Antiviral Efficacy Against Virus Infections in Human-Derived Tracheal/Bronchial Epithelial Cells

USU Study Number:      10-20-21-3D XLEAR
Report date:                    1 December 2021
Test compounds:           Sorbitol, Erythritol, Xylitol, GSE, Chlorpheniramine Maleate
Viruses:                            Influenza A/CA/07/09 (H1N1), Respiratory Syncytial Virus (RSV) strain A2, SARS-CoV-2 strain USA/PHC658/2021 (B.1.617.2; delta)

Study Director:
Kie Hoon Jung, Ph.D.
Institute for Antiviral Research, VSB 105
5600 Old Main Hill
Utah State University
Logan, Utah 84322-5600

Introduction

The antiviral activity of 5 compounds provided by Xlear were evaluated against Influenza A/CA/07/09 (H1N1), Respiratory Syncytial Virus (RSV) strain A2, and SARS-CoV-2 strain USA/PHC658/2021 (B.1.617.2; delta) in a highly differentiated, three-dimensional (3-D), in vitro model of normal, human-derived tracheal/bronchial epithelial (TBE) cells. The compounds were tested at the concentrations indicated in Tables 1-3 in duplicate inserts of the 3D tissue models of the human airway (MatTek Life Sciences). Antiviral activity was measured by virus yield reduction assays on day 3 (H1N1), day 5 (RSV), or day 6 (SARSC- V2) after infection.

Materials and Methods

Compounds: The compounds received as solids were dissolved in the MatTek culture medium (AIR-100-MM) and further diluted to the test dilutions. Sorbitol (45%) and GSE (43%) were received in solution and were further diluted to the test dilutions in the culture medium. Ribavirin (ICN Pharmaceuticals, Inc. Costa Mesa, CA) or Remdesivir (MedChemExpress, cat# HY-104077) were tested as the positive control.

Cell Culture: The EpiAirway^TM Model consists of normal, human-derived tracheal/bronchial epithelial (TBE) cells which have been cultured to form a multi-layered, highly differentiated model which closely resembles the epithelial tissue of the respiratory tract. The cell cultures were made to order by MatTek Life Sciences (https://www.mattek.com) (Ashland, MA) and arrived in kits with either 12- or 24-well inserts each. The TBE cells were grown on 6mm mesh disks in transwell inserts. During transportation, the tissues were stabilized on a sheet of agarose, which was removed upon receipt. One insert was estimated to consist of approximately 1.2 x 10⁶ cells. Kits of cell inserts (EpiAirway^TM AIR-100, AIR-112) originated from a single, healthy, non-smoker donor #9831. Upon arrival, the cell transwell inserts were immediately transferred to individual wells of a 6-well plate according to manufacturer’s instructions, and 1 mL of MatTek’s proprietary culture medium (AIR-100- MM) was added to the basolateral side, whereas the apical side was exposed to a humidified 5% CO₂ environment. The TBE cells were cultured at 37°C for a minimum of one day before the start of the experiment. After the equilibration period, the mucin layer, secreted from the apical side of the cells, was removed by washing with 400 μL prewarmed 30 mM HEPES buffered saline solution 3X. Culture medium was replenished to the basal side following the wash steps. The tissues were then allowed to rest in a 37°C and 5% CO₂ environment for a minimum of 1 hour prior to the assay.

Viruses: The virus stocks were diluted in AIR-100-MM and infected at MOI 0.01 (H1N1), MOI 0.01 (RSV) and MOI 0.02 (SARS-CoV-2) CCID₅₀ per cell, respectively.

Experimental design: Each compound treatment (140 μL) is applied to the apical side, and culture medium only is applied to the basal side (1 mL), for a 2 h incubation. Virus is then added (140 μL) to the apical side for a 2 h infection period. As a virus control, some of the cells were treated with placebo (cell culture medium only). Following the infection, the apical medium was removed, wells are washed once with media, and fresh test compound is added to the apical side. The basal side was replaced with fresh medium. The cells were maintained at the air-liquid interface. On days 3 (H1N1), 5 (RSV), or 6 (SARS-CoV-2) postinfection, the medium was removed and discarded from the basal side. Virus released into the apical compartment of the TBE cells was harvested by the addition of 400 μL of culture medium that was pre-warmed at 37°C. The contents were incubated for 30 min, mixed well, collected, thoroughly vortexed and plated on MDCK (H1N1), MA-104 cells (RSV), or Vero E6 cells (SARS-CoV-2) for VYR titration. Triplicate wells were used for virus controls.

Determination of virus titers from each treated cell culture: MDCK (H1N1), MA-104 cells (RSV), or Vero E6 cells (SARS-CoV-2) cells were seeded in 96-well plates and grown overnight (37°C) to confluence. Samples containing virus were diluted in 10-fold increments in infection medium and 200 μL of each dilution transferred into respective wells of a 96- well microtiter plate. Four microwells were used for each dilution to determine 50% viral endpoints. After 3-7 days of incubation, each well was scored positive for virus if any cytopathic effect (CPE) was observed as compared with the uninfected control. The virus dose that was able to infect 50% of the cell cultures (CCID₅₀ per 0.2 mL) was calculated by the Reed-Muench method (1948). The VYR data and log reduction values (LRV) are summarized in Tables 1-3.

References

Reed, L.J., Muench, H., 1938. A simple method of estimating fifty percent endpoints. The
American Journal of Hygiene 27, 493–497.