Molecular profiling reveals features of clinical immunity and immunosuppression in asymptomatic P. falciparum malaria.

Clinical immunity to P. falciparum malaria is non-sterilizing, with adults often experiencing asymptomatic infection. Historically, asymptomatic malaria has been viewed as beneficial and required to help maintain clinical immunity. Emerging views suggest that these infections are detrimental and constitute a parasite reservoir that perpetuates transmission. To define the impact of asymptomatic malaria, we pursued a systems approach integrating antibody responses, mass cytometry, and transcriptional profiling of individuals experiencing symptomatic and asymptomatic P. falciparum infection. Defined populations of classical and atypical memory B cells and a TH2 cell bias were associated with reduced risk of clinical malaria. Despite these protective responses, asymptomatic malaria featured an immunosuppressive transcriptional signature with upregulation of pathways involved in the inhibition of T-cell function, and CTLA-4 as a predicted regulator in these processes. As proof of concept, we demonstrated a role for CTLA-4 in the development of asymptomatic parasitemia in infection models. The results suggest that asymptomatic malaria is not innocuous and might not support the induction of immune processes to fully control parasitemia or efficiently respond to malaria vaccines.

Evaluation of splenic accumulation and colocalization of immature reticulocytes and Plasmodium vivax in asymptomatic malaria: A prospective human splenectomy study.

BACKGROUND: A very large biomass of intact asexual-stage malaria parasites accumulates in the spleen of asymptomatic human individuals infected with Plasmodium vivax. The mechanisms underlying this intense tropism are not clear. We hypothesised that immature reticulocytes, in which P. vivax develops, may display high densities in the spleen, thereby providing a niche for parasite survival. METHODS AND FINDINGS: We examined spleen tissue in 22 mostly untreated individuals naturally exposed to P. vivax and Plasmodium falciparum undergoing splenectomy for any clinical indication in malaria-endemic Papua, Indonesia (2015 to 2017). Infection, parasite and immature reticulocyte density, and splenic distribution were analysed by optical microscopy, flow cytometry, and molecular assays. Nine non-endemic control spleens from individuals undergoing spleno-pancreatectomy in France (2017 to 2020) were also examined for reticulocyte densities. There were no exclusion criteria or sample size considerations in both patient cohorts for this demanding approach. In Indonesia, 95.5% (21/22) of splenectomy patients had asymptomatic splenic Plasmodium infection (7 P. vivax, 13 P. falciparum, and 1 mixed infection). Significant splenic accumulation of immature CD71 intermediate- and high-expressing reticulocytes was seen, with concentrations 11 times greater than in peripheral blood. Accordingly, in France, reticulocyte concentrations in the splenic effluent were higher than in peripheral blood. Greater rigidity of reticulocytes in splenic than in peripheral blood, and their higher densities in splenic cords both suggest a mechanical retention process. Asexual-stage P. vivax-infected erythrocytes of all developmental stages accumulated in the spleen, with non-phagocytosed parasite densities 3,590 times (IQR: 2,600 to 4,130) higher than in circulating blood, and median total splenic parasite loads 81 (IQR: 14 to 205) times greater, accounting for 98.7% (IQR: 95.1% to 98.9%) of the estimated total-body P. vivax biomass. More reticulocytes were in contact with sinus lumen endothelial cells in P. vivax- than in P. falciparum-infected spleens. Histological analyses revealed 96% of P. vivax rings/trophozoites and 46% of schizonts colocalised with 92% of immature reticulocytes in the cords and sinus lumens of the red pulp. Larger splenic cohort studies and similar investigations in untreated symptomatic malaria are warranted. CONCLUSIONS: Immature CD71+ reticulocytes and splenic P. vivax-infected erythrocytes of all asexual stages accumulate in the same splenic compartments, suggesting the existence of a cryptic endosplenic lifecycle in chronic P. vivax infection. Findings provide insight into P. vivax-specific adaptions that have evolved to maximise survival and replication in the spleen.

Plasmodium falciparum and Plasmodium vivax Demonstrate Contrasting Chloroquine Resistance Reversal Phenotypes.

High-grade chloroquine (CQ) resistance has emerged in both Plasmodium falciparum and P. vivax The aim of the present study was to investigate the phenotypic differences of CQ resistance in both of these species and the ability of known CQ resistance reversal agents (CQRRAs) to alter CQ susceptibility. Between April 2015 and April 2016, the potential of verapamil (VP), mibefradil (MF), L703,606 (L7), and primaquine (PQ) to reverse CQ resistance was assessed in 46 P. falciparum and 34 P. vivax clinical isolates in Papua, Indonesia, where CQ resistance is present in both species, using a modified schizont maturation assay. In P. falciparum, CQ 50% inhibitory concentrations (IC50s) were reduced when CQ was combined with VP (1.4-fold), MF (1.2-fold), L7 (4.2-fold), or PQ (1.8-fold). The degree of CQ resistance reversal in P. falciparum was highly correlated with CQ susceptibility for all CQRRAs (R2 = 0.951, 0.852, 0.962, and 0.901 for VP, MF, L7, and PQ, respectively), in line with observations in P. falciparum laboratory strains. In contrast, no reduction in the CQ IC50s was observed with any of the CQRRAs in P. vivax, even in those isolates with high chloroquine IC50s. The differential effect of CQRRAs in P. falciparum and P. vivax suggests significant differences in CQ kinetics and, potentially, the likely mechanism of CQ resistance between these two species.

Quantification of Plasmodium ex vivo drug susceptibility by flow cytometry.

BACKGROUND: The emergence and spread of multidrug-resistant Plasmodium falciparum and Plasmodium vivax highlights the need for objective measures of ex vivo drug susceptibility. Flow cytometry (FC) has potential to provide a robust and rapid quantification of ex vivo parasite growth. METHODS: Field isolates from Papua, Indonesia, underwent ex vivo drug susceptibility testing against chloroquine, amodiaquine, piperaquine, mefloquine, and artesunate. A single nucleic acid stain (i.e., hydroethidine (HE) for P. falciparum and SYBR Green I (SG) for P. vivax) was used to quantify infected red blood cells by FC-based signal detection. Data derived by FC were compared to standard quantification by light microscopy (LM). A subset of isolates was used to compare single and double staining techniques. RESULTS: In total, 57 P. falciparum and 23 P. vivax field isolates were collected for ex vivo drug susceptibility testing. Reliable paired data between LM and FC was obtained for 88 % (295/334) of these assays. The median difference of derived IC50 values varied from -5.4 to 6.1 nM, associated with 0.83-1.23 fold change in IC50 values between LM and FC. In 15 assays (5.1 %), the derived difference of IC50 estimates was beyond the 95 % limits of agreement; in eleven assays (3.7 %), this was attributable to low parasite growth (final schizont count < 40 %), and in four assays (1.4 %) due to low initial parasitaemia at the start of assay (<2000 µl(-1)). In a subset of seven samples, LM, single and double staining FC techniques generated similar IC50 values. CONCLUSIONS: A single staining FC-based assay using a portable cytometer provides a simple, fast and versatile platform for field surveillance of ex vivo drug susceptibility in clinical P. falciparum and P. vivax isolates.

Longitudinal ex vivo and molecular trends of chloroquine and piperaquine activity against Plasmodium falciparum and P. vivax before and after introduction of artemisinin-based combination therapy in Papua, Indonesia.

Drug resistant Plasmodium parasites are a major threat to malaria control and elimination. After reports of high levels of multidrug resistant P. falciparum and P. vivax in Indonesia, in 2005, the national first-line treatment policy for uncomplicated malaria was changed in March 2006, to dihydroartemisinin-piperaquine against all species. This study assessed the temporal trends in ex vivo drug susceptibility to chloroquine (CQ) and piperaquine (PIP) for both P. falciparum and P. vivax clinical isolates collected between 2004 and 2018, by using schizont maturation assays, and genotyped a subset of isolates for known and putative molecular markers of CQ and PIP resistance by using Sanger and next generation whole genome sequencing. The median CQ IC50 values varied significantly between years in both Plasmodium species, but there was no significant trend over time. In contrast, there was a significant trend for increasing PIP IC50s in both Plasmodium species from 2010 onwards. Whereas the South American CQ resistant 7G8 pfcrt SVMNT isoform has been fixed since 2005 in the study area, the pfmdr1 86Y allele frequencies decreased and became fixed at the wild-type allele in 2015. In P. vivax isolates, putative markers of CQ resistance (no pvcrt-o AAG (K10) insertion and pvmdr1 Y967F and F1076L) were fixed at the mutant alleles since 2005. None of the putative PIP resistance markers were detected in P. falciparum. The ex vivo drug susceptibility and molecular analysis of CQ and PIP efficacy for P. falciparum and P. vivax after 12 years of intense drug pressure with DHP suggests that whilst the degree of CQ resistance appears to have been sustained, there has been a slight decline in PIP susceptibility, although this does not appear to have reached clinically significant levels. The observed decreasing trend in ex vivo PIP susceptibility highlights the importance of ongoing surveillance.

High-dimensional mass cytometry identifies T cell and B cell signatures predicting reduced risk of Plasmodium vivax malaria.

IFN-γ-driven responses to malaria have been shown to modulate the development and function of T follicular helper (TFH) cells and memory B cells (MBCs), with conflicting evidence of their involvement in the induction of antibody responses required to achieve clinical immunity and their association with disease outcomes. Using high-dimensional single-cell mass cytometry, we identified distinct populations of TH1-polarized CD4+ T cells and MBCs expressing the TH1-defining transcription factor T-bet, associated with either increased or reduced risk of Plasmodium vivax (P. vivax) malaria, demonstrating that inflammatory responses to malaria are not universally detrimental for infection. Furthermore, we found that, whereas class-switched but not IgM+ MBCs were associated with a reduced risk of symptomatic malaria, populations of TH1 cells with a stem central memory phenotype, TH17 cells, and T regulatory cells were associated with protection from asymptomatic infection, suggesting that activation of cell-mediated immunity might also be required to control persistent P. vivax infection with low parasite burden.