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1.
Cell ; 165(4): 813-26, 2016 May 05.
Article in English | MEDLINE | ID: mdl-27114034

ABSTRACT

The HIV-1-envelope (Env) trimer is covered by a glycan shield of ∼90 N-linked oligosaccharides, which comprises roughly half its mass and is a key component of HIV evasion from humoral immunity. To understand how antibodies can overcome the barriers imposed by the glycan shield, we crystallized fully glycosylated Env trimers from clades A, B, and G, visualizing the shield at 3.4-3.7 Å resolution. These structures reveal the HIV-1-glycan shield to comprise a network of interlocking oligosaccharides, substantially ordered by glycan crowding, that encase the protein component of Env and enable HIV-1 to avoid most antibody-mediated neutralization. The revealed features delineate a taxonomy of N-linked glycan-glycan interactions. Crowded and dispersed glycans are differently ordered, conserved, processed, and recognized by antibody. The structures, along with glycan-array binding and molecular dynamics, reveal a diversity in oligosaccharide affinity and a requirement for accommodating glycans among known broadly neutralizing antibodies that target the glycan-shielded trimer.


Subject(s)
HIV-1/chemistry , env Gene Products, Human Immunodeficiency Virus/chemistry , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Crystallography, X-Ray , Glycosylation , HIV-1/classification , HIV-1/immunology , Immune Evasion , Models, Molecular , Molecular Dynamics Simulation , Polysaccharides/analysis , Polysaccharides/metabolism
2.
Cell ; 166(3): 609-623, 2016 Jul 28.
Article in English | MEDLINE | ID: mdl-27453470

ABSTRACT

Antibodies capable of neutralizing divergent influenza A viruses could form the basis of a universal vaccine. Here, from subjects enrolled in an H5N1 DNA/MIV-prime-boost influenza vaccine trial, we sorted hemagglutinin cross-reactive memory B cells and identified three antibody classes, each capable of neutralizing diverse subtypes of group 1 and group 2 influenza A viruses. Co-crystal structures with hemagglutinin revealed that each class utilized characteristic germline genes and convergent sequence motifs to recognize overlapping epitopes in the hemagglutinin stem. All six analyzed subjects had sequences from at least one multidonor class, and-in half the subjects-multidonor-class sequences were recovered from >40% of cross-reactive B cells. By contrast, these multidonor-class sequences were rare in published antibody datasets. Vaccination with a divergent hemagglutinin can thus increase the frequency of B cells encoding broad influenza A-neutralizing antibodies. We propose the sequence signature-quantified prevalence of these B cells as a metric to guide universal influenza A immunization strategies.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Influenza A virus/immunology , Influenza Vaccines/immunology , Adult , Amino Acid Sequence , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Viral/chemistry , Antibodies, Viral/genetics , B-Lymphocytes/immunology , Epitopes, B-Lymphocyte , Female , Gene Rearrangement, B-Lymphocyte, Heavy Chain , Humans , Immunologic Memory , Influenza A Virus, H5N1 Subtype/immunology , Male , Middle Aged , Models, Molecular , Protein Structure, Tertiary , Structure-Activity Relationship , Young Adult
3.
Cell ; 165(2): 449-63, 2016 Apr 07.
Article in English | MEDLINE | ID: mdl-26949186

ABSTRACT

Antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. Here, we define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. We integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.


Subject(s)
AIDS Vaccines/immunology , Antibodies, Neutralizing/immunology , HIV Antibodies/immunology , HIV Infections/immunology , Amino Acid Sequence , Antibodies, Neutralizing/chemistry , B-Lymphocytes/immunology , HIV Antibodies/chemistry , HIV Envelope Protein gp120/immunology , HIV Infections/prevention & control , HIV-1/immunology , Humans , Models, Molecular , Molecular Sequence Data , Sequence Alignment
4.
Cell ; 161(3): 470-485, 2015 04 23.
Article in English | MEDLINE | ID: mdl-25865483

ABSTRACT

HIV-1-neutralizing antibodies develop in most HIV-1-infected individuals, although highly effective antibodies are generally observed only after years of chronic infection. Here, we characterize the rate of maturation and extent of diversity for the lineage that produced the broadly neutralizing antibody VRC01 through longitudinal sampling of peripheral B cell transcripts over 15 years and co-crystal structures of lineage members. Next-generation sequencing identified VRC01-lineage transcripts, which encompassed diverse antibodies organized into distinct phylogenetic clades. Prevalent clades maintained characteristic features of antigen recognition, though each evolved binding loops and disulfides that formed distinct recognition surfaces. Over the course of the study period, VRC01-lineage clades showed continuous evolution, with rates of ∼2 substitutions per 100 nucleotides per year, comparable to that of HIV-1 evolution. This high rate of antibody evolution provides a mechanism by which antibody lineages can achieve extraordinary diversity and, over years of chronic infection, develop effective HIV-1 neutralization.


Subject(s)
Antibodies, Neutralizing/genetics , B-Lymphocytes/immunology , Evolution, Molecular , HIV Infections/immunology , HIV-1/immunology , Amino Acid Sequence , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/immunology , Antibody Diversity , Chronic Disease , Humans , Leukocytes, Mononuclear , Models, Molecular , Molecular Sequence Data , Sequence Alignment
5.
Cell ; 161(6): 1280-92, 2015 Jun 04.
Article in English | MEDLINE | ID: mdl-26004070

ABSTRACT

The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures -8 determined here- of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies.


Subject(s)
Antibodies, Neutralizing/chemistry , Antibodies, Viral/chemistry , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/metabolism , HIV-1/physiology , Amino Acid Sequence , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , B-Lymphocytes/immunology , CD4 Antigens/metabolism , Complementarity Determining Regions , Epitopes, B-Lymphocyte , HIV Envelope Protein gp120/immunology , Humans , Models, Molecular , Molecular Sequence Data , Sequence Alignment
6.
J Immunol ; 211(7): 1082-1098, 2023 10 01.
Article in English | MEDLINE | ID: mdl-37647360

ABSTRACT

T cells are implicated in the pathophysiology of preterm labor and birth, the leading cause of neonatal morbidity and mortality worldwide. Specifically, maternal decidual T cells infiltrate the chorioamniotic membranes in chronic chorioamnionitis (CCA), a placental lesion considered to reflect maternal anti-fetal rejection, leading to preterm labor and birth. However, the phenotype and TCR repertoire of decidual T cells in women with preterm labor and CCA have not been investigated. In this study, we used phenotyping, TCR sequencing, and functional assays to elucidate the molecular characteristics and Ag specificity of T cells infiltrating the chorioamniotic membranes in women with CCA who underwent term or preterm labor. Phenotyping indicated distinct enrichment of human decidual effector memory T cell subsets in cases of preterm labor with CCA without altered regulatory T cell proportions. TCR sequencing revealed that the T cell repertoire of CCA is characterized by increased TCR richness and decreased clonal expansion in women with preterm labor. We identified 15 clones associated with CCA and compared these against established TCR databases, reporting that infiltrating T cells may possess specificity for maternal and fetal Ags, but not common viral Ags. Functional assays demonstrated that choriodecidual T cells can respond to maternal and fetal Ags. Collectively, our findings provide, to our knowledge, novel insight into the complex processes underlying chronic placental inflammation and further support a role for effector T cells in the mechanisms of disease for preterm labor and birth. Moreover, this work further strengthens the contribution of adaptive immunity to the syndromic nature of preterm labor and birth.


Subject(s)
Chorioamnionitis , Obstetric Labor, Premature , Pregnancy , Infant, Newborn , Humans , Female , Placenta , Inflammation , Receptors, Antigen, T-Cell
7.
Nature ; 566(7744): 398-402, 2019 02.
Article in English | MEDLINE | ID: mdl-30760926

ABSTRACT

The human genome contains approximately 20 thousand protein-coding genes1, but the size of the collection of antigen receptors of the adaptive immune system that is generated by the recombination of gene segments with non-templated junctional additions (on B cells) is unknown-although it is certainly orders of magnitude larger. It has not been established whether individuals possess unique (or private) repertoires or substantial components of shared (or public) repertoires. Here we sequence recombined and expressed B cell receptor genes in several individuals to determine the size of their B cell receptor repertoires, and the extent to which these are shared between individuals. Our experiments revealed that the circulating repertoire of each individual contained between 9 and 17 million B cell clonotypes. The three individuals that we studied shared many clonotypes, including between 1 and 6% of B cell heavy-chain clonotypes shared between two subjects (0.3% of clonotypes shared by all three) and 20 to 34% of λ or κ light chains shared between two subjects (16 or 22% of λ or κ light chains, respectively, were shared by all three). Some of the B cell clonotypes had thousands of clones, or somatic variants, within the clonotype lineage. Although some of these shared lineages might be driven by exposure to common antigens, previous exposure to foreign antigens was not the only force that shaped the shared repertoires, as we also identified shared clonotypes in umbilical cord blood samples and all adult repertoires. The unexpectedly high prevalence of shared clonotypes in B cell repertoires, and identification of the sequences of these shared clonotypes, should enable better understanding of the role of B cell immune repertoires in health and disease.


Subject(s)
Antibodies/genetics , Antibodies/immunology , B-Lymphocytes/immunology , Clone Cells/immunology , Receptors, Antigen, B-Cell/immunology , Adult , Amino Acid Sequence , Antibodies/chemistry , Antigens/immunology , B-Lymphocytes/cytology , B-Lymphocytes/metabolism , Base Sequence , Clone Cells/cytology , Clone Cells/metabolism , Female , Fetal Blood/cytology , Fetal Blood/immunology , Healthy Volunteers , Humans , Infant, Newborn , Male , Receptors, Antigen, B-Cell/chemistry , Receptors, Antigen, B-Cell/genetics , Sequence Analysis, DNA
8.
Bioinformatics ; 36(24): 5695-5697, 2021 Apr 05.
Article in English | MEDLINE | ID: mdl-33325481

ABSTRACT

SUMMARY: B-cell receptor (BCR) and T-cell receptor (TCR) repertoires are generated through somatic DNA rearrangements and are responsible for the molecular basis of antigen recognition in the immune system. Next-generation sequencing (NGS) of DNA and the falling cost of sequencing due to continued development of these technologies have made sequencing assays an affordable way to characterize the repertoire of adaptive immune receptors (sometimes termed the 'immunome'). Many new workflows have been developed to take advantage of NGS and have placed the resulting immunome datasets in the public domain. The scale of these NGS datasets has made it challenging to search through the Complementarity-determining region 3 (CDR3), which is responsible for imparting specific antibody-antigen interactions. Thus, there is an increasing demand for sequence analysis tools capable of searching through CDR3s from immunome data collections containing millions of sequences. To address this need, we created a software package called ClonoMatch that facilitates rapid searches in bulk immunome data for BCR or TCR sequences based on their CDR3 sequence or V3J clonotype. AVAILABILITY AND IMPLEMENTATION: Documentation, software support and the codebase are all available at https://github.com/crowelab/clonomatch. This software is distributed under the GPL v3 license.

9.
Proc Natl Acad Sci U S A ; 115(48): E11369-E11378, 2018 11 27.
Article in English | MEDLINE | ID: mdl-30420516

ABSTRACT

Hepatitis B virus (HBV)-associated acute liver failure (ALF) is a dramatic clinical syndrome leading to death or liver transplantation in 80% of cases. Due to the extremely rapid clinical course, the difficulties in obtaining liver specimens, and the lack of an animal model, the pathogenesis of ALF remains largely unknown. Here, we performed a comprehensive genetic and functional characterization of the virus and the host in liver tissue from HBV-associated ALF and compared the results with those of classic acute hepatitis B in chimpanzees. In contrast with acute hepatitis B, HBV strains detected in ALF livers displayed highly mutated HBV core antigen (HBcAg), associated with increased HBcAg expression ex vivo, which was independent of viral replication levels. Combined gene and miRNA expression profiling revealed a dominant B cell disease signature, with extensive intrahepatic production of IgM and IgG in germline configuration exclusively targeting HBcAg with subnanomolar affinities, and complement deposition. Thus, HBV ALF appears to be an anomalous T cell-independent, HBV core-driven B cell disease, which results from the rare and unfortunate encounter between a host with an unusual B cell response and an infecting virus with a highly mutated core antigen.


Subject(s)
Antibodies, Viral/immunology , Hepatitis B Core Antigens/immunology , Hepatitis B virus/immunology , Immunity, Humoral , Liver Failure, Acute/immunology , Adult , Animals , B-Lymphocytes/immunology , Female , Hepatitis B/immunology , Hepatitis B/pathology , Hepatitis B/virology , Hepatitis B Core Antigens/genetics , Hepatitis B virus/genetics , Humans , Liver/immunology , Liver/virology , Liver Failure, Acute/pathology , Liver Failure, Acute/virology , Male , Middle Aged , Pan troglodytes , T-Lymphocytes/immunology
10.
Immunol Rev ; 275(1): 108-128, 2017 01.
Article in English | MEDLINE | ID: mdl-28133812

ABSTRACT

Numerous antibodies have been identified from HIV-1-infected donors that neutralize diverse strains of HIV-1. These antibodies may provide the basis for a B cell-mediated HIV-1 vaccine. However, it has been unclear how to elicit similar antibodies by vaccination. To address this issue, we have undertaken an informatics-based approach to understand the genetic and immunologic processes controlling the development of HIV-1-neutralizing antibodies. As DNA sequencing comprises the fastest growing database of biological information, we focused on incorporating next-generation sequencing of B-cell transcripts to determine the origin, maturation pathway, and prevalence of broadly neutralizing antibody lineages (Antibodyomics1, 2, 4, and 6). We also incorporated large-scale robotic analyses of serum neutralization to identify and quantify neutralizing antibodies in donor cohorts (Antibodyomics3). Statistical analyses furnish another layer of insight (Antibodyomics5), with physical characteristics of antibodies and their targets through molecular dynamics simulations (Antibodyomics7) and free energy perturbation analyses (Antibodyomics8) providing information-rich output. Functional interrogation of individual antibodies (Antibodyomics9) and synthetic antibody libraries (Antibodyomics10) also yields multi-dimensional data by which to understand and improve antibodies. Antibodyomics, described here, thus comprise resolution-enhancing tools, which collectively embody an information-driven discovery engine aimed toward the development of effective B cell-based vaccines.


Subject(s)
AIDS Vaccines/immunology , B-Lymphocytes/immunology , Computational Biology , HIV Infections/immunology , HIV-1/immunology , Animals , Antibodies, Neutralizing/metabolism , B-Lymphocytes/virology , HIV Antibodies/metabolism , Humans , Immunity, Humoral
11.
BMC Bioinformatics ; 21(1): 314, 2020 Jul 16.
Article in English | MEDLINE | ID: mdl-32677886

ABSTRACT

BACKGROUND: Recent advances in DNA sequencing technologies have enabled significant leaps in capacity to generate large volumes of DNA sequence data, which has spurred a rapid growth in the use of bioinformatics as a means of interrogating antibody variable gene repertoires. Common tools used for annotation of antibody sequences are often limited in functionality, modularity and usability. RESULTS: We have developed PyIR, a Python wrapper and library for IgBLAST, which offers a minimal setup CLI and API, FASTQ support, file chunking for large sequence files, JSON and Python dictionary output, and built-in sequence filtering. CONCLUSIONS: PyIR offers improved processing speed over multithreaded IgBLAST (version 1.14) when spawning more than 16 processes on a single computer system. Its customizable filtering and data encapsulation allow it to be adapted to a wide range of computing environments. The API allows for IgBLAST to be used in customized bioinformatics workflows.


Subject(s)
Immunoglobulins/genetics , Receptors, Antigen, T-Cell/genetics , Sequence Alignment , Software , Base Sequence , Humans , Sequence Analysis, DNA , Time Factors , User-Computer Interface
12.
Nature ; 514(7523): 455-61, 2014 Oct 23.
Article in English | MEDLINE | ID: mdl-25296255

ABSTRACT

The human immunodeficiency virus type 1 (HIV-1) envelope (Env) spike, comprising three gp120 and three gp41 subunits, is a conformational machine that facilitates HIV-1 entry by rearranging from a mature unliganded state, through receptor-bound intermediates, to a post-fusion state. As the sole viral antigen on the HIV-1 virion surface, Env is both the target of neutralizing antibodies and a focus of vaccine efforts. Here we report the structure at 3.5 Å resolution for an HIV-1 Env trimer captured in a mature closed state by antibodies PGT122 and 35O22. This structure reveals the pre-fusion conformation of gp41, indicates rearrangements needed for fusion activation, and defines parameters of immune evasion and immune recognition. Pre-fusion gp41 encircles amino- and carboxy-terminal strands of gp120 with four helices that form a membrane-proximal collar, fastened by insertion of a fusion peptide-proximal methionine into a gp41-tryptophan clasp. Spike rearrangements required for entry involve opening the clasp and expelling the termini. N-linked glycosylation and sequence-variable regions cover the pre-fusion closed spike; we used chronic cohorts to map the prevalence and location of effective HIV-1-neutralizing responses, which were distinguished by their recognition of N-linked glycan and tolerance for epitope-sequence variation.


Subject(s)
HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/immunology , HIV Envelope Protein gp41/chemistry , HIV Envelope Protein gp41/immunology , AIDS Vaccines/chemistry , AIDS Vaccines/immunology , Amino Acid Sequence , Antibodies, Neutralizing/immunology , Cohort Studies , Crystallography, X-Ray , Genetic Variation , Glycosylation , HIV Antibodies/immunology , HIV Envelope Protein gp120/genetics , HIV Envelope Protein gp41/genetics , HIV Infections/immunology , Humans , Immune Evasion , Membrane Fusion , Models, Molecular , Molecular Sequence Data , Polysaccharides/chemistry , Polysaccharides/immunology , Protein Multimerization , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/immunology , Structural Homology, Protein , Virus Internalization
13.
Nature ; 509(7498): 55-62, 2014 May 01.
Article in English | MEDLINE | ID: mdl-24590074

ABSTRACT

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.


Subject(s)
Antibodies, Neutralizing/immunology , HIV Antibodies/immunology , HIV Envelope Protein gp160/chemistry , HIV Envelope Protein gp160/immunology , AIDS Vaccines/chemistry , AIDS Vaccines/immunology , Amino Acid Sequence , Antibodies, Neutralizing/chemistry , Antibodies, Neutralizing/genetics , Antibodies, Neutralizing/isolation & purification , Antibody Affinity/genetics , Antibody Affinity/immunology , B-Lymphocytes/cytology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , Binding Sites/immunology , CD4 Antigens/immunology , CD4 Antigens/metabolism , Cell Lineage , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/genetics , Complementarity Determining Regions/immunology , Epitope Mapping , Epitopes, B-Lymphocyte/chemistry , Epitopes, B-Lymphocyte/immunology , Evolution, Molecular , HIV Antibodies/chemistry , HIV Antibodies/genetics , HIV Antibodies/isolation & purification , HIV Infections/immunology , HIV-1/chemistry , HIV-1/immunology , Humans , Models, Molecular , Molecular Sequence Data , Neutralization Tests , Protein Structure, Tertiary , Somatic Hypermutation, Immunoglobulin/genetics
14.
BMC Bioinformatics ; 20(1): 513, 2019 Oct 22.
Article in English | MEDLINE | ID: mdl-31640540

ABSTRACT

BACKGROUND: Carbohydrates are a class of large and diverse biomolecules, ranging from a simple monosaccharide to large multi-branching glycan structures. The covalent linkage of a carbohydrate to the nitrogen atom of an asparagine, a process referred to as N-linked glycosylation, plays an important role in the physiology of many living organisms. Most software for glycan modeling on a personal desktop computer requires knowledge of molecular dynamics to interface with specialized programs such as CHARMM or AMBER. There are a number of popular web-based tools that are available for modeling glycans (e.g., GLYCAM-WEB (http:// https://dev.glycam.org/gp/ ) or Glycosciences.db ( http://www.glycosciences.de/ )). However, these web-based tools are generally limited to a few canonical glycan conformations and do not allow the user to incorporate glycan modeling into their protein structure modeling workflow. RESULTS: Here, we present Glycosylator, a Python framework for the identification, modeling and modification of glycans in protein structure that can be used directly in a Python script through its application programming interface (API) or through its graphical user interface (GUI). The GUI provides a straightforward two-dimensional (2D) rendering of a glycoprotein that allows for a quick visual inspection of the glycosylation state of all the sequons on a protein structure. Modeled glycans can be further refined by a genetic algorithm for removing clashes and sampling alternative conformations. Glycosylator can also identify specific three-dimensional (3D) glycans on a protein structure using a library of predefined templates. CONCLUSIONS: Glycosylator was used to generate models of glycosylated protein without steric clashes. Since the molecular topology is based on the CHARMM force field, new complex sugar moieties can be generated without modifying the internals of the code. Glycosylator provides more functionality for analyzing and modeling glycans than any other available software or webserver at present. Glycosylator will be a valuable tool for the glycoinformatics and biomolecular modeling communities.


Subject(s)
Glycoproteins/chemistry , Molecular Dynamics Simulation , Polysaccharides/chemistry , Software , Glycosylation
15.
BMC Bioinformatics ; 20(1): 629, 2019 Dec 04.
Article in English | MEDLINE | ID: mdl-31801472

ABSTRACT

BACKGROUND: Advances in next-generation sequencing (NGS) of antibody repertoires have led to an explosion in B cell receptor sequence data from donors with many different disease states. These data have the potential to detect patterns of immune response across populations. However, to this point it has been difficult to interpret such patterns of immune response between disease states in the absence of functional data. There is a need for a robust method that can be used to distinguish general patterns of immune responses at the antibody repertoire level. RESULTS: We developed a method for reducing the complexity of antibody repertoire datasets using principal component analysis (PCA) and refer to our method as "repertoire fingerprinting." We reduce the high dimensional space of an antibody repertoire to just two principal components that explain the majority of variation in those repertoires. We show that repertoires from individuals with a common experience or disease state can be clustered by their repertoire fingerprints to identify common antibody responses. CONCLUSIONS: Our repertoire fingerprinting method for distinguishing immune repertoires has implications for characterizing an individual disease state. Methods to distinguish disease states based on pattern recognition in the adaptive immune response could be used to develop biomarkers with diagnostic or prognostic utility in patient care. Extending our analysis to larger cohorts of patients in the future should permit us to define more precisely those characteristics of the immune response that result from natural infection or autoimmunity.


Subject(s)
Antibodies/genetics , Principal Component Analysis , Adult , Cohort Studies , Fetal Blood/immunology , HIV Infections/blood , HIV Infections/immunology , High-Throughput Nucleotide Sequencing/methods , Humans , Influenza, Human/blood , Influenza, Human/immunology , Middle Aged , Vaccination
16.
Chembiochem ; 18(8): 764-771, 2017 04 18.
Article in English | MEDLINE | ID: mdl-28166380

ABSTRACT

Man9 GlcNAc2 (Man-9) present at the surface of HIV makes up the binding sites of several HIV-neutralizing agents and the mammalian lectin DC-SIGN, which is involved in cellular immunity and trans-infections. We describe the conformational properties of Man-9 in its free state and when bound by the HIV entry-inhibitor protein microvirin (MVN), and define the minimum epitopes of both MVN and DC-SIGN by using NMR spectroscopy. To facilitate the implementation of 3D 13 C-edited spectra to deconvolute spectral overlap and to determine the solution structure of Man-9, we developed a robust expression system for the production of 13 C,15 N-labeled glycans in mammalian cells. The studies reveal that Man-9 interacts with HIV-binding proteins through distinct epitopes and adopts diverse conformations in the bound state. In combination with molecular dynamics simulations we observed receptor-bound conformations to be sampled by Man-9 in the free state, thus suggesting a conformational selection mechanism for diverse recognition.


Subject(s)
Bacterial Proteins/chemistry , Cell Adhesion Molecules/chemistry , Lectins, C-Type/chemistry , Magnetic Resonance Spectroscopy , Mannans/chemistry , Mannose-Binding Lectin/chemistry , Receptors, Cell Surface/chemistry , A549 Cells , Carbohydrate Conformation , Carbon Radioisotopes , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/metabolism , HIV-1/metabolism , Humans , Mannans/biosynthesis , Microcystis , Molecular Dynamics Simulation , Nitrogen Radioisotopes
17.
Nature ; 463(7281): 689-92, 2010 Feb 04.
Article in English | MEDLINE | ID: mdl-20130653

ABSTRACT

The M2 protein of influenza A virus is a membrane-spanning tetrameric proton channel targeted by the antiviral drugs amantadine and rimantadine. Resistance to these drugs has compromised their effectiveness against many influenza strains, including pandemic H1N1. A recent crystal structure of M2(22-46) showed electron densities attributed to a single amantadine in the amino-terminal half of the pore, indicating a physical occlusion mechanism for inhibition. However, a solution NMR structure of M2(18-60) showed four rimantadines bound to the carboxy-terminal lipid-facing surface of the helices, suggesting an allosteric mechanism. Here we show by solid-state NMR spectroscopy that two amantadine-binding sites exist in M2 in phospholipid bilayers. The high-affinity site, occupied by a single amantadine, is located in the N-terminal channel lumen, surrounded by residues mutated in amantadine-resistant viruses. Quantification of the protein-amantadine distances resulted in a 0.3 A-resolution structure of the high-affinity binding site. The second, low-affinity, site was observed on the C-terminal protein surface, but only when the drug reaches high concentrations in the bilayer. The orientation and dynamics of the drug are distinct in the two sites, as shown by (2)H NMR. These results indicate that amantadine physically occludes the M2 channel, thus paving the way for developing new antiviral drugs against influenza viruses. The study demonstrates the ability of solid-state NMR to elucidate small-molecule interactions with membrane proteins and determine high-resolution structures of their complexes.


Subject(s)
Amantadine/metabolism , Antiviral Agents/metabolism , Influenza A virus/chemistry , Lipid Bilayers/metabolism , Viral Matrix Proteins/chemistry , Viral Matrix Proteins/metabolism , Amantadine/chemistry , Amantadine/pharmacology , Amino Acid Sequence , Antiviral Agents/chemistry , Antiviral Agents/pharmacology , Binding Sites , Crystallography, X-Ray , Dimyristoylphosphatidylcholine/chemistry , Dimyristoylphosphatidylcholine/metabolism , Hydrogen-Ion Concentration , Influenza A virus/drug effects , Lipid Bilayers/chemistry , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Protein Conformation , Structure-Activity Relationship , Temperature , Viral Matrix Proteins/antagonists & inhibitors
18.
Proc Natl Acad Sci U S A ; 110(43): E4088-97, 2013 Oct 22.
Article in English | MEDLINE | ID: mdl-24106303

ABSTRACT

Next-generation sequencing of antibody transcripts provides a wealth of data, but the ability to identify function-specific antibodies solely on the basis of sequence has remained elusive. We previously characterized the VRC01 class of antibodies, which target the CD4-binding site on gp120, appear in multiple donors, and broadly neutralize HIV-1. Antibodies of this class have developmental commonalities, but typically share only ∼50% amino acid sequence identity among different donors. Here we apply next-generation sequencing to identify VRC01 class antibodies in a new donor, C38, directly from B cell transcript sequences. We first tested a lineage rank approach, but this was unsuccessful, likely because VRC01 class antibody sequences were not highly prevalent in this donor. We next identified VRC01 class heavy chains through a phylogenetic analysis that included thousands of sequences from C38 and a few known VRC01 class sequences from other donors. This "cross-donor analysis" yielded heavy chains with little sequence homology to previously identified VRC01 class heavy chains. Nonetheless, when reconstituted with the light chain from VRC01, half of the heavy chain chimeric antibodies showed substantial neutralization potency and breadth. We then identified VRC01 class light chains through a five-amino-acid sequence motif necessary for VRC01 light chain recognition. From over a million light chain sequences, we identified 13 candidate VRC01 class members. Pairing of these light chains with the phylogenetically identified C38 heavy chains yielded functional antibodies that effectively neutralized HIV-1. Bioinformatics analysis can thus directly identify functional HIV-1-neutralizing antibodies of the VRC01 class from a sequenced antibody repertoire.


Subject(s)
Antibodies, Neutralizing/genetics , B-Lymphocytes/metabolism , HIV-1/immunology , High-Throughput Nucleotide Sequencing/methods , Amino Acid Sequence , Antibodies, Neutralizing/immunology , B-Lymphocytes/immunology , Base Sequence , Genetic Variation , Humans , Immunoglobulin Heavy Chains/classification , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Light Chains/classification , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/immunology , Molecular Sequence Data , Phylogeny , Sequence Homology, Amino Acid
19.
J Virol ; 88(21): 12669-82, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25142607

ABSTRACT

UNLABELLED: Over the past 5 years, a new generation of highly potent and broadly neutralizing HIV-1 antibodies has been identified. These antibodies can protect against lentiviral infection in nonhuman primates (NHPs), suggesting that passive antibody transfer would prevent HIV-1 transmission in humans. To increase the protective efficacy of such monoclonal antibodies, we employed next-generation sequencing, computational bioinformatics, and structure-guided design to enhance the neutralization potency and breadth of VRC01, an antibody that targets the CD4 binding site of the HIV-1 envelope. One variant, VRC07-523, was 5- to 8-fold more potent than VRC01, neutralized 96% of viruses tested, and displayed minimal autoreactivity. To compare its protective efficacy to that of VRC01 in vivo, we performed a series of simian-human immunodeficiency virus (SHIV) challenge experiments in nonhuman primates and calculated the doses of VRC07-523 and VRC01 that provide 50% protection (EC50). VRC07-523 prevented infection in NHPs at a 5-fold lower concentration than VRC01. These results suggest that increased neutralization potency in vitro correlates with improved protection against infection in vivo, documenting the improved functional efficacy of VRC07-523 and its potential clinical relevance for protecting against HIV-1 infection in humans. IMPORTANCE: In the absence of an effective HIV-1 vaccine, alternative strategies are needed to block HIV-1 transmission. Direct administration of HIV-1-neutralizing antibodies may be able to prevent HIV-1 infections in humans. This approach could be especially useful in individuals at high risk for contracting HIV-1 and could be used together with antiretroviral drugs to prevent infection. To optimize the chance of success, such antibodies can be modified to improve their potency, breadth, and in vivo half-life. Here, knowledge of the structure of a potent neutralizing antibody, VRC01, that targets the CD4-binding site of the HIV-1 envelope protein was used to engineer a next-generation antibody with 5- to 8-fold increased potency in vitro. When administered to nonhuman primates, this antibody conferred protection at a 5-fold lower concentration than the original antibody. Our studies demonstrate an important correlation between in vitro assays used to evaluate the therapeutic potential of antibodies and their in vivo effectiveness.


Subject(s)
Antibodies, Neutralizing/immunology , HIV Antibodies/immunology , HIV-1/immunology , Immunization, Passive/methods , Simian Acquired Immunodeficiency Syndrome/prevention & control , Animals , Antibodies, Neutralizing/administration & dosage , Antibodies, Neutralizing/genetics , HIV Antibodies/administration & dosage , HIV Antibodies/genetics , HIV-1/genetics , Macaca mulatta , Male , Molecular Sequence Data , Sequence Analysis, DNA
20.
PLoS Comput Biol ; 9(1): e1002878, 2013.
Article in English | MEDLINE | ID: mdl-23359663

ABSTRACT

The PhoQP two-component system is a signaling complex essential for bacterial virulence and cationic antimicrobial peptide resistance. PhoQ is the histidine kinase chemoreceptor of this tandem machine and assembles in a homodimer conformation spanning the bacterial inner membrane. Currently, a full understanding of the PhoQ signal transduction is hindered by the lack of a complete atomistic structure. In this study, an atomistic model of the key transmembrane (TM) domain is assembled by using molecular simulations, guided by experimental cross-linking data. The formation of a polar pocket involving Asn202 in the lumen of the tetrameric TM bundle is crucial for the assembly and solvation of the domain. Moreover, a concerted displacement of the TM helices at the periplasmic side is found to modulate a rotation at the cytoplasmic end, supporting the transduction of the chemical signal through a combination of scissoring and rotational movement of the TM helices.


Subject(s)
Escherichia coli/enzymology , Membrane Proteins/metabolism , Models, Molecular , Protein Kinases/metabolism , Signal Transduction , Histidine Kinase , Membrane Proteins/chemistry , Molecular Dynamics Simulation , Protein Kinases/chemistry
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