Asymptomatic primary Epstein-Barr virus infection occurs in the absence of blood T-cell repertoire perturbations despite high levels of systemic viral load.

Primary infection with the human herpesvirus, Epstein-Barr virus (EBV), may result in subclinical seroconversion or may appear as infectious mononucleosis (IM), a lymphoproliferative disease of variable severity. Why primary infection manifests differently between patients is unknown, and, given the difficulties in identifying donors undergoing silent seroconversion, little information has been reported. However, a longstanding assumption has been held that IM represents an exaggerated form of the virologic and immunologic events of asymptomatic infection. T-cell receptor (TCR) repertoires of a unique cohort of subclinically infected patients undergoing silent infection were studied, and the results highlight a fundamental difference between the 2 forms of infection. In contrast to the massive T-cell expansions mobilized during the acute symptomatic phase of IM, asymptomatic donors largely maintain homeostatic T-cell control and peripheral blood repertoire diversity. This disparity cannot simply be linked to severity or spread of the infection because high levels of EBV DNA were found in the blood from both types of acute infection. The results suggest that large expansions of T cells within the blood during IM may not always be associated with the control of primary EBV infection and that they may represent an overreaction that exacerbates disease.

Crossreactive recognition of viral, self, and bacterial peptide ligands by human class I-restricted cytotoxic T lymphocyte clonotypes: implications for molecular mimicry in autoimmune disease.

The immunodominant, CD8(+) cytotoxic T lymphocyte (CTL) response to the HLA-B8-restricted peptide, RAKFKQLL, located in the Epstein-Barr virus immediate-early antigen, BZLF1, is characterized by a diverse T cell receptor (TCR) repertoire. Here, we show that this diversity can be partitioned on the basis of crossreactive cytotoxicity patterns involving the recognition of a self peptide-RSKFRQIV-located in a serine/threonine kinase and a bacterial peptide-RRKYKQII-located in Staphylococcus aureus replication initiation protein. Thus CTL clones that recognized the viral, self, and bacterial peptides expressed a highly restricted alphabeta TCR phenotype. The CTL clones that recognized viral and self peptides were more oligoclonal, whereas clones that strictly recognized the viral peptide displayed a diverse TCR profile. Interestingly, the self and bacterial peptides equally were substantially less effective than the cognate viral peptide in sensitizing target cell lysis, and also resulted only in a weak reactivation of memory CTLs in limiting dilution assays, whereas the cognate peptide was highly immunogenic. The described crossreactions show that human antiviral, CD8(+) CTL responses can be shaped by peptide ligands derived from autoantigens and environmental bacterial antigens, thereby providing a firm structural basis for molecular mimicry involving class I-restricted CTLs in the pathogenesis of autoimmune disease.

A functional link for major TCR expansions in healthy adults caused by persistent Epstein-Barr virus infection.

Dramatic clonal expansions of unknown functional significance have been documented in the T cell receptor (TCR) alpha beta peripheral blood repertoires of apparently healthy adults. In this study, we provide evidence that persistent infection with the ubiquitous Epstein-Barr virus (EBV) causes major distortions within the memory repertoire of healthy virus carriers. Using complementarity determining region 3 (CDR3) length analysis to measure repertoire diversity, dominant expansions that dramatically skewed the entire TCRBV6 blood repertoire towards oligoclonality were enriched in the CD8(+)CD45RO+CD45RA- subset of HLA B8(+) healthy virus carriers. Evidence of phenotypic heterogeneity between individuals was also observed for these expansions based on their variable coexpression of CD45RO and CD45RA. TCR junctional region sequencing revealed that these expansions were clonal and that they represented commonly selected HLA B8-restricted memory cytotoxic T cells that recognize the immunodominant latent EBV epitope, FLRGRAYGL. Furthermore, the functional identity of these virus-specific CD8(+) T cells was confirmed by their FLRGRAYGL-specific cytotoxicity. Therefore, the functional significance of dramatic clonal expansions in healthy adults can be linked in some cases to virus-specific CD8(+) T cells that play an essential role in immunosurveillance. This first identified link for expansions in the circulation of healthy adults strongly implies that restricted-memory TCR responses to environmental antigens play a pivotal role in expansion development, which should have an important impact on studies interpreting TCR expansion patterns in health and disease.

Targeting a polyepitope protein incorporating multiple class II-restricted viral epitopes to the secretory/endocytic pathway facilitates immune recognition by CD4+ cytotoxic T lymphocytes: a novel approach to vaccine design.

The role of CD4+ and CD8+ cells in the generation of an effective immune response against viral infections is well established. Moreover, there is an increasing realization that subunit vaccines which include both CD4+- and CD8+-T-cell epitopes are highly effective in controlling viral infections, as opposed to those which are designed to activate a CD8+- or CD4+-T-cell response alone. One of the major limitations of epitope-based vaccines designed to stimulate virus-specific CD4+ T cells is that endogenously expressed class II-restricted minimal cytotoxic-T-lymphocyte (CTL) epitopes are poorly recognized by CD4+ CTLs. In the present study we attempted to enhance the efficiency of class II-restricted endogenous presentation of minimal class II-restricted CTL epitopes by specifically targeting a polyepitope protein to class II processing compartments through the endosomal and/or lysosomal pathway. A significantly enhanced stimulation of virus-specific CD4+-T-cell clones by antigen-presenting cells (APC) expressing the recombinant polyepitope protein targeted to the endocytic/secretory pathway was readily demonstrated in cytotoxicity assays. In addition, in vitro activation of Epstein-Barr virus- and influenza virus-specific CD4+ memory CTLs by the recombinant constructs encoding the polyepitope protein, specifically targeted to the lysosomal compartment, was also demonstrated. The enhanced stimulatory capacity of APC expressing a lysosome-targeted polyepitope protein has important implications for vaccine design.

Dominant cytotoxic T lymphocyte response to the immediate-early trans-activator protein, BZLF1, in persistent type A or B Epstein-Barr virus infection.

Five healthy human leukocyte antigen-B8 (HLA-B8)-positive virus carriers were studied to investigate the CD8+ cytotoxic T lymphocyte (CTL) response to an HLA-B8-restricted peptide, RAKFKQLLQ, located in the Epstein-Barr virus (EBV) immediate-early trans-activator protein, BZLF1. Of the 5 virus carriers, 4 were infected with type A and 1 with type B EBV. Using limiting-dilution analysis of peripheral blood mononuclear cells, a high RAKFKQLLQ-specific CTL precursor frequency was demonstrated after specific peptide or autologous lymphoblastoid cell line stimulation in both type A and type B EBV carriers. The RAKFKQLLQ-specific CTL precursor frequencies in all 5 persons were at least as dominant as those observed with two other EBV-associated, HLA-B8-restricted latent epitopes, FLRGRAYGL and QAKWRLQTL. These findings show that healthy virus carriers maintain a high frequency of BZLF1-specific memory T cells, potentially to control virus spread from lytically infected cells.

A case report: immune responses and clinical course of the first human use of granulocyte/macrophage-colony-stimulating-factor-transduced autologous melanoma cells for immunotherapy.

The first use of granulocyte/macrophage-colony-stimulating-factor-transduced, lethally irradiated, autologous melanoma cells as a therapeutic vaccine in a patient, with rapidly progressive, widely disseminated malignant melanoma resulted in the generation of a novel antitumour immune response associated with partial, albeit temporary, clinical benefit. An initially negative reaction to non-transduced, autologous melanoma cells was converted to a delayed-type hypersensitivity (DTH) reaction of increasing magnitude following successive vaccinations. While intradermal vaccine sites showed prominent dendritic cell accrual, DTH sites revealed a striking influx of eosinophils in addition to activated/memory T lymphocytes and macrophages, recalling the histology of challenge tumour cell rejection in immune mice. Cytotoxic T lymphocytes (CTL) reactive with autologous melanoma cells were detectable at high frequency after vaccination, not only in limiting-dilution analysis, but also in bulk culture without added cytokines. Clonal analysis of CTL showed a conversion from a purely CD8+ response to a high proportion of CD4+ clones following vaccination. A prominent acute-phase response manifested by a five- to tenfold increase in C-reactive protein was observed, as was a systemic eosinophila. Vaccination resulted in the regression of axillary lymphatic metastases, stabilisation of pulmonary metastases, and a dramatic, reversible increase in cerebral oedema associated with multiple central nervous system metastases: however, lesions in the adrenal glands, pancreas and spleen proved refractory. The antitumour effects and immune response were not detectable 2 months following the last vaccination. Irradiation of the extensive cerebral metastases resulted in rapid deterioration and death of the patient.

Selection of a diverse TCR repertoire in response to an Epstein-Barr virus-encoded transactivator protein BZLF1 by CD8+ cytotoxic T lymphocytes during primary and persistent infection.

We investigated the CD8+ cytotoxic T lymphocyte (CTL) repertoire to an HLA B8-restricted peptide, RAKFKQLLQ, located in the Epstein-Barr virus (EBV) immediate-early protein, BZLF1. Repertoire selection was monitored by determining the TCR beta chain sequences of RAKFKQLLQ-specific CTL established from primary infected and healthy virus carriers. PCR analysis of spontaneous EBV-transformed lymphoblastoid cell lines (LCL) from three individuals with primary infection showed that two were infected with type A and one with type B EBV. Polyclonal and clonal CTL that were generated by stimulating peripheral blood mononuclear cells with an HLA B8+ homozygous LCL lysed T cell blasts pulsed with the peptide, RAKFKQLLQ; lysis of certain HLA B8+ LCL targets was associated with the abundance of BZLF1 transcripts. TCR beta analysis showed that while there was loop length restriction in the putative peptide contact site of all responding beta chains, diverse and unique (non-recurrent) TCR beta clonotypes were selected in individuals during primary infection and continued to emerge after long-term virus exposure. TCR-contact site heterogeneity was excluded as the selective force in diversity generation since the epitope-encoded sequences were found to be identical within endogenous virus isolates. In this first study of TCR repertoire selection for an EBV lytic antigen, a BZLF1-reactive component of diverse clonotypes was identified in primary type A or type B EBV infection which was sustained in the EBV-specific memory response throughout life-long infection. This diversity selection is likely to play a critical role in maintaining a balanced viral load throughout EBV persistence.

Development of Epstein-Barr virus-specific memory T cell receptor clonotypes in acute infectious mononucleosis.

The importance of cytotoxic T lymphocytes (CTLs) in the immunosurveillance of Epstein-Barr virus (EBV)-infected B cells is firmly established, and the viral antigens of CTL recognition in latent infection are well defined. The epitopes targeted by CTLs during primary infection have not been identified, however, and there is only limited information about T cell receptor (TCR) selection. In the present report, we have monitored the development of memory TCR-beta clonotypes selected in response to natural EBV infection in a longitudinal study of an HLA-B8+ individual with acute infectious mononucleosis (IM). By stimulating peripheral blood lymphocytes with HLA-B8+ EBV-transformed B lymphoblastoid cells, the primary virus-specific CTL response was shown to include specificities for two HLA-B8-restricted antigenic determinants, FLRGRAYGL and QAKWRLQTL, which are encoded within the latent EBV nuclear antigen EBNA-3. TCR-beta sequence analysis of CTL clones specific for each epitope showed polyclonal TCR-beta repertoire selection, with structural restrictions on recognition that indicated antigen-driven selection. Furthermore, longitudinal repertoire analysis revealed long-term preservation of a multiclonal effector response throughout convalescence, with the reemergence of distinct memory T cell clonotypes sharing similar structural restrictions. Tracking the progression of specific TCR-beta clonotypes and antigen-specific TCR-V beta family gene expression in the peripheral repertoire ex vivo using semiquantitative PCR strongly suggested that selective TCR-beta expansions were present at the clonotype level, but not at the TCR-V beta family level. Overall, in this first analysis of antigen-specific TCR development in IM, a picture of polyclonal TCR stimulation is apparent. This diversity may be especially important in the establishment of an effective CTL control during acute EBV infection and in recovery from disease.

Recruitment during infectious mononucleosis of CD3+CD4+CD8+ virus-specific cytotoxic T cells which recognise Epstein-Barr virus lytic antigen BHRF1.

Epstein-Barr virus (EBV) is the aetiological agent of infectious mononucleosis (IM) which is a common sequel to primary EBV infection. Thereafter, the virus is maintained as a lifetime latent infection. Although the proteins expressed during the latent EBV infection provide a rich source of immunogenic epitopes, very little is known about cytotoxic T lymphocyte (CTL) control of primary EBV infection. The present report is based on an analysis of CTL clones derived from a patient suffering from acute IM. An intriguing feature of six CTL clones that displayed an HLA-restricted pattern of cell lysis was their initial coexpression of the T cell markers CD3, CD4, and CD8. Detailed analysis of one of these clones, which was restricted through the class II MHC antigen DR2, revealed reactivity with an epitope within the EBV lytic cycle early antigen, BHRF-1, which corresponds to the C-terminal region of the protein (AGLTLSLLVICSYLFISRG) (residues 171-189). There have been no previously published reports describing a CTL response during acute IM directed against an EBV lytic antigen. Interestingly, the coexpression of CD4 and CD8 by these CTLs during acute IM suggests that CD3+CD4+CD8+ cortical thymocytic precursor cells are recruited in order to overcome the EBV infection.

The ecology and pathology of Epstein-Barr virus.

Epstein-Barr virus achieves its ubiquitous and uniform epidemiological distribution by a dual strategy of latency to guarantee lifelong persistence and intermittent replication to guarantee transmission. These two functions appear to dictate residence in different cell types: latency in B lymphocytes and replication in epithelial cells. Both of these cell compartments are potential sites for EBV-associated malignancies.

T cell receptor repertoire for a viral epitope in humans is diversified by tolerance to a background major histocompatibility complex antigen.

Two unusual characteristics of the memory response to the immunodominant Epstein-Barr virus (EBV) epitope FLRGRAYGL, which associates with HLA B8, have provided an unique opportunity to investigate self tolerance and T cell receptor (TCR) plasticity in humans. First, the response is exceptionally restricted, dominated by cytotoxic T lymphocytes (CTL) with identical TCR protein sequences (Argaet, V. P., C. W. Schmidt, S. R. Burrows, S. L. Silins, M. G. Kurilla, D. L. Doolan, A. Suhrbier, D. J. Moss, E. Kieff, T. B. Sculley, and I. S. Misko. 1994. J. Exp. Med. 180:2335-2340). Second, CTL expressing this receptor are cross-reactive with the alloantigen HLA B* 4402 on uninfected cells (Burrows, S. R., R. Khanna, J. M. Burrows, and D. J. Moss. 1994. J. Exp. Med. 179:1155-1161). No CTL using this conserved public TCR could be reactivated from the peripheral blood of EBV exposed individuals expressing both HLA B8 and B*4402, demonstrating the clonal inactivation of potentially self-reactive T cells in humans. A significant FLRGRAYGL-specific response was still apparent, however, and TCR sequence analysis of multiple CTL clones revealed an oligoclonal TCR repertoire for this determinant within these individuals, using diverse V and J gene segments and CDR3 regions. In addition, a significant public TCR component was identified in which several distinct alpha/beta rearrangements are shared by CTL clones from a number of unrelated HLA B8+, B*4402+ donors. The striking dominance of public TCR in the response to this EBV epitope suggests a strong genetic bias in TCR gene recombination. Fine specificity analysis using peptide analogues showed that, of six different antigen receptors for FLRGRAYGL/HLA B8, none associate closely with the peptide's full array of potential TCR contact residues. Whereas the HLA B*4402-cross-reactive receptor binds amino acids toward the COOH terminus of the peptide, others preferentially favor an NH2-terminal determinant, presumably evading an area that mimics a structure presented on HLA B*4402. Thus, tolerance to a background major histocompatibility antigen can effectively diversify the TCR repertoire for a foreign epitope by deflecting the response away from an immunodominant combination of TCR-binding residues.

Dominant selection of an invariant T cell antigen receptor in response to persistent infection by Epstein-Barr virus.

To examine T cell receptor (TCR) diversity involved in the memory response to a persistent human pathogen, we determined nucleotide sequences encoding TCR-alpha and -beta chains from HLA-B8-restricted, CD8+ cytotoxic T cell clones specific for an immunodominant epitope (FLRGRAYGL) in Epstein-Barr virus (EBV) nuclear antigen 3. Herein, we show that identical TCR protein sequences are used by clones from each of four healthy unrelated virus carriers; a clone from a fifth varied conservatively at only two residues. This dominant selection of alpha and beta chain rearrangements suggest that a persistent viral infection can select for a highly focused memory response and indicates a strong bias in gene segment usage and recombination. A novel double-step semiquantitative polymerase chain reaction (PCR) procedure and direct sequencing of amplified TCR cDNA from fresh lymphocytes derived from three HLA-B8 individuals detected transcripts specific for the conserved beta chain in an EBV-seropositive donor but not in two seronegative donors. This report describes an unprecedented degree of conservation in TCR selected in response to a natural persistent infection.

EBV peptide epitope sensitization restores human cytotoxic T cell recognition of Burkitt's lymphoma cells. Evidence for a critical role for ICAM-2.

The pathogenesis of EBV+ Burkitt's lymphoma (BL) suggests evasion of the CTL response against EBV. Two important features of this tumor have been previously suggested to explain this immune evasion, (a) absence/low expression of cellular adhesion molecules and (b) restricted expression of EBV latent Ag. To determine the relative importance of these features in relation to evasion of EBV-specific CTL, a group of BL cell lines with variable expression of the aforementioned phenotypic characteristics were assayed for specific CTL lysis after exogenous addition of EBV peptide epitopes. In spite of down-regulated expression of the adhesion molecules LFA-1, LFA-3, and/or ICAM-1, peptide-sensitized BL cells were recognized and lysed by EBV-specific CTL. Moreover, there was no significant difference between the CTL lysis of the BL cells and that of adhesion molecule-positive control cells over a wide range of peptide epitope concentrations. Blocking experiments with mAb to individual adhesion molecules suggested that virus-specific CTL recognition of lymphoblastoid cell lines was dependent on an intact LFA-3/CD2 pathway. In contrast, the CTL recognition of peptide-sensitized BL cells was critically dependent on the LFA-1/ICAM pathway, with an insignificant contribution by CD2/LFA-3. The consistently high expression of ICAM-2 on all BL cell lines suggests that the accessory function in CTL recognition of these cells is mediated by the LFA-1/ICAM-2 pathway. Thus, down-regulation of LFA-1, LFA-3, and/or ICAM-1 expression on BL cells does not provide an absolute barrier to tumor cell recognition by virus-specific CTL. The ability of virus-specific CTL to recognize peptide epitope-sensitized BL cells as efficiently as normal cells has demonstrated the importance of latent Ag expression in the CTL control of EBV+ tumors.

Localization of Epstein-Barr virus cytotoxic T cell epitopes using recombinant vaccinia: implications for vaccine development.

There is considerable interest in designing an effective vaccine to the ubiquitous Epstein-Barr virus (EBV). An important role for EBV-specific cytotoxic T lymphocytes (CTLs) in eliminating virus-infected cells is well established. Limited studies using a small number of immune donors have defined target epitopes within the latent antigens of EBV. The present study provides an extensive analysis of the distribution of class I-restricted CTL epitopes within EBV-encoded proteins. Using recombinant vaccinia encoding individual EBV latent antigens (Epstein-Barr nuclear antigen [EBNA] 1, 2, 3A, 3B, 3C, LP, and LMP 1), we have successfully localized target epitopes recognized by CTL clones from a panel of 14 EBV-immune donors. Of the 20 CTL epitopes localized, five were defined at the peptide level. Although CTL clones specific for nine epitopes recognized both type 1 and type 2 transformants, a significant number of epitopes (7/16 epitopes for which EBV type specificity was determined) were detected only on type 1 EBV transformants. Vaccinia recombinants encoding EBNA 3A and EBNA 3C were recognized more frequently than any other vaccinia recombinants used in this study, while no CTL epitopes were localized in EBNA 1. Surprisingly, epitope specificity for a large number of EBV-specific CTL clones could not be localized, although vaccinia recombinants used in this study encoded most of the latent antigens of EBV. These results suggest that any EBV vaccine based on CTL epitopes designed to provide widespread protection will need to include not only latent antigen sequences but also other regions of the genome. The apparent inability of human CTLs to recognize EBNA 1 as a target antigen, often the only latent antigen expressed in Burkitt's lymphoma and nasopharyngeal carcinoma, suggests that EBV-specific CTL control of these tumors will not be feasible unless the down-regulation of latent antigens can be reversed.

Immune surveillance against Epstein-Barr virus.

Following primary infection, EBV retains a life-long latent association with B lymphocytes and a permissive association with stratified epithelium in the oropharynx. This review presents a model for the host-virus relationships in healthy virus carriers, a relationship which, if perturbed, may result in EBV-associated disease. Cytotoxic T cells that recognise virally-determined epitopes on infected cells are the major effector arm and control the persistant infection. A strategy for developing a vaccine to EBV is discussed.

Failure of Epstein-Barr virus-specific cytotoxic T lymphocytes to lyse B cells transformed with the B95-8 strain is mapped to an epitope that associates with the HLA-B8 antigen.

There are two types, A and B, of Epstein-Barr virus (EBV) and B95-8 represents the common type A laboratory strain. Herein, we show in a family study that paternal EBV-specific cytotoxic T lymphocytes (CTL) generated in short-term cultures following stimulation with the autologous B95-8-transformed lymphoblastoid cell line (LCL) or B cells freshly infected with the B95-8 isolate did not lyse haploidentical B95-8 LCL expressing the HLA-A1, -B8, -DR3 paternal haplotype. In contrast, the haploidentical B95-8 LCL expressing the HLA-A11, -B51, -DR7 paternal haplotype was strongly lysed. Moreover, paternal CTL generated in response to stimulation with the B95-8 LCL expressing the haploidentical HLA-A1, -B8, -DR3 paternal haplotype included an allogeneic response against the maternal haplotype but no EBV-specific response as shown by the poor lysis of the autologous LCL target cells. However, stimulation with the haploidentical HLA-A11, -B51, -DR7 paternal haplotype resulted in the generation of both an allogeneic and an EBV-specific response. CTL clones were generated from two HLA-B8+ donors in response to stimulation with the autologous type A LCL transformed with wildtype EBV. The clones were cross-reactive for an immunodominant B95-8-associated peptide epitope that interacted with the HLA-B8 allele but failed to lyse B95-8-transformed LCL targets unless the targets were pre-coated with the exogenous peptide. A CTL clone that was initially stimulated with the autologous BL74 LCL lysed the spontaneous autologous LCL and spontaneous LCL from an HLA-B8+ donor, but failed to lyse the B95-8 LCL from that donor. The observed haplotype preference can be explained in terms of sequence variation between the B95-8 and the corresponding wildtype epitope. Our findings may help to clarify the role of EBV in the pathogenesis of primary Sjögren's syndrome which is closely associated with HLA-B8.

Expression of Epstein-Barr virus nuclear antigens in anti-IgM-stimulated B cells following recombinant vaccinia infection and their recognition by human cytotoxic T cells.

Cytotoxic T lymphocytes (CTL) recognizing Epstein-Barr virus (EBV) nuclear antigens (EBNA) are an important host defence mechanism in restricting the proliferation of EBV-infected B cells. Previously, B-type lymphoblastoid cell lines (LCL) infected with vaccinia recombinants encoding for the EBNA proteins have been used to identify A-type-specific CTL epitopes. However, to localize the CTL epitopes encoded by both A- and B-type transformants, B-type LCL are an inappropriate host for vaccinia. In the present study, an alternative host cell for vaccinia infection is described. Initial studies demonstrated that anti-IgM (mu-chain specific)-stimulated human B cells allowed vaccinia virus to replicate more efficiently than either phytohaemagglutinin-stimulated lymphocytes (PHA blasts) or CTL and expressed EBNA proteins following recombinant vaccinia infection. Furthermore, the presentation and recognition of target epitopes expressed on vaccinia-infected anti-mu-stimulated B cell blasts were comparable to that on similarly infected LCL. Anti-mu-stimulated B cells were used to define the CTL epitopes recognized by a panel of CTL clones from an EBV-immune donor. Using recombinant vaccinia-infected anti-mu-stimulated B cells, the CTL response from this donor was mapped to the EBNA6 protein. Most importantly, in vitro stimulation of unfractionated mononuclear cells with vaccinia-infected anti-mu B cells activated a memory CTL response. Based on the vaccinia results, screening of peptides from EBNA6 localized the epitope for the majority of the EBNA6-specific CTL clones to the sequence EENLLDFVRFM, apparently in association with HLA-B44. This work clearly demonstrates that anti-mu-stimulated B cells not only provide an efficient model for localizing the CTL epitope(s) but also raises the possibility of reactivating a memory T-cell response to any gene product expressed by recombinant vaccinia.

Nonresponsiveness to an immunodominant Epstein-Barr virus-encoded cytotoxic T-lymphocyte epitope in nuclear antigen 3A: implications for vaccine strategies.

An immunodominant Epstein-Barr virus (EBV)-encoded cytotoxic T lymphocyte (CTL) epitope has been mapped to the EBV nuclear antigen 3A. The epitope, represented by the peptide sequence AWNAGFLRGRAYGLD (hereafter termed AWNA), is restricted through the HLA-B8 allele and is expressed by type A but not type B-infected transformants. Herein, we show that EBV-specific memory CTLs from an HLA-B8+ healthy virus carrier, JS, did not respond in vitro to AWNA, even though that individual's endogenously infected transformants processed and presented the natural equivalent of this peptide to AWNA-specific CTLs from another B8+ individual. Instead, an epitope, represented by the peptide sequence QLSDTPLIPLTIFVGENTGV, was the dominant EBV-specific CTL epitope in donor JS. This epitope mapped to EBV nuclear antigen 2A, was restricted by an HLA-A2 subtype, and specifically associated with type A strains of EBV. No AWNA-specific CTL precursors were detected by limiting dilution analysis of peripheral blood mononuclear cells from donor JS whereas the precursor frequency of AWNA-specific CTLs from a responder donor, LC, was estimated at 1:4500. The presentation in vivo of an immunogenic epitope-HLA antigen complex is clearly insufficient to guarantee an effective memory CTL response to that foreign epitope. Thus, vaccination strategies based on peptides inducing CTL responses may need to take into account not only the polymorphism of HLA antigens but also possible allelic variation in the repertoires of T-cell receptors.

Oligopeptide induction of a secondary cytotoxic T-cell response to Epstein-Barr virus in vitro.

Three Epstein-Barr virus (EBV) nuclear antigen (EBNA)-encoded oligopeptide epitopes have been mapped, each capable of acting as a recognition determinant for class I-restricted lysis by CD8+ cytotoxic T lymphocytes (CTL). This report shows that each peptide, when presented on an appropriate autologous antigen-presenting cell (APC), also stimulates EBV-specific memory T cells present in peripheral blood mononuclear cell (PBMC) populations to develop in vitro into peptide-specific CTL. These CTL specifically lysed autologous EBV-infected lymphoblastoid cell lines (LCL) and peptide-sensitized uninfected targets. Identical viral oligopeptides could therefore function as recognition determinants for both the induction and commission of class I-restricted specific cytotoxicity. A model system is described in which autologous phytohaemagglutinin (PHA) blasts present exogenous peptide during the stimulation phase. The magnitude of the peptide-specific CTL response was dependent on the concentration of peptide added to the APC and specific lysis was inhibited by anti-class I monoclonal antibody (MoAb) but not anti-class II MoAb. Cultures depleted of CD8+ T cells by cell separation with immunomagnetic beads prior to stimulation invariably failed to generate a peptide-specific CTL response. However, the effect of CD4 depletion on CTL activity was equivocal and indicated that a need for CD4+ T cells as accessory helper cells may depend on the efficiency of the APC to elaborate their own help. This model has advantages in the analysis of events involved in the development of CTL activity in vitro.

Cytotoxic T lymphocyte discrimination between type A Epstein-Barr virus transformants is mapped to an immunodominant epitope in EBNA 3.

An immunodominant Epstein-Barr virus (EBV)-specific cytotoxic T lymphocyte (CTL) epitope, represented by peptide 68, has been mapped to the EBV nuclear antigen, EBNA 3. The epitope is recognized by class I-restricted CTLs through HLA-B8 and is functionally active on type A but not type B lymphoblastoid cell lines (LCLs). Herein we show that peptide 68 is not expressed as a functional CTL epitope by type A LCLs infected with an EBV B95-8 isolate. CTLs from cultures stimulated with autologous type A IARC-BL74 or QIMR-WIL LCLs lysed autologous cells stimulated with phytohaemagglutinin (PHA blasts) and coated with exogenous peptide 68. No peptide 68-specific CTLs were generated in cultures stimulated with autologous type A B95-8 or type B AG876 LCLs. However, the B95-8 LCL coated with peptide 68 was effective in the induction of a peptide-specific CTL response. A peptide 68-specific CTL clone failed to lyse the B95-8 LCL, type B AG876 LCL and PHA blasts, although such targets were lysed when coated with peptide 68.