A replication competent Plasmodium falciparum parasite completely attenuated by dual gene deletion

Vaccination with infectious Plasmodium falciparum (Pf) sporozoites (SPZ) administered with antimalarial drugs (PfSPZ-CVac), confers superior sterilizing protection against infection when compared to vaccination with replication-deficient, radiation-attenuated PfSPZ. However, the requirement for drug administration constitutes a major limitation for PfSPZ-CVac. To obviate this limitation, we generated late liver stage-arresting replication competent (LARC) parasites by deletion of the Mei2 and LINUP genes ( mei2 – / linup – or LARC2). We show that Plasmodium yoelii (Py) LARC2 sporozoites did not cause breakthrough blood stage infections and engendered durable sterilizing immunity against various infectious sporozoite challenges in diverse strains of mice. We next genetically engineered a PfLARC2 parasite strain that was devoid of extraneous DNA and produced cryopreserved PfSPZ-LARC2. PfSPZ-LARC2 liver stages replicated robustly in liver-humanized mice but displayed severe defects in late liver stage differentiation and did not form liver stage merozoites. This resulted in complete abrogation of parasite transition to viable blood stage infection. Therefore, PfSPZ-LARC2 is the next-generation vaccine strain expected to unite the safety profile of radiation-attenuated PfSPZ with the superior protective efficacy of PfSPZ-CVac. Synopsis Malaria persists as a formidable global health threat, necessitating development of a vaccine that protects against malaria parasite infection. A late liver stage-arresting replication competent (LARC) parasite vaccine was engineered by deletion of the Mei2 and LINUP genes ( mei2 - /linup - or LARC2) in Plasmodium yoelii (PyLARC2) and Plasmodium falciparum (PfSPZ-LARC2). PyLARC2 did not show any breakthrough blood stage infections in mice. PyLARC2 elicited durable sterilizing immunity against diverse sporozoite challenges in a range of mouse strains. PfSPZ-LARC2 liver stages showed robust replication in liver-humanized mice but did not transition to viable blood stage infection. Cryopreserved PfSPZ-LARC2 vaccine was successfully produced. PfSPZ-LARC2 vaccine is the next-generation vaccine strain designed to unite the safety attributes of radiation-attenuated PfSPZ with the superior protective efficacy of PfSPZ-CVac. Malaria persists as a formidable global health threat, necessitating development of a vaccine that protects against malaria parasite infection. A late liver stage-arresting replication competent (LARC) parasite vacci