The rapid spread of SARS-CoV-2 necessitates understanding the qualitative aspects of the immune response, particularly neutralizing antibody activity and the duration of protective immunity. Previous studies show that SARS-CoV-2 infection leads to IgG production and neutralizing antibodies targeting the receptor-binding domain (RBD) of the spike protein. The initial response strength correlates with disease severity, and antibody levels decline over time. However, antibody affinity maturation continues for months after infection, improving the memory B-cell (MBC) compartment. COVID-19 mRNA vaccines offer high protection, but antibody responses wane, requiring boosting. The emergence of SARS-CoV-2 variants of concern (VOCs) that evade antibody responses underscores the need for durable protection. Individuals with hybrid immunity (prior infection followed by vaccination) develop higher antibody titers and broader neutralization against VOCs compared to those vaccinated only. This study aimed to investigate the evolution of the B-cell response following infection and vaccination using high-throughput mAb isolation, deep IgG repertoire sequencing, and mAb lineage tracing.

Extensive research has explored the humoral immune response to SARS-CoV-2, focusing on neutralizing antibody activity and the duration of protection. Studies demonstrate that SARS-CoV-2 infection elicits a rapid IgG response and neutralizing antibodies primarily targeting the receptor-binding domain (RBD) of the spike protein. The strength of this early response is linked to disease severity, with milder symptoms often associated with lower antibody levels. Following viral replication control, serum antibody levels gradually decline as short-lived antibody-producing plasma cells cease production. However, antibody affinity maturation within germinal centers continues for several months post-infection, resulting in a higher-quality memory B-cell (MBC) compartment. Numerous studies have characterized the immune response elicited by COVID-19 vaccines, particularly mRNA vaccines. While these vaccines offer strong protection against severe disease, antibody responses wane over time, necessitating booster doses to maintain protection against symptomatic disease. The emergence of various VOCs, such as Delta and Omicron, which exhibit varying degrees of immune evasion, necessitates further investigation into the durability and breadth of immune protection.

This study employed a high-throughput mAb isolation platform to obtain 459 spike-binding mAbs from two individuals who had recovered from SARS-CoV-2 infection and received a single dose of the mRNA-1273 vaccine approximately five months later. Longitudinal samples were collected at multiple time points: acute infection, convalescent, pre-vaccination, and post-vaccination. The mAbs were characterized genetically (germline gene usage, clonal origin, somatic hypermutation) and functionally (neutralization activity against various SARS-CoV-2 variants). Deep IgG repertoire sequencing and mAb lineage tracing were used to analyze B-cell repertoire diversity and evolution over time. Serum IgG titers against SARS-CoV-2 and HCoV-HKU1 were analyzed using ELISA. Neutralizing antibody titers were determined using a spike-pseudotyped MLV neutralization assay and a VSV-SARS-CoV-2 pseudovirus neutralization assay. Binding profiles of mAbs were assessed using biolayer interferometry. Phylogenetic trees were constructed to analyze antibody lineage evolution. Statistical analysis included Mann-Whitney U test and Wilcoxon signed-rank test.

The study found that prior SARS-CoV-2 infection significantly increased antibody binding and neutralizing titers upon vaccination. The 459 isolated spike-specific mAbs used a broad range of IGHV genes targeting all spike subdomains (RBD, NTD, S2), indicating a polyclonal response. Thirty-one mAbs neutralized one or more SARS-CoV-2 variants, including four that potently neutralized the Omicron BA.2.75 variant. Lineage tracing revealed that vaccination recalled a highly polyclonal MBC repertoire, with increased somatic hypermutation (SHM) observed in lineages traced from acute infection to post-vaccination. The majority of HCoV-HKU1 cross-reactive mAbs were isolated at the acute infection timepoint and showed higher SHM levels than SARS-CoV-2-specific mAbs, suggesting that prior infection with endemic coronaviruses boosted pre-existing cross-reactive MBCs. The study demonstrated that the vaccine-induced recall response engaged a highly polyclonal B cell repertoire, including several neutralizing antibody lineages. For most lineages, a decrease in traceable sequences was observed during the convalescent timepoint compared to the acute timepoint. Several S-specific lineages were present at both acute infection and post-vaccination, indicating distinct differentiation fates for infection-induced B cells. The SHM levels of mAbs isolated from MBCs at the pre-vaccination timepoint were similar to their variants traced at the post-vaccination timepoint, suggesting that affinity maturation occurred before vaccination.

This study provides a detailed analysis of the B-cell response to SARS-CoV-2 infection and subsequent mRNA vaccination in convalescent individuals. The findings underscore the polyclonal nature of the humoral immune response and demonstrate that vaccination efficiently expands a broad repertoire of affinity-matured memory B cells, contributing to the potent antibody responses observed in individuals with hybrid immunity. The identification of mAbs that neutralize Omicron variants highlights the ability of the immune system to generate antibodies against conserved epitopes despite viral evolution. The results suggest that the type and interval between exposures influence the B-cell immunodominance hierarchy. The limitations of sampling only the circulating blood compartment and the lack of later sampling timepoints should be considered when interpreting the results. Future research should investigate the role of tissue-resident MBCs and the extent to which circulating MBCs are recruited back into germinal centers upon vaccine boosting.

This study comprehensively demonstrates that vaccination of SARS-CoV-2 convalescent individuals expands a broad repertoire of affinity-matured antibody lineages, contributing to the robust antibody response observed in hybrid immunity. The identification of neutralizing antibodies targeting Omicron variants highlights the system's ability to adapt to evolving viral epitopes. Future research should focus on further characterizing the role of tissue-resident memory B cells and the dynamics of germinal center recruitment upon boosting.

The study's limitations include the small sample size (two individuals) and the focus on circulating blood MBCs, potentially underestimating the overall diversity of the SARS-CoV-2-specific antibody response. The lack of later sampling timepoints prevented the investigation of whether infection-induced spike-specific MBCs were recruited back to germinal centers upon vaccine boosting. The study did not explore all aspects of the immune response such as T cell involvement and other aspects of the humoral immune response.