Forecyt

To determine the relative concentrations of total IgG, IgG1, IgG2, IgG3, IgG4, and IgM in the sera of the Mamu-B^*17+ RMs at the time of the first SIV intrarectal challenge, we first coupled SIVmac239 gp140 to magnetic beads (Luminex Corporation) using NHS-ester linkages with Sulfo-NHS and EDC (Thermo Fisher). Next, we incubated SIVmac239 gp140-conjugated magnetic beads with diluted serum overnight at 4°C on a shaker. The following day, plates were spun down and washed before adding these reagents to the appropriate wells: mouse anti-monkey IgG PE (SouthernBiotech, #4700-09), to determine total IgG, mouse anti-rhesus Ig (rhIg) G1 (NHPRR, clone 7H11), rhIgG2 (NHPRR, clone 3C10), rhIgG3 (NHPRR, clone 2G11), rhIgG4 (NHPRR, clone 7A8), and rhIgM (Life Diagnostics, #2C11-1-5), to define the relative concentration of each isotype, or PE-conjugated FcγRIIa, FcγRIIb, and FcγRIIIa (R&D Systems, #1330-CD-050, #1875-CD-050, #4325-FC-050, respectively). The plates were incubated for 1 h at room temperature on a shaker. Next, we added goat anti-mouse IgG Fc PE (Invitrogen, #31861) to the corresponding “isotype” wells and incubated the plates for 1 h at room temperature on a shaker. After washing all plates, fluorescence was quantified using an IQue 3 flow cytometer (Intellicyt) and analysis was performed using IntelliCyt ForeCyt (v8.1).

Luminex analhysis was conducted as described previously (Brown et al., 2017 (link)). Briefly, proteins (Spike: D614G, E484K, N501Δ69-70, K417N, B.1.1.7, B.1.351; RBD (ImmuneTech): WT, E484K, B.1.1.7, B.1.351, B.1.128) were carboxy-coupled to magnetic Luminex microplex carboxylated beads (Luminex Corporation) using NHS-ester linkages with Sulfo-NHS and EDC (Thermo Fisher) and then incubated with serum (IgG1, FcγRIIb, FcγRIII 1:3000; IgG2a, G2b, G3, A, FcγRIV 1:1000, IgM 1:500) for 2 h at 37°C. Isotype analysis was perfomed by incubating the immune complexes with secondary goat anti-mouse-PE antibody (IgG1 1070-09, IgG2a 1080-09S, IgG2b 1090-09S, IgG3 1100-09, IgM 1020-09, IgA 1040-09 Southern Biotech) for each isotype. FcγR binding was quantified by incubating immune complexes with biotinylated FcγRs (FcγRIIB, FcγRIII, and FcγRIV, courtesy of Duke Protein Production Facility) conjugated to Steptavidin-PE (Prozyme). Flow cytometry was performed with an IQue (Intellicyt), and analysis was performed on IntelliCyt ForeCyt (v8.1).

Antibody-dependent NK cell degranulation was conducted as previously described^{59 (link)}. NVX-CoV2373 Spike protein was coated on Maxisorp ELISA plate (Thermo Fisher), and then blocked with 5% BSA. Antibodies were then added and incubated for 2 hours at 37°C. Human NK cells were isolated from peripheral blood by negative selection using the RosetteSep Human NK cell enrichment cocktail following the manufacturer’s instructions. Human NK cells were then added to the bound antibody and incubated for 5 hours at 37°C in the presence of RMPI+10%FBS, GolgiStop (BD), Brefeldin A (Sigma), and anti-human CD107a antibody (BD Bioscience). After incubation, cells were washed, stained with CD16, CD56, and CD3 (BD Bioscience), and fixed in 4% PFA for 15 minutes. Intracellular staining was performed using the FIX/PERM Cell fixation and permeabilization kit (Thermo), and cells were stained for interferon-γ and macrophage inflammatory protein-1β (BD bioscience). Flow cytometry was performed with an IQue (Intellicyt) and analysis was performed on IntelliCyt ForeCyt (v8.1).

Antibody-dependent NK cell degranulation was conducted as previously described^{59 (link)}. NVX-CoV2373 Spike protein was coated on Maxisorp ELISA plate (Thermo Fisher), and then blocked with 5% BSA. Antibodies were then added and incubated for 2 hours at 37°C. Human NK cells were isolated from peripheral blood by negative selection using the RosetteSep Human NK cell enrichment cocktail following the manufacturer’s instructions. Human NK cells were then added to the bound antibody and incubated for 5 hours at 37°C in the presence of RMPI + 10%FBS, GolgiStop (BD), Brefeldin A (Sigma), and anti-human CD107a antibody (BD Bioscience). After incubation, cells were washed, stained with CD16, CD56, and CD3 (BD Bioscience), and fixed in 4% PFA for 15 minutes. Intracellular staining was performed using the FIX/PERM Cell fixation and permeabilization kit (Thermo), and cells were stained for interferon-γ and macrophage inflammatory protein-1β (BD bioscience). Flow cytometry was performed with an IQue (Intellicyt) and analysis was performed on IntelliCyt ForeCyt (v8.1).

The kinetic data were converted to MFI versus time using FCSQuery software developed by Dr. Bruce Edwards (University of New Mexico). Analysis of apoptosis was done using ForeCyt software (IntelliCyt). Curve fits and statistics were done using GraphPad Prism software version 5.01 (GraphPad Software), as described in figure captions.