While the carnage wreaked by the current Ebola epidemic in West Africa has played out in the gaze of the world's media, another pandemic has continued virtually unnoticed in the background. Malaria threatens half of the human population and even now kills one child every minute. Yet mortality rates from malaria have been falling steadily since 2000, thanks to insecticide-treated bed nets and spraying programmes targeting the African malaria mosquito (Anopheles gambiae), which spreads the disease. However, the mosquitoes are fighting back. Resistance to the pesticide pyrethroid is increasing, leading scientists to search for other effective insecticides. ‘Ivermectin has arisen as a new candidate,’ says Jacob Meyers from Colorado State University, USA, explaining that the drug kills or disables A. gambiae mosquitoes after consumption in a blood meal. However, little was known about how Ivermectin targets mosquitoes – it was developed to treat diseases in nematodes – so Meyers, Brian Foy and a team of colleagues from Colorado State University, including Meg Gray, Wojtek Kuklinski, Lucas Johnson, Christopher Snow, William Black IV and Kathryn Partin, decided to find out more about the drug's modus operandi.
Explaining that Invermectin targets and opens a key component of the synaptic communication system – the glutamate gated chloride channel (GluCl) – to kill nematodes, the team decided to find out more about the channel's function in the mosquitoes at a genetic and physiological level. Cloning the mosquito gene for the GluCl channel (AgGluCl) and analysing its structure, Gray, Black and Meyers were surprised to find that it could be expressed in four different ways, producing subtly different versions of the chloride channel – known as splice isoforms. However, after months of attempts to measure the minute currents flowing through the four channel isoforms in the presence and absence of Ivermectin, Partin and Meyers were in for a shock. ‘We discovered one channel [AgGluCl-a] was sensitive to Ivermectin, as predicted, but the second isoform tested [AgGluCl-b] was surprisingly insensitive’, says Meyers. He adds that the lack of sensitivity is puzzling, because Ivermectin should bind successfully to both channels to activate them – the region of AgGluCl-a that interacts with the drug should be identical to the Ivermectin binding site in AgGluCl-b.
Next, Kuklinski and Meyers analysed the expression patterns of the different channel isoforms and were pleased to see that the ‘a’ forms of the protein were expressed predominantly, explaining the insect's vulnerability to the drug. However, the team warns that the cunning mosquitoes could develop immunity to the insecticide if they switched expression to produce the Ivermectin-insensitive ‘b’ form of the channel.
Finally, Meyers tested which tissues the channel was expressed in and located it in nerves, including the thoracic ganglia, controlling the mosquitoes’ movements. Recalling that Ivermectin causes paralysis, he says, ‘Our data suggest that this paralysis may be due to disruption of AgGluCl in the motor neurons controlling the leg and flight muscles’.
Having narrowed down how Ivermectin targets the AgGluCl channel to incapacitate malaria-spreading mosquitoes, Foy and Meyers wondered if they could find an even more effective way of defeating the African malaria mosquitoes by targeting the essential channel using another strategy. Could they make blood meals toxic for the voracious mosquitoes by immunising the animals that they feast upon to produce antibodies targeting the AgGluCl channel? And if so, could such a therapy be used to target other disease-carriers, such as yellow fever mosquitoes and West Nile virus mosquitoes? Meyer admits that the strategy was risky. ‘Antibodies against a single mosquito antigen have never been shown to have mosquitocidal properties before and the majority of previous research had focused on midgut antigens, while we were targeting a neuronal antigen expressed only in tissues found outside of the midgut’, he says.
Injecting rabbits with a tiny portion of the surface of the protein channel, Meyers waited for the animals’ immune systems to kick in and begin producing antibodies tailored to the channel. Then he collected the antibodies, mixed them with fresh blood and fed the tasty mixture to all three mosquito species.
Frustratingly, neither the yellow fever nor West Nile virus mosquitoes reacted to the spiked blood. However, significant numbers of the malaria mosquitoes expired after the blood–antibody cocktail, with the highest antibody doses killing over 90% of the insects within a day. And when Meyers and Gray tested why the yellow fever and western encephalitis mosquitoes had been immune to the antibody snack, they found that the antibodies could not pass across the guts into the haemolymph of the yellow fever or West Nile virus mosquitoes, while the antibodies passed into the haemolymph of the malaria-carrying mosquitoes with ease.
Intrigued by the antibodies’ attack mechanism, Meyers fed the insects a blood meal laced with the antibodies and a lethal dose of Ivermectin and monitored their survival. Remarkably, the insects survived much better than insects fed Ivermectin alone. ‘We believe that Ivermectin is still able to bind to AgGluCl, but the antibody keeps the channel from opening, even after Ivermectin binds to it’, he says.
Having shown that antibodies targeted to AgGluCl in blood meals can be effective insecticides, Meyers and Foy are keen to find out if antibody-laced blood meals are equally deadly in real life. ‘The next step… is to immunize cattle against the AgGluCl antigen and directly feed A. gambiae on the immunized cattle in the lab’, explains Meyers. And if the strategy proves successful, Meyers envisages a large-scale cattle immunisation program as part of a combined attack on the parasite. ‘Cattle are a major blood meal source for multiple malaria vectors,’ he says, explaining that any malaria-harbouring mosquito that consumed blood carrying the toxic antibodies during the malaria parasite's incubation period would die, disrupting transmission of the disease and offering hope of a malaria-free future for generations to come.
