Title Promoter Affiliations Abstract "Development and validation of a bona fide iPSC derived human neuronal infection model to evaluate antivirals targeted against neurotropic viral infections." "Peter Delputte" "Centre for Health Economics Research and Modelling of Infectious Diseases (CHERMID), Laboratory for Experimental Hematology (LEH), Laboratory for Microbiology, Parasitology and Hygiene (LMPH)" "Neurotropic viral infections continue to cause major disease and economic burden. Such infections are most commonly caused by herpesviruses, arboviruses and enteroviruses, often leading to severe neurological damage with poor clinical outcomes. The search for interventions to prevent and/or treat these infections is however challenging. The main reason for this is the nature of the target cells, neurons, which are chiefly non-renewable and drastically differ from other cells (or cell lines). Discovery of novel antivirals via the classically performed research with cell lines, is not appropriate for viruses that infect neurons. Highly specialized, bona fide, human neuronal culture models are imperative. With this project, we will develop specialized neuronal cultures aimed at higher throughput antiviral screening." "Valorization of novel strategies for the prevention of viral infection and inhibition of viral replication." "Johan Neyts" "Laboratory of Virology and Chemotherapy (Rega Institute), Intellectual Property" "Antiviral drugs are only available for a handful of viruses. Also in the vaccine field a similar scenario can be seen. For many other viral infections that pose an important treat to human health (including re-emerging & neglected infections), there are no antiviral strategies available. The changing ecosystem increases the pressure on the appearance of (novel) viruses (with epidemic/pandemic potential). With this project, we aim to further play a leading role in the development of therapeutic and prophylactic strategies against viral infections both by the development of (i) small molecule antivirals and of (ii) vaccine candidates against a number of indications. For the latter, we will make use of our revolutionary PLLAV vaccine platform technology, which allows to develop thermostable vaccines that will largely facilitate the logistics of vaccination campaigns in low and middle income countries. This program is entirely in line with the mission and expertise of the laboratory and the Rega Institute that has a track record in the successful development of antiviral drugs." "Role of viral glycans and siglec receptors for infection of macrophages and viral immune evasion" "Hans Nauwynck" "Department of Translational Physiology, Infectiology and Public Health" "The role of different siglec receptors for viral infection of macrophages and the potential role of siglec-specific virus internatlization for immune evasion will be studied." "Developing an in vivo humanized mouse model suitable for the study of hepatotropic viral infection" "Catherine Verfaillie" "Stem Cell and Developmental Biology" "The pharmaceutical industry is in high need of efficient and physiologically relevant in vitro and in vivo models to study hepatotropic viruses, screen for potential antiviral drugs and test for liver toxicity that might be caused by these and other drugs. Current in vitro models rely on primary human hepatocytes (PHHs) and hepatoma cell lines, which have major drawbacks. As an alternative hepatocyte source, the use of human pluripotent stem cell derived hepatocyte-like cells (hPSC-HLCs) to study hepatotropic viruses has been proposed. Previous studies have shown that these hPSC-HLCs are indeed relevant models to study hepatotropic viruses as they are susceptible for hepatitis C, hepatitis B, hepatitis E and dengue virus.In this thesis, I examined whether hPSC-HLCs could also be a relevant model to study zika virus (ZIKV) infection. ZIKV has recently been linked to microcephaly in newborns. However, ZIKV-infected mouse models and non-human primates do not only have a high viral load in the brain, but also in other organs such as the liver. Additionally, as two reports described the association between ZIKV infection and liver injury and as ZIKV belongs to the same viral family of known hepatotropic viruses, such as hepatitis C and dengue virus, I examined whether human hepatocytes are susceptible to ZIKV infection. I demonstrated that hPSC-HLCs and the hepatoma cell line, Huh7, supports the complete ZIKV life cycle, including the entry, viral replication and the production of novel infectious ZIKV virions. Furthermore, treatment of ZIKV-infected hPSC-HLCs and Huh7 cells with 7-deaza-2’-C-methyladenosine, a known viral RNA-dependent RNA polymerase inhibitor, decreased ZIKV replication significantly in a dose-dependent manner, while 2’-C-methylcytidine and 6-fluoro-3-hydroxypyrazine-2-carboxamide only decreased ZIKV replication in ZIKV-infected Huh7 cells. Finally, ZIKV-infected hPSC-HLCs but not Huh7 cells induced an innate immune and NFκβ response, which may explain the more extensive cytopathic effect observed in ZIKV-infected Huh7 cells. These significant differences between hPSC-HLCs and hepatoma cells in the innate immune response against ZIKV and antiviral drug sensitivity highlight the need to assess ZIKV infection as well as antiviral activity not only in cell line models, but also in more physiologically relevant systems.As hPSC-HLCs used to demonstrate the susceptibility of hepatocytes to ZIKV, resemble fetal hepatocytes rather than adult primary hepatocytes and do not have drug metabolizing activity, I tested if metabolically improved hPSC-HLCs created by the Verfaillie lab at the Stem Cell Institute Leuven, consisting of inducible overexpression of 3 transcription factors (TFs)  HNF1α, PROX1 and FOXA3 (termed HC3x-HLCs) and cultured in metabolically optimized differentiation culture medium, would be a suitable model to study hepatitis B virus (HBV) and to test new anti-HBV antivirals that might need to undergo biotransformation to be active. The reason to study HBV infection is that despite an adequate anti-HBV vaccine, 260 million people remain chronically infected with HBV worldwide and available treatments do not cure patients as the covalently closed circular DNA (cccDNA) is retained in the hepatocyte nucleus. Hence, there is an urgent need for novel therapeutics that can eliminate HBV infection in chronically infected patients. I here demonstrated that metabolically improved HC3x-HLCs can efficiently be infected with HBV by immunofluorescence staining of HBV core antigen and surface antigens (HBsAg). Furthermore, I detected the release of HBsAg and HBV e antigen in the supernatant, which increased over time, via ELISA, as well as high titers of infectious virus when back-titrated on HepG2-NTCP cells. This data suggested that functional cccDNA was formed by the HBV-infected HC3x-HLCs. Additionally, I validated the model for use in antiviral drug studies using various known HBV inhibitors.Finally, as the pharmaceutical industry also requires suitable in vivo models to study hepatotropic viruses and test drugs for liver toxicity, I attempted to transplant the metabolically improved HC3x-HLCs and HC6x-HLCs (that overexpress not only HNF1α, PROX1, FOXA3 but also PGC1α, SIRT1 and AMPK) into urokinase plasminogen activator – severe combined immunodeficiency (uPA-SCID) mice to generate a humanized liver mouse model. Six months after transplantation, human cells were detected in the livers of mice transplanted with HC3x-HLCs, but not HC6x-HLCs. However, engraftment efficiencies were low, despite the enhanced maturity of HC3x-HLCs compared to many other HLC populations. Studies will be required to determine if the failure to robustly repopulate murine livers is due to the specific mouse model used or due to failure to home and establish initial engraftment, and/or subsequent expansion of the commonly small numbers of initially engrafted cells." "Exploring viral evolution with respect to disease progression and therapy-response in HCV infection." "Anne-Mieke Vandamme" "Laboratory of Clinical and Epidemiological Virology (Rega Institute), Hepatology" "Chronic hepatitis caused by the hepatitis C virus is a serious public health problem and affects more than 170 million people worldwide. Infection by HCV is defined by a complex interplay between virus and host. Already from the beginning, both virus and host genetics define the disease progression in a patient, ranging from spontaneous clearance to chronic infection potentially leading to liver cancer. Treatment options are limited, but viral clearance can be achieved in a large number of patients, depending on both viral and host characteristics. However, the emergence of resistant viral variants under drug selective pressure severely hampers therapeutic success. The population of closely related viruses in the patient, also known as quasispecies, evolves in response to immune and drug selective pressure. In this project we aim to develop the necessary in vitro and in silico tools to study within-host evolution and explore its relationship with disease progression and therapy response. Furthermore, we will study the emergence of drug resistance mutations under drug selective pressure. These results can be readily implemented in the management of HCV infected patients, giving clinicians an additional tool for individual patient guidance and treatment." "Viral and cellular factors involved in the infection and invasion of alphaherpesviruses in the respiratory and genital mucosae" "Hans Nauwynck" "Department of Translational Physiology, Infectiology and Public Health" "Alphaherpesviruses (aHV) of humans and animals cause respiratory and genital lesions In the promoter’ lab, respiratory and genital mucosa explants and respiratory epithelial cell cultures of several species have been developed to compare the behavior of different aHV All aHV have problems in infecting an intact epithelial cell layer The reason for this in the respiratory mucosa was recently found by the promoter’ team: the viral receptor is present at the basolateral side of the epithelial cells In the present project proposal, it will be analysed if this is also the case for genital epithelial cells Afterwards, the receptor will be identified During the intra-epithelial infection, aHV are destroying the basement membrane via activation of urokinase plasminogen activator and plasmin, which allows them penetrating into the connective tissue, where most aHV infect fibrocytes During this replication, the virus finds sensory nerve endings and spreads towards the neuronal nucleus, where the virus establishes latency It will now be examined if aHV also use proteases in the genital mucosa to cross the basement membrane Further, the intriguing interplay between viral proteins and cellular proteases will be examined The results of this project will lead to better insights in the replication and invasion of aHV in both mucosae, which will allow the oriented development of attenuated vaccines and treatments in order to prevent and cure aHV lesions" "Analysis of pathogenic variants in patients suffering from severe viral infection; unraveling known and novel inborn errors of immunity" "Isabelle Meyts" "Inborn Errors of Immunity" "This pHD thesis centers around genetic variants in inborn errors of immunityy that are yet not fully understood. The main aim of this thesis is the identification abd analysis of new pathogenic variants and their underlying mechanisms. The focus lays on protein and gene expression, protein localization and stability as well as interactions with other proteins. The study of pathogenic variants will be exhibited in both suitable cell lines and primary cells of patients and controls. western blots, PCRs, qPCRs, flow cytometry, and microscopy are the main techniques that will be used during this study." "Introducing mRNA vaccination in chicken: first steps towards a commercial mRNA vaccine against a leading viral infection in chicken" "Niek Sanders" "Department of Pathobiology, Pharmacology and Zoological Medicine, Department of Pharmaceutics, Department of Translational Physiology, Infectiology and Public Health, Department of Veterinary and Biosciences" "In this project we will introduce our self-amplifying mRNA vaccine platform in chicken. To that end we will design and in vitro validate two mRNA vaccines encoding selected antigens of one of the most important pathogens in poultry production. Subsequently, we will formulate the sa-mRNA vaccines with UGent owned lipid nanoparticles and investigate their immunogencity and protection efficacy in a challenge study in chicken." "Investigation of Nanobody-based LYsosome TArgeting Chimeras (nanoLYTACs): a novel concept in therapy of cancer and viral infection." "Nico Callewaert" "Department of Biochemistry and microbiology" "Drugs that mediate active degradation of their protein target are currently one of the most actively investigated areas in medicinal chemistry. Indeed, chimeric small molecule drugs (PROteolysis Targeting Chimeras, or PROTACs) have been invented that link up the target protein to an ubiquitin E3 ligase for subsequent proteasomal degradation, ablating all of the functions of the target protein. However, such strategies have been limited to targets within the intracellular environment, accessible to the proteasome. Here, we introduce a novel biopharmaceutical concept to direct extracellular target proteins for degradation in the lysosome. We make use of unique VHH single-domain antibodies that we developed, directed against the cation-independent mannose-6-phosphate receptor (CI-M6PR). This constitutively endocytosing receptor targets its protein ligands to the lysosome. This is widely used for enzyme replacement therapy of lysosomal storage diseases. Now, by fusing these lysosome-targeting VHHs to other VHHs that bind to extracellularly accessible disease target proteins, we target these for lysosomal degradation. We named this concept ‘Nanobody-based Lysosome-Targeting Chimeras’ (nanoLYTACs) and will test it in two settings. First, we will target receptor tyrosine kinases that frequently drive cancer, with EGFR as a benchmark testcase. Second, we will use our center’s recently generated VHHs against conserved epitopes on SARS-CoV-2 and influenza membrane proteins." "Optimize viral replication in primary human airway epithelial (HAE) cells for use in antiviral research to combat respiratory viral infections" "Johan Neyts" "Laboratory of Virology and Chemotherapy (Rega Institute)" "This project focuses on exploring the use of HAE cultures to study the treatment of viral respiratory infections. HAE cultures will be used to evaluate novel antiviral strategies to combat Respiratory Syncytial Virus (RSV) infection and explore the kinetics of replication of different RSV virus strains. This will result in an optimized culture system to be used to characterize viral strains in a relevant ex vivo system and link viral factors with pathogenesis. Using HAE derived from healthy volunteers and COPD patients (provided by P12 (ES)), the role of RSV in exacerbations of COPD will be studied. Novel antiviral strategies will be evaluated in the RSV/HAE infection model. This will generate important information on the therapeutic potential of the different antiviral strategies, the risk of resistance development and the use of combination strategies. These results will be compared with results from existing animal studies with the aim to reduce the use of these animal models in the future. The role of inflammation in RSV pathology and the effect of antiviral strategies to recover the inflammation will be explored in collaboration with B3 (JU). A co-culture of HAE with immune cells obtained from healthy donors will be implemented and infected with several RSV strains. The cytokine profile of the co-cultures will be determined. During this project, we will focus on the following research objectives: - Optimize HAE culture system to study Respiratory Syncytial Virus (RSV) infections. - Proof-of-principle that the RSV/HAE can be used to characterize the potency of novel antiviral strategies including the characterization of resistance associated mutations. - Proof-of-principle that the co-culture of immune cells and HAE can be used to characterize the potency of novel antiviral strategies against RSV. This would mainly focus on the role of alveolar macrophages in pathology and replication of RSV. - Demonstrate that the HAE system can bring important contributions to the drug discovery/development pipeline for respiratory pathogens."