Cetylpyridinium Chloride Mouthwash to Reduce Shedding of Infectious SARS-CoV-2: A Double-Blind Randomized Clinical Trial.

The airborne transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via respiratory fluids and droplets suggests that mouthwashes containing substances with virucidal activity can help reduce viral spread. We conducted a multicenter, double-blind, placebo-controlled, randomized trial to assess the virucidal activity of cetylpyridinium chloride (CPC) mouthwashes. Outpatients who tested positive for SARS-CoV-2 infection with or without symptoms were randomized to perform washes and gargles for 1 min with 15 mL of either colored distilled water or 0.07% CPC (Vitis CPC Protect) mouthwash. The study outcomes were the SARS-CoV-2 log10 viral RNA load and the nucleocapsid protein levels, both in saliva at 1 and 3 h after the intervention. In total, 118 patients were enrolled and randomized (mean [SD], age 46 [14] y). Thirteen of 118 participants (11%) did not complete follow-up or had insufficient sample volume for testing and were excluded from the analysis. The assessment of the viral load showed no significant differences between groups at any of the investigated points. However, the levels of SARS-CoV-2 nucleocapsid protein of lysed viruses were significantly higher in the CPC group compared with the control group at 1 h (adjusted difference 269.3 pg/mL; 95% confidence interval [CI], 97.1-441.5) and at 3 h postintervention (561.1 pg/mL; 95% CI, 380.0-742.2). In nonhospitalized patients with asymptomatic or mild symptomatic SARS-CoV-2 infection, a 0.07% CPC mouthwash, compared to placebo, was associated with a significant increase of nucleocapsid protein levels in saliva, indicating enhanced disruption of viral particles.

UNRAVELING the ANTIVIRAL ACTIVITY of PLITIDEPSIN by ULTRASTRUCTURAL ANALYSIS

Background: The use of compounds against highly conserved cellular host factors required to complete the replication cycle of distinct viruses such as SARS-CoV-2 offers a common solution to diverse viral threats. This approach is especially relevant for pan-antiviral effects given that viruses converge at intracellular steps such as viral genome replication and protein production. Currently, there are only a limited number of approved drugs involved in targeting intracellular host factors. One of these compounds is plitidiepsin, which has shown a potent preclinical efficacy against SARS-CoV-2 by targeting the host protein eEF1A. Plitidepsin inhibits nucleocapsid viral protein expression and viral induced cytopathic effect in vitro. In addition, it also reduces genomic and subgenomic RNA expression. However, how plitidepsin exerts its antiviral activity remains unknown. Methods: Current models of SARS-CoV-2 replication propose that upon viral fusion, non-structural viral proteins form a replication-transcription complex that associates to compartments with a double membrane vesicle (DMV) morphology that shelters the viral genome replication. Here we have used an electron microcopy analysis to explore the antiviral effect of plitidepsin and its impact on SARS-CoV-2 replication and DMV formation on target Vero E6 cells. Results: This ultrastructural analysis allowed to recapitulate the SARS-CoV-2 infectious life cycle, where evident viral DMV formation was observed as well as viral budding events along with cell-associated viruses. However, in cells treated with plitidepsin at different non-toxic concentrations (0.2 and 0.05 μ M) there was a lack of viral DMV formation and a complete absence of viral particles. Complementary SARS-CoV-2 nucleocapsid and dsRNA immunogold labelling unambiguously confirmed the lack of viral replication in plitidepsin-treated cells. Overall, these data indicate that plitidepsin treatment abrogated the formation of DMVs, and the detection of nucleocapsid or dsRNA viral products. Conclusion: Electron microscopy ultrastructural analysis coupled to immunogold labelling of SARS-CoV-2 products offer a unique approach to understand how antivirals work. This knowledge is key to identify the mechanism of action of promising compounds interfering with host factors whose implication in strategic biological processes can be applied as pan-antiviral strategies.

CETYLPYRIDINIUM CHLORIDE MOUTHWASHES to REDUCE the SHEDDING of VIABLE SARS-CoV-2

Background: SARS-CoV-2 is spread via airborne transmission. Mouthwashes containing virucidal compounds can help reduce viral spread. Here we show that cetylpyridinium chloride (CPC), a quaternary ammonium present in many oral mouthwashes, reduces SARS-CoV-2 infectivity by disrupting viral membranes both in vitro and in vivo. Methods: We tested the capacity of CPC-containing mouthwashes to inhibit SARS-CoV-2 entry into target cells by using a luciferase-based assay with a reporter lentivirus pseudotyped with the SARS-CoV-2 spike protein. The replication-competent SARS-CoV-2 B.1.1.7 and D614G variants were also assayed. Viral envelope disruption by CPC's virucidal effect was measured by dynamic light-scattering analyses (DSL). We confirmed these results by modifying an ELISA that detects the SARS-CoV-2 nucleocapsid (NC), which was used in the absence of its own lysis buffer. The effect of CPC in the saliva of individuals with CoVID-19 was assessed in a double-blind, placebo-controlled, randomized clinical trial. SARS-CoV-2 positive patients were randomized to gargle either water or 0.07% CPC mouthwash. The study outcomes were the SARS-CoV-2 log10 viral RNA load by RT-PCR and the NC protein levels by ELISA, both in saliva at 1h and 3h post-intervention. Results: CPC-containing mouthwashes inhibited SARS-CoV-2 viral fusion in vitro in a dose-dependent manner and decreased more than a 1000 times the viral TCID50 in target cells, regardless of the variant tested. The ELISA and the DSL analyses pointed to the effective disruption of the integrity of viral membranes after treatment with CPC. The clinical study performed with 105 patients showed no significant differences in viral RNA load at 1h and 3h post-treatment in saliva between placebo and CPC-treated groups. However, the levels of SARS-CoV-2 NC protein of lysed viruses were significantly higher in the CPC group at 1h and 3h post-intervention. Conclusion: CPC decreased more than a 1000 times the infectivity of SARS-CoV-2 in vitro and was effective against different SARS-CoV-2 variants. In CoVID-19 patients, the use of a 0.07% CPC mouthwash correlated with a statistically significant increase of NC protein levels in saliva, indicating enhanced disruption of viral particles. CPC-containing mouth rinses can represent a cost-effective measure to reduce SARS-CoV-2 infectivity in saliva, aiding to reduce viral transmission from infected individuals regardless of the variants they are infected with.

Mouthwashes with CPC Reduce the Infectivity of SARS-CoV-2 Variants In Vitro.

Oral mouthwashes decrease the infectivity of several respiratory viruses including SARS-CoV-2. However, the precise agents with antiviral activity in these oral rinses and their exact mechanism of action remain unknown. Here we show that cetylpyridinium chloride (CPC), a quaternary ammonium compound in many oral mouthwashes, reduces SARS-CoV-2 infectivity by inhibiting the viral fusion step with target cells after disrupting the integrity of the viral envelope. We also found that CPC-containing mouth rinses decreased more than a thousand times the infectivity of SARS-CoV-2 in vitro, while the corresponding vehicles had no effect. This activity was effective for different SARS-CoV-2 variants, including the B.1.1.7 or Alpha variant originally identified in United Kingdom, and in the presence of sterilized saliva. CPC-containing mouth rinses could therefore represent a cost-effective measure to reduce SARS-CoV-2 infectivity in saliva, aiding to reduce viral transmission from infected individuals regardless of the variants they are infected with.