Biology subjects

Castellana, N.

Publications and source records attributed to Castellana, N..

4 recordsLinked to original sources

bioRxiv2024

Systematic benchmarking of mass spectrometry-based antibody sequencing reveals methodological biases

The circulating antibody repertoire is crucial for immune protection, holding significant immunological and biotechnological value. While bottom-up mass spectrometry (MS) is the most widely used proteomics technique for profiling the sequence diversity of circulating antibodies (Ab-seq), it has not been thoroughly benchmarked. We quantified the replicability and robustness of Ab-seq using six monoclonal antibodies with known protein sequences in 70 different combinations of concentration and oligoclonality, both with and without polyclonal serum IgG background. Each combination underwent four protease treatments and was analyzed across four experimental and three technical replicates, totaling 3,360 LC-MS/MS runs. We quantified the dependence of MS-based Ab-seq identification on antibody sequence, concentration, protease, background signal diversity, and bioinformatics setups. Integrating the data from experimental replicates, proteases, and bioinformatics tools enhanced antibody identification. De novo peptide sequencing showed similar performance to database-dependent methods for higher antibody concentrations, but de novo antibody reconstruction remains challenging. Our work provides a foundational resource for the field of MS-based antibody profiling.

immunology↗

bioRxiv2024

Serum proteomics reveals high-affinity and convergent antibodies by tracking SARS-CoV2 hybrid immunity to emerging variants of concern

The rapid spread of SARS-CoV2 and continuing impact on human health has prompted the need for effective and rapid development of monoclonal antibody therapeutics. In this study, we interrogate polyclonal antibodies in serum and B cells from whole blood of three donors with SARS-CoV2 immunity to find high-affinity anti-SARS-CoV2 antibodies to escape variants. Serum IgG antibodies were selected by affinity to the receptor-binding domain (RBD) and non-RBD sites on the spike protein of Omicron subvariant B.1.1.529 from each donor. Antibodies were analyzed by bottom-up mass spectrometry, and matched to single- and bulk-cell sequenced repertoires for each donor. Antibodies observed in serum were recombinantly expressed, and characterized to assess domain binding, cross-reactivity between different variants, and capacity to inhibit RBD binding to host protein. Donors infected with early Omicron subvariants had serum antibodies with subnanomolar affinity to RBD that show binding activity to a newer Omicron subvariant BQ.1.1. The donors also showed a convergent immune response. Serum antibodies and other single- and bulk-cell sequences were similar to publicly reported anti-SARS-CoV-2 antibodies, and characterized serum antibodies had the same variant-binding and neutralization profiles as their reported public sequence. The serum antibodies analyzed were a subset of anti-SARS-CoV2 antibodies in the B cell repertoire, which demonstrates significant dynamics between the B cells and circulating antibodies in peripheral blood.

immunology↗

bioRxiv2022

Systematic analysis of human antibody response to ebolavirus glycoprotein reveals high prevalence of neutralizing public clonotypes

Understanding the human antibody response to emerging viral pathogens is key to epidemic preparedness. As the size of the B cell response to a pathogenic virus protective antigen is undefined, we performed deep paired heavy and light chain sequencing in EBOV-GP specific memory B cells, allowing analysis of the ebolavirus-specific antibody repertoire both genetically and functionally. This approach facilitated investigation of the molecular and genetic basis for evolution of cross-reactive antibodies by elucidating germline-encoded properties of antibodies to EBOV and identification of the overlap between antibodies in the memory B-cell and serum repertoire. We identified 73 public clonotypes to EBOV, 20% of which encoded antibodies with neutralization activity and capacity to protect in vivo. This comprehensive analysis of the public and private antibody repertoire provides insight into the molecular basis of the humoral immune response to EBOV-GP, which informs vaccine design of new vaccines and improved therapeutics.

immunology↗

bioRxiv2019

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

AO_SCPLOWBSTRACTC_SCPLOWRabbits are a model for immunology studies, and monoclonal antibodies developed from rabbits have been sought after to empower immunoassays in a variety of applications. High-throughput characterization of circulating serum antibodies in response to specific antigens is highly impactful for both humoral immunology studies and antibody development. A combination of high throughput sequencing of antibody transcripts from B cells and proteomic analysis of serum antibodies, an approach referred to as immunoproteogenomics, is applied to profile the immune response of rabbits to {beta}-galactosidase (Beta-gal) in both recombinant antigen and peptide antigen immunization formats. The use of intact protein antigen resulted in observing 56.3% more heavy chains CDR3s 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 CDR3s, 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 determine antibody protein sequences missed by repertoire sequencing.

immunology↗