Summary Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from CAPRISA-donor CAP256; each antibody contained the protruding tyrosine-sulfated, anionic antigen-binding loop (CDR H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30–38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation, an important vaccine insight.

Molecular understanding of serological immunity to influenza has been confounded by the complexity of the polyclonal antibody response in humans. Here we used high-resolution proteomics analysis of immunoglobulin (referred to as Ig-seq) coupled with high-throughput sequencing of transcripts encoding B cell receptors (BCR-seq) to quantitatively determine the antibody repertoire at the individual clonotype level in the sera of young adults before and after vaccination with trivalent seasonal influenza vaccine. The serum repertoire comprised between 40 and 147 clonotypes that were specific to each of the three monovalent components of the trivalent influenza vaccine, with boosted pre-existing clonotypes accounting for ~60% of the response. An unexpectedly high fraction of serum antibodies recognized both the H1 and H3 monovalent vaccines. Recombinant versions of these H1 + H3 cross-reactive antibodies showed broad binding to hemagglutinins (HAs) from previously circulating virus strains; several of these antibodies, which were prevalent in the serum of multiple donors, recognized the same conserved epitope in the HA head domain. Although the HA-head-specific H1 + H3 antibodies did not show neutralization activity in vitro, they protected mice against infection with the H1N1 and H3N2 virus strains when administered before or after challenge. Collectively, our data reveal unanticipated insights regarding the serological response to influenza vaccination and raise questions about the added benefits of using a quadrivalent vaccine instead of a trivalent vaccine.

Isolation of antigen-specific monoclonal antibodies (mAbs) and antibody fragments relies on high-throughput screening of immortalized B cells or recombinant antibody libraries. We bypassed the screening step by using high-throughput DNA sequencing and bioinformatic analysis to mine antibody variable region (V)-gene repertoires from bone marrow plasma cells (BMPC) of immunized mice. BMPCs, which cannot be immortalized, produce the vast majority of circulating antibodies. We found that the V-gene repertoire of BMPCs becomes highly polarized after immunization, with the most abundant sequences represented at frequencies between approximately 1% and >10% of the total repertoire. We paired the most abundant variable heavy (V(H)) and variable light (V(L)) genes based on their relative frequencies, reconstructed them using automated gene synthesis, and expressed recombinant antibodies in bacteria or mammalian cells. Antibodies generated in this manner from six mice, each immunized with one of three antigens were overwhelmingly antigen specific (21/27 or 78%). Those generated from a mouse with high serum titers had nanomolar binding affinities.

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)...

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)...

Obtaining full-length antibody heavy- and light-chain variable regions from individual B cells at scale remains a challenging problem. Here we use high-throughput single-cell B-cell receptor sequencing (scBCR-seq) to obtain accurately paired full-length variable regions in a massively parallel fashion. We sequenced more than 250,000 B cells from rat, mouse and human repertoires to characterize their lineages and expansion. In addition, we immunized rats with chicken ovalbumin and profiled antigen-reactive B cells from lymph nodes of immunized animals. The scBCR-seq data recovered 81% ( n = 56/69) of B-cell lineages identified from hybridomas generated from the same set of B cells subjected to scBCR-seq. Importantly, scBCR-seq identified an additional 710 candidate lineages not recovered as hybridomas. We synthesized, expressed and tested 93 clones from the identified lineages and found that 99% ( n = 92/93) of the clones were antigen-reactive. Our results establish scBCR-seq as a powerful tool for antibody discovery.

Immunodeficient mice reconstituted with human hematopoietic stem cells enable the in vivo study of human hematopoiesis. In particular, NOD-scid-IL2Rγnull engrafted mice have been shown to have reasonable levels of T and B cell repopulation and can mount T-cell dependent responses; however, antigen-specific B-cell responses in this model are generally poor. We explored whether developmental defects in the immunoglobulin gene repertoire might be partly responsible for the low level of antibody responses in this model. Roche 454 sequencing was used to obtain over 685,000 reads from cDNA encoding immunoglobulin heavy (IGH) and light (IGK and IGL) genes isolated from immature, naïve, or total splenic B cells in engrafted NOD-scid-IL2Rγnull mice, and compared with over 940,000 reads from peripheral B cells of two healthy volunteers. We find that while naïve B-cell repertoires in humanized mice are chiefly indistinguishable from those in human blood B cells, and display highly correlated patterns of immunoglobulin gene segment use, the complementarity-determining region H3 (CDR-H3) repertoires are nevertheless extremely diverse and are specific for each individual. Despite this diversity, preferential DH-JH pairings repeatedly occur within the CDR-H3 interval that are strikingly similar across all repertoires examined, implying a genetic constraint imposed on repertoire generation. Moreover, CDR-H3 length, charged amino-acid content, and hydropathy are indistinguishable between humans and humanized mice, with no evidence of global autoimmune signatures. Importantly, however, a statistically greater usage of the inherently autoreactive IGHV4-34 and IGKV4-1 genes was observed in the newly formed immature B cells relative to naïve B or total splenic B cells in the humanized mice, a finding consistent with the deletion of autoreactive B cells in humans. Overall, our results provide evidence that key features of the primary repertoire are shaped by genetic factors intrinsic to human B cells and are principally unaltered by differences between mouse and human stromal microenvironments.

The vast initial diversity of the antibody repertoire is generated centrally by means of a complex series of V(D)J gene rearrangement events, variation in the site of gene segment joining, and TdT catalyzed N-region addition. Although the diversity is great, close inspection has revealed distinct and unique characteristics in the antibody repertoires expressed by different B cell developmental subsets. In order to illustrate our approach to repertoire analysis, we present an in-depth comparison of V(D)J gene usage, hydrophobicity, length, D H reading frame, and amino acid usage between heavy chain repertoires expressed by immature, transitional, mature, memory IgD + , memory IgD − , and plasmacytes isolated from the blood of a single individual. Our results support the view that in both human and mouse, the H chain repertoires expressed by individual, developmental B cell subsets appear to differ in sequence content. Sequencing of unsorted B cells from the blood is thus likely to yield an incomplete or compressed view of what is actually happening in the immune response of the individual. Our findings support the view that studies designed to correlate repertoire expression with diseases of immune function will likely require deep sequencing of B cells sorted by subset.