Virus titration and neutralization

For molecular clones, ELISA-based influenza MN assay was performed following WHO-recommended protocol. Briefly, influenza viruses were titrated in MDCK-SIAT1 cells plated in 96-well plates at 50,000 cells ml⁻¹ 24 h before infection. Virus dilutions were prepared using OptiMEM supplemented with TPCK-treated trypsin (1 µg ml⁻¹). Infected cells were incubated at 37 °C in a humidified 5% CO₂ atmosphere. After 18 h, cells were fixed with 80% cold acetone and air dried. Virus replication was detected by biotin-conjugated antibodies to influenza virus nucleoprotein (MAB8257B and MAB8258B, Millipore Sigma) and was visualized with horseradish peroxidase-conjugated streptavidin and SureBlue TMB Microwell Peroxidase Substrate (KPL). Absorbance was read at 450 nm (A450) and 650 nm (A650) with the SpectraMax Paradigm microplate reader (Molecular Devices). The A650 was used to subtract plate background. Half-maximal tissue culture infectious dose (TCID₅₀) titer was calculated using Reed–Muench method. Neutralization assays were performed using 100–200 TCID₅₀ units of virus and 4-fold antibody dilutions made in OptiMEM supplemented with TPCK-treated trypsin. Virus and antibody were mixed in equal volumes and incubated 1 h at 37 °C prior to adding to substrate MDCK-SIAT1 cells. Control wells of virus alone (VC) and diluent alone (CC) were included on each plate. Fifty microliters of antibody–virus mixture were then added to wells of pre-washed cells in duplicate and the plates were incubated for 18 h at 37 °C in a humidified 5% CO₂ atmosphere. Infected cells were detected as described above. The percent neutralization was calculated by constraining the VC control as 0% and the CC control as 100% and plotted against antibody concentration. A curve fit was generated by a four-parameter nonlinear fit model in Prism (GraphPad). The 80% (IC₈₀) inhibitory concentrations were obtained from the curve fit for each antibody.

Titer of R3∆PB1 or R3∆HA viruses was measured in PB1-expressing MDCK-SIAT1 cells plated in 96-well black plates with transparent bottom (Greiner) at 18 h post infection and counting fluorescent foci using Celigo Image Cytometer (Nexcelom) with customized red channel to enhance detection of mKate2/tdKatushka2 reporter (EX 540/80 nm, DIC 593 nm and EM 593/LP nm). In Celigo operation and analysis software v4.1, Target 1 protocol was used to detect and count fluorescent foci. Titer was expressed as fluorescent foci per ml. For each neutralization reaction, virus dilution that resulted in cca. 1000 (500–4000) fluorescent foci per well at 18 h post infection was used. Neutralization assays using R3 viruses performed to compare the fluorescence- and ELISA-based assays were done in 96-well black transparent bottom plates. Cells were plated 24 h before the experiment. Neutralization reaction was done as described above. R3 influenza neutralization assay was optimized to be performed in 384-well plate format. PB1-expressing MDCK-SIAT1 cells were washed twice with PBS, re-suspended in OptiMEM, and plated 2 h before the assay in 384-well plates at 150,000 cells ml⁻¹ (20 µl per well). Twenty-five microliters of each neutralization mixture consisted of 2 μg ml⁻¹ TPCK-treated trypsin and equal parts of virus and 4-fold serial dilutions of antibodies were transferred to wells in quadruplicate. Control wells of virus alone (VC) and diluent alone (CC) were included on each plate. Fluorescent foci were counted at 18–24 h post infection using a Celigo instrument. Neutralization titers were calculated using Prism as described above.

Detailed protocols are provided as Supplementary Notes ¹ and ².