Neil Steven

Large clonal expansions of CD8+ T cells in acute infectious mononucleosis.

Primary infection with Epstein–Barr virus often results in the clinical syndrome of acute infectious mononucleosis (glandular fever). This illness is characterized by a striking lymphocytosis, the nature of which has been controversial. We show that large monoclonal or oligoclonal populations of CD8+ T cells account for a significant proportion of the lymphocytosis and provide molecular evidence that these populations have been driven by antigen. The results suggest that the selective and massive expansion of a few dominant clones of CD8+ T cells is an important feature of the primary response to this virus.

Human CD8+ T Cell Responses to EBV EBNA1: HLA Class I Presentation of the (Gly-Ala)–Containing Protein Requires Exogenous Processing

Epstein-Barr virus (EBV)-induced cytotoxic T lymphocyte (CTL) responses have been detected against many EBV antigens but not the nuclear antigen EBNA1; this has been attributed to the presence of a glycine-alanine repeat (GAr) domain in the protein. Here we describe the isolation of human CD8+ CTL clones recognizing EBNA1-specific peptides in the context of HLA-B35.01 and HLA-A2.03. Using these clones, we show that full-length EBNA1 is not presented when expressed endogenously in target cells, whereas the GAr-deleted form is presented efficiently. However, when supplied as an exogenous antigen, the full-length protein can be presented on HLA class I molecules by a TAP-independent pathway; this may explain how EBNA1-specific CTLs are primed in vivo.

A phase II study of adoptive immunotherapy using dendritic cells pulsed with tumor lysate in patients with hepatocellular carcinoma

This is a phase II clinical trial investigating the safety and efficacy of intravenous vaccination with mature autologous dendritic cells (DCs) pulsed ex vivo with a liver tumor cell line lysate (HepG2) in patients with advanced hepatocellular carcinoma (HCC). HCC is an attractive target for immunotherapy as evidenced by an active recruitment of tumor‐infiltrating lymphocytes that are capable of lysing autologous tumor cells in ex vivo studies. DCs are the most potent antigen‐presenting cells, with the capacity to take up, process, and present tumor antigens to T cells and stimulate an immune response, thus providing a rational platform for vaccine development. Thirty‐five patients with advanced HCC and not suitable for radical or loco‐regional therapies received a maximum of six DC vaccinations each at 3‐week intervals. In total, 134 DC infusions were administered with no significant toxicity and no evidence of autoimmunity. Twenty‐five patients who received at least three vaccine infusions were assessed clinically for response. The radiologically determined disease control rate (combined partial response and stable disease ≥3 months) was 28%. In 17 patients the baseline serum α‐fetoprotein (AFP) was ≥ 1,000 ng/mL; in four of these patients, it fell to <30% of baseline following vaccination. In one patient there was a radiological partial response associated with a fall in AFP to <10% of baseline. Immune responses were assessed using an ELIspot assay of interferon‐γ (IFN‐γ) release. In several cases there was induction of T cell responses to the vaccine and/or AFP following vaccination. Conclusion: Autologous DC vaccination in patients with HCC is safe and well tolerated with evidence of antitumor efficacy assessed radiologically and serologically, with generation of antigen‐specific immune responses in some cases. (HEPATOLOGY 2009;49:124‐132.)

Dual Stimulation of Epstein-Barr Virus (EBV)-Specific CD4+- and CD8+-T-Cell Responses by a Chimeric Antigen Construct: Potential Therapeutic Vaccine for EBV-Positive Nasopharyngeal Carcinoma

ABSTRACT Virus-associated malignancies are potential targets for immunotherapeutic vaccines aiming to stimulate T-cell responses against viral antigens expressed in tumor cells. Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma, a high-incidence tumor in southern China, expresses a limited set of EBV proteins, including the nuclear antigen EBNA1, an abundant source of HLA class II-restricted CD4+ T-cell epitopes, and the latent membrane protein LMP2, a source of subdominant CD8+ T-cell epitopes presented by HLA class I alleles common in the Chinese population. We used appropriately modified gene sequences from a Chinese EBV strain to generate a modified vaccinia virus Ankara recombinant, MVA-EL, expressing the CD4 epitope-rich C-terminal domain of EBNA1 fused to full-length LMP2. The endogenously expressed fusion protein EL is efficiently processed via the HLA class I pathway, and MVA-EL-infected dendritic cells selectively reactivate LMP2-specific CD8+ memory T-cell responses from immune donors in vitro. Surprisingly, endogenously expressed EL also directly accesses the HLA class II presentation pathway and, unlike endogenously expressed EBNA1 itself, efficiently reactivates CD4+ memory T-cell responses in vitro. This unscheduled access to the HLA class II pathway is coincident with EL-mediated redirection of the EBNA1 domain from its native nuclear location to dense cytoplasmic patches. Given its immunogenicity to both CD4+ and CD8+ T cells, MVA-EL has potential as a therapeutic vaccine in the context of nasopharyngeal carcinoma.

T CELL SELECTION DURING THE EVOLUTION OF CD8+ T CELL MEMORY IN VIVO

Memory T cell responses are frequently highly restricted in terms of receptor usage. How and when such clonotypic dominance is established remains poorly understood. Here we have investigated the evolution of the T cell responses to an epitope from Epstein‐Barr virus (EBV), (FLRGRAYGL), by analyzing TCR use of clones specific for this epitope, derived from peripheral blood mononuclear cells taken from individuals early during primary EBV infection and up to 3 years later. We show that, in a given individual, particular T cell clonotypes are selected early during the primary response to this epitope and that the same clonotypes dominate the late memory response. In one individual direct analysis of HLA‐B8‐restricted FLRGRAYGL‐specific T cells, isolated from peripheral blood lymphocytes taken during primary EBV infection using a tetrameric MHC‐peptide complex, confirmed the early selection of the dominant clonotypes.

Phase I Trial of Recombinant Modified Vaccinia Ankara Encoding Epstein–Barr Viral Tumor Antigens in Nasopharyngeal Carcinoma Patients

Epstein-Barr virus (EBV) is associated with several malignancies including nasopharyngeal carcinoma, a high incidence tumor in Chinese populations, in which tumor cells express the two EBV antigens EB nuclear antigen 1 (EBNA1) and latent membrane protein 2 (LMP2). Here, we report the phase I trial of a recombinant vaccinia virus, MVA-EL, which encodes an EBNA1/LMP2 fusion protein designed to boost T-cell immunity to these antigens. The vaccine was delivered to Hong Kong patients with nasopharyngeal carcinoma to determine a safe and immunogenic dose. The patients, all in remission more than 12 weeks after primary therapy, received three intradermal MVA-EL vaccinations at three weekly intervals, using five escalating dose levels between 5 × 10(7) and 5 × 10(8) plaque-forming unit (pfu). Blood samples were taken during prescreening, immediately before vaccination, one week afterward and at intervals up to one year later. Immunogenicity was tested by IFN-γ ELIspot assays using complete EBNA1 and LMP2 15-mer peptide mixes and known epitope peptides relevant to patient MHC type. Eighteen patients were treated, three per dose level one to four and six at the highest dose, without dose-limiting toxicity. T-cell responses to one or both vaccine antigens were increased in 15 of 18 patients and, in many cases, were mapped to known CD4 and CD8 epitopes in EBNA1 and/or LMP2. The range of these responses suggested a direct relationship with vaccine dose, with all six patients at the highest dose level giving strong EBNA1/LMP2 responses. We concluded that MVA-EL is both safe and immunogenic, allowing the highest dose to be forwarded to phase II studies examining clinical benefit.

Epstein-Barr virus (EBV) in infectious mononucleosis: detection of the virus in tonsillar B lymphocytes but not in desquamated oropharyngeal epithelial cells

Aims—Despite its well established tropism for B cells, the nature of the cellular compartment(s) mediating primary and persistent Epstein-Barr virus (EBV) infection is still a matter of controversy. In view of the association of EBV with several lymphoid and epithelial malignancies, resolution of this issue is important. Methods—Desquamated oropharyngeal epithelial cells from 10 patients with acute infectious mononucleosis and from seven chronic virus carriers were studied for evidence of EBV infection using in situ hybridisation for the detection of the small EBV encoded RNAs (EBERs) and of the viral genome. In addition, immunocytochemistry was used to detect the BZLF1 transactivator protein of EBV. Results—There was no evidence of latent or replicative EBV infection in oropharyngeal epithelial cells in any of the samples. In contrast, EBV infected B cells were readily identified in a tonsil from a patient with infectious mononucleosis. Conclusions—The results suggest that oropharyngeal epithelial cells are not a major site of EBV infection and provide further support for the notion that B cells mediate primary and persistent EBV infection.

Cytotoxic T lymphocyte responses to Epstein-Barr virus

Epstein-Barr virus induces a potent cytotoxic T lymphocyte response in man that is preferentially directed towards a particular subset of the virus latent cycle antigens; the immunodominance of these proteins, apparent in both primary and memory responses, may reflect some differential access to the HLA class I processing pathway. Effector cells recognizing these immunodominant antigens can reverse the growth of virus-induced lymphoproliferative lesions in immunosuppressed patients; however, responses to some of the subdominant latent proteins will be needed to target other virus-positive tumours.

Retroviral transfer of a dominant TCR prevents surface expression of a large proportion of the endogenous TCR repertoire in human T cells

The latent membrane protein-2 (LMP2) of Epstein–Barr virus is a potential target for T-cell receptor (TCR) gene therapy of Hodgkin lymphoma and nasopharyngeal carcinoma. Here, we modified a human leukocyte antigen-A2-restricted, LMP2-specific TCR to achieve efficient expression following retroviral TCR gene transfer. The unmodified TCR was poorly expressed in primary human T cells, suggesting that it competed inefficiently with endogenous TCR chains for cell surface expression. In order to improve this TCR, we replaced the human constant region with murine sequences, linked the two TCR genes using a self-cleaving 2A sequence and finally, codon optimized the TCR-α-2A-β cassette for efficient translation in human cells. Retroviral transfer of the modified TCR resulted in efficient surface expression and HLA-A2/LMP2 pentamer binding. The transduced cells showed peptide-specific interferon-γ and interleukin-2 production and killed target cells displaying the LMP2 peptide. Importantly, the introduced LMP2-TCR suppressed the cell surface expression of a large proportion of endogenous TCR combinations present in primary human T cells. The design of dominant TCR is likely to improve TCR gene therapy by reducing the risk of potential autoreactivity of endogenous and mispaired TCR combinations.

Proteomic profiling of urine for the detection of colon cancer

BackgroundColorectal cancer is the second most common cause of cancer related death in the developed world. To date, no blood or stool biomarkers with both high sensitivity and specificity for potentially curable early stage disease have been validated for clinical use. SELDI and MALDI profiling are being used increasingly to search for biomarkers in both blood and urine. Both techniques provide information predominantly on the low molecular weight proteome (<15 kDa). There have been several reports that colorectal cancer is associated with changes in the serum proteome that are detectable by SELDI and we hypothesised that proteomic changes would also be detectable in urine.ResultsWe collected urine from 67 patients with colorectal cancer and 72 non-cancer control subjects, diluted to a constant protein concentration and generated MALDI and SELDI spectra. The intensities of 19 peaks differed significantly between cancer and non-cancer patients by both t-tests and after adjusting for confounders using multiple linear regressions. Logistic regression classifiers based on peak intensities identified colorectal cancer with up to 78% sensitivity at 87% specificity. We identified and independently quantified 3 of the discriminatory peaks using synthetic stable isotope peptides (an 1885 Da fragment of fibrinogen and hepcidin-20) or ELISA (β2-microglobulin).ConclusionChanges in the urine proteome may aid in the early detection of colorectal cancer.