Autoantibodies neutralizing type I IFNs are present in ~ 4% of uninfected individuals over 70 years old and account for ~ 20% of COVID-19 deaths

Abstract

Circulating autoantibodies (auto-Abs) neutralizing high concentrations (10 ng/mL, in plasma diluted 1 to 10) of IFN-α and/or -ω are found in about 10% of patients with critical COVID-19 pneumonia, but not in subjects with asymptomatic infections. We detect auto-Abs neutralizing 100-fold lower, more physiological, concentrations of IFN-α and/or -ω (100 pg/mL, in 1/10 dilutions of plasma) in 13.6% of 3,595 patients with critical COVID-19, including 21% of 374 patients > 80 years, and 6.5% of 522 patients with severe COVID-19. These antibodies are also detected in 18% of the 1,124 deceased patients (aged 20 days-99 years; mean: 70 years). Moreover, another 1.3% of patients with critical COVID-19 and 0.9% of the deceased patients have auto-Abs neutralizing high concentrations of IFN-β. We also show, in a sample of 34,159 uninfected subjects from the general population, that auto-Abs neutralizing high concentrations of IFN-α and/or -ω are present in 0.18% of individuals between 18 and 69 years, 1.1% between 70 and 79 years, and 3.4% >80 years. Moreover, the proportion of subjects carrying auto-Abs neutralizing lower concentrations is greater in a subsample of 10,778 uninfected individuals: 1% of individuals <70 years, 2.3% between 70 and 80 years, and 6.3% >80 years. By contrast, auto-Abs neutralizing IFN-β do not become more frequent with age. Auto-Abs neutralizing type I IFNs predate SARS-CoV-2 infection and sharply increase in prevalence after the age of 70 years. They account for about 20% of both critical COVID-19 cases in the over-80s, and total fatal COVID-19 cases.

Fig. 1

Neutralizing auto-Abs against IFN-α2 and/or IFN-ω in patients with life-threatening COVID-19. (A) Gyros (high-throughput automated ELISA) results for auto-Abs against IFN-α2 and/or IFN-ω in patients with critical COVID-19 (N=2,240), severe COVID-19 (N=500), or asymptomatic/mild SARS-CoV-2 infection (N=663). (B) Schematic representation of the neutralization assay developed in HEK293T cells, using a luciferase system. ISRE: interferon-sensitive response elements. (C) Results for the neutralization of 10 ng/mL IFN-α2 or IFN-ω in the presence of plasma 1/10 from patients with critical COVID-19 (N=3,136), severe COVID-19 (N=623), or controls with mild/asymptomatic infection (N=1,076). Relative luciferase activity is shown (ISRE dual luciferase activity, with normalization against Renilla luciferase activity) after stimulation with 10 ng/mL IFN-α2 or IFN-ω in the presence of plasma 1/10. RLA: relative luciferase activity. (D) RLA after stimulation with IFN-α2 at a concentration of 10 ng/mL or 100 pg/mL, with various dilutions of plasma from a positive control (from 1/10 to 1/10⁷) neutralizing 10 ng/mL of type I IFNs (AAB+ pt, 10 ng/mL), a patient neutralizing 100 pg/mL of type I IFNs but not 10 ng/mL (AAB+ pt, 100 pg/mL), and a healthy control (HC). AAB: auto-Ab. Pt: patient. (E) Neutralization of 100 pg/mL IFN-α2 or IFN-ω in the presence of plasma 1/10 from patients with critical COVID-19 (N=3,595), severe COVID-19 (N=522), or controls with asymptomatic/mild infection (N=1,639). (F) Plot showing luciferase induction after stimulation with 10 ng/mL or 100 pg/mL IFN-α2, in the presence of plasma from patients with critical COVID-19. Dotted lines indicate neutralizing levels, defined as induction levels below 15% of the mean value for controls tested the same day. Patients with antibodies neutralizing both 10 ng/mL and 100 pg/mL IFN-α2 are shown in the bottom left corner, whereas the patients in the bottom right corner had antibodies capable of neutralizing only 100 pg/mL IFN-α2. (G) Plot showing luciferase induction after stimulation with 10 ng/mL or 100 pg/mL IFN-ω, for patients with critical COVID-19.

Fig. 2

Enhanced SARS-CoV-2 replication, despite the presence of IFN-α2, in the presence of plasma from patients with auto-Abs neutralizing 100 pg/mL IFN-α2. (A) SARS-CoV-2 replication in Huh-7.5 cells untreated (in dark blue), or treated with ~100 pg/mL or ~400 pg/mL IFN-α2 in the presence of 1/100 plasma from healthy controls without auto-Abs (N=3, in blue), from patients with life-threatening COVID-19 but without auto-Abs against IFN-α2 (N=3, in black), a commercial anti–IFN-α2 antibody (mAb, in red); from a patient with life-threatening COVID-19 and auto-Abs neutralizing 10 ng/mL IFN-α2 in plasma 1/100 (COVID-19 AAB+, N=1, in orange), from patients with life-threatening COVID-19 and auto-Abs neutralizing 100 pg/mL IFN-α2 in plasma 1/100 (N=5, in grey); elderly individuals with auto-Abs neutralizing 100 pg/mL IFN-α2 in plasma 1/100 (N=2, in purple). Each dot represents a technical replicate. All experiments were done in triplicate. (B) ELISA (enzyme-linked immunosorbent assay) for auto-Abs against the 13 IFN-α forms, IFN-ω, IFN-β, IFN-ε, and IFN-κ in patients with life-threatening COVID-19 and auto-Abs neutralizing 100 pg/mL IFN-α2 (N=6), APS-1 patient with life-threatening COVID-19 and auto-Abs neutralizing 10 ng/mL IFN-α2 and IFN-ω (N=1), and healthy controls (N=2). (C) RLA after stimulation with the all individual IFN-α at a concentration of 1ng/mL, with 1/10 plasma from a healthy control (negative control), an APS-1 patient (positive control), patients with life-threatening COVID-19 and neutralizing IFN-α2 and/or IFN-ω, or a monoclonal antibody anti-IFN-α2. (D) Neutralization of 10 ng/mL IFN-β in the presence of plasma 1/10 from patients with critical COVID-19 (N=1,773), severe COVID-19 (N=187), or asymptomatic/mild controls (N=1,044).

Fig. 3

Higher prevalence of neutralizing auto-Abs against type I IFNs in elderly patients with critical COVID-19. (A) Bar plot of the age and sex distribution of the patients with life-threatening COVID-19 included in our expanded cohort (N=3,595). (B) Graph showing the anti-IFN-α2 auto-Ab levels, assessed by Gyros, in patients with life-threatening COVID-19. Men and women are shown separately. The upper section of the Y-axis starts at 3%. (C-J) Proportion by decade of patients with critical COVID-19, and positive for neutralizing auto-Abs (in plasma 1/10) against (C) IFN-α2 and/or IFN-ω, at 10 ng/mL, for both sexes. (D) IFN-α2 and/or IFN-ω, at 10 ng/mL, for men or women. (E) IFN-α2 and IFN-ω, at 10 ng/mL, for both sexes. (F) IFN-α2 and IFN-ω, at 10 ng/mL, for men or women. (G) IFN-α2 and/or IFN-ω, at 100 pg/mL, for both sexes. (H) IFN-α2 and/or IFN-ω, at 100 pg/mL, for men or women. (I) IFN-α2 and IFN-ω, at 100 pg/mL, for both sexes. (J) IFN-α2 and IFN-ω, at 100 pg/mL, for men or women.

Fig. 4

Higher prevalence of neutralizing auto-Abs against type I IFNs in patients who died of COVID-19. (A) Bar plot of the age and sex distribution of the patients who died of COVID-19 included in our cohort (N=1,124). (B) Graph showing the anti-IFN-α2 auto-Ab levels, assessed by Gyros, in patients who died of COVID-19. Men or women are shown separately. The upper section of the Y-axis starts at 3%. (C-J) Proportion by decade of patients who died of COVID-19, and positive for neutralizing auto-Abs (in plasma 1/10) against (C) IFN-α2 and/or IFN-ω, at 10 ng/mL, for both sexes. (D) IFN-α2 and/or IFN-ω, at 10 ng/mL, for men or women. (E) IFN-α2 and IFN-ω, at 10 ng/mL, for both sexes. (F) IFN-α2 and IFN-ω, at 10 ng/mL, for men or women. (G) IFN-α2 and/or IFN-ω, at 100 pg/mL, for both sexes. (H) IFN-α2 and/or IFN-ω, at 100 pg/mL, for men or women. (I) IFN-α2 and IFN-ω, at 100 pg/mL, for both sexes. (J) IFN-α2 and IFN-ω, at 100 pg/mL, for men or women.

Fig. 5

Neutralizing auto-Abs against IFN-α2 and/or IFN-ω at 10 ng/mL are more prevalent in the elderly, in the general population. (A) Bar plot of the age and sex distribution of individuals from the general population (N=34,159). (B) Graph showing the IFN-α2 auto-Ab levels, assessed by Gyros, in individuals from the general population. Men or women are shown separately. The upper section of the Y-axis starts at 3%. (C-H) Proportion by 5 years of individuals from the general population, and positive for neutralizing auto-Abs (in plasma 1/10) against (C) IFN-α2 and/or IFN-ω, at 10 ng/mL, for both sexes. (D) IFN-α2 and/or IFN-ω, at 10 ng/mL, for men or women. (E) IFN-α2 and IFN-ω, at 10 ng/mL, for both sexes. (F) IFN-α2 and IFN-ω, at 10 ng/mL, for men or women. (G) IFN-β, at 10 ng/mL, for both sexes. (H) IFN-β, at 10 ng/mL, for men or women. (I) Plot showing luciferase induction after stimulation with 10 ng/mL or 100 pg/mL IFN-α2, in the presence of plasma from individuals from the general population. Dotted lines indicate neutralizing levels, defined as induction levels below 15% of the mean value for controls tested the same day. Individuals with antibodies neutralizing both 10 ng/mL and 100 pg/mL IFN-α2 are shown in the bottom left corner, whereas the individuals in the bottom right corner had antibodies capable of neutralizing only 100 pg/mL IFN-α2. (J) Plot showing luciferase induction after stimulation with 10 ng/mL or 100 pg/mL IFN-ω, for individuals from the general population.

Fig. 6

Neutralizing auto-Abs against IFN-α2 and/or IFN-ω at 100 pg/mL are more prevalent in the elderly, in the general population. (A-H) Proportion, binned every 5 years, of individuals from the general population, and positive for neutralizing auto-Abs (in plasma 1/10) against (A) IFN-α2 and/or IFN-ω, at 100 pg/mL, for both sexes. (B) IFN-α2 and/or IFN-ω, at 100 pg/mL, for men or women. (C) IFN-α2 and IFN-ω, at 100 pg/mL, for both sexes. (D) IFN-α2 and IFN-ω, at 100 pg/mL, for men or women. (E) IFN-α2, at 100 pg/mL, for both sexes. (F) IFN-α2, at 100 pg/mL, for men or women. (G) IFN-ω, at 100 pg/mL, for both sexes. (H) IFN-ω, at 100 pg/mL, for men or women.