A T-cell HCV vaccine eliciting effective immunity against heterologous virus challenge in chimpanzees.

Three percent of the world's population is chronically infected with the hepatitis C virus (HCV) and at risk of developing liver cancer. Effective cellular immune responses are deemed essential for spontaneous resolution of acute hepatitis C and long-term protection. Here we describe a new T-cell HCV genetic vaccine capable of protecting chimpanzees from acute hepatitis induced by challenge with heterologous virus. Suppression of acute viremia in vaccinated chimpanzees occurred as a result of massive expansion of peripheral and intrahepatic HCV-specific CD8(+) T lymphocytes that cross-reacted with vaccine and virus epitopes. These findings show that it is possible to elicit effective immunity against heterologous HCV strains by stimulating only the cellular arm of the immune system, and suggest a path for new immunotherapy against highly variable human pathogens like HCV, HIV or malaria, which can evade humoral responses.

Stringent control of gene expression in vivo by using novel doxycycline-dependent trans-activators.

The tetracycline (Tet)-dependent regulatory system has been widely used for controlling gene expression. The Tet-on version of the system, in which the reverse Tet-responsive transcriptional activator (rtTA) is positively regulated by Tet or its analogs, such as doxycycline (Dox), is of potential utility for gene therapy applications in humans. However, rtTA may display a high basal activity, especially when delivered in vivo by using episomal vectors such as plasmids. Two novel Dox-inducible activators, called rtTA2(S)-S2 and rtTA2(S)-M2, which have a significantly lower basal activity than rtTA in stably transfected cell lines, have been described. In this study we tested the capability of these trans-activators to control expression of mouse erythropoietin (mEpo) and to modulate hematocrit (Hct) increase in vivo on delivery of plasmids into quadriceps muscles of adult mice by DNA electroinjection. Both rtTA2(S)-M2 and rtTA2(S)-S2 displayed a considerably lower background activity and higher window of induction than rtTA in vivo. Moreover, a stringent control of mEpo gene expression and Hct levels in the absence of any background activity was maintained over a 10-month period by injecting as little as 1 microg of a single plasmid containing the rtTA2(S)-S2 expression cassette and the Tet-responsive mEpo cDNA. This constitutes the first report of a stringent ligand-dependent control of gene expression in vivo obtained by delivering a single plasmid encoding both the trans-activator and the regulated gene. Notably, the rtTA2(S)-S2-based system was induced by oral doses of doxycycline comparable to those normally used in clinical practice in humans.

An adenovirus type 5 (Ad5) amplicon-based packaging cell line for production of high-capacity helper-independent deltaE1-E2-E3-E4 Ad5 vectors.

Production of multiply deleted adenoviral (Ad) vectors with increased cloning capacity and reduced immunogenicity to adenovirus gene products requires the concomitant generation of efficient packaging cell lines. High expression levels of the complementing genes must be achieved in a coordinated fashion with viral replication. This is a particularly difficult task in light of the significant cytotoxicity displayed by adenoviral proteins. To this end, we developed a novel adenovirus-based amplicon with an Epstein-Barr virus origin of replication, Ad type 5 (Ad5) inverted terminal repeats, all Ad5 early region 2 (E2) genes, and the early region 4 (E4) open reading frame 6 (ORF6) under the control of a tetracycline-dependent promoter. The amplicon (pE2) was stably maintained in multiple copies in the nuclei of 293 cells stably expressing the Epstein-Barr virus nuclear antigen 1 (EBNA1) and allowed replication as a linear DNA upon induction of E2 and ORF6 gene expression. A stable cell line (2E2) was generated by introducing pE2 into 293EBNATet cells expressing the tetracycline-dependent transcriptional silencer and the reverse Tet transactivator (rtTA2). Upon induction with doxicycline, 2E2 cells produced higher levels of polymerase, precursor terminal protein (pTP), and DNA binding protein than noninduced 2E2 cells infected with first-generation Ad5 vector and supported efficient amplification of a multiply deleted Ad5 vector lacking E1, E2, E3, and E4 genes (Ad5DeltaE(1-4)). The high cloning capacity of Ad5DeltaE(1-4) (up to 12.6 kb) was exploited to construct a vector encoding the entire hepatitis C virus (HCV) polyprotein. Infection of HeLa cells by the resulting vector showed high levels of correctly processed HCV proteins.

Immune responses against tetracycline-dependent transactivators affect long-term expression of mouse erythropoietin delivered by a helper-dependent adenoviral vector.

BACKGROUND: Helper-dependent adenoviral (HD-Ad) vectors give rise to sustained gene expression after delivery in a variety of organisms. In particular, we previously documented persistent expression of erythropoietin (EPO) in mice after a single intramuscular (i.m.) injection of a HD-Ad vector harboring the mouse EPO cDNA. METHODS: We use the same vector harboring the tetracycline (tet)-dependent transactivator (rtTA2S-M2) and silencer (tTS) and mouse EPO cDNA to analyze the capacity of the dual tet-dependent transactivator system to control long-term EPO gene expression and to study the effect of an eventual immune response against these artificial proteins after i.m. delivery in immuno-competent mice. RESULTS: In the present study we demonstrate that i.m. injection of this vector in immuno-competent mice generates a cellular immune response to the rtTA2S-M2 transcription factor. This response curtails the duration of mouse EPO expression in mice, presumably by destroying the cells that co-express transcription factors and the therapeutic gene. Nonetheless, regulation of mouse EPO secretion was maintained during the entire experimental period, both when the vector dosage was reduced and when the tet-dependent transcription factors were put under the control of a muscle-specific promoter. CONCLUSIONS: Delivery of the tet transactivators using as vehicle a HD-Ad vector induced an immune response directed against the transactivators themselves, causing short-term therapeutic transgene expression. Regulated, long-term therapeutic transgene expression was, however, obtained by reducing the vector dose or expressing the transactivators under the control of a muscle-specific promoter.

Long-term and tight control of gene expression in mouse skeletal muscle by a new hybrid human transcription factor.

Diseases requiring frequent and lifelong injections of recombinant proteins would be more efficaciously treated by intramuscular delivery of genes encoding secretable proteins. However, the success of this approach largely depends on our capability to temporally regulate transcription of delivered genes. Therefore, we sought to generate a humanized transcription factor to regulate transgene expression in muscle. A novel 4-hydroxytamoxifen (4-OHT)-dependent transcriptional regulator (called HEA-3) was constructed by fusing in-frame the DNA binding domain of the human hepatocyte nuclear factor-1alpha (HNF1alpha), which is not expressed in muscle cells, a G(521)R mutant of the ligand binding domain of human estrogen receptor-alpha (ERalpha), and the activation domain derived from human nuclear factor-kappaB p65 subunit (NF-kappaB p65). We demonstrate that an artificial promoter containing multimeric HNF1alpha binding sites is silent in muscles and in cell lines that lack endogenous HNF1alpha. HEA-3 stimulated transcription from this target promoter in a stringent 4-OHT-dependent manner. The dynamic range of transgene regulation was high, because of the low basal activity and high inducibility of the system. Ex vivo, HEA-3 increased expression of the transfected reporter gene by more than 1000-fold in a ligand-dependent manner. In vivo, HEA-3 stimulated by more than 100-fold, the expression of secreted alkaline phosphatase after delivery as plasmid DNA into mouse muscles. Moreover, long-term modulation of the expression of intramuscularly delivered mouse erythropoietin was achieved in immunocompetent mice.