key: cord-0726818-2qlxxt23
authors: Abraham, Roshini S.; Marshall, Joanna M.; Kuehn, Hye Sun; Rueda, Cesar M.; Gibbs, Amber; Guider, Will; Stewart, Claire; Rosenzweig, Sergio D.; Wang, Huanyu; Jean, Sophonie; Peeples, Mark; King, Tiffany; Hunt, W. Garrett; Honegger, Jonathan R.; Ramilo, Octavio; Mustillo, Peter J.; Mejias, Asuncion; Ardura, Monica I.; Shimamura, Masako
title: Severe SARS-CoV-2 Disease In The Context Of A NFκB2 Loss-Of-Function Pathogenic Variant
date: 2020-09-30
journal: J Allergy Clin Immunol
DOI: 10.1016/j.jaci.2020.09.020
sha: c7473c2a2f17fb08f8855d3ce63bea7d09ad64ff
doc_id: 726818
cord_uid: 2qlxxt23

Background SARS-CoV-2 is a novel coronavirus that emerged recently and has created a global pandemic. Symptomatic SARS-CoV-2 infection, termed Coronavirus disease 2019 (COVID-19), has been associated with a host of symptoms affecting numerous organ systems, including lungs, cardiovascular system, kidney, central nervous system, gastrointestinal tract and skin among others. Objective While several risk factors have been identified related to complications from and severity of COVID-19, much about the virus remains unknown. The host immune response appears to affect the outcome of disease. It is not surprising that patients with intrinsic or secondary immune compromise might be particularly susceptible to complications from SARS-CoV-2 infection. Pathogenic loss-of-function (LOF) or gain-of-function (GOF) heterozygous variants in NFκB2 have been reported to be associated with either a combined immunodeficiency (CID) or common variable immunodeficiency (CVID) phenotype. Methods We evaluated the functional consequence and immunological phenotype of a novel NFKB2 LOF variant in a 17y old male patient and describe the clinical management of SARS-CoV-2 infection in this context. Results This patient required a 2-week hospitalization for SARS-CoV-2 infection, including seven days of mechanical ventilation. We used biological therapies to avert potentially fatal acute respiratory distress syndrome and treat hyperinflammatory responses. The patient had an immunological phenotype of B cell dysregulation with decreased switched memory B cells. Despite the underlying immune dysfunction, he recovered from the infection with intense management. Conclusions This clinical case exemplifies some of the practical challenges in management of patients with SARS-CoV-2 infection, especially in the context of underlying immune dysregulation.

Since its discovery more than 30 years ago, the NFκB pathway has come to occupy a pivotal position in the function of the immune system, and mediates a variety of cellular processes including immune response, inflammation, cellular proliferation, and cell survival among others 1 3 . The non-canonical pathway, of which NFκB2 is a key player, involves activation through a specific set of receptors, including BAFF-R, CD40 and other TNFR family members, such as RANK. The NFκB2 pathway involves processing of the p100 precursor molecule to form the p52 protein, which complexes with RelB and results in nuclear translocation. The J o u r n a l P r e -p r o o f canonical pathway of NFκB is integral to the holistic function of the immune response, while the non-canonical pathway appears to play an adjunct role, and is relevant to specific aspects of adaptive immunity. Patients with loss-of-function variants in NFΚB2 have been described to have either a B cell defect with a common variable immunodeficiency (CVID)-like or combined immunodeficiency (CID) phenotype [4] [5] [6] . Though NFκB plays a critical role in B cell development and activation, and was first identified in activated B cells, its role is not exclusive to B cells, and it plays a significant role in multiple components of the immune system, both innate and adaptive 7 .

The newly described SARS-CoV-2 pathogen has rapidly spread worldwide, leading to a global pandemic and over one-half million deaths to date. The virus gains access to epithelial cells in the upper respiratory tract by initially binding to the ACE2 receptor 8, 9 . The early immune response to SARS-CoV-2 likely involves the innate immune system and sensing of viral RNA by Toll-like receptors (TLRs) and other pathogen recognition receptors (PRRs), based on parallels drawn from related viruses, such as SARS-CoV-1 and MERS-CoV (Middle East respiratory syndrome coronavirus) 10 . The activation of PRRs triggers various signaling cascades and production of cytokines. Type 1 interferons are key anti-viral cytokines 11, 12 , but in addition to these, IL-1b, IL-6, IL-18, TNFα and other proinflammatory cytokines and chemokines are secreted. The immune dysregulation observed in patients with severe SARS-CoV-2 infection suggests there is an imbalance between the host immune response and immune-evasion tactics of the pathogen [13] [14] [15] . The kinetics of the immune response and triggering of pro-inflammatory pathways leads to the observed "hyperinflammatory" phenotype or "cytokine storm" described in SARS-CoV-2 infection 16, 17 . Patients with acute SARS-Co-V-2 infection demonstrate pan-J o u r n a l P r e -p r o o f lymphopenia 18 , which is more profound in patients with severe disease compared to mild disease.

It is reasonable to postulate that in patients with primary immunodeficiencies (PIDs) and immune regulatory disorders (PIRDs), the range of disease from mild to severe would depend on the specific immune defect and immune response to the infection. Recently, a study of a small group of patients with humoral immune defects demonstrated that B cells might play a critical role in immune dysregulation in response to SARS-CoV-2 infection 19 . In this study, patients with agammaglobulinemia due to absent B cells (X-linked agammaglobulinemia, XLA or other causes of agammaglobulinemia) appeared to have a milder clinical course in contrast to CVID patients with hypogammaglobulinemia and dysregulated but detectable B cells, in whom disease was more severe necessitating multiple therapeutic agents and other life-saving measures. In a recent study, the use of a BTK inhibitor was shown to improve markers of the inflammatory response in COVID-19 patients 20 , which may represent a pharmacological phenocopy of BTK loss-of-function in XLA patients. However, in other patients with XLA, the disease course appears to be prolonged requiring adjunct therapy (Meyts I et al, manuscript submitted), suggesting that decreased mortality does not imply rapid recovery from infection. The data from XLA and CVID patients suggests that dysregulated B cells might contribute to the pathology of SARS-CoV-2 infection and that the absence of B cells or low immunoglobulins in itself may not necessarily predicate high mortality but may still result in increased morbidity.

There are several reports in the literature on the association of NFΚB2 variants with either CVID or CID. NFΚB2 loss-of-function (LOF) variants, located at the C-terminus of the protein, and associated with an autosomal dominant negative phenotype were shown to result in severe B cell deficiency with hypogammaglobulinemia and alopecia 4, 21, 22 . On the other hand, gain-of-J o u r n a l P r e -p r o o f function (GOF) variants in the NFκB2 gene resulted in a combined immunodeficiency (CID) phenotype in some patients with impaired T cell and B cell function 6 without endocrine anomalies or ectodermal dysplasia. This is in contrast to patients with the LOF variants who have been reported to have endocrine anomalies, manifesting as ACTH and growth hormone (GH) deficiencies, also called DAVID syndrome (anterior pituitary hormone deficiency with variable immunodeficiency) 4, 22 . A recent cohort analysis by Klemann et al. of several NFκB2 patients revealed that adrenal insufficiency was noted in less than half (21/50; 42%) of patients 5 . The penetrance and expressivity of NFκB2 variants has been previously shown to be variable, depending on the specific type of genetic variant present, but in the Klemann study, the overall penetrance of disease was high, as only 2/50 patients were totally asymptomatic 5 . Several of these patients had clear manifestations of CID with associated T cell dysfunction and autoimmunity. NK cell cytotoxic function has been reported to be impaired in NFκB2 p52 haploinsufficiency 23 and has also been shown to be adversely affected in SARS-CoV-2 infection with impaired degranulation and decreased production of key cytotoxic proteins and chemokines, and elevated serum IL-6 [24] [25] [26] . We describe here a patient with a novel NFKB2 LOF variant, who developed severe SARS-CoV-2 infection, which required a multi-pronged approach to effectively manage the disease and its associated complications. We anticipate that these findings will be informative in the management of patients with an underlying intrinsic immunodeficiency, particularly affecting the B and/or T cell compartment.

The patient was consented to an Institutional Review Board-approved protocol (#00000015) and specific consent was obtained for publication of study findings. All dates are written relative to first day of hospitalization (e.g. day#1 etc.). This assay targets two regions of nucleocapsid gene of SARS-CoV-2, N1 and N2, as well as a human housekeeping gene, RNaseP, to assess the quality of the specimen. Viral load data for this assay are described as the "N1" or "N2" RT-PCR, which are reported as "true" cycle threshold 

Serum IL-6 was assessed at multiple time-points using the ProQuantum ® human IL-6 assay (ThermoFisher). Briefly, the ProQuantum ® assay uses two oligonucleotide labels, which bind a pair of antibodies targeted against IL-6. PCR amplification utilizes the proximity ligation assay (PLA) principle, which measures the amount of fluorescent signal that is proportional to the amount of DNA produced after each amplification cycle. The cytokine level was quantitated J o u r n a l P r e -p r o o f using a recombinant IL-6 cytokine standard curve using the fluorescence threshold data at multiple time points.

NFκB pathway protein expression studies: PBMCs isolated from sodium heparin anticoagulated blood were stimulated with or without anti-CD3 antibody at 1µg/mL (eBioscience, #16-0037085) for 48 hours. Cell lysates were prepared and immunoblotted with anti-phospho-NFκB2 (Cell Signaling, #4810, S866/870), anti-NFκB2 (Cell Signaling, #37359), and anti-phospho-NFκBp65 (Cell Signaling, #3033, S536). T cell blasts were generated from PBMCs by stimulation with anti-CD3 and anti-CD28 (microgram/mL) in the presence of IL-2 (10ng/mL) for eight days. IL-2 was added every 2-3 days. Cell lysates were prepared and analyzed for full-length p100 and processed form, p52 of NFκB2. Immunoblotting of beta-actin (Cell Signaling, #4970) was used as a loading control. Images were acquired and analyzed with Image Studio ® software (LI-COR Biosciences, Lincoln, Nebraska). The data is shown as band intensities normalized to the beta-actin control by densitometry. The control data is represented as mean +/-SEM from three healthy controls.

The male patient, currently 18 y old, initially presented at 8 years of age with recurrent upper respiratory infections, pneumonia and recurrent orolabial herpes simplex, confirmed by PCR to be HSV1. Immunological evaluation (Table 1) 

The patient, at age 17 years, contacted his primary care provider 4 days prior to admission (PTA; day# -4) with a one-week history of nasal congestion and cough, and was prescribed amoxicillin for presumed sinusitis. The following day, he developed fever and anosmia. One day PTA, he was evaluated in the Emergency Department (ED), where he underwent viral testing for SARS-CoV-2; however, the test result was not immediately available and he was discharged from the ED. He returned the following day with worsening fever and respiratory distress, by which time the SARS-CoV-2 PCR result was positive, and he was hospitalized due to tachypnea, tachycardia, and hypoxemia with oxygen saturations of 95% mm Hg. Laboratories and microbiologic diagnostics ( showed normal adrenal function. Since the patient missed his maintenance dose of replacement J o u r n a l P r e -p r o o f immunoglobulin (subcutaneous Ig, SCIg) due to his acute illness, he received a dose on day#2, approximately 2.5 weeks from his last maintenance dose. The patient was not eligible for national clinical trials of the antiviral drug, remdesivir, due to his age (<18 years), but a patient emergency investigational new drug (eIND) application was approved by the FDA and treatment (200 mg IV x1, then 100 mg IV q24h) was initiated on day#3. On day#4, the patient's respiratory distress, hypoxemia, lymphopenia and inflammatory markers continued to worsen, and he was intubated and transferred to the pediatric intensive care service. He developed premature ventricular contractions, intermittent bigeminy, and nonspecific ST and T wave abnormalities, with normal cardiac enzymes and no evidence of myocarditis on echocardiogram. On day#5, due to concern for progression to a cytokine release syndrome, including ongoing fevers, new hypotension requiring inotropic support and stress-dose hydrocortisone, and increasing inflammatory markers (CRP, IL-6, and D-dimer), a single dose of the IL-6 receptor antagonist, tocilizumab (8 mg/kg) was administered. With these interventions, the fever resolved, and the CRP and procalcitonin began to improve. The patient received a dose (40g) of intravenous immunoglobulin (IVIG) on day#7. Due to stabilization of his blood pressure, inotropic support was discontinued on day#6, and improvement in respiratory status led to extubation on day#11.

On day#8, he developed a transaminitis of unclear etiology. Doppler ultrasound demonstrated patent hepatic veins with no evidence of thrombi, but abnormally decreased phasicity of flow, suggesting venous outflow resistance. Liver enzymes continued to rise, therefore, no more than the single dose of tocilizumab was given, and remdesivir was discontinued after the eighth dose of therapy (day#10). Liver enzymes peaked on day 16 after hospitalization (ALT=622 U/L) and improved thereafter. He was discharged from the hospital on day#15. identified. The patient received five doses (2 units/dose) of CP in total, which were administered every other day starting on day#13, with three doses administered after discharge, although the patient was improving clinically prior to CP administration, suggesting that the overall clinical management was effective even without CP. At 60+ days post-discharge, the patient is doing well without symptom recrudescence.

The PCR results at each time point from different specimen types are shown in Table 2 . The nasopharyngeal swab (NP) was positive for SARS-CoV-2 the day prior to hospitalization (day#-1). Viral RNA was also detectable from lower respiratory tract (LRT) specimens (day#6) and whole blood samples (days #2 and 5). Over the course of the remdesivir treatment, SARS-CoV-2 PCR cycle threshold (Ct) values increased (indicating decreased viral burden) from NP and whole blood specimens but did not change across two LRT specimens (Figure 3 ). SARS-CoV-2 RNA was no longer detected from blood beginning day#11. PCR was negative from NP samples on days #13-18, but as discussed in the clinical history, was again positive on day#20 with Ct (Table 3) . However, the absolute quantitation (cells/µL) was remarkable in that there was pan-lymphopenia observed during his hospitalization for SARS-CoV-2 at day#4 (Table 3) . By day#15, the CD45+ lymphocyte, and CD3+, CD4+ and CD8+ T cell counts rebounded to normal levels. The CD4/CD8 ratio was relatively skewed only in the acute infection sample (day#4). He had documented B cell lymphopenia at 10 months prior to his hospitalization for COVID-19 (Table 3) . During and after his hospitalization, his B cell count further decreased compared to his pre-infection assessment (Table 3) . His cytotoxic NK cells (CD16++CD56+) are also decreased, and while they plummeted at the time of the infection, they rebounded to baseline levels post-infection.

Detailed T cell subset phenotyping for naïve, memory, activated, exhausted and senescent T cells were unremarkable (data not shown) and did not show any substantive differences from healthy controls, with the exception of the acute T cell lymphopenia related to his infection and hospitalization from which he recovered. Similar findings in certain T cell subsets have also been described previously though there is variability in the patient cohorts and time points studied.

Assessment of T cell function 5 weeks post-hospitalization showed normal T cell proliferation to mitogens, antigens and stimulation with anti-CD3 (soluble) alone or with other co-stimulants (anti-CD28 and IL-2), further confirming a lack of a cellular immune defect in T cells, as has been described in some other patients with NFκB2 variants (Table 1) .

B cell subset phenotyping on the other hand, showed a persistent decrease in total memory, and particularly switched memory B cells, consistent with his underlying humoral immunodeficiency related to the heterozygous NFΚB2 C-terminus LOF variant ( Table 3 (Table   3 ). This result suggests that there is no impairment in expression of FOXP3, CD25 or CD127 proteins on Treg subsets, however, the frequency (%) of these cell populations is reduced, although there is quantitative recovery in the post-hospitalization sample. The decline in IL-6 levels after the initial spike from administration of tocilizumab correlated with a recent report on better prognosis in COVID-19 patients with lower IL-6 levels after treatment 30 .

J o u r n a l P r e -p r o o f

The patient was discharged from the hospital 2 weeks after initial admission for SARS-Co-V-2 infection. The rationale for administration of CP was to mitigate the severity of his acute and critical illness as well as to provide passive immunity against viral recrudescence or reinfection given his underlying humoral immunodeficiency and the presumed lack of anti-SARS-CoV-2 antibodies in contemporary replacement immunoglobulin products. CP donors were identified and the patient received five doses of CP starting at day#13. Assessment for SARS-CoV-2 antibodies was performed using a clinically validated qualitative IgG antibody test at multiple time points, including days #4, 11, 13 and 15 during hospitalization as well as at post-hospital days #20 and 22 after discharge. While the sample from day#4 showed no SARS-CoV-2 IgG antibodies, serologic evidence of antibodies was observed starting at day#11 prior to receipt of CP as well as after administration of CP ( Table 2 ). The positive antibody detection by two independent tests reduces the possibility of false-positive detection. The IgG antibody levels to SARS-CoV-2 were monitored through post-hospital day #22.

With the recent global outbreak of SARS-CoV-2, the concern for high-risk patients, such as those with underlying immune defects or other co-morbidities has been high. The spectrum of immunological anomalies associated with NFκB2 gene defects is varied and can manifest primarily as a humoral immunodeficiency (CVID-like) or a combined immunodeficiency, affecting both T and B cell compartments 5 . Some of the NFκB2-deficient patients with the CVID-like phenotype also had endocrine defects with adrenocorticotropic hormone (ACTH) deficiency and nail dystrophy, described as DAVID syndrome 31 . NFΚB2 gene defects can J o u r n a l P r e -p r o o f present with loss-of-function (LOF) in an autosomal dominant manner, due to a non-processed form of mutant p100 leading to p52 dominant negative or haploinsufficient states, and these appear to be highly penetrant though there is variable expressivity 4, 22, 32, 33 . Most of the LOF variants have been described in the C-terminal region and affect either the serine phosphorylation sites at 866/870 or the ubiquitination site at amino acid 855. All of these variants affect the p100 precursor protein creating a molecule, which cannot be processed, thus reducing p52 nuclear translocation and heterodimer formation, with RelB. In contrast to the LOF variants, gain-of-function NFκB variants are largely clustered in the glycine-rich domain ( Figure   1 ) or the ankyrin-repeat domain. The GOF variants are likely to show incomplete penetrance with variable expressivity. As can be expected, heterozygous GOF variants result in constitutive activation with increased nuclear translocation of p52/RelB heterodimers, but also increased canonical NFκB pathway activity, illustrating that NFκB signaling is not confined to either the canonical or the non-canonical pathways, but is an integrated and interactive signaling mechanism.

NFκB2 protein expression and its phosphorylation upon activation (Figure 2) , was used to establish that the novel LOF variant was indeed likely to have an impact on NFκB signaling, through both the non-canonical and canonical pathways, due to a significant decrease in phosphoNFκB2 and the generation of the processed form, p52. This finding is consistent with other variants reported at position 866, which as previously mentioned, is a critical serine phosphorylation site. This patient had all of the clinical features associated with LOF variants (Table 1) This patient showed evidence of severe COVID-19 disease, as discussed previously, and his therapeutic regimen included compassionate use remdesivir 34 , intensive cardio-respiratory support, tocilizumab and convalescent plasma (Figure 3 ). During the critical phase of illness, a lymphocyte subset quantitation (Table 3) was performed, which revealed a stark panlymphopenia, which has been reported in other COVID-19 patients as well 18, 35 . This result when compared specifically to his pre-COVID-19 immunophenotyping result indicated the lymphopenia was COVID-19-related rather than due to his underlying immunodeficiency. The rebound of the T cell counts to the pre-COVID-19 baseline post-discharge from the hospital provided further evidence of this (Table 3 ). Further immunophenotyping of T and B cell subsets was unrevealing, in contrast to certain other reports 35 , though not all markers used were identical between this study and the published reports. Also, there may be differences in various immune cell populations, depending on the timing of analysis from onset of infection, age of patient, clinical status and other factors. However, switched memory B cells were decreased, as one would expect to find in patients carrying heterozygous NFΚB2 C-terminus LOF variants (Table   3 ). Regulatory T cells revealed no intrinsic defects in Treg subset numbers compared to controls, especially post-recovery (Table 3 ; Suppl. Fig.1 ).

With evidence of respiratory failure in conjunction with CVID, it was imperative to determine whether biologicals should be used to control the "cytokine storm" reported in COVID-19 patients 13, 36, 37 . IL-6 was measured at several time-points both during and following hospitalization with COVID-19, and was elevated early on, which was informative from a clinical management standpoint. Data from other centers have variable cut-offs that define elevated IL-6 and trigger the use of biological therapy. There is data to suggest that an IL-6 level of 80pg/mL is associated with respiratory failure and more severe disease 38 . Some centers have used lower cut-offs in protocols or clinical trials, while other centers do not have a fixed cut-off for IL-6 at which blocking therapy is initiated. Tocilizumab has been used empirically in several centers to treat the "hyper-inflammatory" response in COVID-19 39, 40 . Some centers assess serum IL-6 levels to determine timing of drug administration, while others use inflammatory markers, such as CRP and ferritin to determine the indication for tocilizumab. A study using tocilizumab reported clinical improvement in COVID-19 patients receiving the drug 40 . While analogies can be drawn between the cytokine storm seen in COVID-19, and cytokine release syndrome (CRS) in patients following CAR-T therapy 41 , it is unclear that such analogies are viable or valid, especially in intrinsically immune-compromised patients. The decision to treat with biological therapy should not be solely decided by a single biomarker but by several biomarkers of inflammation along with clinical context. In COVID-19 patients, IL-6 has been shown to be a particularly useful biomarker, in correlation with other biomarkers in predicting severe disease and reduced cytotoxicity, and thus represents a useful therapeutic target with drugs, such as tocilizumab 24, 42 . In this patient, the IL-6 levels peaked at day#4 but began trending down (though this was not known prior to analysis) before the first and only dose of tocilizumab was administered (8mg/kg). The half-life of the drug at this dose is approximately J o u r n a l P r e -p r o o f 13 days, and therefore, additional samples were collected after administration of tocilizumab at three different time points. A notable but not unusual finding was that IL-6 level was increased after tocilizumab was administered. This has been reported previously in patients with rheumatoid arthritis and Castleman disease, in which settings, elevated IL-6 levels persisted for several days, and has been attributed to saturation of the IL-6 receptors by the drug 43 . In our patient, the highest IL-6 level was seen immediately after administration of tocilizumab, with levels gradually decreasing over the course of two weeks though at that time point it had not returned to baseline (either pre-COVID-19 for the patient or comparable to healthy controls).

Tocilizumab can bind either the membrane-bound or the soluble form of the IL-6 receptor and prevent IL-6/IL-6R from dimerizing with membrane-bound gp130 inhibiting downstream signaling. Since IL-6 can bind either membrane-bound IL-6R inducing "classical" signaling, which is primarily anti-inflammatory or soluble IL-6R, forming a complex with gp130 and mediating pro-inflammatory trans-signaling 44 , it seems unlikely IL-6 is competing with tocilizumab, which binds with high affinity to the membrane-bound or soluble form of the receptor, to generate circulating immune complexes. In fact, the levels of IL-6 post-tocilizumab appear to be prognostically informative in COVID-19, and may actually reflect the level of IL-6 production 30 . In addition, in vitro studies in our laboratory with "spiked" IL-6 in healthy control serum samples to which tocilizumab has been added does not show any increase in IL-6 levels,

indicating that the origin of the IL-6 is endogenous. Therefore, serum IL-6 levels after tocilizumab administration should be used for prognostication and determining the necessity for additional dosing of tocilizumab, especially after the half-life of the drug has been reached.

However, it would be reasonable to suggest that the use of additional doses of tocilizumab should be based on multiple factors than serum IL-6 levels alone. It is critical to note when J o u r n a l P r e -p r o o f performing serum or plasma cytokine measurements that results are often not comparable across immunoassay platforms, and thus all serial monitoring of cytokines should be performed within the same laboratory and on the same platform, and account for the presence of heterophile antibodies, which might cause spurious elevation of cytokines, especially in multiplex assays 45, 46 .

The use of convalescent plasma has been described in a few studies to treat severe disease in COVID-19 patients [47] [48] [49] . Since this patient had a humoral immunodeficiency and was receiving immunoglobulin replacement, it seemed logical to use SARS-CoV-2 CP to provide passive therapeutic support to help clear the virus, therefore, he was given five doses of CP (Table 2) . A recent study of anti-SARS-CoV-2 antibodies in 149 convalescent patients revealed that very few of these individuals had high levels of neutralizing antibodies, but the presence of antibodies directed against the receptor-binding-domain (RBD) of the virus was present in many of these patients with antiviral activity, albeit at low levels, suggesting that strategies to increase the concentrations of such antibodies would be effective in the design of a vaccine 50 . The practical utility of administering CP has been limited by the inability to obtain quantitative assessment of SARS-CoV-2-specific IgG antibodies, particularly those with neutralizing capability to determine the best donors for CP. However, the introduction of the IMMUNO-COV TM test, which can measure neutralizing antibodies to the SARS-CoV-2 spike glycoprotein using a pseudovirus will substantially facilitate this aspect of measurement of the serological immune response to SARS-CoV-2 infection as it becomes available in diagnostic laboratories in the future 51 .

Interestingly, assessment of IgG antibodies to SARS-CoV-2 prior to CP infusion showed a positive result, which was unexpected but perhaps not altogether surprising. The most plausible J o u r n a l P r e -p r o o f explanation for this phenomenon is that the patient was capable of mounting a partial humoral immune response (Table 1) , similar to other NFκB2 patients carrying heterozygous NFκB2 Cterminus LOF variants 4, 5, 21 , especially to neoantigens, such as SARS-CoV-2, despite the very low switched memory B cells in blood. In addition, there may be switched memory B cells in secondary lymphoid organs, which cannot be assessed by peripheral analysis, and his intact CD4+ T cell compartment may likely provide adequate help to B cells for antibody production.

Most commercial immunoglobulin products are expected to contain little to no antibodies to SARS-CoV-2 in current preparations, so it is unlikely that the replacement immunoglobulin accounted for this observed positive antibody response. Both SARS-CoV-2 serologic assays demonstrated 100% specificity and no cross-reactivity with seasonal coronaviruses, and therefore this does not account for the SARS-CoV-2 IgG antibody pre-CP. A recent study identified SARS-CoV-2-specific CD4+ and CD8+ T cells in a large proportion of the 20 patients who were recovering from COVID-19 infection but also in approximately half of the "unexposed" individuals (n=20), which was suggested to be due to cross-reactivity with other more common coronaviruses 52, 53 . However, T cell cross-reactivity does not necessarily have to correlate with antibody cross-reactivity.

It is unclear if the quality (affinity and avidity) of the antibody response in these NFκB2 patients is the same as in healthy individuals, and it is likely that these "endogenous" antibodies may be relatively short-lived. Several days after the infusion, the index (ratio of signal/cutoff) of IgG antibodies appeared to be decreasing. In contrast to several other patients reported in the literature, the patient displayed evidence of clinical improvement even prior to initiation of CP, though it may have had an adjunctive effect in consolidating recovery.

The patient tested negative for SARS-CoV-2 by PCR at least three times after discharge from the hospital but tested positive again at day 22 from a nasopharyngeal sample. However, this was not accompanied by any clinical manifestations. A positive PCR test after multiple negative PCR results has been previously reported 54, 55 . Also, Ct values greater than 25-30 have not been associated with culturable virus, further substantiating the hypothesis this represents noninfectious viral RNA 56 . Since this result was not associated with new clinical disease, further action was not taken. Serial monitoring of virus along with other clinical parameters may be useful for prognosis as viral persistence has been shown to be correlative with severe disease 57 .

However, mild and transient viral positivity after recovery from clinical disease does not appear to correlate with adverse clinical outcomes based on this patient. Interestingly, in one study, asymptomatic individuals (n=37) appeared to shed virus for a longer duration than symptomatic individuals and also produced SARS-CoV-2 IgG antibodies as well as neutralizing antibodies relatively early compared to patients with active disease 58 .

In summary, we have characterized the pathogenic impact of a novel heterozygous NFκB2 lossof-function C-terminus variant, which resulted in a CVID-like phenotype in this patient. An important lesson learned from this case is that while patients with inborn errors of immunity are predisposed to infection with higher morbidity and mortality in the context of such infection, patients with milder immunodeficiency (i.e. partially preserved antibody response, intact T cell function) can survive infections, such as SARS-CoV-2 with a multidisciplinary approach to clinical management, and aggressive mitigation strategies including monitoring of biomarkers and early utilization therapies, including biologics and immunomodulators when indicated. were stimulated with soluble anti-CD3. Immunoblotting was performed for NFκB2 precursor (p100) and the processed form, p52, phosphorylated NFκB2, and phosphorylated p65 (RelA) of the canonical pathway in both stimulated and unstimulated samples. There is a distinct inability to phosphorylate NFκB2 and the amount of processed p52 protein is substantially reduced in the patient sample on stimulation. Figure 2B depicts the densitometric analysis of the immunoblot in J o u r n a l P r e -p r o o f

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Mechanisms and pathologic significances in increase in serum interleukin-6 (IL-6) and soluble IL-6 receptor after administration of an anti-IL-6 receptor antibody, tocilizumab, in patients with rheumatoid arthritis and Castleman disease

IL-6 trans-signaling via the soluble IL-6 receptor: importance for the proinflammatory activities of IL-6

Variability in the Laboratory Measurement of Cytokines: A Longitudinal Summary of a College of American Pathologists Proficiency Testing Survey

Heterophile antibody interference in a multiplexed fluorescent microsphere immunoassay for quantitation of cytokines in human serum

The convalescent sera option for containing COVID-19

Effectiveness of convalescent plasma therapy in severe COVID-19 patients

Treatment of 5 Critically Ill Patients With COVID-19 With Convalescent Plasma

Convergent antibody responses to SARS-CoV-2 in convalescent individuals

Development and validation of IMMUNO-COV: a high-throughput clinical assay for detecting antibodies that neutralize SARS-CoV-2

SARS-CoV-2-reactive T cells in healthy donors and patients with COVID-19

Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals

Interpreting Diagnostic Tests for SARS-CoV-2

PCR Assays Turned Positive in 25 Discharged COVID-19 Patients

Predicting infectious SARS-CoV-2 from diagnostic samples

Persistent Viral Presence Determines the Clinical Course of the Disease in COVID-19

Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections

 Fig. 2A . (C) . T cell blasts were generated from PBMCs by stimulation with anti-CD3 and anti-CD28 with IL-2. Blast cell lysates were analyzed for p100 and p52 of NFκB2 and the bar graphs ( Figure 2D ) depict the relative expression levels of these proteins normalized to beta-actin by densitometry. Compared to normal controls, there is a small decrease in p100 but a remarkable reduction in the processed form, p52 protein, which correlates with the PBMC data. The extent of decrease in phosphoNFκB2 and p52 is supportive for a complete deficiency for this variant.