Journal: Frontiers in Immunology

Article Title: Adenovectored RSV prefusion glycoprotein + soluble glycoprotein combination immunization establishes persistent opsonophagocytic antibody response through IgG3

doi: 10.3389/fimmu.2025.1609779

Figure Lengend Snippet: The AD26.preF/SpreF protein induces an acute FcγR-binding repertoire against both RSV/A F and RSV/B F. (A) Univariate comparisons of baseline (black), placebo-treated (gray), or AD26.preF/SpreF protein-vaccinated (gold) participants at the indicated timepoints for FcγRIIA, FcγRIIB, FcγRIIIA, and FcγRIIIB against RSV F subtype A. (B) Same as (A) but for RSV-F subtype B. Statistical comparisons were done using an initial Wilcox test followed by a Bonferroni correction for multiple comparisons. Comparisons were made for recipients of AD26.preF/SpreF protein against placebo at the same timepoint. Bonferroni-corrected p -values ( q -values) were represented above the timepoints with * q < 0.05, ** q < 0.01, and *** q < 0.001; comparisons whose q -value was ≥ 0.05 were left unlabeled. Data was plotted as a box and whisker showing median, interquartile ranges, and ranges. Individual data points were superimposed on each box and whisker plot. Values were plotted as binding mean fluorescence units (MFI) quantified as arbitrary units (A.U.) via multiplexed flow cytometry.

Article Snippet: For FcγR binding, a biotinylated PE-streptavidin coupled recombinant human FcγR protein (Agilent Technologies, Santa Clara, CA, USA) was used as the secondary probe.

Techniques: Binding Assay, Whisker Assay, Fluorescence, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Human Fcγ-receptors selectively respond to C-reactive protein isoforms

doi: 10.3389/fimmu.2025.1598605

Figure Lengend Snippet: Effect of CRP source and addition of LPS on BW5147-FcγRζ activation and binding of epitope-tagged FcγRs to immobilized IgG, pCRP or mCRP: (A) BWCD64 and BWCD32b cell activation by recombinant human pCRP produced in E coli and pCRP purified from human ascites/pleural fluid. pCRP was coated in graded amounts in PBS. A total of 100,000 BW5147 reporter cells were added to each well and incubated overnight. (B) Addition of graded amounts of LPS to a pCRP (5 µg/well) preparation was compared with activation caused by LPS only using BWCD64 reporter cells. LPS was added at the concentrations stated. EU units as stated by supplier: 1 mg/ml=1x10^6 EU/ml. 100,000 BW5147 cells were added to each well and incubated overnight. FcγR-activation shown as OD in sandwich mIL-2-ELISA after subtraction of background. (C–E) Titration of recombinant His-tagged hFcγRs from 0.25 µg to 0 µg in 50 µl PBS; binding to 0.05 µg coated IgG1/pCRP or mCRP/well. ODs for 450–620 nm. Data shown with standard deviation for two individual experiments with three technical replicates each. Calculation of AUCs of the binding curves using GraphPad Prism software. AUC for N=6 with standard error. Ordinary one-way ANOVA and Tukey´s multiple comparisons test carried out using GraphPad Prism software and selected significances are indicated on the graph.

Article Snippet: Binding of recombinant His-tagged FcγRs (Sino Biological, Bejing, China, recombinant, HEK293/ECD, C-terminal polyhistidine tag: 1038-H08H1/10374-H08H1/10374-H08H/10259-H08H/10256-H08H/10389-H08C) to immobilized pCRP (US Biological Life Sciences) or IgG (human IgG1 kappa, # I5154-1MG, Sigma-Aldrich) was investigated by ELISA.

Techniques: Activation Assay, Binding Assay, Recombinant, Produced, Purification, Incubation, Enzyme-linked Immunosorbent Assay, Titration, Standard Deviation, Software

Journal: Frontiers in Immunology

Article Title: Human Fcγ-receptors selectively respond to C-reactive protein isoforms

doi: 10.3389/fimmu.2025.1598605

Figure Lengend Snippet: In solution phase BW5147-FcγRζ reporter assay for sol. ICs, sol. pCRP and sol. pCRP-streptococci complexes and binding of soluble IgG, pCRP and mCRP to coated His-tagged FcγRs: (A) MaxiSorp ELISA plates were saturated with 10% FCS. sICs as well as soluble CRP-streptococci complexes (with S. pneumoniae serotype 27) were allowed to incubate for two hours at RT prior to adding them to the experiment. Upper: sICs were added in 100 µl/well medium and consisted of 25 nM Infliximab (149.1 kDa) and 50 nM TNFα monomer (17.5 kDa) to ensure 1:1 stoichiometry (per ml stock of 25 nM ICs: 0.875 µg TNFα + 2.66 µg Infliximab). Selected values of log2 titration depicted in this graph. Central: pCRP in solution assay without pre-incubation with streptococci. CRP was added in 100 µl medium. Lower: 10 µl of streptococci were added to 20/10/5 µg of CRP. Complexes were added to wells in 100 µl medium. 100,000 BW5147 reporter cells were added to each well in another 100 µl of medium. Activation shown as OD in sandwich mIL-2-ELISA. Data are shown with standard deviation (N=2; N=3 for ICs). (B) BWCD64 activation assay comparing coated pCRP and soluble pCRP/soluble pCRP-streptococci complexes (N=2). Ordinary one-way ANOVA and Tukey´s multiple comparisons test carried out using GraphPad Prism software and selected significances are indicated on the graph. (C) Titration of His-tagged hFcγRs from 0.25 µg to 0 µg and coating to ELISA wells. Addition of 0.1 µg IgG1 (upper), pCRP (central) or mCRP (lower) and detection via goat-anti-hCRP antibody and DAG-POD for CRP and anti-human-IgG-POD for IgG1. ODs for 450–620 nm. Data shown with standard deviation for two individual experiments with three technical replicates each. (D) Coating of goat F(ab) 2 anti-human IgG (Fab-specific) (0.1 µg in 50 µl/well) was followed by blocking and addition of hIgG1 (0.25 µg in 50 µl/well) before addition of soluble human FcyR-His-proteins titrated as stated in the graph. Detection with rabbit anti-His antibody and GAR-POD was performed. Data shown in technical triplicates for two individual experiments.

Article Snippet: Binding of recombinant His-tagged FcγRs (Sino Biological, Bejing, China, recombinant, HEK293/ECD, C-terminal polyhistidine tag: 1038-H08H1/10374-H08H1/10374-H08H/10259-H08H/10256-H08H/10389-H08C) to immobilized pCRP (US Biological Life Sciences) or IgG (human IgG1 kappa, # I5154-1MG, Sigma-Aldrich) was investigated by ELISA.

Techniques: Reporter Assay, Binding Assay, Enzyme-linked Immunosorbent Assay, Titration, Incubation, Activation Assay, Standard Deviation, Software, Blocking Assay