Journal: Clinical Mass Spectrometry

Article Title: Subtyping of hepatitis C virus with high resolution mass spectrometry

doi: 10.1016/j.clinms.2017.08.003

Figure Lengend Snippet: SDS-PAGE gel of deglycosylated recombinant E1 and E2 proteins.

Article Snippet: Deglycosylation and tryptic digestion of recombinant HCV envelope glycoproteins Recombinant envelope glycoproteins E1 (subtype 1b) and E2 (subtype 1a) of hepatitis C virus strains, each expressed with a polyhistidine tag at the N-terminus, were purchased from Sino Biological Inc. (Beijing, China) with predicted molecular weights of approximately 19 and 32 kDa respectively.

Techniques: SDS Page, Recombinant

Journal: Clinical Mass Spectrometry

Article Title: Subtyping of hepatitis C virus with high resolution mass spectrometry

doi: 10.1016/j.clinms.2017.08.003

Figure Lengend Snippet: Number of full-length sequences of E1 for each HCV subtype. *

Article Snippet: Deglycosylation and tryptic digestion of recombinant HCV envelope glycoproteins Recombinant envelope glycoproteins E1 (subtype 1b) and E2 (subtype 1a) of hepatitis C virus strains, each expressed with a polyhistidine tag at the N-terminus, were purchased from Sino Biological Inc. (Beijing, China) with predicted molecular weights of approximately 19 and 32 kDa respectively.

Techniques:

Journal: Clinical Mass Spectrometry

Article Title: Subtyping of hepatitis C virus with high resolution mass spectrometry

doi: 10.1016/j.clinms.2017.08.003

Figure Lengend Snippet: Subtype specific signature and indicator peptides for HCV envelope glycoprotein E1.

Article Snippet: Deglycosylation and tryptic digestion of recombinant HCV envelope glycoproteins Recombinant envelope glycoproteins E1 (subtype 1b) and E2 (subtype 1a) of hepatitis C virus strains, each expressed with a polyhistidine tag at the N-terminus, were purchased from Sino Biological Inc. (Beijing, China) with predicted molecular weights of approximately 19 and 32 kDa respectively.

Techniques: Sequencing

Journal: Clinical Mass Spectrometry

Article Title: Subtyping of hepatitis C virus with high resolution mass spectrometry

doi: 10.1016/j.clinms.2017.08.003

Figure Lengend Snippet: MALDI mass spectrum of the tryptic digest products of recombinant E1 protein of the 1b subtype.

Article Snippet: Deglycosylation and tryptic digestion of recombinant HCV envelope glycoproteins Recombinant envelope glycoproteins E1 (subtype 1b) and E2 (subtype 1a) of hepatitis C virus strains, each expressed with a polyhistidine tag at the N-terminus, were purchased from Sino Biological Inc. (Beijing, China) with predicted molecular weights of approximately 19 and 32 kDa respectively.

Techniques: Recombinant

Journal: Clinical Mass Spectrometry

Article Title: Subtyping of hepatitis C virus with high resolution mass spectrometry

doi: 10.1016/j.clinms.2017.08.003

Figure Lengend Snippet: Subtype signature and indicator peptides for HCV envelope glycoprotein E2.

Article Snippet: Deglycosylation and tryptic digestion of recombinant HCV envelope glycoproteins Recombinant envelope glycoproteins E1 (subtype 1b) and E2 (subtype 1a) of hepatitis C virus strains, each expressed with a polyhistidine tag at the N-terminus, were purchased from Sino Biological Inc. (Beijing, China) with predicted molecular weights of approximately 19 and 32 kDa respectively.

Techniques: Sequencing

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

Journal: Viruses

Article Title: Removal of the C6 Vaccinia Virus Interferon-β Inhibitor in the Hepatitis C Vaccine Candidate MVA-HCV Elicited in Mice High Immunogenicity in Spite of Reduced Host Gene Expression

doi: 10.3390/v10080414

Figure Lengend Snippet: Generation and in vitro characterization of modified vaccinia virus Ankara (MVA)-hepatitis C virus (HCV) ΔC6L. ( A ) Scheme of the MVA-HCV ΔC6L deletion mutant genome map. The different regions of the MVA vector are shown in capital letters. Below the map, the deleted or fragmented vaccinia virus (VACV) genes are depicted as black boxes, with the deletion of C6L being indicated. The organization of the HCV genome (genotype 1a, strain H77), driven by the sE/L VACV promoter and inserted within the VACV thymidine kinase (TK) viral locus (J2R), is indicated. TK-L, TK left; TK-R, TK right. ( B ) Polymerase chain reaction (PCR) analysis of the VACV C6L (left panel) and TK (right panel) loci. DNA products are indicated by an arrow on the right. ( C ) Expression of HCV proteins in DF-1 cells infected with MVA-HCV or MVA-HCV ΔC6L at 24 h post-infection (hpi). ( D ) Viral growth kinetics in DF-1 cells infected with MVA-HCV or MVA-HCV ΔC6L. At different times post-infection, cells were harvested and virus titers in cell lysates were determined by plaque immunostaining assay with anti-VACV antibodies. The mean and standard deviation (SD) from two independent experiments are shown. WT—wild-type; PFU—plaque-forming units.

Article Snippet: Ninety-six-well plates (Nunc MaxiSorp ® ) were coated and left overnight at 4 °C with 2 μg/mL of purified recombinant HCV E2 protein (genotype 1a, isolate H77; Sino Biological, Wayne, PA, USA) or with 10 μg/mL of a soluble extract of BSC-40 cells infected (5 PFU/cell) for 24 h with VACV WR virus strain.

Techniques: In Vitro, Modification, Mutagenesis, Plasmid Preparation, Polymerase Chain Reaction, Expressing, Infection, Immunostaining, Standard Deviation

Journal: Viruses

Article Title: Removal of the C6 Vaccinia Virus Interferon-β Inhibitor in the Hepatitis C Vaccine Candidate MVA-HCV Elicited in Mice High Immunogenicity in Spite of Reduced Host Gene Expression

doi: 10.3390/v10080414

Figure Lengend Snippet: HCV-specific T cell and humoral adaptive immune responses induced in immunized mice. Splenocytes were obtained from mice ( n = 4 per group) immunized with two doses of MVA-WT, MVA-HCV, or MVA-HCV ∆C6L 10 days after the last immunization. Then, HCV-specific CD8 + T cell adaptive immune responses elicited were measured by intracellular cytokine staining (ICS) after stimulation of splenocytes with different HCV peptide pools. Values from unstimulated controls were subtracted in all cases. p values indicate significantly response differences when comparing MVA-HCV with MVA-HCV ΔC6L (*** = p ≤ 0.001). Data is from one experiment representative of four independent experiments. ( A ) Magnitude of total HCV-specific CD8 + T cell adaptive immune responses directed against HCV antigens. The values represent the sum of the percentages of CD8 + T cells producing CD107a and/or IFN-γ and/or TNF-α and/or IL-2 against Core, E1, E2, p7 + NS2, NS3, NS4, and NS5 peptide pools. ( B ) Percentages of Core, E1, E2, p7 + NS2, NS3, NS4, or NS5 HCV-specific CD8 + T cells. Frequencies represent the sum of the percentages of CD8 + T cells producing CD107a and/or IFN-γ and/or TNF-α and/or IL-2 against each HCV peptide pool. ( C ) Polyfunctionality profile of total HCV-specific CD8 + T cell adaptive immune responses directed against HCV antigens and producing CD107a and/or IFN-γ and/or TNF-α and/or IL-2. Responses are grouped and color coded, taking into consideration the number of functions (one, two, three, or four). ( D ) Humoral immune responses induced by MVA-WT, MVA-HCV, and MVA-HCV ∆C6L against HCV E2 protein. Levels of E2-specific total IgG binding antibodies were measured by ELISA in serum from individually mice immunized with two doses of MVA-WT, MVA-HCV, or MVA-HCV ∆C6L at day 10 after the last immunization. Absorbance values (measured at 450 nm) correspond to 1/100 dilution of individual serum, and each mouse is represented by a dot. The mean ± SD are indicated.

Article Snippet: Ninety-six-well plates (Nunc MaxiSorp ® ) were coated and left overnight at 4 °C with 2 μg/mL of purified recombinant HCV E2 protein (genotype 1a, isolate H77; Sino Biological, Wayne, PA, USA) or with 10 μg/mL of a soluble extract of BSC-40 cells infected (5 PFU/cell) for 24 h with VACV WR virus strain.

Techniques: Staining, Binding Assay, Enzyme-linked Immunosorbent Assay

Journal: Viruses

Article Title: Removal of the C6 Vaccinia Virus Interferon-β Inhibitor in the Hepatitis C Vaccine Candidate MVA-HCV Elicited in Mice High Immunogenicity in Spite of Reduced Host Gene Expression

doi: 10.3390/v10080414

Figure Lengend Snippet: Magnitude, breath, polyfunctionality, and phenotype of HCV-specific CD8 + T cell memory immune responses. Splenocytes were obtained from mice ( n = 4 per group) immunized with two doses of MVA-WT, MVA-HCV, or MVA-HCV ∆C6L 53 days after the last immunization. Then, HCV-specific CD8 + T cell memory immune responses elicited were measured by ICS after stimulation of splenocytes with different HCV peptide pools. Values from unstimulated controls were subtracted in all cases. p values indicate significantly response differences when comparing MVA-HCV with MVA-HCV ΔC6L (*** = p ≤ 0.001). Data is from one experiment representative of four independent experiments. ( A ) Percentage of total HCV-specific CD8 + T cell memory immune responses directed against HCV antigens. The values represent the sum of the percentages of CD8 + T cells producing CD107a and/or IFN-γ and/or TNF-α and/or IL-2 against Core, E1, E2, p7 + NS2, NS3, NS4, and NS5 peptide pools. ( B ) Percentages of Core, E1, E2, p7 + NS2, NS3, NS4, or NS5 HCV-specific CD8 + T cells. Frequencies represent the sum of the percentages of CD8 + T cells producing CD107a and/or IFN-γ and/or TNF-α and/or IL-2 against each HCV peptide pool. ( C ) Polyfunctionality profile of total HCV-specific CD8 + T cell memory immune responses directed against HCV antigens and producing CD107a and/or IFN-γ and/or TNF-α and/or IL-2. Responses are grouped and color coded, taking into consideration the number of functions (one, two, three, or four). Each slice in the pie charts corresponds to the proportion of the total HCV-specific CD8 + T cells exhibiting one, two, three, or four functions (CD107a and/or IFN-γ and/or TNF-α and/or IL-2) within the total HCV-specific CD8 + T cells. ( D ) Phenotypic profile of total HCV-specific CD8 + T cell memory immune responses directed against HCV antigens. Frequencies represent percentages of T central memory (TCM), T effector memory (TEM), and T effector (TE) HCV-specific CD8 + T cells producing CD107a and/or IFN-γ and/or TNF-α and/or IL-2 against HCV peptide pools. TCM, TEM, and TE phenotypes are determined on the basis of CD127 and CD62L expression: TCM (CD127 + , CD62L + ), TEM (CD127 + , CD62L − ), and TE (CD127 − , CD62L − ).

Article Snippet: Ninety-six-well plates (Nunc MaxiSorp ® ) were coated and left overnight at 4 °C with 2 μg/mL of purified recombinant HCV E2 protein (genotype 1a, isolate H77; Sino Biological, Wayne, PA, USA) or with 10 μg/mL of a soluble extract of BSC-40 cells infected (5 PFU/cell) for 24 h with VACV WR virus strain.

Techniques: Expressing