The mechanisms by which in vivo electroporation (EP) improves the potency of i.m. DNA vaccination were characterized by using the hepatitis C virus nonstructural (NS) 3/4A gene. Following a standard i.m. injection of DNA with or without in vivo EP, plasmid levels peaked immediately at the site of injection and decreased by 4 logs the first week. In vivo EP did not promote plasmid persistence and, depending on the dose, the plasmid was cleared or almost cleared after 60 days. In vivo imaging and immunohistochemistry revealed that protein expression was restricted to the injection site despite the detection of significant levels of plasmid in adjacent muscle groups. In vivo EP increased and prolonged NS3/4A protein expression levels as well as an increased infiltration of CD3+ T cells at the injection site. These factors most likely additively contributed to the enhanced and broadened priming of NS3/4A-specific Abs, CD4+ T cells, CD8+ T cells, and γ-IFN production. The primed CD8+ responses were functional in vivo, resulting in elimination of hepatitis C virus NS3/4A-expressing liver cells in transiently transgenic mice. Collectively, the enhanced protein expression and inflammation at the injection site following in vivo EP contributed to the priming of in vivo functional immune responses. These localized effects most likely help to insure that the strength and duration of the responses are maintained when the vaccine is tested in larger animals, including rabbits and humans. Thus, the combined effects mediated by in vivo EP serves as a potent adjuvant for the NS3/4A-based DNA vaccine.

Clearance of infections caused by the hepatitis C virus (HCV) correlates with HCV-specific T cell function. We therefore evaluated therapeutic vaccination in 12 patients with chronic HCV infection. Eight patients also underwent a subsequent standard-of-care (SOC) therapy with pegylated interferon (IFN) and ribavirin. The phase I/IIa clinical trial was performed in treatment naive HCV genotype 1 patients, receiving four monthly vaccinations in the deltoid muscles with 167, 500, or 1,500 μg codon-optimized HCV nonstructural (NS) 3/4A-expressing DNA vaccine delivered by in vivo electroporation (EP). Enrollment was done with 2 weeks interval between patients for safety reasons. Treatment was safe and well tolerated. The vaccinations significantly improved IFN-γ–producing responses to HCV NS3 during the first 6 weeks of therapy. Five patients experienced 2–10 weeks 0.6–2.4 log10 reduction in serum HCV RNA. Six out of eight patients starting SOC therapy within 1–30 months after the last vaccine dose were cured. This first-in-man therapeutic HCV DNA vaccine study with the vaccine delivered by in vivo EP shows transient effects in patients with chronic HCV genotype 1 infection. The interesting result noted after SOC therapy suggests that therapeutic vaccination can be explored in a combination with SOC treatment.

Cytomegalovirus (CMV) infection induces a series of cellular responses that resemble those observed in cells activated by growth factors or hormones including: hydrolysis of phosphatidylinositol-4,5-bisphosphate; Ca²⁺ influx and an increase in the cytosolic free [Ca²⁺]; an increase in Na⁺ entry; and, increases in cellular levels of cyclic AMP and cyclic GMP. The time courses for some of these responses appear to be markedly protracted relative to those observed for growth factors. The prolonged physiologic responses in CMV-infected cells appear to be related to modifications in the intracellular environment that are associated with the development of cytomegaly and with the phasing of CMV-directed macromolecular synthesis. For example, as the infected cell enlarges, the rate of CMV DNA synthesis increases by about 4-fold, late nuclear inclusions develop and progeny viruses are formed. When the CMV-induced activation signals are inhibited or their physiologic responses are blocked, then the yields of infectious CMV are substantially reduced. Furthermore, perturbation of the cell cycle resulting from induction of the cell activation process by CMV may be causally related to the induction of cellular damage by CMV, even in the absence of productive infection. Accordingly, the CMV-induced pathophysiologic cell activation responses represent potential targets for novel antiviral therapy.

No abstract

Immunotherapy against infectious agents and malignant tumors requires efficient priming of effector cells through direct expression and/or efficient cross-presentation of antigens by antigen-presenting cells. Electroporation is a new procedure aimed at transiently increasing cell membrane permeability and direct delivery of antigen or antigen-encoding nucleic acids inside targeted cells. We evaluated the tolerability including compliance with repeated electroporation treatments using MedPulser DDS in 24 healthy adults. Pain severity was evaluated at time of electroporation treatment, and at 1, 5, 10, and 20 minutes, and 24 hours thereafter, using two clinically validated questionnaires: McGill Pain Questionnaire (MPQ) (Present Pain Intensity) and Brief Pain Inventory (BPI). Electroporation treatments were generally well tolerated. Twenty-two out of 24 subjects returned for the second electroporation treatment 14 days after first treatment. Only two subjects reported a treatment-related systemic adverse experience following either electroporation application. For both pain assessment tools, maximum pain and/or discomfort were mostly reported immediately (within 5 minutes) after electroporation; Furthermore, no difference was observed when comparing peak-pain scores after first and second electroporation treatments. This study supports the clinical application of MedPulser DDS for the improvement of antigen-induced immune responses for prophylactic or therapeutic vaccines, especially in gene-based therapies for cancer.