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Malaria, the disease caused byPlasmodiumparasites, causes significant mortality and morbidity. Whole parasite vaccination with pre-erythrocytic parasite stages, attenuated through sporozoite irradiation or chemo-attenuation, confers sterilizing immunity against subsequent parasite infection. This provides a rationale for the creation of whole parasite vaccines that are attenuated using gene editing. Here, we report on the creation of a novel genetically attenuated parasite (GAP) by the deletion ofPlasmodium LINUP,encoding aliver stagenuclearprotein.Epitope-tagging of LINUP in the rodent malaria parasitePlasmodium yoeliishowed LINUP expression exclusively in liver stage nuclei after the onset of exo-erythrocytic schizogony.P. yoeliiparasites with a gene deletion ofLINUP(linup—) suffered an exclusive liver stage phenotype with developmental arrested late in exo-erythrocytic schizogony. Liver stages showed incomplete segregation of nuclei and, mitochondria and apicoplast. These cellular perturbations caused a defect in exo-erythrocytic merozoite formation and a concomitant severe attenuation of liver stage-to-blood stage transition.LINUPgene deletion inPlasmodium falciparumalso caused a severe defect in late liver stage differentiation. Importantly,P. falciparum linup—liver stages showed a severe defect in parasite transitioning from liver stage to viable blood stage infection. These results suggest thatP. falciparum LINUPis a useful target for late liver stage attenuation and an additional gene deletion that can be incorporated into a late liver stage-arresting replication competent whole parasite vaccine.

Vaccine-induced sterilizing protection from infection by Plasmodium parasites, the pathogens that cause malaria, will be essential in the fight against malaria as it would prevent both malaria-related disease and transmission. Stopping the relatively small number of parasites injected by the mosquito before they can migrate from the skin to the liver is an attractive means to this goal. Antibody-eliciting vaccines have been used to pursue this objective by targeting the major parasite surface protein present during this stage, the circumsporozoite protein (CSP). While CSP-based vaccines have recently had encouraging success in disease reduction, this was only achieved with extremely high antibody titers and appeared less effective for a complete block of infection (i.e. sterile protection). While such disease reduction is important, these and other results indicate that strategies focusing on CSP alone may not achieve the high levels of sterile protection needed for malaria eradication. Here, we show that monoclonal antibodies (mAbs) recognizing another sporozoite protein, TRAP/SSP2, exhibit a range of inhibitory activity and that these mAbs can augment CSP-based protection despite conferring no sterile protection on their own. Therefore, pursuing a multivalent subunit vaccine immunization is a promising strategy for improving infection-blocking malaria vaccines.