Potent monoclonal antibodies neutralize Omicron sublineages and other SARS-CoV-2 variants

Abstract

The emergence and global spread of the SARS-CoV-2 Omicron variants, which carry an unprecedented number of mutations, raise serious concerns due to the reduced efficacy of current vaccines and resistance to therapeutic antibodies. Here, we report the generation and characterization of two potent human monoclonal antibodies, NA8 and NE12, against the receptor-binding domain of the SARS-CoV-2 spike protein. NA8 interacts with a highly conserved region and has a breadth of neutralization with picomolar potency against the Beta variant and the Omicron BA.1 and BA.2 sublineages and nanomolar potency against BA.2.12.1 and BA.4. Combination of NA8 and NE12 retains potent neutralizing activity against the major SARS-CoV-2 variants of concern. Cryo-EM analysis provides the structural basis for the broad and complementary neutralizing activity of these two antibodies. We confirm the in vivo protective and therapeutic efficacies of NA8 and NE12 in the hamster model. These results show that broad and potent human antibodies can overcome the continuous immune escape of evolving SARS-CoV-2 variants.

Keywords: CP: Immunology; CP: Microbiology; Omicron sublineages BA.1., BA.2, BA.2.12.1, and BA.4; SARS-CoV-2; hamster model; immune escape; neutralization; therapeutic antibodies; variants of concern.

Graphical abstract

Figure 1

Study design and characterization of monoclonal antibodies against the SARS-CoV-2 spike protein (A) Schematic design of the different phases of combinatorial phage-display library production and screening leading to the development of SARS-CoV-2 neutralizing antibodies (mAbs). (B) Normalized binding of 18 mAbs to a stabilized SARS-CoV-2 spike protein trimer, S-2P, in ELISA; OD denotes optical density. (C and D) V-gene usage of (C) heavy chains and (D) light chains of 18 mAbs against the SARS-CoV-2 spike protein. (E) Rate of somatic hypermutation of V genes of heavy chains (VH) and light chains (VL) of 18 mAbs. (F and G) Amino acid length of the CDR3 loop of the (F) heavy (CDR3H) and (G) light chain (CDR3L) of 18 mAbs.

Figure 2

Neutralizing activity of 18 monoclonal antibodies derived from convalescent COVID-19 patients and seven clinically approved monoclonal antibodies against different SARS-CoV-2 variants (A) Heatmap of pseudovirus neutralization activity of 18 mAbs derived from convalescent COVID-19 patients. IC₅₀ values (μg/mL) are shown for neutralization of SARS-CoV-2 pseudoviruses bearing spike proteins from the USA/WA1/2020 isolate (WA-1, lineage A), the Alpha variant, Beta variant, Delta variant, and Omicron BA.1, BA.2, BA.4, and BA.2.12.1 sublineages. The color-coded legend of IC₅₀ ranges is indicated in (C). (B) Neutralizing activity of seven clinically approved mAbs, including REGN-10933, REGN-10987, LY-CoV555, LY-CoV016, COV2-2130, COV2-2196, and S309 (sotrovimab), against the same SARS-CoV-2 variants as in (A). (C) Neutralizing activity of mAbs NA8 and NE12 and six clinically approved mAbs used in paired combinations against the SARS-CoV-2 WA-1 strain and the Omicron BA.1, BA.2, BA.4, and BA.2.12.1 sublineages. (D) Neutralization curves of mAbs NE12 and NA8 and six clinically approved mAbs used in paired combinations against the Omicron BA.1 sublineage. (E) Neutralization curves of mAbs NE12 and NA8 and six clinically approved mAbs used in paired combinations against the Omicron BA.2 sublineage. (F) Neutralization curves of mAbs NE12 and NA8 and six clinically approved mAbs used in paired combinations against the Omicron BA.4 sublineage. (G) Neutralization curves of mAbs NE12 and NA8 and six clinically approved mAbs used in paired combinations against the Omicron BA.2.12.1 sublineage. (H) Neutralization curves of mAbs NE12 and NA8 used in combination against all major SARS-CoV-2 variants of concern. (I) Neutralization curves of mAb NA8 against the major SARS-CoV-2 variants of concern. (J) Neutralization curves of mAb NE12 against the major SARS-CoV-2 variants of concern. Results are the average of two or three independent experiments performed in duplicate using a pseudovirus assay. Values represent mean ± SD. The dotted horizontal lines indicate the IC₉₀ and IC₅₀.

Figure 3

Cryo-EM structures of the Fab fragment of neutralizing monoclonal antibody NE12 in complex with a stabilized SARS-CoV-2 spike protein trimer (A) Cryo-EM map of Fab NE12 in complex with the SARS-CoV-2 S-6P spike trimer with docked spike and Fab atomic models. The heavy and light chains of the mAb are colored in pink and purple, respectively. Spike protomers are colored individually. The Fab fragment binds the receptor-binding domain (RBD) in both the up and down positions. (B) Local refinement of a region containing one RBD with bound Fab resolved atomic details of the RBD-NE12 interactions. (C) Ribbon diagram of the RBD/NE12 contact interface. Coloring is as in panel (B). Only the complementarity-determining regions (CDRs) and RBD fragments participating in the interaction are shown for clarity. Key residues are shown in stick representation. Dashed lines represent salt bridges and hydrogen bonds. (D and E) Surface representation of the RBD with the epitope of NE12 colored in green. Residues mutated in B.1.1.7/Alpha, B.1.617.2/Delta, and B.1.351/Beta (D) or Omicron BA.1 sublineage (E) variants of concern are colored in magenta. The antibody residues are numbered according to the IMGT (http://www.imgt.org) (Giudicelli et al., 2005).

Figure 4

Cryo-EM structures of the Fab fragment of neutralizing monoclonal antibody NA8 in complex with a stabilized SARS-CoV-2 spike protein trimer (A) Cryo-EM map of Fab NA8 in complex with the SARS-CoV-2 S-6P spike trimer with docked spike and Fab atomic models. The heavy and light chains of the mAb are colored in pink and purple, respectively. Spike protomers are colored individually. The Fab fragment binds the receptor-binding domain (RBD) in both the up and down positions. (B) Local refinement of a region containing one RBD with bound Fab resolved atomic details of the RBD-NA8 interactions. (C) Ribbon diagram of the RBD/NA8 contact interface. Coloring is as in (B). Only the complementarity-determining regions (CDRs) and RBD fragment participating in the interaction are shown for clarity. Key residues are shown in stick representation. Dashed lines represent salt bridges and hydrogen bonds. (D and E) Surface representation of the RBD with residues mutated in the B.1.1.7/Alpha, B.1.617.2/Delta, and B.1.351/Beta (D) or Omicron BA.1 sublineage (E) variants of concern are colored in magenta. The antibody residues are numbered according to the IMGT (http://www.imgt.org) (Giudicelli et al., 2005).

Figure 5

Comparison of binding epitopes, genetic characteristics, and neutralizing activity of NA8 and NE12 with clinically approved monoclonal antibodies (A) Amino acid sequences of the receptor-binding domain (RBD) of Omicron sublineages BA.1 and BA.2 with mutations colored in red compared with the sequence of the original North American founder strain WA-1 (GenBank: MN985325); the asterisks indicate residues that contact the ACE receptor. The binding epitopes of different mAbs are presented below and highlighted in different colors. (B) Surface representation of the RBD with the epitopes of NA8 (green line) and NE12 or clinically approved monoclonal antibodies (blue line) highlighted. Surface areas corresponding to the RBD residues mutated in the Omicron BA.1 sublineage are colored in magenta. (C) Characteristics of NA8 and NE12 in comparison with clinically approved mAbs against SARS-CoV2.

Figure 6

Prophylactic efficacy of two neutralizing monoclonal antibodies, NE12 and NA8, against infection with SARS-CoV-2 in the golden Syrian hamster model (A) Design of the study. MAbs NE12 or NA8 were administered intraperitoneally (IP) at the dose of 12 or 4 mg/kg. Control animals received an anti-HIV-1 IgG1 (VRC01) at 12 mg/kg. One day later, each group of 10 animals was challenged with 10^4.5 TCID₅₀ of SARS-CoV-2 WA-1 or B.1.351 instilled intranasally (IN). The animal weight was monitored daily (days 0–3: n = 10 per group; days 4, 5: n = 5 per group) as an indicator of disease progression. Tissues were collected for virus quantification at 3 and 5 days after challenge (n = 5 per group per time point). (B and C) Body weight changes (means ± SE) from baseline in hamsters that received neutralizing mAbs at different doses or isotype control (IgG) after challenge with (B) WA-1 or (C) B.1.351 SARS-CoV-2 strains. (D–G) Viral titers in (D) nasal turbinate and (E) lung tissues at days 3 and 5 post-infection with (F) WA-1 or (G) the B.1.351 variant, as determined using an assay to quantify the TCID₅₀ of infectious virus. Individual titers and means ± SD are shown for each group. Dashed lines indicate the limit of detection of the assay. Statistical comparisons of weight changes between treatment groups were performed using a repeated-measures mixed-effects model with Tukey’s multiple-comparison test (B and C). Comparisons of the viral titers between groups were performed using two-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test. p values between groups treated with neutralizing mAbs and isotype control are indicated. p > 0.05, not significant (ns); ^∗p = 0.0381; ^∗∗∗p = 0.0009; ^∗∗∗∗p < 0.0001.

Figure 7

Therapeutic efficacy of two neutralizing mAbs, NE12 and NA8, against SARS-CoV-2 in the golden Syrian hamster model (A) Design of the study. Each group of 10 animals was first challenged with 10^4.5 TCID₅₀ of SARS-CoV-2 WA-1 or B.1.351/Beta, instilled intranasally (IN). Twenty-four hours later, the animals were injected intraperitoneally (IP) with mAbs NE12 or NA8 at the dose of 12 mg/kg. Control animals received an anti-HIV-1 IgG1 (VRC01) at 12 mg/kg. The animal weight was monitored daily as an indicator of disease progression (days 0–2: n = 10 per group; days 3–11: n = 5 per group, except for the WA-1/NE12 group (days 6–11: n = 3 due to a technical issue). Tissues were collected at day 3 after challenge for virus quantification (n = 5 per group). (B and C) Body weight changes (means ± SE) from baseline in hamsters that were challenged with SARS-CoV-2 (B) WA-1 or (C) B.1.351/Beta. (D–G) Quantification of viral titers at day 3 post-challenge in (D) nasal turbinate and (E) lung tissues of animals infected with WA-1 or in (F) nasal turbinate and (G) lung tissues of animals infected with the B.1.351/Beta variant, as determined using an assay to quantify the TCID₅₀ of infectious virus. Individual titers and means ± SD are shown for each group. Dashed lines indicate the limit of detection of the assay. Statistical comparisons of weight changes between treatment groups were performed using a repeated-measures mixed-effects model with Tukey’s multiple-comparison test. Comparisons of viral titers between the treatment groups were performed using the non-parametric Kruskal-Wallis test with a Benjamini, Krieger, and Yekutieli false discovery test. p values between groups treated with neutralizing mAbs and isotype control are indicated. p > 0.05, not significant (ns); ^∗∗p = 0.0043; ^∗∗∗∗p < 0.0001.