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.

Immunization against proprotein convertase subtilisin-like/kexin type 9 lowers plasma LDL-cholesterol levels in mice.

Successful development of drugs against novel targets crucially depends on reliable identification of the activity of the target gene product in vivo and a clear demonstration of its specific functional role for disease development. Here, we describe an immunological knockdown (IKD) method, a novel approach for the in vivo validation and functional study of endogenous gene products. This method relies on the ability to elicit a transient humoral response against the selected endogenous target protein. Anti-target antibodies specifically bind to the target protein and a fraction of them effectively neutralize its activity. We applied the IKD method to the in vivo validation of plasma PCSK9 as a potential target for the treatment of elevated levels of plasma LDL-cholesterol. We show that immunization with human-PCSK9 in mice is able to raise antibodies that cross-react and neutralize circulating mouse-PCSK9 protein thus resulting in increased liver LDL receptor levels and plasma cholesterol uptake. These findings closely resemble those described in PCSK9 knockout mice or in mice treated with antibodies that inhibit PCSK9 by preventing the PCSK9/LDLR interaction. Our data support the IKD approach as an effective method to the rapid validation of new target proteins.

Synergistic effect of gene-electro transfer and adjuvant cytokines in increasing the potency of hepatitis C virus genetic vaccination.

BACKGROUND: Gene electro-transfer (GET) increases DNA uptake and expression by muscle cells following intramuscular plasmid injection. This technology has been used to increase the production of therapeutic proteins, such as cytokines and growth factors, and to improve immunization efficiency following the injection of antigen-encoding plasmids. METHODS: Hepatitis C virus (HCV) E2 and cytokine encoding plasmids were co-injected in the mouse quadriceps with or without GET and vaccination outcome was monitored by analysis of antigen-specific cellular-mediated or antibody-mediated immunity. RESULTS: GET co-injection of cytokine-encoding and HCV E2-encoding plasmids strongly enhanced T- or B-cell responses to various levels, depending on the particular combination used. CONCLUSIONS: We propose that a cocktail of plasmids followed by GET can be the most efficient and fine-tunable approach for genetic immunization.

Hyaluronidase increases electrogene transfer efficiency in skeletal muscle.

Electrogene transfer (EGT) of plasmid DNA into skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. We report here that preinjecting hyaluronidase (HYAse) significantly increases the gene transfer efficiency of muscle EGT. Three constructs encoding mouse erythropoietin (pCMV/mEPO), secreted alkaline phosphatase (pCMV/SeAP), and luciferase (pGGluc) were electroinjected intramuscularly in BALB/c mice and rabbits with and without HYAse pretreatment. Preinjection 1 or 4 hr before EGT increased EPO gene expression by about 5-fold in mice and maintained higher gene expression than plasmid EGT alone. A similar increment in gene expression was observed on pretreatment with HYAse and electroinjection of pCMV/mEPO into rabbit tibialis muscle. The increment of gene expression in rabbits reached 17-fold on injection of plasmid pCMV/SeAP and 24-fold with plasmid pGGluc. Injection of a plasmid encoding beta-galactosidase (pCMV/beta gal/NLS) and subsequent staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside indicated that HYAse increased the tissue area involved in gene expression. No irreversible tissue damage was observed on histological analysis of treated muscles. HYAse is used in a variety of clinical applications, and thus the combination of HYAse pretreatment and muscle EGT may constitute an efficient gene transfer method to achieve therapeutic levels of gene expression.

ErbB2 genetic cancer vaccine in nonhuman primates: relevance of single nucleotide polymorphisms.

Aberrant Her2/neu expression is associated with the development of epithelial-derived human carcinomas and for this reason it is considered a good target for immunologic intervention. To define methods to circumvent immunologic tolerance and to elicit immunity against the Her2/neu tumor-associated antigen in a suitable animal model, we have isolated the cDNA encoding the rhesus monkey homolog of human Her2/neu (RhErbB2) to construct DNA plasmids and adenoviral vectors for the development of a cancer vaccine against this protein. To further increase the immunogenic potency of these vectors, a synthetic codon-optimized RhErbB2 cDNA (RhErbB2OPT) was constructed and characterized. Genetic vaccination of rhesus monkeys was effective in inducing a response against RhErbB2 in immunized animals; importantly, the elicited immunity was associated with natural RhErbB2 polymorphisms, thus distinguishing responses against "self " and "nonself " epitopes. In particular, the postpriming response recognized mainly nonself epitopes whereas the boosted response cross-reacted with self epitopes. Our findings are particularly relevant in the investigation of the impact of TAA polymorphisms on the efficacy of a cancer vaccine strategy.

Modulation of the immune response induced by gene electrotransfer of a hepatitis C virus DNA vaccine in nonhuman primates.

Induction of multispecific, functional CD4+ and CD8+ T cells is the immunological hallmark of acute self-limiting hepatitis C virus (HCV) infection in humans. In the present study, we showed that gene electrotransfer (GET) of a novel candidate DNA vaccine encoding an optimized version of the nonstructural region of HCV (from NS3 to NS5B) induced substantially more potent, broad, and long-lasting CD4+ and CD8+ cellular immunity than naked DNA injection in mice and in rhesus macaques as measured by a combination of assays, including IFN-gamma ELISPOT, intracellular cytokine staining, and cytotoxic T cell assays. A protocol based on three injections of DNA with GET induced a substantially higher CD4+ T cell response than an adenovirus 6-based viral vector encoding the same Ag. To better evaluate the immunological potency and probability of success of this vaccine, we have immunized two chimpanzees and have compared vaccine-induced cell-mediated immunity to that measured in acute self-limiting infection in humans. GET of the candidate HCV vaccine led to vigorous, multispecific IFN-gamma+CD8+ and CD4+ T lymphocyte responses in chimpanzees, which were comparable to those measured in five individuals that cleared spontaneously HCV infection. These data support the hypothesis that T cell responses elicited by the present strategy could be beneficial in prophylactic vaccine approaches against HCV.

In vivo DNA gene electro-transfer: a systematic analysis of different electrical parameters.

BACKGROUND: Intramuscular plasmid injection followed by electroporation is an efficient method for gene therapy or vaccination. Several protocols have been described that give good transduction levels with several reporter genes. METHODS: In this work we have explored the efficiency of gene delivery upon variation of the different electrical parameters such as pulse length frequency and voltage monitoring both on short- and long-term protein production. RESULTS: Having defined the best performing parameters, we have designed a short electric treatment that gives good levels of plasmid-encoded protein in different species such as mice, rabbits and monkeys.

Gene electro-transfer of an improved erythropoietin plasmid in mice and non-human primates.

BACKGROUND: Anemia due to impaired erythropoietin (EPO) production is associated with kidney failure. Recombinant proteins are commonly administered to alleviate the symptoms of this dysfunction, whereas gene therapy approaches envisaging the delivery of EPO genes have been tried in animal models in order to achieve stable and long-lasting EPO protein production. Naked DNA intramuscular injection is a safe approach for gene delivery; however, transduction levels show high inter-individual variability in rodents and very poor efficiency in non-human primates. Transduction can be improved in several animal models by application of electric pulses after DNA injection. METHODS: We have designed a modified EPO gene version by changing the EPO leader sequence and optimizing the gene codon usage. This modified gene was electro-injected into mice, rabbits and cynomolgus monkeys to test for protein production and biological effect. CONCLUSIONS: The modified EPO gene yields higher levels of circulating transgene product and a more significant biological effect than the wild-type gene in all the species tested, thus showing great potential in clinically developable gene therapy approaches for EPO delivery.

Electro-gene-transfer: a new approach for muscle gene delivery.

Gene transfer into skeletal muscle cells by direct injection of naked plasmid DNA results in sustained gene expression. Intramuscular injection of plasmid DNA might thus be used to correct myopathies, to secrete locally or systematic therapeutic proteins and to elicit an immune response against specific antigens. However, the potential utility of this technique for gene application in humans is limited by the poor transduction efficiency and the low and highly variable level of gene expression. Different methods are thus being developed to increase the efficiency of gene transfer in muscles. It has been recently reported that a dramatic improvement of DNA transfer is achieved by applying an electric field to the muscle fibers subsequent to local DNA injection. Electro-gene-transfer increases gene expression by several orders of magnitude and strongly reduces interindividual variability. Electroinjection of genes encoding for secreted proteins resulted in sustained expression and disease correction in animal models of gene therapy. Moreover, the immunogenicity of DNA vaccines is dramatically increased when antigen-encoding plasmids are delivered by this technique. This technique may thus have broad and important applications in human gene therapy. This review provides a brief overview of the theory of electro-gene-transfer and describes parameters governing its efficiency in muscle. We also summarize the results obtained with electro-gene-transfer in animal models to date and the technical issues that must be solved before its use for human therapy can be considered.

Gene electro-transfer improves transduction by modifying the fate of intramuscular DNA.

BACKGROUND: Intramuscular gene delivery through injection of plasmid DNA has long been considered a promising approach for safe and simple in vivo gene expression for vaccination and gene therapy purposes. Recently, intramuscular gene delivery has been improved by applying low-voltage electric pulses after plasmid injection, a procedure that has been variably called gene electro-transfer, in vivo electroporation or electrical stimulation. Different types of electrical treatments have been used with excellent results both in terms of transgene expression levels and immunization outcome. This approach, therefore, holds promise for safe gene delivery to animals and humans designed for non-viral gene therapy and DNA-based vaccination. The molecular mechanisms underlying this increment in transduction efficiency are, however, still unclear. METHODS: Plasmid DNA status and kinetics following gene electro-transfer was analyzed by different methods (Southern analysis, Q-PCR and transformation into competent bacteria). RESULTS: A large amount of plasmid DNA is degraded in the first 4 h post-injection, with or without electroporation; later, the amount of intramuscular plasmid DNA is higher in electroporated samples. On electroporation, plasmid is partially protected from degradation, presumably by its early compartmentalization into the nuclei of muscle cells. CONCLUSIONS: By investigating the intracellular outcome and persistence of plasmid DNA following simple injection or gene electro-transfer we provide useful information on the mechanisms of plasmid entry and expression and underline some of the steps that could be taken to further improve this methodology.

Construction of an rtTA2(s)-m2/tts(kid)-based transcription regulatory switch that displays no basal activity, good inducibility, and high responsiveness to doxycycline in mice and non-human primates.

The tetracycline (Tc)-dependent system in its "on" version (rtTA system) displays a baseline activity in the uninduced state, severely limiting its potential applicability in human gene therapy. So far, two different strategies to circumvent this limitation have been described. On one side, co-expression of the tetracycline regulated repressor tTS(kid) has proved capable of substantially reducing the baseline activity of rtTA. On the other, novel versions of the activator, namely rtTA2(s)-S2 and rtTA2(s)-M2, with a lower basal activity have been engineered. We have combined these two approaches by co-expressing TS(kid) with the novel transactivators. Bicistronic vectors were constructed that co-express TS(kid) with rtTA, rtTA2(s)-S2, or rtTA2(s) M2, through an internal ribosome entry site (plasmids IRES-A, IRES-S2, and IRES-M2, respectively). IRES-M2 proved to be the most effective construct EX VIVO: it displayed a negligible basal activity, > 1000 fold inducibility, and high responsiveness to doxycycline (Dox). Upon delivery as plasmid DNA in mouse muscles, IRES-M2 facilitated 1000-fold induction of serum alkaline phosphatase (SEAP) gene expression and long-term, stringent, and strictly Dox-dose-dependent regulation of erythropoietin (Epo) gene expression. Tight regulation of the gene encoding SEAP was demonstrated also in non-human primates. Notably, the system was induced in animals by Dox-dosing regimens comparable to those used in humans.