Genetic variation in interleukin-7 is associated with a reduced erythropoietic response in Kenyan children infected with Plasmodium falciparum.

BACKGROUND: Severe malarial anemia (SMA) is a leading cause of malaria-related morbidity and mortality in children. The genetic factors that influence development of SMA and inefficient erythropoiesis, a central pathogenic feature of SMA, are only partially understood. METHODS: We performed a pilot Genome-wide Association Study (GWAS) on children with Plasmodium falciparum. The GWAS was performed using the Illumina® Infinium® HD Super Assay in conjunction with Illumina's® Human Omni2.5-8v1 BeadChip (with > 2.45 M markers). Data were analyzed using single SNP logistic regression analysis with an additive model of inheritance controlling for covariates. Results from our pilot global genomics study identified that variation in interleukin (IL)-7 was associated with enhanced risk of SMA. To validate this finding, we investigated the relationship between genotypes and/or haplotypes of two single nucleotide polymorphisms (SNPs) in IL7 [72194 T/C and - 2440 A/G] and susceptibility to both SMA and inefficient erythropoiesis [i.e., reticulocyte production index (RPI) < 2.0 in anemic children (Hb < 11.0 g/dL). Children presenting with P. falciparum malaria (< 3 years, n = 883) were stratified into two groups: Uncomplicated malaria (UM, n = 718) and SMA (n = 165). RESULTS: Regression modeling, controlling for anemia-related confounders, revealed that carriage of the TC genotype at position 72194 T/C was associated with enhanced susceptibility to inefficient erythropoiesis (OR = 1.90; 95% CI 1.09-3.30; P = 0.02) as was homozygous CC (OR 5.14; 95% CI = 1.20-21.99; P = 0.03). Consistent with this finding, individuals with the CA (72194C/-2440A) haplotype had an increased risk of inefficient erythropoiesis (OR = 1.90; 95% CI = 1.10-3.30; P = 0.02), whereas TA haplotype carriers had marginal protection against inefficient erythropoiesis (OR = 0.24; 95% CI = 0.06-1.21; P = 0.05). These observations were supported by Cochran-Armitage trend test for inefficient erythropoiesis (CA > TA > CG; P < 0.01). Although none of the genotype and/or haplotypic variants were significantly associated with SMA, the direction of the risk profiles were consistent with the erythropoiesis results. CONCLUSION: Taken together, variation in IL7 is associated with erythropoietic responses in children with falciparum malaria, a central physiological feature contributing to development of SMA.

Human antibodies activate complement against Plasmodium falciparum sporozoites, and are associated with protection against malaria in children.

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.

Integrative microRNA and mRNA deep-sequencing expression profiling in endemic Burkitt lymphoma.

BACKGROUND: Burkitt lymphoma (BL) is characterized by overexpression of the c-myc oncogene, which in the vast majority of cases is a consequence of an IGH/MYC translocation. While myc is the seminal event, BL is a complex amalgam of genetic and epigenetic changes causing dysregulation of both coding and non-coding transcripts. Emerging evidence suggest that abnormal modulation of mRNA transcription via miRNAs might be a significant factor in lymphomagenesis. However, the alterations in these miRNAs and their correlations to their putative mRNA targets have not been extensively studied relative to normal germinal center (GC) B cells. METHODS: Using more sensitive and specific transcriptome deep sequencing, we compared previously published small miRNA and long mRNA of a set of GC B cells and eBL tumors. MiRWalk2.0 was used to identify the validated target genes for the deregulated miRNAs, which would be important for understanding the regulatory networks associated with eBL development. RESULTS: We found 211 differentially expressed (DE) genes (79 upregulated and 132 downregulated) and 49 DE miRNAs (22 up-regulated and 27 down-regulated). Gene Set enrichment analysis identified the enrichment of a set of MYC regulated genes. Network propagation-based method and correlated miRNA-mRNA expression analysis identified dysregulated miRNAs, including miR-17~95 cluster members and their target genes, which have diverse oncogenic properties to be critical to eBL lymphomagenesis. Central to all these findings, we observed the downregulation of ATM and NLK genes, which represent important regulators in response to DNA damage in eBL tumor cells. These tumor suppressors were targeted by multiple upregulated miRNAs (miR-19b-3p, miR-26a-5p, miR-30b-5p, miR-92a-5p and miR-27b-3p) which could account for their aberrant expression in eBL. CONCLUSION: Combined loss of p53 induction and function due to miRNA-mediated regulation of ATM and NLK, together with the upregulation of TFAP4, may be a central role for human miRNAs in eBL oncogenesis. This facilitates survival of eBL tumor cells with the IGH/MYC chromosomal translocation and promotes MYC-induced cell cycle progression, initiating eBL lymphomagenesis. This characterization of miRNA-mRNA interactions in eBL relative to GC B cells provides new insights on miRNA-mediated transcript regulation in eBL, which are potentially useful for new improved therapeutic strategies.

Impact of Plasmodium falciparum Coinfection on Longitudinal Epstein-Barr Virus Kinetics in Kenyan Children.

Endemic Burkitt lymphoma is associated with Epstein-Barr virus (EBV) and Plasmodium falciparum coinfection, although how P. falciparum exposure affects the dynamics of EBV infection is unclear. We have used a modeling approach to study EBV infection kinetics in a longitudinal cohort of children living in regions of high and low malaria transmission in Kenya. Residence in an area of high malaria transmission was associated with a higher rate of EBV expansion during primary EBV infection in infants and during subsequent episodes of EBV DNA detection, as well as with longer episodes of EBV DNA detection and shorter intervals between subsequent episodes of EBV DNA detection. In addition, we found that concurrent P. falciparum parasitemia also increases the likelihood of the first and subsequent peaks of EBV in peripheral blood. This suggests that P. falciparum infection is associated with increased EBV growth and contributes to endemic Burkitt lymphoma pathogenesis.

A novel approach to identifying patterns of human invasion-inhibitory antibodies guides the design of malaria vaccines incorporating polymorphic antigens.

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.

Time-to-infection by Plasmodium falciparum is largely determined by random factors.

BACKGROUND: The identification of protective immune responses to P. falciparum infection is an important goal for the development of a vaccine for malaria. This requires the identification of susceptible and resistant individuals, so that their immune responses may be studied. Time-to-infection studies are one method for identifying putative susceptible individuals (infected early) versus resistant individuals (infected late). However, the timing of infection is dependent on random factors, such as whether the subject was bitten by an infected mosquito, as well as individual factors, such as their level of immunity. It is important to understand how much of the observed variation in infection is simply due to chance. METHODS: We analyse previously published data from a treatment-time-to-infection study of 201 individuals aged 0.5 to 78 years living in Western Kenya. We use a mathematical modelling approach to investigate the role of immunity versus random factors in determining time-to-infection in this cohort. We extend this analysis using a modelling approach to understand what factors might increase or decrease the utility of these studies for identifying susceptible and resistant individuals. RESULTS: We find that, under most circumstances, the observed distribution of time-to-infection is consistent with this simply being a random process. We find that age, method for detection of infection (PCR versus microscopy), and underlying force of infection are all factors in determining whether time-to-infection is a useful correlate of immunity. CONCLUSIONS: Many epidemiological studies of P. falciparum infection assume that the observed variation in infection outcomes, such as time-to-infection or presence or absence of infection, is determined by host resistance or susceptibility. However, under most circumstances, this distribution appears largely due to the random timing of infection, particularly in children. More direct measurements, such as parasite growth rate, may be more useful than time-to-infection in segregating patients based on their level of immunity.

Decreased growth rate of P. falciparum blood stage parasitemia with age in a holoendemic population.

In malaria holoendemic settings, decreased parasitemia and clinical disease is associated with age and cumulative exposure. The relative contribution of acquired immunity against various stages of the parasite life cycle is not well understood. In particular, it is not known whether changes in infection dynamics can be best explained by decreasing rates of infection, or by decreased growth rates of parasites in blood. Here, we analyze the dynamics of Plasmodium falciparum infection after treatment in a cohort of 197 healthy study participants of different ages. We use both polymerase chain reaction (PCR) and microscopy detection of parasitemia in order to understand parasite growth rates and infection rates over time. The more sensitive PCR assay detects parasites earlier than microscopy, and demonstrates a higher overall prevalence of infection than microscopy alone. The delay between PCR and microscopy detection is significantly longer in adults compared with children, consistent with slower parasite growth with age. We estimated the parasite multiplication rate from delay to PCR and microscopy detections of parasitemia. We find that both the delay between PCR and microscopy infection as well as the differing reinfection dynamics in different age groups are best explained by a slowing of parasite growth with age.

Sickle cell trait is not associated with endemic Burkitt lymphoma: an ethnicity and malaria endemicity-matched case-control study suggests factors controlling EBV may serve as a predictive biomarker for this pediatric cancer.

Endemic Burkitt lymphoma (eBL) is associated with Epstein-Barr virus (EBV) and Plasmodium falciparum coinfections. Malaria appears to dysregulate immunity that would otherwise control EBV, thereby contributing to eBL etiology. Juxtaposed to human genetic variants associated with protection from malaria, it has been hypothesized that such variants could decrease eBL susceptibility, historically referred to as "the protective hypothesis." Past studies attempting to link sickle cell trait (HbAS), which is known to be protective against malaria, with protection from eBL were contradictory and underpowered. Therefore, using a case-control study design, we examined HbAS frequency in 306 Kenyan children diagnosed with eBL compared to 537 geographically defined and ethnically matched controls. We found 23.8% HbAS for eBL patients, which was not significantly different compared to 27.0% HbAS for controls [odds ratio (OR) = 0.85; 95% confidence interval (CI) 0.61-1.17; p-value = 0.33]. Even though cellular EBV titers, indicative of the number of latently infected B cells, were significantly higher (p-value < 0.0003) in children residing in malaria holoendemic compared to hypoendemic areas, levels were not associated with HbAS genotype. Combined, this suggests that although HbAS protects against severe malaria and hyperparasitemia, it is not associated with viral control or eBL protection. However, based on receiver operating characteristic curves factors that enable the establishment of EBV persistence, in contrast to those involved in EBV lytic reactivation, may have utility as an eBL precursor biomarker. This has implications for future human genetic association studies to consider variants influencing control over EBV in addition to malaria as risk factors for eBL.

Holoendemic malaria exposure is associated with altered Epstein-Barr virus-specific CD8(+) T-cell differentiation.

Coinfection with Plasmodium falciparum malaria and Epstein-Barr virus (EBV) is a major risk factor for endemic Burkitt lymphoma (eBL), still one of the most prevalent pediatric cancers in equatorial Africa. Although malaria infection has been associated with immunosuppression, the precise mechanisms that contribute to EBV-associated lymphomagenesis remain unclear. In this study, we used polychromatic flow cytometry to characterize CD8(+) T-cell subsets specific for EBV-derived lytic (BMFL1 and BRLF1) and latent (LMP1, LMP2, and EBNA3C) antigens in individuals with divergent malaria exposure. No malaria-associated differences in EBV-specific CD8(+) T-cell frequencies were observed. However, based on a multidimensional analysis of CD45RO, CD27, CCR7, CD127, CD57, and PD-1 expression, we found that individuals living in regions with intense and perennial (holoendemic) malaria transmission harbored more differentiated EBV-specific CD8(+) T-cell populations that contained fewer central memory cells than individuals living in regions with little or no (hypoendemic) malaria. This profile shift was most marked for EBV-specific CD8(+) T-cell populations that targeted latent antigens. Importantly, malaria exposure did not skew the phenotypic properties of either cytomegalovirus (CMV)-specific CD8(+) T cells or the global CD8(+) memory T-cell pool. These observations define a malaria-associated aberration localized to the EBV-specific CD8(+) T-cell compartment that illuminates the etiology of eBL.

Humoral and cellular immunity to Plasmodium falciparum merozoite surface protein 1 and protection from infection with blood-stage parasites.

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.

The dynamics of naturally acquired immunity to Plasmodium falciparum infection.

Severe malaria occurs predominantly in young children and immunity to clinical disease is associated with cumulative exposure in holoendemic settings. The relative contribution of immunity against various stages of the parasite life cycle that results in controlling infection and limiting disease is not well understood. Here we analyse the dynamics of Plasmodium falciparum malaria infection after treatment in a cohort of 197 healthy study participants of different ages in order to model naturally acquired immunity. We find that both delayed time-to-infection and reductions in asymptomatic parasitaemias in older age groups can be explained by immunity that reduces the growth of blood stage as opposed to liver stage parasites. We found that this mechanism would require at least two components - a rapidly acting strain-specific component, as well as a slowly acquired cross-reactive or general immunity to all strains. Analysis and modelling of malaria infection dynamics and naturally acquired immunity with age provides important insights into what mechanisms of immune control may be harnessed by malaria vaccine strategists.

Genetic variation in CSF2 (5q31.1) is associated with longitudinal susceptibility to pediatric malaria, severe malarial anemia, and all-cause mortality in a high-burden malaria and HIV region of Kenya.

Plasmodium falciparum infections remain among the leading causes of morbidity and mortality in holoendemic transmission areas. Located within region 5q31.1, the colony-stimulating factor 2 gene (CSF2) encodes granulocyte-macrophage colony-stimulating factor (GM-CSF), a hematopoietic growth factor that mediates host immune responses. Since the effect of CSF2 variation on malaria pathogenesis remains unreported, we investigated the impact of two genetic variants in the 5q31.1 gene region flanking CSF2:g-7032 G > A (rs168681:G > A) and CSF2:g.64544T > C (rs246835:T > C) on the rate and timing of malaria and severe malarial anemia (SMA, Hb < 5.0 g/dL) episodes over 36 months of follow-up. Children (n = 1654, aged 2-70 months) were recruited from a holoendemic P. falciparum transmission area of western Kenya. Decreased incidence rate ratio (IRR) for malaria was conferred by inheritance of the CSF2:g.64544 TC genotype (P = 0.0277) and CSF2 AC/GC diplotype (P = 0.0015). Increased IRR for malaria was observed in carriers of the CSF2 AT/GC diplotype (P = 0.0237), while the inheritance of the CSF2 AT haplotype increased the IRR for SMA (P = 0.0166). A model estimating the longitudinal risk of malaria showed decreased hazard rates among CSF2 AC haplotype carriers (P = 0.0045). Investigation of all-cause mortality revealed that inheritance of the GA genotype at CSF2:g-7032 increased the risk of mortality (P = 0.0315). Higher risk of SMA and all-cause mortality were observed in younger children (P < 0.0001 and P = 0.0015), HIV-1(+) individuals (P < 0.0001 and P < 0.0001), and carriers of HbSS (P = 0.0342 and P = 0.0019). Results from this holoendemic P. falciparum area show that variation in gene region 5q31.1 influences susceptibility to malaria, SMA, and mortality, as does age, HIV-1 status, and inheritance of HbSS.

The company malaria keeps: how co-infection with Epstein-Barr virus leads to endemic Burkitt lymphoma.

PURPOSE OF REVIEW: Co-infection with Plasmodium falciparum malaria and Epstein-Barr virus (EBV) are implicated in the cause of endemic Burkitt lymphoma (eBL), the most prevalent pediatric cancer in equatorial Africa. Although the causal association between EBV and eBL has been established, P. falciparum malaria's role is not as clearly defined. This review focuses on how malaria may disrupt EBV persistence and immunity. RECENT FINDINGS: Two mutually compatible theories have been proposed. One suggests that P. falciparum malaria induces polyclonal B-cell expansion and lytic EBV reactivation, leading to the expansion of latently infected B cells and the likelihood of a c-myc translocation, a hallmark of Burkitt lymphoma tumors. The other advocates that EBV-specific T-cell immunity is impaired during P. falciparum malaria co-infection, either as a cause or consequence of enhanced EBV replication, leading to loss of viral control. Advancements in our ability to query the complexity of human responses to infectious diseases have stimulated interest in eBL pathogenesis. SUMMARY: EBV is necessary but not sufficient to cause eBL. A more dynamic model encompasses incremental contributions from both chronic and acute P. falciparum malaria leading to alterations in EBV persistence and EBV-specific immunity that culminate in eBL. A better understanding of how P. falciparum malaria modifies EBV infections in children may allow us to anticipate reductions in eBL incidence coinciding with malaria control programs.

Mechanisms and targets of Fcγ-receptor mediated immunity to malaria sporozoites.

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.

Allele specificity of gamma interferon responses to the carboxyl-terminal region of Plasmodium falciparum merozoite surface protein 1 by Kenyan adults with naturally acquired immunity to malaria.

Cross-sectional seroepidemiological studies of populations naturally exposed to Plasmodium falciparum suggest an association between protection from malaria and circulating antibodies to the carboxyl terminus of merozoite surface protein 1 (MSP1). Questions remain regarding the significance of cell-mediated immunity to MSP1 in conferring protection and inducing immunologic memory. Vaccine constructs have been based on the 42-kDa recombinant MSP1 protein (MSP1(42)), which includes the 19-kDa (MSP1(19)) and 33-kDa (MSP1(33)) fragments containing the major B- and T-cell epitopes, respectively. To evaluate T-cell responses to the MSP1(33) fragment, two libraries of overlapping 18-mer peptides from the 3D7 and FVO MSP1(33) regions were used to screen a cohort of asymptomatic Kenyan adults. Gamma interferon (IFN-γ) measured by enzyme-linked immunospot assay (ELISPOT) at multiple time points assessed the magnitude and stability of these responses. The percentage of individuals with IFN-γ responses to single MSP1(33) peptides ranged from nil to 24%, were clustered among a subset of peptides, and were not consistently recalled over time. In comparison to peptide responses, IFN-γ ELISPOT responses to recombinant MSP1(42) were more prevalent, more frequently elicited by the 3D7 as opposed to the FVO allele, and more stable over time. The prevailing MSP1(33) genotype infection was 3D7, with few mixed infections and no sole FVO infections. This study demonstrates that immunity against MSP1(33) after cumulative natural infections consists of low-magnitude and difficult-to-detect IFN-γ responses. Although immunity against MSP1 alone will not confer protection against malaria, demonstrating a relative and sustained increase in T-cell immunity to MSP1 after vaccination would be a reasonable measurement of vaccine responsiveness.

Children with endemic Burkitt lymphoma are deficient in EBNA1-specific IFN-gamma T cell responses.

Endemic Burkitt lymphoma (eBL) is the most common childhood cancer in equatorial Africa and is linked to Epstein-Barr virus (EBV) and Plasmodium falciparum coinfections early in life. Epstein-Barr nuclear antigen 1 (EBNA1) is the sole viral latent antigen expressed in BL tumors. Loss of EBNA1-specific immune surveillance could allow eBL emergence. Therefore, EBNA1-specific T cell responses were analyzed by IFN-gamma ELISPOT in Kenyan children with eBL and compared to healthy children with divergent malaria exposure. Significantly fewer children with eBL, 16% (7/44) had EBNA1-specific IFN-gamma responses in contrast to healthy children living in a malaria holoendemic area or in an area with sporadic malaria transmission, 67% (40/60) and 72% (43/60) responders, respectively (p < 0.003). Children with eBL maintained IgG(1) dominated antibody responses to EBNA1 similar to healthy children suggesting a selective loss of IFN-gamma secreting EBNA1-specific T cells in the presence of intact humoral immunity. CD8(+) T cell responses to EBV lytic and latent antigens not expressed in the tumors were similarly robust in eBL patients compared to healthy children. In addition, CD4(+) T cell responses to a malaria protein, merozoite surface protein 1, were present in lymphoma patients. This study demonstrates a selective loss of EBNA1-specific T cell responses in children with eBL and suggests a potential immunotherapeutic target for this EBV-associated lymphoma.

Serological evidence for long-term Epstein-Barr virus reactivation in children living in a holoendemic malaria region of Kenya.

To study the long term the effects of chronic exposure to P. falciparum malaria on Epstein-Barr virus (EBV) reactivation in children, EBV-specific antibody levels were measured in a cross-sectional survey of two groups of Kenyan children with divergent malaria exposure, varying in age from 1 to 14 years. A total of 169 children were analyzed within three age groups (1-4 years, 5-9 years and 10-14 years). Using a Luminex assay, elevated levels of IgG to EBV lytic and latent antigens were observed in children from the holoendemic malaria area; these remained elevated for each age group studied. In comparison, children from the sporadic malaria area had lower levels of EBV-specific IgG antibodies and these levels declined across age groups. These data suggest that chronic exposure to malaria may lead to long-term EBV reactivation.

Temporal stability of naturally acquired immunity to Merozoite Surface Protein-1 in Kenyan adults.

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.

Low Levels of Human Antibodies to Gametocyte-Infected Erythrocytes Contrasts the PfEMP1-Dominant Response to Asexual Stages in P. falciparum Malaria.

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.

Human and Epstein-Barr Virus miRNA Profiling as Predictive Biomarkers for Endemic Burkitt Lymphoma.

Endemic Burkitt lymphoma (eBL) is an aggressive B cell lymphoma and is associated with Epstein-Barr virus (EBV) and Plasmodium falciparum malaria co-infections. Central to BL oncogenesis is the over-expression of the MYC proto-oncogene which is caused by a translocation of an Ig enhancer in approximation to the myc gene. While whole genome/transcriptome sequencing methods have been used to define driver mutations and transcriptional dysregulation, microRNA (miRNA) dysregulation and differential expression has yet to be fully characterized. We hypothesized that both human and EBV miRNAs contribute to eBL clinical presentation, disease progression, and poor outcomes. Using sensitive and precise deep sequencing, we identified miRNAs from 17 Kenyan eBL patient tumor samples and delineated the complement of both host and EBV miRNAs. One human miRNA, hsa-miR-10a-5p was found to be differentially expressed (DE), being down-regulated in jaw tumors relative to abdominal and in non-survivors compared to survivors. We also examined EBV miRNAs, which made up 2.7% of the miRNA composition in the eBL samples. However, we did not find any significant associations regarding initial patient outcome or anatomical presentation. Gene ontology analysis and pathway enrichment of previously validated targets of miR-10a-5p suggest that it can promote tumor cell survival as well as aid in evasion of apoptosis. To examine miR-10a-5p regulatory effect on gene expression in eBL, we performed a pairwise correlation coefficient analysis on the expression levels of all its validated targets. We found a significant enrichment of correlated target genes consistent with miR-10a-5p impacting expression. The functions of genes and their correlation fit with multiple target genes impacting tumor resilience. The observed downregulation of miR-10a and associated genes suggests a role for miRNA in eBL patient outcomes and has potential as a predictive biomarker that warrants further investigation.