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1.
Nat Med ; 22(12): 1456-1464, 2016 12.
Article in English | MEDLINE | ID: mdl-27820605

ABSTRACT

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.


Subject(s)
Antibodies, Viral/immunology , Immunoglobulin G/immunology , Influenza Vaccines/therapeutic use , Influenza, Human/prevention & control , Orthomyxoviridae/immunology , Adult , Animals , B-Lymphocytes/immunology , Chromatography, Liquid , Cross Reactions , Epitopes , Female , Hemagglutinin Glycoproteins, Influenza Virus/immunology , High-Throughput Nucleotide Sequencing , Humans , Immunogenicity, Vaccine , Influenza A Virus, H1N1 Subtype/immunology , Influenza A Virus, H3N2 Subtype/immunology , Male , Mice , RNA, Messenger/genetics , Receptors, Antigen, B-Cell/genetics , Sequence Analysis, RNA , Tandem Mass Spectrometry , Young Adult
2.
Proc Natl Acad Sci U S A ; 113(19): E2636-45, 2016 May 10.
Article in English | MEDLINE | ID: mdl-27114511

ABSTRACT

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.


Subject(s)
Antibodies/chemistry , Antibodies/immunology , Antigen-Antibody Complex/chemistry , Antigen-Antibody Complex/immunology , High-Throughput Nucleotide Sequencing/methods , Sequence Alignment/methods , Amino Acid Sequence , Antibodies/genetics , Antigen-Antibody Complex/genetics , Base Sequence , Computer Simulation , High-Throughput Screening Assays/methods , Humans , Models, Chemical , Models, Genetic , Models, Immunological , Sequence Homology, Amino Acid
3.
ACS Chem Biol ; 10(3): 875-82, 2015 Mar 20.
Article in English | MEDLINE | ID: mdl-25517993

ABSTRACT

Microbial arsenate resistance is known to be conferred by specialized oxidoreductase enzymes termed arsenate reductases. We carried out a genetic selection on media supplemented with sodium arsenate for multicopy genes that can confer growth to E. coli mutant cells lacking the gene for arsenate reductase (E. coli ΔarsC). We found that overexpression of glutathione S-transferase B (GstB) complemented the ΔarsC allele and conferred growth on media containing up to 5 mM sodium arsenate. Interestingly, unlike wild type E. coli arsenate reductase, arsenate resistance via GstB was not dependent on reducing equivalents provided by glutaredoxins or a catalytic cysteine residue. Instead, two arginine residues, which presumably coordinate the arsenate substrate within the electrophilic binding site of GstB, were found to be critical for transferase activity. We provide biochemical evidence that GstB acts to directly reduce arsenate to arsenite with reduced glutathione (GSH) as the electron donor. Our results reveal a pathway for the detoxification of arsenate in bacteria that hinges on a previously undescribed function of a bacterial glutathione S-transferase.


Subject(s)
Arsenate Reductases/deficiency , Arsenates/metabolism , Escherichia coli/metabolism , Glutathione Transferase/metabolism , Glutathione/chemistry , Arsenate Reductases/genetics , Arsenates/toxicity , Arsenites/metabolism , Catalytic Domain , Drug Resistance, Bacterial , Escherichia coli/drug effects , Escherichia coli/genetics , Gene Deletion , Gene Expression , Genetic Complementation Test , Glutaredoxins/metabolism , Glutathione/metabolism , Glutathione Transferase/chemistry , Glutathione Transferase/genetics , Kinetics , Models, Molecular , Oxidation-Reduction , Plasmids/chemistry , Plasmids/metabolism , Protein Binding , Transformation, Bacterial
4.
Nat Biotechnol ; 28(9): 965-9, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20802495

ABSTRACT

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.


Subject(s)
Antibodies, Monoclonal/isolation & purification , High-Throughput Screening Assays/methods , Immunoglobulin Variable Region/genetics , Plasma Cells/immunology , Animals , Antibodies, Monoclonal/immunology , Antigens/immunology , Base Sequence , Complementarity Determining Regions/immunology , High-Throughput Nucleotide Sequencing , Humans , Immunization , Immunoglobulin Heavy Chains/genetics , Mice , Molecular Sequence Data
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