Fig 1.
Neutralization of SARS-CoV-2 pseudotyped lentivirus.
Neutralization assay with iota-carrageenan compared to neutralizing serum and negative serum. Approximately 7500 ACE2-HEK293 cells/well were infected with SARS-CoV-2 Spike pseudotyped lentivirus at an MOI of 0.1 (Luc reporter). Mock infected and mock treated infected cells served as negative control and positive control, respectively. 10 μg/ml iota-carrageenan and serum diluted 1:15 were incubated with the virus before infection. For infection, the virus/polymer or virus/serum inoculum was diluted with 5 volumes of medium. 48 hours after infection the infection efficiency was determined by measuring the luciferase activity. Luciferase data were corrected with metabolic data (Alamar blue) derived from a parallel plate with identical set-up. Data are normalized to % positive control and represent means of triplicates with standard deviation indicated.
Fig 2.
Titration of iota-carrageenan (100 μg/ml to 0.33 μg/ml) and determination of IC50. ACE2-HEK293cells/well were infected with SARS-CoV-2 Spike pseudotyped lentivirus at an MOI of 0.1 (Luc reporter). Mock infected and mock treated infected cells served as negative control (0% infection) and positive control (100% infection control), respectively. The virus was incubated with different concentrations of iota-carrageenan for 30 minutes before infection. 48 hours after infection the infection efficiency was determined by measuring the luciferase activity. Luciferase data were corrected with metabolic data (Alamar blue) derived from a parallel plate with identical set-up. Data represent means of triplicates ± standard deviation. The red line indicates the determination of the IC50.
Fig 3.
Comparison of iota-carrageenan with other polymers and a sulfated sugar.
Neutralization assay with iota-carrageenan compared to other sulfated and non-sulfated polymers as well as a sulfated sugar. ACE2-HEK293cells/well were infected with SARS-CoV-2 Spike pseudotyped lentivirus (Luc reporter). Mock infected and mock treated infected cells served as negative control (0% infection) and positive control (100% infection control; y-axis), respectively. 100 μg/ml and 10 μg/ml sulfated polymers and 100 μg/ml non-sulfated polymers and galactose-4-sulfate were incubated with the virus for 30 minutes before infection. Infection efficiency was determined by measuring the luciferase activity 48 hours post infection. Luciferase data were corrected with metabolic data (Alamar blue) derived from a parallel plate with identical set-up. Data represent means of triplicates ± standard deviation. Abbr.: Iota-carr. (iota-carrageenan), kappa-carr. (kappa-carrageenan), lambda-carr, (lambda-carrageenan), Fuc.U.p. (high molecular weight fucoidan from Undaria pinnatifida), Fuc.F.v. (high molecular weight fucoidan from Fucus vesiculosus), CMC (carboxymethylcellulose), HPMC (hydroxypropylmethylcellulose), and Gal-4-SO4 (galacatose-4-sulfate).
Table 1.
Results of NMR analysis of kappa- and lambda-carrageenan preparations.
Fig 4.
Iota-carrageenan restricts replication of SARS-CoV-2 in Vero B4 cells.
(A): Western blot analysis of virus fractions (NP = Nucleoprotein) after treatment with increasing concentrations of Carrageenan for 3 days. Vero B4 cells were infected with SARS-CoV-2 at a MOI of 0.2. 1 hour after infection and subsequent removal of input virus, cells were treated with different concentrations of carrageenan. After 3 days, cells were harvested and virions from three independent experiments were purified and analyzed by Western blot using SARS-CoV-2 convalescent serum. Densitometric analyses of Western blots were performed using AIDA®. Bars represent mean values of three independent experiments, ±standard deviation. (B): Real time PCR analysis of purified virions. The experiment was performed as described in Fig 4A. The purified virions were analyzed using real time PCR. Shown are the results of three independent experiments ±standard deviation.
Fig 5.
Influence of carrageenan on the cell viability of Vero B4 cells.
Following treatment with different concentrations of carrageenan (iota-carrageenan concentrations are indicated at the x-axis) for three days, the influence on cell viability was measured by water-soluble tetrazolium salt (WST)-1 assays. Bars represent mean values of 3 independent experiments ±SD. Staurosporin was used as a positive control.
Table 2.
Comparison of IC50 values of iota-carrageenan for SSPL and CoV OC43.