Autoantibodies against type I IFNs in patients with critical influenza pneumonia

Abstract

Autoantibodies neutralizing type I interferons (IFNs) can underlie critical COVID-19 pneumonia and yellow fever vaccine disease. We report here on 13 patients harboring autoantibodies neutralizing IFN-α2 alone (five patients) or with IFN-ω (eight patients) from a cohort of 279 patients (4.7%) aged 6-73 yr with critical influenza pneumonia. Nine and four patients had antibodies neutralizing high and low concentrations, respectively, of IFN-α2, and six and two patients had antibodies neutralizing high and low concentrations, respectively, of IFN-ω. The patients' autoantibodies increased influenza A virus replication in both A549 cells and reconstituted human airway epithelia. The prevalence of these antibodies was significantly higher than that in the general population for patients <70 yr of age (5.7 vs. 1.1%, P = 2.2 × 10-5), but not >70 yr of age (3.1 vs. 4.4%, P = 0.68). The risk of critical influenza was highest in patients with antibodies neutralizing high concentrations of both IFN-α2 and IFN-ω (OR = 11.7, P = 1.3 × 10-5), especially those <70 yr old (OR = 139.9, P = 3.1 × 10-10). We also identified 10 patients in additional influenza patient cohorts. Autoantibodies neutralizing type I IFNs account for ∼5% of cases of life-threatening influenza pneumonia in patients <70 yr old.

Graphical abstract

Figure 1.

Auto-Abs neutralizing IFN-α2 and/or IFN-ω in patients with critical influenza pneumonia. (A) Age and sex distribution of the patients with critical influenza pneumonia or mild influenza infection. (B) Luciferase-based neutralization assay to detect auto-Abs neutralizing 10 ng/ml or 100 pg/ml IFN-α2, IFN-ω, or IFN-β. Plasma samples from patients with critical (red) or mild (black) influenza were diluted 1:10 in all tests. HEK293T cells were transfected with the dual luciferase system with IFN-sensitive response elements (ISRE) before treatment with type I IFNs with or without patient plasma, and relative luciferase activity (RLA) was calculated by normalizing firefly luciferase activity against Renilla luciferase activity. An RLA <15% of the value for the mock treatment was considered to correspond to neutralizing activity (dashed line; Bastard et al., 2021a). Experiments were repeated at least twice, and the average was plotted in the figure. (C) Age and sex distribution of patients with auto-Ab neutralizing IFN-α2 and/or IFN-ω (n = 13).

Figure 2.

Enrichment in auto-Ab–positive cases among patients with critical influenza pneumonia. (A) Prevalence of auto-Ab–positive cases among patients with critical influenza (n = 279, red bars) and in the general population (n = 34,159, black bars). *, P < 0.05; ****, P < 10⁻⁵. (B) OR for the presence of auto-Abs, by sex and age, relative to the general population, with adjustment of the comparison by means of Firth’s bias-corrected logistic regression. The horizontal bars indicate the upper and lower limits of the 95% CIs. α + ω, auto-Abs neutralizing both IFN-α2 and IFN-ω; α ± ω, auto-Abs neutralizing IFN-α2 with or without IFN-ω; α, auto-Abs neutralizing IFN-α2 only; *, P < 0.05; **, P < 10⁻²; ***, P < 10⁻³; ****, P < 10⁻⁴.

Figure 3.

Neutralizing auto-Abs block the antiviral function of IFN-α2 in IAV-infected A549 epithelial cells. (A) A549 cells were treated with 20 pg/ml exogenous IFN-α2 with or without patient plasma (titrated to the dilutions indicated on the x axis), anti–IFN-α2 monoclonal antibody, and healthy donor plasma overnight before infection with IAV Cal/09 virus expressing NS1-mCherry (CalNSmCherry) at an MOI of 0.5. The day after infection, the percentage of the cells infected was determined with a Celigo (Nexcelcom) imaging cytometer. The dotted line at 64.98% represents the mean percentage infection in cells treated with 20 pg/ml IFN-α2 in the absence of plasma or anti–IFN-α2 antibody. Experiments were repeated four times. (B) Longitudinal testing of six patients with life-threatening influenza pneumonia (two positive and four negative for auto-Abs), with the assay as described in A.

Figure 4.

Neutralizing auto-Abs block the antiviral function of IFN-α2 in IAV-infected HAE cultures. (A–F) HAE reconstituted from human nasal primary cells and maintained in an air–liquid interface were either left untreated or treated with 2 ng/ml exogenous IFN-α2a (A–C) or 20 ng/ml exogenous IL-29, IL-28A, or IL-28B (D–F), in the presence of inactivated patient plasma (1:100 diluted) for 24 h before IAV infection. Cells were treated again on the basolateral side with same concentration of IFN-α2a or IFN-λ1/IL-29, IFN-λ2/IL-28A, or IFN-λ3/IL-28B in the presence of patient plasma (n = 7) 1 h after IAV infection. These seven patients had auto-Abs neutralizing IFN-α at 10 ng/ml, but not IFN-β or -λ. HAE apical poles were washed, 54 h after infection, and titrated by TCID₅₀ determination (A and D) and quantitative RT-PCR (B and E). Changes in TEER (ΔTEER) were measured as a surrogate for the integrity of HAE (C and F). Previously identified auto-Ab–positive (auto-Ab [+]) or auto-Ab [−] plasma samples were used as controls. Experiments were repeated three times.

Figure 5.

ISG and proinflammatory responses in IAV-infected HAE cultures. HAE reconstituted from human nasal primary cells and maintained in an air–liquid interface were either left untreated or treated with 2 ng/ml exogenous IFN-α2a in the presence of inactivated patient plasma (1:100 dilution) for 24 h before IAV infection. Cells were treated again on the basolateral side with 2 ng/ml IFN-α2a in the presence of patient plasma 1 h after IAV infection. RNA was isolated 54 h after infection, and NanoString analysis was performed with a panel of immune response genes. (A) Heatmap of gene expression profiles from unsupervised analysis (Euclidean distance matrix, Ward’s method) generated by scaling and centering log₁₀-transformed normalized gene expression (expressed as fold-change induction relative to mock conditions) and based on the full 96-gene panel. Gene and sample clustering is indicated by dendrogram trees above and to the left, respectively, of the heatmap. Gene clustering distinguished ISGs (cluster 1) from proinflammatory genes (cluster 2; Table S1). (B and C) Relative expression (mean) levels of two ISGs, IFI44L and IFIT1 (B), and two proinflammatory cytokines, IL-6 and IL1A (C), based on NanoString analysis on total cellular RNA extracted after infection. Gene expression is expressed as a fold-change induction relative to mock conditions (untreated/uninfected). AAb−, auto-Ab–negative plasma; AAb+, auto-Ab–positive plasma.

Figure 6.

Auto-Abs neutralizing IFN-α2 and/or IFN-ω in ELISA-positive patients hospitalized with influenza pneumonia in additional cohorts. (A) Age and sex distribution of patients from Chile, Spain, France, Belgium, and Taiwan hospitalized for influenza pneumonia (n = 130). (B) Patient plasma samples were tested by ELISA for auto-Abs against IFN-α2 and -ω. Patient plasma samples were diluted 1:50 before being added to plates coated with 2 μg/ml rhIFN-α or rhIFN-ω. HRP-conjugated goat antiserum against human IgG or IgA was added to final concentration of 2 μg/ml. OD was measured. Each plasma sample was tested once. (C) Luciferase-based neutralization assay to detect auto-Abs neutralizing 10 ng/ml or 100 pg/ml IFN-α2, IFN-ω, or IFN-β. Plasma samples from ELISA-positive patients were diluted 1:10 in all tests. HEK293T cells were transfected with the dual luciferase system with IFN-sensitive response elements (ISRE) before treatment with type I IFNs with or without plasma from patients, and relative luciferase activity (RLA) was calculated by normalizing firefly luciferase activity against Renilla luciferase activity. An RLA <15% the value for the mock treatment was considered to indicate that the antibodies were neutralizing (dashed line). (D) Age and sex distribution of patients with auto-Ab neutralizing IFN-α2 and/or IFN-ω (n = 10).