Recombinant adeno-associated virus for the generation of autologous, gene-modified tumor vaccines: evidence for a high transduction efficiency into primary epithelial cancer cells.

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.

Engineering thermostability in archaebacterial glyceraldehyde-3-phosphate dehydrogenase. Hints for the important role of interdomain contacts in stabilizing protein conformation.

Construction of hybrid enzymes between the glyceraldehyde-3-phosphate dehydrogenases from the mesophilic Methanobacterium bryantii and the thermophilic Methanothermus fervidus by recombinant DNA techniques revealed that a short C-terminal fragment of the Mt. fervidus enzyme contributes largely to its thermostability. This C-terminal region appears to be homologous to the alpha 3-helix of eubacterial and eukaryotic glyceraldehyde-3-phosphate dehydrogenases which is involved in the contacts between the two domains of the enzyme subunit. Site-directed mutagenesis experiments indicate that hydrophobic interactions play an important role in these contacts.

Recombinant adeno-associated virus vectors.

Recombinant adeno-associated virus (rAAV) is a promising vector for somatic gene therapy due to the ability to transduce terminally-differentiated and non-dividing cells, the lack of any apparent pathogenicity, a low immunogenicity, a relatively high stability of transgene expression, and the potential for targeted integration. Improved methods of rAAV packaging allow the generation of concentrated and highly purified rAAV for clinical trials. Preclinical studies with rAAV are currently in progress for the treatment of a variety of inherited monogenic defects, but also for acquired diseases like HIV infection and cancer. Because of the broad host range of wild type AAV, rAAV is able to transduce a variety of human tissues, preferentially those of epitheloid origin, with high efficiency and therefore may be used for various clinical applications. Whilst several issues including safety, tissue tropism and methods to achieve site-specific integration need further improvement, rAAV certainly has a sufficient number of advantages to be seriously considered as a gene therapy vector.

Epstein-Barr virus and its interaction with the host.

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.

Glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei: characterization of the enzyme, cloning and sequencing of the gene, and expression in Escherichia coli.

The glyceraldehyde-3-phosphate dehydrogenase from the hyperthermophilic archaebacterium Pyrococcus woesei (optimal growth temperature, 100 to 103 degrees 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 degrees 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 (Methanothermus fervidus), 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 of P. woesei was expressed at a high level in Escherichia coli, thus providing an ideal basis for detailed structural and functional studies of that enzyme.

Specific cytotoxic T lymphocytes recognize the immediate-early transactivator Zta of Epstein-Barr virus.

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-Cw6-positive 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.

Different activation of Epstein-Barr virus immediate-early and early genes in Burkitt lymphoma cells and lymphoblastoid cell lines.

Specific expression of the Epstein-Barr virus (EBV) immediate-early and early gene products Zta, Rta, I'ta, and MSta by a recombinant vaccinia virus system allowed us to analyze the first steps in the induction of the lytic cycle in EBV-infected Burkitt lymphoma (BL) cells and lymphoblastoid cell lines (LCLs). Significant differences in the induction of early genes were found between these cell types: whereas in BL cells the trans activator Zta was found to induce key steps of the early lytic cycle, only minor activities of Zta were noted in LCLs. Contrary to Zta, the trans activator Rta was found to be highly effective in LCLs. These observations suggest that Rta may play an important role in the activation of the early lytic cycle in LCLs, although it cannot be activated by Zta. The latter may be a reason for the lower tendency of LCLs to switch into the lytic cycle compared with BL cells or differentiated epithelial cells.

The Epstein-Barr virus-encoded glycoprotein gp 110 (BALF 4) can serve as a target for antibody-dependent cell-mediated cytotoxicity (ADCC).

Antibody-dependent cell-mediated cytotoxicity (ADCC) is thought to play a major role in controlling the spread of the Epstein-Barr virus (EBV) in an infected individual. Recently, the viral membrane protein gp 350/220, which is also expressed at the surface of the virus producing cell, was identified as a target for ADCC reactions. Due to its glycoprotein nature, the EBV protein gp 110 is another possible ADCC target. It is one of the most abundant proteins found during the late phase of viral replication; until now, however, researchers have not been able to localize it on the surface of EBV positive cells. By means of recombinant vaccinia viruses containing the genes for gp 350/220 and gp 110, respectively, we expressed these proteins in lymphoblastoid cells, which were then used as targets in ADCC studies with sera from EBV-positive and -negative individuals. In these experiments we were able to demonstrate the feasibility of our approach for the investigation of EBV-specific ADCC reactions and could confirm the role of gp 350/220 as an ADCC target. Furthermore, we were able to show that gp 110 can also be recognized in an ADCC reaction, proving that at least some gp 110 molecules must be expressed at the cell surface.

The use of recombinant adeno-associated viral vectors for the transduction of epithelial tumor cells.

Using hight-titer recombinant adeno-associated viral vectors (rAAV), we have investigated the feasibility of cancer vaccines from tumor explants. In a first set of experiments, rAAV vectors expressing firefly luciferase reporter genes were used to transduce different human tumor cell lines. At day three post transduction, all of the human tumor cell lines tested showed high levels of luciferase expression. To further evaluate rAAV-mediated gene transfer efficiency into primary tumor cells, we transduced freshly isolated tumor cells from malignant melanoma and ovarian carcinoma patients. As a remarkable result, reporter gene expression in primary tumor cells was significantly higher than in the tested established tumor cell lines. These data could also be reproduced with a rAAV/lacZ vector, since the portion of successfully transduced primary tumor was higher than 90%. Taken together, our data demonstrate that rAAV-mediated gene transfer is a very efficient method for the transduction of freshly isolated human tumor cells and may allow the generation of potent autologous cancer vaccines.

The lytic transition of Epstein-Barr virus is imitated by recombinant B-cells.

Lytic transition of Epstein-Barr virus (EBV) is initiated by distinct immediate early regulators of the viral cycle, in synchronization to temporary, permissive conditions during host cell differentiation. We developed eukaryotic vectors suitable to imitate the processes involved in lytic transition in cell culture systems. Two stable B cell lines were established: R59Z activator cells were used to induce lytic EBV expression in a constitutive manner by the production of the BZLF 1 trans-activator (Zta). R7-57 reporter cells, on the other hand, signaled induced activity of the lytic origin of EBV replication (ori Lyt). Different modes, like chemical induction, lytic superinfection with EBV and single gene trans-activation converted the recombinant ori Lyt element in R7-57 reporter cells. BZLF 1, transiently expressed in R7-57 reporter cells, was the only EBV trans-activator found, sufficient in inducing the viral lytic cycle. Basing on these experiments, trans-cellular activation of EBV was tested by cocultivation of BZLF 1-expressing R59Z activator cells with the R7-57 reporter line. No lytic effect on the reporter cells could be measured, neither by cocultivation of activator cells nor by coincubation of BZLF 1-containing cell lysates. Latency breaking activity, however, was transferred from activator to reporter cells when active, exogenous virus was added. The cell system described in these experiments provides a tool for the detection of EBV reactivation and demonstrates the potential of the lytic regulatory gene BZLF 1.

Efficient foreign gene expression in Epstein-Barr virus-transformed human B-cells.

Epstein-Barr virus (EBV) is a herpesvirus that transforms B-cells (B-LCL) and has undergone intense scrutiny owing to its association with Burkitt's lymphoma, nasopharyngeal carcinoma, and immunoblastic lymphomas. B-LCL have also proven useful in the study of human immunology. We describe a novel system for inducing efficient foreign gene expression in B-LCL using biotinylated adenovirus as an endosome-disrupting agent. Plasmid DNA is coupled to the exterior of viral particles by streptavidin-polylysine chimeric proteins. Up to 67% of B-LCL may be induced to express foreign genes in vitro in transient expression systems, and gene expression lasts for at least 17 days. We have expressed firefly luciferase, beta-galactosidase (beta-gal), chloramphenicol acetyltransferase, HIV gag, and env genes, as well as infectious HIV, and the EBV-specific BZLF gene in B-LCL with this system. In vivo delivery of a beta-gal reporter gene to B-LCL was documented in a SCID mouse model. Potential applications include study of genetic regulation of EBV infection and transformation events, study of potential gene therapies for EBV-related B-cell tumors, and production of antigen-presenting cells for use in immunologic assays. Because of the high percentage of cells transformed and the length of foreign gene expression, the possibility of examining foreign gene expression in transient assays, without selection for clonal populations, exists.