Preemptive discovery of insecticide cross-resistance mechanisms for next generation malaria control products

Malaria is a parasitic tropical disease transmitted by mosquitoes which kills hundreds of thousands of people every year, predominantly children in Sub-Saharan Africa (SSA). Reducing malaria primarily relies on reducing adult mosquito numbers using insecticides, either by spraying the walls of houses (IRS) on which mosquitoes rest or by protecting sleeping humans with insecticide-treated bed nets (ITNs). Insecticide resistance is a major threat, with many populations of the major Anopheles malaria vector species in SSA evolving strong resistance to pyrethroids, for years the most important insecticide class. This is increasingly resulting in reduced effectiveness of control tools, and whilst other insecticides are available for IRS, most programmes have become over-reliant on a single insecticide to which resistance is spreading. However, with very recent availability of new, contrasting insecticide classes for vector control, and more expected in the next 5-10 years, there is an opportunity to effectively manage insecticide resistance in mosquitoes for the first time in decades and sustain control.
Resistance management strategies typically combine or rotate different insecticides to prevent the evolution of resistance to each occurring. For this to be effective we need to understand how insecticides exert their toxic effects, from which mosquito genes resistance is likely to arise, and crucially whether the same genetic mechanisms can cause resistance across different insecticides. We and others are sequencing all genes (DNA) or their expressed transcripts (RNA) in wild mosquitoes subject to insecticidal pressure, to identify resistance genes and mutations. However, this is challenging when resistance is still relatively weak or recent, and especially so for new insecticides, to which resistance does not yet exist. To predict resistance and provide knowledge of resistance mechanisms and associated diagnostic tools for monitoring mosquito populations exposed to newly available-insecticides for malaria control, novel complementary methodologies are urgently required.
We propose to apply a new approach, known as activity based protein profiling (ABPP), which uses chemical probes to identify mosquito enzymes which are targeted by an insecticide, and may be candiates for the development of resistance. We will first use both ABPP and RNA sequencing to compare African Anopheles differing in their resistance to the most important current insecticide for IRS. Integrating results from the two methodologies will identify genes that are both highly expressed in resistant mosquitoes (RNA sequencing) and show different binding activity (ABPP) provided a uniquely powerful platform to incriminate resistance candidates. Our second pipeline will target a wider range of insecticides including those to which there is no known resistance. Here we will apply ABPPs to mosquito extracts in competition with insecticides to identify targets, with repetition across the different insecticides used in current and oncoming control programmes yielding a matrix of enzyme-insecticide interactions. Incrimination of the same enzyme with multiple insecticides will reveal targets through which resistance across insecticides may arise. We will test the capacity of the most promising genes to generate resistance using a third pipeline. This will involve techniques such as expression of candidate enzymes in bacteria, transgenic expression in fruit flies and mosquitoes, and blocking of expression in otherwise resistant mosquitoes to determine whether resistance may be gained or lost.
Our results will provide new understanding of the potential of different insecticides to be used together effectively to manage resistance, and provide diagnostics to screen wild mosquitoes for genetic changes resulting from control programmes. Our pipelines may also be extended to pre-emptively investigate how resistance may arise to future insecticides as available

Malaria control is being impacted by insecticide resistance but newly-available and oncoming insecticides offer the opportunity to effectively manage resistance, provided mechanistic knowledge and monitoring tools become available to inform rational deployment. Genomic and transcriptomic comparisons of resistant and susceptible mosquitoes, which we and others are applying as part of ongoing and recent programmes are powerful for identification of resistance candidates, but lack capacity to pre-emptively identify mechanisms of resistance and cross-resistance for new insecticides. Novel complementary approaches are required to meet this challenge and we propose to combine transcriptomic and functional genomic approaches with a novel chemico-proteomic approach, Activity Based Protein Profiling (ABPP ). We will apply dual pipelines: the first integrates comparative transcriptomic and ABPP analyses to identify differentially expressed genes and correspondent differentially binding enzymes between Anopheles strains with contrasting resistance to the key current IRS insecticide pirimiphos methyl (PM). The second pipeline will apply competitive inhibition of ABPs with insecticide or their reactive metabolites to identify modes of toxicity for each and generate an insecticide-enzyme interaction matrix for identification of cross-resistance candidates. The most promising candidates from each pipeline will be tested using a third functional validation pipeline involving heterologous expression and metabolism, transgenic expression in Drosophila and Anopheles and RNAi to investigate resultant gains and loss of resistance. Our results will provide new knowledge and pre-emptive diagnostic tools for resistance management programmes, and pipelines readily adaptable to future insecticides

The crux of the proposal is development of a pre-emptive approach to identify genes capable of causing cross-resistance to new insecticides used for malaria vector control. This is a major advance in offering proactive decision making for the use of insecticides across the spectrum of insect-borne diseases. This much-needed knowledge will have early impacts (from project year 2) on the research and development sector, with longer term impacts extending to disease control programmes (from project years 3), disease endemic communities, industry and the general UK public. This will be facilitated by the team's extensive links with tropical disease control programs, policy makers and industry as described in 'Pathways to Impact'.
Research and Development: In the short term, research outputs will enable the development of pre-emptive knowledge bases and molecular surveillance diagnostic panels that can be integrated into malaria control decision-making and monitoring systems through our GAARD and GAARDian partnerships and via links with the PMI/Abt VectorLinks IRS programmes. Long term, the ABP pipelines we propose apply to a wide spectrum of diseases transmitted by insects in public health, farming and veterinary practice, with beneficiaries including scientists in academia and industry who study mechanisms of insecticide resistance and pest control. There are also wider applications for our second ABPP pipeline to the pre-emptively identify of insecticide target activity in vulnerable beneficial organisms (e.g. providing environmental services), such as pollinators, which may stimulate new research into the environmental impact of insecticide applications. For example, sensitive molecular surveillance tools directed at insecticide-target interactions could help to monitor changes related to exposure that would improve understanding of the effects of insecticide exposure for off-target species and an evidence base to allow for reactive measures to minimize harm. Our productive collaboration with Rothamstead Research would be an obvious avenue to investigate these environmental impacts.

Disease control programmes: Results from this project will have a critical impact on disease control in providing early identification of potential cross-resistance liabilities, that will be important for control programmes such as the US President Malaria Initiative (and Abt associates), that are responsible for widespread IRS programs. Such diagnostics are a pillar of the Global Plan for Insecticide Resistance Management (WH0, 2012) planning. Moreover, National Malaria Control Programmes are producing bespoke IRM plans, and it is our vision that these markers will be taken up as part of the surveillance regimes to back up new insecticide use. The ability of control programmes to utilise pro-active resistance surveillance activities to manage valuable resources effectively is vital in order to mitigate resistance and extend product life-time, critical to reach the malaria elimination target of 2040.

Disease endemic communities: In the near term, improving the management of insecticides will result in sustaining their effectiveness for vector control, with a consequent continued impact on reducing the morbidity and mortality caused by malaria in disease endemic countries in Africa. Long term, removal of the disease burden leads to improved social welfare, healthy work force, enhanced tourism and wealth creation.

Industry: pre-emptive identification of resistance candidates will greatly impact the industry process of insecticide design and product development enabling early identification of resistance liabilities.

UK: We will capitalize on project outcomes to develop undergrad and postgrad projects including those through the MRC-DTP PhD program that explore basic science applications that will deliver further training and equip UK graduates with key scientific and molecular skills for future employment