Post-Artesunate Delayed Hemolysis in Pediatric Malaria Patients in the United States.

Post-artesunate delayed hemolysis (PADH) occurred in 6 of 24 children treated with artesunate for severe malaria in the United States; however severe hemolysis requiring hospitalization or transfusion was rare. In children in the U.S. treated with artesunate, counseling and symptom monitoring may be preferred to weekly laboratory surveillance for PADH.

Clinical immunity to malaria involves epigenetic reprogramming of innate immune cells.

The regulation of inflammation is a critical aspect of disease tolerance and naturally acquired clinical immunity to malaria. Here, we demonstrate using RNA sequencing and epigenetic landscape profiling by cytometry by time-of-flight, that the regulation of inflammatory pathways during asymptomatic parasitemia occurs downstream of pathogen sensing-at the epigenetic level. The abundance of certain epigenetic markers (methylation of H3K27 and dimethylation of arginine residues) and decreased prevalence of histone variant H3.3 correlated with suppressed cytokine responses among monocytes of Ugandan children. Such an epigenetic signature was observed across diverse immune cell populations and not only characterized active asymptomatic parasitemia but also correlated with future long-term disease tolerance and clinical immunity when observed in uninfected children. Pseudotime analyses revealed a potential trajectory of epigenetic change that correlated with a child's age and recent parasite exposure and paralleled the acquisition of clinical immunity. Thus, our data support a model whereby exposure to Plasmodium falciparum induces epigenetic changes that regulate excessive inflammation and contribute to naturally acquire clinical immunity to malaria.

Gravidity influences distinct transcriptional profiles of maternal and fetal placental macrophages at term.

Introduction: Maternal intervillous monocytes (MIMs) and fetal Hofbauer cells (HBCs) are myeloid-derived immune cells at the maternal-fetal interface. Maternal reproductive history is associated with differential risk of pregnancy complications. The molecular phenotypes and roles of these distinct monocyte/macrophage populations and the influence of gravidity on these phenotypes has not been systematically investigated. Methods: Here, we used RNA sequencing to study the transcriptional profiles of MIMs and HBCs in normal term pregnancies. Results: Our analyses revealed distinct transcriptomes of MIMs and HBCs. Genes involved in differentiation and cell organization pathways were more highly expressed in MIMs vs. HBCs. In contrast, HBCs had higher expression of genes involved in inflammatory responses and cell surface receptor signaling. Maternal gravidity influenced monocyte programming, as expression of pro-inflammatory molecules was significantly higher in MIMs from multigravidae compared to primigravidae. In HBCs, multigravidae displayed enrichment of gene pathways involved in cell-cell signaling and differentiation. Discussion: Our results demonstrated that MIMs and HBCs have highly divergent transcriptional signatures, reflecting their distinct origins, locations, functions, and roles in inflammatory responses. Furthermore, maternal gravidity influences the gene signatures of MIMs and HBCs, potentially modulating the interplay between tolerance and trained immunity. The phenomenon of reproductive immune memory may play a novel role in the differential susceptibility of primigravidae to pregnancy complications.

Differences in phenotype between long-lived memory B cells against Plasmodium falciparum merozoite antigens and variant surface antigens.

Plasmodium falciparum infections elicit strong humoral immune responses to two main groups of antigens expressed by blood-stage parasites: merozoite antigens that are involved in the erythrocyte invasion process and variant surface antigens that mediate endothelial sequestration of infected erythrocytes. Long-lived B cells against both antigen classes can be detected in the circulation for years after exposure, but have not been directly compared. Here, we studied the phenotype of long-lived memory and atypical B cells to merozoite antigens (MSP1 and AMA1) and variant surface antigens (the CIDRα1 domain of PfEMP1) in Ugandan adults before and after local reduction of P. falciparum transmission. After a median of 1.7 years without P. falciparum infections, the percentage of antigen-specific activated B cells declined, but long-lived antigen-specific B cells were still detectable in all individuals. The majority of MSP1/AMA1-specific B cells were CD95+CD11c+ memory B cells, which are primed for rapid differentiation into antibody-secreting cells, and FcRL5-T-bet- atypical B cells. On the other hand, most CIDRα1-specific B cells were CD95-CD11c- memory B cells. CIDRα1-specific B cells were also enriched among a subset of atypical B cells that seem poised for antigen presentation. These results point to differences in how these antigens are recognized or processed by the immune system and how P. falciparum-specific B cells will respond upon re-infection.

Broadly inhibitory antibodies against severe malaria virulence proteins.

Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasite's polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDRα1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria.

Atypical B cells consist of subsets with distinct functional profiles.

Atypical B cells are a population of activated B cells that are commonly enriched in individuals with chronic immune activation but are also part of a normal immune response to infection or vaccination. To better define the role of atypical B cells in the human adaptive immune response, we performed single-cell sequencing of transcriptomes, cell surface markers, and B cell receptors in individuals with chronic exposure to the malaria parasite Plasmodium falciparum, a condition known to lead to accumulation of circulating atypical B cells. We identified three previously uncharacterized populations of atypical B cells with distinct transcriptional and functional profiles and observed marked differences among these three subsets in their ability to produce immunoglobulin G upon T-cell-dependent activation. Our findings help explain the conflicting observations in prior studies regarding the function of atypical B cells and highlight their different roles in the adaptive immune response in chronic inflammatory conditions.

Distinct transcriptional profiles of maternal and fetal placental macrophages at term are associated with gravidity.

Maternal intervillous monocytes (MIMs) and fetal Hofbauer cells (HBCs) are myeloid-derived immune cells at the maternal-fetal interface. Little is known regarding the molecular phenotypes and roles of these distinct monocyte/macrophage populations. Here, we used RNA sequencing to investigate the transcriptional profiles of MIMs and HBCs in six normal term pregnancies. Our analyses revealed distinct transcriptomes of MIMs and HBCs. Genes involved in differentiation and cell organization pathways were more highly expressed in MIMs vs. HBCs. In contrast, HBCs had higher expression of genes involved in inflammatory responses and cell surface receptor signaling. Maternal gravidity influenced monocyte programming, as expression of pro-inflammatory molecules was significantly higher in MIMs from multigravidas compared to primigravidas. In HBCs, multigravidas displayed enrichment of gene pathways involved in cell-cell signaling and differentiation. In summary, our results demonstrated that MIMs and HBCs have highly divergent transcriptional signatures, reflecting their distinct origins, locations, functions, and roles in inflammatory responses. Our data further suggested that maternal gravidity influences the gene signatures of MIMs and HBCs, potentially modulating the interplay between tolerance and trained immunity. The phenomenon of reproductive immune memory may play a novel role in the differential susceptibility of primigravidas to pregnancy complications.

Malaria-specific Type 1 regulatory T cells are more abundant in first pregnancies and associated with placental malaria.

BACKGROUND: Malaria in pregnancy (MIP) causes higher morbidity in primigravid compared to multigravid women; however, the correlates and mechanisms underlying this gravidity-dependent protection remain incompletely understood. We aimed to compare the cellular immune response between primigravid and multigravid women living in a malaria-endemic region and assess for correlates of protection against MIP. METHODS: We characterised the second trimester cellular immune response among 203 primigravid and multigravid pregnant women enrolled in two clinical trials of chemoprevention in eastern Uganda, utilizing RNA sequencing, flow cytometry, and functional assays. We compared responses across gravidity and determined associations with parasitaemia during pregnancy and placental malaria. FINDINGS: Using whole blood RNA sequencing, no significant differentially expressed genes were identified between primigravid (n = 12) and multigravid (n = 11) women overall (log 2(FC) > 2, FDR < 0.1). However, primigravid (n = 49) women had higher percentages of malaria-specific, non-naïve CD4+ T cells that co-expressed IL-10 and IFNγ compared with multigravid (n = 85) women (p = 0.000023), and higher percentages of these CD4+ T cells were associated with greater risks of parasitaemia in pregnancy (Rs = 0.49, p = 0.001) and placental malaria (p = 0.0073). These IL-10 and IFNγ co-producing CD4+ T cells had a genomic signature of Tr1 cells, including expression of transcription factors cMAF and BATF and cell surface makers CTLA4 and LAG-3. INTERPRETATION: Malaria-specific Tr1 cells were highly prevalent in primigravid Ugandan women, and their presence correlated with a higher risk of malaria in pregnancy. Understanding whether suppression of Tr1 cells plays a role in naturally acquired gravidity-dependent immunity may aid the development of new vaccines or treatments for MIP. FUNDING: This work was funded by NIH (PO1 HD059454, U01 AI141308, U19 AI089674, U01 AI155325, U01 AI150741), the March of Dimes (Basil O'Connor award), and the Bill and Melinda Gates Foundation (OPP 1113682).

Highly potent, naturally acquired human monoclonal antibodies against Pfs48/45 block Plasmodium falciparum transmission to mosquitoes.

Malaria transmission-blocking vaccines (TBVs) aim to induce antibodies that interrupt malaria parasite development in the mosquito, thereby blocking onward transmission, and provide a much-needed tool for malaria control and elimination. The parasite surface protein Pfs48/45 is a leading TBV candidate. Here, we isolated and characterized a panel of 81 human Pfs48/45-specific monoclonal antibodies (mAbs) from donors naturally exposed to Plasmodium parasites. Genetically diverse mAbs against each of the three domains (D1-D3) of Pfs48/45 were identified. The most potent mAbs targeted D1 and D3 and achieved >80% transmission-reducing activity in standard membrane-feeding assays, at 10 and 2 µg/mL, respectively. Co-crystal structures of D3 in complex with four different mAbs delineated two conserved protective epitopes. Altogether, these Pfs48/45-specific human mAbs provide important insight into protective and non-protective epitopes that can further our understanding of transmission and inform the design of refined malaria transmission-blocking vaccine candidates.

Potent transmission-blocking monoclonal antibodies from naturally exposed individuals target a conserved epitope on Plasmodium falciparum Pfs230.

Pfs230 is essential for Plasmodium falciparum transmission to mosquitoes and is the protein targeted by the most advanced malaria-transmission-blocking vaccine candidate. Prior understanding of functional epitopes on Pfs230 is based on two monoclonal antibodies (mAbs) with moderate transmission-reducing activity (TRA), elicited from subunit immunization. Here, we screened the B cell repertoire of two naturally exposed individuals possessing serum TRA and identified five potent mAbs from sixteen Pfs230 domain-1-specific mAbs. Structures of three potent and three low-activity antibodies bound to Pfs230 domain 1 revealed four distinct epitopes. Highly potent mAbs from natural infection recognized a common conformational epitope that is highly conserved across P. falciparum field isolates, while antibodies with negligible TRA derived from natural infection or immunization recognized three distinct sites. Our study provides molecular blueprints describing P. falciparum TRA, informed by contrasting potent and non-functional epitopes elicited by natural exposure and vaccination.

Malaria-driven expansion of adaptive-like functional CD56-negative NK cells correlates with clinical immunity to malaria.

Natural killer (NK) cells likely play an important role in immunity to malaria, but the effect of repeated malaria on NK cell responses remains unclear. Here, we comprehensively profiled the NK cell response in a cohort of 264 Ugandan children. Repeated malaria exposure was associated with expansion of an atypical, CD56neg population of NK cells that differed transcriptionally, epigenetically, and phenotypically from CD56dim NK cells, including decreased expression of PLZF and the Fc receptor γ-chain, increased histone methylation, and increased protein expression of LAG-3, KIR, and LILRB1. CD56neg NK cells were highly functional and displayed greater antibody-dependent cellular cytotoxicity than CD56dim NK cells. Higher frequencies of CD56neg NK cells were associated with protection against symptomatic malaria and high parasite densities. After marked reductions in malaria transmission, frequencies of these cells rapidly declined, suggesting that continuous exposure to Plasmodium falciparum is required to maintain this modified, adaptive-like NK cell subset.

Demographic and clinical characteristics of confirmed human monkeypox virus cases in individuals attending a sexual health centre in London, UK: an observational analysis.

BACKGROUND: Historically, human monkeypox virus cases in the UK have been limited to imported infections from west Africa. Currently, the UK and several other countries are reporting a rapid increase in monkeypox cases among individuals attending sexual health clinics, with no apparent epidemiological links to endemic areas. We describe demographic and clinical characteristics of patients diagnosed with human monkeypox virus attending a sexual health centre. METHODS: In this observational analysis, we considered patients with confirmed monkeypox virus infection via PCR detection attending open-access sexual health clinics in London, UK, between May 14 and May 25, 2022. We report hospital admissions and concurrent sexually transmitted infection (STI) proportions, and describe our local response within the first 2 weeks of the outbreak. FINDINGS: Monkeypox virus infection was confirmed in 54 individuals, all identifying as men who have sex with men (MSM), with a median age of 41 years (IQR 34-45). 38 (70%) of 54 individuals were White, 26 (48%) were born in the UK, and 13 (24%) were living with HIV. 36 (67%) of 54 individuals reported fatigue or lethargy, 31 (57%) reported fever, and ten (18%) had no prodromal symptoms. All patients presented with skin lesions, of which 51 (94%) were anogenital. 37 (89%) of 54 individuals had skin lesions affecting more than one anatomical site and four (7%) had oropharyngeal lesions. 30 (55%) of 54 individuals had lymphadenopathy. One in four patients had a concurrent STI. Five (9%) of 54 individuals required admission to hospital, mainly due to pain or localised bacterial cellulitis requiring antibiotic intervention or analgesia. We recorded no fatal outcomes. INTERPRETATION: Autochthonous community monkeypox virus transmission is currently observed among MSM in the UK. We found a high proportion of concomitant STIs and frequent anogenital symptoms, suggesting transmissibility through local inoculation during close skin-to-skin or mucosal contact, during sexual activity. Additional resources are required to support sexual health and other specialist services in managing this condition. A review of the case definition and better understanding of viral transmission routes are needed to shape infection control policies, education and prevention strategies, and contact tracing. FUNDING: None.

In Utero Activation of Natural Killer Cells in Congenital Cytomegalovirus Infection.

BACKGROUND: Congenital cytomegalovirus (CMV) infection is the most common infectious cause of birth defects and neurological damage in newborns. Despite a well-established role for natural killer (NK) cells in control of CMV infection in older children and adults, it remains unknown whether fetal NK cells can sense and respond to CMV infection acquired in utero. METHODS: Here, we investigate the impact of congenital CMV infection on the neonatal NK-cell repertoire by assessing the frequency, phenotype, and functional profile of NK cells in cord blood samples from newborns with congenital CMV and from uninfected controls enrolled in a birth cohort of Ugandan mothers and infants. RESULTS: We find that neonatal NK cells from congenitally CMV infected newborns show increased expression of cytotoxic mediators, signs of maturation and activation, and an expansion of mature CD56- NK cells, an NK-cell subset associated with chronic viral infections in adults. Activation was particularly prominent in NK cell subsets expressing the Fcγ receptor CD16, indicating a role for antibody-mediated immunity against CMV in utero. CONCLUSIONS: These findings demonstrate that NK cells can be activated in utero and suggest that NK cells may be an important component of the fetal and infant immune response against CMV. CLINICAL TRIALS REGISTRATION: NCT02793622.

Genetic variation that determines TAPBP expression levels associates with the course of malaria in an HLA allotype-dependent manner.

HLA class I (HLA-I) allotypes vary widely in their dependence on tapasin (TAPBP), an integral component of the peptide-loading complex, to present peptides on the cell surface. We identified two single-nucleotide polymorphisms that regulate TAPBP messenger RNA (mRNA) expression in Africans, rs111686073 (G/C) and rs59097151 (A/G), located in an AP-2α transcription factor binding site and a microRNA (miR)-4486 binding site, respectively. rs111686073G and rs59097151A induced significantly higher TAPBP mRNA expression relative to the alternative alleles due to higher affinity for AP-2α and abrogation of miR-4486 binding, respectively. These variants associated with lower Plasmodium falciparum parasite prevalence and lower incidence of clinical malaria specifically among individuals carrying tapasin-dependent HLA-I allotypes, presumably by augmenting peptide loading, whereas tapasin-independent allotypes associated with relative protection, regardless of imputed TAPBP mRNA expression levels. Thus, an attenuated course of malaria may occur through enhanced breadth and/or magnitude of antigen presentation, an important consideration when evaluating vaccine efficacy.

Age-dependent changes in circulating Tfh cells influence development of functional malaria antibodies in children.

T-follicular helper (Tfh) cells are key drivers of antibodies that protect from malaria. However, little is known regarding the host and parasite factors that influence Tfh and functional antibody development. Here, we use samples from a large cross-sectional study of children residing in an area of high malaria transmission in Uganda to characterize Tfh cells and functional antibodies to multiple parasites stages. We identify a dramatic re-distribution of the Tfh cell compartment with age that is independent of malaria exposure, with Th2-Tfh cells predominating in early childhood, while Th1-Tfh cell gradually increase to adult levels over the first decade of life. Functional antibody acquisition is age-dependent and hierarchical acquired based on parasite stage, with merozoite responses followed by sporozoite and gametocyte antibodies. Antibodies are boosted in children with current infection, and are higher in females. The children with the very highest antibody levels have increased Tfh cell activation and proliferation, consistent with a key role of Tfh cells in antibody development. Together, these data reveal a complex relationship between the circulating Tfh compartment, antibody development and protection from malaria.

A cytotoxic-skewed immune set point predicts low neutralizing antibody levels after Zika virus infection.

Although generating high neutralizing antibody levels is a key component of protective immunity after acute viral infection or vaccination, little is known about why some individuals generate high versus low neutralizing antibody titers. Here, we leverage the high-dimensional single-cell profiling capacity of mass cytometry to characterize the longitudinal cellular immune response to Zika virus (ZIKV) infection in viremic blood donors in Puerto Rico. During acute ZIKV infection, we identify widely coordinated responses across innate and adaptive immune cell lineages. High frequencies of multiple activated cell types during acute infection are associated with high titers of ZIKV neutralizing antibodies 6 months post-infection, while stable immune features suggesting a cytotoxic-skewed immune set point are associated with low titers. Our study offers insight into the coordination of immune responses and identifies candidate cellular biomarkers that may offer predictive value in vaccine efficacy trials aimed at inducing high levels of antiviral neutralizing antibodies.

Peripheral Plasmodium falciparum Infection in Early Pregnancy Is Associated With Increased Maternal Microchimerism in the Offspring.

BACKGROUND: Placental malaria has been associated with increased cord blood maternal microchimerism (MMc), which in turn may affect susceptibility to malaria in the offspring. We sought to determine the impact of maternal peripheral Plasmodium falciparum parasitemia during pregnancy on MMc and to determine whether maternal cells expand during primary parasitemia in the offspring. METHODS: We conducted a nested cohort study of maternal-infant pairs from a prior pregnancy malaria chemoprevention study. Maternal microchimerism was measured by quantitative polymerase chain reaction targeting a maternal-specific marker in genomic DNA from cord blood, first P falciparum parasitemia, and preparasitemia. Logistic and negative binomial regression were used to assess the impact of maternal peripheral parasitemia, symptomatic malaria, and placental malaria on cord blood MMc. Generalized estimating equations were used to assess predictors of MMc during infancy. RESULTS: Early maternal parasitemia was associated with increased detection of cord blood MMc (adjusted odds ratio = 3.91, P = .03), whereas late parasitemia, symptomatic malaria, and placental malaria were not. The first parasitemia episode in the infant was not associated with increased MMc relative to preparasitemia. CONCLUSIONS: Maternal parasitemia early in pregnancy may increase the amount of MMc acquired by the fetus. Future work should investigate the impact of this MMc on immune responses in the offspring.

HLA Alleles B*53:01 and C*06:02 Are Associated With Higher Risk of P. falciparum Parasitemia in a Cohort in Uganda.

Variation within the HLA locus been shown to play an important role in the susceptibility to and outcomes of numerous infections, but its influence on immunity to P. falciparum malaria is unclear. Increasing evidence indicates that acquired immunity to P. falciparum is mediated in part by the cellular immune response, including NK cells, CD4 and CD8 T cells, and semi-invariant γδ T cells. HLA molecules expressed by these lymphocytes influence the epitopes recognized by P. falciparum-specific T cells, and class I HLA molecules also serve as ligands for inhibitory receptors including KIR. Here we assessed the relationship of HLA class I and II alleles to the risk of P. falciparum infection and symptomatic malaria in a cohort of 892 Ugandan children and adults followed prospectively via both active and passive surveillance. We identified two HLA class I alleles, HLA-B*53:01 and HLA-C*06:02, that were associated with a higher prevalence of P. falciparum infection. Notably, no class I or II HLA alleles were found to be associated with protection from P. falciparum parasitemia or symptomatic malaria. These findings suggest that class I HLA plays a role in the ability to restrict parasitemia, supporting an essential role for the cellular immune response in P. falciparum immunity. Our findings underscore the need for better tools to enable mechanistic studies of the T cell response to P. falciparum at the epitope level and suggest that further study of the role of HLA in regulating pre-erythrocytic stages of the P. falciparum life cycle is warranted.

Malaria and Early Life Immunity: Competence in Context.

Childhood vaccines have been the cornerstone tool of public health over the past century. A major barrier to neonatal vaccination is the "immaturity" of the infant immune system and the inefficiency of conventional vaccine approaches at inducing immunity at birth. While much of the literature on fetal and neonatal immunity has focused on the early life propensity toward immune tolerance, recent studies indicate that the fetus is more immunologically capable than previously thought, and can, in some circumstances, mount adaptive B and T cell responses to perinatal pathogens in utero. Although significant hurdles remain before these findings can be translated into vaccines and other protective strategies, they should lend optimism to the prospect that neonatal and even fetal vaccination is achievable. Next steps toward this goal should include efforts to define the conditions for optimal stimulation of infant immune responses, including antigen timing, dose, and route of delivery, as well as antigen presentation pathways and co-stimulatory requirements. A better understanding of these factors will enable optimal deployment of vaccines against malaria and other pathogens to protect infants during their period of greatest vulnerability.