Volume 39, Issue 3, Pages 405-413 (September 2003) Induction of antibodies to the PreS region of surface antigens of woodchuck hepatitis virus (WHV) in chronic carrier woodchucks by immunizations with WHV surface antigens  Mengji Lu, Ruediger Klaes, Stephan Menne, Wolfram Gerlich, Benno Stahl, Hans-Peter Dienes, Uta Drebber, Michael Roggendorf  Journal of Hepatology  Volume 39, Issue 3, Pages 405-413 (September 2003) DOI: 10.1016/S0168-8278(03)00241-1

Fig. 1 Anti-WHpreS antibody response in woodchucks. (A) The kinetics of anti-WHs and anti-WHpreS antibody responses in woodchucks WH29 and WH30 with acute self-limited WHV infection (○), anti-WHs; (□) anti-WHpreS antibody. WHV DNA was detected during weeks 4–8 p.i. as indicated in the graph. The SDH in these woodchucks were elevated between weeks 7–9 p.i.. The cutoff was set for anti-WHpreS antibody. (B). Detection of anti-WHpreS antibodies. Naive woodchucks (N1–N4), woodchucks immunized tree times with 20 μg of plasma-derived WHsAg (I1–I2), and chronically WHV-infected woodchucks (C1–C4). The cutoff was set for anti-WHpreS antibody. (C) Detection of large and middle WHsAg in plasma-derived WHsAg fraction by Western blotting with anti-WHs. M, molecular weight marker. Five micrograms of plasma-derived WHsAg fraction was subjected to SDS–PAGE and subsequent Western blotting with anti-WHs. Journal of Hepatology 2003 39, 405-413DOI: (10.1016/S0168-8278(03)00241-1)

Fig. 2 Immunization of chronically WHV-infected woodchucks. (A) Anti-WHs antibody response. (B) Anti-WHpreS antibody response. Woodchucks were immunized with 20 μg of WHsAg at day 0, 21, 42, 63, and 84. The antibody response at 1 week later at day 7, 28, 49, 70, and 91 is shown in the figure. (○) Anti-WHs antibody; (□) anti-WHpreS antibody. Journal of Hepatology 2003 39, 405-413DOI: (10.1016/S0168-8278(03)00241-1)

Fig. 3 The kinetic of anti-WHs and anti-WHpreS antibody response in 6 representative woodchucks. These woodchucks received a total of six immunizations with 20 μl of WHsAg (indicated by ?). Sera were taken from these woodchucks and tested for anti-WHs and anti-WHpreS antibodies. (○) Anti-WHs antibody; (□), anti-WHpreS antibody. Journal of Hepatology 2003 39, 405-413DOI: (10.1016/S0168-8278(03)00241-1)

Fig. 4 The reactivity to WHsAg of woodchuck sera with and without a preincubation with GST–WHpreS fusion protein. All tested woodchuck sera were taken at day 98 and were positive for anti-WHs and anti-WHpreS. Serum samples were serially diluted at 1:25, 1:50, 1:100, and 1:500 and incubated for 1 h in microtiter plates coated with purified GST–WHpreS or GST alone, then the samples were tested by anti-WHs ELISA. White bars: preincubated with GST; black bars: preincubated with 10 μg GST–WHpreS fusion protein. The cutoff was set at 0.085 as the OD value 3× standard deviations over mean value of 8 negative serum samples. Journal of Hepatology 2003 39, 405-413DOI: (10.1016/S0168-8278(03)00241-1)

Fig. 5 The reactivity to WHsAg of woodchuck sera with and without a preincubation with WHsAg-derived peptides. All tested woodchuck sera were taken at day 98 and were positive for anti-WHsAg and anti-WHpreS antibodies. Serum samples were serially diluted at 1:50, 1:100, 1:500, and 1:1000. After incubation for 1 h with 1 μg of each specific peptide or control peptide, the samples were tested by anti-WHs ELISA. White bars: without preincubation; black bars: preincubated with peptides. For inhibition: 378: incubated with a mixture of peptides aa 142–161, aa 182–203, and 204–226; 379: incubated aa 122–141 and aa 162–181; 380: incubated with aa 82–101 and aa 204–226; 384: incubated with aa 102–121 and aa 142–161 The numbering of peptides is according the their position in the large WHsAg [33]. The cutoff was set at 0.085 as the OD value 3× standard deviations over mean value of 8 negative serum samples. Journal of Hepatology 2003 39, 405-413DOI: (10.1016/S0168-8278(03)00241-1)

Fig. 6 Detection of anti-WHV PreS antibodies by Western blotting. GST–WHpreS fusion protein (5 μg/lane) was blotted onto a nitrocellulose strips. The nitrocellulose strips were blocked with 5% of skimmed milk powder dissolved in PBS. All three tested woodchuck sera were taken at day 98, positive for anti-WHs and anti-WhpreS, and diluted 1:100 for Western blotting. A peroxidase-conjugated protein A was used for detection of woodchuck antibodies. Journal of Hepatology 2003 39, 405-413DOI: (10.1016/S0168-8278(03)00241-1)

Fig. 7 Serum WHV DNA (A) and WHsAg (B) concentrations in woodchucks before and after immunizations. WHV DNA concentrations were determined by spot-blot hybridization using a 32P-labeled full-length WHV genome. WHsAg concentrations were measured by electroimmunodiffusion described in Section 2. White bars: at day 0; black bars: at day 130. nt, not determined. (C) Fluctuation of WHV DNA concentrations in a chronic carrier woodchuck. A woodchuck was kept without treatment. WHV DNA concentrations in this woodchuck at months 0, 6, 10, and 12 were determined by spot-blot hybridization using a 32P-labeled full-length WHV genome. Journal of Hepatology 2003 39, 405-413DOI: (10.1016/S0168-8278(03)00241-1)

Fig. 8 The changes of WHV DNA and WHsAg titers in six representative woodchucks during the immunizations. WHV DNA concentrations were determined by spot-blot hybridization using a 32P-labeled full-length WHV genome. WHsAg concentrations were measured by electroimmunodiffusion as described in Section 2. Journal of Hepatology 2003 39, 405-413DOI: (10.1016/S0168-8278(03)00241-1)