Virucidal activity and mechanism of action of cetylpyridinium chloride against SARS-CoV-2.

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.

Production of monoclonal shark-derived immunoglobulin new antigen receptor antibodies using Chinese hamster ovary cell expression system.

Cartilaginous fishes such as sharks have adaptive immune systems based on immunoglobulins similar to those in mammals. During their evolution, cartilaginous fishes individually have acquired their adaptive immune system called immunoglobulin new antigen receptor (IgNARs). IgNARs maintain their functions in the harsh environment of shark serum, which contains a high concentration of urea to prevent water loss in seawater. Therefore, IgNARs have high structural stability, and are expected to be used as next-generation antibodies in applications different from those of conventional IgG antibodies. However, no recombinant expression system for IgNAR, which has a molecular weight of approximately 147 kDa as a dimer and multiple N-glycosylation sites, has yet been constructed. This has stalled research into IgNAR development. Here, we constructed a recombinant expression system for IgNAR using Chinese hamster ovary (CHO) cells, widely used as hosts for IgG antibody production. Using this system, IgNAR was successfully expressed and purified as a human IgG Fc fusion protein and showed antigen-binding ability. After Protein A affinity purification, followed by specific cleavage and removal of the human Fc-region, the final yield of IgNAR was 1.07 mg/L-medium. Moreover, this CHO cell expression system modified IgNAR with various N-glycans, including high-mannose and complex types. This expression system will allow us to analyze the structure, physicochemical properties, and biological functions of IgNAR. This fundamental information will advance the development of IgNARs for industrial and biotechnological applications.

Virucidal activity of oral care products against SARS-CoV-2 in vitro.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) caused by infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. Since reducing the amount of virus in saliva is considered to prevent broader infection, the Center for Disease Control (CDC) and American Dental Hygienists' Association (ADHA) have recommended use of CPC- or CHX-containing oral care products before the dental procedure. However, there is no certified evidence. So, we examined inactivation of SARS-CoV-2 by oral care products in several countries in vitro. METHODS: 0.05 % Cetylpyridinium chloride (CPC) mouthwash, 0.05 % CPC toothpaste and 0.30 % CPC spray in Japan; 0.06 % chlorhexidine gluconate (CHX) + 0.05 % CPC mouthwash and 0.12 % CHX + 0.05 % CPC mouthwash in Europe; 0.075 % CPC mouthwash, 0.12 % CHX mouthwash, and 0.20 % delmopinol hydrochloride mouthwash in the USA; and 0.04 % CPC mouthwash in China were assessed for their virucidal activity with ASTM E1052. RESULTS: The virus was inactivated in vitro by the contact time in directions for use of all oral care products containing CPC or delmopinol hydrochloride as anticeptics. CONCLUSIONS: These results suggest that these oral care products in each country may reduce the viral load in the mouth.