Journal: Frontiers in Cellular and Infection Microbiology

Article Title: Peptidyl-prolyl cis/trans isomerase Pin1 interacts with hepatitis B virus core particle, but not with HBc protein, to promote HBV replication

doi: 10.3389/fcimb.2023.1195063

Figure Lengend Snippet: HBV Core Particles, But Not HBc Dimers or Monomers, Interact with Pin1. (A) Schematic diagram and amino acid sequence of the HBV HBc (adw). The positions of the helices ( Wynne et al., 1999 ) are shown by arrows. The CTD-truncated (at amino acid 149) HBc used for this study is denoted as ΔCTD. The S/TP motifs are in bold, italicized, and underlined. Y132 and other phosphoacceptor sites are marked by arrowheads and asterisks, respectively. (B) Myc-HBc WT and Pin1 WT can be coimmunoprecipitated. HEK293T cells were (co)transfected with mock (lane 1), Myc-HBc WT (lane 2), Myc-HBc WT plus Pin1 WT (lane 3), or Pin1 WT (lane 4). Two days after transfection, cell lysates were immunoprecipitated with an in-house rabbit polyclonal anti-HBc antibody ( Jung et al., 2012 ) and then immunoblotted with a mouse monoclonal anti-Pin1 antibody, or vice versa. As a negative control, normal rabbit IgG or normal mouse IgG was used to examine lysates from Myc-HBc WT plus Pin1 WT cotransfected cells (lane 5). Immunoprecipitated proteins were subjected to SDS-PAGE, followed by immunoblotting with anti-HBc and anti-Pin1 antibodies. Lysates were also subjected to SDS-PAGE, followed by immunoblotting with anti-HBc and anti-Pin1 antibodies. Coimmunoprecipitated proteins are marked with a white arrowhead. (C) Pin1 interacts with the core particle. HEK293T cells were (co)transfected as above. Two days after transfection, cell lysates were prepared and subjected to NAGE; transferred to PVDF membranes; and immunoblotted with anti-Pin1, anti-HBc, or mouse monoclonal anti-Myc antibodies. Lysates were also subjected to SDS-PAGE, followed by immunoblotting with anti-HBc, anti-Myc, anti-Pin1, or mouse monoclonal anti-GAPDH antibodies, as described in B. GAPDH was used as a loading control. (D) The interaction between Pin1 and the core particle is more specific than that between the core particle and other PPIases. HEK293T cells were transfected with mock (lane 1), Myc-HBc WT (lane 2), or HA-HBc WT (lane 3), and cell lysates were subjected to NAGE plus immunoblotting and SDS-PAGE plus immunoblotting, as described in (B, C) . Additionally, blots were probed with rabbit monoclonal anti-CypA and mouse monoclonal anti-FKBP12 antibodies. (E) The core particle, not a dimer or monomer of HBc, binds to Pin1 through the CTD phosphoacceptor S/TP motifs of HBc. HEK293T cells were cotransfected with Pin1 WT plus Myc-HBc WT (SST-STSSSS) (lane 1), or with the Myc-HBc-Y132A (lane 2), Myc-HBc-AAA-STSSSS (lane 3), or Myc-HBc-SST-AAAAAA (lane 4) mutants. Lysates were immunoprecipitated and then subjected to SDS-PAGE and immunoblotting as described in B. Core particle immunoblotting after NAGE was also performed as described in (C) . (F) Pin1 does not interact with core particle-defective, dimer-positive HBc mutants. HEK293T cells were (co)transfected with mock (lane 1), Myc-HBc WT (lane 2), Pin1 WT (lane 3), Pin1 WT plus Myc-HBc WT (lane 4), Myc-HBc-R133D (lane 5), Myc-HBc-R133E (lane 6), Myc-HBc-AAA-AAASAS (lane 7), Myc-HBc-AAA-AAAAAA (lane 8), or Myc-HBc-Y132A (lane 9). Lysates were immunoprecipitated and then subjected to SDS-PAGE and immunoblotting as described in (B) . Representative data are shown. Relative levels of core particles and HBc proteins were calculated using ImageJ 1.50b software. Statistical significance was evaluated using Student’s t test. ns , not significant; * P < 0.05; ** P < 0.005, relative to the corresponding control.

Article Snippet: Core particles, virions, Pin1-bound-core particles, and/or subviral particles were detected using rabbit polyclonal anti-HBc (produced in-house; 1:1,000) , mouse monoclonal anti-PreS1 (1:1,000; Santa Cruz), rabbit polyclonal anti-HBs (for NAGE; 1:1,000; Virostat), mouse monoclonal anti-HBs (for SDS-PAGE; 1:1,000; Santa Cruz), or mouse monoclonal anti-Pin1 (1:1,000; Santa Cruz) primary antibodies, followed by HRP–conjugated anti-rabbit (1:5,000; Thermo Fisher Scientific) or anti-mouse (1:5,000; Seracare) secondary antibodies.

Techniques: Sequencing, Transfection, Immunoprecipitation, Negative Control, SDS Page, Western Blot, Control, Software

Journal: Vaccines

Article Title: The Design and Development of a Multi-HBV Antigen Encoded in Chimpanzee Adenoviral and Modified Vaccinia Ankara Viral Vectors; A Novel Therapeutic Vaccine Strategy against HBV

doi: 10.3390/vaccines8020184

Figure Lengend Snippet: Vaccination with MVA-SIi-CP mut TPA-S (sh) 7–8 weeks after ChAdOx1-SP-SIi-CP mut TPA-S (sh) induces HBs-antibody. C57BL6 mice, n = 5 per group, were given intramuscularly injections with 5 × 10 5 IU per mice (low dose) or 5 × 10 7 IU per mice (high dose) of ChAdOx1-SP-SIi-CP mut -TPA-S (sh) at week 0 followed by 2 × 10 6 pfu per mice of MVA-SIi-HBV-CP mut TPA-S sh at week 7–8. The third group of C57BL6 mice ( n = 5), received 5 × 10 7 IU per mice of ChAdOx1-GFP at week 0 followed by 2 × 10 6 pfu per mice of MVA-HBV at week 8. Sera were collected 14 days post MVA vaccination and the levels of anti-HBs induction in response to vaccination was quantified by ELISA. Anti-HBs quantitation based on commercial standard mouse monoclonal antibodies to surface (GeneTex, GTX40707) above background value of naïve un-vaccinated sera are shown. Median and interquartile ranges are shown. The Kruskal–Wallis test and Dunn’s multiple comparisons test were used for statistical comparison of medians between groups. * p < 0.05, ns = not significant.

Article Snippet: After blocking and wash step, cell culture supernatants were added to capture the PreS1 and HBs antigen, followed by wash and detection of captured antigens via HPR conjugated rabbit anti-HBs polyclonal antibody (GeneTex, GTX17448).

Techniques: Enzyme-linked Immunosorbent Assay, Quantitation Assay, Bioprocessing, Comparison

Journal: Clinical and Vaccine Immunology : CVI

Article Title: Development of a Highly Sensitive Bioluminescent Enzyme Immunoassay for Hepatitis B Virus Surface Antigen Capable of Detecting Divergent Mutants

doi: 10.1128/CVI.00186-13

Figure Lengend Snippet: Characterization of the anti-HBs MAbs and the rabbit polyclonal antibody by the inhibition test. Captured HBsAg on rabbit anti-HBs polyclonal antibody-coated magnetic particles was reacted with each unlabeled antibody. Enzyme-labeled anti-HBs antibody was then added to compare the inhibition by each unlabeled antibody. The percent inhibition is shown as the B/B0 ratio on the y axis, where B is bioluminescent intensity when unlabeled antibody is added, and B0 is the bioluminescent intensity when unlabeled antibody is not added.

Article Snippet: The magnetic particles used in the inhibition test were coated with 0.15 mg/ml of anti-HBs rabbit polyclonal antibodies (Biokit, Barcelona, Spain) and prepared in the same way as the MAbs described above.

Techniques: Inhibition, Labeling

Journal: Clinical and Vaccine Immunology : CVI

Article Title: Development of a Highly Sensitive Bioluminescent Enzyme Immunoassay for Hepatitis B Virus Surface Antigen Capable of Detecting Divergent Mutants

doi: 10.1128/CVI.00186-13

Figure Lengend Snippet: Comparison of the detectability during the late phase of infection (days since first bleed) between the BLEIA and PCR. The seroconversion panel PHM935(B) was used to detect HBsAg by the BLEIA. The measurement results of the HBsAg BLEIA are shown in mIU/ml and were compared against various HBV markers, including HBsAg by CLIA2, anti-HBc, anti-HBs, and HBV DNA by PCR, whose data were obtained from the data sheet included with the seroconversion panel. The horizontal dotted line indicates the cutoff value for both the BLEIA (5 mIU/ml) and PCR (400 copies/ml). ▲, HBV PCR-positive data; △, HBV PCR-negative data; ● HBsAg BLEIA-positive data. Top, HBsAg results by CLIA2, anti-HBc, and anti-HBs are shown as + and −.

Article Snippet: The magnetic particles used in the inhibition test were coated with 0.15 mg/ml of anti-HBs rabbit polyclonal antibodies (Biokit, Barcelona, Spain) and prepared in the same way as the MAbs described above.

Techniques: Infection

Journal: Clinical and Vaccine Immunology : CVI

Article Title: Development of a Highly Sensitive Bioluminescent Enzyme Immunoassay for Hepatitis B Virus Surface Antigen Capable of Detecting Divergent Mutants

doi: 10.1128/CVI.00186-13

Figure Lengend Snippet: Results of the analysis of discrepant and nondiscrepant specimens by BLEIA

Article Snippet: The magnetic particles used in the inhibition test were coated with 0.15 mg/ml of anti-HBs rabbit polyclonal antibodies (Biokit, Barcelona, Spain) and prepared in the same way as the MAbs described above.

Techniques:

Journal: Clinical and Vaccine Immunology : CVI

Article Title: Development of a Highly Sensitive Bioluminescent Enzyme Immunoassay for Hepatitis B Virus Surface Antigen Capable of Detecting Divergent Mutants

doi: 10.1128/CVI.00186-13

Figure Lengend Snippet: Detection of preS1/preS2/S proteins by Western blotting. Electrophoresis was conducted in 10 to 20% polyacrylamide gels. The nondiscrepant specimen (P35; see Table 4) was serially diluted 1:2 with 1× sample buffer. The concentration decreases by half going from left to right. The discrepant specimen (P38; see Table 4) was prepared in the same way. (A) A 10-μg/ml preparation of HRP-labeled anti-HBs rabbit polyclonal antibody was used for detection. (B) A 10-μg/ml preparation of anti-preS1 MAb was used as a primary antibody, and 500-fold-diluted HRP-labeled anti-mouse immunoglobulin (IgG) was used as a secondary antibody. (C) A 10-μg/ml preparation of anti-preS2 MAb was used as a primary antibody, and 500-fold-diluted HRP-labeled anti-mouse IgG was used as a secondary antibody. The large HBs proteins (p39 and gp42), middle HBs proteins (gp33 and gp36), and small HBs (S) proteins (p24 and gp27) are marked with arrows.

Article Snippet: The magnetic particles used in the inhibition test were coated with 0.15 mg/ml of anti-HBs rabbit polyclonal antibodies (Biokit, Barcelona, Spain) and prepared in the same way as the MAbs described above.

Techniques: Western Blot, Electrophoresis, Concentration Assay, Labeling

Journal: Clinical and Vaccine Immunology : CVI

Article Title: Development of a Highly Sensitive Bioluminescent Enzyme Immunoassay for Hepatitis B Virus Surface Antigen Capable of Detecting Divergent Mutants

doi: 10.1128/CVI.00186-13

Figure Lengend Snippet: Results of the analysis of nondiscrepant specimens following protease treatment

Article Snippet: The magnetic particles used in the inhibition test were coated with 0.15 mg/ml of anti-HBs rabbit polyclonal antibodies (Biokit, Barcelona, Spain) and prepared in the same way as the MAbs described above.

Techniques: