key: cord-334175-x10bbv7y
authors: Okur, Hacer Kuzu; Yalcin, Koray; Tastan, Cihan; Demir, Sevda; Yurtsever, Bulut; Sir, Gozde; Kancagi, Derya Dilek; Abanuz, Selen; Seyis, Utku; Zengin, Rehile; Hemsinlioglu, Cansu; Kara, Mujdat; Yildiz, Mehmet Erdem; Deliceo, Elif; Birgen, Nur; Pelit, Nil Banu; Cuhadaroglu, Caglar; Kocagoz, Ayse Sesin; Ovali, Ercument
title: Preliminary report of In vitro and In vivo Effectiveness of Dornase alfa on SARS-CoV-2 infection
date: 2020-09-07
journal: New Microbes New Infect
DOI: 10.1016/j.nmni.2020.100756
sha: 
doc_id: 334175
cord_uid: x10bbv7y

Dornase alfa, the recombinant form of the human DNase I enzyme, breaks down neutrophil extracellular traps (NET) that include a vast amount of DNA fragments, histones, microbicidal proteins and oxidant enzymes released from necrotic neutrophils in the highly viscous mucus of cystic fibrosis patients. Dornase alfa has been used for decades in patients with cystic fibrosis to reduce the viscoelasticity of respiratory tract secretions, to decrease the severity of respiratory tract infections, and to improve lung function. Previous studies have linked abnormal NET formations to lung diseases, especially to acute respiratory distress syndrome (ARDS). It is well known that novel Coronavirus disease 2019 (COVID-19) pneumonia progresses to ARDS and even multiple organ failure. High blood neutrophil levels are an early indicator of COVID-19 and predict severe respiratory diseases. Also it is reported that mucus structure of COVID-19 is very similar to cystic fibrosis due to the accumulation of excessive NET in the lungs. In this study, we showed the recovery of three COVID-19 patients after including Dornase alfa in their treatment. We followed clinical improvement in the radiological analysis (2-of-3 cases), oxygen saturation (SpO2), respiratory rate, disappearing of dyspnea, coughing and a decrease in NET formation and SARS-CoV-2 viral load after the treatment. Also here, we share our preliminary results suggesting that Dornase alfa has an anti-viral effect against SARS-CoV-2 infection in a green monkey kidney cell line, Vero, and a bovine kidney cell line, MDBK without determined cytotoxicity on healthy peripheral blood mononuclear cells.

The Coronavirus Disease 2019 (COVID-19) pandemic has affected more than 23 million people around the world, resulting in unprecedented health, social and economic crises. COVID-19 is caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which is manifested by flu-like symptoms and in some patients with acute respiratory distress syndrome (ARDS) and even viral pneumonia that progresses to multiple organ failure 1 . The exudative phase of ARDS is characterized by the productive and excessive immune response of proinflammatory cytokines and chemokines; increased neutrophil infiltration and accumulation in the alveoli; and alveolar epithelial capillary barrier disruption 2 . High blood neutrophil levels are an early indicator of SARS-CoV-2 infection and cause serious respiratory diseases. It has been demonstrated a pathogenic role 3, 4 for neutrophil-derived extracellular traps (neutrophil extracellular traps, NET) in a variety of thrombo-inflammatory conditions, including sepsis 5,6 , thrombosis [6] [7] [8] and respiratory failure 5, 6 . NETs are DNA fragments, histones, microbicidal proteins, and oxidant enzymes that are released by neutrophils during infection. However, in over response, NETs have the potential to initiate and spread inflammation and thrombosis 9, 10 . NETs have been reported in many lung and cardiovascular diseases in association with IL-1β secretion 8, [11] [12] [13] [14] [15] [16] . Although the exact determination of COVID-19 pathophysiology requires the development of model systems, the neutrophilic infiltration relationship has been established many times in the infection sites of Influenza A (H1N1), SARS-CoV and MERS-CoV and the development of ARDS in these patients 17, 18 . It was reported that in-vitro culturing neutrophils from influenza-infected lungs with infected alveolar epithelial cells trigger NETosis and increase endothelial damage. Mice lacking neutrophil cells have a mild response to influenza virus 19 . Also, higher plasma NETs level reported for patients with transfusion-related ARDS 20 . Similarly, J o u r n a l P r e -p r o o f recent studies confirmed the role of immune hyperactivation in the pathogenesis of COVID-19 21 .

Immune cells, especially neutrophils, infiltrate pulmonary capillaries that cause acute fibrin deposition and extravasation into the alveolar space. This excessive accumulation of neutrophils constitutes NETs which makes the mucus thick and viscous. The highly viscous mucus lowers the patient's respiratory function and impairs ventilation 22 suggesting NETs may play a major role in the disease. As a mechanism, double-stranded DNA constitutes the backbone of NETs and Dornase alfa promotes the clearance of NETs from plasma neutralization. Endogenous Deoxyribonucleases (DNases), which physiologically break up this extracellular DNA, may become overwhelmed by a massive influx of NETs 23, 24 . Clinically, recombinant human DNase I (rhDNase, Dornase Alfa) has the identical primary amino acid sequence with the native human enzyme and has been approved for the management of cystic fibrosis. Daily administration of Dornase alfa is effective in the treatment of cystic fibrosis patients improving pulmonary functions 25, 26 . The similarity of mucus secretions in COVID-19 and cystic fibrosis patients by the means of NETs makes Dornase alfa a therapeutic option in COVID-19 27, 28 . In this study, preliminary data is presented about in-vitro and in-vivo anti-viral and nuclease activity of Dornase alfa for the clearance of SARS-CoV-2 viral load and NETs in the lungs of COVID-19 patients.

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

In vitro isolation and propagation of SARS-CoV-2 from diagnosed COVID-19 patients are described in our previous study 29 

This study involves 3 patients diagnosed with COVID-19 specific PCR and clinical/radiological findings in Acibadem Altunizade Hospital, Istanbul. All the patients were dyspneic and supported by oxygen without mechanic ventilation before Dornase alfa treatment. Dornase alfa was always used as add-on therapy combined with other investigational drugs for COVID-19. All patients were followed by clinical findings (oxygen saturation (SpO 2 ), respiratory rate, fever, coughing, and dyspnea) and radiography with computerized tomography of the thorax (thorax CT). Dornase alfa was used daily for the subsequent three days at a dose of 2.5 mg with jet nebulizer at least one hour before chest physiotherapy as recommended 31 . All necessary patient consent has been obtained and the forms have been archived.

Nasopharyngeal and oropharyngeal specimens of three COVID-19 patient were collected on the day before and after 7 days of Dornase alfa treatment. Total RNA isolations from 100 µl patient In the bar graphs, independent two-tailed t-tests were performed using SPSS Statistics software.

No outliers were excluded in any of the statistical tests and each data point represents an independent measurement. Bar plots report the mean and standard deviation. The threshold of significance for all tests was set at * p<0.05.

Recombinant human DNase I (Dornase Alfa) degrades extracellular DNA and helps the clearance of NET that is constituted primarily of DNA fragments 32 . Although SARS-CoV-2 is positivestrand RNA virus 33 , we wanted to determine an anti-viral effect of Dornase alfa co-incubated with 1X TCID 50 dose of SARS-CoV-2 and MDBK cell line. The results show that Dornase alfa even with a small dose (3 U/mL) efficiently inhibits the infectious capacity of SARS-CoV-2, as seen by the cytopathic effect that was lost in higher doses of Dornase alfa (Fig. 1A) . Afterward, we asked whether Dornase alfa can degrade the SARS-CoV-2 positive-strand RNA genome.

Isolated total SARS-CoV-2 RNA was pre-incubated with Dornase alfa at different doses and Dornase alfa was inactivated at 72 o C before RT-PCR. We detected amplifications of the E and Orf1ab gene at higher cycles than the control that included only RNA with MgSO 4 (Fig. 1B) . We determined high doses of Dornase alfa (DA) increased cycle threshold (Ct) of the E gene from 10 in control No DA condition to 14 in 10 U DA treated condition, which was 16 fold difference ( Fig. 1C) . Besides, the Ct of the Orf1ab gene significantly increased from 11 in control No DA condition to 17 in 10U DA treated condition, which was 64 fold difference (Fig. 1C) . This preliminary study suggested that the Dornase alfa may have an anti-viral effect; although the mechanism is yet to be elucidated.

After we determined Dornase alfa may have nuclease activity against the SARS-CoV-2 genome.

We asked whether Dornase alfa can interfere with SARS-CoV-2 infection in a real-time cell Dornase alfa (Fig 2A) . Furthermore, we determined that cell index value in 100 U Dornase alfa & SARS-CoV-2 was significantly higher than that of the control Vero cell condition for 120 hour incubation (Fig 2A) . Between 120 and 144 hours, inhibition of SARS-CoV-2 propagation on the Vero cells that shows cytopathic effect is more evident a possible dose-dependent effect upon Dornase alfa pre-treatment (Fig 2A) . Also, at the end of the 144th hour normalized cell index analysis showed that the SARS-CoV-2 virus incubated with increasing doses of Dornase alfa significantly increased cell index similar to the untreated control Vero cells in a dose-dependent manner (Fig 2B) . Next, we used the same values and transformed them to the cytopathic effect (CPE)% by normalizing cell index value to the CPE value (control only Vero cell condition normalized to 0% CPE and other cell index values were normalized with respect to that value).

As the lower cell index, the higher CPE value. As Fig. 2B shows, the CPE rate was the highest in the only SARS-CoV-2 incubated condition (control No DA) while the value was significantly the lowest in 100 U Dornase alfa treated SARS-CoV-2 incubated wells (Fig 2C) . This real-time cell analyzing data further showed that Dornase alfa may have an anti-viral effect against the SARS-CoV-2 virus. Yet, plaque assay can be perfomed to determine change in TCID50 value in Dornase alfa treatment, which will be more direct approach to show the anti-viral effect.

The Dornase alfa has been used in the treatment of patients with cystic fibrosis for decades in order to prevent the clearance of NET formation 32, 34 . Several studies have showed DNase activity of Dornase alfa by degrading DNA fragment trapped NETs 26, [34] [35] [36] [37] . In this study, we wanted to show a new approach to evaluate in vitro clearance of NET by Dornase alfa as a proof of concept.

We mimicked in vitro NETosis with a clump formation constituting of extracellular DNA fragments, erythrocytes, lymphocytes, and neutrophils 38, 39 . In the test, we thawed adult human mononuclear cells. Following NET formation, we treated the cells with Dornase alfa along with PBMCs. However, after the treatment, the count increased to 12.9 +/-5.45 x10 6 million viable cells.

Also, we determined viability was not affected by Dornase alfa treatment using the dye exclusion test by staining the cells with Trypan blue (Supplementary Figure 2E) . This proof-of-concept study showed similar results with previous studies 26, 34, 36, 37, 40 that Dornase alfa efficiently clear NETs that trapped large amount of mononuclear cells.

PBMCs include most of the immune cell subtypes in the human blood such as lymphocytes, neutrophils, B cells, Natural Killer (NK) T cells, and monocytes. Healthy PBMCs are widely used in toxicology analysis of drug studies because peripheral blood is the front-line of exposure J o u r n a l P r e -p r o o f to chemicals in the body. Therefore, toxicity analysis with PBMCs is very important 39 . Although Dornase alfa is used in clinics for years without serious adverse effect 36 , we wanted to determine cytotoxicity on healthy adult PBMCs. We incubated PBMCs with Dornase alfa in a dosedependent manner for 48 hour and assessed changes in proportions of the immune cell subtypes and upregulation of activation markers (CD25 and CD107a) in the cells using flow cytometry as shown in Fig. 3A . We did not determine a statistically significant increase in the frequency of CD3+ T cells, especially CD3+ CD4+ T helper cells, except CD3+ CD8+ cytotoxic T cells (Fig.   3B& 3C) . Similarly, CD19+ B cell proportion did not change upon increasing the Dornase alfa concentration (Fig. 3C) . However, the proportion of CD3+ CD56+ NK T cells significantly increased in a dose-dependent manner (Fig. 3C) . However, we did not determine a significant increase in CD25 and CD107a expression, suggesting that Dornase alfa does not induce lymphocyte activation (Fig. 3C) . Furthermore, we labeled dead cells in PBMCs that were treated with Dornase alfa in a dose-dependent manner with 7AAD after 72 hour of incubation (Fig. 3D ).

We determined that there was significantly no cytotoxic effect of Dornase alfa up to 100 U although there was 10% cytotoxic at the highest dose, 300 U (Fig. 3D) . These results suggest that Dornase alfa does not show cytotoxicity in PBMC up to 100 U and it may increase proportion of NK-T cells that is yet to be studied what is the mechanism.

Case 1: He was 64 years old man, with type 2 diabetes mellitus using daily metformin ( Table 1) .

He had a fever and was dyspneic at admission. After 5 days on hydroxychloroquine therapy, he was coughing, still dyspneic with SpO 2 97% at room air and respiratory rate 20/min. Favipiravir was added but after 3 days on favipiravir, he was coughing, still dyspneic with SpO 2 decreasing 95% at room air and respiratory rate still 20/min ( Table 1) . He had extensive ground-glass J o u r n a l P r e -p r o o f opacities on thorax CT. Dornase alfa was added on the 8 th day of the disease. After 24 hours of first Dornase alfa administration, cough increased, dyspnea disappeared, the respiratory rate decreased to 18/min and SpO 2 increased to 97% at room air ( Table 1) . On the third day of Dornase alfa therapy, Thorax CT Image was taken with significant improvement (Fig. 4A) . Also, fever, dyspnea, and coughing disappeared, SpO 2 97% without oxygen support (Fig. 5A) with respiratory rate 18/min (Fig. 5B) . Besides, inflammatory biomarkers like C-reactive protein (CRP) and procalcitonin levels decreased after 3 days of the treatment (Fig. 5C & 5D) and (Table 1) . Case 2: She was 33 years old woman, breast cancer still on tamoxifen therapy (Table 1) . At admission, she had a fever, was coughing and dyspneic, SpO 2 99% with oxygen support, and respiratory rate 19/min. After 2 days on hydroxychloroquine and favipiravir treatment, she was still dyspneic and coughing, respiratory rate 19/min, and SpO 2 decreasing 96% with oxygen support ( Table 1) . She had extensive ground-glass opacities on thorax CT. Dornase alfa was added on the 2 nd day of the disease. After Dornase alfa administration, Thorax CT Image was taken and persistent lesions were detected (Fig. 4B) . Coughing increased, dyspnea disappeared, SpO 2 was 96% with oxygen support after 24 hours of the treatment (Fig. 5A) , and respiratory rate 19/min (Fig. 5B) . On the third day of dornase alfa therapy, fever, dyspnea, and coughing disappeared, SpO 2 97% (Fig. 5A) without oxygen support and respiratory rate 18/min (Fig. 5B) .

Also, the CRP level decreased (Fig. 5C ) but the procalcitonin level was stable (Fig. 5D ) after 3 days of the treatment ( Table 1) . Case 3: She was 27 years old woman, without comorbidities (Table 1) . At first admission to another center, she had a fever, was coughing and dyspneic. She had extensive ground glass opacities on thorax CT and hydroxychloroquine was started. After 5 days of hydroxychloroquine J o u r n a l P r e -p r o o f therapy, she deteriorated and favipiravir was started. At admission in our center, she was on the 3 rd day of favipiravir therapy with dyspnea and coughing, her respiratory rate 20/min and SpO 2 91% with oxygen support (4 L/min) ( Table 1) . After dornase alfa administration, Thorax CT Image was taken with a significant improvement (Fig. 4C) . After 24 hours of first dornase alfa administration, cough increased, dyspnea persisted, SpO 2 was 95% with oxygen support (3 L/min) (Fig. 5A) , and respiratory rate 20/min (Fig. 5B) . On the third day of Dornase alfa therapy, fever, dyspnea, and coughing disappeared, SpO 2 99% with oxygen support (1 L/min) (Fig. 5A) and respiratory rate 18/min (Fig. 5B) . Also, the CRP level decreased (Fig. 5C ) but the procalcitonin level was stable (Fig. 5D ) after 3 days of the treatment ( Table 1) .

In addition to a clinical improvement in the Dornase alfa treated patients, we also collected nasopharyngeal and oropharyngeal specimens from the patients on the day before the treatment and after 7 days of the Dornase alfa treatment for assessing SARS-CoV-2 viral load in the patients using quantitative RT-PCR analysis. We determined a decrease in viral load in all three patients after Dornase alfa treatment (Fig. 6) . This study suggests that Dornase alfa participates in the clearance of virus load and NETs in the infected lung (Fig. 7) . Dornase alfa degrades extracellular DNA fragments and promotes clearance of NET formation.

In our daily-process of patient blood apheresis, we frequently use Dornase alfa to clear NET-like clumps that can easily form after cryo-preservation and thawing process. Dornase alfa has been used in patients with cystic fibrosis to clear NETs in respiratory tract. As reported in literature 26, 34, 36, 37, 40 , we recapitulated that NETs were cleared by Dornase alfa in Supplementary   Fig 2. This result gave rise to the thought that Dornase alfa can be used in COVID-19 patients that can have NET-like formations induced by infiltrated neutrophils in their lungs. Therefore, three COVID-19 patients at different ages (64, 33, and 27 years-old) were treated with Dornase alfa.

We observed significant improvement in breathing by the disappearing of dyspnea after dornase alfa therapy. All of the patients had better oxygenation after dornase alfa by the means of SpO 2 values. Increased coughing was observed after the first dornase alfa dose most probably due to the resolving of viscous mucus, but this phenomenon was followed by the disappearing of coughing at the end of therapy. Also after the last dornase alfa dose, decreasing respiratory rates for all patients were accepted as an improvement in pulmonary functions. In addition to the improvement in the clinical data, we also observed a decrease in the inflammatory biomarkers like CRP and procalcitonin. To our knowledge, dornase alfa has no direct anti-inflammatory effects so it is needed to test these laboratory findings in further studies.

Afterward, we detected radiological improvements in thorax CT images that were compatible with better ventilation after dornase alfa therapy and supported our clinical findings. Radiological improvement was significant for Case 1 and Case 3. However, persistent lesions were detected in Case 2, this also might be accepted as a response to therapy in this early phase of the disease that mostly progressed with radiological deterioration. On the other hand, we speculated comorbidities that both cancer and COVID-19 in the same patient may avoid determining similar clinical improvement. Although it is apparent that only three patients are not enough to comment on the efficacy of dornase alfa, two independent clinical trials with Dornase alfa on COVID-19 patients will evaluate efficacy. One of them is led by Agence Nationale de Sécurité du As Dornase alfa is routinely taken drug by cystic fibrosis patients, data about the impact of COVID-19 on cystic fibrosis patients could be helpful to comment on the efficacy of Dornase alfa. There is a recent study just about this subject, reporting very few and less severe COVID-19 positive cystic fibrosis patients across Europe 42 . This data is very interesting because it is not compatible with the reported 8 times higher death rate in chronic respiratory diseases compared to the cases with no comorbidities (COVID-19 Fatality Rate by Comorbidity.

www.worldometers.info). Lower severity of COVID-19 in cystic fibrosis patients could be explained with the long-term antibiotic therapy such as azithromycin or extreme efforts of families to minimize social contacts 42 . Also, we know that Dornase alfa is a regular therapy used by cystic fibrosis patients and it might be the reason for this promising result in COVID-19.

Besides the patients presented in this study, we have the data about two cystic fibrosis patients recovered from COVID-19 without severe symptoms under regular Dornase alfa therapy (personal communication).

All of these data gave rise to the thought that Dornase alfa may have an anti-viral effect against SARS-CoV-2 infection. With this purpose, in our real-time cell analysis assay that Dornase alfa was co-incubated with SARS-CoV-2, we determined that the infection and SARS-CoV-2 viral propogation on the Vero cells did not progress in increasing concentrations of Dornase alfa pretreatment. This preliminary in vitro data proposed that Dornase alfa may have a possible antiviral effect against SARS-CoV-2 which is yet to be studied in detail. Our in vitro tests have shown that the RNA genome can be degraded by pre-incubating the SARS-CoV-2 virus with 

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