The emergence of SARS-CoV-2 and its variants, particularly Omicron, poses a significant challenge to existing vaccines and therapeutics. Omicron's numerous mutations, especially within the receptor-binding domain (RBD) of the spike (S) protein, lead to widespread immune evasion. While mRNA vaccines offer some protection, their effectiveness against emerging VOCs like BA.5, BQ.1.1, and XBB is significantly reduced. Concerns also exist regarding the long-term sequelae of COVID-19. While mRNA therapy is a crucial countermeasure, many approved mAbs and cocktail therapies lose effectiveness against variants like B.1.1.7 and B.1.617.2. Breakthrough infections, however, can elicit more potent and broad antibody responses. This study leveraged single-cell sequencing to isolate and characterize potent neutralizing antibodies from individuals experiencing Omicron breakthrough infections after vaccination, aiming to identify broadly effective therapeutic agents.

The literature review highlights the limitations of existing SARS-CoV-2 vaccines and antibody therapies in the face of emerging variants, specifically Omicron and its sublineages. Studies showed that Omicron’s mutations cause significant immune evasion, reducing the efficacy of vaccines and previously developed monoclonal antibodies (mAbs). The review emphasizes the need for broadly neutralizing antibodies that can overcome this immune evasion, mentioning successful examples of antibody isolation and cocktail development. It supports the hypothesis that breakthrough infections might yield potent, broadly neutralizing antibodies, prompting the investigation of convalescent serum for antibody discovery.

The study employed a multi-step approach. First, single-cell RNA sequencing (scRNA-seq) and single-cell BCR sequencing were performed on B cells from 35 Omicron breakthrough infection patients. Antigen-binding B cells were enriched using biotinylated RBD magnetic beads. scRNA-seq data was analyzed using the Atlas of Blood Cells (ABC) database to identify memory B cells (MBCs). Clonotypes were identified based on heavy and light chain VDJ sequences, filtering for high frequency, IGH1 expression (but not IGHC), SHM rates >2%, and the presence of at least one MBC. A total of 386 antibodies were expressed in HEK293 cells. ELISA was used to screen for broadly binding and blocking antibodies against various SARS-CoV-2 variants. Neutralization assays were performed to assess the potency of selected antibodies against different variants. Surface plasmon resonance (SPR) was used to determine binding affinities. Cryo-EM combined with X-ray crystallography determined the structures of Omicron BA.4/5 spike in complex with selected antibodies. Epitope mapping and sequence analysis were performed to understand the mechanism of neutralization. Finally, in vivo efficacy was evaluated using a K18-hACE2 transgenic mouse model. Statistical analysis using nonparametric Wilcoxon tests was conducted.

The researchers identified 19 potent neutralizing monoclonal antibodies (mAbs) from the Omicron breakthrough infection cohort. These antibodies showed broad neutralizing activity against various SARS-CoV-2 variants, including Wuhan-Hu-1, Delta, BA.1, and BA.2. While neutralization potency against Omicron variants was lower compared to Wuhan-Hu-1 and Delta, it remained within the clinically relevant range. Cryo-EM structural analysis revealed the binding modes of these antibodies, revealing interactions with various epitopes on the RBD, some overlapping but others distinct. Analysis showed that the antibodies belonged to distinct groups based on germline origin and HCDR3 sequences. SPR analysis revealed high binding affinities (KD values from 10⁻⁹ to 10⁻¹⁰ M for 6 out of 7 representative antibodies). Further investigation demonstrated that a rationally designed cocktail of seven elite antibodies exhibited potent neutralizing activity against Omicron BA.2.12.1, BA.4, and BA.5 variants. Structural studies revealed that specific residues on the antibodies were involved in key hydrophobic and hydrogen bond interactions with the RBD, explaining their broad neutralization capabilities. The study also demonstrated that some antibody cocktails maintained good neutralizing effects on BF.7, BQ.1.1, and XBB variants. In vivo studies showed that the TH027 + TH132 cocktail effectively reduced viral load and improved body weight in K18-hACE2 transgenic mice infected with Omicron BA.5, both prophylactically and therapeutically. The TH027 + TH132 cocktail showed improved efficacy compared to the individual antibodies.

The findings address the urgent need for broadly neutralizing antibodies against SARS-CoV-2 variants. The identification of potent pan-variant neutralizing antibodies from individuals with Omicron breakthrough infections demonstrates the potential of leveraging immune responses after infection. The structural insights reveal crucial interactions contributing to broad neutralization, paving the way for rational design of improved therapeutic antibodies. The effectiveness of antibody cocktails further strengthens the potential for therapeutic applications. The in vivo data confirms the clinical relevance of these antibodies, supporting further development for COVID-19 treatment.

This study successfully identified and characterized potent, pan-variant neutralizing antibodies derived from Omicron breakthrough infections. The structural analysis provided insights into the mechanisms of broad neutralization. The developed antibody cocktails showed promising neutralizing activity against multiple SARS-CoV-2 VOCs, including emerging variants. In vivo studies demonstrated the efficacy of the TH027 + TH132 cocktail in reducing viral load and improving disease outcomes in a mouse model. This research lays a foundation for developing effective therapeutics against SARS-CoV-2 and its future variants.

The study used a relatively small sample size of Omicron breakthrough infection patients. The in vivo study was conducted in a mouse model; further research is necessary to validate these findings in humans. The long-term durability and efficacy of the identified antibodies require further investigation. The generalizability of these findings to other populations needs to be determined.