We investigated CD8(+) T cell frequencies of five different Epstein-Barr virus-specific cytotoxic T lymphocyte epitopes located within proteins of the replicative cycle and the latent state in healthy long-term virus carriers with IFN-gamma enzyme-linked immunospot assay. Frequencies of the HLA-A3-restricted epitope RVRAYTYSK (RVR) whose minimal length was mapped in this study to amino acid position 148-156 of the immediate-early protein BRLF1 were compared with those of a further known HLA-A3-restricted epitope within EBNA3A, RLRAEAQVK (RLR). Determination of frequencies of CD8(+) T lymphocytes directed against lytic antigen epitope RVR revealed that only one of eight donors recognized this epitope. Frequency was calculated to be 65 RVR-specific CD8(+) T lymphocytes per 10(6) PBMC. None of the HLA-A3-positive donors exhibited IFN-gamma release after antigenic stimulation with the EBNA3A-specific peptide epitope RLR. Furthermore, we chose three known HLA-B8-restricted epitopes, RAKFKQLL (RAK), FLRGRAYGL (FLR), and QAKWRLQTL (QAK), of the lytic protein BZLF1 and the latent protein EBNA3A. Examination of eight HLA-B8-positive virus carriers revealed that the BZLF1-specific epitope RAK was recognized by all donors with a median frequency of 233 RAK-specific CD8(+) T lymphocytes per 10(6) PBMC. Only 50% of these donors reacted against EBNA3A-specific epitope FLR and a minority (25%) reacted against EBNA3A-specific epitope QAK.

We analyzed the immediate-early transactivator Rta of Epstein-Barr virus (EBV) for its role as a target for specific cytotoxic T lymphocytes (CTL). Panels of overlapping peptides covering the entire amino acid sequence of Rta were synthesized and used to induce and analyze specific CTL responses in EBV-positive donors. Using peptide-pulsed target cells, we found nine different CTL epitopes that are distributed over the entire protein sequence. One epitope restricted by HLA-A24 could be mapped to the decameric sequence DYCNVLNKEF between amino acid positions 28 and 37 of the Rta protein. A second epitope could be assigned to the same region of Rta (residues 25 to 39) and was shown to be restricted by HLA-B18. Another, minimal epitope could be mapped to the nonameric sequence ATIGTAMYK between amino acid positions 134 and 142; this peptide was restricted by HLA-A11. Another four epitopes were proven to be restricted by HLA-A2, -A3, -B61, and -Cw4 and were located between Rta residues 225 and 239, 145 and 159, 529 and 543, and 393 and 407, respectively. For two other epitopes, only the location within the Rta protein is known so far (residues 121 to 135 and 441 to 455); their exact HLA restriction patterns have not yet been identified. Using target cells infected with recombinant vaccinia virus containing the gene for Rta, we showed that six of eight Rta-specific CTL lines recognized the corresponding peptides also after endogenous processing. These data suggest that Rta comprises an important target for EBV-specific cellular cytotoxicity. Together with recent findings of other immediate-early and early proteins also acting as CTL targets, they reveal the role of proteins of the lytic cycle in the immune recognition of EBV-infected cells.

Primary infection with the Epstein‐Barr virus (EBV) results in fatal infectious mononucleosis in up to 70% of males affected by the X‐linked lymphoproliferative syndrome (XLP). This rare disease is often associated with diverse natural killer (NK)‐, B‐ and T‐cell deficiencies. We describe experiments testing whether the B lymphocytes of affected males play a role in the pathogenesis of XLP due to a low susceptibility to T‐cell‐mediated immunity. Using reverse transcription‐polymerase chain reaction (RT‐PCR) and immunohistochemistry we detected in these B cells the expression of viral proteins EBNA‐1, EBNA‐2, EBNA‐3A, EBNA‐3C, LMP‐1 and LMP‐2A, which provide targets for cytotoxic T cells. Major histocompatibility complex (MHC) class I, MHC class II and the B7 costimulatory molecule were present on the cell surface. Accordingly, the EBV‐infected B cells were lysed in 51Cr‐release assays by T lymphocytes sharing MHC determinants with the targets. This MHC‐restricted and specific lysis was confirmed in competition experiments using MHC‐specific monoclonal antibodies (MAbs) and synthetic peptides. XLP‐derived LCLs could also induce MHC class I‐restricted memory and cytotoxic T lymphocytes. Thus, these XLP‐derived B cells resembled normal LCIs in vitro with respect to induction of EBV‐specific cytotoxic T cells (CTL), the ability to present EB viral antigens and the susceptibility to EBV‐specific and MHC‐restricted CTL‐mediated killing. The failure of the immune system to eliminate these virus‐infected B cells in XLP is clearly not caused by a B‐cell‐specific defect. Int. J. Cancer 76:694–701, 1998.© 1998 Wiley‐Liss, Inc.

Non-commercial clinical trials may be of great benefit to the patients concerned. The 12th amendment to the German Drug Law (AMG) changed legal liability of the initiators of investigator-initiated clinical trials with extensive consequences for traditional project leaders. The central point under discussion is the sponsor's responsibility according to the AMG. Presently leading management divisions of university hospitals and universities are developing proceedings to assume sponsor responsibility by institutions (institutional sponsorship), which should enable investigator-initiated clinical trials to be conducted according to legal requirements in the future. Detailed problems and special questions can only be resolved in a single-minded fashion, and if necessary political processes should be catalyzed.

Cure rates in children with cancer exceed 75 % today. Notwithstanding this high success rate, 60-90 % of drugs employed in Paediatric Oncology are used off label. Comprehensive pre-clinical screening of candidate substances may help to generate entity-specific priority lists and to be faster and more selective in feeding promising candidates into the development process of drugs for use in children. The upcoming EU regulation "Better medicine for children" offers professional societies and their trial groups the opportunity to upgrade existing structures and benefit from the expected increase in paediatric clinical trials. Networking joining more national and international institutions and boards may serve to expand the repertoire of clinical trials and to establish powerful structures. Such a network may be an attractive partner for the drug industry and may thus eventually lead to a reduction in off label use in paediatric oncology.