Imprinted antibody responses against SARS-CoV-2 Omicron sublineages

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron sublineages carry distinct spike mutations resulting in escape from antibodies induced by previous infection or vaccination. We show that hybrid immunity or vaccine boosters elicit plasma-neutralizing antibodies against Omicron BA.1, BA.2, BA.2.12.1, and BA.4/5, and that breakthrough infections, but not vaccination alone, induce neutralizing antibodies in the nasal mucosa. Consistent with immunological imprinting, most antibodies derived from memory B cells or plasma cells of Omicron breakthrough cases cross-react with the Wuhan-Hu-1, BA.1, BA.2, and BA.4/5 receptor-binding domains, whereas Omicron primary infections elicit B cells of narrow specificity up to 6 months after infection. Although most clinical antibodies have reduced neutralization of Omicron, we identified an ultrapotent pan-variant-neutralizing antibody that is a strong candidate for clinical development.

Figure 1.. Evaluation of plasma, memory and mucosal antibody responses upon Omicron breakthrough infections in humans.

A, Pairwise neutralizing activity (half-maximum inhibitory dilution; ID₅₀) against Wu-G614, Delta, BA.1, BA.2, BA.2.12.1, BA.4/5 and SARS-CoV S VSV pseudoviruses using plasma from subjects who were infected and vaccinated, vaccinated and experienced breakthrough infection, or vaccinated-only individuals. VeroE6-TMPRSS2 cells were used as target cells (93). Data are the geometric mean of an n = 2 technical replicate and have been performed in at least 2 biologically independent experiments. GMTs are shown with a color-matched bar (and reported in Table S1) with fold change compared to Wu-G614 indicated above it. Demographics of enrolled donors are provided in Data S1. B, Cross-reactivity of IgGs secreted from memory B cells obtained from infected-vaccinated individuals (n=11), primary SARS-CoV-2 infection (n=3 samples collected at 1–3 months and n=2 samples collected at 6–7 months) or breakthrough cases (n=7 samples collected at 1–3 months and n=4 samples collected at 4–6 months) occurring in January-March 2022 when the prevalence of Omicron BA.1/BA.2 exceeded 90% in the region where samples were obtained (Fig. S2). Each dot represents a well containing oligoclonal B cell supernatant screened for the presence of IgGs binding to the SARS-CoV-2 Wuhan-Hu-1 and BA.1 RBDs (top) or to the SARS-CoV-2 Wuhan-Hu-1 and SARS-CoV RBDs (bottom) using ELISA. Red dots indicate inhibition of the interaction with ACE2 (using Wuhan-Hu-1 target antigen) as determined in a separate assay. The percentages are expressed relative to the total of positive hits against any of the antigens tested. Numbers of positive hits relative to individual donors are shown in Fig. S3. C, Frequency analysis of site-specific IgG antibodies derived from memory B cells. RBD sites targeted by IgG derived from memory B cells were defined by a blockade-of-binding assay using mAbs specific for sites Ia (S2E12), Ib (S2X324), IIa (S2X259), IV (S309; parent of sotrovimab) and V (S2H97). Hybrid sites Ia/Ib, Ia/IIa, Ib/IIa, Ib/IV, IIa/V and IV/V were defined by competition with the two corresponding mAbs. Hybrid sites exhibiting competition with more than 2 mAbs are indicated as “Multiple”. Lack of competition is indicated as “Other”. Pie charts show cumulative frequencies of IgGs specific for the different sites among total RBD-directed IgG antibodies (left) and those inhibiting binding of RBD to human ACE2 (right) in 11 infected-vaccinated individuals or 7 breakthrough cases. D, Neutralizing activity against Wu-G614 and BA.1 S VSV pseudoviruses determined from nasal swabs obtained longitudinally upon BA.1 breakthrough infection up to 185 days following positive PCR test (post (+) PCR). E, Neutralizing activity against Wu-G614 and BA.1 S VSV pseudoviruses from nasal swabs obtained longitudinally following a negative PCR (post (−) PCR)_ test of vaccinated-only individuals.

Figure 2:. Identification and characterization of S2X324 as a pan-variant RBD-directed mAb.

(A) mAb-mediated neutralization of BA.1, BA.2, BA.3, BA.4, BA.5, BA.2.12.1, and BA.2.75 S VSV pseudoviruses. Two haplotypes of BA.4 S were tested: BA.4-V3G (orange dots) and BA.4-N658S (white dots) and the IC₅₀ values reported in the text are the averages of both haplotypes. The potency of each mAb or mAb cocktail is represented by their IC₅₀ (top, geometric mean ± SD) or fold change relative to neutralization of the Wuhan-Hu-1 (D614) pseudovirus (bottom, average ± SD). *, not determined. (B) Neutralization of SARS-CoV-2 variant S VSV pseudoviruses mediated by broadly neutralizing sarbecovirus mAbs. Each symbol represents the GMT of at least two independent experiments. (C) Neutralizing activity (left) and fold change relative to WA-1/2020 (right) of S2X324 and sotrovimab against SARS-CoV2 Omicron BA.1, BA.2, BA.4, BA.5, and BA.2.12.1 authentic viruses using VeroE6-TMPRSS2 target cells. Data are representative of at least 2 biological independent experiments. Neutralization of Omicron BA.1 by sotrovimab refers to previously published data (3). (D) Cross-reactivity of S2X324 with sarbecovirus clade 1a and 1b RBDs analyzed by ELISA. (E) Preincubation of serial dilutions of S2X324 or S2E12 with the SARS-CoV-2 RBD prevents binding to the immobilized human ACE2 ectodomain in ELISA. PG-GX: Pangolin-Guangxi, PG-GD: Pangolin-Guangdong. Error bars indicate standard deviation between replicates. (F) S2X324-mediated S₁-shedding from cell surface–expressed SARS-CoV-2 S as determined by flow cytometry. S2E12 and S2X259 were used as positive controls whereas S2M11 and S309 were used as negative controls.

Figure 3:. Structural characterization of the S2X324 pan-variant mAb.

(A) Cryo-EM structure viewed along two orthogonal orientations of the prefusion SARS-CoV-2 Omicron BA.1 S ectodomain trimer with three S2X324 Fab fragments bound. SARS-CoV-2 S protomers are colored light blue, pink, and gold. S2X324 heavy chain and light chain variable domains are colored purple and magenta, respectively. Glycans are rendered as blue spheres. (B) Ribbon diagram of the S2X324-bound SARS-CoV-2 RBD. The N343 glycan is rendered as blue spheres. (C) Zoomed-in view of the contacts formed between S2X324 and the SARS-CoV-2 BA.1 RBD. Selected epitope residues are labeled, and electrostatic interactions are indicated with dotted lines. A few of the escape mutants identified are colored turquoise. (D) Superimposition of the S2X324 -bound (purple and magenta) and ACE2-bound [dark gray, PDB 6M0J (94)] SARS-CoV-2 RBD (light blue) structures showing steric overlap. The N343 glycan is rendered as blue spheres.

Figure 4.. S2X324 protects hamsters against SARS-CoV-2 Delta, BA.2 and BA.5 challenge.

A-C, Dose-dependent (expressed in mg of mAb/kg of body weight) prophylactic protection of S2X324 (blue circles) and S309 (green diamonds) hamster IgG2a (harboring hamster IgG2a constant regions) administered to animals one day before infection with SARS-CoV-2 Delta and evaluated 4 days post infection based on fraction of body weight change (A), replicating viral titers [50% tissue culture infectious dose (TCID₅₀)] (B) and viral RNA load (C). (n=6 animals/dose) *, **, ***, **** p< 0.05, p< 0.01, 0.001, and 0.0001 relative to isotype control (MGH2 mAb against circumsporozoite protein of Plasmodium sporozoites), respectively (Kruskal-Wallis test followed by Dunn’s multiple comparison test). D, Quantification of viral RNA loads in the lung and trachea of Syrian hamsters 4 days after intranasal infection with SARS-CoV-2 Omicron BA.2, which was preceded one day prior by prophylactic intraperitoneal administration of S2X324 hamster IgG2a at 5 mg/kg of body weight. *** p<0.001 relative to control, respectively (Mann-Whitney 2-tail T test). E-F, Quantification of replicating virus titers (TCID₅₀) (E) and viral RNA load (F) in the lung of Syrian hamsters 4 days after intranasal infection with SARS-CoV-2 Omicron BA.5, which was preceded 1 day prior by prophylactic intraperitoneal administration of S309 or S2X324 human IgG1 (HuS309 and HuS2X324) at 5 mg/kg of body weight. G-I, Dose-dependent protection in animals 4 days post infection with SARS-CoV-2 Delta by therapeutic intraperitoneal administration of S2X324 hamster IgG2a (blue symbols) or the S2X324 N297A mutant IgG2a (purple symbols) one day later at 5, 2, 0.5 or 0.1 mg/kg of body weight. *, **, ***, **** p< 0.05, p< 0.01, 0.001, and 0.0001 relative to control, respectively (Mann-Whitney 2-tail T test).