Furin

BG505 SSOIP.664 trimer was produced in HEK 293 GnTI −/− cells via transient transfection of the BG505 SOSIP expressing plasmid with furin and purified as described previously over 2G12-affinity column^{12 (link),14 (link),18 (link)} (see Supplementary Note for additional details, including transient transfection in 96-well plates). The eluted protein was then dialyzed against PBS and set for deglycosylation reaction at 37 °C in the reaction buffer containing 1 mM EDTA, 150 mM NaCl, protease inhibitor cocktail (Roche), 17,000 units of Endo H/ml, and 50 mM sodium acetate, pH 5.8. The deglycosylated BG505 SOSIP was further purified with Superdex 200 16/60 (GE Healthcare) column in the buffer containing 5 mM HEPES 7.5, 150 mM NaCl, and 0.02% NaN₃. The peak corresponding to trimeric HIV-1 Env was identified, pooled and concentrated to ~10 mg/ml using an Amicon Ultra-15 centrifugal filter (MWCO 50,000, Millipore) and screened for crystallization. Similarly purified “crystallization-grade” samples were also used for HDX experiments.
For most antigenicity and stability analyses, after trimers were purified by affinity chromatography and gel filtration over a Superdex 200 16/60 (GE Healthcare) column in buffer containing 5 mM HEPES 7.5, 150 mM NaCl, and 0.02% NaN₃, they were subjected to negative selection^{39 (link)}. This generally involved a V3-antibody 447-52D (PDB ID 4M1D^{61 (link)}) affinity column to remove aberrant trimer species. However, for select antigenic analyses (e.g. Figs. 4a and 4d), an additional column comprising a cocktail of V3-directed antibodies, 1006-15D, 2219, 2557, 2558, 3074 and 50.1 (PDB IDs 3MLW^{42 (link)}, 2B0S^{62 (link)}, 3MLS^{42 (link)}, 3UJI^{63 (link)}, 3MLX^{42 (link)}, 1GGI^{64 (link)}, respectively), was employed (Fig. 3, Supplementary Data Set 1 and Supplementary Note).

The D7324-epitope-tagged version of BG505 SOSIP.664, referred to as SOSIP.664-D7324, and BG505 gp120 with a reconstructed D7324 epitope in C5, were made as described previously (9 (link)). JR-CSF gp120 was prepared by Diane Kubitz at the Scripps Center for Antibody Development and Production (La Jolla, CA) using transient transfection of HEK293F cells, and purified by Galanthus nivalis lectin (Vector Labs, Burlingame, CA) affinity chromatography followed by SEC using a Sephacryl S200HR column. Anti-gp120 and anti-trimer ELISAs using the above proteins as antigens were performed as described previously (9 (link)). The C-terminal His-tagged BG505 SOSIP.664-His trimer was prepared as described previously (16 (link), 37 (link)). His-tagged BG505 gp41 (gp41-His) was produced as follows: A His-tagged version of the BG505 IP.664 gp140 protein, which is based on the SOSIP.664 construct but with the SOS disulfide bond omitted (13 (link)), was expressed in the presence of excess furin in 293F cells. The protein was purified via the His-tag using Ni-NTA chromatography with elution using 250 mM imidazole, followed by three rounds of negative selection using a 2G12 bNAb column to remove any residual gp120 or uncleaved gp140 proteins. The purified gp41 protein bound the gp41-specific non-NAb F240 efficiently, but did not bind 2G12 or VRC01, indicating that contaminant gp120 or gp140 proteins were not present (data not shown). The gp41 protein also did not bind the PGT151 or 3BC315 bNAbs, suggesting that it was not in a pre-fusion conformation (not shown; (37 (link), 47 (link)). Ni-NTA ELISAs using His-tagged trimers and the gp41 protein were performed as described elsewhere (37 (link)).
For bNAb competition ELISA experiments, rabbit or macaque sera (1:100 dilution) were incubated with D7324-captured BG505 SOSIP.664-D7324 trimers for 1 h. A biotinylated bNAb was then added at a concentration sufficient to give ~80% of the maximum binding signal, as assessed in a prior titration experiment (i.e., with no competitor present). The bound bNAb was detected using horseradish peroxidase (HRP)-labeled streptavidin. As the PGT151, 35O22 and 3BC315 bNAbs, and also CD4-IgG2, could not be biotinylated without impairing their binding activity (37 (link)) we detected unlabeled human bNAbs using an HRP-labeled donkey anti-human IgG conjugate that was minimally cross-reactive with rabbit IgG (Jackson Immunoresearch, Westgrove, PA). The latter assay format was unsuitable for macaque sera because the anti-human antibody cross-reacted with macaque IgG. As a result, it was not possible to test the macaque sera for inhibition of PGT151, 35O22, 3BC315 or CD4-IgG2 binding to the trimer.
To analyze the relative titers for serum antibody binding to conformational vs. linear epitopes, trimers (0.1 μg/ml) or monomers (0.03 μg/ml) were denatured by heating for 5 min at 99°C in 50 μl of TBS/10% FCS/1% SDS/50 mM DTT (sodium dodecyl sulfate, SDS; dithiothreitol, DTT). The samples were then diluted 140-fold in TBS/10% FCS to prevent SDS and DTT from interfering with the ELISA. The use of both SDS and DTT ensures that the Env proteins are fully denatured by heat treatment (48 (link)). The native or denatured proteins were then used in a D7324-capture ELISA, essentially as described elsewhere (9 (link), 48 (link)). The relative reduction of binding to the denatured vs. native Env proteins was calculated using the half maximal binding values (EC₅₀).