Zanamivir

MDCK cells were seeded in plain Dulbecco’s modified Eagle medium (Gibco DMEM, Thermo Fisher Scientific) containing 10% FBS in 96-well plates overnight. The cells were washed 3 times with Virus Growth Medium (VGM) (DMEM with 2% BSA and 2 μg/mL TPCK-treated trypsin, MilliporeSigma) and 100 μL of 1 multiplicity of infection (MOI) of A/Shanghai/2/2013 (H7N9)-PR8-IDCDC-RG32A virus in VGM added to the cells and incubated for 3 hours at 37°C in 5% CO₂. The cells were then washed with VGM again and replenished with VGM containing 3-fold serial dilutions of mAbs or zanamivir (GlaxoSmithKline), starting at the highest concentration of mAbs 10 μg/mL or equimolar. The plates were incubated for 21 hours at 37°C in 5% CO₂, and the supernatants were collected for performing the HA assay. For HA assay, we used turkey red blood cells (Rockland Immunochemicals) that were washed and diluted to 0.5% in PBS. A volume of 50 μL of the supernatants was incubated with 50 μL of the 0.5% turkey red blood cells in V-bottom plates for 1 hour at 4°C. The IC₁₀₀ values were defined as the lowest antibody concentration added to infected MDCK cells that corresponded to absence of virus in supernatant according to HA of red blood cells.

Virus binding was measured as previously described (16 (link)). Briefly, binding was measured by biolayer interferometry (BLI) using an Octet Red system (Pall ForteBio Corp., Menlo Park, CA, USA). Streptavidin-coated sensors (Pall ForteBio Corp.) were loaded with biotinylated sialoglycopolymers, which consisted of 20 mol% sugar, either α2,6-sialyl-N-acetyllactosamine (6SLN) or α2,3-sialyl-N-acetyllactosamine (3SLN), attached to a polyacrylamide backbone with 5 mol% biotin (Lectinity Holdings, Moscow, Russia). Sugars were loaded at a range of concentrations for equilibrium binding assays. HA and NA balance measurements (16 (link)) were made using sensors saturated with receptor analogues (loading ≈ 0.6 nm). All experiments were carried out in 10 mM HEPES, pH 7.4, 150 mM NaCl, 0.005% (vol/vol) Tween 20, 4 mM CaCl₂. When present, the NA inhibitors oseltamivir carboxylate (Roche Products Ltd., Welwyn Garden City, UK) and zanamivir (GlaxoSmithKline, Stevenage, UK) were added at concentrations of 100 μM to inhibit NA sialidase activity. Unless stated otherwise, the virus concentration was 100 pM in all binding assays. For equilibrium binding, the total amplitude of virus binding was measured and plotted as a function of sugar loading.

NA from C. perfringens (type V), KDO, 4-MUNANA, complete Freund’s adjuvant (CFA), incomplete Freund’s adjuvant (IFA), and anti-FLAG antibody (clone M2) were purchased from Sigma-Aldrich (St. Louis, MO). Anti-HA tag antibody was purchased from Roche Applied Science (Indianapolis, IN). Anti-NEU1 antibody was purchased from Rockland (Gilbertsville, PA). Zanamivir was purchased from GlaxoSmithKline (Research Triangle Park, NC). Oseltamivir phosphate (Tamiflu) was purchased from Sequoia Research Products (Berkshire, United Kingdom). 2-DN was purchased from Calbiochem (Gibbstown, NJ). Goat antiserum against influenza virus serotypes N1 to N9 was obtained from BEI Resources (Manassas, VA).

H7N9 AIVs were purified as previously described [32 (link)]. Briefly, virus harvested from allantoic fluid of embryonated eggs was pelleted by ultracentrifugation and then purified through a continuous 30–60% (weight/volume) sucrose gradient and resuspended in PBS buffer. The biotinylated α2,3- and α2,6-linked sialyl lactosamine sugars (3SLN and 6SLN, respectively) were purchased from GlycoNZ. Virus was diluted in HBS-EP buffer (TEKnova) containing 10 μM oseltamivir carboxylate (Roche) and 10 μM zanamivir (GSK) to a concentration of 100 pM, and the binding to receptor analogues was measured on an Octet RED instrument (ForteBio). The equilibrium responses for virus binding were plotted as a function of the amount of sugar immobilized on the biosensor calculated from the response during the sugar loading step [38 (link)]. The relative dissociation constant, as a measure of binding to 3SLN and 6SLN was calculated.