Autoantibodies neutralizing type I IFNs underlie severe tick-borne encephalitis in ∼10% of patients

Abstract

Tick-borne encephalitis (TBE) virus (TBEV) is transmitted to humans via tick bites. Infection is benign in >90% of the cases but can cause mild (<5%), moderate (<4%), or severe (<1%) encephalitis. We show here that ∼10% of patients hospitalized for severe TBE in cohorts from Austria, Czech Republic, and France carry auto-Abs neutralizing IFN-α2, -β, and/or -ω at the onset of disease, contrasting with only ∼1% of patients with moderate and mild TBE. These auto-Abs were found in two of eight patients who died and none of 13 with silent infection. The odds ratios (OR) for severe TBE in individuals with these auto-Abs relative to those without them in the general population were 4.9 (95% CI: 1.5-15.9, P < 0.0001) for the neutralization of only 100 pg/ml IFN-α2 and/or -ω, and 20.8 (95% CI: 4.5-97.4, P < 0.0001) for the neutralization of 10 ng/ml IFN-α2 and -ω. Auto-Abs neutralizing type I IFNs accounted for ∼10% of severe TBE cases in these three European cohorts.

Figure 1.

Demographic and phenotypic distributions of the TBE cohort and auto-Abs neutralizing type I IFNs in individuals infected with TBEV. (A) Age and sex distribution of the patients according to TBE severity. (B) Composition of the TBE cohort: number of individuals in each TBE group enrolled at each center. (C) Luciferase-based neutralization assay to detect auto-Abs neutralizing 10 ng/ml IFN-α2, IFN-ω, or IFN-β (left panel) and 100 pg/ml IFN-α2 or IFN-ω, or 1 ng/ml IFN-β (right panel). Plasma samples from asymptomatic TBE cases (gray), patients with mild TBE (brown), patients with moderate TBE (orange), and patients with severe TBE (red) were diluted 1:10. HEK293T cells were transfected with (1) a plasmid containing the firefly luciferase gene under the control of an ISRE-containing promotor and (2) a plasmid containing the Renilla luciferase gene. The cells were then treated with type I IFNs and RLA was calculated by normalizing firefly luciferase activity against Renilla luciferase activity. An RLA <15% of the median RLA for healthy controls was considered to correspond to neutralizing activity (dotted line; Bastard et al., 2021a). Each sample was tested once. (D) Proportions of individuals with auto-Abs neutralizing type I IFNs at a concentration of 10 ng/ml (left) or 1 ng/ml–100 pg/ml (right) in the three groups of TBE patients (mild, moderate, severe), as determined with the luciferase-based neutralization assay. IFN-α, auto-Abs neutralizing IFN-α2 (regardless of their effects on other IFNs); IFN-ω, auto-Abs neutralizing IFN-ω (regardless of their effects on other IFNs); IFN-α ± ω ± β, auto-Abs neutralizing IFN-α2 and/or IFN-ω and/or IFN-β; IFN-α + ω, auto-Abs neutralizing both IFN-α2 and IFN-ω. The bars indicate the upper and lower limits of the 95% CI. (E) Proportion of type I IFNs neutralized in the three groups of TBE patients (mild, moderate, severe) and in individuals with silent infection according to the nature and combination of auto-Abs.

Figure S1.

Correlation between ELISA and neutralization assay results for the detection of auto-Abs neutralizing type I IFNs.

Figure 2.

Comparison of the proportions of individuals positive for auto-Abs neutralizing type I IFNs in the different TBE cohorts and estimation of the risk of severe TBE in auto-Ab-positive individuals relative to the general population. (A) Comparison of auto-Ab prevalence in TBE patients from the Austrian, Czech, and French cohorts (considering auto-Abs neutralizing 10 ng/ml IFN). IFN-α, auto-Abs neutralizing IFN-α2 (regardless of their effects on other IFNs); IFN-ω, auto-Abs neutralizing IFN-ω (regardless of their effects on other IFNs); IFN-α ± ω ± β, auto-Abs neutralizing IFN-α2 and/or IFN-ω and/or IFN-β; IFN-α + ω, auto-Abs neutralizing both IFN-α2 and IFN-ω. (B) Comparison of auto-Ab prevalence in TBE patients from the Austrian, Czech, and French cohorts (considering auto-Abs neutralizing 100 pg/ml [IFN-α2 and IFN-ω] or 1 ng/ml [IFN-β]). IFN-α, auto-Abs neutralizing IFN-α2 (regardless of their effects on other IFNs); IFN-ω, auto-Abs neutralizing IFN-ω (regardless of their effects on other IFNs); IFN-α ± ω ± β, auto-Abs neutralizing IFN-α2 and/or IFN-ω and/or IFN-β; IFN-α + ω, auto-Abs neutralizing both IFN-α2 and IFN-ω. (C) OR for the presence of auto-Abs in individuals with non-severe (gray) or severe (red) TBE relative to the general population, with adjustment for age and sex by logistic regression. The horizontal bars indicate the upper and lower limits of the 95% CI. IFN-α, auto-Abs neutralizing IFN-α2 (regardless of their effects on other IFNs); IFN-ω, auto-Abs neutralizing IFN-ω (regardless of their effects on other IFNs); IFN-α ± ω ± β, auto-Abs neutralizing IFN-α2 and/or IFN-ω and/or IFN-β; IFN-α + ω, auto-Abs neutralizing both IFN-α2 and IFN-ω. (D) OR for the presence of auto-Abs in individuals with severe TBE relative to the general population by age group, with adjustment for sex by logistic regression. ORs were calculated separately for patients with severe TBE aged ≤65 and >65 years. The horizontal bars indicate the upper and lower limits of the 95% CI. IFN-α, auto-Abs neutralizing IFN-α2 (regardless of their effects on other IFNs); IFN-ω, auto-Abs neutralizing IFN-ω (regardless of their effects on other IFNs); IFN-α ± ω ± β, auto-Abs neutralizing IFN-α2 and/or IFN-ω and/or IFN-β; IFN-α + ω, auto-Abs neutralizing both IFN-α2 and IFN-ω. [0,65): individuals aged 0–65 years old; [65,110): individuals aged 65–110 years old; ns: non-significant; **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure S2.

Anti-TBEV IgG and IgM titers in the Austrian and French cohorts of TBE patients, by disease severity. (A and B) The blue arrows show the patients with auto-Abs neutralizing type I IFNs. The dotted lines represent the thresholds for positivity. The assays used to detect anti-TBEV IgG and IgM titers differed between the two cohorts, accounting for the difference in positivity thresholds.

Figure 3.

TBEV infection and IFN treatment in Vero-E6 cells. (A and B) Plaque assay showing TBEV viral titers (PFU/ml) in Vero-E6 cells left untreated or treated with serum samples from patients with or without auto-Abs neutralizing IFN-ω in the presence of 50 pg/ml IFN-ω (A, left panel), 100 pg/ml IFN-ω (A, right panel), 50 pg/ml IFN-α2 (B, left panel), or 100 pg/ml IFN-α2 (B, right panel). For each set of serum conditions, we compared the values obtained in the presence and absence of IFN treatment in an ordinary one-way ANOVA with Bonferroni correction (as implemented in GraphPad Prism version 10.2.3). ns: non-significant; *: P < 0.05; **: P < 0.01; ***: P < 0.001. The dashed line indicates the limit of detection of the plaque assay. (C and D) Fluorescence microscopy images of Vero-E6 cells with and without treatment with 50 pg/ml IFN-ω (C) or 50 pg/ml IFN-α2 (D) in the absence of serum or the presence of serum from a healthy donor (HD), an APS-1 patient (with auto-Abs against IFN-ω and IFN-α2), a TBE patient with auto-Abs against IFN-ω (C) or a TBE patient with auto-Abs against IFN-α2 (D). Vero-E6 cells were then infected with mCherry-TBEV. The nuclei were stained with Hoechst stain before the measurement of fluorescence. The scale bars represent 300 µm. (E and F) Quantification of TBEV-positive Vero-E6 cells normalized against the percentage of cells infected in TBEV-only conditions, for different serum samples, with or without IFN-ω (E) or IFN-α2 (F) treatment. ns: non-significant; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001. For each set of serum conditions, the values obtained in the presence and absence of IFN treatment were compared in an ordinary one-way ANOVA with Bonferroni correction (as implemented in GraphPad Prism version 10.2.3).

Figure 4.

Auto-Abs neutralizing type I IFNs in longitudinal samples. (A–C) Auto-Abs neutralizing IFN-α2 (A), IFN-ω (B), and IFN-β (C), as determined with the luciferase-based neutralization assay, at different time points after infection. Each sample was tested once with an IFN concentration of 100 pg/ml (IFN-α2 and IFN-ω) or 1 ng/ml (IFN-β). I: time of admission to hospital, II: 2 days after admission, III: hospital discharge, and IV: follow-up (1–3 mo).