NFKB2 Haploinsufficiency Identified Via Screening For IFN-a2 Autoantibodies in Children And Adolescents Hospitalized With SARS-CoV-2–related Complications

Background: Autoantibodies against type I IFNs occur in approximately 10% of adults with life-threatening coronavirus disease 2019 (COVID-19). The frequency of anti-IFN autoantibodies in children with severe sequelae of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is unknown.

Objective: We quantified anti–type I IFN autoantibodies in a multicenter cohort of children with severe COVID-19, a multisystem inflammatory syndrome in children (MIS-C), and mild SARS-CoV-2 infections.

cistanche supplement benefits-increase immunity

Methods: Circulating anti–IFN–a2 antibodies were measured by a radioligand binding assay. Whole-exome sequencing, RNA sequencing, and functional studies of peripheral blood mononuclear cells were used to study any patients with levels of anti–IFN–a2 autoantibodies exceeding the assay’s positive control.

Results: Among 168 patients with severe COVID-19, 199 with MIS-C, and 45 with mild SARS-CoV-2 infections, only 1 had high levels of anti–IFN–a2 antibodies. Anti–IFN–a2 autoantibodies were not detected in patients treated with intravenous immunoglobulin before sample collection. Whole exome sequencing identified a missense variant in the ankyrin domain of NFKB2, encoding the p100 subunit of nuclear factor kappa–light-chain enhancer of activated B cells, aka NF-kB, essential for noncanonical NF-kB signaling. The patient’s peripheral blood mononuclear cells exhibited impaired cleavage of p100 characteristic of NFKB2 haploinsufficiency, an inborn error of immunity with a high prevalence of autoimmunity. Conclusions: High levels of anti–IFN–a2 autoantibodies in children and adolescents with MIS-C, severe COVID-19, and mild SARS-CoV-2 infections are rare but can occur in patients with inborn errors of immunity. (J Allergy Clin Immunol 2023;151:926-30.)

cistanche supplement benefits-how to strengthen immune system

Key words: Anti-interferon autoantibody, COVID-19, MIS-C, NFKB2, inborn errors of immunity

INTRODUCTION

Neutralizing autoantibodies against type I IFNs occurs in approximately 10% of adults with life-threatening coronavirus disease 2019 (COVID-19).1,2 Less is known about levels of antiIFN antibodies in pediatric populations. Small studies of 7 to 59 children identified autoantibodies to several tissue antigens in patients with multisystem inflammatory syndrome in children (MIS-C), a postinfectious inflammatory disorder typically occurring within 2 to 6 weeks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.3-6 Although inborn errors of immunity can be associated with autoantibodies,7 it is unknown if anti-IFN autoantibodies are associated with severe COVID-19 or MIS-C in the general pediatric population. Treatment of patients with MIS-C with intravenous immunoglobulin (IVIG), which can contain autoantibodies to tissue antigens, may confound the measurement of endogenous autoantibodies.8 Here we present results from a multicenter study investigating anti–IFN–a2 autoantibodies in a large cohort of children and adolescents with MIS-C, severe COVID-19, or mild SARS-CoV-2 infections.

cistanche tubulosa-improve immune system

RESULTS AND DISCUSSION

This study included 412 patients: 199 patients with MIS-C, 168 patients hospitalized for COVID-19 in an intensive care or step-down unit (henceforth referred to as severe COVID-19), and 45 with SARS-CoV-2 infections managed as outpatients. All but 2 patients were under 21 years of age (Table I). IVIG was administered to 137 patients (68.8%) with MIS-C and to 9 patients (5%) with severe COVID-19 before specimen collection. Critical care was required for 85.4% of patients with MIS-C and 69.6% of patients with severe COVID-19 (Table I). Five patients (3%) who were hospitalized for COVID-19 died; all with MIS-C survived. Anti–IFN–a2 autoantibodies were measured using an established radioligand binding assay (see the Methods in this article’s Online Repository at www.jacionline.org). The antibody index indicates the ratio of IFN-a2 protein precipitated by patient plasma normalized to the assay’s positive control.2 Plasma from 8 healthy adults served as negative controls (mean antibody index of 0.011). Positive disease controls included 6 individuals with anti–IFN–a2 autoantibodies due to autoimmune polyglandular syndrome type 1 (APS-1), a disease known to cause neutralizing anti–type I IFN autoantibodies.9,10 In our cohort, only 1 patient had an antibody index (3.73) exceeding that of the assay’s positive control and the levels previously found in adults with severe COVID-19 due to neutralizing anti–IFN–a2 antibodies2 (Fig 1). This patient’s sample was obtained before treatment with IVIG. Additionally, none of the other 137 patients who received IVIG before specimen collection had high levels of anti–IFN–a2 autoantibody, thus excluding IVIG as a confounding source of autoantibodies. Our study was not designed to quantify the incidence of anti–IFN–a2 autoantibodies in children with COVID-19 or MISC, as not all eligible children consented to enrollment. However, the largest study of anti-IFN autoantibodies in adults identified anti–IFN–a2 autoantibodies in 88 (8.9%) of 987 patients with severe COVID-19,1 which is higher than the 0.5% (n 5 1) with autoantibodies and severe COVID-19 in our cohort. The patient with high levels of anti–IFN–a2 autoantibodies was an adolescent female subject with acute hypoxemic respiratory failure due to severe COVID-19. Neutralizing antibodies to SARS-CoV-2 were not detected until the fifth day of hospitalization, rising to levels comparable to that of other patients with severe COVID-19 by day 12.11 Her respiratory failure resolved after 2 weeks of hospitalization, but she subsequently developed left ventricular dysfunction, which was treated with milrinone in the setting of persistently elevated inflammatory markers, prompting diagnostic consideration of MIS-C. Compared to other patients similarly treated for MIS-C, the patient’s peripheral blood mononuclear cells (PBMCs) exhibited reduced expression of differentially expressed genes in pathways of neutrophil degranulation, innate immune signaling, SARS-CoV-2 IFN-stimulated genes, and oncostatin M, an enhancer of type I IFN signaling (Fig 2, A). Reduced IFN signaling is found in adults with acute COVID-19, particularly those with anti–type I IFN autoantibodies.1,2 However, the reduced neutrophil degranulation and innate immune signaling in our patient contrasts with that of neutrophil and monocyte activation found in adults with severe COVID-196,12,13 and children with MIS-C,4,14 suggesting that additional factors beyond anti–IFN–a2 autoantibodies contributed to her immune dysregulation.

TABLE I. Patient characteristics of children with MIS-C, severe COVID-19 requiring intensive care unit or step-down unit hospital care, and outpatients with mild SARS-CoV-2 infections evaluated for anti–IFN–a2 autoantibodies

FIG 1. High levels of anti–IFN–a2 autoantibodies in children and adolescents with MIS-C, severe COVID-19, and mild SARS-CoV-2 infections are rare. Levels of anti–IFN–a2 were measured by radioligand binding assay. The dotted line represents the antibody index of the anti-Myc assay–positive control. P denotes the patient with high levels of anti–IFN–a2 autoantibodies. APS-1 was used as positive disease control. Medians and interquartile ranges are shown for the control (n 5 8) and APS-1 (n 5 6) cohorts.

In addition to prolonged hospitalization for COVID-19 followed by MIS-C, this patient had a history of influenza A pandemic H1N1/09 viral pneumonia requiring noninvasive ventilation. Immunologic evaluation after recovery from that hospitalization was notable only for reduced levels of IgG and IgA, although titers to tetanus and pneumococcus were normal (see Table E1 in the Online Repository available at www.jacionline.org). Genetic testing and immunoglobulin replacement were not initiated then, as she had no prior significant infections. Immunologic evaluation after recovery from COVID-19 and MIS-C in 2020 revealed panhypogammaglobu linemia, with reduced titers to pneumococcal subtypes. Whole-exome sequencing identified a heterozygous variant in the ankyrin domain of NFKB2 (p.Thr684Pro), encoding the p100 subunit of nuclear factor kappa–light-chain enhancer of activated B cells (aka NF-kB) essential for noncanonical NF-kB signaling. This variant is absent from the gnomAD database (gnomad.broadinstitute.org) and is predicted to be pathogenic, with a CADD score of 27.6. Structural modeling indicates that the Thr684Pro variant causes steric clash (Fig 2, B). After stimulation with anti-CD3, the patient’s PBMCs exhibited increased p100 levels and reduced p52 levels, indicative of impaired cleavage of p100 into its active form (Fig 2, C). This finding is consistent with the importance of the ankyrin domain for p100 ubiquitinylation and cleavage.15 All previously published missense mutations affect the protein’s C-terminal degron domain essential for p100 processing.16,17 The patient’s pan-hypogammaglobulinemia, anti–IFN–a2 autoantibodies, and susceptibility to severe viral infections indicate the deleterious effect of her NFKB2Thr683Pro variant. Her residual levels of p52 likely contributed to the sporadic nature of her infections, which emerged only when exposed to newly emerged pathogens during 2 pandemics. 

cistanche supplement benefits-increase immunity

Click here to view  Cistanche Enhance Immunity products

【Ask for more】 Email:cindy.xue@wecistanche.com /  Whats App:  0086 18599088692 /  Wechat:  18599088692

Similarly, all 3 previously reported patients with NFKB2 haploinsufficiency and COVID-19 required critical care.7,18,19 Autoimmunity, including anti-cytokine antibodies, occurs in up to 80% of patients with NFKB2 haploinsufficiency.17 Although we did not measure the neutralizing capacity of our patient’s anti–IFN–a2 autoantibodies, her antibody index exceeds that of autoantibodies with neutralizing capacity in published studies using the same assay in independent cohorts.2,20 Additionally, differentially expressed genes from her whole-blood transcriptome were reduced in pathways downstream of type I IFN signaling compared to other patients with MIS-C, further supporting a neutralizing effect of her anti–IFN–a2 autoantibodies. At the time of this study, she had no clinical evidence of autoimmunity beyond the anti–IFN–a2 autoantibodies. Because our study is limited by the identification of only a single patient with NFKB2 haploinsufficiency, future studies with larger cohorts are needed to determine the prevalence and levels of anti-IFN autoantibodies in patients with this disease. Our study underscores the rarity of high levels of anti–IFN–a2 antibodies in most children. While the majority of adults with severe COVID-19 and autoantibodies to type 1 IFNs have anti–IFN–a2 autoantibodies, Bastard et al1 have shown that a subset of individuals have only anti–IFN-v antibodies, for which we did not screen. In samples collected before the COVID-19 pandemic, neutralizing autoantibodies to IFN-a were identified in less than 0.3% of individuals younger than 69 years,1,21 compared to 1.1% of adults aged 70 through 79 years, and 3.4% in those over 80 years.21 Neutralizing autoantibodies to IFN occurred in 10% of individuals with severe COVID-19, the majority of whom were over 75 years of age. Other than APS-1, no inborn errors of immunity, including defects in the NFKB2 pathway, have been reported in adults with severe COVID-19 and autoantibodies to type I IFNs. Autoantibodies occur more frequently in the elderly as a result of progressive B-cell dysfunction, differentiation of age-associated B cells into autoantibody-producing plasma cells and the release of self-antigens from tissue damage. In children,anti-IFN autoantibodies may instead reflect early onset B-cell dysfunction with underlying immune dysfunction. In support of this, a study of 31 individuals with known inborn errors of immunity identified neutralizing autoantibodies against IFN-a2 and IFN-v in one child with a combined immunodeficiency and another with immune dysregulation.7 This previously published cohort had 3 additional pediatric patients with antinuclear antibodies, reflecting the spectrum of autoantibodies in patients with inborn errors of immunity. Thus, diagnostic studies for genetic causes of immune dysregulation are merited in children with anti-cytokine antibodies.

FIG 2. A, Differentially expressed genes (>_2-fold change, false discovery rate < 0.05) determined via bulk RNA sequencing of whole blood from the single patient with increased levels of anti–IFN-a2 autoantibodies, compared to 5 disease controls (patients with MIS-C of comparable age, disease severity, and treatment). The patient and one of the disease controls (labeled 2) had 2 samples available at the midpoint of their hoscapitalizations. B, Top, Schematic of NFKB2 with the patient’s variant indicated with a red triangle in the ankyrin repeat domain 6 (ANK6). DD, Death domain, with the degron domain needed for p100 processing noted in orange; RHD, Rel homology domain. Bottom, Steric clash (red disks) is predicted to arise from the patient’s substitution of a bulky proline residue for threonine 684. C, Representative immunoblot of full-length p100 and processed p52 in PBMCs from the patient (P) and 2 healthy controls (C1 and C2) with and without anti-CD3 stimulation for 2 days. Bar graphs show densitometric quantitation of indicated proteins pooled from 2 experiments in PBMCs from the patient and 5 controls, with and without 2 days of anti-CD3 stimulation. *P < .05, **P < .01 by Student t test.

We thank the patients and their families for participating in this study. We thank Michail S. Lionakis (National Institute of Allergy and Infectious Diseases) for providing positive control APS-1 serum, and the New York Blood Center for providing pre–COVID–19 control plasma. 

cistanche benefits for men-strengthen immune system

Support for document creation and format was provided by AuthorArranger (analysistools.cancer.gov/), a tool developed at the National Cancer Institute. The following members of the Overcoming COVID-19 Network Study Group Investigators (presented alphabetically by state) were all closely involved with the design, implementation, and oversight of the Overcoming COVID-19 study as well as collecting patient samples and data. Alabama: Children’s of Alabama, Birmingham: Michele Kong, MD. Arkansas: Arkansas Children’s Hospital, Little Rock: Katherine Irby, MD; Ronald C. Sanders Jr, MD, MS; Masson Yates; Chelsea Smith. California: UCSF Benioff Children’s Hospital Oakland, Oakland: Natalie Z. Cvijanovich, MD; UCSF Benioff Children’s Hospital, San Francisco: Matt S. Zinter, MD. Florida: Holtz Children’s Hospital, Miami: Brandon Chatani, MD; Gwenn McLaughlin, MD, MSPH. Georgia: Children’s Healthcare of Atlanta at Egleston, Atlanta: Keiko M. Tarquinio, MD. Illinois: Ann & Robert H. Lurie Children’s Hospital of Chicago, Chicago: Bria M. Coates, MD. Indiana: Riley Children’s Hospital, Indianapolis: Courtney M. Rowan, MD, MScr. Massachusetts: Boston Children’s Hospital, Boston: Adrienne G. Randolph, MD; Margaret M. Newhams, MPH; Suden Kucukak, MD; Tanya Novak, PhD; Hye Kyung Moon, MA; Takuma Kobayashi BS; Jeni Melo, BS; Cameron Young, BS; Sabrina R. Chen, BS; Janet Chou, MD. Michigan: University of Michigan C. S. Mott Children’s Hospital, Ann Arbor: Heidi R. Flori, MD, FAAP; Mary K. Dahmer, PhD. Minnesota: Mayo Clinic, Rochester: Emily R. Levy, MD, FAAP; Supriya Behl, MSc; Noelle M. Drapeau, BA. Missouri: Children’s Mercy Hospital, Kansas City: Jennifer E. Schuster, MD. Nebraska: Children’s Hospital & Medical Center, Omaha: Melissa L. Cullimore, MD, PhD; Russell J. McCulloh, MD. New Jersey: Cooperman Barnabas Medical Center, Livingston: Shira J. Gertz, MD. North Carolina: University of North Carolina, Chapel Hill: Stephanie P. Schwartz, MD; Tracie C. Walker, MD. Ohio: Akron Children’s Hospital, Akron: Ryan A. Nofziger, MD; Cincinnati Children’s Hospital, Cincinnati: Mary Allen Staat, MD, MPH; Chelsea C. Rohlfs, BS, MBA. Pennsylvania: Children’s Hospital of Philadelphia, Philadelphia: Julie C. Fitzgerald, MD, PhD, MSCE. South Carolina: MUSC Shawn Jenkins Children’s Hospital, Charleston: Elizabeth H. Mack, MD, MS; Nelson Reed, MD. Tennessee: Monroe Carell Jr Children’s Hospital at Vanderbilt, Nashville: Natasha B. Halasa, MD, MPH. Texas: Texas Children’s Hospital and Baylor College of Medicine, Houston: Laura L. Loftis, MD. Utah: Primary Children’s Hospital and University of Utah, Salt Lake City: Hillary Crandall, MD, PhD. Members of the US Centers for Disease Control and Prevention COVID-19 Response Team on Overcoming COVID-19 were Laura D. Zambrano, Ph.D., MPH, Manish M. Patel, MD, MPH, and Angela P. Campbell, MD, MPH.

REFERENCES

1. Bastard P, Rosen LB, Zhang Q, Michailidis E, Hoffmann HH, Zhang Y, et al. Autoantibodies against type I IFNs in patients with life-threatening COVID-19. Science 2020. 

2. van der Wijst MGP, Vazquez SE, Hartoularos GC, Bastard P, Grant T, Bueno R, et al. Type I interferon autoantibodies are associated with systemic immune alterations in patients with COVID-19. Sci Transl Med 2021. 

3. Consiglio CR, Cotugno N, Sardh F, Pou C, Amodio D, Rodriguez L, et al. The immunology of multisystem inflammatory syndrome in children with COVID-19. Cell 2020. 

4. Porritt RA, Binek A, Paschold L, Rivas MN, McArdle A, Yonker LM, et al. The autoimmune signature of hyperinflammatory multisystem inflammatory syndrome in children. J Clin Invest 2021;131:e151520. 

5. Gruber CN, Patel RS, Trachtman R, Lepow L, Amanat F, Krammer F, et al. Mapping systemic inflammation and antibody responses in multisystem inflammatory syndrome in children (MIS-C). Cell 2020;183:982-95.e14. 

6. de Cevins C, Luka M, Smith N, Meynier S, Magerus A, Carbone F, et al. A monocyte/dendritic cell molecular signature of SARS-CoV-2–related multisystem in-flammatory syndrome in children with severe myocarditis. Med (N Y) 2021;2: 1072-92.e7. 

7. Abolhassani H, Delavari S, Landegren N, Shokri S, Bastard P, Du L, et al. Genetic and immunologic evaluation of children with inborn errors of immunity and severe or critical COVID-19. J Allergy Clin Immunol 2022;150:1059-73. 

8. Burbelo PD, Castagnoli R, Shimizu C, Delmonte OM, Dobbs K, Discepolo V, et al. Autoantibodies against proteins previously associated with autoimmunity in adult and pediatric patients with COVID-19 and children with MIS-C. Front Immunol 2022. 

9. Bastard P, Orlova E, Sozaeva L, Levy R, James A, Schmitt MM, et al. Preexisting autoantibodies to type I IFNs underlie critical COVID-19 pneumonia in patients with APS-1. J Exp Med 2021. 

10. Meyer S, Woodward M, Hertel C, Vlaicu P, Haque Y, K€arner J, et al. AIRE-deficient patients harbor unique high-affinity disease-ameliorating autoantibodies. Cell 2016;166:582-95. 

11. Tang J, Novak T, Hecker J, Grubbs G, Zahra FT, Bellusci L, et al. Cross-reactive immunity against the SARS-CoV-2 Omicron variant is low in pediatric patients with prior COVID-19 or MIS-C. Nat Commun 2022;13:2979. 

12. Mann ER, Menon M, Knight SB, Konkel JE, Jagger C, Shaw TN, et al. Longitudinal immune profiling reveals key myeloid signatures associated with COVID-19. Sci Immunol 2020;5:eabd6197. 

13. Schulte-Schrepping J, Reusch N, Paclik D, Baßler K, Schlickeiser S, Zhang B, et al. Severe COVID-19 is marked by a dysregulated myeloid cell compartment. Cell 2020;182:1419-40.e23. 

14. Ramaswamy A, Brodsky NN, Sumida TS, Comi M, Asashima H, Hoehn KB, et al. Immune dysregulation and autoreactivity correlate with disease severity in SARSCoV-2–associated multisystem inflammatory syndrome in children. Immunity 2021;54:1083-95.e7. 

15. Vatsyayan J, Qing G, Xiao G, Hu J. SUMO1 modification of NF-kappaB2/p100 is essential for stimuli-induced p100 phosphorylation and processing. EMBO Rep 2008;9:885-90. 

16. Wirasinha RC, Davies AR, Srivastava M, Sheridan JM, Sng XYX, Delmonte OM, et al. Nfkb2 variants reveal a p100-degradation threshold that defines autoimmune susceptibility. J Exp Med 2021;218:e20200476. 

17. Klemann C, Camacho-Ordonez N, Yang L, Eskandarian Z, Rojas-Restrepo JL, Frede N, et al. Clinical and immunological phenotype of patients with primary immunodeficiency due to damaging mutations in NFKB2. Front Immunol 2019;10: 297. 

18. Meyts I, Bucciol G, Quinti I, Neven B, Fischer A, Seoane E, et al. Coronavirus disease 2019 in patients with inborn errors of immunity: an international study. J Allergy Clin Immunol 2021;147:520-31. 

19. Abraham RS, Marshall JM, Kuehn HS, Rueda CM, Gibbs A, Guider W, et al. Severe SARS-CoV-2 disease in the context of an NF-kB2 loss-of-function pathogenic variant. J Allergy Clin Immunol 2021;147:532-44.e1. 

20. Vazquez SE, Bastard P, Kelly K, Gervais A, Norris PJ, Dumont LJ, et al. Neutralizing autoantibodies to type I interferons in COVID-19 convalescent donor plasma. J Clin Immunol 2021;41:1169-71. 

21. Bastard P, Gervais A, Le Voyer T, Rosain J, Philippot Q, Manry J, et al. Autoantibodies neutralizing type I IFNs are present in;4% of uninfected individuals over 70 years old and account for;20% of COVID-19 deaths. Sci Immunol 2021;6: eabl4340.