Journal: The Journal of Experimental Medicine

Article Title: Type I interferon autoantibody footprints reveal neutralizing mechanisms and allow inhibitory decoy design

doi: 10.1084/jem.20242039

Figure Lengend Snippet: Comparison of IFNα residues targeted by autoAbs or bound by IFNAR1/IFNAR2. (A) Amino acid sequence alignment of all human IFNα subtypes. Residues that are part of the R1 or R2 autoAb footprints are highlighted in bold, and receptor-binding residues are underlined. Amino acid numbering refers to the mature form of IFNα2. (B) Previously determined structure of the IFNα2-IFNAR1-IFNAR2 protein complex (PDB: 3SE3 ). The identified R1 and R2 autoAb footprints are colored green. (C and D) Side-by-side comparison of the IFNα2 structure with important R1-footprint residues colored green (C) and important IFNAR1-binding residues colored orange (D). (E and F) Side-by-side comparison of the IFNα2 structure with important R2-footprint residues colored green (E) and important IFNAR2-binding residues colored orange (F).

Article Snippet: His-tagged IFNAR1 (IF1-H5225; ACROBiosystems), His-tagged IFNAR2 (IF2-H5224; ACROBiosystems), Fc-tagged IFNα1 (IFA-H5258; ACROBiosystems), and IFNω (NBP2-35893; Novus Biologicals) were diluted to the indicated concentrations in assay buffer (0.02% Tween-20, 0.1% BSA in 1x PBS, pH 7.4).

Techniques: Comparison, Sequencing, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Type I interferon autoantibody footprints reveal neutralizing mechanisms and allow inhibitory decoy design

doi: 10.1084/jem.20242039

Figure Lengend Snippet: Mechanism of IFNα neutralization by anti-IFNα autoAbs. (A and B) Optimization of BLI assays to detect association of IFNα1 with IFNAR1 (A) or IFNAR2 (B). NSB: no receptor loaded, association phase with 200 nM IFNα1. Zoom-in images of the association phases are shown. (C) Abilities of rontalizumab and sifalimumab to neutralize IFNα2 at 10 or 0.2 ng/ml. Dashed lines represent neutralization thresholds set at 75% reduction compared with control donor samples. Data are normalized to the luciferase signal from the control group. Mean values and standard deviations are shown and are representative of at least n = 2 independent experiments. (D) IFNα2 avidity indexes as determined for rontalizumab and sifalimumab at different urea concentrations. The dashed line indicates the threshold for high avidity (>0.6). (E) Blocking of the interaction between IFNα1 and IFNAR1 or IFNAR2 by Ronta and Sifa. (F) Blocking of the interaction between IFNα1 and IFNAR1 or IFNAR2 by four plasmas containing neutralizing anti-IFNα autoAbs from the COVID ICU cohort. For all data panels, results shown are representative of at least n = 2 similar experiments. Ronta, rontalizumab; Sifa, sifalimumab; NSB, nonspecific binding control.

Article Snippet: His-tagged IFNAR1 (IF1-H5225; ACROBiosystems), His-tagged IFNAR2 (IF2-H5224; ACROBiosystems), Fc-tagged IFNα1 (IFA-H5258; ACROBiosystems), and IFNω (NBP2-35893; Novus Biologicals) were diluted to the indicated concentrations in assay buffer (0.02% Tween-20, 0.1% BSA in 1x PBS, pH 7.4).

Techniques: Neutralization, Control, Luciferase, Blocking Assay, Binding Assay

Journal: The Journal of Experimental Medicine

Article Title: Type I interferon autoantibody footprints reveal neutralizing mechanisms and allow inhibitory decoy design

doi: 10.1084/jem.20242039

Figure Lengend Snippet: Mechanism of IFNω neutralization by anti-IFNω autoAbs. (A) Anti-IFNω IgG levels in plasma samples from Aged ( n = 10) and COVID ICU ( n = 11) cohorts. Data are expressed as MFI FC values compared with the values of six negative control plasma samples. The dashed line represents the threshold to determine positivity, set as the mean plus 5 standard deviations of the control group. (B) Abilities of plasma samples to functionally neutralize IFNω at 0.2 or 10 ng/ml. Data are normalized to the luciferase signal from the control group. Dashed lines represent neutralization thresholds that were set as the mean minus 5 standard deviations of the control group. Pos indicates data from an anti-IFNω mAb–positive control. The orange circle (A and B) indicates plasma C4 with non-neutralizing anti-IFNω IgG. (C and D) Optimization of the concentration of IFNω required for association with IFNAR1 (C) and IFNAR2 (D) in BLI assays. Zoom-in images of the association phases are shown. (E) BLI assays to demonstrate inhibition of the interaction between IFNω and IFNAR1 or IFNAR2 by two plasmas harboring neutralizing anti-IFNω IgGs from the COVID ICU cohort. For all data panels, results are representative of at least n = 2 independent experiments. Statistical significance between groups was determined by the Mann–Whitney U test (A and B): ns, not significant; *P < 0.05; ***P < 0.001; ****P < 0.0001. FC, fold change.

Article Snippet: His-tagged IFNAR1 (IF1-H5225; ACROBiosystems), His-tagged IFNAR2 (IF2-H5224; ACROBiosystems), Fc-tagged IFNα1 (IFA-H5258; ACROBiosystems), and IFNω (NBP2-35893; Novus Biologicals) were diluted to the indicated concentrations in assay buffer (0.02% Tween-20, 0.1% BSA in 1x PBS, pH 7.4).

Techniques: Neutralization, Clinical Proteomics, Negative Control, Control, Luciferase, Positive Control, Concentration Assay, Inhibition, MANN-WHITNEY

Journal: The Journal of Experimental Medicine

Article Title: Type I interferon autoantibody footprints reveal neutralizing mechanisms and allow inhibitory decoy design

doi: 10.1084/jem.20242039

Figure Lengend Snippet: Non-neutralizing anti-IFN-I autoAbs display relatively low avidities. (A) BLI assays to assess inhibition of the interaction between IFNα1 and IFNAR1 or IFNAR2 by plasma C11 harboring non-neutralizing anti-IFNα IgGs. (B) BLI assays to assess inhibition of the interaction between IFNω and IFNAR1 or IFNAR2 by plasma C4 harboring non-neutralizing anti-IFNω IgGs. (C) Immunostimulatory activity of plasmas C11 and C4 compared with a HD plasma and 1,000 U/ml IFNα2. Data are normalized to the luciferase signal from untreated cells. Mean values from n = 2 replicates are shown. Error bars indicate standard deviations. (D) IFNα2 avidity indexes as determined for Ronta, three plasmas with neutralizing anti-IFNα IgGs (C1–C3), and plasma C11 with non-neutralizing anti-IFNα IgGs at different urea concentrations. (E) IFNω avidity indexes as determined for three plasmas with neutralizing anti-IFNω IgGs (C7, C8, C10) and plasma C4 with non-neutralizing anti-IFNω IgGs at different urea concentrations. The dashed lines (D and E) indicate the threshold for high avidity (>0.6). For all data panels, results are representative of at least n = 2 similar experiments. Ronta, rontalizumab. HD, healthy donor.

Article Snippet: His-tagged IFNAR1 (IF1-H5225; ACROBiosystems), His-tagged IFNAR2 (IF2-H5224; ACROBiosystems), Fc-tagged IFNα1 (IFA-H5258; ACROBiosystems), and IFNω (NBP2-35893; Novus Biologicals) were diluted to the indicated concentrations in assay buffer (0.02% Tween-20, 0.1% BSA in 1x PBS, pH 7.4).

Techniques: Inhibition, Clinical Proteomics, Activity Assay, Luciferase

Journal: The Journal of Experimental Medicine

Article Title: Type I interferon autoantibody footprints reveal neutralizing mechanisms and allow inhibitory decoy design

doi: 10.1084/jem.20242039

Figure Lengend Snippet: Development of a simIFNα to counteract anti-IFNα autoAbs. (A) IFNα2 amino acid sequence with residues targeted by autoAbs in bold, and residues selected for substitution that are bound by IFNAR1/IFNAR2 underlined. Numbering refers to the mature form of IFNα2. (B) Immunostimulatory activity of the indicated IFNα2 mutants compared with IFNα2 WT on AIR cells at 16 h after stimulation. Input IFNα2 amounts were first normalized to 10 7 HiBiT luciferase units before the stimulatory activity of each mutant was titrated out using fourfold serial dilutions. The dilutions at which each protein induced AIR cell activity 10-fold over baseline were calculated by nonlinear regression curve fitting using GraphPad Prism 10. Data represent mean values from n = 2 replicates. ND: not detectable. Blue bars indicate mutants studied further. (C) Immunostimulatory activity of IFNα2 R33A and IFNα2 R120E mutants using HEK293T cell supernatants at the indicated dilutions. Data are normalized to the luciferase signal from unstimulated cells. Data represent mean values from n = 3 replicates. (D) Western blot analysis of Avi-tagged IFNα2 R33A/R120E protein, as compared to IFNα2 WT , in cell and supernatant fractions from transfected HEK293T cells. β-Actin was used as a loading/specificity control. (E) Immunostimulatory activity of IFNα2 R33A/R120E , as compared to IFNα2 WT , on AIR cells at the indicated dilution and at 16 h after stimulation. Data are normalized to the luciferase signal obtained from the unstimulated control. Data represent mean values from n = 3 replicates. (F) Comparison of anti-IFNα IgG autoAb reactivity with IFNα2 WT and IFNα2 R33A/R120E proteins using the HiBiT-based qIP assay for six plasmas. Data represent mean values from n = 3 replicates. (G) Neutralization of 1 ng/ml IFNα2 activity on AIR cells by six plasmas, and inhibition of neutralization by preincubation of plasmas with simIFNα. Data are normalized to the luciferase signals from the mock plasma–treated conditions. (H) Relative levels of anti-IFNα IgG autoAbs and virus-specific IgG antibodies (HIV: BG505 SOSIP; COVID: S2) before (pre-) and after plasma depletion using control microparticle beads, or microparticle beads coupled to simIFNα. Data are expressed as MFI FC values made relative to the values derived from six negative control (healthy donor) plasma samples without anti-IFN-I or anti-virus IgG. (I) IFNα2 neutralization activities of the indicated anti-IFNα IgG autoAb–positive plasma samples before and after depletion as described in H. Data are normalized to the luciferase signal from a healthy control plasma–treated condition. Dashed lines in G and I indicate neutralization thresholds, set at 25% activity relative to the IFN-only condition (G) or healthy donor control (I). For all data panels, results shown are representative of at least n = 2 similar experiments. Statistical significance between groups was determined using unpaired t tests (F, H, and I): ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. See also . FC, fold change. Source data are available for this figure: .

Article Snippet: His-tagged IFNAR1 (IF1-H5225; ACROBiosystems), His-tagged IFNAR2 (IF2-H5224; ACROBiosystems), Fc-tagged IFNα1 (IFA-H5258; ACROBiosystems), and IFNω (NBP2-35893; Novus Biologicals) were diluted to the indicated concentrations in assay buffer (0.02% Tween-20, 0.1% BSA in 1x PBS, pH 7.4).

Techniques: Sequencing, Activity Assay, Luciferase, Mutagenesis, Western Blot, Transfection, Control, Comparison, Neutralization, Inhibition, Clinical Proteomics, Virus, Derivative Assay, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: Type I interferon autoantibody footprints reveal neutralizing mechanisms and allow inhibitory decoy design

doi: 10.1084/jem.20242039

Figure Lengend Snippet: Design of simIFNα retaining the mapped R1 and R2 autoAb footprints. (A) Previously described structure of the IFNα2 protein (PDB: 3SE3 ) with residues in the R1- and R2-footprints colored green. (B) Previously described structure of the IFNα2 protein (PDB: 3SE3 ) with residues selected for mutational analysis, based on their role in mediating interactions with IFNAR1/IFNAR2, colored orange. (C) Schematic overview of the assay to determine whether simIFN-Is can block neutralization of IFN-I by anti-IFN-I autoAbs.

Article Snippet: His-tagged IFNAR1 (IF1-H5225; ACROBiosystems), His-tagged IFNAR2 (IF2-H5224; ACROBiosystems), Fc-tagged IFNα1 (IFA-H5258; ACROBiosystems), and IFNω (NBP2-35893; Novus Biologicals) were diluted to the indicated concentrations in assay buffer (0.02% Tween-20, 0.1% BSA in 1x PBS, pH 7.4).

Techniques: Blocking Assay, Neutralization