Irina Redchenko

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.

Differential Immunogenicity of Epstein-Barr Virus Latent-Cycle Proteins for Human CD4+ T-Helper 1 Responses

ABSTRACT Human CD4+ T-helper 1 cell responses to Epstein-Barr virus (EBV) infection are likely to be important in the maintenance of virus-specific CD8+ memory and/or as antiviral effectors in their own right. The present work has used overlapping peptides as stimulators of gamma interferon release (i) to identify CD4+ epitopes within four EBV latent-cycle proteins, i.e., the nuclear antigens EBNA1 and EBNA3C and the latent membrane proteins LMP1 and LMP2, and (ii) to determine the frequency and magnitude of memory responses to these proteins in healthy virus carriers. Responses to EBNA1 and EBNA3C epitopes were detected in the majority of donors, and in the case of EBNA1, their antigen specificity was confirmed by in vitro reactivation and cloning of CD4+ T cells using protein-loaded dendritic cell stimulators. By contrast, responses to LMP1 and LMP2 epitopes were seen much less frequently. EBV latent-cycle proteins therefore display a marked hierarchy of immunodominance for CD4+ T-helper 1 cells (EBNA1, EBNA3C ≫ LMP1, LMP2) which is different from that identified for the same proteins with respect to CD8+-T-cell responses (EBNA3C > EBNA1 > LMP2 ≫ LMP1). Furthermore, the range of CD4+ memory T-cell frequencies in peripheral blood of healthy virus carriers was noticeably lower and narrower than the corresponding range of latent antigen-specific CD8+-T-cell frequencies.

Vaccination of Colorectal Cancer Patients with Modified Vaccinia Ankara Delivering the Tumor Antigen 5T4 (TroVax) Induces Immune Responses which Correlate with Disease Control: A Phase I/II Trial

Purpose: The highly attenuated strain of vaccinia virus, modified vaccinia Ankara (MVA), encoding the tumor antigen 5T4 (termed TroVax), has been evaluated in an open-label phase I/II study in colorectal cancer patients. The primary objectives were to assess the safety and immunogenicity of ascending doses of TroVax and to determine the biodistribution of the vector. Experimental Design: TroVax was given to 22 patients with metastatic colorectal cancer. Seventeen patients received doses of TroVax ranging from 5 × 107 up to 5 × 108 plaque-forming units at 0, 4, and 8 weeks and were considered to be evaluable for assessment of immunologic responses. Both antibody and cellular responses specific for the tumor antigen 5T4 and the viral vector were monitored throughout the study. Results: TroVax was well tolerated in all patients with no serious adverse events attributed to vaccination. Of 17 evaluable patients, 16 showed 5T4-specific cellular responses whereas 14 had detectable antibody levels following vaccination. TroVax was able to boost 5T4-specific immune responses in the presence of MVA neutralizing antibodies. Periods of disease stabilization ranging from 3 to 18 months were observed in five patients, all of whom mounted 5T4-specific immune responses. Furthermore, statistical analysis showed a positive association between the development of a 5T4 (but not MVA) antibody response and patient survival or time to disease progression. Conclusion: These data indicate that vaccination with TroVax is safe and well tolerated and that immune responses to 5T4 can be induced without any evidence of autoimmune toxicity. Furthermore, 5T4-specific antibody responses correlate with evidence of disease control.

Vaccination of Colorectal Cancer Patients with Modified Vaccinia Ankara Encoding the Tumor Antigen 5T4 (TroVax) Given Alongside Chemotherapy Induces Potent Immune Responses

Purpose: The attenuated strain of vaccinia virus, modified vaccinia Ankara (MVA) encoding the tumor antigen 5T4 (TroVax), has been evaluated in an open-label phase II study in metastatic colorectal cancer patients. The primary objective was to assess the safety and immunogenicity of TroVax injected before, during, and after treatment with cycles of 5-fluorouracil, folinic acid, and oxaliplatin. Experimental Design: TroVax was administered to 17 patients with metastatic colorectal cancer. In total, 11 patients were considered to be evaluable for assessment of immunologic responses having received a total of six injections of TroVax, administered before, during, and following completion of chemotherapy. Antibody and cellular responses specific for 5T4 and MVA were monitored throughout the study. Results: Administration of TroVax alongside 5-fluorouracil, folinic acid, and oxaliplatin was safe and well tolerated with no serious adverse events attributed to TroVax. Ten of the 11 evaluable patients mounted 5T4-specific antibody responses with titers ranging from 10 to >1,000. IFNγ enzyme-linked immunospot responses specific for 5T4 were detected in 10 patients with precursor frequencies exceeding 1 in 1,000 peripheral blood mononuclear cells in 4 patients. Of the 11 evaluable patients, 6 had complete or partial responses. 5T4-specific immune responses, but not MVA-specific immune responses, correlated with clinical benefit. Conclusions: Potent 5T4-specific cellular and/or antibody responses were induced in all evaluable patients and were still detectable during the period in which chemotherapy was administered. These results suggest that TroVax can be added to chemotherapy regimens without any evidence of enhanced toxicity or reduced immunologic efficacy and may provide additional clinical benefit.

Active treatment of murine tumors with a highly attenuated vaccinia virus expressing the tumor associated antigen 5T4 (TroVax) is CD4+ T cell dependent and antibody mediated

Abstract5T4 is a tumor associated antigen that is expressed on the surface of a wide spectrum of human adenocarcinomas. The highly attenuated virus, modified vaccinia Ankara, has been engineered to express human 5T4 (h5T4). In a pre-clinical murine model, the recombinant virus (TroVax) induces protection against challenge with CT26–h5T4 (a syngeneic tumor line expressing h5T4). Anti-tumor activity is long lived, with protection still evident 6 months after the final vaccination. In a therapeutic setting, injection of mice with TroVax results in a reduction in tumor burden of >90%. Depletion of CD8+ T cells has no effect upon therapy in the active treatment model, whereas depletion of CD4+ T cells completely abrogates anti-tumor activity. In a prophylactic setting, depletion of CD4+ and CD8+ T cells after the induction of a h5T4 immune response has no deleterious effect on protection following challenge with CT26–h5T4. In light of these studies, the role of antibodies in protection against tumor challenge was investigated. 5T4 specific polyclonal serum decreased tumor burden by approximately 70%. Thus, we conclude that CD4+ T cells are essential for the induction of a protective immune response and that antibodies are the likely effector moiety in this xenogeneic murine tumor model.

The Human Vaccines Project: A roadmap for cancer vaccine development

A concerted international effort is necessary to achieve clinically effective cancer vaccines. Cancer vaccine development has been vigorously pursued for 40 years. Immunity to tumor antigens can be elicited by most vaccines tested, but their clinical efficacy remains modest. We argue that a concerted international effort is necessary to understand the human antitumor immune response and achieve clinically effective cancer vaccines.

Monitoring of human immunological responses to vaccinia virus.

For the last 30 yr, interest in vaccinia virus immune monitoring has focused on the use of the vaccinia virus as a recombinant vaccine vector and the potential detrimental effect of antivector immunity on subsequent vaccination with a recombinant vaccinia virus. However, interest in this area has intensified after the publication of reports suggesting that smallpox may be a major pathogen selected for bioterrorist activities. Owing to the unacceptably high incidence of complications induced by previous effective smallpox vaccine strains, alternative safer strains (e.g., modified vaccinia Ankara [MVA]) are being assessed for their antigenicity in clinical trials. The exact immune effector mechanism responsible for vaccine-induced protection to smallpox infection has not been fully elucidated, although it is believed that neutralizing antibody plays a major role. This chapter describes a simple enzyme-linked immunosorbent assay (ELISA) to quantify vaccinia virus antibody titer. Additionally, to define serum-neutralizing activity, both a classical plaque reduction assay and a high-throughput 96-well plate method based on reduction of recombinant vaccinia virus expressed beta-galactosidase is described. Furthermore, details are given for a T-cell proliferation assay, primarily for monitoring T-helper CD4 activity and an enzyme-linked immunospot (ELISPOT) assay for CD8 analysis. The use of reliable immunological assays is vital in assessing the potential efficacy of new vaccines to protect against smallpox infection.

Time-dependent changes of LAK cell phenotypes correlate with the secretion of different cytotoxic proteins.

Human lymphokine-activated killer (LAK) cells were generated from peripheral blood lymphocytes (PBL) of normal volunteers by interleukin-2 (IL-2) stimulation for 1-8 days. During the first 3 days the surface marker CD16 characteristic for natural killer (NK) cells was expressed and later the CD3 marker characteristic for cytotoxic T cells became predominant. The conditioned media of LAK cells collected after interaction of LAK cells with K562 target cells was chromatographically separated into two cytotoxic fractions: F1 and F2. It was demonstrated that fraction F1 contained cytotoxic proteins having molecular weights of 30 and 40 kDa, and fraction F2 contained cytotoxic proteins having molecular weights of 22, 38 and 75 kDa. The presence of the proteins in each of these two fractions correlated with the phenotype changes of LAK cells: the F2 cytotoxic proteins were characteristic for NK-like cells, and the F1 proteins for cytotoxic T-lymphocyte (CTL)-like phenotypes.

Identification of a major histocompatibility complex class I-restricted T-cell epitope in the tumour-associated antigen, 5T4

5T4 is a surface glycoprotein expressed on placental trophoblasts and also on a wide range of human carcinomas. Its highly restricted expression on normal tissues and broad distribution on many carcinomas make 5T4 a promising target for cancer immunotherapy. In the current study, we set out to investigate whether a 5T4‐specific cytotoxic T lymphocyte (CTL) repertoire exists in healthy individuals. CD4‐depleted peripheral blood mononuclear cells (PBMCs) from blood donors were screened using an ex vivo interferon‐γ (IFN‐γ) enzyme‐linked immunospot (ELISPOT) assay. A panel of overlapping peptides, spanning the full length of the 5T4 protein, was used as a source of antigen. In the process of screening, one out of 30 blood donors demonstrated a positive ex vivo IFN‐γ ELISPOT response to a single 5T4 peptide. A polyclonal T‐cell line was derived from this donor by culturing PBMCs with autologous peptide‐pulsed dendritic cells (DCs). The resulting polyclonal T‐cell line and clones were tested in a 51Cr‐release assay and by ELISPOT and were shown to be peptide specific. Furthermore, antigen‐presenting cells (APCs), infected with a viral vector expressing 5T4, were able to stimulate IFN‐γ production by the peptide‐specific T‐cell clones. A minimal CD8 epitope, PLADLSPFA, has been identified and found to be restricted through human leucocyte antigen (HLA) Cw7. Subsequently, we have demonstrated that HLA‐Cw7‐positive colorectal cancer patients vaccinated with a recombinant vaccinia viral vector encoding 5T4 (TroVax) are capable of mounting a strong IFN‐γ ELISPOT response to this novel CTL epitope. These findings have potential application in cancer immunotherapy in terms of subunit vaccine design and the monitoring of immune responses induced in patients by 5T4‐based therapies.

Inhibitory effect of calf fetuin on the cytotoxic activity of LAK cell-derived factors and tumor necrosis factor

A protein-inhibiting LAK cell-mediated cytotoxicity was isolated from a LAK cell-conditioned medium. The N-terminal amino acid sequence of this protein appeared to be identical to fetal calf fetuin. Pure isolated fetuin, as well as commercially available preparations of this protein, were shown to inhibit cytotoxic activity of both cytotoxic proteins released by LAK cells and TNF.