Charakterisierung von Virus-Wirt-Interaktionen, Persistenz und Einfluss antiviraler Wirkstoffe

About 15 million people worldwide are chronically infected with the hepatitis D virus (HDV). A chronic HDV infection shows the most severe clinical course of all hepatitis infections leading to liver cirrhosis in up to 70 % and to the development of hepatocellular carcinoma. Virus replication requires the generation of genomic and antigenomic HDV RNAs which are generated in a double rolling circle, while the envelope proteins of the hepatitis B virus (HBV) are needed for assembly and release of infectious virions. The limited availability of HDV infection models has hindered investigations of HDV pathogenesis and interactions between HDV and infected human hepatocytes. Currently an HDV specific therapy does not exist. Our group recently developed an HDV in vivo infection model using humanized uPA/SCID mice, which after transplantation of primary human hepatocytes into mice livers can stably be infected with HBV and HDV. Aims of this study were to characterize interactions of HDV and the innate immune system, to investigate the persistence and cell toxicity of an HDV mono-infection and to determine effects of different antiviral drugs on HDV productivity by establishing and using a novel assay to quantify genomic and antigenomic HDV RNA. By using the uPA/SCID mouse model it could be shown that the development of HDV viremia was accompanied by a clear induction of human-specific interferon stimulated genes (ISGs), which play an important role in the defense of viruses. While an HBV mono-infection only showed moderate inductions of classic ISGs (e.g. hMxA, hOAS1, hHLA-E, hIP10) in human hepatocytes, the expression of these ISGs was significantly stronger in an HBV/HDV co-infection. Also levels of human specific cytokines (hTGF-ß, hIFN-ß, hIFN-λ) were clearly increased in HBV/HDV co-infected mice, while their expression was lower or even below the lower limit of detection in HBV mono- and uninfected mice. This strong activation of the innate immune system might directly lead to liver damage and inflammation providing a rationale for the more severe course of HDV-associated liver disease. Another investigation within this thesis showed that an HDV mono- infection in humanized mice can persist for at least 6 weeks in the absence of HBV. Moreover persistent HDV particles could be rescued upon HBV super- infection and serially passaged into naïve human chimeric mice proofing the maintenance of HDV infectivity after a 6-weeks-phase of intracellular latency. These results explain observations in patients showing detectable intrahepatic HDV markers despite very low HBV activity or even after liver transplantation and emphasize the risk for these patients to develop a productive HBV/HDV infection after re-infection with HBV. Furthermore, a novel, quantitative RT- PCR assay was established, which specifically detects genomic and antigenomic HDV RNA in human chimeric mice livers and human liver patient biopsies. This assay now provides new possibilities to investigate the intrahepatic life cycle of HDV and the impact of different antivirals on HDV productivity in a more detailed way. E.g. a 4-week administration of pegylated interferon alpha not only reduced HDV viremia but also showed a decrease of both intrahepatic genomic and antigenomic HDV RNA levels. In contrast, a therapy with the nucleotide analogue entecavir efficiently suppressed HBV viremia but had no influence on HDV replication. Our interferon alpha studies also indicated that intrahepatic viral clearance could be achievable by suppressing HDV replication. At the same time these findings underline that due to its simplicity in structure HDV offers only very limited targets to eradicate the virus and that new therapeutic approaches are urgently needed.Circa 15 Millionen Menschen sind weltweit chronisch mit dem Hepatitis-D-Virus (HDV) infiziert. Die chronische HDV-Infektion zeigt den schwersten klinischen Verlauf aller viralen Hepatitiden und führt in bis zu 70% der Fälle zu Leberzirrhose und zur Entstehung des hepatozellulären Karzinoms. Für die Replikation des Virus ist das Vorhandensein von genomischer und antigenomischer HDV RNA essentiell, während es für die Bildung und Sekretion von infektiösen Viruspartikeln die Hüllproteine von HBV benötigt. Da geeignete HDV-Infektionsmodelle fehlen, ist bisher wenig über die Pathogenese einer HDV- Infektion und Virus-Wirts-Wechselwirkungen in infizierten humanen Hepatozyten bekannt. Eine HDV-spezifische Therapie existiert derzeit nicht. Kürzlich haben wir ein HDV-Infektionsmodell entwickelt, bei dem uPA-SCID-Mäuse mit primären, humanen Hepatozyten repopuliert und mit HBV und HDV infiziert werden können. Die Ziele dieser Studie waren, Interaktionen zwischen HDV und dem angeborenen Immunsystem zu charakterisieren, die Persistenz und Zellschädigung des Virus in einer HDV-Monoinfektion zu untersuchen und mit Hilfe einer neuen Methode zur Quantifizierung genomischer und antigenomischer HDV, antivirale Effekte verschiedener Virustatika zu bestimmen. Unter Verwendung des uPA/SCID- Mausmodells konnte gezeigt werden, dass die Entwicklung der HDV-Virämie von einer klaren Induktion humaner Interferon-stimulierter Gene (ISGs), die eine wichtige Rolle bei der Abwehr Viren spielen, begleitet wurde. Während eine HBV-Monoinfektion im Vergleich mit uninfizierten Kontrollmäusen nur zu einem moderaten Anstieg der klassischen ISGs (z.B. hMxA, hOAS1, hHLA-E, hIP10) in humanen Hepatozyten führte, war die Induktion der ISGs während einer HBV/HDV- Koinfektion signifikant stärker ausgeprägt. Humane Zytokine (hTGF-ß, hIFN-ß, hIFN-λ) waren in einer HBV/HDV-Koinfektion ebenfalls deutlich induziert, während die Expression dieser Zytokine in HBV-monoinfizierten und uninfizierten Mäusen niedriger oder unterhalb der Messgrenze war. Diese ausgeprägte Aktivierung des angeborenen Immunsystems könnte direkt zu Leberschädigung und Leberentzündung führen und somit den schweren klinischen Verlauf einer HDV-Infektion erklären. Zusätzlich wurde beobachtet, dass eine HDV-Monoinfektion im uPA/SCID-Mausmodell intrahepatisch in der Abwesenheit von HBV für mindestens 6 Wochen persistieren kann und dass persistierende HDV- Partikel durch eine HBV-Superinfektion „gerettet“ werden können. Darüber hinaus bewies eine serielle Übertragung dieser HDV-Partikel in naive, human- chimäre Mäuse, dass deren Infektiosität bestehen bleibt. Diese Ergebnisse erklären Beobachtungen in Patienten, bei denen HDV auch bei sehr niedriger HBV-Replikation und sogar nach vollständiger Transplantation der Leber nachweisbar ist und unterstreichen das Risiko, dass nach erneuter Infektion mit HBV sofort eine aktive HBV/HDV-Koinfektion vorliegen kann. Weiterhin wurde eine neue, quantitative RT-PCR-Methode entwickelt, welche genomische und antigenomische HDV-RNA spezifisch in human chimären Mauslebern und Patientenbiopsien detektiert. Dieses Nachweisverfahren liefert neue Möglichkeiten, den Replikationszyklus von HDV und den Einfluss verschiedener Wirkstoffe detailliert zu untersuchen. So zeigte eine 4-wöchige Behandlung mit pegyliertem Interferon alpha nicht nur eine Erniedrigung der HDV-Virämie, sondern auch eine Reduktion der genomischen und antigenomischen RNA in humanen Hepatozyten, während eine Therapie mit Entecavir keinen Einfluss auf die Aktivität von HDV hatte. Am Beispiel von pegyliertem Interferon alpha ist zu sehen, dass durch erfolgreiche Hemmung der HDV-Replikation eine Viruselimination erreicht werden könnte. Gleichzeitig wird aber auch deutlich, dass HDV wegen seines einfachen Aufbaus sehr wenig Angriffspunkte für eine antivirale Therapie bietet und dringend neue Therapieansätze benötigt werden

In Vivo Models of HDV Infection: Is Humanizing NTCP Enough?

The discovery of sodium taurocholate co-transporting polypeptide (NTCP) as a hepatitis B (HBV) and delta virus (HDV) entry receptor has encouraged the development of new animal models of infection. This review provides an overview of the different in vivo models that are currently available to study HDV either in the absence or presence of HBV. By presenting new advances and remaining drawbacks, we will discuss human host factors which, in addition to NTCP, need to be investigated or identified to enable a persistent HDV infection in murine hepatocytes. Detailed knowledge on species-specific factors involved in HDV persistence also shall contribute to the development of therapeutic strategies

Adaptation and validation of a quantitative vanA/vanB DNA screening assay on a high-throughput PCR system

Abstract Vancomycin resistant enterococci (VRE) are a leading cause of ICU-acquired bloodstream infections in Europe. The bacterial load in enteral colonization may be associated with a higher probability of transmission. Here, we aimed to establish a quantitative vanA/vanB DNA real-time PCR assay on a high-throughput system. Limits of detection (LOD), linear range and precision were determined using serial bacterial dilutions. LOD was 46.9 digital copies (dcp)/ml for vanA and 60.8 dcp/ml for vanB. The assay showed excellent linearity between 4.7 × 101 and 3.5 × 105 dcp/ml (vanA) and 6.7 × 102 and 6.7 × 105 dcp/ml (vanB). Sensitivity was 100% for vanA and vanB, with high positive predictive value (PPV) for vanA (100%), but lower PPV for vanB (34.6%) likely due to the presence of vanB DNA positive anerobic bacteria in rectal swabs. Using the assay on enriched VRE broth vanB PPV increased to 87.2%. Quantification revealed median 2.0 × 104 dcp/ml in PCR positive but VRE culture negative samples and median 9.1 × 104 dcp/ml in VRE culture positive patients (maximum: 107 dcp/ml). The automated vanA/B_UTC assay can be used for vanA/vanB detection and quantification in different diagnostic settings and may support future clinical studies assessing the impact of bacterial load on risk of infection and transmission

Hepatitis Delta co-infection in humanized mice leads to pronounced induction of innate immune responses in comparison to HBV mono-infection

Background & Aims: The limited availability of hepatitis Delta virus (HDV) infection models has hindered studies of interactions between HDV and infected hepatocytes. The aim was to investigate the antiviral state of HDV infected human hepatocytes in the setting of co-infection with hepatitis B virus (HBV) compared to HBV mono-infection using human liver chimeric mice. Methods: Viral loads, human interferon stimulated genes (hISGs) and cytokines were determined in humanized uPA/SCID/beige (USB) mice by qRT-PCR, ELISA and immunofluorescence. Results: Upon HBV/HDV inoculation, all mice developed viremia, which was accompanied by a significant induction of hISGs (i.e. hISG15, hSTATs, hHLA-E) compared to uninfected mice, while HBV mono-infection led to weaker hISG elevations. In the setting of chronic infection enhancement of innate defense mechanisms was significantly more prominent in HBV/HDV infected mice. Also the induction of human-specific cytokines (hIP10, hTGF-beta, hIFN-beta and hIFN-lambda) was detected in HBV/HDV co-infected animals, while levels remained lower or below detection in uninfected and HBV mono-infected mice. Moreover, despite the average increase of hSTAT levels determined in HBV/HDV infected livers, we observed a weaker hSTAT accumulation in nuclei of hepatocytes displaying very high HDAg levels, suggesting that HDAg may in part limit hSTAT signaling. Conclusions: Establishment of HDV infection provoked a clear enhancement of the antiviral state of the human hepatocytes in chimeric mice. Elevated pre-treatment ISG and interferon levels may directly contribute to inflammation and liver damage, providing a rationale for the more severe course of HDV-associated liver disease. Such antiviral state induction might also contribute to the lower levels of HBV activity frequently found in co-infected hepatocytes. (C) 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved

Clinical Evaluation of a Fully-Automated High-Throughput Multiplex Screening-Assay to Detect and Differentiate the SARS-CoV-2 B.1.1.529 (Omicron) and B.1.617.2 (Delta) Lineage Variants

Background: The recently emerged SARS-CoV-2 B.1.1.529 lineage and its sublineages (Omicron variant) pose a new challenge to healthcare systems worldwide due to its ability to efficiently spread in immunized populations and its resistance to currently available monoclonal antibody therapies. RT-PCR-based variant tests can be used to screen large sample-sets rapidly and accurately for relevant variants of concern (VOC). The aim of this study was to establish and validate a multiplex assay on the cobas 6800/8800 systems to allow discrimination between the two currently circulating VOCs, Omicron and Delta, in clinical samples. Methods: Primers and probes were evaluated for multiplex compatibility. Analytic performance was assessed using cell culture supernatant of an Omicron variant isolate and a clinical Delta variant sample, normalized to WHO-Standard. Clinical performance of the multiplex assay was benchmarked against NGS results. Results: In silico testing of all oligos showed no interactions with a high risk of primer-dimer formation or amplification of human DNA/RNA. Over 99.9% of all currently available Omicron variant sequences are a perfect match for at least one of the three Omicron targets included in the multiplex. Analytic sensitivity was determined as 19.0 IU/mL (CI95%: 12.9–132.2 IU/mL) for the A67V + del-HV69-70 target, 193.9 IU/mL (CI95%: 144.7–334.7 IU/mL) for the E484A target, 35.5 IU/mL (CI95%: 23.3–158.0 IU/mL) for the N679K + P681H target and 105.0 IU/mL (CI95%: 80.7–129.3 IU/mL) for the P681R target. All sequence variances were correctly detected in the clinical sample set (225/225 Targets). Conclusion: RT-PCR-based variant screening compared to whole genome sequencing is both rapid and reliable in detecting relevant sequence variations in SARS-CoV-2 positive samples to exclude or verify relevant VOCs. This allows short-term decision-making, e.g., for patient treatment or public health measures