Virucidal and antiviral activity of astodrimer sodium against SARS-CoV-2 in vitro

Abstract

An effective response to the ongoing coronavirus disease (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) will involve a range of complementary preventive modalities. The current studies were conducted to evaluate the in vitro SARS-CoV-2 antiviral and virucidal (irreversible) activity of astodrimer sodium, a dendrimer with broad spectrum antimicrobial activity, including against enveloped viruses in in vitro and in vivo models, that is marketed for antiviral and antibacterial applications. We report that astodrimer sodium inhibits replication of SARS-CoV-2 in Vero E6 and Calu-3 cells, with 50% effective concentrations (EC₅₀) for i) reducing virus-induced cytopathic effect of 0.002-0.012 mg/mL in Vero E6 cells, and ii) infectious virus release by plaque assay of 0.019-0.032 mg/mL in Vero E6 cells and 0.030-0.037 mg/mL in Calu-3 cells. The selectivity index (SI) in these assays was as high as 2197. Astodrimer sodium was also virucidal, irreversibly reducing SARS-CoV-2 infectivity by >99.9% (>3 log₁₀) within 1 min of exposure, and up to >99.999% (>5 log₁₀) shown at astodrimer sodium concentrations of 10-30 mg/mL in Vero E6 and Calu-3 cell lines. Astodrimer sodium also inhibited infection in a primary human airway epithelial cell line. The data were similar for all investigations and were consistent with the potent antiviral and virucidal activity of astodrimer sodium being due to irreversible inhibition of virus-host cell interactions, as previously demonstrated for other viruses. Further studies will confirm if astodrimer sodium binds to SARS-CoV-2 spike protein and physically blocks initial attachment of the virus to the host cell. Given the in vitro effectiveness and significantly high SI, astodrimer sodium warrants further investigation for potential as a topically administered agent for SARS-CoV-2 therapeutic applications.

Keywords: Antiviral; Astodrimer; COVID-19; Dendrimer; SARS-CoV-2; SPL7013.

Fig. 1

Dose-response analysis of SARS-CoV-2 (2019-nCoV/USA-WA1/2020) antiviral activity of astodrimer sodium in Vero E6 cells, as measured by infectious virus release (Log₁₀ pfu/mL), and cytotoxicity on Day 4 post-infection. Astodrimer sodium (0.0005–10 mg/mL) was added to cell cultures 1 h prior to (t = -1 h), at the time of (t = 0), and 1 h post-infection (t = +1 h). Cytotoxicity was assessed by LDH detection (OD @ 490 nm), with 0.5% saponin used as the positive cytotoxic control. Points and error bars represent mean ± SD of triplicate readings.

Fig. 2

Dose-response analysis of SARS-CoV-2 (2019-nCoV/USA-WA1/2020) antiviral activity of astodrimer sodium in Calu-3 cells, as measured by infectious virus release (Log₁₀ pfu/mL), and cytotoxicity on Day 4 post-infection. Astodrimer sodium (0.0005–10 mg/mL) was added to cell cultures 1 h prior to (t = -1 h), at the time of (t = 0), and 1 h post-infection (t = +1 h). Cytotoxicity was assessed by LDH detection (OD @ 490 nm), with 0.5% saponin used as the positive cytotoxic control. Points and error bars represent mean ± SD of triplicate readings.

Fig. 3

Virucidal efficacy of astodrimer sodium against SARS-CoV-2 (2019-nCoV/USA-WA1/2020) measured by a reduction in mean infectious virus (Log₁₀ pfu/mL), at 96 h post-infection in Vero E6 cells. Astodrimer sodium (1–30 mg/mL) was incubated with SARS-CoV-2 (2019-nCoV/USA-WA1/2020) for 5 s up to 15 min. Treated virus was added to Vero E6 cells and the amount of infectious virus in the supernatant was determined by plaque assay 96 h post-infection. Graph shows dose-response of astodrimer sodium virucidal activity using 10⁴ pfu/mL virus inoculum. Points and error bars represent mean ± SD of triplicate readings. Dotted line indicates level of mean infectious virus when untreated virus was added to Vero E6 cells (virus control).

Fig. 4

Virucidal efficacy of astodrimer sodium against SARS-CoV-2 (2019-nCoV/USA-WA1/2020) measured by a reduction in mean infectious virus (Log₁₀ pfu/mL), at 16 h post-infection in Vero E6 cells. Astodrimer sodium (0.0046–30 mg/mL) was incubated with SARS-CoV-2 (2019-nCoV/USA-WA1/2020) for 30 s, 1 min, 5 min and 15 min. Treated virus was added to Vero E6 cells and the amount of infectious virus in the supernatant was determined by plaque assay 16 h post-infection. Graph shows dose-response of astodrimer sodium virucidal activity using 10⁴ pfu/mL virus inoculum. Points and error bars represent mean ± SD of triplicate readings. Dotted line indicates level of mean infectious virus when untreated virus was added to Vero E6 cells (virus control).

Fig. 5

Antiviral efficacy of astodrimer sodium and iota-carrageenan against SARS-CoV-2 (2019-nCoV/USA-WA1/2020) measured by a reduction in nucleocapsid (ng/mL), at Day 4 post-infection in human bronchial epithelial primary cells (HBEpC). Astodrimer sodium (0, 1.1, 3.3 and 10 mg/mL) or iota-carrageenan (0, 6, 60 and 600 μg/mL) were added to cell cultures 1 h prior to infection. A. Dose-response of astodrimer sodium antiviral activity. Points and error bars represent mean ± SD of triplicate readings. B. Dose-response of carrageenan antiviral activity. Points represent one replicate. Dotted lines indicates level of inhibition achieved with positive control, SARS-CoV-2 pAb.