Preliminary report of in vitro and in vivo effectiveness of dornase alfa on SARS-CoV-2 infection

Abstract

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 in COVID-19 is very similar to that in cystic fibrosis due to the accumulation of excessive NET in the lungs. In this study, we showed the recovery of three individuals with COVID-19 after including dornase alfa in their treatment. We followed clinical improvement in the radiological analysis (two of three cases), oxygen saturation (Spo₂), respiratory rate, disappearance of dyspnoea, coughing and a decrease in NET formation and severe acute respiratory syndrome coronavirus 2 (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.

Keywords: Coronavirus disease 2019; NETosis; dornase alfa; neutrophil extracellular traps; severe acute respiratory syndrome coronavirus 2.

FIG. 1

Anti-viral effect of dornase alfa against SARS-CoV-2. (a) Cytopathic effect (CPE) in MDBK cells infected with SARS-CoV-2 (10 × TCID₅₀) along with dornase alfa in a dose-dependent manner for 8 days. (b) Detected cycle of real-time PCR fluorescence intensity of the SARS-CoV-2 RNA genome that were pre-incubated with dornase alfa at different doses. PC, positive control, synthetic E or Orf1ab genes. NC, negative control, dH₂O. (c) The bar graphs showing cycle threshold (Ct) values of the dornase-alfa-treated SARS-CoV-2 samples, PC, and NC in (b). ∗p < 0.05; NS, not significant.

FIG. 2

Cytopathic effect of SARS-CoV-2–dornase alfa incubated Vero cells in real-time cell analysis (RTCA). (a) Representative normalized cell index histogram showing proliferating viable cells that were control only Vero (green line), 100 U (blue line) or 10 U (purple line) dornase alfa-preincubated SARS-CoV-2 (blue line), and only 10 × TCID₅₀ dose of SARS-CoV-2 (red line). Cell index was normalized based on the values at the end of 24-hour pre-culture. (b) Bar graph showing quantification of the normalized cell index value of the conditions treated with the SARS-CoV-2 along with/without dornase alfa. (c) Bar graph showing normalized cytopathic effect (% CPE) using values of normalized cell index. ∗p < 0.05; NS, not significant.

FIG. 3

Drug toxicities of dornase alfa on peripheral blood mononuclear cells (PBMCs). Three healthy adult PBMC samples were inoculated with dornase alfa in a dose-dependent manner for 48 h before flow cytometric analysis. (a) Flow cytometry plots that show immune cell subtypes (CD19⁺ B, CD3⁺ CD4⁺ T helper, CD3⁺ CD8⁺ cytotoxic T, and CD3⁺ CD56⁺ natural killer T (NKT) cells) and their activation (with alfaCD25 and alfaCD107a). (b) The bar graph that shows the change in average frequency of total CD3⁺ T lymphocytes in the PBMCs after treatment. (c) The bar graphs that show average proportions of the immune cell subtypes and their activation. (d) The flow cytometry plots showing viability of the cells analysed with 7aminoactinomycin D staining in flow cytometry. The bar graph showing viable proportion of the PBMCs treated with dornase alfa. ∗p < 0.05; NS, not significant.

FIG. 4

Comparison of thorax CT images before (left) and after (right) dornase alfa therapy for all three patients. In Case 1 (a) and Case 3 (c) significant improvement occurred, in Case 2 (b) persistent lesions were detected.

FIG. 5

Improvement in clinical outcomes following dornase alfa treatment. Temporal changes of (a) Spo₂ per patient, (b) respiratory rate per patient, (c) C-reactive protein per patient and (d) procalcitonin per patient.

FIG. 6

SARS-CoV-2 viral load in three dornase alfa-treated COVID-19 patients. The bar graphs showing SARS-CoV-2 viral copy numbers the day before the treatment and 7 days after dornase alfa treatment.

FIG. 7

Summary of the anti-viral and nuclease activities of dornase alfa. SARS-CoV-2-infected alveoli in infected lungs that induce formation of NETs (NETosis) trapping DNA and histone fragments along with macrophages (blue) and neutrophils (pink). Dornase alfa clears SARS-CoV-2 viral load and NETs in the alveoli.