NS1 Specific CD8(+) T-Cells with Effector Function and TRBV11 Dominance in a Patient with Parvovirus B19 Associated Inflammatory Cardiomyopathy

Background: Parvovirus B19 (B19V) is the most commonly detected virus in endomyocardial biopsies (EMBs) from patients with inflammatory cardiomyopathy (DCMi). Despite the importance of T-cells in antiviral defense, little is known about the role of B19V specific T-cells in this entity. Methodology and Principal Findings: An exceptionally high B19V viral load in EMBs (115,091 viral copies/mg nucleic acids), peripheral blood mononuclear cells (PBMCs) and serum was measured in a DCMi patient at initial presentation, suggesting B19V viremia. The B19V viral load in EMBs had decreased substantially 6 and 12 months afterwards, and was not traceable in PBMCs and the serum at these times. Using pools of overlapping peptides spanning the whole B19V proteome, strong CD8(+) T-cell responses were elicited to the 10-amico-acid peptides SALKLAIYKA (19.7% of all CD8(+) cells) and QSALKLAIYK (10%) and additional weaker responses to GLCPHCINVG (0.71%) and LLHTDFEQVM (0.06%). Real-time RT-PCR of IFN gamma secretion-assay-enriched T-cells responding to the peptides, SALKLAIYKA and GLCPHCINVG, revealed a disproportionately high T-cell receptor Vbeta (TRBV) 11 expression in this population. Furthermore, dominant expression of type-1 (IFN gamma, IL2, IL27 and Tbet) and of cytotoxic T-cell markers (Perforin and Granzyme B) was found, whereas gene expression indicating type-2 (IL4, GATA3) and regulatory T-cells (FoxP3) was low. Conclusions: Our results indicate that B19V Ag-specific CD8(+) T-cells with effector function are involved in B19V associated DCMi. In particular, a dominant role of TRBV11 and type-1/CTL effector cells in the T-cell mediated antiviral immune response is suggested. The persistence of B19V in the endomyocardium is a likely antigen source for the maintenance of CD8(+) T-cell responses to the identified epitopes

Generation of cytomegalovirus specific T-cell lines from healthy and immunocompromised donors for adoptive therapy

Titelblatt und Verzeichnisse Einleitung Ziele der Arbeit Methoden Ergebnisse Diskussion Literaturverzeichnis Abkürzungen Eidesstattliche ErklärungIn der Transplantation stellen Zytomegalovirus- (CMV-) Infektionen ein ernstzunehmendes Problem dar und können sowohl das Überleben des Patienten als auch das des Transplantats negativ beeinflussen. Zur Behandlung von CMV- Komplikationen ist die medikamentöse antivirale Therapie die erste therapeutische Option. Eine langfristige Behandlung durch antivirale Substanzen ist aber aufgrund toxischer Nebenwirkungen sowie viraler Resistenzbildungen nur bedingt möglich. Die einzige Möglichkeit einen langfristigen Schutz vor CMV-Komplikationen zu gewährleisten, ist die Etablierung einer effektiven CMV-spezifischen Immunantwort. Bei transplantierten Patienten ist die natürliche Entwicklung einer protektiven Antwort durch die medikamentöse Immunsuppression, welche die Abstoßung des transplantierten Organs verhindern soll, stark erschwert. Darum setzen neuere Therapieansätze auf den adoptiven Transfer in vitro-manipulierter CMV- spezifischer T-Zelllinien zur Überwindung dieses iatrogenen Effektes. Obwohl klinische Studien die Sicherheit und Effektivität adoptiv transferierter T-Zelllinien in der Bekämpfung von CMV-Komplikationen belegten, kommen diese bis heute lediglich bei wenigen Patienten zur Anwendung. Dies ist vor allem durch den technisch aufwendigen Herstellungsprozess, sowie die mangelnde Übertragbarkeit vieler Generierungsmethoden in die klinische Praxis bedingt. Kürzlich wurde in unserer Arbeitsgruppe ein Protokoll entwickelt, das die Herstellung CMV-spezifischer T-Zelllinien aus gesunden Probanden ermöglicht. Dieses Protokoll basiert auf der Stimulation mit überlappenden Peptidpools, der anschließenden magnetischen Isolierung der Interferon-γ\- sezernierenden Zellen, sowie deren in vitro-Expansion. Dieses Protokoll überwindet viele Limitierungen bisheriger Herstellungsmethoden, da es für jeden HLA-Typ einsetzbar ist und keine Kenntnis von immundominanten Epitopen voraussetzt. Die vorliegende Arbeit beschäftigte sich mit der Weiterentwicklung und Verbesserung dieses Herstellungsprotokolls. Es konnte gezeigt werden, dass die beschriebene Methode für die Herstellung von CMV-spezifischen T-Zelllinien aus immunsupprimierten Patienten genutzt werden kann. Dadurch können Patienten nach solider Organstransplantation behandelt und somit die Anwendung der Methode um ein Vielfaches ausgeweitet werden. Des Weiteren wurden T-Zelllinien gegen mehrere virale Proteine aus demselben Spender generiert. Die dadurch erreichte breitere Antigenreaktivität sollte die Effizienz der adoptiven Immuntherapie weiter erhöhen. Abschließend wurde in dieser Arbeit die Übertragbarkeit der Generierungsmethode in die klinische Praxis demonstriert, und es konnte ein Patient mit CMV-spezifischen T-Zelllinien behandelt werden. Somit stellt das entwickelte Herstellungsprotokoll einen wichtigen Fortschritt in der adoptiven Immuntherapie dar und erlaubt eine bessere Versorgung von Patienten mit schweren CMV-Komplikationen. Des Weiteren ist zukünftig eine Übertragung dieser Methode auf die Behandlung anderer viraler Erkrankungen oder Tumoren denkbar.Transplant recipients are at increased risk for severe human cytomegalovirus (HCMV) infection, which impairs graft survival and increases patient mortality. Prolonged virostatic therapy is limited by its side effects and the development of viral resistance. Reconstitution of cellular immunity by adoptive transfer of HCMV-specific T cells is a safe and effective treatment option but is limited to haematopoietic stem cell transplantation. A novel protocol based on stimulation with overlapping peptide libraries followed by isolation of the IFN- γ secreting cells was developed in our group to generate HCMV specific T-cell lines from healthy volunteers. The aim of this study was to determine whether this protocol is applicable to generate functional HCMV- specific T cells from chronically immunosuppressed solid-organ-transplant (SOT) patients. Autologous CD4+/8+ T-cell lines directed against two immunodominant HCMV proteins were generated from the peripheral blood of SOT patients. Despite chronic immunosuppression, T-cell lines with HCMV-specific killing behavior were generated from 8/10 SOT patients. Cell lines recognized multiple epitope targets of the viral proteins and were oligoclonal at the T-cell receptor V-beta level. Additionally, the feasibility of the protocol to generate T-cell lines under clinical conditions was demonstrated and one patient was treated with HCMV specific T cells. These data indicate the potential of this procedure for application in clinical relevant situations, like treatment of SOT patients with HCMV infection resistant to virostatic therapy

Epidemiology and Risk Factors of Clostridioides difficile Infections in Germany: A Health Claims Data Analysis

Abstract Introduction Clostridioides difficile infection (CDI) is increasingly recognized as a public health threat at the community level in addition to being one of the most common causes of healthcare-associated infections. In Germany, the epidemiology of CDI is primarily informed by national hospital-based CDI surveillance. We used health claims data from Germany to obtain valuable insights on population-level disease burden and risk factors for CDI. Methods This was a retrospective cohort study using a representative sample from the InGef research database. Overall and age- and sex-stratified CDI incidence rates were estimated for German adults from 2013 to 2017 using different case definitions (i.e., main, broad, strict), and further stratified by setting (inpatient versus outpatient). Risk factors for CDI were assessed for the 2013–2016 period. Results The CDI incidence rate was high but declined by 15.3% from 2013 [141 (95% confidence interval, CI 137–145) cases/100,000 person-years] to 2017 [120 (95% CI 116–123)]. Annual CDI incidence rates were higher in female patients and the elderly. The most important risk factors for CDI were chronic inflammatory bowel disease [odds ratio (OR) 4.7, 95% CI 4.0–5.5], chemotherapy (OR 4.7, 95% CI 4.1–5.2), chronic kidney disease (OR 2.9, 95% CI 2.6–3.3), and ciprofloxacin receipt (OR 2.6, 95% CI 2.4–2.8). Conclusions Despite prevention strategies leading to declining incidence, CDI remains an important public health threat in Germany, with a high burden in the hospital setting and an outpatient epidemiology that is poorly understood. These findings, which are relevant both regionally and globally, can be used as a basis for further research on the full burden of CDI in Germany

Clonotype Analysis of Cytomegalovirus-Specific Cytotoxic T Lymphocytes

Cytotoxic T lymphocytes (CTL) control the replication of human cytomegalovirus (CMV). Previous studies assessed the clonotypic composition of CTL specific for individual immunodominant peptides within a certain HLA context. Such an approach has inherent limitations and may not assess the true clonal CTL response in vivo. Here, the clonotypic composition of CMV-specific CTL was determined in HLA-A2, CMV-seropositive kidney transplant recipients and healthy blood donors after stimulation of peripheral blood mononuclear cells with either a pp65 whole-peptide pool or a single immunodominant peptide. Even after stimulation with the whole peptide pool, CMV-specific CTL remained monoclonal or oligoclonal. Regarding intraindividual variation, the CDR3 motifs of the dominant clones were identical to those observed in CTL generated by the single immunodominant peptide. Sequencing of the CDR3 regions demonstrated significant interindividual variation; however, structural homology was observed for immunodominant clonotypes in three individuals. In conclusion, the highly focused T cell receptor repertoire found after stimulation with either a single immunodominant peptide or a peptide pool demonstrates a pivotal role for immunodominant epitopes in the generation of a clonal repertoire. These results provide new insights into the regulation of CMV clonal dominance and may contribute to the design and monitoring of adoptive immunotherapy

Comparative proteome analysis of culture supernatant proteins from virulent Mycobacterium tuberculosis H37Rv and attenuated M-bovis BCG Copenhagen

A comprehensive analysis of culture supernatant (CSN) proteins of Mycobacterium tuberculosis H37Rv was accomplished by combination of two-dimensional electrophoresis (2-DE), mass spectrometry, and N-terminal sequencing by Edman degradation. Analytical 2-DE gels resolved approximately 1250 protein spots from CSN of M. tuberculosis H37Rv, 381 of which were identified by mass spectrometry and/or Edman degradation. This study revealed 137 different proteins, 42 of which had previously been described as secreted. Comparative proteome analysis of CSN from virulent M. tuberculosis H37Rv and attenuated Mycobacterium bovis BCG Copenhagen identified 39 M. tuberculosis-specific spots containing 27 different proteins, representing candidate antigens for novel vaccines and diagnostics in tuberculosis. These included five proteins encoded by open reading frames absent from M. bovis BCG, e.g., early secretory antigen target (Esat6), as well as 22 novel differential proteins, such as acetyl-CoA C-acetyltransferase (Rv0243) and two putative Esat6-like proteins (Rv1198, Rv1793)

Epidemiology of Clostridioides difficile Infections in Germany, 2010–2019: A Review from Four Public Databases

Abstract Introduction Clostridioides difficile infection (CDI) is a recognized global threat especially for vulnerable populations. It is of particular concern to healthcare providers as it is found in both hospital and community settings, with severe courses, frequent recurrence, high mortality and substantial financial impact on the healthcare system. The CDI burden in Germany has been described and compared by analysing data from four different public databases. Methods Data on hospital burden of CDI have been extracted, compared, and discussed from four public databases for the years 2010–2019. Hospital days due to CDI were compared to established vaccine preventable diseases, such as influenza and herpes zoster, and also to CDI hospitalisations in the United States (US). Results All four databases reported comparable incidences and trends. Beginning in 2010, population-based hospitalised CDI incidence increased to a peak of > 137/100,000 in 2013. Then, incidence declined to 81/100,000 in 2019. Hospitalised patients with CDI were predominantly > 50 years of age. The population-based incidence of severe CDI was between 1.4 and 8.4/100,000 per year. Recurrence rates were between 5.9 to 6.5%. More than 1,000 CDI deaths occurred each year, with a peak of 2,666 deaths in 2015. Cumulative CDI patient days (PD) were between 204,596 and 355,466 each year, which exceeded cumulated PD for influenza and herpes zoster in most years, though year-to-year differences were observed. Finally, hospitalized CDI incidence was higher in Germany than in the US, where the disease is well recognized as a public health threat. Conclusions All four public sources documented a decline in CDI cases since 2013, but the disease burden remains substantial and warrants continued attention as a severe public health challenge

Primers and probes of self-designed gene expression assays.

<p>Sequences of primers and fluorescence hybridization probes. For the different TRBV forward primers, one common reverse primer and one common hybridization probe were used. ^* For TRBV5, 6 and 7, wobbled (WBL) forward primers were designed.</p