High Epstein-Barr virus (EBV) DNA loads in HIV-infected patients: correlation with antiretroviral therapy and quantitative EBV serology

OBJECTIVE: To study Epstein-Barr virus (EBV) DNA loads in peripheral blood of HIV carriers to determine base-line values and diagnostic relevance of viral load in relation to quantitative serology; to compare EBV presence in parallel plasma and unfractionated whole blood samples; and to correlate EBV DNA load to HIV, CD4 T-cell counts and HAART. DESIGN: One-hundred and nine random patients receiving highly active antiretroviral therapy (HAART) during 1999 and 99 patients on anti-HIV monotherapy during 1993-1996 were included. METHODS: EBV DNA load was determined by quantitative competitive PCR. EBV serology was determined by immunoblot profile and quantitative enzyme-linked immunosorbent assay for responses against VCA-p18 and EBNA-1. RESULTS: Twenty-two out of 109 patients receiving HAART and 28 out of 99 of patients on anti-HIV monotherapy showed elevated EBV DNA loads in whole blood (> 2000 copies/ml), without elevated loads in parallel plasma. EBV DNA load distribution did not differ between the two groups (P = 0.78) and did not correlate with HIV or CD4 T-cell count. In three patients with high EBV DNA loads EBV RNA was virtually absent. Patients with high EBV DNA loads (3610-89 400 copies/ml) had higher anti-VCA-p18 IgG levels than patients with undetectable EBV DNA (P < 0.0001) but lower anti-EBNA-1 IgG levels (P = 0.005). CONCLUSION: Absolute values of EBV DNA load may have poor diagnostic value for defining HIV patients at risk for developing EBV-associated disease. Elevated EBV DNA loads are cell-associated and are not influenced by HAART. Increased anti-p18-VCA and decreased anti-EBNA-1 IgG levels in patients with high EBV loads indicate impaired latency control and increased lytic replication suggesting disturbed overall immunosurveillance against EBV.     

Safety of allogeneic Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes for patients with refractory EBV-related lymphoma

Epstein-Barr virus (EBV) causes lymphomas in immunocompromised individuals such as recipients of stem cell or organ transplants and patients with acquired immunodeficiency syndrome (AIDS). EBV has also been detected in the Reed-Sternberg cells of approximately 50% of all cases of Hodgkin's disease (HD). The purpose of this study was to examine the safety, and the clinical and immunological effects of infusing allogeneic EBV-specific cytotoxic T lymphocytes (CTL) for patients with refractory EBV-positive malignancies. In this pilot study, we have treated four patients with EBV-related lymphoma using allogeneic EBV-specific CTL. Two patients received EBV-specific CTL derived from partially human leucocyte antigen (HLA)-matched donors and the other two from HLA-matched siblings. No complications were observed as a result of the CTL infusions and all patients showed increased levels of EBV-specific CTL precursors (CTLp) post infusion. Of the two organ transplant patients, one had refractory disease and has sustained a complete remission following the T-cell infusions. The second has also been disease free since T-cell infusions, although the efficacy cannot be definitively attributed to CTL therapy because this patient received local radiation therapy prior to immunotherapy. A patient with AIDS-related, EBV-positive lymphoma had disease progression following CTL infusions. One HD patient received HLA 4/6 matched T cells from an unrelated donor and showed a decrease in the size of affected lymph nodes and resolution of B-symptoms post infusion. In conclusion, adoptive immunotherapy with allogeneic EBV-specific CTL is safe and may have efficacy in patients with high-risk or refractory EBV-related tumours.     

Changing patterns in the Epstein-Barr virus (EBV)and Hodgkin lymphoma association in Tunisia

Annals of Hematology - We compared the features of the Epstein-Barr virus (EBV) and Hodgkin lymphoma (HL) association in Tunisia in two periods of time, 1991–2001 (111 cases) and...     

Epstein-Barr virus (EBV) in lymphoproliferative diseases]

The results of DNA or RNA study in EBV-associated lymphoproliferative diseases were shown. For detecting EBV DNA, Southern blot analysis with Bam HIW probe (Internal Repeat) and non-repeat (least often deleted) probe are used. Probes close to (ex. LMP) or within terminal repeat can indicate clonality in terms of junctional structure. Several such examples were shown, in which benign polyclonal EBV (+) CD3 + 8+ lymphocytes, EBV (+) CD3 + 4 - 8- granulay lymphocytosis, EBV (+) t(14, 22) B-cell lymphoma and EBV (-) follicular lymphoma with reactivation type serology were included. The detectability of EBV DNA was tested in consecutively sampled acute IM peripheral cells by Southern blot analysis with Bam HIW, PCR with Bam HIK (EBNA1) and its Southern re-estimation. The results indicated that EBV DNA was detectable rarely in the earliest samples, and that PCR can increase the sensitivity, as expected. The gene expression of IL-2R alpha (-) IL-2R beta (+) and perforin (-) by IM cells were assessed with Northern blot analysis. The abundant gamma IFN gene expression by IM cells was revealed by reversed PCR.     

Serum IgA antibodies to Epstein-Barr virus (EBV) early lytic antigens are present in primary EBV infection

Primary Epstein-Barr virus (EBV) infection is characterized by the presence of IgM antibodies to viral capsid antigen and the absence of antibodies to EB nuclear antigen. Here, using a flow cytometry-based assay, we investigated whether IgA antibodies are a marker for primary infection. Serum IgA antibodies in 15 individuals with primary EBV infection reacted with 15%-55.6% of HH514-16 Burkitt lymphoma cells expressing early lytic antigens (EAs), whereas IgA antibodies in serum samples from 15 healthy EBV-seropositive individuals reacted with 0.02%-2% of cells with EAs (P<.0001). IgA antibodies in primary infection were directed against the Bam Z Epstein-Barr replication activator (ZEBRA) (BZLF1) and diffuse EA (BMRF1) EAs. Thus, IgA antibodies to EBV EAs are produced during primary EBV infection and are likely to be stimulated as a result of lytic EBV replication in mucosal sites. Detection of IgA antibodies to EA may be developed into a diagnostic tool for primary EBV infection.     

Persistently high Epstein-Barr virus (EBV) loads in peripheral blood lymphocytes from patients with chronic active EBV infection

Chronic active Epstein-Barr virus infection (CAEBV) is a severe illness with unusual EBV activation that persists for years, and its pathogenesis is largely unknown. After the creation of an accurate and reproducible polymerase chain reaction system to quantify EBV DNA, virus loads in peripheral blood lymphocytes (PBL) were determined in 54 children: 15 with CAEBV, 16 with infectious mononucleosis (IM), and 23 healthy children. Children with CAEBV and those with IM had high virus loads. Lower loads were detected in 47% of seropositive healthy donors. There were two distinct differences between children with CAEBV and those with IM: The former had greater viral replication (10(3)-10(7) copies/2.5x10(5) PBL) than those with IM, and viral replication declined in children with IM whereas active replication persisted for years in subjects with CAEBV. Persisting high virus loads are a possible diagnostic criterion for CAEBV. EBV loads may enable classification and prognosis of EBV infections.     

Hemiplegia--a rare complication of acute Epstein-Barr virus (EBV) infection.

A 32-year-old male presented to hospital with a transient hemiplegia associated with a rash and systemic upset. He was found to have an acute Epstein-Barr virus (EBV) infection. Hemiplegia complicating glandular fever has been described but once previously.     

Quantitative analysis of Epstein-Barr virus (EBV)-specific CD8+ T cells in patients with chronic active EBV infection

To clarify the pathogenesis of chronic active Epstein-Barr virus (EBV) infection, EBV-specific CD8+ T cells were enumerated, by use of human leukocyte antigen (HLA)-A*2402-restricted tetramers, in 8 patients with chronic active EBV infection, 10 patients with infectious mononucleosis, and 16 EBV-seropositive healthy control subjects. In most of the patients with chronic active EBV infection, EBV-specific CD8+ T cells were not detected. Of note, latent membrane protein 2-specific CD8+ T cells were not detectable in any patients with chronic active EBV infection. In contrast, EBV-specific CD8+ T cells were detected in patients with infectious mononucleosis and in healthy control subjects. Low frequencies of EBV-specific CD8+ T cells may be one of the immunological features of chronic active EBV infection.     

Epstein-Barr virus (EBV)-positive pyothorax-associated lymphoma (PAL): chromosomal integration of EBV in a novel CD2-positive PAL B-cell line

Pyothorax-associated lymphoma (PAL) is a clinico-pathological entity arising in the pleural cavity of patients with long-standing inflammatory pyothorax. PAL is closely associated with Epstein-Barr virus (EBV), but how this virus contributes to the development of the lymphoma is unknown. We have successfully obtained a novel EBV-infected PAL cell line, designated Pal-1. The cell line and its source coexpressed CD2 and CD20 molecules, but other representative B- and T-cell markers such as CD1, CD3, CD5, CD7, CD10 and CD19 were not found. The B-cell origin of Pal-1 cells was proven by rearrangement of the immunoglobulin heavy- and light-chain genes without rearranged T-cell receptor genes. Both the cell line and primary tumour cells carried monoclonal EBV genome. Although EBV genome is known to be maintained as circular extrachromosomal DNA, neither circular nor linear extrachromosomal EBV DNA was detectable in Pal-1 cells by in situ lysis gel analysis. Fluorescence in situ hybridization demonstrated viral integration at a marker chromosome mostly consisting of the centromere region of chromosome 1. The viral integration event may enhance a chromosomal instability at the insertion site. This cell line represents the first example of EBV integration in PAL and could enable the study of the potential role of integrated viral infection in the development of PAL as well as mechanism of the aberrant phenotype expression.     

Dysfunctional Epstein-Barr virus (EBV)-specific CD8(+) T lymphocytes and increased EBV load in HIV-1 infected individuals progressing to AIDS-related non-Hodgkin lymphoma.

Acquired immunodeficiency syndrome-related non-Hodgkin lymphomas (AIDS-NHL) are thought to arise because of loss of Epstein-Barr Virus (EBV)-specific cellular immunity. Here, an investigation was done to determine whether cellular immunity to EBV is lost because of physical loss or dysfunction of EBV-specific cytotoxic T cells. Data on EBV-specific cellular immunity were correlated with EBV load. For comparison, individuals who progressed to AIDS with opportunistic infections (AIDS-OI) and long-term asymptomatics (LTAs) were studied. The number of virus-specific T cells was detected using tetrameric HLA-EBV-peptide complexes; function of these EBV-specific T cells was determined using the interferon-gamma (IFN-gamma) Elispot assay. It was observed that EBV-specific CD8(+) T cells were present in normal numbers in human immunodeficiency virus (HIV)-infected individuals. However, their functional capacity was decreased compared with HIV(-) individuals. In AIDS-NHL patients, EBV-specific T cells were not physically lost in the course of HIV-1 infection but showed progressive loss of their capability to produce IFN-gamma in response to EBV peptides. This loss of function correlated with lower CD4(+) T-cell numbers and was accompanied by increasing EBV load. In HIV-1-infected LTA individuals, in whom CD4(+) T-cell numbers were maintained, and progressors to AIDS-OI, IFN-gamma-producing EBV-specific T cells were stable and EBV load remained stable or decreased in the course of HIV infection, suggestive of immune control. Our data indicate that functional loss of EBV-specific CD8(+) T cells with a concomitant increase in EBV load may play a role in the pathogenesis of AIDS-NHL.     

Quantitative analysis of circulating cell-free Epstein-Barr virus (EBV) DNA levels in patients with EBV-associated lymphoid malignancies

Cell-free Epstein-Barr virus (EBV) DNA has recently been detected in the plasma and serum of patients with Hodgkin's disease, post-transplant lymphoproliferative disease (PTLD) and acquired immunodeficiency syndrome-related lymphoma. However, no data are available on the temporal variation of plasma/serum EBV DNA levels in patients with EBV-associated lymphoid malignancies during the course of therapy. Using a real-time quantitative polymerase chain reaction assay, we studied the plasma EBV DNA levels in 13 patients with EBV-associated lymphoid malignancies (six patients with Hodgkin's disease, four with nasal natural killer/T-cell lymphoma, two cases of PTLD and one patient with Burkitt's lymphoma) at presentation and during therapy. Plasma EBV DNA was detected in 12 of the 13 patients (median 2,266 copies/ml; interquartile range 181-8,379 copies/ml), but not in any of 35 healthy control subjects (P < 0.0001). The EBV status in tumour cells was also examined in 12 of these patients using in situ hybridization for EBV-encoded small RNAs (EBERs). EBER positivity was observed in 11 patients, all of whom had EBV DNA detectable in plasma. The one patient who had no detectable plasma EBV DNA was also negative for EBERs in tumour tissue. Serial measurements of plasma EBV DNA levels were performed in nine of the patients during the course of therapy. All patients who responded to therapy demonstrated a significant reduction of plasma EBV DNA to low or undetectable levels, whereas in two patients with ineffective therapy, disease progression was associated with a rapid increase in plasma EBV DNA levels. We concluded that plasma EBV DNA is detectable in a wide range of EBV-associated lymphoid malignancies. As plasma EBV DNA levels correlate well with the therapeutic response, such analysis may be a valuable tool for monitoring clinical progress.     

Biological aspects of Epstein-Barr virus (EBV)-infected lymphocytes in chronic active EBV infection and associated malignancies

Most primary Epstein-Barr virus (EBV) infections are clinically inapparent, but occasionally EBV infection can cause acute infectious mononucleosis. EBV has been linked to a variety of hematologic and non-hematologic malignancies. Chronic active EBV (CAEBV) infection designates a recently identified EBV-associated syndrome characterized by a variety of serious hematological disorders, including malignant lymphoma. EBV was found to infect circulating T- and/or NK-cells in patients with CAEBV infection. These EBV-infected T- and/or NK-cells express EBNA-1, LMP-1, and LMP-2A, a type II form of EBV latency, which is also observed in nasopharyngeal carcinoma (NPC), Hodgkin's disease (HD), and peripheral T-cell lymphoma. CAEBV infections may thus represent a subset of EBV-associated T- and/or NK-cell lymphoproliferative disorders.     

Epstein-Barr virus (EBV)-carrying and -expressing T-cell lines established from severe chronic active EBV infection

Four novel Epstein-Barr virus (EBV)-carrying T-cell lines, designated SIS, AIK-T8, AIK-T4, and SKN, were established from peripheral blood lymphocytes (PBL) of patients with severe chronic active EBV infection, in the presence of interleukin-2 and 4-deoxyphorbol ester. AIK-T8 and - T4 were derived from a single patient. Cell marker and genotype analyses showed that SIS, AIK-T8, and AIK-T4 had mature T-cell phenotypes with clonally rearranged T-cell receptor (TCR) genes, whereas SKN had an immature T-cell phenotype without TCR gene rearrangement. None of the cell lines expressed B, natural killer, or myeloid antigens or had Ig gene rearrangement. All lines carried EBV genomes in a single episomal form. SIS, AIK-T8, and SKN showed the same phenotype, TCR gene configuration, and/or EBV clonotype as their source or biopsied materials; therefore, they represented EBV-infected T cells proliferating in the patients. TCR gene and EBV episomal structures similar to those of AIK-T4 were not found in its source PBL, probably due to the few parental clones in vivo. All lines expressed EBV-encoded small RNA (EBER) 1, nuclear antigen (EBNA) 1, and latent membrane protein (LMP) 1, -2A, and -2B, but not other EBNAs that could be recognized by EBV-specific immune T cells. EBV replicative antigens were rarely expressed or induced. Such EBV latency reflects the in vivo situation, in which the T cells may evade immune surveillance and be insensitive to antiherpesvirus drugs. Collectively, the data suggest that EBV can target and latently infect T cells at any stage of differentiation in vivo, thus potentially causing uncontrolled T-cell proliferation. These cell lines will facilitate further analyses of possible EBV-induced oncogenicity in T cells.