Expi293

Plasmids encoding 5C4 Fab heavy and light chains were transiently co-transfected into a suspension of Expi293 (Invitrogen) cells with a previously constructed plasmid encoding RSV F (+) FdTHS based on strain A2^{13 (link)}. After incubation at 37 °C for 6 days with shaking, the cell supernatants were concentrated and buffer exchanged into Ni-NTA binding buffer (20 mM Tris pH 8.0, 20 mM imidazole, 300 mM NaCl) using tangential flow filtration. The 5C4–RSV F complex was purified using manufacturer protocols over a column of Ni-NTA Superflow resin (Qiagen). The Ni-NTA elution was then concentrated and further purified over a column of Strep-Tactin resin (IBA) using the manufacturer’s protocol. Following elution off the Strep-Tactin column, C-terminal purification tags on RSV F were removed by overnight digestion at 4 °C with restriction-grade thrombin (EMD Millipore). The complex was then concentrated and further purified to remove excess 5C4 Fab and purification tags by gel filtration using a Superose 6 column (GE Healthcare Biosciences) with a running buffer of 2 mM Tris pH 8.0, 200 mM NaCl and 0.02% NaN₃. Fractions corresponding to the 315 kDa complex (Supplementary Fig. 2) were pooled and concentrated for crystallization.

The variable heavy- and light-chain sequences of the known anti-SARS CoV-2 antibody H4 and variants were cloned into the full-length IgG1 expression vectors pcDNA3.3 HC and pcDNA3.3 LC (ATUM). The recombinant antibodies were transiently expressed in both ExpiCHO and Expi293 cells according to the manufacturer’s protocol (Invitrogen). The supernatants from 1 mL transient transfections of the antibodies were purified using the AssayMAP BRAVO platform with 5 mL Protein A cartridges (Agilent Technologies). Lager scale transient transfections were purified on the Akta FPLC system using 1 mL of HiTrap MabSelect PrismA™ affinity chromatography resin (Cytiva). The purified recombinant monoclonal antibodies were stored in 1x phosphate-buffered saline at 4 °C until use. Specific site-directed mutations on the H4 antibody sequence were performed using the Quick-Change Site-Directed Mutagenesis Kit II (Agilent technologies).

To produce full-length human IgG antibodies, the heavy and light chain variable regions obtained from phage panning were subcloned into expression vector as described previously (35 (link)). Transient transfection was performed in FreeStyle 293 or Expi 293 cells (Invitrogen). 37.5 µg of plasmid DNA and 75 µg of linear polyethyleneiminie (Polysciences, Warrington, PA, USA) were added in 150 mmol/L NaCl, respectively. DNA and PEI solutions were allowed to stand at room temperature for 5 min. The solutions were mixed gently and allowed to stand at room temperature for another 10 min. DNA-PEI mixture was added into 293 cells and incubated for 4 h. An equal volume of fresh culture medium was added and the cells were cultured for 5 to 7 days. Culture supernatant containing the antibody was affinity-purified using the Montage Antibody Purification kit (Millipore, Billerica, MA, USA).

Recombinant HA proteins for use as flow cytometry probes were derived for A/California/7/2009, A/Switzerland/9715293/2013, A/New Caledonia/20/1999 and A/Hong Kong/1/1968 strains as previously described34 (link). Briefly, synthetic genes encompassing the ectodomain of HA modified to limit sialic acid binding were synthesised (GeneArt) and cloned into mammalian expression vectors. HA proteins were expressed by transient transfection of Expi293 (Life Technologies) suspension cultures and purified by polyhistidine-tag affinity chromatography and gel filtration. Proteins were biotinylated using BirA (Avidity) and stored at −80 °C. Prior to use, biotinylated HA proteins were labelled by the sequential addition of streptavidin (SA) conjugated to phycoerythrin (PE) or allophycocyanin (APC) and stored at 4 °C. A mock probe to control for specificity was generated using SA-BB515 (BD).

Recombinant HA-FL proteins used in immunizations, ELISA, and flow cytometric assays were derived for PR8, A/California/07/2009, and A/Michigan/45/2015 as previously described (21 (link)). HA-FL proteins carry a Y98F mutation in the receptor-binding site, which abolishes binding to cell-surface sialic acids. Stabilized HA stem proteins were engineered for PR8, A/New Caledonia/20/1999, and A/California/07/2009 using methods established previously for the design of Gen6 HA stem in Yassine et al. (6 (link)). Briefly, expression constructs were synthesized (GeneArt) and cloned into mammalian expression vectors. HA-FL and HA stem proteins were expressed by transient transfection of Expi293 (Life Technologies, Thermo Fisher Scientific) suspension cultures and purified by polyhistadine-tag affinity chromatography and gel filtration. Proteins were biotinylated using BirA (Avidity) and stored at –80°C. Prior to use, biotinylated HA proteins were labeled by the sequential addition of streptavidin (SA) conjugated to PE, allophycocyanin (APC), or BV421 and stored at 4°C.

Recombinant HA protein for use as flow cytometry probes was derived for A/Puerto Rico/08/1934 as previously described (24 (link)). Briefly, expression constructs were synthesized (GeneArt) and cloned into mammalian expression vectors. Proteins were expressed by transient transfection of Expi293 (Life Technologies) suspension cultures and purified by polyhistidine-tag affinity chromatography and gel filtration. Proteins were biotinylated using BirA (Avidity) and stored at −80°C. Prior to use, biotinylated HA proteins were labeled by the sequential addition of streptavidin (SA) conjugated to BV421, phycoerythrin (PE), or allophycocyanin (APC), and stored at 4°C. Recombinant influenza A H1N1 NP protein (11675-V08B; Sino Biological) was labeled with PE or APC fluorochromes using commercial conjugation kits, as per manufacturer's protocol (AB102918, AB 201807; Abcam).