A Cryptic Culprit

A saboteur is on the loose and wreaking havoc. Every time the authorities start closing in, the malefactor switches into one of many disguises and slips unnoticed into a new facility, causing still more damage until security forces again catch wise and the pursuit begins anew. But how long can he get away with it before his luck — and disguises — run out?

This same question arises when the malaria-causing pathogen Plasmodium falciparum circulates in the human bloodstream. Every time the immune system is on the verge of wiping out the infection, these parasites employ a sophisticated gene-switching mechanism that masks their presence and resets the clock on the body’s immune response. In the absence of medical treatment, these capabilities turn malaria into a prolonged, chronic condition that produces cycles of symptomatic disease and remission. Even experts in the parasite have been puzzled by P. falciparum’s capacity to maintain a haven in otherwise asymptomatic individuals — and potentially spread surreptitiously to new human hosts — for years on end.

Researchers led by Dr. Kirk Deitsch, professor of microbiology and immunology at Weill Cornell Medicine, are now closing in on an explanation for these immunity-eluding capabilities. Their findings suggest that the parasites are actually going into even deeper cover during chronic, asymptomatic infection than scientists previously understood — turning into “sleeper agents” that patiently wait for the threat from the immune system to die down before they reactivate and proliferate once again.

Beyond illuminating an important medical mystery, this work could have major implications for efforts to monitor, control and eliminate malaria, a disease that kills half a million people annually. “Most eradication plans are to go treat everybody who has symptoms and hope that you wipe out this disease,” says Dr. Deitsch. “It turns out there are probably a ton of people walking around with no symptoms who are carrying parasites and not going to the doctor, and they’re going to be a problem for eradication.”

A quick-change act

Like all parasites, P. falciparum has made trade-offs over the course of evolution to thrive in human hosts. With steady access to nutrients and a stable physiological environment, this organism has shed a lot of genes that would normally be indispensable for independent survival. Dr. Deitsch notes that this has also freed up valuable real estate for the parasite to acquire other essential functions. “They have to expand large parts of their genome in order to deal with things that we don’t — like their host trying to kill them all the time.” Indeed, this seemingly simple single-celled organism has devised sophisticated survival tactics, including a complex mechanism for eluding immunity-mediated detection and elimination.

While sequestered within red blood cells, P. falciparum expresses a protein called PfEMP1, which gets shuttled to the surface of infected cells and causes them to stick to the surface of blood vessels in various parts of the body. This prevents those cells from reaching the spleen — an immune cell-producing organ that would otherwise quickly destroy those abnormal cells — while also allowing the parasite to reproduce and inflict damage on host tissues in the process. But this only buys a temporary reprieve, as circulating immune cells gradually recognize PfEMP1 as a signature of infection.

The onset of an immune response provokes a “wardrobe change” in the surviving parasites. The P. falciparum genome contains roughly 60 different genes referred to as var, each encoding a distinct version of PfEMP1 — and this only scrapes the surface of the total complexity. “Every parasite that you find in the field has a different repertoire of var genes,” says Dr. Deitsch. “We don’t know how big the total number of var genes is, but it’s very, very large.” The parasites within the host employ a complicated regulatory mechanism to undergo a coordinated switch to a new var gene, encoding a distinct PfEMP1 that is unfamiliar to the immune system.

This leads to a new cycle of parasite proliferation and resurgence of symptomatic malaria within the infected individual. “In most mammals and humans, it takes a week to 10 days to mount a high-end antibody response that recognizes a pathogen,” says Dr. Deitsch. This gene-switching tactic enables parasite populations to survive for protracted periods in the humans they infect, thereby weathering the dry season in malaria-endemic regions, when mosquitoes are scarce and transmission to new hosts is effectively halted.

Maintaining radio silence

Malaria is primarily a childhood disease in endemic regions — 75% of malaria deaths occur before the age of five — and Dr. Deitsch says that by adolescence, children in these regions have typically encountered enough PfEMP1 variants during infection to achieve “semi-immune” status. They experience no symptoms but still harbor a persistent population of parasites that can spread to new hosts via mosquito.

Although 60 genes is a lot, sooner or later the parasite’s luck should run out — but it doesn’t. “You’d imagine that you would pretty rapidly exhaust your repertoire and the immune system will say, ‘We’ll just make antibodies to all 60 and off you go,’ yet somehow this does not happen,” says Dr. Lars Hviid, an immunologist at the University of Copenhagen who specializes in malaria. “The problem for us as scientists is: How is this system [of gene switching and hiding] maintained?”

Last summer, the Deitsch lab published a study that provides a potential explanation for this apparent immunological invisibility. This work began as a follow-up to a 2022 study in which his team aimed to characterize the process by which billions of parasites collectively coordinate the var gene switching process. To achieve this, the team obtained a “standardized” P. falciparum strain with a well-defined collection of var genes and studied how gene expression changed in the lab during protracted cultivation in human red blood cells.

Their analysis revealed something unusual: A subset of parasites that appeared to express low levels of many var genes rather than strongly expressing a single var gene. But as a population-scale analysis, it was hard to interpret these results. “Is this actually what each individual parasite looks like or is this just combining all of these together, and it’s giving you a false sense of what the population looks like?” says Dr. Joseph Visone (Ph.D. ’25), who worked on the 2022 study as a graduate student in Dr. Deitsch’s lab. “Single-cell methods were the only way to assess that.”

Dr. Visone and Deitsch lab postdoc Dr. Francesca Florini dug deeper, using sophisticated analytical technologies that enabled them to sensitively profile var expression in individual parasites from their experimental system. “That was the first time that we actually observed that there was a population of parasites that didn’t seem to express any var gene,” says Dr. Florini. The researchers applied multiple technologies to maximize the sensitivity of their expression profiling, and Dr. Visone developed an analytical workflow that was sufficiently rigorous to give both the team and the colleagues who subsequently reviewed their work high confidence in the findings. But their unusual result withstood this trial by fire. “This unexpected population has basically silenced everything and become immunologically invisible entirely,” says Dr. Deitsch.

Secrets of survival

Although this finding was unexpected, there was evidence in the literature that such parasites can sometimes arise during natural infection. For example, a 2009 case report describes a Cameroonian woman who was asymptomatic for malaria but had a highly enlarged spleen and extremely high levels of antibodies against P. falciparum. When her spleen was surgically removed, clinicians subsequently detected a sharp spike in parasite-infected red blood cells — and notably, these parasites were not expressing any var genes at measurable levels.

“At the time, we thought ‘Wow, what’s going on here?’” recalls Dr. Anna Bachmann, a parasitologist at the Bernhard Nocht Institute for Tropical Medicine who was first author on that study. She concluded that the patient’s heightened antibody response was effectively eliminating parasite-infected red cells, creating strong pressure on the parasites to thoroughly mask their presence. “The parasites were really forced not to express anything on the surface because then the red blood cells would have been recognized,” she says. These infected cells accumulated in the spleen but immediately became detectable again when that organ was removed. And notably, when her team cultured the isolated parasites, var gene expression was restored, suggesting this was only a temporary defense mechanism.

There have been other reported cases of people harboring apparently silent malaria infections for many years or even decades, and which re-emerge only when those patients undergo a splenectomy or donate blood to a malaria-naïve recipient. Dr. Florini says the present work provides important molecular evidence that malaria parasites can indeed suppress var gene suppression to cover their tracks. “I feel like it brought together stuff from the past that people didn’t really know how to interpret,” she says.

Dr. Deitsch’s colleagues consider these new findings compelling. “It explains the results we had from the splenectomized patients,” says Dr. Bachmann, noting that the detailed single-cell analysis provides a strong foundation for the work. And Dr. Hviid, who is also deeply engaged with the question of how P. falciparum regulates var expression during infection, is intrigued by the notion of a second “control knob” that the parasite can fiddle with to elude immune detection alongside gene-switching. “If you turn down the number of copies of this molecule you have on the surface, of course you’ll bind less well if you’re in the infected red cell, but you will also be much more difficult to detect,” he says.

Tying it all together

The hard part now will be confirming that what happens in the laboratory actually reflects the biological reality of human infection.

Malaria is fiendishly difficult to replicate in an experimental setting. “The parasites will grow fine in a culture flask in human red blood cells… and it appears to be like what you would find if you’re in the field working with people,” says Dr. Deitsch. “The tricky part is trying to mimic the immune response, because your flask doesn’t have that.” And unfortunately, that component will be critical if one intends to test whether these “var-null” parasites naturally emerge following an aggressive immune response — and if the resulting parasites persist for a meaningful duration under such conditions.

To make things trickier, P. falciparum is a picky parasite. A descendent of species that specifically infect our great ape relatives, it is unable to infiltrate red blood cells from other mammals. Meanwhile, Plasmodium parasites that do infect popular animal models like rats and mice differ in key aspects of their life cycle and lack the relevant var gene family, making them poor stand-ins. One can employ “humanized” mouse models, which produce human-like red blood cells that are accessible to P. falciparum, but these animals must also be engineered to be immune-deficient.

“It becomes a super artificial system,” says Dr. Hviid, who still questions whether fully PfEMP1-deficient parasites could survive long in an immune-competent human host. “If they do not have any PfEMP1, then they have no means of adhering — so that means that they will have to go through the spleen, where they will die,” he says. “I think what is happening really is that instead of having ‘var-null,’ we could call them ‘var-very, very low.’”

Dr. Deitsch is hoping to get to the bottom of this mystery by eventually working with tissues collected from infected donors by collaborators at multiple research universities in Ghana. Once a framework for supporting the research is identified amidst a freeze on NIH funding for many international collaborations, his focus will be on specimens collected from asymptomatic trauma patients in the northern parts of the country, where malaria prevalence is especially high. “We’re particularly interested in bone marrow and spleens as possible reservoirs of these immunologically invisible parasites,” he says.

Stopping a silent killer

Even if the full molecular and immunological details of asymptomatic malaria remain incompletely understood, there is compelling evidence that silent infection is real and poses a serious threat to efforts to stop the spread of this deadly disease.

“To reach elimination… we need to know where these cryptic infections are so that they can be treated as well as the symptomatic infections,” says Dr. Deitsch. He cites a recent trip to Ghana, where he and Dr. Florini visited a transfusion center that mandated antimalarial treatment for all recipients based on the assumption that all donated blood was harboring parasites. Dr. Bachmann concurs about the importance of treating these chronic but asymptomatic patients and says that more rigorous and repeated screening and consistent treatment will be needed to move toward true eradication of malaria.

Drugs and intensive screening programs cost money that many malaria-endemic countries simply don’t have — and there are concerning signs that existing drugs are starting to falter. However, researchers at Weill Cornell Medicine are working on solutions that could extend their utility (see page 43), and Dr. Deitsch even sees theoretical opportunities to shut the var gene-switching mechanism down and essentially take away P. falciparum’s box of disguises. This would be an unconventional approach, however — drugs are typically designed to kill rather than debilitate parasites, and Dr. Deitsch believes drugmakers may hesitate to diverge from that formula.

The best strategy for tackling chronic malaria infection remains to be determined, but the insights uncovered by Dr. Deitsch and colleagues into the gene-switching process will provide a valuable foundation for understanding what these silent parasites are doing within their hosts and how they hide. “We’re learning stuff that I’ve been trying to figure out for decades, and it’s just now starting to come to fruition,” he says, and adds that similar immunity-eluding mechanisms are likely to be relevant in other vector-borne parasitic infections as well. For example, the tick-borne parasite responsible for babesiosis — a potentially fatal disease for which the prevalence has steadily grown in the United States — is also known to use a gene-switching mechanism to evade immune destruction. “What we learn in Plasmodium is going to be applicable to other diseases,” says Dr. Deitsch.

Finding the Path of Least Resistance

The discovery of artemisinin — a plant-derived compound with potent antimalarial properties — was a game changer for public health and won Tu Youyou, the Chinese scientist who identified it, the 2015 Nobel Prize in Medicine and Physiology. Artemisinin combination therapy (ACT) that pairs this drug with other medicines is now the standard intervention against Plasmodium falciparum, and can wipe out infection within days.

The history of malaria is full of painful lessons about this parasite’s resourcefulness, however, and alarming signs of ACT-resistant infection have now emerged in Southeast Asia and East Africa. Unfortunately, the medical community does not have a good plan B. “There’s one or two drugs that are trying to replace it, but nothing is that potent,” says Dr. Laura Kirkman, associate professor of medicine.

Dr. Kirkman and her collaborator Dr. Gang Lin, professor of research in microbiology and immunology, have recently identified a promising opportunity to restore artemisinin’s potency. Dr. Lin’s team is developing compounds that interfere with the proteasome, an essential protein complex that eliminates damaged and defective proteins which would otherwise sicken and kill cells.

“We gave a few compounds to Laura, and it turns out they’re super potent,” says Dr. Lin. As it turns out, the proteasome is a ripe target in artemisinin-treated parasites. This drug chemically modifies and thereby inflicts severe damage on proteins, overwhelming the proteasome’s ability to keep up and thereby killing off the parasite. As such, debilitating the proteasome can amplify artemisinin’s lethality. “We hope that if we combine proteasome inhibitors with artemisinin, we can significantly prolong the life of artemisinin,” says Dr. Lin, “because the parasite would have actually a lot of difficulties to develop resistance to either.”

In a 2023 paper, they tested out a first-generation drug design based on this concept, in which artemisinin was physically linked to a potent proteasome inhibitor. With this approach, every protein modified by artemisinin is also directly tethered to the inhibitor, producing degradation products that subsequently gum up the degradation process. Some of these compounds proved quite effective in laboratory models — and the researchers also observed an unexpected advantage of their two-pronged attack. “[Parasites] resistant to the proteasome inhibitors actually become more sensitive to artemisinin, and resistance mutations to artemisinin become more sensitive to proteasome inhibitors,” says Dr. Lin. This should make it far harder for the parasite to pull its usual escape act. In principle, even currently artemisinin-resistant infections should succumb to this combined approach.

Although these first-generation compounds offer robust proof of concept for proteasome inhibition as an antimalarial strategy, Dr. Lin says that they do not have ideal properties for clinical use. Accordingly, he and Dr. Kirkman are now working on a new generation of candidates based on more clinically appropriate proteasome inhibitors — including one that is already FDA-approved for myeloma therapy.

Considerable work remains on the road to a feasible drug candidate, but Dr. Kirkman is enthusiastic about their approach and the opportunity to refresh artemisinin’s lifespan rather than moving on to an entirely new drug. “If we roll out one inhibitor at a time in a not-thoughtful way, we’re just going to continue the cycle of resistance in the field,” she says. “I think we have to be a little clever.”