Highly efficient induction of protective immunity by a vaccinia virus vector defective in late gene expression.
(9/1328)
Vaccinia viruses defective in the essential gene coding for the enzyme uracil DNA glycosylase (UDG) do not undergo DNA replication and do not express late genes in wild-type cells. A UDG-deficient vaccinia virus vector carrying the tick-borne encephalitis (TBE) virus prM/E gene, termed vD4-prME, was constructed, and its potential as a vaccine vector was evaluated. High-level expression of the prM/E antigens could be demonstrated in infected complementing cells, and moderate levels were found under noncomplementing conditions. The vD4-prME vector was used to vaccinate mice; animals receiving single vaccination doses as low as 10(4) PFU were fully protected against challenge with high doses of virulent TBE virus. Single vaccination doses of 10(3) PFU were sufficient to induce significant neutralizing antibody titers. With the corresponding replicating virus, doses at least 10-fold higher were needed to achieve protection. The data indicate that late gene expression of the vaccine vector is not required for successful vaccination; early vaccinia virus gene expression induces a potent protective immune response. The new vaccinia virus-based defective vectors are therefore promising live vaccines for prophylaxis and cancer immunotherapy. (+info)
Broad-spectrum protection against tombusviruses elicited by defective interfering RNAs in transgenic plants.
(10/1328)
We have designed a DNA cassette to transcribe defective interfering (DI) RNAs of tomato bushy stunt virus (TBSV) and have investigated their potential to protect transgenic Nicotiana benthamiana plants from tombusvirus infections. To produce RNAs with authentic 5' and 3' termini identical to those of the native B10 DI RNA, the DI RNA sequences were flanked by ribozymes (RzDI). When RzDI RNAs transcribed in vitro were mixed with parental TBSV transcripts and inoculated into protoplasts or plants, they became amplified, reduced the accumulation of the parental RNA, and mediated attenuation of the lethal syndrome characteristic of TBSV infections. Analysis of F1 and F2 RzDI transformants indicated that uninfected plants expressed the DI RNAs in low abundance, but these RNAs were amplified to very high levels during TBSV infection. By two weeks postinoculation with TBSV, all untransformed N. benthamiana plants and transformed negative controls died. Although infection of transgenic RzDI plants initially induced moderate to severe symptoms, these plants subsequently recovered, flowered, and set seed. Plants from the same transgenic lines also exhibited broad-spectrum protection against related tombusviruses but remained susceptible to a distantly related tombus-like virus and to unrelated viruses. (+info)
Mrvi1, a common MRV integration site in BXH2 myeloid leukemias, encodes a protein with homology to a lymphoid-restricted membrane protein Jaw1.
(11/1328)
Ecotropic MuLVs induce myeloid leukemia in BXH2 mice by insertional mutagenesis of cellular proto-oncogenes or tumor suppressor genes. Disease genes can thus be identified by viral tagging as common sites of viral integration in BXH2 leukemias. Previous studies showed that a frequent common integration site in BXH2 leukemias is the Nf1 tumor suppressor gene. Unexpectedly, about half of the viral integrations at Nf1 represented a previously undiscovered defective nonecotropic virus, termed MRV. Because other common integration sites in BXH2 leukemias encoding proto-oncogenes contain ecotropic rather than MRV viruses, it has been speculated that MRV viruses may selectively target tumor suppressor genes. To determine if this were the case, 21 MRV-positive BXH2 leukemias were screened for new MRV common integration sites. One new site, Mrvi1 was identified that was disrupted by MRV in two of the leukemias. Ecotropic virus did not disrupt Mrvi1 in 205 ecotropic virus-positive leukemias, suggesting that Mrvi1 is specifically targeted by MRV. Mrvi1 encodes a novel protein with homology to Jaw1, a lymphoid restricted type II membrane protein that localizes to the endoplasmic reticulum. MRV integration occurs at the 5' end of the gene between two differentially used promoters. Within hematopoietic cells, Mrvi1 expression is restricted to megakaryocytes and some myeloid leukemias. Like Jaw1, which is down-regulated during lymphoid differentiation, Mrv1 is downregulated during monocytic differentiation of BXH2 leukemias. Taken together, these data suggest that MRV integration at Mrvi1 induces myeloid leukemia by altering the expression of a gene important for myeloid cell growth and/or differentiation. Experiments are in progress to test whether Mrvi1 is a tumor suppressor gene. (+info)
Efficient gene delivery to the inflamed colon by local administration of recombinant adenoviruses with normal or modified fibre structure.
(12/1328)
BACKGROUND/AIMS: Replication deficient recombinant adenoviruses represent an efficient means of transferring genes in vivo into a wide variety of dividing and quiescent cells from many different organs. Although the gastrointestinal tract is a potentially attractive target for gene therapy approaches, only a few studies on the use of viral gene transfer vehicles in the gut have been reported. The prospects of using recombinant adenoviruses for gene delivery into epithelial and subepithelial cells of the normal and inflamed colon are here analysed. METHODS: An E1/E3 deleted recombinant adenovirus (denoted AdCMVbetaGal) and an adenovirus with modified fibre structure (denoted AdZ.F(pk7)) both expressing the bacterial lacZ gene under the control of a human cytomegalovirus promoter were used for reporter gene expression in vitro and in vivo. beta-Galactosidase activity was determined by specific chemiluminescent reporter gene assay. RESULTS: Intravenous or intraperitoneal injection of AdCMVbetaGal into healthy Balb/c mice caused strong reporter gene expression in the liver and spleen but not in the colon. In contrast, local administration of AdCMVbetaGal resulted in high reporter gene expression in colonic epithelial cells and lamina propria mononuclear cells. A local route of adenovirus administration in mice with experimental colitis induced by the hapten reagent trinitrobenzenesulphonic acid was next evaluated. Interestingly, rectal administration of AdCMVbetaGal caused a higher beta-galactosidase activity in isolated lamina propria cells from infected mice with experimental colitis than in those from controls. Furthermore, isolated lamina propria cells from mice with colitis infected in vitro showed a significant increase in reporter gene activity compared with controls. Finally, AdZ.F(pk7) adenoviruses with modified fibre structure produced 10- to 40-fold higher reporter gene activity in spleen T cells and lamina propria mononuclear cells of colitic mice compared with standard AdCMVbetaGal vectors. CONCLUSIONS: Local administration of recombinant adenoviruses with normal or modified fibre structure could provide a new reliable method for targeted gene expression in the inflamed colon. Such gene delivery could be used to specifically express signal transduction proteins with therapeutic potential in inflamed colonic tissue. In particular, adenoviruses with modified fibre structure may be useful in T cell directed therapies in intestinal inflammation. (+info)
Induction of genital immunity by DNA priming and intranasal booster immunization with a replication-defective adenoviral recombinant.
(13/1328)
Mice immunized through different routes such as i.m., intradermally, or intratracheally with a DNA vaccine to rabies virus developed high titers of serum Ab but only borderline levels of mucosal Abs determined from vaginal secretions. DNA vaccines given by either route enhanced vaginal IgA and IgG2a secretion upon a subsequent intranasal booster immunization with an E1-deleted adenoviral recombinant expressing the same Ag of rabies virus. DNA vaccine priming reduced the Ab response to the adenoviral Ags and counterbalanced the impaired B cell response to the rabies virus Ag expressed by the adenoviral recombinant in mice preimmune to adenovirus. The vaginal B cell response could further be enhanced by using the Th2-type cytokines IL-4 or IL-5 as genetic adjuvants concomitantly with the DNA vaccine before intranasal booster immunization with the recombinant vaccine. (+info)
Fibroblast growth factor 2 retargeted adenovirus has redirected cellular tropism: evidence for reduced toxicity and enhanced antitumor activity in mice.
(14/1328)
Adenovirus (Ad) have been used as vectors to deliver genes to a wide variety of tissues. Despite achieving high expression levels in vivo, Ad vectors display normal tissue toxicity, transient expression, and antivector immune responses that limit therapeutic potential. To circumvent these problems, several retargeting strategies to abrogate native tropism and redirect Ad uptake through defined receptors have been attempted. Despite success in cell culture, in vivo results have generally not shown sufficient selectivity for target tissues. We have previously identified (C. K. Goldman et al., Cancer Res., 57: 1447-1451, 1997) the fibroblast growth factor (FGF) ligand and receptor families as conferring sufficient specificity and binding affinity to be useful for targeting DNA in vivo. In the present studies, we retargeted Ad using basic FGF (FGF2) as a targeting ligand. Cellular uptake is redirected through high-affinity FGF receptors (FGFRs) and not the more ubiquitous lower-affinity Ad receptors. Initial in vitro experiments demonstrated a 10- to 100-fold increase in gene expression in numerous FGFR positive (FGFR+) cell lines using FGF2-Ad when compared with Ad. To determine whether increased selectivity could be detected in vivo, FGF2-Ad was administered i.v. to normal mice. FGF2-Ad demonstrates markedly decreased hepatic toxicity and liver transgene expression compared with Ad treatment. Importantly, FGF2-Ad encoding the herpes simplex virus thymidine kinase (TK) gene transduces Ad-resistant FGFR+ tumor cells both ex vivo and in vivo, which results in substantially enhanced survival (180-260%) when the prodrug ganciclovir is administered. Because FGFRs are up-regulated on many types of malignant or injured cells, this broadly useful method to redirect native Ad tropism and to increase the potency of gene expression may offer significant therapeutic advantages. (+info)
Intravenous administration of ONYX-015, a selectively replicating adenovirus, induces antitumoral efficacy.
(15/1328)
Replication-incompetent viral vectors are being developed for the gene therapy of cancer. Although some of these may eventually be proven to have significant localized antitumoral activity, none to date have been shown to infect and cause regression of established tumors following i.v. administration. Because cancer is a systemic disease in almost all fatal cases, the lack of i.v. efficacy is a major limitation to treatment with replication-incompetent viral vectors. ONYX-015 (d11520) is an attenuated adenovirus that replicates in and causes selective lysis of cancer cells. We carried out i.v. efficacy and distribution studies in nude mice with s.c. and intraparenchymal tumor xenografts. ONYX-015 infected and replicated efficiently within tumors following i.v. administration. Viral titers in livers were relatively high 3 h after administration but decreased rapidly, becoming undetectable after 24 h. Effective antitumor doses were not associated with hepatic toxicity. Viral replication within tumors was associated with regressions in several tumor models. Selectively replicating viruses like ONYX-015 hold promise as agents to treat metastatic cancer. (+info)
Formation of virus assembly intermediate complexes in the cytoplasm by wild-type and assembly-defective mutant human immunodeficiency virus type 1 and their association with membranes.
(16/1328)
We have previously identified two distinct forms of putative viral assembly intermediate complexes, a detergent-resistant complex (DRC) and a detergent-sensitive complex (DSC), in human immunodeficiency virus type 1 (HIV-1)-infected CD4(+) T cells (Y. M. Lee and X. F. Yu, Virology 243:78-93, 1998). In the present study, the intracellular localization of these two viral assembly intermediate complexes was investigated by use of a newly developed method of subcellular fractionation. In wild-type HIV-1-infected H9 cells, the DRC fractionated with the soluble cytoplasmic fraction, whereas the DSC was associated with the membrane fraction. The DRC was also detected in the cytoplasmic fraction in H9 cells expressing HIV-1 Myr- mutant Gag. However, little of the unmyristylated Gag and Gag-Pol proteins was found in the membrane fraction. Furthermore, HIV-1 Gag proteins synthesized in vitro in a rabbit reticulocyte lysate system in the absence of exogenous lipid membrane were able to assemble into a viral Gag complex similar to that of the DRC identified in infected H9 cells. The density of the viral Gag complex was not altered by treatment with the nonionic detergent Triton X-100, suggesting a lack of association of this complex with endogenous lipid. Formation of the DRC was not significantly affected by mutations in assembly domains M and L of the Gag protein but was drastically inhibited by a mutation in the assembly I domain. Purified DRC could be disrupted by high-salt treatment, suggesting electrostatic interactions are important for stabilizing the DRC. The Gag precursor proteins in the DRC were more sensitive to trypsin digestion than those in the DSC. These findings suggest that HIV-1 Gag and Gag-Pol precursors assemble into DRC in the cytoplasm, a process which requires the protein-protein interaction domain (I) in NCp7; subsequently, the DRC is transported to the plasma membrane through a process mediated by the M domain of the matrix protein. It appears that during this process, a conformational change might occur in the DRC either before or after its association with the plasma membrane, and this change is followed by the detection of virus budding structure at the plasma membrane. (+info)