Mechanisms Causing Lethal Cerebral Malaria Revealed
November 2017 — Researchers at Seattle Children’s Center for Global Infectious Disease Research (CGIDR), Albert Einstein College of Medicine and Michigan State University, in collaboration with the Blantyre Malaria Project, have found a mechanism by which malaria parasites cause highly lethal swelling of the brain in cerebral malaria cases. Cerebral malaria is the deadliest form of malaria, and 90% of its victims are children. The mechanisms leading to the breakdown of the blood brain barrier in these severe cases have long been a mystery; now that this biological pathway is understood, researchers are better equipped to develop new therapies to save the lives of hundreds of thousands of children.
The new findings substantiate a hypothesis that when large numbers of parasite-infected red blood cells accumulate in the brain, they interfere with a receptor that plays a major role in the health of the blood brain barrier, creating a vulnerability that leads to dangerous brain swelling and high risk of death.
Plasmodium falciparum has certain qualities that give malaria its reputation as one of the world’s most challenging diseases – it can hide from the immune system, rapidly develop drug resistance, and cause highly variable disease presentation, ranging from mild symptoms to coma and death. It is the severe manifestation associated with coma and brain swelling that researchers from Malawi, New York, Michigan, England, Australia and Seattle joined forces to investigate.
Within humans, malaria parasites infect red blood cells. P. falciparum malaria is especially dangerous because infected red blood cells have a unique ability to attach to the inner walls of blood vessels. In patients with cerebral malaria, parasite-infected red blood cells accumulate within blood vessels of the brain. Previous work has suggested that a protein on human blood vessels called endothelial protein C receptor (EPCR) is an important receptor for infected red blood cells – especially in children with severe malaria illness. EPCR is found on endothelial cells of blood vessels throughout the body and plays a major role in controlling blood clotting, inflammation, and the health of the blood brain barrier. In the current study, the team studied a unique clinical cohort of African children suffering from cerebral malaria to better understand the disease processes that lead to brain swelling episodes.
Multiple Organizations Partner for Multi-Step Process
Because the brain is such an inaccessible organ to study in life, researchers combined three methods to obtain information to study what is going on in the brain: eye exams, MRIs, and blood work. The Blantyre Malaria Project, based in the Queen Elizabeth Central Hospital in Blantyre, Malawi, has been treating and studying children with cerebral malaria for over 25 years. Led by Drs. Terrie Taylor and Karl Seydel, the Blantyre Malaria Project has the capacity to study brain and eye findings associated with cerebral malaria.
The eye exams were used to detect characteristic features associated with the presence of infected red blood cells in blood vessels of the eye and were carried out by ophthalmologists from the University of Liverpool. In addition, MRI scans were carried out in Malawi, and were analyzed and ranked for the extent of brain swelling by radiologists at the University of Rochester and in Blantyre. The presence of these clinical features allowed the team to stringently classify children based on the eye findings and the extent of brain swelling.
In parallel, a small volume of blood was collected from children with cerebral malaria to compare with blood from children with mild malaria symptoms. Anne Kessler and Kami Kim at Albert Einstein College of Medicine and researchers at the Center for Infectious Disease Research conducted experiments to study the types of parasites present in infected red blood cells. All of this information became data points that were analyzed by machine learning approaches to identify parasite and host features that predicted worse disease outcomes. This analysis showed that EPCR-binding parasites were highly elevated in patients with cerebral malaria and brain swelling.
In addition to those profiles, the team also studied a particular parasite variant isolated from a brain autopsy provided by the team in Blantyre. The EPCR binding domain from this parasite was expressed as a recombinant protein and used in a laboratory assay designed to mimic a blood-brain barrier. This analysis showed that when the parasite cytoadhesion domain attaches to EPCR it interferes with its ability to function. These comparisons showed that EPCR-binding parasites were common to both severe brain swelling cases and fatal cerebral malaria cases and was another piece of evidence that this variant played a role in interrupting the functions of the blood brain barrier.
Applying Computation and Deep Sequencing to Experimental Biology
By pairing experimental biology and computational biology, scientists at CGIDR harnessed the power of machine learning to identify biological markers that are most predictive of cerebral malaria.
Bringing together the variety of expertise required for a project of this magnitude, three CGIDR labs, the Smith Lab, the Sather Lab and the Aitchison Lab, worked together to perform detailed analyses of the parasites in children suffering from cerebral malaria.
The Smith Lab, experts in the pathobiology of malaria, performed experiments to learn more about the parasite cytoadhesion binding traits and mechanisms leading to brain swelling.
The Sather Lab adapted next-generation deep sequencing techniques that allowed for in-depth examinations of the types of parasites affecting sick children. The researchers leveraged this capability to look at the parasite cytoadhesion proteins expressed in the Malawi cases.
“Once we determined how infected red blood cells were attaching to blood vessels in cerebral malaria patients, we recreated the specific protein in the lab to make it easier to study how this process leads to disruption of the blood brain barrier and brain swelling,” says Dr. Selasi Dankwa, postdoctoral scientist in the Smith Lab. Then in a unique approach to test experimental models against unbiased computer models, the Smith Lab turned to the Aitchison Lab.
The Aitchison Lab specializes in machine learning analysis, used here to find predictive profiles among cerebral malaria cases from the Malawi cohort, compared to cases of milder malaria.
“There are thousands of variants of the malaria cytoadhesion proteins. By using machine learning, we were able to identify specific patterns of variants responsible for the most lethal symptoms in these children,” says Fergal Duffy, postdoctoral scientist in the Aitchison Lab.
Starting with stringently classified children with varying disease symptomology, the international team of scientists pieced together the data gleaned from the autopsy sample, along with the deep sequencing data and the machine-learning predictions to compile a picture of the parasite’s effects on the brain.
The new findings corroborate a hypothesis that when large numbers of infected erythrocytes accumulate in the brain, they interfere with the normal function of EPCR, creating a vulnerability that leads to dangerous brain swelling and high risk of death.
Basic Research Moving Towards an Intervention
With the details of this complex mechanism revealed, the target for finding an effective treatment is significantly clearer. Researchers hope to find a way to interrupt this process and drastically reduce the number of lives lost to cerebral malaria. “Now we’re in a stronger position to address and correct the damage the parasite causes in the brain,” says Dr. Maria Bernabeu, postdoctoral scientist in the Smith Lab.
Already, the Smith Lab is collaborating with another lab at CGIDR, the Kaushansky Lab, to screen compounds that can strengthen or restore blood brain barrier integrity, which is disrupted in cerebral malaria but also in many other diseases.
“All these partnerships – across different countries and within our own institute – enable us to bring the newest technologies and best scientific processes together to tackle the world's toughest health problems, which particularly benefit resource-poor settings. This is what science should be like,” says Joe Smith, principal investigator at CGIDR.
The study “Linking EPCR-binding PfEMP1 to Brain Swelling in Pediatric Cerebral Malaria” was published in Cell Host & Microbe.