Four naive woodchucks (no. 58060, 58062, 58064, and 58066) were immunized with pcDNA3p24 and pwIFN�� i.m. (500 ��g/500 ��l each in a total volume of 1 ml) in the musculi tibiales Wortmannin mTOR anteriores and with pcDNA3p24 intradermally via gene gun (Helios gene gun; Bio-Rad, Hercules, CA) in weeks 0, 3, and 6 (Fig. 2). Woodchucks were pretreated with 250 ��l of cardiotoxin (10 ��M in PBS; Latoxan) 1 week before the first i.m. immunization. For the gene gun immunization, the plasmid was used to coat gold microcarriers as recommended by the manufacturer and as described previously (15). Booster immunizations (i.m.) with Ad5p27 followed in weeks 10 and 13 and with Ad5F35p27 in week 16. A second set of experiments was conducted to confirm and possibly improve the results. Three naive woodchucks (no.

37668, 37670, and 37671) were immunized at weeks 0, 2, and 4 with pcDNA3p27 intradermally with the gene gun. In weeks 10 and 13, gene gun immunization was repeated and combined with i.m. injections of pcDNA3p27 and pwIFN�� after cardiotoxin pretreatment. After their hibernation period, the woodchucks were given boosters of Ad5p27 in weeks 62 and 65 and Ad5F35p27 in week 68. Fig 2 Immunization schedule. Altogether seven naive woodchucks were immunized with a DNA prime and adenoviral boost regimen, three of them with a prolonged protocol (bottom). Two weeks after the last immunization, the woodchucks were challenged with WHV and … WHV/HDV challenge experiment. To establish simultaneous WHV/HDV infection, two naive woodchucks (no. 37669 and 46950) were infected intravenously with 109 copies of HDV and 105 or 109 copies of WHV, respectively.

The HDV inoculum had been preliminarily passaged eight times in woodchucks and has been used for HDV superinfection before (15). The WHV inoculum was described previously (7). The 7 immunized woodchucks were challenged with 105 copies of HDV and 109 copies of WHV 2 weeks after the last immunization. Four unvaccinated woodchucks (no. 46955, 48160, 58058, and 58065) were challenged with the same dose and served as controls. Preparation, in vitro stimulation, and staining of murine splenocytes. Single-cell suspensions of murine splenocytes were prepared according to the procedure described previously (18). Splenic lymphocytes were stimulated for 7 days with a panel of 27 16-mer peptides (8-mer overlapping) spanning the whole HDAg at a final concentration of 2 ��g/ml per peptide (EMC Microcollections, T��bingen, Germany).

Unstimulated cells and cells stimulated with a cytomegalovirus (CMV)-derived peptide (YILEETSVM) served as negative controls. Cell surface and intracellular IFN-�� staining were performed as described in detail before (7). Analyses were performed using GSK-3 FlowJo software (Tree Star, Ashland, OR). CD107a degranulation assay of woodchuck PBMCs.