The emergence of SARS-CoV-2 Omicron variants posed a significant challenge due to their immune escape capabilities. While a third dose of the BNT162b2 mRNA vaccine significantly improves neutralization, the underlying mechanism remains unclear. The spike protein, particularly its RBD, is a primary target of neutralizing antibodies. Omicron variants harbor numerous RBD mutations, leading to reduced neutralization by antibodies induced after two doses of the BNT162b2 vaccine. Although bivalent vaccines are being developed, most individuals have received only the monovalent vaccine. Previous studies showed increased somatic hypermutation (SHM) in virus-specific antibodies after a third dose of the BNT162b2 vaccine, but the process of generating Omicron-neutralizing antibodies remains poorly understood. This study aimed to analyze the chronological B-cell receptor (BCR) repertoires of BNT162b2 vaccinees to trace the development of Omicron variant-neutralizing antibodies.

Extensive research has focused on SARS-CoV-2 variants and their mutations. The spike protein's RBD is crucial for viral entry and a major target for neutralizing antibodies. Omicron variants' multiple RBD mutations result in significantly reduced neutralization by antibodies elicited after two BNT162b2 vaccine doses. The development of bivalent vaccines addresses this issue, but the majority of the population has only received the monovalent vaccine. The third dose of the BNT162b2 vaccine demonstrates improved neutralization against Omicron variants, associated with increased SHM in antibodies. However, the precise mechanisms underlying the generation of Omicron-neutralizing antibodies following repeated exposure to the ancestral spike protein remain unclear. This study builds upon previous findings demonstrating increased SHM after the third dose, aiming to elucidate the mechanism of Omicron variant-neutralizing antibody generation.

Forty-one healthcare workers who received three doses of the BNT162b2 mRNA vaccine were enrolled. Blood samples were collected six times: pre-vaccination and at various time points after each dose. Plasma antibody levels against ancestral and Omicron RBDs were measured by ELISA. Single-chain variable fragment (scFv) phage display libraries were constructed from the sixth blood samples of six selected participants. These libraries were used to select B.A.1 RBD-reactive clones based on bioinformatics analysis and phage ELISA. The selected clones were characterized, including sequencing of their BCR heavy chain (HC) clonotypes, analysis of somatic hypermutations (SHM), and assessment of their binding affinities to different RBDs using ELISA. Next-generation sequencing (NGS) was used to analyze BCR repertoires at each time point, focusing on the evolution of BA.1 RBD-reactive BCRs. Recombinant scFv-hFc-HA fusion proteins were expressed and their affinity for ancestral and Omicron RBDs was determined by ELISA. Microneutralization assays were performed to assess the neutralization capacity of the selected clones against various SARS-CoV-2 variants. The study also investigated the expansion of the BCR repertoire's reactivity to Omicron subvariants, focusing on SHM's role in this process. Statistical analyses were conducted to compare the frequency and SHM levels of BCR sequences across different time points.

Plasma levels of IgA and IgG against the ancestral RBD increased significantly after the second dose, but a third dose was necessary for elevated levels of antibodies reactive to BA.1 and BQ.1 RBDs. Nine BA.1 RBD-reactive scFv clones were selected. One prominent cluster of scFv clones (27-60) shared IGHV3-33/3-66 and IGHJ6 genes. Analysis of BCR HC sequences revealed a dramatic increase in the average number of SHMs after the third dose, along with increased diversity. The 27-60 clonotype showed increased affinity for both ancestral and BA.1 RBDs due to SHM. Back-mutation of SHMs reduced the affinity. Analysis of other selected clones (35-15, 35-46, 43-09, and 43-34) showed similar patterns of SHM accumulation and increased affinity for the ancestral RBD. Interestingly, the diversification of BCRs through SHM expanded their reactivity to RBDs of Omicron subvariants. Clones generated after the third dose showed significant reactivity to multiple Omicron subvariants, including BQ.1.1, XBB.1.5, and XBB.1.16. The findings suggest that the accumulation of SHM in antibodies is a key mechanism for broadening the BCR repertoire's specificity to escape viral immune escape.

The study's findings demonstrate that the third dose of the ancestral SARS-CoV-2 mRNA vaccine induces the accumulation of SHMs in antibodies, leading to increased affinity and broader reactivity against Omicron variants. The expansion of BCR repertoire specificity via SHM represents a crucial protective immune mechanism against viral immune escape. This highlights the importance of booster vaccination, even with monovalent vaccines, in providing broader protection against emerging variants. The identified BCR clonotypes and their SHMs could provide valuable insights for designing next-generation vaccines and therapeutic antibodies.

The study conclusively shows that the third dose of the ancestral SARS-CoV-2 vaccine triggers the generation of Omicron-neutralizing antibodies through the accumulation of somatic hypermutations. This process broadens the BCR repertoire's specificity, acting as a protective mechanism against viral immune escape. The findings underscore the importance of booster doses and offer valuable information for future vaccine and therapeutic antibody development.

The study focused on a specific population (healthcare workers) and a single vaccine (BNT162b2), limiting the generalizability of the findings. The relatively small number of selected clones for detailed analysis might not fully represent the overall BCR repertoire dynamics. Further research is needed to validate these findings in larger, more diverse populations and with other vaccine types. The long-term durability of the observed antibody response needs further investigation.