A replication-competent late liver stage–attenuated human malaria parasite
Debashree Goswami, … , Ashley M. Vaughan, Stefan H.I. Kappe
Debashree Goswami, … , Ashley M. Vaughan, Stefan H.I. Kappe
Published June 2, 2020
Citation Information: JCI Insight. 2020;5(13):e135589. https://doi.org/10.1172/jci.insight.135589.
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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 10

Generation of marker-free P. falciparum mei2–.

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Generation of marker-free P. falciparum mei2–. (A) Cartoon showing the b...
(A) Cartoon showing the binding of primers specific for S. pyogenes Cas9 in the pFC-PlasMei2 plasmid. The PCR amplicon is 525 bp. Agarose gel electrophoresis shows the Cas9 PCR product amplified from genomic DNA of 4 P. falciparum mei2– clones: F2, F5, C7, and B7. DNA from pFC-PlasMei2 was used as a positive control, and genomic DNA from P. falciparum NF54 was used as a negative control for the PCR. (B) The schematic depicts the generation of marker-free P. falciparum mei2– using CRISPR/Cas9-mediated gene editing using the pFC-yFCU-PlasMei2 plasmid, carrying yFCU negative selection cassette. Primers used to verify gene deletion are indicated, and the sizes of the PCR products are shown below in kilobases. (C) Agarose gel electrophoresis shows the Cas9 PCR product amplified from genomic DNA of 2 independent transfections before limiting dilution cloning and before (column 1) and after (column 2) undergoing 2 rounds of negative selection. The Cas9 primers and the amplicon size are the same as in A. 18S rRNA was used as a positive control for the PCR reaction. (D) Agarose gel electrophoresis shows the PCR products corresponding to the gene deletion P. falciparum mei2– clones A6 and C5 after positive and negative selection. PCR amplicons are shown in B. (E) Agarose gel electrophoresis shows absence of Cas9 PCR product amplified from genomic DNA of P. falciparum mei2– clones A6 and C5, indicating no retention of plasmid. DNA from pFC-yFCU-PlasMei2 was used as a positive control, and genomic DNA from P. falciparum NF54 was used as a negative control for the PCR. The Cas9 primers and the amplicon size are the same as in A.