key: cord-280960-88hzovg2
authors: Galani, I. E.; Rovina, N.; Lampropoulou, V.; Triantafyllia, V.; Manioudaki, M.; Pavlos, E.; Koukaki, E.; Fragkou, P. C.; Panou, V.; Rapti, V.; Koltsida, O.; Mentis, A.; Koulouris, N.; Tsiodras, S.; Koutsoukou, A.; Andreakos, E.
title: Untuned antiviral immunity in COVID-19 revealed by temporal type I/III interferon patterns and flu comparison
date: 2020-08-24
journal: nan
DOI: 10.1101/2020.08.21.20179291
sha: 
doc_id: 280960
cord_uid: 88hzovg2

A central paradigm of immunity is that interferon (IFN) mediated antiviral responses precede the pro-inflammatory ones, optimizing host protection and minimizing collateral damage. Here, we report that for COVID-19 this does not apply. By investigating temporal IFN and inflammatory cytokine patterns in 32 COVID-19 patients hospitalized for pneumonia and longitudinally followed for the development of respiratory failure and death, we reveal that IFN-{lambda} and type I IFN production is both diminished and delayed, induced only in a fraction of patients as they become critically ill. On the contrary, pro-inflammatory cytokines such as TNF, IL-6 and IL-8 are produced before IFNs, in all patients, and persist for a prolonged time. By comparison, in 16 flu patients hospitalized for pneumonia with similar clinicopathological characteristics to COVID-19 and 24 milder non-hospitalized flu patients IFN-{lambda} and type I IFN are robustly induced, earlier, at higher levels and independently of disease severity, while pro-inflammatory cytokines are only acutely and transiently produced. Notably, higher IFN-{lambda} levels in COVID-19 patients correlate with lower viral load in bronchial aspirates and faster viral clearance, and a higher IFN-{lambda}:type I IFN ratio with improved outcome of critically ill patients. Moreover, altered cytokine patterns in COVID-19 patients correlate with longer hospitalization time and higher incidence of critical disease and mortality compared to flu. These data point to an untuned antiviral response in COVID-19 contributing to persistent viral presence, hyperinflammation and respiratory failure.

disease severity, while pro-inflammatory cytokines are only acutely and transiently produced. Notably, 41

higher IFN- levels in COVID-19 patients correlate with lower viral load in bronchial aspirates and 42 faster viral clearance, and a higher IFN-:type I IFN ratio with improved outcome of critically ill 43 patients. Moreover, altered cytokine patterns in COVID-19 patients correlate with longer 44 hospitalization time and higher incidence of critical disease and mortality compared to flu. These data 45 point to an untuned antiviral response in COVID-19 contributing to persistent viral presence, 46 hyperinflammation and respiratory failure. of the worst pandemics of our time, causing high incidence of pneumonia, acute respiratory distress 52 syndrome (ARDS) and death 3,4 . One of the most notable features of SARS-CoV2 infection is that it goes 53 unnoticed for a remarkably prolonged period of time, running a course of a mild or uncomplicated 54 illness for weeks until sudden and severe symptoms develop, in a subgroup of patients, requiring 55 hospitalization, oxygen support and/or admission to an intensive care unit (ICU) 3,4 . This is consistent 56

with an unusually long incubation period of the virus, ranging from 2 to 14 days, and an unusually long 57 presence of it in the respiratory tract, often being detectable for over a month after initial infection by 58 conventional molecular diagnostic tests 5,6 . By comparison, influenza virus infection, the main 59 respiratory virus accounting for pneumonia hospitalizations till now, has an incubation time of 1 to 4 60 days, a short window of virus positivity of a few days, and an abrupt onset of symptoms causing 61 pneumonia within 1-3 days 7,8 . Other frequent respiratory viruses such as respiratory syncytial viruses, 62 rhinoviruses, parainfluenza viruses, metapneumonoviruses and regular human coronaviruses have also 63 shorter incubation times (ranging from 1-5 days) and more rapid and acute manifestation of 64 symptoms 9 , rendering SARS-CoV2 quite unique in that respect. The basis of this is unknown but is likely 65

to be a key driver of the pathophysiology of COVID-19 underlying its distinctive disease course and 66 clinical manifestations. 67

The hallmark of COVID-19 is the development of a hyper-inflammatory response, also known as 68 'cytokine storm', impairing the gas-exchange function and leading to acute respiratory distress 69 syndrome (ARDS), multi-organ failure and death 10-12 . We and others have previously shown that a 70 finely tuned antiviral response, orchestrated by IFN- (type III IFN) and type I IFN is critical for 71

balancing immunity for optimal protection and minimal damage [13] [14] [15] . Deviation from this can unleash a 72 detrimental 'cytokine storm' with devastating consequences for human health. A recent study 73

suggested that in COVID-19 patients type I IFN and IFN- are not produced as they could not be 74 detected in the sera of a small COVID-19 cohort of otherwise unspecified clinical characteristics 16 . In 75

contrast, another one reported that type I IFN is induced in COVID-19 patients, and indicated that their 76 levels might be reduced in those that are critically ill 17 . Such discrepancy could be due to the fact that 77 each of these studies focuses on a single and likely distinct snapshot of an apparently heterogeneous 78 disease process. Therefore, pursuing kinetic analyses is pertinent to delineating the course of the 79 immune response, especially given that cytokines are transiently produced . This is particularly true for  80  IFNs which are expressed early during infection and are rapidly down-regulated thereafter.  81   Here, we have performed a comprehensive temporal analysis of type I and type III IFN, and major  82  inflammatory cytokine patterns in 32 COVID-19 and 16 influenza A virus infected (flu) patients  83 hospitalized for community acquired pneumonia and longitudinally followed up according to current 84 WHO guidelines 18 . Both groups of patients exhibited similar clinicopathological characteristics and 85 comparable disease severity on admission (Table 1) . We have also analyzed 24 milder flu patients with 86 no radiological findings of pneumonia and no need for hospitalization (referred to as mild flu; Table 1),  87 as well as 10 healthy individuals. Using high sensitivity Luminex and ELISA assays, we quantified 18 88 cytokines and chemokines relevant to antiviral immunity and hyperinflammation in patient sera 89 collected at defined time intervals following hospital admission ( Fig. 1a and S1aa). This aligns patients 90 on the basis of the same clinical criteria of disease symptoms and severity, mainly the presence of 91 pneumonia and the requirement for oxygen support. 92

We found that COVID-19 patients had profoundly impaired induction of both IFN- and type I IFNs. 93

Median levels of IFN- and type I IFNs were not detectable in most COVID-19 patients (Fig. 1b Interestingly, despite their limited ability to make IFNs, COVID-19 patients robustly expressed pro-99 inflammatory cytokines such as TNF, IL-6, IL-7, IL-8, IL-10, IFN- and CCL3 that were maintained at high 100 levels for a prolonged time (Fig. 1b) . Other cytokines such as IL-1, IL-12, IL-23 and CCL4 were also 101 significantly up-regulated at specific time intervals compared to healthy individuals reflecting the 102 heterogeneity of the disease course (Fig. S2) . 103 A similar pattern emerged when comparisons were made according to disease symptoms onset (Fig.  104 S1b). COVID-19 patients exhibited markedly delayed and reduced IFN- and type I IFN levels which  105 were detectable only in a fraction of the patients and from days 7-10 onwards of symptoms onset ( Fig.  106 S3, a-b). By comparison, all flu patients exhibited high levels during the first 6 days ( Fig. S3 , a-b). 107

Although COVID-19 patients made little IFN during the first 6 days of symptom onset, they potently 108 produced pro-inflammatory cytokines and chemokines such as TNF, IL-6, IL-8, IL-10 and CCL3 at levels 109 similar to flu (Fig. S3 , b-c). Moreover, they exhibited prolonged expression of pro-inflammatory 110 mediators, with high levels of TNF, IL-6, IL-7, IL-8, IL-10 and CCL4 remaining detectable for over three 111

weeks of onset, whereas in flu patients a number of these were by that time down-regulated (Fig. S3 ). 112

Notably, COVID-19 patients were admitted to hospital with similar markers of systemic inflammation 113 such as CRP levels, white blood cell (WBC) and neutrophil counts, and neutrophil/lymphocyte (N/L) 114 ratio to flu patients (Table 1 and Fig. S4 , a-f). They even had lower fever and a lower CURB-65 score, a 115 commonly used measure of pneumonia severity 19 (Fig. S4 , g-h). However, during follow up COVID-19 116 patients developed a much higher incidence of ARDS requiring ICU support. In our cohort, 16 out of 32 117 patients (50%) developed critical disease, 3 of which died, compared to only 3 out of 16 flu patients 118

(18.7%) none of which died (Fig. 2, a-b) . Strikingly, COVID-19 patients became critically ill over a much 119 broader time window (up to nine days after admission) than flu patients which manifested critical 120 disease within the first day post admission. This is in agreement with the high incidence and protracted 121 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 24, 2020. . https://doi.org/10.1101/2020.08.21.20179291 doi: medRxiv preprint course of severe respiratory failure described for COVID-19 4,12 . Interestingly, among COVID-19 patients 122 those who became critically ill had higher CRP levels, WBC and neutrophil counts, and N/L ratio on 123 admission (Fig. S4 , a-f), but not CURB-65 or fever (Fig. S4 , g-h and Table S1 ). Critically ill flu patients 124 also had a tendency for higher WBC and neutrophil counts and a N/L ratio, as well as significantly 125 raised CURB-65, whereas non-hospitalized flu patients did not exhibit any of these increases (Fig S4, a-126 h). 127

We thus examined whether temporal cytokine patterns differ between the various patient groups.

128

Surprisingly, we observed that although COVID-19 patients that do not become critically ill produce 129 little type I or III IFN, the ones that become critically ill make IFN- which are significantly higher at the 130 day 1-3 time interval compared to healthy and non-critically ill patients ( Fig. 2c and S5 ). Some of the 131 critically ill patients also make IFN-α ( Fig. 2d and S5 ), albeit at significantly lower levels to mild non-132 hospitalized flu patients (Fig. 2d ) or the total of hospitalized flu patients (both critically and non-133 critically ill; p<0.05). On the contrary, all COVID-19 patients make pro-inflammatory cytokines such as 134 TNF, IL-6, IL-8, IL-10 and IFN-, with critically ill patients exhibiting also higher levels of IL-6, IL-7 and 135 TNF at specific time intervals, and a tendency for higher IFN- consistent with the increased hyper-136 inflammatory state they are in (Fig. 2e , S5 and S6). Individual patient data further confirmed these 137 trends (Fig. S7 ). Interestingly, CCL3 is significantly higher than healthy controls in non-critically ill 138 COVID-19 patients but not in the critically ill ones (Fig. S6 ). By comparison, critically ill and non-139 critically ill flu patients did not differ in their ability to make type I and type III IFNs nor pro-140 inflammatory cytokines such as TNF, IL-6 or IL-7 (Fig. 2 , c-e, S5 and S6). Similarly, non-hospitalized flu 141 patients with mild disease exhibited strong production of type I and type III IFNs, indicating that across 142 the spectrum of flu disease severity the antiviral response remains robust. Visualizing these patterns 143 on radar plot reveals a major imbalance in the induction of antiviral and pro-inflammatory responses 144

of COVID-19 patients that does not occur in flu (Fig. 2e) . 145

We next sought to determine whether imbalanced cytokine patterns in COVID-19 patients are related  147 to systemic immune effects, and parameters linked to disease severity. To that end, we obtained 148 temporal white blood cell transcriptomes from 5 healthy individuals and 9 COVID-19 patients, 5 non-149 critically and 4 critically ill, starting from day 1 of entry to the ward or ICU and at different timepoints 150 thereafter. In total, 24 comprehensive RNAseq gene expression datasets were analyzed, clustering 151

according to the clinical phenotype and indicating this as the main source of variation ( Fig. 3a and S8a). 152

Focusing at day 1 as the most relevant timepoint, we found that 4225 genes were differentially 153 expressed (DEGs) in COVID-19 patients compared to healthy individuals (Table S2) . When critically and 154 non-critically ill patients were compared separately to healthy controls, 4225 and 4902 DEGs were 155 observed, respectively, of which 1979 were common whereas the rest were uniquely found in one or 156 the other patient group (Fig. S8b , Table S2 and Table S3 ). Volcano plots pointed out notable 157 differences in the most highly regulated genes between the groups with critically ill patients exhibiting 158 a stronger immune and antiviral response gene patterns (Fig. S8 , c-e). Pathway analysis of DEGs indeed 159 revealed that the most significant pathways over-represented in critically ill patients were related to 160 the positive regulation of the immune system, the activation of the innate immune response, the 161 defense response to virus and the cellular response to IFN ( Fig. 3b and Table S4 ). By contrast, in non-162 critically ill patients these pathways were not significant; the ones over-represented instead included 163 the regulation of the cellular component size, IL-1 production and NK cell cytotoxicity (Fig. 3b) . 164 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 24, 2020. Fcgr1a) in the critically ill group that was milder and not significant in non-critically ill patients (Fig. 3,  169 c-d). On the contrary, T and B lymphocyte lineage and related genes (Cd3d, Cd3e, Cd4, Cd8a, Cd19, 170

Cd22) were markedly down-regulated in critically ill patients. These data are consistent with 171 lymphopenia, high neutrophil counts, and a high N/L ratio also present in these patients (Fig. S4 ) and 172 previously reported to be associated with more severe disease and worse outcomes in COVID-19 173 patients 3,4 . Cytokines such as TNF, IL-6 and IL-8 may directly account for these effects, as they are well 174 known to trigger the mobilization and activation of neutrophils, the development of lymphopenia and 175

the induction of innate immune responses and systemic inflammation 20,21 . Notably, a long set of IFN-176 stimulated genes (ISGs) was also strongly induced in critically ill patients compared to only a fraction of 177 them being up-regulated in the non-critically ill group (Fig 3e and S8f ), in agreement with the patterns 178 of IFN- and type I IFN production in these patients. This cannot be attributed to differential 179 expression of IFN receptors as no differences between Ifnlr1, Il10rb, Ifnar1 and Ifnar2 mRNA levels 180

were observed among patient groups and healthy individuals with the exception of a 2-fold up-181 regulation of the already high levels of Ifnar2 in critically ill patients (Table S2) . 182

Interestingly, imbalanced cytokine patterns in COVID-19 patients with pneumonia were associated 183 with a much worse disease outcome compared to flu. First, the COVID-19 group exhibited higher 184 incidence of critical disease and mortality (Fig. 2b) . Second, COVID-19 patients overall, as well as when 185

grouped as critically and non-critically ill, required longer hospitalization time than their flu 186 counterparts (Fig. 4 , a-c). For non-critically and critically ill COVID-19 patients, median time was 14 and 187 23 days, respectively, compared to flu that was 7 and 19 days (Fig. 4 , b-c). Prolonged hospitalization 188 could be attributed to the untuned antiviral responses, leading to a more protracted clinical course of 189 COVID-19 relative to flu and a need for longer recovery even for the non-critically ill group. To identify 190

cytokines and cytokine combinations that can predict hospitalization time and therefore be of 191 prognostic value, we generated a correlation matrix of the cytokine levels at admission (days 1-3 192 interval) and the duration of hospital stay (Fig. 4d ). We found that higher IL-6 and IL-10, and lower 193 CCL3 levels, are directly proportional to the duration of hospitalization (Fig. 4 , d-f). The value of TNF 194 and IL-6 as biomarkers for monitoring COVID-19 severity has been reported 4,22,23 but for CCL3 this is 195 new. Interestingly, IFN- levels also correlated with higher IL-6, and longer hospitalization, consistent 196

with their almost exclusive induction in critically but not non-critically ill patients ( Fig. 4d and 2c) . 197

A question that arises is whether IFN levels induced in critically ill patients are beneficial as delayed 198 type I or type III IFN production has been shown in animal models to cause immunopathology 13,14,24 or 199 interfere with epithelial repair 25,26 , respectively. We found that higher IFN- concentrations during ICU 200 entry were associated with lower SARS-CoV2 viral load in the respiratory tract and faster viral 201 clearance (Fig. 4, g-h) . Moreover, a higher IFN- to type I IFN ratio at that time was linked to a shorter 202 stay in the ICU (Fig. 4i) , with the two patients with the highest IFN- levels also exhibiting the longest 203 stay (both 23 days over a median of 17 days). These data suggest that delayed IFN- induction may still 204 be protective in critically ill COVID-19 patients whereas IFN- may do more harm than good, at least in 205 a subset of patients. 206

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 24, 2020. . https://doi.org/10.1101/2020.08.21.20179291 doi: medRxiv preprint Taken together, our findings demonstrate that SARS-CoV2 infection does not follow the conventional 207 paradigm of antiviral immunity. Instead of activating first the antiviral response followed by the pro-208 inflammatory process as a second line of protection, it does the opposite; it triggers the pro-209 inflammatory response long before IFN-mediated antiviral defenses are induced-if at all. This is a 210 major paradox and helps explain many of the unique or unusual features of COVID-19. The long virus 211 incubation time and persistence in the respiratory tract, giving positive SARS-CoV2 tests for weeks, can 212 be attributed to the delayed and/or reduced production of type I and III IFNs. The absent or very mild 213 symptoms of patients for an unusually extended period of time, can be attributed to the lack or 214 impaired and delayed expression of type I IFNs, principal mediators of flu-like disease and symptoms 215 such as runny nose, coughing, fatigue, dyspnea and fever in humans 27 . Finally, the early and persistent 216 expression of pro-inflammatory cytokines culminating into prolonged hyper-inflammation can 217

promote the sudden development of respiratory failure requiring hospitalization and frequently ICU 218

admission. Noteworthy, in flu the swift induction of the type I and III IFN response, across the 219 spectrum of disease severity, correlates with quicker recovery, and markedly lower incidence of critical 220 disease or mortality 13,24 . The recent demonstration in a retrospective cohort study of 446 COVID-19 221 patients that early administration of IFN- (interferon-a2b) is linked to reduced in-hospital mortality 222

whereas late IFN- therapy leads to increased mortality and delayed recovery leaves little doubt that 223 the timing of IFN production is also crucial in COVID-19 patients 28 . Conceivably, late production of type 224 I or III IFN production might confer no viral resistance, but instead promote immunopathology. 225

Whether this unique clinical course of COVID-19 is related to the presence of SARS-CoV2-derived IFN 226

inhibitors as previously proposed for SARS-CoV 29,30 and MERS-CoV 31 is not known but is a possibility. As 227

with other viruses, inhibition may be overcome once higher viral loads are reached, e.g. after 228

incubation of the virus and eventual spread in susceptible individuals. In our study, we did not see 229 significant differences in virus levels between non-critically and critically ill patients at the time IFNs 230

were measured (Fig. S9) . However, higher virus load in severe over mild disease has been described in 231 one study but not been confirmed in another 32,33 . Moreover, higher virus load can overcome SARS-232

CoV2 dose-dependent suppression of IFN production in cultured respiratory epithelial cells 16 . 233

Our study is not without caveats. First, it characterizes cytokine patterns in the circulation, and 234 although these are commonly used to analyze 'cytokine storms' in response to infection, how well they 235 correlate to immune responses in the respiratory tract is difficult to know. Second, it is relatively small, 236 and our findings await validation in other cohorts. Still, our study is uniquely informative as it 237

addresses the production of IFNs and the activation of the 'cytokine storm' in COVID-19 in a temporal 238 manner, from hospital admission to ICU entry, and should therefore be particularly useful for the 239 design of clinical trials testing IFN therapies. Finally, it provides a side-by-side comparison of COVID-19 240 with flu, studying patient populations with similar genetic, demographic and clinicopathological 241 characteristics, and therefore uncovers important differences in the antiviral immune response 242 between these two diseases that have not been previously suspected. 243 244

Study participants 247

In this non-interventional study thirty two patients with diagnosis of COVID-19 pneumonia according to 248

WHO guidelines and positive SARS-CoV-2 reverse transcription polymerase chain reaction (RT-PCR) 249 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 24, 2020. COVID-19 patients upon admission (Table 1 ). All subjects included in the study were clinically 278 evaluated and followed longitudinally during the whole period of hospitalization (from admission to 279 discharge). All blood specimens were processed immediately for serum collection and aliquots were 280 stored at -80 o C. 281

The study conforms to the principles outlined in the Declaration of Helsinki, and received approval by perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 24, 2020. 

Data were analyzed on GraphPad Prism software. Statistical significance of differences was assessed 335

using the Mann-Whitney U (MWW) test for non-parametric data. Associations between cytokines and 336 hospitalization time (in days) were tested using Spearman rank-order correlation coefficient and 337 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 24, 2020. perpetuity.

preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in

The copyright holder for this this version posted August 24, 2020. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 24, 2020. . https://doi.org/10.1101/2020.08.21.20179291 doi: medRxiv preprint time intervals. For flu, n=16, 14 and 11, respectively. For healthy, n=10. Grey shading marks the limit of 367 quantification of the assay. P values were determined by a two tailed Mann-Whitney U test for non-368 parametric comparisons. *P < 0.05, **P < 0.01 and ***P < 0.001 show significance over healthy 369 controls. # P < 0.05, ## P < 0.01 and ### P < 0.001 show significance between COVID-19 and flu groups. shown in the healthy control. For days 1-3 n=9, 7, 24, 13 and 3 for each of the five consecutive groups, 384

respectively. For days 7-10 n=8, 13, 15, 12 and 2, respectively. For healthy individuals, n=10. P values 385

were determined by a two tailed Mann-Whitney U test for non-parametric comparisons. *P < 0.05, 386 **P < 0.01 and ***P < 0.001 show significance over healthy controls. # P < 0.05, ## P < 0.01 and ### P < 387 0.001 show significance between COVID-19 and flu subgroups. Correlation of IFN-1 concentration levels with time required for viral clearance assessed as the first -407 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in correlation coefficient for non-parametric data. *P < 0.05, **P < 0.01 and ***P < 0.001 411 412 493 All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted August 24, 2020. IFI27  SOCS3  GBP1P1  SIGLEC1  IFI44L  OAS1  OAS3  EIF2AK2  IFIT1  IFI6  LY6E  RSAD2  IFIT3  IFI44  CMPK2  OAS2  OASL  MX1  BATF2  GBP5  IRF1  IRF3   Innate immune 

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