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Immediate early and early lytic cycle proteins are frequent targets of the Epstein-Barr virus-induced cytotoxic T cell response.

Steven NM, Annels NE, Kumar A, Leese AM, Kurilla MG, Rickinson AB - J. Exp. Med. (1997)

Bottom Line: In several cases, the peptide epitope and HLA-restricting determinant recognized by these CTLs has been defined, one unusual feature being the number of responses restricted through HLA-C alleles.The work strongly suggests that EBV-replicative lesions are subject to direct CTL control in vivo and that immediate early and early proteins are frequently the immunodominant targets.The unique capacity of gamma-herpesvirus to amplify the viral load in vivo through a latent growth-transforming infection may have rendered these agents less dependent upon viral replication as a means of successfully colonizing their hosts.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Campaign Institute for Cancer Studies, University of Birmingham, U.K.

ABSTRACT
Epstein-Barr virus (EBV), a human gamma-herpesvirus, can establish both nonproductive (latent) and productive (lytic) infections. Although the CD8+ cytotoxic T lymphocyte (CTL) response to latently infected cells is well characterized, very little is known about T cell controls over lytic infection; this imbalance in our understanding belies the importance of virus-replicative lesions in several aspects of EBV disease pathogenesis. The present work shows that the primary CD8+ CTL response to EBV in infectious mononucleosis patients contains multiple lytic antigen-specific reactivities at levels at least as high as those seen against latent antigens; similar reactivities are also detectable in CTL memory. Clonal analysis revealed individual responses to the two immediate early proteins BZLF1 and BRLF1, and to three (BMLF1, BMRF1, and BALF2) of the six early proteins tested. In several cases, the peptide epitope and HLA-restricting determinant recognized by these CTLs has been defined, one unusual feature being the number of responses restricted through HLA-C alleles. The work strongly suggests that EBV-replicative lesions are subject to direct CTL control in vivo and that immediate early and early proteins are frequently the immunodominant targets. This contrasts with findings in alpha- and beta-herpesvirus systems (herpes simplex, cytomegalovirus) where viral interference with the antigen-processing pathway during lytic infection renders immediate early and early proteins much less immunogenic. The unique capacity of gamma-herpesvirus to amplify the viral load in vivo through a latent growth-transforming infection may have rendered these agents less dependent upon viral replication as a means of successfully colonizing their hosts.

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Screening of (A) IM69 and (B) IM61 primary effectors immediately ex vivo for evidence of cytotoxicity against the HLA-A2.01– restricted epitope BMLF1 280-288. Assays were conducted on autologous or on HLA-A2.01, B7-matched PHA blast targets pretreated with  the BMLF1 280-288 peptide, with another A2.01-restricted epitope peptide LMP2 329-337, with the B7-restricted epitope peptides EBNA3A  379-387 and EBNA3C 881-891, or with DMSO alone as a “no peptide”  control. Results are expressed as in Fig. 1 and both assays were conducted  at E/T ratios of 100:1 (▪) and 50:1 (▨ ).
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Figure 5: Screening of (A) IM69 and (B) IM61 primary effectors immediately ex vivo for evidence of cytotoxicity against the HLA-A2.01– restricted epitope BMLF1 280-288. Assays were conducted on autologous or on HLA-A2.01, B7-matched PHA blast targets pretreated with the BMLF1 280-288 peptide, with another A2.01-restricted epitope peptide LMP2 329-337, with the B7-restricted epitope peptides EBNA3A 379-387 and EBNA3C 881-891, or with DMSO alone as a “no peptide” control. Results are expressed as in Fig. 1 and both assays were conducted at E/T ratios of 100:1 (▪) and 50:1 (▨ ).

Mentions: We subsequently went on to test a number of IM patients who did not have the HLA-B8 allele to look for evidence of other immunodominant reactivities to lytic cycle antigens. Analysis of IM69 (HLA-A2.01, A32, B7, B63), whose predominant latent antigen-specific response had earlier been mapped to the HLA-B7–restricted epitope EBNA3A 379-387 (7), yielded a number of CTL clones which strongly recognized the vacc-BMLF1–infected autologous LCL in the original screening assays and which proved to be HLA-A2.01 restricted on extension of the work to vacc-BMLF1–infected allogeneic targets (Fig. 4 A). Peptide sensitization assays with the BMLF1 panel of synthetic peptides first mapped recognition to the 15-mer BMLF1 276-290 and subsequent assays identified the minimal epitope as BMLF1 residues 280-288, GLCTLVAML (Fig. 4 B); again, this accords well to the consensus sequence for A2.01-binding peptides (39). In view of the number of clones from IM69 which recognized the epitope, we tested cryopreserved effectors from this individual and from a second HLA-A2.01, B7-positive patient IM61 for evidence of BMLF1 280288–specific lysis in ex vivo assays. The results are shown in Fig. 5. In both cases, the response to this A2.01-restricted BMLF1 epitope was easily detectable in the primary CTL population ex vivo and, in fact, appeared to be as strong as the immunodominant latent antigen-specific response recognizing the B7-restricted EBNA3A 379-387 epitope.


Immediate early and early lytic cycle proteins are frequent targets of the Epstein-Barr virus-induced cytotoxic T cell response.

Steven NM, Annels NE, Kumar A, Leese AM, Kurilla MG, Rickinson AB - J. Exp. Med. (1997)

Screening of (A) IM69 and (B) IM61 primary effectors immediately ex vivo for evidence of cytotoxicity against the HLA-A2.01– restricted epitope BMLF1 280-288. Assays were conducted on autologous or on HLA-A2.01, B7-matched PHA blast targets pretreated with  the BMLF1 280-288 peptide, with another A2.01-restricted epitope peptide LMP2 329-337, with the B7-restricted epitope peptides EBNA3A  379-387 and EBNA3C 881-891, or with DMSO alone as a “no peptide”  control. Results are expressed as in Fig. 1 and both assays were conducted  at E/T ratios of 100:1 (▪) and 50:1 (▨ ).
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Related In: Results  -  Collection

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Figure 5: Screening of (A) IM69 and (B) IM61 primary effectors immediately ex vivo for evidence of cytotoxicity against the HLA-A2.01– restricted epitope BMLF1 280-288. Assays were conducted on autologous or on HLA-A2.01, B7-matched PHA blast targets pretreated with the BMLF1 280-288 peptide, with another A2.01-restricted epitope peptide LMP2 329-337, with the B7-restricted epitope peptides EBNA3A 379-387 and EBNA3C 881-891, or with DMSO alone as a “no peptide” control. Results are expressed as in Fig. 1 and both assays were conducted at E/T ratios of 100:1 (▪) and 50:1 (▨ ).
Mentions: We subsequently went on to test a number of IM patients who did not have the HLA-B8 allele to look for evidence of other immunodominant reactivities to lytic cycle antigens. Analysis of IM69 (HLA-A2.01, A32, B7, B63), whose predominant latent antigen-specific response had earlier been mapped to the HLA-B7–restricted epitope EBNA3A 379-387 (7), yielded a number of CTL clones which strongly recognized the vacc-BMLF1–infected autologous LCL in the original screening assays and which proved to be HLA-A2.01 restricted on extension of the work to vacc-BMLF1–infected allogeneic targets (Fig. 4 A). Peptide sensitization assays with the BMLF1 panel of synthetic peptides first mapped recognition to the 15-mer BMLF1 276-290 and subsequent assays identified the minimal epitope as BMLF1 residues 280-288, GLCTLVAML (Fig. 4 B); again, this accords well to the consensus sequence for A2.01-binding peptides (39). In view of the number of clones from IM69 which recognized the epitope, we tested cryopreserved effectors from this individual and from a second HLA-A2.01, B7-positive patient IM61 for evidence of BMLF1 280288–specific lysis in ex vivo assays. The results are shown in Fig. 5. In both cases, the response to this A2.01-restricted BMLF1 epitope was easily detectable in the primary CTL population ex vivo and, in fact, appeared to be as strong as the immunodominant latent antigen-specific response recognizing the B7-restricted EBNA3A 379-387 epitope.

Bottom Line: In several cases, the peptide epitope and HLA-restricting determinant recognized by these CTLs has been defined, one unusual feature being the number of responses restricted through HLA-C alleles.The work strongly suggests that EBV-replicative lesions are subject to direct CTL control in vivo and that immediate early and early proteins are frequently the immunodominant targets.The unique capacity of gamma-herpesvirus to amplify the viral load in vivo through a latent growth-transforming infection may have rendered these agents less dependent upon viral replication as a means of successfully colonizing their hosts.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research Campaign Institute for Cancer Studies, University of Birmingham, U.K.

ABSTRACT
Epstein-Barr virus (EBV), a human gamma-herpesvirus, can establish both nonproductive (latent) and productive (lytic) infections. Although the CD8+ cytotoxic T lymphocyte (CTL) response to latently infected cells is well characterized, very little is known about T cell controls over lytic infection; this imbalance in our understanding belies the importance of virus-replicative lesions in several aspects of EBV disease pathogenesis. The present work shows that the primary CD8+ CTL response to EBV in infectious mononucleosis patients contains multiple lytic antigen-specific reactivities at levels at least as high as those seen against latent antigens; similar reactivities are also detectable in CTL memory. Clonal analysis revealed individual responses to the two immediate early proteins BZLF1 and BRLF1, and to three (BMLF1, BMRF1, and BALF2) of the six early proteins tested. In several cases, the peptide epitope and HLA-restricting determinant recognized by these CTLs has been defined, one unusual feature being the number of responses restricted through HLA-C alleles. The work strongly suggests that EBV-replicative lesions are subject to direct CTL control in vivo and that immediate early and early proteins are frequently the immunodominant targets. This contrasts with findings in alpha- and beta-herpesvirus systems (herpes simplex, cytomegalovirus) where viral interference with the antigen-processing pathway during lytic infection renders immediate early and early proteins much less immunogenic. The unique capacity of gamma-herpesvirus to amplify the viral load in vivo through a latent growth-transforming infection may have rendered these agents less dependent upon viral replication as a means of successfully colonizing their hosts.

Show MeSH
Related in: MedlinePlus