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

Plasmodium falciparum remodels infected erythrocytes using exporting 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 largely unclear, yet they may serve important 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 Maurer’s 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 not detected beyond this membrane. Finally, genetic disruption of LSA3 in P. falciparum NF54 attenuated fitness at the liver stage, manifesting as a 40% reduction in parasite liver load by day 5 postinfection of humanized mice. The identification of LSA3 as a member of the P. falciparum exportome and important for liver-stage development confirms the hypothesized potential of exported proteins as promising vaccine candidates, underscoring the need for their continued discovery and biological characterization, including those expressed at the liver stage.