Immunosequencing and Profiling of T Cells at the Maternal-Fetal Interface of Women with Preterm Labor and Chronic Chorioamnionitis.

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.

Computational identification of HCV neutralizing antibodies with a common HCDR3 disulfide bond motif in the antibody repertoires of infected individuals.

Despite recent success in hepatitis C virus (HCV) treatment using antivirals, an HCV vaccine is still needed to prevent reinfections in treated patients, to avert the emergence of drug-resistant strains, and to provide protection for people with no access to the antiviral therapeutics. The early production of broadly neutralizing antibodies (bNAbs) associates with HCV clearance. Several potent bNAbs bind a conserved HCV glycoprotein E2 epitope using an unusual heavy chain complementarity determining region 3 (HCDR3) containing an intra-loop disulfide bond. Isolation of additional structurally-homologous bNAbs would facilitate the recognition of key determinants of such bNAbs and guide rational vaccine design. Here we report the identification of new antibodies containing an HCDR3 disulfide bond motif using computational screening with the Rosetta software. Using the newly-discovered and already-known members of this antibody family, we review the required HCDR3 amino acid composition and propose determinants for the bent versus straight HCDR3 loop conformation observed in these antibodies.

AIRR Community Guide to Planning and Performing AIRR-Seq Experiments.

The development of high-throughput sequencing of adaptive immune receptor repertoires (AIRR-seq of IG and TR rearrangements) has provided a new frontier for in-depth analysis of the immune system. The last decade has witnessed an explosion in protocols, experimental methodologies, and computational tools. In this chapter, we discuss the major considerations in planning a successful AIRR-seq experiment together with basic strategies for controlling and evaluating the outcome of the experiment. Members of the AIRR Community have authored several chapters in this edition, which cover step-by-step instructions to successfully conduct, analyze, and share an AIRR-seq project.

Bulk Sequencing from mRNA with UMI for Evaluation of B-Cell Isotype and Clonal Evolution: A Method by the AIRR Community.

During the course of an immune response to a virus such as influenza, B cells undergo activation, clonal expansion, isotype switching, and somatic hypermutation (SHM). Members of an antigen-experienced B-cell clone can have different sequence features including SHM in the immunoglobulin heavy-chain V (IGHV) gene and can use the same IGVH gene in combination with different constant regions or isotypes (e.g., IgM, IgG, IgA). To study these features of expanded clones in an immune response by AIRR-seq, we provide a bulk RNA-based sequencing experimental procedure with unique molecular identifiers (UMIs) and the accompanying bioinformatics analytical workflow.

Rapid discovery of diverse neutralizing SARS-CoV-2 antibodies from large-scale synthetic phage libraries.

Coronavirus disease 2019 (COVID-19) is an evolving global public health crisis in need of therapeutic options. Passive immunization of monoclonal antibodies (mAbs) represents a promising therapeutic strategy capable of conferring immediate protection from SARS-CoV-2 infection. Herein, we describe the discovery and characterization of neutralizing SARS-CoV-2 IgG and VHH antibodies from four large-scale phage libraries. Each library was constructed synthetically with shuffled complementarity-determining region loops from natural llama and human antibody repertoires. While most candidates targeted the receptor-binding domain of the S1 subunit of SARS-CoV-2 spike protein, we also identified a neutralizing IgG candidate that binds a unique epitope on the N-terminal domain. A select number of antibodies retained binding to SARS-CoV-2 variants Alpha, Beta, Gamma, Kappa and Delta. Overall, our data show that synthetic phage libraries can rapidly yield SARS-CoV-2 S1 antibodies with therapeutically desirable features, including high affinity, unique binding sites, and potent neutralizing activity in vitro, and a capacity to limit disease in vivo.

Canonical features of human antibodies recognizing the influenza hemagglutinin trimer interface.

Broadly reactive antibodies targeting the influenza A virus hemagglutinin (HA) head domain are thought to be rare and to require extensive somatic mutations or unusual structural features to achieve breadth against divergent HA subtypes. Here we describe common genetic and structural features of protective human antibodies from several individuals recognizing the trimer interface (TI) of the influenza A HA head, a recently identified site of vulnerability. We examined the sequence of TI-reactive antibodies, determined crystal structures for TI antibody-antigen complexes, and analyzed the contact residues of the antibodies on HA to discover common genetic and structural features of TI antibodies. Our data reveal that many TI antibodies are encoded by a light chain variable gene segment incorporating a shared somatic mutation. In addition, these antibodies have a shared acidic residue in the heavy chain despite originating from diverse heavy chain variable gene segments. These studies show that the TI region of influenza A HA is a major antigenic site with conserved structural features that are recognized by a common human B cell public clonotype. The canonical nature of this antibody-antigen interaction suggests that the TI epitope might serve as an important target for structure-based vaccine design.

Biological controls for standardization and interpretation of adaptive immune receptor repertoire profiling.

Use of adaptive immune receptor repertoire sequencing (AIRR-seq) has become widespread, providing new insights into the immune system with potential broad clinical and diagnostic applications. However, like many high-throughput technologies, it comes with several problems, and the AIRR Community was established to understand and help solve them. We, the AIRR Community's Biological Resources Working Group, have surveyed scientists about the need for standards and controls in generating and annotating AIRR-seq data. Here, we review the current status of AIRR-seq, provide the results of our survey, and based on them, offer recommendations for developing AIRR-seq standards and controls, including future work.

ClonoMatch: a tool for identifying homologous immunoglobulin and T-cell receptor sequences in large databases.

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.

Integrated pipeline for the accelerated discovery of antiviral antibody therapeutics.

The emergence and re-emergence of highly virulent viral pathogens with the potential to cause a pandemic creates an urgent need for the accelerated discovery of antiviral therapeutics. Antiviral human monoclonal antibodies (mAbs) are promising candidates for the prevention and treatment of severe viral diseases, but their long development timeframes limit their rapid deployment and use. Here, we report the development of an integrated sequence of technologies, including single-cell mRNA-sequence analysis, bioinformatics, synthetic biology and high-throughput functional analysis, that enables the rapid discovery of highly potent antiviral human mAbs, the activity of which we validated in vivo. In a 78-d study modelling the deployment of a rapid response to an outbreak, we isolated more than 100 human mAbs that are specific to Zika virus, assessed their function, identified that 29 of these mAbs have broadly neutralizing activity, and verified the therapeutic potency of the lead candidates in mice and non-human primate models of infection through the delivery of an antibody-encoding mRNA formulation and of the respective IgG antibody. The pipeline provides a roadmap for rapid antibody-discovery programmes against viral pathogens of global concern.

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.

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.

Diverse patterns of antibody variable gene repertoire disruption in patients with amyloid light chain (AL) amyloidosis.

Immunoglobulin light chain amyloidosis is the most common form of systemic amyloidosis. AL amyloidosis is caused by a misfolded light chain produced by a clonal population of plasma cells. Disease status currently is defined by measuring the absolute quantity of serum free light chain protein, but this measurement often fails to identify the subclinical presence of clonal cells that may merit additional therapy. Next generation sequencing has the sensitivity to measure the relative amount of dominating light chains within the repertoire of a patient, and this technique is in clinical use to identify clonal populations of plasma cells for multiple myeloma, a related disorder. In this proof-of-concept study, we used bone marrow aspirates of AL amyloidosis positive patients and used reverse transcription of the antibody transcriptome followed by next generation sequencing to identify antibody variable-diversity-joining gene sequences for patients with immunoglobulin light chain amyloidosis, and demonstrate that this technology can be used to identify the dominant clone. The data also reveal differing patterns of overall antibody repertoire disruption in different patients. This method merits further study in larger prospective studies to establish its utility in detecting residual disease for patients with immunoglobulin light chain amyloidosis.

Human-likeness of antibody biologics determined by back-translation and comparison with large antibody variable gene repertoires.

The antibody (Ab) germline gene rearrangement of variable (V), diversity (D), and joining (J) gene segments, as well as somatic hypermutation, give rise to the human Ab variable gene sequence repertoire. It is common to characterize single nucleotide frequencies of the variable region by alignment to species-specific wildtype germline genes. The increasing application of next-generation sequencing to immune repertoire studies has led to the compilation of increasing large adaptive immunome receptor repertoire datasets. We have developed a method that maps the sequence of a target Ab onto an immunome dataset of 326 million human Ab sequences. For this purpose, we created a position- and gene-specific scoring matrix (PGSSM) and its corresponding antibody similarity score. We characterized our PGSSM score and found that it strongly correlated with the phylogenetic distance of 181,355 Ab sequences from GenBank across 20 species. The most likely human nucleotide back-translation was obtained given only PGSSMs and the amino acid sequence of an Ab achieving a nucleotide sequence recovery of 95.9% and 97.2% for human heavy and light chains, respectively. In conclusion, the scoring of our back-translation is a valuable estimate for the similarity of an Ab sequence to the natural human repertoire. As expected, Ab therapeutic molecules developed from a human source showed a higher similarity to the repertoire than engineered Abs. Thus, the PGSSM metric introduced here can be used to engineer human-like Ab therapeutics.

Immune repertoire fingerprinting by principal component analysis reveals shared features in subject groups with common exposures.

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.

Glycosylator: a Python framework for the rapid modeling of glycans.

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.

Human VH1-69 Gene-Encoded Human Monoclonal Antibodies against Staphylococcus aureus IsdB Use at Least Three Distinct Modes of Binding To Inhibit Bacterial Growth and Pathogenesis.

Staphylococcus aureus is an important human pathogen that infects nearly every human tissue. Like most organisms, the acquisition of nutrient iron is necessary for its survival. One route by which it obtains this metal is through the iron-regulated surface determinant (Isd) system that scavenges iron from the hemoglobin of the host. We show that the heavy chain variable region IGHV1-69 gene commonly encodes human monoclonal antibodies (mAbs) targeting IsdB-NEAT2. Remarkably, these antibodies bind to multiple antigenic sites. One class of IGHV1-69-encoded mAbs blocks S. aureus heme acquisition by binding to the heme-binding site of NEAT2, while two additional classes reduce the bacterial burden in vivo by an alternative Fc receptor-mediated mechanism. We further identified clonal lineages of IGHV1-69-encoded mAbs using donor samples, showing that each lineage diversifies during infection by somatic hypermutation. These studies reveal that IGHV1-69-encoded antibodies contribute to a protective immune response, furthering our understanding of the correlates of protection against S. aureus infection.IMPORTANCE The human pathogen Staphylococcus aureus causes a wide range of infections, including skin abscesses and sepsis. There is currently no licensed vaccine to prevent S. aureus infection, and its treatment has become increasingly difficult due to antibiotic resistance. One potential way to inhibit S. aureus pathogenesis is to prevent iron acquisition. The iron-regulated surface determinant (Isd) system has evolved in S. aureus to acquire hemoglobin from the human host as a source of heme-iron. In this study, we investigated the molecular and structural basis for antibody-mediated correlates against a member of the Isd system, IsdB. The association of immunoglobulin heavy chain variable region IGHV1-69 gene-encoded human monoclonal antibodies with the response against S. aureus IsdB is described using structural and functional studies to define the importance of this antibody class. We also determine that somatic hypermutation in the development of these antibodies hinders rather than fine-tunes the immune response to IsdB.

Prolonged evolution of the memory B cell response induced by a replicating adenovirus-influenza H5 vaccine.

Induction of an antibody response capable of recognizing highly diverse strains is a major obstacle to the development of vaccines for viruses such as HIV and influenza. Here, we report the dynamics of B cell expansion and evolution at the single-cell level after vaccination with a replication-competent adenovirus type 4 recombinant virus expressing influenza H5 hemagglutinin. Fluorescent H1 or H5 probes were used to quantitate and isolate peripheral blood B cells and their antigen receptors. We observed increases in H5-specific antibody somatic hypermutation and potency for several months beyond the period of active viral replication that was not detectable at the serum level. Individual broad and potent antibodies could be isolated, including one stem-specific antibody that is part of a new multidonor class. These results demonstrate prolonged evolution of the B cell response for months after vaccination and should be considered in efforts to evaluate or boost vaccine-induced immunity.

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.

Role of humoral immunity against hepatitis B virus core antigen in the pathogenesis of acute liver failure.

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.

Structural basis for broad neutralization of ebolaviruses by an antibody targeting the glycoprotein fusion loop.

The severity of the 2014-2016 ebolavirus outbreak in West Africa expedited clinical development of therapeutics and vaccines though the countermeasures on hand were largely monospecific and lacked efficacy against other ebolavirus species that previously emerged. Recent studies indicate that ebolavirus glycoprotein (GP) fusion loops are targets for cross-protective antibodies. Here we report the 3.72 Å resolution crystal structure of one such cross-protective antibody, CA45, bound to the ectodomain of Ebola virus (EBOV) GP. The CA45 epitope spans multiple faces of the fusion loop stem, across both GP1 and GP2 subunits, with ~68% of residues identical across > 99.5% of known ebolavirus isolates. Extensive antibody interactions within a pan-ebolavirus small-molecule inhibitor binding cavity on GP define this cavity as a novel site of immune vulnerability. The structure elucidates broad ebolavirus neutralization through a highly conserved epitope on GP and further enables rational design and development of broadly protective vaccines and therapeutics.