N octyl β d glucopyranoside

Acetonitrile (ACN), methanol (MeOH), acetic acid (HAc), formic acid (FA), ammonium bicarbonate (NH₄HCO₃), tri-n-butylphosphate (TBP), sodium chloride (NaCl) were obtained from Merck (Darmstadt, Germany). Acetone, ethylenediaminetetraacetic acid tetrasodium salt dihydrate (EDTA), protease inhibitor cocktail, phosphate buffered saline (PBS), Tris-HCl, diethylamide (DEA), trifluoroacetic acid (TFA), n-octyl-β-D-glucopyranoside, triethyl ammonium bicarbonate (TEAB), and formaldehyde CH₂O (37% (vol/vol)) were purchased from Sigma Aldrich (St. Louis, MO, USA). For tryptic digestion, iodoacetamide (IAA), urea and dithiothreitol (DTT) were obtained from Sigma Aldrich and trypsin (sequencing grade from bovine pancreas 1418475; Roche diagnostic, Basel, Switzerland) were used. Formaldehyde (¹³CD₂O) (20% (vol/vol), 99% 13C, 98% D) and sodium cyanoborodeuteride (NaBD₃CN) (96% D) were purchased from Isotec (Miamisburg, OH). Sodium cyanoborohydride (NaBH₃CN) was obtained from Fluka (Buchs, Switzerland). Sucrose was purchased from Fisher Scientific Company (Göteborg, Sweden). Triton X-114 was obtained from KEBO Lab (Stockholm, Sweden). Ultrapure water was prepared by Milli-Q water purification system (Millipore, Bedford, MA, USA).

Myc-tagged DRA1/DRB1*0301 HLA class II and untagged DRA1/DRB1*0301, DRA1/DRB1*1101 and DRA1/DRB1*1501 monomer reagents were generated essentially as previously described^{29 (link),35 (link)}. In brief, transfected S2 cells were expanded to a 2  L volume in spinner flasks (Bellco, Vineland NJ) and induced for 5 days with 1 mM copper sulfate, adding 2 μg mL⁻¹ biotin to ensure efficient protein biotinlyation. Supernatants were separated from intact cells by centrifugation (11,000g), separated from debris using a 0.2 μm filter (ThermoFisher, Waltham, MA) and then affinity purified using L243 coupled with CNBr-Activated Sepharose 4B (GE Healthcare, Pittsburgh, PA). Class II protein was eluted at pH 11.5, equilibrated using pH 4.0 Tris buffer and exchanged into a pH 6.0 storage buffer (0.2 M sodium phosphate). Class II monomers were loaded with individual peptides by incubating for 72 h at 37°°C in the presence of 2.5 mg mL⁻¹n-octyl-β-d-glucopyranoside (Sigma, St Louis, MO). Tetramers were formed by individually incubating class II molecules with metal labeled streptavidin or PE-labeled streptavidin (Thermo Fisher Scientific) for 6–18 h at room temperature at a molar ratio of 8:1. Metal labeled streptavidin were produced as described earlier^{36 (link),37 (link)}.

HLA-DRB1*04:01 protein used within this study was recombinantly produced in S2 insect cells as previously described [20 ]. All peptides were synthesised at > 95% purity by GenScript (Piscataway, NJ, USA). The biotinylated monomer was loaded with the different peptides (Table 1) by incubation in the presence of n-octyl-β-D-glucopyranoside and Pefabloc SC (Sigma-Aldrich) and subsequently tetramerised using streptavidin conjugated to PE, PE-Cy5, PE-CF594 (BD Biosciences) or APC (Biolegend), respectively. Every single tetramer was loaded with one specific peptide during this individual assembly, so we ended up having ten different tetramers. Three of these were conjugated to PE and PE-CF594, respectively and two conjugated to APC and PE-Cy5, respectively (Table 1).

Bioneedles (15 mm long and 1 mm wide, internal volume of 5 μl) were obtained from the Bioneedles Technologies Group. Influenza A/PR/8/34 whole inactivated virus was produced by Intravacc. The process was based on egg virus propagation and β-propiolactone virus inactivation [12] (link). Split and subunit vaccines were produced by solubilization of WIV with n-octyl-β-D-glucopyranoside (Sigma-Aldrich) as described previously [8] (link). Virosome vaccine was produced as described previously [13] (link). All vaccines were concentrated with Centriprep centrifugal filters (Millipore) with a molecular weight cut-off (MWCO) of 10 kDa, and formulated in HBS (20 mM HEPES, 125 mM NaCl, 9 mM CaCl₂, 5 mM MgCl₂). Vaccine formulations for bioneedles contained 2.5% (w/w) D-trehalose dihydrate (Sigma-Aldrich) as a stabilizer. Influenza vaccine containing bioneedles (subunit, split, virosomal and WIV vaccine) were prepared by filling bioneedles with 5 μL of 1 mg/mL (HA content) liquid vaccines from the hollow back of the bioneedle using specially designed filling apparatus, and frozen on a metal plate at minus 50°C [11] (link). Next, bioneedles were freeze-dried using a Zirbus Sublimator 3×4×7 (Zirbus Technology). Lyophilized bioneedles were stored in glass vials with rubber stoppers under ambient air and relative humidity.

Sifuvirtide (SWETWEREIENYTRQIYRILEESQEQQDRNERDLLE, MW 4727) and enfuvirtide (YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF, MW 4492) were synthesized by GL Biochem, Ltd. (Shanghai, China). 1-Palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPC) and SM were purchased from Avanti Polar-Lipids (Alabaster, AL, USA). HBS-N buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES] + NaCl), 0.2-M NaOH and the Biacore Maintenance Kit were purchased from General Electric (CT, USA). N-octyl β-D-glucopyranoside and bovine serum albumin (BSA) were purchased from Sigma-Aldrich.

MIL77-1, MIL77-2, and MIL77-3 were gifts from Dr. Ming Lv from the Laboratory of Immunology, Institute of Basic Medical Sciences. 1-Palmitoyl-2-oleyl-sn-glycero-3-phosphocholine(POPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero- 3-phospho- (1′-rac- glycerol) (POPG) and cholesterol (Chol) were purchased from Avanti Polar-Lipids (Alabaster, AL, USA). N-octyl β-D-glucopyranoside and bovine serum albumin (BSA) were purchased from Sigma-Aldrich. HBS-N buffer (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES] + NaCl), 0.2-M NaOH, L1 sensor chip and the Biacore Maintenance Kit were purchased from General Electric (CT, USA).