T0440 il

Ninety-six-well plates were coated with 100 μL SARS-CoV-2 S protein (2 μg mL⁻¹) overnight at 4 °C. Plates were washed with phosphate-buffered saline with 0.05% Tween (PBS-T) buffer and blocked with TBS Startblock blocking buffer (Thermo Fisher Scientific). Histidine-tagged hACE2 and hDPP4 receptors were threefold serially diluted (5–0.0001 μg mL⁻¹) and added to coated wells for 2 h at room temperature. The plates were washed with PBS-T. Optimally diluted (1:4000) horseradish peroxidase (HRP) conjugated mouse anti-histidine was added and color developed by addition of and 3,3ʹ,5,5ʹ-tetramethylbenzidine (TMB) peroxidase substrate (T0440-IL, Sigma, St. Louis, MO, USA). Plates were read at an OD of 450 nm with a SpectraMax Plus plate reader (Molecular Devices, Sunnyvale, CA, USA) and data analyzed with SoftMax software. EC₅₀ values were calculated by 4-parameter fitting using GraphPad Prism 7.05 software (San Diego, CA, USA).

An ELISA was used to determine anti-SARS-CoV-2 S IgG titers. Briefly, 96-well microtiter plates (Thermo Fisher Scientific, Rochester, NY, USA) were coated with 1.0 µg mL⁻¹ of SARS-CoV-2 S protein. Plates were washed with PBS-T and blocked with TBS Startblock blocking buffer (Thermo Fisher Scientific). Mouse, baboon, or human serum samples were serially diluted (10⁻² to 10⁻⁸) and added to the blocked plates before incubation at room temperature for 2 h. Following incubation, plates were washed with PBS-T and HRP-conjugated goat anti-mouse IgG (1:5000) or goat anti-human IgG (1:2000) (Southern Biotech, Birmingham, AL, USA) added for 1 h. Plates were washed with PBS-T and TMB peroxidase substrate (T0440-IL, Sigma, St. Louis, MO, USA) was added. Reactions were stopped with TMB stop solution (ScyTek Laboratories, Inc. Logan, UT). Plates were read at OD 450 nm with a SpectraMax Plus plate reader (Molecular Devices, Sunnyvale, CA, USA) and data analyzed with SoftMax software. EC₅₀ values were calculated by 4-parameter fitting using SoftMax Pro 6.5.1 GxP software. Individual animal anti-SARS-CoV-2 S IgG titers and group GMTs and 95% confidence intervals (±95% CI) were plotted using GraphPad Prism 7.05 software.

An ELISA was used to determine anti-RSV F IgG titers. Briefly, 96-well microtiter plates (Thermo Fisher Scientific, Rochester, NY, USA) were coated with 2.0 µg mL⁻¹ of prefusogenic F (BV1184), prefusion F (BV2129), DS-Cav1 (BV2280), or postfusion F (BV2128). Plates were washed with PBS-T and blocked with blocking buffer (Quality Biologicals, Gaithersburg, MD, USA). Serum samples were serially diluted (10⁻² to 10⁻⁸) and the plates incubated at room temperature for 2 h. Following incubation, plates were washed with PBS-T and HRP-conjugated goat anti-mouse IgG or anti-rat IgG (Southern Biotech, Birmingham, AL, USA) added for 1 h. Plates were washed with PBS-T and TMB peroxidase substrate (T0440-IL, Sigma, St Louis, MO, USA) was added. Reactions were stopped with TMB stop solution (ScyTek Laboratories, Inc. Logan, UT). Plates were read at OD 450 nm with a SpectraMax plus plate reader (Molecular Devices, Sunnyvale, CA, USA) and data analyzed with SoftMax software. EC₅₀ values were calculated by 4-parameter fitting using GraphPad Prism 7.05 software (San Diego, CA, USA). Individual animal anti-RSV F protein IgG titers and group geometric mean titers (GMT) and 95% confidence interval (±95% CI) were plotted.