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Despite increased funding towards malaria research and vaccine development, the number of deaths from the parasitic disease returned over the past three years to the high numbers seen in 2012, with more than 600,000 deaths annually. That adds up to more than four times as many total deaths in sub-Saharan Africa as COVID-19 caused in 2020 and 2021. To combat the disease, scientists agree they need to improve the production process of malaria vaccines, which began rolling out in 2022. To that end, a group of researchers has documented the first successful cultivation of the necessary vaccine ingredients in vitro. The innovation, reported December 7 in Nature, could improve vaccine production efficiency by tenfold, the study authors say, and make malaria research cheaper and faster.

“This is a major breakthrough,” says Alon Warburg, a microbiologist and entomologist at the Hebrew University of Jerusalem who wasn’t involved in the research, but who has offered advice to the authors in the past. “It will completely revolutionize the production [of vaccines].”

As of now, only one malaria vaccine has been recommended for use by the World Health Organization (WHO), explains study coauthor and physician scientist Stephen Hoffman, who owns the biotechnology and vaccine development company Sanaria Inc. To meet the rising demand, Sanaria is currently working on new vaccines that use at least part of the sporozoite life stage of the protozoan Plasmodium falciparum, the deadliest malaria-inducing Plasmodium species, as the antigenic agent to induce an immune memory of the parasite.

Sporozoites, which are the sporelike infective form of the protozoan (hence the name), do not cause malaria themselves; once injected into a human host’s bloodstream by a mosquito, they colonize the liver and mature into fully fledged parasites, which infect red blood cells to cause disease as well as produce the gametocytes that mosquitoes slurp up when they feed. Still, since they are the parasite’s form upon invading a host, if a vaccine can teach a person’s immune system to spot and kill sporozoites, it could provide robust protection against malaria.

The sporozoites needed for developing and producing vaccines like Sanaria’s are usually manufactured using specially cultivated “aseptic” mosquitoes that are fed cultured blood infected with P. falciparum gametocytes. Scientists extract each mosquito’s salivary glands, where sporozoites accumulate, then purify the sporozoites before using them. Although Hoffman and other researchers are working to speed up this extraction through robotics, the process is still time and resource intensive. It’s also, Warburg adds, somewhat risky: Should a P. falciparum–infected mosquito escape the lab, it could potentially bite and infect someone nearby. To achieve the efficiency and cost-effectiveness necessary for widespread dissemination of their malaria vaccine, Hoffman and his team wanted to expedite the process, something they figured they could do by taking mosquitoes out of the equation and producing sporozoites in vitro.

One 1992 paper by Warburg had suggested that in vitro culturing of P. falciparum sporozoites was possible. There was a reason, however, that little progress had been made since: P. falciparum doesn’t start to produce the necessary sporozoites until two to three weeks from the gametocyte stage, and getting them to grow to maturity without a mosquito host wasn’t easy.

If you’re a malaria biologist, being able to study the parasite in all of its different stages in the lab is a huge potential development. But of course, for us trying to make a vaccine, it’s beyond enormous.

-Stephen Hoffman, Sanaria Inc.

In the new study, Hoffman and his team started with the blood mixture used to feed the mosquitoes, which contains P. falciparum gametocytes. Going step by step, the team started by seeing if they could grow the gametocytes to the 3-day-old stage, at which point the gametocytes become early oocysts. They eventually figured out the correct matrix and feeder cells to use, which allowed the oocysts to survive for 8 days. At this point, the researchers could see the sporozoites forming. Finally, they found the right conditions that allowed the oocysts to survive for 2 to 3 weeks, at which point the now-infectious sporozoites could be extracted. Unfortunately, the matrix gel that supported these P. falciparum in vitro contained rat sarcoma cells and was therefore not suitable for use in vaccine production.

After much tinkering over the course of several years, Hoffman and his team were able to cultivate the parasites without the use of the matrix and can now extract hundreds of millions of 3-week-old P. falciparum sporozoites.

“It was one of these wonderful, exhilarating moments where you feel like you’ve really done something that’s never been done before,” Hoffman says. “If you’re a malaria biologist, being able to study the parasite in all of its different stages in the lab is a huge potential development. But of course, for us trying to make a vaccine, it’s beyond enormous.” He estimates that in vitro P. falciparum cultivation could increase the efficiency and cost-effectiveness of malaria vaccine production by ten times.

Hoffman tested the sporozoite’s infectiousness by injecting them into mice engineered to have humanized livers. The team was excited to find that they worked: The sporozoites produced parasites in the liver, and when the mice were injected with human blood, the parasites grown in the liver infected the red blood cells, albeit at a lesser rate to the sporozoites extracted from mosquitoes. This meant that they would theoretically generate an immune response when included in a vaccine, protecting the vaccine’s recipient from future malaria infection.

Hoffman states that they are still quite far from using the process for vaccine production. Although the cultured sporozoites did produce an immune response in the mice, it was a smaller response than that induced by the mosquito-cultured sporozoites, and the number of infectious liver-stage parasites generated in the mice by in vitro sporozoites was around half of the number generated by the mosquito sporozoites. Moreover, the process through which the cultured sporozoites are cleaned still needs to be fine-tuned before any vaccines including them could be tested in humans. Nonetheless, Hoffman claims the culturing process will make its way into Sanaria’s vaccine manufacturing process in about two years.

Warburg emphasizes the importance of in vitro production for decreasing costs, especially for a vaccine mostly used in countries that can’t afford a higher price tag. “The lower the price, the more useful the vaccine is in endemic areas,” he says, adding that without cost reduction, malaria vaccines could end up going primarily to travelers and military personnel from wealthier countries instead of to the communities that desperately need them.

“At the end of the day,” Hoffman says, “we’re here because today, there’ll be maybe 2,000 people that die of malaria. We need a vaccine; We need to get it to people all over the world, and that’s why we work so hard on developing this system.”