Transient anti-interferon autoantibodies in the airways are associated with recovery from COVID-19

Abstract

Preexisting anti-interferon-α (anti-IFN-α) autoantibodies in blood are associated with susceptibility to life-threatening COVID-19. However, it is unclear whether anti-IFN-α autoantibodies in the airways, the initial site of infection, can also determine disease outcomes. In this study, we developed a multiparameter technology, FlowBEAT, to quantify and profile the isotypes of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and anti-IFN-α antibodies in longitudinal samples collected over 20 months from the airways and blood of 129 donors spanning mild to severe COVID-19. We found that nasal IgA1 anti-IFN-α autoantibodies were induced after infection onset in more than 70% of mild and moderate COVID-19 cases and were associated with robust anti-SARS-CoV-2 immunity, fewer symptoms, and efficient recovery. Nasal anti-IFN-α autoantibodies followed the peak of host IFN-α production and waned with disease recovery, revealing a regulated balance between IFN-α and anti-IFN-α response. In contrast, systemic IgG1 anti-IFN-α autoantibodies appeared later and were detected only in a subset of patients with elevated systemic inflammation and worsening symptoms. These data reveal a protective role for nasal anti-IFN-α in the immunopathology of COVID-19 and suggest that anti-IFN-α autoantibodies may serve a homeostatic function to regulate host IFN-α after viral infection in the respiratory mucosa.

Figure 1.. FlowBEAT reveals the breadth of antibody response to COVID-19.

(A) Graphical overview of FlowBEAT sampling from different tissues to reveal antibody isotype and subclasses and antigen specificity. asp., aspirate. (B) Dot plot showing the linear range of the anti-RBD median fluorescence intensity (MFI) signal by FlowBEAT serial dilution of seropositive NIH standard (n = 3 independent replicates) and monoclonal human IgG1 anti-RBD. (C) Graphical summary of study cohort spanning mild, moderate, and severe COVID-19 with multi-tissue collection and longitudinal follow-up. See cohort details in tables S3-S4 and data files S1-S2. (D) Sunburst plots of FlowBEAT showing the breadth of antibody response against SARS-CoV-2 antigens and autoantigens (8 antibody isotypes against 18 antigens) in the airway and systemic samples, grouped by disease severity over the first 90 days post-onset. Individual points represent the per-donor maximum FlowBEAT MFI signal on the Y-axis moving outward from the center. Each outward circle corresponds to the MFI signal noted on the scale. The radial X-axis corresponds to the antigens described on the outermost circle. The color of each dot represents the individual antibody isotypes. The pie chart inserts in the center of the plot indicate the antigen type, as described in panel (A). The mean value for the most prevalent isotype in each tissue (IgA1 in airways and IgG1 in blood) was plotted as a shaded line on the top of each sunburst to provide contrast. Mild donors: n = 17 nasal and 28 systemic; Moderate donors: n = 19 nasal and 24 systemic; Severe donors: n = 22 endotracheal aspirates and 23 systemic.

Figure 2.. The type and specificity of antibodies against SARS-CoV-2 structural and non-structural proteins change with disease severity, vaccination, and source.

(A) Longitudinal plots showing isotype-specific anti-spike protein MFI signal (Y-axis) across infection and up to four doses of mRNA vaccination (X-axis) in the nasal mucosa and blood of patients with mild and moderate COVID-19 (n = 36 nasal, 107 longitudinal samples; n = 52 systemic, 116 longitudinal samples). Solid lines connect longitudinal samples from individual patients and an overlaid Loess regression smooth curve. The shaded area represents the confidence interval around the smoothed curve. (B) Bar plots show the maximum anti-spike protein signal by individual patients (one point per patient) and are grouped by disease severity (fill texture) and antibody isotype (color). Statistics by Wilcoxon rank-sum test (mild donors: n = 17 nasal and 28 systemic; moderate donors: n = 19 nasal and 24 systemic; severe donors: n = 22 ETA and 23 systemic). (C) Tissue-specific antibody signature against SARS-CoV-2 non-structural proteins (NSP), including open reading frame (ORF) proteins (NSP signature). The gray shaded area highlights the 0-14 days post-onset. Pie charts show the percentage of donors with detectable anti-SARS-CoV-2 NSP signal, defined by MFI > 10². The gradient shading in the pie chart corresponds to peak antibody MFI signal during infection. Colored shaded boxes drawn on the systemic IgG1 anti-NSP plots represent gates to include only donors who generate anti-NSP IgG1 > 10² MFI. These donors were analyzed for maximum (Max.) C-reactive protein (CRP) concentration (mg/L; healthy < 8.1 mg/L) in blood (n = 37) and peak viral load (copies/mL) in the nasal swabs (n = 35). Statistics by Wilcoxon rank-sum test, p = 2x10⁻⁴.

Figure 3.. Anti-IFN-α autoantibodies are transiently induced in the nasal mucosa after infection and are associated with viral load and local IFN-α secretion.

(A) Longitudinal plot showing the post-onset induction of nasal IgA1, IgA2, and IgG1 anti-IFN-α2a autoantibodies in nasal swabs and blood samples from individuals with mild and moderate COVID-19. Days post-onset on the X-axis, and longitudinal samples from individual donors are linked by solid lines. Loess regression smooth curves of the same data are overlaid with the confidence interval shown as a shaded area, and color corresponding to the antibody isotype. The gray shaded area highlights the 0-14 days post-onset. Pie charts show the percentage of donors with detectable anti-IFN-α2a in nasal swabs (orange) and blood (green) samples. The gradient shading in the pie chart corresponds to the peak antibody MFI signal during acute infection. Mild + Moderate donors: n = 36 with matching nasal and systemic samples; 102 nasal and 112 systemic longitudinal samples. Naïve-vaccinated donors (n = 7) were included as a control. (B) Longitudinal viral load (viral copies per mL, qPCR) in nasal swabs of patients were grouped by the presence (IgA producers, orange) or absence (IgA non-producers, gray) of anti-IFN-α2a autoantibodies as in (A). Statistic by Wilcoxon rank-sum test, p = 0.001. (C) Longitudinal nasal IFN-α2a cytokine concentrations were plotted as pg/mL of nasal swab supernatant (n = 41 swabs from 31 donors) and grouped by the presence or absence of anti-IFN-α2a as in (B). Statistic by Wilcoxon rank-sum test, p = 3.0x10⁻⁴. (D) Longitudinal plots binned by week (7-day increments) show peak viral load, IFN-α2a secretion, and anti-IFN-α2a autoantibodies in the nasal mucosa. (E) In vitro IFN-α2a neutralization assay using a type I IFN-reporter cell line. The IFN-induced response signal following exogenous IFN-α2a (60 units/mL) stimulation is plotted as arbitrary units (AU). Lower AU values indicate neutralization of exogenous IFN-α2a in the presence of systemic serum or plasma (n = 22 donors, 27 samples) or nasal samples (n = 29 donors, 51 samples). Samples were grouped by antibody detection based on the FlowBEAT MFI signals. Samples with antibody MFI < 10² were considered as not detected (N.D.). Horizontal lines represent group means and standard error. A thin black line connects groups tested by one-way ANOVA, p = 0.007. (F) Pearson correlation between FlowBEAT MFI signal (X-axis) and cell response to exogenous IFN-α stimulation (Y-axis) as in (E) for systemic (n = 16 donors, 21 samples) and nasal samples (n = 16 donors, 18 samples). (G) CXCL10 measurements in systemic (n = 10 donors, 17 samples) and nasal samples (n = 13 donors, 14 samples) of IFN-α2a cytokine-positive samples. Statistics by Wilcoxon rank-sum test.

Figure 4.. Nasal IgA1 anti-IFN-α autoantibodies are associated with fewer symptoms, less systemic inflammation, and increased anti-SARS-CoV-2 antibody responses.

(A) Biaxial plot of nasal IgA1 anti-IFN-α2a (Y-axis) and systemic anti-IFN-α2a IgG1 (X-axis) autoantibody signal. Points represent the patient’s peak antibody FlowBEAT signal (MFI) within the first six weeks post-onset. Patient populations (n = 36 donors with matched nasal swabs and systemic samples) are grouped (dashed lines) as nasal producers (pink), blood-only producers (green), and non-producers (black). (B) Heatmap of nasal and blood anti-IFN-α autoantibodies, demographics, airway cytokines, and frequency of symptoms (Chi-square test, with p-value reported) of patients grouped as in (A). Rows correspond to individual patients; the columns describe patient features as labeled. The pie chart shows the frequency of “shortness of breath” (SOB) symptoms between pink and green groups as in (A). (C) The scatterplot shows the maximum systemic C-reactive protein (CRP) concentration (healthy CRP < 8.1 mg/L) for each patient (n = 30) grouped as in (A), with non-producers indicated as double negative (DN). The thin black line connects group means tested by one-way ANOVA (p = 9.3x10⁻⁴). (D) Pearson correlation between anti-IFN-α2a (X-axis) and composite anti-SARS-CoV-2 NSP signal (Y-axis). All samples plotted (n = 57) are from donors in the pink group (nasal producers, n = 21 donors) as in (A). For IgA1 anti-NSP (NSP1, NSP7, ORF3a, ORF8) against IgA1 anti-IFN-α: Pearson’s r = 0.75, p = 9.0x10^-12. For IgA2 anti-NSP (NSP1, NSP2, ORF3a, ORF8) against IgA2 anti-IFN-α: Pearson’s r = 0.97, p = 2.2x10⁻¹⁶. (E) Maximum IgA1 (nasal) or IgG1 (blood) anti-SARS-CoV-2 NSP signal (as in D, composite signal of anti-NSP1, −NSP7, −ORF3a, −ORF8) grouped by gates displayed in (A): nasal IgA producers (nasal producers, pink gate), systemic IgG producers (blood-only producers, green gate), and double-negative (DN) (non-producers, gray gate). Statistics by Wilcoxon rank-sum test.

Figure 5.. Patients with severe COVID-19 show persistent IFN-α and anti-IFN-α responses associated with viral load, hyperinflammation, and lower anti-SARS-CoV-2 humoral immunity.

(A) Pie charts of IgA1+IgA2 (orange) and IgG1 (green) anti-IFN-α2a autoantibody frequency and peak FlowBEAT signal (shaded gradient) in the endotracheal aspirate (ETA) and blood of hospitalized patients (n = 23 donors) with severe COVID-19. (B) Pearson correlation between ETA IgA1 and IgA2 anti-IFN-α autoantibodies and IFN-α2a cytokine. Pearson’s r = 0.59, p = 0.0063 (IgA1), and r = 0.61, p = 0.0035 (IgA2). (C) Bar chart comparing the relative IgA1 and IgA2 subclass usage in the airways of mild/moderate (nasal swabs) and severe (ETA) patients. Statistics by Wilcoxon rank-sum test, p = 0.02 (anti-IFN-α2a IgA1), p = 9.1x10⁻⁴ (anti-IFN-α2a IgA2), and p = 5.9x10⁻⁵ (anti-spike protein IgA2). (D) Biaxial plot of individual patients showing antibody signals corresponding to systemic IgG1 and airway IgA1 + IgA2 anti-IFN-α2a. Patients are grouped into airway IgA producers (pink) and non-producers (gray). The bar chart shows the viral load (viral copies/mL by qPCR) in the ETA of each individual in the biaxial plot. Wilcoxon rank-sum test, p = 0.028. (E) Heatmap of the airway (ETA IgA1+IgA2) and blood (IgG1) anti-IFN-α2a and airway (ETA) cytokine concentrations (n = 20 donors). Displayed cytokines showed a significant Pearson correlation with anti-IFN-α2a (IgA1+IgA2) in the ETA (*p < 0.05; **p < 0.01; ***p < 0.001). Rows correspond to individual patients as in (D), and columns indicate features as labeled. (F) Correlation plots between anti-IFN-α2a (X-axis) and the composite anti-SARS-CoV-2 NSP signal (Y-axis) as described in Fig. 4D. All samples plotted are from donors in the pink group (nasal producers) as in (D). Pearson’s product-moment correlation coefficient tests did not reach significance for either IgA1 or IgA2 (correlation not statistically significant, p > 0.05).