Project Summary/Abstract We confirm that the scope of the parent grant (R01AI168163) will remain unchanged. Malaria parasites infect red blood cells (RBCs), within which they proliferate to cause disease in a continuous cycle of invasion, growth, and egress from one host RBC to another. Free merozoite parasites use ligand- receptor interactions to invade RBCs and escape the humoral immune system after egress from an infected RBC. Plasmodium vivax, a parasite that causes malaria, exclusively invades reticulocytes, the youngest RBCs, through these ligand-receptor interactions. The Duffy antigen receptor for chemokines (DARC) is a blood group antigen on the RBC surface. DARC was the first identified receptor for P. vivax, binding the parasite protein P. vivax Duffy Binding Protein (PvDBP). Genetic variations in the DARC gene, which determines the presence or absence of Duffy antigens, affect malaria control efforts. Duffy-negative populations of African ancestry were initially thought to resist P. vivax invasion. However, recent studies showed that Duffy-negative cells allow a low level of DARC expression, primarily on immature reticulocytes, the presumed target of invasion by P. vivax parasites. There have been increasing reports of antimalarial drug resistance with P. vivax, which poses a significant public health concern and impedes malaria control efforts and elimination programs. The mechanisms of drug resistance in P. vivax are poorly understood, and we hypothesize that drug resistance will be influenced by invasion into reticulocytes of different ages, cells undergoing dynamic changes to surface receptor presentation, sub-cellular organelle composition, and metabolic capacity. Given the observation regarding Duffy-negative invasion into immature reticulocytes, I hypothesize that the invasion of parasites into younger reticulocytes alters sensitivity to standard antimalarial drugs. Here, I propose to (Aim 1) explore whether P. vivax invasion into reticulocytes of different ages promotes drug resistance (in reticulocytes with and without DARC); (Aim 2) identify reticulocyte host receptors for P. vivax using a CRISPR/Cas9-based RBC knockout screen of candidate membrane receptors. This work is crucial for advancing 1) our knowledge of P. vivax invasion in Duffy-negative reticulocyte populations and drug resistance mechanisms and 2) identifying novel reticulocyte-tropic invasion pathways.