ABSTRACT Here we present evidence for previously unappreciated B-cell immune dysregulation during acute Epstein-Barr virus (EBV)-associated infectious mononucleosis (IM). Longitudinal analyses revealed that patients with acute IM have undetectable EBV-specific neutralizing antibodies and gp350-specific B-cell responses, which were associated with a significant reduction in memory B cells and no evidence of circulating antibody-secreting cells. These observations correlate with dysregulation of tumor necrosis factor family members BAFF and APRIL and increased expression of FAS on circulating B cells.

Acute infectious mononucleosis (IM) is associated with altered expression of inflammatory cytokines and disturbed T-cell homeostasis, however, the precise mechanism of this immune dysregulation remains unresolved. In the current study we demonstrated a significant loss of circulating myeloid and plasmacytoid dendritic cells (DCs) during acute IM, a loss correlated with the severity of clinical symptoms. In vitro exposure of blood DCs to acute IM plasma resulted in loss of plasmacytoid DCs, and further studies with individual cytokines showed that exposure to interleukin 10 could replicate this effect. Our data provide important mechanistic insight into dysregulated immune homeostasis during acute IM.

Epstein-Barr virus (EBV) is associated with nasopharyngeal carcinoma (NPC). We assess the safety and tolerability of adoptive transfer of autologous cytotoxic T lymphocytes (CTLs) specific for the EBV latent membrane protein (LMP) in a patient with recurrent NPC. After infusion, the majority of pulmonary lesions were no longer evident, although the primary tumor did not regress.

Abstract Nasopharyngeal carcinoma (NPC) is endemic in China and Southeast Asia where it is tightly associated with infections by Epstein-Barr virus (EBV). The role of tumor-associated viral antigens in NPC renders it an appealing candidate for cellular immunotherapy. In earlier preclinical studies, a novel adenoviral vector–based vaccine termed AdE1-LMPpoly has been generated that encodes EBV nuclear antigen-1 (EBNA1) fused to multiple CD8+ T-cell epitopes from the EBV latent membrane proteins, LMP1 and LMP2. Here, we report the findings of a formal clinical assessment of AdE1-LMPpoly as an immunotherapeutic tool for EBV-associated recurrent and metastatic NPC. From a total of 24 patients with NPC, EBV-specific T cells were successfully expanded from 16 patients with NPC (72.7%), whereas six patients with NPC (27.3%) showed minimal or no expansion of virus-specific T cells. Transient increase in the frequencies of LMP1&2- and EBNA1-specific T-cell responses was observed after adoptive transfer to be associated with grade I flu-like symptoms and malaise. The time to progression in these patients ranged from 38 to 420 days with a mean time to progression of 136 days. Compared with patients who did not receive T cells, the median overall survival increased from 220 to 523 days. Taken together, our findings show that adoptive immunotherapy with AdE1-LMPpoly vaccine is safe and well tolerated and may offer clinical benefit to patients with NPC. Cancer Res; 72(5); 1116–25. ©2012 AACR.

Human herpesviruses have coevolved with humans over millions of years, and adaptation of latent infection within the cells of the immune system is a unique characteristic of many of these viruses. Following primary infection, these herpesviruses establish an asymptomatic-persistent infection in healthy individuals that is strictly controlled by virus-specific CD8 + and CD4 + T cells. Here, we provide a brief overview of how the human immune system interacts with these latent viruses and regulates the lifelong host–virus relationship in healthy virus carriers. Extensive studies on T-cell-mediated immune regulation over the last decade has allowed researchers to successfully translate these findings into the clinical setting to treat various herpesvirus-associated diseases in transplant patients and individuals with virus-associated malignancies. It is highly likely that these newly emerging T-cell-based therapeutic and diagnostic technologies will revolutionize the clinical management of patients with herpesvirus-associated diseases.

Human herpesviruses have coevolved with humans over millions of years, and adaptation of latent infection within the cells of the immune system is a unique characteristic of many of these viruses. Following primary infection, these herpesviruses establish an asymptomatic-persistent infection in healthy individuals that is strictly controlled by virus-specific CD8(+) and CD4(+) T cells. Here, we provide a brief overview of how the human immune system interacts with these latent viruses and regulates the lifelong host-virus relationship in healthy virus carriers. Extensive studies on T-cell-mediated immune regulation over the last decade has allowed researchers to successfully translate these findings into the clinical setting to treat various herpesvirus-associated diseases in transplant patients and individuals with virus-associated malignancies. It is highly likely that these newly emerging T-cell-based therapeutic and diagnostic technologies will revolutionize the clinical management of patients with herpesvirus-associated diseases. Immunology and Cell Biology (2011) 89, 352-358; doi:10.1038/icb.2010.136; published online 8 February 2011

Human herpesviruses have coevolved with humans over millions of years, and adaptation of latent infection within the cells of the immune system is a unique characteristic of many of these viruses. Following primary infection, these herpesviruses establish an asymptomatic‐persistent infection in healthy individuals that is strictly controlled by virus‐specific CD8 + and CD4 + T cells. Here, we provide a brief overview of how the human immune system interacts with these latent viruses and regulates the lifelong host–virus relationship in healthy virus carriers. Extensive studies on T‐cell‐mediated immune regulation over the last decade has allowed researchers to successfully translate these findings into the clinical setting to treat various herpesvirus‐associated diseases in transplant patients and individuals with virus‐associated malignancies. It is highly likely that these newly emerging T‐cell‐based therapeutic and diagnostic technologies will revolutionize the clinical management of patients with herpesvirus‐associated diseases.

αβ T cell receptors (TCRs) are genetically restricted to corecognize peptide antigens bound to self-major histocompatibility complex (pMHC) molecules; however, the basis for this MHC specificity remains unclear. Despite the current dogma, evaluation of the TCR–pMHC-I structural database shows that the nongermline-encoded complementarity-determining region (CDR)-3 loops often contact the MHC-I, and the germline-encoded CDR1 and -2 loops frequently participate in peptide-mediated interactions. Nevertheless, different TCRs adopt a roughly conserved docking mode over the pMHC-I, in which three MHC-I residues (65, 69, and 155) are invariably contacted by the TCR in one way or another. Nonetheless, the impact of mutations at these three positions, either individually or together, was not uniformly detrimental to TCR recognition of pHLA-B*0801 or pHLA-B*3508. Moreover, when TCR–pMHC-I recognition was impaired, this could be partially restored by expression of the CD8 coreceptor. The structure of a TCR–pMHC-I complex in which these three (65, 69, and 155) MHC-I positions were all mutated resulted in shifting of the TCR footprint relative to the cognate complex and formation of compensatory interactions. Collectively, our findings reveal the inherent adaptability of the TCR in maintaining peptide recognition while accommodating changes to the central docking site on the pMHC-I.

Primary infection or recrudescence of latent virus infection in transplant recipients can be manifested either as asymptomatic or symptomatic disease. Here we show that symptomatic human cytomegalovirus (HCMV) or Epstein–Barr virus (EBV) infection or recrudescence following solid organ transplantation (SOT) was coincident with a dramatic skewing of T-cell receptor beta variable (TRBV) repertoire, with expansions of monoclonal/oligoclonal clonotypes. As the clinical symptoms resolved, the peripheral blood repertoire reverted to a more diverse distribution. In contrast, SOT recipients with asymptomatic or no viral infection or recrudescence showed minimal or no skewing of the T-cell receptor repertoire to maintain peripheral blood repertoire diversity. More importantly, we show that large monoclonal/oligoclonal repertoire expansions are associated with the loss of HCMV-specific T-cell function observed in SOT patients undergoing symptomatic viral infection or recrudescence, whereas SOT recipients who maintain peripheral blood TRBV repertoire diversity and functional antigen-specific T-cell responses can resist clinical symptomatic disease in spite of high levels of viral load.

Immunosuppression resulting in impaired Epstein-Barr virus (EBV)-specific T-cell immunity is involved in the pathogenesis of EBV-positive post-transplantation lymphoproliferative disorder (EBV(+) PTLD). Restoration of EBV-specific T-cell immunity by adoptive immunotherapy can induce remission. EBV-nuclear antigen-1 (EBNA1) is unique in being expressed in all cases of EBV(+) PTLD. Recent data demonstrate that EBNA1 is not immunologically silent and can be exploited as a T-cell target. There are no data on EBNA1-specific T cells in PTLD. EBNA1-specific T cells capable of proliferation, interferon-γ release, and CD107a/b degranulation were assayed in 14 EBV(+) PTLD diagnostic blood samples and 19 healthy controls. EBNA1-specific CD4(+) T cells predominated and were expanded in 10 of 14 patients and 19 of 19 controls. Although human leukocyte antigen class I alleles influenced the magnitude of the response, EBNA1-specific CD8(+) effector T cells were successfully generated in 9 of 14 EBV(+) PTLD patients and 16 of 19 controls. The majority of PTLD patients had a polymorphism in an EBNA1 epitope, and T-cell recognition was greatly enhanced when EBNA1 peptides derived from the polymorphic epitope were used. These results indicate that EBNA1-specific T cells should be included in adoptive immunotherapy for PTLD. Furthermore, expansion protocols should use antigenic sequences from relevant EBV strains.

ABSTRACT Epstein-Barr virus (EBV) is associated with several malignant diseases including nasopharyngeal carcinoma (NPC), a common neoplasm throughout southeast Asia. Radiotherapy and chemotherapy can achieve remission, but a reemergence of disease is not uncommon. Therefore, there is a need for specific therapies that target the tumor through the recognition of EBV antigens. In NPC, latent membrane protein 1 (LMP1) and LMP2 offer the best opportunity for specific targeting since they are typically expressed and T-cell determinants in each of these proteins have been defined. We have attempted to maximize the opportunity of incorporating every possible CD4 and CD8 determinant in a single formulation. We have achieved this by generating a scrambled protein incorporating random overlapping peptide sets from EBNA1, LMP1, and LMP2, which was then inserted into a replication-deficient strain of adenovirus (adenovirus scrambled antigen vaccine [Ad-SAVINE]). This report describes the construction of this Ad-SAVINE construct, its utility in generating LMP1 and LMP2 responses in healthy individuals as well as NPC patients, and its capacity to define new epitopes. This formulation could have a role in NPC immunotherapy for all ethnic groups since it has the potential to activate all possible CD4 and CD8 responses within EBNA1 and LMPs.

Primary infection or recrudescence of latent virus infection in transplant recipients can be manifested either as asymptomatic or symptomatic disease. Here we show that symptomatic human cytomegalovirus (HCMV) or Epstein–Barr virus (EBV) infection or recrudescence following solid organ transplantation (SOT) was coincident with a dramatic skewing of T-cell receptor beta variable (TRBV) repertoire, with expansions of monoclonal/oligoclonal clonotypes. As the clinical symptoms resolved, the peripheral blood repertoire reverted to a more diverse distribution. In contrast, SOT recipients with asymptomatic or no viral infection or recrudescence showed minimal or no skewing of the T-cell receptor repertoire to maintain peripheral blood repertoire diversity. More importantly, we show that large monoclonal/oligoclonal repertoire expansions are associated with the loss of HCMV-specific T-cell function observed in SOT patients undergoing symptomatic viral infection or recrudescence, whereas SOT recipients who maintain peripheral blood TRBV repertoire diversity and functional antigen-specific T-cell responses can resist clinical symptomatic disease in spite of high levels of viral load.

Nasopharyngeal carcinoma (NPC) is Epstein–Barr virus (EBV) positive in all undifferentiated cases, expressing the latency II phenotype of latent membrane proteins (LMPs) 1 and 2, in addition to EBV nuclear antigen (EBNA) 1. Several studies have attempted to treat NPC with EBV‐specific cytotoxic T lymphocyte (CTL) with a partial response. To improve this therapy, there is a need to expand CTL targeted to the latency II antigens of EBV, rather than the immunodominant EBV nuclear antigens 3–6 peptides typically expanded by lymphoblastoid cells. In order to maximize the expansion of LMP‐specific CTL in vitro for use in adoptive immunotherapy of nasopharyngeal carcinoma patients, we used lymphoblastoid cell lines coated with synthetic peptides corresponding to CTL determinants from the LMP proteins. We investigated several issues pertaining to the expansion of an immunologically weak CTL response, including peptide and interleukin‐2 concentration, and screening assays for selecting the optimal peptide for use in expansion of LMP‐specific CTL. Although screening of ex vivo peripheral blood mononuclear cells did not prove to be useful in the selection of an LMP peptide for use in CTL cultures, the peptide and interleukin‐2 concentrations were critical for the maximum expansion of CTL. Therefore, it is imperative that stimulation protocols are optimized for the expansion of LMP‐specific CTL.

Nasopharyngeal carcinoma (NPC) is Epstein–Barr virus (EBV) positive in all undifferentiated cases, expressing the latency II phenotype of latent membrane proteins (LMPs) 1 and 2, in addition to EBV nuclear antigen (EBNA) 1. Several studies have attempted to treat NPC with EBV-specific cytotoxic T lymphocyte (CTL) with a partial response. To improve this therapy, there is a need to expand CTL targeted to the latency II antigens of EBV, rather than the immunodominant EBV nuclear antigens 3–6 peptides typically expanded by lymphoblastoid cells. In order to maximize the expansion of LMP-specific CTL in vitro for use in adoptive immunotherapy of nasopharyngeal carcinoma patients, we used lymphoblastoid cell lines coated with synthetic peptides corresponding to CTL determinants from the LMP proteins. We investigated several issues pertaining to the expansion of an immunologically weak CTL response, including peptide and interleukin-2 concentration, and screening assays for selecting the optimal peptide for use in expansion of LMP-specific CTL. Although screening of ex vivo peripheral blood mononuclear cells did not prove to be useful in the selection of an LMP peptide for use in CTL cultures, the peptide and interleukin-2 concentrations were critical for the maximum expansion of CTL. Therefore, it is imperative that stimulation protocols are optimized for the expansion of LMP-specific CTL.

Epstein-Barr virus (EBV) nuclear antigen (EBNA) 1 is perhaps the most widely studied EBV protein, because of its critical role in maintaining the EBV episome and its expression in all EBV-associated malignancies. Much of this research has focused exclusively on the EBV wild-type (wt) strain (B95-8). Sequence analysis of the gene encoding for EBNA1 in EBV isolates from 43 Caucasians has now revealed considerable EBNA1 sequence divergence from the EBV wt strain in the majority of isolates from this population group. Importantly, T cell recognition of an endogenously processed HLA-B8 - binding EBNA1 epitope was greatly influenced by this sequence polymorphism.

Head and neck cancer is a broad term used to describe malignancies that arise in the nasal and oral cavities, pharynx and larynx, as well as the paranasal sinuses. Head and neck squamous cell carcinoma (HNSCC) affects the squamous epithelium of the oral cavity, tongue and oropharynx, excluding the nasopharynx. Recent advances in molecular technology, including gene expression and proteomic profiling appear to offer the potential for the development of specific biomarkers including diagnostic tools which may act as an aid to guide therapy for this malignancy. The other human head and neck cancer included in this review, nasopharyngeal carcinoma (NPC) is a malignancy derived from the undifferentiated epithelium of the nasopharyngeal cavity, and is considered here as a separate entity because its strong association with Epstein-Barr virus (EBV) presents the opportunity for the development of virus related and unrelated biomarkers. In particular, IgA antibodies to EBV and high levels of EBV DNA in serum samples of NPC patients have been recorded. This review aims to summarize some current and also potential new biomarkers that could be used for screening, diagnosis, monitoring and prognostic prediction for cancers of the head and neck, including NPC and HNSCC.

The use of cellular therapy in Australia has been limited by the number of facilities deemed acceptable for the preparation of T cells for immunotherapy. In spite of this, a number of trials using cellular immunotherapy have been conducted and a number are underway or are in the planning stage, and selected examples of these are summarized in this review.

The biology and immunology of Epstein–Barr virus (EBV) has continued to fascinate researchers because the lessons learnt provide a platform for understanding the interplay between the biology of this ubiquitous infection, the immune system seeking to restrict its spread and the emergence of a variety of malignancies. As with other gamma herpes viruses, EBV encodes a large set of lytic cycle genes together with a number of latent genes which are associated with expansion of the latent EBV pool in B-lymphocytes. Current evidence suggests that the virus gains entry into the body by infection of B-lymphocytes in the oral cavity via an interaction between the major viral glycoprotein gp340 and the complement receptor CR2 which is expressed on B-cells, although a role for CR2-expressing or non-expressing epithelial and/or T-cells cannot be totally discounted. In either case, evidence suggests that the earliest detectable event following primary infection is the expression of lytic cycle proteins resulting in the release of infectious virus into the oral cavity followed by a generalized seeding of latently infected B-lymphocytes throughout the body. This primary infection results in symptoms of acute infectious mononucleosis (IM) in about 50% of adolescents and is coincident with a marked lymphocytosis (dominated by EBV-specific cytotoxic T-cells) and the appearance of an IgM response to a variety of EBV proteins, most notably the viral capsid antigen, VCA.