Investigation of the impact of the mosquito immune system on shaping the transmitted malaria parasite populations

Malaria is a devastating disease transmitted by mosquitoes. It infects about 220 million and kills about 430,000 people every year, mostly children in sub-Saharan Africa. This is a much better situation than 15 years ago, when deaths were almost twice as many, and is due to the combination of more effective medicines, improved health care and, above all, enhanced mosquito control. However, the latest data indicate that these measures can have no further impact as numbers have remain unchanged in the past few years. Importantly, widespread resistance to insecticides has seriously hampered mosquito control. Therefore, it has become evident that research that could lead to new interventions must be intensified. While efforts to manufacture an effective vaccine continues, focus is now placed in identifying new ways to halt malaria parasites before they are transmitted to humans. To this purpose, genetic modification of mosquitoes preventing them from transmitting the disease and manufacturing vaccines that act against parasites inside mosquitoes have received great attention and funding. This project aims to map the molecular and evolutionary landscape in which mosquito-parasite interactions take place and provide an informed list of targets of new antimalarial interventions.

Our research is based on earlier findings that only few of the malaria parasites entering a mosquito upon blood feeding on an infected human survive to be transmitted to a new host. Most parasites are eliminated by mosquito immune responses before infection is established. Recently, we discovered that a specialised mosquito immune response, called the complement-like system, attacks and removes already compromised and unfit parasites. Therefore, we hypothesised that this response can act as an evolutionary sieve that purifies malaria parasite populations from compromising mutations.

To examine our hypothesis, we will use a library of over 150 rodent malaria parasite lines, each carrying a deleterious mutation in a gene expressed during early stages of mosquito infection. Pools of these mutant parasites will infect a mouse and be transmitted to mosquitoes that are normal or have parts of their immune system disrupted. This includes the complement-like pathway and another pathway that attacks parasites soon after they enter the mosquito. Parasites that manage to survive will be transmitted back to mice through mosquito bites, and the mouse-mosquito-mouse transmission will continue for four additional cycles. In each cycle, the population of parasites in the mice will be characterised to identify genes of which mutations did or did not affect transmission. We expect that some parasites carrying deleterious mutations will be transmitted only when the mosquito immune system is disrupted. A proof-of-concept experiment with parasites carrying mutations in six such genes corroborated our hypothesis. The outcome of these experiments will be twofold: it will reveal novel parasite genes important in interactions with the mosquito, which can be targets of interventions aiming to block disease transmission, and it will identify the impact the mosquito immune system can have on shaping parasite populations transmitted between hosts. The latter can be very important in delineating the forces that determine the composition of the malaria parasite populations circulating among people.

In Africa, some mosquitoes have more robust immune systems than others, and in some cases these mosquitoes occupy different geographic regions; hence, we hypothesise that they can transmit different parasites. A second research line will directly investigate this question using species of mosquitoes with different geographic distributions and playing different roles in malaria epidemiology. Determining which gene mutations can be transmitted by some mosquito species but not others can provide unprecedented insights into the malaria transmission landscape.

The Anopheles gambiae immune system forms a barrier to infection of the malaria parasite Plasmodium. Soon after entering the mosquito via a blood meal, parasites are attacked by an array of immune responses that greatly impact on their population size. In nature, only a handful of parasites survive these responses to establish infection. Prior to ookinete traversal of the mosquito midgut epithelium, the antibacterial Imd pathway significantly reduces the number of parasites. However, most losses are recorded after ookinetes traverse the midgut epithelium and encounter the complement-like response. We recently identified several genes that appear to be important for parasite protection from this response. Disruption of any of these genes leads to a variable number of ookinetes that traverse the mosquito midgut epithelium but are eliminated by complement reactions upon reaching the basal sub-epithelial space. Silencing key components of the complement-like system restores parasite development and transmission to a new host. These results led us hypothesise that either all of these genes have essential functions in parasite immune evasion or that loss-of-function of any of them bears a fitness cost exceeding a certain threshold required for parasites to endure the mosquito immune response. With the latter hypothesis, the mosquito immune system acts as a purifying selection filter for parasite populations permitting only the fittest parasites to survive and be transmitted to a new host. This project will further investigate this hypothesis by: (1) examining how the mosquito immune system shapes the parasite populations transmitted between hosts, (2) investigating the impact these responses may have on parasite populations transmitted by different vectors, and (3) characterising the function of prioritised parasite genes and proteins found to play important roles in mosquito infection and examining their potential as targets of transmission blocking interventions.

Malaria is a global disease affecting half of the world population. The estimated number of cases in 2017 was 219 million, a 2 and 7 million increase compared to 2016 and 2015, respectively. These figures make it evident that current measures for malaria control are no longer effective and new tools are needed. It is also evident that any new tools must target transmission rather than therapeutics. This project will produce new knowledge on malaria transmission and vector-parasite interactions and contribute to developing novel interventions that could save millions of lives and reduce the global economic burden of the disease.

The realisation that the mosquito passage is a major bottleneck for parasite populations was instrumental in introducing and communicating the concept of transmission blocking that now spearheads the efforts for malaria elimination. The discovery, by us and others, that the mosquito immune response is largely responsible for this bottleneck provided a biological framework to this concept and highlighted the fact that the mosquito is not just a vector of the parasite but its definitive host and that studying the mosquito-parasite interactions is essential for understanding malaria transmission. Here, we will investigate the impact that this bottleneck has on the observed genetic structure of parasite populations. This project will be the first step towards this goal by examining whether this response acts to remove deleterious mutations from parasite populations. This, together with the reported parasite immune evasion through directional selection of genes, will be the framework to explain co-adaptation and arms race evolution between mosquitoes and parasites. What is more, investigating whether mosquito species displaying varying geographic distributions, importance in malaria transmission and antimicrobial defences can transmit different parasite populations will shed new light into the genetic structure of and gene flow within malaria parasites across Africa. This would help us understand and design interventions targeting transmission that are effective across the continent.

The observation that most mosquitoes block parasite infection and malaria transmission was central in reviving the concept of mosquito population replacement, first introduced in the 1990's. This concept involves genetic modification of mosquitoes that are refractory to the parasite and can spread this feature in wild populations. The discovery of effective gene drive engines such as CRISPR/Cas9 sparked new hopes for this technology that requires minimum intervention and steering once a drive is released. Indeed, our Bill and Melinda Gates Foundation funded programme aims to achieve that. One approach that is to boost the mosquito immune system will be heavily informed by this project, as boosting a purifying selection system is unlikely to give results whereas increasing parasite damage prior to this can be a better strategy. The second approach is to express and drive nanobodies that target specific parasite proteins in the mosquito midgut, thus decreasing parasite fitness and blocking infection. Indeed, we are currently engineering mosquitoes expressing nanobodies against Pfs47 and PfPIMMS43. This project will identify additional targets and further inform this approach. The same proteins can also be targets of transmission blocking vaccines, a strategy that also gained momentum in recent years. While others are pursuing the generation of Pfs47 and Pfs48/45 vaccines, we showed that a PfPIMMS43 vaccine is even more effective in blocking mosquito infection and can provide an additional natural antibody titre booster since PIMMS43 is also expressed in sporozoites. Additional vaccine targets characterised in this project will be explored.