Combined prophylactic and therapeutic use maximizes hydroxychloroquine anti-SARS-CoV-2 effects in vitro

Nicola Clementi; Elena Criscuolo; Roberta Antonia Diotti; Roberto Ferrarese; Matteo Castelli; Roberto Burioni; Massimo Clementi; Nicasio Mancini

Vero E6 cells (4 × 10 5 cells/mL) were seeded into 96 wells plates and treated with HCQ (10 μM) at different stages of virus infection. For full-time treatment, cells were pre-treated with the drug for 1 h prior to virus infection at 37°C, followed by virus adsorption for 1 h in the presence of the molecule. Then, cells were washed with PBS, and further cultured at 37°C with the molecule-containing medium until the end of the experiment. For pre-adsorption treatment, the agent was added to the cells for 1 h at 37°C before virus infection and maintained during virus adsorption. Then, the mixture was replaced with fresh medium without molecule till the end of the experiment. For postadsorption assays, the drug-containing medium was added to cells only after virus adsorption and maintained until the end of the experiment. Then, cells were washed with PBS, and further cultured at 37°C with the molecule-containing medium until the end of the experiment. BFLA (100 nM) was tested as control of inhibition of viral infectivity at a phagolysosome level only in a pre-adsorption treatment, alone or in combination with preadsorption HCQ treatment. Uninfected cells were included in all experimental settings to exclude possible drug-toxicity CPE. For all the experimental groups, cells were infected with 50 TCID50/mL (98 PFU/mL) SARS-CoV-2 and absorption was performed for 1h at 37°C or 4°C. Live images were acquired (Olympus CKX41 inverted phase-contrast microscopy) and cell supernatants were collected for real-time PCR (RT PCR) analysis at 72 hpi. All conditions were tested in quadruplicate.