Erforschung der Einsatzmöglichkeiten von B Zellen für die Diagnose der Borreliose : Investigation of the possible use of B cells for the diagnosis of acute Lyme disease

Die Fehldiagnose der Borreliose bleibt noch immer ein relevantes Problem. Große Auseinandersetzungen über die Definition und Ursachen des chronischen Verlaufs der Krankheit sind noch immer aktuell. Die nur geringe Anzahl an Borrelien in Patientenproben erschwert den direkten Nachweis des Krankheitserregers. Aus diesem Grund basieren diagnostische Tests auf indirekten Nachweismethoden. Der beste und einzig offiziell anerkannte Test zur Unterstützung der Diagnose der Borreliose bleibt der Nachweis von spezifischen Antikörpern. Obwohl heutige Versionen sehr gut optimiert wurden und ziemlich zuverlässig sind, ermöglichen es diese Tests nicht, eine akute von einer geheilten Krankheit zu unterscheiden. Wegen ihrer Spezifität für Antigene sind B Zellen sehr interessante Kandidaten für neue Biomarker. Im Gegensatz zu den Antikörpern, die noch lange nach dem Immunkontakt nachweislich bleiben können, sollten die B Zellen nur während einer akuten Infektion erhöht sein. Das Ziel dieser Studie war es zu zeigen, dass durch Borrelien hervorgerufene B Zellen im peripheren Blut von akuten Patienten nachweislich sind, und zu testen, inwiefern ähnliche Zellen in verschiedenen Patienten vorkommen. Unsere Resultate stimmen mit denen von anderen Studien überein und zeigen, dass im Vergleich zu zufällig gewählten Individuen eine größere Wahrscheinlichkeit besteht, überlappende B Zell Klone zwischen Patienten, die dem gleichen Immun Stimulus ausgesetzt waren, zu finden. Obwohl wir die Patienten an unterschiedlichen Zeitpunkten untersucht haben, konnten wir eine größere Überlappung zwischen unseren Patienten feststellen. Unsere Daten zeigen allerdings, dass die überlappenden Klone in sehr unterschiedlichem Ausmaß in den verschiedenen Patienten vorkommen. Die Klone, die in den einzelnen Proben dominierten, waren eher Patienten spezifisch. Dies stimmt ganz gut mit der dynamischen Natur der B Zell Immunantwort überein. Da das Auftreten von identischen B Zell Rezeptor Sequenzen in verschiedenen Patienten eher selten war, wollten wir testen, ob weniger restriktive Gruppierungskriterien es ermöglichen, Sequenzen in Kategorien nach der gleichen Epitop Reaktivität einzuteilen. Es stellte sich heraus, dass die Sequenzen wahrscheinlich zu unterschiedlich sind, als dass Sie in Gruppen mit der gleichen Epitop Reaktivität eingeteilt werden können. Um dies zu erreichen müssen ausgefeiltere bioinformatische Skripte entwickelt werden. Diese müssten Mutationen an Schlüsselstellen in der Antikörpersequenz sowie die dreidimensionale Struktur des Antikörpermoleküls mit einbeziehen. Unsere Befunde deuten darauf hin, dass die gesuchten B Zell Signaturen komplexer sind als initial erwartet. Es wird wahrscheinlich nicht möglich sein, ein paar Schlüssel CDR3 Aminosäure Sequenzen zu isolieren. Wir erwarten uns eher eine Liste an verschiedenen Borrelia-spezifischen B Zell Klonen, die durch Einzelmutationen an Schlüsselstellen von Klonen anderer Reaktivität unterscheidbar sind. Wegen der Polyspezifität und der dynamischen Natur der B Zell Immunantwort wird wahrscheinlich eine Kombination von verschiedenen Klonen gleichzeitig nachgewiesen werden müssen. Das Matchen von B Zell Klonen mit ihren jeweiligen Antigenen, zusammen mit der Analyse vorkommender Mutationen und des B Zell Rezeptor Isotyps, sowie die phylogenetische Verwandtschaft der einzelnen Sequenzen dieser Klone hat das Potenzial, laufende (primäre und sekundäre) von vergangenen oder autoimmun Reaktionen zu unterscheiden. Dies könnte sehr hilfreich sein um einige der Kontroversen über den chronischen Verlauf der Borreliose aufzuklären.Misdiagnosis of acute Lyme disease still remains a huge problem. Also many debates are going on about the definition and underlying causes of the chronic course of this disease. Due to the scarcity of Borrelia in patient samples, direct detection of the pathogen is challenging. Diagnostic laboratories need to work with indirect approaches to prove the presence of Borrelia inside patients. The best and currently only accepted test to support the diagnosis of acute Lyme disease infection is the detection of Borrelia-reactive antibodies. Although extensive optimization has led to the development of specific and quite reliable tests to prove the presence of these molecules, they have the limitation that they do not enable to directly distinguish an ongoing from a previous infection. Since B cells are the cells of our immune system that react most specifically to immune challenges, they are very interesting candidates to be explored for biomarker research. As opposed to the antibodies, which can stay in peripheral blood for prolonged times even after clearance of the infection, Borrelia-reactive B cells should only be elevated when the pathogen is present. The main goal of this study was to prove the presence of Borrelia-reactive B cells in peripheral blood of acute Lyme disease patients and to assess to what extent expanded clones overlap between patients. Our results are in line with other B cell repertoire studies, indicating that individuals that have been challenged with the same immune stimulus have a higher chance to present overlapping clones as compared to other randomly selected donors. Since we were not able to use precise timepoints in the case of acute disease, it was quite surprising to find an increased overlap of B cell clones in our patients. Our data indicate, that overlapping clones appear at heterogeneous levels between donors. Rather patient unique B cell clones are predominating individual repertoires. This is in accordance with the rather dynamic nature of B cell immune responses. Since the occurrence of identical B cell receptor sequences in different patients was a rare event, we explored whether sequence clustering using less stringent parameters would allow to extract B cell clones reacting towards the same epitope. Differences in sequences between clones were however too large to allow reliable grouping of sequences according to epitope-reactivity. For this purpose, more sophisticated bioinformatics clustering tools need to be developed. Those need to take mutations at key residues and three dimensional structures of antibody molecules into account. From our findings, we conclude, that B cell repertoire signatures might be more complex than initially imagined. We will probably not be able to isolate a few key CDR3 amino acid signature candidates, but we will end up with a rather large list of Borrelia-reactive B cell clones that can be distinguished from B cells reactive towards other antigens by the presence of single key mutations. The polyspecific and dynamic nature of the B cell immune response will probably require to prove the presence of certain combinations of clones. Matching of B cell clones with their corresponding antigens and analysis of mutation levels, isotypes and phylogenetic relationships among sequences from these clones has the potential to allow to distinguish ongoing (primary and secondary) from past immune responses and autoimmune diseases. This might help to solve some of the controversies about post treatment Lyme disease

Diagnosing Borreliosis

Borrelia species fall into two groups, the Borrelia burgdorferi sensu lato (Bbsl) complex, the cause of Lyme borreliosis (also known as Lyme disease), and the relapsing fever group. Both groups exhibit inter- and intraspecies diversity and thus have variations in both clinical presentation and diagnostic approaches. A further layer of complexity is derived from the fact that ticks may carry multiple infectious agents and are able to transmit them to the host during blood feeding, with potential overlapping clinical manifestations. Besides this, pathogens like Borrelia have developed strategies to evade the host immune system, which allows them to persist within the host, including humans. Diagnostics can be applied at different times during the clinical course and utilize sample types, each with their own advantages and limitations. These differing methods should always be considered in conjunction with potential exposure and compatible clinical features. Throughout this review, we aim to explore different approaches providing the reader with an overview of methods appropriate for various situations. This review will cover human pathogenic members of Bbsl and relapsing fever borreliae, including newly recognized Borrelia miyamotoi spirochetes

Detection of a Low Level and Heterogeneous B Cell Immune Response in Peripheral Blood of Acute Borreliosis Patients With High Throughput Sequencing

The molecular diagnosis of acute Borreliosis is complicated and better strategies to improve the diagnostic processes are warranted. High Throughput Sequencing (HTS) of human B cell repertoires after e.g., Dengue virus infection or influenza vaccination revealed antigen-associated “CDR3 signatures” which may have the potential to support diagnosis in infectious diseases. The human B cell immune response to Borrelia burgdorferi sensu lato—the causative agent of Borreliosis—has mainly been studied at the antibody level, while less attention has been given to the cellular part of the humoral immune response. There are indications that Borrelia actively influence the B cell immune response and that it is therefore not directly comparable to responses induced by other infections. The main goal of this study was to identify B cell features that could be used to support diagnosis of Borreliosis. Therefore, we characterized the B cell immune response in these patients by combining multicolor flow cytometry, single Borrelia-reactive B cell receptor (BCR) sequencing, and B cell repertoire deep sequencing. Our phenotyping experiments showed, that there is no significant difference between B cell subpopulations of acute Borreliosis patients and controls. BCR sequences from individual epitope-reactive B cells had little in common between each other. HTS showed, however, a higher complementarity determining region 3 (CDR3) amino acid (aa) sequence overlap between samples from different timepoints in patients as compared to controls. This indicates, that HTS is sensitive enough to detect ongoing B cell immune responses in these patients. Although each individual's repertoire was dominated by rather unique clones, clustering of bulk BCR repertoire sequences revealed a higher overlap of IgG BCR repertoire sequences between acute patients than controls. Even if we have identified a few Borrelia-associated CDR3aa sequences, they seem to be rather unique for each patient and therefore not suitable as biomarkers

Applying Unique Molecular Identifiers in Next Generation Sequencing Reveals a Constrained Viral Quasispecies Evolution under Cross-Reactive Antibody Pressure Targeting Long Alpha Helix of Hemagglutinin

To overcome yearly efforts and costs for the production of seasonal influenza vaccines, new approaches for the induction of broadly protective and long-lasting immune responses have been developed in the past decade. To warrant safety and efficacy of the emerging crossreactive vaccine candidates, it is critical to understand the evolution of influenza viruses in response to these new immune pressures. Here we applied unique molecular identifiers in next generation sequencing to analyze the evolution of influenza quasispecies under in vivo antibody pressure targeting the hemagglutinin (HA) long alpha helix (LAH). Our vaccine targeting LAH of hemagglutinin elicited significant seroconversion and protection against homologous and heterologous influenza virus strains in mice. The vaccine not only significantly reduced lung viral titers, but also induced a well-known bottleneck effect by decreasing virus diversity. In contrast to the classical bottleneck effect, here we showed a significant increase in the frequency of viruses with amino acid sequences identical to that of vaccine targeting LAH domain. No escape mutant emerged after vaccination. These results not only support the potential of a universal influenza vaccine targeting the conserved LAH domains, but also clearly demonstrate that the well-established bottleneck effect on viral quasispecies evolution does not necessarily generate escape mutants