Interferon-a2b treatment for COVID-19

Summary Background The global pandemic of COVID-19 cases caused by infection with SARS-CoV-2 is ongoing. We describe here the clinical course of COVID-19 in a cohort of confirmed cases in Wuhan, China, treated with the repurposed potential experimental therapeutics IFN-α2b, arbidol or a combination of IFN-α2b plus arbidol. Methods 77 adults with confirmed COVID-19 were treated with either nebulized IFN-α2b (5mU,b.i.d.), arbidol (200mg dispersible tablet, t.i.d.) or a combination of IFN-α2b plus arbidol. Serial SARS-CoV-2 testing along with hematological measurements, including cell counts and blood biochemistry, serum cytokine levels, temperature and blood oxygen saturation levels were recorded for each patient during their hospital stay. Findings Treatment with IFN-α2b with or without arbidol significantly reduced the duration of detectable virus in the upper respiratory tract and in parallel reduced duration of elevated blood levels for the inflammatory markers IL-6 and CRP. Interpretation IFN-α2b should be investigated as therapy in COVID-19 cases.


Introduction
In December 2019, an outbreak of pneumonia was reported in Wuhan, Hubei province, China, resulting from infection with a novel coronavirus (CoV), severe acute respiratory syndrome (SARS)-CoV-2. SARS-CoV-2 is a novel, enveloped betacoronavirus with phylogenetic similarity to SARS-CoV. 1 Unlike the coronaviruses HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU, that are pathogenic in humans and are associated with mild clinical symptoms, SARS-CoV-2 resembles both SARS-CoV and Middle East respiratory syndrome (MERS), with the potential to cause more severe disease. A critical distinction is that CoVs that infect the upper respiratory tract tend to cause a mild disease, whereas CoVs that infect both upper and lower respiratory tracts (such as SARS-CoV-2 appears to be) may cause more severe disease. Coronavirus disease (COVID)-19, the disease caused by SARS-CoV-2, has since spread around the globe as a pandemic.
In the absence of a SARS-CoV-2-specific vaccine or an approved antiviral, a number of antivirals are currently being evaluated for their therapeutic effectiveness. Type I IFNsα/β are broad spectrum antivirals, exhibiting both direct inhibitory effects on viral replication and supporting an immune response to clear virus infection. 2  indole-3-carboxylate hydrochloride monohydrate), a broad spectrum direct-acting antiviral, induces IFN production and phagocyte activation. ARB displays antiviral activity against respiratory viruses, including coronaviruses. 4 All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
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Patients and treatments
Individuals with suspected COVID-19 were admitted to Union Hospital, Tongii Medical College, Wuhan, China, during the period January 16 -February 20, 2020, based on initial symptoms that included fever, chills, cough, sore throat, headache, nasal discharge, myalgia, fatigue, shortness of breath and/or diarrhea. We retrospectively examined the outcomes of patients with laboratory confirmed COVID-19 who received antiviral treatment with either IFN-α2b (Tianjin Sinobloway Biology, 5mIU/ml), arbidol (ARB) (arbidol hydrochloride; Jiangsu Simcere Pharm. Co.,100mg dispersible tablets), or a combination of IFN-α2b plus ARB, at the discretion of the treating physician, in accordance with the current practice guidelines. 5mIU IFN-α2b (1ml) were added to 2ml of sterile water and introduced as an aerosol by use of a nebulizer and mask. IFN-α2b treatment was bid, i.e. 10mIU/day. ARB treatment was 200mg (2 tablets) tid, i.e. 600mg/day. Additional COVID-19 confirmed cases from Wuhan Temporary Shelter Hospital (February 2-17, 2020), who were transferred to Union Hospital and treated with only ARB, were also included in this retrospective study. Ethics approval for analysis of all data collected was waived by hospital Institutional Review Boards, since all patient data collected conformed with the policies for a public health outbreak investigation of emerging infectious diseases issued by the National Health Commission of the People's Republic of China.

Laboratory tests
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Time-to-event analysis
Time-to-viral clearance, defined as the number of days elapsed from the onset of symptoms to the time of the first of two consecutive negative PCR tests at least 24 hours apart, was compared between the treatment groups using time-to-event analysis. Time-toviral clearance as a function of treatment was analyzed using Cox proportional hazards All rights reserved. No reuse allowed without permission.
author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.06.20042580 doi: medRxiv preprint 7 regression modelling, with age, coded as either a continuous variable or using categorical cut-offs of either >50 or >60 years of age, included as a covariate or not.

Time course analysis
Time course data were aligned to date of symptom onset and aggregated over 2-4-day intervals (depending on the analyte) to account for data not being available for all patients at all time points during disease course. For each interval and analyte, the statistical means were compared between treatment groups using analysis of variance (ANOVA).
All analyses were carried out using R version 3.6.0. 6 All rights reserved. No reuse allowed without permission.
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The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.06.20042580 doi: medRxiv preprint Table 1 summarizes the patient demographics and clinical characteristics of the cohort of COVID-19 cases evaluated in this retrospective study. 77 adults with confirmed COVID-19 admitted to Union Hospital, Wuhan, and at the discretion of the admitting physician, were treated with nebulized IFN-α2b (7) Figure 1). While all patients showed some radiographic abnormalities on chest computer tomography (CT) that were interpreted by local radiologists as 'consistent with viral pneumonia,' detailed evaluation of the CT findings were not performed due to the All rights reserved. No reuse allowed without permission.

Results
author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.06.20042580 doi: medRxiv preprint 9 overwhelming workload at Union Hospital at the time of this study. Serial laboratory measurements of blood levels for haemoglobin, glucose, total bilirubin, direct bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinas (CK), blood urea nitrogen (BUN), albumin (Alb), creatinine, and troponin 1 were also conducted (Supplementary Appendix, Figure 2). Clinical course of the COVID-19 cases was also assessed in relation to age, sex and comorbidities. With the exception of haemoglobin, which was lower in females, for each of the other measurements listed above we found no effect of age, sex or co-morbidities on disease course or laboratory measurements.
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The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.06.20042580 doi: medRxiv preprint Viral clearance was defined as two consecutive negative PCR tests at least 24 hours apart as previously described. 5 Figure 6). Notable and significant exceptions were IL-6 and CRP. As disease course progressed and prior to resolution, we observed a clear All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.06.20042580 doi: medRxiv preprint distinction of serum IL-6 levels between cases treated with IFN (i.e. IFN alone or IFN & ARB) and cases treated with ARB alone. More specifically, whereas circulating levels of IL-6 remained low for all patients who received IFN, those who received ARB alone (i.e. with no IFN) exhibited a significant spike in circulating IL-6 levels ( Figure 2A, Figure 7). We also noted elevated levels of CRP over approximately the same time that IL-6 was elevated (Supplementary Appendix, Figure   7). Similar to IL-6, CRP also returned to within normal range as disease resolved. These data suggested that treatment with IFN, whether alone or in combination with ARB, limited the circulating CRP level ( Figure 2B).
Sex and co-morbidities had no effect on the effects of ARB and IFN treatment on time to viral clearance, or IL-6 and CRP levels. Cognizant that the ARB-only treatment group consisted generally of older patients, we adjusted for age in the statistical analyses.
Regardless of whether age was considered as a continuous variable or a categorical variable (<50 yrs vs >50 yrs; <60 yrs vs >60 yrs), the effects of IFN treatment on IL-6, CRP, and time to viral clearance remained statistically significant. For those cases treated with ARB alone, IL-6 levels were significantly higher than for those treated with IFN from day 12-47 (p=2.2 x 10 -9 ). Similarly, for the ARB alone treatment group, CRP levels were significantly higher than for those cases treated with IFN from day 0-20 (p=0.0033).
Time to viral clearance was significantly shorter for those cases treated with IFN-α2b compared to those treated only with ARB (p=0.003).
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Discussion
This retrospective study provides several important and novel insights into COVID-19 disease. Most importantly, IFN-α2b therapy appears to shorten duration of viral shedding. Notably, reduction of markers of acute inflammation such as CRP and IL6 correlated with this shortened viral shedding, suggesting IFN-α2b acted along a functional cause-effect chain where virally induced inflammation represents a pathophysiological driver. Taken together, these findings elevate the biological plausibility of IFN-α2b representing a therapy for COVID-19 disease.
As the SARS-CoV-2 pandemic takes an ever-increasing toll, the urgent search for effective prophylactic and therapeutic interventions is rapidly accelerating. This includes lopinavir/ritonavir, 7,8 chloroquine, 9 remdesivir ,10 as well as interferon (IFN)-α 2 and arbidol (ARB) 4 and combinations of these. Most of these antivirals only have in vitro data to support consideration for coronavirus targets prior to clinical testing; as such, while unfortunate, it is not surprising that there is a high chance of failure. 11 However, we had shown during the SARS-CoV-1 outbreak in Canada that IFN-α treatment could hasten resolution of coronavirus-mediated human disease. 3 This prompted us to evaluate IFN-α therapy that was administered for COVID-19 disease in the early stages of the outbreak in Wuhan, Hubei province, China. Indeed, our analysis suggests that inhaled IFN-α2b accelerated viral clearance from the respiratory tract and hastened resolution of systemic inflammatory processes when compared to ARB treatment alone. While we recognize that these data are at best suggestive, given the urgency, the findings indicate that a follow-up randomized placebo controlled clinical trial (RCT) is now warranted. Success All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.06.20042580 doi: medRxiv preprint 13 could not only benefit the individual infected patient but, by reducing duration of viral shedding even in moderate cases (such as this cohort), assist in slowing the population spread.
The reduction of the inflammatory biomarker IL-6 following inhaled IFN-α2b therapy not only supported a clinically relevant impact of this approach, but also hinted at likely functional connections between viral infection and host end organ damage. IL-6 has been shown to provide prognostic value in acute respiratory distress syndrome (ARDS), which is the most severe form of COVID-19 disease. 12 If this were indeed the case, then targeting interventions such as interleukin-6 (IL-6) receptor inhibitors (e.g. toclizumab or sarilumab) towards this axis may prove a useful therapeutic adjunct, at least in those most severely ill. This form of therapy has recently been approved by China's National Health Commission 13 and is currently under consideration by the Italian Medical Agency. 14 The advantage of IFN-α2b over blocking IL-6 rests in IFN targeting the cause (SARS-CoV-2), not only the symptoms (IL-6).
This retrospective study has several significant limitations. Most obvious is the fact that the study cohort was small, non-randomized, with unbalanced demographics between treatment arms that were of unequal size. However, we considered this an exploratory study only, with the objective of determining in as rapid a manner as possible if a full trial should be considered. The results indicate that an IFN-α RCT is now warranted.
Furthermore, since the entire cohort consisted only of moderate cases of COVID-19 disease, our findings may not be indicative of what occurs in more severely ill patients; All rights reserved. No reuse allowed without permission.
author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.06.20042580 doi: medRxiv preprint 14 such caution about generalizability is indeed further supported by the lack of impact of age, sex and comorbidities on the course of COVID-19 disease in our cohort, as all these have been shown to influence clinical course. 15 Irrespective of these significant limitations, to our knowledge, the findings presented here are the first to suggest therapeutic efficacy in COVID-19 disease of IFN-α2b, an available antiviral intervention. Furthermore, beyond clinical benefit to the individual patient, treatment with IFN-α2b may also benefit public health measures aimed at slowing the tide of this pandemic, in that duration of viral shedding appears shortened.
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Contributors
QZ was responsible for patient care and treatment, clinical oversight and clinical data collection. VC and CPS analysed the data and generated the figures. X-SW, XX, XW and Z-HW collected laboratory and radiographic data. SJT analysed data. TRK, ENF conducted data analysis, data interpretation, literature searches and manuscript writing.

Declaration of interests
We declare no competing interests All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

Figure 2 Reduced inflammatory markers with IFN-α2b treatment
The same patients as in Figure 1 were serially sampled for assessment of interleukin-6 (IL-6; panel A) and C-reactive protein (CRP; panel B) from the day of symptom onset.
Values recorded were aggregated across 3 day intervals and shown as the mean +/-SE All rights reserved. No reuse allowed without permission. author/funder, who has granted medRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.06.20042580 doi: medRxiv preprint author/funder, who has granted medRxiv a license to display the preprint in perpetuity.