Multimodal, broadly neutralizing antibodies against SARS-CoV-2 identified by high-throughput native pairing of BCRs from bulk B cells

Abstract

TruAB Discovery is an approach that integrates cellular immunology, high-throughput immunosequencing, bioinformatics, and computational biology in order to discover naturally occurring human antibodies for prophylactic or therapeutic use. We adapted our previously described pairSEQ technology to pair B cell receptor heavy and light chains of SARS-CoV-2 spike protein-binding antibodies derived from enriched antigen-specific memory B cells and bulk antibody-secreting cells. We identified approximately 60,000 productive, in-frame, paired antibody sequences, from which 2,093 antibodies were selected for functional evaluation based on abundance, isotype and patterns of somatic hypermutation. The exceptionally diverse antibodies included RBD-binders with broad neutralizing activity against SARS-CoV-2 variants, and S2-binders with broad specificity against betacoronaviruses and the ability to block membrane fusion. A subset of these RBD- and S2-binding antibodies demonstrated robust protection against challenge in hamster and mouse models. This high-throughput approach can accelerate discovery of diverse, multifunctional antibodies against any target of interest.

Keywords: Antibodies; Antibody secreting cells; BCR pairSEQ; Broadly neutralizing antibodies; Cell fusion; High throughput sequencing; S2 antibodies; SARS CoV-2; TruAB Discovery.

Graphical abstract

Figure 1

Characteristics of antibodies from enriched antigen-specific memory B cells (A) Every synthesized antibody was tested in duplicate by ELISA for binding against four spike antigens: RBD, S1 including RBD, S2, and the full spike protein in its trimeric form (Tri). Non-specific IgG controls were included in each ELISA and are denoted. (B) Diversity of heavy (left) and light chain (right) of spike-reactive antibodies. Numbers above bars indicate the total antibodies represented. Only genes represented by at least 1.5% of antibodies in an individual category are shown. Where multiple antibodies per lineage were tested, only a representative single lineage is included. (C) Proportion of sites in heavy chain V genes in spike-reactive antibodies that have experienced somatic hypermutation relative to germline. Red dots = antibodies that bind and do not neutralize pseudovirus and blue = antibodies that bind and neutralize pseudovirus. See also Figure S1 and Table S2.

Figure 2

Functional characterization of selected spike-specific antibodies (A) Screening of ACE2/RBD blockade by competitive ELISA, depicted as % blockade. Antibodies that blocked this interaction by at least 50% were advanced (triangles). Only antibodies considered positive binders (OD₄₅₀ > 0.2) by ELISA are shown and spike region specificity (RBD, S1, S2, and Trimer [Tri.]) is defined based on Figure 1A. (B) Pseudovirus neutralization screening of all antibodies expressed as recombinant IgG₁. Target cells used were an ACE2/TMPRSS2-expressing cell line and all antibodies were tested at 10 μg/mL. Neutralization cut-off of over 70% was used to advance antibodies (blue). Only antibodies considered positive binders to spike are shown. (C) Heatmap showing degree of binding interference between paired antibodies as determined by Octet competition experiments. “Class” assignments are inferred from the epitope groups identified and critical binding residues identified by shotgun mutagenesis. (D) Critical binding residues are highlighted on RBD structure (PDB: 7BZ5).(E) Binding affinity measured by SPR of selected antibodies to RBD (antibodies labeled with “Class”), non-RBD S1 or the full spike trimer. (F) Pseudovirus neutralization shown by IC₅₀ of selected antibodies against SARS-CoV-2 variants (WT = WA1). IC₅₀ shown is the average of two independent replicates. (G) IC₅₀ of antibodies against live WT and Omicron variant SARS-CoV-2 viruses. IC₅₀ values were calculated using the Plaque reduction neutralization test (PRNT) assay. IC₅₀ shown is the average of two independent replicates. (H) Results from bead-based effector function assay measuring complement activation (ADCD), antibody mediated phagocytosis (ADCP and ADNP), and cytotoxicity (ADNKA CD107⁺ and MIP1b⁺). The y axis shows normalized assay scores where higher scores indicate higher effector function. Also see Figures S2 and S3 and Table S1.

Figure 3

Distinctive antibody repertoire from the ASCs of an individual with a recent SARS-CoV-2 infection (A) Depicts ELISA on donor (ADIRP0003238) serum showing binding to nucleocapsid, RBD, S2, and S1 proteins. (B) ELISA binding to RBD, S2, S1, and Trimer (Tri.) proteins by antibodies selected for synthesis and screening from ADIRP0003238. (C and D) (C) V-gene usage of selected antibodies, and (D) SHM in ADIRP0003238 donor’s ASC-derived monoclonal antibodies. (E) Competitive ELISA screening of antibodies for ACE2/RBD blockade shown as % blockade. (F) Pseudovirus neutralization screening of all antibodies expressed as recombinant IgG₁. Target cells used were an ACE2/TMPRSS2-expressing cell line and all antibodies were tested at 10 μg/mL. Neutralizing antibodies indicated in blue. Also see Figure S1.

Figure 4

Neutralization, breadth, and mechanism of action for S2-binding antibodies (A) Pseudovirus neutralization IC₅₀ of ASC-derived S2 antibodies as described in Figure 2F. Neutralization IC₅₀ of pseudoviruses of various SARS-CoV-2 strains as well as SARS-CoV-1 is shown. (B) Dilution curves of antibody binding by ELISA to spike trimer and S2 protein from betacoronaviruses (SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-HKU1, and HCoV-OC43) and one alphacoronavirus (HCoV-229E). (C) Schematic of the cell-cell fusion assay. Figure created using BioRender. (D) Dilution curves of selected antibodies blocking cell-cell fusion. LCB1, a peptide previously shown to block fusion, was used as a positive control. Data shown is representative of 3 experiments. (E) Results from bead-based effector function assay measuring ADCD, ADCP, ADNP, and cytotoxicity via ADNKA CD107⁺ and MIP1b⁺ measurement, as described in Figure 2H. Also see Figures S4 and S5.

Figure 5

RBD-, non-RBD S1-, and trimer-directed antibodies provide effective in vivo protection (A) Schema of the in vivo experiments. K-18-hACE2 mice and hamsters were treated with antibodies at 1.5 or 5.0 mg/kg, respectively, via intraperitoneal (IP) administration. Animals were then inoculated with SARS-CoV-2 intranasally (dose as indicated), after either 24 h (mice) or 48 h (hamsters). Mock-infected and untreated animals were used as controls. (B) Mean ± SEM percent change in mouse body weight after inoculation of each experimental group (N = 10 mice in each group). (C) Survival curve of mice after passive transfer of SARS-CoV-2 antibodies. Animals were euthanized when body weight was less than 75% starting weight. (D) Percent weight change in Syrian hamsters treated with 5 mg/kg of antibody IP 48 h prior to infection with SARS-CoV-2 intranasally (N = 5 hamsters per group). Data points represent Mean ± SEM. Human IgG₁ was used as isotype control.

Figure 6

Prophylactic protection of both mice and hamsters by S2-targeting antibodies at varying doses (A–C) S2-binding antibodies also showed strong protection in K-18-hACE2 mice (A and B) and Syrian hamsters (C). Percent body weight data points represent Mean ± SEM. Experiments were conducted as detailed in Figure 5. Antibody dose varied per group as indicated in the individual plots.