Auto-Abs neutralizing type I IFNs in patients with severe Powassan, Usutu, or Ross River virus disease

Abstract

Arboviral diseases are a growing global health concern. Pre-existing autoantibodies (auto-Abs) neutralizing type I interferons (IFNs) can underlie encephalitis due to West Nile virus (WNV) (∼40% of patients) and tick-borne encephalitis (TBE, due to TBE virus [TBEV]) (∼10%). We report here that these auto-Abs can also underlie severe forms of rarer arboviral infections. Auto-Abs neutralizing high concentrations of IFN-α2, IFN-β, and/or IFN-ω are present in the single case of severe Powassan virus (POWV) encephalitis studied, two of three cases of severe Usutu virus (USUV) infection studied, and the most severe of 24 cases of Ross River virus (RRV) disease studied. These auto-Abs are not found in any of the 137 individuals with silent or mild infections with these three viruses. Thus, auto-Abs neutralizing type I IFNs underlie an increasing list of severe arboviral diseases due to Flaviviridae (WNV, TBEV, POWV, USUV) or Togaviridae (RRV) viruses transmitted to humans by mosquitos (WNV, USUV, RRV) or ticks (TBEV, POWV).

Figure 1.

Auto-Abs neutralizing type I IFNs in individuals infected with POWV. (A) Age and sex distribution of the patients according to POWV disease severity. (B and C) Luciferase-based neutralization assay to detect auto-Abs neutralizing 10 ng/ml IFN-α2, IFN-ω, or IFN-β (B), or 100 pg/ml IFN-α2 and IFN-ω or 1 ng/ml IFN-β (C). The positive control (blue) was plasma from a patient with RAG1 deficiency known to carry auto-Abs neutralizing IFN-α2, IFN-ω, and IFN-β at a concentration of 10 ng/ml. Plasma samples from healthy donors (black) were obtained from individuals from the general population without auto-Abs neutralizing type I IFNs. 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 in the presence of 10% plasma or serum from patients or controls, and RLA was calculated by dividing firefly luciferase activity by 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). The samples of the POWV patients were tested twice and the associated datapoints represent the mean RLA of these independent duplicates.

Figure 2.

Auto-Abs neutralizing type I IFNs in individuals infected with USUV. (A) Age and sex distribution of the patients according to USUV disease severity. (B and C) Luciferase-based neutralization assay to detect auto-Abs neutralizing 10 ng/ml IFN-α2, IFN-ω, or IFN-β (B), or 100 pg/ml IFN-α2 and IFN-ω or 1 ng/ml IFN-β (C). The positive control (blue) was plasma from a patient with RAG1 deficiency known to carry auto-Abs neutralizing IFN-α2, IFN-ω, and IFN-β at a concentration of 10 ng/ml. Healthy donor plasma (black dots) and whole blood (black stars) samples were tested as negative controls; whole blood was tested because we had only whole-blood samples available for two of the three severe USUV cases. The asymptomatic cases (gray) tested positive for anti-USUV Abs during a blood donation but did not report symptomatic disease. 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 in the presence of 10% plasma or serum from patients or controls, and RLA was calculated by dividing firefly luciferase activity by 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) Correlation between ELISA and neutralization assay results for the detection of auto-Abs neutralizing type I IFNs.

Figure 3.

Auto-Abs neutralizing type I IFNs in individuals infected with RRV. (A) Age and sex distribution of the patients according to RRV disease severity. (B and C) Luciferase-based neutralization assay to detect auto-Abs neutralizing 10 ng/ml IFN-α2, IFN-ω, or IFN-β (B), or 100 pg/ml IFN-α2 and IFN-ω or 1 ng/ml IFN-β (C) in patients with mild (brown), moderate (orange) or severe (red) RRV disease. 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 in the presence of 10% plasma or serum from patients or controls, and RLA was calculated by dividing firefly luciferase activity by 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) Correlation between IFN-α2 neutralization and the severity score of the patients. The severity score was based on the evaluation of symptoms, such as muscle pain after activity, needing to sleep longer, prolonged tiredness after activity, tired muscles after activity, headache, pains in the arms/legs, waking up tired, arms/legs feeling heavy, fevers, back pain, joint pain, and weak muscles. (E) Correlation between ELISA and neutralization assay results for the detection of auto-Abs neutralizing IFN-α2. (F) Neutralization of 10 ng/ml IFN-α2 by the original plasma sample (T1) and a longitudinal plasma sample (T2) from P7 obtained 1 year later. The HDs tested (black) were healthy individuals tested at the time of collection of each of the patient samples, but the HD samples are not longitudinal.