Role of Sickle-Cell Trait, Malaria and Epstein Barr Virus Coinfection On Ebna-L-Specific T-Cell Immunity in the Etiology Of Endemic Burkitt Lymphoma in Children Aged 4-9 Years From Western Kenya

Abstract

Etiological mechanism underlying endemic Burkitt lymphoma (eBL), remain unknown despite the long-standing epidemiological link between Plasmodium Jalciparum and Epstein Barr Virus (EBV) co-infections. Although past studies have linked malaria and EBV in eBL etiology none has investigated the role of sickle cell trait (HbAS) and malaria transmission on the quality of T-cell memory phenotype and function. Furthermore, there, is paucity of data on the role of common infectious agents in eBL etiology. Previous studies~have shown an agedependent deficiency in EBV-specific CD8+ T-cell-mediated IFN-y immunosurveillance in children from malaria holoendemic regions. Further, deficiency in Epstein-Barr nuclear antigen-l (EBNA-I)-specific IFN-y responses has been associated with eBL diagnosis in children aged 4 to 9 years. EBNA-I is the sole antigen expressed by EBV-infected B cells as well as EBV-associated turnours. This case-control study hypothesized that qualitative differences in EBNA-I- specific T-cell phenotype and function as well as serology existed in malaria-exposed children and, thus in part, explains their increased risk for eBL. Moreover, the study examined the relationship between HbAS and EBV titers and how it relates to anti- EBV immunity. Thus to test this hypothesis, healthy children from western Kenya with divergent malaria exposure histories: Kisumu (n=24), a malaria holoendemic region and Nandi (n=23), a hypoendemic region; were age-matched to eBL patients (n=31). T-cell memory phenotype and function was evaluated by multiparameter flow cytometry against T-cell lineage markers and functional phenotype based on IFN-y, IL-lO, IL-17 and PD-I expression after ex vivo stimulation with EBNA-l peptides. Multiplex ELISA was used to determine serological profiles. EBV titers were determined by qPCR, while PCR-based Restriction Fragment Length Polymorphism (RFLP) was used to determine HbAS genotype. Pairwise comparisons were done using Mann-Whitney U test while multiple comparisons were done using Kruskal-Wallis tests. Logistic regression analysis was used to determine the association between HbAS trait and viral titers while Chi square was used to test for differences in proportions of HbAS genotypes. Results show that EBNA-l-specific IFN-y responses were significantly lower in eBL and Kisumu compared to Nandi children (p=0.01). IFN-y was generated from CD4+ effector memory T cells and a heterogeneous combination of CD8+ T cells (jJ<0.0001). The frequencies ofPD-1 expressing T cells were low in eBL patients and highest in Kisumu children than Nandi children for both CD4+ T cells (p=0.0237) and CD8+ T cells (p=0.0423). The expression of PD-I and IFN-y in response to EBNA-I was mutually exclusive. Further, Kisumu and eBL children had higher cellular and plasma EBV titers compared to Nandi children (p=0.000 1). Although HBAS genotype was more common in Kisumu children than other groups (X~ 23.42, df 2, p<O.OOOl), it was neither associated with EBV titers nor eBL (OR=0.5473, 95% CI, 0.2835-1.057, p=0.0996). This suggests that EBNA-I-specific deficiencies in eBL may be due to a deletion of EBNA-l-specific T-cell population as a consequence of T-cell exhaustion. These results show that HbAS trait does not offer protective advantage against eBL and hence all children in malaria holoendemic regions should be treated as vulnerable to eBL risk irrespective of HbAS genotype. Further, results show that EBV peptide such as EBNA-I can be exploited further as a possible vaccine candidate against EBV associated turnours. This study recommends a paradigm shift from use of total EBV viral loads as a prognostic biomarker to use for cellular viral loads in clinical management of eBL. In addition, there is need for development of novel immunotherapeutic agents such as adoptive autologous EBNA-I specific T cells as well as PD-I mediated blockade in clinical management of eBL.