During the second funding period, we have analysed the consequences of integration of subgenomic HBV sequences for HDV persistence, characterized the mechanism of HDV RNA recognition by innate immune sensors and obtained initial insights into the mechanism of cell division-mediated spread of HDV RNA . Considering that the recently approved entry inhibitor BLV, in combination with IFN, may lead to a cure of HDV infection, we will focus now on HDV with three complementary objectives:
First, we will investigate the multiple roles of the large HDV antigen (L-HDAg), i.e. virion secretion and modulation of replication, for HDV persistence. L-HDAg becomes prenylated within its C-terminus to hook up to the self-assembly competent HBV envelope proteins, promoting HDV secretion. Besides, prenylated L-HDAg inhibits replication by a poorly understood mechanism. Thus, inhibition of farnesyltrans-ferase prevents secretion, but concurrently enhances intracellular RNA replication. We will investigate in detail the roles of L-HDAg farnesylation (e.g. its modes of regulating RNA synthesis) and its contribution to HDV persistence (with Schütz/Seitz, Bartenschlager). We will apply a set of HDV L-HDAg mutants affect-ing L-HDAg synthesis and prenylation as well as novel systems for non-human HDV-like agents that lack L-HDAg. These novel RNA-elements are expected to propagate predominantly via mitosis. The results of these studies will help to predict clinical consequences of using prenylation inhibitors like Lonafarnib (LNF) as therapeutic agents.
Second, we will identify and characterize IFN-stimulated genes (ISGs) participating in the regulation of HDV cell-division-mediated spread (with Binder/Beisel, Bartenschlager, Pichlmair, Protzer/Wettengel). Following the identification of MDA5 and LGP2 as the two major sensors of HDV RNA, we direct our stud-ies at the identification of specific ISGs that are responsible to prevent HDV RNA propagation via mitosis. We already performed a pilot screen with an expression library of ISGs and identified first candidates (with Charlie Rice, New York and Lefteris Michailidis, Atlanta). This screen shall be refined, implementing a CRISPR/Cas9 ISG knock-out library and a newly developed HDV replicon-based split-GFP reporter system that allows to track viral spread and its inhibition. For the most promising candidates, we will investigate their mode of action on HDV replication, evaluate the druggability of the respective pathways and investi-gate synergisms with the standard of care BLV.
Third, we will expand the scope beyond innate cytokine responses and specify the role of the adap-tive immune response in patients (with Neumann-Haefelin/Luxenburger, Thimme, Bengsch, Hofmann, Cerwenka; Z02 and Z03). This is important because IFNs restrict not only HDV cell division-mediated spread, but also enhance the effector function of adaptive immune cells. Such immune-mediated killing may be an additional mechanism augmented by virus-induced and therapeutic IFNs that most likely con-tributes to the synergism of BLV and pegIFN-α as indicated by the higher level of inflammation seen in HBV/HDV coinfected patients as compared to HBV monoinfection. To address this aspect, we will use bio-banked human liver samples and characterize and immunoprofile HDV-infected hepatocyte clusters. We will evaluate cell division-mediated spread in humans, and define transcriptional and protein expression changes in hepatocytes and surrounding immune cells that can be attributed to HDV infection using im-munostaining, imaging mass cytometry, and spatial transcriptomics (with Z02 and Z03). Results will be connected to the multiomics cell atlas of HBV- and HBV/HDV-infected patients, thus allowing comparisons of innate and adaptive immune responses in the infected human liver tissue. Although triggering type I and type III IFN responses, HDV obviously developed escape mechanisms from this antiviral system, at least in non-dividing cells. However, both in chronic HBV and HBV/HDV coinfection, non-specific liver-resident bystander cells have been identified to mediate liver damage by killing hepatocytes, thereby likely also reducing the number of already infected cells. A main focus will therefore be on the activation of non-virus-specific T cells; as well as virus-specific T-cells (with Thimme, Neumann-Haefelin/Luxenburger, and Hofmann) and NK cells (with Cerwenka) and their role in HDV persistence.
The overall aim of this project is a detailed understanding of HDV cell-division-mediated spread, its sensi-tivity to IFN and immune cells and the role in viral persistence, with the goal to define therapeutic targets and strategies to improve therapy success of chronic HDV infection.