Construction, cloning, and purification of the plasmids pcDNA3p24 and pcDNA3p27 selleck EPZ-5676 encoding the gene for HDAgp24 or p27 were described previously (15). Biologically active woodchuck-specific gamma interferon (IFN-��) was cloned and characterized previously. The 560-bp woodchuck IFN-�� cDNA fragment was obtained from the plasmid pwIFN�� (16). The plasmids were dissolved in phosphate-buffered saline (PBS) at a concentration of 1 mg/ml. Construction of recombinant adenoviral vectors expressing HDAg. The adenoviral vectors Ad5p27 and Ad5F35p27 expressing HDAgp27 were constructed using the AdEasy system (Qbiogene, Carlsbad, CA) (Fig. 1A). For the construction of a pShuttle plasmid expressing HDAgp27 the pcDNA3p27 plasmid was employed.

The insert was amplified by PCR using specific primers introducing BglII (5��-CGCTAGAGATCTATGAGCCGGTCCGAGTCGAGG-3��) and HindIII (5��-ATCTTATCTAGAAGCTTTCACTGGGGTCGACAACTCTGGGGAG-3��) restriction sites. Recombinant Ad5- and Ad35-based vectors were obtained by homologous recombination of pShuttlep27 with pAdEasy-1 and pAdEasy-1/F35 using the AdEasy system, respectively, transfected into 293 cells, and purified with Vivapure AdenoPACK 100 kit (Vivascience, Hannover, Germany). The adenovirus particle concentrations were determined by spectrophotometry as described previously and expressed as number of viral particles/ml (17). Fig 1 Construction and expression of Ad5p27 and AdF35p27. (A) HDVp27 was inserted under the control of the CMV immediate early promoter. The two vectors express different fibers (top, Ad5.Fiber; bottom, Ad35.Fiber).

(B) Expression of HDAgp24 and HDAgp27 by … Transient expression of HDAg and detection of HDAg by Western blotting. The human embryonic kidney (HEK) cell line 293 (Microbix Biosystems, Toronto, Ontario, Canada) was propagated as presented previously followed by infection with 5 �� 107 PFU of Ad5p27 or Ad5F35p27 (multiplicity of infection [MOI], 10) (7). BHK-21 cells (baby hamster kidney cells; ATCC CCL-10) were handled as described previously (7), and 104 cells were transfected with 1 ��g of pcDNA3p24 or pcDNA3p27 using the Lipofectamine reagent (Invitrogen, Karlsruhe, Germany) according to the manufacturers’ instructions. For Western blot analysis, cultured cells were handled as usual (7). Expression of HDAgp27 was verified using a polyclonal human anti-HDV serum (Fig. 1B).

Immunization of mice by intramuscular injection of pcDNA3 plasmid. Mice were pretreated by intramuscular (i.m.) injection of 50 ��l of cardiotoxin (10 ��M in PBS; Latoxan, Valence, France) into the musculi tibiales anteriores, followed by three plasmid immunizations (100 ��g of pcDNA3p27, 50 ��g each) at 2-week intervals, according to the protocol described previously (6). Mice in the control group received sterile PBS i.m. (same volume). Mice were sacrificed 2 weeks after the Anacetrapib last immunization.