Characteristics of rabbit hapten-specific and germline-based BCR repertoires following repeated immunization

The rabbit is well known for producing diverse antibodies against various antigens including small molecules such as drugs and toxins, due to a robust immune response. Elucidating how hapten repeated immunization shapes the rabbit B cell receptor (BCR) repertoire is crucial to understanding rabbit immune response to small molecules and assisting rare antibody discovery/engineering. In this study, we enriched and sequenced chloramphenicol (CAP)-specific rabbit B cells following repeated immunization, and analyzed both CAP-specific repertoires combined with the structure and affinity features of V1S69/V1S37 germline-based BCRs. The length of rabbit complementarity-determining region 3 of heavy chain (CDRH3) increased after hapten immunization. Repeated immunization significantly reduced the diversity of CAP-specific rabbit BCR clonotypes, and changed the frequency of VDJ usage and the type of V(D)J recombination. The average number of mutations among VL is notably higher than that of VH genes in rabbits, however, they are both not changed along with repeated immunization. Moreover, repeated immunization resulted in an increase surface charge and a decrease in solvent accessible surface area, leading to improvement in the stability of the most abundant V1S69/V1S37 germline-based BCR, along with an affinity increase from an IC 50 of 898.2 ng mL −1 at the 1st immunization to 4.16 ng mL −1 at the 6th immunization. The study provides a benchmark for rabbit repertoire-scale analyses and offers a method for antibody discovery of small molecules.