A key step in fighting malaria is understanding the biology behind the parasites’ invasion of their human hosts. Two recent studies from Harvard T.H. Chan School of Public Health researchers and colleagues have identified key essential proteins on the surface of red blood cells that two species of malaria parasite need in order to enter the cells for replication and transmission.
These discoveries — that the Plasmodium vivax parasite needs the TfR1 protein and the Plasmodium falciparum parasite needs the CD44 protein — open promising new avenues for the development of vaccines against the clinical blood-stage of the disease, when symptoms occur.
“P. falciparum is still a major scourge of humanity and P. vivax has been long-neglected in studies,” said Dr. Usheer Kanjee, a research fellow in the department of immunology and infectious Diseases and a lead author of both studies. “We believe that this work is a major step forward in the field. Our findings have identified targets that can be used to inform vaccine development.”
The P. vivax study was published in Science on January 5, 2018. It builds on a study published in PNAS on October 31, 2017 of P. falciparum, which also established a new method for studying genes in red blood cells.
The P. falciparum parasite causes the deadliest form of malaria, and is responsible for most of the cases in sub-Saharan Africa. P. vivax malaria is more widely distributed worldwide and less virulent, although it can lead to severe disease. Both parasites — and the four other Plasmodium species known to cause malaria — infect humans by binding to receptors on red blood cells. Researchers are working to discover which molecules on the surface of both the parasites and their red blood hosts are necessary for invasion to occur. Only a handful of these pathways have been identified.
A key obstacle in malaria biology research is that human red blood cells do not naturally possess a nucleus, which prevents researchers from performing direct genetic manipulation. But Dr. Kanjee and colleagues were able to generate nucleated red blood cells suited for parasite invasion studies through the use of an “immortal” cell line — human cancer cells that don’t die after multiple divisions. Using a genome editing tool known as CRISPR/Cas9, they created mutated versions of the cells with particular genes “knocked out” (made inoperative) in order to study their effects.
This technique enabled the researchers to identify the CD44 protein — critically required by all strains for invasion — as a key pathway into red blood cells for P. falciparum. In order for this pathway to become a vaccine target, researchers must also identify the molecule on the parasite that binds to this protein. In an additional finding from the PNAS study, the researchers validated previous evidence around another parasite and protein interaction. They found that the red blood cell protein BSG is an essential receptor for the parasite protein Rh5, making this pathway a major vaccine candidate. The study also found that BSG and CD44 work together, suggesting that they could be jointly targeted.
In the Science study, the researchers generated red blood cells that were mutated in the surface protein TfR1. These mutant cells were used by Dr. Kanjee and colleagues to directly demonstrate that TfR1 is needed for P. vivax parasites to invade red blood cells, and that it acts an essential receptor for the P. vivax protein PvRBP2b, only the second such molecule identified. The researchers say that more study is needed on P. vivax, a parasite that is very difficult to study due to the lack of culture system in the laboratory, and very likely contains at least half a dozen additional proteins that could be involved in red blood cell invasion.
Dr. Manoj Duraisingh, John LaPorte Given Professor of Immunology and Infectious Diseases, was senior author on the PNAS study and a co-author of the Science study. “The approach for generating genetic knockouts in red blood cells developed in these studies is fast and easy, and can be powerfully exploited for numerous biological and medical studies in the diverse fields of hematology, human genetics, and the study of malaria infection,” he said.
The Science study was conducted in collaboration with an international research group led by Dr. Wai-Hong Tham, a professor at the Walter and Eliza Hall Institute of Medical Research in Melbourne, Australia.