Tbs buffer

Prometheus NT.48 NanoDSF instrument (NanoTemper Technologies) was used for the DSF experiments, as described previously.⁶⁰ (link) GPC, GPC-I53-50A and GPC-I53-50NP samples were diluted to 1 mg/mL in the TBS buffer (Alfa Aesar) and ∼10 μL of each diluted sample (in duplicates) was loaded into NanoDSF capillaries (NanoTemper Technologies). The temperature was raised from 20°C to 95°C at 1°C/min rate. The T_m value was determined from the first derivative curves using the NT.48 NanoDSF instruments software. The average value from the duplicate measurements is reported as the T_m value in the manuscript.

To further stabilize the trimeric conformation, purified GPC-I53-50A was chemically cross-linked using glutaraldehyde. A concentration of 0.75 μg/mL GPC-I53-50A in PBS was mixed in a 1:1 volume ratio with PBS, 60 mM glutaraldehyde and incubated for 5 min at RT. The cross-linking reaction was then stopped by adding Tris, pH 7.4 to a final concentration of 75 mM, followed by an incubation step of 10 min at RT. Subsequently, GPC-I53-50A was dialysed twice to TBS and then twice to PBS using the Slide-A-Lyzer Mini dialysis devices with a 10 kDa molecular weight cutoff (Thermo Scientific). Finally, GPC-I53-50A was concentrated to at least 2 μg/mL using Vivaspin centrifugal filters 10 kDa molecular weight cutoff (Sigma-Aldrich). To generate complexes, 400 μg of LAVA01 antibody (as Fab fragment) was incubated with 200 μg of cross-linked GPC-I53-50A trimers for 1 h at room temperature. The complex was purified from excess/unassembled material using size-exclusion chromatography on a HiLoad® 16/600 Superdex® pg200 (Sigma-Aldrich) column running TBS buffer (Alfa Aesar), and concentrated to 3 mg/mL using the Amicon ultrafiltration units with a 10 kDa molecular weight cutoff (Sigma-Aldrich).

Truncated human MBL-2 was purchased from MyBioSource (Cat# MBS2086086). Five micrograms of MBL-2 was incubated with 5 µg of BG505 SOSIP or BG505 SOSIP-T33_dn2 nanoparticle for 4 h at 37 °C in TBS buffer (Alfa Aesar) containing 2 mM CaCl₂. Following incubation, the samples were diluted to 40 µg/mL, loaded onto carbon-coated copper EM grids, negatively stained with uranyl-formate, and imaged as described in the “Negative stain-electron microscopy” section. Imaging was also performed on grids containing free, noncomplexed MBL-2, BG505 SOSIP, and BG505 SOSIP-T33_dn2 NP for comparison. For SEC experiments, 30 µg of MBL-2 was mixed with 15 µg of BG505 SOSIP or BG505 SOSIP-T33_dn2 nanoparticle samples and incubated for 4 h at 37 °C in TBS + 2 mM CaCl₂. For comparison, equivalent amounts of free MBL-2, BG505 SOSIP, and BG505 SOSIP-T33_dn2 nanoparticle were incubated under identical conditions. For the SEC step, we used a Superose 6 Increase 10/300 column, running in TBS + 2 mM CaCl₂. SEC traces of the assembly and control reactions are shown in Supplementary Fig. 4b, c.

Top-scoring heavy and light chain sequences from the corresponding NGS database searches were subcloned into the AbVec-hIgG1 and AbVec-hIgKappa expression vectors, respectively (40 (link), 41 (link)). Sanger sequencing was applied to verify the final DNA vectors. Heavy chain (500 μg) and 250 μg of the light chain DNA expression vectors were applied for cotransfection of 1 liter of human embryonic kidney 293F cells to produce Rh.33104 mAb.1 and Rh.33172 mAb.1 (as full IgG and Fab fragments). Polyethylenimine (PEI MAX, Polysciences Inc.) was used as a transfection reagent at threefold mass excess with respect to the total DNA amount. Antibodies were purified from cell supernatants using the MAbSelect Xtra (GE Healthcare Life Sciences) and CaptureSelect IgG-CH1 (Thermo Fisher Scientific) columns for IgG and Fab purification, respectively. Antibody samples were concentrated, buffer exchanged to TBS buffer (Alfa Aesar), and then subjected to SEC purification (HiLoad 16/600 Superdex S200 pg column; GE Healthcare Life Sciences).