1. Volume 117, Issue 6, Pages 1397-1407 (December 1999)
Hepatitis C virus-like particles synthesized in insect cells as a potential vaccine candidate 
Thomas F. Baumert, John Vergalla, Jujin Satoi, Michael Thomson, Martin Lechmann, David Herion, Harry B. Greenberg, Susumu Ito, T.Jake Liang 
Gastroenterology 
Volume 117, Issue 6, Pages (December 1999)
DOI: /S (99)
Copyright © 1999 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Generation of HCV-LPs in insect cells. (A) Electron microscopy of HCV-LPs. The partially purified HCV-LPs were fixed and visualized by electron microscopy (arrows show contaminating baculoviral particles; bar = 100 nm) as described in Materials and Methods. The insets show labeling of partially purified particles with anticore (C1/C2), anti-E1 (mAb A4), and anti-E2 (Ab9482) antibodies and serum from an HCV-infected human (HUM) (bars = 50 nm). (B) Molecular composition of HCV-LPs. Insect cells were infected with either BVGUS or BVHCV.S, metabolic-labeled, and then harvested for HCV-LP purification as described. The partially purified HCV-LPs were immunoprecipitated using the anti-E2 antibody (Ab9482) or nonimmune serum (IgG). The immunoprecipitated proteins were analyzed by SDS-PAGE and autoradiography using a phosphorimager (left panel). The identity of HCV structural proteins was confirmed by parallel immunoblotting with anticore (C1/C2), anti-E1 (mAb 159), and anti-E2 (mAb 917) antibodies (right panel). (C) Glycosylation of HCV envelope proteins in HCV-LPs. HCV-LPs derived from either BVHCV.S (lanes 5 and 6) or BVHCV.Sp7− (lanes 7 and 8) were purified and subjected to digestion with Endo H (ENDO) as described. Mock- and endoglycosidase-digested proteins were analyzed by SDS-PAGE and immunoblotted with anti-E1 (mAb 159)(left panel) or -E2 (mAb 917)(right panel) antibodies. A similarly prepared sample of BVGUS-infected insect cells served as the negative control (lane 4). For analysis of HCV envelope proteins expressed in mammalian cells, BSC-1 cells were infected with either a wild-type vaccinia virus (vvWT, lane 1) or a recombinant vaccinia virus containing the same HCV cDNA as the BVHCV.S (vvHCV.S, lanes 2 and 3). The cells were lysed and subjected to the same endoglycosidase digestion as the HCV-LPs.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

3. Fig. 1
Generation of HCV-LPs in insect cells. (A) Electron microscopy of HCV-LPs. The partially purified HCV-LPs were fixed and visualized by electron microscopy (arrows show contaminating baculoviral particles; bar = 100 nm) as described in Materials and Methods. The insets show labeling of partially purified particles with anticore (C1/C2), anti-E1 (mAb A4), and anti-E2 (Ab9482) antibodies and serum from an HCV-infected human (HUM) (bars = 50 nm). (B) Molecular composition of HCV-LPs. Insect cells were infected with either BVGUS or BVHCV.S, metabolic-labeled, and then harvested for HCV-LP purification as described. The partially purified HCV-LPs were immunoprecipitated using the anti-E2 antibody (Ab9482) or nonimmune serum (IgG). The immunoprecipitated proteins were analyzed by SDS-PAGE and autoradiography using a phosphorimager (left panel). The identity of HCV structural proteins was confirmed by parallel immunoblotting with anticore (C1/C2), anti-E1 (mAb 159), and anti-E2 (mAb 917) antibodies (right panel). (C) Glycosylation of HCV envelope proteins in HCV-LPs. HCV-LPs derived from either BVHCV.S (lanes 5 and 6) or BVHCV.Sp7− (lanes 7 and 8) were purified and subjected to digestion with Endo H (ENDO) as described. Mock- and endoglycosidase-digested proteins were analyzed by SDS-PAGE and immunoblotted with anti-E1 (mAb 159)(left panel) or -E2 (mAb 917)(right panel) antibodies. A similarly prepared sample of BVGUS-infected insect cells served as the negative control (lane 4). For analysis of HCV envelope proteins expressed in mammalian cells, BSC-1 cells were infected with either a wild-type vaccinia virus (vvWT, lane 1) or a recombinant vaccinia virus containing the same HCV cDNA as the BVHCV.S (vvHCV.S, lanes 2 and 3). The cells were lysed and subjected to the same endoglycosidase digestion as the HCV-LPs.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

4. Fig. 1
Generation of HCV-LPs in insect cells. (A) Electron microscopy of HCV-LPs. The partially purified HCV-LPs were fixed and visualized by electron microscopy (arrows show contaminating baculoviral particles; bar = 100 nm) as described in Materials and Methods. The insets show labeling of partially purified particles with anticore (C1/C2), anti-E1 (mAb A4), and anti-E2 (Ab9482) antibodies and serum from an HCV-infected human (HUM) (bars = 50 nm). (B) Molecular composition of HCV-LPs. Insect cells were infected with either BVGUS or BVHCV.S, metabolic-labeled, and then harvested for HCV-LP purification as described. The partially purified HCV-LPs were immunoprecipitated using the anti-E2 antibody (Ab9482) or nonimmune serum (IgG). The immunoprecipitated proteins were analyzed by SDS-PAGE and autoradiography using a phosphorimager (left panel). The identity of HCV structural proteins was confirmed by parallel immunoblotting with anticore (C1/C2), anti-E1 (mAb 159), and anti-E2 (mAb 917) antibodies (right panel). (C) Glycosylation of HCV envelope proteins in HCV-LPs. HCV-LPs derived from either BVHCV.S (lanes 5 and 6) or BVHCV.Sp7− (lanes 7 and 8) were purified and subjected to digestion with Endo H (ENDO) as described. Mock- and endoglycosidase-digested proteins were analyzed by SDS-PAGE and immunoblotted with anti-E1 (mAb 159)(left panel) or -E2 (mAb 917)(right panel) antibodies. A similarly prepared sample of BVGUS-infected insect cells served as the negative control (lane 4). For analysis of HCV envelope proteins expressed in mammalian cells, BSC-1 cells were infected with either a wild-type vaccinia virus (vvWT, lane 1) or a recombinant vaccinia virus containing the same HCV cDNA as the BVHCV.S (vvHCV.S, lanes 2 and 3). The cells were lysed and subjected to the same endoglycosidase digestion as the HCV-LPs.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

5. Fig. 2
Detection of anti-HCV antibodies in HCV-infected humans using HCV-LP ELISA. HCV-LPs were coated onto 96-well immunoplates and incubated with serially diluted human serum as described in Materials and Methods. Seroreactivities of (A) patients with chronic hepatitis B, primary biliary cirrhosis (PBC), and healthy controls (CTRL) (n = 3 of each); (B) patients with chronic hepatitis C infected with genotype 1a (n = 2) and 1b (n = 3); and (C) HCV patients with genotypes 2 and 3 (n = 2 of each). The cutoff OD of a signal-to-noise (S/N) ratio of 2 is shown (dotted line).
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

6. Fig. 3
Induction of anti-HCV antibodies in HCV-LP–immunized mice. Balb/c mice were immunized intraperitoneally 3 times (as indicated) with control preparation (n = 3) or HCV-LPs (n = 3). Blood was sampled at various time points before and after immunization and analyzed for anticore immunoreactivities by an immunoassay using 2 different core antigens. (A) For the first assay, a synthetic core peptide (aa 10–53) was used as the antigen (mouse sera diluted 1:200). (B) The second assay used the full-length core protein (sera diluted 1:1000). The cutoff OD for the first assay was 0.08 and the second The immunoreactivities could be blocked by a recombinant core protein purified from human embryonal kidney 293 cells transfected with a core expression construct. (C) Four HCV-positive human serum samples and 4 negative controls were assayed in parallel. (D) Serum samples from mice after 3 immunizations with HCV-LPs (4 positive ones are shown) or control preparation (n = 4) were collected and diluted 1:1000 and analyzed by antienvelope ELISA (with a cutoff OD of 0.21). Human sera (diluted 1:1000) from noninfected controls (n = 4) and HCV-infected individuals (genotype 1, n = 2 each for types 1a and 1b; nongenotype 1, n = 2 each for types 2 and 3) served as comparison. For each ELISA, the cutoff OD (dotted line) for positive immunoreactivity is determined by a signal-to-noise ratio of 2 with the noise value as the average OD of 6 negative control samples.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

7. Fig. 4
Detection of anti-HCV envelope antibodies by Western immunoblotting. (A) E1 and E2 proteins. Twenty nanograms of purified C-terminally truncated E1 (type 1a) or E2 (type 1b) and 50 ng of full-length E1 and E2 of our laboratory strain (type 1b) were analyzed by Western immunoblotting with the immunized mouse serum (3 examples of HCV-LP– and 1 of control-immunized mice are shown). The left panel represents positive control blotting using anti-E2 (mAb 917; top) and anti-E1 (mAb 159; bottom). Lane 1, truncated E1 (type 1b); lane 2, truncated E2 (type 1a); lane 3, truncated E1 (type 1a); lane 4, full-length E1 and E2 (type 1b). (B) GST-E2 domain fusion proteins. E2 domains A, B, and C were purified as GST fusion proteins and analyzed by Western immunoblotting with the immunized mouse serum. GST protein was purified similarly and used as control. The left panel was probed with anti-GST antibodies, the second panel with anti-E2 (mAb 917) whose epitope was mapped to domain A. The next 3 panels represent 3 examples of HCV-LP–immunized serum, and the right panel a control-immunized mouse.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

8. Fig. 4
Detection of anti-HCV envelope antibodies by Western immunoblotting. (A) E1 and E2 proteins. Twenty nanograms of purified C-terminally truncated E1 (type 1a) or E2 (type 1b) and 50 ng of full-length E1 and E2 of our laboratory strain (type 1b) were analyzed by Western immunoblotting with the immunized mouse serum (3 examples of HCV-LP– and 1 of control-immunized mice are shown). The left panel represents positive control blotting using anti-E2 (mAb 917; top) and anti-E1 (mAb 159; bottom). Lane 1, truncated E1 (type 1b); lane 2, truncated E2 (type 1a); lane 3, truncated E1 (type 1a); lane 4, full-length E1 and E2 (type 1b). (B) GST-E2 domain fusion proteins. E2 domains A, B, and C were purified as GST fusion proteins and analyzed by Western immunoblotting with the immunized mouse serum. GST protein was purified similarly and used as control. The left panel was probed with anti-GST antibodies, the second panel with anti-E2 (mAb 917) whose epitope was mapped to domain A. The next 3 panels represent 3 examples of HCV-LP–immunized serum, and the right panel a control-immunized mouse.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions

9. Fig. 5
Analysis of anti-E2 immunoreactivity by immunofluorescence. Human hepatoma (HuH-7) cells were cotransfected with (A–F) a plasmid expressing the E2 protein from a CMV promotor (pCDE2) and a GFP-expression construct (pEGFP-N1) or with (G and H) pEGFP-N1 alone. Forty-eight hours after transfection, the cells were fixed with methanol/acetone (50%/50%, vol/vol) and incubated either with (A and B) an anti-E2 antibody (mAb 3E5-1), (C, D, G, and H) serum (diluted 1:100 in PBS/1%BSA) from mice immunized with HCV-LPs, or (E and F) serum from control-immunized mice (dilution 1:100), (B, D, F, and H) followed by Cy3-conjugated anti-mouse IgG antibody. (A, C, E, and G) To identify and localize transfected individual cells, the cells were coincubated with a polyclonal anti-GFP rabbit antibody followed by a fluorescein isothiocyanate–conjugated anti-rabbit IgG antibody.
Gastroenterology  , DOI: ( /S (99) )
Copyright © 1999 American Gastroenterological Association Terms and Conditions