Similarly Efficacious Anti-Malarial Drugs SJ733 and Pyronaridine Differ in Their Ability to Remove Circulating Parasites in Mice

BACKGROUND: Artemisinin-based combination therapy (ACT) has been a mainstay for malaria prevention and treatment. However, emergence of drug resistance has incentivised development of new drugs. Defining the kinetics with which circulating parasitized red blood cells (pRBC) are lost after drug treatment, referred to as the parasite clearance curve , has been critical for assessing drug efficacy; yet underlying mechanisms remain partly unresolved. The clearance curve may be shaped both by the rate at which drugs kill parasites, and the rate at which drug-affected parasites are removed from circulation. METHODS: In this context, two anti-malarials, SJ733, and an ACT partner drug, pyronaridine were compared against sodium artesunate in mice infected with Plasmodium berghei (strain ANKA). To measure each compound\u27s capacity for pRBC removal in vivo, flow cytometric monitoring of a single cohort of fluorescently-labelled pRBC was employed, and combined with ex vivo parasite culture to assess parasite maturation and replication. RESULTS: These three compounds were found to be similarly efficacious in controlling established infection by reducing overall parasitaemia. While sodium artesunate acted relatively consistently across the life-stages, single-dose SJ733 elicited a biphasic effect, triggering rapid, partly phagocyte-dependent removal of trophozoites and schizonts, followed by arrest of residual ring-stages. In contrast, pyronaridine abrogated maturation of younger parasites, with less pronounced effects on mature parasites, while modestly increasing pRBC removal. CONCLUSIONS: Anti-malarials SJ733 and pyronaridine, though similarly efficacious in reducing overall parasitaemia in mice, differed markedly in their capacity to arrest replication and remove pRBC from circulation. Thus, similar parasite clearance curves can result for anti-malarials with distinct capacities to inhibit, kill and clear parasites

Transcriptional profiling and immunophenotyping show sustained activation of blood monocyte in subpatent Plasmodium falciparum infection

Objectives Malaria, caused by Plasmodium infection, remains a major global health problem. Monocytes are integral to the immune response, yet their transcriptional and functional responses in primary Plasmodium falciparum infection and in clinical malaria are poorly understood. Methods The transcriptional and functional profiles of monocytes were examined in controlled human malaria infection with P. falciparum blood stages and in children and adults with acute malaria. Monocyte gene expression and functional phenotypes were examined by RNA sequencing and flow cytometry at peak infection and compared to pre‐infection or at convalescence in acute malaria. Results In subpatent primary infection, the monocyte transcriptional profile was dominated by an interferon (IFN) molecular signature. Pathways enriched included type I IFN signalling, innate immune response and cytokine‐mediated signalling. Monocytes increased TNF and IL‐12 production upon in vitro toll‐like receptor stimulation and increased IL‐10 production upon in vitro parasite restimulation. Longitudinal phenotypic analyses revealed sustained significant changes in the composition of monocytes following infection, with increased CD14+CD16− and decreased CD14−CD16+ subsets. In acute malaria, monocyte CD64/FcγRI expression was significantly increased in children and adults, while HLA‐DR remained stable. Although children and adults showed a similar pattern of differentially expressed genes, the number and magnitude of gene expression change were greater in children. Conclusions Monocyte activation during subpatent malaria is driven by an IFN molecular signature with robust activation of genes enriched in pathogen detection, phagocytosis, antimicrobial activity and antigen presentation. The greater magnitude of transcriptional changes in children with acute malaria suggests monocyte phenotypes may change with age or exposure

Loss of complement regulatory proteins on red blood cells in mild malarial anaemia and in Plasmodium falciparum induced blood-stage infection

Background: Anaemia is a major consequence of malaria, caused by the removal of both infected and uninfected red blood cells (RBCs) from the circulation. Complement activation and reduced expression of complement regulatory proteins (CRPs) on RBCs are an important pathogenic mechanism in severe malarial anaemia in both Plasmodium falciparum and Plasmodium vivax infection. However, little is known about loss of CRPs on RBCs during mild malarial anaemia and in low-density infection. Methods: The expression of CRP CR1, CD55, CD59, and the phagocytic regulator CD47, on uninfected normocytes and reticulocytes were assessed in individuals from two study populations: (1) P. falciparum and P. vivax-infected patients from a low transmission setting in Sabah, Malaysia; and, (2) malaria-naïve volunteers undergoing P. falciparum induced blood-stage malaria (IBSM). For clinical infections, individuals were categorized into anaemia severity categories based on haemoglobin levels. For IBSM, associations between CRPs and haemoglobin level were investigated. Results: CRP expression on RBC was lower in Malaysian individuals with P. falciparum and P. vivax mild malarial anaemia compared to healthy controls. CRP expression was also reduced on RBCs from volunteers during IBSM. Reduction occurred on normocytes and reticulocytes. However, there was no significant association between reduced CRPs and haemoglobin during IBSM. Conclusions: Removal of CRPs occurs on both RBCs and reticulocytes during Plasmodium infection even in mild malarial anaemia and at low levels of parasitaemia

Plasmodium-specific antibodies block in vivo parasite growth without clearing infected red blood cells.

Plasmodium parasites invade and multiply inside red blood cells (RBC). Through a cycle of maturation, asexual replication, rupture and release of multiple infective merozoites, parasitised RBC (pRBC) can reach very high numbers in vivo, a process that correlates with disease severity in humans and experimental animals. Thus, controlling pRBC numbers can prevent or ameliorate malaria. In endemic regions, circulating parasite-specific antibodies associate with immunity to high parasitemia. Although in vitro assays reveal that protective antibodies could control pRBC via multiple mechanisms, in vivo assessment of antibody function remains challenging. Here, we employed two mouse models of antibody-mediated immunity to malaria, P. yoelii 17XNL and P. chabaudi chabaudi AS infection, to study infection-induced, parasite-specific antibody function in vivo. By tracking a single generation of pRBC, we tested the hypothesis that parasite-specific antibodies accelerate pRBC clearance. Though strongly protective against homologous re-challenge, parasite-specific IgG did not alter the rate of pRBC clearance, even in the presence of ongoing, systemic inflammation. Instead, antibodies prevented parasites progressing from one generation of RBC to the next. In vivo depletion studies using clodronate liposomes or cobra venom factor, suggested that optimal antibody function required splenic macrophages and dendritic cells, but not complement C3/C5-mediated killing. Finally, parasite-specific IgG bound poorly to the surface of pRBC, yet strongly to structures likely exposed by the rupture of mature schizonts. Thus, in our models of humoral immunity to malaria, infection-induced antibodies did not accelerate pRBC clearance, and instead co-operated with splenic phagocytes to block subsequent generations of pRBC

Age-dependent changes in circulating Tfh cells influence development of functional malaria antibodies in children.

T-follicular helper (Tfh) cells are key drivers of antibodies that protect from malaria. However, little is known regarding the host and parasite factors that influence Tfh and functional antibody development. Here, we use samples from a large cross-sectional study of children residing in an area of high malaria transmission in Uganda to characterize Tfh cells and functional antibodies to multiple parasites stages. We identify a dramatic re-distribution of the Tfh cell compartment with age that is independent of malaria exposure, with Th2-Tfh cells predominating in early childhood, while Th1-Tfh cell gradually increase to adult levels over the first decade of life. Functional antibody acquisition is age-dependent and hierarchical acquired based on parasite stage, with merozoite responses followed by sporozoite and gametocyte antibodies. Antibodies are boosted in children with current infection, and are higher in females. The children with the very highest antibody levels have increased Tfh cell activation and proliferation, consistent with a key role of Tfh cells in antibody development. Together, these data reveal a complex relationship between the circulating Tfh compartment, antibody development and protection from malaria

Age-dependent changes in circulating Tfh cells influence development of functional malaria antibodies in children

Despite being key drivers of protective antibodies against malaria, little is known regarding the host and parasite factors that influence CD4 T-follicular helper cell and antibody development. Authors utilise samples from a study of children living in an area of high malaria transmission in Uganda, to characterize Tfh cells and functional antibodies to multiple parasites stages