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Simple paired heavy- and light-chain antibody repertoire sequencing using endoplasmic reticulum microsomes.

ABSTRACT: Existing methods for paired antibody heavy- and light-chain repertoire sequencing rely on specialized equipment and are limited by their commercial availability and high costs. Here, we report a novel simple and cost-effective emulsion-based single-cell paired antibody repertoire sequencing method that employs only basic laboratory equipment. We performed a proof-of-concept using mixed mouse hybridoma cells and we also showed that our method can be used for discovery of novel antigen-specific monoclonal antibodies by sequencing human CD19+ B cell IgM and IgG repertoires isolated from peripheral whole blood before and seven days after Td (Tetanus toxoid/Diphtheria toxoid) booster immunization. We anticipate broad applicability of our method for providing insights into adaptive immune responses associated with various diseases, vaccinations, and cancer immunotherapies.

SUBMITTER: Devulapally PR

PROVIDER: S-EPMC5921987 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Paired antibody heavy-light chain sequencing

Project description:Paired antibody heavy-light chain sequencing

| PRJEB25901 | ENA

High-throughput sequencing of the paired human immunoglobulin heavy and light chain repertoire.

Project description:Each B-cell receptor consists of a pair of heavy and light chains. High-throughput sequencing can identify large numbers of heavy- and light-chain variable regions (V(H) and V(L)) in a given B-cell repertoire, but information about endogenous pairing of heavy and light chains is lost after bulk lysis of B-cell populations. Here we describe a way to retain this pairing information. In our approach, single B cells (>5 × 10(4) capacity per experiment) are deposited in a high-density microwell plate (125 pl/well) and lysed in situ. mRNA is then captured on magnetic beads, reverse transcribed and amplified by emulsion V(H):V(L) linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of V(H):V(L) pairs identified by this approach and used the method to sequence the repertoire of three human cell subsets-peripheral blood IgG(+) B cells, peripheral plasmablasts isolated after tetanus toxoid immunization and memory B cells isolated after seasonal influenza vaccination.

| S-EPMC3910347 | biostudies-literature

Paired heavy- and light-chain signatures contribute to potent SARS-CoV-2 neutralization in public antibody responses.

Project description:Understanding mechanisms of protective antibody recognition can inform vaccine and therapeutic strategies against SARS-CoV-2. We report a monoclonal antibody, 910-30, targeting the SARS-CoV-2 receptor-binding site for ACE2 as a member of a public antibody response encoded by IGHV3-53/IGHV3-66 genes. Sequence and structural analyses of 910-30 and related antibodies explore how class recognition features correlate with SARS-CoV-2 neutralization. Cryo-EM structures of 910-30 bound to the SARS-CoV-2 spike trimer reveal binding interactions and its ability to disassemble spike. Despite heavy-chain sequence similarity, biophysical analyses of IGHV3-53/3-66-encoded antibodies highlight the importance of native heavy:light pairings for ACE2-binding competition and SARS-CoV-2 neutralization. We develop paired heavy:light class sequence signatures and determine antibody precursor prevalence to be ∼1 in 44,000 human B cells, consistent with public antibody identification in several convalescent COVID-19 patients. These class signatures reveal genetic, structural, and functional immune features that are helpful in accelerating antibody-based medical interventions for SARS-CoV-2.

| S-EPMC8479507 | biostudies-literature

Reprogramming of the heavy-chain CDR3 regions of a human antibody repertoire.

Project description:B cells have been engineered ex vivo to express an HIV-1 broadly neutralizing antibody (bNAb). B cell reprograming may be scientifically and therapeutically useful, but current approaches limit B cell repertoire diversity and disrupt the organization of the heavy-chain locus. A more diverse and physiologic B cell repertoire targeting a key HIV-1 epitope could facilitate evaluation of vaccines designed to elicit bNAbs, help identify more potent and bioavailable bNAb variants, or directly enhance viral control in vivo. Here we address the challenges of generating such a repertoire by replacing the heavy-chain CDR3 (HCDR3) regions of primary human B cells. To do so, we identified and utilized an uncharacterized Cas12a ortholog that recognizes PAM motifs present in human JH genes. We also optimized the design of 200 nucleotide homology-directed repair templates (HDRT) by minimizing the required 3'-5' deletion of the HDRT-complementary strand. Using these techniques, we edited primary human B cells to express a hemagglutinin epitope tag and the HCDR3 regions of the bNAbs PG9 and PG16. Those edited with bNAb HCDR3 efficiently bound trimeric HIV-1 antigens, implying they could affinity mature in vivo in response to the same antigens. This approach generates diverse B cell repertoires recognizing a key HIV-1 neutralizing epitope.

| S-EPMC8753427 | biostudies-literature

Single cell paired heavy- and light-chain immunoglobulin sequencing of sorted Lyme disease plasmablasts

Project description:Lyme disease (Borrelia burgdorferi infection) is increasingly recognized as a significant source of morbidity world-wide. Here, we investigated B cell responses to Lyme disease through barcode-enabled single cell sequencing of activated B cells (plasmablasts) sorted from PBMCs. Bulk immunglobulin heavy-chain sequencing from this project has also been separately deposited. Additional information regarding patient characteristics and overlap with other data from the SLICE study is available upon request.

2018-12-21 | GSE114309 | GEO

Genetic variation in the immunoglobulin heavy chain locus shapes the human antibody repertoire.

Project description:Variation in the antibody response has been linked to differential outcomes in disease, and suboptimal vaccine and therapeutic responsiveness, the determinants of which have not been fully elucidated. Countering models that presume antibodies are generated largely by stochastic processes, we demonstrate that polymorphisms within the immunoglobulin heavy chain locus (IGH) impact the naive and antigen-experienced antibody repertoire, indicating that genetics predisposes individuals to mount qualitatively and quantitatively different antibody responses. We pair recently developed long-read genomic sequencing methods with antibody repertoire profiling to comprehensively resolve IGH genetic variation, including novel structural variants, single nucleotide variants, and genes and alleles. We show that IGH germline variants determine the presence and frequency of antibody genes in the expressed repertoire, including those enriched in functional elements linked to V(D)J recombination, and overlapping disease-associated variants. These results illuminate the power of leveraging IGH genetics to better understand the regulation, function, and dynamics of the antibody response in disease.

| S-EPMC10362067 | biostudies-literature

Heavy chain-only antibody genes in fish evolved to generate unique CDR3 repertoire.

Project description:In addition to conventional immunoglobulin, camelids and cartilaginous fish express a special class of antibody that consists only of heavy (H) chain (HCAbs). In the holocephalan elephantfish, there are two HCAb classes, one of which has evolved surprising features. The H-chain genes in cartilaginous fish are organized as 20-200 minigenes, or clusters, each consisting of VH, 1-3 DH, JH gene segments with one set of constant region exons. We report that HHC2 (holocephalan H-chain antibody 2) evolved from IgM H-chain clusters, but its DH gene segments have diverged considerably. The three DH in HHC2 clusters are A-rich, so that one to three potential reading frames for each DH encode lysine and arginine. All three are incorporated into the rearranged VDJ, ensuring that the ligand-binding site carries multiple basic residues, as cDNA sequences demonstrate. The electropositive character in HHC2 CDR3 is accompanied by a paucity of aromatic amino acids, the latter feature at variance to the established, interactive role of tyrosine not only in ligand-binding but generally at interfaces of protein complexes. The selection for these divergent HHC2 features challenges currently accepted ideas on what determines antibody reactivity and molecular recognition.

| S-EPMC9590256 | biostudies-literature

Inferring B cell phylogenies from paired heavy and light chain BCR sequences with Dowser.

Project description:Antibodies are vital to human immune responses and are composed of genetically variable heavy and light chains. These structures are initially expressed as B cell receptors (BCRs). BCR diversity is shaped through somatic hypermutation and selection during immune responses. This evolutionary process produces B cell clones, cells that descend from a common ancestor but differ by mutations. Phylogenetic trees inferred from BCR sequences can reconstruct the history of mutations within a clone. Until recently, BCR sequencing technologies separated heavy and light chains, but advancements in single cell sequencing now pair heavy and light chains from individual cells. However, it is unclear how these separate genes should be combined to infer B cell phylogenies. In this study, we investigated strategies for using paired heavy and light chain sequences to build phylogenetic trees. We found incorporating light chains significantly improved tree accuracy and reproducibility across all methods tested. This improvement was greater than the difference between tree building methods and persisted even when mixing bulk and single cell sequencing data. However, we also found that many phylogenetic methods estimated significantly biased branch lengths when some light chains were missing, such as when mixing single cell and bulk BCR data. This bias was eliminated using maximum likelihood methods with separate branch lengths for heavy and light chain gene partitions. Thus, we recommend using maximum likelihood methods with separate heavy and light chain partitions, especially when mixing data types. We implemented these methods in the R package Dowser: https://dowser.readthedocs.io.

| S-EPMC10592837 | biostudies-literature

Rapid quantitative analysis of monoclonal antibody heavy and light chain charge heterogeneity.

Project description:An alternative method to traditional 2-dimensional gel electrophoresis (2D-PAGE) and its application in characterizing the inherent charge heterogeneity of chromatographically isolated monoclonal antibody heavy and light chains is described. This method, referred to as ChromiCE, utilizes analytical size-exclusion chromatography (SEC), performed under reducing and denaturing conditions, followed by imaged capillary isoelectric focusing (icIEF) of the chromatographically separated heavy and light chains. Under conditions suitable for the subsequent icIEF analysis, the absolute and relative SEC elution volumes of the heavy and light chains were found to be highly pH dependent, a phenomenon that can be exploited in optimizing chromatographic separation. Compared to 2D-PAGE, the ChromiCE method substantially decreases the time and labor needed to complete the analysis, improves reproducibility, and provides fully quantitative assessment of charge heterogeneity. The ChromiCE methodology was applied to a set of diverse monoclonal antibodies to demonstrate suitability for quantitative charge variant analysis of heavy and light chains. A typical application of ChromiCE in extended characterization and stability studies of a purified antibody is shown.

| S-EPMC4966340 | biostudies-literature

The intrinsic hypermutability of antibody heavy and light chain genes decays exponentially.

Project description:Somatic hypermutation, essential for the affinity maturation of antibodies, is restricted to a small segment of DNA. The upstream boundary is sharp and is probably related to transcription initiation. However, for reasons unknown, the hypermutation domain does not encompass the whole transcription unit, notably the C-region exon. Since analysis of the downstream decay of hypermutation is obscured by sequence-dependent hot and cold spots, we describe a strategy to minimize these fluctuations by computing mutations of different sequences located at similar distances from the promoter. We pool large databases of mutated heavy and light chains and analyse the decay of mutation frequencies. We define an intrinsic decay of probability of mutation that is remarkably similar for heavy and light chains, faster than anticipated and consistent with an exponential fit. Indeed, quite apart from hot spots, the intrinsic probability of mutation at CDR1 can be almost twice that of CDR3. The analysis has mechanistic implications for current and future models of hypermutation.

| S-EPMC125579 | biostudies-literature

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