Plasmodium vivax parasites preferentially invade reticulocyte cells in a multistep process that is still poorly understood. In this study, we used ex vivo invasion assays and population genetic analyses to investigate the involvement of complement receptor 1 (CR1) in P . vivax invasion. First, we observed that P . vivax invasion of reticulocytes was consistently reduced when CR1 surface expression was reduced through enzymatic cleavage, in the presence of naturally low-CR1-expressing cells compared with high-CR1-expressing cells, and with the addition of soluble CR1, a known inhibitor of P . falciparum invasion. Immuno-precipitation experiments with P . vivax Reticulocyte Binding Proteins showed no evidence of complex formation. In addition, analysis of CR1 genetic data for worldwide human populations with different exposure to malaria parasites show significantly higher frequency of CR1 alleles associated with low receptor expression on the surface of RBCs and higher linkage disequilibrium in human populations exposed to P . vivax malaria compared with unexposed populations. These results are consistent with a positive selection of low-CR1-expressing alleles in vivax-endemic areas. Collectively, our findings demonstrate that CR1 availability on the surface of RBCs modulates P . vivax invasion. The identification of new molecular interactions is crucial to guiding the rational development of new therapeutic interventions against vivax malaria.

The Plasmodium vivax reticulocyte invasion process is still poorly understood, with only a few receptor-ligand interactions identified to date. Individuals with the Southeast Asian ovalocytosis (SAO) phenotype have a deletion in the band 3 protein on the surface of erythrocytes, and are reported to have a lower incidence of clinical P. vivax malaria. Based on this observation, band 3 has been put forward as a receptor for P. vivax invasion, although direct proof is still lacking. In this study, we combined functional ex vivo invasion assays and transcriptome sequencing to uncover a band 3–mediated invasion pathway in P. vivax and potential band 3 ligands. Invasion by P. vivax field isolates was 67%-71% lower in SAO reticulocytes compared with non-SAO reticulocytes. Reticulocyte invasion was decreased by 40% and 27%-31% when blocking with an anti-band 3 polyclonal antibody and a PvTRAg38 peptide, respectively. To identify new band 3 receptor candidates, we mRNA-sequenced schizont-stage isolates used in the invasion assays, and observed high transcriptional variability in multigene and invasion-related families. Transcriptomes of isolates with low or high dependency on band 3 for invasion were compared by differential expression analysis, which produced a list of band 3 ligand candidates with high representation of PvTRAg genes. Our ex vivo invasion assays have demonstrated that band 3 is a P. vivax invasion receptor and confirm previous in vitro studies showing binding between PvTRAg38 and band 3, although the lower and variable inhibition levels observed suggest the involvement of other ligands. By coupling transcriptomes and invasion phenotypes from the same isolates, we identified a list of band 3 ligand candidates, of which the overrepresented PvTRAg genes are the most promising for future research.

BackgroundWhole-genome sequencing (WGS) is becoming an increasingly popular tool to study the population genetics and drug resistance ofPlasmodiumspp. However, the predominance of human DNA in a malaria patient blood sample requires time-consuming lab procedures to filter out human DNA or enrichPlasmodiumDNA. Here, we investigated the potential of adaptive sampling to enrich forPlasmodiumDNA while sequencing unenriched patient blood samples on a minION device.ResultsTo compare adaptive sampling versus regular sequencing, a dilution series consisting of 0% up to 100%P. falciparumDNA in human DNA was sequenced. Half of the flowcell channels were run in adaptive sampling mode, enriching for theP. falciparumreference genome, resulting in a 3.2 fold enrichment ofP. falciparumbases on average. Samples with a lower concentration of parasite DNA had a higher enrichment potential. We confirmed these findings by sequencing twoP. falciparumpatient blood samples with common levels of parasitaemia (0.1% and 0.2%). The estimated enrichment was 3.9 and 5.8, which was sufficient to cover at least 97% of theP. falciparumreference genome at a median depth of 20 (highest parasitaemia) or 5 (lowest parasitaemia). A comparison of 38 drug resistance variants (WHO) obtained via adaptive sequencing or Sanger sequencing showed a high concordance between the two methods, suggesting that the obtained sequencing data is of sufficient quality to address common clinical research questions for patients with parasitaemias of 0.1% and higher.ConclusionsOur results demonstrate that adaptive Nanopore sequencing has the potential to replace more time-consumingPlasmodium-enrichment protocols and sequence directly from patient blood, given further improvements in cost-efficiency.