High frequency of shared clonotypes in human B cell receptor repertoires.

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.

PyIR: a scalable wrapper for processing billions of immunoglobulin and T cell receptor sequences using IgBLAST.

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.

Capsid antibodies to different adeno-associated virus serotypes bind common regions.

Interactions between viruses and the host antibody immune response are critical in the development and control of disease, and antibodies are also known to interfere with the efficacy of viral vector-based gene delivery. The adeno-associated viruses (AAVs) being developed as vectors for corrective human gene delivery have shown promise in clinical trials, but preexisting antibodies are detrimental to successful outcomes. However, the antigenic epitopes on AAV capsids remain poorly characterized. Cryo-electron microscopy and three-dimensional image reconstruction were used to define the locations of epitopes to which monoclonal fragment antibodies (Fabs) against AAV1, AAV2, AAV5, and AAV6 bind. Pseudoatomic modeling showed that, in each serotype, Fabs bound to a limited number of sites near the protrusions surrounding the 3-fold axes of the T=1 icosahedral capsids. For the closely related AAV1 and AAV6, a common Fab exhibited substoichiometric binding, with one Fab bound, on average, between two of the three protrusions as a consequence of steric crowding. The other AAV Fabs saturated the capsid and bound to the walls of all 60 protrusions, with the footprint for the AAV5 antibody extending toward the 5-fold axis. The angle of incidence for each bound Fab on the AAVs varied and resulted in significant differences in how much of each viral capsid surface was occluded beyond the Fab footprints. The AAV-antibody interactions showed a common set of footprints that overlapped some known receptor-binding sites and transduction determinants, thus suggesting potential mechanisms for virus neutralization by the antibodies.

Three-dimensional reconstruction of icosahedral particles from single micrographs in real time at the microscope.

Single particle analysis is a valuable tool in cryo-electron microscopy for determining the structure of biological complexes. However, the conformational state and the preparation of the sample are factors that play a critical role in the ultimate attainable resolution. In some cases extensive analysis at the microscope of a sample under different conditions is required to derive the optimal acquisition conditions. Currently this analysis is limited to raw micrographs, thus conveying only limited information on the structure of the complex. We are developing a computing system that generates a three-dimensional reconstruction from a single micrograph acquired under cryogenic and low dose conditions, and containing particles with icosahedral symmetry. The system provides the microscopist with immediate structural information from a sample while it is in the microscope and during the preliminary acquisition stage. The system is designed to run without user intervention on a multi-processor computing resource and integrates all the processing steps required for the analysis. Tests performed on experimental data sets show that the probability of obtaining a reliable reconstruction from one micrograph is primarily determined by the quality of the sample, with success rates close to 100% when sample conditions are optimal, and decreasing to about 60% when conditions are sub-optimal. The time required to generate a reconstruction depends significantly on the diameter of the particles, and in most instances takes about 1min. The proposed approach can provide valuable three-dimensional information, albeit at low resolution, on conformational states, epitope binding, and stoichiometry of icosahedral multi-protein complexes.

Structural analysis of coxsackievirus A7 reveals conformational changes associated with uncoating.

Coxsackievirus A7 (CAV7) is a rarely detected and poorly characterized serotype of the Enterovirus species Human enterovirus A (HEV-A) within the Picornaviridae family. The CAV7-USSR strain has caused polio-like epidemics and was originally thought to represent the fourth poliovirus type, but later evidence linked this strain to the CAV7-Parker prototype. Another isolate, CAV7-275/58, was also serologically similar to Parker but was noninfectious in a mouse model. Sequencing of the genomic region encoding the capsid proteins of the USSR and 275/58 strains and subsequent comparison with the corresponding amino acid sequences of the Parker strain revealed that the Parker and USSR strains are nearly identical, while the 275/58 strain is more distant. Using electron cryomicroscopy and three-dimensional image reconstruction, the structures of the CAV7-USSR virion and empty capsid were resolved to 8.2-Å and 6.1-Å resolutions, respectively. This is one of the first detailed structural analyses of the HEV-A species. Using homology modeling, reconstruction segmentation, and flexible fitting, we constructed a pseudoatomic T = 1 (pseudo T = 3) model incorporating the three major capsid proteins (VP1 to VP3), addressed the conformational changes of the capsid and its constituent viral proteins occurring during RNA release, and mapped the capsid proteins' variable regions to the structure. During uncoating, VP4 and RNA are released analogously to poliovirus 1, the interfaces of VP2 and VP3 are rearranged, and VP1 rotates. Variable regions in the capsid proteins were predicted to map mainly to the surface of VP1 and are thus likely to affect the tropism and pathogenicity of CAV7.

Structural insight into the unique properties of adeno-associated virus serotype 9.

Adeno-associated virus serotype 9 (AAV9) has enhanced capsid-associated tropism for cardiac muscle and the ability to cross the blood-brain barrier compared to other AAV serotypes. To help identify the structural features facilitating these properties, we have used cryo-electron microscopy (cryo-EM) and three-dimensional image reconstruction (cryo-reconstruction) and X-ray crystallography to determine the structure of the AAV9 capsid at 9.7- and 2.8-Å resolutions, respectively. The AAV9 capsid exhibits the surface topology conserved in all AAVs: depressions at each icosahedral two-fold symmetry axis and surrounding each five-fold axis, three separate protrusions surrounding each three-fold axis, and a channel at each five-fold axis. The AAV9 viral protein (VP) has a conserved core structure, consisting of an eight-stranded, ß-barrel motif and the αA helix, which are present in all parvovirus structures. The AAV9 VP differs in nine variable surface regions (VR-I to -IX) compared to AAV4, but at only three (VR-I, VR-II, and VR-IV) compared to AAV2 and AAV8. VR-I differences modify the raised region of the capsid surface between the two-fold and five-fold depressions. The VR-IV difference produces smaller three-fold protrusions in AAV9 that are less "pointed" than AAV2 and AAV8. Significantly, residues in the AAV9 VRs have been identified as important determinants of cellular tropism and transduction and dictate its antigenic diversity from AAV2. Hence, the AAV9 VRs likely confer the unique infection phenotypes of this serotype.

Use of accounting concepts to study research: return on investment in XSEDE, a US cyberinfrastructure service.

This paper uses accounting concepts-particularly the concept of Return on Investment (ROI)-to reveal the quantitative value of scientific research pertaining to a major US cyberinfrastructure project (XSEDE-the eXtreme Science and Engineering Discovery Environment). XSEDE provides operational and support services for advanced information technology systems, cloud systems, and supercomputers supporting non-classified US research, with an average budget for XSEDE of US$20M+ per year over the period studied (2014-2021). To assess the financial effectiveness of these services, we calculated a proxy for ROI, and converted quantitative measures of XSEDE service delivery into financial values using costs for service from the US marketplace. We calculated two estimates of ROI: a Conservative Estimate, functioning as a lower bound and using publicly available data for a lower valuation of XSEDE services; and a Best Available Estimate, functioning as a more accurate estimate, but using some unpublished valuation data. Using the largest dataset assembled for analysis of ROI for a cyberinfrastructure project, we found a Conservative Estimate of ROI of 1.87, and a Best Available Estimate of ROI of 3.24. Through accounting methods, we show that XSEDE services offer excellent value to the US government, that the services offered uniquely by XSEDE (that is, not otherwise available for purchase) were the most valuable to the facilitation of US research activities, and that accounting-based concepts hold great value for understanding the mechanisms of scientific research generally.

Atomic structure reveals the unique capsid organization of a dsRNA virus.

For most dsRNA viruses, the genome-enclosing capsid comprises 120 copies of a single capsid protein (CP) organized into 60 icosahedrally equivalent dimers, generally identified as 2 nonsymmetricallyinteracting CP molecules with extensive lateral contacts. The crystal structure of a partitivirus, Penicillium stoloniferum virus F (PsV-F), reveals a different organization, in which the CP dimer is related by almost-perfect local 2-fold symmetry, forms prominent surface arches, and includes extensive structure swapping between the 2 subunits. An electron cryomicroscopy map of PsV-F shows that the disordered N terminus of each CP molecule interacts with the dsRNA genome and probably participates in its packaging or transcription. Intact PsV-F particles mediate semiconservative transcription, and transcripts are likely to exit through negatively charged channels at the icosahedral 5-fold axes. Other findings suggest that the PsV-F capsid is assembled from dimers of CP dimers, with an arrangement similar to flavivirus E glycoproteins.

Controlling and switching the morphology of micellar nanoparticles with enzymes.

Micelles were prepared from polymer-peptide block copolymer amphiphiles containing substrates for protein kinase A, protein phosphatase-1, and matrix metalloproteinases 2 and 9. We examine reversible switching of the morphology of these micelles through a phosphorylation-dephosphorylation cycle and study peptide-sequence directed changes in morphology in response to proteolysis. Furthermore, the exceptional uniformity of these polymer-peptide particles makes them amenable to cryo-TEM reconstruction techniques lending insight into their internal structure.

Interaction of alphaVbeta3 and alphaVbeta6 integrins with human parechovirus 1.

Human parechovirus (HPEV) infections are very common in early childhood and can be severe in neonates. It has been shown that integrins are important for cellular infectivity of HPEV1 through experiments using peptide blocking assays and function-blocking antibodies to alpha(V) integrins. The interaction of HPEV1 with alpha(V) integrins is presumably mediated by a C-terminal RGD motif in the capsid protein VP1. We characterized the binding of integrins alpha(V)beta(3) and alpha(V)beta(6) to HPEV1 by biochemical and structural studies. We showed that although HPEV1 bound efficiently to immobilized integrins, alpha(V)beta(6) bound more efficiently than alpha(V)beta(3) to immobilized HPEV1. Moreover, soluble alpha(V)beta(6), but not alpha(V)beta(3), blocked HPEV1 cellular infectivity, indicating that it is a high-affinity receptor for HPEV1. We also showed that HPEV1 binding to integrins in vitro could be partially blocked by RGD peptides. Using electron cryo-microscopy and image reconstruction, we showed that HPEV1 has the typical T=1 (pseudo T=3) organization of a picornavirus. Complexes of HPEV1 and integrins indicated that both integrin footprints reside between the 5-fold and 3-fold symmetry axes. This result does not match the RGD position predicted from the coxsackievirus A9 X-ray structure but is consistent with the predicted location of this motif in the shorter C terminus found in HPEV1. This first structural characterization of a parechovirus indicates that the differences in receptor binding are due to the amino acid differences in the integrins rather than to significantly different viral footprints.

Human bocavirus capsid structure: insights into the structural repertoire of the parvoviridae.

Human bocavirus (HBoV) was recently discovered and classified in the Bocavirus genus (family Parvoviridae, subfamily Parvovirinae) on the basis of genomic similarity to bovine parvovirus and canine minute virus. HBoV has been implicated in respiratory tract infections and gastroenteric disease in children worldwide, yet despite numerous epidemiological reports, there has been limited biochemical and molecular characterization of the virus. Reported here is the three-dimensional structure of recombinant HBoV capsids, assembled from viral protein 2 (VP2), at 7.9-A resolution as determined by cryo-electron microscopy and image reconstruction. A pseudo-atomic model of HBoV VP2 was derived from sequence alignment analysis and knowledge of the crystal structure of human parvovirus B19 (genus Erythrovirus). Comparison of the HBoV capsid structure to that of parvoviruses from five separate genera demonstrates strong conservation of a beta-barrel core domain and an alpha-helix, from which emanate several loops of various lengths and conformations, yielding a unique surface topology that differs from the three already described for this family. The highly conserved core is consistent with observations for other single-stranded DNA viruses, and variable surface loops have been shown to confer the host-specific tropism and the diverse antigenic properties of this family.

Infectious myonecrosis virus has a totivirus-like, 120-subunit capsid, but with fiber complexes at the fivefold axes.

Infectious myonecrosis virus (IMNV) is an emerging pathogen of penaeid shrimp in global aquaculture. Tentatively assigned to family Totiviridae, it has a nonsegmented dsRNA genome of 7,560 bp and an isometric capsid of the 901-aa major capsid protein. We used electron cryomicroscopy and 3D image reconstruction to examine the IMNV virion at 8.0-A resolution. Results reveal a totivirus-like, 120-subunit T = 1 capsid, 450 A in diameter, but with fiber complexes protruding a further 80 A at the fivefold axes. These protrusions likely mediate roles in the extracellular transmission and pathogenesis of IMNV, capabilities not shared by most other totiviruses. The IMNV structure is also notable in that the genome is centrally organized in five or six concentric shells. Within each of these shells, the densities alternate between a dodecahedral frame that connects the threefold axes vs. concentration around the fivefold axes, implying certain regularities in the RNA packing scheme.

A tale of two symmetrons: rules for construction of icosahedral capsids from trisymmetrons and pentasymmetrons.

The capsids of large, icosahedral dsDNA viruses are built from well-ordered aggregates of capsomers, known as trisymmetrons and pentasymmetrons, which are centered on the icosahedral 3-fold and 5-fold axes, respectively. We derive the complete set of rules for constructing an icosahedral structure from these symmetrons when the T lattice symmetry is odd and show that there are three classes of solutions, each of which follows from a different relationship between the size of the pentasymmetron and the values of the h and k icosahedral lattice parameters. Together, these three classes account for all possible ways of building an icosahedral structure solely from trisymmetrons and pentasymmetrons. Also, every icosahedral lattice with odd T number has solutions from exactly two of these three classes, with the set of allowed classes dependent on which of the two lattice parameters is odd. For symmetric lattices (if h=k or h=0), the two solutions yield the same symmetron sizes, but when the lattice parameters are equal (h=k) the solutions can be distinguished by the relative orientations of the symmetrons. We discuss these results in the context of known virus structures and explore the implications for viruses whose structures have not yet been solved.

Cryo-reconstructions of P22 polyheads suggest that phage assembly is nucleated by trimeric interactions among coat proteins.

Bacteriophage P22 forms an isometric capsid during normal assembly, yet when the coat protein (CP) is altered at a single site, helical structures (polyheads) also form. The structures of three distinct polyheads obtained from F170L and F170A variants were determined by cryo-reconstruction methods. An understanding of the structures of aberrant assemblies such as polyheads helps to explain how amino acid substitutions affect the CP, and these results can now be put into the context of CP pseudo-atomic models. F170L CP forms two types of polyhead and each has the CP organized as hexons (oligomers of six CPs). These hexons have a skewed structure similar to that in procapsids (precursor capsids formed prior to dsDNA packaging), yet their organization differs completely in polyheads and procapsids. F170A CP forms only one type of polyhead, and though this has hexons organized similarly to hexons in F170L polyheads, the hexons are isometric structures like those found in mature virions. The hexon organization in all three polyheads suggests that nucleation of procapsid assembly occurs via a trimer of CP monomers, and this drives formation of a T = 7, isometric particle. These variants also form procapsids, but they mature quite differently: F170A expands spontaneously at room temperature, whereas F170L requires more energy. The P22 CP structure along with scaffolding protein interactions appear to dictate curvature and geometry in assembled structures and residue 170 significantly influences both assembly and maturation.

Partitivirus structure reveals a 120-subunit, helix-rich capsid with distinctive surface arches formed by quasisymmetric coat-protein dimers.

Two distinct partitiviruses, Penicillium stoloniferum viruses S and F, can be isolated from the fungus Penicillium stoloniferum. The bisegmented dsRNA genomes of these viruses are separately packaged in icosahedral capsids containing 120 coat-protein subunits. We used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structure of Penicillium stoloniferum virus S at 7.3 A resolution. The capsid, approximately 350 A in outer diameter, contains 12 pentons, each of which is topped by five arched protrusions. Each of these protrusions is, in turn, formed by a quasisymmetric dimer of coat protein, for a total of 60 such dimers per particle. The density map shows numerous tubular features, characteristic of alpha helices and consistent with secondary structure predictions for the coat protein. This three-dimensional structure of a virus from the family Partitiviridae exhibits both similarities to and differences from the so-called "T = 2" capsids of other dsRNA viruses.

High Frequency of Shared Clonotypes in Human T Cell Receptor Repertoires.

The collection of T cell receptors (TCRs) generated by somatic recombination is large but unknown. We generate large TCR repertoire datasets as a resource to facilitate detailed studies of the role of TCR clonotypes and repertoires in health and disease. We estimate the size of individual human recombined and expressed TCRs by sequence analysis and determine the extent of sharing between individual repertoires. Our experiments reveal that each blood sample contains between 5 million and 21 million TCR clonotypes. Three individuals share 8% of TCRß- or 11% of TCRα-chain clonotypes. Sorting by T cell phenotypes in four individuals shows that 5% of naive CD4+ and 3.5% of naive CD8+ subsets share their TCRß clonotypes, whereas memory CD4+ and CD8+ subsets share 2.3% and 0.4% of their clonotypes, respectively. We identify the sequences of these shared TCR clonotypes that are of interest for studies of human T cell biology.

A bioinformatics roadmap for the human vaccines project.

Biomedical research has become a data intensive science in which high throughput experimentation is producing comprehensive data about biological systems at an ever-increasing pace. The Human Vaccines Project is a new public-private partnership, with the goal of accelerating development of improved vaccines and immunotherapies for global infectious diseases and cancers by decoding the human immune system. To achieve its mission, the Project is developing a Bioinformatics Hub as an open-source, multidisciplinary effort with the overarching goal of providing an enabling infrastructure to support the data processing, analysis and knowledge extraction procedures required to translate high throughput, high complexity human immunology research data into biomedical knowledge, to determine the core principles driving specific and durable protective immune responses.

Components of the antigen processing and presentation pathway revealed by gene expression microarray analysis following B cell antigen receptor (BCR) stimulation.

BACKGROUND: Activation of naïve B lymphocytes by extracellular ligands, e.g. antigen, lipopolysaccharide (LPS) and CD40 ligand, induces a combination of common and ligand-specific phenotypic changes through complex signal transduction pathways. For example, although all three of these ligands induce proliferation, only stimulation through the B cell antigen receptor (BCR) induces apoptosis in resting splenic B cells. In order to define the common and unique biological responses to ligand stimulation, we compared the gene expression changes induced in normal primary B cells by a panel of ligands using cDNA microarrays and a statistical approach, CLASSIFI (Cluster Assignment for Biological Inference), which identifies significant co-clustering of genes with similar Gene Ontology annotation. RESULTS: CLASSIFI analysis revealed an overrepresentation of genes involved in ion and vesicle transport, including multiple components of the proton pump, in the BCR-specific gene cluster, suggesting that activation of antigen processing and presentation pathways is a major biological response to antigen receptor stimulation. Proton pump components that were not included in the initial microarray data set were also upregulated in response to BCR stimulation in follow up experiments. MHC Class II expression was found to be maintained specifically in response to BCR stimulation. Furthermore, ligand-specific internalization of the BCR, a first step in B cell antigen processing and presentation, was demonstrated. CONCLUSION: These observations provide experimental validation of the computational approach implemented in CLASSIFI, demonstrating that CLASSIFI-based gene expression cluster analysis is an effective data mining tool to identify biological processes that correlate with the experimental conditional variables. Furthermore, this analysis has identified at least thirty-eight candidate components of the B cell antigen processing and presentation pathway and sets the stage for future studies focused on a better understanding of the components involved in and unique to B cell antigen processing and presentation.

Fast determination of structurally cohesive subgroups in large networks.

Structurally cohesive subgroups are a powerful and mathematically rigorous way to characterize network robustness. Their strength lies in the ability to detect strong connections among vertices that not only have no neighbors in common, but that may be distantly separated in the graph. Unfortunately, identifying cohesive subgroups is a computationally intensive problem, which has limited empirical assessments of cohesion to relatively small graphs of at most a few thousand vertices. We describe here an approach that exploits the properties of cliques, k-cores and vertex separators to iteratively reduce the complexity of the graph to the point where standard algorithms can be used to complete the analysis. As a proof of principle, we apply our method to the cohesion analysis of a 29,462-vertex biconnected component extracted from a 128,151-vertex co-authorship data set.