A Potently Neutralizing Antibody Protects Mice against SARS-CoV-2 Infection

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for millions of infections and hundreds of thousands of deaths globally. There are no widely available licensed therapeutics against SARS-CoV-2, highlighting an urgent need for effective interventions. The virus enters host cells through binding of a receptor-binding domain within its trimeric spike glycoprotein to human angiotensin-converting enzyme 2. In this article, we describe the generation and characterization of a panel of murine mAbs directed against the receptor-binding domain. One mAb, 2B04, neutralized wild-type SARS-CoV-2 in vitro with remarkable potency (half-maximal inhibitory concentration of <2 ng/ml). In a murine model of SARS-CoV-2 infection, 2B04 protected challenged animals from weight loss, reduced lung viral load, and blocked systemic dissemination. Thus, 2B04 is a promising candidate for an effective antiviral that can be used to prevent SARS-CoV-2 infection.

Fig. 1.. Isolation of SARS-CoV-2 RBD-specific murine mAbs

A. Immunization regimen. C57BL/6J mice were immunized with 10 μg SARS-CoV-2 RBD i.m. and boosted with 5 μg S protein 14 and 24 days later. Serum and dLNs were harvested 5 days after the second boost. B. IgG serum Ab ELISA for SARS-CoV-2 S protein (left panel) and RBD (right panel). C. Serum neutralization activity against SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020 using a focus reduction neutralization test (FRNT). Values normalized to serum from a PBS-immunized mouse. D. Sorting strategies for total PBs (grey gate) and RBD⁺ PBs (red gate) from dLNs pooled from both mice. Total PBs were bulk-sorted for single-cell RNA sequencing, and RBD⁺ PBs were single-cell sorted for mAb cloning. E. mAb screening ELISA for binding to SARS-CoV-2 RBD.

Fig. 2.. SARS-CoV-2 RBD-binding plasmablast response is clonally diverse

A. Clonal diversity of single-cell sorted RBD-binding PB sequences. Each slice represents one clone; width represents frequency distribution. B. Minimum positive concentrations of clonally unique mAbs as determined by SARS-CoV-2 RBD ELISA of mammalian cell-expressed RBD; positive binding defined as greater than 3x background. Representative of 3 independent experiments. Dotted line represents limit of detection. C. Gene expression-based clustering visualized via t-distributed stochastic neighbor embedding. D. Plasmablasts found in clones containing RBD⁺ (red) and RBD⁻ (gray) mAbs. E. Isotypes of plasmablasts found in clones containing RBD⁺ mAbs. IgG are shown in pink, IgM in blue, and IgE in green. F. IGHV nucleotide mutation frequency of plasmablasts found in clones containing RBD⁺ (red; n=657) and RBD⁻ (gray; n=5263) mAbs. Lines represent medians. P-value from two-sided Mann-Whitney.

Fig. 3.. Cross-reactivity, ACE2 competition, and neutralization capacity of RBD-binding mAbs

A–C. Minimum positive concentrations of clonally unique mAbs as determined by SARS-CoV-2 (A), SARS-CoV (B), and MERS-CoV (C) S protein ELISA; positive binding defined as greater than 3x background. Representative of 3 independent experiments. D. Half maximal infection inhibitory concentrations of clonally distinct anti-RBD mAbs against SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020 in an FRNT. Mean ±SEM from 2 (1B10) or 3 (all other mAbs) independent experiments. Daggers indicate mAbs that compete with hACE2 binding to RBD; see also Fig. S3E. Dotted lines represent limit of detection.

Fig. 4.. In vivo protection by mAb 2B04

A. SARS-CoV-2 challenge model. BALB/c mice received αIFNAR1 mAb i.p. 24 hours prior to i.n. administration of AdV-hACE2. Mice received 10mg/kg mAb 2B04 or isotype i.p. 4 days later, followed by i.n. challenge with SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020 one day later. Mice were weighed daily, and tissues were collected 4 days post-challenge. B–D. Percent of baseline weight (B) and viral titers measured in the indicated tissue by RT-qPCR (C) and plaque assay (D) 4 days post-challenge of mice that received isotype (open circles) or 2B04 (closed circles). Data from 3 independent experiments with 14–15 mice per group (B, C) or 1 experiment with 6 mice per group (D). Mean ±SEM shown in B. *, P<0.05, **, P<0.005; P-values from two-sided Mann-Whitney.