fcγriv  (Sino Biological)

Journal: Frontiers in Immunology

Article Title: Selective Engagement of FcγRIV by a M2e-Specific Single Domain Antibody Construct Protects Against Influenza A Virus Infection

doi: 10.3389/fimmu.2019.02920

Figure Lengend Snippet: Characterization of the isolated FcγRIV-specific VHH. (A) The predicted amino acid residue sequence of FcγRIV-VHH-7m, isolated after panning on recombinant mouse FcγRIV protein of a VHH library derived from a llama immunized with immature mouse dendritic cells. Above the sequences the Kabat numbering is indicated. CDR1, −2, and −3 are boxed. (B) Flow cytometric analysis showing binding of FcgRIV-VHH-7m and M2e-VHH-23m to HEK 293T cells transiently transfected with expression vectors coding for mouse FcγRI, FγRIIb, FcγRIII, and FcγRIV along with the common γ-chain for the activating FcγRs and a GFP expression plasmid. The lower graph depicts binding to Mf4/4 cells. The cells were incubated with fourfold serial dilution of Alexa Fluor™ 647 labeled FcγRIV-VHH-7m or M2e-VHH-23m, starting at a concentration of 0.2 μM, binding of the VHHs to the GFP positive cells was analyzed. Data points represent averages of triplicates and error bars standard deviations.

Article Snippet: VHH Binding to FcγRs Expressing Cells Human Embryonic Kidney (HEK) 293T cells were transiently transfected with full length mouse FcγRI (MG50086-CF), FcγRIIb (MG50030-CY, SinoBiological Inc.), FcγRIII (MG50326, SinoBiological Inc.) or FcγRIV (MG50036-CF, SinoBiological Inc.) expression constructs along with the common γ-chain for the activating FcγRs (MG50935-CF) by polyethylenimine (PEI)-based transfection.

Techniques: Isolation, Sequencing, Recombinant, Derivative Assay, Flow Cytometry, Binding Assay, Transfection, Expressing, Plasmid Preparation, Incubation, Serial Dilution, Labeling, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Selective Engagement of FcγRIV by a M2e-Specific Single Domain Antibody Construct Protects Against Influenza A Virus Infection

doi: 10.3389/fimmu.2019.02920

Figure Lengend Snippet: Bispecific fusion construct of a FcγRIV- and M2e-specific VHH protects mice against a potentially lethal influenza A virus infection. (A) Groups of 16 (bispecific VHHs) or 14 (PBS) BALB/c mice were intranasally treated with 50 μg of the bispecific VHHs or PBS 4 h before and 24 h after challenge with 2xLD 50 of A/X47 (H3N2) influenza virus. Body weight change (left) and survival (right) were monitored for 14 days. The mean relative changes in body weight together with their standard errors, are represented. The difference in body weight loss between FcγRIV VHH-M2e VHH and the negative-control groups was statistically significant (*** P

Article Snippet: VHH Binding to FcγRs Expressing Cells Human Embryonic Kidney (HEK) 293T cells were transiently transfected with full length mouse FcγRI (MG50086-CF), FcγRIIb (MG50030-CY, SinoBiological Inc.), FcγRIII (MG50326, SinoBiological Inc.) or FcγRIV (MG50036-CF, SinoBiological Inc.) expression constructs along with the common γ-chain for the activating FcγRs (MG50935-CF) by polyethylenimine (PEI)-based transfection.

Techniques: Construct, Mouse Assay, Infection, Negative Control

Journal: Frontiers in Immunology

Article Title: Selective Engagement of FcγRIV by a M2e-Specific Single Domain Antibody Construct Protects Against Influenza A Virus Infection

doi: 10.3389/fimmu.2019.02920

Figure Lengend Snippet: Bispecific fusion construct of anti-mouse FcγRIV VHH with M2e-VHH-23m selectively activates FcγRIV in vitro . (A) Schematic representation of the bispecific VHHs and ELISA on human H3N2 M2e peptide and recombinant FcγRIV protein is shown on the right. Wells of microtiter plates were coated with 100 ng peptide or protein. Dilution series of the bispecific VHH fusion constructs were added to the coated plates. Binding was detected with a mouse anti-His tag MAb, followed by a secondary sheep anti-mouse IgG Ab conjugated to HRP for the peptide ELISA. In the ELISA with coated recombinant FcγRIV protein, binding was detected with a HRP-conjugated rabbit anti-camelid VHH antibody. (B) Serial dilutions of the bispecific VHH fusion construct or monoclonal antibodies were added to HEK293T cells stably transfected with an influenza M2 expression plasmid. Thirty minutes later, FcγR-ζ BW5147 reporter cells were added to the HEK293T cells. After overnight incubation produced mIL-2 was measured in a sandwich-ELISA, which served as an indicator for the magnitude of FcγR activation. (C) MDCK cells were infected with A/Puerto Rico/8/1934 (H1N1) virus for 1 h. Unbound virus particles were washed away and serial dilutions of the bispecific VHHs or monoclonal antibodies were added and incubated for 30 min, followed by the addition of the FcγRIV-ζ BW5147 reporter cells (C) or human FcγRIIIa-ζ BW5147 reporter cells (D) . After overnight incubation supernatants were analyzed by an anti mIL-2 sandwich ELISA. Data points represent averages of triplicates and error bars represent standard deviations. The graphs are a representative of one out of three repeat experiments.

Article Snippet: VHH Binding to FcγRs Expressing Cells Human Embryonic Kidney (HEK) 293T cells were transiently transfected with full length mouse FcγRI (MG50086-CF), FcγRIIb (MG50030-CY, SinoBiological Inc.), FcγRIII (MG50326, SinoBiological Inc.) or FcγRIV (MG50036-CF, SinoBiological Inc.) expression constructs along with the common γ-chain for the activating FcγRs (MG50935-CF) by polyethylenimine (PEI)-based transfection.

Techniques: Construct, In Vitro, Enzyme-linked Immunosorbent Assay, Recombinant, Binding Assay, Peptide ELISA, Protein Binding, Stable Transfection, Transfection, Expressing, Plasmid Preparation, Incubation, Produced, Sandwich ELISA, Activation Assay, Infection