Infection of fibroblast cell cultures with human cytomegalovirus (HCMV) leads to the production of significant amounts of defective enveloped particles, termed dense bodies (DB). These noninfectious structures contain major antigenic determinants which are responsible for induction of both the humoral and the cellular immune response against HCMV. We tested the hypothesis that, by virtue of their unique antigenic and structural properties, DB could induce a significant immune response in the absence of infectious virus. Mice were immunized with gradient-purified DB, which were either left untreated or subjected to sequential rounds of sonication and freeze-thawing to prevent cellular entry. Titers of neutralizing antibodies induced by DB were in a range comparable to levels present in convalescent human sera. The virus-neutralizing antibody response was surprisingly durable, with neutralizing antibodies detected 12 months following primary immunization. The HCMV-specific major histocompatibility complex class I-restricted cytolytic T-cell (CTL) response was assayed using mice transgenic for the human HLA-A2 molecule. Immunization with DB led to high levels of HCMV-specific CTL in the absence of de novo viral protein synthesis. Maximal total cytolytic activity in mice immunized with DB was nearly as efficient as the cytolytic activity induced by a standard immunization with murine cytomegalovirus. Furthermore, DB induced a typical T-helper 1 (Th1)-dominated immune response in mice, as determined by cytokine and immunoglobulin G isotype analysis. Induction of humoral and cellular immune responses was achieved without the concomitant use of adjuvant. We thus propose that DB can serve as a basis for the future development of a recombinant nonreplicating vaccine against HCMV. Finally, such particles could be engineered for efficient delivery of antigens from other pathogens to the immune system.
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.
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