Journal: Virus Research
Article Title: Identification of neutralizing epitopes in the preS2 domain of the hepatitis B virus
doi: 10.1016/j.virusres.2022.199014
Figure Lengend Snippet: Immunization of mice with plasmids encoding HBV preS1 and preS2. (A) Schematic diagram of the structure of HBsAg and of plasmids encoding N-terminal preS1 and preS2, showing the position of the CAG promoter (CAG) and of sequences encoding the murine Ig κ-chain leader sequence (S), Myc tag (myc), spacer, and GroEL. The amino acid sequences of preS1/2–47 and preS2 are derived from HBV genotype-C. (B) Expression of preS1/2–47 and preS2. Lysates and supernatants of 293T cells transfected with either plasmid were harvested at 3 days post-transfection and subjected to immunoblotting using anti-preS1 and anti-preS2; anti-actin antibodies were used as a loading control. (C) Detection of serum antibodies binding to the preS1/2–47 peptide, preS2 peptide, HBs-L. HBs-M, and HBs-S proteins. Individual sera from mice (n=4) immunized with the indicated plasmids were diluted 100-fold for peptides and 250-fold for HBs proteins; the diluted sera then were added to ELISA microtiter wells containing each antigen. Bound antibody was detected using HRP-conjugated anti-mouse secondary antibody. The vertical axis for each serum and antigen shows absorbance in individual wells. (D and E) Neutralizing activities of sera from mice immunized with preS1 (D) or preS2 (E) expression plasmids. HBV/NL derived from genotype-C was preincubated with pooled sera (at 100-, 200-, 400-, 800-, and 1600-fold dilutions) derived from mice immunized with the indicated plasmids; the mixtures then were then used to infect G2/NT-18C cells for 16 h. Luciferase activity was determined at 7 days post-infection and is expressed relative to activity in reactions without serum. The statistical significance of differences between groups was evaluated using a Student's t-test (* P < 0.05, ** P < 0.01 vs. serum from empty vector-immunized control group). (F) HBVcc was mixed with indicated serum (100-fold dilutions) and used to inoculate HepG2-NTCPsec+ cells. HBsAg titers in the culture medium were measured. Statistical significance was evaluated using the Student's t-test (** P < 0.01 vs. no serum control). (G) HBV-positive cells were visualized by staining with an anti-HBc antibody, and nuclei were visualizedby staining with DAPI.
Article Snippet: The HBVcc-infected cells were fixed, permeabilized, and treated with rabbit polyclonal anti-HBc IgG (Austral Biologicals) followed by staining with Alexa Fluor 555-conjugated anti-rabbit IgG (Thermo Fisher Scientific).
Techniques: Sequencing, Derivative Assay, Expressing, Transfection, Plasmid Preparation, Western Blot, Binding Assay, Enzyme-linked Immunosorbent Assay, Luciferase, Activity Assay, Infection, Staining
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