key: cord-0969311-ospf0do1
authors: van Laarhoven, Arjan; Kurver, Lisa; Overheul, Gijs J.; Kooistra, Emma J.; Abdo, Wilson F.; van Crevel, Reinout; Duivenvoorden, Raphaël; Kox, Matthijs; ten Oever, Jaap; Schouten, Jeroen; van de Veerdonk, Frank L.; van der Hoeven, Hans; Rahamat-Langendoen, Janette; van Rij, Ronald P.; Pickkers, Peter; Netea, Mihai G.
title: Interferon gamma immunotherapy in five critically ill COVID-19 patients with impaired cellular immunity: a case series
date: 2021-09-21
journal: Med (N Y)
DOI: 10.1016/j.medj.2021.09.003
sha: 3cd57414178fc2321b1c672a6741d05fc4ace2f8
doc_id: 969311
cord_uid: ospf0do1

Background Prolonged SARS-CoV-2 shedding has been described in immunocompromised COVID-19 patients, resulting in protracted disease and poor outcome. Specific therapy to improve viral clearance and outcome for this group of patients is currently unavailable. Methods Five critically ill COVID-19 patients with severe defects in cellular immune responses, high SARS-CoV-2 viral RNA loads, and no respiratory improvement were treated with interferon gamma, 100 μg subcutaneously, thrice weekly. Bronchial secretion was collected every 48 hours for routine diagnostic SARS-CoV-2 RT-PCR and viral culture. Findings Interferon gamma administration was followed by a rapid decline in SARS-CoV-2 load and a positive to negative viral culture conversion. Four patients recovered and no signs of hyperinflammation were observed. Conclusions Interferon gamma may be considered as adjuvant immunotherapy in a subset of immunocompromised COVID-19 patients. Funding AvL and RvC are supported by National Institute of Health [R01AI145781]. GJO and RPvR are supported by a VICI grant [016.VICI.170.090] from the Dutch Research Council (NWO). WFA is supported by Clinical Fellowship grant [#9071561]) of Netherlands Organization for Health Research and Development. MGN is supported by an ERC Advanced Grant [#833247] and a Spinoza Grant of the Netherlands Organization for Scientific Research.

1 2 Summary 25 Background: Prolonged SARS-CoV-2 shedding has been described in immunocompromised patients, resulting in protracted disease and poor outcome. Specific therapy to improve viral 27 clearance and outcome for this group of patients is currently unavailable. 28

Methods: Five critically ill COVID-19 patients with severe defects in cellular immune responses, high 29 SARS-CoV-2 viral RNA loads, and no respiratory improvement were treated with interferon gamma, 30 100 μg subcutaneously, thrice weekly. Bronchial secretion was collected every 48 hours for routine 31 diagnostic SARS-CoV-2 RT-PCR and viral culture. 32

Findings: Interferon gamma administration was followed by a rapid decline in SARS-CoV-2 load and a 33 positive to negative viral culture conversion. Four patients recovered and no signs of 34 hyperinflammation were observed. 35

Conclusions: Interferon gamma may be considered as adjuvant immunotherapy in a subset of 36 immunocompromised COVID-19 patients. 37

Funding: AvL and RvC are supported by National Institute of Health [R01AI145781] . GJO However, in patients with pre-existent immunodeficiencies, replication-competent Severe Acute 48

Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) may be shed well after 21 days after disease 49 onset. 3,4 Convalescent plasma or remdesivir is used in immunocompromised patients, but has not 50 been shown to promote viral clearance. Our patients had conditions reducing their interferon 51 response, which is likely important for viral clearance. 4 In addition, interferon-mediated immunity is 52 known to be impaired by 6 Clinical data however failed to demonstrate positive 53 effects of systemic interferon beta-1a in COVID-19 patients, 7 while interferon gamma has been thus 54 far avoided due to its potential proinflammatory effects. However, in patients with severe cellular 55 immune defects, therapeutic stimulation of the antiviral host defence may benefit SARS-CoV-2 56 clearance. 57 58

Five immunocompromised COVID-19 patients were treated with adjuvant interferon gamma, 100 μg 60 subcutaneously, thrice weekly. The patients were monitored for SARS-CoV-2 viral load, culture 61 conversion (patient 1-3), and signs of hyperinflammation. 62 63 Patient 1. A 50-year old woman with a history of splenectomy and common variable 64 immunodeficiency (Table 1) and 90, she recovered with preserved renal function (eGFR CKD-EPI > 90ml/min/1.73m 2 ) and started 92 weaning from mechanical ventilation through a tracheostomy tube and was discharged at day 107. 93

After discharge, the creatinine steadily increased, for which a kidney biopsy was performed on day 94 211, showing signs of an antibody-mediated rejection. It cannot be excluded that this is related to 95 the discontinuation of the mycophenolate mofetil and tacrolimus and the treatment with interferon 96 gamma during the hospital admission for COVID-19. 97 98 Patient 3. A 45-year old woman was admitted 7 days after COVID-19 symptom onset. One month 99 earlier a blood group-incompatible renal transplantation had been performed, for which she had 100 received rituximab, immunoadsorption, intravenous immune globulin and basiliximab. She used 101 tacrolimus, mycophenolate mofetil and prednisone as maintenance therapy, and immune 102 suppression was adjusted during admission. She developed end-stage renal failure with thrombotic 103 microangiopathy and tubular damage, but no signs of rejection on biopsy. Because of negative SARS-104

CoV-2 serology, she received convalescent plasma at day 12. High-flow nasal oxygen therapy could 105 be stopped at day 20 and she was discharged at her own request despite persistent requirement for 106 oxygen therapy. At day 25 she was re-admitted and at day 30, a suspected bacterial pneumonia was 107 treated. On day 38, renal replacement therapy and mechanical ventilation were initiated, a second 108 dose of convalescent plasma was administered, and remdesivir was started without clinical 109 improvement. At day 42 the persistent lymphopenia, high SARS-CoV-2 load, and lack of clinical 110 improvement prompted us to start interferon gamma, after which the viral load swiftly declined and 111 respiratory function improved. Virus culture was negative before initiation of interferon gamma 112 shown. Remdesivir has not demonstrated a clinical antiviral effect and it is therefore doubtful 155 whether its administration in two of our patients contributed to viral clearance. In critically ill COVID-156 19 cases, type I interferon pathways are impaired, 6 and although these mediators are likely 157 important for viral clearance, 4 subcutaneous interferon beta-1a was not associated with survival 158 benefit. 7 Because of the lack of effectivity of adjuvant type I interferons, and because type I 159 interferons can have suppressive effects on T-helper function, 9 we deemed administration of type I 160 interferons undesirable in our patients, as Th1 immunity was already impaired in our patients as 161 illustrated by their Aspergillus and herpes simplex virus infections. 162

Type II interferons are less well studied in COVID-19. Although circulating concentrations are not 163 altered in COVID-19 patients, the interferon-gamma production upon ex-vivo stimulation is reduced, 164 5,10 as is the interferon gamma responsive gene signature in critically ill COVID-19 patients. 6 165

Interferon gamma immunotherapy has been applied previously in patients with pulmonary 166 tuberculosis 11 and pulmonary aspergillosis, 12 but no published data were available for use in COVID-167 19. We considered application of adjuvant interferon gamma therapy a logical choice of last-resort 168 therapy because of its potent immunostimulatory effects including on tissue macrophages that are 169 likely important for COVID-19 immunity. 1 In our patients, interferon gamma administration was 170 indeed followed by viral clearance and clinical improvement in four out of five patients. Inherent to a case series, limitations of the study include the small sample size of five patients with 194 the specific phenotype of high SARS-CoV-2 viral loads and impaired cellular immunity. Moreover, 195 because interferon gamma was used off-label as last-resort therapy instead of in a randomised 196 setting, we cannot attribute the viral clearance with certainty to interferon gamma immunotherapy. 197

Lastly, functional immunological data was not available for these patients. 

as Ct-value. Interferon gamma was administered as 100 μg subcutaneously, thrice weekly. 221

Convalescent plasma and C-IVIG were provided by the Dutch national blood supply Sanquin, and was 222 administered intravenously in doses of approximately 200 mL (plasma) and 50 to 100 mL (C-IVIG 

Patients were admitted to the ICU department and received additional treatment before and during 260 interferon gamma therapy as discussed. Interferon gamma (Immukine, Clinigen Healthcare Ltd) was 261 administered as 100 μg subcutaneously, thrice weekly. During interferon gamma immunotherapy, 262 J o u r n a l P r e -p r o o f 13 bronchial secretion was collected every 48 hours for routine diagnostic SARS-CoV-2 RT-PCR using 263 commercially available systems targeting the E gene, and in patient 1-3 for inoculation of Vero E6 264 cells using methods adapted from Wölfel et al 14 . Bronchial secretion was diluted and mixed 1:1 in 265 viral transport medium, of which 100 µl was used to infect Vero E6 cells, which were seeded in 266 24 well plates at a density of 2.5x10 5 cells/well. After 1 hour, the inoculum was replaced with 1 267 ml of Dulbecco's Modified Eagle Medium (DMEM, Gibco) containing 2% fetal bovine serum 268 (Sigma), 100 μg/ml gentamycin (Gibco), 2.5 μg/ml amphotericin-B (Gibco), 100 μg/ml 269 streptomycin and 100 U/ml penicillin (Gibco) and culture medium was collected directly and at 270 48 hours and at 96 hours post infection for RNA isolation and RT-qPCR using primers targeting 271 the E gene, 14 as described previously. 15 

AvL and RvC are supported by National Institute of Health for a project

Tryptophan Metabolism and Response to Corticosteroids to Define New Therapeutic Targets for 202

Tuberculosis Meningitis: Integration of Large Scale Clinical, Metabolomic, and Genomic Data

WFA is supported by Clinical Fellowship grant

Organization for Health Research and Development. MGN is supported by an ERC Advanced Grant 206

Dexamethasone in 289 Hospitalized Patients with Covid-19

Case Study: Prolonged Infectious SARS-292

CoV-2 Shedding from an Asymptomatic Immunocompromised Individual with Cancer

Autoantibodies against type I IFNs in patients 296 with life-threatening COVID-19

Dysregulated Innate and Adaptive

Immune Responses Discriminate Disease Severity in COVID-19. The Journal of Infectious 300 Diseases 223

Impaired type I interferon activity and 303 inflammatory responses in severe COVID-19 patients

Repurposed Antiviral Drugs for Covid-19 -Interim WHO Solidarity Trial Results

Casirivimab and 310 imdevimab in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, 311 controlled, open-label

Type I interferons in 313 infectious disease

Hydroxychloroquine 316 Inhibits the Trained Innate Immune Response to Interferons

Immunomodulation with Recombinant Interferon-γ1b 319 in Pulmonary Tuberculosis

Exogenous Interferon-γ Immunotherapy for Invasive Fungal Infections in 322 Kidney Transplant Patients

Hemophagocytic lymphohistiocytosis (HLH): 324 A heterogeneous spectrum of cytokine-driven immune disorders. Cytokine & Growth Factor 325 Reviews 26

Virological assessment of hospitalized patients 328 with COVID-2019

Obatoclax Inhibit SARS-Cov-2 Replication in Primary Human Nasal Epithelial Cells In Vitro. 332 Viruses 13

The authors thank Bart van den Bosch for SARS-CoV-2 PCR measurements and dr. Maarten Cobussen 200

The authors declare no competing interests. 217