Researchers found that cetylpyridinium chloride, found in most mouthwashes, lowers infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro, likely by inhibiting viral fusion with target cells. With more tests to prove validity, mouthwashes could be a simple, cheap way of curbing transmission.
Studies in the past have shown that mouthwashes can have antiviral properties. They can decrease airborne respiratory coronavirus infections, including infections from the flu virus and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic.
If more tests prove the antiviral properties of mouthwashes, they may be a potent and economical strategy to combat the pandemic. Using mouthwashes would be a cheap and easily accessible strategy that can be quickly implemented. Reducing viral loads in the mouth can help curb the transmission chain. Mouthwashes are also produced as oral sprays, so can be used easily by the elderly population.
Although studies have reported in vitro antiviral activity of various oral products, it is not known which components in mouthwashes are responsible for the antiviral activity and what is their mechanism of action.
Understanding effect of mouthwash on SARS-CoV-2 infectivity
In a new study published in the bioRxiv* preprint server, researchers report the effect of cetylpyridinium chloride (CPC) used in many mouthwashes and sprays. CPC is a quaternary ammonium compound with antiseptic and antimicrobial activity.
The authors tested the ability of CPC-containing mouthwashes to prevent entry of SARS-CoV-2 into cells. They used HEK293T cells expressing the angiotensin-converting enzyme 2 (ACE2) receptor as target cells and pseudotyped lentivirus expressing the SARS-CoV-2 spike protein. A luciferase-based assay allowed them to detect viral binding to the target cells.
The pseudovirus was mixed with different concentrations of mouthwashes and added to the target cells. The authors tested two mouthwashes from Dentaid: Perio Aid Intensive Care and Vitis CPC Protec. To control for cytotoxicity, the authors also mixed the mouthwashes with the target cells without the pseudovirus. In addition, they also used the same mouthwash formulation but without CPC as a control.
They found that the mouthwashes inhibited viral fusion to host cells, with their inhibition activity increasing with increasing concentration of mouthwash. They did not see any inhibition using the mouthwash formulation without CPC, suggesting the antiviral activity is because of CPC. In addition, the researchers suspended CPC in water and they found that this suspension also inhibited virus entry into the target cells.
The team also isolated SARS-CoV-2 from a clinical sample collected from an 89-year old male patient. They added equal amounts of the virus and CPC or CPC-containing mouthwash and mixed them.
They found a high dose of CPC was effective in reducing virus infection on Vero E6 cells. A 2-minute treatment with CPC mouthwash decreased the Tissue Culture Infectious Dose 50% (TCID50)/ml 1000 times. The formulations without CPC had no effect of virus infectivity. The doses of CPC that inhibited viral fusion to the host cell were not toxic to the host cells.
Cetylpyridinium chloride reduces SARS-CoV-2 infectivity
It is likely that the antiviral activity of CPC arises from its ability to disrupt the viral envelope, similar to that seen before for the flu virus. So, mouthwashes with CPC could protect against infection of the oral mucus membrane.
However, since SARS-CoV-2 mainly enters hosts via the upper respiratory tract, nasal sprays containing CPC could be more effective in preventing or reducing infection.
The experiments the authors performed were using highly infectious viral stock, whereas virus present in about 1–2 ml saliva likely will have lower infectivity. Mouth rinses are usually done with about 10 ml of mouthwash, so the CPC to virus ratio will likely be more compared to those used in the lab experiments, the authors think it is possible that it may be more effective in the mouth than in the in vitro tests.
Antiviral activity of CPC-containing mouthwashes inhibiting SARS-CoV-2 entry. Percentage of viral entry inhibition on target HEK-293T cells expressing ACE2 exposed to a fixed concentration of SARS-CoV-2 in the presence of increasing concentrations of oral formulations (A and C), their vehicles (B and D) and CPC diluted in water (E). Non-linear fit to a variable response curve from one experiment with two replicates is shown (red lines), excluding data from drug concentrations with associated toxicity. When calculated, the particular IC50 value of the graph is indicated. Cytotoxic effect on HEK-293T cells expressing ACE2 cells exposed to increasing concentrations of mouthwashes or vehicles in the absence of virus is also shown (grey lines).
“Thus, CPC-containing mouthwashes could be a cost-effective measure to reduce SARS-CoV-2 infectivity in saliva, aiding to reduce viral transmission from infected individuals,” write the authors. Virus transmission has been seen when infected people talk, shout, or sing; viable virus has been recovered from the saliva of infected people.
The authors write that future work should investigate if mouthwashes with CPC can reduce viral loads and infectivity in the mouths of infected persons. In addition, more work is needed to understand how long the antiviral activity of CPC lasts in the mouth. All this will help us understand how to use mouthwashes as a cheap measure to reduce SARS-CoV-2 infectivity in the saliva.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.