CD4+ T-cell responses to Epstein-Barr virus nuclear antigen EBNA1 in Chinese populations are highly focused on novel C-terminal domain-derived epitopes.

Epstein-Barr virus nuclear antigen EBNA1, the one viral protein uniformly expressed in nasopharyngeal carcinoma (NPC), represents a prime target for T-cell-based immunotherapy. However, little is known about the EBNA1 epitopes, particularly CD4 epitopes, presented by HLA alleles in Chinese people, the group at highest risk for NPC. We analyzed the CD4+ T-cell responses to EBNA1 in 78 healthy Chinese donors and found marked focusing on a small number of epitopes in the EBNA1 C-terminal region, including a DP5-restricted epitope that was recognized by almost half of the donors tested and elicited responses able to recognize EBNA1-expressing, DP5-positive target cells.

CD4+ T-cell responses to Epstein-Barr virus (EBV) latent-cycle antigens and the recognition of EBV-transformed lymphoblastoid cell lines.

There is considerable interest in the potential of Epstein-Barr virus (EBV) latent antigen-specific CD4+ T cells to act as direct effectors controlling EBV-induced B lymphoproliferations. Such activity would require direct CD4+ T-cell recognition of latently infected cells through epitopes derived from endogenously expressed viral proteins and presented on the target cell surface in association with HLA class II molecules. It is therefore important to know how often these conditions are met. Here we provide CD4+ epitope maps for four EBV nuclear antigens, EBNA1, -2, -3A, and -3C, and establish CD4+ T-cell clones against 12 representative epitopes. For each epitope we identify the relevant HLA class II restricting allele and determine the efficiency with which epitope-specific effectors recognize the autologous EBV-transformed B-lymphoblastoid cell line (LCL). The level of recognition measured by gamma interferon release was consistent among clones to the same epitope but varied between epitopes, with values ranging from 0 to 35% of the maximum seen against the epitope peptide-loaded LCL. These epitope-specific differences, also apparent in short-term cytotoxicity and longer-term outgrowth assays on LCL targets, did not relate to the identity of the source antigen and could not be explained by the different functional avidities of the CD4+ clones; rather, they appeared to reflect different levels of epitope display at the LCL surface. Thus, while CD4+ T-cell responses are detectable against many epitopes in EBV latent proteins, only a minority of these responses are likely to have therapeutic potential as effectors directly recognizing latently infected target cells.

Clonal expansions in acute EBV infection are detectable in the CD8 and not the CD4 subset and persist with a variable CD45 phenotype.

We have applied a sensitive global analysis of TCR heterogeneity to compare clonal dynamics of CD4(+) and CD8(+) T cells in acute infectious mononucleosis. Using this approach, we are able to identify a broad representation of the total virus-specific population without the bias of in vitro culture and then to track their phenotype and fate by their unique molecular footprint. We demonstrate a large number of Ag-driven clones using different TCRs in the acute phase, all CD8(+). The diverse large clones generated in the CD8 subset in response to this virus contrast with the complete lack of detectable clonal expansion in the CD4 compartment. Many of the same clones remain detectable in directly ex vivo CD8(+) T cells for at least a year after resolution of infectious mononucleosis, although the clone size is reduced. Thus, memory CD8 cells following EBV infection persist at relatively high circulating frequency and represent a subset of the large range of clonotypes comprising the acute effectors. Separation of samples into CD45RA (naive) and CD45RO (memory) fractions shows the accumulation of identical CDR3 region defined clonotypes in both CD45RO and CD45RA fractions and sequencing confirms that dominant long-lived monoclonal expansions can reside in the CD45RA pool.

EBV-specific CD8+ T cell memory: relationships between epitope specificity, cell phenotype, and immediate effector function.

EBV infection in humans induces CD8+ T cell memory to viral epitopes derived from both lytic and latent cycle Ags. We have analyzed the relationship between the phenotype and function of the memory pool of T cells specific for these Ags. Lytic epitope-specific populations were heterogeneous in terms of CD45RO/RA and CD28 expression, whereas latent epitope-specific populations were uniformly CD45RO+ and CD28+, consistent with the higher antigenic challenge from lytic epitopes driving some memory cells toward a CD45RA+, CD28- phenotype. However, both types of memory population showed immediate epitope-specific cytotoxicity and type 1 cytokine production in ex vivo assays. Cytotoxic function was not associated with preactivated T cells, as EBV-specific populations were negative for activation markers such as CD69 or CD38, nor could cytotoxic function be ascribed to CD27- or CD56+ subsets, as such cells were not detected in EBV-specific memory. Furthermore, cytotoxicity was not limited to CD45RA+ and/or CD28- fractions, but also was observed in CD45RO+, CD28+ populations in lytic and latent epitope-specific memory. Cytokine (IFN-gamma, TNF-alpha) responses, measured by intracytoplasmic staining after peptide stimulation, also were detectable in CD45RO+ and RA+ subsets as well as CD28+ and CD28- subsets. Of other markers that were heterogeneous in both lytic and latent epitope populations, CCR7 gave the best discrimination of functionality; thus, CCR7+ cells consistently failed to give an IFN-gamma or TNF-alpha response, whereas many CCR7- cells were responsive. Our data are consistent with effector functions having a broad distribution among phenotypically distinct subsets of "effector memory" cells that have lost the CCR7 marker.

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.

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.

A re-evaluation of the frequency of CD8+ T cells specific for EBV in healthy virus carriers.

EBV is a gammaherpesvirus that can establish both nonproductive (latent) and productive (lytic) infections within the cells of its host. Although T cell responses to EBV latent proteins have been well characterized, little is known about the importance of responses to lytic proteins in long term virus carriers. Here we have compared the frequencies of CD8+ T cells specific for EBV latent and lytic Ags in healthy virus carriers, using three techniques: limiting dilution analysis, enzyme-linked immunospot assay, and FACS staining with tetrameric MHC-peptide complexes. T cells specific for EBV lytic protein epitopes were readily detectable in all donors and were usually more abundant than those specific for latent epitopes. We infer that direct T cell control of viral replicative lesions is maintained in long term carriers of EBV and is an important component of the immune response to this virus. Estimates of CD8+ T cell frequencies varied considerably according to methodology; values obtained from MHC-peptide tetramer staining were, on the average, 4.4-fold higher than those obtained from enzyme-linked immunospot assays, which were, in turn, on the average, 5.3-fold higher than those obtained from limiting dilution analysis. Tetramer staining showed that as many as 5.5% circulating CD8+ T cells in a virus carrier were specific for a single EBV lytic protein epitope. Such values are much greater than previously imagined and illustrate how antigenic challenge from a persistent herpesvirus can influence the composition of the host's CD8+ T cell pool.