Natural malaria infection elicits rare but potent neutralizing antibodies to the blood-stage antigen RH5.

Plasmodium falciparum reticulocyte-binding protein homolog 5 (RH5) is the most advanced blood-stage malaria vaccine candidate and is being evaluated for efficacy in endemic regions, emphasizing the need to study the underlying antibody response to RH5 during natural infection, which could augment or counteract responses to vaccination. Here, we found that RH5-reactive B cells were rare, and circulating immunoglobulin G (IgG) responses to RH5 were short-lived in malaria-exposed Malian individuals, despite repeated infections over multiple years. RH5-specific monoclonal antibodies isolated from eight malaria-exposed individuals mostly targeted non-neutralizing epitopes, in contrast to antibodies isolated from five RH5-vaccinated, malaria-naive UK individuals. However, MAD8-151 and MAD8-502, isolated from two malaria-exposed Malian individuals, were among the most potent neutralizers out of 186 antibodies from both cohorts and targeted the same epitopes as the most potent vaccine-induced antibodies. These results suggest that natural malaria infection may boost RH5-vaccine-induced responses and provide a clear strategy for the development of next-generation RH5 vaccines.

Autoantibodies inhibit Plasmodium falciparum growth and are associated with protection from clinical malaria.

Many infections, including malaria, are associated with an increase in autoantibodies (AAbs). Prior studies have reported an association between genetic markers of susceptibility to autoimmune disease and resistance to malaria, but the underlying mechanisms are unclear. Here, we performed a longitudinal study of children and adults (n = 602) in Mali and found that high levels of plasma AAbs before the malaria season independently predicted a reduced risk of clinical malaria in children during the ensuing malaria season. Baseline AAb seroprevalence increased with age and asymptomatic Plasmodium falciparum infection. We found that AAbs purified from the plasma of protected individuals inhibit the growth of blood-stage parasites and bind P. falciparum proteins that mediate parasite invasion. Protected individuals had higher plasma immunoglobulin G (IgG) reactivity against 33 of the 123 antigens assessed in an autoantigen microarray. This study provides evidence in support of the hypothesis that a propensity toward autoimmunity offers a survival advantage against malaria.

A Molecular Signature in Blood Reveals a Role for p53 in Regulating Malaria-Induced Inflammation.

Immunity that controls parasitemia and inflammation during Plasmodium falciparum (Pf) malaria can be acquired with repeated infections. A limited understanding of this complex immune response impedes the development of vaccines and adjunctive therapies. We conducted a prospective systems biology study of children who differed in their ability to control parasitemia and fever following Pf infection. By integrating whole-blood transcriptomics, flow-cytometric analysis, and plasma cytokine and antibody profiles, we demonstrate that a pre-infection signature of B cell enrichment, upregulation of T helper type 1 (Th1) and Th2 cell-associated pathways, including interferon responses, and p53 activation associated with control of malarial fever and coordinated with Pf-specific immunoglobulin G (IgG) and Fc receptor activation to control parasitemia. Our hypothesis-generating approach identified host molecules that may contribute to differential clinical outcomes during Pf infection. As a proof of concept, we have shown that enhanced p53 expression in monocytes attenuated Plasmodium-induced inflammation and predicted protection from fever.

Subcutaneous Administration of a Monoclonal Antibody to Prevent Malaria.

BACKGROUND: Subcutaneous administration of the monoclonal antibody L9LS protected adults against controlled Plasmodium falciparum infection in a phase 1 trial. Whether a monoclonal antibody administered subcutaneously can protect children from P. falciparum infection in a region where this organism is endemic is unclear. METHODS: We conducted a phase 2 trial in Mali to assess the safety and efficacy of subcutaneous administration of L9LS in children 6 to 10 years of age over a 6-month malaria season. In part A of the trial, safety was assessed at three dose levels in adults, followed by assessment at two dose levels in children. In part B of the trial, children were randomly assigned, in a 1:1:1 ratio, to receive 150 mg of L9LS, 300 mg of L9LS, or placebo. The primary efficacy end point, assessed in a time-to-event analysis, was the first P. falciparum infection, as detected on blood smear performed at least every 2 weeks for 24 weeks. A secondary efficacy end point was the first episode of clinical malaria, as assessed in a time-to-event analysis. RESULTS: No safety concerns were identified in the dose-escalation part of the trial (part A). In part B, 225 children underwent randomization, with 75 children assigned to each group. No safety concerns were identified in part B. P. falciparum infection occurred in 36 participants (48%) in the 150-mg group, in 30 (40%) in the 300-mg group, and in 61 (81%) in the placebo group. The efficacy of L9LS against P. falciparum infection, as compared with placebo, was 66% (adjusted confidence interval [95% CI], 45 to 79) with the 150-mg dose and 70% (adjusted 95% CI, 50 to 82) with the 300-mg dose (P<0.001 for both comparisons). Efficacy against clinical malaria was 67% (adjusted 95% CI, 39 to 82) with the 150-mg dose and 77% (adjusted 95% CI, 55 to 89) with the 300-mg dose (P<0.001 for both comparisons). CONCLUSIONS: Subcutaneous administration of L9LS to children was protective against P. falciparum infection and clinical malaria over a period of 6 months. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT05304611.).

Safety and Efficacy of a Monoclonal Antibody against Malaria in Mali.

BACKGROUND: CIS43LS is a monoclonal antibody that was shown to protect against controlled Plasmodium falciparum infection in a phase 1 clinical trial. Whether a monoclonal antibody can prevent P. falciparum infection in a region in which the infection is endemic is unknown. METHODS: We conducted a phase 2 trial to assess the safety and efficacy of a single intravenous infusion of CIS43LS against P. falciparum infection in healthy adults in Mali over a 6-month malaria season. In Part A, safety was assessed at three escalating dose levels. In Part B, participants were randomly assigned (in a 1:1:1 ratio) to receive 10 mg of CIS43LS per kilogram of body weight, 40 mg of CIS43LS per kilogram, or placebo. The primary efficacy end point, assessed in a time-to-event analysis, was the first P. falciparum infection detected on blood-smear examination, which was performed at least every 2 weeks for 24 weeks. At enrollment, all the participants received artemether-lumefantrine to clear possible P. falciparum infection. RESULTS: In Part B, 330 adults underwent randomization; 110 were assigned to each trial group. The risk of moderate headache was 3.3 times as high with 40 mg of CIS43LS per kilogram as with placebo. P. falciparum infections were detected on blood-smear examination in 39 participants (35.5%) who received 10 mg of CIS43LS per kilogram, 20 (18.2%) who received 40 mg of CIS43LS per kilogram, and 86 (78.2%) who received placebo. At 6 months, the efficacy of 40 mg of CIS43LS per kilogram as compared with placebo was 88.2% (adjusted 95% confidence interval [CI], 79.3 to 93.3; P<0.001), and the efficacy of 10 mg of CIS43LS per kilogram as compared with placebo was 75.0% (adjusted 95% CI, 61.0 to 84.0; P<0.001). CONCLUSIONS: CIS43LS was protective against P. falciparum infection over a 6-month malaria season in Mali without evident safety concerns. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT04329104.).

Neither the African-Centric S47 Nor P72 Variant of TP53 Is Associated With Reduced Risk of Febrile Malaria in a Malian Cohort Study.

BACKGROUND: TP53 has been shown to play a role in inflammatory processes, including malaria. We previously found that p53 attenuates parasite-induced inflammation and predicts clinical protection to Plasmodium falciparum infection in Malian children. Here, we investigated whether p53 codon 47 and 72 polymorphisms are associated with differential risk of P. falciparum infection and uncomplicated malaria in a prospective cohort study of malaria immunity. METHODS: p53 codon 47 and 72 polymorphisms were determined by sequencing TP53 exon 4 in 631 Malian children and adults enrolled in the Kalifabougou cohort study. The effects of these polymorphisms on the prospective risk of febrile malaria, incident parasitemia, and time to fever after incident parasitemia over 6 months of intense malaria transmission were assessed using Cox proportional hazards models. RESULTS: Confounders of malaria risk, including age and hemoglobin S or C, were similar between individuals with or without p53 S47 and R72 polymorphisms. Relative to their respective common variants, neither S47 nor R72 was associated with differences in prospective risk of febrile malaria, incident parasitemia, or febrile malaria after parasitemia. CONCLUSIONS: These findings indicate that p53 codon 47 and 72 polymorphisms are not associated with protection against incident P. falciparum parasitemia or uncomplicated febrile malaria.

Accumulation of Neutrophil Phagocytic Antibody Features Tracks With Naturally Acquired Immunity Against Malaria in Children.

BACKGROUND: Studies have demonstrated the protective role of antibodies against malaria. Young children are known to be particularly vulnerable to malaria, pointing to the evolution of naturally acquired clinical immunity over time. However, whether changes in antibody functionality track with the acquisition of naturally acquired malaria immunity remains incompletely understood. METHODS: Using systems serology, we characterized sporozoite- and merozoite-specific antibody profiles of uninfected Malian children before the malaria season who differed in their ability to control parasitemia and fever following Plasmodium falciparum (Pf) infection. We then assessed the contributions of individual traits to overall clinical outcomes, focusing on the immunodominant sporozoite CSP and merozoite AMA1 and MSP1 antigens. RESULTS: Humoral immunity evolved with age, with an expansion of both magnitude and functional quality, particularly within blood-stage phagocytic antibody activity. Moreover, concerning clinical outcomes postinfection, protected children had higher antibody-dependent neutrophil activity along with higher levels of MSP1-specific IgG3 and IgA and CSP-specific IgG3 and IgG4 prior to the malaria season. CONCLUSIONS: These data point to the natural evolution of functional humoral immunity to Pf with age and highlight particular antibody Fc-effector profiles associated with the control of malaria in children, providing clues for the design of next-generation vaccines or therapeutics.

Plasmodium falciparum malaria drives epigenetic reprogramming of human monocytes toward a regulatory phenotype.

In malaria-naïve children and adults, Plasmodium falciparum-infected red blood cells (Pf-iRBCs) trigger fever and other symptoms of systemic inflammation. However, in endemic areas where individuals experience repeated Pf infections over many years, the risk of Pf-iRBC-triggered inflammatory symptoms decreases with cumulative Pf exposure. The molecular mechanisms underlying these clinical observations remain unclear. Age-stratified analyses of uninfected, asymptomatic Malian individuals before the malaria season revealed that monocytes of adults produced lower levels of inflammatory cytokines (IL-1ß, IL-6 and TNF) in response to Pf-iRBC stimulation compared to monocytes of Malian children and malaria-naïve U.S. adults. Moreover, monocytes of Malian children produced lower levels of IL-1ß and IL-6 following Pf-iRBC stimulation compared to 4-6-month-old infants. Accordingly, monocytes of Malian adults produced more IL-10 and expressed higher levels of the regulatory molecules CD163, CD206, Arginase-1 and TGM2. These observations were recapitulated in an in vitro system of monocyte to macrophage differentiation wherein macrophages re-exposed to Pf-iRBCs exhibited attenuated inflammatory cytokine responses and a corresponding decrease in the epigenetic marker of active gene transcription, H3K4me3, at inflammatory cytokine gene loci. Together these data indicate that Pf induces epigenetic reprogramming of monocytes/macrophages toward a regulatory phenotype that attenuates inflammatory responses during subsequent Pf exposure. Trial Registration: ClinicalTrials.gov NCT01322581.

Evidence for exposure dependent carriage of malaria parasites across the dry season: modelling analysis of longitudinal data.

BACKGROUND: In malaria endemic regions, transmission of Plasmodium falciparum parasites is often seasonal with very low transmission during the dry season and high transmission in the wet season. Parasites survive the dry season within some individuals who experience prolonged carriage of parasites and are thought to 'seed' infection in the next transmission season. METHODS: Dry season carriers and their role in the subsequent transmission season are characterized using a combination of mathematical simulations and data analysis of previously described data from a longitudinal study in Mali of individuals aged 3 months-12 years (n = 579). RESULTS: Simulating the life-history of individuals experiencing repeated exposure to infection predicts that dry season carriage is more likely in the oldest, most exposed and most immune individuals. This hypothesis is supported by the data from Mali, which shows that carriers are significantly older, experience a higher biting rate at the beginning of the transmission season and develop clinical malaria later than non-carriers. Further, since the most exposed individuals in a community are most likely to be dry season carriers, this is predicted to enable a more than twofold faster spread of parasites into the mosquito population at the start of the subsequent wet season. CONCLUSIONS: Carriage of malaria parasites over the months-long dry season in Mali is most likely in the older, more exposed and more immune children. These children may act as super-spreaders facilitating the fast spread of parasites at the beginning of the next transmission season.

Dendritic cell responses to Plasmodium falciparum in a malaria-endemic setting.

BACKGROUND: Plasmodium falciparum causes the majority of malaria cases worldwide and children in sub-Saharan Africa are the most vulnerable group affected. Non-sterile clinical immunity that protects from symptoms develops slowly and is relatively short-lived. Moreover, current malaria vaccine candidates fail to induce durable high-level protection in endemic settings, possibly due to the immunomodulatory effects of the malaria parasite itself. Because dendritic cells play a crucial role in initiating immune responses, the aim of this study was to better understand the impact of cumulative malaria exposure as well as concurrent P. falciparum infection on dendritic cell phenotype and function. METHODS: In this cross-sectional study, the phenotype and function of dendritic cells freshly isolated from peripheral blood samples of Malian adults with a lifelong history of malaria exposure who were either uninfected (n = 27) or asymptomatically infected with P. falciparum (n = 8) was assessed. Additionally, plasma cytokine and chemokine levels were measured in these adults and in Malian children (n = 19) with acute symptomatic malaria. RESULTS: With the exception of lower plasmacytoid dendritic cell frequencies in asymptomatically infected Malian adults, peripheral blood dendritic cell subset frequencies and HLA-DR surface expression did not differ by infection status. Peripheral blood myeloid dendritic cells of uninfected Malian adults responded to in vitro stimulation with P. falciparum blood-stage parasites by up-regulating the costimulatory molecules HLA-DR, CD80, CD86 and CD40 and secreting IL-10, CXCL9 and CXCL10. In contrast, myeloid dendritic cells of asymptomatically infected Malian adults exhibited no significant responses above the uninfected red blood cell control. IL-10 and CXCL9 plasma levels were elevated in both asymptomatic adults and children with acute malaria. CONCLUSIONS: The findings of this study indicate that myeloid dendritic cells of uninfected adults with a lifelong history of malaria exposure are able to up-regulate co-stimulatory molecules and produce cytokines. Whether mDCs of malaria-exposed individuals are efficient antigen-presenting cells capable of mounting an appropriate immune response remains to be determined. The data also highlights IL-10 and CXCL9 as important factors in both asymptomatic and acute malaria and add to the understanding of asymptomatic P. falciparum infections in malaria-endemic areas.

Atypical activation of dendritic cells by Plasmodium falciparum.

Dendritic cells (DCs) are activated by pathogens to initiate and shape immune responses. We found that the activation of DCs by Plasmodium falciparum, the main causative agent of human malaria, induces a highly unusual phenotype by which DCs up-regulate costimulatory molecules and secretion of chemokines, but not of cytokines typical of inflammatory responses (IL-1ß, IL-6, IL-10, TNF). Similar results were obtained with DCs obtained from malaria-naïve US donors and malaria-experienced donors from Mali. Contact-dependent cross-talk between the main DC subsets, plasmacytoid and myeloid DCs (mDCs) was necessary for increased chemokine and IFN-α secretion in response to the parasite. Despite the absence of inflammatory cytokine secretion, mDCs incubated with P. falciparum-infected erythrocytes activated antigen-specific naïve CD4+ T cells to proliferate and secrete Th1-like cytokines. This unexpected response of human mDCs to P. falciparum exhibited a transcriptional program distinct from a classical LPS response, pointing to unique P. falciparum-induced activation pathways that may explain the uncharacteristic immune response to malaria.

The Regulation of Inherently Autoreactive VH4-34-Expressing B Cells in Individuals Living in a Malaria-Endemic Area of West Africa.

Plasmodium falciparum malaria is a deadly infectious disease in which Abs play a critical role in naturally acquired immunity. However, the specificity and nature of Abs elicited in response to malaria are only partially understood. Autoreactivity and polyreactivity are common features of Ab responses in several infections and were suggested to contribute to effective pathogen-specific Ab responses. In this article, we report on the regulation of B cells expressing the inherently autoreactive VH4-34 H chain (identified by the 9G4 mAb) and 9G4+ plasma IgG in adults and children living in a P. falciparum malaria-endemic area in West Africa. The frequency of 9G4+ peripheral blood CD19+ B cells was similar in United States adults and African adults and children; however, more 9G4+ B cells appeared in classical and atypical memory B cell compartments in African children and adults compared with United States adults. The levels of 9G4+ IgG increased following acute febrile malaria but did not increase with age as humoral immunity is acquired or correlate with protection from acute disease. This was the case, even though a portion of 9G4+ B cells acquired phenotypes of atypical and classical memory B cells and 9G4+ IgG contained equivalent numbers of somatic hypermutations compared with all other VHs, a characteristic of secondary Ab repertoire diversification in response to Ag stimulation. Determining the origin and function of 9G4+ B cells and 9G4+ IgG in malaria may contribute to a better understanding of the varied roles of autoreactivity in infectious diseases.

Treatment of Chronic Asymptomatic Plasmodium falciparum Infection Does Not Increase the Risk of Clinical Malaria Upon Reinfection.

Background: Chronic asymptomatic Plasmodium falciparum infections are common in endemic areas and are thought to contribute to the maintenance of malaria immunity. Whether treatment of these infections increases the subsequent risk of clinical episodes of malaria is unclear. Methods: In a 3-year study in Mali, asymptomatic individuals with or without P. falciparum infection at the end of the 6-month dry season were identified by polymerase chain reaction (PCR), and clinical malaria risk was compared during the ensuing 6-month malaria transmission season. At the end of the second dry season, 3 groups of asymptomatic children were identified: (1) children infected with P. falciparum as detected by rapid diagnostic testing (RDT) who were treated with antimalarials (n = 104), (2) RDT-negative children whose untreated P. falciparum infections were detected retrospectively by PCR (n = 55), and (3) uninfected children (RDT/PCR negative) (n = 434). Clinical malaria risk during 2 subsequent malaria seasons was compared. Plasmodium falciparum-specific antibody kinetics during the dry season were compared in children who did or did not harbor asymptomatic P. falciparum infections. Results: Chronic asymptomatic P. falciparum infection predicted decreased clinical malaria risk during the subsequent malaria season(s); treatment of these infections did not alter this reduced risk. Plasmodium falciparum-specific antibodies declined similarly in children who did or did not harbor chronic asymptomatic P. falciparum infection during the dry season. Conclusions: These findings challenge the notion that chronic asymptomatic P. falciparum infection maintains malaria immunity and suggest that mass drug administration during the dry season should not increase the subsequent risk of clinical malaria.

The V gene repertoires of classical and atypical memory B cells in malaria-susceptible West African children.

Immunity to Plasmodium falciparum malaria is naturally acquired in individuals living in malaria-endemic areas of Africa. Abs play a key role in mediating this immunity; however, the acquisition of the components of Ab immunity, long-lived plasma cells and memory B cells (MBCs), is remarkably inefficient, requiring years of malaria exposure. Although long-lived classical MBCs (CD19(+)/CD20(+)/CD21(+)/CD27(+)/CD10(-)) are gradually acquired in response to natural infection, exposure to P. falciparum also results in a large expansion of what we have termed atypical MBCs (CD19(+)/CD20(+)/CD21(-)/CD27(-)/CD10(-)). At present, the function of atypical MBCs in malaria is not known, nor are the factors that drive their differentiation. To gain insight into the relationship between classical and atypical IgG(+) MBCs, we compared the Ab H and L chain V gene repertoires of children living in a malaria-endemic region in Mali. We found that these repertoires were remarkably similar by a variety of criteria, including V gene usage, rate of somatic hypermutation, and CDR-H3 length and composition. The similarity in these repertoires suggests that classical MBCs and atypical MBCs differentiate in response to similar Ag-dependent selective pressures in malaria-exposed children and that atypical MBCs do not express a unique V gene repertoire.

Exposure-dependent control of malaria-induced inflammation in children.

In malaria-naïve individuals, Plasmodium falciparum infection results in high levels of parasite-infected red blood cells (iRBCs) that trigger systemic inflammation and fever. Conversely, individuals in endemic areas who are repeatedly infected are often asymptomatic and have low levels of iRBCs, even young children. We hypothesized that febrile malaria alters the immune system such that P. falciparum re-exposure results in reduced production of pro-inflammatory cytokines/chemokines and enhanced anti-parasite effector responses compared to responses induced before malaria. To test this hypothesis we used a systems biology approach to analyze PBMCs sampled from healthy children before the six-month malaria season and the same children seven days after treatment of their first febrile malaria episode of the ensuing season. PBMCs were stimulated with iRBC in vitro and various immune parameters were measured. Before the malaria season, children's immune cells responded to iRBCs by producing pro-inflammatory mediators such as IL-1ß, IL-6 and IL-8. Following malaria there was a marked shift in the response to iRBCs with the same children's immune cells producing lower levels of pro-inflammatory cytokines and higher levels of anti-inflammatory cytokines (IL-10, TGF-ß). In addition, molecules involved in phagocytosis and activation of adaptive immunity were upregulated after malaria as compared to before. This shift was accompanied by an increase in P. falciparum-specific CD4+Foxp3- T cells that co-produce IL-10, IFN-γ and TNF; however, after the subsequent six-month dry season, a period of markedly reduced malaria transmission, P. falciparum-inducible IL-10 production remained partially upregulated only in children with persistent asymptomatic infections. These findings suggest that in the face of P. falciparum re-exposure, children acquire exposure-dependent P. falciparum-specific immunoregulatory responses that dampen pathogenic inflammation while enhancing anti-parasite effector mechanisms. These data provide mechanistic insight into the observation that P. falciparum-infected children in endemic areas are often afebrile and tend to control parasite replication.

Asymptomatic Multiclonal Plasmodium falciparum Infections Carried Through the Dry Season Predict Protection Against Subsequent Clinical Malaria.

BACKGROUND: Immunity to the antigenically diverse parasite Plasmodium falciparum is acquired gradually after repeated exposure. Studies in areas of high malaria transmission have shown that asymptomatic individuals infected with multiclonal infections are at reduced risk of febrile malaria during follow-up. METHODS: We assessed the relationship between the genetic diversity of clones in P. falciparum infections that persist through the dry season and the subsequent risk of febrile malaria in 225 individuals aged 2-25 years in Mali, where the 6-month malaria and dry seasons are sharply demarcated. Polymerase chain reaction-based genotyping of the highly polymorphic merozoite surface protein 2 gene was performed on blood samples collected at 5 cross-sectional surveys. RESULTS: In an age-adjusted analysis, individuals with multiclonal P. falciparum infections before the rainy season were at reduced risk of febrile malaria, compared with individuals who were uninfected (hazard ratio [HR], 0.28; 95% confidence interval [CI], .11-.69). In contrast, there was no significant association between risk of malaria and having 1 clone at baseline (HR, 0.71; 95% CI, .36-1.40). CONCLUSIONS: The results suggest that persistent multiclonal infections carried through the dry season contribute to protection against subsequent febrile malaria, possibly by maintaining protective immune responses that depend on ongoing parasite infection.

Plasmodium falciparum Gametocyte-Specific Antibody Profiling Reveals Boosting through Natural Infection and Identifies Potential Markers of Gametocyte Exposure.

Malaria elimination efforts would benefit from vaccines that block transmission of Plasmodium falciparum gametocytes from humans to mosquitoes. A clear understanding of gametocyte-specific antibody responses in exposed populations could help determine whether transmission-blocking vaccines (TBV) would be boosted by natural gametocyte exposure, and also inform the development of serologic tools to monitor gametocyte exposure in populations targeted for malaria elimination. To this end, plasma was collected from Malian children and adults before and after the 6-month malaria season and probed against a microarray containing 1,204 P. falciparum proteins. Using publicly available proteomic data, we classified 91 proteins as gametocyte specific and 69 as proteins not expressed by gametocytes. The overall breadth and magnitude of gametocyte-specific IgG responses increased during the malaria season, although they were consistently lower than IgG responses to nongametocyte antigens. Notably, IgG specific for the TBV candidates Pfs48/45 and Pfs230 increased during the malaria season. In addition, IgGs specific for the gametocyte proteins Pfmdv1, Pfs16, PF3D7_1346400, and PF3D7_1024800 were detected in nearly all subjects, suggesting that seroconversion to these proteins may be a sensitive indicator of gametocyte exposure, although further studies are needed to determine the specificity and kinetics of these potential serologic markers. These findings suggest that TBV-induced immunity would be boosted through natural gametocyte exposure, and that antibody responses to particular antigens may reliably indicate gametocyte exposure.

Naturally acquired antibodies specific for Plasmodium falciparum reticulocyte-binding protein homologue 5 inhibit parasite growth and predict protection from malaria.

BACKGROUND: Plasmodium falciparum reticulocyte-binding protein homologue 5 (PfRH5) is a blood-stage parasite protein essential for host erythrocyte invasion. PfRH5-specific antibodies raised in animals inhibit parasite growth in vitro, but the relevance of naturally acquired PfRH5-specific antibodies in humans is unclear. METHODS: We assessed pre-malaria season PfRH5-specific immunoglobulin G (IgG) levels in 357 Malian children and adults who were uninfected with Plasmodium. Subsequent P. falciparum infections were detected by polymerase chain reaction every 2 weeks and malaria episodes by weekly physical examination and self-referral for 7 months. The primary outcome was time between the first P. falciparum infection and the first febrile malaria episode. PfRH5-specific IgG was assayed for parasite growth-inhibitory activity. RESULTS: The presence of PfRH5-specific IgG at enrollment was associated with a longer time between the first blood-stage infection and the first malaria episode (PfRH5-seropositive median: 71 days, PfRH5-seronegative median: 18 days; P = .001). This association remained significant after adjustment for age and other factors associated with malaria risk/exposure (hazard ratio, .62; P = .02). Concentrated PfRH5-specific IgG purified from Malians inhibited P. falciparum growth in vitro. CONCLUSIONS: Naturally acquired PfRH5-specific IgG inhibits parasite growth in vitro and predicts protection from malaria. These findings strongly support efforts to develop PfRH5 as an urgently needed blood-stage malaria vaccine. CLINICAL TRIALS REGISTRATION: NCT01322581.

A nested real-time PCR assay for the quantification of Plasmodium falciparum DNA extracted from dried blood spots.

BACKGROUND: As public health efforts seek to eradicate malaria, there has been an emphasis on eliminating low-density parasite reservoirs in asymptomatic carriers. As such, diagnosing submicroscopic Plasmodium infections using PCR-based techniques has become important not only in clinical trials of malaria vaccines and therapeutics, but also in active malaria surveillance campaigns. However, PCR-based quantitative assays that rely on nucleic acid extracted from dried blood spots (DBS) have demonstrated lower sensitivity than assays that use cryopreserved whole blood as source material. METHODS: The density of Plasmodium falciparum asexual parasites was quantified using genomic DNA extracted from dried blood spots (DBS) and the sensitivity of two approaches was compared: quantitative real-time PCR (qPCR) targeting the P. falciparum 18S ribosomal RNA gene, either with an initial conventional PCR amplification prior to qPCR (nested qPCR), or without an initial amplification (qPCR only). Parasite densities determined by nested qPCR, qPCR only, and light microscopy were compared. RESULTS: Nested qPCR results in 10-fold higher sensitivity (0.5 parasites/µl) when compared to qPCR only (five parasites/ul). Among microscopy-positive samples, parasite densities calculated by nested qPCR correlated strongly with microscopy for both asymptomatic (Pearson's r=0.58, P<0.001) and symptomatic (Pearson's r=0.70, P<0.0001) P. falciparum infections. CONCLUSION: Nested qPCR improves the sensitivity for the detection of P. falciparum blood-stage infection from clinical DBS samples. This approach may be useful for active malaria surveillance in areas where submicroscopic asymptomatic infections are prevalent.

Susceptibility to febrile malaria is associated with an inflammatory gut microbiome.

Malaria is a major public health problem, but many of the factors underlying the pathogenesis of this disease are not well understood. Here, we demonstrate in Malian children that susceptibility to febrile malaria following infection with Plasmodium falciparum is associated with the composition of the gut microbiome prior to the malaria season. Gnotobiotic mice colonized with the fecal samples of malaria-susceptible children had a significantly higher parasite burden following Plasmodium infection compared to gnotobiotic mice colonized with the fecal samples of malaria-resistant children. The fecal microbiome of the susceptible children was enriched for bacteria associated with inflammation, mucin degradation, gut permeability and inflammatory bowel disorders (e.g., Ruminococcus gauvreauii, Ruminococcus torques, Dorea formicigenerans, Dorea longicatena, Lachnoclostridium phocaeense and Lachnoclostridium sp. YL32). However, the susceptible children also had a greater abundance of bacteria known to produce anti-inflammatory short-chain fatty acids and those associated with favorable prognosis and remission following dysbiotic intestinal events (e.g., Anaerobutyricum hallii, Blautia producta and Sellimonas intestinalis). Metabolomics analysis of the human fecal samples corroborated the existence of inflammatory and recovery-associated features within the gut microbiome of the susceptible children. There was an enrichment of nitric oxide-derived DNA adducts (deoxyinosine and deoxyuridine) and long-chain fatty acids, the absorption of which has been shown to be inhibited by inflamed intestinal epithelial cells, and a decrease in the abundance of mucus phospholipids. Nevertheless, there were also increased levels of pseudouridine and hypoxanthine, which have been shown to be regulated in response to cellular stress and to promote recovery following injury or hypoxia. Overall, these results indicate that the gut microbiome may contribute malaria pathogenesis and suggest that therapies targeting intestinal inflammation could decrease malaria susceptibility.