elisa e2 protein  (Sino Biological)

Journal: Journal of Virology

Article Title: Potent Anti-hepatitis C Virus (HCV) T Cell Immune Responses Induced in Mice Vaccinated with DNA-Launched RNA Replicons and Modified Vaccinia Virus Ankara-HCV

doi: 10.1128/JVI.00055-19

Figure Lengend Snippet: Generation and analysis of HCV protein expression of the different DREP-based HCV vaccine candidates. (A) Scheme of the four novel DREP-based HCV vaccine candidates generated in this study, expressing either core-E1-E2 or p7-NS2-NS3 HCV genes. DREP-C-E1-E2 and DREP-p7-NS2-NS3 were grouped to form the DREP-HCV vaccine candidate, while DREP-e-C-E1-E2 and DREP-e-p7-NS2-NS3 were grouped to form the DREP-e-HCV vaccine candidate. The DREP replicons contain the alphavirus replicase placed under the control of the cytomegalovirus (CMV) promoter, and the HCV genes (either core-E1-E2 or p7-NS2-NS3) were placed under the control of the alphavirus subgenomic promoter (SP). In DREP-e-C-E1-E2 and DREP-e-p7-NS2-NS3, the translational enhancer (e) is placed in frame and upstream of the HCV genes, as indicated. (B) PCR analysis. Primers hybridizing in the DREP-vector regions flanking the place where the HCV genes were inserted were used to confirm their correct insertion. (C) Expression of HCV proteins in human HEK293T cells mock transfected or transfected with DREP-HCV (mixture of DREP-C-E1-E2 and DREP-p7-NS2-NS3), DREP-e-HCV (mixture of DREP-e-C-E1-E2 and DREP-e-p7-NS2-NS3), or empty DREP-Ø at 48 h posttransfection.

Article Snippet: Serum samples from immunized mice were used to analyze by ELISA total IgG binding antibody levels against HCV E2 protein (genotype 1a, isolate H77; Sino Biological Wayne, PA, USA) and isotypes IgG1, IgG2c, and IgG3, as previously described ( 34 ).

Techniques: Expressing, Generated, Plasmid Preparation, Transfection

Journal: Journal of Virology

Article Title: Potent Anti-hepatitis C Virus (HCV) T Cell Immune Responses Induced in Mice Vaccinated with DNA-Launched RNA Replicons and Modified Vaccinia Virus Ankara-HCV

doi: 10.1128/JVI.00055-19

Figure Lengend Snippet: HCV-specific CD4+ and CD8+ T cell adaptive immune responses elicited in immunized mice. Five mice per group were sacrificed at 10 days postboost, and the splenic HCV-specific CD4+ and CD8+ T cell immune responses were analyzed by ICS, as described in Materials and Methods. P values indicate significantly differences between results for DREP-HCV/MVA-HCV, DREP-e-HCV/MVA-HCV, and MVA-HCV/MVA-HCV, as indicated (*, P < 0.05; ***, P < 0.001). (A) Magnitude of total HCV-specific CD4+ and CD8+ T cell adaptive immune responses directed against all HCV antigens. Percentages of CD4+ or CD8+ T cells expressing CD107a and/or producing IFN-γ and/or TNF-α and/or IL-2 against a mixture of core, E1, E2, p7, NS2, NS3, NS4, and NS5 genotype 1a HCV peptide pools are represented. (B) Breadth of HCV-specific CD4+ and CD8+ T cell adaptive immune responses. Percentages of core-, E1-, E2-, p7-, NS2-, NS3-, NS4-, or NS5-specific CD4+ and CD8+ T cells expressing CD107a and/or producing IFN-γ and/or TNF-α and/or IL-2 against each specific HCV peptide pool are represented. (C) Polyfunctionality of total HCV-specific CD4+ and CD8+ T cell adaptive immune responses directed against all HCV antigens. Responses are divided according to function in combined production of CD107a, IFN-γ, TNF-α, and/or IL-2 and grouped according to the color-coded pie charts, taking into consideration the number of functions (one, two, three, or four).

Article Snippet: Serum samples from immunized mice were used to analyze by ELISA total IgG binding antibody levels against HCV E2 protein (genotype 1a, isolate H77; Sino Biological Wayne, PA, USA) and isotypes IgG1, IgG2c, and IgG3, as previously described ( 34 ).

Techniques: Expressing

Journal: Journal of Virology

Article Title: Potent Anti-hepatitis C Virus (HCV) T Cell Immune Responses Induced in Mice Vaccinated with DNA-Launched RNA Replicons and Modified Vaccinia Virus Ankara-HCV

doi: 10.1128/JVI.00055-19

Figure Lengend Snippet: HCV-specific CD4+ and CD8+ T cell memory immune responses elicited in immunized mice. Five mice per group were sacrificed at 53 days postboost, and the splenic HCV-specific CD4+ and CD8+ T cell immune responses were analyzed by ICS, as described in Materials and Methods. P values indicate significantly differences between results for DREP-HCV/MVA-HCV, DREP-e-HCV/MVA-HCV, and MVA-HCV/MVA-HCV, as indicated (***, P < 0.001). (A) Magnitude of total HCV-specific CD4+ and CD8+ T cell memory immune responses directed against all HCV antigens. Percentages of CD4+ or CD8+ T cells expressing CD107a and/or producing IFN-γ and/or TNF-α and/or IL-2 against a mixture of core, E1, E2, p7, NS2, NS3, NS4, and NS5 genotype 1a HCV peptide pools are represented. (B) Breadth of HCV-specific CD4+ and CD8+ T cell memory immune responses. Percentages of core-, E1-, E2-, p7-, NS2-, NS3-, NS4-, or NS5-specific CD4+ and CD8+ T cells expressing CD107a and/or producing IFN-γ and/or TNF-α and/or IL-2 against each specific HCV peptide pool are represented. (C) Polyfunctionality of total HCV-specific CD4+ and CD8+ T cell memory immune responses directed against all HCV antigens. Responses are divided according to function in combined production of CD107a, IFN-γ, TNF-α, and/or IL-2 and grouped according to the color-coded pie charts, taking into consideration the number of functions (one, two, three, or four).

Article Snippet: Serum samples from immunized mice were used to analyze by ELISA total IgG binding antibody levels against HCV E2 protein (genotype 1a, isolate H77; Sino Biological Wayne, PA, USA) and isotypes IgG1, IgG2c, and IgG3, as previously described ( 34 ).

Techniques: Expressing

Journal: Journal of Virology

Article Title: Potent Anti-hepatitis C Virus (HCV) T Cell Immune Responses Induced in Mice Vaccinated with DNA-Launched RNA Replicons and Modified Vaccinia Virus Ankara-HCV

doi: 10.1128/JVI.00055-19

Figure Lengend Snippet: HCV-specific humoral immune responses elicited in immunized mice. (A) Levels of HCV E2-specific total IgG binding antibodies were measured by ELISA in serial 2-fold dilutions of pooled serum samples (n = 10 per group) obtained from immunized mice at 10 days postboost. Absorbance values were measured at 450 nm. The mean and standard deviations are indicated. Blue asterisks indicate significant differences between results with DREP-HCV/MVA-HCV and MVA-HCV/MVA-HCV, while red asterisks indicate significant differences between results with DREP-e-HCV/MVA-HCV and MVA-HCV/MVA-HCV (*, P < 0.05). (B) Levels of HCV E2-specific IgG1, IgG2c, and IgG3 isotype antibodies in diluted 1/50 individual serum samples (n = 10 per group) obtained from immunized mice at 10 days postboost. Absorbance (optical density [OD]) values were measured at 450 nm. The mean and standard deviations are indicated.

Article Snippet: Serum samples from immunized mice were used to analyze by ELISA total IgG binding antibody levels against HCV E2 protein (genotype 1a, isolate H77; Sino Biological Wayne, PA, USA) and isotypes IgG1, IgG2c, and IgG3, as previously described ( 34 ).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay