Imprinted antibody responses against SARS-CoV-2 Omicron sublineages

Abstract

SARS-CoV-2 Omicron sublineages carry distinct spike mutations and represent an antigenic shift resulting in escape from antibodies induced by previous infection or vaccination. We show that hybrid immunity or vaccine boosters result in potent plasma neutralizing activity against Omicron BA.1 and BA.2 and that breakthrough infections, but not vaccination-only, induce neutralizing activity 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 and BA.2 receptor-binding domains whereas Omicron primary infections elicit B cells of narrow specificity. While most clinical antibodies have reduced neutralization of Omicron, we identified an ultrapotent pan-variant antibody, that is unaffected by any Omicron lineage spike mutations and 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 dose; ID₅₀) against Wu-G614, Delta, BA.1, BA.2 and SARS-CoV S VSV pseudoviruses using plasma from subjects who were infected and vaccinated, vaccinated and experienced breakthrough infection, or vaccinated-only individuals. Vero E6-TMPRSS2 cell were used as target cells (34). Data are the geometric mean of an nlJ=lJ2 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 Table S2. B, Cross-reactivity of IgGs secreted from memory B cells obtained from infected-vaccinated individuals (left, n=11), subjects who experienced a primary infection (center, n=3) or a breakthrough infection in January-March 2022 (right, n=7) when the prevalence of Omicron BA.1/BA.2 exceeded 90% in the region where samples were obtained (Table S3). 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 antigen 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 monoclonal antibodies specific for sites Ia (S2E12), Ib (S2X324), IIa (S2X259) and IV (S309). Lack of competition is indicated as “Other”. Pie charts show cumulative frequencies of IgGs specific for the different sites among total RBD-specific 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 in nasal swabs obtained longitudinally upon BA.1 breakthrough infection up to 28 days following symptom onset (pso). E, Neutralizing activity against Wu-G614 and BA.1 S VSV pseudoviruses in nasal swabs obtained longitudinally following a negative PCR test of vaccinated-only individuals.

Figure 2:. Identification and characterization of S2X324 as a pan-variant RBD-targeted 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. 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). Two haplotypes of BA.4 S were tested: BA.4-V3G (orange dots) and BA.4-N658S (white dots). (B) Neutralization of SARS-CoV-2 VSV pseudoviruses mediated by broadly neutralizing sarbecovirus mAbs. Each symbol represents the Geometric mean of IC₅₀ values of at least two independent experiments. (C) Neutralization of SARS-CoV-2 authentic viruses by sotrovimab and S2X324 expressed as the geometric mean of IC₅₀ values (left) and the average fold change relative to neutralization of the WA1/2020 virus (right). (D) Cross-reactivity of S2X324 with sarbecovirus clades 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. 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 RBD. Selected epitope residues are labeled, and electrostatic interactions are indicated with dotted lines. (D) Superimposition of the S2X324 -bound (purple and magenta) and ACE2-bound [dark gray, PDB 6M0J (44)] 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 and BA.2 challenge.

(A-C) A-C, Dose-dependent (expressed in mg/kg of body weight) prophylactic protection of S2X324 (blue circles) vs S309 (green diamonds) hamster IgG2a (harboring hamster IgG2a constant regions) in animals infected with SARS-CoV-2 Delta evaluated 4 days post infection based on % of body weight change (A), % of lung weight vs body weight ratio (B), viral RNA load (C). (n=6 animals/dose) *, **, ***, **** p< 0.05, p< 0.01, 0.001, and 0.0001 relative to isotype control (MGH2 antibody against circumsporozoite protein of Plasmodium sporozoites), respectively (Mann-Whitney 2-tail T test). D, Quantification of viral RNA load in the lung and trachea of Syrian hamsters 4 days after intranasal infection with SARS-CoV-2 Omicron BA.2 which was preceded 1 day prior by prophylactic intraperitoneal administration of S2X324 hamster IgG2a at 5 mg/kg of body weight. **, ***, **** p< 0.01, 0.001, and 0.0001 relative to control, respectively (Kruskal-Wallis test). E-F, Quantification of replicating virus titers [50% tissue culture infectious dose (TCID₅₀)] (E) and viral RNA load (F) in the lung of Syrian hamsters 4 days after intranasal infection with SARS-CoV-2 Delta followed by therapeutic intraperitoneal administration of S2X324 hamster IgG2a (blue symbols) or N297A mutant IgG2a (purple symbols) at four different doses: 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).