JP5913202 | New immunoglobulin binding protein which has the improved singularity |
WO/2013/081540 | AFFINITY CHROMATOGRAPHY MATRIX |
JP2016538267 | Antibody purification |
INST NAT SANTE RECH MED (FR)
ASSIST PUBLIQUE HOPITAUX PARIS APHP (FR)
UNIV PARIS CITE (FR)
FOND IMAGINE (FR)
WHAT IS CLAIMED IS: 1. A method for evaluating the presence of anti-type I IFN specific auto-antibodies (auto-Abs) in a patient or individual positive for or at risk for SARS-CoV-2 infection, having COVID-19 disease, prior to vaccination with live attenuated vaccine (LAV), or having vaccine-associated disease and thereby determining treatment and treating the patient or individual comprising: (a) isolating a blood or serum sample from said patient or individual; (b) evaluating the blood or serum sample for auto-antibodies specific for one or more type I IFN selected from: (i) IFN-α2 and IFN-ω; (ii) IFN-α2, IFN-ω and IFN-β; (iii) IFN-α2, IFN-ω, IFN-β and IFN-ε; (iv) IFN-α2, IFN-ω, IFN-β, IFN-ε and IFN-κ; (v) IFN-α2, IFN-ω, IFN-β and IFN-α1/13; (vi) IFN-α2, IFN-ω, IFN-β, IFN- α1/13 and IFN-α14; (vii) IFN-α2, IFN-ω, IFN-β, IFN- α1/13, IFN-α14 and IFN-α7; (viii) IFN-α2, IFN-ω, IFN- α1/13, IFN-α14, IFN-α7 (ix) IFN-α2, IFN-ω, IFN-β, IFN-ε, IFN-α1, IFN-α2, IFN-α6, IFN-α13, IFN-α14 and IFN-α16; and (x) IFN-α2, IFN-ω, IFN-α1, IFN-α2, IFN-α6, IFN-α13, IFN-α14 and IFN-α16; (c) wherein a patient or individual having one or more auto-antbody is not administered the vaccine or wherein a patient or individual positive for or at risk for SARS-CoV-2 infection, having COVID-19 disease, or having vaccine-associated disease is treated to remove or deplete the auto-Abs and/or is administered a type I IFN against which they do not have auto-Abs and/or is administered an immune-modulatory agent that increases or facilitates type I IFN-mediated response. 2. The method of claim 1 for evaluating the presence of anti-type I IFN specific auto-antibodies (auto-Abs) in a patient or individual positive for or at risk for SARS-CoV-2 infection or having COVID-19 disease and thereby determining treatment and treating the patient or individual comprising: (a) isolating a blood or serum sample from said patient or individual; (b) evaluating the blood or serum sample for auto-antibodies specific for one or more type I IFN selected from: (i) IFN-α2 and IFN-ω; (ii) IFN-α2, IFN-ω and IFN-β; (iii) IFN-α2, IFN-ω, IFN-β and IFN-ε; (iv) IFN-α2, IFN-ω, IFN-β, IFN-ε and IFN-κ; (v) IFN-α2, IFN-ω, IFN-β, IFN-ε, IFN-α1, IFN-α2, IFN-α6, IFN-α13, IFN-α14 and IFN-α16; and (vi) IFN-α2, IFN-ω, IFN-α1, IFN-α2, IFN-α6, IFN-α13, IFN-α14 and IFN-α16; (c) wherein a patient or individual having one or more auto-antbody is treated to remove or deplete the auto-Abs and/or is administered a type I IFN against which they do not have auto-Abs and/or is administered an immune-modulatory agent that increases or facilitates type I IFN-mediated response. 3. The method of claim 1 for evaluating the presence of anti-type I IFN specific auto-antibodies (auto-Abs) in a patient or individual prior to vaccination with live attenuated vaccine (LAV) against yellow fever virus or having yellow fever vaccine-associated disease and thereby determining whether the patient or individual can safely receive YFV LAV and/or treating the patient or individual comprising: (a) isolating a blood or serum sample from said patient or individual; (b) evaluating the blood or serum sample for auto-antibodies specific for one or more type I IFN selected from: (i) IFN-α2 and IFN-ω; (ii) IFN-α2, IFN-ω and IFN-β; (iii) IFN-α2, IFN-ω, IFN-β and IFN-α1/13; (iv) IFN-α2, IFN-ω, IFN-β, IFN- α1/13 and IFN-α14; (v) IFN-α2, IFN-ω, IFN-β, IFN- α1/13, IFN-α14 and IFN-α7; and (vi) IFN-α2, IFN-ω, IFN- α1/13, IFN-α14, IFN-α7; (c) wherein a patient or individual having one or more auto-antibody is not administered the vaccine or wherein a patient or individual having yellow fever vaccine-associated disease is treated to remove or deplete the auto-Abs and/or is administered a type I IFN against which they do not have auto-Abs and/or is administered an immune-modulatory agent that increases or facilitates type I IFN-mediated response. 4. The method of claim 1, 2 or 3, wherein the blood or serum sample is evaluated for neutralizing antibodies. 5. The method of claim 1, 2 or 3, wherein plasmapheresis is conducted on the patient or individual to deplete the antibodies, or wherein B cells, such as auto-reactive B cells, and/or plasmacytes or plasmablasts are depleted in the patient or individual. 6. The method of claim 1, 2 or 3, wherein a patient or individual having auto-Abs against one or more of IFN-α2, IFN-ω, IFN-ε, IFN-α1, IFN-α2, IFN-α6, IFN-α13, IFN-α14 or IFN-α16 and not having auto-Abs against IFN-β is treated by administering IFN-β. 7. The method of claim 1, 2 or 3, wherein a patient or individual having auto-Abs against one or more of IFN-α2, IFN-ω, IFN-ε, IFN-α1/13, IFN-α14, or IFN-α7 and not having auto-Abs against IFN- β is treated by administering IFN-β. 8. The method of claim 1, 2 or 3, wherein a patient or individual having auto-Abs against one or more Type I IFN selected from IFN-ω, IFN-ε, IFN-β, IFN-κ, and not having auto-Abs against IFN-α2 is treated by administering IFN-α2. 9. The method of claim 1, 2 or 3, wherein the blood or serum sample is additionally or alternatively evaluated for auto-antibodies specific for one or more type I IFN selected from IFN-α4, IFN-α5, IFN- α6, IFN-α8, IFN-α10, IFN-α16, IFN-α17 and IFN-α21. 10. The method or claim 1, 2, 3 or 9, wherein the blood or serum sample is additionally or alternatively evaluated for auto-antibodies specific for one or more type I IFN selected from IFN-κ and IFN-ε. 11. The method of claim 9 or 10, wherein a patient or individual not having auto-Abs against IFN-β is treated by administering IFN-β. 12. The method of claim 1, 2 or 3, wherein a patient or individual having auto-Abs against one or more Type I IFN is treated by administering an IFN subtype which is not neutralized by the patient’s or individual’s auto-Abs. 13. The method of claim 9 or 10, wherein a patient or individual having auto-Abs against one or more of Type I IFN is treated by administering an IFN-α subtype which is not neutralized by the patient’s or individual’s auto-Abs. 14. The method of any of claims 1-13, wherein the LAV is a COVID-19/SARsCoV-2 vaccine or is a yellow fever vaccine. 15. The method of any of claims 1-13, wherein the vaccine-associated disease is COVID- 19/SARsCoV-2 vaccine-associated disease or is yellow fever virus (YFV) vaccine-associated disease. 16. An assay for evaluating the presence of auto-antibodies directed against one or more Type I IFN in a patient or individual positive for or at risk for SARS-CoV-2 infection or having COVID-19 disease or prior to vaccination with live attenuated vaccine (LAV) or having vaccine-associated disease comprising: (a) contacting a sample of blood or serum from the patient or individual with one or more recombinant type I IFN protein selected from IFN-α2, IFN-ω, IFN-β, IFN-ε and IFN-κ, wherein each IFN protein is labeled with a distinct detectable tag or marker to form an antibody-protein complex; (b) contacting any antibody-protein complex of (a) with one or more labeled anti-human immune globulin molecule that will bind and label auto-antibody bound to any one or more IFN protein; (c) specifically and selectively detecting each type I IFN protein bound by specific auto- Ab thereto. 17. The assay of claim 16, wherein one or more recombinant type I IFN protein is labeled with a fluorescent marker. 18. The assay of claim 16 or 17, wherein one or more recombinant type I IFN protein is covalently coupled to a magnetic bead with a fluorescent marker. 19. The assay of claim 16, wherein each of the one or more recombinant type I IFN proteins is covalently coupled to a magnetic bead with a distinct and differential fluorescent marker. 20. The assay of claim 16, wherein the one or more labeled anti-human immune globulin molecule that will bind and label bound auto-antibody is a labeled non-human animal derived anti- human IgG. 21. The assay of claim 16, wherein the one or more recombinant type I IFN protein is selected from IFN-α2, IFN-ω and IFN-β. 22. The assay of claim 16, wherein the one or more recombinant type I IFN protein is selected from IFN-α2 and IFN-ω. 23. The assay of any of claims 16-22, wherein the presence of neutralizing auto-antibodies is determined. 24. A kit for for evaluating the presence of auto-antibodies directed against one or more Type I IFN in a patient or individual at risk of, suspected of or determined to have coronavirus or in a patient or individual prior to vaccination with a live attenuated vaccine (LAV) or having vaccine-associated disease comprising: (a) one or more recombinant type I IFN protein selected from IFN-α2, IFN-ω, IFN-β, IFN-ε and IFN-κ, wherein each IFN protein is labeled with a distinct detectable tag or marker; (b) one or more labeled anti-human immune globulin molecule that will bind and label auto-antibody bound to any one or more IFN protein; (c) a means for specific and selective detection of each type I IFN protein bound by specific auto-Ab thereto. 25. The kit of claim 24, wherein one or more recombinant type I IFN protein is labeled with a fluorescent marker. 26. The kit of claim 24, wherein one or more recombinant type I IFN protein is covalently coupled to a magnetic bead with a fluorescent marker. 27. The kit of claim 24 or 26, wherein each of the one or more recombinant type I IFN proteins is covalently coupled to a magnetic bead with a distinct and differential fluorescent marker. 28. The kit of any of claims 24-27, wherein the one or more labeled anti-human immune globulin molecule that will bind and label bound auto-antibody is a labeled non-human animal derived anti- human IgG. 29. The kit of any of claims 24-28, wherein the one or more recombinant type I IFN protein is selected from IFN-α2, IFN-ω and IFN-β. 30. The kit of any of claims 24-28, wherein the one or more recombinant type I IFN protein is selected from IFN-α2 and IFN-ω. 31. The kit of any of claims 24-30, wherein the LAV is a COVID-19/SARsCoV-2 vaccine or is a yellow fever vaccine. 32. The kit of any of claims 24-30, wherein the vaccine-associated disease is COVID- 19/SARsCoV-2 vaccine-associated disease or is yellow fever virus (YFV) vaccine-associated disease. 33. A method for evaluating a patient or individual positive for or at risk for SARS-CoV-2 infection or having COVID-19 disease or prior to vaccination with live attenuated vaccine or having vaccine-associated disease for the presence of an autosomal recessive IFN deficiency or loss-of- function (LOF) variation or mutation in a type I IFN pathway or response relevant gene and thereby determining treatment or whether the patient or individual can be safely vaccinated or should be vaccinated and/or treating the patient or individual comprising: (a) isolating a blood or serum sample from said patient or individual; (b) evaluating the blood or serum sample for a LOF variation or mutation in one or more type I IFN pathway gene selected from: (i) TLR7, IFNAR1, IRF7, IFIH1, TLR3, TBK1, IRF3, TICAM1, UNC93B1, IFNAR2, STAT1, STAT2, TRAF3 or IRF9; (ii) TLR7, IFNAR1, IRF7, IFIH1, TLR3, TBK1, IRF3, TICAM1, UNC93B1, IFNAR2, STAT1, STAT2 or TRAF3; (iii) TLR7, IFNAR1, IRF7, IFIH1, TLR3, TBK1, IRF3, TICAM1, UNC93B1 and IFNAR2; (iv) TLR7, IFNAR1, IRF7, IFIH1, TLR3, TBK1, IRF3, TICAM1 and UNC93B1; and (v) IFNAR1, IRF7, IFIH1, TLR3, TBK1, IRF3, TICAM1 and UNC93B1 (c) wherein a patient or individual determined to have an autosomal recessive IFN deficiency or LOF mutation is administered one or more Type I IFN to replace the function lost due to the mutation and/or is administered an immune-modulatory agent that increases or facilitates type I IFN- mediated response. 34. The method of claim 33, wherein the patient or individual determined to have a LOF mutation is administered a Type I IFN. 35. The method of claim 33, wherein the patient or individual determined to have a LOF mutation is administered IFN-α2 or IFN-β. 36. The method of any of claims 33-35, wherein the variation or mutation in one or more type I IFN pathway gene is selected from a mutation provided in TABLE 1, TABLE 2, TABLE 11 or TABLE 15. 37. The method of claim 36, wherein the variation or mutation in one or more type I IFN pathway gene selected from TABLE 1, TABLE 2, TABLE 11 or TABLE 15 is determined via a primer or oligonucleotide specific for the mutation. 38. The method of claim 36, wherein the variation or mutation in one or more type I IFN pathway gene selected from TABLE 1, TABLE 2, TABLE 11 or TABLE 15 is determined via a detectably labeled primer or oligonucleotide specific for the mutation, wherein hybridization and detection of the labeled primer or oligonucleotide is diagnostic for the presence of the mutation and LOF of the type I IFN in the patient or individual. 39. The method of claim 36, wherein the variation or mutation in one or more type I IFN pathway gene selected from TABLE 1, TABLE 2, TABLE 11 or TABLE 15 is determined via whole genome or whole exome sequencing. 40. The method of claim 23, wherein the variation or mutation in one or more type I IFN pathway gene is determined via whole genome or whole exome sequencing. 41. The method of claim 23, wherein the LAV is a COVID-19/SARsCoV-2 vaccine or is a yellow fever vaccine. 42. The method of claim 23, wherein the vaccine-associated disease is COVID-19/SARsCoV-2 vaccine-associated disease or is yellow fever virus (YFV) vaccine-associated disease. 43. A method for evaluating a patient or individual positive for or at risk for SARS-CoV-2 infection or having COVID-19 disease for response to SARS-CoV-2 infection comprising: (h) isolating plasmacytoid dendritic cells (pDCs) from the patient or individual; (i) contacting the isolated pDCs with SARS-CoV-2 virus; and (j) assessing the production of type I IFNs by the pDCs in response to SARS-CoV-2; wherein reduced production of type I IFNs indicates that the patient or individual is altered in response to virus and is at high risk for severe COVID-19 disease. 44. The method of claim 43, wherein the method further includes thereby determining treatment and treating the patient or individual. 45. The method of claim 43, wherein the method further includes administering to the patient or individual the type I IFN or one or more type I IFN for which production is reduced. 46. The method of claim 43, wherein the patient is suspected of or first determined to carry or have family history of a variation or mutation in one or more type I IFN pathway or response relevant gene selected from TABLE 1, TABLE 2, TABLE 11 or TABLE 15. |
TABLE 2 Candidate Variants found in Severe COVID-19 Patients
TABLE 3 HA Abbreviation Key
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Nucleic Acids Res 42, e97 (2014). 40. G. Kerner et al., Homozygosity for TYK2 P1104A underlies tuberculosis in about 1% of patients in a cohort of European ancestry. Proc Natl Acad Sci U S A 116, 10430-10434 (2019). 41. S. Belkaya et al., Autosomal Recessive Cardiomyopathy Presenting as Acute Myocarditis. J Am Coll Cardiol 69, 1653-1665 (2017). 42. P. Meade et al., Influenza Virus Infection Induces a Narrow Antibody Response in Children but a Broad Recall Response in Adults. mBio 11, (2020). EXAMPLE 2 AUTO-ANTIBODIES AGAINST TYPE I IFNs IN PATIENTS WITH SEVERE COVID-19 [000197] Interindividual clinical variability in the course of SARS-CoV-2 infection is immense, ranging from silent infection to lethal disease. We report that 55 of 603 patients (9.1%) with life- threatening COVID-19 pneumonia had high titers of IgG auto-Abs against 7 of the 17 type I IFNs, including 6 of the 11 widely functional IFNs (5 IFN-α and IFN-ε), at the onset of their critical illness. The auto-Abs neutralize IFN-α2 in vitro, including against anti-SARS-CoV-2, while the 13 IFN-α, of which 7 are widely functional, are undetectable in the serum in vivo. No such auto-Abs are found in 370 individuals with asymptomatic or mild SARS-CoV-2 infection and 400 healthy controls. All but seven of the patients with auto-Abs are male (85%), and older than male patients without auto-Abs. Moreover, one of the female patients has X-linked incontinentia pigmenti (IP) and a third of uninfected women with IP tested also have such auto- Abs. Finally, four patients with other, life-threatening viral diseases display such auto-Abs. At least 9% of patients and 10.5% of men with life-threatening COVID-19 pneumonia have an X- linked, age-dependent auto-immune phenocopy of autosomal inborn errors of type I IFN immunity. Treatment with IFN-β, plasmapheresis, B-cell depletion, or the inhibition of type I IFN-reactive B cells may benefit these patients. [000198] Three life-threatening infectious diseases can be driven by monogenic inborn errors of cytokine immunity or their auto-immune phenocopies (1). Like inborn errors of IFN-γ immunity, auto- Abs against IFN-γ underlie mycobacterial disease (2–4). Both inborn errors of IL-17A/F immunity and auto-Abs against IL-17A or IL-17F underlie chronic mucocutaneous candidiasis (5, 6). Patients with inborn errors of IL-6 immunity or auto-Abs against IL-6 suffer from staphylococcal disease (7). Anti- cytokine auto-Abs can be genetically driven, as illustrated by auto-Abs against IL-17A/F, which are driven by mono- or biallelic mutations in AIRE and autoimmune polyendocrine syndrome type 1 (APS- 1) (5, 6, 8, 9), and by auto-Abs against IFN-γ, which are driven with lower penetrance by heterozygosity or homozygosity for HLA-DRB11502 or 1602 (10–12). Neutralizing auto-Abs against type I IFNs have long been known to be present in patients treated with IFN-α (13, 14) and in almost all patients with APS- 1 (15, 16). They are also seen in women with systemic lupus erythematosus (17, 18, 18, 19) and patients with Down syndrome (unpublished). It is intriguing that these patients do not apparently suffer from unusually severe viral infections, as Ion Gresser described a patient with unexplained auto-Abs against type I IFNs suffering from severe varicella and zoster in 1985 (20). Moreover, auto-Abs against type I IFNs in patients homozygous for hypomorphic RAG1 or RAG2 mutations have been associated with severe viral disease, including varicella, and RSV or adenovirus pneumonia (21). Finally, human inborn errors of type I IFNs underlie severe viral diseases, respiratory and otherwise (22–29). In this context, our attention was recently drawn to three APS-1 patients with life-threatening COVID-19 pneumonia (30) (unpublished data). Moreover, patients with Down syndrome (DS), who often carry auto-Abs against type I IFNs (31), were also recently reported to suffer from severe COVID-19 pneumonia (32). While searching for inborn errors of type I IFNs in patients with severe COVID-19 pneumonia (33) (accompanying report), we hypothesized that neutralizing auto-Abs against type I IFNs might also underlie severe COVID-19 pneumonia. [000199] We searched for auto-Abs against type I IFNs in 603 hospitalized patients with life- threatening COVID-19 pneumonia (as defined in M&M), 400 healthy controls not infected with SARS- CoV-2, 370 individuals infected with SARS-CoV-2 and asymptomatic or with mild disease, and 120 patients hospitalized for other severe viral infections (TABLE 4). Plasma or serum samples were collected from patients with severe COVID-19 on the first days of hospitalization and during the acute phase of the disease. Multiplex particle-based flow cytometry revealed a high fluorescence intensity (FI; >4,000, for IFN-α2 and >1,000 for IFN-ω, definitions based on correlation with neutralizing effect from previous experiments) of IgG auto-Abs against IFN-α2 and/or IFN-ω in 62 patients (10.2%) with severe forms of COVID-19 (Figure 9, A). In the same assay, some patients also had auto-Abs against other cytokines (IFN-γ, IL-6, IL-10, IL-22, IL-17F and/or G-CSF), but none of these Abs were neutralizing (Figure 10A, B and C). Of the 62 patients with high titers of anti-IFN-α2 and/or anti-IFN-ω auto-Abs, 30 were positive for antibodies against both IFN-α2 and IFN-ω, ten were positive for antibodies against IFN-α2 only and 22 were positive for antibodies against IFN-ω only. Six women (excluding a woman with IP) tested positive for auto-Abs: 4 had both anti-IFN-α2 and anti-IFN-ω antibodies, whereas two had only anti-IFN-ω auto-Abs. None of the 770 individuals from any of the two control groups (asymptomatic/mild infected or healthy) were positive for either type of auto-Ab, except for one woman who tested positive for auto-Abs against IFN-ω (Figure 9B). TABLE 4 Age and Gender of the patients and controls cohorts [000200] For IFN-α2 and IFN-ω, we also ran classical enzyme-linked immunosorbent assays (ELISA), with plasma samples with a dilution factor twice that used in the multiplex assay. Of the 350 patients tested, 10% were positive for IFN-α2 and/or IFN-ω, yielding results matching those of the other assay, and the discrepancy rate was 5% (Figure 10D, E). We also found that most patients tested (N=22) had low titers of IgA auto-Abs against IFN-α2 and/or IFN-ω, while some had IgM auto-Abs (Figure 10F, G). Finally, we investigated whether the patients with auto-Abs against IFN-α2 and/or IFN-ω also had auto- Abs against some of the other 15 type I IFNs. Interestingly all the patients tested (N=5) with auto-Abs against IFN-α2 also had auto-Abs against 6 other IFN-α subtypes (IFN-α1, -α2, -α6, - α13, -α14, -α16) (Figure 9C). Of note, 4 of these are among the 7/11 type I IFNs that are widely functional in the general population, having evolved under strong purifying selection (34). They also cluster in two main phylogenetic branches (Fig 10H). Only 1/62 patient also had auto-Abs against IFN-β while 7/62 (11%) had auto-Abs against IFN-ε (Fig 10A). We did not detect auto-Abs against IFN-α4, IFN-α5, IFN- α7, IFN-α8, IFN-α10, IFN-α17 and IFN-α21 in the patients tested (Figure 9C). Overall, we found that at least 10.2% of patients with severe COVID-19 pneumonia had high levels of IgG auto-Abs against at least one type I IFN, while all tested had auto-Abs against several of the 17 type I IFNs. [000201] In two unrelated patients for whom we had plasma drawn prior infection with SARS- CoV- 2, we detected the auto-Abs against type I IFNs, indicating that they were not triggered by, but pre- existed infection with SARS-CoV-2. We further tested this possibility, by screening for auto-Abs against type I IFNs in 120 patients with other severe viral infections, including severe viral respiratory infections. Interestingly, we found that four (2 men, 2 women) displayed auto-Abs against IFN-α2 and IFN-ω (TABLE 5). Two children had suffered from severe viral pneumonia, including acute respiratory distress syndrome caused by influenza virus in one and recurrent severe pneumonia caused by various viruses, including common coronavirus, respiratory syncytial virus (RSV), and rhinovirus in another. Two unrelated adults had suffered from severe clinical disease following inoculation with live attenuated yellow fever virus vaccine (YFV). One of these patients reacting to YFV is a woman who was later diagnosed with systemic lupus erythematosus (SLE). SLE patients are known to have high levels of IFN- α (35), and auto-Abs against type I IFN have been described in SLE (17–19, 36). The observation of these 4 patients is important for several reasons. First, it suggests that various severe viral diseases, other than severe COVID-19 pneumonia, can be favored by auto-Abs against type I IFNs. Second, it suggests that the auto-Abs seen in COVID-19 patients were probably present before SARS- CoV-2 or any other viral infection, and not triggered by these viruses, as neatly illustrated by the adverse reaction to YFV, which occurred in the days following vaccination. Third, it validates the notion that auto-Abs against type I IFN can neatly phenocopy disorders of the three IFN-related pathways selected for the search for autosomal inborn errors of IFN immunity in the accompanying report: the TLR3 pathway for severe influenza pneumonia (28), the MDA5 pathway for RSV and related viruses, including common coronaviruses (37, 38), and the IFNAR1/IFNAR2 pathway for YFV-associated disease (22, 23). TABLE 5 [000202] We then tested whether the auto-Abs against IFN-α2 and IFN-ω were neutralizing. We incubated PBMCs from healthy controls with 10 ng/mL IFN-α2 or IFN-ω in the presence of plasma from healthy individuals or from patients with auto-Abs. Complete abolition of pSTAT1 induction was observed in all patients with high titers of auto-Abs against IFN-α2 tested (N=31) confirming the relevance of the threshold that was used (Figure 9D, E) and that the 9 remaining patients to be tested should be neutralizing and were thus considered as such. We also screened 58 patients with intermediate titers of auto-Abs against IFN-α2 and identified an additional 5 patients with neutralizing auto-Abs against IFN-α2. For IFN-ω, 32 out of 61 (52%) patients that we tested had neutralizing auto-Abs. Overall, we identified 55 patients with neutralizing auto- Abs (9.2% of severe COVID patients) against IFN-α2 and IFN-ω (N=42), IFN-α2 only (N=11) or IFN-ω only (N=2) (TABLE 6). We also analyzed interferon- stimulated gene (ISG) induction after 2 h of stimulation with IFN-α2 or IFN-γ. With plasma of twelve patients with auto-Abs against IFN-α2, the induction of CXCL10 was abolished after IFN-α2 stimulation but maintained after stimulation with IFN-γ (Figure 9F). Interestingly, serum from the patient with high titers auto-Abs against IFN-β showed neutralizing activity against IFN-β. Finally, we tested if the plasmas from patients with auto-Abs against type I IFN were neutralizing for their anti-viral activity. We found that all 5 tested plasmas of patients with neutralizing auto-Abs neutralized the protective activity of IFN-α2 in vero cells infected with vesicular stomatitis virus. Moreover, we found that the two plasma of patients with neutralizing auto-Abs tested did neutralize the activity of IFN-α2 in ACE2- expressing human fibroblasts infected with YFV- 17D or SARS-CoV-2 (Figure 9G, H). Conversely, the plasmas of healthy controls or SARS- CoV-2 infected patients without auto-Abs did not block the protective effect of IFN-α2. These data provided compelling evidence that the patients’ blood carried titers of auto-Abs that were neutralizing of the corresponding type I IFNs for their activity against SARS-CoV-2. [000203] Neutralizing antibodies against both IFN-α2 and IFN-ω were detected in 52 of 101 patients (51%), against only IFNα2 in 36 patients (36%), and against only IFNω in 13 patients (13%) at the IFN- α2 and IFN-ω concentrations tested. IgG depletion from patients with auto-Abs restored normal pSTAT1 induction after IFN-α2 and IFN-ω stimulation, whereas the purified IgG fully neutralized this induction (Figure 9D and data not shown). Furthermore, these auto-Abs neutralized high amounts of IFN-α2 and were neutralizing at high dilutions. Notably, 15 patients with lifethreatening COVID-19 and auto-Abs against IFN-α2 and/or IFN-ω also had auto-Abs against other cytokines [IFN-γ, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, IL-10, IL-12p70, IL-22, IL-17A, IL-17F, and/or tumor necrosis factor–β (TNFβ)], only three of which (IL-12p70, IL-22, and IL-6) were neutralizing (in four patients). Similar proportions were observed in the other cohorts (data not shown). [000204] We also analyzed ISG induction after 2 hours of stimulation with IFN-α2, IFN-β, or IFN-γ in the presence of plasma from healthy individuals or from patients with auto-Abs. With plasma from eight patients with auto-Abs against IFN-α2, the induction of ISG CXCL10 was abolished after IFN- α2 stimulation but maintained after stimulation with IFN-γ.We then found that plasma from the five patients with neutralizing auto-Abs neutralized the protective activity of IFN-α2 in Madin–Darby bovine kidney (MDBK) cells infected with vesicular stomatitis virus (VSV). Overall, we found that 101 of 987 patients (10.2%)—including 95 men (94%)—with life-threatening COVID-19 pneumonia had neutralizing IgG auto-Abs against at least one type I IFN. By contrast, auto-Abs were detected in only 4 of 1227 healthy controls (0.33%) (Fisher exact test, P < 10 −16 ) and in none of the 663 patients with asymptomatic or mild SARS-CoV-2 infection tested (Fisher exact test, P < 10 −16 ). TABLE 6
CVID: Common variable immunodeficiency; HTA: Hypertension; Cardiovascular: cardiovascular disease; Respiratory: respiratory disease; Obese: Body-mass index >30; NA: Non- applicable. [000205] Recent studies have reported that some patients with COVID-19, including patients with severe disease, have low or undetectable IFN-α levels during SARS-CoV-2 infection, as shown by Simoa digital ELISA, which measures the levels of the 13 IFN-α types (39, 40) (unpublished). Low levels of these IFN may be a cause or consequence of disease or severe disease. We thus assessed 12 patients who were studied here and previously found to have low or undetectable levels of serum IFN-α, including some of the patients reported in these papers (39, 41); we found that 42% (N=5) had auto-Abs against type I IFNs. We are investigating whether and how many of the remaining 7 have inborn errors of type I IFNs (as described in Example 1). Moreover, we found that all patients with neutralizing auto- Abs against type I IFNs tested (N=25) also had undetectable levels of IFN-α in their plasma (Figure 9I). Another 5 patients, with positive auto-Abs against IFN-α2, not yet tested for their neutralization capacity, had undetectable serum IFN-α levels, while one was low. The presence of circulating neutralizing auto-Abs against type I IFNs is therefore strongly associated with low serum IFN-α levels (p<10 -6 ). Other patients with low or undetectable levels of serum IFN-α may still have auto-Ab levels but below the limit of detection in this study, or undetectable because bound to IFN-α, or have genetic defects affecting IFN production or amplification other than those reported in the accompanying paper. Our findings indicate that the auto-Abs against type I IFNs present in these patients were also neutralizing in vivo. [000206] We performed whole-exome sequencing (WES) or whole-genome sequencing (WGS) on 35 of the 55 patients with high levels of neutralizing auto-Abs against type I IFNs. Various HLA alleles have been associated with autoimmune diseases, including the production of anti- IFN-g auto-Abs (10– 12). We therefore inferred HLA data from WES or WGS data (42). We were unable to identify a specific haplotype either common to all patients with auto-Abs against type I IFNs or even enriched in these individuals. Nevertheless, there was a strong excess of male patients (49 of 55, 89%) with severe COVID-19 pneumonia and auto-Abs against type I IFNs. This proportion of males was slightly higher than that observed in severe patients without auto-Abs (76%; p=0.028), yet, importantly, much greater than that of male patients within the asymptomatic or pauci-symptomatic cohort (28%, p<10 -6 ). Moreover, one of the female patients had X-linked incontinentia pigmenti (IP) due to a large heterozygous deletion in NEMO. All cells in women with IP, whose prevalence is about 1/1,000,000, express the same single X chromosome (43–45). We tested 13 other women with IP who had not been infected with SARS-CoV-2. Strikingly, we found high titers of auto-Abs against IFN-α2 in 30% of these women (Fig 11E). This suggested that the auto-Abs against type I IFNs in the woman with IP and severe COVID-19 were present prior to infection with SARS-CoV-2, and that women with IP, like women with SLE or patients with DS, are at much higher risk of displaying such auto-Abs than the general population. The high male preponderance in auto- Ab production and their frequent occurrence in women with IP (46, 47) suggest that the production of auto-Abs against type I IFNs can be X-linked recessive, at least in a subset of patients. [000207] Using the WGS data of male patients with (N=29) and without (N=242) auto-Abs, we found no single-nucleotide variants (SNV) or copy-number variants (CNV) located on the X significantly associated, at the chromosome-wide level, with the presence of auto-Abs. There was no variant significantly associated on the Y chromosome either. This result suggests that the pathogenesis of auto- Ab production if X-linked, is not monogenic with homogeneity. Alternative plausible genetic models of X-linked inheritance include (i) monogenic inheritance with heterogeneity, and (ii) digenic inheritance with heterogeneity. A gene dosage mechanism involving a large number of loci on the X chromosome may also be involved, as suggested by the presence of auto-Abs in four of the 13 women with IP tested. Women (XX) appear to be protected, with the exception of women with IP, SLE, of DS, and perhaps those with early skewed X inactivation in the course of development, whereas a larger fraction of men (XY) develop auto-Abs against type I IFNs. These findings contrast with the usual epidemiological distribution of autoimmunity. Based on the absence of auto-Abs in 200 healthy males, the prevalence of auto-Abs against type I IFNs in the general male population is probably <1%, lower than that in this population of patients with severe COVID-19 pneumonia by a factor of at least 10. The patients tested positive for auto-Abs were slightly older than the rest of our cohort (64% of patients’ positives for auto-Abs were over 60 years old versus 42% in the rest of the cohort, p=0.0064), suggesting that the frequency of auto-Ab production increases with age. Auto-Abs were however found from age 25 to 88 years old. Of the 36 patients for whom we performed PCA analysis, 32 were European, 1 was North-African, 1 South-Asian and 1 Latino. Large-scale studies will be required to determine the frequency of such auto-Abs in humans of different ages and ancestries. [000208] We provide evidence that the auto-Abs against type I IFNs observed in these 55 patients were not triggered by SARS-CoV-2, but preceded the infection with this virus, and made a significant contribution to the severity of the disease. First, we obtained a plasma sample taken before COVID-19 infection for two patients, which showed that the auto-Abs were already present before infection. Second, 30% of uninfected women with IP had auto-Abs against type I IFNs, including one who developed life-threatening COVID-19 upon infection. Third, three patients with AR APS-1 and high titers of neutralizing auto-Abs had severe COVID-19. Fourth, patients with DS more frequently suffer from severe COVID-19 than the general population (32) (unpublished). Fifth, many of our patients also had auto-Abs against other cytokines, suggestive of an underlying auto-immune process, rather than a response to the virus, although past and present viral infections may have increased anti-IFN auto-Ab production. Sixth, IFN- α were undetectable in the serum of the patients with these auto-Abs on the first days of hospitalization, suggesting a pre-existing biological impact in vivo. Seventh, it is difficult to conceive that patients could both break self-tolerance and mount high titers of neutralizing IgG and IgA auto-Abs against type I IFN within only one week of infection with the virus, or even two weeks. Eighth, inborn errors of type I IFNs underlying severe COVID-19 in other previously healthy adult patients are reported in an accompanying paper. Ninth, four patients with other severe viral infections related to type I IFN immunity also have auto-Abs against type I IFNs, including a woman with SLE. Tenth, there is a strong bias in favor of men (p<10 - 6 ), suggesting that the production of auto- Abs to type I IFNs is genetically driven and X-linked recessive, at least in some patients. Collectively, these findings provide compelling evidence that auto-Abs to type I IFNs are a cause, and not a consequence of severe SARS-Cov-2 infection. [000209] We thus report that at least 10% of patients with life-threatening COVID-19 pneumonia have X-linked neutralizing auto-Abs against type I IFNs. The prior Example 1 shows that another 10% of patients carry known or novel forms of autosomal inborn errors of 13 type I IFN-related genes. These two studies highlight the crucial role of type I IFNs in protective immunity against SARS-CoV-2, and other viruses, including respiratory viruses in particular. Our study also reveals a fourth example of auto- Abs against cytokines mimicking inborn errors of the corresponding cytokine, after IFN-γ, IL-6, and IL- 17A/F (1). The auto-Abs against type I IFNs were probably clinically silent until the patients were infected with SARS-CoV-2, which has been shown to be a poor inducer of type I IFN (48, 49), making the small amounts of IFN produced even more important for protective immunity. The neutralizing auto- Abs against type I IFNs, like autosomal inborn errors of type I IFN production, tip the balance in favor of the virus, resulting in a devastating pneumonia. This report also provides a first compelling explanation for the major sex bias observed in patients with severe COVID-19, and perhaps also the increased risk with age, while offering a possible explanation for geographic disparities in the severity of COVID-19. The penetrance of neutralizing auto-Abs against type I IFNs for severe COVID-19 is probably high upon infection with SARS-CoV-2 and inoculation with the YFV vaccine, while it is much lower upon infection with other, less virulent respiratory viruses. Our findings thus already have clinical implications. First, it is possible to screen SARS-CoV-2-infected patients to identify individuals at risk. Second, this observation paves the way for therapeutic intervention. Although high-dose intravenous IgG apparently helped some patients with severe COVID-19 pneumonia (50), this immunosuppressive approach might be irrelevant to the pathogenic auto-Abs against type I IFNs (51), and pharmaceutical immunoglobulins could also contain certain amounts of anti-cytokine neutralizing antibodies (52). Plasmapheresis or monoclonal Abs depleting plasmablasts (daratumumab) would be better options, if SARS-CoV-2-specific plasma or monoclonal Abs can be used to compensate for the loss of the patient’s Abs against the virus (53). Alternatively, one may consider a specific inhibition of type I IFN-reactive B cells by cross-linking their IFN-specific surface Ig with their inhibitory FcRIIb (51). By contrast, early treatment with IFN-α is unlikely to be beneficial, given the high titers of neutralizing auto-Abs, and it might even select B cells producing Auto- Abs with higher affinity to type I IFNs; yet, treatment with IFN-β in patients without auto-Abs against IFN-β could be beneficial. Convalescent plasma therapy (CPT) has also been proposed and administered to a small number of COVID-19 patients (54, 55). We suggest that anti-type I IFN auto-Ab levels should be measured in the convalescent plasma preparations currently being tested in clinical trials, or at least that patients with severe COVID-19 should be excluded. Finally, recombinant IFN-α2, intramuscularly injected or inhaled, may be beneficial in a second step, once the auto-Abs have been depleted by plasmapheresis or the auto-reactive B cells inhibited or depleted. [000210] MATERIALS AND METHODS [000211] 603 patients with proven severe COVID-19 infection, 370 asymptomatic or pauci- symptomatic individuals with proven COVID-19 infection, 350 healthy controls and 120 patients with other severe viral diseases were enrolled in this study, with informed consent and approval obtained from the Necker Hospital and Medical School Institutional Review Board (IRB), the Rockefeller University IRB, the IRB of ASST Ospedale San Gerardo – University of Milano-Bicocca, Monza (Italy) and the IRB of Fondazione IRCCS Policlinico San Matteo, Pavia (Italy). Some patients were enrolled in the French COVID cohort (clinicaltrials.gov NCT04262921). Ethics approval was obtained from the CPP IDF VI (ID RCB: 2020-A00256- 33). Some contact subjects were enrolled in the Cov-Contact cohort (clinicaltrials.gov NCT04259892). De-identified samples were studied at the NIAID under non- human subjects research conditions; no additional IRB consent was required at the NIH. [000212] Plasma and serum samples from the patients and controls were frozen at -20°C immediately after collection. The fluid-phase LIPS assay was used to determine the levels of antibodies against the SARS-CoV-2 nucleoprotein and spike protein, as has been previously described (Burbelo, PD et al (2020) J Infect Dis 222, 206-213). [000213] Serum/plasma samples were screened for autoantibodies against 25 targets in a multiplex particle-based assay, in which magnetic beads with differential fluorescence were covalently coupled to recombinant human protein (2.5 µg/reaction). Beads were combined and incubated with 1:100 diluted serum/plasma samples for 30 minutes. They were then washed and incubated with PE-labeled goat anti- human IgG (1 µg/mL) for an additional 30 minutes. Beads were then washed again and run on a BioPlex X200 instrument in a multiplex assay. Patients with an FI of >1500 for IFN-α2 or IFN-β or >1000 for IFN-ω were tested for blocking activity, as were patients positive for another cytokine. [000214] ELISA was performed as previously described (Puel et al., 2008). In brief, 96-well ELISA plates (MaxiSorp; Thermo Fisher Scientific) were coated by incubation overnight at 4°C with 2 µg/mL rIL- 17F, rIL-22, rhIFN-α, and rhIFN-ω (R&D Systems). Plates were then washed (PBS/Tween 0.005%), blocked by incubation with the same buffer supplemented with 5% nonfat milk powder, washed, and incubated with 1:50 dilutions of plasma samples from the patients or controls for 2 h at room temperature (or with specific mAbs as positive controls). Plates were thoroughly washed. Horseradish peroxidase (HRP)–conjugated Fc-specific IgG fractions from polyclonal goat antiserum against human IgG or IgA (Nordic Immunological Laboratories) were added to a final concentration of 2 µg/mL. Plates were incubated for 1 h at room temperature and washed. Substrate was added and OD was measured. A similar protocol wass used when testing for the 21 subtypes of IFN-α, except that the plates were coated using cytokines from PBL assay science (catalog #11002-1). [000215] LIPS - Levels of auto-Abs against IFN-a subtypes were measured with LIPS, as previously described (Meyer S et al (2016) Cell 166:582-595). IFN-a1, IFN-a2, IFN-a4, IFN-a5, IFN-a6, IFN-a7, IFN-a8, IFN-a10, IFN-a14, IFN-a16, IFN-a17, and IFN-a21 sequences were transfected in HEK293 cells, and the IFN-a-luciferase fusion proteins were collected in the tissue culture supernatant. For autoantibody screening, serum samples were incubated with protein G agarose beads, and we then added 2 × 10 6 luminescence units (LU) of antigen and incubated. Luminescence intensity was measured. The results are expressed in arbitrary units (AU), as a fold-difference relative to the mean of the negative control samples. [000216] The blocking activity of anti-IFNα and anti-IFNω autoantibodies was determined by assessing STAT1 phosphorylation in healthy control cells following stimulation with the appropriate cytokines in the presence of 10% healthy control or patient serum/plasma. Surface- stained healthy control PBMCs (350,000/reaction) were cultured in serum-free RPMI medium with 10% healthy control or patient serum/plasma and were either left unstimulated or stimulated with IFNα or IFNω (10 ng/mL) for 15 minutes at 37°C. Cells were fixed, permeabilized, and stained for intranuclear phopsho- STAT1 (Y701). Cells were acquired on a BD LSRFortessa cytometer with gating on CD14+ monocytes and analyzed with FlowJo software. [000217] The blocking activity of anti-IFNγ, -IL-6, -IL-10, and -G-CSF antibodies was determined in a similar manner, by flow cytometry, with the specifications indicated in TABLE 7 below. TABLE 7 [000218] We demonstrated that the IFN-α and IFN-ω blocking activity observed was due to auto- Abs and not another plasma factor, by depleting IgG fromthe plasma with a protein G column. Without eluting the IgG, the flow-through fraction (IgG-depleted) was then collected and compared with total plasma in the phospho-STAT1 assay. [000219] The blocking activity of anti–IFN-γ, –GMCSF, –IFN-λ1, –IFN-λ2, –IFN-l3, –IL-6, –IL- 10, –IL-12p70, –IL-22, –IL-17A, –IL-17F, -TNFα, and -TNFβ antibodies was assessed with the assays outlined, as previously reported (Walter JE et al (2015) J Clin Invest 125:413504148). For the neutralization of ISG induction, PBMCs were left unstimulated or were stimulated for 2 hours with 10 ng/mL IFN-α or 10 ng/mL IFN-γ in a final volume of 100 mL. Realtime quantitative polymerase chain reaction (RT-qPCR) analysis was performed with Applied Biosystems Taqman assays for CXCL10, and the b-glucuronidase (GUS) housekeeping gene for normalization. Results are expressed according to the ∆∆Ct method, as described by the manufacturer’s kit. [000220] For the neutralization of ISG induction, peripheral blood mononuclear cells (PBMCs) were thawed and kept overnight in X-VIVO20 (Lonza) at 37°C under an atmosphere containing 5% CO 2 . We added 0.75 x 10 6 PBMCs in 40 µL of X-VIVO20 medium to each well of a 96- well U-bottomed plate with 10 µL of the indicated plasma, and the contents of each well were mixed. PBMCs were then left unstimulated or stimulated for two hours with 10 ng/mL IFNa or 10 ng/mL IFNg in a final volume of 100 µL. We extracted mRNA from the cells with a kit, according to manufacturer’s instructions (Zymo Research). We used 250 ng of each mRNA preparation for reverse transcription with oligo-DT primers and Superscript II (Thermo Fisher Scientific). Quantitative real-time PCR (RT-qPCR) was performed with Applied Biosystems Taqman assays for CXCL10 and IFIT1, and the β-glucuronidase (GUS) housekeeping gene for normalization. Results are expressed according to the ∆∆Ct method, as described by the kit manufacturer. [000221] Serum-IFNα concentrations were determined with Simoa technology as described in (35, 56) with reagents and procedures obtained from Quanterix Corporation (Quanterix Simoa TM IFNα Reagent Kit, Lexington, MA, USA), according to the manufacturer’s instructions. The dynamic range of the assay was 0 to 54.6 pg/mL with a lower limit of detection of 16 fg/mL and a lower limit of quantification of 64 fg/mL. [000222] The seroneutralization assay was performed as previously described (57). In brief, IFN- α2 incubated with Madin–Darby bovine kidney protects cultured cells against the cytopathic effect of vesicular stomatitis virus (VSV). Serial two-fold dilutions of patients’ serum was added in the incubation medium prior to viral challenge. The titer of anti IFN alpha antibodies was defined as the last dilution causing 50% cell death. [000223] The SARS-CoV-2 infection experiments were performed as follows. Huh7.5 cells were seeded in 96-well plates. 24h later, recombinant IFN-α2 was incubated together with plasma for 1h at 37°C, using 2 concentrations of IFN-α2 (2pM and 10pM). Starting plasma dilution was 1/50 and 3-fold serial dilutions were done. For the commercial anti-IFN-α2 antibody, dilutions started from 1/500. Then, media from the cells was removed and cells were incubated overnight with the plasma + IFN-α2 mixture. The next day, cells were washed to remove anti- SARS-CoV-2 neutralizing antibodies and fresh media was added. They were then infected with SARS-CoV-2. 48h later, the cells were fixed and stained for SARS-CoV-2 N protein as described elsewhere (53). A similar method was used for YFV-17D, except that cells were fixed at 3 days post-infection. [000224] SARS-CoV-2 strain USA-WA1/2020 was obtained from BEI Resources and amplified in Huh7.5 hepatoma cells at 33°C. Viral titers were measured on Huh7.5 cells in a standard plaque assay. Plasma samples or a commercial anti– IFN-α2 antibody were serially diluted and incubated with 20 pM recombinant IFN-α2 for 1 hour at 37°C (starting concentrations: plasma samples = 1/100 and anti–IFN- α2 antibody = 1/1000). The cell culture medium was then removed and replaced with the plasma– or antibody–IFN-α2mixture. The plates were incubated overnight, and the plasma– or antibody–IFN-α2 mixture was removed by aspiration. The cells were washed once with phosphate-buffered saline (PBS) to remove potential anti–SARS-CoV-2 neutralizing antibodies, and fresh medium was then added. Cells were then infected with SARSCoV-2 by directly adding the virus to the wells. 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These antibodies were recently shown to underlie at least 10% of cases of lifethreatening COVID-19 pneumonia. The auto-Abs were neutralizing in vitro, blocking the protective effect of IFN-α2 against YFV vaccine strains. AR IFNAR1 or IFNAR2 deficiency and neutralizing auto-Abs against type I IFNs thus accounted for more than half the cases of life-threatening YFV vaccine-associated disease studied here. Previously healthy subjects could be tested for both predispositions before anti-YFV vaccination. [000227] Introduction [000228] The 17D live-attenuated vaccine against yellow fever virus (YFV) was approved for use in humans by the World Health Organization in 1945. It has since been used to vaccinate more than 600 million people worldwide, with very high rates of seroconversion following the administration of a single dose, providing long-term protection (Monath, 2005; Monath et al., 2005). About half the vaccine recipients develop transient low-level viremia detectable four to six days after inoculation, a timing similar to that for viremia after wild-type YFV infection. All live attenuated YFV vaccines in current use are derivatives of the 17D strain and produced by amplification in embryonated chicken eggs. Although initially considered to be the world’s safest live virus vaccine, rare cases of life-threatening disease following vaccination with YFV-17D were subsequently detected, from 2001 onward (Chan et al., 2001; Martin et al., 2001; Seligman, 2014; Vasconcelos et al., 2001). Systemic disease with clinical manifestations of organ dysfunction is often reported as yellow fever vaccine associated viscerotropic disease (YEL-AVD) (Seligman, 2014), and cases with neurological manifestations are referred to as yellow fever vaccine-associated neurological disease (YELAND). The prevalences of these conditions are about 0.3 and 0.8 per 100,000 vaccinees, respectively (Lindsey et al., 2016). However, prevalence estimates vary, ranging, for YELAVD, from 0-0.01 per 100,000 vaccinees in Africa, to 0.02-0.31 per 100,000 vaccinees in Brazil, and 0.35 per 100,000 vaccinees in USA, which is considered to be the most accurate estimate. The incidence of YEL-AND is estimated at 0.39 per 10 5 administered vaccine doses (range 0.02–1.5) (Lecomte et al., 2020). Mortality rates vary depending on age, but approximatively two thirds of individuals die (Seligman, 2014). The rate of severe adverse events seems to increase with age, particularly after the age of 55 years, and is higher in men (Lindsey et al., 2016; Seligman, 2014). Women in their prime child-bearing years and patients with thymoma are also at risk (Seligman, 2014). Severe adverse reactions have also occurred in association with various autoimmune diseases, including systemic lupus erythematosus (SLE), Addison’s disease, pernicious anemia, and myasthenia gravis, suggesting an immunological mechanism (de Menezes Martins et al., 2014; Seligman, 2014; Seligman and Casanova, 2016). [000229] We previously described and provided an explanation for life-threatening disease following YFV-17D vaccination in 2019, with the discovery of autosomal recessive (AR), complete IFNAR1 deficiency in a 14-year-old Brazilian girl with no prior history of severe viral illness, who suffered from YEL-AVD (Hernandez et al., 2019). This study also highlighted the crucial role of type I interferons (IFNs) in controlling live attenuated YFV-17D. Four other patients with IFNAR1 deficiency have been reported: a nine-year-old child from Iran with measles-mumpsrubella (MMR) live vaccine-associated disease (Hernandez et al., 2019), a two-year-old child from Palestine with herpes simplex encephalitis (HSE) (Bastard et al., 2020a), and two adults, aged 26 and 38 years, from Saudi Arabia and Turkey, with life-threatening COVID-19 pneumonia (Zhang et al., 2020b). A case of YEL-AVD was also reported in a one-year-old patient with a suspected AR deficiency of IRF9, a component of the ISGF3 complex activated by both type I and III IFNs (Bravo Garcia-Morato et al., 2019). Another child, with severe influenza pneumonia, had proven AR IRF9 deficiency but was not vaccinated against yellow fever (Hernandez et al., 2018). Thus, inborn errors of type I IFN immunity can underlie life threatening disease following vaccination against YFV. [000230] We studied seven other unrelated and previously healthy patients, currently aged 35 to 80 years, who had suffered from YEL-AVD (N = 5) and/or YEL-AND (N = 3) (Table 1) (Lecomte et al., 2020; Pulendran et al., 2008; Slesak et al., 2017). All had suffered life-threatening complications following vaccination with YFV-17D. In this report, we tested the hypothesis that some of these patients carry deleterious variants of genes of the type I IFN pathway, including IFNAR2, encoding the second chain of the type I IFN receptor (Duncan et al., 2015), JAK1 and TYK2, encoding the two kinases constitutively associated with the receptors (Eletto et al., 2016; Kreins et al., 2015; Lazear et al., 2019; Russell-Harde et al., 2000; Wilmes et al., 2015), and STAT1, STAT2, and IRF9, encoding the three components of ISGF-3 (Dupuis et al., 2003; Hambleton et al., 2013; Hernandez et al., 2018). We also tested the hypothesis that auto-Abs against type I IFNs may be causal for disease, as recently reported in 10% of patients suffering from severe COVID-19 (Bastard et al., 2020b; Zhang et al., 2020a) [000231] RESULTS [000232] A rare homozygous essential splice variant of IFNAR2 in one patient [000233] In addition to the patient with proven AR IFNAR1 deficiency (Hernandez et al., 2019), we studied seven patients who had experienced life-threatening disease following vaccination with the YFV-17D vaccine (Table 8). We performed whole-exome sequencing (WES) in all patients. Principal component analysis (PCA) confirmed that the patients were of different ancestries (Fig. 14A). We hypothesized that the patients developed YEL-AVD and/or YEL-AND because of monogenic inborn errors of immunity (IEI) compromising the cellular response to type I IFNs (IFNAR1, IFNAR2, STAT1, STAT2, IRF9 genes) (Bastard et al., 2020a; Duncan et al., 2015; Dupuis et al., 2003; Hambleton et al., 2013; Hernandez et al., 2019; Hernandez et al., 2018). We also considered possible defects of JAK1 and TYK2, although both encode proteins that are much more pleiotropic than the components of the ISGF3 complex (Eletto et al., 2016; Kreins et al., 2015). We tested this hypothesis by searching for homozygous single-nucleotide variants (SNVs) or large deletions (copy number variants, CNVs). We filtered out common variants (minor allele frequency [MAF] >0.01) and variants predicted to be benign (combined annotation-dependent depletion [CADD] score below the mutation significance cutoff [MSC] within the 99% confidence interval) (Itan et al., 2016; Kircher et al., 2014) (Fig.14B). We also analyzed all genes underlying known inborn errors of immunity (IEI) (Bousfiha et al., 2020; Notarangelo et al., 2020; Tangye et al., 2020). We found that one patient (P1) carried a homozygous essential splicing variant of IFNAR2 (c.840+1G>T). P1 is a 35-year-old woman from Brazil, who suffered from YFV-AVD at the age of 13-years-old. She had no previous history of severe viral infections. Three days after vaccination with YFV-17D, P1 presented with fever and digestive symptoms. She was admitted to the hospital four days later for epistaxis, hepatitis, and hypotension. She recovered with supportive care. Her sister died from YEL-AVD at the age of 19 years, but no samples were available for this study (Fig.11A). There were no rare variants in the other six candidate genes or in known IEI genes. The patient’s homozygosity rate was 0.88% and there were only seven other homozygous rare non-synonymous variants in her exome, none of which was connected to anti- viral immunity (Fig.14C). The IFNAR2 variant was confirmed by Sanger sequencing and was found to be present in the heterozygous state in both of P1’s parents and in several other members of her family (Fig.11B). This variant was present in the gnomADv3.1 public database but was extremely rare (MAF= 1.3x10-5) and found only in the heterozygous state (Fig.14D). Given the crucial role of human IFNAR2 in the type I IFN pathway (Duncan et al., 2015) and the previously described IFNAR1-deficient patient with YFV-AVD (Hernandez et al., 2019), we hypothesized that the homozygous essential splice site variant of IFNAR2 might underlie the pathogenesis of the adverse reaction to YFV 17D in P1. TABLE 8: Clinical and epidemiological characteristics of the eight patients included in the study with adverse events following YFV-17D vaccination. IFN: interferon, YFV: yellow-fever virus, M: male, F: female; YEL-AVD: yellow fever vaccine-associated viscerotropic disease; YEL-AND: yellow fever vaccineassociated neurological disease. NT: non tested, yo: year old, CSF: cerebrospinal fluid § Coding regions of IFNAR1, IFNAR2, TYK2, JAK1, STAT1, STAT2, and IRF9
[000234] Autosomal recessive complete IFNAR2 deficiency [000235] The IFNAR2 variant was predicted in silico to alter splicing and to lead to the loss of exon 8 (Fig. 14E). We thus performed exon trapping on the patient’s genomic DNA (gDNA) to test the impact of the variant. As predicted, a complete loss of exon 8 was observed in analyses of messenger RNA (mRNA) from the patient but not in analyses of mRNA from a healthy control (Fig. 11C, D and 4F). We also performed mutagenesis on gDNA to restore the patient’s variant to the WT form, which restored normal splicing (Fig. 14G). Human IFNAR2 is a ubiquitously expressed transmembrane protein that constitutively binds JAK1 (Russell-Harde et al., 2000; Wilmes et al., 2015). The loss of exon 8 is predicted to lead to a frameshift and a premature stop codon (p.Ser238Phefs*3) (Fig.11E, F). We first studied the expression of the mutant (MT) IFNAR2 by plasmid-mediated overexpression in HEK293T cells. Following the transient transfection of cells with plasmids containing the WT or MT IFNAR2 cDNA (isoform c), similar levels of IFNAR2 mRNA were detected for the WT, MT and the previously published variant (p.E104fs110*) when using a probe spanning exons 1-2 (Fig. 11G). Western-blot (WB) analysis of these cell extracts with a C-terminal (C-ter) tag showed the protein to be absent, consistent with the loss of exon 8 leading to a frameshift without translation reinitiation (Fig. 11H). We then performed flow cytometry to analyze the cell surface expression of WT and MT IFNAR2 in transfected HEK293T cells. The MT protein was not detected on the cell surface (Fig.11I). Finally, we used the WT or MT IFNAR2 cDNA to transfect IFNAR2 knock-out (KO) HEK293T cells, which we created by CRISPR/Cas9-mediated gene editing. Upon stimulation with IFN-α2 and transfection with a reporter gene, the cells expressing WT IFNAR2 displayed luciferase activity, unlike those expressing MT IFNAR2 (Fig. 11J). We did not test the response of the patient’s cells to type I IFNs, but previous reports have indicated that the fibroblasts from patients with IFNAR2 deficiency are unresponsive to IFN-α2 and IFN-β (Bastard et al., 2020a; Duncan et al., 2015). P1 had AR complete IFNAR2 deficiency. Together with our previous report of a patient with AR IFNAR1 deficiency underlying YEL-AVD (Hernandez et al., 2019), these findings show that two of the eight patients in our cohort with severe adverse reactions to YFV-17D vaccination had an AR complete deficiency of one of the chains of the type I IFN receptor. They were surprisingly healthy prior to vaccination with YFV 17D at 12 and 13 years of age. [000236] Auto-Abs against IFN-α2 and IFN-ω in three unrelated patients [000237] No non-synonymous variants of IFNAR1, IFNAR2, or of the five genes controlling the type I IFN response pathway (JAK1, TYK2, STAT1, STAT2, and IRF9) (Bastard et al., 2020a; Dupuis et al., 2003; Eletto et al., 2016; Gothe et al., 2020; Hambleton et al., 2013; Hernandez et al., 2018; Kreins et al., 2015; Watford and O'Shea, 2006) were found in the other six patients. We then hypothesized that these patients might have an autoimmune phenocopy of inborn errors of type I IFN immunity, as recently shown in patients with life-threatening COVID-19 pneumonia (Bastard et al., 2020b; Ku et al., 2020; Zhang et al., 2020a; Zhang et al., 2020b). We therefore searched for IgG auto-Abs against IFN- α2 and IFN-ω by ELISA and with a Luminex assay. We found high titers in three (P2, P3, and P4) of the six patients tested (Fig. 12A and 15A). P2 is a 62-year-old man originating from and living in Germany, with no prior history of severe viral infections. At the age of 57 years, five days after YFV- 17D vaccination, before traveling to Zanzibar, he developed neurological symptoms consistent with YEL-AND, and his liver enzyme levels increased (Slesak et al., 2017). He was also hospitalized two years later, at the age of 59 years, for influenza B infection with bilateral interstitial pneumonia. The blood sample positive for auto-Abs against type I IFNs was taken six months after YFV-17D disease. Patient 3 is a 50-year-old Brazilian woman with no history of severe viral infections. She presented with YFV-AVD following YFV-17D vaccination at the age of 47 years. Three days after vaccination, she reported fever, headache, and myalgia, which rapidly worsened. She was subsequently admitted to hospital with shock, acute renal and hepatic insufficiency, and thrombopenia. She was hospitalized in the intensive care unit (ICU) for two months. Of note, she reported a history of three months of polyarthralgia before hospitalization, and a history of miscarriage and preterm birth for hertwo pregnancies. She was diagnosed with systemic lupus erythematosus (SLE) during her hospital stay. Not coincidentally, women with SLE have been reported to be both at risk of adverse reactions to YFV-17D vaccination (Seligman, 2014) and to be prone to producing auto-Abs against type I IFN (Gupta et al., 2016; Howe and Leung, 2019; Mathian et al., 2019; Panem et al., 1982). The blood sample positive for auto-Abs against type I IFNs was taken when the patient was 49 years old, two years after YFV-17D disease. P4 is an 80-year-old man originating from and living in the United States, with no prior history of severe viral infections. At the age of 64, two days after YFV-17D vaccination, he developed fever and digestive symptoms. His condition quickly deteriorated, and he was hospitalized two days later for hypotension, skin rash, high fever, renal insufficiency, cytolysis and thrombopenia (Pulendran et al., 2008). The blood sample positive for auto-Abs was taken 16 days after the onset of YFV-17D disease. Interestingly, all patients tested (including P2 and P3) had been exposed to several common viruses (Fig.15B) and P2 and P3 were positive for anti-nuclear auto-Abs (Table 9). Three of the eight patients with adverse reactions to YFV vaccination studied therefore had high titers of auto-Abs to type I IFNs. [000238] Table 9 contains data on autoantibodies against other targets, used in clinical practice in two patients positive for auto-Abs against type I IFNs (P2 and P3). Table 9: Autoantibodies against other targets, used in clinical practice in two patients positive for anuto-Abs to type I IFNs (P2 and P3) Anti-Sm: anti-Smith; anti-RNP: anti-ribonucleoprotein; Anti-Scl70: anti-topoisomerase I. Anti-JO1: anti-Histidyl-tRNA synthetase; Anti-DFS: anti-dense fine speckled; Anti-PM/Scl: anti-poly- myositis/scleroderma; Anti-PCNA: anti-proliferating cell nuclear antigen
(a) Threshold: 1/80; (b) Threshold: 20UI/ml; (c) Threshold: 1 (Optical density) [000239] The auto-Abs are neutralizing and recognize most of the 17 subtypes of type I IFN [000240] We then investigated whether these auto-Abs were neutralizing in vitro (i.e., if they could block the activity of recombinant type I IFNs in an experimental assay). We incubated PBMCs derived from a healthy control individual with 10% plasma from a healthy control or from the three patients, and stimulated the cells with IFN-α2 or IFN-ω. We found that the presence of plasma from the three patients completely abolished type I IFN signaling, as shown by analyses of pSTAT1 induction following treatment with IFN-α2 or IFN-ω (Fig. 12B and 15C). For P2 and P3, we also analyzed the induction of interferon-stimulated genes (ISG) after 2 h of stimulation with IFN-α2 or IFN-ω, using type II IFN (IFN-γ) as a control. The incubation of cells with plasma from either patient led to the abolition of ISG induction in response to either of the individual type I IFNs tested, but not in response to type II IFN. By contrast, induction in response to both type I and II IFNs remained normal in the presence of plasma from healthy control individuals (Fig. 12C). We tested the three patients for auto- Abs against the other 15 individual type I IFNs, by performing ELISA with recombinant proteins for the 17 individual type I IFNs. We found that P2, P3, and P4 had auto-Abs recognizing at least 14 subtypes (Fig. 12D), a finding similar to that for most of the patients with life threatening COVID-19 carrying auto-Abs against type I IFNs and autoimmune polyendocrine syndrome type I (APS-1) (see Example 2; Bastard et al., 2020b), although two patients (P2 and P3) also had neutralizing auto-Abs against IFN-β (Fig. 15C). The plasma samples from P2, P3 and P4 did not recognize IFN-κ or IFN-ε, at least in the experimental conditions tested. These two type I IFNs were not, however, sufficient to protect the patients from YFV-17D infection. Overall, we found that three patients presenting severe adverse reactions to YFV-17D vaccination had high titers of neutralizing auto-Abs against type I IFNs and that these auto-Abs recognized most of the 17 individual type I IFN subtypes. [000241] The auto-Abs neutralize IFN-α2-mediated protection against YFV 17D in vitro [000242] Finally, we investigated whether these auto-Abs blocking type I IFN in biochemical assays were also able to block type I IFN-mediated protection against YFV-17D infection in cells in vitro. Plasma from P2 and P3, who had auto-Abs against type I IFNs, clearly blocked the ability of IFN-α2 to protect Huh-7.5 cells from infection with YFV-17D (Fig. 13). By contrast, plasma from two healthy donors without auto-Abs or from two other patients with severe YFV infection but without auto-Abs did not block the protective effect of IFN-α2 (Fig. 13). Furthermore, plasma from eight previously reported patients with life-threatening COVID-19 and auto-Abs against IFN-α2 also blocked the protective effect of IFN-α2 against YFV17D (Fig.16), in addition to that against SARS-CoV-2 (Bastard et al., 2020b). Interestingly, the three patients reported here belong to two groups known to be at higher risk of displaying auto-Abs against type I IFNs: P2 and P4 are men over the age of 55 years (Bastard et al., 2020b; Seligman, 2014), and P3 is a 47 year old woman with SLE (Panem et al., 1982). Finally, we found that the prevalence of auto-Abs against type I IFNs in our cohort of patients with severe adverse events following YFV-17D vaccination and without known IEI was significantly higher than the estimated prevalence in the general population (Bastard et al., 2020b) (37.5% vs. 0.3%; Fisher’s exact test, P=6.2 x 10-6). It is also probably not coincidental that the two patients with inherited IFNAR1 and IFNAR2 deficiency were younger, at 14 and 13 years, and did not display auto-Abs against type I IFNs. This suggests that the two mechanisms disrupting type I IFN immunity and resulting in YFV-17D disease are related but independent. This finding is reminiscent of our recent reports of inborn errors of type I IFNs and auto-Abs against type I IFNs in non-overlapping groups of patients with life-threatening COVID-19 pneumonia (Bastard et al., 2020b; Zhang et al., 2020a; Zhang et al., 2020b). Overall, these findings strongly suggest that the three patients reported here suffered adverse reactions to YFV-17D because of pre-existing neutralizing auto-Abs against type I IFNs, which targeted most of the individual subtypes of type I IFN, blocking their protective effect against YFV-17D. [000243] DISCUSSION [000244] Impaired type I IFN immunity underlies yellow fever vaccine adverse events [000245] Two members of our cohort of eight patients with YFV-AVD or YFV-AND have AR IFNAR1 or IFNAR2 deficiency, and another three have neutralizing auto-Abs against type I IFNs. These results suggest that at least half the patients with severe adverse reactions to YFV-17D have inadequate type I IFN immunity. This may be the case for the 78 reported patients with YFV-AVD or YEL-AND, including our five patients (de Menezes Martins et al., 2014; Hernandez et al., 2019; Lindsey et al., 2016; Seligman, 2014; Seligman and Casanova, 2016; Slesak et al., 2017). These findings unambiguously place human type I IFNs at the core of protective immunity to YFV-17D. They have important clinical implications. First, patients with known inborn errors of, or auto-Abs against type I IFN should not be vaccinated with YFV-17D. Second, patients with adverse reactions to YFV-17D should be tested for inborn errors of type I IFN immunity and for the presence of auto-Abs against type I IFNs. Third, both types of patients may benefit from treatment with recombinant IFN-β in the course of YFV-17D disease, provided that they do not have auto-Abs against IFN-β (such antibodies were present in two of the three patients reported here but were absent from 99 of 101 patients from a previous report on severe COVID-19) (Bastard et al., 2020b). Fourth, patients with autoimmune manifestations, including SLE and thymoma in particular, should be screened for auto- Abs against type I IFNs before vaccination with YFV-17D, as, perhaps, should men over the age of 55 years. Younger patients may nevertheless also be at risk of adverse reactions to YFV-17D vaccination due to pre-existing auto-Abs against type I IFNs, as suggested by the case of an eight- year-old patient homozygous for a hypomorphic RAG1 mutation who had defective adaptive immunity, multiple severe infectious diseases, auto-Abs against type I IFNs, and encephalitis after YFV-17D vaccination (Walter et al., 2015) (Table 2: JCI80477sdt1). Moreover, the youngest patient with life threatening COVID-19 pneumonia and auto-Abs against type I IFNs reported to date was 25 years old (Bastard et al., 2020b), younger than the two patients with inherited IFNAR1 deficiency and critical COVID-19, aged 26 and 38 years (Zhang et al., 2020b). Although difficult in some regions, screening for both inborn errors of type I IFN immunity and auto-Abs against type I IFNs could, therefore, be considered for patients of all ages before vaccination with YFV-17D. Moreover, patients with auto-Abs against type I IFNs are likely to be at risk of developing an adverse event if vaccinated with the newly developed vaccine against SARS-CoV-2 that uses the YFV live attenuated vaccine as carrier (Sanchez-Felipe et al., 2020). Sanchez-Felipe L et al 2020 describe the development of a candidate vaccine (YF-S0) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that uses live-attenuated yellow fever 17D (YF17D) vaccine as a vector to express a noncleavable prefusion form of the SARS-CoV-2 spike antigen. [000246] Pre-existing auto-Abs against type I IFNs can underlie different viral diseases [000247] Our findings also have implications for the pathogenic role of auto-Abs against type I IFNs. Our previous study on life-threatening COVID-19 suggested that the auto-Abs against type I IFNs were causal of disease, rather than triggered by SARS-CoV-2 (see Example 2; Bastard et al., 2020b). Indeed, they were already present in blood samples from two of the 101 patients collected before the onset of COVID-19. Moreover, they were detected early in the course of SARS-CoV-2 infection in the other patients, making it highly unlikely that these auto-Abs of the IgG type with a high affinity for type I IFNs were triggered by infection. Finally, these antibodies are common in patients with two genetic diseases underlying severe COVID-19: APS-1 (Meager et al., 2006) and incontinentia pigmenti (Bastard et al., 2020b). The three patients with auto-Abs reported here add weight to this conclusion. Indeed, they developed YFV-17D disease within five days of vaccination. We did not test their blood for the presence of auto-Abs before vaccination, or even during YFV-17D disease, but the presence of these antibodies six months (P2), two years (P3) and 16 days (P4) later provides compelling evidence for an association with disease (Fisher’s exact test, P=6.2 x 10-6), even without taking into account a previously reported patient whose YFV-17D disease may have been triggered by her many profound deficits of adaptive immunity other than auto-Abs against type I IFNs (Walter et al., 2015). Assuming that the three patients had auto-Abs at the time of hospital admission for YFV-17D disease, it would have been almost impossible for them to mount a neutralizing IgG response against a self-antigen so quickly. Furthermore, one of the patients (P3) was diagnosed with SLE during hospitalization and her SLE symptoms began three months before admission, consistent with the occurrence of such auto-Abs, as reported in about 5-10% of patients with SLE (Gupta et al., 2016; Mathian et al., 2019; Panem et al., 1982). The sex of the patient and age at the time of vaccination are similar to those in the other three known cases of SLE with YFV-17D disease (Seligman, 2014). Auto-Abs against type I IFNs can therefore underlie severe COVID-19 or YFV 17D disease. Intriguingly, children with APS-I and auto- Abs to type I IFNs have been vaccinated with MMR without any reported adverse reaction, despite the occurrence of MMR disease in several patients with inherited IFNAR1 or IFNAR2 deficiency (Bastard et al., 2020a; Duncan et al., 2015; Gothe et al., 2020; Hernandez et al., 2019). This may be due to the activity of IFN-β, IFN-κ, or IFN-ε in APS-I patients, whose auto-Abs typically neutralize the 13 individual IFN-α2 and IFN-ω (Bastard et al., 2020b; Meager et al., 2006). It is nevertheless tempting to speculate that other severe, unexplained viral illnesses, such as shingles and influenza, particularly, but not exclusively, in patients with autoimmune conditions or in elderly men, may be caused by auto-Abs against type I IFNs. [000248] METHODS [000249] Patients and study approval [000250] All cases of YEL-AVD and YEL-AND satisfied the Brighton Collaboration criteria (Gershman et al., 2012). The Brazilian patients were recruited by a collaboration with BioManguinhos/Fiocruz, as indicated in the acknowledgments. Additional patients were found from listings on ProMED (promedmail.org) and PubMed (pubmed.ncbi.nlm.nih.gov/). Written informed consent was obtained from patients in the country in which they were followed, in accordance with local regulations, and the study was approved by the institutional review boards of The Rockefeller University and Institut National de la Santé et de la Recherche Médicale (INSERM). Experiments were conducted in the United States of America and France, in accordance with local regulations and with the approval of the institutional review boards of The Rockefeller University and INSERM, respectively. [000251] Whole-exome sequencing [000252] Exome capture was performed with the SureSelect Human All Exon 50 Mb kit (Agilent Technologies). Paired-end sequencing was performed on a HiSeq 2000 (Illumina) generating 100-base reads. We aligned the sequences with the GRCh37 reference build of the human genome with the BWA (Li and Durbin, 2009). Downstream processing and variant calling were performed with the Genome Analysis Toolkit, SAMtools, and Picard (Li et al., 2009; McKenna et al., 2010). Substitution and InDel calls were made with GATK Unified Genotyper. All variants were annotated with an annotation software system developed inhouse (Adzhubei et al., 2010; Kircher et al., 2014; Ng and Henikoff, 2001). The patients reported here did not consent to the deposition of their genomic data. [000253] Statistical analysis [000254] Comparison of proportions were performed using a Fisher exact test, as implemented in R (cran.r-project.org/). PCA was performed with Plink v1.9 software on wholeexome and whole-genome sequencing data with the 1000 Genomes (1kG) Project phase 3 public data- base as a reference. [000255] Cells [000256] Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque density gradient (GE Life Science, USA) centrifugation. Primary fibroblasts, SV40-immortalized dermal fibroblasts, HEK293T kidney epithelial cells (H. sapiens), VeroE6 kidney epithelial cells (Chlorocebus sabaeus) and Huh-7.5 hepatoma cells (H. sapiens) were maintained in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) at 37°C under an atmosphere containing 5% CO2. EBV-B cells were maintained in Roswell Park Memorial Institute medium (RPMI, Thermo Fisher Scientific) supplemented with 10% FBS. All cells tested negative for mycoplasma contamination. [000257] Exon trapping [000258] DNA segments encompassing the IFNAR2 exon 8 region were amplified from genomic DNA and inserted into the pSPL3 vector, between the EcoRI and BamHI sites. Wildtype, mutant (IFNAR2 c.840+1G>T) or mutagenesis rescue plasmids were used to transfect COS-7 cells. After 24 h, total RNA was extracted and reverse-transcribed. The IFNAR2 splicing products were amplified with flanking HIV-TAT sequences from the pSPL3 vector and ligated into the pCR4-TOPO vector (Invitrogen). Stellar cells (Takara) were transformed with the resulting plasmids. Colony PCR and sequencing with primers binding to the flanking HIV-TAT sequences of pSPL3 were performed to determine the splicing products produced by the WT and mutated alleles. [000259] Plasmids [000260] A plasmid containing the cDNA of IFNAR2 was used (generously provided by Sandra Pellegrini) and site-directed mutagenesis was performed to obtain the indicated mutant constructs. [000261] IFNAR2 overexpression by plasmid transfection and the generation of stably reconstituted cell lines [000262] The IFNAR2 plasmid was used to transfect HEK293T cells by incubation for 48 h, in the presence of X-tremeGene 9 transfection reagent (Sigma Aldrich). We used 1 µg of plasmid to transfect 0.5 x 106 cells. [000263] Western blotting [000264] HEK293T cells were transfected for 36 h with WT or MT IFNAR2. Cells were lysed in NP40 lysis buffer (280 mM NaCl, 50 mM Tris pH 8, 0.2 mM EDTA, 2 mM EGTA, 10% glycerol, 0.5% NP40) supplemented with 1 mM DTT, PhosSTOP (Roche, Mannheim, Germany) and complete protease inhibitor cocktail (Roche, Mannheim, Germany). The protein lysate was subjected to SDS- PAGE and the bands obtained were transferred to a nitrocellulose membrane. For detection of the protein overproduced following transfection, we used an HRP-conjugated anti-V5 antibody purchased from commercial suppliers. An anti-GAPDH (Santa Cruz) antibody was used as a loading control. The membrane was incubated overnight at 4°C with the primary antibodies. SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific) was used to visualize HRP activity after incubation with secondary antibody, and this signal was detected with an Amersham Imager 600 (GE Life Sciences). [000265] Flow cytometry [000266] The cell surface expression of IFNAR2 was assessed with a PE-conjugated mouse anti- IFNAR2 (#21385-3PBL Assay Science, Piscataway, NJ, USA) antibody. Cells were stained and then washed twice with PBS and analyzed by flow cytometry. Data were acquired on a Gallios flow cytometer. [000267] Generation of IFNAR2-deficient HEK293T cells [000268] IFNAR2-deficient HEK293T cells were generated with the CRISPR/Cas9 system. Guide RNAs were designed with the Benchling design tool, and inserted into lentiCRISPR v2, which was a gift from Feng Zhang (Addgene plasmid # 52961). The three guide RNAs were designed to bind and cut at different places in the IFNAR2 gene, one in exon 3 (FOR: CACCGCATATGAAATACCAAACACG (SEQ ID NO:1); REV: AAACCGTGTTTGGTATTTCATATGC (SEQ ID NO:2)), one in exon 4 (FOR: CACCGCATTGCTGTATACAATCATG (SEQ ID NO:3); REV: AAACCATGATTGTATACAGCAATGC (SEQ ID NO:4)), one in exon 5 (FOR: CACCGTGAGTGGAGAAGCACACACG (SEQ ID NO:5); REV: AAACCGTGTGTGCTTCTCCACTCAC (SEQ ID NO:6)). Using X-tremeGENE 9 DNA Transfection Reagent (Roche), we transiently transfected WT HEK293T cells with the resulting plasmids and cultured them for seven days before sorting IFNAR2-deficient cells by flow cytometry after staining with an antibody against IFNAR2 (Miltenyi Biotec, REA124). The three resulting cell lines were subsequently tested to check for a lack of IFNAR2 expression at the cell surface and were used in the luciferase assays. [000269] Luciferase reporter assays [000270] IFNAR2 -/- HEK293T cells generated by CRISPR/Cas9 system were transfected with the indicated expression plasmids, firefly luciferase plasmids under the control of WT or Mut IFNAR2, or human ISRE promoters in the pGL4.45 backbone, and a constitutively expressing Renilla luciferase plasmid for normalization (pRL-SV40). Cells were transfected in the presence of the X-tremeGene 9 transfection reagent (Sigma Aldrich) for 36 hours. Luciferase levels were measured with the Dual-Glo reagent, according to the manufacturer’s protocol (Promega). Firefly luciferase values were normalized against Renilla luciferase values, and fold induction is shown relative to controls transfected with empty plasmids. [000271] Quantitative RT-PCR [000272] RNA was isolated from PBMCs, or from plasmid-transfected or untransfected HEK293T cells, with a kit, according to the manufacturer’s protocol (Zymo Research, Irvine, CA), and treated with DNase (Zymo Research, Irvine, CA). Reverse transcription was performed with random hexamers and the Superscript III reverse-strand synthesis kit, according to the manufacturer’s instructions (Thermo Fisher Scientific, Springfield Township, NJ). Quantitative real-time PCR (qPCR) was performed with Applied Biosystems Taqman assays for IFNAR2, and the β glucuronidase (GUS) housekeeping gene for normalization. Results are expressed according to the ∆∆Ct method, as described by the kit manufacturer. [000273] Detection of anti-cytokine autoantibodies in a multiplex particle-based assay [000274] Serum/plasma samples were screened for autoantibodies against IFN-α2 and IFN-ω targets in a multiplex particle-based assay, in which magnetic beads with differential fluorescence were covalently coupled to recombinant human proteins (2.5 µg/reaction). Beads were combined and incubated with 1:100 diluted serum/plasma samples for 30 minutes. Each sample was tested once. The beads were then washed and incubated with PE-labeled goat anti-human IgG (1 µg/mL) for 30 minutes. They were washed again and used in a multiplex assay run on a BioPlex X200 instrument. Patients with a fluorescence intensity (FI) > 1500 for IFN-α2 or IFN-β, or > 1000 for IFN-ω were tested for blocking activity. [000275] Enzyme-linked immunosorbent assays (ELISA) for anti-cytokine autoantibodies [000276] ELISA was performed as previously described (Bastard et al., 2020b). In brief, 96- well ELISA plates (MaxiSorp; Thermo Fisher Scientific) were coated by incubation overnight at 4°C with 2 µg/mL rhIFN-α, and rhIFN-ω (R&D Systems). Plates were then washed (PBS/0.005% Tween), blocked by incubation with 5% nonfat milk powder in the same buffer, washed, and incubated with 1:50 dilutions of plasma from the patients or controls for 2 h at room temperature (or with specific mAbs as positive controls). Each sample was tested once. Plates were thoroughly washed. Horseradish peroxidase (HRP)–conjugated Fc-specific IgG fractions from polyclonal goat antiserum against human IgG or IgA (Nordic Immunological Laboratories) were added to a final concentration of 2 µg/mL. Plates were incubated for 1h at room temperature and washed. Substrate was added and the optical density (O.D.) was measured. A similar protocol was used to test for antibodies against 12 subtypes of IFN-α, except that the plates were coated with cytokines from PBL Assay Science (catalog #11002-1). [000277] Clinical screening for other autoantibodies [000278] The assay for ANA was performed using an indirect immunofluorescence on Hep-2 cells (Novalite ref# 704320, Inova Diagnostics San Diego, CA, distributed by Werfen, Le Pré-Saint-Gervais France). Dilutions of 1:80 were performed with phosphate-buffered saline for screening test. In brief, 30 µL of each diluted serum was incubated on one well with fixed Hep-2 cells. After the cells were incubated and washed, they were incubated with DAPI-IgG conjugated antibody. The results were analyzed using a Nikon Eclipse fluorescence microscope with a magnification ×400. Extractable Nuclear Antigen (ENA) Antibodies were detected by ELISA (RNP, Sm, SSA/Ro, SSB/la, Scl70 and JO-1) using the ANA 8 screen Kit (Euroimmun Bussy-Saint-Martin, France). Immunoblotting was performed for a larger ENA panel detection (RNP, Sm, SSA/Ro, SSA/Ro 52 kD, SSB/la, Scl70, JO-1, Centrome B, PCNA, Nucleosome, Histones, Ribosomes P, Type 2 mitochondria, DFS 70) using the ANA Profil 3 Dot (Euroimmun Bussy-Saint-Martin, France). Anti-native DNA detection was performed by indirect immunofluorescence on the flagellate organism Crithidia luciliae using the KIT Theradiag (Croissy-Beaubourg, France) and ELISA for antibodies quantification using the anti-dsDNA IgG KIT on ETI-MAX 3000 Equipment from Diasorin (Antony, France). [000279] Functional evaluation of anti-cytokine autoantibodies [000280] The blocking activity of anti-IFN-α and anti-IFN-ω autoantibodies was determined by assessing STAT1 phosphorylation in healthy control cells following stimulation with the appropriate cytokines in the presence of 10% serum/plasma from a healthy control or a patient. Surface-stained healthy control PBMCs (350,000/reaction) were cultured in serumfree RPMI medium supplemented with 10% healthy control or patient serum/plasma and were either left unstimulated or were stimulated with IFN-α or IFN-ω (10 ng/mL) for 15 minutes at 37°C. Each sample was tested once. Cells were fixed, permeabilized, and stained for intranuclear phospho-STAT1 (Y701). Cells were acquired on a BD LSRFortessa cytometer with gating on CD14+ monocytes and analyzed with FlowJo software. [000281] YFV-17D experiment [000282] The generation of virus stocks for the YFV 17D reporter virus expressing the Venus fluorescent protein (YFV-Venus) (derived from YF17D-5′C25Venus2AUbi) has been described elsewhere (Yi et al., 2011). Virus stock titers were determined by standard plaque assays on Huh-7.5 cells. YFV-Venus experiments were performed as follows: Huh-7.5 cells were used to seed 96-well plates at a density of 5 x 103 cells/well, with triplicate wells for each sample. 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Inborn errors of type I IFN immunity in patients with life-threatening COVID-19. Science 370(6515):eabd4570. doi: 10.1126/science.abd4570. EXAMPLE 4 Autoantibodies Neutralizing type I IFNs Are Common in the Elderly and Account for At Least 20% of COVID-19 Deaths [000284] Circulating autoantibodies (auto-Abs) neutralizing very high concentrations (10 ng/mL, in plasma 1/10) of IFN-α and/or -ω have been found in about 10% of patients with critical COVID-19 pneumonia, but not in subjects with asymptomatic infections. We detected auto-Abs neutralizing 100- fold lower, more physiological, concentrations of IFN-α and/or -ω (100 pg/mL, in plasma 1/10) in at least 15% of 2,248 patients with critical COVID-19 pneumonia, including 22% of 290 patients over the age of 80 years. These antibodies were also detected in 21% of 865 individuals aged 20-99 (mean: 70 years) who died of COVID-19 pneumonia. We also show, in a sample of 33,352 uninfected subjects, that the prevalence of auto-Abs neutralizing high concentrations of type I IFNs sharply increases with age, with such antibodies present in 0.16% of individuals between 18 and 69 years, 1% of individuals between 70 and 79 years, and 2.9% of individuals after 80 years of age. Moreover, in a subsample of 816 individuals, the proportion of subjects carrying auto-Abs neutralizing 100-fold lower concentrations was even greater, at 0.53% of individuals between 60 and 70 years, 1.3% between 70 and 80 years, and 3.7% over 80 years of age. Auto-Abs neutralizing type I IFNs predate SARS-CoV-2 infection, sharply increase in prevalence after the age of 70 years, and account for more than 20% of cases of both critical COVID-19 in subjects over the age of 80 years and fatal COVID-19 at all ages. [000285] Introduction [000286] Since the start of the COVID-19 pandemic, in December 2019, more than 150 million people have been infected with SARS-CoV-2, resulting in at least three million deaths worldwide, and more likely six to eight million. Inter-individual clinical variability in the course of acute infection is vast, extending from silent or mild infection in about 90% of subjects to pneumonia and respiratory failure, in less than 10% and 2% of cases, respectively, who require hospitalization. Age is the major epidemiological risk factor for hospitalization or death from pneumonia, the risk doubling with every five years of age (1, 2). The frequencies of critical disease and death from COVID-19 are higher in men than in women (3). With the COVID Human Genetic Effort (4), we previously reported that inborn errors of TLR3- and IRF7-dependent type I IFN induction and amplification can underlie life- threatening COVID-19 pneumonia in a small subset of patients (5). Autosomal dominant disorders were found in 19 patients, but our cohort also included four previously healthy unrelated adults aged 25 to 50 years with autosomal recessive, complete IRF7 (N = 2) or IFNAR1 (N = 2) deficiency. These findings indicate that type I IFN immunity is essential for protective immunity to respiratory infection with SARS-CoV-2 but can be surprisingly redundant otherwise. We also reported that an autoimmune phenocopy of inborn errors of type I IFN immunity can underlie critical COVID-19 pneumonia (6). Indeed, auto-Abs neutralizing 10 ng/mL IFN-α2 and/or -ω were found in the blood of at least 10% of an international cohort of patients with life-threatening COVID-19 pneumonia, but in none of the individuals with asymptomatic or paucisymptomatic infection tested (6). These auto-Abs were detected in serum or plasma diluted 1/10. The auto-Abs in the patients’ undiluted blood are probably, therefore, able to neutralize as much as 100 ng/mL IFN-α2 and/or -ω. These auto-Abs were mostly found in men (95%) and in the elderly (half the patients with antibodies being over the age of 65 years) (6). These findings were later replicated in independent cohorts from Amsterdam, Lyon, Madrid, New Haven, and San Francisco (7-12). [000287] These IgG auto-Abs against type I IFNs were found in about 0.3% of a general population sample of 1,227 subjects collected before the pandemic and aged 20 to 65 years, suggesting that they predated SARS-CoV-2 infection and caused critical COVID-19 rather than being triggered by it (6). Moreover, the production of these antibodies can be genetically driven, and can begin during early childhood, as attested by their presence in almost all patients with autoimmune polyendocrine syndrome type-1 (APS-1) due to germline mutations of AIRE (13-15). These patients are, therefore, at very high risk of developing severe or critical COVID-19 pneumonia (16). These auto-Abs are also found in patients with combined immunodeficiency and hypomorphic mutations of RAG1 or RAG2 (17), in men with immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) and mutations of FOXP3 (18), and in women with incontinentia pigmenti and heterozygous null mutations of X-linked NEMO (6). They are also seen in patients treated with IFN-α or IFN-β (19, 20), in patients with systemic lupus erythematosus (21), thymoma (22), or with myasthenia gravis (23). Finally, they underlie a third of adverse reactions to the live attenuated YFV-17D vaccine, further suggesting that they were present in patients with critical COVID-19 before SARS-CoV-2 infection (24). Remarkably, the auto-Abs neutralized the protective effect of ~384 pg/mL IFN-α2 against SARS-CoV-2 or YFV-17D in vitro, even when diluted by >1/1,000, for all patients tested (6). As blood IFN-α concentrations during acute asymptomatic or paucisymptomatic SARS-CoV-2 infection typically range from 1 to 100 pg/mL (25, 26), and IFN-α levels in the respiratory tract may be even lower, we hypothesized that auto-Abs neutralizing concentrations of type I IFNs below 10 ng/mL (from plasma diluted 1/10) may underlie life-threatening COVID-19 pneumonia in more than 10% of cases. We also hypothesized that the prevalence of auto-Abs against type I IFNs in the general, uninfected, population may increase with age and that these antibodies may be more common in men than in women. [000288] High and intermediate levels of IgG auto-Abs against type I IFNs in ~20% of critical COVID-19 patients [000289] We searched for auto-Abs against IFN-α2 and -ω in a cohort of patients hospitalized with life-threatening COVID-19 pneumonia (hereafter referred to as “patients”), including 566 of the initial 987 patients with critical COVID-19 pneumonia for whom residual samples were available (6), and 588 individuals with asymptomatic or paucisymptomatic SARS-CoV-2 infection (the “controls”), including 136 samples from the initial control cohort of 663 individuals (6). We did not test patients with moderate or severe pneumonia (5, 6). We first set up novel, sensitive, and robust assays for the detection of circulating IgG auto-Abs. We used Gyros technology (27), a high-throughput automated ELISA (enzyme-linked immunosorbent assay) yielding reproducible results (Fig. 22A), and screened all patients and controls from our COVID-19 cohort (Fig. 17A). We confirmed that the Gyros technique was as sensitive as the techniques previously used (ELISA and Luminex), and that all patients with high levels of anti-IFN-α2 and/or anti-IFN-ω auto-Abs on ELISA, as reported in our previous studies (defined as an optical density > 0.5) had high levels of auto-Abs when assessed with Gyros (defined as levels >100) (Fig.22B). We found high levels of anti-IFN-α2 and/or anti-IFN-ω auto-Abs in 9% of the patients in our cohort of critical patients (203 of 2,248), whereas none of the 588 asymptomatic or paucisymptomatic controls had high titers of auto-Abs against IFN-α2 and only one had high titers of auto-Abs against IFN-ω (Fisher’s exact test, P < 10 -16 ) (Figure 17A). We also found that another 11.5% of patients with critical COVID-19 (259 of 2,248) had intermediate levels of anti-IFN-α2 and/or IFN- ω auto-Abs in Gyros assays (defined as levels >30 and <100, based on the distribution observed in healthy controls), whereas this was the case for only 3% (17 of 588) of the individuals in our control cohort (Fisher exact test, P = 9.10 -11 ). Importantly, none of the patients with high or intermediate auto- Abs carried inborn errors of TLR3- or TLR7-dependent type I IFN (5) (Asano et al, accompanying report submitted). Collectively, these findings replicate and extend our previous results and those of other groups (6-12), while also suggesting that intermediate levels of auto-Abs against type I IFNs may be neutralizing and underlie critical disease. [000290] Auto-Abs neutralizing 10 ng/mL IFN-α2 and/or -ω in 10% of the patients [000291] We investigated the ability of these auto-Abs to neutralize high concentrations of type I IFNs, as defined in our previous reports (10 ng/mL in medium containing 10% plasma or serum, the equivalent of 100 ng/mL in undiluted plasma). We tested not only the patients with high levels of auto- Abs, as in our previous study (6), but all the available patients (N = 2,036) and controls (N = 342) from our expanded cohort. We designed a high-throughput luciferase assay in which we transfected human embryonic kidney (HEK)-293T cells with a plasmid containing five interferon-sensitive response element (ISRE) repeats and a luciferase reporter. We stimulated these cells with an individual recombinant type I IFN (IFN-α2 or IFN-ω), in the presence of 10% plasma from patients or controls. We then measured firefly luciferase induction, normalized against Renilla luciferase activity (Fig.17B). We confirmed the robustness of this assay by comparing its results with our previous pSTAT1 flow cytometry data (6). Consistent results were obtained for all 50 patients tested with both techniques (Fig. 22C, D). We then tested all patients and controls. We found that 10% (204 of 2,036) of the patients tested had auto-Abs that neutralized 10 ng/mL IFN-α2 and/or IFN-ω in 10% plasma, whereas none of the 342 controls had auto-Abs neutralizing IFN-α2 and only two had auto-Abs neutralizing IFN-ω (Fisher’s exact test, P < 10 -12 ) (Fig.17C) (Table 10). Plasma samples with high auto-Ab levels (>100) against IFN-α2 according to the Gyros assay were all neutralizing (Fig. 22E). In the patients with neutralizing auto-Abs, they were able to neutralize both IFN-α2 and IFN-ω in 112 of the 204 patients (55%), IFN-α2 alone in 75 patients (37%), and IFN-ω alone in 17 patients (8%), after stimulation with 10 ng/mL IFN-α2 or IFN-ω. [000292] Auto-Abs neutralizing 100 pg/mL IFN-α2 and/or -ω in >15% of the patients [000293] As the amounts of circulating type I IFNs in infected individuals are 100 to 1,000 times lower than the amounts tested previously (25, 26), we investigated the neutralization of more physiological concentrations of type I IFNs, by performing assays with 100 pg/mL type I IFN. We observed a robust response in our luciferase system, in the presence of 10% control plasma (Fig.22F). The plasma or serum was diluted 1/10, so the concentration neutralized corresponds to 1 ng/mL IFN in circulating whole blood. With diluted plasma samples from a positive control, we gained at least 2 orders of magnitude of sensitivity in terms of neutralizing activity, providing proof-of-concept that these auto-Abs can neutralize lower, more physiological, amounts of type I IFNs (Fig.17D, 22G), 100 times lower than the concentrations previously tested (6). We then retested all the available samples from our extended cohort. Overall, 15.7% of all patients tested (N = 353 of 2,248) had circulating auto-Abs that neutralized 100 pg/mL IFN-α2 and/or IFN-ω in 10% plasma (Fig. 17E) (Table 10). These patients had a mean age of 63.5 years, and 73% were men. The auto-Abs of these patients neutralized both cytokines in 171 of cases (49%), only IFN-ω in 104 (29%) and only IFN-α2 in 78 (22%). All samples (N = 177) tested that had previously been found to neutralize 10 ng/mL IFN-α2 or IFN-ω also neutralized 100 pg/mL IFN, as expected (Fig.17F, G). Further dilution of a plasma sample from one patient neutralizing 100 pg/mL of type I IFNs led to a loss of neutralizing activity (Fig. 17D, 22G). None of the plasma samples from the 588 asymptomatic controls neutralized IFN-α2 and only five (0.85%) neutralized IFN- ω in these conditions (Fisher’s exact test, P < 10 -16 ) (Fig. 17E) (Table 10). These five controls with auto-Abs were all under 60 years old, one was a woman and four were men. Among the 129 samples tested that were neutralizing at 100 pg/mL but not at 10 ng/mL, 31 neutralized IFN-α2 at 100 pg/mL, 84 neutralized IFN-ω at 100 pg/mL, and 14 neutralized both (Fig.17F, G), further suggesting that auto- Abs neutralizing 100 pg/mL are causal for critical disease. None of the patients with these auto-Abs had inborn errors of TLR3- or TLR7-dependent type I IFN immunity (5) (Asano et al, accompanying report submitted). The serum/plasma samples being diluted 1/10 in these assays, these findings therefore suggest that more than 15% of patients with life-threatening COVID-19 have circulating auto- Abs neutralizing 1 ng/mL IFN-α2 and/or IFN-ω in vivo, a significantly greater proportion than the 10% of patients with auto-Abs neutralizing 100 ng/mL reported in previous studies (6-12). [000294] The auto-Abs neutralize low concentrations of IFN-α2 protective against SARS-CoV- 2 [000295] We previously reported that plasma diluted 1/100 from patients with auto-Abs against type I IFNs neutralized the ability of IFN-α2 (at a concentration of 20 pM, approximately 384 pg/mL) to block SARS-CoV-2 and YFV-17D replication in Huh-7.5 cells (see Example 2; 6, 24). Strikingly, this neutralization was seen in all patients tested, even for a 1,000-fold dilution, and, in most patients, it was more potent than the neutralizing effect of a commercially available neutralizing monoclonal Ab (mAb) against IFN-α2. These auto-Abs against type I IFNs were, therefore, able to neutralize IFN-α2 at concentrations well beyond physiological levels. We therefore hypothesized that patients with lower titers of auto-Abs against type I IFNs, which can neutralize 100 pg/mL but not 10 ng/mL in plasma diluted 1/10, would also neutralize the protective effect of IFN-α2 against SARS-CoV-2. We therefore performed our SARS-CoV-2 assay with 5 pM (~96 pg/mL) or 20 pM (~384 pg/mL) IFN-α2, on five samples from patients with life-threatening COVID-19 and two samples from uninfected elderly individuals with auto-Abs neutralizing 100 pg/mL but not 10 ng/mL IFN-α2. As controls, we tested a commercial mAb against IFN-α2, a sample from a patient with auto-Abs neutralizing 10 ng/mL IFN- α2, and samples from three patients with life-threatening COVID-19 and three healthy controls without detectable auto-Abs against type I IFNs. We found that the 1/100 dilutions of plasma from five of the seven patients or elderly individuals with auto-Abs neutralizing 100 pg/mL IFN were able to neutralize the protective effect of ~384 pg/mL IFN-α2 against SARS-CoV-2, while samples from the seven patients fully or partially neutralized ~96 pg/m IFN-α2 (Fig. 18A). No such neutralizing effect was observed for any of the auto-Ab-negative controls. Overall, our findings indicate that auto-Abs against type I IFNs capable of neutralizing 100 pg/mL IFN in 1% plasma can block the protective effect of ~96 pg/mL or ~384 pg/mL IFN-α2 against SARS-CoV-2. These findings raise the possibility that even 100- fold lower levels of auto-Abs against type I IFNs, capable of neutralizing lower, physiological concentrations of 10 pg/mL IFN-α2, may be present in an even larger proportion of patients. The testing of this hypothesis will require the development of new, more sensitive methods for screening for neutralization. [000296] Neutralization of type I IFNs in the absence of detectable auto-Abs against IFN-α2 or -ω [000297] The neutralization assays performed on all patients and controls revealed that some patients (15/186, 8%) with neutralizing activity against 10 ng/mL IFN-α2 and/or IFN-ω, as shown in luciferase assays, did not have high, or even intermediate levels of IgG auto-Abs in Gyros assays (Fig. 22E). Similar results were observed when using the Luminex assay for the titer and pSTAT1 induction (Fig. 22H). For these individuals, we assessed the prevalence of IgA and IgM auto-Abs against type I IFNs; we found that none of the patients tested (N = 12) had detectable titers of IgA or IgM auto-Abs. We then tested the alternative hypothesis that these auto-Abs were directed against the IFNAR1 or IFNAR2 chain of type I IFN receptors, assessing the ability of plasma samples from these patients to neutralize IFN-β. None of the samples from these patients neutralized IFN-β, suggesting that the auto-Abs in these patients were not directed against IFNAR1 or IFNAR2. An alternative, plausible hypothesis is that the epitope recognized by the auto-Ab might be concealed by the binding of the cytokine to the plate (ELISA), biotin (Gyros), or the bead (Luminex) in the assays used. The auto-Abs in these patients might also potentially recognize an epitope concealed by the coupling of the cytokines to biotin (for the Gyros and Luminex assays). This hypothesis was supported by an in silico analysis, as the Luminex beads bind to lysine residue, and could thus block the access to a common epitope (15). This observation has important clinical implications, suggesting that a lack of detection of auto-Abs against type I IFNs does not rule out the possibility of such antibodies being present and having neutralization capacity. [000298] The auto-Abs typically neutralize the 13 IFN-α forms and/or IFN-ω, and rarely IFN- β [000299] In six patients with auto-Abs neutralizing 100 pg/mL but not 10 ng/mL IFN-α2 and/or IFN- ω, we tested the reactivity of the antibodies against the 17 type I IFNs (the 13 IFN-α forms, IFN-ω, IFN-β, IFN-ε, and IFN-κ). Like patients with auto-Abs neutralizing 10 ng/mL type I IFNs (6), those capable of neutralizing only 100 pg/mL had detectable auto-Abs against most of the 13 IFN-α forms and/or IFN-ω, albeit at lower levels (Fig.18B). Of the six patients with auto-Abs against IFN-α and/or IFN-ω tested, only one also had auto-Abs against IFN-β and none had detectable auto-Abs against IFN- ε or IFN-κ. Given the potential therapeutic use of IFN-β (28, 29), we also tested a larger number of patients and controls, including patients without auto-Abs against IFN-α or IFN-ω, for auto-Abs against this cytokine, assessing levels and neutralizing activity of auto-Abs against 10 ng/mL IFN-β. Of 50 patients with auto-Abs neutralizing 10 ng/mL and/or 100 pg/mL IFN-α2 and/or IFN-ω tested, only one (2%) had auto-Abs capable of neutralizing 10 ng/mL IFN-β (Fig.18C). We then screened 642 patients without auto-Abs to IFN-α2 or IFN-ω neutralizing 100 pg/mL for neutralization activity against IFN- β. We found that only three patients (0.7%) had auto-Abs capable of neutralizing 10 ng/mL IFN-β, with no neutralizing activity in any of the asympto- or pauci-symptomatic infected controls tested (N = 109) (Fig. 18C). By ELISA, two of the 642 patients tested had high levels of auto-Abs, while none of the controls tested had (N = 109) (Fig. 23A). The third patient with auto-Abs neutralizing IFN-β had no detectable auto-Abs by ELISA. As these three patients with neutralizing auto-Abs against IFN-β did not have neutralizing auto-Abs against IFN-α2 or IFN-ω, this suggests that auto-Abs against IFN-β alone may also underlie life-threatening COVID-19 in rare cases. Overall, the patients with auto-Abs against IFN-α2 and/or IFN-ω capable of neutralizing 100 pg/mL IFN displayed patterns of reactivity to the 17 type I IFNs similar to those reported in previously described patients with auto-Abs neutralizing 10 ng/mL (6), as only a few (2%) had auto-Abs neutralizing only IFN-β. Finally, a few rare patients (0.7%) had auto-Abs that appeared to neutralize IFN-β only, and no such antibodies were detected in any of the controls tested. [000300] Neutralizing auto-Abs against type I IFNs in at least 20% of patients over 80 years of age [000301] We investigated the risk factors for critical COVID-19, by stratifying the percentage of patients with auto-Abs against type I IFNs by age and sex. In our previous report, half the patients with auto-Abs and life-threatening COVID-19 were more than 65 years old and 95% were men (6). These results were confirmed by other groups, although the proportion of men was smaller (7-11). In our expanded cohort of patients with critical COVID-19 pneumonia (N = 2,248), the mean age was 61 years and 73% were men (Fig.19A). We assessed the percentage of individuals positive for neutralizing auto- Abs per decade of life and by sex, in patients with life-threatening COVID-19 (Fig.19B-I, Fig.24A-J) (Table 10). The mean age of patients with auto-Abs was 64 years old, and 74% were males. The proportion of auto-Abs significantly increased with age in both men and women (Wald test, P = 7.10- 5 ), and the proportion of patients with auto-Abs was higher in men. In patients with auto-Abs neutralizing 10 ng/mL IFN, the increase was continuous, with auto-Abs against IFN-α2 and/or IFN-ω detected in 4.8% of patients under the age of 39 years, 5.9% of those between 40 and 49 years of age, 7.6% in those between 50 and 59 years of age, 11.2% in those between 60 and 69 years of age, and more than 13% in those over the age of 70 years (Fig. 19C-F, Fig. 24B-E). When compared with the critical patients without auto-Abs, the effect of sex on the proportion of patients with auto-Abs was not significant (Fisher exact test, P = 0.13), while it was highly significant when compared with the controls (Fisher exact test, P < 10 -16 ). Similar results were obtained for patients with auto-Abs neutralizing 100 pg/mL IFN, but with even higher proportions (Fig.19G-J, S3F-I) (Table 10). Indeed, the proportion of patients with auto-Abs ranged from 12% of patients below the age of 40 years, to more than 21% of those over 80 years of age again having a significant increase with age (Wald test, P = 0.0004) (Fig. 19G-J, Fig.24F-I). The increase in age was even more marked in men, with 24% of men over 80 years of age positive for these antibodies, versus only 20% of women in this age group. Overall, the prevalence of auto-Abs neutralizing 10 ng/ml and/or 100 pg/mL type I IFN increased sharply with age in all patients, particularly in men. A striking enrichment in patients with neutralizing auto-Abs against type I IFNs was observed in the elderly, with more than 20% of patients, and 24% of men, over the age of 80 years with critical COVID-19 having neutralizing auto-Abs against type I IFNs. [000302] Neutralizing auto-Abs against type I IFNs in at least 20% of deceased patients [000303] The prevalence of auto-Abs against type I IFNs in patients dying from COVID-19 pneumonia is unknown. We also analyzed a subset of 865 individuals who died from COVID-19 in five different countries (France, Italy, Spain, Turkey, and the United States). These patients were aged 20 to 99 years (mean age: 70 years), 73% were male, and all had confirmed SARS-CoV-2 infection and critical COVID-19 pneumonia before death (Fig. 20A). In these patients, we analyzed the presence of neutralizing auto-Abs against type I IFNs at concentrations of 10 ng/mL and 100 pg/mL (Fig. 20B-J, Fig.25A-I). We found that 12.5% of the samples from deceased patients carried auto-Abs neutralizing 10 ng/mL IFN-α2 and/or IFN-ω (Fig. 20B-F, Fig. 25A-E). Strikingly, 21% contained auto-Abs neutralizing 100 pg/mL of either or both cytokines (Fig. 20G-J, Fig. 25F-I). An analysis of the prevalence of neutralizing auto-Abs against type I IFNs in these patients who died of COVID-19 by decade of age revealed no significant increase of the prevalence of these auto-Abs with age for auto- Abs neutralizing 10 ng/mL (Wald test, P = 0.17) or those neutralizing 100 pg/mL (Wald test, P = 0.16). The prevalence of these antibodies exceeded 20% in most age groups (Fig. 20G). For a type I IFN concentration of 100 pg/mL, the prevalence of auto-Abs neutralizing IFN-α2 and/or IFN-ω was 20% below 40 years, 21.2% for individuals between 40 and 49 years old, 13.1% for those between 50 and 60 years old, 20.6% for those between 60 and 69 years old and greater than 22% for those over the age of 70 years. Thus, at least 20% of patients dying from COVID-19 pneumonia have auto-Abs capable of neutralizing 100 pg/mL type I IFNs, in plasma 1/10. [000304] Neutralizing auto-Abs against type I IFNs in 0.2% of individuals under the age of 70 years [000305] We previously tested a sample of 1,227 individuals aged 20 to 65 years from the general population collected in 2015-2017. This sample had an equal distribution of the two sexes, and we identified four individuals with auto-Abs against type I IFNs among the 1,227 tested (0.3%) (see Example 2; 6). These findings were replicated at the University of California San Francisco (UCSF) in a sample of 4,041 subjects aged four to 90 years (0.2%) (8). We tested a much larger cohort of 27,738 individuals aged 20 to 70 years from the general population, with an equal distribution in both sexes. All samples were collected in 2017, from blood donors at the French Blood Bank (20,093 individuals), participants in the French Constances cohort (5,952), or donors at the US-based IgG producer ADMA pharmaceuticals (1,693) (Fig. 21A). We used Gyros to screen this whole cohort for IgG auto-Abs against IFN-α2 and IFN-ω. We found that only 0.05% and 1.2% had anti-IFN-α2 and/or anti-IFN-ω auto-Abs above the thresholds of 100 and 30, respectively (Fig. 21B, Fig. 26A). We then assessed the ability of these antibodies to neutralize 10 ng/mL IFN-α2 or IFN-ω, for all individuals with a high or intermediate level of IgG auto-Abs against IFN-α2 or IFN-ω. We found 44 individuals with neutralizing auto-Abs, for whom 1/10 dilutions of plasma neutralized 10 ng/mL IFN-α2 and/or IFN-ω, giving an overall prevalence in individuals under the age of 60 years of 0.16% (Fig. 21C-F, Fig. 26B-E) (Table 10), consistent with our two previous reports (6, 8). The prevalence of auto-Abs was not higher in men than in women in this age group, and we instead observed a slightly higher frequency in younger women. In addition, there seemed to be a slightly higher prevalence of neutralizing anti-IFN-ω auto- Abs in younger individuals, especially women (Fig.26B-E). [000306] Sharp increase in the prevalence of auto-Abs against type I IFNs in subjects over 70 years of age [000307] We assessed the prevalence of auto-Abs against type I IFNs in elderly individuals — the group most affected by critical COVID-19 pneumonia — by recruiting another cohort from the general population, of 5,611 individuals over the age of 70 years, equally distributed between the sexes (Fig. 5A). These individuals were recruited from the 3-C cohort (N = 980), the Constances cohort (N = 2,631), and Cerba Health Care (N = 2,000). We performed serological tests for SARS-CoV-2 on the samples collected after 2019 (N = 2,2631), and we included only those individuals who had not been infected with SARS-CoV-2 in the sample. Anti-IFN-α2 and IFN-ω auto-Abs were also tested with Gyros in all samples (Fig.21B, Fig.26A). We then assessed in all samples the ability of the plasma/serum samples (diluted 1/10) to neutralize 10 ng/mL and 100 pg/mL type I IFN in the luciferase assay. Strikingly, we noted that the prevalence of auto-Abs neutralizing 10 ng/mL type I IFN was more than 10 times higher in individuals over the age of 70 years than in those below this age (Wald test, P < 2.10 -16 ) (Fig. 21C- F, Fig.26B-E) (Table 10). [000308] TABLE 10 below provides total numbers of patients, controls and individuals from the general population tested for neutralizing auto-Abs against type I IFNs. All is shown by gender, age, type of type I IFN auto-Ab and dose of type I IFN neutralized. TABLE 10
1119-69PCT only anti-IFNa2 All [60,70) 541 25 4.62 225 12 5.33 25 0 0.00 6877 5 0.07 auto-Abs (10 ng/mL) 0 0.00 3482 12 0.34 0 0.00 2285 22 0.96 0 0.00 823 9 1.09 0 0.00 0 0 0.00 0 0.00 0 0 0.00 0 0.00 0 0 0.00 0 0.00 190 1 0.53 0 0.00 232 1 0.43 0 0.00 401 7 1.75 2 0.58 33599 52 0.15 0 0.00 9106 11 0.12 1 0.99 5405 5 0.09 0 0.00 6419 5 0.08
[000309] The prevalence of auto-Abs capable of neutralizing 10 ng/mL IFN-α2 and/or IFN-ω was 1% in individuals between 70 and 80 years of age, and 2.6% in individuals over the age of 80 years. This trend was even more striking in men, with 1.3% positive between the ages of 70 and 80 years, and more than 3% after 80 years. These findings were replicated independently in two cohorts of 704 and 376 elderly individuals from Estonia and Japan, with LIPS and ELISA assays, respectively (Fig. 26F, G). Finally, we tested a random subsample of these elderly individuals for auto-Abs capable of neutralizing 100 pg/mL type I IFN and, again, the prevalence of neutralizing auto-Abs was even higher (Fig. 21G-H, S5H-M), and more than doubling with each decade of age. Indeed, 0.53% of individuals between the ages of 60 and 70 years were positive, 1.3% of those between 70 and 80 years, and 3.8% of those over 80 years. Interestingly, there was no significant difference between men and women (Fisher exact test, P = 0.8). Overall, these findings indicate that there is an exponential increase in the prevalence of auto-Abs neutralizing type I IFNs with age in elderly individuals. [000310] Concluding remarks [000311] We report that at least 20% of patients over 80 years of age with life-threatening COVID- 19 pneumonia carry circulating auto-Abs neutralizing 100 pg/mL IFN-α2 and/or IFN-ω, and that such antibodies are present in more than 15% of patients of all ages with this condition. Some of these auto- Abs are hidden and only detectable by a neutralization assay. In addition, at least 20% of deceased individuals in most age groups were found to have such auto-Abs. Importantly, auto-Abs capable of neutralizing high or low concentrations of type I IFNs have been found in patients without inborn errors of TLR3- or TLR7-dependent type I IFN immunity (5) (Asano et al, accompanying report submitted), suggesting that both inborn errors and auto-Abs are independently causal of critical disease. It is also striking that inborn errors are more common in patients below the age of 70 years, whereas auto-Abs are more common in patients over the age of 70 years. We also report that the prevalence of auto-Abs against type I IFNs increases significantly with age in the general population, reaching 0.1%, 1%, and 2.9% in individuals older than 60, 70, and 80 years of age, respectively, for antibodies neutralizing 10 ng/mL type I IFNs, and 0.5%, 1.3%, and 3.8%, respectively, for antibodies neutralizing 100 pg/mL IFN. These auto-Abs provide an explanation for the major increase in the risk of critical COVID-19 in the elderly. This increase with age is consistent with studies of various auto-Abs since the 1960s (30- 34). These auto-Abs appear to have remained clinically silent in these individuals until SARS-CoV-2 infection. It is tempting to speculate that an even greater proportion of life-threatening COVID-19 cases are due to auto-Abs neutralizing lower, physiological concentrations of type I IFNs. In vitro, concentrations of type I IFN as low as 100 pg/mL can impair SARS-CoV-2 replication in epithelial cells (Fig.2A). Moreover, the levels of type I IFN detected in the blood and respiratory tract of patients with acute and benign SARS-CoV-2 infections are in the range of 1 to 100 pg/mL (25, 26). We have shown that antibodies neutralizing 100 pg/mL type I IFN in plasma diluted 1/10, corresponding to the neutralization of 1 ng/mL IFN in vivo, can account for at least 20% of deaths across all ages and of critical cases in the elderly > 80 years. We speculate that auto-Abs neutralizing concentrations of type I IFN 10 or 100 times lower than this may be even more common in the general population, and perhaps also pathogenic upon infection with SARS-CoV-2. [000312] Our findings have immediate clinical applications. First, it is easy and rapid to test for auto- Abs against type I IFNs in patients infected with SARS-CoV-2. Screening for these antibodies is even possible in the general population prior to infection. The type I IFN-neutralizing activity of these antibodies is a better read-out than their mere detection, which can be falsely negative. Particular attention should be paid to elderly individuals, and patients with known auto-immune or genetic conditions associated with auto-Abs against type I IFNs (13-18, 21-23). Second, patients with auto-Abs against type I IFN should be vaccinated against COVID-19 as a priority. Third, live-attenuated vaccines, including YFV-17D and vaccines using the YFV-17D backbone against SARS-CoV-2, should not be given to patients with auto-Abs (24, 35). Fourth, these patients appeared to be healthy before SARS- CoV-2 infection, but they should also be carefully followed for other viral illnesses, as exemplified by the adverse reactions to YFV-17D (24). Fifth, in cases of SARS-CoV-2 infection in unvaccinated individuals with auto-Abs against type I IFNs, the patients should be hospitalized for prompt management. Early treatment with monoclonal antibodies (36, 37) can be administered to patients who do not have symptoms of severe COVID-19 pneumonia, and IFN-β can be administered in the absence of both pneumonia and auto-Abs against IFN-β (28, 29). Rescue treatment by plasma exchange is another therapeutic option in patients that already have pneumonia (38). Sixth, blood products should be screened for anti-IFN auto-Abs and any products containing such antibodies should be excluded from donation (10). Plasma from donors convalescing from COVID-19 should be tested for such auto- Abs (10). Seventh, given the documented innocuity and potential efficacy of a single injection, early therapy with IFN-β may be considered for the contacts of contagious subjects or during the first week after infection, even in the absence of, or before the documentation of auto-Abs against type I IFNs, in elderly patients, who have a higher risk of auto-Abs against type I IFN and, therefore, of critical COVID-19 pneumonia (39). Finally, it will be important to decipher the mechanism underlying the development of these auto-Abs, which may be genetic (germline or somatic), epigenetic and/or involve thymic dysfunction. Overall, our findings show that auto-Abs neutralizing concentrations of type I IFN lower than previously reported, but still higher than physiological concentrations, are common in the elderly population. They underlie at least 20% of cases of critical COVID-19 pneumonia in patients over the age of 80 years, at least 20% of COVID-19 deaths at all ages, and their prevalence increases with age in the uninfected general population, reaching at least 3% of individuals after the age of 70 years. [000313] Materials and methods [000314] Subjects and samples [000315] We enrolled 2,248 patients with proven life-threatening (critical) COVID-19, 588 asymptomatic or paucisymptomatic individuals with proven COVID-19, and 33,352 healthy controls in this study. All subjects were recruited according to protocols approved by local institutional review boards (IRBs). For patients enrolled in the Italian cohort, ethics approval was obtained from the University of Milano-Bicocca School of Medicine, San Gerardo Hospital, Monza – Ethics Committee of the National Institute of Infectious Diseases Lazzaro Spallanzani (84/2020) (Italy), the IRB of Fondazione IRCCS Policlinico San Matteo, Pavia (Italy), and the Comitato Etico Provinciale (NP 4000 – Studio CORONAlab). STORM-Health care workers were enrolled in the STudio OsseRvazionale sullo screening dei lavoratori ospedalieri per COVID- 19 (STORM-HCW) study, which was approved by the local IRB on June 18, 2020. For the COV-Contact study, ethics approval was obtained from the CPP IDF VI (ID RCB: 2020-A00280- 39). For patients were enrolled in the French COVID cohort (clinicaltrials.gov NCT04262921), ethics approval was obtained from the CPP IDF VI (ID RCB: 2020- A00256-33). Some contact subjects were enrolled in the Cov-Contact cohort (clinicaltrials.gov NCT04259892). Anonymized samples were studied at the NIAID under non-human subject research conditions; no additional IRB consent was required at the NIH. Protocols in San Raffaele were approved by San Raffaele Hospital Ethical Committee. Samples were obtained from the Milieur Intérieur Cohort, which was approved by the Comité de Protection des Personnes – Ouest 6 (Committee for the protection of persons) on June 13, 2012 and by the French Agence nationale de sécurité du médicament (ANSM) on June 22, 2012. The Milieur Intérieur Cohort study is sponsored by Institut Pasteur (Pasteur ID-RCB Number: 2012-A00238-35), and was conducted as a single-center interventional study without an investigational product. The original protocol was registered under ClinicalTrials.gov (study# NCT01699893). The samples and data used in this study were formally established as the Milieu Interieur biocollection (NCT03905993), with approval from the Comité de Protection des Personnes – Sud Méditerranée and the Commission nationale de l'informatique et des libertés (CNIL), obtained on April 11, 2018. The content of the manuscript is the sole responsibility of the authors and does not necessarily represent the official views of any of the funding sources. The patients of the Amsterdam UMC Covid-19 cohort were enrolled prospectively at the Amsterdam UMC. Ethics approval was obtained from the Amsterdam UMC Biobank Committee (202_065#A202029). All protocols conformed to local ethics recommendations and informed consent was obtained when required. [000316] The severity of COVID-19 was assessed for each patient as follows (5, 6). “Life-threatening COVID-19 pneumonia” was defined as pneumonia developing in patients with critical disease, whether pulmonary, with mechanical ventilation (CPAP, BIPAP, intubation, high-flow oxygen), septic shock, or with damage to any other organ requiring admission to the ICU. The controls were individuals infected with SARS-CoV-2 (as demonstrated by a positive PCR and/or serological test and/or displaying typical symptoms, such as anosmia/ageusia after exposure to a confirmed COVID-19 case) who remained asymptomatic or developed mild, self-healing, ambulatory disease with no evidence of pneumonia. [000317] Detection of anti-cytokine autoantibodies [000318] Gyros [000319] Cytokines, recombinant human (rh)IFN-α2 (Milteny Biotec, ref. number 130-108-984) or rhIFN-ω (Merck, ref. number SRP3061), were first biotinylated with EZ-Link Sulfo-NHS-LC-Biotin (Thermo Fisher Scientific, cat. number A39257), according to the manufacturer’s instructions, with a biotin-to-protein molar ratio of 1:12. The detection reagent contained a secondary antibody (Alexa Fluor 647 goat anti-human IgG (Thermo Fisher Scientific, ref. number A21445) diluted in Rexip F (Gyros Protein Technologies, ref. number P0004825; 1/500 dilution of the 2 mg/mL stock to yield a final concentration of 4 µg/mL). Buffer PBS-T 0.01% and Gyros Wash buffer (Gyros Protein Technologies, ref. number P0020087) were prepared according to the manufacturer’s instructions. Plasma or serum samples were then diluted 1/100 in PBS-T 0.01% and tested with the Bioaffy 1000 CD (Gyros Protein Technologies, ref. number P0004253), and the Gyrolab X-Pand (Gyros Protein Technologies, ref. number P0020520). Cleaning cycles were performed in 20% ethanol. [000320] Multiplex particle-based assay [000321] Serum/plasma samples were screened for autoantibodies against IFN-α2 and IFN-ω in a multiplex particle-based assay, in which magnetic beads with differential fluorescence were covalently coupled to recombinant human proteins (2.5 µg/reaction). Beads were combined and incubated with 1/100-diluted serum/plasma samples for 30 minutes. Each sample was tested once. The beads were then washed and incubated with PE-labeled goat anti-human IgG (1 µg/mL) for an additional 30 minutes. They were then washed again and used for a multiplex assay on a BioPlex X200 instrument. [000322] Enzyme-linked immunosorbent assays (ELISA) [000323] ELISA was performed as previously described (6). In brief, 96-well ELISA plates (MaxiSorp; Thermo Fisher Scientific) were coated by incubation overnight at 4°C with 2 µg/mL rhIFN- α2 (Milteny Biotec, ref. number 130-108-984), and rhIFN-ω (Merck, ref. number SRP3061). Plates were then washed (PBS 0.005% Tween), blocked by incubation with 5% nonfat milk powder in the same buffer, washed, and incubated with 1:50 dilutions of plasma from the patients or controls for 2 h at room temperature (or with specific mAbs as positive controls). Each sample was tested once. Plates were thoroughly washed. Horseradish peroxidase (HRP)–conjugated Fc-specific IgG fractions from polyclonal goat antiserum against human IgG, IgM or IgA (Nordic Immunological Laboratories) were added to a final concentration of 2 µg/mL. Plates were incubated for 1 h at room temperature and washed. Substrate was added and the optical density (OD) was measured. A similar protocol was used to test for antibodies against 12 subtypes of IFN-α, except that the plates were coated with cytokines from PBL Assay Science (catalog #11002-1), or IFN-β (Milteny Biotech, ref. number: 130-107-888). [000324] LIPS [000325] Levels of autoantibodies against IFN-α subtypes were measured in luciferase-based immunoprecipitation assay (LIPS), as previously described (15). IFNA1, IFNA2, IFNA8, and IFNA21 sequences were inserted into a modified pPK-CMV-F4 fusion vector (PromoCell GmbH, Germany), in which the firefly luciferase replaced the NanoLuc luciferase (Promega, USA). The resulting constructs were used to transfect HEK293 cells and the IFNA-luciferase fusion proteins were collected in the tissue culture supernatant. For autoantibody screening, we combined 2x10 6 luminescence units (LU) of IFNA1, IFNA2, IFNA8 and IFNA21 in a single IP reaction mixture (pool 1), and IFNA4, IFNA5, IFNA6 and IFNA7 in another IP reaction mixture (pool 2). Serum samples were incubated with Protein G agarose beads (Exalpha Biologicals, USA) at room temperature for 1 h in a 96-well microfilter plate (Merck Millipore, Germany), and we then added 2x10 6 luminescence units (LU) of antigen and incubated for another hour. Each sample was tested once. The plate was washed with a vacuum system and Nano-Glo® Luciferase Assay Reagent (Promega, USA) was added. Luminescence intensity was measured with a VICTOR X Multilabel Plate Reader (PerkinElmer Life Sciences, USA). The results are expressed in arbitrary units (AU), as a fold-difference relative to the mean of the negative control samples. [000326] Functional evaluation of anti-cytokine autoantibodies [000327] pSTAT1 induction in PBMC [000328] The blocking activity of anti-IFN-α2 and anti-IFN-ω auto-Abs was determined by assessing STAT1 phosphorylation in healthy control cells following stimulation with the appropriate cytokines in the presence of 10% healthy control or patient serum/plasma. Surface-stained healthy control PBMCs (350,000/reaction) were cultured in serum-free RPMI medium with 10% healthy control or patient serum/plasma and were either left unstimulated or were stimulated with IFNa or IFNw (10 ng/mL) for 15 minutes at 37°C. Each sample was tested once. Cells were fixed, permeabilized, and stained for intranuclear phospho-STAT1 (Y701). Cells were acquired on a BD LSRFortessa cytometer with gating on CD14 + monocytes and the data were analyzed with FlowJo software. [000329] Luciferase reporter assays [000330] The blocking activity of anti-IFN-α2 and anti-IFN-ω auto-Abs was determined with a reporter luciferase activity. Briefly, HEK293T cells were transfected with a plasmid containing the firefly luciferase gene under the control of the human ISRE promoter in the pGL4.45 backbone, and a plasmid constitutively expressing Renilla luciferase for normalization (pRL-SV40). Cells were transfected in the presence of the X-tremeGene 9 transfection reagent (Sigma Aldrich, ref. number 6365779001) for 36 hours. Cells in Dulbecco’s modified Eagle medium (DMEM, Thermo Fisher Scientific) supplemented with 2% fetal calf serum (FCS) and 10% healthy control or patient serum/plasma were either left unstimulated or were stimulated with IFN-α2 (Milteny Biotec, ref. number 130-108-984), IFN-ω (Merck, ref. number SRP3061), at 10 ng/mL or 100 pg/mL, or IFN-β (Milteny Biotech, ref. number: 130-107-888) at 10 ng/mL, for 16 hours at 37°C. Each sample was tested once for each cytokine and dose. Finally, cell were lysed for 20 minutes at room temperature and luciferase levels were measured with the Dual-Luciferase® Reporter 1000 assay system (Promega, ref. number E1980), according to the manufacturer’s protocol. Luminescence intensity was measured with a VICTOR X Multilabel Plate Reader (PerkinElmer Life Sciences, USA). Firefly luciferase activity values were normalized against Renilla luciferase activity values. These values were then normalized against the median induction level for non-neutralizing samples, and expressed as a percentage. Samples were considered to be neutralizing if luciferase induction, normalized against Renilla luciferase activity, was below 15% of the median values for controls tested the same day. [000331] Statistical analysis [000332] Comparison of proportions of individuals with neutralizing autoantibodies according to categorical variables were performed using a Fisher exact test, as implemented in R (https://cran.r- project.org/). Impact of age in years on the presence of neutralizing autoantibodies was tested by means of logistic regression using the Wald test and adjusting on sex. [000333] Schematic representation [000334] Schematic representations (Fig.1B) were created with BioRender.com. [000335] SARS-CoV-2 experiment [000336] SARS-CoV-2 strain USA-WA1/2020 was obtained from BEI Resources and amplified in Caco-2 cells at 37°C. Viral titers were measured on Huh-7.5 hepatoma cells in a standard plaque assay. Caco-2 (H. sapiens, sex: male, colon epithelial) and Huh-7.5 cells (H. sapiens, sex: male, liver epithelial) were cultured in DMEM supplemented with 1% nonessential amino acids (NEAA) and 10% fetal bovine serum (FBS) at 37°C, under an atmosphere containing 5% CO2. Both cell lines have been tested negative for contamination with mycoplasma. [000337] SARS-CoV-2 experiments were performed as follows. Huh-7.5 cells were used to seed 96- well plates at a density of 7.5x10 3 cells/well. The following day, plasma samples or a commercial anti- IFN-α2 antibody (catalog number 21100-1; R&D Systems) were diluted to 1% and incubated with 5 pM (~96 pg/mL) or 20 pM (~384 pg/mL) recombinant IFN-α2 (catalog number 11101-2; R&D systems) for 1 h at 37°C (dilutions: plasma samples = 1/100 and anti-IFN-α2 antibody = 1/1,000). Molar ratio was calculated according to the manufacturer’s datasheet and with molbiol.ru/eng/scripts/01_04.html. Following this incubation period, the cell culture medium was removed from the 96-well plates by aspiration and replaced with the plasma/anti-IFN-α2 antibody and IFN-α2 mixture. Each sample was tested once, in triplicate. The plates were incubated overnight and the plasma/anti-IFN-α2 antibody plus IFN-α2 mixture was removed by aspiration. The cells were washed once with PBS to remove potential anti-SARS-CoV-2-neutralizing antibodies and fresh medium was then added. Cells were then infected with SARS-CoV-2 by directly adding the virus to the wells. 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Sanchez-Felipe et al., A single-dose live-attenuated YF17D-vectored SARS-CoV-2 vaccine candidate. Nature 590, 320-325 (2021). 36. P. Chen et al., SARS-CoV-2 Neutralizing Antibody LY-CoV555 in Outpatients with Covid- 19. N Engl J Med 384, 229-237 (2021). 37. D. M. Weinreich et al., REGN-COV2, a Neutralizing Antibody Cocktail, in Outpatients with Covid-19. N Engl J Med 384, 238-251 (2021). 38. N. de Prost et al., Plasma Exchange to Rescue Patients with Autoantibodies Against Type I Interferons and Life-Threatening COVID-19 Pneumonia. J Clin Immunol, (2021). 39. D. C. Vinh, L. Abel, P. Bastard, C. JL., I. Meyts, Harnessing type I IFN immunity against SARS-CoV-2 with early administration of IFN-beta. JoCI In Press, (2021). EXAMPLE 5 Human TLR7 and Plasmacytoid Dendritic Cells are Essential for Type I IFN Pulmonary Immunity to SARS-CoV-2 [000339] Autosomal inborn errors of type I IFN immunity and autoantibodies against these cytokines underlie at least 10% of critical COVID-19 pneumonia cases. We report very rare, biochemically deleterious X-linked TLR7 variants in 14 unrelated male individuals aged 13 to 71 years (mean: 36.7 years) from a cohort of 1,005 male individuals aged 0.5 to 99 years (mean: 52.9 years) with unexplained critical COVID-19 pneumonia. None of the 326 asymptomatically infected male individuals aged 1.3 to 102 years (mean: 41.1 years) tested carried such TLR7 variants (p = 6x10 -6 ). The cumulative allele frequency for deleterious TLR7 variants in the male general population was < 6.5x10 -4 . We also found that blood lymphoid cell lines and myeloid cell subsets from the patients failed to respond to TLR7 stimulation, a phenotype rescued with wild-type TLR7. Finally, the patients’ blood plasmacytoid dendritic cells (pDCs) produced low levels of type I IFNs in response to SARS-CoV-2. X-linked recessive TLR7 deficiency is a genetic etiology of life-threatening COVID-19 pneumonia in about 1.6% of male patients below the age of 70 years. Human TLR7 and pDCs are essential for protective type I IFN immunity against SARS-CoV-2 in the lungs. [000340] Introduction [000341] Inter-individual clinical variability in the course of SARS-CoV-2 infection is vast, ranging from silent infection to lethal disease (1). The greatest risk factor for life-threatening COVID-19 pneumonia is age, with a doubling in risk every five years from the age of five years onward, and a sharp rise after the age of 65 years (2, 3). Other epidemiological risk factors, including common genetic variants, have only modest effects, with odds ratios (ORs) < 2 and typically < 1.5 (2). One intriguing observation is the approximately 1.5-fold higher risk in men, which seems to be age-independent (2-4). COVID Human Genetic Effort (covidhge.com) has enrolled an international cohort of patients, with the aim of investigating genetic and immunological causes of life-threatening COVID-19 pneumonia. We tested the hypothesis that critical influenza and critical COVID-19 can be allelic (5-7), and showed that life-threatening COVID-19 pneumonia can be caused by rare inborn errors of autosomal genes controlling TLR3- and IRF7-dependent type I IFN immunity (8). These disorders were found in 23 men and women aged 17 to 77 years (mean: 48 years). Remarkably, four unrelated patients aged 25 to 50 years had autosomal recessive IFNAR1 (n=2) or IRF7 (n=2) deficiency. These patients had no previous history of severe viral illness, including influenza pneumonia, implying that these genetic disorders unexpectedly show incomplete penetrance for critical influenza. These findings revealed that TLR3- and IRF7-dependent type I IFN immunity is essential for host defense against SARS-CoV-2 infection in the respiratory tract. [000342] We found pre-existing neutralizing auto-Abs against type I IFN in at least 10% of the patients from this same cohort (9). These auto-Abs were found in 101 patients, mostly men (95%), and older than the rest of the cohort, including patients with inborn errors, as they were aged 25 to 87 years (mean: 65 years). These findings have been replicated in five other cohorts (10-15). These auto-Abs predated SARS-CoV-2 infection and were causal for critical COVID-19 pneumonia, because (i) they were found in samples drawn before infection in some patients (9), (ii) they were found in about 0.3% of the general population before the age of 65 years (9), (iii) they were absent from patients with asymptomatic or paucisymptomatic (mild) SARS-CoV-2 infection (9), (iv) they were of childhood onset in patients with various disorders, including autoimmune polyendocrinopathy type I (APS-1), known to be at very high risk of life-threatening COVID-19 (16), and (v) they have been shown to underlie a third of adverse reactions to the live attenuated viral vaccine for yellow fever (17). Collectively, these studies showed that type I IFNs are essential for protective immunity to SARS-CoV- 2 in the respiratory tract, but otherwise surprisingly redundant. Auto-Abs against type I IFNs also provide a first explanation for both the biased sex ratio and the higher risk of critical COVID-19 in patients over the age of 65 years. We tested the hypothesis that critical and unexplained COVID-19 pneumonia in younger men may be due to rare variants on the X-chromosome. [000343] Enrichment for very rare TLR7 non-synonymous variants in male patients [000344] We tested the hypothesis of genetic homogeneity for X-linked recessive disorders in male individuals with life-threatening COVID-19 pneumonia (hereafter referred to as “patients”). We analyzed an international cohort of 1,005 male individuals aged 6 months to 99 years (mean: 52.9 years) with no known inborn errors of TLR3- and IRF7-dependent type I IFN immunity (8) and without neutralizing auto-Abs against type I IFNs (9) (accompanying paper by Bastard P. et al.) (Table 12). We also analyzed 326 asymptomatic or paucisymptomatic infected patients aged 1.3 to 102 years (mean: 41.1 years), with positive results for PCR and/or serological screening for SARS-CoV-2 infection (the controls) (Table 12). We sequenced the exomes (n=934) or genomes (n=397) of these patients. We selected in-frame and out-of-frame non-synonymous variants of protein-coding exons that are very rare, that is, with a MAF below 10 -4 in the full gnomAD database (v2.1.1) containing sequences from both male and female individuals. We compared the proportions of patients and controls carrying at least one qualifying variant, by logistic regression adjusted for age and ethnicity (Fig. 31A). We found 190 of 731 genes on the X chromosome with at least five patients carrying non-synonymous variants (Table 13). TLR7 was the highest ranked of these genes (P-value = 6×10 -6 ), with 19 patients carrying one very rare (n=4 patients), two very rare (n=1 patient), or one private (n=14 patients) non-synonymous variants (P-value = 6×10 −6 ) (Fig. 27A - Table 14). One variant (L988S) was recurrent, found in three patients, including a patient carrying two very rare variants (M854I;L988S). No such variants were found in the controls. The same analysis performed on very rare (MAF<10 -4 ) synonymous TLR7 variants showed no enrichment in patients (one carrier) relative to controls (three carriers). Human TLR7 is an endosomal receptor of ribonucleic acids expressed by B cells and myeloid subsets (18-22), the stimulation of which in plasmacytoid dendritic cells (pDCs) results in the production of large amounts of type I IFN (23-25). We observed no significant enrichment for coding non-synonymous variants of the X-linked gene TLR8 (P-value = 0.27, Table 14), the product of which, TLR8, is endosomal and can be stimulated by some synthetic TLR7 agonists, with an expression pattern and signaling pathway overlapping those of TLR7 (26, 27). Unlike TLR7, TLR8 is expressed on granulocytes but not pDCs, possibly accounting for its gain-of-function mutations underlying a phenotype different from type I interferonopathies (28-30). Overall, we found an enrichment in very rare or private non-synonymous TLR7 variants among male patients with critical COVID-19 pneumonia (n=19, 1.9% of our cohort, including only one man over the age of 70 years). [000345] The TLR7 mutant alleles of 14 of the 19 patients are biochemically deleterious [000346] The 19 patients carried 18 different TLR7 alleles. We expressed the 18 TLR7 mutant proteins in human embryonic kidney (HEK) 293T cells, which have no endogenous TLR7 and TLR8 expression (31), by transient transfection with the corresponding cDNAs. Immunoblotting of protein extracts with a TLR7-specific mAb showed an absence of TLR7 protein for p.N158Tfs*11 and p.L227fs* and a truncated protein for K684* (Fig. 27B). The other mutant TLR7 proteins were produced in normal amounts (Fig. 27B). We tested their function by cotransfection with an NF-κB- specific reporter. We measured luciferase activity upon stimulation with R848, an agonist of both TLR7 and TLR8 (Fig. 27C). Eleven of the 18 alleles were loss-of-function (LOF) (including L988S in two patients, and M854I;L988S in another), two (p.I657T and p.P715S) were hypomorphic (activity < 25%), and the remaining five were neutral (Fig.27C, Table 15). Similar results were obtained with imiquimod and CL264, two TLR7-specific agonists (Fig. 31B, 31C). We also tested eight other private (p.S301P, p.Q710Rfs*18, p.V795F), very rare (MAF <10 -4 ; p.A288V) or rare (MAF between 10 -4 and 10 -2 ; p.V219I, p.A448V, p.R920K, p.A1032T) TLR7 variants previously reported in patients with critical COVID-19 but without biochemical characterization (32, 33). These variants were expressed as truncated or full-length proteins (Fig. 31D). The proteins encoded by the three private variants were found to be LOF, that encoded by the very rare variant (p.A288V) was hypomorphic, and those encoded by the four rare variants were neutral (Fig.27C, Fig.31B). Collectively, these findings suggest that 14 of the 19 patients in our cohort (Table 11) and six of the previously reported 12 patients carry deleterious TLR7 variants. TABLE 11. X-linked TLR7 deleterious variants in unrelated male patients with life-threatening COVID-19 pneumonia TABLE 12 - Characteristic of the cohort of life-threatening COVID-19 pneumonia and the cohort control of asymptomatic or paucisymptomatic individuals 1119-69PCT TABLE 13 - Selection of genes on chromosome X with 5 or more hemizygous carriers. CHR Models model Ncases Nctrl Ncases_carriers Nctrl_carriers N OR P-value P Bonferroni corrected X hemizygous TLR7 1005 326 19 0 19 12025656 5.96E-06 1.13E-03 0.063837 0.00563332 1.00 3285287 0.00869198 1.00 2495162 0.01377879 1.00 2182535 0.01764981 1.00 1642866 0.02045208 1.00 1458377 0.02194455 1.00 1252448 0.0326101 1.00 1014444 0.04412556 1.00 1826922 0.04546455 1.00 1485836 0.04917914 1.00 0.201194 0.06200739 1.00 1774796 0.06458935 1.00 1461520 0.07771555 1.00 552742.6 0.0912534 1.00 765016.8 0.09265719 1.00 4.365769 0.0955476 1.00 889670.1 0.09558978 1.00 997431.4 0.09592991 1.00 1130604 0.09773911 1.00 0.243808 0.10078192 1.00 646383 0.10115663 1.00 1113882 0.10238587 1.00 0.340619 0.10368151 1.00 0.226707 0.10497478 1.00
TABLE 14 - Statistical analysis of non-synonymous TLR7 and TLR8 rare variants in our cohorts
Coding non-synonymous (MAF< 10-4)
TABLE 15 - TLR7 variant activity reported in this study, in previous studies and in gnomAD (v2.1.1)
[000347] The cumulative MAF of deleterious TLR7 alleles is < 6.5x10 -4 [000348] We also investigated the production and function of all 100 remaining non-synonymous TLR7 variants identified in the general population (141,456 individuals in gnomAD v2.1) that had been reported in men or had a general MAF > 10 -5 (Fig.27D and Fig.31E, 31F). In total, 96 of these variants were missense and three were in-frame small deletions, one of which was not expressed, 17 were weakly expressed, and the others were normally expressed (Fig. 31E, 31F, Table 15), and one variant was a small deletion creating a frameshift found in one man and resulting in an absence of protein production. Seven of the 100 variants were LOF and 15 were hypomorphic (< 25% activity) (Table 15). There were thus 24 deleterious TLR7 variants, including the L988S and A288V variants found in four patients with critical COVID-19 pneumonia. Each of these 24 deleterious variants had an individual MAF < 1.3x10 -4 in men and their cumulative MAF in men was 6.5 x10 -4 (Table 15, Table 16). The cumulative MAF of strictly LOF TLR7 alleles (excluding hypomorphic alleles) in men is about 2.2 x10 -4 (Table 15). Overall, we found 11 LOF and two hypomorphic TLR7 alleles in 14 unrelated men with critical COVID-19 pneumonia, whereas deleterious alleles were not found in men with asymptomatic or paucisymptomatic infection. Moreover, deleterious TLR7 alleles in the general population had individual and collective MAF values in men of < 1.3x10 -4 and < 6.5x10 -4 , respectively (Fig. 27E, Table 15). These findings suggest that X-linked recessive (XR) TLR7 deficiency is a genetic etiology of life-threatening COVID- 19 pneumonia in men.
TABLE 16 - Summary of TLR7 variants.
TABLE 17 - TLR7 deficient patients with severe/critical COVID-19 and hemizygous relative in our cohort (clinical information, laboratory findings, and immunological findings).
[000349] Incomplete clinical penetrance of inherited TLR7 deficiency [000350] The 14 patients were of three major ethnic origins, as confirmed by principal component analysis (PCA) of their exomes or genomes (34), and they were resident in six countries (France n=2, Spain n=3, Italy n=3, Turkey n=2, Iran n=3, Colombia n=1) (Fig. 28A, Fig 28B, Fig. 31, Table 11, Table 17). The patients were hospitalized for critical COVID-19 between March 2020 and May 2021. Blood samples (diluted 1/10) from these 14 patients did not carry auto-Abs neutralizing 10 ng/mL (9) or even 100 pg/mL IFN-α2 or -ω (Bastard P.; Examples 2 and 4). They were aged 13 to 71 years and their mean age was lower than that of the total cohort (mean age of 36.7 years, versus 52.9 years for the total cohort, in which age ranged from 0.5 to 99 years). TLR7-deficient patients accounted for about 1.6% of the patients below the age of 70 years (13 patients) and 1.4% of the entire cohort (14 patients). Two patients died and 12 survived (Fig. 28A - Table 11). None had previously been hospitalized for a severe viral illness, including influenza pneumonia. Sanger sequencing of the TLR7 locus in the relatives of these patients identified the deleterious alleles in 13 heterozygous women from nine families and six hemizygous men from six families (Fig. 28A). None of the TLR7 variants were de novo in the index cases. Four of the six hemizygous relatives of the index cases had antibodies against SARS-CoV- 2 (Fig.28A, Table 17). One was a 38-year-old adult with no relevant clinical history, another was a 27- year-old adult hospitalized for moderate pneumonia, and the remaining two were five-year-old boys, one of whom had been hospitalized for moderate COVID-19 pneumonia, the other having no relevant clinical history (Table 17). The clinical penetrance of critical COVID-19 in men is therefore high, but not complete, particularly in young patients. Blood pDC counts decrease with age (35-37), and this may contribute to the apparent increase in penetrance with age. [000351] Deleterious TLR7 alleles abolish B-cell responses to TLR7 agonists [000352] As a first approach to testing the impact of deleterious TLR7 alleles in the patients’ cells, we tested Epstein-Barr virus-transformed B-cell lines (EBV-B cells) from healthy controls and patients carrying the hemizygous p.K684* (P9) or p.H781L (P11) variants. The endogenous expression of the p.H781L TLR7 protein was normal, whereas p.K684* generated a truncated protein (Fig. 28C). In response to agonists of TLR7 (imiquimod) or TLR7 plus TLR8 (R848), the EBV-B-cell lines carrying these two mutations failed to produce TNF (Fig. 28D, Fig. 32A, 32B). The lentiviral transduction of these TLR7-deficient EBV-B cells with a WT TLR7 cDNA was unsuccessful, despite numerous attempts, and this was also the case for control EBV-B cells, perhaps because the overproduction of TLR7 is toxic in B cells (38). Consistent this view, we were able to express this cDNA in IRAK4- or MyD88-deficient EBV-B cells. We therefore investigated whether the addition of an IRAK4 inhibitor (PF06650833) would permit the expression of WT TLR7 in control and TLR7-mutated EBV-B cells. This approach was successful, and WT TLR7 expression restored responses to TLR7 agonists (after removal of the inhibitor) (Fig. 28E). Hemizygosity for LOF TLR7 alleles thus abolished responses to TLR7 stimulation in EBV-B cells, a phenotype that was rescued by WT TLR7 expression. Collectively, these findings further suggest that XR TLR7 deficiency is a genetic etiology of critical COVID-19 pneumonia. [000353] The patients’ myeloid cells, including pDCs, do not respond to TLR7 agonists [000354] Human TLR7 is only known to be expressed and functional in leukocyte subsets: plasmacytoid and classical dendritic cells (pDCs and mDCs), monocytes (classical, intermediate, and non-classical), and B cells (26, 31, 39). TLR8 is expressed in mDCs but not pDCs, monocytes but not B cells, and neutrophils (unlike TLR7) (26, 31, 39). Neither TLR7 nor TLR8 mRNAs have been detected in the lung or pulmonary epithelial cells (40). Deep immunophenotyping by CyTOF in seven patients with TLR7 deficiency revealed no major abnormalities in 18 leukocyte subsets, including pDCs, mDCs, monocytes, and B cells (Fig. 29A, Fig. 33A). We previously reported inherited IRF7 deficiency in a child with critical influenza pneumonia (5) and two unrelated adults with critical COVID-19 pneumonia (8). This defect disrupts the amplification of type I IFNs in all cell types, including pDCs, which are normally the main producers of type I IFN upon blood cell stimulation with TLR7 agonists or viruses, due to their constitutive expression of IRF7 (26, 41-43). We hypothesized that TLR7 deficiency in pDCs impairs the production of type I IFN by these cells in response to ssRNA. We confirmed that TLR7 was expressed on pDCs, and that TLR8 was not (Fig.29B, 33B, 33C). We measured the production of type I IFNs by purified leukocyte subsets (pDCs, mDCs, monocytes, B cells, T cells), in response to TLR7, TLR8 and TLR9 agonists (Fig. 29C, Fig. 33D). We confirmed that pDCs produced 100-1,000 times more type I IFN per cell than other leukocyte subsets upon TLR7 stimulation (Fig.29C, Fig.33D). We purified pDCs from P7 and P11 and analyzed their production of type I IFNs in response to CL264 and class C CpG oligonucleotide (CpG-c), relative to that of pDCs from healthy relatives, using a cytometric bead array (CBA) (Fig. 29D). pDCs from P7 and P11 did not produce type I IFNs upon stimulation with a TLR7 agonist, whereas they responded to a TLR9 agonist (Fig. 29D, 29E). Moreover, agonist- induced upregulation of PD-L1 and CD80 defines the maturation of pDCs into the S1 (PD- L1 high/ CD80 low ), S2 (PD-L1 high/ CD80 high ), and S3 (PD-L1 low/ CD80 high ) subsets (44). This maturation was not observed in the pDCs of P7 and P11, but was detected in the pDCs of heathy relatives and controls (Fig.29E, Fig.33E). Thus, pDCs from patients with TLR7 mutations do not respond to TLR7 agonists in terms of maturation into specialized subsets and type I IFN production. [000355] The patients’ pDCs respond poorly to SARS-CoV-2 [000356] A plausible mechanism accounting for the severity of COVID-19 in TLR7-deficient patients is the impairment of type I IFN production by pDCs upon stimulation with SARS-CoV-2, which can enter these cells, but cannot replicate productively within them (44, 45). Indeed, we previously showed that the activation of human pDCs by SARS-CoV-2 depends on IRAK4 and UNC- 93B, but not TLR3 (44). We tested the hypothesis that TLR7 is an essential pDC sensor of SARS-CoV- 2, upstream from IRAK4 and UNC-93B, by infecting pDCs and pDC-depleted leukocytes from healthy controls and TLR7-deficient patients with SARS-CoV-2 for 24 hours. Control pDC-depleted leukocytes infected with SARS-CoV-2 displayed no significant up- or downregulation of gene expression (Fig. 34A). By contrast, transcriptomic analysis showed a strong upregulation of the type I IFN transcriptional module in pDCs from healthy controls, which was greatly reduced in pDCs from TLR7- deficient patients (Fig.30A). Induction of the 17 type I IFN genes in pDCs from TLR7-deficient patients was 10 to 100 times weaker than that in pDCs from healthy individuals (Fig. 30B, 34B). We also analyzed the functional specialization of pDC subsets (S1-, S2-, and S3-pDC subsets) in response to SARS-CoV-2 activation (44, 46). pDCs from P11 cultured with SARS-CoV-2 for 24 hours displayed abnormally low levels of maturation into the S1-subset —the pDC subset principally responsible for IFN-α production upon SARS-CoV-2 infection (Fig. 34C). Finally, we evaluated the amount of type I IFN secreted by SARS-CoV-2-infected pDCs. All 13 individual IFN-α forms were produced in significantly smaller amounts by TLR7-deficient pDCs than by control pDCs (Fig. 30C, 34D). However, IFN-α production by TLR7-deficient pDCs upon SARS-CoV-2 was impaired, but not entirely abolished, as in UNC93B- or IRF7-deficient pDCs (8, 44), implying that there are also TLR7- independent sensors of SARS-CoV-2 in pDCs. Thus, SARS-CoV-2 triggers type I IFN induction in pDCs in a manner that is dependent on TLR7, but not exclusively so. As pDCs are normally the main leukocytes producing type I IFN in such conditions, and type I IFN is essential for protective immunity to SARS-CoV-2 (8, 9), these findings suggest that XR TLR7 deficiency underlies critical COVID-19 pneumonia by disrupting TLR7-and pDC-dependent type I IFN production. [000357] Concluding remarks [000358] We report XR TLR7 deficiency as a genetic etiology of critical COVID-19 pneumonia in 14 unrelated male patients, aged 13 to 71 years, from six countries. Only one of these 14 patients (7%) was older than 70 years, consistent with our previous observation that only two of 23 patients (8.7%) with inborn errors of TLR3-dependent type I IFN immunity were older than 65 years (8). This suggests that these genetic defects are mostly found in the youngest patients. This contrasts with the auto-Abs to type I IFNs, which are found mostly in patients older than 70 years, and not in patients with inborn errors of TLR3- or TLR7-dependent type I IFN ( (8, 9); Bastard P. et al. accompanying report, Examples 2 and 4). TLR7-deficient patients accounted for about 1.6% of the male patients with critical COVID- 19 pneumonia below the age of 70 years in our cohort. This discovery provides a first explanation for the higher risk of critical disease in men than in women under the age of 70 years, complementing our previous observation of a much higher frequency of neutralizing auto-Abs against type I IFNs in men than in women with critical COVID-19 pneumonia for patients over the age of 65 years (9). Previously reports of patients with critical COVID-19 pneumonia due to inborn errors of TLR3-dependent type I IFN immunity (8), including autosomal recessive IRF7 or IFNAR1 deficiency (5, 6), or due to auto- Abs neutralizing type I IFNs (9, 11-14, 16, 17), strongly suggest that critical disease in TLR7-deficient patients is a consequence of impaired type I IFN production upon SARS-CoV-2 infection. The absence of biochemically deleterious X-linked TLR8 variants in our cohort of patients (Fig. 35) suggests that TLR8 is not essential for host defense against SARS-CoV-2. This is consistent with the modest capacity of TLR8 to induce type I IFN and its lack of expression on pDCs (26), and with the inflammatory phenotype of TLR8 gain-of-function mutations, which do not underlie a type I interferonopathy (28- 30). Patients with inherited IRAK4 or MyD88 deficiency, whose cells do not respond to the stimulation of IL-1Rs and TLRs other than TLR3, including TLR7, are predicted to be vulnerable to SARS-CoV-2 (47, 48). However, they have not been reported to display any severe viral illness over the almost 20 years since the discovery of IRAK-4 deficiency (49-51). This is intriguing, as strong negative selection operates at the human TLR7, TLR8, and TLR9 loci (49, 52). Our study provides a first answer to this riddle, by establishing that TLR7 is essential for protective immunity to SARS-CoV-2. As critical COVID-19 and seasonal influenza can be caused by inborn errors of TLR3-dependent type I IFN immunity (5-8), it is tempting to speculate that TLR7 might also be essential for host defense against more virulent, pandemic influenza viruses. [000359] Through the discovery of the essential nature of TLR7 for the induction of type I IFN in response to SARS-CoV-2, our study also reveals the essential function of human pDCs in host defense. The constitutively high levels of IRF7 expression of these cells make them the most potent producers of type I IFN in the blood, and perhaps in the entire human body, and this has long suggested a possible key role in antiviral immunity (24). However, the essential and redundant roles of this particular leukocyte subset have yet to be determined, in the absence of human pDC-specific deficiencies causally underlying a clinical phenotype. It has long been suspected, but never proved, that pDCs are essential for host defense (25, 53-55). Inherited IRF7 deficiency, which underlies critical influenza or COVID- 19 pneumonia, disrupts the production of type I IFNs not only by pDCs (5, 8), but also by all other cell types, not only in the blood, but also in other tissues, including fibroblasts and pulmonary epithelial cells (5). Likewise, patients with GATA2 deficiency, who are prone to critical influenza (56), lack pDCs, but these patients also lack many other blood myeloid and lymphoid cell subsets, including monocytes, NK, and B cells (57-60). It therefore remains unclear whether IRF7 and GATA2 deficiencies confer a predisposition to critical viral pneumonia via their impact on pDCs or through effects on other leukocytes or pulmonary cells. Our study clarifies this matter, as TLR7 is expressed by only a few leukocyte subsets, including pDCs, which are the only potent type I IFN producers among these subsets, and TLR7 is not expressed by pulmonary epithelial cells. The expression of both TLR7 and IRF7 is a feature unique to pDCs (61, 62). While inborn errors of TLR3 underlie critical COVID- 19 pneumonia by impairing the production of type I IFNs by cells other than pDCs, such as pulmonary epithelial cells (5-8, 63), inborn errors of TLR7 are pathogenic by impairing the production of type I IFNs by pDCs. Inborn errors of IRF7 and IFNAR1 have a broader impact, on both pulmonary cells and pDCs (5, 6, 8). pDCs express other viral sensors, including TLR9 (for DNA), MDA5 and RIG-I (for dsRNA) (64), but TLR7 deficiency impairs their capacity to respond to SARS-CoV-2 by producing sufficient quantities of type I IFNs. Our findings therefore indicate that both human TLR7 and pDCs are essential for type I IFN-dependent protective immunity to SARS-CoV-2. [000360] Methods [000361] Cohort recruitment and consent [000362] This study included 1,005 male patients with life-threatening COVID-19 pneumonia, defined as patients with pneumonia who developed critical disease, whether pulmonary with high-flow oxygen or mechanical ventilation (CPAP, BIPAP, intubation), septic shock, or any other type of organ damage requiring ICU admission. Patients who developed Kawasaki-like syndrome were excluded. The age of the patients ranged from 0.5-99 years, with a mean age of 52.9 years (SD 15.6 years). Asymptomatic or paucisymptomatic individuals were recruited on the basis of positive PCR or serological tests for SARS-CoV-2 in the absence of symptoms. These individuals were close contacts of patients or were recruited after clinical screening. The age of the asymptomatic or paucisymptomatic individuals ranged from 1.3-102 years, with a mean age of 41.1 years (SD: 16.1 years). [000363] All the enrolled subjects provided written informed consent and were collected through protocols conforming to local ethics requirements. For patients enrolled in the French COVID cohort (clinicaltrials.gov NCT04262921), ethics approval was obtained from the CPP IDF VI (ID RCB: 2020- A00256-33) or the Ethics Committee of Erasme Hospital (P2020/203). For subjects enrolled in the COV-Contact study (clinicaltrials.gov NCT04259892), ethics approval was obtained from the CPP IDF VI (ID RCB: 2020-A00280-39). For patients enrolled in the Italian cohort, ethics approval was obtained from the University of Milano-Bicocca School of Medicine, San Gerardo Hospital, Monza – Ethics Committee of the National Institute of Infectious Diseases Lazzaro Spallanzani (84/2020) (Italy), and the Comitato Etico Provinciale (NP 4000 – Studio CORONAlab). STORM-Health care workers were enrolled in the STudio OsseRvazionale sullo screening dei lavoratori ospedalieri per COVID-19 (STORM-HCW) study, with approval from the local IRB obtained on June 18, 2020. Patients and relatives from San Raffaele Hospital (Milan) were enrolled in protocols COVID-BioB/Gene-COVID and, for additional studies, TIGET-06, which were approved by local ethical committee. For patients enrolled in Spain, the study was approved by the Committee for Ethical Research of the Infanta Leonor University Hospital, code 008-20, Committee for Ethical Research of the University Hospital 12 de Octubre, code 16/368 and the Bellvitge University Hospital code PR127/20, the University Hospital of Gran Canaria Dr. Negrín code 2020-200-1 COVID-19 and the Vall d’Hebron University Hospital, code PR(AMI)388/2016. Anonymized samples were sequenced at the NIAID through USUHS/TAGC under non-human subject research conditions; no additional IRB consent was required at the NIH. [000364] Next-generation sequencing [000365] Genomic DNA was extracted from whole blood. For the 1,331 patients included, the whole exome (n=934) or whole genome (n=397) was sequenced at several sequencing centers, including the Genomics Core Facility of the Imagine Institute (Paris, France), the Yale Center for Genome Analysis (USA), the New-York Genome Center (NY, USA), and the American Genome Center (TAGC, USUHS, Bethesda, USA), and the Genomics Division-ITER of the Canarian Health System sequencing hub (Canary Islands, Spain). [000366] For WES, libraries were generated with the Twist Bioscience kit (Twist Human Core Exome Kit), the xGen Exome Research Panel from Integrated DNA Technologies (IDT xGen), the Agilent SureSelect V7 kit or the SeqCap EZ MedExome kit from Roche, and the Nextera Flex for Enrichment-Exome kit (Illumina). Massively parallel sequencing was performed on a HiSeq4000 or NovaSeq6000 system (Illumina). For WES analysis performed at CNAG Barcelona, Spain, capture was performed with the SeqCap EZ Human Exome Kit v3.0 (Roche Nimblegen, USA) and 100-bp paired- end read sequences were obtained on a HiSeq 2000-4000 platform (Illumina, Inc. USA). For the OSR Italian cohort, WES was performed with the Agilent SureSelect V7 kit on a NovaSeq6000 system (Illumina). [000367] For WGS on patients of the Italian cohort (TAGC), genomic DNA samples were dispensed into the wells of a Covaris 96 microTUBE plate (1,000 ng per well) and sheared with the Covaris LE220 Focused-ultrasonicator, at settings targeting a peak size of 410 bp (t:78; Duty:18; PIP:450; 200 cycles). Sequencing libraries were generated from fragmented DNA with the Illumina TruSeq DNA PCR-Free HT Library Preparation Kit, with minor modifications for automation (Hamilton STAR Liquid Handling System), with IDT for Illumina TruSeq DNA UD Index (96 indices, 96 samples) adapters. Library size distribution was assessed and the absence of free adapters or adapter dimers was checked by automated capillary gel electrophoresis (Advanced Analytical Fragment Analyzer). Library concentration was determined by qPCR with the KAPA qPCR Quantification Kit (Roche Light Cycler 480 Instrument II). Sequencing libraries were normalized and combined as 24-plex pools and quantified as above, before dilution to 2.9 nM and sequencing on an Illumina NovaSeq 6000 with the S4 Reagent Kit (300 cycles) and 151+8+8+151 cycle run parameters. Primary sequencing data were demultiplexed with the Illumina HAS2.2 pipeline and sample-level quality control was performed for base quality, coverage, duplicates and contamination (FREEMIX < 0.05 by VerifyBamID). [000368] We used the Genome Analysis Software Kit (GATK) (version 3.4-46 or 4) best-practice pipeline to analyze our WES data (65). We aligned the reads obtained with the human reference genome (hg19), using the maximum exact matches algorithm in the Burrows–Wheeler Aligner (BWA) (66). PCR duplicates were removed with Picard tools (picard.sourceforge.net). The GATK base quality score recalibrator was applied to correct sequencing artifacts. Genotyping was performed with GATK GenotypeGVCFs in the interval intersecting all the capture kits ± 50 bp. Sample genotypes with a coverage < 8X, a genotype quality (GQ) < 20, or a ratio of reads for the less covered allele (reference or variant allele) over the total number of reads covering the position (minor read ratio, MRR) < 20% were filtered out. We filtered out variant sites (i) with a call rate <50% in gnomAD genomes and exomes, (ii) a non-PASS filter in the gnomAD database, (iii) falling in low-complexity or decoy regions, (iv) that were multi-allelic with more than four alleles, (v) with more than 20% missing genotypes in our cohort, and (vi) spanning more than 20 nucleotides. Variant effects were predicted with the Ensembl Variant Effect Predictor (VEP) (67) and the Ensembl GRCh37.75 reference database, retaining the most deleterious annotation obtained from Ensembl canonical transcripts overlapping with RefSeq transcripts. [000369] Statistical analysis [000370] We performed an enrichment analysis focusing on X chromosome genes on our cohort of 1,005 male patients with life-threatening COVID-19 pneumonia without known inborn errors of TLR3- and IRF7-dependent type I IFN immunity (8) and without neutralizing auto-Abs against type I IFNs (9), and 326 male individuals with asymptomatic or paucisymptomatic infection (Table 12). We considered variants that were predicted to be loss-of-function or missense, with a MAF below 0.0001 (gnomAD v2.1.1). We compared the proportion of patients and controls carrying at least one nonsynonymous variant by logistic regression and likelihood ratio tests. We accounted for the ethnic heterogeneity of the cohorts by including the first five principal components of the principal component analysis (PCA) in the logistic regression model. Analyses were also adjusted for age. We checked that our adjusted burden test was well-calibrated by also performing an analysis of enrichment in rare (MAF < 0.0001) synonymous variants. PCA was performed with Plink v1.9 software on whole-exome and whole-genome sequencing data, with the 1000 Genomes (1kG) Project phase 3 public database as a reference, using 18,917 exonic variants with a minor allele frequency > 0.01 and a call rate > 0.99. [000371] Cell culture [000372] EBV-transformed B-lymphocyte (B-EBV) cell lines derived from the patients were grown in complete RPMI 1640 (Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum (FBS). HEK293T cells, derived from the human embryonic kidney 293 cell line, which expresses a mutant version of the SV40 large T antigen, were grown in complete DMEM (Life Technologies) supplemented with 10% FBS. Cells were incubated at 37°C in the presence of 5% CO2. [000373] Expression vectors and transfection experiments [000374] All the TLR7 variants in our analysis were generated by site-directed mutagenesis (Table 18). The WT or variant alleles were re-introduced into a Myc-DDK-pCMV6 vector (Origene). HEK293T cells, which have no endogenous TLR7 or TLR8 expression, were transfected with the Myc- DDK-pCMV6 vector, empty or containing the WT or a variant allele, in the presence of X- tremeGENE™ 9 DNA Transfection Reagent (Sigma-Aldrich), according to the manufacturer’s instructions.
1119-69PCT TABLE 18. TLR7 Variant Primer Sequences for the Site-Directed Mutagenesis. Variant Sequence 5' to 3' Forward primer Reverse primer CTTCTTGGATTTTTGCAATC (SEQ ID NO:8) CTTCTTGGATTTTTGCAATC (SEQ ID NO:10) TGGTCCTGCTGGTGG (SEQ ID NO:12) ATTTTAGCAGATTCTTGAATAATTG(SEQ ID ATCTTGGGGGCACATGCTG (SEQ ID NO:16) TCCTCTGAATTCCAGAGTTC (SEQ ID NO:18) AGCAACATCTTC (SEQ ID NO:20) TTTTTACTTAGATCCAAGGTCTG (SEQ ID GTCTAATTCCTCTAATTTTAGCAGATTC(SEQ ID CCAAAGAGAGATTCTTTAGATTTGG(SEQ ID TAAAGGGATTTTAAATAAG(SEQ ID NO:28) AAGTAGATGGCAAAACAGTAGG(SEQ ID NO:30) ATCACATCAAC(SEQ ID NO:32) ACACGGCGCACAAGGAAATGG(SEQ ID NO:34) CAAGGCTGAGAAGCTG(SEQ ID NO:36) GACAGTGGTCAGTTGGTTGTG(SEQ ID NO:38) GCCCCACACAAG(SEQ ID NO:40) GTGGCTGAGGTCCAAAG(SEQ ID NO:42) CCACACAGCATCACAGGTG(SEQ ID NO:44)
[000375] Western blotting [000376] For whole-cell extracts, the cells were lysed by incubation in the following buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 % NP40), supplemented with a mixture of protease inhibitors (Sigma-Aldrich), for 30 minutes at 4 °C. The lysates were then centrifuged at 21,000 x g for 20 minutes at 4 °C. The supernatants were processed directly for western blotting. Western blotting was performed on 10 µg of total extract from transfected HEK293T cells, with monoclonal antibodies specific for the leucine-rich repeats within the human TLR7 protein (Cell Signaling Technology). [000377] RNA extraction and reverse transcription-quantitative PCR (RT-qPCR) [000378] Total RNA was extracted with the RNeasy Mini Kit (Qiagen), according to the manufacturer’s instructions. Reverse transcription was performed on 1 µg of RNA with random primers and the SuperScript ® III reverse transcriptase (Invitrogen), according to the manufacturer’s protocol. Quantitative PCR was then performed with the TaqMan™ Fast Universal PCR Master Mix (2X) and the FAM-MGB TaqMan TNF exons 1-2 (Hs99999-43_m1) probes. The VIC-TAMRA probe for GUSB (Applied Biosystems, Cat: 4310888E) was used as an endogenous control. Real-time PCR amplification was monitored with the 7500 Fast Real-Time PCR System (Applied Biosystems). Relative expression levels were determined according to the ∆Ct method. [000379] Luciferase reporter assay [000380] HEK293T cells, which have no endogenous TLR7 expression, were transfected with the pCMV6 vector bearing wild-type or variant TLR7 (50 ng), the reporter construct pGL4.32 (100 ng), and an expression vector for Renilla luciferase (10 ng), with the X-tremeGENE™ 9 DNA Transfection Reagent kit (Sigma-Aldrich). The pGL4.32 [luc2P/NF-κB-RE/Hygo] (Promega) reporter vector contains five copies of the NF-κB-responsive element (NF-κB-RE) linked to the luciferase reporter gene luc2P. After 24 hours, the transfected cells were left unstimulated or were stimulated with 1 µg/mL R848 (Resquimod), for activation via TLR7/8 (Invivogen), or 5 µg/mL R837 (Imiquimod) (Invivogen), or 5 µg/mL CL264 (Invivogen), human TLR7-specific agonists, for 24 hours. Relative luciferase activity was then determined by normalizing the values against the firefly:Renilla luciferase signal ratio. [000381] ELISA analysis of TNF production in B-EBV cells [000382] ELISA was performed as previously described (68). We suspended 1x10 6 EBV-B cells per well in RPMI 1640 supplemented with 10% FBS. The cells were activated by incubation with 1 µg/mL R848, and 5 µg/mL imiquimod for 24 hours. The supernatants were harvested after 24 hours of activation. ELISA determinations of TNF in cell culture supernatants were performed with a kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. [000383] Stable transduction [000384] The WT coding sequence of TLR7 was inserted into pTRIP-CMV-puro-2A. For lentivirus production, HEK293T cells were transfected with 1.6 µg pTRIP-CMV-puro-2A-TLR7-WT (or Mutant: K684*), 0.2 µg pCMV-VSV-G (Addgene), 0.2 µg pHXB2 (NIH-AIDS Reagent 22 Program) and 1 µg psPAX2 (Addgene), with X-treme gene 9 (Roche), according to the manufacturer's instructions. Supernatants were harvested after 24 hours and 8 µg/mL protamine sulfate was added. The lentiviral suspension obtained was used to transduce 2x10 5 EBV-B cells by spinoculation at 1,200 x g for 2 hours. The transduced cells were selected by incubation on medium containing 1 µg/mL puromycin for two days. The cells were then selected by incubation for a further two days on medium containing 2 µg/mL puromycin. During viral transduction, the cells were cultured with 5 µM IRAK4 inhibitor (PF06650833) (Bio-techne) to prevent cell death due to the overproduction of TLR7. Selected transduced cells were then stimulated with 1 µg/mL R848 or 5 µg/mL imiquimod for 24 hours without IRAK4 inhibitor. The supernatants were harvested after 24 hours of activation. ELISA determinations of TNF in cell culture supernatants were performed with a kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. [000385] Deep immunophenotyping by mass cytometry (CyTOF) [000386] CyTOF was performed on whole blood with the Maxpar Direct Immune Profiling Assay (Fluidigm), according to the manufacturer’s instructions. Cells were frozen at -80 °C after overnight staining to eliminate dead cells, and acquisition was performed on a Helios machine (Fluidigm). All the samples were processed within 24 hours of sampling. Data analysis was performed with OMIQ software. [000387] PBMC enrichment using MACS system [000388] Blood were collected from two healthy individuals and separated by the concentration gradient method with Ficoll® Paque Plus (Cytiva). After isolations of PBMCs, leucocyte subset (T cell, B cell, monocyte, pDC, and mDC) were purified by negative selection using MACS beads system (Milteni Biotec). Cells were plated into a U-bottomed 96-well plate at a density of 2×10 4 cells/well for T cells, B cells, monocytes, pDCs, or mDCs in 200 µL/well RPMI-1640 with GlutaMAX supplemented with 10% FBS or 10×10 4 cells/well for whole blood and PBMCs. Cells were left unstimulated or stimulated with 1µg/mL CL264, 100ng/ml TL8-506 (Invivogen), 1µg/mL R848, 2µM CpG-c (Invivogen), or 12.5ng/ml PMA and 0.125µM ionomycin for 24 hours. The supernatants were harvested after 24 hours of activation. Cytokines production were determined by ELISA (IFN-α - PBL Assay Science, IFN-β- PBL Assay Science, IFN- λ1 (IL-29) - Invivogen, IFN-ω- Invitrogen or IL-8 - R&D SYSTEMS); according to the manufacturer’s instructions. [000389] Analysis for TLR7 and TLR8 expression pattern in PBMCs by flow cytometry [000390] Freshly thawed PBMCs from healthy donors were dispensed into a V-bottomed 96-well plate at a density of 1×10 6 cells/well, in 200 µL PBS/well. In brief, cells were stained by incubation with the LIVE/DEAD fixable blue dead-cell staining kit (Thermo Fisher Scientific, 1:800) and FcR blocking reagent (Miltenyi Biotec, 1:25) on ice for 15 min. For surface staining, cells were incubated with anti-γδTCR-BUV611 (BD Biosciences, 1:50), anti-CD183-BV750 (BD Biosciences, 1:20), and anti-CD194-BUV615 (BD Biosciences, 1:20) antibodies on ice for 30 min in 0.1% BSA and 0.01% sodium azide in PBS. They were then incubated with anti-CD141-BB515 (BD Biosciences, 1:40), anti- CD57-FITC (Biolegend, 1:83), anti-TCR Vδ2-PerCP (Biolegend, 1:166), anti-TCR Vα7.2- PerCP/Cyanine5.5 (Biolegend, 1:40), anti-TCR Vd1-PerCP-Vio 700 (Miltenyi Biotec, 1:100), anti- CD14-Spark Blue 550 (Biolegend, 1:40), anti-CD1c-Alexa Fluor 647 (Biolegend, 1:50), anti-CD38- APC/Fire 810 (Biolegend, 1:30), anti-CD27-APC-H7 (BD Biosciences, 1:50), anti-CD127-APC-R700 (BD Biosciences, 1:50), anti-CD19-Spark NIR 685 (Biolegend, 1:83), anti-CD45RA-BUV395 (BD Biosciences, 1:83), anti-CD16-BUV496 (BD Biosciences, 1:166), anti-CD11b-BUV563 (BD Biosciences, 1:100), anti-CD56-BUV737 (BD Biosciences, 1:83), anti-CD8-BUV805 (BD Biosciences, 1:83), anti-hMR1-BV421 (NIH tetramer facility, 1:100), anti-CD11c-BV480 (BD Biosciences, 1:40), anti-CD45-BV510 (Biolegend, 1:83), anti-CD33-BV570 (Biolegend, 1:83), anti- iNKT-BV605 (Biolegend, 1:25), anti-CD161-BV650 (BD Biosciences, 1:25), anti-CCR6-BV711 (Biolegend, 1:83), anti-CCR7- BV785 (Biolegend, 1:40), anti-CD3-Pacific Blue (Biolegend, 1:83), anti-CD20-Pacific Orange (Life Technologies, 1:50), anti-CD123-Super Bright 436 (Invitrogen, 1:40), anti-CD24-PE-Alexa Fluor 610 (Life Technologies, 1:25), anti-CD25-PE-Alexa Fluor 700 (Life Technologies, 1:25), anti-CD294-Biotin (Invitrogen, 1:50), anti-CD209-PE/Cyanine7 (Biolegend, 1:25), anti-CD117-PE/Dazzle 594 (Biolegend, 1:83), anti-HLA-DR-PE/Dazzle 810 (Biolegend, 1:50), and anti-CD4-cFluor TM YG584 (Cytek, 1:83) antibodies on ice for at least 30 min. The cells were then washed and stained by incubation with streptavidin-PE/Cy5 (Biolegend, 1:3000) on ice for 30 min. The cells were then fixed and permeabilized for intracellular staining with anti-hTLR7-PE (R&D Systems) and anti-TLR8-APC (Biolegend) antibodies, with the eBioscience Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), according to the manufacturer’s instructions. The cells were then washed and acquired with a five-laser Cytek Aurora (Cytek) flow cytometer. [000391] pDC activation [000392] Freshly sorted pDCs were cultured in 96-well plates at a concentration of 5 x 10 5 cells per mL in the presence of medium alone (RPMI 1640 Medium with GlutaMAX, 10% FBS, 1% MEM NEAA, 1% sodium pyruvate, and 1% penicillin/streptomycin), CL264 (Invivogen, 1 µg/mL), or the SARS-CoV-2 primary strain 220_95 (44) at a multiplicity of infection (MOI) of 1. After 24 h of culture, the pDC supernatant was collected for cytokine quantification, and the PDCs were collected for diversification assessment by flow cytometry. In some experiments, RNA was purified from the pDCs with the RNeasyMicro kit (Qiagen) and IFN responses were analyzed by RNAseq (low-input SMARTer kit (Takara) and sequencing on an Illumina NextSeq 500). [000393] Flow cytometry analysis for human pDCs [000394] For assessments of pDC diversification, cells were stained with Zombie Violet fixable viability dye (Biolegend), BV711 anti-CD123 (Biolegend, clone 6H6), PE anti-CD80 (BD, clone L307.4), and PerCP-efluor 710 anti-PD-L1 (eBioscience, clone MIH1) antibodies. Data were acquired with an LSR Fortessa (BD Biosciences) flow cytometer, and analyzed with FlowJo software (Tree Star). Flow cytometry analyses were performed at the flow cytometry core facility of IRSL (Paris, France). 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[000398] This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein. [000399] Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety.
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