Mouthrinses against SARS-CoV-2 - High antiviral effectivity by membrane disruption in vitro translates to mild effects in a randomized placebo-controlled clinical trial

Abstract

The emergence of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) represents an unprecedented threat for the human population, necessitating rapid and effective intervention measures. Given the main infection route by airborne transmission, significant attention has been bestowed upon the use of antiseptic mouthrinses as a way to possibly reduce infectious viral titers. However, clinical evaluations are still sparse. Thus, we evaluated a wide variety of antiseptic agents that can be used as mouthrinses for their antiviral effects in vitro and their respective mode of action. One of the most promising antiseptic agents (benzalkoniumchloride, BAC) was used in a randomized placebo-controlled clinical trial with subsequent analysis of viral loads by RT-qPCR and virus rescue in cell culture. Mechanistic analysis revealed that treatment with BAC and other antiseptic agents efficiently inactivated SARS-CoV-2 in vitro by primarily disrupting the viral envelope, without affecting viral RNA integrity. However, the clinical application only resulted in a mild reduction of viral loads in the oral cavity. These results indicate that gargling with mouthrinses comprising single antiseptic agents may play a minor role towards a potential reduction of transmission rates and thus, these findings are of utmost importance when considering alternative COVID-19 prevention strategies.

Keywords: Antiseptic agents; Benzalkonium chloride; Capsid protection assay; Mouthrinse; Mouthwash; SARS-CoV-2.

Fig. 1

Quantitative suspension test of commercially available mouthrinses. (A) Product A and (B) Product B of different compositions (white bars) or medium (grey bar) were incubated with SARS-CoV-2 and an interfering substance for 30 s. Viral titers were obtained by end point dilution on Vero E6 cells. Cytotoxicity is indicated as lower limit of quantification (LLOQ, dotted line). 50% tissue culture infectious dose (TCID₅₀/mL) was calculated according to Spearman and Kärber. Data are represented as mean ± SD of three independent experiments.

Fig. 2

Quantitative suspension test of antiseptic agents. (A-J) Antiseptic agents in various concentrations (white bars) or medium (grey bar) were incubated with SARS-CoV-2 and an interfering substance for 30 s. Viral titers were obtained by end point dilution on Vero E6 cells. Cytotoxicity is indicated as lower limit of quantification (LLOQ, dotted line). 50% tissue culture infectious dose (TCID₅₀/mL) was calculated according to Spearman and Kärber. Red numbers indicate concentrations that appear in commercially available mouthrinses. Data are represented as mean ± SD of three independent experiments.

Fig. 3

Mode of action of antiseptic agents. SARS-CoV-2 was either incubated with DMEM (A-C), UV-inactivated (D-F), treated with 70% EtOH (G-I) or 0.1% BAC (J-L) and an interfering substance for 30 s. RNA integrity (A, D, G and J) for each treatment (white bar) was investigated by RT-qPCR and compared to DMEM (grey bar). Sucrose step gradient ultracentrifugation was performed to evaluate viral envelope integrity (B, E, H, K) and infectivity (blue line, B). RNA copy numbers in each fraction were determined by RT-qPCR (black line) and compared to DMEM (grey line). The viral envelope was further assessed by a capsid protection assay (C, F, I, L). Therefore, one replicate was left untreated, one part was treated with proteinase K for 1 h at 4 °C, and another part was lysed in 5% Triton X-100 prior to proteinase K treatment. The amount of protease-resistant nucleocapsid protein was quantified by Western blot. Data indicate averages.

Fig. 4

In vivo activity of BAC against SARS-CoV-2. BAC was applied in a randomized placebo-controlled clinical trial in COVID-19 patients. RNA copy numbers were determined before gargling (baseline) as well as 15 and 30 min after gargling (A) comparing the placebo group (grey) to the BAC group (black). At similar time points SARS-CoV-2 antigen (B and C) and infectivity (D and E) was assessed for the BAC and placebo group, respectively. Data are shown as medians including 1st and 3rd quartiles.

Fig. 5

Schematic modes of action of antiseptic agents on SARS-CoV-2 particles. Selected agents disrupt the viral envelope without affecting RNA integrity. Figure created with BioRender.com.