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Biological relevance of the multiple infection unit as a novel target for antiviral development

Periodic Reporting for period 2 - CM_GF (Biological relevance of the multiple infection unit as a novel target for antiviral development)

Reporting period: 2021-11-01 to 2022-10-31

Enteric viruses are a prevalent group of human pathogens with a great clinical and socio-economic impact. Recently, it has been described that in vivo infection by enteric viruses is dependent on the model of multiple infection unit (MIU), whereby multiple viral particles enter the target cell, and the chance of successful infection by complementation of defective particles is increased. The mechanisms by which enteric viruses achieve the MIU is i) by clustering in vesicles, ii) by binding indigenous bacteria of the gastrointestinal tract and iii) by aggregation. The aim of my project is to determine whether the multiple infectious unit model is a druggable target in vitro and in vivo by focusing on blocking the interaction between enteric viruses and indigenous bacteria.
The positive impact of this work is the development of pan-viral small molecules that could curb infection of enteric viruses by blocking the MIU.
The main objectives during the outgoing phase were the development of i) the MIU model in human intestinal enteroids and ii) the virus-bacteria screening platform.
This project started in the laboratory of Prof. Wobus at the University of Michigan in February 2020. By then, we had experienced issues with the biomimetic model of intestine, the human intestinal enteroids (HIE) that was supposed to be implemented for the MIU model. In particular, human norovirus (HNoV) infection of HIE in 2D monolayers was not consistently reproducible. For this reason, we implemented a protocol of infection in 3D HIE. Unfortunately, the model development was halted due to the COVID-19 pandemics. Starting from April 2020, I was involved in a COVID-19 task force at the University of Michigan for the setup of a BSL3 facility, bottom-up implementation of protocol of SARS-CoV-2 infection and viral inactivation, development of a high-content imaging screening for the identification and repurposing of FDA-approved drug with in vitro activity against SARS-CoV-2. The pivot on SARS-CoV-2 related research was promptly communicated to the program officer and for about 8 months, I was involved in SARS-CoV-2 collaborative research and training of new personnel for the BSL3 facility. Overall, we performed:
1. studies of SARS-CoV-2 inactivation with UV (to reuse N95 masks during the shortage)
2. high-content imaging screening of over 1400 FDA-approved drugs and found that bovine lactoferrin retains activity against SARS-CoV-2 in vitro with multiple mode of action: at the cell entry level by blocking interaction with heparan sulfate and the at the post-entry level by modulate innate immune responses
3. molecular studies on host-SARS-CoV-2 chimeric mRNA as an artifact for RNA sequencing
4. clinical studies on prolonged SARS-CoV-2 infection in an immune compromised patient
5. investigation on complement activation upon SARS-CoV-2 infection
6. studies on viral biology: involvement of ARF6 in viral entry
7. host factor identification, in particular we looked for modifiers of viral receptor (ACE2) expression by CRISPR screening.

This effort resulted in many collaborative publications and an undoubtful social impact. Data were disseminated as manuscripts in open access platforms (bioRxiv and medRxiv) and journals with open access, to conferences (World Virology Symposium) and department seminars.

In the eight months left at the University of Michigan (January-August 2021), I also succeeded in bringing forward the proposed project. In particular, we characterized the model of infection of HNoV in 3D-HIE and we implemented a screening platform for virus-bacteria interaction by flow cytometry, and not by pulldown assay. We also identified compounds, glyco-oligomers that holds promise to block the interactCion between HNoV and indigenous bacteria by interacting with histo blood group antigen (HBGA) binding site on the viral particle.
The 3D HIE model of infection for human norovirus is currently being used to optimize co-culture with indigenous bacteria, This model is amenable to co-culture because upon differentiation, 3D HIE undego polarity reversion, and by consequence allow colonization with microbes and/or infection with HNoV without the burden of microinjection. In addition, the enteroids are kept in basal membrane extract (Matrigel), therefore multiple washing with fresh bacteria inoculum could avoid the toxicity of co-culture by bacteria overgrowth. By the end of the proposal, we expect to establish a robust co-culture system and disseminate these finding to the scientific community.

We have also identified a group of synthetic glycooligomers that could be used to block the interaction between E. cloacae and HNoV in the HIE model. We will test them in the HIE model and run toxicity assay in mice to assess translatability. We expect to propose at least one hit compound for follow up studies.

Lastly, we will collect data on the response of other indigenous bacteria to HNoV binding and assess how specific/general the findings on the E. cloacae and HNoV are. These data will be foundational for my future research program as an independent researcher.
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