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.

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.

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.

The immune response to malaria in utero.

Malaria causes tremendous early childhood morbidity and mortality, providing an urgent impetus for the development of a vaccine that is effective in neonates. However, the infant immune response to malaria may be influenced by events that occur well before birth. Placental malaria infection complicates one quarter of all pregnancies in Africa and frequently results in exposure of the fetus to malaria antigens in utero, while the immune system is still developing. Some data suggest that in utero exposure to malaria may induce immunologic tolerance that interferes with the development of protective immunity during childhood. More recently, however, a growing body of evidence suggests that fetal malaria exposure can prime highly functional malaria-specific T- and B-cells, which may contribute to postnatal protection from malaria. In utero exposure to malaria also impacts the activation and maturation of fetal antigen presenting cells and innate lymphocytes, which could have implications for global immunity in the infant. Here, we review recent advances in our understanding of how various components of the fetal immune system are altered by in utero exposure to malaria, discuss factors that may tilt the critical balance between tolerance and adaptive immunity, and consider the implications of these findings for malaria prevention strategies.

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.

Opsonized antigen activates Vδ2+ T cells via CD16/FCγRIIIa in individuals with chronic malaria exposure.

Vγ9Vδ2 T cells rapidly respond to phosphoantigens produced by Plasmodium falciparum in an innate-like manner, without prior antigen exposure or processing. Vδ2 T cells have been shown to inhibit parasite replication in vitro and are associated with protection from P. falciparum parasitemia in vivo. Although a marked expansion of Vδ2 T cells is seen after acute malaria infection in naïve individuals, repeated malaria causes Vδ2 T cells to decline both in frequency and in malaria-responsiveness, and to exhibit numerous transcriptional and phenotypic changes, including upregulation of the Fc receptor CD16. Here we investigate the functional role of CD16 on Vδ2 T cells in the immune response to malaria. We show that CD16+ Vδ2 T cells possess more cytolytic potential than their CD16- counterparts, and bear many of the hallmarks of mature NK cells, including KIR expression. Furthermore, we demonstrate that Vδ2 T cells from heavily malaria-exposed individuals are able to respond to opsonized P.falciparum-infected red blood cells through CD16, representing a second, distinct pathway by which Vδ2 T cells may contribute to anti-parasite effector functions. This response was independent of TCR engagement, as demonstrated by blockade of the phosphoantigen presenting molecule Butyrophilin 3A1. Together these results indicate that Vδ2 T cells in heavily malaria-exposed individuals retain the capacity for antimalarial effector function, and demonstrate their activation by opsonized parasite antigen. This represents a new role both for Vδ2 T cells and for opsonizing antibodies in parasite clearance, emphasizing cooperation between the cellular and humoral arms of the immune system.

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.

Association of Inhibitory Killer Cell Immunoglobulin-like Receptor Ligands With Higher Plasmodium falciparum Parasite Prevalence.

Killer cell immunoglobulin-like receptors (KIRs) and their HLA ligands influence the outcome of many infectious diseases. We analyzed the relationship of compound KIR-HLA genotypes with risk of Plasmodium falciparum infection in a longitudinal cohort of 890 Ugandan individuals. We found that presence of HLA-C2 and HLA-Bw4, ligands for inhibitory KIR2DL1 and KIR3DL1, respectively, increased the likelihood of P. falciparum parasitemia in an additive manner. Individuals homozygous for HLA-C2, which mediates strong inhibition via KIR2DL1, had the highest odds of parasitemia, HLA-C1/C2 heterozygotes had intermediate odds, and individuals homozygous for HLA-C1, which mediates weaker inhibition through KIR2DL2/3, had the lowest odds of parasitemia. In addition, higher surface expression of HLA-C, the ligand for inhibitory KIR2DL1/2/3, was associated with a higher likelihood of parasitemia. Together these data indicate that stronger KIR-mediated inhibition confers a higher risk of P. falciparum parasitemia and suggest that KIR-expressing effector cells play a role in mediating antiparasite immunity.

The impact of gravidity, symptomatology and timing of infection on placental malaria.

BACKGROUND: Placental malaria is associated with increased risk of adverse perinatal outcomes. While primigravidity has been reported as a risk factor for placental malaria, little is known regarding the relationship between gravidity, symptomatology and timing of Plasmodium falciparum infection and the development of placental malaria. METHODS: The aim of this study was to investigate the relationship between the development of placental malaria and gravidity, timing of infection, and presence of symptoms. This is a secondary analysis of data from a double-blind randomized control trial of intermittent preventive therapy during pregnancy in Uganda. Women were enrolled from 12 to 20 weeks gestation and followed through delivery. Exposure to malaria parasites was defined as symptomatic (fever with positive blood smear) or asymptomatic (based on molecular detection of parasitaemia done routinely every 4 weeks). The primary outcome was placental malaria diagnosed by histopathology, placental blood smear, and/or placental blood loop-mediated isothermal amplification. Multivariate analyses were performed using logistic regression models. Subgroup analysis was performed based on the presence of symptomatic malaria, gravidity, and timing of infection. RESULTS: Of the 228 patients with documented maternal infection with malaria parasites during pregnancy, 101 (44.3%) had placental malaria. Primigravidity was strongly associated with placental malaria (aOR 8.90, 95% CI 4.34-18.2, p < 0.001), and each episode of malaria was associated with over a twofold increase in placental malaria (aOR 2.35, 95% CI 1.69-3.26, p < 0.001). Among multigravid women, the odds of placental malaria increased by 14% with each advancing week of gestation at first documented infection (aOR 1.14, 95% CI 1.02-1.27, p = 0.02). When stratified by the presence of symptoms, primigravidity was only associated with placental malaria in asymptomatic women, who had a 12-fold increase in the odds of placental malaria (aOR 12.19, 95% CI 5.23-28.43, p < 0.001). CONCLUSIONS: Total number of P. falciparum infections in pregnancy is a significant predictor of placental malaria. The importance of timing of infection on the development of placental malaria varies based on gravidity. In primigravidas, earlier asymptomatic infections were more frequently identified in those with placental malaria, whereas in multigravidas, parasitaemias detected later in gestation were associated with placental malaria. Earlier initiation of an effective intermittent preventive therapy may help to prevent placental malaria and improve birth outcomes, particularly in primigravid women.

Dihydroartemisinin-Piperaquine for the Prevention of Malaria in Pregnancy.

BACKGROUND: Intermittent treatment with sulfadoxine-pyrimethamine is widely recommended for the prevention of malaria in pregnant women in Africa. However, with the spread of resistance to sulfadoxine-pyrimethamine, new interventions are needed. METHODS: We conducted a double-blind, randomized, controlled trial involving 300 human immunodeficiency virus (HIV)-uninfected pregnant adolescents or women in Uganda, where sulfadoxine-pyrimethamine resistance is widespread. We randomly assigned participants to a sulfadoxine-pyrimethamine regimen (106 participants), a three-dose dihydroartemisinin-piperaquine regimen (94 participants), or a monthly dihydroartemisinin-piperaquine regimen (100 participants). The primary outcome was the prevalence of histopathologically confirmed placental malaria. RESULTS: The prevalence of histopathologically confirmed placental malaria was significantly higher in the sulfadoxine-pyrimethamine group (50.0%) than in the three-dose dihydroartemisinin-piperaquine group (34.1%, P=0.03) or the monthly dihydroartemisinin-piperaquine group (27.1%, P=0.001). The prevalence of a composite adverse birth outcome was lower in the monthly dihydroartemisinin-piperaquine group (9.2%) than in the sulfadoxine-pyrimethamine group (18.6%, P=0.05) or the three-dose dihydroartemisinin-piperaquine group (21.3%, P=0.02). During pregnancy, the incidence of symptomatic malaria was significantly higher in the sulfadoxine-pyrimethamine group (41 episodes over 43.0 person-years at risk) than in the three-dose dihydroartemisinin-piperaquine group (12 episodes over 38.2 person-years at risk, P=0.001) or the monthly dihydroartemisinin-piperaquine group (0 episodes over 42.3 person-years at risk, P<0.001), as was the prevalence of parasitemia (40.5% in the sulfadoxine-pyrimethamine group vs. 16.6% in the three-dose dihydroartemisinin-piperaquine group [P<0.001] and 5.2% in the monthly dihydroartemisinin-piperaquine group [P<0.001]). In each treatment group, the risk of vomiting after administration of any dose of the study agents was less than 0.4%, and there were no significant differences among the groups in the risk of adverse events. CONCLUSIONS: The burden of malaria in pregnancy was significantly lower among adolescent girls or women who received intermittent preventive treatment with dihydroartemisinin-piperaquine than among those who received sulfadoxine-pyrimethamine, and monthly treatment with dihydroartemisinin-piperaquine was superior to three-dose dihydroartemisinin-piperaquine with regard to several outcomes. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development; ClinicalTrials.gov number, NCT02163447.).

Dihydroartemisinin-piperaquine for intermittent preventive treatment of malaria during pregnancy and risk of malaria in early childhood: A randomized controlled trial.

BACKGROUND: Intermittent preventive treatment of malaria in pregnancy (IPTp) with dihydroartemisinin-piperaquine (IPTp-DP) has been shown to reduce the burden of malaria during pregnancy compared to sulfadoxine-pyrimethamine (IPTp-SP). However, limited data exist on how IPTp regimens impact malaria risk during infancy. We conducted a double-blinded randomized controlled trial (RCT) to test the hypothesis that children born to mothers given IPTp-DP would have a lower incidence of malaria during infancy compared to children born to mothers who received IPTp-SP. METHODS AND FINDINGS: We compared malaria metrics among children in Tororo, Uganda, born to women randomized to IPTp-SP given every 8 weeks (SP8w, n = 100), IPTp-DP every 8 weeks (DP8w, n = 44), or IPTp-DP every 4 weeks (DP4w, n = 47). After birth, children were given chemoprevention with DP every 12 weeks from 8 weeks to 2 years of age. The primary outcome was incidence of malaria during the first 2 years of life. Secondary outcomes included time to malaria from birth and time to parasitemia following each dose of DP given during infancy. Results are reported after adjustment for clustering (twin gestation) and potential confounders (maternal age, gravidity, and maternal parasitemia status at enrolment).The study took place between June 2014 and May 2017. Compared to children whose mothers were randomized to IPTp-SP8w (0.24 episodes per person year [PPY]), the incidence of malaria was higher in children born to mothers who received IPTp-DP4w (0.42 episodes PPY, adjusted incidence rate ratio [aIRR] 1.92; 95% CI 1.00-3.65, p = 0.049) and nonsignificantly higher in children born to mothers who received IPT-DP8w (0.30 episodes PPY, aIRR 1.44; 95% CI 0.68-3.05, p = 0.34). However, these associations were modified by infant sex. Female children whose mothers were randomized to IPTp-DP4w had an apparently 4-fold higher incidence of malaria compared to female children whose mothers were randomized to IPTp-SP8w (0.65 versus 0.20 episodes PPY, aIRR 4.39, 95% CI 1.87-10.3, p = 0.001), but no significant association was observed in male children (0.20 versus 0.28 episodes PPY, aIRR 0.66, 95% CI 0.25-1.75, p = 0.42). Nonsignificant increases in malaria incidence were observed among female, but not male, children born to mothers who received DP8w versus SP8w. In exploratory analyses, levels of malaria-specific antibodies in cord blood were similar between IPTp groups and sex. However, female children whose mothers were randomized to IPTp-DP4w had lower mean piperaquine (PQ) levels during infancy compared to female children whose mothers received IPTp-SP8w (coef 0.81, 95% CI 0.65-1.00, p = 0.048) and male children whose mothers received IPTp-DP4w (coef 0.72, 95% CI 0.57-0.91, p = 0.006). There were no significant sex-specific differences in PQ levels among children whose mothers were randomized to IPTp-SP8w or IPTp-DP8w. The main limitations were small sample size and childhood provision of DP every 12 weeks in infancy. CONCLUSIONS: Contrary to our hypothesis, preventing malaria in pregnancy with IPTp-DP in the context of chemoprevention with DP during infancy does not lead to a reduced incidence of malaria in childhood; in this setting, it may be associated with an increased incidence of malaria in females. Future studies are needed to better understand the biological mechanisms of in utero drug exposure on drug metabolism and how this may affect the dosing of antimalarial drugs for treatment and prevention during infancy. TRIAL REGISTRATION: ClinicalTrials.gov number NCT02163447.

FCRL5 Delineates Functionally Impaired Memory B Cells Associated with Plasmodium falciparum Exposure.

Exposure to Plasmodium falciparum is associated with circulating "atypical" memory B cells (atMBCs), which appear similar to dysfunctional B cells found in HIV-infected individuals. Functional analysis of atMBCs has been limited, with one report suggesting these cells are not dysfunctional but produce protective antibodies. To better understand the function of malaria-associated atMBCs, we performed global transcriptome analysis of these cells, obtained from individuals living in an area of high malaria endemicity in Uganda. Comparison of gene expression data suggested down-modulation of B cell receptor signaling and apoptosis in atMBCs compared to classical MBCs. Additionally, in contrast to previous reports, we found upregulation of Fc receptor-like 5 (FCRL5), but not FCRL4, on atMBCs. Atypical MBCs were poor spontaneous producers of antibody ex vivo, and higher surface expression of FCRL5 defined a distinct subset of atMBCs compromised in its ability to produce antibody upon stimulation. Moreover, higher levels of P. falciparum exposure were associated with increased frequencies of FCRL5+ atMBCs. Together, our findings suggest that FCLR5+ identifies a functionally distinct, and perhaps dysfunctional, subset of MBCs in individuals exposed to P. falciparum.

Decline of FoxP3+ Regulatory CD4 T Cells in Peripheral Blood of Children Heavily Exposed to Malaria.

FoxP3+ regulatory CD4 T cells (Tregs) help to maintain the delicate balance between pathogen-specific immunity and immune-mediated pathology. Prior studies suggest that Tregs are induced by P. falciparum both in vivo and in vitro; however, the factors influencing Treg homeostasis during acute and chronic infections, and their role in malaria immunopathogenesis, remain unclear. We assessed the frequency and phenotype of Tregs in well-characterized cohorts of children residing in a region of high malaria endemicity in Uganda. We found that both the frequency and absolute numbers of FoxP3+ Tregs in peripheral blood declined markedly with increasing prior malaria incidence. Longitudinal measurements confirmed that this decline occurred only among highly malaria-exposed children. The decline of Tregs from peripheral blood was accompanied by reduced in vitro induction of Tregs by parasite antigen and decreased expression of TNFR2 on Tregs among children who had intense prior exposure to malaria. While Treg frequencies were not associated with protection from malaria, there was a trend toward reduced risk of symptomatic malaria once infected with P. falciparum among children with lower Treg frequencies. These data demonstrate that chronic malaria exposure results in altered Treg homeostasis, which may impact the development of antimalarial immunity in naturally exposed populations.

Both inflammatory and regulatory cytokine responses to malaria are blunted with increasing age in highly exposed children.

BACKGROUND: Young children are at greatest risk for malaria-associated morbidity and mortality. The immune response of young children differs in fundamental ways from that of adults, and these differences likely contribute to the increased susceptibility of children to severe malaria and to their delayed development of immunity. Elevated levels of pro-inflammatory cytokines and chemokines in the peripheral blood during acute infection contribute to the control of parasitaemia, but are also responsible for much of the immunopathology seen during symptomatic disease. Clinical immunity to malaria may depend upon the ability to regulate these pro-inflammatory responses, possibly through mechanisms of immunologic tolerance. In order to explore the effect of age on the immune response to malaria and the development of clinical immunity, cytokines and chemokines were measured in the plasma of children at day 0 of an acute malaria episode and during convalescence. RESULTS: Younger children presenting with acute malaria exhibited much higher levels of TNF, IL2, and IL6, as well as increased Th1 associated chemokines IP10, MIG, and MCP1, compared to older children with acute malaria. Additionally, the regulatory cytokines IL10 and TNFRI were dramatically elevated in younger children compared to older children during acute infection, indicating that regulatory as well as pro-inflammatory cytokine responses are dampened in later childhood. CONCLUSIONS: Together these data suggest that there is a profound blunting of the cytokine and chemokine response to malaria among older children residing in endemic settings, which may be due to repeated malaria exposure, intrinsic age-based differences in the immune response, or both.

Frequent Malaria Drives Progressive Vδ2 T-Cell Loss, Dysfunction, and CD16 Up-regulation During Early Childhood.

γδ T cells expressing Vδ2 may be instrumental in the control of malaria, because they inhibit the replication of blood-stage parasites in vitro and expand during acute malaria infection. However, Vδ2 T-cell frequencies and function are lower among children with heavy prior malaria exposure. It remains unclear whether malaria itself is driving this loss. Here we measure Vδ2 T-cell frequency, cytokine production, and degranulation longitudinally in Ugandan children enrolled in a malaria chemoprevention trial from 6 to 36 months of age. We observed a progressive attenuation of the Vδ2 response only among children incurring high rates of malaria. Unresponsive Vδ2 T cells were marked by expression of CD16, which was elevated in the setting of high malaria transmission. Moreover, chemoprevention during early childhood prevented the development of dysfunctional Vδ2 T cells. These observations provide insight into the role of Vδ2 T cells in the immune response to chronic malaria.

Effective Antimalarial Chemoprevention in Childhood Enhances the Quality of CD4+ T Cells and Limits Their Production of Immunoregulatory Interleukin 10.

BACKGROUND: Experimental inoculation of viable Plasmodium falciparum sporozoites administered with chemoprevention targeting blood-stage parasites results in protective immunity. It is unclear whether chemoprevention similarly enhances immunity following natural exposure to malaria. METHODS: We assessed P. falciparum-specific T-cell responses among Ugandan children who were randomly assigned to receive monthly dihydroartemisinin-piperaquine (DP; n = 87) or no chemoprevention (n = 90) from 6 to 24 months of age, with pharmacologic assessments for adherence, and then clinically followed for an additional year. RESULTS: During the intervention, monthly DP reduced malaria episodes by 55% overall (P < .001) and by 97% among children who were highly adherent to DP (P < .001). In the year after the cessation of chemoprevention, children who were highly adherent to DP had a 55% reduction in malaria incidence as compared to children given no chemoprevention (P = .004). Children randomly assigned to receive DP had higher frequencies of blood-stage specific CD4(+) T cells coproducing interleukin-2 and tumor necrosis factor α (P = .003), which were associated with protection from subsequent clinical malaria and parasitemia, and fewer blood-stage specific CD4(+) T cells coproducing interleukin-10 and interferon γ (P = .001), which were associated with increased risk of malaria. CONCLUSIONS: In this setting, effective antimalarial chemoprevention fostered the development of CD4(+) T cells that coproduced interleukin 2 and tumor necrosis factor α and were associated with prospective protection, while limiting CD4(+) T-cell production of the immunoregulatory cytokine IL-10.

IFNγ/IL-10 co-producing cells dominate the CD4 response to malaria in highly exposed children.

Although evidence suggests that T cells are critical for immunity to malaria, reliable T cell correlates of exposure to and protection from malaria among children living in endemic areas are lacking. We used multiparameter flow cytometry to perform a detailed functional characterization of malaria-specific T cells in 78 four-year-old children enrolled in a longitudinal cohort study in Tororo, Uganda, a highly malaria-endemic region. More than 1800 episodes of malaria were observed in this cohort, with no cases of severe malaria. We quantified production of IFNγ, TNFα, and IL-10 (alone or in combination) by malaria-specific T cells, and analyzed the relationship of this response to past and future malaria incidence. CD4(+) T cell responses were measurable in nearly all children, with the majority of children having CD4(+) T cells producing both IFNγ and IL-10 in response to malaria-infected red blood cells. Frequencies of IFNγ/IL10 co-producing CD4(+) T cells, which express the Th1 transcription factor T-bet, were significantly higher in children with ≥2 prior episodes/year compared to children with <2 episodes/year (P<0.001) and inversely correlated with duration since malaria (Rho = -0.39, P<0.001). Notably, frequencies of IFNγ/IL10 co-producing cells were not associated with protection from future malaria after controlling for prior malaria incidence. In contrast, children with <2 prior episodes/year were significantly more likely to exhibit antigen-specific production of TNFα without IL-10 (P = 0.003). While TNFα-producing CD4(+) T cells were not independently associated with future protection, the absence of cells producing this inflammatory cytokine was associated with the phenotype of asymptomatic infection. Together these data indicate that the functional phenotype of the malaria-specific T cell response is heavily influenced by malaria exposure intensity, with IFNγ/IL10 co-producing CD4(+) T cells dominating this response among highly exposed children. These CD4(+) T cells may play important modulatory roles in the development of antimalarial immunity.

B cell sub-types following acute malaria and associations with clinical immunity.

BACKGROUND: Repeated exposure to Plasmodium falciparum is associated with perturbations in B cell sub-set homeostasis, including expansion atypical memory B cells. However, B cell perturbations immediately following acute malaria infection have been poorly characterized, especially with regard to their relationship with immunity to malaria. METHODS: To better understand the kinetics of B cell sub-sets following malaria, the proportions of six B cell sub-sets were assessed at five time points following acute malaria in four to 5 years old children living in a high transmission region of Uganda. B cell sub-set kinetics were compared with measures of clinical immunity to malaria-lower parasite density at the time of malaria diagnosis and recent asymptomatic parasitaemia. RESULTS: Atypical memory B cell and transitional B cell proportions increased following malaria. In contrast, plasmablast proportions were highest at the time of malaria diagnosis and rapidly declined following treatment. Increased proportions of atypical memory B cells were associated with greater immunity to malaria, whereas increased proportions of transitional B cells were associated with evidence of less immunity to malaria. CONCLUSIONS: These findings highlight the dynamic changes in multiple B cell sub-sets following acute, uncomplicated malaria, and how these sub-sets are associated with developing immunity to malaria.

Timing of in utero malaria exposure influences fetal CD4 T cell regulatory versus effector differentiation.

BACKGROUND: In malaria-endemic areas, the first exposure to malaria antigens often occurs in utero when the fetal immune system is poised towards the development of tolerance. Children exposed to placental malaria have an increased risk of clinical malaria in the first few years of life compared to unexposed children. Recent work has suggested the potential of pregnancy-associated malaria to induce immune tolerance in children living in malaria-endemic areas. A study was completed to evaluate the effect of malaria exposure during pregnancy on fetal immune tolerance and effector responses. METHODS: Using cord blood samples from a cohort of mother-infant pairs followed from early in pregnancy until delivery, flow cytometry analysis was completed to assess the relationship between pregnancy-associated malaria and fetal cord blood CD4 and dendritic cell phenotypes. RESULTS: Cord blood FoxP3+ Treg counts were higher in infants born to mothers with Plasmodium parasitaemia early in pregnancy (12-20 weeks of gestation; p = 0.048), but there was no association between Treg counts and the presence of parasites in the placenta at the time of delivery (by loop-mediated isothermal amplification (LAMP); p = 0.810). In contrast, higher frequencies of activated CD4 T cells (CD25+FoxP3-CD127+) were observed in the cord blood of neonates with active placental Plasmodium infection at the time of delivery (p = 0.035). This population exhibited evidence of effector memory differentiation, suggesting priming of effector T cells in utero. Lastly, myeloid dendritic cells were higher in the cord blood of infants with histopathologic evidence of placental malaria (p < 0.0001). CONCLUSION: Together, these data indicate that in utero exposure to malaria drives expansion of both regulatory and effector T cells in the fetus, and that the timing of this exposure has a pivotal role in determining the polarization of the fetal immune response.

IFNγ Responses to Pre-erythrocytic and Blood-stage Malaria Antigens Exhibit Differential Associations With Past Exposure and Subsequent Protection.

BACKGROUND: The malaria-specific T-cell response is believed to be important for protective immunity. Antimalarial chemoprevention may affect this response by altering exposure to malaria antigens. METHODS: We performed interferon γ (IFNγ) ELISpot assays to assess the cellular immune response to blood-stage and pre-erythrocytic antigens longitudinally from 1 to 3 years of age in 196 children enrolled in a randomized trial of antimalarial chemoprevention in Tororo, Uganda, an area of high transmission intensity. RESULTS: IFNγ responses to blood-stage antigens, particularly MSP1, were frequently detected, strongly associated with recent malaria exposure, and lower in those adherent to chemoprevention compared to nonadherent children and those randomized to no chemoprevention. IFNγ responses to pre-erythrocytic antigens were infrequent and similar between children randomized to chemoprevention or no chemoprevention. Responses to blood-stage antigens were not associated with subsequent protection from malaria (aHR 0.96, P = .83), but responses to pre-erythrocytic antigens were associated with protection after adjusting for prior malaria exposure (aHR 0.52, P = .009). CONCLUSIONS: In this high transmission setting, IFNγ responses to blood-stage antigens were common and associated with recent exposure to malaria but not protection from subsequent malaria. Responses to pre-erythrocytic antigens were uncommon, not associated with exposure but were associated with protection from subsequent malaria.