Discovery and characterization of potent pan-variant SARS-CoV-2 neutralizing antibodies from individuals with Omicron breakthrough infection

Abstract

The SARS-CoV-2 Omicron variant evades most currently approved neutralizing antibodies (nAbs) and caused drastic decrease of plasma neutralizing activity elicited by vaccination or prior infection, urging the need for the development of pan-variant antivirals. Breakthrough infection induces a hybrid immunological response with potentially broad, potent and durable protection against variants, therefore, convalescent plasma from breakthrough infection may provide a broadened repertoire for identifying elite nAbs. We performed single-cell RNA sequencing (scRNA-seq) and BCR sequencing (scBCR-seq) of B cells from BA.1 breakthrough-infected patients who received 2 or 3 previous doses of inactivated vaccine. Elite nAbs, mainly derived from the IGHV2-5 and IGHV3-66/53 germlines, showed potent neutralizing activity across Wuhan-Hu-1, Delta, Omicron sublineages BA.1 and BA.2 at picomolar NT₅₀ values. Cryo-EM analysis revealed diverse modes of spike recognition and guides the design of cocktail therapy. A single injection of paired antibodies cocktail provided potent protection in the K18-hACE2 transgenic female mouse model of SARS-CoV-2 infection.

Fig. 1. Identification of high-confidence clonotypes by single-cell transcriptomic and BCR profiling.

a Schematic diagram of the experimental design. Single-cell RNA-seq and single-cell BCR-seq were conducted for CD19⁺ RBD⁺ B cells derived from 38 Omicron patients, followed by clonotype detection and experimental verification. b UMAP display of 277,630 B cells. Orange and blue correspond to memory B cells and naïve B cells, which are characterized by high expression of CD27/GPR183 and TCL1A/IL4R/IGHD, respectively. c Filter criteria for the selection of high-confidence clonotypes. d Distribution of frequency, memory B-cell number, and SHM for 286 high-confidence candidates. SHM somatic hypermutation rate. e VDJ gene rearrangements for heavy chains of the 286 high-confidence candidates. f VDJ gene rearrangements for light chains of the 286 high-confidence candidates.

Fig. 2. Discovery and characterization of isolated neutralizing antibodies.

a Evaluation of the binding of 286 antibodies to SARS-CoV-2 variants, measured by ELISA. The OD₄₅₀ values of each mAb against WT S protein, Omicron BA.1.1 S protein, and Omicron BA.1.1 RBD are shown. The balls in purple indicate 119 mAbs selected for further study (left). Evaluation of the blocking abilities of 119 antibodies against SARS-CoV-2 variants, measured by ELISA. The IC₅₀ values of each mAb against WT RBD, Delta RBD, Omicron BA.1.1 RBD are shown. The red balls refer to 44 potent neutralizing mAbs for further experiments. The ball size represents the number of antibodies. Sixty-four mAbs had no blocking ability against any variants, represented by the large gray ball (the IC₅₀ values shown as NA) (middle). Evaluation of the neutralizing activity of 44 antibodies against SARS-CoV-2 variants. The NT₅₀ values of each mAb against the WT strain, Delta strain, and Omicron BA.2 are shown. The balls in orange indicate 19 mAbs selected for further study (right). b Neutralization curves for the 19 selected antibodies against WT virus or VOCs. (n = 3 technical replicates). Data are presented as mean values ± SD. All experiments were performed in duplicate. c Neutralization potency of the 19 selected antibodies against different SARS-CoV-2 strains.

Fig. 3. Evaluation of elite antibodies against Omicron BA.2.12.1 and BA.4 & 5.

a Details of the 19 selected antibodies. The seven groups of antibodies are shown in different colors, and the asterisks indicate representative antibodies. b Binding kinetics of the seven candidate antibodies (TH003, TH027, TH048, TH236, TH257, TH272, and TH281) with the S protein of the Omicron BA.1 and BA.4/5 variants were measured by the SPR assay. c Omicron BA.2.12.1 or BA.4 & 5 SARS-CoV-2-S pseudotyped virus neutralization. (n = 3 technical replicates). Data are presented as mean values ± SD.

Fig. 4. Cryo-EM structures of the Omicron BA.4/5 spike in complex with elite nAbs.

a The structures of the BA.4/5 S trimer in complex with TH003, TH027, TH236, TH272, TH132, and TH281 are displayed in cartoon representation. TH003, TH027, TH236, TH272, TH132, and TH281 are colored pink, purple blue, purple, green, yellow, and orange, respectively, and the S trimer is colored gray. Side view (top), top view (bottom). b The epitopes of antibodies are displayed in surface representation. The colors are the same as in a, and the hACE2 interface is circled in red.

Fig. 5. Rational pairings of noncompeting antibodies show promising therapeutic potential for antibody therapy against emerging VOC.

a The overall structure of TH027-BA.4/5 RBD. The TH027 heavy chain (colored deep blue) and light chain (colored yellow) are displayed in cartoon representation. The BA.4/5 RBD is colored gray and displayed in surface representation (left). The epitope of TH027 is shown in surface representation. The only residue V445, which contacts both the heavy chain and light chain, is colored pink (right). b The interface of TH027-BA.4/5 RBD. Hydrogen bond interactions are shown as yellow dashed lines. Salt bridge interactions are shown as green dashed lines. The residues are shown in stick representation with the same colors as in a. c The overall structure of TH132-BA.4/5 RBD. The TH132 heavy chain (colored orange) and light chain (colored green) are displayed in cartoon representation. BA.4/5 RBD is colored in gray and displayed in surface representation (left). The epitope of TH132 is shown in surface representation. The only residue that contacts both the heavy chain and light chain, N417, is colored yellow (right). d The interface of TH132-BA.4/5 RBD. Hydrogen bond interactions are shown as yellow dashed lines. The residues are shown in stick representation with identical colors to c. e The structure of the BA.4/5 S trimer in complex with the TH027 + TH132 cocktail, and neutralization potency of the TH027 + TH132 cocktail against different sublineages of Omicron strains. S trimer is displayed in gray surface representation. RBD is colored pink. TH027 + TH132 are displayed in cartoon representation. TH027 is colored in deep blue. TH132 is colored yellow. Side view (left). Top view (right). f The structure of the BA.4/5 S trimer in complex with the TH272 + TH281 cocktail, and neutralization potency of the TH272 + TH281 cocktail against different sublineages of Omicron strains. S trimer is displayed in gray surface representation. RBD is colored pink. TH272 + TH281 are displayed in cartoon representation. TH272 is colored green. TH281 is colored orange. Side view (left). Top view (right).

Fig. 6. Evaluation of in vivo efficacy of prophylactic and therapeutic TH027 + TH132 cocktail.

a Experimental design for TH027 + TH132 cocktail efficacy evaluation in K18-hACE2 transgenic mice. b Mice were intraperitoneally injected with a single injection of 5 or 20 mg/kg of TH027, TH132, or TH027 + TH132 cocktail 2 h before or after an intranasal challenge of 1 × 10⁴ TCID₅₀ BA.5 virus. Body weight of mice were monitored daily until 6 days post-infection (d.p.i.). (PBS and TH027 + TH132/20 mpk on before 2 d.p.i.: n = 6 mice, other groups: n = 5 mice. PBS and TH027 + TH132/20 mpk on after 2 d.p.i.: n = 3 mice, other groups: n = 2 mice). c Viral RNA levels in the lung tissue were measured by qRT–PCR assay at 2 d.p.i. (n = 3 mice). d Viral titers in the lung tissue were measured by fluorescence focus assay at 2 d.p.i. (n = 3 mice). e Histopathological and immunofluorescence analyses of TH027, TH132, or TH027 + TH132 cocktail treated or untreated mice infected with Omicron BA.5. Representative images of lung sections stained by HE in antibodies prophylactic and therapeutic groups at 6 d.p.i. The images of bronchioles, blood vessels, and alveoli of the lungs are indicated by black dotted squares with numbers 1, 2, and 3, respectively. The images on the right (bars = 50 μm) are enlarged regions in the dashed boxes of the left images (bars = 500 μm). Red, blue, green, yellow, black, and purple arrows indicate necrotic epithelial cells in the alveoli, dead cell debris in the bronchioles, severe bleeding in the alveoli, a large amount of protein mucus in the lungs, mononuclear cell infiltrations in the blood vessels and inflammatory infiltrations in the lungs, respectively. Representative images of the N protein expression in the lungs at 6 d.p.i. (bars = 50 μm). Three mice were sampled in each group and 5 sections from each animal were used for histology analysis. All data are presented as mean values ± SD. Statistical differences were determined by two-way ANOVA in c, d. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.