Discovery of ultrapotent broadly neutralizing antibodies from SARS-CoV-2 elite neutralizers

Abstract

A fraction of COVID-19 convalescent individuals mount a potent antibody response to SARS-CoV-2 with cross-reactivity to SARS-CoV-1. To uncover their humoral response in detail, we performed single B cell analysis from 10 SARS-CoV-2 elite neutralizers. We isolated and analyzed 126 monoclonal antibodies, many of which were sarbecovirus cross-reactive, with some displaying merbecovirus- and embecovirus-reactivity. Several isolated broadly neutralizing antibodies were effective against B.1.1.7, B.1.351, B.1.429, B.1.617, and B.1.617.2 variants and 19 prominent potential escape sites. Furthermore, assembly of 716,806 SARS-CoV-2 sequences predicted emerging escape variants, which were also effectively neutralized. One of these broadly neutralizing potent antibodies, R40-1G8, is a IGHV3-53 RBD-class-1 antibody. Remarkably, cryo-EM analysis revealed that R40-1G8 has a flexible binding mode, targeting both "up" and "down" conformations of the RBD. Given the threat of emerging SARS-CoV-2 variants, we demonstrate that elite neutralizers are a valuable source for isolating ultrapotent antibody candidates to prevent and treat SARS-CoV-2 infection.

Keywords: COVID-19; SARS-CoV-2; broadly neutralizing antibodies; cryo-EM; emerging variants; ultrapotent monoclonal antibodies; variants of concern.

Graphical abstract

Figure 1

Identification of SARS-CoV-2 elite neutralizers (A) Heatmap depicting the IgG neutralization IC₅₀ values against the SARS-CoV-2 Wu01 pseudovirus in the COVID-19 convalescent cohort studied (Vanshylla et al., 2021). Pie chart shows the fraction of elite neutralizers in the cohort (3.3%). (B) Neutralization curves depicting IgG neutralization from n = 10 donor elite neutralizers against SARS-CoV-2 and SARS-CoV-1 pseudovirus. Mean of two measurements plotted and dotted line represents 50% neutralization. (C) Schematic of study design used to identify and isolate monoclonal antibodies from SARS-CoV-2 elite neutralizers.

Figure 2

Single B cell analysis of the antibody response against SARS-CoV-2 in elite neutralizers (A) Pie charts depicting distribution of clonal (shades of blue) and non-clonal (gray) single B cell-derived heavy chain sequences from each elite neutralizer. (B) Pie chart illustrating overall clonality of all productive (n = 1,361) SARS-CoV-2 reactive heavy chain B cell sequences. Total numbers of sequences analyzed shown in center of pie charts in (A) and (B). (C) Frequencies of heavy chain V-gene distribution from SARS-CoV-2 elite neutralizers (upper panel) and analysis of heavy chain germline identity and CDRH3 length (lower panels) of IGHV sequences derived from elite neutralizers. (D) Analysis of rates of sequence similarity in the heavy chain CDRH3 from the SARS-CoV-2 antibody repertoire of elite neutralizers. Top to bottom: analysis of size (number) of B cell clusters, V-gene information and individuals included in the cluster, length of the CDRH3s (bars show min. to max. with line at median), and the median CDRH3 distance. Reference in panels (C) and (D) (gray) refers to IGHV sequences derived from naive donors.

Figure 3

CoV-cross-reactive and potent monoclonal antibodies derived from elite neutralizers (A) Heatmap illustrating ELISA binding (AUC) against indicated CoV-spikes, pseudovirus neutralization (IC₅₀) (SARS-2, SARS-1, and WIV-1 PSV column), authentic virus neutralization (IC₁₀₀) (SARS-2 AV column), and clonality and germline identity of n = 126 elite neutralizer mAbs. iGL, inferred germline. (B) Annotated P0DTC2 (6XKL) model of the SARS-CoV-2 spike (left) and pie chart showing epitope-binding distribution of the mAbs determined by ELISA (right). (C) Bar graph presenting fraction of neutralizing mAbs (n = 122) binding to SARS-CoV-2 spike epitopes. (D) Pearson correlation matrix of binding and neutralization data from (A) in order to study relationships between binding epitopes, SARS-2-specific neutralization, β-CoV cross-reactivity (SARS-1, MERS, HKU1, and OC43), and sarbecovirus cross-neutralization (SARS-1 and WiV-1). (E) Pie chart depicting fraction of cross-reactive mAbs (left) and plot depicting IC₅₀ values of sarbecovirus cross-neutralizing mAbs (right). (F) Pie chart depicting fraction of NAbs, based on potency (left), and plot showing IC₅₀s of NAbs against the SARS-CoV-2 Wu01 pseudovirus (right); black bar denotes geometic mean. Gray area in (E) and (F) highlights values below 0.02 μg/mL.

Figure 4

Broadly neutralizing next-generation SARS-CoV-2 bNAbs (A) Neutralization escape map profile of n = 57 elite neutralizer NAbs against 25 SARS-CoV-2 spike pseudovirus variants. Average IC₅₀ values, relative neutralization breadth across the variants, the spike epitope determined by ELISA, and cross-neutralizing capacity, as well as IGHV3-53 usage, are depicted in columns to the right. (B) Dot plot depicting average IC₅₀s and IC₅₀s against Wu01 for all isolated bNAbs (n = 23) with the IGHV3-53/IGKV1-9 bNAbs highlighted in blue. Pie chart shows fraction of IGHV3-53/IGKV1-9 bNAbs among nBAns obtained from elite neutralizers. (C) Plot evaluating the IC₅₀ values of the broadest (100%) and most potent NAbs against SARS-CoV-2 pseudovirus variants with spike sequence of B.1, B.1.1.7, B.1.351, B.1.429, B.1.617, and B.1.617.2 are compared to published monoclonal antibodies. REGN antibodies tested up to 5 μg/mL. Gray area in (C) highlights values below 0.02 μg/mL, and black bars denote geometric means.

Figure 5

Maintenance of bNAb potency amidst emerging escape variants (A) Schematic of study design used to analyze emerging escape variants. (B) A full phylogenetic tree with leaves corresponding to isolated sequences collected after January 01, 2020, highlighting the 4 RBD mutations: T478K (red), R346S (green), Q414H (blue), and N440K (purple). The number of leaves for each clade reflects the frequency of that clade in 2021, where the 4 RBD mutations are upweighted. (C) Neutralization analysis of the 11 most potent and broad bNAbs along with published antibodies tested against B.1 pseudoviruses carrying the 4 emerging spike mutations, R346S, Q414H, N440K, and T478K.

Figure 6

Structural basis of SARS-CoV-2 neutralization breadth of R40-1G8 (A and B) Cryo-EM density maps for R40-1G8 Fab-SARS-CoV-2 S protein complexes at 3.2 Å (state 1) (A) and 3.7 Å (state 2) (B), revealing binding of R40-1G8 Fab to both up and down RBDs as indicated by the orange arrows. (C) Locally refined cryo-EM map of the R40-1G8 Fab-RBD complex from which the R40-1G8 Fab was built. (D) Surface representation of the R40-1G8 Fab epitope on the surface of RBD. Epitope residues are shown as sticks in blue (for interactions with the R40-1G8 heavy chain) and light blue (interactions with the R40-1G8 light chain). (E) Close-up showing interactions between the heavy and light chains of R40-1G8 and RBD with the contact residues involved in key interactions shown in sticks. (F) Structural alignment of C102 (PDB 7K8M), C002 (PDB 7K8T), and R40-1G8 on the RBD, revealing that R40-1G8 binds at a similar location as the class 1 anti-RBD antibody C102.