Establishment and operation of a Good Manufacturing Practice-compliant allogeneic Epstein-Barr virus (EBV)-specific cytotoxic cell bank for the treatment of EBV-associated lymphoproliferative disease.

Epstein-Barr virus (EBV) is associated with several malignancies, including post-transplant lymphoproliferative disorder (PTLD). Conventional treatments for PTLD are often successful, but risk organ rejection and cause significant side effects. EBV-specific cytotoxic T lymphocytes (CTLs) generated in vitro from peripheral blood lymphocytes provide an alternative treatment modality with few side effects, but autologous CTLs are difficult to use in clinical practice. Here we report the establishment and operation of a bank of EBV-specific CTLs derived from 25 blood donors with human leucocyte antigen (HLA) types found at high frequency in European populations. Since licensure, there have been enquiries about 37 patients, who shared a median of three class I and two class II HLA types with these donors. Cells have been infused into ten patients with lymphoproliferative disease, eight of whom achieved complete remission. Neither patient with refractory disease was matched for HLA class II. Both cases of EBV-associated non-haematopoietic sarcoma receiving cells failed to achieve complete remission. Thirteen patients died before any cells could be issued, emphasizing that the bank should be contacted before patients become pre-terminal. Thus, this third party donor-derived EBV-specific CTL cell bank can supply most patients with appropriately matched cells and most recipients have good outcomes.

Hypomorphic promoter mutation in PIGM causes inherited glycosylphosphatidylinositol deficiency.

Attachment to the plasma membrane by linkage to a glycosylphosphatidylinositol (GPI) anchor is a mode of protein expression highly conserved from protozoa to mammals. As a clinical entity, deficiency of GPI has been recognized as paroxysmal nocturnal hemoglobinuria, an acquired clonal disorder associated with somatic mutations of the X-linked PIGA gene in hematopoietic cells. We have identified a novel disease characterized by a propensity to venous thrombosis and seizures in which deficiency of GPI is inherited in an autosomal recessive manner. In two unrelated kindreds, a point mutation (c --> g) at position -270 from the start codon of PIGM, a mannosyltransferase-encoding gene, disrupts binding of the transcription factor Sp1 to its cognate promoter motif. This mutation substantially reduces transcription of PIGM and blocks mannosylation of GPI, leading to partial but severe deficiency of GPI. These findings indicate that biosynthesis of GPI is essential to maintain homeostasis of blood coagulation and neurological function.

Soluble CD30: a serum marker for Epstein-Barr virus-associated lymphoproliferative diseases.

The soluble form of CD30 (sCD30), a member of tumor necrosis factor receptor superfamily, has been used as a marker of disease activity in various lymphomas. Epstein-Barr virus (EBV) is a potent stimulator of CD30 expression. The study aims to evaluate whether sCD30 can be used as a diagnostic marker for EBV-associated infectious mononucleosis (IM) and post-transplant lymphoproliferative disease (PTLD). Plasma from EBV seropositive healthy controls (N = 90), acute IM patients (n = 90), non-PTLD heart/lung transplant recipients (N = 30) and EBV-positive PTLD patients (N = 23) was tested for sCD30 using a commercially available ELISA kit. EBV DNA was tested by real time quantitative polymerase chain reaction assay. Significantly higher sCD30 levels were observed in acute IM patients (median 242.9 ng/ml) compared to EBV seropositive controls (median 15.7 ng/ml; P < 0.0001). These levels were highest in IM patients within 14 days of onset of illness. PTLD patients had significantly higher sCD30 levels (median 94 ng/ml) than healthy controls (P < 0.0001) and transplant patients (median 27 ng/ml; P = 0.0007). EBV DNA was detected mostly in acute IM and PTLD patients. In both cases there was a significant correlation between sCD30 and EBV DNA levels in plasma (P < 0.0001). This study demonstrates that sCD30 and EBV DNA levels can be used as potential markers for diagnosis of IM and PTLD.

Epitope specificity and clonality of EBV-specific CTLs used to treat posttransplant lymphoproliferative disease.

In a recent phase II clinical trial using banked allogeneic CTL lines to treat EBV-associated posttransplant lymphoproliferative disease, a response rate of 52% was recorded 6 mo posttreatment. Tumor response was associated with an increase in both CTL/recipient HLA matches and CD4(+) T cells within the infused CTL lines. The present study was undertaken to correlate tumor response with CTL specificity. The majority of CTL lines infused recognized EBV-encoded nuclear Ag-3 proteins, but CTL protein specificity itself did not correlate with tumor response. Specificity in conjunction with donor/recipient functional HLA matching as opposed to HLA matching alone, however, was important for tumor response. CTL receptor TCR beta-chain variable gene subfamilies were polyclonal, with no preferential use of a particular family. However, tumor response was improved in those receiving CTL lines with polyclonal vs clonal distribution for subfamilies 2, 3, and 9. Interestingly, in five of six tumors (five Hodgkin's-like and one Burkitt's-like posttransplant lymphoproliferative disease) with restricted viral gene expression a complete response was recorded, although in some cases the tumor cells did not express the proteins recognized by the infused CTL. Thus CTL were advantageous when functionally HLA matched but for certain tumor types complete responses occurred in the absence of detectable specific CTL/tumor recognition. We suggest that either the allogenic CTL contained small, undetectable, EBV-specific, HLA-matched T cell populations or perhaps they stimulated nonspecific inflammatory responses in vivo, which were beneficial for tumor regression. These observations should be considered when designing and implementing CTL therapies.

Infectious mononucleosis in university students in the United kingdom: evaluation of the clinical features and consequences of the disease.

BACKGROUND: Infectious mononucleosis (IM) is common among university students. We undertook to analyze the clinical features and sequelae of the disease in a cohort of students at Edinburgh University. METHODS: Consecutive IM case patients were recruited from 2000 through 2002 at the University Health Service after diagnosis of IM. RESULTS: IM resulted in marked reductions in student study time, physical exercise, and non-exercise-related social activities, and sustained increases in reported number of hours of sleep. The disease profile differed between the sexes, with significantly more females reporting fatigue, which was more likely to be prolonged (P = .003) and to lead to loss of study time (P = .013). Female case patients were more likely to discontinue their studies following IM (16% vs 0%; P = .056). Within the typically elevated lymphocyte counts in IM, we identified an elevated gammadelta T cell component that may contribute to the disease pathogenesis. CONCLUSIONS: IM results in substantial morbidity among university students, reported as more profound in females, and affecting academic studies, physical exercise, and social activities. Immunization to prevent IM and strategies to reduce post-IM disability would be beneficial in this population.

HLA class I polymorphisms are associated with development of infectious mononucleosis upon primary EBV infection.

Infectious mononucleosis (IM) is an immunopathological disease caused by EBV that occurs in young adults and is a risk factor for Hodgkin lymphoma (HL). An association between EBV-positive HL and genetic markers in the HLA class I locus has been identified, indicating that genetic differences in the HLA class I locus may alter disease phenotypes associated with EBV infection. To further determine whether HLA class I alleles may affect development of EBV-associated diseases, we analyzed 2 microsatellite markers and 2 SNPs located near the HLA class I locus in patients with acute IM and in asymptomatic EBV-seropositive and -seronegative individuals. Alleles of both microsatellite markers were significantly associated with development of IM. Specific alleles of the 2 SNPs were also significantly more frequent in patients with IM than in EBV-seronegative individuals. IM patients possessing the associated microsatellite allele had fewer lymphocytes and increased neutrophils relative to IM patients lacking the allele. These patients also displayed higher EBV titers and milder IM symptoms. The results of this study indicate that HLA class I polymorphisms may predispose patients to development of IM upon primary EBV infection, suggesting that genetic variation in T cell responses can influence the nature of primary EBV infection and the level of viral persistence.

Cytolytic mechanisms and T-cell receptor Vbeta usage by ex vivo generated Epstein-Barr virus-specific cytotoxic T lymphocytes.

Ex-vivo-generated Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) have been used for cellular adoptive immunotherapy of EBV-associated lymphomas. Here we investigated the phenotypes, cytolytic mechanisms, polyfunctionality and T-cell receptor (TCR) usage in growing and established CTL, generated by weekly stimulation with an EBV-transformed autologous lymphoblastoid cell line (LCL). Our results showed that phenotypically mature CTL developed within the first 4 weeks of culture, with an increase in CD45RO and CD69, and a decrease in CD45RA, CD62L, CD27 and CD28 expression. Spectratyping analysis of the variable beta-chain of the TCR revealed that TCR repertoire remained diverse during the course of culture. Cytotoxicity of CTL was significantly inhibited by concanamycin A (P < 0.0001) and ethylene glycol-bis tetraacetic acid (P < 0.0001), indicating that a calcium and perforin-mediated exocytosis pathway with the release of granzyme B was the principal cytotoxic mechanism. The CTL mainly produced interferon-gamma (IFN-gamma) or tumour necrosis factor-alpha (TNF-alpha) upon restimulation with autologous LCL, although there were some polyfunctional cells producing IFN-gamma and TNF-alpha. Granzyme B, perforin and Fas ligand were detected in CD8(+) and CD4(+) cells in all CTL; however, a greater proportion of CD8(+) than CD4(+) T cells expressed granzyme B (P < 0.0001) and more granzyme B was detected in CD8(+) T cells than in CD4(+) T cells (P = 0.001). This difference was not observed with Fas ligand or perforin expression. Our results provide insight into the basic characteristics of ex-vivo-generated CTL.

A cohort study among university students: identification of risk factors for Epstein-Barr virus seroconversion and infectious mononucleosis.

BACKGROUND: A vaccine against Epstein-Barr virus (EBV) infection is in clinical trials. Up-to-date information on risk factors for EBV infection and infectious mononucleosis (IM) among young adults is required to inform a vaccination strategy. METHODS: We carried out a prospective study on a cohort of university students. All EBV-seronegative students were asked to report symptoms of IM and were followed up 3 years later to undergo repeat EBV testing and to complete a lifestyle questionnaire. EBV typing was performed for these subjects, as well as for students who were EBV seropositive at enrollment and for additional students with IM. RESULTS: A total of 510 students (25%) who took part in the study were EBV seronegative when they entered the university; of the 241 who donated a second blood sample 3 years later, 110 (46%) had seroconverted to EBV, 27 (25%) of whom developed IM [corrected] Penetrative sexual intercourse was a risk factor for EBV seroconversion (P = .004), but neither condom use nor oral sex significantly altered the rate of seroconversion. EBV type 1 was significantly overrepresented in IM, compared with silent seroconversion (P = .001). CONCLUSIONS: Our findings suggest that acquisition of EBV is enhanced by penetrative sexual intercourse, although transmission could occur through related sexual behaviors, such as "deep kissing." We also found that EBV type 1 infection is significantly more likely to result in IM. Overall, the results suggest that a large EBV type 1 load acquired during sexual intercourse can rapidly colonize the B cell population and induce the exaggerated T cell response that causes IM. Thus, IM could, perhaps, be prevented with a vaccine that reduces the viral load without necessarily inducing sterile immunity.

Epstein-Barr virus-specific cytotoxic T-lymphocytes for adoptive immunotherapy of post-transplant lymphoproliferative disease.

Post-transplant lymphoproliferative disease (PTLD) refers to a collection of clinically and pathologically diverse tumours associated with iatrogenic immunosuppression following transplantation. In most cases, tumourigenesis results from a deficit in Epstein-Barr virus (EBV)-specific cytotoxic T-lymphocyte (CTL) activity that leads to uncontrolled EBV-driven outgrowth of latently infected B-lymphocytes. Conventional treatment for PTLD typically involves a reduction in immunosuppression, but this approach is frequently unsuccessful and mortality remains high. An alternative, adoptive immunotherapy, involving the administration of EBV-specific CTLs cultured in vitro has been developed with the aim of selectively reconstituting EBV-directed immunity and effecting targeted tumour destruction. This approach has been the subject of several clinical studies, and these provide encouraging evidence of its clinical efficacy. This review presents an overview of the pathogenesis of PTLD and examines current progress in the use of adoptive immunotherapy for its treatment.

Epstein-Barr virus-recent advances.

Epstein-Barr virus is a tumorigenic herpes virus that is ubiquitous in the adult population. The virus is generally spread to and between young children through salivary contact, and only causes clinical illness where primary infection is delayed until adolescence or beyond, when an intense immunopathological reaction leads to the symptoms of infectious mononucleosis in roughly 50% of cases. More than 90% of the world's population carry Epstein-Barr virus as a life-long, latent infection of B lymphocytes. Recent data show that by mimicking B-cell antigen-activation pathways the virus enters the long-lived memory B lymphocyte pool where it evades immune elimination by severely restricting its own gene expression. By influencing B-cell survival mechanisms Epstein-Barr virus may induce tumours such as B lymphoproliferative disease and Hodgkin's disease. Vaccines are being developed to prevent and/or treat these conditions, but an animal model is required to study pathogenesis before a rational vaccine strategy can be formulated.

Viruses and tumours--an update.

In the 40 years since the publication of the article by Harris (Eur J Cancer 1965, 1, 183-188), considerable progress has been made towards the understanding of the contribution of infectious agents to the development of tumours. It is estimated that 15-20% of human malignancies may have an infectious aetiology. This article attempts to summarise the current level of our knowledge with respect to tumour-associated viruses, and to place this in the context of the earlier article by Harris (Eur J Cancer 1965, 1, 183-188).

Risk of lymphoid neoplasia after cardiothoracic transplantation: the influence of underlying disease and human leukocyte antigen type and matching.

BACKGROUND: It has been known for more than 20 years that there is an increased risk of lymphoid neoplasia after cardiothoracic transplantation. Recent studies have demonstrated the importance of primary Epstein-Barr virus (EBV) infection and type of immunosuppressive therapy to the cause of these neoplasms, but the contribution of other factors remains equivocal. METHODS: The authors followed 1,562 patients undergoing cardiothoracic transplantation at Harefield Hospital, United Kingdom, and used standard cohort methods of analysis to examine whether posttransplant lymphoma risk was related to the underlying disease requiring transplantation or the human leukocyte antigen (HLA) type and matching. Lymphomas were categorized into EBV-associated lymphoproliferative disease (LPD) and EBV-negative non-Hodgkin's lymphoma (NHL), and the authors carried out separate analyses of these. RESULTS: The authors found no significant association between the underlying disease necessitating transplantation and the risk of lymphoid neoplasia. There was also no evidence of a relation of lymphoma risk with the presence or absence of any particular HLA antigen, although significant protective effects of HLA-B14 and -B57 were found when analyses were conducted without adjustment for multiple testing. Risk of LPD was not associated with degree of HLA mismatching, but there was a significant effect of mismatching on risk of EBV-negative tumors. CONCLUSIONS: The differential effect of HLA mismatching on the risks of LPD and EBV-negative NHL provides further evidence that these two tumors are distinct etiologic entities. The authors' results suggest that the immunologic cause of EBV-negative NHL may be different from that of LPD. Investigation of the relation of risk of EBV-negative NHL to degree of immunosuppression is needed.

Persistent Epstein-Barr virus infection: unrestricted latent and lytic viral gene expression in healthy immunosuppressed transplant recipients.

BACKGROUND: Posttransplant lymphoproliferative disease (PTLD) is a common Epstein-Barr virus (EBV)-associated complication of transplantation which, despite treatment, is often fatal. This study was undertaken to monitor persistent EBV infection in transplant recipients, to compare EBV load and gene expression in healthy individuals and EBV-associated diseases, and to highlight differences in PTLD that could be used to define those at risk of the disease. METHODS: A cohort of 96 cardiothoracic transplant recipients was monitored posttransplant for up to 1110 days (median 268 days). Levels of EBV DNA and viral mRNA transcripts in peripheral blood mononuclear cells (PBMs) were measured at regular intervals and compared with those found in healthy individuals, infectious mononucleosis (IM) patients, and 12 PTLD patients bled at the time of diagnosis. Overall posttransplant levels were significantly higher than pretransplant and healthy subjects, and correlate with dose of immunosuppression. EBV DNA levels in both IM and PTLD were significantly higher than in healthy recipients, with the highest levels in PTLD patients. Individual measurements in 12 healthy transplant recipients reached levels seen in PTLD, and thus single estimations are not of predictive significance for PTLD development. RESULTS: Analysis of viral gene expression in peripheral blood mononuclear cells showed a restricted (LMP 2 only) pattern in healthy subjects, and an unrestricted (latency 3) pattern with lytic replication in 14% of IM blood and 45% of cases of PTLD. A total of 55% of healthy transplant recipients had additional transcripts in one or more blood samples, and this finding correlated with high viral load. Analysis of the 12 samples from healthy recipients with viral loads equivalent to those seen in PTLD showed additional transcripts in all cases and latency 3 with lytic replication in 33%. Thus, an isolated finding of high viral load and/or unrestricted latent and lytic gene expression is not indicative of PTLD.

Role of the HLA system in the association between multiple sclerosis and infectious mononucleosis.

OBJECTIVE: To determine whether multiple sclerosis (MS) and infectious mononucleosis (IM) share common HLA associations. DESIGN: A prospective cohort study was conducted from October 1, 1999, through September 30, 2003. SETTING: University of Edinburgh Richard Verney Health Centre, Edinburgh, Scotland. PATIENTS: Participants included 179 individuals who underwent asymptomatic Epstein-Barr virus seroconversion and 175 patients who developed IM. INTERVENTION: Genotyping for 5 classical HLA loci (HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1). MAIN OUTCOME MEASURE: Diagnosis of IM and allele frequency. RESULTS: Allelic analysis showed that HLA-DRB1*01:01 was significantly associated with the development of IM (odds ratio, 3.2; P < .001). Patients with IM and HLA-DRB1*01:01 had a lower Epstein-Barr virus viral load compared with those without the allele (median, 783 vs 7366 copies/10(6) peripheral blood mononuclear cells; P = .03). CONCLUSION: HLA-DRB1*01:01 is protective against developing MS; thus, a common genetic basis between IM and MS is not supported.

A study of risk factors for acquisition of Epstein-Barr virus and its subtypes.

BACKGROUND: Risk factors for primary infection with Epstein-Barr virus (EBV) and its subtypes have not been fully investigated. METHODS: Questionnaires and serum samples from a total of 2006 students who entered Edinburgh University in 1999-2000 were analyzed to examine risk factors for EBV seropositivity, both overall and by EBV type. RESULTS: The prevalence of EBV seropositivity was significantly increased among females, older students, those who had lived in tropical countries, those with siblings, and those who were sexually active, particularly if they had had numerous sex partners. Risk was lower (1) among students who always used a condom than among those who had sexual intercourse without one and (2) among female oral-contraceptive users than among sexually active nonusers. Risk factors for type 1 EBV infection were similar to those for EBV overall. No associations were found between nonsexual risk factors and type 2 infection. Sexual activity increased the risk of type 2 infection, but the increase in risk with number of sex partners was less consistent than for type 1 infections. Dual infection was uncommon, but the patterns of risk appeared to be similar to those of type 1 infection. CONCLUSION: This study provides further evidence that EBV may be sexually transmitted and some suggestion that the risk factors for type 1 and type 2 infection differ.

Allogeneic cytotoxic T-cell therapy for EBV-positive posttransplantation lymphoproliferative disease: results of a phase 2 multicenter clinical trial.

We present the results of a multicenter clinical trial using Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTLs) generated from EBV-seropositive blood donors to treat patients with EBV-positive posttransplantation lymphoproliferative disease (PTLD) on the basis of the best HLA match and specific in vitro cytotoxicity. Thirty-three PTLD patients who had failed on conventional therapy were enrolled. No adverse effects of CTL infusions were observed and the response rate (complete or partial) in 33 patients was 64% at 5 weeks and 52% at 6 months. Fourteen patients achieved a complete remission, 3 showed a partial response, and 16 had no response at 6 months (5 died before completing treatment). At 5 weeks, there was a significant trend toward better responses with higher numbers of CD4(+) cells in infused CTL lines (P = .001) that were maintained at 6 months (P = .001). Patients receiving CTLs with closer HLA matching responded better at 6 months (P = .048). Female patients responded better than male patients, but the differences were not statistically significant. Our results show that allogeneic CTLs are a safe and rapid therapy for PTLD, bypassing the need to grow CTLs for individual patients. The response rate in this poor prognosis patient group is encouraging.

Epstein-Barr virus: the impact of scientific advances on clinical practice.

Epstein-Barr virus (EBV) is a tumorigenic herpes virus that infects and persists in B lymphocytes in the majority of humans, generally without causing disease. However, in a few individuals the virus is associated with significant pathology, particularly benign and malignant lymphoproliferations. Recently acquired knowledge on the mechanisms of EBV persistence, immune control of primary and persistent infection, and disease pathogenesis is now being translated into the clinic with novel methods of diagnosis, prevention and treatment contributing to improved patient care. This review concentrates on these recent advances in the field of hematology/oncology.

Evidence of shared Epstein-Barr viral isolates between sexual partners, and low level EBV in genital secretions.

Epstein-Barr virus is present in the saliva of most persistently infected individuals and is generally thought to be spread by close oral contact. However, there are now several reports of EBV in genital secretions, suggesting the possibility of sexual transmission between adults. The present study was undertaken to investigate the risk of sexual transmission of EBV. PCR analysis was used to examined the degree to which a group (n = 11) of patients with infectious mononucleosis (IM) shared the same viral isolates as their sexual partners, and compare this to the extent of isolate sharing among a different group (n = 18) of IM patients and their non-sexual contacts. There was significantly more sharing of EBV isolates among the IM/sexual-contact pairs than among the IM/non-sexual-contact pairs (P = 0.0012). Female cervical (n = 84), male urethral (n = 55), and semen (n = 30) samples from asymptomatic, unselected volunteers were analyzed for the presence of EBV DNA, revealing 7%, 5%, and 3% to be EBV positive, respectively. Fractionation of cervical and urethral samples into cellular and supernatant fluid components showed EBV to be mainly cell-associated. Quantitation of EBV in these samples gave levels of below 10 EBV genomes per microg of DNA. Overall the findings support the possibility that EBV could on occasions be transmitted sexually, however, the low levels detected in genital secretions compared to saliva suggest that this is not a major transmission route. The finding of small quantities of cell-associated virus suggests a latent infection; thus EBV is probably in the B lymphocyte rather than in the epithelial cell component of the secretions.

A mouse monoclonal antibody against Epstein-Barr virus envelope glycoprotein 350 prevents infection both in vitro and in vivo.

A mouse monoclonal antibody (MAb) against Epstein-Barr virus (EBV) envelope glycoprotein 350, 72A1, inhibited EBV infection of B lymphocytes in vitro. When severe combined immunodeficient mice were injected with EBV-seronegative donors' peripheral-blood mononuclear cells and challenged with EBV, 72A1 MAb prevented development of EBV-positive tumors: none of the test mice (0/12) developed EBV-positive tumors. In contrast, 67% (8/12) of control mice developed EBV-positive tumors (P=.001). Purified 72A1 MAb was infused into 1 healthy adult and 4 EBV-seronegative children after liver transplant. No adverse reactions were seen in the adult or in 3 of the transplant recipients. The remaining patient developed a hypersensitivity reaction, thus underlining the need to humanize the MAb.