The vaccine candidate Liver Stage Antigen 3 is exported during Plasmodium falciparum infection and required for liver-stage development

Plasmodium falciparum , which causes the most severe malaria, remodels infected erythrocytes by exporting several hundred effector proteins. Parasites express the aspartyl protease plasmepsin V that processes proteins containing a PEXEL motif and the PTEX translocon to successfully export proteins. During liver-stage infection, PTEX is required for P. falciparum development, but which proteins are exported remain unknown; these proteins may serve important biological functions and be presented by MHC-I molecules, thereby representing potential vaccine candidates. Here, we investigated liver stage antigen 3 (LSA3), an immunogenic protein of the Laverania subgenus of Plasmodium . We show that LSA3 possesses a PEXEL motif processed by plasmepsin V and is targeted to one or more membranes surrounding the blood-stage parasite, suggestive of the parasitophorous vacuole membrane (PVM). A subset of LSA3 also localizes in the erythrocyte, where it forms punctate structures that are not Maurers clefts but are soluble in biochemical fractionation assays reminiscent of J-dot proteins. During infection of human hepatocytes, antibodies to LSA3 co-localize with EXP1 and EXP2 at the PVM, yet these antibodies were rarely detected beyond this membrane. Finally, genetic disruption of LSA3 in P. falciparum NF54 attenuated fitness at the liver stage, manifest as a 40% reduction in parasite liver load by day 5 postinfection of humanized mice. The identification of LSA3 as a previously unrecognized member of the P. falciparum exportome, essential for normal liver-stage development and capable of eliciting protective pre-erythrocytic immunity, confirms the hypothesized potential of exported proteins as promising malaria vaccine candidates, underscoring the need for continued investigation into their discovery and biological characterization.