Search

The humoral immune response to Epstein–Barr virus (EBV) in classical Hodgkin lymphoma (cHL) stratified by EBV tumor status is unclear. We examined IgG and IgA antibody responses against 202 protein sequences representing 86 EBV proteins using a microarray and sera from 139 EBV-positive cHL cases, 70 EBV-negative cHL cases and 141 population-based controls frequency matched to EBV-positive cHL cases on sex and age by area (UK, Denmark and Sweden). We leveraged existing data on the proportion of circulating B-cells infected by EBV and levels of serum CCL17, a chemokine secreted by cHL tumor cells, from a subset of the cHL cases in the UK. Total IgG but not IgA response level was significantly different between EBV-positive cHL cases and controls. The distinct serological response included significant elevations in 16 IgG antibodies and 2 IgA antibodies, with odds ratioshighest vs. lowest tertile > 3 observed for the following EBV proteins: LMP1 (oncogene), BcLF1 (VCAp160, two variants) and BBLF1 (two variants). Our cHL IgG signature correlated with the proportion of circulating EBV-infected B-cells, but not serum CCL17 levels. We observed no differences in the anti-EBV antibody profile between EBV-negative cHL cases and controls. BdRF1(VCAp40)-IgG and BZLF1(Zta)-IgG were identified as the serological markers best able to distinguish EBV-positive from EBV-negative cHL tumors. Our results support the hypothesis that differences in the EBV antibody profile are specific to patients with EBV-positive cHL and are not universally observed as part of a systematically dysregulated immune response present in all cHL cases.

Familial aggregation, coupled with ethnic variation in incidence, suggests that inherited susceptibility plays a role in the development of lymphoma, and the search for genetic risk factors has highlighted the contribution of the human leukocyte antigen (HLA) complex. In a landmark study published almost 50 years ago, Hodgkin lymphoma (HL) was the first disease to be associated with HLA variation. It is now clear that Epstein-Barr virus (EBV)-positive and -negative HL are strongly associated with specific HLA polymorphisms but these differ by EBV status of the tumours. HLA class I alleles are consistently associated with EBV-positive HL while a polymorphism in HLA class II is the strongest predictor of risk of EBV-negative HL. Recent investigations, particularly genome-wide association studies (GWAS), have also revealed associations between HLA and common types of non-Hodgkin lymphoma (NHL). Follicular lymphoma is strongly associated with two distinct haplotypes in HLA class II whereas diffuse large B-cell lymphoma is most strongly associated with HLA-B*08. Although chronic lymphocytic leukaemia is associated with variation in HLA class II, the strongest signals in GWAS are from non-HLA polymorphisms, suggesting that inherited susceptibility is explained by co-inheritance of multiple low risk variants. Associations between B-cell derived lymphoma and HLA variation suggest that antigen presentation, or lack of, plays an important role in disease pathogenesis but the precise mechanisms have yet to be elucidated.

SUMMARYLittle is known about the main routes of human cytomegalovirus (HCMV) transmission in young adult populations. This study investigated risk factors for HCMV transmission in young adults attending university over a 3-year period. Blood samples were tested for HCMV specific viral capsid antigen IgG by enzyme immunoassay. Being born in a developing country and having lived in Africa were associated with HCMV seropositivity at study onset. No risk factors were associated with HCMV seroconversion over the 3-year follow-up. In contrast to previous reports, sexual activity was not associated with HCMV seroprevalence or seroconversion.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that persists in the body for life after primary infection. The primary site of EBV persistence is the memory B lymphocyte, but whether the virus initially infects naïve or memory B cells is still disputed. We have analyzed EBV infection in nine cases of X-linked hyper-immunoglobulin M (hyper-IgM) syndrome who, due to a mutation in CD40 ligand gene, do not have a classical, class-switched memory B-cell population (IgD − CD27 + ). We found evidence of EBV infection in 67% of cases, which is similar to the infection rate found in the general United Kingdom population (60 to 70% for the relevant age range). We detected EBV DNA in peripheral blood B cells and showed in one case that the infection was restricted to the small population of nonclassical, germinal center-independent memory B cells (IgD + CD27 + ). Detection of EBV small RNAs, latent membrane protein 2, and EBV nuclear antigen 3C expression in peripheral blood suggests full latent viral gene expression in this population. Analysis of EBV DNA in serial samples showed variability over time, suggesting cycles of infection and loss. Our results demonstrate that short-term EBV persistence can occur in the absence of a germinal center reaction and a classical memory B-cell population.

Cardiac myxoma and diffuse large B-cell lymphoma are uncommon tumors, yet four composite tumors have been reported since 2009. We are reporting on the fifth case providing detailed immunohistochemical and FISH analyses. The lymphoma was present as superficially located nests of large cells with patchy necrosis in the background of a typical atrial myxoma. It displayed features of DLBCL with non-germinal center phenotype, expressed EBER, LMP1, EBNA2 and shared the following features with the previously reported cases: B-cell lineage, high-grade cytology, high proliferation rate, EBV infection in latency type 3 with one tested case and an excellent outcome. The lymphomas arising within myxoma may follow a pathogenic pathway driven by EBV, whose transformation potential is unleashed in the cytokine-rich milieu of a myxoma, presumably accentuating age-related decline of adaptive immunity known as immune senescence. DLBCL arising within atrial myxoma grouped together with EBV+ DLBCL associated with valve prosthesis and with an atrial thrombus differs in the immunocompetent patients from primary cardiac DLBCL, not otherwise specified, in clinical presentation, pathological features and a course of the disease. Distinction between these groups may have important therapeutic consequences.

Regulatory T cells (Treg) provide a balance to immune T cell activation thereby protecting the body from pathogen-induced immunopathology. Several persistent viruses induce Treg that subvert protective immune mechanisms and promote viral persistence. Epstein–Barr virus (EBV) generally infects children subclinically and persists thereafter, but primary infection in early adulthood may cause immunopathological damage manifest as infectious mononucleosis. In this study the role of Treg was investigated in acute infectious mononucleosis and healthy EBV seropositive donors. The proportion of CD4+CD25high T cells in blood from infectious mononucleosis patients was significantly lower than in seropositive donors (P = 0.05). Using the FOXP3 marker for Treg the same frequency and extra-follicular distribution of Treg was noted in infectious mononucleosis and control tonsils. Regulatory cytokines, interleukin (IL)-10 and transforming growth factor (TGF)-β, were significantly raised in infectious mononucleosis compared to seropositive donor plasma (P = 0.0001, P = 0.0004 respectively) although levels of IL-10 peaked earlier in infectious mononucleosis than TGF-β. Previous studies identified EBV latent membrane protein (LMP)-1-induced Treg activity [Marshall et al. (2003): J Immunol 170:6183–6189; Marshall et al. (2007): Brit J Haematol 139:81–89], and in this study a significant reduction in interferon-γ production was found from infectious mononucleosis but not seropositive donor lymphocytes after stimulation with a recall antigen when LMP-1 peptide PRG was added (P = 0.03). It is possible that Treg are important in controlling primary EBV infection to a subclinical level in most cases and that infectious mononucleosis represents a failure of this protective mechanism. J. Med. Virol. 81:870–877, 2009. © 2009 Wiley-Liss, Inc.

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.

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.

Lymphoma is the most common haematopoietic malignancy in dogs but little is known about the aetiology of this heterogeneous group of cancers. In humans, the Epstein-Barr virus (EBV) is associated with several lymphoma subtypes. Recently, it has been suggested that EBV or an EBV-like virus is circulating in dogs. We therefore investigated whether EBV, or a novel herpesvirus, is associated with canine lymphoma using both serological and molecular techniques. In an assay designed to detect antibodies to EBV viral capsid antigens, 41% of dogs were positive. Dogs with cancers, including lymphoma, were more frequently positive than controls, but no particular association with B-cell lymphoma was noted. EBV-specific RNA and DNA sequences were not detected in lymphoma tissue by in situ hybridisation or PCR and herpesvirus genomes were not detected using multiple degenerate PCR assays with the ability to detect novel herpesviruses. We therefore found no evidence that herpesviruses are directly involved in common types of canine lymphoma although cannot exclude the presence of an EBV-like virus in the canine population.

Early in 2002, three nineteenth-century Scottish flute manuscripts came to light in the Whittaker Library at the Royal Scottish Academy of Music and Drama (RSAMD). The manuscripts are inscribed with the name of James Simpson of Dundee. The two slimmer volumes are dated 1828 and 1830. The third undated manuscript is a more handsomely bound volume and, judging by the content and handwriting, was likely to have been started at around the same time. Each manuscript consists almost entirely of flute duets and trios, and untexted psalm tunes for three and four voices. The history of the manuscripts is unknown, but it can be deduced that they were acquired by the RSAMD sometime after 1958. The manuscripts offer a colourful ‘snapshot’ of music-making in Dundee in the nineteenth century, with their cross-section of Scottish tunes and more widely-used drawing-room music, not to mention their church connections.

The symptoms of infectious mononucleosis (IM) are thought to be caused by T cell activation and cytokine production. Surface lymphocyte activation marker (SLAM)-associated protein (SAP) regulates lymphocyte activation via signals from cell-surface CD244 (2B4) and SLAM (CD150). We followed T cell activation via this SAP/SLAM/CD244 pathway in IM and analyzed whether the results were associated with clinical severity. At diagnosis, SAP, SLAM, and CD244 were significantly up-regulated on CD4 and CD8 T cells; expression decreased during IM, but CD244 and SLAM levels remained higher on CD8 cells 40 days later. There were significantly more lymphocytes expressing CD8 and CD244/CD8 in patients with severe sore throat. The expression of CD8 alone and CD244 on CD8 cells correlated with increased virus load. We suggest that T cells expressing CD244 and SLAM are responsible for the clinical features of IM but that the control of activation is maintained by parallel increased expression of SAP.

Background & Aims: Studies have indicated that stem cells have unexpected plasticity and can differentiate down a multitude of nonhematopoietic cell lineages in rodents. Our aim was to identify whether human cord blood cells, which are a rich source of stem cells, would be able to differentiate into hepatocytes when infused into nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. We also wanted to test whether such differentiated cells were the result of cellular fusion or true stem cell transdifferentiation. Methods: Unsorted mononuclear cell preparations of human cord blood were infused into sublethally irradiated NOD-SCID mice. After death, immunohistologic analysis of murine livers was performed using human specific hepatocyte, biliary, and endothelial markers. Fluorescent in situ hybridization (FISH) for mouse and human DNA was also performed. Results: We show that human cord blood cells have the ability to engraft into NOD-SCID liver and become mature hepatocytes. We were unable to identify any biliary or endothelial differentiation. Furthermore, we do not detect any evidence of cell fusion in any of the human cells found in the mouse liver, suggesting that human cord blood cells are capable of true transdifferentiation into hepatocytes in vivo. Conclusions: We conclude that hepatocytes can derive from human cord blood cells when infused into NOD-SCID mice in the absence of fusion. The demonstration that human stem cell differentiation can occur in this murine model permits comprehensive study of human stem cell plasticity in vivo.

To determine the role of sexual contact in transmission of Epstein-Barr virus (EBV) and occurrence of infectious mononucleosis (IM), a cross-sectional study was undertaken of EBV serologic testing and histories of IM and sexual behavior among 1006 new students at Edinburgh University. Prevalence of EBV seropositivity was significantly greater among women (79.2%) than among men (67.4%; P

HLA genotyping and genome wide association studies provide strong evidence for associations between Human Leukocyte Antigen (HLA) alleles and classical Hodgkin lymphoma (cHL). Analysis of these associations is complicated by the extensive linkage disequilibrium within the major histocompatibility region and recent data suggesting that associations with EBV‐positive and EBV‐negative cHL are largely distinct. To distinguish independent and therefore potentially causal associations from associations confounded by linkage disequilibrium, we applied a variable selection regression modeling procedure to directly typed HLA class I and II genes and selected SNPs from EBV‐stratified patient subgroups. In final models, HLA‐A*01:01 and B*37:01 were associated with an increased risk of EBV‐positive cHL whereas DRB1*15:01 and DPB1*01:01 were associated with decreased risk. Effects were independent of a prior history of infectious mononucleosis. For EBV‐negative cHL the class II SNP rs6903608 remained the strongest predictor of disease risk after adjusting for the effects of common HLA alleles. Associations with “all cHL” and differences by case EBV status reflected the subgroup analysis. In conclusion, this study extends previous findings by identifying novel HLA associations with EBV‐stratified subgroups of cHL, highlighting those alleles likely to be biologically relevant and strengthening evidence implicating genetic variation associated with the SNP rs6903608., What's new? Strong evidence exists for associations between HLA alleles and classical Hodgkin lymphoma (cHL). Analysis is however complicated by the linkage disequilibrium within the MHC region and data suggesting that associations with Epstein‐Barr virus (EBV)‐positive and negative cHL are distinct. In the largest study to date to investigate associations between EBV‐stratified cHL subgroups and directly typed HLA alleles, the authors extend associations with EBV‐positive cHL to novel HLA class II alleles, which are associated with decreased disease risk. For EBV‐negative disease, the class II SNP rs6903608 remains the strongest predictor of risk after adjusting for the effects of common HLA alleles.

Summary Background Vaccination of health-care workers has been claimed to prevent nosocomial influenza infection of elderly patients in long-term care. Data are, however, limited on this strategy. We aimed to find out whether vaccination of healthcare workers lowers mortality and the frequency of virologically proven influenza in such patients. Methods In a parallel-group study, health-care workers in 20 long-term elderly-care hospitals (range 44–105 patients) were randomly offered or not offered influenza vaccine (cluster randomisation, stratified for policy for vaccination of patients and hospital size). All deaths among patients were recorded over 6 months in the winter of 1996–97. We selected a random sample of 50% of patients for virological surveillance for influenza, with combined nasal and throat swabs taken every 2 weeks during the epidemic period. Swabs were tested by tissue culture and PCR for influenza viruses A and B. Findings Influenza vaccine uptake in health-care workers was 50·9% in hospitals in which they were routinely offered vaccine, compared with 4·9% in those in which they were not. The uncorrected rate of mortality in patients was 102 (13·6%) of 749 in vaccine hospitals compared with 154 (22·4%) of 688 in no-vaccine hospitals (odds ratio 0·58 [95% CI 0·40–0·84], p=0·014). The two groups did not differ for proportions of patients positive for influenza infection (5·4% and 6·7%, respectively); at necropsy, PCR was positive in none of 17 patients from vaccine hospitals and six (20%) of 30 from novaccine hospitals (p=0·055). Interpretation Vaccination of health-care workers was associated with a substantial decrease in mortality among patients. However, virological surveillance showed no associated decrease in non-fatal influenza infection in patients.

Objectives: To compare the conventional virus isolation method for diagnosis of influenza infection with reverse-transcription polymerase chain reaction (RT-PCR) in prospectively collected nose and throat swabs from elderly patients during the winter influenza season. The use of a denaturing buffer as an alternative to viral transport medium (VTM) for submission of combined nose and throat swabs to the laboratory for PCR was then investigated in a second study. Methods: Virus was cultured in microtitre plates using two different cell lines and detected using monoclonal antibody staining. A multiplex, matrix gene PCR assay was optimized to increase the sensitivity and specificity of detection of influenza A (H3 and H1) and B nucleic acid. Results: The multiplex assay detected all viruses with equal sensitivity to individual assays. In a large, multicentre field study PCR detected twice as many influenza infections compared with virus isolation. No positive culture was missed. PCR has a rapid turn around time ( Conclusions: PCR significantly increased the sensitivity and clinical utility of influenza A (H3 and H1) and B diagnosis. there were a number of advantages in using denaturing buffer for submission of samples, including high sensitivity, rapidity, ease of use and no requirement for the virus to be viable on arrival at the laboratory. Therefore, PCR is a rapid, sensitive and user-friendly alternative for influenza diagnosis. Virus isolation technology should be limited to referral centres for further epidemiological characterization.

International audience; The soluble form of CD30 (sCD30), a member of tumour 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

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 gamma delta 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.

BACKGROUND: Epstein-Barr virus-positive post-transplant lymphoproliferative disease (PTLD) is a potentially lethal complication of iatrogenic immunosupression after transplantation. Predicting the development of PTLD allowing early and effective intervention is therefore of importance. Polymorphisms within cytokine genes are implicated in susceptibility to, and progression of, disease however the published data are often conflicting. We undertook investigation of polymorphic alleles within cytokine genes in PTLD and non-PTLD transplant cohorts to determine risk factors for disease.METHODS: SSP-PCR was used to analyse single nucleotide polymorphism within tumour necrosis factor (TNF)-alpha, interleukin-1,-6,-10 and lymphotoxin-alpha genes. The TNF-alpha levels were measured by standard enzyme-linked immuno-absorbant assay.RESULTS: We show an association between variant alleles within the TNF-a promoter (-1031C (P = 0.005)); -863A (P = 0.0001) and TNF receptor I promoter regions (-201T (P = 0.02)); -1135C (P = 0.03) with the development of PTLD. We also show an association with TNF-alpha promoter haplotypes with haplotype-3 significantly increased (P = 0.0001) and haplotype-1 decreased (P 0.02) in PTLD patients compared to transplant controls. Furthermore, we show a significant increase (P 0.02) in the level of TNF-a in PTLD patient plasma (range 0-97.97 pg ml(-1)) compared to transplant controls (0-8.147 pg ml(-1)), with the highest levels found in individuals carrying the variant alleles.CONCLUSION: We suggest that genetic variation within TNF-a loci and the level of plasma cytokine could be used as a predictive risk factor for the development of PTLD. British Journal of Cancer (2009) 101, 1019-1027. doi:10.1038/sj.bjc.6605278 www.bjcancer.com (C) 2009 Cancer Research UK

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.

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 β-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 (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.

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.

The role of antigen-specific CD3(+)CD8(+) cytotoxic T cells in the control of primary Epstein-Barr Virus (EBV) infection is well established. However, time is required for the antigen-specific immune response to develop and expand. In contrast, innate immune responses, such as natural killer (NK) cells, are considered vital early in the infection process. We analysed the scale, phenotype and function of the NK cell response during symptomatic primary EBV infection, infectious mononucleosis (IM) and showed that NK cell numbers were significantly elevated both at diagnosis of IM and in the first month following diagnosis. There were also significant changes in cell phenotype and function, an increase in the proportion of CD56(bright) cells at diagnosis, and freshly isolated cells showing an enhanced ability to kill EBV-infected cell lines. Moreover, in our cohort of IM patients higher NK cell counts were associated with significantly lower viral load in peripheral blood. Our results suggest NK cells have an important role in the control of primary EBV infection by eliminating infected B cells and augmenting the antigen-specific T cell response via release of immunomodulatory cytokines. The magnitude of the NK cell response may ultimately determine whether primary EBV infection has a clinical outcome.

Injecting severe combined immunodeficient (SCID) mice with peripheral blood mononuclear cells (PBMCs) from Epstein-Barr virus (EBV)-positive donors regularly produces EBV-positive B-lymphoproliferative disease, which closely resembles posttransplantation lymphoproliferative disease (PTLD) in humans

Abstract 3635 Background: Class I HLA genes are associated with risk of developing EBV-associated classical Hodgkin Lymphoma (EBV-cHL). HLA-A*01:01 is associated with increased risk and HLA-A*02:01 with decreased risk, resulting in an almost 10-fold difference in odds of developing EBV-cHL between HLA-A*02:01 and HLA-A*01:01 homozygotes with a gradation of risk in intermediate genotypes. Class I HLA genes are involved in EBV-specific cytotoxic T cell (CTL) responses, keeping infection under tight control in healthy hosts. HLA associations with EBV-cHL suggest that EBV-specific immune responses at time of initial infection, during chronic latent infection or at time of oncogenesis are critical in the pathogenesis of EBV-cHL. Many immunodominant EBV peptides are known to be presented by HLA-A*02:01. Since HLA-A*01:01 appears to increase risk of disease even in those individuals with HLA-A*02:01, we investigated whether the presence of an HLA-A*01:01 allele modifies the CTL response to HLA-A*02:01-restricted EBV epitopes. Methods: Individuals with relevant HLA-A genotypes were selected from a pool of healthy adults who agreed to take part in this study; A*01:01/A*02:01 heterozygotes (n=15), A*02:01 homozygotes (n=12) and A*02:01/× heterozygotes (n=15), where x is neither A*01:01 or A*02:01. Assessment of A*02:01 restricted EBV response used IFN-γ ELISPOT and all 31 reported A*02:01 restricted EBV peptides. PBMCs were exposed to single EBV peptides and controls in duplicate. The highest negative control plus two standard deviations was taken as the cut-off for a “positive” response. Measures of response were: the number of peptides recognised; maximal response to any single peptide; and summative response to EBV peptides. Analyses examined all peptides, lytic peptides, latent peptides and peptides expressed in type II latency. Assessment of cytotoxicity by degranulation was performed by flow cytometric analysis of CD107 expression after exposure to peptide pools. Supernatants from these stimulations were used to measure TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 and IL-17. Results were compared by Kruskall-Wallis analysis across groups and Mann-Whitney analysis between pairs of groups, using SPSS. Results: Satisfactory ELISPOTs were obtained from 31 donors. No difference in response to A*02:01 restricted EBV peptides was observed between A*01:01/A*02:01 heterozygotes and A*02:01 homozygotes. A*02:01/x heterozygotes recognised more EBV peptides than either A*01:01/A*02:01 heterozygotes (p=0.023) or A*02:01/A*02:01 homozygotes (p=0.049), an effect which was greatest for lytic peptides (p= 0.012). The single maximal response to any EBV peptide was higher in A*02:01/x heterozygotes, reaching significance for lytic peptide responses (p=0.042). The summative response was also higher in A*02:01/x heterozygotes, and for lytic peptides these differences were significant (p=0.022). A*02:01/x heterozygotes also demonstrated greater cytotoxicity assessed by CD107 than either A*01:01/A*02:01 heterozygotes or A*02:01 homozygotes. This effect was seen in response to latent and lytic peptides, with the differences greatest between A*02:01/x heterozygotes and A*02:01/A*01:01 heterozygotes. A*02:01/A*01:01 heterozygotes produced significantly higher levels of IL-10 (mean 1427 vs 155 pg/ml p= 0.03), IL-17 (84 vs 70 pg/ml p=0.02) and IL-5 (35 vs 29 pg/ml p= 0.04) in response to lytic peptides compared to both other HLA-A groups. IFN-g secretion was higher in A*02:01/x heterozygotes (2552 vs 552 pg/ml, p=0.007). Conclusions: The null hypothesis of this study, that all HLA-A*02:01 carriers should have the same response to HLA-A*02:01 restricted EBV peptides was disproved. A*02:01/x heterozygotes had CTL responses of greater breadth and magnitude than either A*01:01/A*02:01 heterozygotes or A*02:01 homozygotes, as measured by three different methods. A*01:01 did not inhibit CTL responses to A*02:01-restricted EBV peptides; however, in response to EBV lytic peptides, A*02:01/*01:01 heterozygotes demonstrated significant increases in cytokines associated with a Th2 response. These data suggest that overall HLA-A phenotype is important in determining CTL response through a single HLA allele. The cytokine profiles observed in the three groups following stimulation with EBV peptides may begin to explain the HLA-associated differences in risk of developing EBV-cHL. Disclosures: No relevant conflicts of interest to declare.

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.