Jefferson Digital Commons quarterly report: October-December 2019

This quarterly report includes: Articles Dean\u27s Research Development Lunch Conference Dissertations Educational Materials From the Archives Grand Rounds and Lectures Journals and Newsletters Population Health Presentation Materials Posters Reports Symposiums What People are Saying About the Jefferson Digital Common

Stereoselective synthesis of a new carba sulfonamide pseudomannobioside as possible DC-SIGN ligand.

Enveloped (Env) viral particles usually contain different types of surface proteins the majority of which is modified by the addition of N-linked, O-linked carbohydrate chains or both. The key role of these carbohydrates in protein function and immune recognition is that to mask the antigenicity of the polypeptide backbone of the Env. At the same time carbohydrates guide the presentation of the whole glycoprotein to antigen-presenting cells. Glycans on gp120 interacts particularly with two host proteins: the endocytosis receptor of human macrophage membranes and serum lectin known as mannose-binding protein. Viral Env interacts first of all with a C-type lectin, dendritic cells-specific intercellular adhesion molecule 3 (ICAM-3-grabbing nonintegrin (DC-SIGN) also known as CD209. Interaction of DC-SIGN with viral envelope is mediated by a CRD (Carbohydrate Recognition Domain) of the protein, which uses a Ca2+ ion to link the oxygen atoms of carbohydrates hydroxyl groups. Indeed, this domain can play a significant role in the stereospecific recognition of carbohydrates via the relative stereochemistry (cis or trans) of two adjacent hydroxyl groups: for instance, hydroxyl groups on C(3)- and C(4) position of mannose serve as coordination ligands for the Ca2+. Many studies on the binding affinity and antiviral activity demonstrated that analogous compounds (compounds 1a/1b) of natural oligosaccharide ligand, Man9(GlcNAc)2, bind efficiently DC-SIGN protein. The design of DC-SIGN high affinity ligands which mimics the terminal two mannose residues of the natural ligand, Manα1,2-Man, is one of the issues pursue in laboratory where I carried out my thesis. The main purpose of my thesis project was to perform a stereoselective synthesis of the new pseudodisaccharide 2, characterized by the presence of a protected amino group on C(4) position of the carbamannose unit. This synthetic approach starts with the transformation of the commercially available tri-O-acetyl-D-glucal (+)-3 into the primary alcohol (+)-4 and the switch of them into the corresponding carba analogue (-)-5, in which a metilene group replaces the endocyclic oxygen present in (+)-4. Then, after appropriate elaborations, the carba skeleton of (-)-5 was transformed into vinyl epoxide (-)-6, precursor of the key vinyl aziridine (+)-7. Ring opening of aziridine (+)-7 and appropriate elaborations allow the formation of protected trans amino-alcohol 8. Then, stereoselective epoxidation of residual C(1)-C(2) double bond of 8 give pivotal epoxy derivative 9β. The new oxirane moiety represents a new carbaglycosylating agent and was suitable to nucleophilic ring opening with azido ethanol to produce the glycosyl acceptor 10. Glycosylation reaction of glycosyl acceptor 10 by typical glycosyl donor, the trichloroacetimidate TCA (-)-11, was successfully carried out to afford the glycoconjugate 12 in a completely stereoselective fashion. The resulting pseudodisaccharide 12 was finally subjected to different steps of deprotection in order to obtain the desired fully-O-deprotected pseudomannobiosides 2. Interesting the aromatic ring of the tosyl group on nitrogen in C(4) position could represent a useful moiety of interaction with DC-SIGN binding site through additional lipophilic van der Walls connections. Pseudodisaccharide 2 will be send to Professor Fieschi at Institute de Biologie Structurale in Grenoble in order to evaluate its affinity with DC-SIGN lectin and its activity. References • N. Varga, I. Sutkeviciute, C. Guzzi, J. McGeagh, I. Petit-Haertlein, S. Gugliotta, J. Weiser, J. Angulo, F. Fieschi, A. Bernardi. Chem. Eur. J., 19 (2013), pp. 4786–4797. • Y. Guo, H. Feinberg, E. Conroy, D. A. Mitchell, R. Alvarez, O. Blixt, M. E. Taylor, W. I. Weis, K. Drickamer, Nat. Struct. Mol. Biol. 2004, 11, 591–598/A. E. Cho, V. Guallar, B. J. Berne, R. Friesner, J. Comput. Chem. 2005, 26, 915–931. • Reina J.J., Sattin S., Invernizzi D., Mari S., Martínez-Prats L., Tabarani G., Fieschi F., Delgado R., Nieto PM., Rojo J., Bernardi A.. ChemMedChem. 2007 Jul; 2(7):1030-6

Relationship between HLA and T cell responses to Ebola virus

Ebola virus disease (EVD) is a severe illness caused by infection with Ebola virus (EBOV) which causes sporadic outbreaks in African countries, the last one taking place in West Africa and affecting over 28000 people. Even though several decades have passed since the description of the first EVD cases, there are still many unanswered questions regarding the involvement of the human immune response in the pathophysiology of EVD. Research in this field is challenging because of the requirement of biosafety level 4 containment and the scarcity of human data. During the last EVD outbreak in West Africa, we had the opportunity to establish an immunology lab at Donka hospital in Conakry, Guinea where we collected and analysed leftover blood samples from patients diagnosed by the European Mobile Laboratory. With the use of benchtop multiparametric flow cytometry and subsequent analysis in the biosafety level 4 laboratory in Hamburg, we evaluated the kinetics and phenotype of antigen-presenting cells as well as T cells with the goal to identify immune biomarkers of disease outcome. Our approach was to utilize flow cytometry to characterize cell profiles in blood as well as immunogenetics, in particular analysis of the T-cell receptor clonotypes and HLA polymorphisms. Immune data was then correlated with clinical and epidemiological findings to try to detect potential predictors of outcome and targets for immunotherapy. Our observations highlight the importance of CD16 monocytes in the innate immune response to EBOV infection and in viral clearance. Additionally, we could identify a dysregulation of the adaptive immune response in fatal cases, characterised by the upregulation of the T cell inhibitory markers CTLA-4 and PD-1 and the inability to control viral replication. These results identify CD16 monocytes and the regulatory pathways of T cell responses as potential targets for post-exposure EVD immunotherapy. Moreover, our immunogenetics study evidenced the relevance of the expression of various HLA alleles and the activation of a T cell response through a broad and diverse T cell receptor repertoire in improved disease outcome

Advancing antiviral strategies against emerging RNA viruses by phenotypic drug discovery

Pathogenic RNA viruses can emerge from unexpected sources at unexpected times and cause severe disease in humans, as exemplified by the ongoing coronavirus disease 19 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Ebola virus (EBOV), Crimean-Congo hemorrhagic fever virus (CCHFV) and Zika virus (ZIKV) outbreaks from the past decade. Despite the increasing impact of emerging viruses to health and economy worldwide, our preparedness to stand against these diseases is hampered by the lack of approved and effective antiviral therapies. Thus, the development of novel antivirals is of urgent need. To date, antiviral drug discovery has primarily focused on targeting specific viral proteins, but these treatments often suffer from viral resistance and are limited to only one or few viruses. Instead, phenotypic drug discovery enables the identification of drug candidates that are active in the disease-relevant model and not restricted to previously characterized biological processes. As RNA viruses are highly dependent on the host cell pathways due to their relatively small genome, targeting virus vulnerabilities within the host cell has been a promising antiviral strategy for broad spectrum antivirals, but is relatively unexplored so far. In fact, phenotypic approaches can additionally identify host-directed antivirals due to the unbiased nature. The focus of this doctoral thesis was to identify novel antiviral compounds with broad spectrum activity and investigate the compound mechanism of action and target pathways from the host cell and virus perspective. To achieve these goals, multiple cutting-edge phenotype-based methodologies were implemented that additionally advanced the antiviral drug discovery landscape. In Paper I, we developed an image-based phenotypic antiviral assay and screened our in-house chemical library targeting cellular oxidative stress and nucleotide metabolism pathways in Hazara virus (HAZV)-infected cells. Screening hit compounds TH3289 and TH6744 activity was validated by their therapeutic window and both compounds were also active beyond HAZV, especially TH3289 that was tested and displayed activity against EBOV, CCHFV, SARS-CoV-2 and a common cold coronavirus 229E (CoV-229E). We also excluded the intended target 8-oxoguanine DNA glycosylase (OGG1) protein to be responsible for TH6744 antiviral activity and characterized host cell chaperone and co-chaperone network as target pathways of TH6744 by implementing thermal proteome profiling methodology. In Paper II, we transferred our image-based phenotypic assay to ZIKV-infected brain cells in order to screen structural analogs of TH3289 and TH6744 against a pathogenic RNA virus. TH3289 and TH6744 again appeared among the screening hits and presented a promising therapeutic window in various cellular models, further confirming their broad activity. Moreover, TH6744 reduced ZIKV infection and progeny release in cerebral organoid model and impressively rescued ZIKV-induced cytotoxicity in organoids. Additionally, treatment with TH6744 rapidly diminished ZIKV progeny release during late replication cycle stages, elucidating the antiviral mechanism of action. In Paper III, we established an untargeted morphological profiling method to provide in-depth host cell responses during antiviral screening. We combined the Cell Painting protocol with antibody-based virus detection in a single assay followed by automated image analysis pipeline providing segmentation and classification of infected cells and extraction of cell morphological features. We demonstrated how our assay reliably distinguished CoV-229E infected human lung fibroblasts from non-infected controls based on cellular morphological features. Furthermore, our method can be applied in phenotypic drug screening as validated by nine host- and virus-targeting antivirals. Effective antivirals Remdesivir and E-64d treatment reversed the infection-specific signatures in host cells. Thereby, the developed method can be implemented for antiviral phenotypic drug discovery by morphological profiling of drug candidates

Comprehensive Analysis of Monkey Pox Outbreak Dynamics and Prevention Strategies

Monkey pox, a zoonotic viral illness, has lately undergone a global revival, prompting considerable public health concerns. This abstract seeks to deliver a thorough summary of the outbreak, encompassing its transmission, symptoms, prevention efforts, and the significance of international collaboration. Most cases of monkey pox spread through intimate contact with an infected person or animal. This may happen via direct contact with lesions, respiratory droplets, or contaminated objects. Common symptoms encompass fever, headache, tiredness, myalgia, lymphadenopathy, and a rash. Although the majority of instances are minor, serious sickness may manifest in specific populations, including children, pregnant women, and immunocompromised individuals. Several techniques are needed to avert the dissemination of monkey pox. These measures encompass refraining from close contact with diseased persons or animals, maintaining proper hygiene, and receiving vaccinations. Vaccination is especially crucial for high-risk groups, including healthcare professionals and those with compromised immune systems. Global cooperation is essential for properly managing the monkey pox outbreak. The World Health Organisation (WHO) and other international health entities have been instrumental in orchestrating global responses, delivering technical support, and facilitating information exchange. Collaborative initiatives have concentrated on monitoring, case management, and the creation and dissemination of vaccines and treatments. As the monkey pox outbreak progresses, it is crucial to maintain vigilance and execute effective preventive strategies. Comprehending the transmission pathways, symptoms, and preventive measures enables people and communities to significantly reduce the effect of this disease

Studies on the neurotropism of the highly pathogenic Nipah and Ebola virus

Nipah and Ebola viruses are highly pathogenic, zoonotic agents that are associated with high case fatality rates. While Nipah virus (NiV) is known to predominantly target the central nervous system (CNS), neurological complications from Ebola virus (EBOV) infections became apparent during the outbreak in West Africa (2013-2016). Due to their pathogenicity and the need for biosafety level 4 facilities for research, our understanding of underlying neuropathogenic mechanisms is limited. Especially how these viruses initially establish infection in the CNS is unclear. Limited autopsy data also restricts our knowledge of neuropathology in humans, necessitating reliance on animal models. However, the use of such models is challenging and costly to perform under adequate safety conditions, prompting the exploration of alternative methods. This project employed primary murine tissue to investigate NiV and EBOV neurotropism. In vitro and ex vivo key model systems were established to investigate the hematogenous pathway as a potential entry route into the central nervous system and to evaluate the susceptibility of different brain cells and regions. Furthermore, by preparing cultures from either wildtype or interferon receptor alpha/beta knockout mice (IFNAR-/-), the role of type I interferons was investigated, as they seem to play a crucial role for disease outcome after systemic infection as well as for the local brain-specific immune responses. In addition, in vitro models using human immortalized cell lines were established to investigate the transferability to infections in humans. To explore the hematogenous route as a potential entry path, primary murine brain endothelial cells were isolated and characterized along with murine and human in vitro models of the blood-brain barrier. Endothelial cells derived from human and murine sources exhibited susceptibility to NiV and EBOV infections, resulting in cytopathic effects and impaired barrier function. These findings suggest that NiV and EBOV may utilize this route to penetrate the CNS. Interestingly, no disparities in susceptibility to NiV and EBOV were observed among endothelial cells isolated from either wild type or IFNAR-/- mice. This suggests that, at the cellular level, a functional type I interferon response alone is insufficient to constrain NiV and EBOV infections in brain endothelial cells. To identify potential target cells for NiV and EBOV within the CNS and to assess their role during infection, a primary murine neuron culture (N), and a neuroglial mixed culture (NG) was isolated. Moreover, ex vivo organotypic brain slices (BS) were established to investigate viral spread in a neuronal network. The suitability of these models for neurotropic virus infections was first evaluated under biosafety level 2 conditions using vesicular stomatitis virus, which exerts a high neurotropism. The virus was able to infect the primary cultures and replicated efficiently, which proved the suitability of the models. However, virus replication in BS was only efficient in tissue from IFNAR-/- mice and led to the release of several cyto- and chemokines and significant tissue damage. This indicates that in BS, where the neuronal network is preserved, more effective antiviral responses can occur. NiV was able to infect the N and NG cultures from both mouse strains. Notably, viral replication was comparatively more efficient in the N culture, accompanied by pronounced cytotoxicity, suggesting a potential role of other brain cell types in offering protection or highlighting neurons as important drivers of NiV replication. In BS, infection primarily manifested in small foci, although areas of widespread infection were also observed. Viral replication was more robust in slices derived from IFNAR-/- mice, underscoring the significance of type I interferons in controlling viral spread. Over time, NiV infection induced tissue damage and secretion of numerous mediators, with generally higher levels observed in slices from IFNAR-/- mice. Similarly, EBOV exhibited infection in N and NG cultures, as well as in BS. In BS, infection was sporadic, forming small foci, often accompanied by astrogliosis near infected areas. Viral replication was constrained in slices from both mouse strains, nevertheless infection prompted tissue damage and the release of cytokines and chemokines, which was more pronounced in slices from IFNAR-/- mice. Altogether, the relatively confined infection of NiV and EBOV in these cultures could contribute to their persistence in the CNS. Surprisingly, the choroid plexus appeared highly susceptible to both viruses, emphasizing the blood-liquor barrier as a potential entry and persistence site for these pathogens. Interestingly, IL-17a and IL-9 were found in increased concentrations following infections with all tested viruses in BS derived from both mouse strains. As both mediators have been associated with neuroinflammatory diseases, further analysis whether they play a decisive role in NiV and EBOV neuropathology should be undertaken. Overall, these findings confirm the appropriateness of the models for investigating the neuropathogenicity of NiV and EBOV. The insights gleaned from this study align with previous observations from human autopsies and animal models, highlighting the utility of these models as alternative methods for investigating NiV and EBOV neurotropism. Nevertheless, further in-depth analyses are necessary to gain a comprehensive understanding of how the viruses enter the brain and how immune responses promote or diminish infection and tissue damage. The established models lay a groundwork for future investigations, such as visual spatial genomics to further explore the neurotropism and neuropathology of NiV and EBOV

Multidisciplinary approaches to combat emerging viruses: diagnostics, therapeutic gene and vaccine delivery, and nanotherapeutics

Emerging viruses, such as filoviruses (Ebola, Marburg), SARS and MERS coronaviruses, and Zika, pose significant threats to global public health, particularly for individuals with co-morbidities. To address these challenges, this review article explores multidisciplinary strategies for combatting emerging viruses. We emphasize the importance of developing accurate diagnostics, innovative therapeutic gene and vaccine delivery systems, and long-acting nanotherapeutics. These approaches are designed to enhance the safety and efficacy of treatments against these deadly pathogens. We discuss the collaborative efforts of virologists, geneticists, formulation scientists, clinicians, immunologists, and medicinal chemists in advancing these therapeutic modalities