Journal: The FASEB Journal

Article Title: Identification of cyclic peptides able to mimic the functional epitope of IgG1-Fc for human FcγRI

doi: 10.1096/fj.08-117069

Figure Lengend Snippet: Peptide binding specificity for FcγRs assessed by AlphaScreen and rosette formation on FcγR bearing cells. A ) Each biotinylated peptide was incubated with streptavidin donor beads, while the FcγRs were preincubated with nickel chelate acceptor beads. Signal due to peptide complexes added over a range of concentrations of 37, 111, and 333 nM was normalized relative to the signal due to IgG1 binding (equivalent to 100%) at 5, 15, and 46 nM for FcγRI, FcγRIIa, and FcγRIIb; and 15, 46, and 137 nM for FcγRIIIa and FcγRIIIb. Graph shows binding results for FcγRI, with results for FcγRIIIa shown as an example. Results shown were means of 4 independent experiments. B ) Microbeads were sensitized with 10 μM peptide (22, 29, 30 or 33) or 400 nM IgG1 and incubated with 2 × 10 5 primary NK, Daudi, K562, or U937 cells at a ratio of 100:1 (molecule-bead complexes:leukocyte cells). Peptide or IgG-bead complex binding to cells was measured as rosette formation using a fluorescence microscope. Rosettes were defined as at least 5 beads clustered around 1 cell. Percentage rosetting indicates the ratio of rosetted cells to total cells counted. Control beads were not conjugated with any peptide. Results shown are 1 of 5 comparable experiments. C ) Rosette formation with peptide 22 and a U937 cell visualized by light microscopy (×400).

Article Snippet: To express the other human FcγR in soluble form, DNA was purchased that encoded human FcγRIIa, FcγRIIb, and FcγRIIIa extracellular domains (Geneservice, Cambridge, UK), cloned in fusion with a Flag-His-tag DNA sequence in pDEST12.2 (Invitrogen, Paisley, UK) and transfected into HEK-293 EBNA cells (6×10 6 cells).

Techniques: Binding Assay, Amplified Luminescent Proximity Homogenous Assay, Incubation, Fluorescence, Microscopy, Control, Light Microscopy

Journal: Journal of Virology

Article Title: Human Monoclonal Antibodies Potently Neutralize Zika Virus and Select for Escape Mutations on the Lateral Ridge of the Envelope Protein

doi: 10.1128/JVI.00405-19

Figure Lengend Snippet: Neutralizing antibodies cannot elicit ADCC but can elicit ADE in vitro. We examined the ability of neutralizing antibodies to elicit Fc-mediated effector functions either on the surface of infected cells or against the virion itself. (A) Vero cells were infected with MR766 ZIKV and used as targets for measuring Fc-FcγR effector functions with a genetically modified Jurkat cell line expressing human FcγRIIIa with an inducible luciferase reporter gene. Fold induction was measured in relative light units, and results were compared to infected cells with no antibody added. Error bars represent standard error of the means (SEM). (B) To examine whether enhancement of flavivirus infection in vitro is observed, monoclonal antibodies were incubated with ZIKV and added to FcγR-bearing K562 cells. All monoclonal antibodies were tested at a starting concentration of 3.3 μg per ml and serially diluted 4-fold. Both assays were run in duplicate, and fold induction was measured as the percentage of infected cells as determined by flow cytometry divided by the percentage of infected cells with no antibody added (virus alone). (C) Representative flow cytometry plots for antibody AD5 and control IgG are shown.

Article Snippet: Genetically modified Jurkat cells expressing the human FcγRIIIa with a luciferase reporter gene under the transcriptional control of a nuclear factor of activated T cells (NFAT) promoter were added at 7.5 × 10 4 cells in 25 μl per well, which is approximately a 1:2 ratio of target cells to effector cells (Promega).

Techniques: In Vitro, Infection, Genetically Modified, Expressing, Luciferase, Bioprocessing, Incubation, Concentration Assay, Flow Cytometry, Virus, Control

Journal: mAbs

Article Title: Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX

doi: 10.1080/19420862.2021.1999194

Figure Lengend Snippet: ADCC response of (a) chimeric (human Fc portion) antibodies (c2C7, c11H9, c12H8 and c2D7) and (b) murine antibodies (m4A2 and m9B6) using SK-RC-52 cells in conjunction with Promega’s Promega’s human FcγRIIIa or murine FcγRIV Jurkat-Luc bioreporter assay, respectively. Shown are the results of a representative experiment (± s.e.m.) carried out in triplicate and repeated twice; ADCC response was expressed as fold-induction of non-treated control cells, and data was analyzed using Graphpad Prism v8 software

Article Snippet: To evaluate the ADCC response of our antibodies, we used SK-RC-52 target cells (high CAIX) in conjunction with Promega’s human FcγRIIIa (for c11H9, c12H8, c2C7, c2D7; and murine FcγRIV (for m4A2, m9B6; ) in a Jurkat-Luc bioreporter assay.

Techniques: Control, Software

Journal: Science immunology

Article Title: HVEM signaling promotes protective antibody-dependent cellular cytotoxicity (ADCC) vaccine responses to herpes simplex viruses

doi: 10.1126/sciimmunol.aax2454

Figure Lengend Snippet: HaCAT cells were infected with HSV-2 (333-ZAG) (expressing GFP) for 4 hours, incubated with immune serum from ΔgD or VD60 control-immunized mice (1:5 dilution) and then cultured with either (A) total bone marrow or (B) bone marrow-derived CD11c+ cells (cultured with GM-CSF) isolated from WT or Hvem−/− mice as effector cells. ADCC was measured by quantifying dead GFP+ infected target cells by flow cytometry and is expressed as percentage change in dead cells compared to a “no serum” control. The line represents the median of individual results; data was analyzed by Mann-Whitney Test (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001); n=10–15 per group, 3 independent experiments. Immune serum normalized to 750 μg total IgG from ΔgD-2 or VD60-control vaccinated mice was transferred to (C) naïve WT or CD11c-DTR mice treated or not with diphtheria toxin 24 hours before skin with an LD90 dose of HSV-2 (4674) or (D) naïve WT or FcγRIV−/− mice. The asterisks, ****, indicate significant protection (p=0.0001) relative to WT mice that received VD60 control-immune serum (Gehan-Breslow-Wilcoxon test). N = 5–10/group, two independent experiments.

Article Snippet: The authors would like to thank John Kim and Bing Chen for technical assistance; David Knipe, Harvard Medical School, for the dl5–29 and SD90 viruses; Richard Longnecker, Northwestern University, for the Hvem −/− mice and the viral variants W176 and W260; and Mei Cong, Vanessa Ott and Aileen Paguio at Promega for the mFcγRIV and human FcγRIIIa ADCC Reporter Bioassay kits.

Techniques: Infection, Expressing, Incubation, Control, Cell Culture, Derivative Assay, Isolation, Flow Cytometry, MANN-WHITNEY

Journal: Science immunology

Article Title: HVEM signaling promotes protective antibody-dependent cellular cytotoxicity (ADCC) vaccine responses to herpes simplex viruses

doi: 10.1126/sciimmunol.aax2454

Figure Lengend Snippet: (A) Immune serum obtained one week after the second vaccine dose from intramuscularly (right and left panels) or subcutaneously (middle panel) ΔgD-2-immunized wild-type mice was assayed for FcγRIV activation against HSV-2 (SD90)-infected Vero target cells in the presence of 5 or 10 μg of gD protein (left), 5 μg of modified gD-2 protein lacking the HVEM binding domain (Δ7–32) (center) or 10 μg anti-HVEM antibody (right). (B) Murine FcγRIV-activation assays were conducted with ΔgD-2 immune serum and Vero cells infected with HSV-2 (SD90), ΔgD-2 (in the absence or presence of 5 μg soluble gD protein), W260 (HSV-2 virus lacking the HVEM-binding domain of gD) and HSV-2 W176 (repaired W260) as target cells. (C) Immune sera from five HSV-2 seropositive individuals were assayed for human FcγRIIIa activation in the absence or presence of increasing doses of soluble gD protein or anti-HVEM antibody. (D) Increasing amounts of anti-HVEM antibody were added to the positive control Raji cells with anti-CD20 antibody in the murine FcγRIV-activation assay. Results are from two independent experiments, n=5–7/group, *p<0.05, **p<0.01, ***p<0.001, by paired Student’s t-test.

Article Snippet: The authors would like to thank John Kim and Bing Chen for technical assistance; David Knipe, Harvard Medical School, for the dl5–29 and SD90 viruses; Richard Longnecker, Northwestern University, for the Hvem −/− mice and the viral variants W176 and W260; and Mei Cong, Vanessa Ott and Aileen Paguio at Promega for the mFcγRIV and human FcγRIIIa ADCC Reporter Bioassay kits.

Techniques: Activation Assay, Infection, Modification, Binding Assay, Virus, Positive Control

Journal: mAbs

Article Title: Isolation and characterization of monoclonal antibodies against human carbonic anhydrase-IX

doi: 10.1080/19420862.2021.1999194

Figure Lengend Snippet: ADCC response of (a) chimeric (human Fc portion) antibodies (c2C7, c11H9, c12H8 and c2D7) and (b) murine antibodies (m4A2 and m9B6) using SK-RC-52 cells in conjunction with Promega’s Promega’s human FcγRIIIa or murine FcγRIV Jurkat-Luc bioreporter assay, respectively. Shown are the results of a representative experiment (± s.e.m.) carried out in triplicate and repeated twice; ADCC response was expressed as fold-induction of non-treated control cells, and data was analyzed using Graphpad Prism v8 software

Article Snippet: ADCC response of (a) chimeric (human Fc portion) antibodies (c2C7, c11H9, c12H8 and c2D7) and (b) murine antibodies (m4A2 and m9B6) using SK-RC-52 cells in conjunction with Promega’s Promega’s human FcγRIIIa or murine FcγRIV Jurkat-Luc bioreporter assay, respectively.

Techniques: Control, Software

Journal: The Journal of Infectious Diseases

Article Title: Failure of Herpes Simplex Virus Glycoprotein D Antibodies to Elicit Antibody-Dependent Cell-Mediated Cytotoxicity: Implications for Future Vaccines

doi: 10.1093/infdis/jiac284

Figure Lengend Snippet: Antibody responses in chronic herpes simplex virus (HSV) 2 infection in human immunodeficiency virus (HIV)–uninfected (HIV−) and HIV-infected (HIV+) women. A, Samples from HIV− and HIV+ women were assayed for total IgG. B, HSV-specific immunoglobulin (Ig) G, IgG1, IgG3, and C1q-binding antibodies assayed by enzyme-linked immunosorbent assay. Abbreviation: OD450, optical density at 450 nm C, Neutralization titers assayed by plaque reduction assay. Abbreviation: IC50, serum dilution that inhibited 50% of infection. D, Antibody-dependent cell-mediated cytotoxicity (ADCC) assessed using a Promega human FcγRIIIa (Fc gamma receptor IIIa) ADCC Reporter Bioassay and expressed as fold induction. Open symbols represent women who reported HSV-2 recurrences within the previous 12 months. *P < 0.5; **P < 0.01 (Mann-Whitney U test).

Article Snippet: D, Human Fc gamma receptor (FcγR) IIIa activation was measured using a Promega human FcγRIIIa ADCC Reporter Bioassay with cells infected with HSV-2 as targets and serum diluted 1:5.

Techniques: Virus, Infection, Binding Assay, Enzyme-linked Immunosorbent Assay, Neutralization, Bioassay, MANN-WHITNEY

Journal: The Journal of Infectious Diseases

Article Title: Failure of Herpes Simplex Virus Glycoprotein D Antibodies to Elicit Antibody-Dependent Cell-Mediated Cytotoxicity: Implications for Future Vaccines

doi: 10.1093/infdis/jiac284

Figure Lengend Snippet: Glycoprotein D (gD/)ASO4 vaccination and primary infection elicit neutralizing but not antibody-dependent cell-mediated cytotoxicity (ADCC) responses. A, Herpes simplex virus (HSV)–specific neutralization was performed using a microneutralization or plaque reduction assay (triangles only), and results are presented as the serum dilution that inhibited 50% of infection (IC50). (See the legend to Figure 1 for definitions of time A and time B.) B, Neutralization titers for the 2 assays were compared for a subset of samples (n = 6). C, C1q-binding immunoglobulin (Ig) G was quantified by enzyme-linked immunosorbent assay. Abbreviation: OD450, optical density at 450 nm. D, Human Fc gamma receptor (FcγR) IIIa activation was measured using a Promega human FcγRIIIa ADCC Reporter Bioassay with cells infected with HSV-2 as targets and serum diluted 1:5. Paired t tests were performed on the subset of paired samples as in Figure 1 (open symbols; n = 5), and Mann-Whitney U tests were performed to compare responses to gD/AS04 versus acute infection at time B for all samples (open and closed symbols). *P < .05; **P < .01; ***P < .001.

Article Snippet: D, Human Fc gamma receptor (FcγR) IIIa activation was measured using a Promega human FcγRIIIa ADCC Reporter Bioassay with cells infected with HSV-2 as targets and serum diluted 1:5.

Techniques: Infection, Virus, Neutralization, Binding Assay, Enzyme-linked Immunosorbent Assay, Activation Assay, Bioassay, MANN-WHITNEY

Journal: The Journal of Infectious Diseases

Article Title: Failure of Herpes Simplex Virus Glycoprotein D Antibodies to Elicit Antibody-Dependent Cell-Mediated Cytotoxicity: Implications for Future Vaccines

doi: 10.1093/infdis/jiac284

Figure Lengend Snippet: Comparison of Humoral Responses in Women With Acute Versus Chronic Herpes Simplex Virus 2 Infection

Article Snippet: D, Human Fc gamma receptor (FcγR) IIIa activation was measured using a Promega human FcγRIIIa ADCC Reporter Bioassay with cells infected with HSV-2 as targets and serum diluted 1:5.

Techniques: Comparison, Virus, Infection, Neutralization, Binding Assay

Journal: Cell reports

Article Title: Protective effect and molecular mechanisms of human non-neutralizing cross-reactive spike antibodies elicited by SARS-CoV-2 mRNA vaccination

doi: 10.1016/j.celrep.2024.114922

Figure Lengend Snippet: (A) FcγRIIIa reporter activity of the RBD-, NTD-, S2-, and unknown-epitope-binding mAbs. (B) FcγRIIa reporter activity of the RBD-, NTD-, S2-, and unknown-epitope-binding mAbs. (C) Binding of complement protein C1q to the Fc portion of the mAbs in the presence of wild-type SARS-CoV-2 spike protein. Dotted lines denote the limit of detection. Each assay was performed at least twice, and the geometric mean and geometric standard deviation of these replicates is shown. The control groups for each assay are shared and are displayed in all panels belonging to the respective assay.

Article Snippet: 3×10 6 ADCC and ADCP Bioassay Effector Cells expressing the human FcγRIIIa or FcγRIIa receptors (Promega) were added per well and the cells were incubated at 37°C with 5% CO 2 for 6 h. 75 μL of Bio-Glo luciferase reagent (Promega) was then added to each well and the luminescence was read in a Synergy 4 (BioTek) plate reader.

Techniques: Activity Assay, Binding Assay, Standard Deviation, Control

Journal: Cell reports

Article Title: Protective effect and molecular mechanisms of human non-neutralizing cross-reactive spike antibodies elicited by SARS-CoV-2 mRNA vaccination

doi: 10.1016/j.celrep.2024.114922

Figure Lengend Snippet: (A) FcγRIIIa reporter activity of the seven most protective mAbs and their LALA and LALAPG mutant counterparts. (B) FcγRIIa reporter activity of the wild-type, LALA, and LALAPG mAbs. (C) Binding of complement protein C1q by the wild-type, LALA, and LALAPG mAbs in the presence of wild-type SARS-CoV-2 spike protein. In (A)–(C), dotted lines denote the limit of detection. Each assay was performed twice, and the geometric mean and geometric standard deviation of these replicates is shown. (D) Weight loss and survival of BALB/cAnNHsd mice ( n = 4, mean ± SD) treated intraperitoneally with a 10 mg/kg dose of the seven most protective mAbs and their LALA and LALAPG counterparts following lethal challenge with a 5×LD 50 dose of mouse-adapted SARS-CoV-2. The negative control was shared across experimental groups and is shown in each respective panel.

Article Snippet: 3×10 6 ADCC and ADCP Bioassay Effector Cells expressing the human FcγRIIIa or FcγRIIa receptors (Promega) were added per well and the cells were incubated at 37°C with 5% CO 2 for 6 h. 75 μL of Bio-Glo luciferase reagent (Promega) was then added to each well and the luminescence was read in a Synergy 4 (BioTek) plate reader.

Techniques: Activity Assay, Mutagenesis, Binding Assay, Standard Deviation, Negative Control

Journal: Cell reports

Article Title: Protective effect and molecular mechanisms of human non-neutralizing cross-reactive spike antibodies elicited by SARS-CoV-2 mRNA vaccination

doi: 10.1016/j.celrep.2024.114922

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: 3×10 6 ADCC and ADCP Bioassay Effector Cells expressing the human FcγRIIIa or FcγRIIa receptors (Promega) were added per well and the cells were incubated at 37°C with 5% CO 2 for 6 h. 75 μL of Bio-Glo luciferase reagent (Promega) was then added to each well and the luminescence was read in a Synergy 4 (BioTek) plate reader.

Techniques: Virus, Recombinant, Luciferase, Electron Microscopy, Enzyme-linked Immunosorbent Assay, Microneutralization Assay, Bioassay, Variant Assay, Software