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Monoclonal antibodies with broad reactivity against antigens on the parasite that causes malaria, Plasmodium falciparum , are isolated from two subjects and are found to have an unusual insertion of an immunoglobulin-like domain from a different chromosome, illustrating a new mechanism of antibody diversification. Broadly reactive anti-malarial antibodies This paper reports the isolation of monoclonal antibodies with broad reactivity against Plasmodium falciparum antigens from two subjects living in a malaria-endemic region in Kilifi, Kenya. The antibodies are unusual in that they carry large insertions of an immunoglobulin-like domain from LAIR1, an Ig superfamily inhibitory receptor encoded on chromosome 19. The antibodies bind to polymorphic surface antigens on the parasite surface; binding depends on the mutated form of the insert. These findings illustrate a novel mechanism of antibody diversification, and the existence of conserved epitopes that may be suitable candidates for the development of a malaria vaccine. Plasmodium falciparum antigens expressed on the surface of infected erythrocytes are important targets of naturally acquired immunity against malaria, but their high number and variability provide the pathogen with a powerful means of escape from host antibodies 1 , 2 , 3 , 4 . Although broadly reactive antibodies against these antigens could be useful as therapeutics and in vaccine design, their identification has proven elusive. Here we report the isolation of human monoclonal antibodies that recognize erythrocytes infected by different P. falciparum isolates and opsonize these cells by binding to members of the RIFIN family. These antibodies acquired broad reactivity through a novel mechanism of insertion of a large DNA fragment between the V and DJ segments. The insert, which is both necessary and sufficient for binding to RIFINs, encodes the entire 98 amino acid collagen-binding domain of LAIR1, an immunoglobulin superfamily inhibitory receptor encoded on chromosome 19. In each of the two donors studied, the antibodies are produced by a single expanded B-cell clone and carry distinct somatic mutations in the LAIR1 domain that abolish binding to collagen and increase binding to infected erythrocytes. These findings illustrate, with a biologically relevant example, a novel mechanism of antibody diversification by interchromosomal DNA transposition and demonstrate the existence of conserved epitopes that may be suitable candidates for the development of a malaria vaccine.

Diverse compounds target the Plasmodium falciparum Na + pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4 G358S parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na + regulation. The G358S mutation reduces the affinity of PfATP4 for Na + and is associated with an increase in the parasite’s resting cytosolic [Na + ]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4 G358S parasites, and that their combination with unrelated antimalarials may mitigate against resistance development. In a recent clinical trial for oral administration of cipargamin in individuals with malaria, there was an emergence of recrudescent parasites with a G358S mutation in PfATP4. In this work, the authors investigate the effect of this mutation on the function of the ATPase, on parasite growth and susceptibility to antimalarial drugs.

We report an analysis of the propensity of the antimalarial agent cabamiquine, a Plasmodium -specific eukaryotic elongation factor 2 inhibitor, to select for resistant Plasmodium falciparum parasites. Through in vitro studies of laboratory strains and clinical isolates, a humanized mouse model, and volunteer infection studies, we identified resistance-associated mutations at 11 amino acid positions. Of these, six (55%) were present in more than one infection model, indicating translatability across models. Mathematical modelling suggested that resistant mutants were likely pre-existent at the time of drug exposure across studies. Here, we estimated a wide range of frequencies of resistant mutants across the different infection models, much of which can be attributed to stochastic differences resulting from experimental design choices. Structural modelling implicates binding of cabamiquine to a shallow mRNA binding site adjacent to two of the most frequently identified resistance mutations. Authors utilize a number of models (mathematical, in vitro and in vivo infection) to analyse pre-clinical and Phase I clinical trial data, in regard to potential risk of resistance associated with a Plasmodium falciparum inhibitor, cabamiquine.

Novel antimalarials are needed to address emerging resistance to artemisinin and partner drugs. We did two trials to evaluate safety, tolerability, pharmacokinetics, and activity against blood stage Plasmodium falciparum for the drug candidate MMV533. A phase 1a first-in-human (FIH) trial was conducted at Nucleus Network (Melbourne, VIC, Australia). Part 1 was a double-blind, randomised, placebo-controlled, sequential ascending dose study and part 2 was an open-label, randomised, two-period crossover, pilot food effect study. A phase 1b, open-label, volunteer infection study (VIS) was conducted at Nucleus Network (Herston, QLD, Australia). Eligible participants were adults aged 18–55 years, with a bodyweight of at least 50 kg and BMI of 18–32 kg/m2 and participants in the VIS were malaria-naive. In part 1 of the FIH study, six cohorts of up to eight participants were randomly assigned (3:1) to a single oral MMV533 dose (5, 10, 20, 50, 100, and 160 mg) or placebo using an automated system, with study staff and participants masked to treatment allocation, and follow-up until day 28. In part 2, MMV533 30 mg was administered open-label to one cohort of nine participants assigned by simple randomisation (1:1) to the fasted–fed (n=4) or fed–fasted (n=5) groups. After a 21-day washout period, fed and fasted groups crossed over with follow-up until day 42. In the VIS, seven participants were assigned using simple randomisation (1:1:1) to three dosing groups of 20 mg (n=3), 35 mg (n=2), or 100 mg (n=2) after parasitaemia was detected, with follow-up until day 28. The primary outcomes were treatment emergent adverse events and relationship to MMV533 for the FIH study assessed in the safety population, and in the VIS primary outcomes were parasite reduction ratio over 48 h (log10PRR48), parasite clearance half-life (PCT1/2), and lag phase assessed in the pharmacodynamic population. MMV533 pharmacokinetics was a secondary outcome for both studies, evaluated in the pharmacokinetic population. The studies are registered with ClinicalTrials.gov, NCT04323306 and NCT05205941 (completed). The FIH study was conducted between July 31, 2020, and Sept 27, 2022, and the VIS between March 31 and Aug 9, 2022. 335 adults were assessed for eligibility, 71 enrolled, and 69 randomly assigned (53 in part 1 and nine in part 2 of the FIH study, and seven in the VIS). 32 (45%) of 71 participants were female and 39 (55%) were male. In part 1, 24 (63%) of 38 participants had an adverse event after MMV533 administration with no apparent relationship to dose versus six (50%) of 12 after placebo. Treatment-related adverse events were reported for four (11%) participants receiving MMV533 and one (8%) receiving placebo, with no relationship to dose. In part 2, adverse events were reported for three (38%) of eight participants when fasted and four (44%) of nine when fed, with no apparent influence of food. Time to maximum plasma concentration was 4·0–6·0 h, and apparent half-life was 103·8–127·2 h. After a high-fat meal, the geometric mean ratio (fed:fasted) of MMV533 AUC0-last was 112·0 (90% CI 89·6–140·0). In the VIS for MMV533 100 mg, log10PRR48 was 2·27 (1·99–2·56), PCT1/2 was 6·36 h (5·64–7·28), and lag phase was 2 h. An acceptable safety and tolerability profile, confirmed parasiticidal activity, and a long half-life support progression of MMV533 into clinical trials in patients with malaria as a component of new antimalarial combination therapies. MMV Medicines for Malaria Venture and Bill & Melinda Gates Foundation.

Background Important regulation occurs at the level of transcription in Plasmodium falciparum and growing evidence suggests that these apicomplexan parasites have complex regulatory networks. Recent studies implicate long noncoding RNAs (lncRNAs) as transcriptional regulators in P. falciparum . However, due to limited research and the lack of necessary experimental tools, our understanding of their role in the malaria-causing parasite remains largely unelucidated. In this work, we address one of these limitations, the lack of an updated and improved lncRNA annotation in P. falciparum . Results We generated long-read RNA sequencing data and integrated information extracted and curated from multiple sources to manually annotate lncRNAs. We identified 1119 novel lncRNAs and validated and refined 1250 existing annotations. Utilising the collated datasets, we generated evidence-based ranking scores for each annotation and characterised the distinct genomic contexts and features of P. falciparum lncRNAs. Certain features indicated subsets with potential biological significance such as 25 lncRNAs containing multiple introns, 335 lncRNAs lacking mutations in piggyBac mutagenic studies and lncRNAs associated with specific biologic processes including two new types of lncRNAs found proximal to var genes. Conclusions The insights and the annotation presented in this study will serve as valuable tools for researchers seeking to understand the role of lncRNAs in parasite biology through both bioinformatics and experimental approaches.

The malaria parasite Plasmodium falciparum survives fever during human infection by using a transcription factor to regulate its heat-shock response.

Global efforts to eliminate malaria depend on the continued success of artemisinin-based combination therapies (ACTs) that target Plasmodium asexual blood-stage parasites. Resistance to ACTs, however, has emerged, creating the need to define the underlying mechanisms. Mutations in the P. falciparum multidrug resistance protein 1 (PfMDR1) transporter constitute an important determinant of resistance. Applying gene editing tools combined with an analysis of a public database containing thousands of parasite genomes, we show geographic selection and expansion of a pfmdr1 gene amplification encoding the N86/184F haplotype in Southeast Asia. Parasites expressing this PfMDR1 variant possess a higher transport capacity that modulates their responses to antimalarials. These data could help tailor and optimize antimalarial drug usage in different regions where malaria is endemic by taking into account the regional prevalence of pfmdr1 polymorphisms. Artemisinin-based combination therapies (ACTs) have been vital in reducing malaria mortality rates since the 2000s. Their efficacy, however, is threatened by the emergence and spread of artemisinin resistance in Southeast Asia. The Plasmodium falciparum multidrug resistance protein 1 (PfMDR1) transporter plays a central role in parasite resistance to ACT partner drugs through gene copy number variations (CNV) and/or single nucleotide polymorphisms (SNPs). Using genomic epidemiology, we show that multiple pfmdr1 copies encoding the N86 and 184F haplotype are prevalent across Southeast Asia. Applying genome editing tools on the Southeast Asian Dd2 strain and using a surrogate assay to measure transporter activity in infected red blood cells, we demonstrate that parasites harboring multicopy N86/184F PfMDR1 have a higher Fluo-4 transport capacity compared with those expressing the wild-type N86/Y184 haplotype. Multicopy N86/184F PfMDR1 is also associated with decreased parasite susceptibility to lumefantrine. These findings provide evidence of the geographic selection and expansion of specific multicopy PfMDR1 haplotypes associated with multidrug resistance in Southeast Asia. IMPORTANCE Global efforts to eliminate malaria depend on the continued success of artemisinin-based combination therapies (ACTs) that target Plasmodium asexual blood-stage parasites. Resistance to ACTs, however, has emerged, creating the need to define the underlying mechanisms. Mutations in the P. falciparum multidrug resistance protein 1 (PfMDR1) transporter constitute an important determinant of resistance. Applying gene editing tools combined with an analysis of a public database containing thousands of parasite genomes, we show geographic selection and expansion of a pfmdr1 gene amplification encoding the N86/184F haplotype in Southeast Asia. Parasites expressing this PfMDR1 variant possess a higher transport capacity that modulates their responses to antimalarials. These data could help tailor and optimize antimalarial drug usage in different regions where malaria is endemic by taking into account the regional prevalence of pfmdr1 polymorphisms.

Immunoepidemiological studies typically reveal slow, age-dependent acquisition of immune responses against sporozoites. Naturally acquired immunity against preerythrocytic stages is considered inadequate to confer protection against clinical malaria. To explore previously unrecognized antisporozoite responses, we measured serum levels of naturally acquired antibodies to whole sporozoites ( spz) and the immunodominant (NANP) repeats of the major sporozoite surface protein, circumsporozoite protein, in a well-characterized Kenyan cohort. Sera were sampled at the start of the malaria transmission season, and all subjects were prospectively monitored for uncomplicated clinical malaria in the ensuing 6 months. We used Kaplan-Meier analysis and multivariable regression to investigate the association of antisporozoite immunity with incidence of clinical malaria. Although naturally acquired humoral responses against spz and (NANP) were strongly correlated (  < 0.0001), 37% of spz responders did not recognize (NANP) . The prevalence and magnitude of antisporozoite responses increased with age, although some high spz responders were identified among children. Survival analysis revealed a reduced risk of and increased time to first or only episode of clinical malaria among spz or (NANP) responders carrying microscopically detectable ( ) parasitemia at the start of the transmission season (  < 0.03). Our Cox regression interaction models indicated a potentially protective interaction between high anti- spz (  = 0.002) or anti-(NANP) (  = 0.001) antibody levels and microscopically detectable parasitemia on the risk of subsequent clinical malaria. Our findings indicate that robust antisporozoite immune responses can be naturally acquired already at an early age. A potentially protective role of high levels of anti- spz antibodies against clinical episodes of uncomplicated malaria was detected, suggesting that antibody-mediated preerythrocytic immunity might indeed contribute to protection in nature.