Dynamics of Epstein-Barr Virus (EBV) in human immunodeficiency virus (HIV)-1 infected adults and children

Epstein-Barr Virus (EBV) is involved in a wide range of malignancies, particularly in immunocompromised subjects. Besides immunodepression, chronic immune activation may induce B-cell stimulation leading to an expansion of EBV-infected cells, thus increasing the risk of EBV-related malignancies. The factors that may contribute to HIV-1-induced B cell activation and expansion of EBV-infected cells are largely unknown. In Africa EBV primary infection occurs during infancy and early childhood and EBV-associated lymphomas represent an important cause of morbidity and mortality in children. High levels of EBV represent a risk factor for the onset of EBV-related malignancies. To date, no data are available about EBV-infection and related malignancies in the context of HIV-1 infection in African children; this may be partly due to lack of access of laboratory analyses in developing countries. The use of Dried Blood Spot (DBS) may represent an easy method to collect and store blood samples and allow their reliable transport to specialized laboratories. My PhD research focused on the following themes: 1) the relationship between markers of immune activation and EBV load in HIV-1 infected patients. 2) EBV infection in HIV-1 infected and uninfected children in African countries. 3) a new application of Dried Blood Spot to type and quantify EBV and to identify B cell clonality in order to diagnose and monitor B-cell malignancies. 1) EBV load and immune activation in HIV-1-infected patients A total of 156 HIV-1-infected patients were included in this study, 85 of which were under antiretroviral therapy (ART). EBV-DNA was detected in 114 patients, and in all but 3 it was EBV type 1. The median [interquartile] EBV-DNA load was 43[1-151] copies/105 cells and it was higher in patients with detectable HIV-1 plasma viremia, despite good immunological status (CD4>500 cells/µl), than in patients with undetectable HIV-1 plasma viremia regardless of immunological status (46[5-136] vs 17[1-56] copies/105 cells, p=0.008). Patients with high EBV-DNA load (>median value) presented higher levels of LPS and proinflammatory cytokines (IL-6, IL-10 and TNF-α) than patients with low EBV load. Furthermore, percentages of activated B-cells correlated with EBV-DNA load (r=0.754; p<0.001). Overall, these findings indicate a strong association between HIV-1 viremia, markers of immune activation and EBV load, and suggest that persistence of HIV-1 viremia and immune activation, regardless of peripheral CD4 cell depletion/repopulation, may favour the expansion of EBV-infected cells. 2) Association between NHL and blood levels of EBV in children in Tanzania, and dynamics of EBV in HIV-1-infected children in Uganda A matched case-control study was performed in children with Non-Hodgkin’s Lymphoma (NHL) admitted to three clinical centers in Tanzania, and their age-matched controls. Blood samples were collected on DBS. 21 out of 35 (60%) NHL patients and only 21 out of 70 (30%) controls presented EBV detectable in peripheral blood, thus showing a significant association between NHL and EBV in blood (OR=4.77 [95% CI 1.71–13.33], p=0.003). Furthermore, EBV-DNA levels were higher in cases compared to EBV-positive controls (p=0.024). Overall, these findings indicate that EBV-DNA load in peripheral blood might have diagnostic relevance. In a second study, blood samples from 213 HIV-1-infected children were collected on DBS at the Nsambya Home Care, Kampala, Uganda. 92 out of 140 (66%) children on ART and 57 out of 73 (78%) ART-naive children were found to be EBV-positive. After adjusting for CD4 Z-score, age and WHO stage, children on ART presented less odds of having detectable EBV than ART-naive children (OR=0.39 [95% CI 0.16-0.94], p=0.036). EBV load was significantly higher in ART-naive children than those on ART (4.22 [3.87-4.58] vs 3.99 [3.55-4.33] log10 copies/ml; p=0.016). Levels of 16S rDNA, a marker of microbial translocation, were significantly higher in ART-naive children than those on ART (2.16 [2.11-2.28] vs 2.09 [2.02-2.22] log10 copies/μl; p=0.007) and correlated with EBV-DNA levels (r=0.382, p=0.016), suggesting that circulating microbial products lead to B cell activation and expansion of EBV-infected B cells. Treatment with ART, likely by limiting HIV-1 load and thus the HIV-1-driven immune activation, may restrict EBV replication and expansion of EBV-infected cells. 3) Detection of clonal B-cell populations from DBS sampling: a tool for the diagnosis and monitoring of B-cell malignancies Firstly, through blood samples from donors, we ensured that DBS contains sufficient lymphocytes to perform a clonality assay without yielding false positive results. Using Namalwa cells that contain 2 EBV copies/cell, we found a good relationship between the expected and detected EBV-DNA copies (r=0.987, p