Einleitung: Laut Schätzungen der WHO sind 150.000.000 Menschen mit einer chronischen Hepatitis C infiziert und 700.000 sterben jährlich an den Folgen der Erkrankung. Die antivirale Therapie der Vergangenheit wurde mittels Interferon alpha und Ribavirin durchgeführt. Diese Therapie war für die Patienten mit unerwünschten Arzneimittelwirkungen verbunden und führte in vielen Fällen nicht zu einem anhaltenden virologischen Ansprechen (SVR) nach Therapieende. Seit Einführung der Direct Acting Antivirals (DAA) können in der Therapie der chronischen Hepatitis C SVR-Raten von über 90 % erreicht werden. Therapieversagen sind auf Resistance Associated Mutations (RAM) in der NS3-, NS5A- oder NS5B-Region zurückzuführen. Fragestellung: Die Entwicklung einer Methode zur genotypspezifischen Resistenztestung, die neben Forschungsaspekten vor allem auch für den Einsatz in der virologischen Diagnostik geeignet ist, ist Gegenstand dieser Arbeit. Aufgrund der Häufigkeitsverteilung der HCV-Genotypen in Deutschland wurde die Entwicklung einer Resistenztestungsmethode auf die Genotypen 1b, 1a und 3a eingegrenzt. Im Fokus dieser Arbeit standen die NS3-, NS5A- und NS5B-Regionen. An diesen Regionen greifen die DAAs in den Replikationszyklus ein. Material und Methoden: Zur Durchführung der Resistenztestung wurde sich für die nested- bzw. seminested-PCR entschieden. Nach reverser Transkription der viralen RNA und anschließender Amplifikation der entstandenen cDNA wurde eine präparative Gelelektrophorese durchgeführt. Spezifische Banden wurden manuell extrahiert, aufbereitet und nach der Sanger-Methode sequenziert. Die Sequenzen wurden mit Geneious dargestellt und ggf. manuell nachbearbeitet. Mittels geno2pheno-Onlinedatenbank wurden die Sequenzen auf vorhandene Resistenzmutationen gegenüber DAA untersucht. Es wurden 45 archivierte Serumproben der Genotypen 1a, 1b und 3a mit sich unterscheidender Viruslast und Vortherapie ausgewählt, um die Methode unter möglichst realistischen Untersuchungsbedingungen zu testen. Ergebnisse: Es wurden genotyp- und genlocusspezifische Primersequenzen und PCR-Protokolle für die drei viralen Genregionen NS3, NS5A und NS5B entworfen. Eine single-step-PCR war in vielen Fällen zur erfolgreichen Amplifikation nicht ausreichend, aus diesem Grund wurde die Resistenztestung mittels (semi)nested-PCR durchgeführt. Je nach Genotyp und Region konnten 86 – 100 % der Proben erfolgreich amplifiziert und sequenziert werden. Eine anschließend durchgeführte Resistenzanalyse erlaubte die Untersuchung von Trends bezüglich des Zusammenhangs zwischen der Art der Vortherapie und dem Auftreten von Resistenzmutationen. Eine statistische Auswertung war nicht Ziel dieser Arbeit und wurde aufgrund der geringen Probenanzahl nicht durchgeführt. Diskussion: Es gelang in dieser Arbeit ein Protokoll zu entwickeln, welches sich für HCV-Resistenzanalysen mit Einsatz in der klinisch-virologischen Diagnostik der in Deutschland häufigsten Genotypen 1a, 1b und 3a eignet. Erfreulicherweise wird dieses Protokoll zur HCV-Resistenztestung im Institut für Virologie des Universitätsklinikums Leipzig angewandt. Durch die nested-PCR können die Anteile der NS3-, NS5A- und NS5B-Regionen, die Resistenz-assoziierten Mutationen enthalten, sicher amplifiziert und sequenziert werden. Fehlamplifikationen sind am ehesten aufgrund von Punktmutationen an den Primerbindungsstellen zu erklären. Durch Selektionsdruck, fehlende proof-reading-Mechanismen und die Ungenauigkeit der Replikation durch die RNA-abhängige-RNA-Polymerase ist die Rate an Neumutationen bei HCV hoch. Eine wesentliche Option zur Verbesserung der HCV-Resistenztestung wäre die Entwicklung eines Protokolls, welches universell für alle Genotypen anwendbar ist.:1 Abkürzungsverzeichnis 4 2 Einleitung 5 2.1 Epidemiologie des Hepatitis C Virus (HCV) 5 2.2 Taxonomie und HCV-Genotypen 6 2.3 Genom, Replikation, Eintritt und Freisetzung 7 2.4 Klinische Symptomatik 10 2.5 Diagnostik 11 2.6 Therapie 12 2.6.1 Virologisches Ansprechen während und nach Therapie 12 2.6.2 Interferon: Therapie der Vergangenheit 13 2.6.3 Direct Acting Antiviral Agents 14 2.6.4 Unerwünschte Arzneimittelreaktionen, Arzneimittelinteraktionen und HCC-Risiko 16 2.6.5 DAA Resistenzmutationen 17 2.6.6 Indikationen zur Resistenzanalyse 18 3 Fragestellung 19 4 Material und Methoden 20 4.1 Geräte und Verbrauchsmittel 20 4.2 Reaktionskits 21 4.3 Reagenzien 21 4.4 Primer 22 4.5 Patienten 22 4.5.1 Proben 22 4.5.2 Ethikvotum 24 4.6 RNA Extraktion aus Serum oder Plasma 25 4.7 RT Reaktion 26 4.8 PCR 27 4.9 Agarosegelelektrophorese 29 4.10 Gelextraktion 31 4.11 Sequenzierung 31 5 Ergebnisse 33 5.1 Primerdesign 33 5.2 Optimierung des PCR Protokolls 36 5.2.1 Optimierung der RT-Reaktion 36 5.2.2 Optimierung der PCR: nested und seminested PCR 37 5.3 Amplifikation der Proben 39 5.4 Sequenzauswertung und Resistenzanalyse 42 5.5 Vorhandene Resistenzmutationen 46 5.5.1 Mutationen sortiert nach Region 46 5.5.2 Mutationen sortiert nach Gruppen 48 6 Diskussion 50 6.1 Einsatz der Methode in der klinischen Diagnostik 50 6.1.1 Erfolgreiche Amplifikation von Therapieresistenzen 50 6.1.2 Auswahl der Nachweismethode 50 6.1.3 Kriterien einer guten diagnostischen Nachweismethode 51 6.1.4 Verbesserungsmöglichkeiten 52 6.1.5 Zeitpunkt des Einsatzes der Hepatitis C Resistenztestung 53 6.2 Probleme und Fehleranalyse 54 6.2.1 Probleme bei der Recherche 54 6.2.2 Gründe für eine erfolglose Amplifikation 54 6.2.3 Primerannealing 55 6.2.4 Genotypen 56 6.3 Prävalenzen von Therapieresistenzen 57 6.4 Ethische Problematik: Hohe Therapiekosten nach Zulassung verhinderten den Zugang zu adäquater DAA-Therapie 59 7 Zusammenfassung 62 8 Literaturverzeichnis 65 9 Tabellenverzeichnis 69 10 Abbildungsverzeichnis 69 11 Anlagen 70 11.1 Resistenzmutationen 70 12 Selbstständigkeitserklärung 73 13 Danksagung 74

This study aimed to assess the prevalence of and factors associated with tobacco use among patients living with HIV/HCV co-infection. Patient reported outcomes (PROs) were analyzed of patients living with HIV/HCV co-infection (n = 313) who presented for clinical evaluation and treatment of HCV between 2013 and 2017 at a university-affiliated HIV/HCV Co-infection Clinic. The prevalence of tobacco use in patients living with HIV/HCV co-infection was 48%. Compared to non-smokers, a higher proportion of tobacco smokers had substance use disorders and concurrent alcohol and substance use. In the multivariate analysis, concurrent alcohol and substance use was positively associated with tobacco use. The findings suggest clinical interventions are urgently needed to reduce tobacco use among patients living with HIV/HCV co-infection—a doubly-vulnerable immunocompromised population. Otherwise, failed efforts to dedicate resources and targeted behavioral interventions for this respective population will inhibit survival—especially considering the recent and evolving COVID-19 pandemic.

T cells play a crucial role in viral clearance and vaccine responses; however, the mechanisms that regulate their homeostasis during viral infections remain unclear. In this study, we investigated the machineries of T-cell homeostasis and telomeric DNA damage using a human model of hepatitis C virus (HCV) infection. We found that naïve CD4 T cells in chronically HCV-infected patients (HCV T cells) were significantly reduced due to apoptosis compared with age-matched healthy subjects (HSs). These HCV T cells were not only senescent, as demonstrated by overexpression of aging markers and particularly shortened telomeres; but also DNA damaged, as evidenced by increased dysfunctional telomere-induced foci (TIF). Mechanistically, the telomere shelterin protein, in particular telomeric repeat binding factor 2 (TRF2) that functions to protect telomeres from DNA damage, was significantly inhibited posttranscriptionally via the p53-dependent Siah-1a ubiquitination. Importantly, knockdown of TRF2 in healthy T cells resulted in increases in telomeric DNA damage and T-cell apoptosis, whereas overexpression of TRF2 in HCV T cells alleviated telomeric DNA damage and T-cell apoptosis. To the best of our knowledge, this is the first report revealing that inhibition of TRF2 promotes T-cell telomere attrition and telomeric DNA damage that accelerates T-cell senescent and apoptotic programs, which contribute to naïve T-cell loss during viral infection. Thus, restoring the impaired T-cell telomeric shelterin machinery may offer a new strategy to improve immunotherapy and vaccine response against human viral diseases.

Virus-infected cells can regulate non-permissive bystander cells, but the precise mechanisms remain incompletely understood. Here we report that this process can be mediated by transfer of viral RNA-loaded exosomes shed from infected cells to myeloid-derived suppressor cells (MDSCs), which in turn regulate the differentiation and function of T cells during viral infection. Specifically, we demonstrated that patients with chronic hepatitis C virus (HCV) infection exhibited significant increases in T follicular regulatory (TFR) cells and decreases in T follicular helper (TFH) cells. These MDSC-mediated T-cell dysregulations resulted in an increased ratio of TFR/TFH and IL-10 production in peripheral blood. Specifically, co-culture of MDSCs derived from HCV patients with healthy peripheral blood mononuclear cells (PBMCs) induced expansion of TFR, whereas depletion of MDSCs from PBMCs of HCV patients reduced the increases in TFR frequency and IL-10 production, and promoted the differentiation of IFN-γ-producing TFH cells. Importantly, we found that exosomes isolated from the plasma of HCV patients and supernatant of HCV-infected hepatocytes could drive monocytic myeloid cell differentiation into MDSCs. These exosomes were enriched in tetraspanins, such as CD63 and CD81, and contained HCV RNA, but exosomes isolated from patients with antiviral treatment contained no HCV RNA and could not induce MDSC differentiation. Notably, these HCV RNA-containing exosomes (HCV-Exo) were sufficient to induce MDSCs. Furthermore, incubation of healthy myeloid cells with these HCV-Exo inhibited the expression of miR−124, whereas reconstitution of PBMCs with miR−124 abolished the effects of HCV−Exo on MDSC induction. Taken together, these results indicate that HCV-associated exosomes can transfer immunomodulatory viral RNA from infected cells to neighboring immune cells and trigger MDSC expansion, which subsequently promotes TFR differentiation and inhibits TFH function. This study reveals a previously unrecognized path that represents a novel mechanism of immune dysregulation during chronic viral infection.

Host innate and adaptive immune responses must be tightly regulated by an intricate balance between positive and negative signals to ensure their appropriate onset and termination while fighting pathogens and avoiding autoimmunity; persistent pathogens may usurp these regulatory machineries to dampen host immune responses for their persistence in vivo. Here, we demonstrate that miR146a is up‐regulated in monocytes from hepatitis C virus (HCV )‐infected individuals compared to control subjects. Interestingly, miR146a expression in monocytes without HCV infection increased, whereas its level in monocytes with HCV infection decreased, following Toll‐like receptor (TLR ) stimulation. This miR146a induction by HCV infection and differential response to TLR stimulation were recapitulated in vitro in monocytes co‐cultured with hepatocytes with or without HCV infection. Importantly, inhibition of miR146a in monocytes from HCV ‐infected patients led to a decrease in IL ‐23, IL ‐10 and TGF ‐β expressions through the induction of suppressor of cytokine signalling 1 (SOCS 1) and the inhibition of signal transducer and activator transcription 3 (STAT 3), and this subsequently resulted in a decrease in regulatory T cells (Tregs) accumulated during HCV infection. These results suggest that miR146a may regulate SOCS 1/STAT 3 and cytokine signalling in monocytes, directing T‐cell differentiation and balancing immune clearance and immune injury during chronic viral infection.

Hepatitis C virus (HCV) or human immunodeficiency virus (HIV) infection leads to a phenomenon of inflammaging, in which chronic infection or inflammation induces an immune aged phenotype with T cell dysfunction. Thus, HCV or HIV infection has been deemed as a model to study the mechanisms of T cell infammaging and viral persistence in humans. In this dissertation, T cell homeostasis, DNA damage and repair machineries were investigated in patients with chronic HCV or HIV infection at risk for inflammaging. We found a significant depletion in CD4 T cells, which was correlated with their apoptosis in chronically HCV/HIVinfected patients, compared to age-matched healthy subjects. In addition, virus-infected patients’ CD4 T cells were prone to DNA damage that extended to chromosome ends (telomeres), leading to accelerated telomere erosion - a hallmark of senescence. Mechanistically, the DNA doublestrand break (DSB) sensor MRE11, RAD50, and NBS1 (MRN) remained intact, but the DNA damage checkpoint kinase ataxia telangiectasia mutated (ATM) and its downstream checkpoint kinase 2 (CHK2) were significantly suppressed in T cells from HCV/HIV-infected individuals. Consistently, ATM/CHK2 activation, DNA repair, and cellular functions were also impaired in primary CD4 T cells following ATM knockdown, or exposure to the ATM inhibitor (KU60019), as well as in CD4 T cells co-cultured with HCV-infected hepatocytes, or a T cell line infected with HIV-1 in the presence of raltegravir in vitro, which recapitulates the biological effects observed in T cells in the setting of HCV/HIV infection in vivo. Importantly, ectopic expression of ATM was essential and sufficient to reduce the DNA damage, survival deficit, and cellular dysfunction in T cells from both HCV and HIV-infected individuals. These results demonstrate that failure of DSB repair due to ATM deficiency leads to unrepaired DNA damage and renders virally infected patients’ T cells prone to senescence and apoptosis, thus contributing to CD4 T cell loss or dysfunction during chronic HCV or HIV infection. This study reveals a novel mechanism by which ATM deficiency promotes telomeric DNA damage and premature T cell aging, and provides a new therapeutic target for inflammaging-induced immune dysfunction during chronic viral infection.

Advanced liver fibrosis in chronic hepatitis C infection (HCV) is associated with a generalized impaired immune system. Many immune cells are affected in chronic liver disease, including CD8+ T cells. The Crawley lab reported CD8+ T cell hyperfunction in cirrhotic HCV-infected individuals that persisted after effective antiviral therapy. To evaluate the link between CD8+ T cell dysfunction in advanced fibrosis, we adapted a hepatotoxic carbon tetrachloride (CCl4) murine model. We consistently observed severe fibrosis in CCl4-treated mice resembling fibrosis in chronic HCV infected individuals. After stimulation of PBMC, the proportion of granzyme B+, and IFN-γ+ CD8+ T cells in fibrotic mice was significantly higher than the controls, particularly naïve and central memory CD8+ T cells. This state of hyperfunction was sustained after liver insult removal and significant fibrosis regression to near normal tissue integrity. Sex differences were also studied in this model and were apparent after prolonged exposure to CCl4 and in the capacity to repair liver fibrosis. Following an ectopic challenge with cancer cells, tumor growth was significantly greater in fibrotic mice. Moreover, the response to immunotherapy was significantly delayed in CCl4-treated mice. In summary, we reported for the first time that circulating CD8+ T cells are hyperfunctional in a murine model of advanced liver fibrosis in response to a hepatotoxin. In this context, affected mice failed to control the growth of a tumor whose growth is known to be controlled by a robust CD8+ T cell response. In addition, the reduced responses to immunotherapeutic effects suggest deficiencies in antigen-specific CD8+ T cell responses. Therefore, this animal model might be useful to identify mechanistic targets with translational potential for immune restoring treatments in human chronic liver diseases with advanced liver fibrosis.

(1) Background: Low rates of hepatitis C virus (HCV) diagnosis and sub-optimal linkage to care constitute barriers toward eliminating the infection. In 2012/2013, we showed that HCV screening in primary care detects unknown cases. However, hepatitis C patients may not receive further diagnostics and therapy because they drop out during the referral pathway to secondary care. Thus, we used an existing network of primary care physicians and a practice of gastroenterology to investigate the pathway from screening to therapy. (2) Methods: HCV screening was prospectively included in a routine check-up of primary care physicians who cooperated regularly with a private gastroenterology practice. Anti-HCV-positive patients were referred for further specialized diagnostics and treatment if indicated. (3) Results: Seventeen primary care practices screened 1875 patients. Twelve individuals were anti-HCV-positive (0.6%), six of them reported previous antiviral HCV therapy, and one untreated patient was HCV-RNA-positive (0.05% of the population). None of the 12 anti-HCV-positive cases showed up at the private gastroenterology practice. Further clinical details of the pathway from screening to therapy could not be analyzed. (4) Conclusions: The linkage between primary and secondary care appears to be problematic in the HCV setting even among cooperating partners, but robust conclusions require larger datasets.

T cells play an important role in adaptive immune system against viral infections, while premature aging and dysfunction of T cells induced by unrepaired DNA damages are always non-negligible snags during the long-term of fighting with chronic viral infections, such as Hepatitis B virus (HBV), Hepatitis C virus (HCV) or Human Immunodeficiency Virus (HIV) infection. In this dissertation, we investigated the role of topological DNA damage in reprogramming telomeric DNA damage responses (DDR), mitochondrial metabolisms, and T cell functions using CD4+ T cells derived from individuals with chronic viral infections or healthy subjects treated with topoisomerase inhibitors. The healthy human T cells were treated with camptothecin (CPT) for mitochondrial topoisomerases I (Top1mt) or ICRF-193 or etoposide (ETP) for topoisomerases IIα (Top2α) as models. We found a significant suppression of Top2α and Top1mt protein levels and enzymatic activity in CD4+ T cells in chronically HCV/HIV-infected patients compared to age and gender-matched healthy subjects, along with an accumulation of the topoisomerase cleavage complex (Topcc) in genomic DNA as well as mitochondrial DNA (mtDNA). Mechanistically, topoisomerase inhibition in healthy CD4+ T cells caused topological DNA damage, telomere attrition, mitochondrial metabolic disorder and T cell apoptosis or dysfunction via inducing Topcc accumulation, PARP1 cleavage and failure in DNA repair, thus recapitulating T cell dysregulation in the setting of chronic viral infections. In addition, T cells from virally infected subjects with lower topoisomerase levels were vulnerable to the inhibitor-induced cell apoptosis, indicating an important role for Top2α and Top1mt in preventing DNA topological disruption and cell death. These results demonstrate that accumulation of Topcc and topoisomerase deficiency lead to unrepaired DNA damage and render virally infected patients’ T cells prone to senescence and apoptosis, thus contributing to mitochondrial metabolic disturbance or dysfunction in CD4+ T cell during chronic HCV or HIV infection. This study reveals a novel mechanism by which topoisomerase deficiency promotes telomeric DNA or mtDNA damage and premature T cell aging, and provides a new therapeutic target for restoring the DNA topologic machinery protecting T cells from unwanted DNA damage and to maintain immune competence.

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