UI $4.5–$9.0 billion)) by 2020, $7.7 billion (95% UI $5.4–$10.9 billion) by 2025 and $8.7 billion (95% UI $6.0–$12.3 billion) by 2030 will be required to reach the targets set in the Global Technical Strategy. These are equivalent to annual investment per person at risk of malaria of US$3.90 by 2020, US$4.30 by 2025 and US$4.40 by 2030, compared with US$2.30 if interventions were sustained at current coverage levels. The 20 countries with the highest burden in 2015 will require 88% of the total investment.Conclusions Given the challenges in increasing domestic and international funding, the efficient use of currently available resources should be a priority
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Journal articleBrady OJ, Slater HC, Pemberton-Ross P, et al., 2017,
Role of mass drug administration in elimination of Plasmodium falciparum malaria: a consensus modelling study
, The Lancet Global Health, Vol: 5, Pages: E680-E687, ISSN: 2214-109XBackgroundMass drug administration for elimination of Plasmodium falciparum malaria is recommended by WHO in some settings. We used consensus modelling to understand how to optimise the effects of mass drug administration in areas with low malaria transmission.MethodsWe collaborated with researchers doing field trials to establish a standard intervention scenario and standard transmission setting, and we input these parameters into four previously published models. We then varied the number of rounds of mass drug administration, coverage, duration, timing, importation of infection, and pre-administration transmission levels. The outcome of interest was the percentage reduction in annual mean prevalence of P falciparum parasite rate as measured by PCR in the third year after the final round of mass drug administration.FindingsThe models predicted differing magnitude of the effects of mass drug administration, but consensus answers were reached for several factors. Mass drug administration was predicted to reduce transmission over a longer timescale than accounted for by the prophylactic effect alone. Percentage reduction in transmission was predicted to be higher and last longer at lower baseline transmission levels. Reduction in transmission resulting from mass drug administration was predicted to be temporary, and in the absence of scale-up of other interventions, such as vector control, transmission would return to pre-administration levels. The proportion of the population treated in a year was a key determinant of simulated effectiveness, irrespective of whether people are treated through high coverage in a single round or new individuals are reached by implementation of several rounds. Mass drug administration was predicted to be more effective if continued over 2 years rather than 1 year, and if done at the time of year when transmission is lowest.InterpretationMass drug administration has the potential to reduce transmission for a limited time, but is not an
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Journal articleKoch M, Wright KE, Otto O, et al., 2017,
Plasmodium falciparum erythrocyte-binding antigen 175 triggers a biophysical change in the red blood cell that facilitates invasion.
, Proc Natl Acad Sci U S A, Vol: 114, Pages: 4225-4230Invasion of the red blood cell (RBC) by the Plasmodium parasite defines the start of malaria disease pathogenesis. To date, experimental investigations into invasion have focused predominantly on the role of parasite adhesins or signaling pathways and the identity of binding receptors on the red cell surface. A potential role for signaling pathways within the erythrocyte, which might alter red cell biophysical properties to facilitate invasion, has largely been ignored. The parasite erythrocyte-binding antigen 175 (EBA175), a protein required for entry in most parasite strains, plays a key role by binding to glycophorin A (GPA) on the red cell surface, although the function of this binding interaction is unknown. Here, using real-time deformability cytometry and flicker spectroscopy to define biophysical properties of the erythrocyte, we show that EBA175 binding to GPA leads to an increase in the cytoskeletal tension of the red cell and a reduction in the bending modulus of the cell's membrane. We isolate the changes in the cytoskeleton and membrane and show that reduction in the bending modulus is directly correlated with parasite invasion efficiency. These data strongly imply that the malaria parasite primes the erythrocyte surface through its binding antigens, altering the biophysical nature of the target cell and thus reducing a critical energy barrier to invasion. This finding would constitute a major change in our concept of malaria parasite invasion, suggesting it is, in fact, a balance between parasite and host cell physical forces working together to facilitate entry.
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Journal articleSundriyal S, Chen P, Lubin A, et al., 2017,
Identification of diaminoquinazoline histone lysine methyltransferase structure activity relationships that allow for segregation of human G9a inhibition and anti-Plasmodium activity
, MedChemComm, Vol: 8, Pages: 1069-1092, ISSN: 2040-2511Plasmodium falciparum HKMTs (PfHKMTs) play a key role in controlling Plasmodium gene expression and represent exciting new anti-malarial epigenetic targets. Using an inhibitor series derived from the diaminoquinazoline HKMT inhibitory chemotype, we have previously identified compounds with highly promising antimalarial activity, including irreversible asexual cycle blood stage-independent cytotoxic activity at nM concentrations, oral efficacy in in vivo models of disease, and the unprecedented ability to reactivate dormant liver stage parasites (hypnozoites). However, future development of this series will need to address host versus parasite selectivity, where inhibitory activity against human G9a is removed from the lead compounds, while maintaining potent anti-Plasmodium activity. Herein, we report an extensive study of the SAR of this series against both G9a and P. falciparum. We have identified key SAR features which demonstrate that high parasite vs. G9a selectivity can be achieved by selecting appropriate substituents at position 2, 4 and 7 of the quinazoline ring. We have also, in turn, discovered that potent G9a inhibitors can be identified by employing a 6-carbon ‘Nle mimic’ at position 7. Together, this data suggests that while broadly similar, the G9a and potential PfHKMT target(s) binding pockets and/or binding modes of the diaminoquinazoline analogues exhibit clear and exploitable differences. Based on this, we believe this scaffold to have clear potential for development into a novel anti-malarial therapeutic.
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Journal articleWong W, Bai XC, Sleebs B, et al., 2017,
Mefloquine targets the Plasmodium falciparum 80S ribosome to inhibit protein synthesis
, Nature Microbiology, Vol: 2, Pages: 1-9, ISSN: 2058-5276Malaria control is heavily dependent on chemotherapeutic agents for disease prevention and drug treatment. Defining the mechanism of action for licensed drugs, for which no target is characterized, is critical to the development of their second-generation derivatives to improve drug potency towards inhibition of their molecular targets. Mefloquine is a widely used antimalarial without a known mode of action. Here, we demonstrate that mefloquine is a protein synthesis inhibitor. We solved a 3.2 Å electron cryo-microscopy structure of the Plasmodium falciparum 80S-ribosome with the (+)-mefloquine enantiomer bound to the ribosome GTPase-associated center. Mutagenesis of mefloquine-binding residues generates parasites with increased resistance, confirming the parasite-killing mechanism. Furthermore, structure-guided derivatives with an altered piperidine group, predicted to improve binding, show enhanced parasiticidal effect. These data reveal one possible mode of action for mefloquine and demonstrate the vast potential of cryo-EM to guide the development of mefloquine derivatives to inhibit parasite protein synthesis.
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Journal articleWinskill P, Walker P, Griffin J, et al., 2017,
Modelling the cost-effectiveness of introducing the RTS,S malaria vaccine relative to scaling up other malaria interventions in sub-Saharan Africa
, BMJ Global Health, Vol: 2, ISSN: 2059-7908Objectives: To evaluate the relative cost-effectiveness of introducing the RTS,S malaria vaccine in sub-Saharan Africa compared with further scale-up of existing interventions.Design: A mathematical modelling and cost-effectiveness study.Setting: Sub-Saharan Africa.Participants: People of all ages.Interventions: The analysis considers the introduction and scale-up of the RTS,S malaria vaccine and the scale-up of long lasting insecticide treated bed nets (LLINs), indoor residual spraying (IRS) and seasonal malaria chemoprevention (SMC).Main outcome measure: The number of Plasmodium falciparum cases averted in all age groups over a ten year period.Results: Assuming access to treatment remains constant, increasing coverage of LLINs was consistently the most cost-effective intervention across a range of transmission settings and was found to occur early in the cost-effectiveness scale-up pathway. IRS, RTS,S and SMC entered the cost-effective pathway once LLIN coverage had been maximised. If non-linear production functions are included to capture the cost of reaching very high coverage, the resulting pathways become more complex and result in selection of multiple interventions.Conclusions: RTS,S was consistently implemented later in the cost-effectiveness pathway than the LLINs, IRS and SMC but was still of value as a fourth intervention in many settings to reduce burden to the levels set out in the international goals.
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Journal articleBeaghton A, Hammond A, Nolan T, et al., 2017,
Requirements for Driving Antipathogen Effector Genes into Populations of Disease Vectors by Homing
, Genetics, Vol: 205, Pages: 1587-1596 -
Journal articleSimoes ML, Dong Y, Hammond A, et al., 2017,
The Anopheles FBN9 immune factor mediates Plasmodium species-specific defense through transgenic fat body expression
, Developmental and Comparative Immunology, Vol: 67, Pages: 257-265 -
Journal articleOkell LC, Churcher TS, 2016,
Generating the evidence base for malaria elimination: the situation in Haiti
, Lancet Global Health, Vol: 5, Pages: e16-e17, ISSN: 2214-109X -
Journal articleParr JB, Verity R, Doctor SM, et al., 2016,
Pfhrp2-deleted Plasmodium falciparum parasites in the Democratic Republic of the Congo: a national cross-sectional survey
, Journal of Infectious Diseases, Vol: 216, Pages: 36-44, ISSN: 0022-1899Background.Rapid diagnostic tests (RDTs) account for more than two-thirds of malaria diagnoses in Africa. Deletions of the Plasmodium falciparum hrp2 (pfhrp2) gene cause false-negative RDT results and have never been investigated on a national level. Spread of pfhrp2-deleted P. falciparum mutants, resistant to detection by HRP2-based RDTs, would represent a serious threat to malaria elimination efforts.Methods.Using a nationally representative cross-sectional study of 7,137 children under five years of age from the Democratic Republic of Congo (DRC), we tested 783 subjects with RDT-/PCR+ results using PCR assays to detect and confirm deletions of the pfhrp2 gene. Spatial and population genetic analyses were employed to examine the distribution and evolution of these parasites.Results.We identified 149 pfhrp2-deleted parasites, representing 6.4% of all P. falciparum infections country-wide (95% confidence interval 5.1–8.0%). Bayesian spatial analyses identified statistically significant clustering of pfhrp2 deletions near Kinshasa and Kivu. Population genetic analysis revealed significant genetic differentiation between wild-type and pfhrp2-deleted parasite populations (GST = .046, p ≤ .00001).Conclusions.Pfhrp2-deleted P. falciparum is a common cause of RDT-/PCR+ malaria among asymptomatic children in the DRC and appears to be clustered within select communities. Surveillance for these deletions is needed, and alternatives to HRP2-specific RDTs may be necessary.
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Journal articleBargieri DY, Thiberge S, Tay CL, et al., 2016,
Plasmodium merozoite TRAP family protein Is essential for vacuole membrane disruption and gamete egress from erythrocytes
, Cell Host & Microbe, Vol: 20, Pages: 618-630, ISSN: 1934-6069Surface-associated TRAP (thrombospondin-related anonymous protein) family proteins are conserved across the phylum of apicomplexan parasites. TRAP proteins are thought to play an integral role in parasite motility and cell invasion by linking the extracellular environment with the parasite submembrane actomyosin motor. Blood stage forms of the malaria parasite Plasmodium express a TRAP family protein called merozoite-TRAP (MTRAP) that has been implicated in erythrocyte invasion. Using MTRAP-deficient mutants of the rodent-infecting P. berghei and human-infecting P. falciparum parasites, we show that MTRAP is dispensable for erythrocyte invasion. Instead, MTRAP is essential for gamete egress from erythrocytes, where it is necessary for the disruption of the gamete-containing parasitophorous vacuole membrane, and thus for parasite transmission to mosquitoes. This indicates that motor-binding TRAP family members function not just in parasite motility and cell invasion but also in membrane disruption and cell egress.
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Journal articleJohnson S, Rahmani R, Drew DR, et al., 2016,
Truncated latrunculins as actin inhibitors targeting plasmodium falciparum motility and host-cell invasion
, Journal of Medicinal Chemistry, Vol: 59, Pages: 10994-11005, ISSN: 0022-2623Polymerization of the cytosolic protein actin is critical to cell movement and host-cell invasion by the malaria parasite, Plasmodium falciparum. Any disruption to actin polymerization dynamics will render the parasite incapable of invading a host cell and thereby unable to cause infection. Here, we explore the potential of using truncated latrunculins as potential chemotherapeutics for the treatment of malaria. Exploration of the binding interactions of the natural actin inhibitor latrunculins, with actin revealed how a truncated core of the inhibitor could retain its key interaction features with actin. This truncated core was synthesised and subjected to preliminary structure-activity relationship studies to generate a focused set of analogues. Biochemical analyses of these analogues demonstrate their 6-fold increased activity compared with latrunculin B against Plasmodium falciparum and a 16-fold improved selectivity ex vivo. These data establish the latrunculin core as a potential focus for future structure-based drug design of chemotherapeutics against malaria.
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Journal articleBaum J,
Plasmodium MTRAP is essential for vacuole membrane disruption and host cell egress by malaria gametes
, Cell Host & Microbe, ISSN: 1934-6069Surface-associated TRAP (thrombospondin-related anonymous protein) family proteins are conservedacross the phylum of apicomplexan parasites. TRAP proteins are thought to play an integral role inparasite motility and cell invasion by linking the extracellular environment with the parasitesubmembrane actomyosin motor. Blood-stage forms of the malaria parasite Plasmodium express aTRAP family protein called merozoite-TRAP (MTRAP) that has been implicated in erythrocyteinvasion. Using MTRAP deficient mutants of the rodent-infecting P. berghei and human-infecting P.falciparum parasites, we show that MTRAP is dispensable for erythrocyte invasion. Instead, MTRAPis essential for gamete egress from erythrocytes, where it is necessary for the disruption of the gametecontainingparasitophorous vacuole membrane, and thus for parasite transmission to mosquitoes. Thisindicates that motor-binding TRAP family members function not just in parasite motility and cellinvasion but also in membrane disruption and cell egress.
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Journal articleSlater HC, Okell LC, Ghani AC, 2016,
Mathematical Modelling to Guide Drug Development for Malaria Elimination
, Trends in Parasitology, Vol: 33, Pages: 175-184, ISSN: 1471-5007Mathematical models of the dynamics of a drug within the host are now frequently used to guide drug development. These generally focus on assessing the efficacy and duration of response to guide patient therapy. Increasingly, antimalarial drugs are used at the population level, to clear infections, provide chemoprevention, and to reduce onward transmission of infection. However, there is less clarity on the extent to which different drug properties are important for these different uses. In addition, the emergence of drug resistance poses new threats to longer-term use and highlights the need for rational drug development. Here, we argue that integrating within-host pharmacokinetic and pharmacodynamic (PK/PD) models with mathematical models for the population-level transmission of malaria is key to guiding optimal drug design to aid malaria elimination.
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Journal articleTardieux I, Baum J, 2016,
Reassessing the mechanics of parasite motility and host-cell invasion
, Journal of Cell Biology, Vol: 214, Pages: 507-515, ISSN: 1540-8140The capacity to migrate is fundamental to multicellular and single-celled life. Apicomplexan parasites, an ancient protozoan clade that includes malaria parasites (Plasmodium) and Toxoplasma, achieve remarkable speeds of directional cell movement. This rapidity is achieved via a divergent actomyosin motor system, housed within a narrow compartment that lies underneath the length of the parasite plasma membrane. How this motor functions at a mechanistic level during motility and host cell invasion is a matter of debate. Here, we integrate old and new insights toward refining the current model for the function of this motor with the aim of revitalizing interest in the mechanics of how these deadly pathogens move.
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Journal articleLamas Oliveira Marques S, 2016,
Routine in vitro culture of P. falciparum gametocytes to evaluate novel transmission-blocking interventions.
, Nature Protocols, Vol: 11, Pages: 1668-1680, ISSN: 1754-2189The prevention of parasite transmission from the human host to the mosquito has been recognized as a vital tool for malaria eradication campaigns. However, transmission-blocking antimalarial drug and/or vaccine discovery and development is currently hampered by the expense and difficulty of producing mature Plasmodium falciparum gametocytes in vitro-the parasite stage responsible for mosquito infection. Current protocols for P. falciparum gametocyte culture usually require complex parasite synchronization and addition of stimulating and/or inhibitory factors, and they may not have demonstrated the essential property of mosquito infectivity. This protocol details all the steps required for reliable P. falciparum gametocyte production and highlights common factors that influence culture success. The protocol can be completed in 15 d, and particular emphasis is placed upon operating a gametocyte culture facility on a continuous cycle. In addition, we show how functionally viable gametocytes can be used to evaluate transmission-blocking drugs both in a field setting and at high throughput (HTP) for drug discovery.
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Journal articleGalizi R, Hammond A, Kyrou K, et al., 2016,
A CRISPR-Cas9 sex-ratio distortion system for genetic control.
, Scientific Reports, Vol: 6, ISSN: 2045-2322Genetic control aims to reduce the ability of insect pest populations to cause harm via the release of modified insects. One strategy is to bias the reproductive sex ratio towards males so that a population decreases in size or is eliminated altogether due to a lack of females. We have shown previously that sex ratio distortion can be generated synthetically in the main human malaria vector Anopheles gambiae, by selectively destroying the X-chromosome during spermatogenesis, through the activity of a naturally-occurring endonuclease that targets a repetitive rDNA sequence highly-conserved in a wide range of organisms. Here we describe a CRISPR-Cas9 sex distortion system that targets ribosomal sequences restricted to the member species of the Anopheles gambiae complex. Expression of Cas9 during spermatogenesis resulted in RNA-guided shredding of the X-chromosome during male meiosis and produced extreme male bias among progeny in the absence of any significant reduction in fertility. The flexibility of CRISPR-Cas9 combined with the availability of genomic data for a range of insects renders this strategy broadly applicable for the species-specific control of any pest or vector species with an XY sex-determination system by targeting sequences exclusive to the female sex chromosome.
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Journal articleBane KS, Lepper S, Kehrer J, et al., 2016,
The Actin Filament-Binding Protein Coronin Regulates Motility in Plasmodium Sporozoites
, PLOS Pathogens, Vol: 12, ISSN: 1553-7366Parasites causing malaria need to migrate in order to penetrate tissue barriers and enter host cells. Here we show that the actin filament-binding protein coronin regulates gliding motility in Plasmodium berghei sporozoites, the highly motile forms of a rodent malaria-causing parasite transmitted by mosquitoes. Parasites lacking coronin show motility defects that impair colonization of the mosquito salivary glands but not migration in the skin, yet result in decreased transmission efficiency. In non-motile sporozoites low calcium concentrations mediate actin-independent coronin localization to the periphery. Engagement of extracellular ligands triggers an intracellular calcium release followed by the actin-dependent relocalization of coronin to the rear and initiation of motility. Mutational analysis and imaging suggest that coronin organizes actin filaments for productive motility. Using coronin-mCherry as a marker for the presence of actin filaments we found that protein kinase A contributes to actin filament disassembly. We finally speculate that calcium and cAMP-mediated signaling regulate a switch from rapid parasite motility to host cell invasion by differentially influencing actin dynamics.
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Journal articleWalker PG, Griffin JT, Ferguson NM, et al., 2016,
Estimating the most efficient allocation of interventions to achieve reductions in Plasmodium falciparum malaria burden and transmission in Africa: a modelling study
, Lancet Global Health, Vol: 4, Pages: e474-e484, ISSN: 2214-109XBACKGROUND: Reducing the burden of malaria is a global priority, but financial constraints mean that available resources must be allocated rationally to maximise their effect. We aimed to develop a model to estimate the most efficient (ie, minimum cost) ordering of interventions to reduce malaria burden and transmission. We also aimed to estimate the efficiency of different spatial scales of implementation. METHODS: We combined a dynamic model capturing heterogeneity in malaria transmission across Africa with financial unit cost data for key malaria interventions. We combined estimates of patterns of malaria endemicity, seasonality in rainfall, and mosquito composition to map optimum packages of these interventions across Africa. Using non-linear optimisation methods, we examined how these optimum packages vary when control measures are deployed and assessed at national, subnational first administrative (provincial), or fine-scale (5 km(2) pixel) spatial scales. FINDINGS: The most efficient package in a given setting varies depending on whether disease reduction or elimination is the target. Long-lasting insecticide-treated nets are generally the most cost-effective first intervention to achieve either goal, with seasonal malaria chemoprevention or indoor residual spraying added second depending on seasonality and vector species. These interventions are estimated to reduce malaria transmission to less than one case per 1000 people per year in 43·4% (95% CI 40·0-49·0) of the population at risk in Africa. Adding three rounds of mass drug administration per year is estimated to increase this proportion to 90·9% (95% CI 86·9-94·6). Further optimisation can be achieved by targeting policies at the provincial level, achieving an estimated 32·1% (95% CI 29·6-34·5) cost saving relative to adopting country-wide policies. Nevertheless, we predict that only 26 (95% CI 22-29) of 41 countries could reduce transmissio
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Journal articleMarshall JM, Touré M, Ouédraogo AL, et al., 2016,
Key traveller groups of relevance to spatial malaria transmission: a survey of movement patterns in four sub-Saharan African countries
, Malaria Journal, Vol: 15, ISSN: 1475-2875 -
Journal articleHall AB, Papathanos P-A, Sharma A, et al., 2016,
Radical remodeling of the Y chromosome in a recent radiation of malaria mosquitoes
, Proceedings of the National Academy of Sciences of the United States of America, Vol: 113, Pages: E2114-E2123 -
Journal articleWhite MT, Shirreff G, Karl S, et al., 2016,
Variation in relapse frequency and the transmission potential of Plasmodium vivax malaria
, Proceedings of the Royal Society of London. Series B, Biological Sciences, Vol: 283, ISSN: 0080-4649There is substantial variation in the relapse frequency of Plasmodium vivax malaria, with fast-relapsing strains in tropical areas, and slow-relapsing strains in temperate areas with seasonal transmission. We hypothesize that much of the phenotypic diversity in P. vivax relapses arises from selection of relapse frequency to optimize transmission potential in a given environment, in a process similar to the virulence trade-off hypothesis. We develop mathematical models of P. vivax transmission and calculate the basic reproduction number R0 to investigate how transmission potential varies with relapse frequency and seasonality. In tropical zones with year-round transmission, transmission potential is optimized at intermediate relapse frequencies of two to three months: slower-relapsing strains increase the opportunity for onward transmission to mosquitoes, but also increase the risk of being outcompeted by faster-relapsing strains. Seasonality is an important driver of relapse frequency for temperate strains, with the time to first relapse predicted to be six to nine months, coinciding with the duration between seasonal transmission peaks. We predict that there is a threshold degree of seasonality, below which fast-relapsing tropical strains are selected for, and above which slow-relapsing temperate strains dominate, providing an explanation for the observed global distribution of relapse phenotypes.
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Journal articleChen PB, Ding S, Zanghì G, et al., 2016,
Plasmodium falciparum PfSET7: enzymatic characterization and cellular localization of a novel protein methyltransferase in sporozoite, liver and erythrocytic stage parasites.
, Scientific Reports, Vol: 6, ISSN: 2045-2322Epigenetic control via reversible histone methylation regulates transcriptional activation throughout the malaria parasite genome, controls the repression of multi-copy virulence gene families and determines sexual stage commitment. Plasmodium falciparum encodes ten predicted SET domain-containing protein methyltransferases, six of which have been shown to be refractory to knock-out in blood stage parasites. We have expressed and purified the first recombinant malaria methyltransferase in sufficient quantities to perform a full enzymatic characterization and reveal the ill-defined PfSET7 is an AdoMet-dependent histone H3 lysine methyltransferase with highest activity towards lysines 4 and 9. Steady-state kinetics of the PfSET7 enzyme are similar to previously characterized histone methyltransferase enzymes from other organisms, however, PfSET7 displays specific protein substrate preference towards nucleosomes with pre-existing histone H3 lysine 14 acetylation. Interestingly, PfSET7 localizes to distinct cytoplasmic foci adjacent to the nucleus in erythrocytic and liver stage parasites, and throughout the cytoplasm in salivary gland sporozoites. Characterized recombinant PfSET7 now allows for target based inhibitor discovery. Specific PfSET7 inhibitors can aid in further investigating the biological role of this specific methyltransferase in transmission, hepatic and blood stage parasites, and may ultimately lead to the development of suitable antimalarial drug candidates against this novel class of essential parasite enzymes.
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Journal articlePothin E, Ferguson NM, Drakeley CJ, et al., 2016,
Estimating malaria transmission intensity from Plasmodium falciparum serological data using antibody density models
, Malaria Journal, Vol: 15, ISSN: 1475-2875Background: Serological data are increasingly being used to monitor malaria transmission intensity and havebeen demonstrated to be particularly useful in areas of low transmission where traditional measures such as EIR andparasite prevalence are limited. The seroconversion rate (SCR) is usually estimated using catalytic models in whichthe measured antibody levels are used to categorize individuals as seropositive or seronegative. One limitationof this approach is the requirement to impose a fixed cut-off to distinguish seropositive and negative individuals.Furthermore, the continuous variation in antibody levels is ignored thereby potentially reducing the precision of theestimate.Methods: An age-specific density model which mimics antibody acquisition and loss was developed to make fulluse of the information provided by serological measures of antibody levels. This was fitted to blood-stage antibodydensity data from 12 villages at varying transmission intensity in Northern Tanzania to estimate the exposure rate asan alternative measure of transmission intensity.Results: The results show a high correlation between the exposure rate estimates obtained and the estimated SCRobtained from a catalytic model (r = 0.95) and with two derived measures of EIR (r = 0.74 and r = 0.81). Estimates ofexposure rate obtained with the density model were also more precise than those derived from catalytic models.Conclusion: This approach, if validated across different epidemiological settings, could be a useful alternative frameworkfor quantifying transmission intensity, which makes more complete use of serological data.
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