Ann M. Moormann

Background: During the COVID-19 pandemic, concerns emerged that hospital-based care could increase the risk of SARS-CoV-2 infection among pediatric patients with cancer, especially in resource-limited settings where the capacity to isolate patients would be challenging. Yet, infection rates in this population remain poorly documented, and it is unclear whether prior common human coronavirus (HCoV) exposures influence SARS-CoV-2 antibody responses. Here, we used serology to estimate SARS-CoV-2 exposure in children with and without cancer from Western Kenya and evaluated responses to common HCoVs to explore cross-reactivity. Methods: We performed multiplex antibody profiling to assess SARS-CoV-2 and HCoV-specific responses in plasma from children with and without cancer sampled between 2014 and 2022. Unsupervised clustering identified participants with elevated SARS-CoV-2 seroreactivity relative to pre-pandemic samples. Seropositivity was further defined using a multi-antigen approach based on IgG levels to nucleocapsid and receptor-binding domain antigens to improve specificity. Cross-reactivity and cross-boosting by common HCoVs were evaluated through correlation analyses and groupwise comparisons of antibody levels, respectively. Results: Of 564 children, 474 were healthy (184 pre-pandemic; 290 post-pandemic) and 90 had cancer (16 pre-pandemic; 74 post-pandemic). Post-pandemic, 69% (200) of healthy controls exhibited elevated SARS-CoV-2 seroreactivity, with 51% (148) meeting the criteria for seropositivity. Children with cancer showed similar rates of seroreactivity (67%) and seropositivity (57%) after adjusting for sampling year, with incidence increasing annually from 2020 to 2022. Pre-pandemic samples exhibited weak cross-reactivity to HCoV-OC43 which increased following SARS-CoV-2 infection; however, no significant boosting of HCoV antibodies was observed. Conclusions: These findings indicate widespread SARS-CoV-2 exposure among children in Western Kenya and provide no evidence that hospitalization heightened infection risk for pediatric patients with cancer.

/ Summary: Survival outcomes for pediatric Burkitt lymphoma (BL) substantially vary depending on geography (50-90%), which also serves as a proxy for the prevalence of Epstein-Barr virus (EBV) within the tumors. Although BL is considered an immunologically “cold” tumor with few tumor-infiltrating lymphocytes (TILs), their functional status has not been fully evaluated, especially for EBV-positive disease. Here, we characterize the exhaustion and activation profiles of T cells in the tumor microenvironment (TME) of EBV-positive BL using orthogonal methods, single-cell gene expression analysis, spectral flow cytometry, and immuno-histochemistry staining (IHC). We found that CD8+ TILs displayed a mosaic of immune inhibitory gene expression encoding, PD1 , TIGIT , LAG3 and HAVCR2/ TIM3. IHC validated the expression of PD1 and TIGIT on CD8+ TILs, as well as their respective ligands, PDL-1, PVR, and Nectin-2 on malignant B cells. Despite exhaustion-associated signatures, CD8+ TILs retain cytotoxic potential, expressing granules (i.e. Granzyme A, Perforin) and cytokines (i.e. IFNγ) and demonstrate an increased uptake of metabolites such as glucose, arginine, and methionine. In peripheral blood, pediatric BL patients exhibited a significantly higher abundance of PD1+TIGIT+ CD8+ T cells compared to healthy children. Notably, these circulating T cells from BL patients express significantly lower levels of TOX, suggesting they are not irreversibly dysfunctional. Together, our results indicate that CD8+ T cells both in the TME and in circulation of children with BL are not terminally exhausted but remain poised for functional re-invigoration. These findings support the potential integration of immune checkpoint inhibitors into combination chemotherapeutic regimens to improve outcomes for these children. Significance: EBV-positive BL tumors contain functional, metabolically active CD8+ T cells. Circulating PD1+TIGIT+CD8+ T cells found in BL patients’ blood are a biomarker for those in the tumor microenvironment.

Terminally differentiated CD56negCD16pos NK cells have been described after chronic viral and malaria infections, and in children diagnosed with Burkitt lymphoma (BL). Despite CD56neg NK cells appearing to be poor at direct cytotoxicity, they express high levels of cytotoxic granules (i.e. granzymes, perforin), activation markers, and Fc-γ receptors (CD32 and CD16) that are typically engaged in antibody-dependent cell cytotoxicity (ADCC). In addition, the abundance of CD56neg NK cells strongly correlates with IgG1 and IgG3 plasma levels, which are essential subclasses for ADCC. To determine whether CD56neg NK cells have superior ADCC capacity relative to CD56dim NK cells, we performed ADCC assays using effector cells from pediatric cancer patients and healthy children from malaria endemic regions of Kenya, targeting in vitro rituximab-treated commercial and newly established BL cell lines. We found that CD56neg NK cells were indeed capable of in vitro ADCC, showing a significant increase of CD107a-mediated degranulation in the presence of rituximab; however, they were not as efficient as CD56dim NK cells. Moreover, we found that the ADCC magnitude was significantly lower against EBV-Type 2 (EBV-T2) BL lines compared to EBV-Type 1 (EBV-T1). EBV-T2 tumor cell lines expressed significantly more lytic viral proteins than EBV-T1, making them more sensitive to direct cytotoxicity. Results from this study highlight the importance of assessing inter-patient variation in NK cell profiles in conjunction with ADCC sensitivity and EBV type within tumor cells when evaluating clinical outcomes for NK-mediated immunotherapies.

Burkitt lymphoma (BL) remains a prevalent pediatric cancer in sub-Saharan Africa and was the first human cancer identified with a virus when Epstein-Barr virus (EBV) was discovered in a Ugandan BL tumor in 1964. The impact of EBV in BL is highlighted by a new molecular tumor classification of EBV positivity versus negativity which is starting to supersede longstanding epidemiologic classifications. The high incidence of EBV-positive BL in Africa and Papua New Guinea has been linked to Plasmodium falciparum (Pf) malaria coinfections in young children. Epidemiologic studies have yielded insight into early-age EBV infections and have demonstrated direct impacts of Pf malaria infections on EBV reactivation and disruptions in EBV persistence. Moreover, when children residing in malaria holoendemic regions are contending with chronic Pf malaria infections, they undergo immune adaptations to mitigate life-threatening immunopathology. We postulate that this malaria-induced immune conditioning leads to diminished EBV-specific cellular immune surveillance, when combined with higher B cell proliferation, and EBV load creates a permissive environment for BL tumorigenesis.

The germinal center model (GCM) of Epstein-Barr virus (EBV) persistence in humans proposes that EBV infects and drives naïve B cells through the GC to establish a quiescent lifelong persistent infection in memory B cells. EBV causes several cancers, and the normal in vivo counterparts of major EBV-associated cancers have been identified in the GCM. Yet, the normal in vivo counterparts of immunosuppression-related/immunoblastic B-cell lymphoma (IL), which occurs in immunosuppressed transplant and HIV patients and resembles lymphoblastoid cell lines (LCLs), remain elusive. We performed a comparative analysis of human tonsillar B cells to identify the closest normal in vivo counterparts of ILs/LCLs. Here we show that EBV-infected tonsillar marginal zone (MZ) B cells, which are GC-independent, are the closest in vivo correlates of ILs/LCLs. Like LCLs, EBV-infected MZ B cells express the EBV growth program, which includes high expression of the oncogenic LMP1 and the DNA-mutating enzyme activation-induced cytidine deaminase (AID), in the absence of the transcription factor BCL6. Furthermore, these EBV-infected MZ B cells are proliferating extensively and are Ki67 ⁺ . AID expression, rapid proliferation, and LMP1 expression put these cells at high risk for tumor development. Of therapeutic importance, the high expression of LMP1 could prove to be the Achilles’ heel of IL/LCL-like EBV-infected MZ B cells to be targeted. Consequently, we now propose that there are two pathways of EBV persistence in vivo : (i) the established GC model and (ii) the GC-independent MZ model, and EBV-infected MZ B cells are plausibly the in vivo precursors of IL. IMPORTANCE: Epstein-Barr virus (EBV) causes several cancers, and the normal counterparts of major EBV-associated cancers have been identified in the germinal center model (GCM) of EBV persistence in humans. We searched for the normal in vivo counterparts of immunoblastic B-cell lymphoma (IL) that occurs in immunosuppressed individuals. IL resembles in vitro EBV-infected B cells driven by EBV to become indefinitely proliferating lymphoblastoid cell lines (LCLs). Here, we demonstrate that EBV-infected tonsillar marginal zone (MZ) B cells, which are GC-independent, are the in vivo correlates of LCLs/ILs and the plausible precursors of IL in humans. EBV-infected MZ B cells express the EBV growth program with high expression of the oncogenic LMP1 and the DNA-mutating enzyme AID and are proliferating extensively. AID expression, rapid proliferation, and LMP1 expression put these cells at high risk for cancer development. Clinical interventions that target LMP1-expressing MZ B cells should dramatically decrease the incidence of IL.

Circulating T-follicular helper (cT_FH) cells have the potential to provide an additional correlate of protection against Plasmodium falciparum (Pf) as they are essential to promote B-cell production of long-lasting antibodies. Assessing the specificity of cT_FH subsets to individual malaria antigens is vital to understanding the variation observed in antibody responses and identifying promising malaria vaccine candidates. Using spectral flow cytometry and unbiased clustering analysis, we assessed antigen-specific cT_FH cell recall responses in vitro to malaria vaccine candidates Pf-schizont egress antigen-1 (PfSEA-1A) and Pf-glutamic acid-rich protein (PfGARP) within a cross-section of children and adults living in a malaria-holoendemic region of western Kenya. In children, a broad array of cT_FH subsets (defined by cytokine and transcription factor expression) were reactive to both malaria antigens, PfSEA-1A and PfGARP, while adults had a narrow profile centering on cT_FH17- and cT_FH1/17-like subsets following stimulation with PfGARP only. Because T_FH17 cells are involved in the maintenance of memory antibody responses within the context of parasitic infections, our results suggest that PfGARP might generate longer-lived antibody responses compared to PfSEA-1A. These findings have intriguing implications for evaluating malaria vaccine candidates as they highlight the importance of including cT_FH profiles when assessing interdependent correlates of protective immunity.

Background Circulating T-follicular helper (cTFH) cells have the potential to provide an additional correlate of protection against Plasmodium falciparum (Pf) as they are essential to promote B-cell production of long-lasting antibodies. Assessing the specificity of cTFH subsets to individual malaria antigens is vital to understanding the variation observed in antibody responses and identifying promising malaria vaccine candidates. Methods Using spectral flow cytometry and unbiased clustering analysis, we assessed antigen-specific cTFH cell recall responses in vitro to malaria vaccine candidates Pf-schizont egress antigen-1 (PfSEA-1A) and Pf-glutamic acid-rich protein (PfGARP) within a cross-section of children and adults living in a malaria-holoendemic region of western Kenya. Findings In children, a broad array of cTFH subsets (defined by cytokine and transcription factor expression) were reactive to both malaria antigens, PfSEA-1A and PfGARP, while adults had a narrow profile centering on cTFH17- and cTFH1/17-like subsets following stimulation with PfGARP only. Interpretation Because TFH17 cells are involved in the maintenance of memory antibody responses within the context of parasitic infections, our results suggest that PfGARP might generate longer-lived antibody responses compared to PfSEA-1A. These findings have intriguing implications for evaluating malaria vaccine candidates as they highlight the importance of including cTFH profiles when assessing interdependent correlates of protective immunity.

Endemic Burkitt lymphoma (eBL) is one of the most prevalent cancer in children in sub-Saharan Africa, and while prior studies have found that Epstein-Barr virus (EBV) type and variation may alter the tumor driver genes necessary for tumor survival, the precise relationship between EBV variation and EBV-associated tumorigenesis remains unclear due to lack of scalable, cost-effective, viral whole-genome sequencing from tumor samples. This study introduces a rapid and cost-effective method of enriching, sequencing, and assembling accurate EBV genomes in BL tumor cell lines through a combination of selective whole genome amplification (sWGA) and subsequent 2-tube multiplex polymerase chain reaction along with long-read sequencing with a portable sequencer. The method was optimized across a range of parameters to yield a high percentage of EBV reads and sufficient coverage across the EBV genome except for large repeat regions. After optimization, we applied our method to sequence 18 cell lines and 3 patient tumors from fine needle biopsies and assembled them with median coverages of 99.62 and 99.68%, respectively. The assemblies showed high concordance (99.61% similarity) to available Illumina-based assemblies. The improved method and assembly pipeline will allow for better understanding of EBV variation in relation to BL and is applicable more broadly for translational research studies, especially useful for laboratories in Africa where eBL is most widespread.

While Epstein-Barr virus (EBV) plays a role in Burkitt lymphoma (BL) tumorigenesis, it is unclear if EBV genetic variation impacts clinical outcomes. From 130 publicly available whole-genome tumor sequences of EBV-positive BL patients, we used least absolute shrinkage and selection operator (LASSO) regression and Bayesian variable selection models within a Cox proportional hazards framework to select the top EBV variants, putative driver genes, and clinical features associated with patient survival time. These features were incorporated into survival prediction and prognostic subgrouping models. Our model yielded 22 EBV variants including seven in LMP1 as most associated with patient survival time. Using the top EBV variants, driver genes, and clinical features, we defined three prognostic subgroups that demonstrated differential survival rates, laying the foundation for incorporating EBV variants such as those in LMP1 as predictive biomarker candidates in future studies.

The Zanzibar archipelago of Tanzania has become a low-transmission area for Plasmodium falciparum. Despite being considered an area of pre-elimination for years, achieving elimination has been difficult, likely due to a combination of imported infections from mainland Tanzania, and continued local transmission. To shed light on these sources of transmission, we applied highly multiplexed genotyping utilizing molecular inversion probes to characterize the genetic relatedness of 282 P. falciparum isolates collected across Zanzibar and in Bagamoyo District on the coastal mainland from 2016-2018. Overall, parasite populations on the coastal mainland and Zanzibar archipelago remain highly related. However, parasite isolates from Zanzibar exhibit population microstructure due to rapid decay of parasite relatedness over very short distances. This, along with highly related pairs within shehias, suggests ongoing low level local transmission. We also identified highly related parasites across shehias that reflect human mobility on the main island of Unguja and identified a cluster of highly related parasites, suggestive of an outbreak, in the Micheweni district on Pemba island. Parasites in asymptomatic infections demonstrated higher complexity of infection than those in symptomatic infections, but have similar core genomes. Our data support importation as a main source of genetic diversity and contribution to the parasite population on Zanzibar, but they also show local outbreak clusters where targeted interventions are essential to block local transmission. These results highlight the need for preventive measures against imported malaria and enhanced control measures in areas that remain receptive for malaria reemergence due to susceptible hosts and competent vectors.