Search

Background: Changing cell-type proportions can confound studies of differential gene expression or DNA methylation (DNAm) from peripheral blood mononuclear cells (PBMCs). We examined how cell-type proportions derived from the transcriptome versus the methylome (DNAm) influence estimates of differentially expressed genes (DEGs) and differentially methylated positions (DMPs)., Methods: Transcriptome and DNAm data were obtained from PBMC RNA and DNA of Kenyan children (n = 8) before, during, and 6 weeks following uncomplicated malaria. DEGs and DMPs between time points were detected using cell-type adjusted modeling with Cibersortx or IDOL, respectively., Results: Most major cell types and principal components had moderate to high correlation between the two deconvolution methods (r = 0.60-0.96). Estimates of cell-type proportions and DEGs or DMPs were largely unaffected by the method, with the greatest discrepancy in the estimation of neutrophils., Conclusion: Variation in cell-type proportions is captured similarly by both transcriptomic and methylome deconvolution methods for most major cell types., (© 2024. The Author(s).)

Background: Previous studies show increased morbidity in children who are HIV-exposed but uninfected (HEU) compared to children who are HIV-unexposed uninfected (HUU). We sought to evaluate the effects of prenatal HIV exposure on clinical and immunological outcomes in the first 24 months of life., Methods: Eighty-five HEU and 168 HUU children from Kenya were followed from birth to 24 months. All mothers living with HIV received combination antiretroviral therapy. Children who were HEU received standard-of-care cotrimoxazole prophylaxis through 18 months. Episodes of acute illness were identified through a combination of active and passive follow up. Trajectories of plasma cytokines, vaccine-specific antibodies, and antimalarial antibodies were examined., Results: Children who were HEU and children who were HUU had similar growth curves. Children who were HEU had lower rates of malaria (rate ratio 0.54, 95% CI 0.38, 0.77) and respiratory illness (rate ratio 0.80, 95% CI 0.68, 0.93). Trajectories of plasma cytokines and vaccine-specific antibodies were similar in children who were HEU and HUU. There were subtle differences in antimalarial antibody dynamics, in which children who were HEU had overall lower antibody levels against five of the 14 malaria antigens tested., Conclusions: Children who were HEU and born to optimally treated mothers living with HIV had similar growth characteristics and immune profiles compared to children who were HUU. Children who were HEU had reduced risk for malaria and respiratory illness, which may be secondary to cotrimoxazole prophylaxis., (© 2024. The Author(s).)

Ebola disease is a severe disease with extremely high case-fatality rates ranging from 28-100%. Observations made during the 2013-2016 West African epidemic improved our understanding of the clinical course of Ebola disease and accelerated the study of therapeutic and preventative strategies. The epidemic also highlighted the unique challenges associated with providing optimal care for children during Ebola disease outbreaks. In this review, we outline current understanding of Ebola disease epidemiology, pathogenesis, management, and prevention, highlighting data pertinent to the care of children. IMPACT: In this review, we summarize recent advancements in our understanding of Ebola disease epidemiology, clinical presentation, and therapeutic and preventative strategies. We highlight recent data pertinent to the care of children and pregnant women and identify research gaps for this important emerging viral infection in children., (© 2023. The Author(s), under exclusive licence to the International Pediatric Research Foundation, Inc.)

Food insecurity, recurrent famine, and poverty threaten the health of millions of African residents. Construction of dams and rural irrigation schemes is key to solving these problems. The sub-Saharan Africa International Center of Excellence for Malaria Research addresses major knowledge gaps and challenges in Plasmodium falciparum and Plasmodium vivax malaria control and elimination in malaria-endemic areas of Kenya and Ethiopia where major investments in water resource development are taking place. This article highlights progress of the International Center of Excellence for Malaria Research in malaria vector ecology and behavior, epidemiology, and pathogenesis since its inception in 2017. Studies conducted in four field sites in Kenya and Ethiopia show that dams and irrigation increased the abundance, stability, and productivity of larval habitats, resulting in increased malaria transmission and a greater disease burden. These field studies, together with hydrological and malaria transmission modeling, enhance the ability to predict the impact of water resource development projects on vector larval ecology and malaria risks, thereby facilitating the development of optimal water and environmental management practices in the context of malaria control efforts. Intersectoral collaborations and community engagement are crucial to develop and implement cost-effective malaria control strategies that meet food security needs while controlling malaria burden in local communities.

Children under the age of 5 years living in areas of moderate to high malaria transmission are highly susceptible to clinical malaria with fever that prompts treatment of blood stage infection with anti-malarial drugs. In contrast, older school age children frequently experience subclinical malaria, i.e. chronic Plasmodium falciparum parasitemia without fever or other clinical symptoms. The role of innate immune cells in regulating inflammation at a level that is sufficient to control the parasite biomass, while at the same time maintaining a disease-tolerant clinical phenotype, i.e., subclinical malaria, is not well understood. Recent studies suggest that host epigenetic mechanisms underlie the innate immune homeostasis associated with subclinical malaria. This Current Opinion article presents evidence supporting the notion that modifications of the host monocyte/macrophage epigenome regulate innate immune functions pertinent to subclinical malaria., (Copyright © 2022 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

The Plasmodium falciparum circumsporozoite protein (CSP) forms the basis of leading subunit malaria vaccine candidates. However, the mechanisms and specific targets of immunity are poorly defined. Recent findings suggest that antibody-mediated complement-fixation and activation play an important role in immunity. Here, we investigated the regions of CSP targeted by functional complement-fixing antibodies and the antibody properties associated with this activity. We quantified IgG, IgM, and functional complement-fixing antibody responses to different regions of CSP among Kenyan adults naturally exposed to malaria (n=102) and using a series of rabbit vaccination studies. Individuals who acquired functional complement-fixing antibodies had higher IgG, IgM and IgG1 and IgG3 to CSP. Acquired complement-fixing antibodies targeted the N-terminal, central-repeat, and C-terminal regions of CSP, and positive responders had greater antibody breadth compared to those who were negative for complement-fixing antibodies (p<0.05). Using rabbit vaccinations as a model, we confirmed that IgG specific to the central-repeat and non-repeat regions of CSP could effectively fix complement. However, vaccination with near full length CSP in rabbits poorly induced antibodies to the N-terminal region compared to naturally-acquired immunity in humans. Poor induction of N-terminal antibodies was also observed in a vaccination study performed in mice. IgG and IgM to all three regions of CSP play a role in mediating complement-fixation, which has important implications for malaria vaccine development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Kurtovic, Drew, Dent, Kazura and Beeson.)

Background: Interferon (IFN)- λ4, a type III IFN, production is controlled by a dinucleotide frameshift variant (rs368234815-dG/TT) within the first exon of the IFNL4 gene. Carriers of the IFNL4-dG allele but not the IFNL4-TT allele are able to produce the IFN-λ4 protein. Patients with hepatitis C virus that do not produce the IFN-λ4 protein have higher rates of viral clearance suggesting a potential inhibitory role of IFN-λ4 in liver-tropic infections., Methods: In this study, it was investigated whether children infected with Plasmodium falciparum, which has a well-characterized liver stage infection, would be more susceptible to clinical malaria relative to their IFNL4-rs368234815 allele. A cohort of 122 children from a malaria holoendemic region of Kenya was analysed. Episodes of clinical malaria and upper respiratory tract infections (URTIs) were determined using information collected from birth to 2 years of age. The dinucleotide frameshift variant IFNL4-rs368234815-dG/TT was genotyped using a TaqMan assay., Results: In this cohort, 33% of the study participants had the dG/dG genotype, 45% had the dG/TT genotype, and 22% had TT/TT genotype. The number and time to first episode of clinical malaria and URTIs with respect to the IFNL4-rs368234815 allele was evaluated. It was found that children that carried the IFNL4-rs368234815-dG allele had an increased number of clinical malaria episodes. In addition, there was a significant association between earlier age of first malaria infection with carriers of the IFNL4-dG allele (p-value: 0.021)., Conclusion: The results suggest that the ability to produce IFN-λ4 negatively affects host immune protection against P. falciparum malaria in Kenyan children.

A highly protective vaccine will greatly facilitate achieving and sustaining malaria elimination. Understanding mechanisms of antibody-mediated immunity is crucial for developing vaccines with high efficacy. Here, we identify key roles in humoral immunity for Fcγ-receptor (FcγR) interactions and opsonic phagocytosis of sporozoites. We identify a major role for neutrophils in mediating phagocytic clearance of sporozoites in peripheral blood, whereas monocytes contribute a minor role. Antibodies also promote natural killer cell activity. Mechanistically, antibody interactions with FcγRIII appear essential, with FcγRIIa also required for maximum activity. All regions of the circumsporozoite protein are targets of functional antibodies against sporozoites, and N-terminal antibodies have more activity in some assays. Functional antibodies are slowly acquired following natural exposure to malaria, being present among some exposed adults, but uncommon among children. Our findings reveal targets and mechanisms of immunity that could be exploited in vaccine design to maximize efficacy.

Background: Age-related changes in adaptive and innate immune cells have been associated with a decline in effective immunity and chronic, low-grade inflammation. Epigenetic, transcriptional, and functional changes in monocytes occur with aging, though most studies to date have focused on differences between young adults and the elderly in populations with European ancestry; few data exist regarding changes that occur in circulating monocytes during the first few decades of life or in African populations. We analyzed DNA methylation profiles, cytokine production, and inflammatory gene expression profiles in monocytes from young adults and children from western Kenya., Results: We identified several hypo- and hyper-methylated CpG sites in monocytes from Kenyan young adults vs. children that replicated findings in the current literature of differential DNA methylation in monocytes from elderly persons vs. young adults across diverse populations. Differentially methylated CpG sites were also noted in gene regions important to inflammation and innate immune responses. Monocytes from Kenyan young adults vs. children displayed increased production of IL-8, IL-10, and IL-12p70 in response to TLR4 and TLR2/1 stimulation as well as distinct inflammatory gene expression profiles., Conclusions: These findings complement previous reports of age-related methylation changes in isolated monocytes and provide novel insights into the role of age-associated changes in innate immune functions.

Background: Transmission stemming from asymptomatic infections is increasingly being recognized as a threat to malaria elimination. In many regions, malaria transmission is seasonal. It is not well understood whether Plasmodium falciparum modulates its investment in transmission to coincide with seasonal vector abundance., Methods: We sampled 1116 asymptomatic individuals in the wet season, when vectors are abundant, and 1743 in the dry season, in two sites in western Kenya, representing different transmission intensities (Chulaimbo, moderate transmission, and Homa Bay, low transmission). Blood samples were screened for P. falciparum by qPCR, and gametocytes by pfs25 RT-qPCR., Results: Parasite prevalence by qPCR was 27.1% (Chulaimbo, dry), 48.2% (Chulaimbo, wet), 9.4% (Homabay, dry), and 7.8% (Homabay, wet). Mean parasite densities did not differ between seasons (P = 0.562). pfs25 transcripts were detected in 119/456 (26.1%) of infections. In the wet season, fewer infections harbored detectable gametocytes (22.3% vs. 33.8%, P = 0.009), but densities were 3-fold higher (wet: 3.46 transcripts/uL, dry: 1.05 transcripts/uL, P < 0.001). In the dry season, 4.0% of infections carried gametocytes at moderate-to-high densities likely infective (> 1 gametocyte per 2 uL blood), compared to 7.9% in the wet season. Children aged 5-15 years harbored 76.7% of infections with gametocytes at moderate-to-high densities., Conclusions: Parasites increase their investment in transmission in the wet season, reflected by higher gametocyte densities. Despite increased gametocyte densities, parasite density remained similar across seasons and were often below the limit of detection of microscopy or rapid diagnostic test, thus a large proportion of infective infections would escape population screening in the wet season. Seasonal changes of gametocytemia in asymptomatic infections need to be considered when designing malaria control measures.

Monocytes are innate immune cells essential for host protection against malaria. Upon activation, monocytes function to help reduce parasite burden through phagocytosis, cytokine production, and antigen presentation. However, monocytes have also been implicated in the pathogenesis of severe disease through production of damaging inflammatory cytokines, resulting in systemic inflammation and vascular dysfunction. Understanding the molecular pathways influencing the balance between protection and pathology is critical. In this review, we discuss recent data regarding the role of monocytes in human malaria, including studies of innate sensing of the parasite, immunometabolism, and innate immune training. Knowledge gained from these studies may guide rational development of novel antimalarial therapies and inform vaccine development., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Altered neonatal immune responses may contribute to the increased morbidity observed in HIV-exposed but uninfected (HEU) infants compared with HIV-unexposed uninfected (HUU) infants. We sought to examine the effects of prenatal HIV and malaria exposure on maternal and neonatal plasma cytokine profiles and transplacental antibody transfer., Methods: Forty-nine HIV+ and 50 HIV- women and their HIV-uninfected neonate pairs from Kenya were assessed. All HIV+ mothers received combination antiretroviral therapy. Maternal plasma and cord blood plasma samples at delivery were tested for 12 cytokines, total IgG, and IgG specific to 4 vaccine antigens and 14 Plasmodium falciparum antigens., Results: HIV+ mothers had lower levels of all 12 plasma cytokines at delivery compared with HIV- mothers, but there were no differences between HEU and HUU neonates. There were no differences in the cord-to-maternal ratios (CMRs) of vaccine-specific IgG between HIV+/HEU and HIV-/HUU maternal-neonate pairs. HIV+/HEU maternal-neonate pairs had significantly lower CMRs for 3 antimalarial IgGs-merozoite surface protein 9, circumsporozoite protein, and erythrocyte binding antigen 181-which remained statistically significant after adjustment for malaria in pregnancy., Conclusions: In a cohort of optimally treated HIV-infected pregnant women, maternal HIV infection was associated with reduced transplacental transfer of antimalarial antibodies.

Background: Studies of the association between the level of anti-malarial antibody and protection from malaria infection can yield conflicting results if they fail to take into account differences in the malaria transmission rate. This can occur because high malaria exposure may drive high antibody responses, leading to an apparent positive association between immune response and infection rate. The neonatal period provides a unique window to study the protective effects of antibodies, because waning maternally-derived antibodies lead to different levels of protection with time., Methods: This study uses data from two well-defined infant cohorts in Western Kenya with different burdens of malaria transmission. Survival models were used to assess how the magnitude of maternally derived malaria-specific IgG antibody (to 24 malaria antigens measured using Luminex beads) affected the time-to-first Plasmodium falciparum infection (detected by PCR). In addition, mathematical models were used to assess how the frequency of malaria infection varied between the cohorts with different exposure levels., Results: Despite differences in underlying malaria incidence in the two regions, there was no difference in time-to-first malaria infection between the cohorts. However, there was a significant period of protection observed in children with high initial MSP1 (42 kDa fragment)-specific antibody levels, but this protection was not observed in children with low antibody levels. Children from the high transmission cohort had both longer initial periods of protection from malaria (attributable to higher initial antibody levels), but more rapid time-to-first-infection once malaria specific maternal antibodies declined below protective levels (attributable to higher exposure rates)., Conclusion: This study demonstrates the complex interaction between passive (maternally-derived) immunity and the degree of malaria exposure in infants. Children from regions of high malaria transmission had higher levels of maternally-derived antibodies in early life, which led to a significant protection for several months. However, once this immunity waned, the underlying higher frequency of infection was revealed. A better understanding of the interaction between malaria exposure, immunity, and transmission risk will assist in identifying protective immune responses in P. falciparum infection.

Vaccines that target Plasmodium falciparum gametocytes have the potential to reduce malaria transmission and are thus attractive targets for malaria control. However, very little is known about human immune responses to gametocytes present in human hosts. We evaluated naturally-acquired antibodies to gametocyte-infected erythrocytes (gametocyte-IEs) of different developmental stages compared to other asexual parasite stages among naturally-exposed Kenyan residents. We found that acquired antibodies strongly recognized the surface of mature asexual-IEs, but there was limited reactivity to the surface of gametocyte-IEs of different stages. We used genetically-modified P. falciparum with suppressed expression of PfEMP1, the major surface antigen of asexual-stage IEs, to demonstrate that PfEMP1 is a dominant target of antibodies to asexual-IEs, in contrast to gametocyte-IEs. Antibody reactivity to gametocyte-IEs was similar to asexual-IEs lacking PfEMP1. Significant antibody reactivity to the surface of gametocytes was observed when outside of the host erythrocyte, including recognition of the major gametocyte antigen, Pfs230. This indicates that there is a deficiency of acquired antibodies to gametocyte-IEs despite the acquisition of antibodies to gametocyte antigens and asexual IEs. Our findings suggest that the acquisition of substantial immunity to the surface of gametocyte-IEs is limited, which may facilitate immune evasion to enable malaria transmission even in the face of substantial host immunity to malaria. Further studies are needed to understand the basis for the limited acquisition of antibodies to gametocytes and whether vaccine strategies can generate substantial immunity.

Background: Antibodies targeting Plasmodium falciparum sporozoites play a key role in human immunity to malaria. However, antibody mechanisms that neutralize sporozoites are poorly understood. This has been a major constraint in developing highly efficacious vaccines, as we lack strong correlates of protective immunity., Methods: We quantified the ability of human antibodies from malaria-exposed populations to interact with human complement, examined the functional effects of complement activity against P. falciparum sporozoites in vitro, and identified targets of functional antibodies. In children and adults from malaria-endemic regions, we determined the acquisition of complement-fixing antibodies to sporozoites and their relationship with antibody isotypes and subclasses. We also investigated associations with protective immunity in a longitudinal cohort of children (n = 206) residing in a malaria-endemic region., Results: We found that antibodies to the major sporozoite surface antigen, circumsporozoite protein (CSP), were predominately IgG1, IgG3, and IgM, and could interact with complement through recruitment of C1q and activation of the classical pathway. The central repeat region of CSP, included in leading vaccines, was a key target of complement-fixing antibodies. We show that antibodies activate human complement on P. falciparum sporozoites, which consequently inhibited hepatocyte cell traversal that is essential for establishing liver-stage infection, and led to sporozoite death in vitro. The natural acquisition of complement-fixing antibodies in malaria-exposed populations was age-dependent, and was acquired more slowly to sporozoite antigens than to merozoite antigens. In a longitudinal cohort of children, high levels of complement-fixing antibodies were significantly associated with protection against clinical malaria., Conclusions: These novel findings point to complement activation by antibodies as an important mechanism of anti-sporozoite human immunity, thereby enabling new strategies for developing highly efficacious malaria vaccines. We also present evidence that complement-fixing antibodies may be a valuable correlate of protective immunity in humans.

Malarial infection in naive individuals induces a robust innate immune response. In the recently described model of innate immune memory, an initial stimulus primes the innate immune system to either hyperrespond (termed training) or hyporespond (tolerance) to subsequent immune challenge. Previous work in both mice and humans demonstrated that infection with malaria can both serve as a priming stimulus and promote tolerance to subsequent infection. In this study, we demonstrate that initial stimulation with Plasmodium falciparum -infected RBCs or the malaria crystal hemozoin induced human adherent PBMCs to hyperrespond to subsequent ligation of TLR2. This hyperresponsiveness correlated with increased H3K4me3 at important immunometabolic promoters, and these epigenetic modifications were also seen in Kenyan children naturally infected with malaria. However, the use of epigenetic and metabolic inhibitors indicated that the induction of trained immunity by malaria and its ligands may occur via a previously unrecognized mechanism(s)., (Copyright © 2018 by The American Association of Immunologists, Inc.)

We sought to identify a subset of Plasmodium falciparum antibody targets that would inform monitoring efforts needed to eliminate malaria in high transmission settings. IgG antibodies to 28 recombinant Pf antigens were measured in residents of two communities in western Kenya examined in 2003 and 2013, when the respective prevalence of asymptomatic parasitemia among children was 81 and 15 percent by microscopy. Annual seroconversion rates based on a sero-catalytic model that dichotomised antibody values to negative versus positive showed that rates were higher in 2003 than 2013 for 1 pre-erythrocytic and 7 blood-stage antigens. Antibody acquisition models that considered antibody levels as continuous variables showed that age-related antibody levels to Circumsporozoite Protein and 10 merozoite proteins increased at different rates with age in 2003 versus 2013. Both models found that antibodies to 5 proteins of the Merozoite Surface Protein 1 complex were differentially acquired between the cohorts, and that changes in antibody levels to Apical Membrane Antigen 1 suggested a decrease in transmission that occurred ~10 years before 2013. Further studies evaluating antibodies to this subset of Pf antigens as biomarkers of malaria exposure and naturally acquired immunity are warranted in endemic settings where transmission has been reduced but persists.

Background: Inflammation and monocytes are thought to be important to human malaria pathogenesis. However, the relationship of inflammation and various monocyte functions to acute malaria, recovery from acute malaria, and asymptomatic parasitemia in endemic populations is poorly understood., Methods: We evaluated plasma cytokine levels, monocyte subsets, monocyte functional responses, and monocyte inflammatory transcriptional profiles of 1- to 10-year-old Kenyan children at the time of presentation with acute uncomplicated malaria and at recovery 6 weeks later; these results were compared with analogous data from asymptomatic children and adults in the same community., Results: Acute malaria was marked by elevated levels of proinflammatory and regulatory cytokines and expansion of the inflammatory "intermediate" monocyte subset that returned to levels of healthy asymptomatic children 6 weeks later. Monocytes displayed activated phenotypes during acute malaria, with changes in surface expression of markers important to innate and adaptive immunity. Functionally, acute malaria monocytes and monocytes from asymptomatic infected children had impaired phagocytosis of P. falciparum-infected erythrocytes relative to asymptomatic children with no blood-stage infection. Monocytes from both acute malaria and recovery time points displayed strong and equivalent cytokine responsiveness to innate immune agonists that were independent of infection status. Monocyte transcriptional profiles revealed regulated and balanced proinflammatory and antiinflammatory and altered phagocytosis gene expression patterns distinct from malaria-naive monocytes., Conclusion: These observations provide insights into monocyte functions and the innate immune response during uncomplicated malaria and suggest that asymptomatic parasitemia in children is not clinically benign., Funding: Support for this work was provided by NIH/National Institute of Allergy and Infectious Diseases (R01AI095192-05), the Burroughs Wellcome Fund/American Society of Tropical Medicine and Hygiene, and the Rainbow Babies & Children's Foundation.

Background: The 2 strains of Epstein-Barr virus (EBV), EBV type 1 (EBV-1) and EBV-2, differ in latency genes, suggesting that they use distinct mechanisms to establish latency. We previously reported that EBV-2 infects T cells in vitro. In this study, we tested the possibility that EBV-2 infects T cells in vivo., Methods: Purified T-cell fractions isolated from children positive for EBV-1 or EBV-2 and their mothers were examined for the presence of EBV and for EBV type., Results: We detected EBV-2 in all T-cell samples obtained from EBV-2-infected children at 12 months of age, with some children retaining EBV-2-positive T cells through 24 months of age, suggesting that EBV-2 persists in T cells. We were unable to detect EBV-2 in T-cell samples from mothers but could detect EBV-2 in samples of their breast milk and saliva., Conclusions: These data suggest that EBV-2 uses T cells as an additional latency reservoir but that, over time, the frequency of infected T cells may drop below detectable levels. Alternatively, EBV-2 may establish a prolonged transient infection in the T-cell compartment. Collectively, these novel findings demonstrate that EBV-2 infects T cells in vivo and suggest EBV-2 may use the T-cell compartment to establish latency., (© The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

Maternal plasma 25-hydroxyvitamin D (25(OH)D) status and its association with pregnancy outcomes in malaria holoendemic regions of sub-Saharan Africa is poorly defined. We examined this association and any potential interaction with malaria and helminth infections in an ongoing pregnancy cohort study in Kenya. The association of maternal plasma 25(OH)D status with pregnancy outcomes and infant anthropometric measurements at birth was determined in a subset of women ( n = 63). Binomial and linear regression analyses were used to examine associations between maternal plasma 25(OH)D and adverse pregnancy outcomes. Fifty-one percent of the women had insufficient (<75 nmol/L) and 21% had deficient (<50 nmol/L) plasma 25(OH)D concentration at enrollment. At birth, 74.4% of the infants had insufficient and 30% had deficient plasma 25(OH)D concentrations, measured in cord blood. Multivariate analysis controlling for maternal age and body mass index (BMI) at enrollment and gestational age at delivery found that deficient plasma 25(OH)D levels were associated with a four-fold higher risk of stunting in neonates ( p = 0.04). These findings add to the existing literature about vitamin D and its association with linear growth in resource-limited settings, though randomized clinical trials are needed to establish causation., Competing Interests: S.M. Is an unpaid board member of a start-up focused on bringing the results of his research on point-of-care diagnostics for nutritional status as a faculty member at Cornell University to market. The other authors declare no conflict of interest.

Background: The polymorphic nature of many malaria vaccine candidates presents major challenges to achieving highly efficacious vaccines. Presently, there is very little knowledge on the prevalence and patterns of functional immune responses to polymorphic vaccine candidates in populations to guide vaccine design. A leading polymorphic vaccine candidate against blood-stage Plasmodium falciparum is apical membrane antigen 1 (AMA1), which is essential for erythrocyte invasion. The importance of AMA1 as a target of acquired human inhibitory antibodies, their allele specificity and prevalence in populations is unknown, but crucial for vaccine design., Methods: P. falciparum lines expressing different AMA1 alleles were genetically engineered and used to quantify functional antibodies from two malaria-exposed populations of adults and children. The acquisition of AMA1 antibodies was also detected using enzyme-linked immunosorbent assay (ELISA) and competition ELISA (using different AMA1 alleles) from the same populations., Results: We found that AMA1 was a major target of naturally acquired invasion-inhibitory antibodies that were highly prevalent in malaria-endemic populations and showed a high degree of allele specificity. Significantly, the prevalence of inhibitory antibodies to different alleles varied substantially within populations and between geographic locations. Inhibitory antibodies to three specific alleles were highly prevalent (FVO and W2mef in Papua New Guinea; FVO and XIE in Kenya), identifying them for potential vaccine inclusion. Measurement of antibodies by standard or competition ELISA was not strongly predictive of allele-specific inhibitory antibodies. The patterns of allele-specific functional antibody responses detected with our novel assays may indicate that acquired immunity is elicited towards serotypes that are prevalent in each geographic location., Conclusions: These findings provide new insights into the nature and acquisition of functional immunity to a polymorphic vaccine candidate and strategies to quantify functional immunity in populations to guide rational vaccine design.

Malaria is one of the most serious infectious diseases with most of the severe disease caused by Plasmodium falciparum (Pf). Naturally acquired immunity develops over time after repeated infections and the development of antimalarial antibodies is thought to play a crucial role. Neonates and young infants are relatively protected from symptomatic malaria through mechanisms that are poorly understood. The prevailing paradigm is that maternal antimalarial antibodies transferred to the fetus in the last trimester of pregnancy protect the infant from early infections. These antimalarial antibodies wane by approximately 6 months of age leaving the infant vulnerable to malaria, however direct evidence supporting this epidemiologically based paradigm is lacking. As infants are the target population for future malaria vaccines, understanding how they begin to develop immunity to malaria and the gaps in their responses is key. This review summarizes the antimalarial antibody responses detected in infants and how they change over time. We focus primarily on Pf antibody responses and will briefly mention Plasmodium vivax responses in infants.

IgG antibodies to Plasmodium falciparum are transferred from the maternal to fetal circulation during pregnancy, wane after birth, and are subsequently acquired in response to natural infection. We examined the dynamics of malaria antibody responses of 84 Kenyan infants from birth to 36 months of age by (i) serology, (ii) variant surface antigen (VSA) assay, (iii) growth inhibitory activity (GIA), and (iv) invasion inhibition assays (IIA) specific for merozoite surface protein 1 (MSP1) and sialic acid-dependent invasion pathway. Maternal antibodies in each of these four categories were detected in cord blood and decreased to their lowest level by approximately 6 months of age. Serologic antibodies to 3 preerythrocytic and 10 blood-stage antigens subsequently increased, reaching peak prevalence by 36 months. In contrast, antibodies measured by VSA, GIA, and IIA remained low even up to 36 months. Infants sensitized to P. falciparum in utero, defined by cord blood lymphocyte recall responses to malaria antigens, acquired antimalarial antibodies at the same rate as those who were not sensitized in utero, indicating that fetal exposure to malaria antigens did not affect subsequent infant antimalarial responses. Infants with detectable serologic antibodies at 12 months of age had an increased risk of P. falciparum infection during the subsequent 24 months. We conclude that serologic measures of antimalarial antibodies in children 36 months of age or younger represent biomarkers of malaria exposure rather than protection and that functional antibodies develop after 36 months of age in this population., (Copyright © 2016 Dent et al.)

Background: We previously reported that infants in Kenya were infected with Epstein-Barr virus (EBV) at <6 months of age, suggesting that mothers were the likely source of transmissible virus to the infant. In this study, we investigated whether breast milk contained infectious EBV and the role of malaria in EBV shedding in breast milk., Methods: Breast milk samples were obtained from Kenyan mothers at postpartum weeks 6, 10, 14, and 18 and analyzed for presence of infectious EBV., Results: We found that the prevalence of EBV DNA and the mean EBV load were significantly higher at 6 weeks and decreased through postpartum week 18 (P < .0001). High EBV load in breast milk correlated with mothers who had Plasmodium falciparum malaria at delivery. To determine whether viral DNA was encapsidated, breast milk samples were treated with DNAse before DNA extraction. Sixty percent of samples were DNAse resistant, suggesting that the viral DNA in breast milk was encapsidated. Next, we exposed peripheral blood mononuclear cells to breast milk supernatant, which resulted in the generation of EBV-positive lymphoblastoid cell lines, indicating that the virus in breast milk was infectious., Conclusions: Our data suggest that breast milk contains infectious EBV and is a potential source of viral transmission to infants living in malaria-endemic regions., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Over 35% of children in a region of malaria endemicity are infected with Epstein-Barr virus (EBV) by 6 months of age. This susceptibility may be linked to impaired transplacental transfer of antibodies. In this study, we determined the effect of malaria exposure during pregnancy on the transfer of EBV-specific maternal antibodies in a region of western Kenya that experiences endemic malaria. Pregnant mothers were recruited and followed up until delivery to determine levels of neonatal malaria exposure. Levels of EBV lytic (viral capsid antigen [VCA], Z transcriptional activator [Zta], and early diffuse antigen complex [EAd]) and EBV latent (EBV nuclear antigen-1 (EBNA1]) and tetanus-specific IgG antibodies were measured in 70 paired maternal and cord blood samples using a Luminex-bead-based assay. A high proportion (63%) of the infants were exposed to malaria in utero. Levels of EBV- and tetanus-specific antibodies were similar in malaria-infected mothers and in mothers who had no detectable malaria infection. Malaria-exposed neonates had significantly lower levels of anti-EBNA1, anti-Zta, and anti-EAd antibodies than were seen in their mothers. In utero malaria exposure resulted in significant reductions in transplacental transfer of anti-VCA-p18 and anti-EBNA1 antibodies of 13% and 22%, respectively. Neonates received significantly low levels of anti-Zta and anti-EAd antibodies irrespective of malaria exposure levels. In multivariate analysis, in utero malaria exposure was associated with a significant reduction in the transfer of anti-VCA-p18 and anti-EBNA1 antibodies to the neonates (P = 0.0234 and P = 0.0017, respectively). Malaria during pregnancy results in differential levels of transfer of EBV-specific antibodies from the mother to the fetus. The impaired transplacental transfer of some antibodies may lead to the malaria-exposed neonates being susceptible to early EBV infection., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Immunoglobulin G antibodies (Abs) to Plasmodium falciparum antigens have been associated with naturally acquired immunity to symptomatic malaria., Methods: We probed protein microarrays covering 824 unique P. falciparum protein features with plasma from residents of a community in Kenya monitored for 12 weeks for (re)infection and symptomatic malaria after administration of antimalarial drugs. P. falciparum proteins recognized by Abs from 88 children (aged 1-14 years) and 86 adults (aged ≥ 18 years), measured at the beginning of the observation period, were ranked by Ab signal intensity., Results: Abs from immune adults reacted with a total 163 of 824 P. falciparum proteins. Children gradually acquired Abs to the full repertoire of antigens recognized by adults. Abs to some antigens showed high seroconversion rates, reaching maximal levels early in childhood, whereas others did not reach adult levels until adolescence. No correlation between Ab signal intensity and time to (re)infection was observed. In contrast, Ab levels to 106 antigens were significantly higher in children who were protected from symptomatic malaria compared with those who were not. Abs to antigens predictive of protection included P. falciparum erythrocyte membrane protein 1, merozoite surface protein (MSP) 10, MSP2, liver-stage antigen 3, PF70, MSP7, and Plasmodium helical interspersed subtelomeric domain protein., Conclusions: Protein microarrays may be useful in the search for malaria antigens associated with protective immunity., (© The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objective: To use ultrasound to explore the impact of malaria in pregnancy on fetal growth and newborn outcomes among a cohort of women enrolled in an intermittent presumptive treatment in pregnancy (IPTp) with sulfadoxine/pyrimethamine (SP) program in coastal Kenya., Methods: Enrolled women were tested for malaria at first prenatal care visit, and physical and ultrasound examinations were performed. In total, 477 women who had term, live births had malaria tested at delivery and their birth outcomes assessed, and were included in the study., Results: Peripheral malaria was detected via polymerase chain reaction among 10.9% (n=87) at first prenatal care visit and 8.8% (n=36) at delivery. Insecticide-treated bed nets (ITNs) were used by 73.6% (n=583) and were associated with decreased malaria risk. There was a trend for impaired fetal growth and placental blood flow in malaria-infected women in the second trimester, but not later in pregnancy. Among women with low body mass index (BMI), malaria was associated with reduced birth weight (P=0.04); anthropometric measures were similar otherwise., Conclusion: With IPTp-SP and ITNs, malaria in pregnancy was associated with transient differences in utero, and reduced birth weight was restricted to those with low BMI., (Copyright © 2014 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.)

Background: Relative contribution of these infections on anemia in pregnancy is not certain. While measures to protect pregnant women against malaria have been scaling up, interventions against helminthes have received much less attention. In this study, we determine the relative impact of helminthes and malaria on maternal anemia., Methods: A prospective observational study was conducted in coastal Kenya among a cohort of pregnant women who were recruited at their first antenatal care (ANC) visit and tested for malaria, hookworm, and other parasitic infections and anemia at enrollment. All women enrolled in the study received presumptive treatment with sulfadoxine-pyrimethamine, iron and multi-vitamins and women diagnosed with helminthic infections were treated with albendazole. Women delivering a live, term birth, were also tested for maternal anemia, fetal anemia and presence of infection at delivery., Principal Findings: Of the 706 women studied, at the first ANC visit, 27% had moderate/severe anemia and 71% of women were anemic overall. The infections with highest prevalence were hookworm (24%), urogenital schistosomiasis (17%), trichuria (10%), and malaria (9%). In adjusted and unadjusted analyses, moderate/severe anemia at first ANC visit was associated with the higher intensities of hookworm and P. falciparum microscopy-malaria infections. At delivery, 34% of women had moderate/severe anemia and 18% of infants' cord hemoglobin was consistent with fetal anemia. While none of the maternal infections were significantly associated with fetal anemia, moderate/severe maternal anemia was associated with fetal anemia., Conclusions: More than one quarter of women receiving standard ANC with IPTp for malaria had moderate/severe anemia in pregnancy and high rates of parasitic infection. Thus, addressing the role of co-infections, such as hookworm, as well as under-nutrition, and their contribution to anemia is needed.

Transplacental transfer of antibodies from clinically malaria immune pregnant women to their fetuses is thought to provide passive protection against malaria during infancy. However, the presences and duration of functional antibodies against Plasmodium falciparum (Pf) in newborns has not been described. We used growth inhibition assays (GIA) to measure total anti-malaria functional antibodies present at birth and over the following year. Samples were drawn from cord blood (n=86) and in infants at six and 12 months of life (n=86 and 65 respectively). Three laboratory Pf strains (D10, W2mef, 3D7) and a field isolate (Msambweni 2006) were used in the assays. Median (ranges) GIA levels for cord plasma differed between laboratory parasite strains: D10, 0% (0-81); W2mef, 6% (0-80); 3D7, 18% (0-88); Msambweni 2006, 6% (0-43) (P<0.001, Wilcoxon signed-rank test). GIA levels against all Pf strains were found to decline in infants from birth to six months (P<0.01, Wilcoxon, signed-rank test). Functional antibodies as measured by GIA are transferred to the fetus and wane in the infants over time. Infant protection from clinical malaria disease may in part be mediated by these functional anti-malaria antibodies., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background:  Acquired immunity to malaria develops with increasing age and repeated infections. Understanding immune correlates of protection from malaria would facilitate vaccine development and identification of biomarkers that reflect changes in susceptibility resulting from ongoing malaria control efforts., Methods:  The relationship between immunoglobulin G (IgG) antibody and both interferon γ (IFN-γ) and interleukin 10 (IL-10) responses to the 42-kD C-terminal fragment of Plasmodium falciparum merozoite surface protein 1 (MSP142) and the risk of (re)infection were examined following drug-mediated clearance of parasitemia in 94 adults and 95 children in an area of holoendemicity of western Kenya., Results:  Positive IFN-γ enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunosorbent spot assay (ELISPOT) responses to MSP142 3D7 were associated with delayed time to (re)infection, whereas high-titer IgG antibodies to MSP142 3D7 or FVO alleles were not independently predictive of the risk of (re)infection. When IFN-γ and IL-10 responses were both present, the protective effect of IFN-γ was abrogated. A Cox proportional hazard model including IFN-γ, IL-10, MSP142 3D7 IgG antibody responses, hemoglobin S genotype, age, and infection status at baseline showed that the time to blood-stage infection correlated positively with IFN-γ responses and negatively with IL-10 responses, younger age, and asymptomatic parasitemia., Conclusions:  Evaluating combined allele-specific cellular and humoral immunity elicited by malaria provides a more informative measure of protection relative to evaluation of either measure alone.

Background: Malaria in pregnancy is a significant contributor to adverse pregnancy outcome, especially in Sub-Saharan Africa. Prevention with sulfadoxine/pyrimethamine (SP) during pregnancy has been recommended in malaria-endemic areas but concerns remain about its benefit., Objectives: To evaluate the association between recommended preventative SP programs in pregnancy and low birth weight (LBW) and maternal anemia through available clinical trial, observational, and programmatic evaluation studies., Search Strategy: Systematic review of published studies on malaria in pregnancy and pregnancy outcomes., Selection Criteria: Clinical studies from Sub-Saharan Africa from the past 10 years were included., Data Collection and Analysis: English articles published since 2002 and listed in PubMed were identified using defined keywords, and their source documents were reviewed. Thirty-three studies involving malaria in pregnancy that recorded treatment rates and birth outcomes were included., Main Results: SP use among primigravidae was consistently associated with decreased LBW and anemia rates in clinical trials. Effects were less consistent in observational studies., Conclusions: Although randomized trials have demonstrated the efficacy of SP, studies evaluating scale-up programs found less consistent reductions in LBW and maternal anemia. Additional strategies to improve SP coverage may reduce the LBW and maternal anemia associated with malaria in pregnancy., (Copyright © 2013 International Federation of Gynecology and Obstetrics. Published by Elsevier Ireland Ltd. All rights reserved.)

In highland areas of unstable, low malaria transmission, the extent to which immunity to uncomplicated malaria develops with age and intermittent parasite exposure has not been well characterized. We conducted active surveillance for clinical malaria during April 2003-March 2005 in two highland areas of western Kenya (Kapsisiywa and Kipsamoite). In both sites, annual malaria incidence was significantly lower in persons ≥ 15 years of age than in persons < 5 years of age (Kapsisiywa: incidence = 382.9 cases/1,000 persons among persons < 1-4 years of age versus 135.1 cases/1,000 persons among persons ≥ 15 years of age; Kipsamoite: incidence = 233.0 cases/1,000 persons in persons < 1-4 years of age versus 43.3 cases/1,000 persons in persons ≥ 15 years of age). In Kapsisiywa, among persons with malaria, parasite density and axillary body temperature were also significantly lower in persons ≥ 15 years of age than in persons < 5 years of age. Even in highland areas of unstable and low malaria transmission, age is associated with development of clinical immunity to malaria.

Background: The 19 kDa C-terminal region of Plasmodium falciparum Merozoite Surface Protein-1 is a known target of naturally acquired humoral immunity and a malaria vaccine candidate. MSP-119 has four predominant haplotypes resulting in amino acid changes labelled EKNG, QKNG, QTSR and ETSR. IgG antibodies directed against all four variants have been detected, but it is not known if these variant specific antibodies are associated with haplotype-specific protection from infection., Methods: Blood samples from 201 healthy Kenyan adults and children who participated in a 12-week treatment time-to-infection study were evaluated. Venous blood drawn at baseline (week 0) was examined for functional and serologic antibodies to MSP-119 and MSP-142 variants. MSP-119 haplotypes were detected by a multiplex PCR assay at baseline and weekly throughout the study. Generalized linear models controlling for age, baseline MSP-119 haplotype and parasite density were used to determine the relationship between infecting P. falciparum MSP-119 haplotype and variant-specific antibodies., Results: A total of 964 infections resulting in 1,533 MSP-119 haplotypes detected were examined. The most common haplotypes were EKNG and QKNG, followed by ETSR and QTSR. Children had higher parasite densities, greater complexity of infection (>1 haplotype), and more frequent changes in haplotypes over time compared to adults. Infecting MSP-119 haplotype at baseline (week 0) had no influence on haplotypes detected over the subsequent 11 weeks among children or adults. Children but not adults with MSP-119 and some MSP-142 variant antibodies detected by serology at baseline had delayed time-to-infection. There was no significant association of variant-specific serology or functional antibodies at baseline with infecting haplotype at baseline or during 11 weeks of follow up among children or adults., Conclusions: Variant transcending IgG antibodies to MSP-119 are associated with protection from infection in children, but not adults. These data suggest that inclusion of more than one MSP-119 variant may not be required in a malaria blood stage vaccine.

In utero priming to malaria antigens renders cord blood mononuclear cells (CBMC) more susceptible to productive HIV infection in vitro in the absence of exogenous stimulation. This provides a unique model to better understand mechanisms affecting lymphocyte susceptibility to HIV infection in vivo. Effector memory CD3(+)CD4(+) T cells (T(EM)) were the exclusive initial targets of HIV with rapid spread to central memory cells. HIV susceptibility correlated with increased expression of CD25 and HLA-DR on T(EM). Virus entered all samples equally, however gag/pol RNA was only detected in HIV susceptible samples, suggesting regulation of proviral gene transcription. Targeted analysis of human genes in memory T cells showed greater expression of IFNG, NFATc1, IRF1, FOS, and PPIA and decreased expression YY1 and TFCP2 in HIV susceptible samples. Thus fetal priming to exogenous antigens enhances specific proviral gene transcription pathways in effector memory cells that may increase risk of vertical transmission of HIV., (Published by Elsevier Inc.)

Apical Membrane Antigen 1 (AMA1) is a leading malaria vaccine candidate and a target of naturally-acquired human immunity. Plasmodium falciparum AMA1 is polymorphic and in vaccine trials it induces strain-specific protection. This antigenic diversity is a major roadblock to development of AMA1 as a malaria vaccine and understanding how to overcome it is essential. To assess how AMA1 antigenic diversity limits cross-strain growth inhibition, we assembled a panel of 18 different P. falciparum isolates which are broadly representative of global AMA1 sequence diversity. Antibodies raised against four well studied AMA1 alleles (W2Mef, 3D7, HB3 and FVO) were tested for growth inhibition of the 18 different P. falciparum isolates in growth inhibition assays (GIA). All antibodies demonstrated substantial cross-inhibitory activity against different isolates and a mixture of the four different AMA1 antibodies inhibited all 18 isolates tested, suggesting significant antigenic overlap between AMA1 alleles and limited antigenic diversity of AMA1. Cross-strain inhibition by antibodies was only moderately and inconsistently correlated with the level of sequence diversity between AMA1 alleles, suggesting that sequence differences are not a strong predictor of antigenic differences or the cross-inhibitory activity of anti-allele antibodies. The importance of the highly polymorphic C1-L region for inhibitory antibodies and potential vaccine escape was assessed by generating novel transgenic P. falciparum lines for testing in GIA. While the polymorphic C1-L epitope was identified as a significant target of some growth-inhibitory antibodies, these antibodies only constituted a minor proportion of the total inhibitory antibody repertoire, suggesting that the antigenic diversity of inhibitory epitopes is limited. Our findings support the concept that a multi-allele AMA1 vaccine would give broad coverage against the diversity of AMA1 alleles and establish new tools to define polymorphisms important for vaccine escape.

Background: Naturally acquired immunity to blood-stage Plasmodium falciparum infection develops with age and after repeated infections. In order to identify immune surrogates that can inform vaccine trials conducted in malaria endemic populations and to better understand the basis of naturally acquired immunity it is important to appreciate the temporal stability of cellular and humoral immune responses to malaria antigens., Methods: Blood samples from 16 adults living in a malaria holoendemic region of western Kenya were obtained at six time points over the course of 9 months. T cell immunity to the 42 kDa C-terminal fragment of Merozoite Surface Protein-1 (MSP-1(42)) was determined by IFN-gamma ELISPOT. Antibodies to the 42 kDa and 19 kDa C-terminal fragments of MSP-1 were determined by serology and by functional assays that measure MSP-1(19) invasion inhibition antibodies (IIA) to the E-TSR (3D7) allele and growth inhibitory activity (GIA). The haplotype of MSP-1(19) alleles circulating in the population was determined by PCR. The kappa test of agreement was used to determine stability of immunity over the specified time intervals of 3 weeks, 6 weeks, 6 months, and 9 months., Results: MSP-1 IgG antibodies determined by serology were most consistent over time, followed by MSP-1 specific T cell IFN-gamma responses and GIA. MSP-1(19) IIA showed the least stability over time. However, the level of MSP-1(19) specific IIA correlated with relatively higher rainfall and higher prevalence of P. falciparum infection with the MSP-1(19) E-TSR haplotype., Conclusion: Variation in the stability of cellular and humoral immune responses to P. falciparum blood stage antigens needs to be considered when interpreting the significance of these measurements as immune endpoints in residents of malaria endemic regions.

Background: Antibodies that impair Plasmodium falciparum merozoite invasion and intraerythrocytic development are one of several mechanisms that mediate naturally acquired immunity to malaria. Attempts to correlate anti-malaria antibodies with risk of infection and morbidity have yielded inconsistent results. Growth inhibition assays (GIA) offer a convenient method to quantify functional antibody activity against blood stage malaria., Methods: A treatment-time-to-infection study was conducted over 12-weeks in a malaria holoendemic area of Kenya. Plasma collected from healthy individuals (98 children and 99 adults) before artemether-lumefantrine treatment was tested by GIA in three separate laboratories., Results: Median GIA levels varied with P. falciparum line (D10, 8.8%; 3D7, 34.9%; FVO, 51.4% inhibition). The magnitude of growth inhibition decreased with age in all P. falciparum lines tested with the highest median levels among children <4 years compared to adults (e.g. 3D7, 45.4% vs. 30.0% respectively, p = 0.0003). Time-to-infection measured by weekly blood smears was significantly associated with level of GIA controlling for age. Upper quartile inhibition activity was associated with less risk of infection compared to individuals with lower levels (e.g. 3D7, hazard ratio = 1.535, 95% CI = 1.012-2.329; p = 0.0438). Various GIA methodologies had little effect on measured parasite growth inhibition., Conclusion: Plasma antibody-mediated growth inhibition of blood stage P. falciparum decreases with age in residents of a malaria holoendemic area. Growth inhibition assay may be a useful surrogate of protection against infection when outcome is controlled for age.