Brandon J. DeKosky

Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies

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 donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (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 and potency 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. These data provide important insights relevant to HIV-1 vaccine development.

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

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.

Identification and characterization of the constituent human serum antibodies elicited by vaccination.

Significance Most vaccines confer immunity by eliciting long-term production of antibodies that bind to and neutralize the vaccine antigen. Remarkably, very little is known regarding the identities, sequence diversity, relative concentrations, or binding functionalities of the mAbs that comprise the serum repertoire elicited by vaccination. Here, we have delineated the constituent antibodies of the human serum IgG repertoire after vaccination and examined their relationship to the antibody V gene repertoire encoded by circulating B cells. The results detail the molecular composition and characteristics of the vaccine-specific serum antibody repertoire and demonstrate differences between the end-point response (the serum antibodies) and the peripheral B cells responding to the vaccine. 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 Kd ∼ 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 Kd). Collectively, our results reveal the nature and dynamics of the serological response to vaccination with direct implications for vaccine design and evaluation.

Molecular-level analysis of the serum antibody repertoire in young adults before and after seasonal influenza vaccination

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.

Induction of HIV Neutralizing Antibody Lineages in Mice with Diverse Precursor Repertoires

The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2(∗)02 segment to undergo normal V(D)J recombination and, thereby, leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies, IGHV1-2(∗)02-rearranging mice, which also express a VRC01-antibody precursor light chain, can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages.

Ultra-high-throughput sequencing of the immune receptor repertoire from millions of lymphocytes

High-throughput sequencing of the variable domains of immune receptors (antibodies and T cell receptors (TCRs)) is of key importance in the understanding of adaptive immune responses in health and disease. However, the sequencing of both immune receptor chains (VH+VL or TCRβ/δ+TCRα/γ) at the single-cell level for typical samples containing >104 lymphocytes is problematic, because immune receptors comprise two polypeptide chains that are encoded by separate mRNAs. Here we present a technology that allows rapid and low-cost determination of a paired immune receptor repertoire from millions of cells with high precision (>97%). Flow focusing is used to encapsulate single cells in emulsions containing magnetic beads for mRNA capture. The mRNA transcripts are then reverse-transcribed, physically linked to their partners by overlap extension PCR, and interrogated by high-throughput paired-end Illumina sequencing. This protocol describes the construction and operation of the flow-focusing device in detail, as well as the bioinformatics pipeline used to interpret the data. The entire procedure can be performed by a single researcher in under 12 h of effort per sample.

Large-scale sequence and structural comparisons of human naive and antigen-experienced antibody repertoires

Significance We applied a very recently developed experimental strategy for high-throughput sequencing of paired antibody heavy and light chains along with large-scale computational structural modeling to delineate features of the human antibody repertoire at unprecedented scale. Comparison of antibody repertoires encoded by peripheral naive and memory B cells revealed (i) preferential enrichment or depletion of specific germline gene combinations for heavy- and light-chain variable regions and (ii) enhanced positive charges, higher solvent-accessible surface area, and greater hydrophobicity at antigen-binding regions of mature antibodies. The data presented in this report provide fundamental new insights regarding the biological features of antibody selection and maturation and establish a benchmark for future studies of antibody responses to disease or to vaccination. Elucidating how antigen exposure and selection shape the human antibody repertoire is fundamental to our understanding of B-cell immunity. We sequenced the paired heavy- and light-chain variable regions (VH and VL, respectively) from large populations of single B cells combined with computational modeling of antibody structures to evaluate sequence and structural features of human antibody repertoires at unprecedented depth. Analysis of a dataset comprising 55,000 antibody clusters from CD19+CD20+CD27− IgM-naive B cells, >120,000 antibody clusters from CD19+CD20+CD27+ antigen–experienced B cells, and >2,000 RosettaAntibody-predicted structural models across three healthy donors led to a number of key findings: (i) VH and VL gene sequences pair in a combinatorial fashion without detectable pairing restrictions at the population level; (ii) certain VH:VL gene pairs were significantly enriched or depleted in the antigen-experienced repertoire relative to the naive repertoire; (iii) antigen selection increased antibody paratope net charge and solvent-accessible surface area; and (iv) public heavy-chain third complementarity-determining region (CDR-H3) antibodies in the antigen-experienced repertoire showed signs of convergent paired light-chain genetic signatures, including shared light-chain third complementarity-determining region (CDR-L3) amino acid sequences and/or Vκ,λ–Jκ,λ genes. The data reported here address several longstanding questions regarding antibody repertoire selection and development and provide a benchmark for future repertoire-scale analyses of antibody responses to vaccination and disease.

Facile Discovery of a Diverse Panel of Anti-Ebola Virus Antibodies by Immune Repertoire Mining

The ongoing evolution of Ebolaviruses poses significant challenges to the development of immunodiagnostics for detecting emergent viral variants. There is a critical need for the discovery of monoclonal antibodies with distinct affinities and specificities for different Ebolaviruses. We developed an efficient technology for the rapid discovery of a plethora of antigen-specific monoclonal antibodies from immunized animals by mining the VH:VL paired antibody repertoire encoded by highly expanded B cells in the draining popliteal lymph node (PLN). This approach requires neither screening nor selection for antigen-binding. Specifically we show that mouse immunization with Ebola VLPs gives rise to a highly polarized antibody repertoire in CD138+ antibody-secreting cells within the PLN. All highly expanded antibody clones (7/7 distinct clones/animal) were expressed recombinantly, and shown to recognize the VLPs used for immunization. Using this approach we obtained diverse panels of antibodies including: (i) antibodies with high affinity towards GP; (ii) antibodies which bound Ebola VLP Kissidougou-C15, the strain circulating in the recent West African outbreak; (iii) non-GP binding antibodies that recognize wild type Sudan or Bundibugyo viruses that have 39% and 37% sequence divergence from Ebola virus, respectively and (iv) antibodies to the Reston virus GP for which no antibodies have been reported.

Low CD21 expression defines a population of recent germinal center graduates primed for plasma cell differentiation

A distinct population of B cells that respond to vaccination serve as potential plasma cell precursors. Plasma cell precursors Memory B cells are critical players in the rapid secondary immune response to pathogens; however, little is known about B cell subsets that are phenotypically different from classical memory populations. Now, Lau et al. report that CD21lo B cells in healthy humans are predisposed to differentiate into long-lived plasma cells. CD21lo B cells were induced during the peak of germinal center (GC) activity after influenza vaccination but formed distinct clades from memory B cells and plasmablasts. These cells were primed for plasma cell differentiation but resistant to further GC differentiation. These data suggest that CD21lo cells are a distinct population of memory B cells that may contribute to plasma cell formation. In this study, we report that antigen-specific CD19+CD27+CD21lo (CD21lo) B cells are transiently induced 14 to 28 days after immunization, at the time germinal centers (GCs) peak. Although clonally related to memory B cells and plasmablasts, CD21lo cells form distinct clades within phylogenetic trees based on accumulated variable gene mutations, supporting exit from active GCs. CD21lo cells express a transcriptional program, suggesting that they are primed for plasma cell differentiation and are refractory to GC differentiation, although they do not spontaneously secrete antibody. In addition, CD21lo cells differentially express multiple cell surface markers and have elevated intracellular levels of Blimp-1 and T-bet protein compared with memory B cells. Together, these data support a model in which CD21lo cells are recent GC graduates that represent a distinct population from CD27+ classical memory cells, are refractory to GC reentry, and are predisposed to differentiate into long-lived plasma cells.

Functional interrogation and mining of natively paired human V H :V L antibody repertoires

We present a technology to screen millions of B cells for natively paired human antibody repertoires. Libraries of natively paired, variable region heavy and light (VH:VL) amplicons are expressed in a yeast display platform that is optimized for human Fab surface expression. Using our method we identify HIV-1 broadly neutralizing antibodies (bNAbs) from an HIV-1 slow progressor and high-affinity neutralizing antibodies against Ebola virus glycoprotein and influenza hemagglutinin.