Creation and preclinical evaluation of genetically attenuated malaria parasites arresting growth late in the liver.

Whole-sporozoite (WSp) malaria vaccines induce protective immune responses in animal malaria models and in humans. A recent clinical trial with a WSp vaccine comprising genetically attenuated parasites (GAP) which arrest growth early in the liver (PfSPZ-GA1), showed that GAPs can be safely administered to humans and immunogenicity is comparable to radiation-attenuated PfSPZ Vaccine. GAPs that arrest late in the liver stage (LA-GAP) have potential for increased potency as shown in rodent malaria models. Here we describe the generation of four putative P. falciparum LA-GAPs, generated by CRISPR/Cas9-mediated gene deletion. One out of four gene-deletion mutants produced sporozoites in sufficient numbers for further preclinical evaluation. This mutant, PfΔmei2, lacking the mei2-like RNA gene, showed late liver growth arrest in human liver-chimeric mice with human erythrocytes, absence of unwanted genetic alterations and sensitivity to antimalarial drugs. These features of PfΔmei2 make it a promising vaccine candidate, supporting further clinical evaluation. PfΔmei2 (GA2) has passed regulatory approval for safety and efficacy testing in humans based on the findings reported in this study. ; We acknowledge the critical contribution of the late Dr Shahid Khan to the design of these studies. We are grateful to Pr Jude Przyborski for the anti- P. falciparum EXP-1 antibody. This work was supported by LUMC internal funds. CM-M was, in part, supported by Colciencias Ph.D. fellowship (Call 568 from 2012 Resolution 01218 Bogotá, Colombia) and LUF project grant, Den Dulk-Moermans Fonds (grant reference number: W19374-2-32, 2019-2021). A.O. is supported by a Skim Latihan Akademik IPTA-SLAI (Ministry of Higher Education, Malaysia). A.P. acknowledges the Faculty Baseline funding (BAS/1/1020-01-01) from King Abdullah University of Science and Technology (KAUST).