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Ex vivo susceptibility to new antimalarial agents differs among human-infecting Plasmodium species.

ABSTRACT: Several promising antimalarial drugs are currently being tested in human trials, such as artefenomel, cipargamin, ferroquine and ganaplacide. Many of these compounds were identified using high throughput screens against a single species of human malaria, Plasmodium falciparum, under the assumption that effectiveness against all malaria species will be similar, as has been observed for other antimalarial drugs. However, using our in vitro adapted line, we demonstrated recently that P. knowlesi is significantly less susceptible than P. falciparum to some new antimalarial drugs (e.g., cipargamin and DSM265), and more susceptible to others (e.g., ganaplacide). There is, therefore, an urgent need to determine the susceptibility profile of all human malaria species to the current generation of antimalarial compounds. We obtained ex vivo malaria samples from travellers returning to the United Kingdom and, using the [³H]hypoxanthine incorporation method, compared susceptibility to select established and experimental antimalarial agents among all major human infective Plasmodium species. We demonstrate that P. malariae and P. ovale spp. are significantly less susceptible than P. falciparum to cipargamin, DSM265 and AN13762, but are more susceptible to ganaplacide. Preliminary ex vivo data from single isolates of P. knowlesi and P. vivax demonstrate a similar profile. Our findings highlight the need to ensure cross species susceptibility profiles are determined early in the drug development pipeline. Our data can also be used to inform further drug development, and illustrate the utility of the P. knowlesi in vitro model as a scalable approach for predicting the drug susceptibility of non-falciparum malaria species in general.

SUBMITTER: van Schalkwyk DA

PROVIDER: S-EPMC8327131 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Plasmodium knowlesi is one of five Plasmodium species known to cause malaria in humans and can result in severe illness and death. While a zoonosis in humans, this simian malaria parasite species infects macaque monkeys and serves as an experimental model for in vivo, ex vivo and in vitro studies. It has underpinned malaria discoveries relating to host-pathogen interactions, the immune response and immune evasion strategies. This study investigated differences in P. knowlesi gene expression in samples from ex vivo and in vitro cultures.Gene expression profiles were generated using microarrays to compare the stage-specific transcripts detected for a clone of P. knowlesi propagated in the blood of a rhesus macaque host and then grown in an ex-vivo culture, and the same clone adapted to long-term in vitro culture. Parasite samples covering one blood-stage cycle were analysed at four-hour intervals. cDNA was generated and hybridized to an oligoarray representing the P. knowlesi genome. Two replicate experiments were developed from in vitro cultures. Expression values were filtered, normalized, and analysed using R and Perl language and applied to a sine wave model to determine changes in equilibrium and amplitude. Differentially expressed genes from ex vivo and in vitro time points were detected using limma R/Bioconductor and gene set enrichment analysis (GSEA).Major differences were noted between the ex vivo and in vitro time courses in overall gene expression and the length of the cycle (25.5 hours ex vivo; 33.5 hours in vitro). GSEA of genes up-regulated ex vivo showed an enrichment of various genes including SICAvar, ribosomal- associated and histone acetylation pathway genes. In contrast, certain genes involved in metabolism and cell growth, such as porphobilinogen deaminase and tyrosine phosphatase, and one SICAvar gene, were significantly up-regulated in vitro.This study demonstrates how gene expression in P. knowlesi blood-stage parasites can differ dramatically depending on whether the parasites are grown in vivo, with only one cycle of development ex vivo, or as an adapted isolate in long-term in vitro culture. These data bring emphasis to the importance of studying the parasite, its biology and disease manifestations in the context of the host.

Project description:Malaria, especially Plasmodium falciparum infection, remains an enormous problem, and its treatment and control are seriously challenged by drug resistance. New antimalarial drugs are needed. To characterize the Medicines for Malaria Venture pipeline of antimalarials under development, we assessed the ex vivo drug susceptibilities to 19 compounds targeting or potentially impacted by mutations in P. falciparum ABC transporter I family member 1, acetyl-CoA synthetase, cytochrome b, dihydroorotate dehydrogenase, elongation factor 2, lysyl-tRNA synthetase, phenylalanyl-tRNA synthetase, plasmepsin X, prodrug activation and resistance esterase, and V-type H+ ATPase of 998 fresh P. falciparum clinical isolates collected in eastern Uganda from 2015 to 2022. Drug susceptibilities were assessed by 72-h growth inhibition (half-maximum inhibitory concentration [IC50]) assays using SYBR green. Field isolates were highly susceptible to lead antimalarials, with low- to midnanomolar median IC50s, near values previously reported for laboratory strains, for all tested compounds. However, outliers with decreased susceptibilities were identified. Positive correlations between IC50 results were seen for compounds with shared targets. We sequenced genes encoding presumed targets to characterize sequence diversity, search for polymorphisms previously selected with in vitro drug pressure, and determine genotype-phenotype associations. We identified many polymorphisms in target genes, generally in <10% of isolates, but none were those previously selected in vitro with drug pressure, and none were associated with significantly decreased ex vivo drug susceptibility. Overall, Ugandan P. falciparum isolates were highly susceptible to 19 compounds under development as next-generation antimalarials, consistent with a lack of preexisting or novel resistance-conferring mutations in circulating Ugandan parasites. IMPORTANCE Drug resistance necessitates the development of new antimalarial drugs. It is important to assess the activities of compounds under development against parasites now causing disease in Africa, where most malaria cases occur, and to determine if mutations in these parasites may limit the efficacies of new agents. We found that African isolates were generally highly susceptible to the 19 studied lead antimalarials. Sequencing of the presumed drug targets identified multiple mutations in these genes, but these mutations were generally not associated with decreased antimalarial activity. These results offer confidence that the activities of the tested antimalarial compounds now under development will not be limited by preexisting resistance-mediating mutations in African malaria parasites.

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Ex vivo susceptibility to new antimalarial agents differs among human-infecting Plasmodium species.

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