key: cord-252950-eiphxwmn
authors: Trouillet-Assant, Sophie; Viel, Sebastien; Gaymard, Alexandre; Pons, Sylvie; Richard, Jean-Christophe; Perret, Magali; Villard, Marine; Brengel-Pesce, Karen; Lina, Bruno; Mezidi, Mehdi; Bitker, Laurent; Belot, Alexandre
title: Type I IFN immunoprofiling in COVID-19 patients
date: 2020-04-29
journal: J Allergy Clin Immunol
DOI: 10.1016/j.jaci.2020.04.029
sha: 
doc_id: 252950
cord_uid: eiphxwmn

COVID patients in ICU present a high mortality rate and immunoprofiling reveals heterogeneous IFN-α2 production with about 20% of critically-ill patients unable to produce IFN-α2, highlighting the immune response heterogeneity and opening avenues for targeted therapies.

Sophie Trouillet-Assant 1,2* , PhD, Sebastien Viel 2,3,4,5* , PharmD, PhD, Alexandre Gaymard Merazga for their excellent work. We thank Fabien Subtil for his helpful advice for statistical analysis. 41 We also thank the LIFE (Lyon Immunopathology FEderation) community for fruitful discussion. 42 Capsule summary: 43 COVID patients in ICU present a high mortality rate and immunoprofiling reveals heterogeneous α2 production with about 20% of critically-ill patients unable to produce IFN-α2, highlighting the 45 immune response heterogeneity and opening avenues for targeted therapies. 46

To the editor, 48 49 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection (COVID-19) is characterized 50 by a wide spectrum of disease encompassing asymptomatic carriage, mild to severe upper 51 respiratory tract illness that can evolve into respiratory failure or rapidly progressing severe viral 52 pneumonia with acute respiratory distress syndrome (ARDS). Disease severity depends on viral strain 53 and host risk factors have been identified such as age and male gender. In addition, an excessive 54 immune response has been identified in patients showing a cytokine storm associated with ARDS 1 . 55

Various immunosuppressive drugs, including IL-6 blockers or JAK-STAT signaling inhibitors have been 56 suggested for the treatment of SARS-COV-2 infection 2 whereas additional clinical trials are evaluating 57 the use of recombinant interferon to foster host antiviral response. (Clinicaltrials NCT04315948, 58 NCT04293887). Type I interferons (IFN-I) are major components of the innate immune system and 59 represent critical antiviral molecules 3 . To date, IFN-I response has not been evaluated in COVID-19 60 patients and its contribution to the viral control and inflammation is unknown. 61

In this study, we assessed the kinetics of plasma IFN-I in COVID-19 patients with a spectrum of 62 severity degree. This study was approved by an ethical committee for biomedical research (Comité 63 de Protection des Personnes HCL). (Supplemental material and method of this article Online 64 Repository). 65

Firstly, we explored three patients issued from the first COVID cluster diagnosed in France (Les 66

Contamines, Haute Savoie, France) in February 2020. We took advantage of the new digital ELISA 67 technology single-molecule arrays (Simoa) 4 and analyzed the kinetics of plasma inflammatory 68 cytokines. Interleukin (IL)-6, C-reactive protein (CRP) and interferon γ-induced protein 10 (IP-10) 69

were elevated in the two symptomatic patients (Pt1, 3) (supplementary Figure 1 in the Online 70 Repository). Strikingly, no IFN-α2 was detectable in these two patients. In contrast, IL-6, CRP and IP-71 elevation of plasmatic IFN-α2 was observed. Viral loads were low with no obvious quantitative 73 difference between all three patients. 74

We further explored a larger cohort of 26 critically ill COVID patients from one of the intensive care 75 unit (ICU) at Hospices Civils de Lyon (Lyon, France). Of note, all the patients were treated with 76 standard of care and none received antiviral or immunotherapies. Considering the first 28 days of 77 infection, more than half of critically ill patients required invasive mechanical ventilation (14/26). We 78 observed that patients demonstrated a peak in IFN-α2 at day 8-10 of symptoms onset corresponding 79

to the viral replication phase, that decreased overtime to low but still detectable IFN-α2 The timing of interferon exposition may be critical to control the virus and avoid 98 immunopathogenesis. Channappavanar et al. have shown that delayed IFN-I expression can be 99 detrimental in mice in the context of SARS-CoV-1 infection 6 . Our data suggests that screening 100 patients for IFN production is instrumental to select those who could benefit from early intervention 101 with IFN. Following day 10, IL-6 remains increased while IFN-α tapered. This kinetics highlight that 102 cytokine inhibitors could be helpful at the second phase of the disease following IFN-I decrease. Viral 103 characteristic or individual genetic susceptibility should be explored to understand the defect of IFN-104 α production in some COVID patients. Some IFN-α2 positive patients also experienced fatal outcome 105 highlighting the multifactorial causes of disease severity. We acknowledge limitations of this study, 106 related to the small number of included patients and the technical limitation for the measurement of 107 IFN-β and IFN-λ, in this proof of concept study. 108

Here, we provide new argues for an early intervention with recombinant IFN-α2 and we also 109 highlight the window of opportunity for immunosuppressors at the second phase of the disease, 110 

Delay between symptom onset and ICU admission (days) 7 [1-11] 7[0-15] 0.769

Bacterial co-infection during ICU stay (n (%)) 3 (60%) 7(33%)

Diabetes (n (%)) 1 (20%) 3(14%)

Chronic obstructive pulmonary disease (n (%)) 0 (0%) 3(14%)

Cardiovascular disease (n (%)) 2(40%) 9 (43%)

Hypertension (n (%)) 3 (60%) 7 (33%)

Cancer (n (%)) 1 (20%) 3 (14%)

Active Smokers (n (%)) 0 (0%) 1(5%) 

Mortality at D28 after symptom onset(n(%)) 2 (40%) 8 (38%)

1.000 CRP -C-Reactive Protein, ICU -Intensive Care Unit, BMI -Body Mass Index Table 1 -Clinical characteristics of COVID-19 patients in intensive care unit 130 P-value are calculated using Mann-Whitney test for quantitative values and using Fisher-exact test for qualitative ones. A. Plasma IFN-α concentrations (fg/ml) were determined by single molecule array (Simoa) B.C.D. IL-6, CRP and IP-10 concentrations were measured using a multiplexed assay with the Ella platform. E. Viral load is represented as cycle threshold of IP2 RT-qPCR using assay designed by Pasteur Institut in Paris.

IFN-Interferon ; IL-6 -Interleukin 6 ; CRP -C-Reactive Protein ; IP-10 -interferon γ-induced protein 10 A. IFN score is a transcriptionnal signature defined by 6 interferon-stimulated gene (ISG) quantified using nanostring technology and obtained from Paxgene tubes in 4 COVID-19 patients. B-D. Normal values for healthy volunteers was indicated by grey area. Vertical bar indicates median delay between symptom onset and ICU admission. Concentrations of IFN-γ were quantified in only 16/26 patients because of lack of material.

Clinical features of patients infected with 114 2019 novel coronavirus in Wuhan, China

COVID-19: 116 consider cytokine storm syndromes and immunosuppression

TYPE I INTERFERONS (α/β) IN IMMUNITY 118 AND AUTOIMMUNITY

/679 and Directive 95/46/EC) and the French data protection law (Law n°78-17 on 06/01/1978 and Décret n°2019-536 on 29/05/2019), we obtained consent from each patient or his next of kin

USA) on plasma samples of COVID-19 patients. The assay was based on a 3-step protocol using an HD-1 Analyzer (Quanterix). IL-6, CRP and interferon γ-induced protein 10 (IP-10) concentrations were measured using a multiplexed assay with the Ella platform (Protein simple©

CA, USA), according to manufacturer's instructions. Plasma IL28A/B and IL-29 (Type III interferon) have been quantified by ELISA (PBL Laboratories

RNA integrity was then evaluated by Agilent RNA microarray (Agilent Technologies©

Data standardization was obtained using the geometric mean of internal control and housekeeping genes count number. Interferon score was calculated as previously described 1 . Virus quantification load Viral load was quantified from nasopharyngeal swabs or endotracheal aspirates. RNA extraction was performed by the automated NucliSENS® easyMAG® (BioMérieux, Marcy l'Etoile, France) using manufacturer's instructions. A 25 μL reaction contained 5 μL of RNA

P-value were calculated using Mann-Whitney test for quantitative values and using Fisher-exact test for qualitative ones

Comparison of RT-qPCR and Nanostring in the measurement of blood interferon response for the diagnosis of type I interferonopathies

Walzer International Center of Research in Infectiology, Lyon University, INSERM U1111, CNRS UMR 5308, ENS, UCBL, Lyon, France

We explored the first three SARS-COV-2 positive patients diagnosed in France (Les Contamines, France) in February 2020. Patient 3 : A high risk contact (a 54-year-old man) initially negative for SARS-CoV-2 developed fever and cough with respiratory crackles at auscultation on the fifth day of hospital isolation. A bilateral interstitial syndrome at the CT-scan with bilateral ground-glass opacification predominant on the left. SARS-CoV2 was detected from endotracheal aspirates (ETA), all nasopharyngeal swabs were always negative. The daily follow-up revealed a short-lasting excretion with only two successive ETA For these three patients, no other respiratory pathogens were detected. These patients did not need oxygenation, nor antibiotics, steroids or antiviral agents.

Plasma samples and Paxgene® tubes were collected from COVID-19 patients hospitalized in the university hospital of Lyon (Hospices Civils de Lyon), France. Diagnosis of COVID-19 was confirmed in all patients by RT-PCR.All critically ill patients, admitted to ICU, were included in the MIR-COVID study. This study was registered to the French National Data Protection Agency under the number 20-097 and was approved by an ethical committee for biomedical research (Comité de Protection des Personnes HCL) under the number N°20-41. In agreement with the General Data Protection Regulation (Regulation