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Summary Background WHO recently approved a partially effective vaccine that reduces clinical malaria in children, but increased vaccine activity is required to pursue malaria elimination. A phase 1 clinical trial was done in Mali, west Africa, to assess the safety, immunogenicity, and protective efficacy of a three-dose regimen of Plasmodium falciparum sporozoite (PfSPZ) Vaccine (a metabolically active, non-replicating, whole malaria sporozoite vaccine) against homologous controlled human malaria infection (CHMI) and natural P falciparum infection. Methods We recruited healthy non-pregnant adults aged 18–50 years in Doneguebougou, Mali, and surrounding villages (Banambani, Toubana, Torodo, Sirababougou, Zorokoro) for an open-label, dose-escalation pilot study and, thereafter, a randomised, double-blind, placebo-controlled main trial. Pilot study participants were enrolled on an as-available basis to one group of CHMI infectivity controls and three staggered vaccine groups receiving: one dose of 4·5 × 105, one dose of 9 × 105, or three doses of 1·8 × 106 PfSPZ via direct venous inoculation at approximately 8 week intervals, followed by homologous CHMI 5 weeks later with infectious PfSPZ by direct venous inoculation (PfSPZ Challenge). Main cohort participants were stratified by village and randomly assigned (1:1) to receive three doses of 1·8 × 106 PfSPZ or normal saline at 1, 13, and 19 week intervals using permuted block design by the study statistician. The primary outcome was safety and tolerability of at least one vaccine dose; the secondary outcome was vaccine efficacy against homologous PfSPZ CHMI (pilot study) or against naturally transmitted P falciparum infection (main study) measured by thick blood smear. Combined artesunate and amodiaquine was administered to eliminate pre-existing parasitaemia. Outcomes were analysed by modified intention to treat (mITT; including all participants who received at least one dose of investigational product; safety and vaccine efficacy) and per protocol (vaccine efficacy). This trial is registered with ClinicalTrials.gov , number NCT02627456 . Findings Between Dec 20, 2015, and April 30, 2016, we enrolled 56 participants into the pilot study (five received the 4·5 × 105 dose, five received 9 × 105, 30 received 1·8 × 106, 15 were CHMI controls, and one withdrew before vaccination) and 120 participants into the main study cohort with 60 participants assigned PfSPZ Vaccine and 60 placebo in the main study. Adverse events and laboratory abnormalities post-vaccination in all dosing groups were few, mainly mild, and did not differ significantly between vaccine groups (all p>0·05). Unexpected severe transaminitis occured in four participants: one participant in pilot phase that received 1·8 × 106 PfSPZ Vaccine, one participant in main phase that received 1·8 × 106 PfSPZ Vaccine, and two participants in the main phase placebo group. During PfSPZ CHMI, approximately 5 weeks after the third dose of 1·8 × 106 PfSPZ, none of 29 vaccinees and one of 15 controls became positive on thick blood smear; subsequent post-hoc PCR analysis for submicroscopic blood stage infections detected P falciparum parasites in none of the 29 vaccine recipients and eight of 15 controls during CHMI. In the main trial, 32 (58%) of 55 vaccine recipients and 42 (78%) of 54 controls became positive on thick blood smear during 24-week surveillance after vaccination. Vaccine efficacy (1–hazard ratio) was 0·51 per protocol (95% CI 0·20–0·70; log-rank p=0·0042) and 0·39 by mITT (0·04–0·62; p=0·033); vaccine efficacy (1–risk ratio) was 0·24 per-protocol (0·02–0·41; p=0·031) and 0·22 mITT (0·01–0·39; p=0·041). Interpretation A three-dose regimen of PfSPZ Vaccine was safe, well tolerated, and conferred 51% vaccine efficacy against intense natural P falciparum transmission, similar to 52% vaccine efficacy reported for a five-dose regimen in a previous trial. Funding US National Institute of Allergy and Infectious Diseases, National Institutes of Health, Sanaria. Translation For the French translation of the abstract see Supplementary Materials section.

In vitro and ex vivo cultivation of Plasmodium (P) falciparum has facilitated active research into the malaria parasite toward the quest for basic knowledge and the discovery of effective drug treatments. Such a drug discovery program is currently difficult for P. malariae simply because of the absence of in vitro and ex vivo cultivation system for its asexual blood stages supporting antimalarial evaluation. Despite availability of artemisinin combination therapies effective on P. falciparum, P. malariae is being increasingly detected in malaria endemic countries. P. malariae is responsible for chronic infections and is associated with a high burden of anemia and morbidity. Here, we optimized and adapted ex vivo conditions under which P. malariae can be cultured and used for screening antimalarial drugs. Subsequently, this enabled us to test compounds such as artemether, chloroquine, lumefantrine, and quinine for ex vivo antimalarial activity against P. malariae.

Plasmodium falciparumerythrocyte membrane protein-1s (PfEMP1s), diverse malaria proteins expressed on the infected erythrocyte surface, play an important role in pathogenesis, mediating adhesion to host vascular endothelium. Antibodies to particular non-CD36-binding PfEMP1s are associated with protection against severe disease. We hypothesized that given lifelongP. falciparumexposure, Malian adults would have broad PfEMP1 serorecognition and high seroreactivity levels during follow-up, particularly to non-CD36-binding PfEMP1s such as those that attach to endothelial protein C receptor (EPCR) and intercellular adhesion molecule-1 (ICAM-1). Using a protein microarray, we determined serologic responses to 166 reference PfEMP1 fragments during a dry and subsequent malaria transmission season in Malian adults. Malian adult sera had PfEMP1 serologic responses throughout the year, with decreased reactivity to a small subset of PfEMP1 fragments during the dry season and increases in reactivity to a different subset of PfEMP1 fragments during the subsequent peak malaria transmission season, especially for intracellular PfEMP1 domains. For some individuals, PfEMP1 serologic responses increased after the dry season, suggesting antigenic switching during asymptomatic infection. Adults were more likely to experience variable serorecognition of CD36-binding PfEMP1s than non-CD36-binding PfEMP1s that bind EPCR or ICAM-1, which remained serorecognized throughout the year. Sustained seroreactivity to non-CD36-binding PfEMP1s throughout adulthood amid seasonal fluctuation patterns may reflect underlying protective severe malaria immunity and merits further investigation.

Many African countries have reported declines in malaria incidence, attributed to the implementation of control strategies. In Mali, artemisinin-based combination therapy (ACT) was introduced in 2004, and long-lasting insecticide-treated nets (LLINs) have been partially distributed free of charge since 2007. In the Malian town of Bandiagara, a study conducted from 2009 to 2013 showed a stable incidence of malaria compared with 1999, despite the implementation of ACTs and LLINs. Since 2016, seasonal malaria chemoprevention has been scaled up across the country. In addition to these strategies, the population of Bandiagara benefited from indoor residual spray implementation in 2017 and 2018 and continued universal bed net coverage. This study aimed to measure the incidence of malaria in Bandiagara, given this recent scaling up of control strategies. A cohort of 300 children aged 6 months to 15 years was followed up from October 2017 to December 2018. We performed monthly cross-sectional surveys to measure anemia and the prevalence of malaria infection by microscopy. The overall incidence of symptomatic malaria was 0.5 episodes/person-year. Malaria incidence in children up to 5 years old significantly declined since 2012 and since 1999 (incidence rate ratio estimates: 6.7 [95% CI: 4.2–11.4] and 13.5 [95% CI: 8.4–22.7]), respectively. The average prevalence of malaria parasitemia was 6.7%. Malaria incidence was higher in children older than 5 years than in those younger than 5 years, highlighting the need to extend malaria control efforts to these older children.

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.

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. Electronic supplementary material The online version of this article (10.1186/s12936-019-2905-9) contains supplementary material, which is available to authorized users.

BackgroundEvaluation of local transmission epidemiology to characterize malaria risk is essential for planning malaria control and elimination programmes. The use of geographical information systems (GIS) has been a major asset to this approach. This study aimed to characterize the local spatio-temporal pattern of malaria infection and clinical disease after implementation of Seasonal Malaria Chemoprevention (SMC) and Indoor Residual Spraying (IRS) in Bandiagara, a Malian town.MethodsFrom October 2017 to December 2018, an active and passive surveillance system was established in a cohort study of three hundred children aged from 6 months to 15 years old. Weekly time-series of clinical malaria and monthly time-series of asymptomatic Plasmodium carriage and rainfall were plotted. Numbers of clinical malaria episodes and asymptomatic parasite carriers were mapped using Quantum Geographic Information System (QGIS). Landscape features of Bandiagara were obtained from Google earth. Clusters of high or low risk were identified under SaTScan® software using a Bernoulli model. ResultsFrom October 2017 to December 2018, 167 clinical malaria cases were recorded, mostly from July to December, while asymptomatic parasite carriage was observed during the entire study period. Three clusters of clinical episodes were found. All were hotspots. They were located in the north-east, south and west. No low risk cluster was identified. Three significant high-risk clusters of asymptomatic parasite carriage were identified in the south, north-east and north. These clusters were located near standing water. ConclusionThis study confirms the seasonality of clinical malaria in Bandiagara. The continued presence of asymptomatic parasite carriers maintains malaria transmission. To advance malaria elimination, control strategies must also target hotspots of asymptomatic parasite carriage. There was a spatial heterogeneity of clinical and asymptomatic malaria. Despite the implementation of additional preventives strategies, the locations of high-risk clusters were stable.

We show that dual RNA-seq from patient blood samples allows characterization of host/parasite interactions during malaria infections and can provide a solid framework to study the acquisition of antimalarial immunity, as well as the adaptations of P. falciparum to malaria-experienced hosts., Children are highly susceptible to clinical malaria, and in regions where malaria is endemic, their immune systems must face successive encounters with Plasmodium falciparum parasites before they develop immunity, first against severe disease and later against uncomplicated malaria. Understanding cellular and molecular interactions between host and parasites during an infection could provide insights into the processes underlying this gradual acquisition of immunity, as well as to how parasites adapt to infect hosts that are successively more malaria experienced. Here, we describe methods to analyze the host and parasite gene expression profiles generated simultaneously from blood samples collected from five consecutive symptomatic P. falciparum infections in three Malian children. We show that the data generated enable statistical assessment of the proportions of (i) each white blood cell subset and (ii) the parasite developmental stages, as well as investigations of host-parasite gene coexpression. We also use the sequences generated to analyze allelic variations in transcribed regions and determine the complexity of each infection. While limited by the modest sample size, our analyses suggest that host gene expression profiles primarily clustered by individual, while the parasite gene expression profiles seemed to differentiate early from late infections. Overall, this study provides a solid framework to examine the mechanisms underlying acquisition of immunity to malaria infections using whole-blood transcriptome sequencing (RNA-seq). IMPORTANCE We show that dual RNA-seq from patient blood samples allows characterization of host/parasite interactions during malaria infections and can provide a solid framework to study the acquisition of antimalarial immunity, as well as the adaptations of P. falciparum to malaria-experienced hosts.

To prevent premature dismissal of promising vaccine programs, it is critical to determine if lack of efficacy in the field is due to allele specific-efficacy, rather than to the lack of immunogenicity of the candidate antigen. Here we use samples collected during a field trial of the AMA1-based FMP2.1/AS02A malaria vaccine, which incorporates the AMA1 variant encoded by the reference Plasmodium falciparum 3D7 strain, to assess the usefulness of epitope-based sieve analysis for the detection of vaccine-induced allele-specific immune responses. The samples used are from volunteers who received the malaria vaccine FMP2.1/AS02A or a control (rabies vaccine), during a vaccine efficacy field trial, and who later developed malaria. In a previous study, P. falciparum DNA was extracted from all samples, and the ama1 locus amplified and sequenced. Here, a sieve analysis was used to measure T and B-cell escape, and difference in 3D7-like epitopes in the two treatment arms. Overall, no difference was observed in mean amino acid distance to the 3D7 AMA1 variant between sequences from vaccinees and controls in B-cell epitopes. However, we found a significantly greater proportion of 3D7-like T-cell epitopes that map to the AMA1 cluster one loop (c1L) region in the control vs. the vaccinee group (p = 0.02), consistent with allele-specific vaccine efficacy. Interestingly, AMA1 epitopes in infections from vaccinees had higher mean IC50, and consequently lower binding affinity, than epitopes generated from the control group (p = 0.01), suggesting that vaccine-induced selection impacted the immunological profile of the strains that pass through the sieve imposed by the vaccine-induced protection. These findings are consistent with a vaccine-derived sieve effect on the c1L region of AMA1 and suggest that sieve analyses of malaria vaccine trial samples targeted to epitopes identified in silico can help identify protective malaria antigens that may be efficacious if combined in a multivalent vaccine.

Variant surface antigens (VSAs) play a critical role in severe malaria pathogenesis. Defining gaps, or “lacunae”, in immunity to these Plasmodium falciparum antigens in children with severe malaria would improve our understanding of vulnerability to severe malaria and how protective immunity develops. Using a protein microarray with 179 antigen variants from three VSA families as well as more than 300 variants of three other blood stage P. falciparum antigens, reactivity was measured in sera from Malian children with cerebral malaria or severe malarial anaemia and age-matched controls. Sera from children with severe malaria recognized fewer extracellular PfEMP1 fragments and were less reactive to specific fragments compared to controls. Following recovery from severe malaria, convalescent sera had increased reactivity to certain non-CD36 binding PfEMP1s, but not other malaria antigens. Sera from children with severe malarial anaemia reacted to fewer VSAs than did sera from children with cerebral malaria, and both of these groups had lacunae in their seroreactivity profiles in common with children who had both cerebral malaria and severe malarial anaemia. This microarray-based approach may identify a subset of VSAs that could inform the development of a vaccine to prevent severe disease or a diagnostic test to predict at-risk children.

The recent World Malaria report shows that progress in malaria elimination has stalled. Current data acquisition by NMCPs depend on passive case detection and clinical reports focused mainly on Plasmodium falciparum (Pf). In recent times, several countries in sub-Saharan Africa have reported cases of Plasmodium vivax (Pv) with a considerable number being Duffy negative. The burden of Pv and Plasmodium ovale (Po) appear to be more than acknowledged. Similarly, the contribution of asymptomatic malaria in transmission is hardly considered by NMCPs in Africa. Inclusion of these as targets in malaria elimination agenda is necessary to achieve elimination goal, as these harbor hypnozoites. The Pan African Vivax and Ovale Network (PAVON) is a new consortium of African Scientists working in Africa on the transmission profile of Pv and Po. The group collaborates with African NMCPs to train in Plasmodium molecular diagnostics, microscopy, and interpretation of molecular data from active surveys to translate into policy. Details of the mission, rational and modus operandi of the group are outlined.

Summary Background Plasmodium falciparum sporozite (PfSPZ) Vaccine is a metabolically active, non-replicating, whole malaria sporozoite vaccine that has been reported to be safe and protective against P falciparum controlled human malaria infection in malaria-naive individuals. We aimed to assess the safety and protective efficacy of PfSPZ Vaccine against naturally acquired P falciparum in malaria-experienced adults in Mali. Methods After an open-label dose-escalation study in a pilot safety cohort, we did a double-blind, randomised, placebo-controlled trial based in Doneguebougou and surrounding villages in Mali. We recruited 18–35-year-old healthy adults who were randomly assigned (1:1) in a double-blind manner, with stratification by village and block randomisation, to receive either five doses of 2·7 × 10 5 PfSPZ or normal saline at days 0, 28, 56, 84, and 140 during the dry season (January to July inclusive). Participants and investigators were masked to group assignments, which were unmasked at the final study visit, 6 months after receipt of the last vaccination. Participants received combined artemether and lumefantrine (four tablets, each containing 20 mg artemether and 120 mg lumefantrine, given twice per day over 3 days for a total of six doses) to eliminate P falciparum before the first and last vaccinations. We collected blood smears every 2 weeks and during any illness for 24 weeks after the fifth vaccination. The primary outcome was the safety and tolerability of the vaccine, assessed as local and systemic reactogenicity and adverse events. The sample size was calculated for the exploratory efficacy endpoint of time to first P falciparum infection beginning 28 days after the fifth vaccination. The safety analysis included all participants who received at least one dose of investigational product, whereas the efficacy analyses included only participants who received all five vaccinations. This trial is registered at ClinicalTrials.gov, number NCT01988636. Findings Between Jan 18 and Feb 24, 2014, we enrolled 93 participants into the main study cohort with 46 participants assigned PfSPZ Vaccine and 47 assigned placebo, all of whom were evaluable for safety. We detected no significant differences in local or systemic adverse events or laboratory abnormalities between the PfSPZ Vaccine and placebo groups, and only grade 1 (mild) local or systemic adverse events occurred in both groups. The most common solicited systemic adverse event in the vaccine and placebo groups was headache (three [7%] people in the vaccine group vs four [9%] in the placebo group) followed by fatigue (one [2%] person in the placebo group), fever (one [2%] person in the placebo group), and myalgia (one [2%] person in each group). The exploratory efficacy analysis included 41 participants from the vaccine group and 40 from the placebo group. Of these participants, 37 (93%) from the placebo group and 27 (66%) from the vaccine group developed P falciparum infection. The hazard ratio for vaccine efficacy was 0·517 (95% CI 0·313–0·856) by time-to-infection analysis (log-rank p=0·01), and 0·712 (0·528–0·918) by proportional analysis (p=0·006). Interpretation PfSPZ Vaccine was well tolerated and safe. PfSPZ Vaccine showed significant protection in African adults against P falciparum infection throughout an entire malaria season. Funding US National Institutes of Health Intramural Research Program, Sanaria.

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/. Electronic supplementary material The online version of this article (doi:10.1186/s13073-017-0422-4) contains supplementary material, which is available to authorized users.

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., 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.

Sickle hemoglobin trait protects against severe and uncomplicated Plasmodium falciparum malaria [1–3], but at a cost, in that hemoglobin (Hb) SS disease is associated with significant morbidity and mortality [4]. In contrast, hemoglobin C trait, which is primarily found in West Africa [5], appears to offer protection against severe malaria [6], but hemoglobin CC disease does not have the degree of morbidity and mortality associated with hemoglobin SS disease [7]. Early work demonstrating the protective effects of hemoglobin C trait against severe malaria involved studies of the predominantly Dogon population in east-central Mali [8]. Subsequently, a case-control study in Burkina Faso found that hemoglobin C trait may be protective against both severe and uncomplicated forms of malaria in children [9]. In contrast, a recent longitudinal study of children less than 2 years of age in Ghana found that hemoglobin S trait protected against uncomplicated malaria and anemia (hemoglobin

Parasite antigen diversity poses an obstacle to developing an effective malaria vaccine. A protein microarray containing Plasmodium falciparum apical membrane antigen 1 (AMA1, n = 57) and merozoite surface protein 1 19-kD (MSP119, n = 10) variants prevalent at a malaria vaccine testing site in Bandiagara, Mali, was used to assess changes in seroreactivity caused by seasonal and lifetime exposure to malaria. Malian adults had significantly higher magnitude and breadth of seroreactivity to variants of both antigens than did Malian children. Seroreactivity increased over the course of the malaria season in children and adults, but the difference was more dramatic in children. These results help to validate diversity-covering protein microarrays as a promising tool for measuring the breadth of antibody responses to highly variant proteins, and demonstrate the potential of this new tool to help guide the development of malaria vaccines with strain-transcending efficacy.

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family mediates parasite sequestration in small capillaries through tissue-specific cytoadherence. The best characterized of these proteins is VAR2CSA, which is expressed on the surface of infected erythrocytes that bind to chondroitin sulfate in the placental matrix. Antibodies to VAR2CSA prevent placental cytoadherence and protect against placental malaria. The size and complexity of the VAR2CSA protein pose challenges for vaccine development, but smaller constitutive domains may be suitable for subunit vaccine development. A protein microarray was printed to include five overlapping fragments of the 3D7 VAR2CSA extracellular region. Malian women with a history of at least one pregnancy had antibody recognition of four of these fragments and had stronger reactivity against the two distal fragments than did nulliparous women, children, and men from Mali, suggesting that the C-terminal extracellular VAR2CSA domains are a potential focus of protective immunity. With carefully chosen sera from longitudinal studies of pregnant women, this approach has the potential to identify seroreactive VAR2CSA domains associated with protective immunity against pregnancy-associated malaria.

International audience; These authors contributed equally to this work. Abstract. In areas of seasonal malaria transmission, the incidence rate of malaria infection is presumed to be near zero at the end of the dry season. Asymptomatic individuals may constitute a major parasite reservoir during this time. We conducted a longitudinal analysis of the spatio-temporal distribution of clinical malaria and asymptomatic parasitemia over time in a Malian town to highlight these malaria transmission dynamics. For a cohort of 300 rural children followed over 2009–2014, periodicity and phase shift between malaria and rainfall were determined by spectral analysis. Spatial risk clusters of clinical episodes or carriage were identified. A nested-case-control study was conducted to assess the parasite carriage factors. Malaria infection persisted over the entire year with seasonal peaks. High transmission periods began 2–3 months after the rains began. A cluster with a low risk of clinical malaria in the town center persisted in high and low transmission periods. Throughout 2009–2014, cluster locations did not vary from year to year. Asymptomatic and gametocyte carriage were persistent, even during low transmission periods. For high transmission periods, the ratio of asymptomatic to clinical cases was approximately 0.5, but was five times higher during low transmission periods. Clinical episodes at previous high transmission periods were a protective factor for asymptomatic carriage, but carrying parasites without symptoms at a previous high transmission period was a risk factor for asymptomatic carriage. Stable malaria transmission was associated with sustained asymptomatic carriage during dry seasons. Control strategies should target persistent low-level parasitemia clusters to interrupt transmission.

Plasmodium vivax was thought to infect only the erythrocytes of Duffy blood group positive people. In the last decade, P. vivax has appeared throughout Africa, both in areas where Duffy positive and negative people live side by side as in Madagascar and Ethiopia and in areas where people are primarily Duffy negative, such as in western Kenya. We performed quantitative polymerase chain reaction on blood samples dried onto filter paper to determine the prevalence of P. vivax and Plasmodium falciparum in a cohort of 300 children (newborn to 6 years of age) in Bandiagara, a Sahelian area of Mali, west Africa, where the people are Duffy negative. We report 1-3 occurrences of P. vivax in each of 25 Duffy-negative children at six time points over two rainy seasons and the beginning of the third season. The prevalence of P. vivax infection was 2.0-2.5% at every time point (June 2009 to June 2010). All children with P. vivax infections were asymptomatic and afebrile, and parasite densities were extremely low. Anemia, however, was the main burden of infection. Plasmodium vivax could become a burden to sub-Saharan Africa, and the evidence of P. vivax existence needs to be taken into consideration in designing malaria control and elimination strategies in Africa.

Many human genetic associations with resistance to malaria have been reported, but few have been reliably replicated. We collected data on 11,890 cases of severe malaria due to Plasmodium falciparum and 17,441 controls from 12 locations in Africa, Asia and Oceania. We tested 55 SNPs in 27 loci previously reported to associate with severe malaria. There was evidence of association at P < 1 × 10(-4) with the HBB, ABO, ATP2B4, G6PD and CD40LG loci, but previously reported associations at 22 other loci did not replicate in the multicenter analysis. The large sample size made it possible to identify authentic genetic effects that are heterogeneous across populations or phenotypes, with a striking example being the main African form of G6PD deficiency, which reduced the risk of cerebral malaria but increased the risk of severe malarial anemia. The finding that G6PD deficiency has opposing effects on different fatal complications of P. falciparum infection indicates that the evolutionary origins of this common human genetic disorder are more complex than previously supposed.

The blood-stage malaria vaccine FMP2.1/AS02A, comprised of recombinant Plasmodium falciparum apical membrane antigen 1 (AMA1) and the adjuvant system AS02A, had strain-specific efficacy against clinical malaria caused by P. falciparum with the vaccine strain 3D7 AMA1 sequence. To evaluate a potential correlate of protection, we measured the ability of participant sera to inhibit growth of 3D7 and FVO strains in vitro using high-throughput growth inhibition assay (GIA) testing. Sera from 400 children randomized to receive either malaria vaccine or a control rabies vaccine were assessed at baseline and over two annual malaria transmission seasons after immunization. Baseline GIA against vaccine strain 3D7 and FVO strain was similar in both groups, but more children in the malaria vaccine group than in the control group had 3D7 and FVO GIA activity ≥15% 30 days after the last vaccination (day 90) (49% vs. 16%, p

Background The host response to infection by Plasmodium falciparum, the parasite most often responsible for severe malaria, ranges from asymptomatic parasitaemia to death. The clinical trajectory of malaria is influenced by host genetics and parasite load, but the factors determining why some infections produce uncomplicated malaria and some proceed to severe disease remain incompletely understood. Methods To identify molecular markers of severe falciparum malaria, human gene expression patterns were compared between children aged 6 months to 5 years with severe and uncomplicated malaria who were enrolled in a case–control study in Bandiagara, Mali. Microarrays were used to obtain expression data on severe cases and uncomplicated controls at the time of acute disease presentation (five uncomplicated and five severe), 1 week after presentation (three uncomplicated and three severe) and treatment initiation, and in the subsequent dry season (late convalescence, four uncomplicated and four severe). This is a pilot study for the first use of microarray technology in Mali. Results Complement and toll-like receptor (TLR) pathways were differentially expressed, with severe cases showing higher expression of the C1q, TLR2, TLR4, TLR8, and CR1 genes. Other genes previously associated with malaria pathogenesis, GZMB, FOS and HSPA6, were also higher among severe cases. TLR2, TLR4, TLR8, CR1, GZMB, FOS, and HSPA6 genes were expressed at lower levels in severe cases at late convalescence. Conclusions Overexpression of genes previously associated with uncomplicated malaria was associated with severe disease. Low baseline expression of these genes may represent candidate markers for severe malaria. Despite the small sample size, results of this pilot study offer promising targets for follow-up analyses. Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1189-6) contains supplementary material, which is available to authorized users.

The disappointing efficacy of blood-stage malaria vaccines may be explained in part by allele-specific immune responses that are directed against polymorphic epitopes on blood-stage antigens. FMP2.1/AS02(A), a blood-stage candidate vaccine based on apical membrane antigen 1 (AMA1) from the 3D7 strain of Plasmodium falciparum, had allele-specific efficacy against clinical malaria in a phase II trial in Malian children. We assessed the cross-protective efficacy of the malaria vaccine and inferred which polymorphic amino acid positions in AMA1 were the targets of protective allele-specific immune responses. FMP2.1/AS02(A) had the highest efficacy against AMA1 alleles that were identical to the 3D7 vaccine-type allele at 8 highly polymorphic amino acid positions in the cluster 1 loop (c1L) but differed from 3D7 elsewhere in the molecule. Comparison of the incidence of vaccine-type alleles before and after vaccination in the malaria vaccine and control groups and examination of the patterns of allele change at polymorphic positions in consecutive malaria episodes suggest that the highly polymorphic amino acid position 197 in c1L was the most critical determinant of allele-specific efficacy. These results indicate that a multivalent AMA1 vaccine with broad efficacy could include only a limited set of key alleles of this extremely polymorphic antigen.

Background The safety and immunogenicity of PfAMA1, adjuvanted with Alhydrogel® was assessed in malaria–experienced Malian adults. The malaria vaccine, PfAMA1-FVO [25-545] is a recombinant protein Pichia pastoris-expressed AMA-1 from Plasmodium falciparum FVO clone adsorbed to Alhydrogel®, the control vaccine was tetanus toxoid produced from formaldehyde detoxified and purified tetanus toxin. Methods A double blind randomized controlled phase 1 study enrolled and followed 40 healthy adults aged 18–55 years in Bandiagara, Mali, West Africa, a rural setting with intense seasonal transmission of P. falciparum malaria. Volunteers were randomized to receive either 50 µg of malaria vaccine or the control vaccine. Three doses of vaccine were given on Days 0, 28 and 56, and participants were followed for 1 year. Solicited symptoms were assessed for seven days and unsolicited symptoms for 28 days after each vaccination. Serious adverse events were assessed throughout the study. The titres of anti-AMA-1 antibodies were measured by ELISA and P. falciparum growth inhibition assays were performed. Results Commonest local solicited adverse events were the injection site pain and swelling more frequent in the PfAMA1 group. No vaccine related serious adverse events were reported. A significant 3.5-fold increase of anti-AMA-1 IgG antibodies was observed in malaria vaccine recipients four weeks after the third immunization compared to the control group. Conclusion The PfAMA1 showed a good safety profile. Most adverse events reported were of mild to moderate intensity. In addition, the vaccine induced a significant though short-lived increase in the anti-AMA1 IgG titres. Registered on www.clinicaltrials.gov with the number NCT00431808 Electronic supplementary material The online version of this article (doi:10.1186/s12936-016-1466-4) contains supplementary material, which is available to authorized users.

Based on Plasmodium falciparum (Pf) apical membrane antigen 1 (AMA1) from strain 3D7, the malaria vaccine candidate FMP2.1/AS02A showed strain-specific efficacy in a Phase 2 clinical trial in 400 Malian children randomized to 3 doses of the AMA1 vaccine candidate or control rabies vaccine on days 0, 30 and 60. A subset of 10 Pf(-) (i.e., no clinical malaria episodes) AMA1 recipients, 11 Pf(+) (clinical malaria episodes with parasites with 3D7 or Fab9-type AMA1 cluster 1 loop [c1L]) AMA1 recipients, and 10 controls were randomly chosen for analysis. Peripheral blood mononuclear cells (PBMCs) isolated on days 0, 90 and 150 were stimulated with full-length 3D7 AMA1 and c1L from strains 3D7 (c3D7) and Fab9 (cFab9). Production of IFN-γ, TNF-α, IL-2, and/or IL-17A was analyzed by flow cytometry. Among AMA1 recipients, 18/21 evaluable samples stimulated with AMA1 demonstrated increased IFN-γ, TNF-α, and IL-2 derived from CD4(+) T cells by day 150 compared to 0/10 in the control group (p

Background Artemisinin-based combination therapies (ACTs) are the main treatment for malaria in endemic countries. Plasmodium falciparum resistance to artemisinins is described as delayed parasite clearance, which is associated with mutations on the parasite K13 propeller gene. Both the mechanisms of action and mechanisms of resistance to artemisinins are poorly understood. Transcriptomic studies can help in improving our understanding of these processes. Here we explore P. falciparum in vivo RNA expression profile after a curative dose of artesunate monotherapy. Methods During a prospective study of the efficacy of artesunate in monotherapy in children aged 1–10 years and presenting uncomplicated P. falciparum malaria in Bougoula-Hameau, Mali, venous blood was collected on PAXgen blood RNA tubes before treatment (H0) and one (H1), two (H2) and three hours (H3) after treatment. RNA was extracted from these respective blood samples and used for microarray experiments with Plasmodium/Anopheles GeneChips and the Affymetrix® platform. Results A total of 23 samples from 6 patients were included in the final analysis after quality control using Affimetrix® and Qlucore® softwares. With a 2-groups comparison of H0/H after treatment, 236 genes were identified as differentially expressed. Overall 42 genes were up-regulated including a knob-associated histidine-rich protein, rifins (pf.12.409.0, pf.13_399.0), stevors (pf.3.184.0), RESA-like proteins with DNAJ domain and thioredoxins. Heat shock protein (Pf.5.258.0), a number of AP2 domain-containing genes (Pf.6.27.0, Pf.11.99.0), an ABC transporter (Pf.12.250.0), genes involved in cell cycle regulation and many exported protein genes with unknown function and membrane proteins genes were among the 194 down-regulated genes. Conclusions Our data support a role for these genes in the in vivo response of P. falciparum to artesunate administration.

Background The FMP2.1/AS02A candidate malaria vaccine was tested in a Phase 2 study in Mali. Based on results from the first eight months of follow-up, the vaccine appeared well-tolerated and immunogenic. It had no significant efficacy based on the primary endpoint, clinical malaria, but marginal efficacy against clinical malaria in secondary analyses, and high allele-specific efficacy. Extended follow-up was conducted to evaluate extended safety, immunogenicity and efficacy. Methods A randomized, double-blinded trial of safety, immunogenicity and efficacy of the candidate Plasmodium falciparum apical membrane antigen 1 (AMA1) vaccine FMP2.1/AS02A was conducted in Bandiagara, Mali. Children aged 1–6 years were randomized in a 1∶1 ratio to receive FMP2.1/AS02A or control rabies vaccine on days 0, 30 and 60. Using active and passive surveillance, clinical malaria and adverse events as well as antibodies against P. falciparum AMA1 were monitored for 24 months after the first vaccination, spanning two malaria seasons. Findings 400 children were enrolled. Serious adverse events occurred in nine participants in the FMP2.1/AS02A group and three in the control group; none was considered related to study vaccination. After two years, anti-AMA1 immune responses remained significantly higher in the FMP2.1/AS02A group than in the control group. For the entire 24-month follow-up period, vaccine efficacy was 7.6% (p = 0.51) against first clinical malaria episodes and 9.9% (p = 0.19) against all malaria episodes. For the final 16-month follow-up period, vaccine efficacy was 0.9% (p = 0.98) against all malaria episodes. Allele-specific efficacy seen in the first malaria season did not extend into the second season of follow-up. Interpretation Allele-specific vaccine efficacy was not sustained in the second malaria season, despite continued high levels of anti-AMA1 antibodies. This study presents an opportunity to evaluate correlates of partial protection against clinical malaria that waned during the second malaria season. Trial Registration Clinicaltrials.gov NCT00460525 NCT00460525

Blood-stage malaria vaccines are intended to prevent clinical disease. The malaria vaccine FMP2.1/AS02(A), a recombinant protein based on apical membrane antigen 1 (AMA1) from the 3D7 strain of Plasmodium falciparum, has previously been shown to have immunogenicity and acceptable safety in Malian adults and children.In a double-blind, randomized trial, we immunized 400 Malian children with either the malaria vaccine or a control (rabies) vaccine and followed them for 6 months. The primary end point was clinical malaria, defined as fever and at least 2500 parasites per cubic millimeter of blood. A secondary end point was clinical malaria caused by parasites with the AMA1 DNA sequence found in the vaccine strain.The cumulative incidence of the primary end point was 48.4% in the malaria-vaccine group and 54.4% in the control group; efficacy against the primary end point was 17.4% (hazard ratio for the primary end point, 0.83; 95% confidence interval [CI], 0.63 to 1.09; P=0.18). Efficacy against the first and subsequent episodes of clinical malaria, as defined on the basis of various parasite-density thresholds, was approximately 20%. Efficacy against clinical malaria caused by parasites with AMA1 corresponding to that of the vaccine strain was 64.3% (hazard ratio, 0.36; 95% CI, 0.08 to 0.86; P=0.03). Local reactions and fever after vaccination were more frequent with the malaria vaccine.On the basis of the primary end point, the malaria vaccine did not provide significant protection against clinical malaria, but on the basis of secondary results, it may have strain-specific efficacy. If this finding is confirmed, AMA1 might be useful in a multicomponent malaria vaccine. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00460525.).

Background Extensive genetic diversity in vaccine antigens may contribute to the lack of efficacy of blood stage malaria vaccines. Apical membrane antigen-1 (AMA1) is a leading blood stage malaria vaccine candidate with extreme diversity, potentially limiting its efficacy against infection and disease caused by Plasmodium falciparum parasites with diverse forms of AMA1. Methods Three hundred Malian children participated in a Phase 2 clinical trial of a bivalent malaria vaccine that found no protective efficacy. The vaccine consists of recombinant AMA1 based on the 3D7 and FVO strains of P. falciparum adjuvanted with aluminum hydroxide (AMA1-C1). The gene encoding AMA1 was sequenced from P. falciparum infections experienced before and after immunization with the study vaccine or a control vaccine. Sequences of ama1 from infections in the malaria vaccine and control groups were compared with regard to similarity to the vaccine antigens using several measures of genetic diversity. Time to infection with parasites carrying AMA1 haplotypes similar to the vaccine strains with respect to immunologically important polymorphisms and the risk of infection with vaccine strain haplotypes were compared. Results Based on 62 polymorphic AMA1 residues, 186 unique ama1 haplotypes were identified among 315 ama1 sequences that were included in the analysis. Eight infections had ama1 sequences identical to 3D7 while none were identical to FVO. Several measures of genetic diversity showed that ama1 sequences in the malaria vaccine and control groups were comparable both at baseline and during follow up period. Pre- and post-immunization ama1 sequences in both groups all had a similar degree of genetic distance from FVO and 3D7 ama1. No differences were found in the time of first clinical episode or risk of infection with an AMA1 haplotype similar to 3D7 or FVO with respect to a limited set of immunologically important polymorphisms found in the cluster 1 loop of domain I of AMA1. Conclusion This Phase 2 trial of a bivalent AMA1 malaria vaccine found no evidence of vaccine selection or strain-specific efficacy, suggesting that the extreme genetic diversity of AMA1 did not account for failure of the vaccine to provide protection.

Vaccines directed against the blood stages of Plasmodium falciparum malaria are intended to prevent the parasite from invading and replicating within host cells. No blood-stage malaria vaccine has shown clinical efficacy in humans. Most malaria vaccine antigens are parasite surface proteins that have evolved extensive genetic diversity, and this diversity could allow malaria parasites to escape vaccine-induced immunity. We examined the extent and within-host dynamics of genetic diversity in the blood-stage malaria vaccine antigen apical membrane antigen-1 in a longitudinal study in Mali. Two hundred and fourteen unique apical membrane antigen-1 haplotypes were identified among 506 human infections, and amino acid changes near a putative invasion machinery binding site were strongly associated with the development of clinical symptoms, suggesting that these residues may be important to consider in designing polyvalent apical membrane antigen-1 vaccines and in assessing vaccine efficacy in field trials. This extreme diversity may pose a serious obstacle to an effective polyvalent recombinant subunit apical membrane antigen-1 vaccine.

Background The recent decline in malaria incidence in many African countries has been attributed to the provision of prompt and effective anti-malarial treatment using artemisinin-based combination therapy (ACT) and to the widespread distribution of long-lasting, insecticide-treated bed nets (LLINs). At a malaria vaccine-testing site in Bandiagara, Mali, ACT was introduced in 2004, and LLINs have been distributed free of charge since 2007 to infants after they complete the Expanded Programme of Immunization (EPI) schedule and to pregnant women receiving antenatal care. These strategies may have an impact on malaria incidence. Methods To document malaria incidence, a cohort of 400 children aged 0 to 14 years was followed for three to four years up to July 2013. Monthly cross-sectional surveys were done to measure the prevalence of malaria infection and anaemia. Clinical disease was measured both actively and passively through continuous availability of primary medical care. Measured outcomes included asymptomatic Plasmodium infection, anaemia and clinical malaria episodes. Results The incidence rate of clinical malaria varied significantly from June 2009 to July 2013 without a clear downward trend. A sharp seasonality in malaria illness incidence was observed with higher clinical malaria incidence rates during the rainy season. Parasite and anaemia point prevalence also showed seasonal variation with much higher prevalence rates during rainy seasons compared to dry seasons. Conclusions Despite the scaling up of malaria prevention and treatment, including the widespread use of bed nets, better diagnosis and wider availability of ACT, malaria incidence did not decrease in Bandiagara during the study period.