The Future of Blood Testing Is the Immunome

It is increasingly clear that an extraordinarily diverse range of clinically important conditions-including infections, vaccinations, autoimmune diseases, transplants, transfusion reactions, aging, and cancers-leave telltale signatures in the millions of V(D)J-rearranged antibody and T cell receptor [TR per the Human Genome Organization (HUGO) nomenclature but more commonly known as TCR] genes collectively expressed by a person's B cells (antibodies) and T cells. We refer to these as the immunome. Because of its diversity and complexity, the immunome provides singular opportunities for advancing personalized medicine by serving as the substrate for a highly multiplexed, near-universal blood test. Here we discuss some of these opportunities, the current state of immunome-based diagnostics, and highlight some of the challenges involved. We conclude with a call to clinicians, researchers, and others to join efforts with the Adaptive Immune Receptor Repertoire Community (AIRR-C) to realize the diagnostic potential of the immunome

Computational strategies for dissecting the high-dimensional complexity of adaptive immune repertoires

The adaptive immune system recognizes antigens via an immense array of antigen-binding antibodies and T-cell receptors, the immune repertoire. The interrogation of immune repertoires is of high relevance for understanding the adaptive immune response in disease and infection (e.g., autoimmunity, cancer, HIV). Adaptive immune receptor repertoire sequencing (AIRR-seq) has driven the quantitative and molecular-level profiling of immune repertoires thereby revealing the high-dimensional complexity of the immune receptor sequence landscape. Several methods for the computational and statistical analysis of large-scale AIRR-seq data have been developed to resolve immune repertoire complexity in order to understand the dynamics of adaptive immunity. Here, we review the current research on (i) diversity, (ii) clustering and network, (iii) phylogenetic and (iv) machine learning methods applied to dissect, quantify and compare the architecture, evolution, and specificity of immune repertoires. We summarize outstanding questions in computational immunology and propose future directions for systems immunology towards coupling AIRR-seq with the computational discovery of immunotherapeutics, vaccines, and immunodiagnostics.Comment: 27 pages, 2 figure

sumrep: a summary statistic framework for immune receptor repertoire comparison and model validation

In studying the binding of host antibodies to the surface antigens of pathogens, the structural and functional characterization of antibody–antigen complexes by X-ray crystallography and binding assay is important. However, the characterization requires experiments that are typically time consuming and expensive: thus, many antibody–antigen complexes are under-characterized. For vaccine development and disease surveillance, it is often vital to assess the impact of amino acid substitutions on antibody binding. For example, are there antibody substitutions capable of improving binding without a loss of breadth, or antigen substitutions that lead to antigenic escape? The questions cannot be answered reliably from sequence variation alone, exhaustive substitution assays are usually impractical, and alanine scans provide at best an incomplete identification of the critical residue–residue interactions. Here, we show that, given an initial structure of an antibody bound to an antigen, molecular dynamics simulations using the energy method molecular mechanics with Generalized Born surface area (MM/GBSA) can model the impact of single amino acid substitutions on antibody–antigen binding energy. We apply the technique to three broad-spectrum antibodies to influenza A hemagglutinin and examine both previously characterized and novel variant strains observed in the human population that may give rise to antigenic escape. We find that in some cases the impact of a substitution is local, while in others it causes a reorientation of the antibody with wide-ranging impact on residue–residue interactions: this explains, in part, why the change in chemical properties of a residue can be, on its own, a poor predictor of overall change in binding energy. Our estimates are in good agreement with experimental results—indeed, they approximate the degree of agreement between different experimental techniques. Simulations were performed on commodity computer hardware; hence, this approach has the potential to be widely adopted by those undertaking infectious disease research. Novel aspects of this research include the application of MM/GBSA to investigate binding between broadly binding antibodies and a viral glycoprotein; the development of an approach for visualizing substrate–ligand interactions; and the use of experimental assay data to rescale our predictions, allowing us to make inferences about absolute, as well as relative, changes in binding energy

The Global Impact of Science Gateways, Virtual Research Environments and Virtual Laboratories

Science gateways, virtual laboratories and virtual research environments are all terms used to refer to community-developed digital environments that are designed to meet a set of needs for a research community. Specifically, they refer to integrated access to research community resources including software, data, collaboration tools, workflows, instrumentation and high-performance computing, usually via Web and mobile applications. Science gateways, virtual laboratories and virtual research environments are enabling significant contributions to many research domains, facilitating more efficient, open, reproducible research in bold new ways. This paper explores the global impact achieved by the sum effects of these programs in increasing research impact, demonstrates their value in the broader digital landscape and discusses future opportunities. This is evidenced through examination of national and international programs in this field

Highly multiplexed immune repertoire sequencing links multiple lymphocyte classes with severity of response to COVID-19

BACKGROUND: Disease progression of subjects with coronavirus disease 2019 (COVID-19) varies dramatically. Understanding the various types of immune response to SARS-CoV-2 is critical for better clinical management of coronavirus outbreaks and to potentially improve future therapies. Disease dynamics can be characterized by deciphering the adaptive immune response. METHODS: In this cross-sectional study we analyzed 117 peripheral blood immune repertoires from healthy controls and subjects with mild to severe COVID-19 disease to elucidate the interplay between B and T cells. We used an immune repertoire Primer Extension Target Enrichment method (immunoPETE) to sequence simultaneously human leukocyte antigen (HLA) restricted T cell receptor beta chain (TRB) and unrestricted T cell receptor delta chain (TRD) and immunoglobulin heavy chain (IgH) immune receptor repertoires. The distribution was analyzed of TRB, TRD and IgH clones between healthy and COVID-19 infected subjects. Using McFadden's Adjusted R2 variables were examined for a predictive model. The aim of this study is to analyze the influence of the adaptive immune repertoire on the severity of the disease (value on the World Health Organization Clinical Progression Scale) in COVID-19. FINDINGS: Combining clinical metadata with clonotypes of three immune receptor heavy chains (TRB, TRD, and IgH), we found significant associations between COVID-19 disease severity groups and immune receptor sequences of B and T cell compartments. Logistic regression showed an increase in shared IgH clonal types and decrease of TRD in subjects with severe COVID-19. The probability of finding shared clones of TRD clonal types was highest in healthy subjects (controls). Some specific TRB clones seems to be present in severe COVID-19 (Figure S7b). The most informative models (McFadden´s Adjusted R2=0.141) linked disease severity with immune repertoire measures across all three cell types, as well as receptor-specific cell counts, highlighting the importance of multiple lymphocyte classes in disease progression. INTERPRETATION: Adaptive immune receptor peripheral blood repertoire measures are associated with COVID-19 disease severity

Enhancement of the intestinal epithelial permeability of peripherally acting opioid analgesics by chitosan

Die schmerzstillende Wirkung von Opiaten wird über Opioidrezeptoren im zentralen und peripheren Nervensystem vermittelt. Die Schmerzlinderung kann jedoch mit sehr starken Nebenwirkungen einhergehen, die das Patientenwohlbefinden beeinträchtigen. Dies legt die Bedeutung von neuen Opioidanalgetika nahe, die ihre schmerzstillende Wirkung ausschließlich über Opioidrezeptoren im PNS entfalten, ohne unerwünschte zentrale Nebenwirkungen zu induzieren. Die orale Gabe von Medikamenten minimiert Unannehmlichkeiten für den Patienten, jedoch müssen die Substanzen die intestinale Barriere passieren können, um in die Blutzirkulation eintreten zu können. Die intestinale Permeabilität von zwei peripher wirksamen Opiaten (AS006 und Loperamid) wurde in Ussing-Kammer Experimenten untersucht. Um die Darmepithelpermeabilität für beide Opiate zu erhöhen, wurde der Absorptionsverstärker Chitosan verwendet. Chitosan bewirkte nach 30 Minuten bei HT29/B6 und Caco-2 Zelllinien eine Abnahme des epithelialen Widerstands in vitro. Die Permeabilität für AS006 war bei beiden Zelllinien erhöht, für Loperamid nur bei HT29/B6, jedoch nicht bei Caco-2 Zellmonolayern. Verhaltensexperimente zur Messung des antinozizeptiven Effektes von oral appliziertem Loperamid auf Entzündungsschmerz wurden an Ratten durchgeführt. Die orale Gabe von Loperamid induzierte eine Dosis-abhängige antinozizeptive Wirkung in der entzündeten Hinterpfote. Bei oraler Gabe von Loperamid in Kombination mit Chitosan wurde keine signifikante Verstärkung des maximalen antinozizeptiven Effekts von Loperamid beobachtet. Zusammenfassend ist Chitosan ein geeigneter Absorptionsverstärker für intestinale Permeabilitätsstudien von peripher wirksamen Opioidanalgetika in vitro. Die in vitro Ergebnisse haben gezeigt, dass der Effekt von Chitosan auf Loperamid möglicherweise schwächer ist als auf AS006. Dementsprechend fiel die Wirkung des Absorptionsverstärkers auf Loperamid-induzierte Analgesie im Verhaltensversuch eher gering aus.Analgesic effects of opioids are mediated by opioid receptors that are widely distributed in the central and peripheral nervous systems (CNS and PNS, respectively). Although opioids are the most powerful analgesics, severe side effects restrict their use and affect patient convalescence. This suggests an advantage of new analgesic opioids which selectively bind to opioid receptors in the PNS. After oral administration however, peripherally restricted opioids first have to cross the intestinal epithelial barrier before absorption into the circulation and distribution to opioid receptors in peripheral tissues. Here, the transport across intestinal epithelia of two opioid ligands (AS006 and loperamide) that selectively activate peripheral opioid receptors without entering the CNS were investigated. To increase the intestinal passage of these drugs, the absorption enhancer chitosan was used. Chitosan significantly decreased the transepithelial resistance of HT29/B6 and Caco-2 cell monolayers after 30 min in vitro. The permeability values for AS006 increased from < 0.3 × 10-6 cm/s up to 10 × 10-6 cm/s in the presence of chitosan. In contrast, HT29/B6 monolayers showed moderate loperamide permeability in the presence of chitosan, and chitosan had no effect on the permeability of loperamide using Caco-2 monolayers. Oral administration of loperamide induced a dose-depended elevation of paw pressure thresholds in inflamed paws that lasted for 60 min. Oral administration of loperamide combined with chitosan slightly but nonsignificantly enhanced the antinociceptive effect of loperamide. In conclusion, chitosan is a suitable absorption enhancer for in vitro intestinal permeability studies. Future in vivo experiments might investigate different formulations and application schedules, and further address the effects of chitosan on the antinociceptive efficacy of hydrophilic opioids

Erforschung des humanen Immunglobulinrepertoires mittels Hochdurchsatz- Sequenzierung

The main role of the adaptive immune system is to protect the body against pathogens. Immunoglobulins are an essential part of this system. The great diversity of immunoglobulins is obtained by a complex mechanism of genetic reorganization from a predetermined set of gene segments on chromosomal level and subsequent affinity maturation. Thereby a specific affinity for a particular pathogen is finally achieved. For a successful immune response to occur, the effector functions of the constant region of the immunoglobulin are also essential - which are different in all immunoglobulin classes. The latest innovations in DNA sequencing technology allow nowadays to analyze and evaluate immunological questions in respect to cell-bound (i.e. B-cell receptors) and secreted immunoglobulins from human donors on cDNA level. The technological innovation, which is generally referred to as "next generation sequencing" and especially the so-called pyro-sequencing laid the basis to the present study. The aim was to develop a method for sequencing antibody cDNA by pyro-sequencing, which allows all genes and all immunoglobulin classes to be quantitatively and qualitatively measurable. With this novel approach the immunoglobulin repertoire from peripheral blood samples of healthy donors of different ages and gender ought to be analyzed. The newly developed immunoglobulin specific cDNA amplification method established during this thesis is independent of the use of V-gene specific primers. It further allows the verification of the corresponding immunoglobulin classes and sub-groups in the obtained sequences. Through the introduction of a newly developed emulsion PCR method, the influences of the sample preparation and the cDNA amplification procedure on the representative quality of the obtained sequences were minimized. Furthermore, it was necessary to create a new amplicon processing protocol for the 454 Roche GS FLX sequencer to achieve compatibility with the amplicons generated in the previous antibody amplification process. Using the novel immunoglobulin-amplification and sequencing method, more than 3.5 million sequences were obtained from a representative group of 14 healthy individuals of different age and gender. At first, a comprehensive analysis of the distribution of V(D)J genes in the donor was performed. Some genes, which were not covered in previous studies, could be included in the study and their relative occurrence evaluated. Moreover, for the first time, the VDJ gene distribution in conjunction with their immunoglobulin class has been analyzed. The results of the analyses show that there are differences in the immunoglobulin repertoire of young (19-30 years) and elderly people (> 50 years). A reduction in the ability to change the classes of antibodies is observed and this correlates with age. This is in line with current studies, where the reduction in class switch is discussed as a cause for decreased vaccine efficacy in the elderly population. In this work, it is shown that the influence of age on the ability to change the immunoglobulin classes can be analyzed by cDNA sequencing. The results also strongly suggest that the senescence of the immune system begins in an age range between 50 and 60 years. A correlation between gender and the ability to change the classes of antibodies could, however, not be detected in this study. The developed methods for sequencing immunoglobulin repertoire allow entirely new insights into the immune system. Due to the improved experimental set-up, the complexity of the variables that can be analyzed from a single sample increases considerably. In future, changes of the immune system in healthy and diseased individuals can be measured and analyzed on a new, unrivaled level of complexity. The methods developed during my PhD and the obtained results provide a solid basis for future research addressing the analysis and verification of disease-specific changes in the immune system.Die wichtigste Rolle des adaptiven Immunsystems ist der Schutz des Körpers vor Pathogenen. Immunglobuline sind ein wesentlicher Bestandteil dieses Systems. Die große Vielfalt von Immunglobulinen wird durch einen komplexen Mechanismus der genetischen Reorganisation aus einem vorgegebenen Satz von Gensegmenten auf chromosomaler Ebene und anschließender Affinitätsreifung erhalten. Dadurch wird letztlich eine spezifische Affinität für ein bestimmtes Pathogen erreicht. Für eine erfolgreiche Immunantwort sind außerdem die Effektor- Funktionen der konstanten Region des Immunoglobulins, die in allen Immunglobulin-Klassen unterschiedlich sind, entscheidend. Die jüngsten Innovationen in der Sequenz-Analyse von DNA erlauben seit kurzem, zellgebundene (B-Zell-Rezeptoren) und sezernierte Immunglobuline von menschlichen Spendern im großen Umfang auf cDNA-Ebene zu sequenzieren und immunologisch zu bewerten. Diese technologischen Innovationen, die generell als „Next-Generation-Sequencing“ bezeichnet werden und speziell die der sogenannten Pyro-Sequenzierung bildeten die Basis der vorliegenden Untersuchung. Ziel war die Entwicklung eines Verfahrens zur Sequenzierung von Antikörpern durch Pyro-Sequenzierung von cDNA, das alle Gene sowie alle Immunglobulin-Klassen quantitativ und qualitativ messbar macht. Dafür sollte das Immunglobulin-Repertoire aus peripheren Blutproben von gesunden Spendern unterschiedlichen Alters und Geschlechts analysiert werden. Die in dieser Arbeit neu entwickelte Immunglobulin-spezifische cDNA-Amplifikationsmethode ist unabhängig von der Verwendung von Primern, die für V-Gene spezifisch sind. Sie ermöglicht zudem die Verifizierung der zugehörigen Immunglobulin-Klassen und ihrer Untergruppen in den erhaltenen Sequenzen. Der Einfluss der Vorbereitung auf die repräsentative Qualität der Proben und deren cDNA- Vervielfältigung wird durch eine entwickelte Emulsions-PCR-Methode minimiert. Des Weiteren war es notwendig, ein neues Amplicon-Aufarbeitungsverfahren für den 454 Roche GS FLX Sequenzer zu erstellen, um Kompatibilität mit den vorangegangenen Schritten der Amplicon-Generierung zu erreichen. Mittels der neuartigen Immunglobulin-Amplifikations- und Sequenzier-Methode konnten aus einer repräsentativen Gruppe von 14 gesunden Individuen unterschiedlichen Alters und Geschlechts mehr als 3,5 Millionen Sequenzen gewonnen werden. Im ersten Schritt wurde eine umfassende Analyse der Verteilung der V(D)J-Genen innerhalb der Spender durchgeführt. Dabei konnten auch Gene, die in früheren Studien nicht abgedeckt wurden, in die Untersuchung einbezogen werden und deren relatives Vorkommen evaluiert werden. Überdies wurde erstmalig die VDJ- Gen-Verteilung im Zusammenhang mit deren Immunglobulin-Klasse analysiert. Das Ergebnis der Analyse zeigt, dass junge (19-30 Jahre) und ältere Menschen (>50 Jahre) sich in Bezug auf ihr jeweiliges Immunglobulin-Repertoir unterschiedlich verhalten. Die Reduktion der Fähigkeit zum Wechsel der Antikörperklassen korreliert mit dem Alter – in aktuellen Untersuchungen wird dies als Ursache für verringerte Impfstoffwirksamkeit bei älteren Menschen diskutiert. In dieser Arbeit wird deutlich, dass der Einfluss des Alters auf die Fähigkeit zum Wechsel der Antikörperklassen durch Immunglobulin- Sequenzierung analysiert werden kann. Die Ergebnisse lassen zudem darauf schließen, dass in einem Alter zwischen 50 und 60 Jahren die Seneszenz des Immunsystems beginnt. Eine Korrelation zwischen Geschlecht und Fähigkeit zum Wechsel der Antikörperklassen konnte in dieser Untersuchung dagegen nicht nachgewiesen werden. Durch die hier entwickelten Verfahren zur Immunglobulin- Sequenzierung ergeben sich ganz neue Erkenntnisse in Bezug auf das Immunsystem. Aufgrund des verbesserten Versuchsaufbaus hat sich die Komplexität an Messgrößen, die aus einer einzigen Probe analysiert werden können, deutlich erhöht. In Zukunft können die Veränderungen des Immunsystems in Gesunden und Erkrankten in einer neuen, vorher unerreichten Komplexität gemessen und analysiert werden. Die im Rahmen meiner Promotion entwickelten Verfahren und die damit erhaltenen Ergebnisse bieten eine solide Grundlage für zukünftige wissenschaftliche Fragestellungen, die der Entdeckung und Verifizierung krankheitsspezifischer Veränderungen des Immunsystems dienen