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* Division of Clinical Microbiology, F68, Karolinska Institutet at Karolinska University Hospital Huddinge, Stockholm, Sweden;
Inovio Biomedical Corp., San Diego, CA 92121;
Inovio AS, Gaustadalleen, Oslo, Norway;
Visionar AB, Uppsala, Sweden; and
¶ Vecura and Clinical Research Center, Karolinska University Hospital, Stockholm, Sweden
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 
-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.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This study was supported by grants from The Swedish Science Council, The Cancer Foundation, and the Stockholm County Council (to M.S.). L.F. was supported by grants from Lars Hiertas Memorial fund, Goljes Memorial fund, and from Karolinska Institutet. Additional funding was provided by Tripep AB and Inovio Biomedical Corp.
2 Address correspondence and reprint requests to Prof. Matti Sällberg, Division of Clinical Microbiology, F68, Karolinska Institutet at Karolinska University Hospital, Huddinge, Stockholm, Sweden. E-mail address: matti.sallberg{at}ki.se
3 Abbreviations used in this paper: HCV, hepatitis C virus; BHK, baby hamster kidney; coNS3/4A, codon-optimized NS3/4A; EP, electroporation; NS, nonstructural; qPCR, quantitative PCR; rNS3, recombinant NS3; SFC, spot-forming cell; S/N, sample to negative (ratio); TA, tibialis anterior.
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