Projects

Funding: SprinD, 684.523 EUR

Project Details:
Current antiviral agents mainly target stages in the viral life cycle such as viral attachment to host cell or replication of viral RNA and DNA. Most of the available antiviral agents are effective against replicating viruses only, and due to the lack of specificity, many have adverse side effects. Particularly in endemic and pandemic disease outbreaks, there is additional challenge through virus mutagenesis and the development of viral variants. Therefore, approaches, which target various virus variants are urgently needed. Current paradigms in antiviral treatment involve usage of small molecules and/or therapeutic antibodies. Small molecules often have secondary targets and thus can cause side effects. Antibodies are expensive, their administration is mostly limited to clinical settings, and also they are affected by mutations. In an endemic or pandemic situation, therapies which have broad coverage within virus families, prevent transmission, and can be safely applied during mild and moderate illness are especially valuable.

Our novel approach aims to fill this need through the use of CRISPR/Cas13, an enzyme from bacteria, which cleaves RNA, including the viral genome (of RNA viruses) and of viral mRNA, thereby blocking viral replication and generation of viral proteins. Our therapeutic strategy has no secondary targets, and can be produced at GMP level at low cost. Through a specific combination of so-called crRNAs, Cas13 is directed to different viral mRNAs and to different sites within the viral genome. The crRNAs are selected such that no human RNA is targeted, and therefore no adverse side effects can be expected. This technology can easily be adapted for any single stranded RNA virus. Through targeting different and highly conserved regions, this approach antagonizes also emerging variants of the original virus.

Cooperation Partners:

Prof. Dr. Elisabeth Zeisberg, Universitätsmedizin Göttingen

Funding: MWK Niedersachsen, 69.925 EUR

Project Details:
Since the first description of SARS-CoV2 and the related disease COVID-19 in December 2019 various in vitro and in vivo models were utilized to investigate viral life cycle and pathophysiology as basis for effective treatment strategies. In vivo models used to investigate SARS-CoV2 or respiratory viral infections in general range from small animals models to non-human primate models (Munoz-Fontela et al., 2020). But next to the ethical questions accompanying animal models in scientific research also not all questions related to human respiratory epithelium can be examined with (small) animal models. Immortalized human cell lines like Caco-2, Calu-3 or HEK293T which are regularly used to investigate respiratory viruses like influenza or Human Respiratory Syncytial virus (RSV) and also Corona Virus infections (SARS, MERS) are frequently applied to investigate infection and replication of SARS-CoV2; however, although these cell lines are comparatively easy and cost effective to grow, they only poorly recapitulate physiological conditions of the respiratory epithelium. Even cell lines such as A549 cells barely reflect the phenotype of lung epithelium. Human primary bronchial epithelial cells (hBECs) represent a more physiological in vitro model and are also utilized in SARS-CoV2 research (Hoffmann et al., 2020). These cells can be isolated from donor lung tissue, expanded in vitro and matured in Air-Liquid-Interface (ALI) cultures (Hoffmann et al., 2020). While the main focus of our group is scalable generation and utilization of cardiovascular and respiratory derivatives from induced pluripotent stem cells for disease modelling, drug screening and cellular therapies (Merkert et al., 2019; Katsirntaki et al., 2015; Kempf et al., 2014; Schmeckebier et al., 2013; Zweigert et al., 2011), we have recently also established isolation of primary human airway cells of consistent high quality: Hannover Medical School is the leading German lung transplantation center with about 100 lung transplantation per year. In close collaboration with our transplant surgeons and the group of Prof. Danny Jonigk (Pathology, MHH), we are routinely preparing hBECs from bronchial tissue that can be culture-expanded and cryopreserved for later applications. Also maturation of these hBECs is already well established in our group and matured proximal epithelial cells are already provided to partners for investigation of SARS-CoV2 as well as other respiratory viral infections (e.g. RSV). While these primary hBECs represent a superior in vitro model for SARS-CoV2 infection studies and COVID disease modelling, they do represent only one respiratory compartment. Infection by SARS-CoV2 not only occurs in the upper throat and nasal tract, trachea and bronchi, but also in the alveolar epithelium. Therefore also organotypic in vitro models that reflect the distal lung compartment are urgently required. While maintenance and expansion of isolated type II alveolar epithelial cells (AT2 cells) was so far impossible, substantial progress was very recently made, and protocols that enable production of such cells in larger numbers have been developed. So called Alveolospheres, organoids consisting of alveolar type 1 and 2 cells, allow expansion and maturation of isolated distal epithelial cell in vitro and were recently used to model SARS-CoV2 infection (Karsura et al., 2020; Salahudeen et al., 2020). Although organoid cultures enabled in vitro culture of AT1 and AT2 cells, we believe ALI cultures would represent a more physiological system with air exposure of the epithelial cells and could subsequently allow co-culture systems with endothelial cells as well as macrophages to create more complex organotypic in vitro models of the alveolar lung compartment. By utilizing our experience from differentiation of induced pluripotent stem cells (Katsirntaki et al., 2015; Schmeckebier et al., 2013) we aim to establish isolation, 2D expansion and cryopreservation of human AT2 cells, and further maturation and differentiation in type I alveolar epithelial (AT1) cells in an ALI culture system for in vitro modelling of SARS-CoV-2 infections.

Cooperation Partners:

Prof. Dr. Ulrich Martin, Dr. rer. nat. Ruth Olmer

Leibniz-Forschungslaboratorien für Biotechnologie und künstliche Organe, Klinik für Herz-, Thorax-, Transplantations- und Gefäßchirurgie - Medizinische Hochschule Hannover

Funding: BMBF, 261.911 EUR

Project Details:
Inflammation and associated lung injury, such as the acute respiratory distress syndrome (ARDS) are major causes of morbidity and and mortality in COVID-19. The Nrf2 transcription factor activates a comprehensive cell-protective and anti-inflammatory program. It is known from mouse models that pharmacological activation of Nrf2 and induction of this program protects animals from LPS-induced ARDS. In a cell line-based screen for agents against COVID-19, we found 2 Nrf2 activators that prevent the cytopathic effect of SARS-CoV-2 at nontoxic doses. Therefore, the aims of the present project are,
- the establishment of infection models based on
human respiratory epithelial cells, a model for the alveolar
epithelial cell barrier, and ex vivo culture of lung explants,
- Evaluation of the importance of the endogenous Nrf2 program for
the cellular response against SARS-CoV-2, and
- Verification of the 2 identified Nrf2 activators and
identification of additional protective agents via a
comprehensive screening of known Nrf2 activators.

Cooperation Partners:

PD Dr. Frank Pessler, Helmholtz-Zentrum für Infektionsforschung, Hannover

Funding: DFG Sonderforschungsbereich 900 "Mirkobielle Persistenz und seine Kontrolle", 430.000 EUR

Project Details:
The early steps of hepatitis C virus (HCV) cell entry involve a complex interplay between the incoming virion and host cell expressed factors. Key host factors in the early phase of HCV cell entry include scavenger receptor class B member 1 (SR-BI), low-density lipoprotein receptor (LDLR) and CD81. Work from the pre-vious funding period has revealed how genetic variation in one host factor, the high-density lipoprotein (HDL) receptor SR-BI, can modulate HCV infection, while highlighting at the same time that to fully under-stand the mechanisms of early HCV-cell interactions a broader view of the host cell molecules involved is required. In particular, others and we demonstrated that HCV host factors interact with each other and with cholesterol and this critically determines susceptibility. Moreover, we have identified CD81-binding partners calpain-5 (CAPN5) and casitas B-lineage lymphoma proto-oncogene b (CBLB) as novel modulators of HCV entry.
The proposed project for the next funding period has the overarching goal to better characterize how the host factors involved in the early phase of HCV entry interact with the virus and each other, and how these interactions are modulated by genetic and pharmacological factors. The project has four parts: (1) We will evaluate coding SR-BI and CD81 variants in a LDLR/SR-BI negative or - respectively - CD81 negative con-text for their effect on the HCV replication cycle and especially the early phase of cell entry in vitro. (2) We will determine how selected CD81 variants influence the interaction between CD81 and SR-BI and to what extent this mechanistically explains cell entry phenotypes. Here we will cooperate with project A6 (Pietsch-mann) which focusesl conversely on variation in SR-BI and membrane cholesterol content. (3) We will inves-tigate how statins and other lipid lowering drugs including the new and highly potent monoclonal antibodies directed at proprotein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of LDLR subcellular locali-zation and function, impact HCV infection. Specifically, we will address the impact on the expression level of SR-BI, LDLR and CD81 and on membrane and cellular cholesterol content of hepatocytes as well as the impact of statin therapy on viral load and hepatic inflammation in patients with chronic hepatitis C. (4) Final-ly, we will extend our analysis to newly identified CD81 interaction partners CAPN5 and CBLB and their role in HCV cell entry and receptor complex regulation beginning with the generation of human variants to probe whether they impact CD81 and SR-BI interactions as well as HCV infection. Taken together, this work holds the promise of a deeper understanding of the mechanistic role of established and newly discovered HCV entry factors during the early phase of HCV cell entry, its modulation by host genetic variants and non-genetic host factors such as exposure to lipid metabolism-modulating drugs and thus of interindividual variability in the course of chronic hepatitis C.

Funding: DFG, 215.000 EUR

Project Details:
The early steps of hepatitis C virus (HCV) cell entry involve a complex interplay between the incoming virion and host cell expressed factors. Key host factors in the early phase of HCV cell entry include scavenger receptor class B member 1 (SR-BI), low-density lipoprotein receptor (LDLR) and CD81. Work from the previous funding period has revealed how genetic variation in one host factor, the high-density lipoprotein (HDL) receptor SR-BI, can modulate HCV infection, while highlighting at the same time that to fully understand the mechanisms of early HCV-cell interactions a broader view of the host cell molecules involved is required. In particular, others and we demonstrated that HCV host factors interact with each other and with cholesterol and this critically determines susceptibility. Moreover, we have identified CD81-binding partners calpain-5 (CAPN5) and casitas B-lineage lymphoma proto-oncogene b (CBLB) as novel modulators of HCV entry.
The proposed project for the next funding period has the overarching goal to better characterize how the host factors involved in the early phase of HCV entry interact with the virus and each other, and how these interactions are modulated by genetic and pharmacological factors. The project has four parts: (1) We will evaluate coding SR-BI and CD81 variants in a LDLR/SR-BI negative or - respectively - CD81 negative context for their effect on the HCV replication cycle and especially the early phase of cell entry in vitro. (2) We will determine how selected CD81 variants influence the interaction between CD81 and SR-BI and to what extent this mechanistically explains cell entry phenotypes. Here we will cooperate with project A6 (Pietschmann) which focusesl conversely on variation in SR-BI and membrane cholesterol content. (3) We will investigate how statins and other lipid lowering drugs including the new and highly potent monoclonal antibodies directed at proprotein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of LDLR subcellular localization and function, impact HCV infection. Specifically, we will address the impact on the expression level of SR-BI, LDLR and CD81 and on membrane and cellular cholesterol content of hepatocytes as well as the impact of statin therapy on viral load and hepatic inflammation in patients with chronic hepatitis C. (4) Finally, we will extend our analysis to newly identified CD81 interaction partners CAPN5 and CBLB and their role in HCV cell entry and receptor complex regulation beginning with the generation of human variants to probe whether they impact CD81 and SR-BI interactions as well as HCV infection. Taken together, this work holds the promise of a deeper understanding of the mechanistic role of established and newly discovered HCV entry factors during the early phase of HCV cell entry, its modulation by host genetic variants and non-genetic host factors such as exposure to lipid metabolism-modulating drugs and thus of interindividual variability in the course of chronic hepatitis C.

Cooperation Partners:

Prof. Dr. Thomas von Hahn

Klinik für Gastroenterologie, Hepatologie und Endokrinologie und Institut für Molekularbiologie - Medizinische Hochschule Hannover

Funding: DFG Sonderforschungsbereich 900 "Mirkobielle Persistenz und seine Kontrolle", 215.000 EUR

Project Details:
The early steps of hepatitis C virus (HCV) cell entry involve a complex interplay between the incoming virion and host cell expressed factors. Key host factors in the early phase of HCV cell entry include scavenger receptor class B member 1 (SR-BI), low-density lipoprotein receptor (LDLR) and CD81. Work from the previous funding period has revealed how genetic variation in one host factor, the high-density lipoprotein (HDL) receptor SR-BI, can modulate HCV infection, while highlighting at the same time that to fully understand the mechanisms of early HCV-cell interactions a broader view of the host cell molecules involved is required. In particular, others and we demonstrated that HCV host factors interact with each other and with cholesterol and this critically determines susceptibility. Moreover, we have identified CD81-binding partners calpain-5 (CAPN5) and casitas B-lineage lymphoma proto-oncogene b (CBLB) as novel modulators of HCV entry.
The proposed project for the next funding period has the overarching goal to better characterize how the host factors involved in the early phase of HCV entry interact with the virus and each other, and how these interactions are modulated by genetic and pharmacological factors. The project has four parts: (1) We will evaluate coding SR-BI and CD81 variants in a LDLR/SR-BI negative or - respectively - CD81 negative context for their effect on the HCV replication cycle and especially the early phase of cell entry in vitro. (2) We will determine how selected CD81 variants influence the interaction between CD81 and SR-BI and to what extent this mechanistically explains cell entry phenotypes. Here we will cooperate with project A6 (Pietschmann) which focusesl conversely on variation in SR-BI and membrane cholesterol content. (3) We will investigate how statins and other lipid lowering drugs including the new and highly potent monoclonal antibodies directed at proprotein convertase subtilisin/kexin type 9 (PCSK9), a key regulator of LDLR subcellular localization and function, impact HCV infection. Specifically, we will address the impact on the expression level of SR-BI, LDLR and CD81 and on membrane and cellular cholesterol content of hepatocytes as well as the impact of statin therapy on viral load and hepatic inflammation in patients with chronic hepatitis C. (4) Finally, we will extend our analysis to newly identified CD81 interaction partners CAPN5 and CBLB and their role in HCV cell entry and receptor complex regulation beginning with the generation of human variants to probe whether they impact CD81 and SR-BI interactions as well as HCV infection. Taken together, this work holds the promise of a deeper understanding of the mechanistic role of established and newly discovered HCV entry factors during the early phase of HCV cell entry, its modulation by host genetic variants and non-genetic host factors such as exposure to lipid metabolism-modulating drugs and thus of interindividual variability in the course of chronic hepatitis C.

Results:

https://www.sfb900.de/en/

Cooperation Partners:

Prof. Dr. Thomas von Hahn, Klinik für Gastroenterologie, Hepatologie und Endokrinologie und Institut für Molekularbiologie - Medizinische Hochschule Hannover

Funding: DFG, 243.950 EUR

Project Details:
CD81 is a transmembrane protein and the receptor for two human pathogens, hepatitis C virus (HCV) and Plasmodium, the causative agent of malaria. Despite substantial differences in their molecular makeup, their transmission and pathology, both pathogens require CD81 to enter liver cells and replicate in these. Also, HCV and the human pathogen Plasmodium falciparum both display a narrow host tropism by naturally only infecting humans and CD81 is one of several host tropism determinants. Since CD81 lacks signaling domains, we had hypothesized that it coordinates HCV and Plasmodium uptake through protein-protein-interactions (PPI). Our previous work identified 68 CD81 PPIs in human hepatoma cells and could show that at least 10 of the CD81 interactors are required for HCV and Plasmodium infection. Currently, the full set of host factors required for CD81-dependent entry of HCV and Plasmodium remains elusive. Moreover, we lack knowledge on how the proteins guide pathogen entry. Here, we propose to confirm HCV and Plasmodium entry host factors based on our previous CD81 receptor interactomics and to characterize the host factors molecular function in depth.

Specifically, this work aims at (1) confirming the relevance of CD81 interactors for HCV and Plasmodium infection; (2) evaluating the specificity of discovered host factors for diverse enveloped viruses, HCV genotypes and Plasmodium species; (3) mechanistically characterizing selected host factors; (4) determining the contribution of the host factors to the narrow tissue and host tropism of HCV and Plasmodium falciparum.

In the first phase of the project we will confirm knockdown phenotypes of all 68 CD81 interactors in hepatoma cells and evaluate the cells susceptibility to HCV and Plasmodium. Genes, which score in this RNA interference assay, will be knocked out by CRISPR/Cas9, a technique, which we successfully applied to hepatoma cells recently. To evaluate the specificity of the host factors, we will infect knockout cells with three enveloped viruses (vesicular stomatitis virus, coronavirus 229E, respiratory syncytial virus), seven HCV genotypes and three Plasmodium species. Broad HCV and Plasmodium host factors will be mechanistically analyzed in terms of the specific entry step they support, their subcellular localization, if applicable their enzymatic activity during pathogen uptake and critical domains in the protein. In the last project phase tissue expression analysis of host factors will reveal possible contributions to liver tropism of HCV and Plasmodium sporozoites. Lastly, complementation of knockout cells with mouse and macaque orthologues of the host factors will highlight possible species restrictions for HCV and Plasmodium. In sum, this work will shed light on the entry of HCV and Plasmodium into liver cells and reveal important aspects of cellular membrane trafficking and signaling.