Most vaccines confer protection via the elicitation of serum antibodies, yet more than 100 y after the discovery of antibodies, the molecular composition of the human serum antibody repertoire to an antigen remains unknown. Using high-resolution liquid chromatography tandem MS proteomic analyses of serum antibodies coupled with next-generation sequencing of the V gene repertoire in peripheral B cells, we have delineated the human serum IgG and B-cell receptor repertoires following tetanus toxoid (TT) booster vaccination. We show that the TT + serum IgG repertoire comprises ∼100 antibody clonotypes, with three clonotypes accounting for >40% of the response. All 13 recombinant IgGs examined bound to vaccine antigen with K d ∼ 10 −8 -10 −10 M. Five of 13 IgGs recognized the same linear epitope on TT, occluding the binding site used by the toxin for cell entry, suggesting a possible explanation for the mechanism of protection conferred by the vaccine. Importantly, only a small fraction (<5%) of peripheral blood plasmablast clonotypes (CD3 − CD14 − CD19 + CD27 ++ CD38 ++ CD20 − TT + ) at the peak of the response (day 7), and an even smaller fraction of memory B cells, were found to encode antibodies that could be detected in the serological memory response 9 mo postvaccination. This suggests that only a small fraction of responding peripheral B cells give rise to the bone marrow long-lived plasma cells responsible for the production of biologically relevant amounts of vaccine-specific antibodies (near or above the K d ). Collectively, our results reveal the nature and dynamics of the serological response to vaccination with direct implications for vaccine design and evaluation.B-cell repertoire | proteomics M ost approved vaccines confer protection against infectious diseases by the induction of long-lived plasma cells (LLPCs), which secrete antibodies that serve to neutralize and opsonize the pathogen for many years or decades (1-3). Additionally, the generation of memory B cells (mBCs) provides both a mechanism for the rapid synthesis of affinity matured, antigenspecific antibodies following rechallenge and a means to diversify the humoral immune response to confer protection against rapidly evolving viruses or bacteria (4). Although some vaccines elicit antibody titers that remain virtually constant for many decades, for others, including the tetanus toxoid (TT) vaccine, antibody titers wane monotonically over time (5). Booster immunization triggers the rapid expansion and differentiation of cognate B cells, generating antigen-specific plasmablasts that peak in concentration in peripheral blood after 6-7 d and subsequently rapidly decline to nearly undetectable levels (6, 7). Some, but not all, of these peak-wave plasmablasts migrate to specialized niches overwhelmingly located in the bone marrow (BM) and survive as LLPCs (8), which constitute the major source of all classes of Ig in the serum (9).The establishment of serological memory following either primary or booster vaccination is not understood well (10-14)...

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 (VH and VL) 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 × 104 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 VH:VL linkage PCR. The linked transcripts are analyzed by Illumina high-throughput sequencing. We validated the fidelity of VH:VL 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.

We have developed and validated a methodology for determining the antibody composition of the polyclonal serum response after immunization. Pepsin-digested serum IgGs were subjected to standard antigen-affinity chromatography, and resulting elution, wash, and flow-through fractions were analyzed by bottom-up, liquid chromatography-high-resolution tandem mass spectrometry. Identification of individual monoclonal antibodies required the generation of a database of IgG variable gene (V-gene) sequences constructed by NextGen sequencing of mature B cells. Antibody V-gene sequences are characterized by short complementarity determining regions (CDRs) of high diversity adjacent to framework regions shared across thousands of IgGs, greatly complicating the identification of antigen-specific IgGs from proteomically observed peptides. By mapping peptides marking unique V H CDRH3 sequences, we identified a set of V-genes heavily enriched in the affinity chromatography elution, constituting the serum polyclonal response. After booster immunization in a rabbit, we find that the antigenspecific serum immune response is oligoclonal, comprising antibodies encoding 34 different CDRH3s that group into 30 distinct antibody V H clonotypes. Of these 34 CDRH3s, 12 account for ∼60% of the antigen-specific CDRH3 peptide mass spectral counts. For comparison, antibodies with 18 different CDRH3s (12 clonotypes) were represented in the antigen-specific IgG fraction from an unimmunized rabbit that fortuitously displayed a moderate titer for BSA. Proteomically identified antibodies were synthesized and shown to display subnanomolar affinities. The ability to deconvolute the polyclonal serum response is likely to be of key importance for analyzing antibody responses after vaccination and for more completely understanding adaptive immune responses in health and disease.antibody proteomics | antibody repertoire | serum immunoprofiling | B-cell response | humoral response T he first Nobel Prize in Medicine was awarded to Emil von Behring, who in collaboration with Kitasato Shibasaburo and Paul Ehrlich discovered serum antitoxins (1, 2). Remarkably, after more than 100 y of intense research in immunology, little is known about the clonality, relative concentrations, and binding properties of the monoclonal antibodies that constitute the antigen-specific Ig pool in serum.At steady state, circulating antibodies are produced by terminally differentiated B lymphocytes (plasma cells) within the bone marrow, and thus cannot be accessed in living individuals (3). Although recent single B-cell cloning methods (4, 5) have led to the identification of peripheral antigen-specific B memory and/or antibody-secreting cells (plasmablasts), it is generally unknown whether the Igs encoded by peripheral blood B cells correspond to the antibodies present in circulation and especially whether they are present at physiologically relevant levels (i.e., at serum concentrations above K D corresponding to >1 μg/mL for an average affinity of individual antibodies of 5 nM)...

Glutaraldehyde has been used for several decades as an effective crosslinking agent for many applications including sample fixation for microscopy, enzyme and cell immobilization, and stabilization of protein crystals. Despite of its common use as a crosslinking agent, the mechanism and chemistry involved in glutaraldehyde crosslinking reaction is not yet fully understood. Here we describe feasibility study and results obtained from a new approach to investigate the process of protein crystals stabilization by glutaraldehyde crosslinking. It involves exposure of a model protein crystal (Lysozyme) to glutaraldehyde in alkaline or acidic pH for different incubation periods and reaction arrest by medium exchange with crystallization medium to remove unbound glutaraldehyde. The crystals were subsequently incubated in diluted buffer affecting dissolution of un-crosslinked crystals. Samples from the resulting solution were subjected to protein composition analysis by gel electrophoresis and mass spectroscopy while crosslinked, dissolution resistant crystals were subjected to high resolution X-ray structural analysis. Data from gel electrophoresis indicated that the crosslinking process starts at specific preferable crosslinking site by lysozyme dimer formation, for both acidic and alkaline pH values. These dimer formations were followed by trimer and tetramer formations leading eventually to dissolution resistant crystals. The crosslinking initiation site and the end products obtained from glutaraldehyde crosslinking in both pH ranges resulted from reactions between lysine residues of neighboring protein molecules and the polymeric form of glutaraldehyde. Reaction rate was much faster at alkaline pH. Different reaction end products, indicating different reaction mechanisms, were identified for crosslinking taking place under alkaline or acidic conditions.

Monoclonal antibodies (mAbs) are a crucial asset for human health and modern medicine, however, the repeated administration of mAbs can be highly immunogenic. Drug immunogenicity manifests in the generation of anti-drug antibodies (ADAs), and some mAbs show immunogenicity in up to 70% of patients. ADAs can alter a drug’s pharmacokinetic and pharmacodynamic properties, reducing drug efficacy. In more severe cases, ADAs can neutralize the drug’s therapeutic effects or cause severe adverse events to the patient. While some contributing factors to ADA formation are known, the molecular mechanisms of how therapeutic mAbs elicit ADAs are not completely clear. Accurate ADA detection is necessary to provide clinicians with sufficient information for patient monitoring and clinical intervention. However, ADA assays present unique challenges because both the analyte and antigen are antibodies, so most assays are cumbersome, costly, time consuming, and lack standardization. This review will discuss aspects related to ADA formation following mAb drug administration. First, we will provide an overview of the prevalence of ADA formation and the available diagnostic tools for their detection. Next, we will review studies that support possible molecular mechanisms causing the formation of ADA. Finally, we will summarize recent approaches used to decrease the propensity of mAbs to induce ADAs.

High-throughput sequencing (HTS) of immunoglobulin (B-cell receptor, antibody) and T-cell receptor repertoires has increased dramatically since the technique was introduced in 2009 (1–3). This experimental approach explores the maturation of the adaptive immune system and its response to antigens, pathogens, and disease conditions in exquisite detail. It holds significant promise for diagnostic and therapy-guiding applications. New technology often spreads rapidly, sometimes more rapidly than the understanding of how to make the products of that technology reliable, reproducible, or usable by others. As complex technologies have developed, scientific communities have come together to adopt common standards, protocols, and policies for generating and sharing data sets, such as the MIAME protocols developed for microarray experiments. The Adaptive Immune Receptor Repertoire (AIRR) Community formed in 2015 to address similar issues for HTS data of immune repertoires. The purpose of this perspective is to provide an overview of the AIRR Community’s founding principles and present the progress that the AIRR Community has made in developing standards of practice and data sharing protocols. Finally, and most important, we invite all interested parties to join this effort to facilitate sharing and use of these powerful data sets (join@airr-community.org).

Characterizing the in vivo dynamics of the polyclonal antibody repertoire in serum, such as that which might arise in response to stimulation with an antigen, is difficult due to the presence of many highly similar immunoglobulin proteins, each specified by distinct B lymphocytes. These challenges have precluded the use of conventional mass spectrometry for antibody identification based on peptide mass spectral matches to a genomic reference database. Recently, progress has been made using bottom-up analysis of serum antibodies by nanoflow liquid chromatography/high-resolution tandem mass spectrometry combined with a sample-specific antibody sequence database generated by high-throughput sequencing of individual B cell immunoglobulin variable domains (V genes). Here, we describe how intrinsic features of antibody primary structure, most notably the interspersed segments of variable and conserved amino acid sequences, generate recurring patterns in the corresponding peptide mass spectra of V gene peptides, greatly complicating the assignment of correct sequences to mass spectral data. We show that the standard method of decoy-based error modeling fails to account for the error introduced by these highly similar sequences, leading to a significant underestimation of the false discovery rate. Because of these effects, antibody-derived peptide mass spectra require increased stringency in their interpretation. The use of filters based on the mean precursor ion mass accuracy of peptide-spectrum matches is shown to be particularly effective in distinguishing between “true” and “false” identifications. These findings highlight important caveats associated with the use of standard database search and error-modeling methods with nonstandard data sets and custom sequence databases.

The ensemble of antibodies found in serum and secretions represents the key adaptive component of B-cell mediated humoral immunity. The antibody repertoire is shaped by the historical record of exposure to exogenous factors such as pathogens and vaccines, as well as by endogenous host-intrinsic factors such as genetics, self-antigens, and age. Thanks to very recent technology advancements it is now becoming possible to identify and quantify the individual antibodies comprising the serological repertoire. In parallel, the advent of high throughput methods for antigen and immunosignature discovery opens up unprecedented opportunities to transform our understanding of numerous key questions in adaptive humoral immunity, including the nature and dynamics of serological memory, the role of polyspecific antibodies in health and disease and how protective responses to infections or vaccine challenge arise. Additionally, these technologies also hold great promise for therapeutic antibody and biomarker discovery in a variety of settings