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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., Antibodies plays critical roles in the adaptive immune response to infectious agents including malaria. Here the authors defined antibody interactions with -Fcγ-receptors expressed on immune cells with sporozoites of Plasmodium falciparum, and identified specific target epitopes of antibodies.

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. Electronic supplementary material The online version of this article (10.1186/s12885-017-3711-9) contains supplementary material, which is available to authorized users.

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) TA > CG; P

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.

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. Electronic supplementary material The online version of this article (10.1186/s12916-018-1054-2) contains supplementary material, which is available to authorized users.

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.

MiRNAs significantly enriched by the network propagation-based method (network perturbation effect score (NPES) >2, adjusted p-value

ATM and NLK downregulation in BL cell lines. A.) Hierarchical clustering of BL cell lines and germinal center (GC) B cells based on the expression of MYC, NLK and ATM genes. B.) Expression changes of ATM and NLK in BL cell line compared to GC B cells. (PDF 403 kb)

miRNA-mRNA pairs permutation test results. (PDF 302 kb)

Principal Component analysis (PCA) using mRNA expression profile. A) PCA plot showing clustering of GC B cells and eBL tumor cells before batch effect removal. B) PCA plot showing clustering of GC B cells and eBL tumor cells after batch effect/noise removal using svaseq. We now observe better clustering of GC B cells based on cell type and not clustering based the previous studies we obtained the data from. (PDF 126 kb)

Differentially expressed Genes between eBL tumor cells and GC B cells (logFC > 2, p-value

Expression of B-cell differentiation markers and eBL diagnostic surface markers. A) Expression of eBL diagnostic surface markers (CD79, CD10, CD20, and CD19). B) Expression of key transcription factors involved in B-cell differentiation (BLIMP1, IRF4, BCL6 and PAX5). (PDF 106 kb)

The devastating effect of cancer is a worldwide concern. Unfortunately current chemotherapeutic and radio therapeutic modalities have been found to possess side effects coupled with the emergence of drug resistance. This has necessitated the search for novel therapeutic products with better efficacy, safety and affordability through identification of anti-tumor agents from natural products. In this study the antineoplastic activity and phytochemical screening of methanol extracts of four medicinal plants that are native to Lake Victoria Basin, including Piptadiniastrum africanum, Kigelia africana, Centella asciatica and Chaemocrista nigricans was investigated. Extraction and concentration of the collected and dried plant samples to obtain crude extracts as well as phytochemical screening of the crude extracts was done following standard procedures. Lung adenocarcinoma cell line, from American type cell culture (ATCC), was exposed to the extracts and antiproliferative analysis using 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2-tetrazolium bromide (MTT) colorimetric assay was performed. Phytochemical analysis of each of the four plants showed presence of terpenoids, alkaloids, saponins, tannins and steroids. Steroids were higher in the bark of P. africanum and the fruits of K. africana. Flavonoid was absent only in extracts of C. nigricans and coumarins present only in the extracts of K. africana. The methanolic extracts of the fruits of K. africana, bark of P. africanum, leaves of C. asciatica and leaves C. nigricans had inhibitory effects with IC50 28.86 ug/ml, 26.57 ug/ml, 15.69 ug/ml and 8.07 ug/ml (p values of 0.079, 0.069, and 0.042 and 0.055, ANOVA) respectively. Statistical differences among fractions of the extract were determined by one way ANOVA and considered significant at P

In malaria holoendemic settings, decreased parasitemia and clinical disease is associated with age and cumula- tive 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 dynam- ics of Plasmodium falciparum infection after treatment in a cohort of 197 healthy study participants of differ- ent 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, consis- tent 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.

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

Recent studies of Plasmodium berghei malaria in mice show that high blood-stage parasitemia levels inhibit the development of subsequent liver-stage infections. Whether a similar inhibitory effect on liver-stage Plasmodium falciparum by blood-stage infection occurs in humans is unknown. We have analyzed data from a treatment-time-to-infection cohort of children < 10 years of age residing in a malaria holoendemic area of Kenya where people experience a new blood-stage infection approximately every 2 weeks. We hypothesized that if high parasitemia blocked the liver stage, then high levels of parasitemia should be followed by a “skipped” peak of parasitemia. Statistical analysis of “natural infection” field data and stochastic simulation of infection dynamics show that the data are consistent with high P. falciparum parasitemia inhibiting liver-stage parasite development in humans.

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.

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.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.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.

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.

BACKGROUND: Malaria and Epstein-Barr virus (EBV) infection are cofactors in the pathogenesis of endemic Burkitt lymphoma (eBL). The mechanisms by which these pathogens predispose to eBL are not known. METHODS: Healthy Kenyan children with divergent malaria exposure were measured for responses to EBV latent and lytic antigens by interferon (IFN)- gamma enzyme-linked immunospot (ELISPOT) assay and interleukin (IL)-10 ELISA. Phytohemagglutinin (PHA), purified protein derivative (PPD), and T cell epitope peptides derived from merozoite surface protein (MSP)-1, a malaria blood-stage antigen, were also evaluated. RESULTS: Children 5-9 years old living in an area holoendemic for malaria had significantly fewer EBV-specific IFN- gamma responses than did children of the same age living in an area with unstable malaria transmission. This effect was not observed for children 9 years old. In contrast, IFN- gamma responses to PHA, PPD, and Plasmodium falciparum MSP-1 peptides did not significantly differ by age. IL-10 responses to EBV lytic antigens, PPD, and PHA correlated inversely with malaria exposure regardless of age. CONCLUSIONS: Children living in malaria-holoendemic areas have diminished EBV-specific T cell immunosurveillance between the ages of 5 and 9 years, which coincides with the peak age incidence of eBL.

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.

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.

Background: Antibodies are important in protection against infection and disease caused by Plasmodium falciparum, but the frequencies of antibodies to multiple P. falciparum antigens in children are not well-characterized. Methods: IgG and IgM antibodies to the vaccine candidate antigens circumsporozoite protein, thrombospondin-related adhesive protein, liver stage antigen-1, apical membrane antigen-1, erythrocyte-binding antigen-175 and merozoite surface protein-1 were measured by enzyme-linked immunosorbent assay in 110 children 0–50 months of age in a malaria holoendemic area of Kenya. Results: A similar pattern was seen for IgG antibodies to circumsporozoite protein, thrombospondin-related adhesive protein, apical membrane antigen-1 and erythrocyte-binding antigen-175: high frequencies (70–90%) in children 0–4 months of age; a decrease in children 5–20 months of age (35–71%); and progressive increases in children 21–36 and 37–50 months of age (53–80% and 60–100%, respectively). In contrast, IgG antibodies to liver stage antigen-1 were infrequent in children 0–4 months of age (5%) and increased with age to 64%, and IgG antibody frequencies to merozoite surface protein-1 were similar across age groups (26–52%). IgG antibodies to all antigens were predominantly of the IgG1 and IgG3 subclasses. Frequencies of IgM antibodies to all antigens were low in children 0–4 months of age (0–15%) and increased with age (24–56% in the oldest children). Conclusion: In children in a malaria-holoendemic area, IgM antibody to all P. falciparum antigens is infrequent in the first 4 months of life but increases with age and increased exposure. The pattern of age-related IgG response frequencies to P. falciparum antigens varies significantly by antigen.

Overexpression of interleukin-6 (IL-6) and IL-10 in endemic Burkitt lymphoma (eBL) may facilitate tumorigenesis by providing a permissive cytokine milieu. Promoter polymorphisms influence interindividual differences in cytokine production. We hypothesized that children genetically predisposed for elevated cytokine levels may be more susceptible to eBL. Using case-control samples from western Kenya consisting of 117 eBL cases and 88 ethnically matched healthy controls, we tested for the association between eBL risk and IL-10 (rs1800896, rs1800871, and rs1800872) and IL-6 (rs1800795) promoter single nucleotide polymorphisms (SNPs) as well as IL-10 promoter haplotypes. In addition, the association between these variants and Epstein Barr Virus (EBV) load was examined. Results showed that selected IL-10 and IL-6 promoter SNPs and IL-10 promoter haplotypes were not associated with risk eBL or EBV levels in EBV-seropositive children. Findings from this study reveal that common variants within the IL-10 and IL-6 promoters do not independently increase eBL risk in this vulnerable population.

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 Epstein Barr virus (EBV) is a gammaherpesvirus that is associated with nasopharyngeal carcinoma (NPC) and endemic Burkitt lymphoma (eBL). EBV carries several latent genes that contribute to oncogenesis including the latent membrane protein 1 (LMP-1), a known oncogene and constitutively active CD40 homolog. Variation in the C terminal region of LMP-1 has been linked to NPC pathogenesis, but little is known regarding LMP-1 variation and eBL. Results In the present study, peripheral blood samples were obtained from 38 eBL patients and 22 healthy controls in western Kenya, where the disease is endemic. The LMP-1 C-terminal region from these samples was sequenced and analyzed. The frequency of a 30 base pair deletion of LMP-1 previously linked to NPC was not associated with eBL compared to healthy controls. However a novel LMP-1 variant was identified, called K for Kenya and for the G318K mutation that characterizes it. The K variant LMP-1 was found in 40.5% of eBL sequences and 25.0% of healthy controls. All K variant sequences contained mutations in both of the previously described minimal T cell epitopes in the C terminal end of LMP-1. These mutations occurred in the anchor residue at the C-terminal binding groove of both epitopes, a pocket necessary for MHC loading. Conclusions Overall, our results suggest that there is a novel K variant of LMP-1 in Kenya that may be associated with eBL. Further studies are necessary to determine the functional implications of the LMP-1 variant on early events in eBL genesis.

Plasmodium falciparum malaria (Pf-malaria) and Epstein Barr Virus (EBV) infections coexist in children at risk for endemic Burkitt's lymphoma (eBL); yet studies have only glimpsed the cumulative effect of Pf-malaria on EBV-specific immunity. Using pooled EBV lytic and latent CD8+ T-cell epitope-peptides, IFN-γ ELISPOT responses were surveyed three times among children (10 months to 15 years) in Kenya from 2002-2004. Prevalence ratios (PR) and 95% confidence intervals (CI) were estimated in association with Pf-malaria exposure, defined at the district-level (Kisumu: holoendemic; Nandi: hypoendemic) and the individual-level. We observed a 46% decrease in positive EBV lytic antigen IFN-γ responses among 5-9 year olds residing in Kisumu compared to Nandi (PR: 0.54; 95% CI: 0.30-0.99). Individual-level analysis in Kisumu revealed further impairment of EBV lytic antigen responses among 5-9 year olds consistently infected with Pf-malaria compared to those never infected. There were no observed district- or individual-level differences between Pf-malaria exposure and EBV latent antigen IFN-γ response. The gradual decrease of EBV lytic antigen but not latent antigen IFN-γ responses after primary infection suggests a specific loss in immunological control over the lytic cycle in children residing in malaria holoendemic areas, further refining our understanding of eBL etiology.

Naturally acquired immunity to Plasmodium falciparum malaria in malaria holoendemic areas is characterized by the gradual, age-related development of protection against high-density parasitemia and clinical malaria. Animal studies, and less commonly, observations of humans with malaria, suggest that T-cell responses are important in the development and maintenance of this immunity, which is mediated primarily by antibodies that slow repeated cycles of merozoites through erythrocytes. To advance our rather limited knowledge on human T-cell immunity to blood stage malaria infection, we evaluated CD4 and CD8 T-cell effector memory subset responses to the 42 kDa C-terminal fragment of Merozoite Surface Protein 1 (MSP1(42)), a malaria vaccine candidate, by 49 healthy 0.5 to ≥18 year old residents of a holoendemic area in western Kenya. The proportion of individuals with peripheral blood mononuclear cell MSP1(42) driven IFN-γ ELISPOT responses increased from 20% (2/20) among 0.5-1 year old children to 90% (9/10) of adults ≥18 years (P = 0.01); parallel increases in the magnitude of IFN-γ responses were observed across all age groups (0.5, 1, 2, 5 and ≥18 years, P = 0.001). Less than 1% of total CD4 and CD8 T-cells from both children and adults produced IFN-γ in response to MSP1(42). However, adults had higher proportions of MSP1(42) driven IFN-γ secreting CD4 and CD8 effector memory (CD45RA(-) CD62L(-)) T-cells than children (CD4: 50.9% vs. 28.8%, P = 0.036; CD8: 52.1% vs. 18.3%, respectively P = 0.009). In contrast, MSP1(42) driven IFN-γ secreting naive-like, transitional (CD45RA(+) CD62L(+)) CD4 and CD8 cells were the predominant T-cell subset among children with significantly fewer of these cells in adults (CD4: 34.9% vs. 5.1%, P = 0.002; CD8: 47.0% vs. 20.5%, respectively, P = 0.030). These data support the concept that meaningful age-related differences exist in the quality of T-cell immunity to malaria antigens such as MSP1.

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.

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-142) was determined by IFN-γ 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-119 invasion inhibition antibodies (IIA) to the E-TSR (3D7) allele and growth inhibitory activity (GIA). The haplotype of MSP-119 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-γ responses and GIA. MSP-119 IIA showed the least stability over time. However, the level of MSP-119 specific IIA correlated with relatively higher rainfall and higher prevalence of P. falciparum infection with the MSP-119 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.

Perennial and intense malaria transmission (holoendemic malaria) and Epstein-Barr virus (EBV) infection are 2 cofactors in the pathogenesis of endemic Burkitt lymphoma (eBL). In the present study, we compared EBV loads in children living in 2 regions of Kenya with differing malaria transmission intensities: Kisumu District, where malaria transmission is holoendemic, and Nandi District, where malaria transmission is sporadic. For comparison, blood samples were also obtained from US adults, Kenyan adults, and patients with eBL. Extraction of DNA from blood and quantification by polymerase chain reaction give an EBV load estimate that reflects the number of EBV-infected B cells. We observed a significant linear trend in mean EBV load, with the lowest EBV load detected in US adults and increasing EBV loads detected in Kenyan adults, Nandi children, Kisumu children, and patients with eBL, respectively. In addition, EBV loads were significantly higher in Kisumu children 1-4 years of age than in Nandi children of the same age. Our results support the hypothesis that repeated malaria infections in very young children modulate the persistence of EBV and increase the risk for the development of eBL.

Etiological mechanism of eBL remain largely unknown despite long-standing epidemiological link between malaria and EBV co-infection. Age-dependent deficiency in EBV-specific CD8+ T cell-mediated IFN-γ immunosurveillance has been reported in malaria holoendemic exposed children which has been associated with eBL diagnosis. We sought to investigate whether qualitative differences in EBNA-1 specific T cells existed in malaria-exposed children to explain their increased risk for eBL. We conducted a cross-sectional study of healthy children from western Kenya with divergent malaria exposure:Kisumu (holoendemic) and Nandi (hypoendemic) and eBL age-matched patients. T cell effector function was evaluated by multiparameter flow cytometry against T cell lineage markers(CD3,4,8,45RA & CCR7) and functional phenotype (IFN-γ, IL10, IL17 & PD1) to EBNA-1. EBNA-1 specific responses were compared to MSP-1, EBNA2 and EBNA3A. We found significantly higher IFN-γ responses to MSP1 in the eBL & Kisumu compared to Nandi children (p=0.03), in contrast to EBNA1, which were significantly lower in eBL and Kisumu compared to Nandi children (p=0.01). Responses to EBNA2 and EBNA3A did not differ between groups. IFN-γ was generated from CD4 TEM and a heterogeneous combination of CD8 T-cells. PD-1 expressing T-cells were less frequent in eBL patients and highest in Kisumu children.This suggests that the EBNA1-specific deficiencies are due to deletion of T-cell population due to malaria induced exhaustion.

Background: In areas of high-level, year-round malaria transmission, morbidity and mortality due to malaria decrease after the first two to three years of life. This reduction may be related to the development of cellular immunity to specific antigens expressed in the different life-cycle stages of Plasmodium falciparum. Methods: A cross sectional study was conducted to evaluate T cell cytokine responses to the P. falciparum pre-erythrocytic antigen liver-stage antigen-1 (LSA-1) and the blood-stage antigen merozoite-surface protein-1 (MSP-1) in children under five years of age residing in a malaria holoendemic region of western Kenya. Interferon-γ (IFN-γ) and interleukin-10 (IL-10) responses to the LSA-1 T3 peptide (aa 1813–1835) and the MSP-1 aa20–39 peptide were tested in 48 children. Results: The proportion of children producing IFN-γ to LSA-1 and to MSP-1 increased with age: in the 0–12, 13–24, 25–36 and 37–48 month age groups, zero, 11.1, 36.4 and 40% of children had IFN-γ responses to LSA-1 (p = 0.019), and 10, 10, 27.7 and 40% of children had IFN-γ responses to MSP-1 (p = 0.07), respectively. In contrast, the proportion of children producing IL-10 to LSA-1 and MSP-1 was similar in all age groups. Conclusion: The data suggest that development of IFN-γ responses to LSA-1 and MSP-1 requires increased age and/or repeated exposure, whereas IL-10 responses to these antigens may occur at any age and with limited exposure. The data also demonstrate that by the age of 4 years, children in a malaria holoendemic area develop frequencies of IFN-γ responses to LSA-1 and MSP-1 similar to those seen in adults in the area.

MiRNAs significantly enriched by the network propagation-based method (network perturbation effect score (NPES) >2, adjusted p-value