Temporal dynamics of the serological compartment of rabbit antibody repertoires

Abstract

Abstract Understanding the serum antibody repertoire is an essential first step in improving vaccine design, understanding autoimmune diseases, and discovering drug candidates. Next-generation sequencing (NGS) of peripheral blood mononuclear cells is a common approach to approximate the repertoire; however it is a poor proxy for circulating antibodies. Sampling serum antibodies with mass spectrometry provides orthogonal data - directly querying the repertoire of interest. In this study, we integrated next-generation sequencing of the B-cell repertoire with mass spectrometry-based proteomic analysis of circulating antibodies in a hyperimmunized New Zealand white rabbit. During a 14 week immunization with 4 boosts, we sampled the B-cell repertoire of blood at multiple time points, as well as the bone marrow and spleen at week 14. At week 14, serum antibodies were enriched for immunogen-specificity using affinity chromatography and subjected to tandem mass spectrometry analysis. We mapped the tandem mass spectra to the antibody sequences in the repertoire to derive a set of clonal lineages and antibodies that are target specific and abundant in serum. We then tracked those lineages across time points and tissues throughout the immunization. Finally, we sought to determine the fraction of antigen-specific antibodies present in serum but absent from the B-cell derived repertoires. We employed spectral networking to identify novel clonal lineages, and estimate that at least 1/3 of the abundant clones in serum are missing from the repertoire.