GoKawiilHuman samples
Serum and peripheral blood mononuclear cell (PBMC) samples were obtained from participants enrolled in two prospective community-based studies in Nicaragua: The Household Influenza Transmission Study (HITS) and the Nicaraguan Pediatric Influenza Cohort Study (NPICS). HITS is an intensive transmission study that enrols households after identification of a laboratory-confirmed influenza case and conducts intensive follow-up of household members. NPICS is an ongoing, longitudinal cohort of children aged 0–14 years, established to study the development of immunity to influenza from birth through annual serosurveys and active surveillance for acute respiratory illness. The sample sizes were not predetermined by statistical methods. These are observational studies, so no blinding or randomization was used.
PBMCs were isolated from venous blood by density-gradient centrifugation and cryopreserved for subsequent cellular immunology assays, as previously described41. NPICS samples were collected during the 2017–2018, 2018–2019, 2019–2020 and 2022–2023 seasons, whereas HITS samples were collected during the 2015–2016 season in Nicaragua, which typically spans June to December each year. These samples represent both the pre-infection (0–3 days between symptom onset) and post-infection (4–8 weeks after symptom onset) timepoints, enabling characterization of immune responses across the course of infection in a well-defined community setting. Written informed consent was obtained from parents or legal guardians of all participants, with assent from children as appropriate. Both studies received ethical approval from institutional review boards in Nicaragua and at the University of Michigan (HUM00091392 and HUM00088895).
Healthy infants for analysis of immune responses to primary influenza vaccination were recruited at paediatric primary clinics at Nationwide Children’s Hospital (NCH), Columbus, Ohio. Sera and PBMCs were collected after 1 month of vaccination as described above. The sample sizes were not predetermined by statistical methods, and no blinding or randomization was used. Written informed consent was obtained from parents or guardians before any of the study procedures. The study was approved by the NCH IRB (18-00591).
Cell lines and viruses
Madin–Darby canine kidney (MDCK) cells were purchased from ATCC and maintained in culture at 37 °C with 5% CO 2 in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% FBS (Gibco), 1% l-glutamine (Gibco) and 1% penicillin–streptomycin (final concentration, 100 units ml−1 penicillin, 100 μg ml−1 streptomycin, Gibco). Humanized MDCK (hCK) cells42 (in-house cell line generated by the Kawaoka laboratory) were maintained in culture at 37 °C with 5% CO 2 in minimum essential medium (MEM, Gibco) containing 5% (v/v) newborn calf serum (Sigma-Aldrich), 0.225% sodium bicarbonate (Corning), 1× amino acids (Gibco), 1× vitamins (Gibco), 1× anti-anti (Gibco), 4 mM l-glutamine (Gibco), 2 μg ml−1 puromycin (InvivoGen) and 10 μg ml−1 blasticidin (InvivoGen). Expi293F suspension cells were purchased from Thermo Fisher Scientific and maintained in culture at 37 °C with 8% CO 2 in Expi293F Expression Medium (Gibco) with shaking at 125 rpm. Cell lines were not authenticated or tested for mycoplasma at the lab level. Influenza viruses were grown in-house in MDCK cells or specific-pathogen free (SPF) eggs, collected, purified and titred (Supplementary Table 9).
Recombinant antigen and probe production
HA ectodomain sequences were synthesized by Integrated DNA Technologies (IDT) and cloned into a mammalian protein expression vector with a C-terminal foldon trimerization domain followed by AviTag and 6×His tag. Constructs were transfected using the ExpiFectamine 293 kit (Gibco) according to the manufacturer’s protocol. Supernatants were collected on day 5 after transfection and incubated with Ni2+-nitrilotriacetic acid (Ni-NTA) agarose (Qiagen). Agarose was then loaded onto a polypropylene gravity flow column (Thermo Fisher Scientific), washed with 20 mM imidazole in PBS and eluted with a solution of 500 mM imidazole, 20 mM Tris and 150 mM NaCl at pH 7.4. The eluate was buffer exchanged with PBS using a 100 kDa Amicon centrifugal column (Millipore). Purified HA proteins were stored at −80 °C (Supplementary Table 8).
For probes used in cell sorting, a Y98F mutation was introduced into all HA constructs, and constructs of the SARS-CoV-2 WT (D614G) receptor-binding domain (RBD) and chimeric HAs were generated as previously described43,44. All probes were synthesized, cloned, expressed and purified as described above. The purified probes were biotinylated (AviTag-specific) with BirA enzyme according to the manufacture’s protocol (Avidity). The unreacted biotin was removed by passage through a 7 kDa molecular mass cut-off desalting column (Zeba spin, Thermo Fisher Scientific). Biotinylated HAs were conjugated to TotalSeq-C PE streptavidin (PE-SA, BioLegend) and BV421 streptavidin (BV421-SA, BioLegend), whereas SARS-CoV-2 WT RBD was conjugated to APC streptavidin (APC-SA, BioLegend). Chimeric HAs (cH8/1 or cH4/3) and an empty control (no antigen, PBS only) were conjugated to TotalSeq-C non-fluorescent streptavidin (NF-SA, BioLegend). The amount of antigen needed for conjugation was calculated based on a 4:1 molar ratio of antigen to a fixed amount of 0.5 μg PE-SA, BV421-SA, APC-SA or NF-SA. Antigens were diluted in PBS to a final volume of 10 μl and SAs were added gradually to antigens five times on ice, 1 μl SA (0.1 mg ml−1 stock) every 10 min for a total of 5 μl (0.5 μg). The reaction was quenched with 5 μl 4 mM Pierce Biotin (Thermo Fisher Scientific) for 30 min for a total volume of 20 μl. Probes used for each sorting experiment were prepared on the same day.
Cell sorting for 10x Genomics single-cell sequencing
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