Journal: Antiviral research

Article Title: A stable hepatitis D virus-producing cell line for host target and drug discovery.

doi: 10.1016/j.antiviral.2022.105477

Figure Lengend Snippet: Fig. 1. Approach and virological characterization of the HuH7-2C8D cell line. A. Experimental strategy. HuH7 cells were first transduced with pLX304-HB2.7 lentiviral vector. From the HuH7.HB2.7 cell pool, HuH7-2C8 clone was isolated, expanded and transfected with HDV-expressing pSVLD3-neo plasmid, leading to the production of the HuH7-2C8D pool cell line. B. Intracellular HBsAg levels in the indicated cell lines assessed by flow cytometry. One representative experiment is shown. C. Extracellular HBsAg expression levels determined by CLIA in the supernatants of the different cell lines. Results are expressed as means ± SD secreted HBsAg (UI/mL) from three independent experiments (n = 15). D-E. Intracellular (D) and extracellular (E) HBsAg expression level from HuH7-2C8D cells assessed by Western blot. HuH7-2C8D cells were seeded and supernatants were harvested and cell lysed at the indicated time points post seeding. For intracellular HBsAg levels, β-tubulin expression was detected in parallel as a control (D). One representative blot is shown. F-G. Intracellular (F) and extracellular (G) HDAg expression level from HuH7-2C8D cells assessed by Western blot. HuH7-2C8D cells were seeded, supernatants were harvested, and cell were lysed at the indicated time points post seeding. For intracellular HDAg levels, β-actin expression was detected in parallel as a control (D). One representative blot is shown. H-J. Iodixanol-based density gradient. Supernatant from HuH7-2C8 (H) and HuH7-2C8D (I) cells were concentrated with a 30% sucrose cushion and purified through a 10–45% iodixanol density gradient. Ten fractions were collected. HBsAg levels in each fraction were assessed by CLIA. Alternatively, total RNA was extracted and HDV RNA levels in each fraction were quantified by RT-qPCR. 200 μL of each fraction were weighted to assess the density (J). One representative experiment out of three independent experiments is shown. ***p < 0.001.

Article Snippet: Intracellular expression of HBsAg in the different cell lines was assessed by flow cytometry as described (Eller et al., 2020) using a specific mouse monoclonal anti-HBsAg antibody (clone 1044/329, Novus) and a secondary AF647-labelled goat antibody targeting mouse IgGs (Jackson ImmunoResearch, 115-605-003).

Techniques: Transduction, Plasmid Preparation, Isolation, Transfection, Expressing, Flow Cytometry, Western Blot, Control, Purification, Quantitative RT-PCR

Journal: Antiviral research

Article Title: A stable hepatitis D virus-producing cell line for host target and drug discovery.

doi: 10.1016/j.antiviral.2022.105477

Figure Lengend Snippet: Fig. 2. Infection of HuH7-NTCP cells with HDV-containing supernatants from HuH7-2C8D cells. A. Experimental strategy. HuH7-2C8D cells were seeded and cultured in production medium. Supernatants were regularly harvested, pooled, and HDV RNA was quantified by RT-qPCR. B–C. HuH7-NTCP cells were seeded one day prior to incubation or not for 1h with preS1 peptide at 37 ◦C. HuH7-NTCP were then inoculated with HDV-containing supernatants at the indicated Vge/cell. HDV infection was assessed at 6 days post infection (dpi) by intracellular immunodetection of HDAg. The number of HDAg-positive cells was quantified using a plate cytometer (B). Results are expressed as means ± SD % HDV infection compared to control HuH7-NTCP cells infected with HDV at 1000 Vge/cell (set at 100%) from three independent experiments (n = 9). Representative IF pictures for each condition are presented (C). scale bar: 100 μm. D-F. Impact of shHBs expression on production of HDV infectious particles. HuH7-2C8D cells were seeded one day prior to transduction with shHBs- or shCtrl-expressing lentiviral vector. Cells were selected with puromycin and supernatants were harvested. shRNA efficacy was assessed by quantification of HBsAg by CLIA (D). Results are expressed as means ± SD % HBsAg secretion compared to shCtrl-treated cells (set at 100%) from three independent experiments (n = 10). HuH7-NTCP cells were then inoculated with the supernatants at adjusted volume. HDV infection was assessed at 6 dpi by RT-qPCR quantification of HDV RNA (E). Results are expressed as means ± SD % HDV RNA compared to HuH7-NTCP infected with supernatant from shCtrl-treated HuH7-2C8D cells (set at 100%) from three independent experiments (n = 8). Alternatively, infection was assessed by quantification of HDAg-positive cells by IF using a plate cytometer (E-F). Results are expressed as means ± SD % HDAg-positive cells compared to HuH7-NTCP infected with supernatant from shCtrl-treated HuH7-2C8D cells (set at 100%) from three independent experiments (n = 10). Representative IF pictures are presented. NT: non transfected cells. scale bar: 100 μm ***p < 0.001.

Article Snippet: Intracellular expression of HBsAg in the different cell lines was assessed by flow cytometry as described (Eller et al., 2020) using a specific mouse monoclonal anti-HBsAg antibody (clone 1044/329, Novus) and a secondary AF647-labelled goat antibody targeting mouse IgGs (Jackson ImmunoResearch, 115-605-003).

Techniques: Infection, Cell Culture, Quantitative RT-PCR, Incubation, Immunodetection, Cytometry, Control, Expressing, Transduction, Plasmid Preparation, shRNA, Transfection

Journal: AMB Express

Article Title: Production of bioactive liver-targeting interferon Mu-IFN-CSP by soluble prokaryotic expression

doi: 10.1186/s13568-017-0493-z

Figure Lengend Snippet: Effect of IFN-CSP or mu-IFN-CSP on HBV antigens secretion and HBV-DNA replication in HepG2.2.15 cells. HepG2.2.15 cells were cultured in the presence of IFN-CSP or mu-IFN-CSP for 6 days. Hepatitis B surface antigen (HBsAg; a ) and hepatitis B e antigen (HBeAg; b ) in the culture supernatants were analyzed by enzyme-linked immunosorbant assay (ELISA). Supernatant HBV-DNA ( c ) and intracellular HBV-DNA ( d ) were measured by real-time quantitative PCR. Data represent the mean ± SEM of three experiments. ***P < 0.001 drug group vs control group; # P < 0.05, ## P < 0.01 IFN-CSP group vs mu-IFN-CSP group

Article Snippet: The treated HepG2.2.15 cells were seeded on coverslip and the primary antibody and the second antibody were goat polyclonal anti-HBsAg antibody (1:200; Novus Biologicals, CO, USA) and Alexa Fluor Cy3-conjugated donkey anti-goat IgG (1:200; Beyotime, Guangzhou, China), respectively.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Control

Journal: AMB Express

Article Title: Production of bioactive liver-targeting interferon Mu-IFN-CSP by soluble prokaryotic expression

doi: 10.1186/s13568-017-0493-z

Figure Lengend Snippet: Effect of IFN-CSP or mu-IFN-CSP on HBsAg expression in HepG2.2.15 cells. HepG2.2.15 cells stained by immunofluorescent staining with anti-HBsAg antibody and representative photographs are captured by microscope. A Controls; B IFN-CSP; C mu-IFN-CSP; 1 red stained with HBsAg; 2 blue nuclear stained with DAPI; 3 merged images of 1 and 2. Bar, 100 μm and is the same for all photomicrographs

Article Snippet: The treated HepG2.2.15 cells were seeded on coverslip and the primary antibody and the second antibody were goat polyclonal anti-HBsAg antibody (1:200; Novus Biologicals, CO, USA) and Alexa Fluor Cy3-conjugated donkey anti-goat IgG (1:200; Beyotime, Guangzhou, China), respectively.

Techniques: Expressing, Staining, Microscopy

Journal: Virology Journal

Article Title: Detection of Hepatitis B virus in serum and liver of chickens

doi: 10.1186/1743-422x-9-2

Figure Lengend Snippet: Figure 2 Immunohistochemical analysis of HBsAg and HBcAg in liver tissues of chickens. HBsAg was distributed mostly in cytoplasm of hepatocytes (A, 400×), and HBcAg was distributed mostly in the nucleus of hepatocytes (B, 400×).

Article Snippet: Sections were incubated with blocking buffer (Zymed Laboratories Inc., San Diego, USA) containing 20% normal goat serum (Gibco) and 80% PBS (0.01 M, pH 7.4) at room temperature for 30 min. After discarding the goat serum, sections were incubated in primary monoclonal antibodies against HBsAg and HBcAg (Zhongshan Golden Bridge Biotech Co. Ltd., Beijing, China) diluted in PBS, for 2 h at 37°C.

Techniques: Immunohistochemical staining

Journal: Vaccines

Article Title: Specific Antibodies Induced by Immunization with Hepatitis B Virus-Like Particles Carrying Hepatitis C Virus Envelope Glycoprotein 2 Epitopes Show Differential Neutralization Efficiency

doi: 10.3390/vaccines8020294

Figure Lengend Snippet: Expression and characterization of the chimeric proteins. ( a ) Western blot analysis of the chimeric proteins expressed in L. tarentolae . Cell lysates were separated using SDS-PAGE and detected with the anti-HBsAg antibody. The lysate from wild-type L. tarentolae cells was used as the negative control (NC). Bands of higher molecular mass correspond with the multimeric forms of the proteins. On the left protein ladder, the molecular weight in kDa is given. ( b ) In order to concentrate and partially purify the chimeric particles, lysates from the L. tarentolae cell cultures expressing chimeric proteins were placed on top of an OptiPrep density gradient. Seventeen fractions of 0.5 mL were harvested from top to bottom. The aliquots of fractions 2–15 were then analyzed using a western blot with anti-HBsAg antibodies. On the left protein ladder, the molecular weight in kDa is given. ( c ) Electron micrographs of chimeric sHBsAg-based particles. After concentration on the Optiprep density gradient, the chimeric particles were stained with uranyl acetate and analyzed using electron microscopy. Observed particles were approximately 20–30 nm in diameter. Black arrows: Chimeric virus-like particles (VLPs). Scale bar: 50 nm.

Article Snippet: Then, 500 μL fractions were harvested and analyzed by a western blot using anti-HBsAg rabbit polyclonal antibodies (OriGene, Rockville, MD, USA).

Techniques: Expressing, Western Blot, SDS Page, Negative Control, Molecular Weight, Concentration Assay, Staining, Electron Microscopy, Virus

Journal: Scientific Reports

Article Title: Minicircle-based vaccine induces potent T-cell and antibody responses against hepatitis C virus

doi: 10.1038/s41598-024-78049-3

Figure Lengend Snippet: ( A ) Schematic diagram of the parental plasmids (PP) used in the study. DNA sequences encoding sHBsAg_412–425, NS3/4A and NS5B were cloned into pMC.CMV-MCS-SV40polyA, containing attB and attP recombination sites. KpnI restriction sites are marked with black lines. ( B ) Analysis of MC DNA. Lanes contain pMC.CMV-MCS-SV40polyA plasmid with NS3/4A, NS5B and sHBsAg_412–425 sequences. Plasmids were purified from bacteria cultures pre-induction ([−] L-Arabinose) and after induction ([+] L-Arabinose) and were digested using KpnI enzyme. M—Gene O’Ruler DNA ladder mix. ( C ) Transfection of HEK293T cells with MC. HEK 293 cells were transfected with 2 µg of NS3/4A, NS5B and sHBsAg_412–425 MCs. For the detection of NS3/4A, NS5B and sHBsAg_412–425 recombinant proteins, anti-NS3, anti-NS5 antibodies and AP33 monoclonal antibody binding 412–423 region of HCV E2 glycoprotein were used. ( D ) Western blot analysis of the proteins expressed in HEK293T cells after transfection with PP (parental plasmid) and MC (minicircle). Cell lysates were separated using SDS-PAGE and detected with the anti-HBsAg, anti-NS3 and anti-NS5 antibodies. The lysate from HEK293T cells was used as the negative control (NC). On the right, the results of transfection with empty MC (eMC) are shown. On the left, protein ladder, the molecular weight in kDa is given. The original blots are presented in Supplementary Fig. .

Article Snippet: Similarly, the antibody response against sHBsAg protein was evaluated using ELISA plates coated with P. pastoris -derived sHBsAg protein (yHBsAg) at 5 µg/mL (OriGene).

Techniques: Clone Assay, Plasmid Preparation, Purification, Bacteria, Transfection, Recombinant, Binding Assay, Western Blot, SDS Page, Negative Control, Molecular Weight

Journal: Scientific Reports

Article Title: Minicircle-based vaccine induces potent T-cell and antibody responses against hepatitis C virus

doi: 10.1038/s41598-024-78049-3

Figure Lengend Snippet: Analysis of cellular response in BALB/c mice immunized with MC. Box plot of antigen-specific IFN-γ ELISPOT responses in MC groups. Splenocytes from six immunized BALB/c mice per group were stimulated with overlapping peptides spanning the sequences of sHBsAg, NS3, NS4 and NS5. Additionally for the mice immunized with sHBsAg_412–425 MC, sHBsAg peptides were divided into three groups (P1, P2, P3), corresponding to sHBsAg amino acid 1–99 (P1), 100–188 (P2) and 189–240 (P3). The splenocytes were collected 4 weeks after the last immunization. The box marks 25–75th percentile; the horizontal line indicates the median; whiskers extend to minimal and maximal values. Data was analyzed using two-way analysis of variance (ANOVA) (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Article Snippet: Similarly, the antibody response against sHBsAg protein was evaluated using ELISA plates coated with P. pastoris -derived sHBsAg protein (yHBsAg) at 5 µg/mL (OriGene).

Techniques: Enzyme-linked Immunospot

Journal: Scientific Reports

Article Title: Minicircle-based vaccine induces potent T-cell and antibody responses against hepatitis C virus

doi: 10.1038/s41598-024-78049-3

Figure Lengend Snippet: Analysis of antibodies elicited in mice following immunization with MC. ( A ) Analysis of binding of immune sera to 412–425 peptide. For analysis of binding of immune sera, streptavidin-coated microplates were coated with 5 µg/mL of biotinylated synthetic peptides covering HCV E2 epitope 412–425. Serum titer calculated as A 450 value above cutoff value (three times the mean background value) is shown on axis y. Dot plot of the individual mouse serum titers against 412–425 peptide. Each dot represents a single mouse. Mean titer calculated for the group is marked with a solid line. Data were analyzed using unpaired t -test (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). ( B ) Analysis of immune sera binding to yeast-derived sHBsAg (yHBsAg). ELISA plates were coated with 5 µg/mL of purified sHBsAg protein derived from P. pastoris . The dilution factors of the mouse sera are shown on axis x. The mean A 450 values are shown on axis y. The dashed horizontal line represents the cutoff value (three times the mean background value). Data were analyzed using two-way analysis of variance (ANOVA) Data were analyzed using two-way analysis of variance (ANOVA) (ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001). The data represent the results from two independent experiments performed in duplicate, and error bars indicate standard deviations. The background signal from the negative control mouse sera was subtracted from the obtained results.

Article Snippet: Similarly, the antibody response against sHBsAg protein was evaluated using ELISA plates coated with P. pastoris -derived sHBsAg protein (yHBsAg) at 5 µg/mL (OriGene).

Techniques: Binding Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Purification, Negative Control