Functional Enrichment and Analysis of Antigen-Specific Memory B Cell Antibody Repertoires in PBMCs

Waltari, Eric; McGeever, Aaron; Friedland, Natalia; Kim, Peter; McCutcheon, Krista

doi:10.3389/fimmu.2019.01452

Cited by 15 publications

(21 citation statements)

References 29 publications

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“…Accordingly, we obtained 8-fold more unique VH sequences from stimulated PBMCs with a greater representation of IgG over IgM clonal families compared to unstimulated PBMCs (Table S2). Compared to previously described healthy BCR repertoire data (Waltari et al, 2019), VH1-69, VH3-30, VH3-30-3, VH4-34, VH4-39 and VH4-59 were the most dominant across both unstimulated and stimulated PBMC VH families in patient 013 (average >= 5% of repertoire; Figure S10).…”

Section: Lineage Analysis Reveals Memory Origin and Divergent Evoluticontrasting

confidence: 48%

“…Given its greater junctional diversity compared to light chain, we focused our analysis on the heavy chain repertoire, which is sufficient to identify clonal relationships (Zhou & Kleinstein, 2019). We have recently shown that PBMC stimulation in a polyclonal, BCR-independent manner can selectively expand antigen-specific memory B cells (Waltari, McGeever, Friedland, Kim, & McCutcheon, 2019). Accordingly, we obtained 8-fold more unique VH sequences from stimulated PBMCs with a greater representation of IgG over IgM clonal families compared to unstimulated PBMCs (Table S2).…”

Section: Lineage Analysis Reveals Memory Origin and Divergent Evolutimentioning

confidence: 99%

“…Plasmablast-derived variable heavy or light chain sequences (Zanini et al, 2018) were synthesized as gene fragments (Genewiz, San Francisco, CA; Integrated DNA Technologies, Coralville, IA) to include at least a 15 basepair overlap with the 5' signal sequence and 3' constant region of our human IgG1, kappa or lambda expression vectors described elsewhere (Waltari et al, 2019). For pilot ELISAs and neutralization assays using crude IgG-containing supernatant, paired heavy and light chain plasmids for each mAb were expressed in Expi293F cells (Cat# A14527, ThermoFisher Scientific, Waltham, MA) in a 96-well format.…”

Section: Monoclonal Antibodiesmentioning

confidence: 99%

“…For pilot ELISAs and neutralization assays using crude IgG-containing supernatant, paired heavy and light chain plasmids for each mAb were expressed in Expi293F cells (Cat# A14527, ThermoFisher Scientific, Waltham, MA) in a 96-well format. IgG levels were quantified by ELISA as described (Waltari et al, 2019). Antibodies with crude IgG expression levels < 0.5 ng/mL were excluded from further characterization.…”

Section: Monoclonal Antibodiesmentioning

confidence: 99%

“…The cells were resuspended in 1 mL of growth media and filtered through a 5 ml polystyrene tube with a cell strainer cap (Thomas Scientific, Swedesboro, NJ). One half of the PBMCs were transferred to the T25 flask with feeder cells and B cell stimulation media as described previously (Waltari et al, 2019) and the other half was spun down in a 1.5 ml Eppendorf tube at 8000 rpm for 5 min, resuspended in 600 µl RLT (Qiagen, Hilden, Germany) + beta-mercaptoethanol, allowed to lyse for 5 min, snap frozen on dry ice and stored at -80°C until RNA purification with the Qiagen AllPrep RNA/DNA kit (Qiagen). Immunoglobulin amplicon preparation, sequencing and BCR analysis were previously described (Waltari et al, 2019).…”

Section: Preparation Of Pbmcs For Bcr Repertoire Analysismentioning

confidence: 99%

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Functional characterization and lineage analysis of broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

Durham

Agrawal

Waltari

et al. 2019

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Eliciting broadly neutralizing antibodies (bNAbs) against the four dengue virus serotypes (DENV1-4) that are spreading into new territories is an important goal of vaccine design. To delineate bNAb targets, we characterized 28 monoclonal antibodies belonging to expanded and hypermutated clonal families identified by transcriptomic analysis of single plasmablasts from DENV-infected individuals. Among these, we identified two somatically related bNAbs that potently neutralized DENV1-4. Mutagenesis studies revealed that the major recognition determinants of these bNAbs are in E protein domain I, distinct from the only known class of human bNAbs against flaviviruses with a well-defined epitope. B cell repertoire analysis from acute-phase peripheral blood suggested a memory origin and divergent somatic hypermutation pathways for these bNAbs, and a limited number of mutations was sufficient for neutralizing activity. Our study suggests multiple B cell evolutionary pathways leading to DENV bNAbs targeting a novel epitope that can be exploited for vaccine design. E monomeric subunits within the dimer. A subset of these E dimer epitope (EDE)-specific bNAbs potently neutralize not only DENV1-4, but also ZIKV, owing to the high conservation of the EDE, which overlaps the prM binding site on E ( Barba-Spaeth et al., 2016;Dejnirattisai et al., 2015;Rouvinski et al., 2015). The exciting discovery of the EDE class of bNAbs highlights the potential for an epitope-focused flavivirus vaccine strategy.As multiple specificities are likely required to provide maximum coverage of diverse circulating viral variants (Bell et al., 2019;Doria-Rose et al., 2012;Goo, Jalalian-Lechak, Richardson, & Overbaugh, 2012;Katzelnick et al., 2015; Keeffe et al., 2018; Kong et al., 2015), in this study, we aimed to define novel sites on the flavivirus E protein that can be targeted by bNAbs. By characterizing 28 monoclonal antibodies from the plasmablasts of two DENV-infected individuals, we identified J8 and J9, clonally related bNAbs that neutralized DENV1-4 in the low picomolar range. The major recognition determinants for J8 and J9 were in E protein DI, distinct from previously characterized bNAbs. Analysis of the corresponding B cell repertoire revealed divergent evolution of J8 and J9, suggesting multiple evolutionary pathways to generate bNAbs within this lineage. Our work identifies both viral and host determinants of the development of DENV bNAbs that can guide immunogen design and evaluation. Results Identification of cross-reactive neutralizing antibodies from clonally expanded plasmablasts of DENV-infected individualsWe previously profiled the single-cell transcriptomics of peripheral blood mononuclear cells (PBMCs) from six dengue patients and four healthy individuals (Zanini et al., 2018). In two DENV-infected patients (013 and 020), we identified 15 clonal families comprising a total of 38 unique paired heavy (VH) and light (VL) chain IgG1 plasmablast sequences, some of which were hypermutated (1.67% to 10.77% for VH, 0.67% to 7.22% for...

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Section: Lineage Analysis Reveals Memory Origin and Divergent Evoluticontrasting

confidence: 48%

Section: Lineage Analysis Reveals Memory Origin and Divergent Evolutimentioning

confidence: 99%

Section: Monoclonal Antibodiesmentioning

confidence: 99%

Section: Monoclonal Antibodiesmentioning

confidence: 99%

Section: Preparation Of Pbmcs For Bcr Repertoire Analysismentioning

confidence: 99%

See 3 more Smart Citations

Functional characterization and lineage analysis of broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

Durham

Agrawal

Waltari

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

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Analysis of Antigen‐Specific Human Memory B Cell Populations Based on In Vitro Polyclonal Stimulation

et al. 2020

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Antigen-specific memory B cell (MBC) populations mediate the rapid, strong, and high-affinity secondary antibody responses that play a key role in combating infection and generating protective responses to vaccination. Recently, cell staining with fluorochrome-labeled antigens together with sequencing methods such as Drop-seq and CITE-seq have provided information on the specificity, phenotype, and transcriptome of single MBCs. However, characterization of MBCs at the level of antigen-reactive populations remains an important tool for assessing an individual's B cell immunity and responses to antigen exposure. This is readily performed using a long-established method based on in vitro polyclonal stimulation of MBCs to induce division and differentiation into antibody-secreting cells (ASCs). Post-stimulation antigen-specific measurement of the MBC-derived ASCs (or the secreted antibodies) indicates the size of precursor MBC populations. Additional information about the character of antigen-reactive MBC populations is provided by analysis of MBC-derived antibodies of particular specificities for binding avidity and functionality. This article outlines a simple and reliable strategy for efficient in vitro MBC stimulation and use of the ELISpot assay as a post-stimulation readout to determine the size of antigen-specific MBC populations. Other applications of the in vitro stimulation technique for MBC analysis are discussed. The following protocols are included.

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Deep Mining of Human Antibody Repertoires: Concepts, Methodologies, and Applications

Tian

et al. 2020

Small Methods

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of V-J genes in the immunoglobulin light chains variable regions (V L), during the early stages of B cell maturation in the bone marrow; [1] 2) junctional diversity resulting from exonuclease trimmings and the random addition of palindrome (P) or non-template encoded (N) nucleotides in the process of rearrangement; [2] and 3) B cell proliferation after antigen stimulation in order to produce high-affinity antibodies and eliminate the antigen through subsequent class switch recombination (CSR) and somatic hypermutation (SHM). [3,4] It is estimated that there are over 10 13 BCRs in the human antibody repertoire. [5] This astronomical size prevents a global analysis of the antibody repertoire using conventional DNA sequencing. However, advancements in highthroughput sequencing (HTS) for Ig-Seq have improved the ability to characterize the antibody repertoire on a large scale. [6] Over a decade of development, a variety of new technologies and approaches have improved Ig-Seq methods, resulting in improved understanding of B-cell immunology and accumulation of massive antibody sequence data. In this review, an overview of Ig-Seq and advanced methodologies applied to HTS of antibody repertoires will be discussed. We will describe the wide applications of Ig-Seq in basic and clinical immunology, and strategies for functional antibody discovery from antibody repertoires. Finally, we will provide our perspective on the future directions and challenges in human antibody repertoire deep mining. 2. Overview of Ig-Seq Pipeline Although multiple strategies have been used for studying antibody repertoires, each strategy follows a similar workflow including sample collection, library preparation and sequencing, data cleaning, sequence alignment, and high-level processing (Figure 1). For sample collection, peripheral blood mononuclear cells (PBMCs) and total B cells have been generally used because of their accessibility. [9-11] However, because antibody repertoire properties are divergent between antigen-specific B cells and plasma cells, [12] specific B cell populations or B cells located in targeted tissues should be enriched for more precise studies. Considering that as few as 2% of total B cells are circulating in The ability of the human adaptive immune system to respond to antigens relies upon the tremendous diversity of T cell receptors (TCR) and B cell receptors (BCR). The entirety of an individual's BCRs, often referred to as an antibody repertoire, shapes the humoral immune system. Therefore, technologies to identify and characterize antibody repertoires are critical for understanding fundamental aspects of the development and maintenance of the humoral immune system. Recently, innovative methodologies and technologies devoted to high-throughput sequencing of antibody repertoires (Ig-Seq) have broadened the understanding of humoral immunity. This review provides an overview of the Ig-Seq pipeline from sample collection, library preparation, and sequencing, to data cleaning, sequence alignment, and highlevel processing....

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Functional Enrichment and Analysis of Antigen-Specific Memory B Cell Antibody Repertoires in PBMCs

Cited by 15 publications

References 29 publications

Functional characterization and lineage analysis of broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

Functional characterization and lineage analysis of broadly neutralizing human antibodies against dengue virus identified by single B cell transcriptomics

Analysis of Antigen‐Specific Human Memory B Cell Populations Based on In Vitro Polyclonal Stimulation

Deep Mining of Human Antibody Repertoires: Concepts, Methodologies, and Applications

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