12 research outputs found
Dynamics of Epstein-Barr Virus (EBV) in human immunodeficiency virus (HIV)-1 infected adults and children
No full textEpstein-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<0.0001) and we established that a clonal B-cell population on DBS was detected when there were at least 200 clonal cells in the analysed sample. Moreover, very similar clonal results were obtained between DNA from DBS and fresh whole blood from patients with chronic lymphocytic leukemia. This study demonstrated the possibility to perform clonality testing on DBS sampling, thus improving the diagnostic and monitoring options.
Conclusions
In these studies, we found a significant relationship between markers of microbial translocation, levels of pro-inflammatory cytokines and EBV-DNA levels in both HIV-1 infected adults and children, suggesting that cell activation driven by HIV-1 antigens may result in chronic B cell stimulation and expansion of EBV-infected B cells, a risk factor for the development of EBV-related malignancies. Of interest, we found that EBV-DNA levels were higher in patients with a gain in CD4 lymphocytes, but incomplete suppression of HIV-1 viremia than in patients with undetectable plasma viremia, regardless their CD4 cell number.
The relationship between immune activation and EBV levels was also supported by the findings in HIV-1 infected children in Uganda. Moreover, we found that EBV-DNA and 16S rDNA levels were significantly lower in children on ART than in those ART-naive, suggesting that treatment with ART, likely by limiting immune activation, may prevent B cell stimulation and expansion of EBV-infected B cells. These observations may become particularly interesting to plan future therapeutic strategies in HIV-1 infected patients.
We also found that NHLs are strongly associated with EBV load in peripheral blood, suggesting that high levels of EBV in blood might have diagnostic and prognostic relevance for the diagnosis and monitoring of EBV-related B cell malignancies. In this context we assessed that DBS sampling was also suitable to identify the presence of a B cell clonal population. Our results showed it is possible to perform clonality testing on DBS sampling, thereby improving the diagnostic and monitoring capability of B cell lymphomas in resource-limited settings
Relationship Between Non-Hodgkin's Lymphoma and Blood Levels of Epstein-Barr Virus in Children in North-Western Tanzania: A Case Control Study.
No full textNon-Hodgkin's Lymphomas (NHL) are common in African children, with endemic Burkitt's lymphoma (BL) being the most common subtype. While the role of Epstein-Barr Virus (EBV) in endemic BL is known, no data are available about clinical presentations of NHL subtypes and their relationship to Human Immunodeficiency Virus (HIV) infection and Epstein Barr Virus (EBV) load in peripheral blood of children in north-western, Tanzania. A matched case control study of NHL subtypes was performed in children under 15 years of age and their respective controls admitted to Bugando Medical Centre, Sengerema and Shirati district designated hospitals in north-western, Tanzania, between September 2010 and April 2011. Peripheral blood samples were collected on Whatman 903 filter papers and EBV DNA levels were estimated by multiplex real-time PCR. Clinical and laboratory data were collected using a structured data collection tool and analysed using chi-square, Fisher and Wilcoxon rank sum tests where appropriate. The association between NHL and detection of EBV in peripheral blood was assessed using conditional logistic regression model and presented as odds ratios (OR) and 95% confidence intervals (CI). A total of 35 NHL cases and 70 controls matched for age and sex were enrolled. Of NHLs, 32 had BL with equal distribution between jaw and abdominal tumour, 2 had large B cell lymphoma (DLBCL) and 1 had NHL-not otherwise specified (NHL-NOS). Central nervous system (CNS) presentation occurred only in 1 BL patient; 19 NHLs had stage I and II of disease. Only 1 NHL was found to be HIV-seropositive. Twenty-one of 35 (60%) NHL and 21 of 70 (30%) controls had detectable EBV in peripheral blood (OR = 4.77, 95% CI 1.71 - 13.33, p = 0.003). In addition, levels of EBV in blood were significantly higher in NHL cases than in controls (p = 0.024). BL is the most common childhood NHL subtype in north-western Tanzania. NHLs are not associated with HIV infection, but are strongly associated with EBV load in peripheral blood. The findings suggest that high levels of EBV in blood might have diagnostic and prognostic relevance in African children
EPSTEIN-BARR VIRUS-DRIVEN LYMPHOMAGENESIS IN THE CONTEXT OF HUMAN IMMUNODEFICIENCY VIRUS TYPE 1 INFECTION
No full textEpstein–Barr Virus (EBV) is a ubiquitous human γ-herpes virus which establishes a life-long asymptomatic infection in immunocompetent hosts. In HIV-1 infected patients, the impaired immunosurveillance against EBV may favor the development of EBV-related diseases, ranging from lymphoproliferative disorders to B-cell Non-Hodgkin's lymphomas (NHL). Antiretroviral therapy (ART) has significantly modified the natural course of HIV-1 infection, resulting in decreased HIV-1 plasmaviremia, increased CD4 lymphocytes, and decreased opportunistic infections, indicating a restoration of immune functions. However, the impact of ART appears to be less favorable on EBV-related malignancies than on other AIDS-defining tumors (ADC), such as Kaposi’s Sarcoma, and NHL remains the most common cancer during the ART era. EBV-driven tumors are associated with selective expression of latent oncogenic proteins, but uncontrolled lytic cycle with virus replication and/or reactivation may favor cell transformation, at least in the early phases. Several host's factors may promote EBV reactivation and replication; besides immunodepression, inflammation/chronic immune stimulation may play an important role. Microbial pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs), through Toll-like receptors (TLR), activate the immune system and may promote EBV reactivation and/or polyclonal expansion of EBV-infected cells. A body of evidence suggests that chronic immune stimulation is a hallmark of HIV-1 pathogenesis and may persist even in ART-treated patients. This review focuses on lymphomagenesis driven by EBV both in the context of the natural history of HIV-1 infection and in ART-treated patients. Understanding the mechanisms involved in the expansion of EBV-infected cells is a premise for the identification of prognostic markers of EBV-associated malignancies
Immune activation, immune senescence and levels of Epstein Barr Virus in kidney transplant patients: Impact of mTOR inhibitors
No full textPost-transplant lymphoproliferative disorders (PTLD) represent a severe complication in transplanted patients and Epstein-Barr Virus (EBV) is the main driver. Besides immunodepression, immune activation/chronic inflammation play an important role in both virus reactivation and expansion of EBV-positive B cells. The aim of this study was to assess the impact of immunosuppressive strategies on factors involved in the PTLD's pathogenesis. 124 kidney transplanted patients were enrolled in this study: 71 were treated with mycophenolic acid (MPA) and 53 treated with mTOR inhibitor (mTORi), both in combination with different doses of calcineurin inhibitor. At the time of the transplant (T0), profile of inflammation/immune activation and immune senescence didn't differ between the two groups, but after one year of treatment (T1) markers were significantly higher in MPA-treated patients; their immunosenescence process was supported by the greater erosion of telomeres despite their younger age. Percentages of activated B cells and levels of EBV-DNA significantly increased in MPA-treated patients, and at T1 were significantly higher in MPA- than in mTORi-treated patients. Overall, these findings indicate that mTOR inhibitors constrain the inflammation/immune activation and senescence status, thus reducing the expansion of EBV-infected B cells and the risk of virus-associated PTLD in kidney transplant recipients. © 2019 The Author
Epstein-Barr virus load in children infected with human immunodeficiency virus type 1 in Uganda.
No full textBACKGROUND: Epstein-Barr Virus (EBV) is involved in a wide range of malignancies, particularly in immunocompromised subjects. In Africa, EBV primary infection occurs during early childhood, but little is known about the EBV load in Human Immunodeficiency Virus type 1 (HIV-1)-infected children. METHODS: Blood samples from 213 HIV-1-infected children, 140 of whom were receiving antiretroviral therapy (ART), were collected at the Nsambya Hospital in Kampala, Uganda, and obtained for dried blood spot analysis. Nucleic acids were extracted and analyzed for quantification of EBV types 1 and 2; 16S ribosomal DNA (rDNA), a marker of microbial translocation; and HIV-1 RNA. RESULTS: Ninety-two of 140 children (66%) receiving ART and 57 of 73 ART-naive children (78%) had detectable EBV DNA levels. Coinfection with both EBV types was less frequent in ART-treated children than in ART-naive children (odds ratio, 0.54 [95% confidence interval {CI}, .30-.98]; P = .042). Mean EBV DNA levels (±standard deviation) were lower in the former (3.99 ± 0.59 vs 4.22 ± 0.54 log10 copies/mL; P = .006) and tended to be inversely associated with ART duration. EBV DNA levels were higher in children with an HIV-1 RNA load of > 3 log10 copies/mL of blood (regression coefficient, 0.32 [95% CI, .05-.59]; P = .020) and correlated with circulating 16S rDNA levels (rs = 0.25 [95% CI, .02-.46]; P = .031). CONCLUSIONS: These findings suggest that ART, by limiting HIV-1 replication, microbial translocation, and related immune activation, prevents superinfection with both EBV types and keeps EBV viremia down, thus potentially reducing the risk of EBV-associated lymphomas
Immune Activation, Exhaustion and Senescence Profiles as Possible Predictors of Cancer in Liver Transplanted Patients
No full textLiver transplanted (LT) patients for hepatocellular carcinoma (LT-HCC) or for other causes (LT-no-HCC) may develop post-transplantation malignancies. Although immune activation and senescence are frequently implicated in cancer development, no data is available on their possible role as biomarkers predictive of tumor onset in this setting. A total of 116 patients were investigated: the 45 LT-HCC patients were older than the 71 LT-non-HCC (p=0.011), but comparable for sex, HCV, HBV infection and immunosuppressive treatment. At baseline, the numbers of activated and senescent-like circulating cells were significantly higher in LT-HCC patients than in LT-no-HCC ones. After a median follow-up of 26.8 months, 6 post-transplant malignancies (PTM) occurred: 4 in LT-HCC (8.9%) and 2 in LT-no-HCC (2.8%) patients. Overall, subjects with high percentages of activated and exhausted T and B cells at baseline were at higher risk of PTM. Notably, within the LT-HCC group, a higher percentage of senescence-like T cells was also associated with cancer development. Moreover, patients with PTM had higher telomere erosion and higher levels of circulating PAMPs (16S rDNA) and DAMPs (mtDNA) when compared with matched patients without PTM. Overall, these findings suggest that immune activation and exhaustion may be useful to predict the risk of PTM occurrence, regardless of the cause of transplantation. In LT-HCC, T-cell senescence represents an additional risk factor for tumor onset
Risk of virus and non-virus related malignancies following immunosuppression in a cohort of liver transplant recipients. Italy, 1985–2014
No full textThis cohort study assessed, in Italy, the overall pattern of risk of de novo malignancies following liver transplantation (LT). The study group included 2,832 individuals who underwent LT between 1985 and 2014 in nine centers all over Italy. Person–years (PYs) at cancer risk were computed from 30 days after LT to the date of cancer diagnosis, to the date of death or to the end of follow-up. Excess cancer risk, as compared to the general population, was estimated using standardized incidence ratios (SIRs) and 95% confidence intervals (CIs). During 18,642 PYs, 246 LT recipients developed 266 de novo malignancies, corresponding to a 1.8-fold higher cancer risk (95% CI: 1.6–2.0). SIRs were particularly elevated for virus-related malignancies, including Kaposi's sarcoma (SIR = 53.6, 95% CI: 30.0–88.5), non-Hodgkin lymphomas (SIR = 7.1, 95% CI: 4.8–10.1) and cervix uteri (SIR = 5.4, 95% CI: 1.1–15.8). Among virus-unrelated malignancies, elevated risks emerged for head and neck (SIR = 4.4, 95% CI: 3.1–6.2), esophagus (SIR = 6.7, 95% CI: 2.9–13.3) and adrenal gland (SIR = 22.9, 95% CI: 2.8–82.7). Borderline statistically significant elevated risks were found for lung cancer (SIR = 1.4, 95% CI: 1.0–2.1) and skin melanoma (SIR = 2.6, 95% CI: 1.0–5.3). A reduced risk emerged for prostate cancer (SIR = 0.1, 95% CI: 0.0–0.5). These findings underline the need of preventive interventions and early detection of malignancies, specifically tailored to LT recipients
Survival after the diagnosis of de novo malignancy in liver transplant recipients
No full textIn the setting of liver transplant (LT), the survival after the diagnosis of de novo malignancies (DNMs) has been poorly investigated. In this study, we assessed the impact of DNMs on survival of LT recipients as compared to corresponding LT recipients without DNM. A nested case–control study was conducted in a cohort of 2,818 LT recipients enrolled in nine Italian centres between 1985 and 2014. Cases were 244 LT recipients who developed DNMs after LT. For each case, two controls matched for gender, age, and year at transplant were selected by incidence density sampling among cohort members without DNM. The survival probabilities were estimated using the Kaplan–Meier method. Hazard ratios (HRs) of death and 95% confidence intervals (CIs) were estimated using Cox proportional hazard models. The all-cancer 10-year survival was 43% in cases versus 70% in controls (HR = 4.66; 95% CI: 3.17–6.85). Survival was impaired in cases for all the most frequent cancer types, including lung (HR = 37.13; 95% CI: 4.98–276.74), non-Hodgkin lymphoma (HR = 6.57; 95% CI: 2.15–20.01), head and neck (HR = 4.65; 95% CI: 1.81–11.95), and colon-rectum (HR = 3.61; 95% CI: 1.08–12.07). The survival gap was observed for both early and late mortality, although the effect was more pronounced in the first year after cancer diagnosis. No significant differences in survival emerged for Kaposi's sarcoma and nonmelanoma skin cancers. The survival gap herein quantified included a broad range of malignancies following LT and prompts close monitoring during the post-transplant follow-up to ensure early cancer diagnosis and to improve survival
Stronger and durable SARS-CoV-2 immune response to mRNA vaccines in 5–11 years old children with prior COVID-19
No full textbackground and objectives: mRNA vaccines elicit a durable humoral response to SARS-CoV-2 in adults, whereas evidence in children is scarce. this study aimed to assess the early and long-term immune response to the mRNA vaccine in children with or without previous SARS-CoV-2 infection. Methods: In a multicentre prospective observational study, we profiled the immune response to the Pfizer BioNTech (BNT162b2) vaccine in 5-11-year-old children attending the university pediatric hospital of padua and bambino-gesù hospital in rome (Italy) from december-2021 to february-2023. blood samples were collected pre-, 1-, and 6-months after vaccination. Neutralizing antibodies (NAbs) and anti-spike-receptor-binding-domain (anti-S-RBD) IgG titers were analyzed through plaque reduction neutralization test (PRNT) and chemiluminescent immune-enzymatic assay (CLIA), respectively. Immune cell phenotypes were analyzed by flow cytometry. results: sixty children (26 [43 %] female, median age = 8 years [IQR = 7-10.7]) were enrolled in the study, including 46 children with a laboratory-confirmed previous COVID-19 (SARS-CoV-2-recovered) and 14 SARS-CoV-2-naïve participants defined as the absence of antigen-specific antibodies before vaccination. SARS-CoV-2-recovered participants recorded higher anti-S-RBD IgG and Wild-type and omicron BA.2 NAbs titers than SARS-CoV-2-naïve participants at both 1- and 6-months after vaccination. antibody titers correlated with T (Tregs) and B (Bregs) regulatory cell frequencies in SARS-CoV-2-recovered children. both SARS-CoV-2-recovered and SARS-CoV-2-naïve participants decreased antibody titers by approximately 100 to 250 % from 1 to 6 months. while children with immunocompromising underlying conditions developed immune responses comparable to those of healthy children, solid organ transplant recipients exhibited lower levels of NAbs and anti-S-RBD IgG titers, as well as reduced frequencies of tregs and bregs. conclusions: mRNA vaccination triggered a higher production of specific anti-SARS-CoV-2 antibodies along with increased levels of regulatory cells in children with previous SARS-CoV-2 infection up to the following 6 months. these findings provide insights into boosting pre-existing immunity
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Early inflammation as a footprint of increased mortality risk in infants living with HIV from three African countries
No full textIn this work our aim was to identify early biomarkers in plasma samples associated with mortality in children with perinatal HIV treated early in life, to potentially inform early intervention targeting this vulnerable group. 20/215 children (9.3%) with perinatal HIV, enrolled within 3 months of age died prematurely within the first year of the study, despite early ART initiation. Using a propensity score, we selected 40 alive study participants having similar clinical and virological records compared to the deceased group. 13 HIV unexposed (HU) healthy children were additionally used as controls. Baseline plasma samples were analyzed using a targeted proteomic approach, and to assess pathogen-associated and damage-associated molecular patterns (PAMPs, DAMPs) levels. Data from deceased participants were compared to both control groups, with multivariate logistic regression models used to evaluate the association between mortality and plasma proteins. We developed a machine learning model to predict mortality risk, finding that IL-6 and CXCL11 not only were higher in deceased children than Matched-children with HIV (p < 0.001 and p = 0.0034) but also predictive of mortality (accuracy of 77%); levels of PAMPs were higher in deceased children (p = 0.0016). Thus, measuring early inflammatory biomarkers, particularly IL-6, could help mortality risk prediction and potentially guide targeted interventions
