Immunization of Woodchucks with Plasmids Expressing Woodchuck Hepatitis Virus (WHV) Core Antigen and Surface Antigen Suppresses WHV Infection

Abstract

DNA vaccination can induce humoral and cellular immune response to viral antigens and confer protection to virus infection. In woodchucks, we tested the protective efficacy of immune response to woodchuck hepatitis core antigen (WHcAg) and surface antigen (WHsAg) of woodchuck hepatitis virus (WHV) elicited by DNA-based vaccination. Plasmids pWHcIm and pWHsIm containing WHV c- or pre-s2/s genes expressed WHcAg and WHsAg in transient transfection assays. Pilot experiments in mice revealed that a single intramuscular injection of 100 μg of plasmid pWHcIm DNA induced an anti-WHcAg titer over 1:300 that was enhanced by boost injections. However, two injections of 100 μg of pWHcIm did not induce detectable anti-WHcAg in woodchucks. With an increase in the dose to 1 mg of pWHcIm per injection, transient anti-WHcAg response and WHcAg-specific proliferation of peripheral mononuclear blood cells (PMBCs) appeared in woodchucks after repeated immunizations. Four woodchucks vaccinated with pWHcIm were challenged with 104or 105 of the WHV 50% infective dose. They remained negative for markers of WHV replication (WHV DNA and WHsAg) in peripheral blood and developed anti-WHs in week 5 after challenge. In contrast, woodchucks not immunized or immunized with the control vector pcDNA3 developed acute WHV infection. Two woodchucks immunized with 1 mg of pWHsIm developed WHsAg-specific proliferative response of PBMCs but no measurable anti-WHsAg response. A rapid anti-WHsAg response developed during week 2 after virus challenge. Neither woodchuck developed any signs of WHV infection. These data indicate that DNA-based vaccination with WHcAg and WHsAg can elicit immunity to WHV infection.

DNA Prime-Adenovirus Boost Immunization Induces a Vigorous and Multifunctional T-Cell Response against Hepadnaviral Proteins in the Mouse and Woodchuck Model

“…Construction of pCGWHc plasmid for DNA vaccination. The WHV strain 8 WHcAg gene was obtained from the WHcAg-encoding pWHcIm plasmid (41). The insert was cut out of the pWHcIm plasmid by using BamHI and XbaI restriction enzymes (New England BioLabs, Ipswich, MA) and then introduced between a ␤-globin intron sequence and polyadenylation signal into the BamHI/XbaI site of pCG vector (69).…”

“…For immunofluorescence (IF) staining, the cells were fixed with 50% methanol (20 min at 4°C). The expressed WHcAg was detected by indirect staining with rabbit antiserum and fluorescein isothiocyanate (FITC)-conjugated antibody as described previously (41). For Western blotting, cultured BHK-21 or HEK-293A cells were lysed in 1ϫ SDS sample buffer supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany).…”

“…Mice were pretreated by intramuscular injection of 50 l of cardiotoxin (10 M in phosphate-buffered saline [PBS]; Latoxan, Valence, France) into tibialis anterior muscle 1 week before the plasmid immunization. Animals were then intramuscularly vaccinated three times with 100 g of pWHcIm or pCGWHc (50 g per muscle) at 2-week intervals, according to a previously described protocol (41). Mice of the control group received intramuscularly 100 l of sterile PBS (50 l per muscle).…”

“…Two naïve woodchucks (animals 58063 and 70096) were pretreated by intramuscular injection of 250 l of cardiotoxin (10 M in PBS; Latoxan) into tibialis anterior muscle. One week later, animals were subsequently immunized three times with 1 mg of pCGWHc plasmid at 2-week intervals, according to a previously described protocol (41). Two naïve woodchucks (animals 46949 and 46957) were intramuscularly immunized with 5 ϫ 10 9 PFU Ad5WHc and 4 weeks later boosted with 1 ϫ 10 10 PFU of Ad35WHc.…”

The Woodchuck, a Nonprimate Model for Immunopathogenesis and Therapeutic Immunomodulation in Chronic Hepatitis B Virus Infection

“…From a medical point of view, the woodchuck model has been used to develop new strategies for prevention of infection (Lu et al 1999;Garcia-Navarro et al 2001)-post-expo-sure prophylaxis of hepatitis B and therapy of chronic hepadnaviral infection, including nucleoside analogs (Cullen et al 1997;Mason et al 1998;Genovesi et al 2000;Peek et al 2001), non-nucleoside analogs (Block et al 1998), therapeutic vaccination (Hervas-Stubbs et al 2001Lu and Roggendorf 2001;Menne et al 2002a), and gene-therapeutic approaches for treatment of HCC (Putzer et al 2001). Liver transplantation has recently been established for woodchucks to study early events in reinfection and adoptive immune transfer (Dahmen et al 2004).…”