System to link the antigen-binding feature with the Ig repertoire genetic information. A. Single cell-sorted B cells were subjected to two processes: Ig-seq database building and Ig-expressing transformant library preparation. Antigen-binding Ig transformants were collected by sorting in bulk and the sequences of the unique CDR3 regions and clones of interest were identified by referring to the Ig-seq database. B. To express both Ig heavy and kappa/lambda chains in a single-expression vector, we generated a dual-expression vector. Four gene fragments were assembled by the Golden Gate method using the BsaI restriction enzyme. They included: 1) the destination vector containing the Ig kappa constant gene and the ccdB Chloramphenicol cassette for negative selection, 2) fragments containing the Ig heavy constant gene fused to Venus derived from the donor vector, 3) the Ig heavy variable gene fragment, and 4) the Ig light variable gene fragment. C. Individually purified plasmids were transiently transfected to the floating human FreeStyle 293 cell line. Igs were expressed on the cell surface in 2 days, and expression levels were confirmed and normalized by Venus expression, since the Ig heavy chain was fused to Venus at the cytoplasmic domain tail.

Isolation of H2 and H1-reactive B cells. A. Mixture of Ig-expressing transformant libraries stained with HA probes. B. Three prominent populations, H1+H2+ (cross), H2+ (H2), and H1+ (H1), were selected and collected in a bulk fashion. Then, the three prominent populations were sorted and sequenced. C. The bulk Ig-seq data were referred to as the single-cell Ig-seq data. Wells containing crossed antibodies were determined by comparing the sequence of the heavy chains of the populations included in H1+H2+ (cross) with the Ig-seq data of single cells. D. Cross antibodies obtained by bulk sorting were examined by single-cell presentation. E. A summary of the results to check whether a candidate mAb was cross-reactive.

Competition assay using various mAbs for binding to H2 and PR8 was performed, including two previously reported antibodies, C179, a broadly-reactive mAb binding to the Stem region of HA, and NSP2 (Narita strain-specific mAb), a strain-specific mAb that binds to the head region of HA.

Affinities (Kd) of the antibodies were determined to 6 HA antigens from strains A/Okuda/1957(H2N2) named for H2, A/Puerto Rico/8/1934(H1N1) for PR8, A/California/2009 (X-179A) [H1N1] Pdm09 for Cal, A/Texas/50/2012 (X-223) [H3N2] for H3, A/Egypt/N03072/2010(H5N1) for H5, and A/Brisbane/59/2007 (H1N1) for Stem using surface plasmon resonance analysis. H2, PR8, Cal, and H5 belong to group 1, and H3 belongs to group 2.

* The affinity of D11p4 for H5 was low and inaccurate (Supplementary Figure 2).

Oligonucleotides

Oligonucleotides

The Ig repertoires were visualized using the in-house software. A, Overview of heavy (V-D-J) and light (V-J) chain usages. B, the repertoire clonality from single cells. C, the mutation rates of heavy chains in three cell populations (PR8+, H2+, PR8+H2+) were compared. D, the lengths of CDR3 of heavy chains of three cell populations (PR8+, H2+, PR8+H2+) were compared.

Kinetics analysis was performed using a BIAcore 3000 machine (GE Healthcare). A6p4, B10p2, C10p2, A2p1, D11p4, E11p2, G6p2, D4p4 or G12p4 antibodies were immobilized on a CM5 sensor chip (GE Healthcare) using an amine-coupling kit according to the supplier’s instructions. HA probes were serially diluted at five different concentrations in HBS-EP buffer.

Two hundred and eighty-four clones are plotted in a two-dimensional map of phylogeny trees for heavy and light chain genes. Nine mAb clones are overlaid in green. Six “cross” clones are located in a major cluster. B10p2 and C10p2 are co-localized and distant from the cluster. A6p4 is uniquely located in the map.

The affinity of antigen-antibody binding (in this case, probe and membrane-bound antibody expressing cell) was inferred from the fluorescence intensity (flow cytometry analysis).