Iota-carrageenan and xylitol inhibit SARS-CoV-2 in Vero cell culture

Abstract

Last year observed a global pandemic caused by SARS-CoV-2 (severe acute respiratory syndrome-coronavirus 2) infection affecting millions of individuals worldwide. There is an urgent unmet need to provide an easily producible and affordable medicine to prevent transmission and provide early treatment for this disease. Since the nasal cavity and the rhinopharynx are the sites of initial replication of SARS-CoV-2, a nasal spray may be an effective option to target SARS-CoV-2 infection. In this study, we tested the antiviral action of three candidate nasal spray formulations against SARS-CoV-2 in vitro. We determined that iota-carrageenan in concentrations as low as 6 μg/mL inhibits SARS-CoV-2 in vitro. The concentrations of iota-carrageenan with activity against SARS-CoV-2 in vitro may be easily achieved through the application of nasal sprays as commonly used in several countries. Recently a double-blind, placebo-controlled study showed that iota-carrageenan in isotonic sodium chloride reduces ca. five times the risk of infection by SARS-CoV-2 in health care personnel. Further, xylitol at a concentration of 50 mg/mL (ca. 329 mM) was found to exert some antiviral action, though this preliminary finding needs further confirmation.

Fig 1. SARS-CoV-2 viral titer after treatment with Samples 1 and P1.

A) Infection assay. Sample 1 composition: 1.2mg/mL iota-carrageenan, 9 mg/mL sodium chloride, pH 6–7. Vero E6 were pre-treated for two hours with dilutions of Sample 1 with Sample P1 (diluent without iota-carrageenan) to obtain 600 μg/mL, 60 μg/mL, 6 μg/mL, and 0.6 μg/mL iota-carrageenan final concentration. After this pretreatment, cells were infected with SARS-CoV-2 and incubated for 48 hours in the presence of the same dilutions of Sample 1. Supernatants were harvested and virus yield was determined using an end point dilution assay (TCID₅₀). Controls consisted of untreated infected cells or infected cells treated with P1 (no iota-carrageenan). Results were determined using the Reed and Muench formula and expressed as log TCID₅₀/mL. The dotted line shows the limit of detection (LOD). Testing of samples was performed in triplicate, and the p-values are p≤0.00025 (****). B) Cellular viability assays. Vero-E6 cells were treated with iota-carrageenan or vehicle (600 μg/mL to 0 μg/mL) for 48 h at 37°C. After incubation, cellular viability was analyzed, and no statistically significant difference was found between the groups compared to the untreated control group (Group 600 μg/ml, p = 0.7464, Group 60 μg/ml, p = 0.0908, Group 6 μg/ml, p = 0.1208, and Group 0.6 μg/ml, p = 0.8938). Data are expressed as mean ± SD. Therefore, these compositions do not adversely affect cell viability. For this reason, cell lysis and death detected in the reported experiments after infection must be attributed to the action of the virus.

Fig 2. SARS-CoV-2 viral titer after treatment with Samples 2 and P2.

A) Infection assay. Sample 2 composition: 1.2mg/mL iota-carrageenan, 5 mg/mL sodium chloride, pH 6–7. Vero E6 were pre-treated with dilutions of Sample 2 with Sample P2 (diluent without iota-carrageenan) to get 600 μg/mL, 60 μg/mL, 6 μg/mL, and 0.6 μg/mL final iota-carrageenan concentration for two hours. After this pretreatment, cells were infected with SARS-CoV-2 and incubated for 48 hours in the presence of the same dilutions of Sample 2. Supernatants were harvested and virus yield was determined using an end point dilution assay (TCID₅₀). Controls consisted of untreated infected cells or infected cells treated with P2 (no iota-carrageenan). Results were determined using the Reed and Muench formula and expressed as log TCID₅₀/mL. The dotted line shows the limit of detection (LOD). Testing of samples was performed in triplicate, and the p-values are p≤0.00074 (***) and p≤0.00001. B) Cellular viability assays. Vero-E6 cells were treated with iota-carrageenan or vehicle (600 μg/mL to 0 μg/mL) for 48 h at 37°C. After incubation, cellular viability was analyzed, and no statistically significant difference was found between the groups compared to the untreated control group (Group 600 μg/ml, p = 0.9880, Group 60 μg/ml, p = 0.0683, Group 6 μg/ml, p = 0.9993, and Group 0.6 μg/ml, p = 0.1957). Data are expressed as mean ± SD.

Fig 3. SARS-CoV-2 viral titer after treatment with Samples 3 and P3.

A) Infection assay. Sample 3 composition: 1.2mg/mL iota-carrageenan, 5% m/V xylitol, pH 6–7. Vero E6 were pre-treated with dilutions of Sample 3 and Sample P3 (placebo without iota-carrageenan) to get 600 μg/mL, 60 μg/mL, 6 μg/mL, and 0.6 μg/mL final iota-carrageenan concentration for two hours. After this pretreatment, cells were infected with SARS-CoV-2 and incubated for 48 hours in the presence of the same dilutions of Sample 3. Supernatants were harvested, and virus yield was determined by an endpoint dilution assay (TCID₅₀). Controls consisted of untreated infected cells or infected cells treated with P3 (no iota-carrageenan). Results were determined using the Reed and Muench formula and expressed as log TCID₅₀/mL. The dotted line shows the limit of detection (LOD). Testing of samples was performed in triplicate and the p-value indicates that the groups are significantly different p = 0.0045, nevertheless no post-hoc analysis could be performed. B) Cellular viability assays. Vero-E6 cells were treated with iota-carrageenan or vehicle (600 μg/mL to 0 μg/mL) for 48 h at 37°C. After incubation, cellular viability was analyzed. No statistically significant difference was found between compositions at low iota-carrageenan concentrations, 6 μg/mL (p = 0.6904) and 0.6 μg/mL (p > 0.9999) compared to the untreated control group (Group 6 μg/ml, p = 0.6904, and Group 0.6 μg/ml, p > 0.9999). Compositions with higher concentrations of iota-carrageenan tend to show a significant difference increasing cell viability (Group 600 μg/ml, p = 0.0031 (***), Group 60 μg/ml, p = 0.0417 (*), They are certainly not toxic, but may exert some cytoprotective effect.). Data are expressed as mean ± SD.