We evaluated the virucidal activity of OSCN− and OSCN−/LF against SARS-CoV-2. In our experimental conditions, OSCN− had the main virucidal effect in-vitro against SARS-CoV-2. Three components are mixed in airway lumen to produces this biocidal compound: the enzymatically active lactoperoxidase (LPO), secreted by serous cells of the submucosal glands and by goblet cells; the anion thiocyanate (SCN−), delivered by duct cells of submucosal gland; and hydrogen peroxide (H2O2), made by epithelial cells . Unlike in the trachea, the main bronchi, third and fifth generation bronchi, LPO mRNA was nearly absent in lung parenchyma suggesting absence of OSCN− production in the alveoli . Furthermore, due to the need of enhancing gas exchange, epithelial alveolar cells cannot contain strong protective structures and hence are weak, fragile and more vulnerable to viral attacks. The biocidal effect of OSCN− takes place in the airways lumen. Interestingly, it was found that a solution of the two molecules combined (ALX-009, developed by Alaxia SAS for the treatment of bacterial infection in cystic fibrosis patients ) was easily administered by inhalation of aerosol, using a nebulizer as delivery device, in a Phase I Clinical Trial (NCT02598999 of 2018) to healthy volunteers and patients affected by cystic fibrosis. Thereafter, on May 2009, the combination of OSCN− and LF for treatment of cystic fibrosis was designated as orphan drug by the US Food and Drug Administration and the European Medicines Agency . Topical administration of OSCN− and LF combined, in the form of aerosol, might effectively inactivate free SARS-CoV-2 virions nearing the epithelium from outside or released from infected cells, thus mitigating or preventing the spread of the infection in the host tissues as well as its propagation to further susceptible individuals.
Despite strong evidence of SARS-CoV-2 inactivation, the exact virucidal mechanism of OSCN− is still unknown. Ozone at high doses has been shown to inactivate SARS-CoV-2 by oxidative stress both directly and indirectly through reactive oxygen species produced by ozone decomposition and/or by oxidation of double bonds of viral lipids, proteins, and amino acids that lead to the formation of reactive radicals (RCOO·) . Indirect modes of action further propagate the oxidation through a chain reaction . Enveloped viruses - as vesicular stomatitis Indiana virus, vaccinia virus, influenza A virus and certain strains of type 1 herpes simplex virus - are more sensitive to ozone, whereas non-enveloped adenovirus type 2 was more resistant to this gas [18; 19]. Irreversible oxidative damage of the lipid components of the viral envelope or the nucleoproteins could also be the mechanism of virucidal activity against SARS-CoV-2 of OSCN−, which is a less potent (but also less damaging) oxidizing agent than ozone. As already shown, LPO gives OSCN− in presence of SCN−; OSCN− in turn reacts with the thiol moiety of peptides or proteins (R-SH) generating sulfenyl thiocyanate (R-S-SCN), which by adding one molecule of H2O produces sulfenic acid (R-S-OH) as well as SCN− at the end of the cycle . The LPO cycle thus extends the duration of effects of OSCN− beyond the limited half-life (about 1 hour) of this compound . Sulphydryl oxidation determines inhibition of numerous enzymes if the proteins contain the amino acid cysteine, which is abundant in the coronavirus proteins such as those in the spikes of the envelope .
The OSCN− concentration used in our experimental conditions was up to 100 µM, higher than that reported in human saliva (20–60 µM) or in resting (31 µM), stimulated whole saliva (25 µM) and parotid saliva (30 µM), respectively [21, 22]. Our data suggested that physiological OSCN− concentrations could inhibit at least the 50% of SARS-CoV-2 infection. However, OSCN− and bovine LF concentrations in ALX-009 were higher than those used in our experimental setting, reaching values of 3,600 µM and 8 g/L respectively, which therefore should likely maximize their respective virucidal activity .
Moreover, we have previously demonstrated that OSCN− inhibits A(H1N1)pdm09 influenza virus infection, when the virus was challenged with OSCN− for 60 min at 37 °C before infection, with a similar IC50 than the one determined for rVSV-S . Thereafter the antiviral activity of OSCN− was tested also against several other types of influenza virus, confirming a strain independent virucidal effect [24; 25]. Overall, these results indicate a potent and potentially wide-range antiviral activity of OSCN−.
Lactoferrin, one of the most abundant antimicrobial proteins in normal airway secretions , seems to improve the inhibition of the viral infection only at lower OSCN− concentrations. The antiviral mechanism of LF is based on the ability to prevent the virus from anchoring with targeted cells . In particular, LF binds with heparan sulfate proteoglycans (HSPGs), which are cell-surface and extracellular matrix macromolecules acting as an attachment factor for many viruses including SARS-CoV-1. LF blocks the infection of SARS-CoV-1 by competing with the virus for HSPGs, therefore preventing the viral concentration on the surface of target cells . Given this mechanism of action, the antiviral activity of LF could be better displayed with an in-vivo study.
In conclusion, our results indicate that OSCN− and LF have a virucidal activity against SARS-CoV-2, alone or in combination. Although the in vitro results require an in-vivo validation, the LF and OSCN− combination may have a relevant clinical impact reducing the diffusion of infection in the host tissues as well as the spread of SARS-CoV-2 particles to new susceptible hosts. The latter, can have important positive effects to control the COVID-19 pandemic.