Malaria research is not a nine-to-five job

Sometimes researchers are in the lab at BPRC at two o’clock in the morning. To feed mosquitoes, explains researcher Annemarie van der Wel. “This particular mosquito species takes up blood best deep in the night.” She laughs: “Unfortunately, malaria research is not a nine-to-five job. When the moment arrives, you have to be ready. Everything revolves around timing.”

That timing already starts with the eggs. Building up a colony of this specific mosquito species ourselves is not yet possible. “It is almost like IVF for mosquitoes”, Annemarie says. That is why a package of eggs is shipped from the United States to Rijswijk once a month.

“It is a complex logistical chain”, Annemarie explains. “Everything is interconnected. One mistake in the planning and you have to start all over again.” That level of precision is necessary because the research focuses on a crucial but largely invisible stage of malaria: the liver stage.

Worldwide, malaria is still a major problem, particularly because of child mortality in Africa. There is a vaccine, but it is not yet optimally effective and it is very expensive. To understand the disease, work in the lab often starts with something very small: mosquitoes.

Delicate work with mosquitoes

In the lab, the eggs are raised into hundreds of mosquitoes. At exactly the right moment, the mosquitoes are offered warm blood containing malaria parasites. The blood comes from an animal and has to be fresh. “You cannot store that in a refrigerator. It has to be collected by animal care staff at precisely the right time.” The mosquito bites through a membrane and takes up the blood. The parasites enter the mosquito, develop in its gut, and after about two weeks move to the salivary glands. Only then does the real delicate work begin. Researchers isolate the salivary glands and extract the parasites. These are used to infect liver cells in a culture dish.

What makes malaria so complicated is that after infection the parasites do not immediately multiply in the blood. They first develop in the liver. Only when the parasites move from the liver into the bloodstream people become ill.

There are experimental vaccines that use weakened parasites which stall during their development in the liver and therefore never reach the bloodstream. Researchers at Leiden University Medical Center (LUMC) previously showed that one of these vaccines provides strong protection in humans, Annemarie explains. Unfortunately, these experimental vaccines cannot easily be used in practice. “Because you have to extract the parasites from mosquitoes”, she says. “If you want to scale that up worldwide, you are talking about enormous numbers of mosquitoes that have to be dissected by hand. Logistically and practically, that is almost impossible.”

Still, these vaccines are valuable because they show that protection is possible.“What makes the parasite in that early stage so effective at triggering protection?” The trail leads to the liver. “It seems that with this experimental vaccine the real protection arises in the stage when you are not yet sick”, Annemarie explains. “That is when the parasite is in the liver.”

Monkey model

To investigate what happens in the liver, researchers work with a monkey model. The research group uses monkey malaria parasites that closely resemble the malaria parasites that causes disease in humans. They have genetically modified these parasites in the same way as the weakened human parasites used in the experimental vaccine.

“An essential component has been removed. As a result, the parasite can still develop in the liver, but it should stop afterwards.” The idea is that the immune system is activated precisely during that liver stage, Annemarie says. “If we can see what happens in the liver in the animal model, we can more precisely determine which building blocks of the parasite are responsible for protection. Those components could then be incorporated into a new vaccine that would be much easier to use than a live attenuated parasite.”

The technique to genetically manipulate monkey malaria parasites is not new. It was developed at BPRC decades ago. “In recent years, this liver work has really accelerated. A few years ago we received funding, and we spent two years working intensively to create this specific weakened parasite.”

“First understand, then improve, and only then translate”

In a culture dish, researchers have examined how the parasite grows: does it really stop where it is supposed to stop? “In vitro you can only see so much”, Annemarie says. “At a certain point the image simply ‘disappears’. That is why we are now moving to the next stage: looking in the animal to see whether we still observe blood-stage parasites. That is exciting, because the answer determines the next step.”

“We want to investigate in a vaccination experiment whether this weakened parasite provides protection against infection with malaria parasites. We compare animals infected with the weakened parasite to a control group”, she continues. “Before you have data from that, you are easily six months further along. And if you think about the bigger goal, you are talking about years. First understand, then improve, and only then translate.”

A piece of the puzzle

There are different malaria models: humans, monkeys, rodents, and cell culture systems. Each has advantages and disadvantages. Together, all these models form pieces of the puzzle of the perfect model. The monkey is an important model because the physiology and immune system of monkeys closely resemble those of humans. “The parasite species in monkeys are also very similar to those in humans, or can even infect humans.”

There is something else as well: the combination of expertise. “This parasite requires exactly this mosquito species”, Annemarie says. “And you need knowledge of genetic modification of the parasite, of working with these mosquitoes, of liver cells, and of the animal model. That whole package of expertise only comes together in a few places.”

Within the malaria group, a small team works on this research, each with their own role. One colleague knows everything about the mosquitoes and is brought in at the moment they need to be fed. Another performs the genetic modification of the parasite. Someone else manages an experiment or analyses the results. At peak moments, everything revolves around that one exact moment in time.

That is why sometimes, in the middle of the night, researchers are in the lab and animal care staff are in the animal facilities. “Because progress in this field starts with planning, patience, and the courage to keep trying again. And because when it works, you are one step closer to the question that drives it all: what happens in that silent liver stage, and how can we use that knowledge to prevent malaria? So that hopefully, in the future, far fewer people will die from malaria.”