key: cord-0981187-lf6s9nza
authors: Okamoto, Nako; Saito, Akatsuki; Okabayashi, Tamaki; Komine, Akihiko
title: Virucidal activity and mechanism of action of cetylpyridinium chloride against SARS-CoV-2
date: 2022-01-31
journal: bioRxiv
DOI: 10.1101/2022.01.27.477964
sha: 0c2eb1259639b0ffa6b6fbe3292c563ea8bffcc1
doc_id: 981187
cord_uid: lf6s9nza

Objective Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen causing the coronavirus disease 2019 (COVID-19) global pandemic. Recent studies have shown the importance of the throat and salivary glands as sites of virus replication and transmission. The viral host receptor, angiotensin-converting enzyme 2 (ACE2), is broadly enriched in epithelial cells of the salivary glands and oral mucosae. Oral care products containing cetylpyridinium chloride (CPC) as a bactericidal ingredient are known to exhibit antiviral activity against SARS-CoV-2 in vitro. However, the exact mechanism of action remains unknown. Methods This study examined the antiviral activity of CPC against SARS-CoV-2 and its inhibitory effect on the interaction between the viral spike (S) protein and ACE2 using an enzyme-linked immunosorbent assay. Results CPC (0.05%, 0.1% and 0.3%) effectively inactivated SARS-CoV-2 within the contact times (20 and 60 s) in directions for use of oral care products in vitro. The binding ability of both the S protein and ACE2 were reduced by CPC. Conclusions Our results suggest that CPC inhibits the interaction between S protein and ACE2, and thus, reduces infectivity of SARS-CoV-2 and suppresses viral adsorption.

Coronavirus disease 2019 has spread worldwide since the end of 2019, and a pandemic was declared by the World Health Organization (WHO) in March 2020. More than 352 million confirmed cases and 5.6 million deaths have been reported as of January 2021 [1] .

The novel disease pathogen, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a single-stranded RNA-enveloped virus. Glycosylated spike (S) proteins cover the surface of SARS-CoV-2, and viral cell entry is mediated by binding to the host cell receptor, angiotensin-converting enzyme 2 (ACE2) [2] .

Recent reports have shown that epithelial cells of salivary glands and the throat are major sites of viral replication and release [3, 4] , as well as infection of gingival epithelial cells in the oral cavity, based on studies of COVID-19 patients [5] . In addition, the Centers for Disease Control and Prevention (CDC) have recommended use of preprocedural oral care products, which may reduce the level of oral microorganisms in aerosols and spatter generated during dental procedures [6] . Many studies have also shown that oral care products containing antiseptic agents decrease the infectivity of SARS-CoV-2 [7] [8] [9] . Use of such products may reduce the risk of viral transmission by removal or inactivation of infectious viral particles in the oral cavity [10] [11] [12] [13] [14] .

Cetylpyridinium chloride (CPC) is a quaternary ammonium compound that is commonly used as a broad-spectrum antimicrobial agent in oral care products. CPC was recently shown to have antiviral activity against SARS-CoV-2 in vitro [7, 8, 15] . The mechanism of action is thought to be viral lipid membrane disruption [16, 17] , but the effects of CPC on SARS-CoV-2 S protein and ACE2 are unknown. In this study, we evaluated the effects of CPC on reduction of SARS-CoV-2 infection in vitro within the contact times for use of oral care products and on the binding ability of S protein and ACE2. Virus suspension mixed with phosphate-buffered saline (PBS(-), Nacalai tesque, Kyoto, Japan) without CPC was used as a negative control. To calculate plaque-forming units (PFUs), cells were fixed with 10% formalin (Fujifilm Wako Pure Chemical, Osaka, Japan) for 30 min, followed by staining with 0.1% methylene blue (Fujifilm Wako Pure Chemical).

The antiviral effects of CPC were assessed using the logPFU ratio. Povidone-iodine (PI, final concentration 0.1%) was used as a positive control. All experiments were performed in a BSL-3 laboratory. Three independent experiments were performed for each sample (n = 3). 

The interaction between the receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 was evaluated using a COVID-19 Spike-ACE2 Binding Assay Kit II An enzyme-linked immunosorbent assay (ELISA) was performed following the manufacturer's instructions [18] . Spike RBD protein was mixed with CPC and added to wells coated with ACE2 at final concentrations of CPC of 0.3%, 0.05% and 0.0125%. The mixture in the ACE2-coated wells was incubated for 2.5 h at room temperature. Next, to evaluate the effect of CPC on the RBD, a different assay was performed. Equal amounts of 0.6%, 0.1% or 0.025% CPC solution and RBD solution were mixed, and after 20 s, the mixture was diluted 10-fold with a neutralizer. The solution was then diluted 10-fold with MEM containing 2% FBS and added to the ACE2-coated wells. These samples were then subjected to an ELISA.

Finally, another assay was performed to evaluate the effect of CPC on ACE2. 0.3% CPC was added to the ACE2-coated wells and incubated for 20 or 60 s. After removing the CPC solution, neutralizer and MEM containing 2% FBS were added in turn. After removing the liquid in the wells, RBD was added and an ELISA was performed. Assay diluent was used as control. The AV of the sample treated with CPC was compared with the control AV, and the % binding for the sample was calculated.

Data are presented as means ± S.E.M. (n=3). Significance was evaluated by a two-sided Dunnett test for comparison with untreated controls. *p < 0.05 and **p < 0.001 indicate a significant difference. IBM SPSS Statistics 27 was used for statistical analysis.

To investigate whether CPC has antiviral activity against SARS-CoV-2 to VeroE6/TMPRSS2 cells, a PFA was performed after mixing SARS-CoV-2 with CPC. This revealed that CPC treatment inactivated SARS-CoV-2 within contact times of 20 and 60 s (Table 1 ). An infectious titer reduction rate of 91.9% was obtained by treatment of virus stock with 0.05% CPC for 20 s. This rate was >97.0% (maximum level of this experiment) with treatment with 0.1% CPC for 20 s, and was also >97.0% with 0.05% CPC treatment for 60 s. These results indicate that CPC has dose-and time-dependent antiviral activity.

The effect of CPC on SARS-CoV-2 S protein was investigated to examine the virus inactivation mechanism. First, we examined whether CPC caused proteolytic cleavage of the recombinant S protein. In Western blotting after SDS-PAGE under reducing conditions, the molecular weights of four forms of the S protein were unchanged (Fig. 1 ).

To evaluate the effect of CPC on the activity of S protein, the interaction between the RBD of S protein and ACE2 was analyzed by ELISA in three different assays. First, the RBD and ACE2 were incubated for 2.5 h in the presence of CPC. The % binding rate was significantly reduced with 0.3% and 0.05% CPC ( Fig. 2A) . Next, a RBD-ACE2 binding assay after CPC treatment of the RBD and neutralization showed that % binding was significantly reduced with 0.3% and 0.05% CPC (Fig. 2B) . In this assay, the influence of neutralized CPC solution on binding of ACE2 was also investigated (Fig. 2B: " Interference").

Under these conditions, binding was equivalent to that with 0% CPC (control), indicating no interference of neutralized CPC solution on binding of ACE2. Finally, a RBD-ACE2 binding assay after CPC treatment of ACE2 and neutralization showed that the % binding of CPCtreated ACE2 decreased by about 70% compared to the untreated control (Fig. 2C) .

This study showed that CPC reduces the infectivity of SARS-CoV-2 below the detection limit at a concentration of CPC above 0.05% in treatment within 60 s. CPC inhibited the interaction between S protein and ACE2 without cleavage of S protein. These results indicate that CPC has dose-and time-dependent antiviral activity. Viral envelope disruption by CPC has been proposed as a mechanism of inactivation for an enveloped-virus [19] and this may also occur in SARS-CoV-2 inactivation [16, 17] . In the current study we considered other 9 mechanisms through examining the effect of CPC on the viral S protein. Western blot analysis showed that cleavage of S protein was not induced by CPC, and an ELISA showed reduced binding of S protein to ACE2. The results suggest that CPC may influence the S protein through conformational change, rather than proteolytic cleavage.

An evaluation of the interaction of CPC with human serum albumin (HSA) using isothermal titration calorimetry indicated that HSA was unfolded in two steps depending on the CPC concentration, resulting in loss of secondary and tertiary structures [20] . In the current study, the CPC-treated S protein showed reduced binding to ACE2 without proteolytic cleavage, which suggests that the mechanism of action of CPC involves denaturation of S protein. Thus, the antiviral activity of CPC against SARS-CoV-2 may be attributable to this mechanism. Our results also suggest that CPC may suppress the activity of human ACE2 and reduce the efficiency of infection of host cells without proteolytic cleavage.

The results for binding of CPC-treated RBD and ACE2 (Fig. 2B) indicated that the effect of CPC on binding was not dose-dependent. These findings may be due to a change in micelle morphology in aqueous solution that depends on the CPC concentration. CPC is a cationic surfactant with strong antibacterial and antifungal activity [21] , and tends to form micelles by association via hydrophobic chains at high aqueous concentrations above the critical micelle concentration (CMC). The mechanism of protein denaturation by ionic surfactants involves binding of a monomeric surfactant to specific sites of the protein via electrostatic and hydrophobic interactions, rather than binding as micelles [22] . The amount of monomers binding to the protein is constant even at free surfactant concentrations beyond the CMC, and thus, the rate of unfolding tends to level off around the CMC [22] . Given that the CMC of aqueous CPC solution is 0.9-1.0 mM [23] (approximately 0.03% w/v) at 25°C, the number of CPC monomers that bind to proteins is about constant at a concentration above 0.03%.Therefore, the absence of dose-dependent binding inhibition is due to the high 10 concentration of CPC above its CMC, at which binding of monomers was saturated and the effect of CPC reached a plateau.

CPC exhibited virucidal activity against SARS-CoV-2 at a concentration above 0.05%.

One mechanism of this phenomenon may be related to denaturation of the viral S protein (RBD) and reduction in binding to the host ACE2 receptor. Our results also suggest that CPC has an influence on human ACE2 and suppresses viral adsorption. 

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