Polysaccharides

The BG505 (BG505.W6M.ENV.C2) env gene (GenBank accession nos. ABA61516 and DQ208458) is derived from a subtype A T/F virus isolated from a 6-week old, HIV-1-infected infant [28] (link). It has 73% identity to the proposed PG9-sensitive progenitor virus from the PG9 bNAb donor, based on computational analysis of the most recent common ancestor sequence [29] (link). The BG505 gp120 monomer binds PG9, which is unusual given the quaternary nature of the PG9-Env interaction [29] (link). To make the BG505 SOSIP.664 gp140 construct, we introduced the following sequence changes (Fig. 1A): A501C and T605C (gp120-gp41_ECTO disulfide bond [5] (link)); I559P in gp41_ECTO (trimer-stabilizing [6] (link)); REKR to RRRRRR in gp120 (cleavage enhancement [31] (link)); T332N in gp120 (introduction of epitopes dependent on glycan-332); stop codon at gp41_ECTO residue 664 (improvement of homogeneity and solubility [23] (link), [24] (link)). The codon-optimized gene for BG505 SOSIP.664 gp140 was obtained from Genscript (Piscataway, NJ) and cloned into pPPI4 using PstI and NotI[5] (link).
Variants of the BG505 SOSIP.664 gp140 trimers bearing either a His-tag or a D7324 epitope-tag sequence at the C-terminus of gp41_ECTO were also made by adding the amino acid sequences GSGSGGSGHHHHHHHH or GSAPTKAKRRVVQREKR, respectively, after residue 664 in gp41_ECTO and preceding the stop codon. These proteins are designated SOSIP.664-His gp140 and SOSIP.664-D7324 gp140. We also made a His-tagged gp140 with the C501 and C605 cysteines replaced by their original residues, and with P559 similarly reverted to the original isoleucine (BG505 WT.664-His gp140). When expressed in the presence of excess furin to ensure efficient precursor cleavage, the absence of the SOS disulfide bond means the gp140 trimer is unstable and dissociates to gp120 and a trimeric form of His-tagged gp41_ECTO (BG505 gp41_ECTO-His); the latter can be used in a NiNTA-capture enzyme-linked immunosorbent assay (ELISA; see below).
A monomeric BG505 gp120 with a similar sequence to the gp120 components of the gp140 trimers was designed by: introducing a stop codon into the SOSIP.664 construct at residue 512; reverting the optimized cleavage site to wild type (RRRRRR→REKR at residues 508–511); reverting the A501C change; introducing the D7324 epitope into the C5 region (R500K+G507Q); and making a L111A substitution to decrease gp120 dimer formation [29] (link), [63] (link). A slightly modified version of BG505 gp120 that has been described previously [25] (link) was used in DSC experiments. For this modification, the BG505 gp120 gene was cloned downstream of an IgK secretion signal in a phCMV3 plasmid and upstream of a His-tag. The cleavage site was mutated to prevent the His-tag from being cleaved off, leading to the following C-terminal sequence: RAKRRVVGSEKSGHHHHHH.
The BG505 gp160 clone for generating Env-pseudoviruses for neutralization assays has been described elsewhere [29] (link). We modified this clone by inserting the same T332N substitution that is present in the BG505 SOSIP.664 trimers, and refer to the resulting virus as BG505.T332N.

Three biological replicates from each time point were analyzed. Frozen cell pellets containing ∼2.0 × 10⁶ cells were resuspended in 100 μl of lysis buffer containing Tris–HCl, EDTA, sodium chloride, and protease inhibitor cocktail. The cells were lysed using a Branson sonicator rod at 1.5 output power, and 25 μl of the suspension (containing ∼5 × 10⁵ cells) were loaded onto the preconditioned PVDF membrane plate wells. BSM (10 μg) was blotted in three different wells of the same PVDF membrane plate. The denaturation as well as the N- and O-glycan release were performed as described previously (31 (link), 33 (link)). Briefly, the proteins were denatured on membrane using guanidine hydrochloride and DTT at 60 °C. Upon removal of denaturing agents, the N-glycans were released by PNGase F digestion overnight at 37 °C and recovered in MQ water. A total of 2 units of PNGase F was added to each well of the PVDF membrane plate containing lysates from approximately 0.5 million cells. Upon recovery of the N-glycans, the O-glycans were released from the same wells by reductive β-elimination, using 50 μl of 0.5 M sodium borohydride in 50 mM potassium hydroxide incubating at 50 °C for 16 h. Samples were desalted by performing Dowex cation exchange resin (50W-X8) and graphitized carbon solid phase extraction in self-packed 96-well filter plates. The samples were dried after cleanup and stored at −20 °C until analysis.

The O-glycan samples were then reconstituted in 20 μl of MQ water, and 2 μl were injected for analysis. Analysis was performed using a PGC nano-LC Ultimate 3000 UHPLC system (Thermo Fisher Scientific) coupled to an amaZon ETD speed ion trap (Bruker Daltonics). The samples were loaded using 100% buffer A (10 mM ABC) at a loading flow of 6 μl/min on a custom-made trap column (size 30 × 0.32 mm) packed with 5 μm particle size PGC stationary phase from Hypercarb PGC analytical column (size 100 × 4.6 mm, 5 μm particle size; Thermo Fisher Scientific). Afterward, the O-glycans were separated at a 0.6 μl/min flow rate on a custom-made PGC column (100 × 0.1 mm, 3 μm particle size obtained from Thermo Fisher Scientific) by applying a linear gradient from 1% to 50% buffer B (MeCN, 10 mM ABC) over 73 min. During the procedures, a constant column temperature of 45 °C was maintained. To continue, the LC system was coupled to an amaZon ETD speed electrospray ionization (ESI) ion trap MS using the CaptiveSpray source (Bruker Daltonics) with an applied capillary voltage of 1000 V in negative-ionization mode. The drying gas (N₂) flow rate was set to 3 l/min, and the temperature was set at 280 ˚C. The nebulizer gas pressure was kept at 3 psi. The nanoBooster bottle (Bruker Daltonics) was filled with methanol, as a dopant solvent (34 (link)). MS spectra were acquired in enhanced mode within a mass to charge ratio (m/z) range of 380 to 1850. The maximum acquisition time was set to 200 ms, the ion charge control (ICC) to 40,000, and the target mass of smart parameter setting was set to m/z 900. MS/MS spectra were generated by collision-induced dissociation of the three most abundant precursors, applying an isolation width of 3 Thomson. In addition, ICC was set to 150,000, and the fragmentation cutoff was set to 27% with a 100% fragmentation amplitude using the Enhanced SmartFrag option (30–120% in 32 ms). To integrate area under the curve for each individual glycan isomer, extracted ion chromatograms of the first three isotopes were used in Bruker Compass DataAnalysis software (version 5.0). Peaks were manually picked and integrated. Total area normalization was employed for relative quantification of O-glycan species. Identification of O-glycan species was performed by comparison with PGC retention time, MS/MS spectra, and the BSM standard.