The glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei (optimal growth temperature, 100 to 103°C) was purified to homogeneity. This enzyme was strictly phosphate dependent, utilized either NAD+ or NADP+, and was insensitive to pentalenolactone like the enzyme from the methanogenic archaebacterium Methanothermus fervidus. The enzyme exhibited a considerable thermostability, with a 44-min half-life at 100°C. The amino acid sequence of the glyceraldehyde-3-phosphate dehydrogenase from P. woesei was deduced from the nucleotide sequence of the coding gene. Compared with the enzyme homologs from mesophilic archaebacteria (Methanobacterium bryantii, Methanobacterium formicicum) and an extremely thermophilic archaebacterium (Methanothermusfervidus), the primary structure of the P. woesei enzyme exhibited a strikingly high proportion of aromatic amino acid residues and a low proportion of sulfur-containing residues. The coding gene ofP. woesei was expressed at a high level in Escherichia coli, thus providing an ideal basis for detailed structural and functional studies of that enzyme.The extraordinary ability to grow above 100°C is exclusively restricted to certain members of archaebacteria. These hyperthermophilic strains with optimal growth temperatures ranging from 100 to 105°C are represented by four genera: Pyrodictium (41), Pyrococcus (19), Pyrobaculum (26), and Methanopyrus (27).Although the hyperthermophily brings up evident questions concerning the molecular background of thermoadaptation, knowledge of the macromolecular cell constituents of these organisms is scarce. Thus, with the exception of the reports on hydrogenase and ferredoxin from Pyrococcus furiosus (5, 10), no description of any protein from these hyperthermophiles has yet been published.Here we characterize some phenotypic properties of the glyceraldehyde-3-phosphate dehydrogenase from Pyrococcus woesei (optimal growth temperature, 100 to 103°C [47]) and report on the cloning and sequencing of the coding gene as well as on its expression in Escherichia coli.To get indications about the structural adaptation to the extreme growth temperatures, we compared the sequence of the glyceraldehyde-3-phosphate dehydrogenase of P. woesei with the structures of the enzyme homologs from mesophilic and thermophilic archaebacteria (17). MATERIALS AND METHODSBacterial strains. Cells of P. woesei Vul4 (DSM 3773) were grown as described previously (47). For cloning and expression of the glyceraldehyde-3-phosphate dehydrogenase gene, the E. coli K-12 strains JM83 [ara A(lac-proAB) strA thi 4)80dlacZAM15 (33)] and DH5a [F-endAl hsdRJ7 (rK-MK+) supE44 thi-J X-recAl gyrA96 relAl A(lacZYAargF)U169 4.80dlacZAM15 (28)] were used.Plasmids, enzymes, chemicals. The vectors for cloning and sequencing were pUC18 and M13mpl8/19 (34), respectively; the expression plasmid was pJF118EH (20).

We identified the immediate-early transactivator Zta of Epstein-Barr virus as a target for specific cytotoxic T lymphocytes (CTL). Cells pulsed with overlapping synthetic peptides representing the entire amino acid sequence of Zta proved to be efficient for the in vitro stimulation of Zta-specific CTL in several donors. With peptide-pulsed target cells, we found that CTL from several donors recognize a peptide comprising 15 amino acids. The immune response against this peptide exerted by CTL lines from different donors was found to be restricted by two different molecules of the major histocompatibility complex: HLA-B8 and HLA-Cw6. The latter molecule could for the first time be identified as a restricting element for a CTL response. The epitope of the HLA-B8-restricted CTL could be mapped to an octameric sequence between amino acid positions 190 and 197 of the Zta protein, whereas the minimal epitope of HLA-Cw6-restricted CTL consists of 11 to 15 residues between positions 187 and 201. Thus, the HLA-B8 and HLA-Cw6 epitopes widely overlap but are not completely identical. In vitro stimulation of blood lymphocytes from a panel of HLA-B8-positive or HLA-Cw6positive virus carriers, using autologous cells pulsed with the Zta peptides comprising the HLA-B8 or HLA-Cw6 epitope, respectively, revealed in both cases that most of these donors developed a Zta-specific cytotoxic activity. These data, as well as the high spread of the major histocompatibility complex molecules HLA-B8 and HLA-Cw6 in most populations, suggest that an efficient CTL response directed against gene products of the immediate-early group of the lytic cycle exists in vivo in a considerable portion of virus carriers. A CTL response against proteins expressed immediately after the switch into the lytic cycle could eliminate lytically activated cells at an early stage and would thus efficiently prevent the production and release of progeny virions.

Epstein-Barr virus (EBV) as a member of the herpesvirus family persists lifelong in the human body and causes diseases associated with virus replication (infectious mononucleosis, oral hairy leukoplakia) as well as neoplastic conditions such as nasopharyngeal carcinoma, B-cell lymphoma, Hodgkin’s disease associated with viral latency. This complex biology relates to a highly regulated control of the persisting virus. Still, EBV is lytically produced in certain compartments of the human body. Epithelial cells were found to be of key importance for this. Various routes (cell fusion, IgA receptor-mediated uptake) were described for EBV to enter epithelial cells in the absence of CR2 receptor. Viral entry into cells, however, via CR2 receptor fusion or IgA mediated was not found to be sufficient for viral production. The molecular mechanisms for the lack of viral production in most target cells are primarily the presence of silencer activities and the early elimination of cells entering the lytic cycle. Only terminally differentiated epithelial cells are capable of supporting an efficient lytic cycle of EBV replication. EBV-mediated suppression of apoptosis as well as down-regulation of cellular and viral gene products, such as HLA molecules, which mediate recognition by the immune system, are important contributing factors to the development of these neoplasias where viral genes, possibly via interaction with anti-oncogenes, such as p53, in context with genetic and environmental factors play a key role. Novel diagnostic tools and a vaccine have been developed which could help to control EBV-related diseases.

To explore the potential of recombinant vectors based on recombinant adeno-associated virus (rAAV) for cancer vaccination, we investigated the transduction efficiency of rAAV into cancer cells ex vivo. Infection of human epithelial cancer cell lines with rAAV carrying reporter genes encoding beta-galactosidase (rAAV/LacZ) or luciferase (rAAV/Luc) resulted in high levels of reporter gene expression (>90% positive cells). In marked contrast, rAAV poorly transduced all murine tumor cell lines, as well as human hematopoietic cell lines. Either irradiation or adenovirus infection of tumor cells prior to rAAV infection induced a 10- to 100-fold increase of reporter gene expression. To determine the transduction efficiency of rAAV into primary cancer cells, freshly isolated, irradiated tumor cells from malignant melanoma and ovarian carcinoma patients were infected with rAAV/Luc, resulting in up to 6.9-fold higher levels of gene expression than in a HeLa tumor cell line. Time course experiments with freshly isolated tumor cells infected with rAAV/Luc showed maximal levels of luciferase expression between days 3 and 9 posttransduction. Simultaneous infection of primary tumor cells with up to three rAAV vectors containing genes encoding the immunostimulatory proteins B7-2 (CD86), p35 subunit of IL-12, and p40 subunit of IL-12 resulted in high expression of B7-2 in more than 90% of the tumor cells and in the secretion of high levels of IL-12. Taken together, our results demonstrate that rAAV efficiently transduces freshly isolated human, epithelial tumor cells and might therefore be a potent tool to produce improved, gene-modified cancer vaccines.