Combination of an Antiviral Drug and Immunomodulation against Hepadnaviral Infection in the Woodchuck Model

Abstract

Approximately 400 million people worldwide are chronically infected with hepatitis B virus (HBV). Therapy with interferon or nucleoside/tide analogs is not satisfactory due to the low responder rate and resistance development (16,18,20,33). To date, immunotherapy against chronic hepatitis B has not yet achieved satisfactory results (7,14,17,31,32,41,44). Given the crucial role of cytotoxic T lymphocytes in the control of HBV infections, new therapeutic vaccines with the ability to stimulate vigorous, broad HBV-specific cytotoxic T lymphocyte responses are needed (3,11,26,38,39).Several studies of therapeutic vaccinations have been carried out in the woodchuck model (reviewed in references 24, 25, 34, and 35) and demonstrated collectively that B-or T-cell responses to viral antigens could be induced in chronic woodchuck hepatitis virus (WHV) carriers (12,13,23,30). Yet, none of these studies have demonstrated the capability of vaccines to suppress viral replication. Here, we carried out a proof-of-principle experiment using DNA vaccines alone and DNA vaccines combined with immune complexes (IC) in the woodchuck model. IC of HBsAg and anti-HBs have been tested in patients and in transgenic mice (42,43,45,46). IC are more efficiently taken up by antigen-presenting cells than free antigens, leading to an improved presentation to T cells. DNA vaccines are potent inducers of T-cell responses. They could stimulate HBV-specific immune responses in humans and prevent hepadnaviral infections in the animal model (21,22,37). In addition, woodchucks were pretreated with lamivudine, a potent antiviral drug against HBV with the ability to enhance T-cell responses in chronically HBV-infected patients (1, 2).A total of 10 chronically WHV-infected woodchucks (from Northeastern Wildlife, Ithaca, NY) were first treated with 15 mg of lamivudine daily and randomly divided into three groups: the control group (n ϭ 2), a group vaccinated with WHV surface antigen (WHsAg)-IC and DNA (n ϭ 4), and a group vaccinated with DNA (n ϭ 4). The immunization schedule is presented in Fig. 1.The DNA vaccines consisted of an equimolar mixture of three plasmids, pWHsIm, pWHcIm, and pWIFN, expressing WHsAg, WHV core antigen (WHcAg), and woodchuck gamma interferon, respectively, as described previously (21, 37). To produce IC for woodchucks, antibodies to WHs (antiWHs) and WHsAg were titrated by the checkerboard method to determine the stoichiometry of the antigen and antibodies. The appropriate concentration of WHsAg was chosen and incubated with antibodies at 37°C for 30 min and then incubated overnight at 4°C, resulting in a preparation with a final concentration of 80 g WHsAg/ml in complex with anti-WHs. The vaccine consisted of 20 g WHsAg-IC and 250 g plasmid pWHsIm DNA per 0.5-ml dose. The DNA vaccines and IC-DNA vaccines were administered by intramuscular injections (21,22).The following parameters were determined by the indicated methods: serum WHV DNA concentrations as genome equivalents (GE) by real-time PCR with the LightCycler DNA Master S...

Novel Woodchuck Hepatitis Virus (WHV) Transgene Mouse Models Show Sex-Dependent WHV Replicative Activity and Development of Spontaneous Immune Responses to WHV Proteins

“…This finding is consistent with the low level of expression of the WHV x gene in the woodchuck liver during natural infection (27). The detection of WHV proteins has failed so far, as no suitable antibodies were available (15,25,28,29).…”

“…Primer pairs used for amplification of the WHV core region were wc1 (5=-TGG GGC ATG GAC ATA GAT CCC TA-3=) (nt 2015) and wc-149s (5=-AAG ATC TCT AAA TGA CTG TAT GTT CCG-3=) (nt 2467), with numbering according to the reference sequence under GenBank accession number M19183. PCR was carried out as described previously (15,20).…”

“…Serum WHV DNA was extracted by using the QIAamp DNA Minikit and detected by real-time PCR, as described previously (15), with primers wc1 and wc-149s.…”

“…WHV causes acute and chronic infections in woodchucks (Marmota monax) similar to HBV infections in humans. The woodchuck model has been proven to be an informative model for studies of hepadnaviral infection and pathogenesis and for the screening and development of antiviral drugs (15). Despite the establishment of immunological assays and tools for the woodchuck model during the last years, immunological studies in outbred and immunogenetically uncharacterized woodchucks are still difficult (16,17).…”

Immune therapy for hepatitis B

“…HBsAg-expressing DNA vaccines and combination of DNA vaccines with antiviral drugs have induced reduction or clearance of DHBV DNA and clearance of duck HBsAg (38,39). Immunizations of woodchucks with HBsAg-based vaccines or combination of vaccines and antiviral agents have also shown anti-HBs response and significant reductions of viral load (40). However, HBsAgbased immunization did not exhibit proper therapeutic effects in HBV-infected chimpanzees (41).…”