Journal: Molecular Therapy. Methods & Clinical Development

Article Title: CRISPR-Cas9 gene editing of hepatitis B virus in chronically infected humanized mice

doi: 10.1016/j.omtm.2020.11.014

Figure Lengend Snippet: Characterization of HBV-infected and AAV- Sa Cas9-treated humanized FRG mouse livers Serial liver sections from humanized liver FRG mice were stained with hematoxylin and eosin (H&E), subjected to RNAscope for the presence of HBV and Sa Cas9 RNA, or co-labeled by immunohistochemistry for human cytokeratin 18 (hCK18) in combination with HBV surface antigen (HBsAg), activated caspase-3, or Ki67. Representative serial sections are shown for animal GFP-L3, which received control GFP6 and GFP7 sgRNAs. Scale bars, 1 mm.

Article Snippet: For some mice, 5-μm paraffin sections were treated with citrate antigen retrieval buffer after rehydration and stained using antibodies against human FAH (hFAH) (Sigma-Aldrich, AV41681), hCK18 (Agilent Technologies, DC10 #M701029-2), HBsAg (Bio-Rad, OBt0990), Ki67 (Cell Signaling Technology, D3B5 #12202), or cleaved caspase-3-Asp175 (Cell Signaling Technology, 9661).

Techniques: Infection, Staining, RNAscope, Labeling, Immunohistochemistry, Control

Journal: Vaccines

Article Title: Evaluating the Compatibility of New Recombinant Protein Antigens (Trivalent NRRV) with a Mock Pentavalent Combination Vaccine Containing Whole-Cell Pertussis: Analytical and Formulation Challenges

doi: 10.3390/vaccines12060609

Figure Lengend Snippet: Overview of the key assays and antibody reagents used in this study to analyze the compatibility and stability of t-NRRV and pentavalent antigens in mock combination formulations containing aluminum-salt adjuvants.

Article Snippet: , Hepatitis B surface antigen (HepB) , mAb 4940-1404 surface sntigen (Bio-Rad) , Conformational epitopes on Hep B surface antigen , [ ] .

Techniques: Antigen Assay, Enzyme-linked Immunosorbent Assay, Immunopeptidomics

Journal: Emerging Microbes & Infections

Article Title: Down-regulation of cell membrane localized NTCP expression in proliferating hepatocytes prevents hepatitis B virus infection

doi: 10.1080/22221751.2019.1625728

Figure Lengend Snippet: Elevated cell membrane expression of NTCP in HepG2-NTCP-tet cells increases HBV susceptibility. (A) The schematic diagram of treating HepG2-NTCP-tet cells in HCM medium for different time points. (B) Percentage of DOX-treated HepG2-NTCP-tet cells in each phase of the cell cycle (tested by flow cytometry cell cycle assays) at the different time points of HCM culture. (C) Immunofluorescent staining for NTCP of DOX-treated HepG2-NTCP-tet cells cultured in HCM for different times. HepG2-NTCP-tet cells without DOX treatment as negative control. (D) HepG2-NTCP cells were cultured in DMEM or HCM respectively for 24 h, and then treated with or without 10 nM Baf-A1 for another 24 h. The NTCP protein was tested using anti-flag-tag by western blot. “*” represents different bands of NTCP protein. (E, F) Changes of HBsAg and HBeAg in cell culture supernatant of HepG2-NTCP-tet cells at different time points after infected with the HBV particles concentrated from the HepAD38 cell culture supernatant. NC: negative control, standing for uninfected cells; DMEM MOI = 200 or DMEM MOI = 500: cells cultured in DMEM with the infection MOI of 200 or 500; HCM MOI = 200 or HCM MOI = 500: cells cultured in HCM with the infection MOI of 200 or 500.

Article Snippet: The antibodies used are anti-NTCP (P17-39, 1:4000); [ ] anti-Ki67 (Origene, ZM-0166, 1:200); anti-HBsAg (Origene, ZM-0122, 1:50).

Techniques: Expressing, Flow Cytometry, Staining, Cell Culture, Negative Control, FLAG-tag, Western Blot, Infection

Journal: Emerging Microbes & Infections

Article Title: Down-regulation of cell membrane localized NTCP expression in proliferating hepatocytes prevents hepatitis B virus infection

doi: 10.1080/22221751.2019.1625728

Figure Lengend Snippet: FNH tissues contain more Ki67-positive hepatocytes, along with significantly lower NTCP and HBsAg expression. (A) Immunohistochemical double staining of Ki67, NTCP and HBsAg in FNH tissues and adjacent non-FNH tissues. The top row showed the double staining of NTCP (brown) and Ki67 (red), and the bottom row showed the double staining of HBsAg (brown) and Ki67 (red). (B) The percentage of Ki67-positive hepatocytes (calculated by the average of five counted fields in every tissue) and the histochemistry scores of NTCP and HBsAg (calculated by positive hepatocyte ratio multiplied with the staining intensity). (C) Spearman's correlation of NTCP histochemistry scores and the percentage of Ki67-positive hepatocytes, the histochemistry scores of NTCP and HBsAg (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: The antibodies used are anti-NTCP (P17-39, 1:4000); [ ] anti-Ki67 (Origene, ZM-0166, 1:200); anti-HBsAg (Origene, ZM-0122, 1:50).

Techniques: Expressing, Immunohistochemical staining, Double Staining, Staining

Journal: Microbial Cell Factories

Article Title: Immunogenicity of Leishmania -derived hepatitis B small surface antigen particles exposing highly conserved E2 epitope of hepatitis C virus

doi: 10.1186/s12934-016-0460-4

Figure Lengend Snippet: Characterization of the particles expressed in the L. tarentolae system. a Immunofluorescence of recombinant L. tarentolae cells transfected with plasmids expressing 412–425_sHBsAg and sHBsAg. Cells transfected with empty pLEXSY_I - blecherry3 plasmid were used as negative control (NC). The staining was carried out using AP33 mAbs ( blue ) and anti-HBsAg Abs ( green ), scale bar = 5 µm. b Western blot analysis of the Leishmania -derived particles in reducing conditions. sHBsAg and 412–425_sHBsAg were treated with PNGase F and detected using the anti-HBsAg specific antibody. G represents the glycosylated and DG deglycosylated form of protein. c Recognition of particles with anti-HBsAg and AP33 Abs in the ELISA tests. ELISA plates were coated with serial dilutions of recombinant L. tarentolae cell lysates containing sHBsAg and 412–425_sHBsAg particles. The dilution factor is depicted on x -axis. For each ELISA assay, the mean from three independent experiments performed in duplicate is shown. The mean A 450 values and standard deviations are shown on the y -axis. The background from the L. tarentolae wild-type cell lysate in each dilution was subtracted from the obtained results

Article Snippet: Antibody response was measured by direct solid-phase ELISA using immunogens 10 µg/mL, 412–425 synthetic peptide (QLINTNGSWHINST) 20 µg/mL (JPT-Innovative Peptide Solutions), commercial vaccine against HBV 5 µg/mL (Engerix-B, GlaxoSmithKline) or yeast-derived HBsAg protein 5 µg/mL (Acris Antibodies).

Techniques: Immunofluorescence, Recombinant, Transfection, Expressing, Plasmid Preparation, Negative Control, Staining, Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: Microbial Cell Factories

Article Title: Immunogenicity of Leishmania -derived hepatitis B small surface antigen particles exposing highly conserved E2 epitope of hepatitis C virus

doi: 10.1186/s12934-016-0460-4

Figure Lengend Snippet: OptiPrep density gradient centrifugation of recombinant particles. Seventeen fractions of 0.5 mL each were harvested from the top of the OptiPrep gradient after centrifugation. The aliquots of each fraction were analyzed by SDS-PAGE Coomassie blue staining (CB) and western-blot (WB) with anti-HBsAg Abs. The figure represents the results of fractions 8–10 where the highest amount of particles was detected

Article Snippet: Antibody response was measured by direct solid-phase ELISA using immunogens 10 µg/mL, 412–425 synthetic peptide (QLINTNGSWHINST) 20 µg/mL (JPT-Innovative Peptide Solutions), commercial vaccine against HBV 5 µg/mL (Engerix-B, GlaxoSmithKline) or yeast-derived HBsAg protein 5 µg/mL (Acris Antibodies).

Techniques: Gradient Centrifugation, Recombinant, Centrifugation, SDS Page, Staining, Western Blot

Journal: Microbial Cell Factories

Article Title: Immunogenicity of Leishmania -derived hepatitis B small surface antigen particles exposing highly conserved E2 epitope of hepatitis C virus

doi: 10.1186/s12934-016-0460-4

Figure Lengend Snippet: Analysis of the humoral response induced by Leishmania -derived particles in BALB/c mice. a Analysis of the antibody endpoint titers of the pooled mouse antisera specific to the recombinant particles. ELISA plates were coated with 412–425_sHBsAg ( right ) or sHBsAg ( left ) particles. b Analysis of the interaction of immune sera with yeast-derived HBsAg proteins. ELISA plates were coated with 5 µg/mL of purified HBsAg protein from P. pastoris (yHBsAg) ( right ) or commercially available vaccine against HBV (Engerix-B) ( left ). c Analysis of the antibody response to the 412–425 synthetic peptide. ELISA plates were coated with 20 µg of 412–425 peptide. AP33 mAb was used to estimate concentration of Abs specific to the 412–425 region in serum. Dilution factor of the pooled 412–425_sHBsAg sera and concentration of the AP33 antibody are shown on x -axis ( bottom and top , respectively). The 412–425_sHBsAg sera values are represented with bars , the AP33 values are marked with solid line . Mean A 450 values and standard deviations are shown on the y -axis. The background from the negative control serum in each dilution was subtracted from the obtained results ( a , b , c ). For each ELISA assay, the mean from three independent experiments performed in duplicate is shown. Asterisks indicate statistical significance (* P < 0.05, paired two-tailed t -test) ( a , b ). The solid horizontal line ( a , b ) indicates the cutoff value (three times the mean background value). d Analysis of cross-reactivity of the 412–425_sHBsAg sera to the E1E2 complex from different HCV genotypes. The figure represents western blotting in reducing conditions with 412–425_sHBsAg sera diluted 1:500. As an antigen, extracts of HEK293 cells transfected with plasmids expressing E1E2 glycoproteins from different HCV genotypes were used. Non-transfected HEK293 cell lysate was used as negative control (NC)

Article Snippet: Antibody response was measured by direct solid-phase ELISA using immunogens 10 µg/mL, 412–425 synthetic peptide (QLINTNGSWHINST) 20 µg/mL (JPT-Innovative Peptide Solutions), commercial vaccine against HBV 5 µg/mL (Engerix-B, GlaxoSmithKline) or yeast-derived HBsAg protein 5 µg/mL (Acris Antibodies).

Techniques: Derivative Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Purification, Peptide ELISA, Concentration Assay, Negative Control, Two Tailed Test, Western Blot, Transfection, Expressing