Volume 116, Issue 3, Pages 658-665 (March 1999) Protective and therapeutic effect of DNA-based immunization against hepadnavirus large envelope protein  Christine Rollier, Claire Sunyach, Luc Barraud, Nora Madani, Catherine Jamard, Christian Trepo, Lucyna Cova  Gastroenterology  Volume 116, Issue 3, Pages 658-665 (March 1999) DOI: 10.1016/S0016-5085(99)70188-5 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 DNA vaccination vector and DHBV large protein expression. (A) Schematic representation of the pCI-preS/S plasmid encoding the DHBV large protein. (B) Expression of the DHBV large protein from pCI-preS/S plasmid in transiently transfected LMH cells. Proteins from cell lysates were probed in immunoblotting analysis with rabbit anti-DHBpreS antibody. Arrow indicates the 36-kilodalton DHBV large protein. Lane 1, concentrated serum-derived DHBV particles; lane 2, mock (LMH cells); lane 3, pCMV-DHBV–transfected LMH cells; lane 4, pCI-transfected LMH cells; lane 5, pCI-preS/S–transfected LMH cells. Gastroenterology 1999 116, 658-665DOI: (10.1016/S0016-5085(99)70188-5) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 DNA vaccination vector and DHBV large protein expression. (A) Schematic representation of the pCI-preS/S plasmid encoding the DHBV large protein. (B) Expression of the DHBV large protein from pCI-preS/S plasmid in transiently transfected LMH cells. Proteins from cell lysates were probed in immunoblotting analysis with rabbit anti-DHBpreS antibody. Arrow indicates the 36-kilodalton DHBV large protein. Lane 1, concentrated serum-derived DHBV particles; lane 2, mock (LMH cells); lane 3, pCMV-DHBV–transfected LMH cells; lane 4, pCI-transfected LMH cells; lane 5, pCI-preS/S–transfected LMH cells. Gastroenterology 1999 116, 658-665DOI: (10.1016/S0016-5085(99)70188-5) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Kinetics of humoral response in ducks after immunization with (A) pCI-preS/S plasmid: 100 μg (ducks 301 and 302), 200 μg (duck 303), and 400 μg (ducks 305 and 306; duck 305 accidentally died at week 5). (B) DHBpreS polypeptide (100 μg) emulsified in Freund's adjuvant. Detection of anti-preS end point titer antibodies, expressed as log10, was performed using a direct ELISA test. Graphs show data from individual responders ducks. The first injection was administered at time 0; filled arrows represent DNA or protein boosts for all ducks. The open arrow represents the delayed DNA boost of 100 μg administered to ducks 301 and 302. Gastroenterology 1999 116, 658-665DOI: (10.1016/S0016-5085(99)70188-5) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Recognition in immunoblotting assay of the recombinant DHBpreS protein, used in the ELISA test, by (A) serum from pCI-preS/S–immunized duck and (B) serum from duck immunized with concentrated DHBV particles. Arrowheads show the p21 DHBpreS protein, p33 DHBpreS precursor, and its p28 degradation product. Gastroenterology 1999 116, 658-665DOI: (10.1016/S0016-5085(99)70188-5) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Recognition in immunoblotting test of DHBV particles. VP lanes, concentrated serum-derived viral particles; Mock lanes, concentrated uninfected duck serum. The lanes were shown with (A) serum from pCI-preS/S–immunized duck, (B) polyclonal anti-DHBpreS rabbit antiserum, and (C) control duck serum from pCI-immunized duck. Arrowhead indicates the 36-kilodalton DHBV large protein. Gastroenterology 1999 116, 658-665DOI: (10.1016/S0016-5085(99)70188-5) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 Recognition in immunoblotting assay of proteins from DHBV-infected and -uninfected duck livers shown with (A) serum from pCI-preS/S–immunized duck, (B) polyclonal anti-DHBpreS rabbit antiserum, and (C) control duck serum from pCI-immunized duck. Arrowheads indicate the p36 DHBV large protein, its p28 cleavage product, and the migration location of p18 DHBV small protein, based on its detection by anti-S monoclonal antibody in this immunoblotting assay (not shown). Gastroenterology 1999 116, 658-665DOI: (10.1016/S0016-5085(99)70188-5) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 In vitro neutralization of DHBV infectivity with immunized duck sera. Primary duck hepatocytes were infected with DHBV inoculum preincubated either with phosphate-buffered saline (– –), PCI-immunized control duck serum (- - - -), or pCI-preS/S–immunized duck serum (—). The release of viral particles was quantified in supernatants of cell culture by dot-blot hybridization. Means of duplicate determinations are presented. Samples of day 8 of phosphate-buffered saline control were not available. Gastroenterology 1999 116, 658-665DOI: (10.1016/S0016-5085(99)70188-5) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 7 In vivo protection of ducklings from DHBV infection. Southern blot analysis of total liver DNA (20 μg/lane) extracted from ducklings inoculated with DHBV preincubated with the following: (A) control duck serum (lanes 1 and 2); (B) 100 μg pCI-preS/S–immunized duck serum (lanes 3–6); (C) 200 μg pCI-preS/S–immunized duck serum (lanes 7–10); and (D) 400 μg pCI-preS/S–immunized duck serum (lanes 11–14). Each lane represents liver DNA of 1 inoculated duckling. Lane 15, control DHBV DNA linearized by EcoRI. L, linear; RC, relaxed circular; SS, single-stranded DHBV DNA forms. Gastroenterology 1999 116, 658-665DOI: (10.1016/S0016-5085(99)70188-5) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 8 Effect of DNA immunization on duck viremia. Viremia was followed up for 42 weeks in 6 pCI-preS/S–immunized, 3 unimmunized, and 3 pCI-immunized DHBV-carrier ducks. The level of mean serum DHBV DNA for each group of ducks, based on individual viremia titers, is represented. Arrows show DNA injections (100 μg/duck) at weeks 4, 7, 11, and 28. Gastroenterology 1999 116, 658-665DOI: (10.1016/S0016-5085(99)70188-5) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 9 Effect of DNA immunization on intrahepatic DHBV DNA. Southern blot analysis of liver DNA (20 μg/lane) extracted from (A) 5 control ducks and (B) 6 pCI-preS/S–immunized ducks. Relative band intensity for total DHBV DNA was quantified by densitometry. Lower panel shows the specific detection of DHBV cccDNA pool. L, linear; RC, relaxed circular; SS, single-stranded DHBV DNA forms. Gastroenterology 1999 116, 658-665DOI: (10.1016/S0016-5085(99)70188-5) Copyright © 1999 American Gastroenterological Association Terms and Conditions