Hypothiocyanite and Hypothiocyanite/Lactoferrin Mixture Exhibit Virucidal Activity In Vitro against SARS-CoV-2

Abstract

SARS-CoV-2 replicates efficiently in the upper airways during the prodromal stage, resulting in environmental viral shedding from patients with active COVID-19 as well as from asymptomatic individuals. There is a need to find pharmacological interventions to mitigate the spread of COVID-19. Hypothiocyanite and lactoferrin are molecules of the innate immune system with a large spectrum cidal activity. The Food and Drug Administration and the European Medicines Agency designated the hypothiocyanite and lactoferrin combination as an orphan drug. We report an in vitro study showing that micromolar concentrations of hypothiocyanite exhibit dose- and time-dependent virucidal activity against SARS-CoV-2 and that the latter is slightly enhanced by the simultaneous presence of lactoferrin.

Keywords: ALX-009; COVID-19; SARS-CoV-2; hypothiocyanite; lactoferrin; lactoperoxidase system.

Figure 1

OSCN⁻ and OSCN⁻/LF virucidal activity against the pseudovirus VSV-S diluted in Minimum Essential Medium (MEM). Infection of Vero cells was evaluated measuring the activity of the VSV-S encoded luciferase. (a) Efficiency of pseudovirus infection at MOI 0.065 and 0.015 FFU/mL after preincubation with different OSCN⁻ concentrations for 1 h at 37 °C; (b) evaluation of the virucidal activity of OSCN⁻ after pseudovirus treatment for 0, 20, 40, and 60 min at 37 °C before the infection of target cells at MOI 0.015; (c) comparison between OSCN⁻ and OSCN⁻ + LF virucidal activity after 1 h of preincubation of VSV-S and before cell infection at MOI 0.015. Data (mean ± SD, N = 3, experiments in duplicate) are percentages of no drug, set as 100% (* = p < 0.05; *** = p < 0.001).

Figure 2

Cytotoxicity of OSCN⁻ and LF. The cytotoxicity of (a) OSCN⁻, (b) LF, and (c) OSCN⁻+ LF (4 g/L) diluted in MEM was evaluated on Vero cells after 24 h of treatment using the MTT assay. Data (mean ± SD, N = 3, experiments in quadruplicate) are percentages of no drug, set as 100%.

Figure 3

Virucidal activity of OSCN⁻ and OSCN⁻/LF against SARS-CoV-2. SARS-CoV-2 was diluted in MEM and incubated for 1 h at 37 °C with only OSCN⁻ or supplemented with LF before infection of Vero-E6 cells. The reduction of infectivity was evaluated by plaque assay. (a) Virucidal effect of OSCN⁻; (b) comparison between different times of virus-OSCN⁻ exposure on the efficiency of the virucidal activity; (c) evaluation of the combination OSCN⁻+ LF on the virucidal activity. Data (mean ± SD, N = 3, experiments in duplicate) are percentages of no drug, set as 100%.

Figure 4

OSCN⁻ extemporaneous production via a two-step biocatalytic pathway.