Serum proteomics expands on high-affinity antibodies in immunized rabbits than deep B-cell repertoire sequencing alone

Abstract

Rabbits are a model for immunology studies, and monoclonal antibodies developed from rabbits have been highly sought after to empower immunoassays in a variety of other applications. Highthroughput characterization of circulating serum antibodies in response to specific antigens is highly impactful for both immunology studies and antibody development. A combination of high throughput sequencing of antibody transcripts from B cells and proteomic analysis of serum antibodies, referred to as immunoproteogenomics, is applied to profile the immune response of rabbits to β-galactosidase (Beta-gal) in both recombinant antigen and peptide antigen immunization formats. The use of recombinant antigen resulted in observing 56.3% more heavy chains clones in serum than immunization with peptide antigens. Additionally, sampling peripheral blood mononuclear cells (PBMCs) for B-cell repertoire sequencing at different time points throughout the immunization was found to capture 47.8%-72.8% of total proteomically observed heavy chain clones, and would serve well in replacing sequencing the B cell rich, but more difficult to access spleen or bone marrow compartments. Despite B-cell repertoire sequencing to depths of 2M to 10M reads, we found proteomic evidence supporting at least 10% of serum antibodies are still missed. Further improvements to proteomic analysis techniques would enable more precise characterization of antibodies circulating in serum and better estimates of antibodies missed by repertoire sequencing.

Progress and challenges in mass spectrometry-based analysis of antibody repertoires

“…Safonova and colleagues could show that adapting the analysis from quantification of identical antibody sequences to that of antibody clones (antibody sequences belonging to the same lineage) at least partially restored the correlation between genomics-and proteomics-based quantification [46]. Nevertheless, Bonissone and colleagues provided evidence supporting the claims by Cheung and colleagues showing that serum antigen-specific clones are not among the most abundant BCR clones as sequenced from blood, bone marrow, or spleen [42]. Follow-up studies are necessary to elucidate to what extent each lymphoid organ contributes to the blood, mucosal, and central nervous system (CNS) antibody repertoire [39,47].…”

“…Acquiring these statistics of antibody repertoire concentration dynamics is crucial as antibodies provide long-lived protection [40] and their concentration has been linked to antibody neutralization efficacy [41]. Interestingly, Bonissone and colleagues showed that, in rabbits, ~27% of heavy and ~15% of light-chain clones were exclusively observed in bone marrow or spleen, and not in the blood-borne B cell population [42]. Nevertheless, they showed that the lack of access to major ASC locales such as the bone marrow and spleen, and thus the lack of sequence coverage of the main ASC subsets responsible for the secretion of the antigen-specific antibody repertoire, may at least partly be remedied by assembling a sequence DB that encompasses BCR-seq data at several timepoints throughout immunization [42].…”

“…The use of additional (more than one) proteases can further increase peptide coverage of the CDR3 repertoire as well as the analysis of both heavy-and light-chain sequences [17,18,29,42,76]. Swaney and colleagues showed that, instead of using only one protease (trypsin), multiple proteases (trypsin, LysC, ArgC, AspN, and GluC) improved both protein identification and characterization in yeast [76].…”

Proteo-Genomic Analysis Identifies Two Major Sites of Vulnerability on Ebolavirus Glycoprotein for Neutralizing Antibodies in Convalescent Human Plasma

“…We used the proprietary proteogenomic platform Alicanto for data analysis and visualization (25). 1,512 paired heavy and light chain antibody sequences derived from antigen-sorted memory B cells were analyzed directly by Alicanto.…”

“…We recently described a proteo-genomic approach for identifying sequences of antigen-specific polyclonal antibodies in animal serum (25) and reported methods for large-scale antiviral human mAb discovery from the memory B cell repertoire (26). In this study, we characterize the circulating antibody response to ebolavirus GPs at the molecular level by identifying mAbs that are present in convalescent plasma collected from an EVD survivor (Figure 1).…”