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Abstract The selection and combination of dose regimens for antimalarials involve complex considerations including pharmacokinetic and pharmacodynamic interactions. In this study, we use immediate ex vivo P. falciparum field isolates to evaluate the effect of cabamiquine and pyronaridine as standalone treatments and in combination therapy. We feed the data into a pharmacometrics model to generate an interaction map and simulate meaningful clinical dose ratios. We demonstrate that the pharmacometrics model of parasite growth and killing provides a detailed description of parasite kinetics against cabamiquine-susceptible and resistant parasites. Pyronaridine monotherapy provides suboptimal killing rates at doses as high as 720 mg. In contrast, the combination of a single dose of 330 mg cabamiquine and 360 mg pyronaridine provides over 90% parasite killing in most of the simulated patients. The described methodology that combines a rapid, 3R-compliant in vitro method and modelling to set meaningful doses for new antimalarials could contribute to clinical drug development.

Abstract 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.

ABSTRACT Antibody responses to variant surface antigens (VSAs) produced by the malaria parasite Plasmodium falciparum may contribute to age-related natural immunity to severe malaria. One VSA family, P. falciparum erythrocyte membrane protein-1 (PfEMP1), includes a subset of proteins that binds endothelial protein C receptor (EPCR) in human hosts and potentially disrupts the regulation of inflammatory responses, which may lead to the development of severe malaria. We probed peptide microarrays containing segments spanning five PfEMP1 EPCR-binding domain variants with sera from 10 Malian adults and 10 children to determine the differences between adult and pediatric immune responses. We defined serorecognized peptides and amino acid residues as those that elicited a significantly higher antibody response than malaria-naïve controls. We aimed to identify regions consistently serorecognized among adults but not among children across PfEMP1 variants, potentially indicating regions that drive the development of immunity to severe malaria. Adult sera consistently demonstrated broader and more intense serologic responses to constitutive PfEMP1 peptides than pediatric sera, including peptides in EPCR-binding domains. Both adults and children serorecognized a significantly higher proportion of EPCR-binding peptides than peptides that do not directly participate in receptor binding, indicating a preferential development of serologic responses at functional residues. Over the course of a single malaria transmission season, pediatric serological responses increased between the start and the peak of the season, but waned as the transmission season ended. IMPORTANCE Severe malaria and death related to malaria disproportionately affect sub-Saharan children under 5 years of age, commonly manifesting as cerebral malaria and/or severe malarial anemia. In contrast, adults in malaria-endemic regions tend to experience asymptomatic or mild disease. Our findings indicate that natural immunity to malaria targets specific regions within the EPCR-binding domain, particularly peptides containing EPCR-binding residues. Epitopes containing these residues may be promising targets for vaccines or therapeutics directed against severe malaria. Our approach provides insight into the development of natural immunity to a binding target linked to severe malaria by characterizing an “adult-like” response as recognizing a proportion of epitopes within the PfEMP1 protein, particularly regions that mediate EPCR binding. This “adult-like” response likely requires multiple years of malaria exposure, as increases in pediatric serologic response over a single malaria transmission season do not appear significant.

Studying malaria transmission biology using scRNA-sequencing provides information on within-host strain diversity and transcriptional states. Here, we comment on our collaborative efforts at establishing single-cell capacities in sub-Saharan Africa and the challenges encountered in Mali’s endemic setting.

OBJECTIVES/GOALS: Recent in vitro evidence suggests that diverse parasite protein families called RIFINs and STEVORs are displayed on the surface of infected red blood cells and may have a role in severe malaria, but they remain sparsely studied in natural infections. We measured the RNA expression of these antigens in Malian children with severe or mild malaria illness. METHODS/STUDY POPULATION: We collected blood samples from Malian children aged six months to five years, including 14 with cerebral malaria, 10 with severe malarial anemia, and demographic-matched controls with mild, uncomplicated malaria. We extracted total RNA from each patient and used a custom capture array to selectively enrich Plasmodium falciparum parasite RNA. We then performed Illumina next-generation RNA sequencing and reconstructed parasite transcriptomes using reference-free de novo assembly. We identified RNA encoding RIFINs and STEVORs using an in-house classifier, then measured the diversity and abundance of gene expression for each infection. Expression diversity was defined as the number of unique variants transcribed. Expression abundance was calculated as transcripts per million (TPM). RESULTS/ANTICIPATED RESULTS: Cerebral malaria cases, but not severe malarial anemia cases, had higher diversity and abundance of RIFIN expression compared to mild infections. Type A RIFINs predominated over Type B RIFINs, and the same two RIFINs were predominantly expressed in all disease phenotypes. We anticipate that predominantly expressed RIFINs share high sequence homology with variants previously shown to bind blood antigens or immune inhibitory receptors. STEVOR expression was also higher in cerebral malaria compared to mild malaria, but STEVOR transcripts were sparse overall. DISCUSSION/SIGNIFICANCE: Elevated RIFIN expression in cerebral malaria over mild malaria supports a role for these antigens in pathogenesis. Severe malarial anemia may progress through a different pathogenic mechanism. Predominantly expressed RIFIN variants may be promising targets for vaccines and therapeutics to protect children against cerebral malaria.

Plasmodium falciparum malaria cases in Africa represent over 90% of the global burden with Mali being amongst the 11 highest burden countries that account for 70% of this annual incidence. The persistence of P. falciparum despite massive global interventions is because of its genetic diversity that drives its ability to adapt to environmental changes, develop resistance to drugs, and evade the host immune system. Knowledge on P. falciparum genetic diversity across populations and intervention landscape is thus critical for the implementation of new strategies to eliminate malaria. This study assessed genetic variation with 12,177 high-quality SNPs from 830 Malian P. falciparum isolates collected between 2007 and 2017 from seven locations. The complexity of infections remained high, varied between sites, and showed a trend toward overall decreasing complexity over the decade. Though there was no significant substructure, allele frequencies varied geographically, partly driven by temporal variance in sampling, particularly for drug resistance and antigen loci. Thirty-two mutations in known drug resistance markers (pfcrt, pfdhps, pfdhfr, pfmdr1, pfmdr2, and pfk13) attained a frequency of at least 2% in the populations. SNPs within and around the major markers of resistance to quinolines (pfmdr1 and pfcrt) and antifolates (pfdhfr and pfdhps) varied temporally and geographically, with strong linkage disequilibrium and signatures of directional selection in the genome. These geo-temporal populations also differentiated at alleles in immune-related loci, including, protein E140, pfsurfin8, pfclag8, and pfceltos, as well as pftrap, which showed signatures of haplotype differentiation between populations. Several regions across the genomes, including five known drug resistance loci, showed signatures of differential positive selection. These results suggest that drugs and immune pressure are dominant selective forces against P. falciparum in Mali, but their effect on the parasite genome varies temporally and spatially. Interventions interacting with these genomic variants need to be routinely evaluated as malaria elimination strategies are implemented.

Abstract Background Artemisinin-based combination therapy (ACT) was deployed in 2005 as an alternative to chloroquine and is considered the most efficacious treatment currently available for uncomplicated falciparum malaria. While widespread artemisinin resistance has not been reported to date in Africa, recent studies have reported partial resistance in Rwanda. The purpose of this study is to provide a current systematic review and meta-analysis on ACT at Mali study sites, where falciparum malaria is highly endemic. Methods A systematic review of the literature maintained in the bibliographic databases accessible through the PubMed, ScienceDirect and Web of Science search engines was performed to identify research studies on ACT occurring at Mali study sites. Selected studies included trials occurring at Mali study sites with reported polymerase chain reaction (PCR)-corrected adequate clinical and parasite response rates (ACPRcs) at 28 days. Data were stratified by treatment arm (artemether–lumefantrine (AL), the first-line treatment for falciparum malaria in Mali and non-AL arms) and analysed using random-effects, meta-analysis approaches. Results A total of 11 studies met the inclusion criteria, and a risk of bias assessment carried out by two independent reviewers determined low risk of bias among all assessed criteria. The ACPRc for the first-line AL at Mali sites was 99.0% (95% CI (98.3%, 99.8%)), while the ACPRc among non-AL treatment arms was 98.9% (95% CI (98.3%, 99.5%)). The difference in ACPRcs between non-AL treatment arms and AL treatment arms was not statistically significant (p = .752), suggesting that there are potential treatment alternatives beyond the first-line of AL in Mali. Conclusions ACT remains highly efficacious in treating uncomplicated falciparum malaria in Mali. Country-specific meta-analyses on ACT are needed on an ongoing basis for monitoring and evaluating drug efficacy patterns to guide local malaria treatment policies, particularly in the wake of observed artemisinin resistance in Southeast Asia and partial resistance in Rwanda.

Up-to-date knowledge of key epidemiological aspects of each Plasmodium species is necessary for making informed decisions on targeted interventions and control strategies to eliminate each of them. This study aims to describe the epidemiology of plasmodial species in Mali, where malaria is hyperendemic and seasonal. Data reports collected during high-transmission season over six consecutive years were analyzed to summarize malaria epidemiology. Malaria species and density were from blood smear microscopy. Data from 6870 symptomatic and 1740 asymptomatic participants were analyzed. The median age of participants was 12 years, and the sex ratio (male/female) was 0.81. Malaria prevalence from all Plasmodium species was 65.20% (95% CI: 60.10–69.89%) and 22.41% (CI: 16.60–28.79%) for passive and active screening, respectively. P. falciparum was the most prevalent species encountered in active and passive screening (59.33%, 19.31%). This prevalence was followed by P. malariae (1.50%, 1.15%) and P. ovale (0.32%, 0.06%). Regarding frequency, P. falciparum was more frequent in symptomatic individuals (96.77% vs. 93.24%, p = 0.014). In contrast, P. malariae was more frequent in asymptomatic individuals (5.64% vs. 2.45%, p < 0.001). P. ovale remained the least frequent species (less than 1%), and no P. vivax was detected. The most frequent coinfections were P. falciparum and P. malariae (0.56%). Children aged 5–9 presented the highest frequency of P. falciparum infections (41.91%). Non-falciparum species were primarily detected in adolescents (10–14 years) with frequencies above 50%. Only P. falciparum infections had parasitemias greater than 100,000 parasites per µL of blood. P. falciparum gametocytes were found with variable prevalence across age groups. Our data highlight that P. falciparum represented the first burden, but other non-falciparum species were also important. Increasing attention to P. malariae and P. ovale is essential if malaria elimination is to be achieved.

Malaria elimination may never succeed without the implementation of transmission-blocking strategies. The transmission of Plasmodium spp. parasites from the human host to the mosquito vector depends on circulating gametocytes in the peripheral blood of the vertebrate host. Once ingested by the mosquito during blood meals, these sexual forms undergo a series of radical morphological and metabolic changes to survive and progress from the gut to the salivary glands, where they will be waiting to be injected into the vertebrate host. The design of effective transmission-blocking strategies requires a thorough understanding of all the mechanisms that drive the development of gametocytes, gametes, sexual reproduction, and subsequent differentiation within the mosquito. The drastic changes in Plasmodium falciparum shape and function throughout its life cycle rely on the tight regulation of stage-specific gene expression. This review outlines the mechanisms involved in Plasmodium falciparum sexual stage development in both the human and mosquito vector, and zygote to oocyst differentiation. Functional studies unravel mechanisms employed by P. falciparum to orchestrate the expression of stage-specific functional products required to succeed in its complex life cycle, thus providing us with potential targets for developing new therapeutics. These mechanisms are based on studies conducted with various Plasmodium species, including predominantly P. falciparum and the rodent malaria parasites P. berghei. However, the great potential of epigenetics, genomics, transcriptomics, proteomics, and functional genetic studies to improve the understanding of malaria as a disease remains partly untapped because of limitations in studies using human malaria parasites and field isolates.

Structural biology is an essential tool for understanding the molecular basis of diseases, which can guide the rational design of new drugs, vaccines, and the optimisation of existing medicines. However, most African countries do not conduct structural biology research due to limited resources, lack of trained persons, and an exodus of skilled scientists. The most urgent requirement is to build on the emerging centres in Africa – some well-established, others growing. This can be achieved through workshops that improve networking, grow skills, and develop mechanisms for access to light source beamlines for defining X-ray structures across the continent. These would encourage the growth of structural biology, which is central to understanding biological functions and developing new antimicrobials and other drugs. In this light, a hands-on training workshop in structural biology series 4 was organised by BioStruct-Africa and the Malaria Research and Training Center (MRTC) in Bamako, Mali, to help bridge this gap. The workshop was hosted by MRTC from the 25th to 28th of April 2022. Through a series of lectures and practicals, the workshop enlightened the participants on how structural biology can be utilised to find solutions to the prevalent diseases in Africa. The short training gave them an overview of target selection, protein production and purification, structural determination techniques, and analysis in combination with high-throughput, structure-guided, fragment-based drug design.

ABSTRACT var genes encode Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens. These highly diverse antigens are displayed on the surface of infected erythrocytes and play a critical role in immune evasion and sequestration of infected erythrocytes. Studies of var expression using non-leukocyte-depleted blood are challenging because of the predominance of host genetic material and lack of conserved var segments. Our goal was to enrich for parasite RNA, allowing de novo assembly of var genes and detection of expressed novel variants. We used two overall approaches: (i) enriching for total mRNA in the sequencing library preparations and (ii) enriching for parasite RNA with a custom capture array based on Roche’s SeqCap EZ enrichment system. The capture array was designed with probes based on the whole 3D7 reference genome and an additional >4,000 full-length var gene sequences from other P. falciparum strains. We tested each method on RNA samples from Malian children with severe or uncomplicated malaria infections. All reads mapping to the human genome were removed, the remaining reads were assembled de novo into transcripts, and from these, var-like transcripts were identified and annotated. The capture array produced the longest maximum length and largest numbers of var gene transcripts in each sample, particularly in samples with low parasitemia. Identifying the most-expressed var gene sequences in whole-blood clinical samples without the need for extensive processing or generating sample-specific reference genome data is critical for understanding the role of PfEMP1s in malaria pathogenesis. IMPORTANCE Malaria parasites display antigens on the surface of infected red blood cells in the human host that facilitate attachment to blood vessels, contributing to the severity of infection. These antigens are highly variable, allowing the parasite to evade the immune system. Identifying these expressed antigens is critical to understanding the development of severe malarial disease. However, clinical samples contain limited amounts of parasite genetic material, a challenge for sequencing efforts further compounded by the extreme diversity of the parasite surface antigens. We present a method that enriches for these antigen sequences in clinical samples using a custom capture array, requiring minimal processing in the field. While our results are focused on the malaria parasite Plasmodium falciparum, this approach has broad applicability to other highly diverse antigens from other parasites and pathogens such as those that cause giardiasis and leishmaniasis.

Abstract Background Plasmodium falciparum (Pf) whole-organism sporozoite vaccines have been shown to provide significant protection against controlled human malaria infection (CHMI) in clinical trials. Initial CHMI studies showed significantly higher durable protection against homologous than heterologous strains, suggesting the presence of strain-specific vaccine-induced protection. However, interpretation of these results and understanding of their relevance to vaccine efficacy have been hampered by the lack of knowledge on genetic differences between vaccine and CHMI strains, and how these strains are related to parasites in malaria endemic regions. Methods Whole genome sequencing using long-read (Pacific Biosciences) and short-read (Illumina) sequencing platforms was conducted to generate de novo genome assemblies for the vaccine strain, NF54, and for strains used in heterologous CHMI (7G8 from Brazil, NF166.C8 from Guinea, and NF135.C10 from Cambodia). The assemblies were used to characterize sequences in each strain relative to the reference 3D7 (a clone of NF54) genome. Strains were compared to each other and to a collection of clinical isolates (sequenced as part of this study or from public repositories) from South America, sub-Saharan Africa, and Southeast Asia. Results While few variants were detected between 3D7 and NF54, we identified tens of thousands of variants between NF54 and the three heterologous strains. These variants include SNPs, indels, and small structural variants that fall in regulatory and immunologically important regions, including transcription factors (such as PfAP2-L and PfAP2-G) and pre-erythrocytic antigens that may be key for sporozoite vaccine-induced protection. Additionally, these variants directly contributed to diversity in immunologically important regions of the genomes as detected through in silico CD8+ T cell epitope predictions. Of all heterologous strains, NF135.C10 had the highest number of unique predicted epitope sequences when compared to NF54. Comparison to global clinical isolates revealed that these four strains are representative of their geographic origin despite long-term culture adaptation; of note, NF135.C10 is from an admixed population, and not part of recently formed subpopulations resistant to artemisinin-based therapies present in the Greater Mekong Sub-region. Conclusions These results will assist in the interpretation of vaccine efficacy of whole-organism vaccines against homologous and heterologous CHMI.

Abstract Background Drug resistance is one of the greatest challenges of malaria control programme in Mali. Recent advances in next-generation sequencing (NGS) technologies provide new and effective ways of tracking drug-resistant malaria parasites in Africa. The diversity and the prevalence of Plasmodium falciparum drug-resistance molecular markers were assessed in Dangassa and Nioro-du-Sahel in Mali, two sites with distinct malaria transmission patterns. Dangassa has an intense seasonal malaria transmission, whereas Nioro-du-Sahel has an unstable and short seasonal malaria transmission. Methods Up to 270 dried blood spot samples (214 in Dangassa and 56 in Nioro-du-Sahel) were collected from P. falciparum positive patients in 2016. Samples were analysed on the Agena MassARRAY® iPLEX platform. Specific codons were targeted in Pfcrt, Pfmdr1, Pfdhfr, and Pfdhps, Pfarps10, Pfferredoxin, Pfexonuclease and Pfmdr2 genes. The Sanger’s 101-SNPs-barcode method was used to assess the genetic diversity of P. falciparum and to determine the parasite species. Results The Pfcrt_76T chloroquine-resistance genotype was found at a rate of 64.4% in Dangassa and 45.2% in Nioro-du-Sahel (p = 0.025). The Pfdhfr_51I-59R-108N pyrimethamine-resistance genotype was 14.1% and 19.6%, respectively in Dangassa and Nioro-du-Sahel. Mutations in the Pfdhps _S436-A437-K540-A581-613A sulfadoxine-resistance gene was significantly more prevalent in Dangassa as compared to Nioro-du-Sahel (p = 0.035). Up to 17.8% of the isolates from Dangassa vs 7% from Nioro-du-Sahel harboured at least two codon substitutions in this haplotype. The amodiaquine-resistance Pfmdr1_N86Y mutation was identified in only three samples (two in Dangassa and one in Nioro-du-Sahel). The lumefantrine-reduced susceptibility Pfmdr1_Y184F mutation was found in 39.9% and 48.2% of samples in Dangassa and Nioro-du-Sahel, respectively. One piperaquine-resistance Exo _E415G mutation was found in Dangassa, while no artemisinin resistance genetic-background were identified. A high P. falciparum diversity was observed, but no clear genetic aggregation was found at either study sites. Higher multiplicity of infection was observed in Dangassa with both COIL (p = 0.04) and Real McCOIL (p = 0.02) methods relative to Nioro-du-Sahel. Conclusions This study reveals high prevalence of chloroquine and pyrimethamine-resistance markers as well as high codon substitution rate in the sulfadoxine-resistance gene. High genetic diversity of P. falciparum was observed. These observations suggest that the use of artemisinins is relevant in both Dangassa and Nioro-du-Sahel.

Abstract Background Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) antigens play a critical role in host immune evasion. Serologic responses to these antigens have been associated with protection from clinical malaria, suggesting that antibodies to PfEMP1 antigens may contribute to natural immunity. The first N-terminal constitutive domain in a PfEMP1 is the Duffy binding-like alpha (DBL-α) domain, which contains a 300 to 400 base pair region unique to each particular protein (the DBL-α “tag”). This DBL-α tag has been used as a marker of PfEMP1 diversity and serologic responses in malaria-exposed populations. In this study, using sera from a malaria-endemic region, responses to DBL-α tags were compared to responses to the corresponding entire DBL-α domain (or “parent” domain) coupled with the succeeding cysteine-rich interdomain region (CIDR). Methods A protein microarray populated with DBL-α tags, the parent DBL-CIDR head structures, and downstream PfEMP1 protein fragments was probed with sera from Malian children (aged 1 to 6 years) and adults from the control arms of apical membrane antigen 1 (AMA1) vaccine clinical trials before and during a malaria transmission season. Serological responses to the DBL-α tag and the DBL-CIDR head structure were measured and compared in children and adults, and throughout the season. Results Malian serologic responses to a PfEMP1’s DBL-α tag region did not correlate with seasonal malaria exposure, or with responses to the parent DBL-CIDR head structure in either children or adults. Parent DBL-CIDR head structures were better indicators of malaria exposure. Conclusions Larger PfEMP1 domains may be better indicators of malaria exposure than short, variable PfEMP1 fragments such as DBL-α tags. PfEMP1 head structures that include conserved sequences appear particularly well suited for study as serologic predictors of malaria exposure.

Abstract Background Artemether–lumefantrine (AL) and artesunate–amodiaquine are first-line treatment for uncomplicated malaria in many endemic countries, including Mali. Dihydroartemisinin–piperaquine (DHA–PQ) is also an alternative first-line artemisinin-based combination therapy, but only few data are available on DHA–PQ efficacy in sub-Saharan Africa. The main aim of this study was to compare clinical efficacy of DHA–PQ versus AL, using the World Health Organization (WHO) 42-day in vivo protocol. Methods The efficacy of three-dose regimens of DHA–PQ was compared to AL combination in a randomized, comparative open label trial using the WHO 42-day follow-up protocol from 2013 to 2015 in Doneguebougou and Torodo, Mali. The primary endpoint was to access the PCR-corrected Adequate Clinical and Parasitological Responses at day 28. Results A total of 317 uncomplicated malaria patients were enrolled, with 159 in DHA–PQ arm and 158 in AL arm. The parasite positivity rate decreased from 68.4% (95% CI 60.5–75.5) on day 1 to 3.8% (95% CI 1.4–8.1) on day 2 for DHA–PQ and 79.8% (95% CI 72.3–85.7) on day 1 to 9.5% (95% CI 5.4–15.2) on day 2 for AL, (p = 0.04). There was a significant difference in the uncorrected ACPR between DHA–PQ and AL, both at 28-day and 42-day follow-up with 97.4% (95% CI 93.5–99.3) in DHA–PQ vs 84.5% (95% CI 77.8–89.8) in AL (p

Abstract Background VAR2CSA, a member of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family, mediates the binding of P. falciparum-infected erythrocytes to chondroitin sulfate A, a surface-associated molecule expressed in placental cells, and plays a central role in the pathogenesis of placental malaria. VAR2CSA is a target of naturally acquired immunity and, as such, is a leading vaccine candidate against placental malaria. This protein is very polymorphic and technically challenging to sequence. Published var2csa sequences, mostly limited to specific domains, have been generated through the sequencing of cloned PCR amplicons using capillary electrophoresis, a method that is both time consuming and costly, and that performs poorly when applied to clinical samples that are commonly polyclonal. A next-generation sequencing platform, Pacific Biosciences (PacBio), offers an alternative approach to overcome these issues. Methods PCR primers were designed that target a 5 kb segment in the 5′ end of var2csa and the resulting amplicons were sequenced using PacBio sequencing. The primers were optimized using two laboratory strains and were validated on DNA from 43 clinical samples, extracted from dried blood spots on filter paper or from cryopreserved P. falciparum-infected erythrocytes. Sequence reads were assembled using the SMRT-analysis ConsensusTools module. Results Here, a PacBio sequencing-based approach for recovering a segment encoding the majority of VAR2CSA’s extracellular region is described; this segment includes the totality of the first four domains in the 5′ end of var2csa (~5 kb), from clinical malaria samples. The feasibility of the method is demonstrated, showing a high success rate from cryopreserved samples and more limited success from dried blood spots stored at room temperature, and characterized the genetic variation of the var2csa locus. Conclusions This method will facilitate a detailed analysis of var2csa genetic variation and can be adapted to sequence other hypervariable P. falciparum genes.

ABSTRACT The repetitive interspersed family (RIFIN) and the subtelomeric variable open reading frame (STEVOR) family represent two of three major Plasmodium falciparum variant surface antigen families involved in malaria pathogenesis and immune evasion and are potential targets in the development of natural immunity. Protein and peptide microarrays populated with RIFINs and STEVORs associated with severe malaria vulnerability in Malian children were probed with adult and pediatric sera to identify epitopes that reflect malaria exposure. Adult sera recognized and reacted with greater intensity to all STEVOR proteins than pediatric sera did. Serorecognition of and seroreactivity to peptides within the semiconserved domain of STEVORs increased with age and seasonal malaria exposure, while serorecognition and seroreactivity increased for the semiconserved and second hypervariable domains of RIFINs only with age. Serologic responses to RIFIN and STEVOR peptides within the semiconserved domains may play a role in natural immunity to severe malaria. IMPORTANCE Malaria, an infectious disease caused by the parasite Plasmodium falciparum, causes nearly 435,000 deaths annually worldwide. RIFINs and STEVORs are two variant surface antigen families that are involved in malaria pathogenesis and immune evasion. Recent work has shown that a lack of humoral immunity to these proteins is associated with severe malaria vulnerability in Malian children. This is the first study to have compared serologic responses of children and adults to RIFINs and STEVORs in settings of malaria endemicity and to examine such serologic responses before and after a clinical malaria episode. Using microarrays, we determined that the semiconserved domains in these two parasite variant surface antigen families harbor peptides whose seroreactivity reflects malaria exposure. A similar approach has the potential to illuminate the role of variant surface antigens in the development of natural immunity to clinical malaria. Potential vaccines for severe malaria should include consideration of peptides within the semiconserved domains of RIFINs and STEVORs.

Abstract Background Encoded by the var gene family, highly variable Plasmodium falciparum erythrocyte membrane protein-1 (PfEMP1) proteins mediate tissue-specific cytoadherence of infected erythrocytes, resulting in immune evasion and severe malaria disease. Sequencing and assembling the 40–60 var gene complement for individual infections has been notoriously difficult, impeding molecular epidemiological studies and the assessment of particular var elements as subunit vaccine candidates. Methods We developed and validated a novel algorithm, Exon-Targeted Hybrid Assembly (ETHA), to perform targeted assembly of var gene sequences, based on a combination of Pacific Biosciences and Illumina data. Results Using ETHA, we characterized the repertoire of var genes in 12 samples from uncomplicated malaria infections in children from a single Malian village and showed them to be as genetically diverse as vars from isolates from around the globe. The gene var2csa, a member of the var family associated with placental malaria pathogenesis, was present in each genome, as were vars previously associated with severe malaria. Conclusion ETHA, a tool to discover novel var sequences from clinical samples, will aid the understanding of malaria pathogenesis and inform the design of malaria vaccines based on PfEMP1. ETHA is available at: https://sourceforge.net/projects/etha/ .

Previous studies have shown that a single season of intermittent preventive treatment in schoolchildren (IPTsc) targeting the transmission season has reduced the rates of clinical malaria, all-cause clinic visits, asymptomatic parasitemia, and anemia. Efficacy over the course of multiple years of IPTsc has been scantly investigated. Methods: An open, randomized-controlled trial among schoolchildren aged 6–13 years was conducted from September 2007 to January 2010 in Kolle, Mali. Students were included in three arms: sulphadoxine-pyrimethamine+artesunate (SP+AS), amodiaquine+artesunate (AQ+AS), and control (C). All students received two full doses, given 2 months apart, and were compared with respect to the incidence of clinical malaria, all-cause clinic visits, asymptomatic parasitemia, and anemia. Results: A total of 296 students were randomized. All-cause clinic visits were in the SP+AS versus control (29 (20.1%) vs. 68 (47.2%); 20 (21.7%) vs. 41 (44.6%); and 14 (21.2%) vs. 30 (44.6%); p < 0.02) in 2007, 2008, and 2009, respectively. The prevalence of asymptomatic parasitemia was lower in the SP+AS compared to control (38 (7.5%) vs. 143 (28.7%); and 47 (12.7%) vs. 75 (21.2%); p < 0.002) in 2007 and 2008, respectively. Hemoglobin concentration was significantly higher in children receiving SP+AS (11.96, 12.06, and 12.62 g/dL) than in control children (11.60, 11.64, and 12.15 g/dL; p < 0.001) in 2007, 2008, and 2009, respectively. No impact on clinical malaria was observed. Conclusion: IPTsc with SP+AS reduced the rates of all-cause clinic visits and anemia during a three-year implementation.