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Malaria and iron deficiency (ID) are common and interrelated public health problems in African children. Observational data suggest that interrupting malaria transmission reduces the prevalence of ID1. To test the hypothesis that malaria might cause ID, we used sickle cell trait (HbAS, rs334 ), a genetic variant that confers specific protection against malaria2, as an instrumental variable in Mendelian randomization analyses. HbAS was associated with a 30% reduction in ID among children living in malaria-endemic countries in Africa (n = 7,453), but not among individuals living in malaria-free areas (n = 3,818). Genetically predicted malaria risk was associated with an odds ratio of 2.65 for ID per unit increase in the log incidence rate of malaria. This suggests that an intervention that halves the risk of malaria episodes would reduce the prevalence of ID in African children by 49%. A genetic link suggests that interventions that halve the risk of malaria episodes could reduce the prevalence of iron deficiency in African children by nearly 50%.

After decades of research, our understanding of when and why individuals infected with Plasmodium falciparum develop clinical malaria is still limited. Correlates of immune protection are often sought through prospective cohort studies, where measured host factors are correlated against the incidence of clinical disease over a set period of time. However, robustly inferring individual-level protection from these population-level findings has proved difficult due to small effect sizes and high levels of variance underlying such data. In order to better understand the nature of these inter-individual variations, we analysed the long-term malaria epidemiology of children ≤12 years old growing up under seasonal exposure to the parasite in the sub-location of Junju, Kenya. Despite the cohort’s limited geographic expanse (ca. 3km x 10km), our data reveal a high degree of spatial and temporal variability in malaria prevalence and incidence rates, causing individuals to experience varying levels of exposure to the parasite at different times during their life. Analysing individual-level infection histories further reveal an unexpectedly high variability in the rate at which children experience clinical malaria episodes. Besides exposure to the parasite, measured as disease prevalence in the surrounding area, we find that the birth time of year has an independent effect on the individual’s risk of experiencing a clinical episode. Furthermore, our analyses reveal that those children with a history of an above average number of episodes are more likely to experience further episodes during the upcoming transmission season. These findings are indicative of phenotypic differences in the rates by which children acquire clinical protection to malaria and offer important insights into the natural variability underlying malaria epidemiology.

Background Children living in sub-Saharan Africa have a high burden of rickets and infectious diseases, conditions that are linked to vitamin D deficiency. However, data on the vitamin D status of young African children and its environmental and genetic predictors are limited. We aimed to examine the prevalence and predictors of vitamin D deficiency in young African children. Methods We measured 25-hydroxyvitamin D (25(OH)D) and typed the single nucleotide polymorphisms, rs4588 and rs7041, in the GC gene encoding the vitamin D binding protein (DBP) in 4509 children aged 0–8 years living in Kenya, Uganda, Burkina Faso, The Gambia and South Africa. We evaluated associations between vitamin D status and country, age, sex, season, anthropometric indices, inflammation, malaria and DBP haplotypes in regression analyses. Results Median age was 23.9 months (interquartile range [IQR] 12.3, 35.9). Prevalence of vitamin D deficiency using 25(OH)D cut-offs of Conclusions Approximately 0.6% and 7.8% of young African children were vitamin D deficient as defined by 25(OH)D levels

Background: Studies of long-term malaria cohorts have provided essential insights into how Plasmodium falciparum interacts with humans, and influences the development of antimalarial immunity. Immunity to malaria is acquired gradually after multiple infections, some of which present with clinical symptoms. However, there is considerable variation in the number of clinical episodes experienced by children of the same age within the same cohort. Understanding this variation in clinical symptoms and how it relates to the development of naturally acquired immunity is crucial in identifying how and when some children stop experiencing further malaria episodes. Where variability in clinical episodes may result from different rates of acquisition of immunity, or from variable exposure to the parasite. Methods: Using data from a longitudinal cohort of children residing in an area of moderate P. falciparum transmission in Kilifi district, Kenya, we fitted cumulative episode curves as monotonic-increasing splines, to 56 children under surveillance for malaria from the age of 5 to 15. Results: There was large variability in the accumulation of numbers of clinical malaria episodes experienced by the children, despite being of similar age and living in the same general location. One group of children from a particular sub-region of the cohort stopped accumulating clinical malaria episodes earlier than other children in the study. Despite lack of further clinical episodes of malaria, these children had higher asymptomatic parasite densities and higher antibody titres to a panel of P. falciparum blood-stage antigens. Conclusions: This suggests development of clinical immunity rather than lack of exposure to the parasite, and supports the view that this immunity to malaria disease is maintained by a greater exposure to P. falciparum, and thus higher parasite burdens. Our study illustrates the complexity of anti-malaria immunity and underscores the need for analyses which can sufficiently reflect the heterogeneity within endemic populations.

The FPN Q248H mutation protects children from anemia, hemolysis, and iron deficiency, but not malaria or bacterial infection., Iron acquisition is critical for life. Ferroportin (FPN) exports iron from mature erythrocytes, and deletion of the Fpn gene results in hemolytic anemia and increased fatality in malaria-infected mice. The FPN Q248H mutation (glutamine to histidine at position 248) renders FPN partially resistant to hepcidin-induced degradation and was associated with protection from malaria in human studies of limited size. Using data from cohorts including over 18,000 African children, we show that the Q248H mutation is associated with modest protection against anemia, hemolysis, and iron deficiency, but we found little evidence of protection against severe malaria or bacteremia. We additionally observed no excess Plasmodium growth in Q248H erythrocytes ex vivo, nor evidence of selection driven by malaria exposure, suggesting that the Q248H mutation does not protect from malaria and is unlikely to deprive malaria parasites of iron essential for their growth.

Background Human infection studies (HIS) that involve deliberately infecting healthy volunteers with a pathogen raise important ethical issues, including the need to ensure that benefits and burdens are understood and appropriately accounted for. Building on earlier work, we embedded social science research within an ongoing malaria human infection study in coastal Kenya to understand the study benefits and burdens experienced by study stakeholders in this low-resource setting and assess the wider implications for future research planning and policy. Methods Data were collected using qualitative research methods, including in-depth interviews (44), focus group discussions (10) and non-participation observation. Study participants were purposively selected (key informant or maximal diversity sampling), including volunteers in the human infection study, study staff, community representatives and local administrative authorities. Data were collected during and up to 18 months following study residency, from sites in Coastal and Western Kenya. Voice recordings of interviews and discussions were transcribed, translated, and analysed using framework analysis, combining data- and theory-driven perspectives. Findings Physical, psychological, economic and social forms of benefits and burdens were experienced across study stages. Important benefits for volunteers included the study compensation, access to health checks, good residential living conditions, new learning opportunities, developing friendships and satisfaction at contributing towards a new malaria vaccine. Burdens primarily affected study volunteers, including experiences of discomfort and ill health; fear and anxiety around aspects of the trial process, particularly deliberate infection and the implications of prolonged residency; anxieties about early residency exit; and interpersonal conflict. These issues had important implications for volunteers’ families, study staff and the research institution’s reputation more widely. Conclusion Developing ethically and scientifically strong HIS relies on grounded accounts of volunteers, study staff and the wider community, understood in the socioeconomic, political and cultural context where studies are implemented. Recognition of the diverse, and sometimes perverse, nature of potential benefits and burdens in a given context, and who this might implicate, is critical to this process. Prior and ongoing stakeholder engagement is core to developing these insights.

Atypical memory B cells (aMBCs) are found in elevated numbers in individuals exposed to malaria. A key question is whether malaria induces aMBCs as a result of exposure to Ag, or non-Ag-specific mechanisms. We identified Plasmodium and bystander tetanus toxoid (TT) specific B cells in individuals from areas of previous and persistent exposure to malaria using tetramers. Malaria-specific B cells were more likely to be aMBCs than TT-specific B cells. However, TT-specific B cells from individuals with continuous exposure to malaria were more likely to be aMBCs than TT-specific B cells in individuals from areas where transmission has ceased. Finally, sequences of BCRs specific for a blood stage malaria-Ag were more highly mutated than sequences from TT-specific BCRs and under strong negative selection, indicative of ongoing antigenic pressure. Our data suggest both persistent Ag exposure and the inflammatory environment shape the B-cell response to malaria and bystander Ags.

Malaria is caused by apicomplexan parasites of the genus Plasmodium. While infection continues to pose a risk for the majority of the global population, the burden of disease mainly resides in Sub-Saharan Africa. Although immunity develops against disease, this requires years of persistent exposure and is not associated with protection against infection. Repeat infections occur due to the parasite's ability to disrupt or evade the host immune responses. However, despite many years of study, the mechanisms of this disruption remain unclear. Previous studies have demonstrated a parasite-induced failure in dendritic cell (DCs) function affecting the generation of helper T cell responses. These T cells fail to help B cell responses, reducing the production of antibodies that are necessary to control malaria infection. This review focuses on our current understanding of the effect of Plasmodium parasite on DC function, DC-T cell interaction, and T cell activation. A better understanding of how parasites disrupt DC-T cell interactions will lead to new targets and approaches to reinstate adaptive immune responses and enhance parasite immunity.

The World Health Organization’s Global Malaria Program implemented a multi-country study to assess the entomological and epidemiological impact of long-lasting insecticide treated nets (LLINs) and indoor residual spray (IRS). Study activities included home visits to perform a LLIN survey and malaria testing of children in the household. In Kenya, Fionet™, a technology for automated malaria Rapid Diagnostic Test (mRDT) processing and interpretation at point-of-care, was evaluated against traditional paper-based methods and manual mRDT processing. Objective: To measure and compare the accuracy of diagnosis, completeness, and timeliness of data transmission between a digital mobile solution (Fionet™) and a paper-based system. Methodology: A randomised cluster sampling design of two cohorts: 1) an Active Infection Detection cohort, and 2) an Active Case Detection cohort was undertaken between November 2013 and April 2014. Community Health Workers (CHWs) visited rural households to: 1) measure malaria prevalence in children under the age of five using mRDTs, and 2) survey the use and physical status of LLINs in the household. Ten clusters were randomly assigned to Fionet™ to perform automated testing, interpretation, and survey data capture. Fionet™ transmitted all the tests and survey data to a cloud-based database. Results: A total of 1770 households were visited, 437 children tested, and 742 LLINs inspected. Fionet™ significantly improved the quality of data gathered; a two-fold increase in adherence to study protocols using Fionet™ resulted in more accurate data, data completeness was 10 times higher than with paper-based collection methods, and 87% of data were available in less than one day. Fionet™ significantly improved data quality and management, which enhanced the health system’s ability to meet the research objectives. This technology can help ensure accurate, complete, and timely availability of data. Future studies should incorporate mobile technologies such as Fionet™ to improve RDT based diagnostics of malaria and data quality.

Background Iron deficiency (ID) is a major public health burden in African children and accurate prevalence estimates are important for effective nutritional interventions. However, ID may be incorrectly estimated in Africa because most measures of iron status are altered by inflammation and infections such as malaria. Through the current study, we have assessed different approaches to the prediction of iron status and estimated the burden of ID in African children. Methods We assayed iron and inflammatory biomarkers in 4853 children aged 0–8 years from Kenya, Uganda, Burkina Faso, South Africa, and The Gambia. We described iron status and its relationship with age, sex, inflammation, and malaria parasitemia. We defined ID using the WHO guideline (ferritin Results The prevalence of ID was highest at 1 year of age and in male infants. Inflammation and malaria parasitemia were associated with all iron biomarkers, although transferrin saturation was least affected. Overall prevalence of WHO-defined ID was 34% compared to 52% using the inflammation and malaria regression-corrected estimate. This unidentified burden of ID increased with age and was highest in countries with high prevalence of inflammation and malaria, where up to a quarter of iron-deficient children were misclassified as iron replete. Transferrin saturation Conclusions The prevalence of ID is underestimated in African children when defined using the WHO guidelines, especially in malaria-endemic populations, and the use of transferrin saturation may provide a more accurate approach. Further research is needed to identify the most accurate measures for determining the prevalence of ID in sub-Saharan Africa.

Background Iron deficiency (ID) and malaria are common causes of ill-health and disability among children living in sub-Saharan Africa. Although iron is critical for the acquisition of humoral immunity, little is known about the effects of ID on antibody responses to Plasmodium falciparum malaria. Methods The study included 1794 Kenyan and Ugandan children aged 0–7 years. We measured biomarkers of iron and inflammation, and antibodies to P. falciparum antigens including apical merozoite antigen 1 (anti-AMA-1) and merozoite surface antigen 1 (anti-MSP-1) in cross-sectional and longitudinal studies. Results The overall prevalence of ID was 31%. ID was associated with lower anti-AMA-1 and anti-MSP-1 antibody levels in pooled analyses adjusted for age, sex, study site, inflammation, and P. falciparum parasitemia (adjusted mean difference on a log-transformed scale (β) −0.46; 95 confidence interval [CI], −.66, −.25 P, Iron deficiency is associated with reduced P falciparum-specific antibody levels. Ferritin, transferrin saturation, hepcidin, and soluble transferrin receptor levels were associated with malaria-specific antibody levels. Iron deficiency in early life may influence subsequent malaria antibody levels.

Background: Vitamin D deficiency is linked to infections, rickets, and poor child development. However, data on the vitamin D status of African children are limited. We examined the prevalence and predictors of low vitamin D status in young African children. Methods: We measured 25-hydroxyvitamin D (25OHD), C-reactive protein (CRP), malaria parasitaemia, and anthropometry among 3880 children aged 0-8 years living in Kenya, Uganda, Burkina Faso, and South Africa. We evaluated associations between vitamin D status and country, age, sex, anthropometric indices, inflammation and malaria parasitaemia in regression analyses and structural equation models. Findings: The overall prevalence of vitamin D deficiency using 25OHD cut-offs of

Summary B‐cell and antibody responses to Plasmodium spp., the parasite that causes malaria, are critical for control of parasitemia and associated immunopathology. Antibodies also provide protection to reinfection. Long‐lasting B‐cell memory has been shown to occur in response to Plasmodium spp. in experimental model infections, and in human malaria. However, there are reports that antibody responses to several malaria antigens in young children living with malaria are not similarly long‐lived, suggesting a dysfunction in the maintenance of circulating antibodies. Some studies attribute this to the expansion of atypical memory B cells (AMB), which express multiple inhibitory receptors and activation markers, and are hyporesponsive to B‐cell receptor (BCR) restimulation in vitro. AMB are also expanded in other chronic infections such as tuberculosis, hepatitis B and C, and HIV, as well as in autoimmunity and old age, highlighting the importance of understanding their role in immunity. Whether AMB are dysfunctional remains controversial, as there are also studies in other infections showing that AMB can produce isotype‐switched antibodies and in mouse can contribute to protection against infection. In light of these controversies, we review the most recent literature on either side of the debate and challenge some of the currently held views regarding B‐cell responses to Plasmodium infections.

Background: Iron deficiency (ID) is a major public health burden in African children and accurate prevalence estimates are important for effective nutritional interventions. However, ID may be commonly underestimated in Africa because most measures of iron status are altered by inflammation and infections such as malaria. Methods: We assayed iron and inflammatory biomarkers in 4,853 children aged 0-8 years from Kenya, Uganda, Burkina Faso, South Africa, and The Gambia. We described iron status and its relationship with age, sex, malaria parasitaemia, inflammation, stunting, underweight, and wasting. We defined ID using the WHO ferritin-based definition (ferritin 5mg/L)) and then used regression modelling, correcting for inflammation and malaria, to estimate 'true' prevalence of ID. We further investigated the utility of other iron biomarkers in predicting ID. Findings: The prevalence of ID was highest at one year of age and in male children. Stunting and underweight were associated with anaemia and iron deficient erythropoiesis. Inflammation and malaria parasitaemia significantly influenced all iron biomarkers, although transferrin saturation was least affected. The WHO definition of ID did not identify 27·0% of Burkinabe, 21·4% of Kenyan, 20·0% of Ugandan, 16·8% of Gambian, and 8·5% of South African children with 'true' iron deficiency. This 'hidden burden' increased with age and with increasing prevalence of inflammation and malaria. We observed similar prevalences of ID using regression modelling and transferrin saturation

Background Gut microbiota were recently shown to impact malaria disease progression and outcome, and prior studies have shown that Plasmodium infections increase the likelihood of enteric bacteria causing systemic infections. Currently, it is not known whether Plasmodium infection impacts human gut microbiota as a prelude to bacteremia or whether antimalarials affect gut microbiota. Our goal was to determine to what degree Plasmodium infections and antimalarial treatment affect human gut microbiota. Methods One hundred Kenyan infants underwent active surveillance for malaria from birth to 10 months of age. Each malaria episode was treated with artemether-lumefantrine (AL). Any other treatments, including antibiotics, were recorded. Stool samples were collected on an approximately biweekly basis. Ten children were selected on the basis of stool samples having been collected before (n = 27) or after (n = 17) a malaria episode and without antibiotics having been administered between collections. These samples were subjected to 16S ribosomal ribonucleic acid gene (V3–V4 region) sequencing. Results Bacterial community network analysis revealed no obvious differences in the before and after malaria/AL samples, which was consistent with no difference in alpha and beta diversity and taxonomic analysis at the family and genus level with one exception. At the sequence variant (SV) level, akin to bacterial species, only 1 of the top 100 SVs was significantly different. In addition, predicted metagenome analysis revealed no significant difference in metagenomic capacity between before and after malaria/AL samples. The number of malaria episodes, 1 versus 2, explained significant variation in gut microbiota composition of the infants. Conclusions In-depth bioinformatics analysis of stool bacteria has revealed for the first time that human malaria episode/AL treatment have minimal effects on gut microbiota in Kenyan infants., Although malaria-induced dysbiosis of gut microbiota has been postulated to contribute towards increased susceptibility to Non-typhoid Salmonella infections, this report demonstrates that Plasmodium infections and oral treatment with antimalarial drugs do not alter stool bacteria populations in Kenyan infants.

We present data from 3 cohorts in 1 geographical area with varying levels of malaria transmission. The results show that asymptomatic parasitemia is modified by transmission level and age, which alter the risk of developing febrile malaria., Background Plasmodium falciparum infections lead to febrile illness unless the host has sufficient immunity, in which case infection may cause no immediate symptoms (ie, “asymptomatic parasitemia”). Previous studies are conflicting on the role of asymptomatic parasitemia in determining the risk of developing febrile malaria. Methods We monitored 2513 children (living in Kilifi, Kenyan Coast) by blood smears in 17 cross-sectional surveys to identify asymptomatic parasitemia and used active surveillance over 11325 child-years of follow-up to detect febrile malaria. We evaluated the interaction between transmission intensity, age, and asymptomatic parasitemia in determining the risk of developing febrile malaria. Results In the moderate and high transmission intensity settings, asymptomatic parasitemia was associated with a reduced risk of febrile malaria in older children (> 3 years), while in the lower transmission setting, asymptomatic parasitemia was associated with an increased risk of febrile malaria in children of all ages. Additionally, the risk associated with asymptomatic parasitemia was limited to the first 90 days of follow-up. Conclusions Asymptomatic parasitemia is modified by transmission intensity and age, altering the risk of developing febrile episodes and suggesting that host immunity plays a prominent role in mediating this process.

Background It remains unclear whether improving iron status increases malaria risk, and few studies have looked at the effect of host iron status on subsequent malaria infection. We therefore aimed to determine whether a child’s iron status influences their subsequent risk of malaria infection in sub-Saharan Africa. Methods We assayed iron and inflammatory biomarkers from community-based cohorts of 1309 Kenyan and 1374 Ugandan children aged 0–7 years and conducted prospective surveillance for episodes of malaria. Poisson regression models were fitted to determine the effect of iron status on the incidence rate ratio (IRR) of malaria using longitudinal data covering a period of 6 months. Models were adjusted for age, sex, parasitemia, inflammation, and study site. Results At baseline, the prevalence of iron deficiency (ID) was 36.9% and 34.6% in Kenyan and Ugandan children, respectively. ID anemia (IDA) affected 23.6% of Kenyan and 17.6% of Ugandan children. Malaria risk was lower in children with ID (IRR, 0.7; 95% confidence interval [CI], 0.6, 0.8; P < .001) and IDA (IRR, 0.7; 95% CI, 0.6, 0.9; P = .006). Low transferrin saturation (, Decreased ferritin and transferrin saturation are associated with protection against malaria in African children. Hepcidin, soluble transferrin receptor, and hemoglobin concentrations are not associated with malaria protection. These findings may reflect differences in parasite iron acquisition.

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

Background: To restrict trial endpoints to infections acquired after vaccination in Phase IIb trials of candidate malaria vaccines, participants are treated with anti-malarial drugs to clear existing infections. Anti-malarial drugs with a long half-life may inhibit the acquisition of new infections. This study evaluated the effects of three anti-malarial drug regimens on the clearance of existing infections and acquisition of new infections. Methods: An open-label randomised controlled trial (MalPaC) was conducted between November 2013 and February 2014. Ninety adults were randomised 1:1:1 to receive one of three treatments: atovaquone/proguanil and artesunate (AP+AS); artesunate (AS); or sulphadoxine-pyrimethamine, artesunate, and primaquine (SP+AS+PQ). Parasite monitoring was determined over 84-day follow-up by assessing Plasmodium falciparum positivity by 18s qPCR, live and sexual stage parasites by RT-PCR, and recrudescence of infections by msp2 genotyping. Results: At enrolment, parasite prevalence by qPCR was 44% (40/90, day 0), which fell to 10% (9/90, day 16), then rose to almost the initial rates by day 84 (39%, 35/90). Individuals treated with AS and SP+AS+PQ were more likely to have higher qPCR positive rates compared to participants treated with AP+AS in the immediate post-treatment phase (days 16-28) (OR=7.7 [95%CI 4.6-12.8] p Conclusion: Falciparum DNA remained detectable by PCR post-treatment with incomplete parasite clearance regardless of drug regimen. Though AP+AS drug regimen may also have partially suppressed the acquisition of new infections during post-treatment follow-up. Trial registration: Pan African Clinical Trials Registry, 22nd of August 2013, PACTR201309000625311.

Antibodies (Abs) are critical for immunity to malaria. However, Plasmodium falciparum specific Abs decline rapidly in absence of reinfection, suggesting impaired immunological memory. This study determines whether residents of Sweden that were treated for malaria following international travel maintained long-lasting malaria-specific Abs and memory B cells (MBCs). We compared levels of malaria-specific Abs and MBCs between 47 travelers who had been admitted with malaria at the Karolinska University Hospital between 1 and 16 years previously, eight malaria-naïve adult Swedes without histories of travel, and 14 malaria-immune adult Kenyans. Plasmodium falciparum-lysate-specific Ab levels were above naïve control levels in 30% of the travelers, whereas AMA-1, merozoite surface protein-142 , and merozoite surface protein-3-specific Ab levels were similar. In contrast, 78% of travelers had IgG-MBCs specific for at least one malaria antigen (59, 45, and 28% for apical merozoite antigen-1, merozoite surface protein-1, and merozoite surface protein-3, respectively) suggesting that malaria-specific MBCs are maintained for longer than the cognate serum Abs in the absence of re-exposure to parasites. Five travelers maintained malaria antigen-specific MBC responses for up to 16 years since the diagnosis of the index episode (and had not traveled to malaria-endemic regions in the intervening time). Thus P. falciparum can induce long-lasting MBCs, maintained for up to 16 years without reexposure.

The study received funding from the UK Medical Research Council, (MRC Programme grant #: MR/M003906/1). MB and AR are supported by the Wellcome Trust (Grant #: WT 206194). Background There are over 200 million reported cases of malaria each year, and most children living in endemic areas will experience multiple episodes of clinical disease before puberty. We set out to understand how frequent clinical malaria, which elicits a strong inflammatory response, affects the immune system and whether these modifications are observable in the absence of detectable parasitaemia. Methods We used a multi-dimensional approach comprising whole blood transcriptomic, cellular and plasma cytokine analyses on a cohort of children living with endemic malaria, but uninfected at sampling, who had been under active surveillance for malaria for 8 years. Children were categorised into two groups depending on the cumulative number of episodes experienced: high (≥ 8) or low (

In two previously described donors, the extracellular domain of LAIR1, a collagen-binding inhibitory receptor encoded on chromosome 19 (ref. 1), was inserted between the V and DJ segments of an antibody. This insertion generated, through somatic mutations, broadly reactive antibodies against RIFINs, a type of variant antigen expressed on the surface of Plasmodium falciparum-infected erythrocytes. To investigate how frequently such antibodies are produced in response to malaria infection, we screened plasma from two large cohorts of individuals living in malaria-endemic regions. Here we report that 5-10% of malaria-exposed individuals, but none of the European blood donors tested, have high levels of LAIR1-containing antibodies that dominate the response to infected erythrocytes without conferring enhanced protection against febrile malaria. By analysing the antibody-producing B cell clones at the protein, cDNA and gDNA levels, we characterized additional LAIR1 insertions between the V and DJ segments and discovered a second insertion modality whereby the LAIR1 exon encoding the extracellular domain and flanking intronic sequences are inserted into the switch region. By exon shuffling, this mechanism leads to the production of bispecific antibodies in which the LAIR1 domain is precisely positioned at the elbow between the VH and CH1 domains. Additionally, in one donor the genomic DNA encoding the VH and CH1 domains was deleted, leading to the production of a camel-like LAIR1-containing antibody. Sequencing of the switch regions of memory B cells from European blood donors revealed frequent templated inserts originating from transcribed genes that, in rare cases, comprised exons with orientations and frames compatible with expression. These results reveal different modalities of LAIR1 insertion that lead to public and dominant antibodies against infected erythrocytes and suggest that insertion of templated DNA represents an additional mechanism of antibody diversification that can be selected in the immune response against pathogens and exploited for B cell engineering.

Memory B cells (MBCs) are a key component of long term humoral immunity to many human infectious diseases. Despite their importance, we know little about the generation or maintenance of antigen-(Ag)-specific MBCs in humans in response to infection. A frequently employed method for quantifying Ag-specific MBCs in human peripheral blood (Crotty et al., 2004) relies on the ability of MBCs but not naïve B cells to differentiate into antibody secreting cells (ASCs) in response to polyclonal activators and Toll-like receptor agonists in vitro and the measurement of Ag-specific ASCs by ELISPOT assays. Here we report on studies to optimize the efficiency of this ELISPOT-based assay and to apply this assay to the detection of Plasmodium falciparum (Pf)-specific MBCs in adults living in a malaria endemic area where immunity to Pf is acquired through natural infection. We show that the addition of IL-10 to in vitro cultures of human peripheral blood mononuclear cells increased the efficiency of the assay from 10% to over 90% without increasing the ASC burst size and without any substantial increase in background from naïve B cells or plasma cells (PCs). Using this assay we were able to quantify the frequency of Pf-specific MBCs in peripheral blood of adults living in a malaria endemic area. Thus, this highly efficient assay appears to be well suited to field studies of the generation and maintenance of MBCs where the volumes of blood obtainable are often limiting. © 2011 Elsevier B.V.

We have studied the human CD4 T cell response to a functionally conserved domain of Plasmodium falciparum erythrocyte membrane protein-1, cysteine interdomain region-1α (CIDR-1α). Responses to CIDR-1α were striking in that both exposed and nonexposed donors responded. The IFN-γ response to CIDR-1α in the nonexposed donors was partially independent of TCR engagement of MHC class II and peptide. Contrastingly, CD4 T cell and IFN-γ responses in malaria-exposed donors were MHC class II restricted, suggesting that the CD4 T cell response to CIDR-1α in malaria semi-immune adults also has a TCR-mediated component, which may represent a memory response. Dendritic cells isolated from human peripheral blood were activated by CIDR-1α to produce IL-12, IL-10, and IL-18. IL-12 was detectable only between 6 and 12 h of culture, whereas the IL-10 continued to increase throughout the 24-h time course. These data strengthen previous observations that P. falciparum interacts directly with human dendritic cells, and suggests that the interaction between CIDR-1α and the host cell may be responsible for regulation of the CD4 T cell and cytokine responses to P. falciparum-infected erythrocytes reported previously.

Antibodies and B cells are critical in the protective immune response to the blood stage of the malaria parasite, Plasmodium chabaudi. However, little is known about the development of memory B cells and their differentiation into plasma cells during infection or after re-infection. Here we have shown that B cells with phenotypic characteristics of memory cells (CD19(+)IgD(-) CD38(+), IgG1(+)) are generated in a primary Plasmodium chabaudi chabaudi infection of mice. In addition, we observed that germinal centre cells (CD19(+), GL7(+), MHCII(hi)) and Marginal Zone B cells (CD19(+)CD23(-)IgD(-)) show faster expansion on re-infection than in the primary, though other subsets do not. Interestingly, though both IgM(-) and IgM(+) memory cells are produced, IgM(+) memory cells do not expand on second infection. The second infection quickly produced mature bone marrow plasma cells (intracellular Ig(hi), CD138(hi), CD9(+), B220(-)), compared to primary infection; which generates a very large population of immature splenic plasma cells (B220+). This analysis suggests that a memory B cell population is generated after a single infection of malaria, which on re-infection responds quickly producing germinal centres and generating long-lived plasma cells making the second encounter with parasite more efficient.

During pregnancy, specific variants of Plasmodium falciparum-infected erythrocytes (IEs) can accumulate in the placenta through adhesion to chondroitin sulfate A (CSA) mediated by expression of PfEMP1 encoded by var2csa-type genes. Antibodies against these variants are associated with protection from maternal malaria. We evaluated antibodies among Kenyan, Papua New Guinean, and Malawian men and Kenyan children against two different CSA-binding P. falciparum isolates expressing var2csa variants. Specific IgG was present at significant levels among some men and children from each population, suggesting exposure to these variants is not exclusive to pregnancy. However, the level and prevalence of antibodies was substantially lower overall than exposed multigravidas. IgG-binding was specific and did not represent antibodies to subpopulations of non-CSA-binding IEs, and some sera inhibited IE adhesion to CSA. These findings have significant implications for understanding malaria pathogenesis and immunity and may be significant for understanding the acquisition of immunity to maternal malaria.

Background The distribution of Plasmodium falciparum clinical malaria episodes is over-dispersed among children in endemic areas, with more children experiencing multiple clinical episodes than would be expected based on a Poisson distribution. There is consistent evidence for micro-epidemiological variation in exposure to P. falciparum. The aim of the current study was to identify children with excess malaria episodes after controlling for malaria exposure. Methods We selected the model that best fit the data out of the models examined and included the following covariates: age, a weighted local prevalence of infection as an index of exposure, and calendar time to predict episodes of malaria on active surveillance malaria data from 2,463 children of under 15 years of age followed for between 5 and 15 years each. Using parameters from the zero-inflated negative binomial model which best fitted our data, we ran 100 simulations of the model based on our population to determine the variation that might be seen due to chance. Results We identified 212 out of 2,463 children who had a number of clinical episodes above the 95th percentile of the simulations run from the model, hereafter referred to as “excess malaria (EM)”. We then identified exposure-matched controls with “average numbers of malaria” episodes, and found that the EM group had higher parasite densities when asymptomatically infected or during clinical malaria, and were less likely to be of haemoglobin AS genotype. Conclusions Of the models tested, the negative zero-inflated negative binomial distribution with exposure, calendar year, and age acting as independent predictors, fitted the distribution of clinical malaria the best. Despite accounting for these factors, a group of children suffer excess malaria episodes beyond those predicted by the model. An epidemiological framework for identifying these children will allow us to study factors that may explain excess malaria episodes. Electronic supplementary material The online version of this article (doi:10.1186/s12916-015-0422-4) contains supplementary material, which is available to authorized users.

To gain insight into why antibody responses to malarial antigens tend to be short lived, we studied antigen-specific memory B cells from donors in an area where malaria is endemic. We compared antibody and memory B cell responses to tetanus toxoid with those to 3 Plasmodium falciparum candidate vaccine antigens: the C-terminal portion of merozoite surface protein 1 (MSP1(19)), apical membrane antigen 1 (AMA1), and the cysteine-rich interdomain region 1 alpha (CIDR1 alpha ) of a protein from the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family. These data are the first to be generated on memory B cells in children who are in the process of acquiring antimalarial immunity, and they reveal defects in B cell memory to P. falciparum antigens. Compared with the results for tetanus toxoid, more donors who were positive for antibody to AMA1 and CIDR1 alpha were negative for memory B cells. These data imply that some exposures to malaria do not result in the establishment of stable populations of circulating antigen-specific memory B cells, suggesting possible mechanisms for the short-lived nature of many anti-malarial antibody responses.

Antibodies to the highly diverse variant surface antigens (VSAs) expressed on Plasmodium falciparum-infected erythrocytes are thought to play a role in the development of naturally acquired immunity to malaria. It has been suggested that children gradually acquire immunity through the piecemeal acquisition of antibodies to a large number of VSAs over several years of exposure. However, in a cross-sectional survey of Kenyan children before the malaria-transmission season, the proportion of children with antibodies recognizing randomly sampled VSAs was found to be strikingly higher among children with microscopically detectable P. falciparum infection, compared with those without detectable infection. We suggest that parasitization status may be an important consideration in longitudinal assessments of the protective role of some anti-parasite immune responses and support this suggestion with data from a prospective study of VSA antibodies in a group of children who subsequently had severe malaria.