High-throughput synthesis and specificity characterization of natively paired influenza hemagglutinin antibodies with oPool+ display

Abstract

Antibody discovery is crucial for developing therapeutics and vaccines and for understanding adaptive immunity. However, the lack of approaches to synthesize antibodies with defined sequences in a high-throughput manner represents a major bottleneck in antibody discovery. Here, we present oPool⁺ display, a high-throughput cell-free platform that combined oligo pool synthesis and mRNA display to rapidly construct and characterize hundreds to thousands of natively paired antibodies in parallel. As a proof of concept, we applied oPool⁺ display to probe the binding specificity of more than 300 uncommon influenza hemagglutinin-specific antibodies against nine hemagglutinin variants through 16 screens. More than 5000 binding tests were performed in 3 to 5 days of hands-on time with further scaling potential. Follow-up structural and functional analysis of two antibodies revealed the versatility of the human immunoglobulin gene segment D3-3 (IGHD-3-3) in recognizing the hemagglutinin stem. Overall, this study established an experimental platform that not only accelerates antibody characterization but also enables unbiased discovery of recurring molecular signatures among antibodies with the same specificity.

Figure 1.. Oligo pool-based PCR assembly enables parallel synthesis of the natively paired HA antibody library.

(A) The overall breakdown of the HA antibody (Ab) library. (B) Design of oligos for single chain variable fragment (scFv) assembly. Each given scFv construct contains a T7 promoter and a start codon at the N-terminal as well as a FLAG tag at the C-terminal. The scFv sequences were then split into 4 fragments at the selected complementary determining region (CDR), with overlap between adjacent fragments. Through an overlap PCR, oligos of the same construct would preferably anneal to each other, ensuring the assembly of natively paired scFvs. (C) Synthesis of the natively paired HA antibody library. Synthesized oligo pools containing scFv fragments were assembled using a two-stage PCR. (D) The unassembled and assembled oligo pools were compared by agarose gel electrophoresis. “U”: unassembled oligo pool. “Rep1”: replicate 1. “Rep2”: replicate 2. The red arrow indicates the target size (800–900 bp) for full length scFvs. (E and F) The reproducibility (E) and coverage (F) of the scFv assembly using varying numbers of scFvs per PCR, ranging from 25 to 200. The Pearson correlation coefficient (R) of the occurrence frequencies of individual scFvs between the two replicates are indicated. Micro-tube icon by Servier https://smart.servier.com/ is licensed under CC-BY 3.0 Unported, available from Bio Icons.

Figure 2.. mRNA display enables rapid specificity characterization of the natively paired antibodies.

The screening results of each scFv are shown as a heatmap. The X-axis labels represent the scFv names. The Y-axis labels represent each individual screen. Screens against CR9114 IgG pre-bound HAs are indicated by “+CR9114”. Binding scores for each screen was calculated based on results from two biological replicates (Materials and Methods). Individual cutoffs were set for each screen to determine positive hits (table S4). Black circles at the top of the heatmap indicate head antibody controls, whereas empty circles indicate stem antibody controls.

Figure 3.. Systematic validation reveals the robust performance of oPool⁺ display.

(A and B) Binding activity of 25 antibodies against different HAs was measured by BLI with antibodies in Fab format (A) and ELISA with antibodies in IgG format (B). The response signals during the association phase (A) and the average OD₄₅₀ values across two technical replicates (B) are shown as heatmaps. The dots represent the positive HA binding in oPool⁺ display. The X-axis labels represent antibody names. The Y-axis labels represent antigen names. (C) Binary confusion matrices based on BLI and ELISA validations are shown. Pos., positive; Neg., negative. (D) Binding affinity of selected HA stem antibody candidates in both scFv and Fab format was measured by BLI. Their dissociation constants (K_D) against H1 HA stem and H3 HA stem are shown as heatmaps. 31.a.55 was a positive control for binding to both H1 HA stem and H3 HA stem(8), AG11–2F01 was a positive control for binding to H1 HA stem (15), and 042-100809-2F04 was a positive control for binding to H3 HA stem (16).

Figure 4.. oPool⁺ display result infers CR9114 competition.

(A) CR9114 competition (Comp.) indices of validated antibodies. The competition indices calculated from oPool⁺ display are shown. High positive values indicate CR9114 competition, low positive and high negative values indicate no CR9114 competition. (B) CR9114 competition indices of validated antibodies based on BLI results are shown as scatter plot against their oPool⁺ display competition indices. The response of each antibody binding to the HA antigen were measured during the association step of BLI with or without prior saturation binding of CR9114. The percentage of competition is shown with the range of 0% (no competition) to 100% (complete competition). The Pearson correlation coefficient (R) between the screening and validation results is indicated. The linear fit lines are shown as red dash lines. Representative antibodies are labelled.

Figure 5.. Structural analysis of AG11–2F01 and 16.ND.92 reveals distinct binding mode of IGHD3–3 HA stem antibodies.

(A) Cryo-EM structures of AG11–2F01 and 16.ND.92 in complex with H1/SI06 HA. HA1 is in light gray. HA2 is in dark gray. Heavy chain variable domain (V_H) is in orange. Light chain variable domain (V_L) is in pink. (B) Epitopes of AG11–2F01 and 16.ND.92 are shown. V_H contacts are in orange. V_L contacts are in pink. Contacts shared by both V_H and V_L are in purple. (C) Interactions between light chain and HA are shown. H-bonds are represented by black dashed lines. (D) Interactions between CDR H3 and HA are shown. (E) Overlay of the CDR H3 loops from IGHD3–3 HA stem-reactive antibodies. HA is in surface representation. (F) Contributions of CDR H3 (orange), non-CDR H3 V_H (yellow), and V_L (pink) to the paratope buried surface areas (BSA) of the indicated antibodies. (G) Contributions of IGHD3–3-encoded residues to V_H paratope BSA of the indicated antibodies.

Figure 6.. AG11–2F01 and 16.ND.92 confer in vitro neutralization and in vivo protection.

(A) The binding activities of AG11–2F01 and 16.ND.92 against recombinant HA proteins from the indicated H1 and H5 strains were measured by ELISA. The half-maximal effective concentration (EC₅₀) values are shown as a heatmap. Technical duplicates were performed. (B) The neutralization activity of AG11–2F01 and 16.ND.92 against different recombinant H1N1 viruses was measured by a microneutralization assay. The half-maximal inhibitory concentration (IC₅₀) values are shown as a heatmap. For (A) and (B) Strain names are abbreviated as follows: H1N1 A/Puerto Rico/8/1934 (H1/PR8), H1N1 A/USSR/90/1977 (H1/USSR77), H1N1 A/Chile/1/1983 (H1/CH83), H1N1 A/Taiwan/01/1986 (H1/TW86), H1N1 A/Beijing/262/1995 (H1/BJ95), H1N1 A/Solomon Island/3/2006 (H1/SI06), H1N1 A/Brisbane/59/2007 (H1/BR07), H1N1 A/California/04/2009 (H1/CA09), H1N1 A/Michigan/45/2015 (H1/MI15), H1N1 A/Brisbane/02/2018 (H1/BR18), H5N8 A/northern pintail/WA/40964/2014 (H5/WA14), H5N2 A/snow goose/Missouri/CC15–84A/2015 (H5/MO15), and H5N1 A/bald eagle/Florida/W22–134-OP/2022 (H5/FL22). H1 HA stem and H3 HA stem represent the HA stem constructs designed based on H1N1 A/Brisbane/59/2007 HA and H3N2 A/Finland/486/2004 HA, respectively (28, 29). Technical duplicates were performed. (C to H) The in vivo protective activity of AG11–2F01 (C, E, and G) and 16.ND.92 (D, F, and H) given 4 hours before (Protection) or after (Therapeutic) infection was assessed using lethal challenge of PR8 virus (n= 5 for all groups in C to F; n=3 for all groups in G and H; separate groups of mice were followed for weight loss and survival versus lung viral titers). Shown are weight loss profiles (C and D), Kaplan-Meier survival curves (E and F), and lung viral titers at day 3 post-infection (G and H). dpi, days post infection. Data in (C and D) represent mean ± standard deviation. Bars in (G) and (H) represent mean values. Two-way ANOVA followed by Tukey’s post hoc tests were performed in (C), (D), (G), and (H). Mantel-Cox log rank tests were performed in (E) and (F). n.s.: not significant; *:P < 0.05; **:P < 0.01; ***:P < 0.001.