IFNγ, TNFα polymorphisms and IFNγ serum levels are associated with the clearance of drug-resistant P. falciparum in Malian children.

Host immunity has been suggested to clear drug-resistant parasites in malaria-endemic settings. However, the immunogenetic mechanisms involved in parasite clearance are poorly understood. Characterizing the host's immunity and genes involved in controlling the parasitic infection can inform the development of blood-stage malaria vaccines. This study investigates host regulatory cytokines and immunogenomic factors associated with the clearance of Plasmodium falciparum carrying a chloroquine resistance genotype. Biological samples from participants of previous drug efficacy trials conducted in two Malian localities were retrieved. The P. falciparum chloroquine resistance transporter (Pfcrt) gene was genotyped using parasite DNA. Children carrying parasites with the mutant allele (Pfcrt-76T) were classified based on their ability to clear their parasites. The levels of the different cytokines were measured in serum. The polymorphisms of specific human genes involved in malaria susceptibility were genotyped using human DNA. The prevalence of the Pfcrt-76T was significantly higher in Kolle than in Bandiagara (81.6 % vs 38.6 %, p < 10-6). The prevalence of children who cleared their mutant parasites was significantly higher in Bandiagara than in Kolle (82.2 % vs 67.4 %, p < 0.05). The genotyping of host genes revealed that IFN-γ -874 T and TNF-α -308A alleles were positively associated with parasite clearance. Cytokine profiling revealed that IFN-γ level was positively associated with parasite clearance (p = 0.04). This study highlights the role of host's immunity and immunogenetic factors to clear resistant parasites, suggesting further characterization of these polymorphisms may help to develop novel approaches to antiparasitic treatment strategies.

Successful Profiling of Plasmodium falciparum var Gene Expression in Clinical Samples via a Custom Capture Array.

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.

Spatio-Temporal Dynamics of Asymptomatic Malaria: Bridging the Gap Between Annual Malaria Resurgences in a Sahelian Environment.

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.

Strain-specific Plasmodium falciparum growth inhibition among Malian children immunized with a blood-stage malaria vaccine.

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<0.0001; and 71.8% vs. 60.4%, p = 0.02). From baseline to day 90, 3D7 GIA in the vaccine group was 7.4 times the mean increase in the control group (p<0.0001). In AMA1 vaccinees, 3D7 GIA activity subsequently returned to baseline one year after vaccination (day 364) and did not correlate with efficacy in the extended efficacy time period to day 730. In Cox proportional hazards regression models with time-varying covariates, there was a slight suggestion of an association between 3D7 GIA activity and increased risk of clinical malaria between day 90 and day 240. We conclude that vaccination with this AMA1-based malaria vaccine increased inhibition of parasite growth, but this increase was not associated with allele-specific efficacy in the first malaria season. These results provide a framework for testing functional immune correlates of protection against clinical malaria in field trials, and will help to guide similar analyses for next-generation malaria vaccines. Clinical trials registry: This clinical trial was registered on clinicaltrials.gov, registry number NCT00460525.

New var reconstruction algorithm exposes high var sequence diversity in a single geographic location in Mali.

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/ .

Phase 1 randomized controlled trial to evaluate the safety and immunogenicity of recombinant Pichia pastoris-expressed Plasmodium falciparum apical membrane antigen 1 (PfAMA1-FVO [25-545]) in healthy Malian adults in Bandiagara.

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.

Strain-specific Plasmodium falciparum multifunctional CD4(+) T cell cytokine expression in Malian children immunized with the FMP2.1/AS02A vaccine candidate.

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<0.0001). Among AMA1 vaccines, CD4(+) cells expressing both TNF-α and IL-2 were increased in Pf(-) children compared to Pf(+) children. When PBMCs were stimulated with c3D7 and cFab9 separately, 4/18 AMA1 recipients with an AMA1-specific CD4(+) response had a significant response to one or both c1L. This suggests that recognition of the AMA1 antigen is not dependent upon c1L alone. In summary, AMA1-specific T cell responses were notably increased in children immunized with an AMA1-based vaccine candidate. The role of CD4(+)TNF-α(+)IL-2(+)-expressing T cells in vaccine-induced strain-specific protection against clinical malaria requires further exploration. Clinicaltrials.gov Identifier: NCT00460525.

Protection of Malian children from clinical malaria is associated with recognition of multiple antigens.

BACKGROUND: Naturally acquired immunity to clinical malaria is thought to be mainly antibody-mediated, but reports on antigen targets are contradictory. Recognition of multiple antigens may be crucial for protection. In this study, the magnitude of antibody responses and their temporal stability was assessed for a panel of malaria antigens in relation to protection against clinical Plasmodium falciparum malaria. METHODS: Malian children aged two to 14 years were enrolled in a longitudinal study and followed up by passive and active case detection for seven months. Plasma was collected at enrolment and at the beginning, in the middle and after the end of the transmission season. Antibody titres to the P. falciparum-antigens apical membrane protein (AMA)-1, merozoite surface protein (MSP)-119, MSP-3, glutamine-rich protein (GLURP-R0) and circumsporozoite antigen (CSP) were assessed by enzyme-linked immunosorbent assay (ELISA) for 99 children with plasma available at all time points. Parasite carriage was determined by microscopy and nested PCR. RESULTS: Antibody titres to all antigens, except MSP-119, and the number of antigens recognized increased with age. After malaria exposure, antibody titres increased in children that had low titres at baseline, but decreased in those with high baseline responses. No significant differences were found between antibody titers for individual antigens between children remaining symptomatic or asymptomatic after exposure, after adjustment for age. Instead, children remaining asymptomatic following parasite exposure had a broader repertoire of antigen recognition. CONCLUSIONS: The present study provides immune-epidemiological evidence from a limited cohort of Malian children that strong recognition of multiple antigens, rather than antibody titres for individual antigens, is associated with protection from clinical malaria.

α-Thalassaemia trait is associated with antibody prevalence against malaria antigens AMA-1 and MSP-1.

A longitudinal study was conducted in a low endemic area in northern Tanzania to examine the influence of the α-thalassaemia trait on malaria incidence and antibody responses to malaria apical membrane antigen-1 (AMA-1) and merozoite surface protein1-19 (MSP-119). Out of 394 children genotyped for α-thalassaemia trait, 4.1% (16 of 394) and 30.7% (121 of 394) were homozygous and heterozygous, respectively. During the 1 year follow-up, four incidents of malaria cases were detected without an evident association with α-thalassaemia. Being heterozygous or homozygous for α-thalassaemia was associated with an increased prevalence of antibodies to AMA-1 [odds ratio (OR): 1.83, 95% confidence interval (CI): 1.07-3.12, p = 0.027] and MSP-1 (OR: 2.04, 95% CI: 1.16-3.60, p = 0.013) after adjustment for age and reported bednet use. The observed association between α-thalassaemia and malaria antibody responses may reflect longer-term differences in antigen exposure or differences in antibody acquisition upon exposure in this low endemic setting.

Stable malaria incidence despite scaling up control strategies in a malaria vaccine-testing site in Mali.

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.

Extended safety, immunogenicity and efficacy of a blood-stage malaria vaccine in malian children: 24-month follow-up of a randomized, double-blinded phase 2 trial.

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.

Spatio-temporal analysis of malaria within a transmission season in Bandiagara, Mali.

BACKGROUND: Heterogeneous patterns of malaria transmission are thought to be driven by factors including host genetics, distance to mosquito breeding sites, housing construction, and socio-behavioural characteristics. Evaluation of local transmission epidemiology to characterize malaria risk is essential for planning malaria control and elimination programmes. The use of geographical information systems (GIS) techniques has been a major asset to this approach. To assess time and space distribution of malaria disease in Bandiagara, Mali, within a transmission season, data were used from an ongoing malaria incidence study that enrolled 300 participants aged under six years old". METHODS: Children's households were georeferenced using a handheld global position system. Clinical malaria was defined as a positive blood slide for Plasmodium falciparum asexual stages associated with at least one of the following signs: headache, body aches, fever, chills and weakness. Daily rainfall was measured at the local weather station.Landscape features of Bandiagara were obtained from satellite images and field survey. QGIS™ software was used to map malaria cases, affected and non-affected children, and the number of malaria episodes per child in each block of Bandiagara. Clusters of high or low risk were identified under SaTScan(®) software according to a Bernoulli model. RESULTS: From June 2009 to May 2010, 296 clinical malaria cases were recorded. Though clearly temporally related to the rains, Plasmodium falciparum occurrence persisted late in the dry season. Two "hot spots" of malaria transmission also found, notably along the Yamé River, characterized by higher than expected numbers of malaria cases, and high numbers of clinical episodes per child. Conversely, the north-eastern sector of the town had fewer cases despite its proximity to a large body of standing water which was mosquito habitat. CONCLUSION: These results confirm the existence of a marked spatial heterogeneity of malaria transmission in Bandiagara, providing support for implementation of targeted interventions.

Antigen-specific B memory cell responses to Plasmodium falciparum malaria antigens and Schistosoma haematobium antigens in co-infected Malian children.

Polyparasitism is common in the developing world. We have previously demonstrated that schistosomiasis-positive (SP) Malian children have age-dependent protection from malaria compared to matched schistosomiasis-negative (SN) children. Evidence of durable immunologic memory to malaria antigens is conflicting, particularly in young children and the effect of concomitant schistomiasis upon acquisition of memory is unknown. We examined antigen-specific B memory cell (MBC) frequencies (expressed as percentage of total number of IgG-secreting cells) in 84 Malian children aged 4-14 to malaria blood-stage antigens, apical membrane antigen 1 (AMA-1) and merozoite surface protein 1 (MSP-1) and to schistosomal antigens, Soluble Worm Antigenic Preparation (SWAP) and Schistosoma Egg Antigen (SEA), at a time point during the malaria transmission season and a follow-up dry season visit. We demonstrate, for the first time, MBC responses to S. haematobium antigens in Malian children with urinary egg excretion and provide evidence of seasonal acquisition of immunologic memory, age-associated differences in MBC acquisition, and correlation with circulating S. haematobium antibody. Moreover, the presence of a parasitic co-infection resulted in older children, aged 9-14 years, with underlying S. haematobium infection having significantly more MBC response to malaria antigens (AMA1 and MSP1) than their age-matched SN counterparts. We conclude that detectable MBC response can be measured against both malaria and schistosomal antigens and that the presence of S. haematobium may be associated with enhanced MBC induction in an age-specific manner.

Reduced T regulatory cell response during acute Plasmodium falciparum infection in Malian children co-infected with Schistosoma haematobium.

BACKGROUND: Regulatory T cells (Tregs) suppress host immune responses and participate in immune homeostasis. In co-infection, secondary parasite infections may disrupt the immunologic responses induced by a pre-existing parasitic infection. We previously demonstrated that schistosomiasis-positive (SP) Malian children, aged 4-8 years, are protected against the acquisition of malaria compared to matched schistosomiasis-negative (SN) children. METHODS AND FINDINGS: To determine if Tregs contribute to this protection, we performed immunologic and Treg depletion in vitro studies using PBMC acquired from children with and without S. haematobium infection followed longitudinally for the acquisition of malaria. Levels of Tregs were lower in children with dual infections compared to children with malaria alone (0.49 versus 1.37%, respectively, P = 0.004) but were similar months later, during a period with negligible malaria transmission. The increased levels of Tregs in SN subjects were associated with suppressed serum Th1 cytokine levels, as well as elevated parasitemia compared to co-infected counterparts. CONCLUSIONS: These results suggest that lower levels of Tregs in helminth-infected children correlate with altered circulating cytokine and parasitologic results which may play a partial role in mediating protection against falciparum malaria.

A field trial to assess a blood-stage malaria vaccine.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.).

Persistence of full-length caspase-12 and its relation to malaria in West and Central African populations.

BACKGROUND: The full-length (L-) variant of caspase-12 is believed to predispose to sepsis. It has been replaced in the genome of most human populations by the (S-) variant, which leads to premature termination of translation. Strikingly, the L-allele is still widely prevalent in African populations, presumably due to a counterbalancing selective force specific to this continent, for which malaria is a prime candidate. METHODS: We investigated associations between caspase-12 genotype and malarial parameters in three West-African populations, in studies encompassing immunological, clinical and obstetric data. RESULTS: The caspase-12 L-allele was found at frequencies of 11-34%. Plasmodium falciparum-stimulated mononuclear cells from S/L heterozygote donors produced stronger interferon-gamma and interleukin-10 responses than S/S homozygotes (p = 0.011 and p = 0.023 in uninfected and infected donors respectively). Nevertheless, we found no association between caspase-12 genotype and either the presentation of severe malaria or individual clinical parameters in sick children. Amongst pregnant women, the caspase-12 genotype did not influence peripheral or placental malaria infection, or basic obstetric parameters. Interestingly, perinatal mortality was more frequent in children of both S/S and L/L than S/L mothers, independent of placental P. falciparum-infection. CONCLUSION: We find little clinical or epidemiological evidence that malaria has contributed to the persistence of functional caspase-12 in Africa, suggesting either that alternative selective forces are at work or that genetic drift underlies its current global distribution.

In Tanzania, hemolysis after a single dose of primaquine coadministered with an artemisinin is not restricted to glucose-6-phosphate dehydrogenase-deficient (G6PD A-) individuals.

The current interest in malaria elimination has led to a renewed interest in drugs that can be used for mass administration to minimize malaria transmission. Primaquine (PQ) is the only generally available drug with a strong activity against mature Plasmodium falciparum gametocytes, the parasite stage responsible for transmission. Despite concerns about PQ-induced hemolysis in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals, a single dose of PQ may be safe and efficacious in clearing gametocytes that persist after conventional treatment. As part of a mass drug intervention, we determined the hemolytic effect of sulfadoxine-pyrimethamine (SP) plus artesunate (AS) plus a single dose of primaquine (PQ; 0.75 mg/kg of body weight) in children aged 1 to 12 years. Children were randomized to receive SP+AS+PQ or placebo; those with a hemoglobin (Hb) level below 8 g/dl were excluded from receiving PQ and received SP+AS. The Hb concentration was significantly reduced 7 days after SP+AS+PQ treatment but not after placebo or SP+AS treatment. This reduction in Hb was most pronounced in G6PD-deficient (G6PD A-) individuals (-2.5 g/dl; 95% confidence interval [95% CI], -1.2 to -3.8 g/dl) but was also observed in heterozygotes (G6PD A) (-1.6 g/dl; 95% CI, -0.9 to -2.2 g/dl) and individuals with the wild-type genotype (G6PD B) (-0.5 g/dl; 95% CI, -0.4 to -0.6 g/dl). Moderate anemia (Hb level of <8 g/dl) was observed in 40% (6/15 individuals) of the G6PD A-, 11.1% (3/27 individuals) of the G6PD A, and 4.5% (18/399 individuals) of the G6PD B individuals; one case of severe anemia (Hb level of <5 g/dl) was observed. PQ may cause moderate anemia when coadministered with artemisinins, and excluding individuals based on G6PD status alone may not be sufficient to prevent PQ-induced hemolysis.

Safety and immunogenicity of an AMA1 malaria vaccine in Malian children: results of a phase 1 randomized controlled trial.

BACKGROUND: The objective was to evaluate the safety and immunogenicity of the AMA1-based malaria vaccine FMP2.1/AS02(A) in children exposed to seasonal falciparum malaria. METHODOLOGY/PRINCIPAL FINDINGS: A Phase 1 double blind randomized controlled dose escalation trial was conducted in Bandiagara, Mali, West Africa, a rural town with intense seasonal transmission of Plasmodium falciparum malaria. The malaria vaccine FMP2.1/AS02(A) is a recombinant protein (FMP2.1) based on apical membrane antigen 1 (AMA1) from the 3D7 clone of P. falciparum, formulated in the Adjuvant System AS02(A). The comparator vaccine was a cell-culture rabies virus vaccine (RabAvert). One hundred healthy Malian children aged 1-6 years were recruited into 3 cohorts and randomized to receive either 10 microg FMP2.1 in 0.1 mL AS02(A), or 25 microg FMP2.1 in 0.25 mL AS02(A), or 50 microg FMP2.1 50 microg in 0.5 mL AS02(A), or rabies vaccine. Three doses of vaccine were given at 0, 1 and 2 months, and children were followed for 1 year. Solicited symptoms were assessed for 7 days and unsolicited symptoms for 30 days after each vaccination. Serious adverse events were assessed throughout the study. Transient local pain and swelling were common and more frequent in all malaria vaccine dosage groups than in the comparator group, but were acceptable to parents of participants. Levels of anti-AMA1 antibodies measured by ELISA increased significantly (at least 100-fold compared to baseline) in all 3 malaria vaccine groups, and remained high during the year of follow up. CONCLUSION/SIGNIFICANCE: The FMP2.1/AS02(A) vaccine had a good safety profile, was well-tolerated, and induced high and sustained antibody levels in malaria-exposed children. This malaria vaccine is being evaluated in a Phase 2 efficacy trial in children at this site. TRIAL REGISTRATION: ClinicalTrials.gov NCT00358332 [NCT00358332].

Early interferon-gamma response against Plasmodium falciparum correlates with interethnic differences in susceptibility to parasitemia between sympatric Fulani and Dogon in Mali.

INTRODUCTION: Interethnic differences in susceptibility to malaria provide a unique opportunity to explore immunological correlates of protection. The Fulani of Sahelian Africa are known for their reduced susceptibility to Plasmodium falciparum, compared with surrounding tribes, yet the immunology underlying this is still poorly understood. METHODS AND RESULTS: Here, we show that mononuclear cells from Fulani elicit >10-fold stronger interferon (IFN)-gamma production following a 24-h in vitro coincubation with asexual parasites than cells from sympatric Dogon. This response appears to be specific for P. falciparum among a panel of other human pathogens and is independent of the lower number of regulatory T cell counts present in Fulani. IFN-gamma responses in both tribes were inversely correlated with peripheral parasite density as quantified by nucleic acid sequenced-based amplification, but responses of Fulani remained significantly stronger than those of Dogon after adjustment for concurrent parasitemia, suggesting that hard-wired immunological differences underlie the observed protection. CONCLUSIONS: These results underscore the value of early IFN-gamma responses to P. falciparum as a correlate of anti-parasite immunity, not only in this setting but also in the wider context of malaria, and support the development of malaria vaccines aimed at inducing such responses.

Caspase-12 and the inflammatory response to Yersinia pestis.

BACKGROUND: Caspase-12 functions as an antiinflammatory enzyme inhibiting caspase-1 and the NOD2/RIP2 pathways. Due to increased susceptibility to sepsis in individuals with functional caspase-12, an early-stop mutation leading to the loss of caspase-12 has replaced the ancient genotype in Eurasia and a significant proportion of individuals from African populations. In African-Americans, it has been shown that caspase-12 inhibits the pro-inflammatory cytokine production. METHODOLOGY/PRINCIPAL FINDINGS: We assessed whether similar mechanisms are present in African individuals, and whether evolutionary pressures due to plague may have led to the present caspase-12 genotype population frequencies. No difference in cytokine induction through the caspase-1 and/or NOD2/RIP2 pathways was observed in two independent African populations, among individuals with either an intact or absent caspase-12. In addition, stimulations with Yersinia pestis and two other species of Yersinia were preformed to investigate whether caspase-12 modulates the inflammatory reaction induced by Yersinia. We found that caspase-12 did not modulate cytokine production induced by Yersinia spp. CONCLUSIONS: Our experiments demonstrate for the first time the involvement of the NOD2/RIP2 pathway for recognition of Yersinia. However, caspase-12 does not modulate innate host defense against Y. pestis and alternative explanations for the geographical distribution of caspase-12 should be sought.