A replication-competent late liver stage–attenuated human malaria parasite
Debashree Goswami, William Betz, Navin K. Locham, Chaitra Parthiban, Carolyn Brager, Carola Schäfer, Nelly Camargo, Thao Nguyen, Spencer Y. Kennedy, Sean C. Murphy, Ashley M. Vaughan, Stefan H.I. Kappe
Debashree Goswami, William Betz, Navin K. Locham, Chaitra Parthiban, Carolyn Brager, Carola Schäfer, Nelly Camargo, Thao Nguyen, Spencer Y. Kennedy, Sean C. Murphy, Ashley M. Vaughan, Stefan H.I. Kappe
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Research Article Infectious disease Vaccines

A replication-competent late liver stage–attenuated human malaria parasite

Abstract

Whole-sporozoite vaccines engender sterilizing immunity against malaria in animal models and importantly, in humans. Gene editing allows for the removal of specific parasite genes, enabling generation of genetically attenuated parasite (GAP) strains for vaccination. Using rodent malaria parasites, we have previously shown that late liver stage–arresting replication-competent (LARC) GAPs confer superior protection when compared with early liver stage–arresting replication-deficient GAPs and radiation-attenuated sporozoites. However, generating a LARC GAP in the human malaria parasite Plasmodium falciparum (P. falciparum) has been challenging. Here, we report the generation and characterization of a likely unprecedented P. falciparum LARC GAP generated by targeted gene deletion of the Mei2 gene: P. falciparum mei2–. Robust exoerythrocytic schizogony with extensive cell growth and DNA replication was observed for P. falciparum mei2– liver stages in human liver-chimeric mice. However, P. falciparum mei2– liver stages failed to complete development and did not form infectious exoerythrocytic merozoites, thereby preventing their transition to asexual blood stage infection. Therefore, P. falciparum mei2– is a replication-competent, attenuated human malaria parasite strain with potentially increased potency, useful for vaccination to protect against P. falciparum malaria infection.

Authors

Debashree Goswami, William Betz, Navin K. Locham, Chaitra Parthiban, Carolyn Brager, Carola Schäfer, Nelly Camargo, Thao Nguyen, Spencer Y. Kennedy, Sean C. Murphy, Ashley M. Vaughan, Stefan H.I. Kappe

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Figure 3

P.falciparum mei2– parasites have no defects in blood stage and mosquito stage development.

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P.falciparum mei2– parasites have no defects in blood stage and mosquit...
(A) The schematic depicts the generation of the P. falciparum mei2– parasite using CRISPR/Cas9-mediated gene editing. Primers used to verify the gene deletion are indicated, and the sizes of the PCR products are shown in kilobases. Agarose gel electrophoresis shows the PCR products corresponding to the gene deletion P. falciparum mei2– clones F2 and F5. hDHFR, human dihydrofolate reductase. (B) The schematic (upper right) and the corresponding Southern blot for the P. falciparum NF54 and P. falciparum mei2– locus. The genomic DNA for P. falciparum NF54 and P. falciparum mei2– clones F2 and F5 was digested with XhoI, HindIII, and AflIII and probed with the 3′ Mei2 probe. (C) Asexual blood stage parasitemia is compared between P. falciparum NF54 (gray line) and P. falciparum mei2– clones F2 (red line) and F5 (blue line) over 3 replication cycles. Data represent mean ± SD. n = 3 biological replicates. Statistical analysis was carried out using 2-way ANOVA. ns, not significant. P > 0.05 is taken as ns. Graphs comparing (D) the counts for oocyst/midgut, (E) oocyst prevalence, and (F) counts for sporozoites/mosquito for P. falciparum NF54 (gray) and P. falciparum mei2– clones F2 (red) and F5 (blue). Data represent mean ± SD. n = 3 biological replicates. Statistical analysis was carried out using 1-way ANOVA. P > 0.05 is taken as ns.