Periodic Reporting for period 4 - MAD-CoV 2 (Modern approaches for developing antivirals against SARS-CoV 2)
Reporting period: 2023-08-01 to 2024-07-31
The MAD-CoV 2 project is a collaboration of world-leading researchers and innovative industry partners that aims to develop and deliver treatment for COVID-19 patients, significantly increasing the capacity to handle the outbreak of SARS-CoV-2. Additionally, this project will also identify and characterize new targets for the development of antiviral therapeutics by using state-of-the-art technology.
The general objectives has been, i) to establish state-of-the-art infection model system; ii) to verify the antiviral activity of recombinant soluble human Angiotensin-converting enzyme 2 (srhACE-2), iii) to identify and characterize new essential host cell factors for SARS-CoV-2 and other Coronaviruses with pandemic potential, iv) to develop novel antiviral therapies to target novel host factor essential for SARS-CoV-2 infections, v) to exploit project outputs and disseminate the results to the scientific community, public health bodies, NGOs, outbreak management teams.
The general objectives has been, i) to establish state-of-the-art infection model system; ii) to verify the antiviral activity of recombinant soluble human Angiotensin-converting enzyme 2 (srhACE-2), iii) to identify and characterize new essential host cell factors for SARS-CoV-2 and other Coronaviruses with pandemic potential, iv) to develop novel antiviral therapies to target novel host factor essential for SARS-CoV-2 infections, v) to exploit project outputs and disseminate the results to the scientific community, public health bodies, NGOs, outbreak management teams.
Within WP1, the management and coordination of MAD-CoV 2 has been organised around a Project Management Team (PMT), composed of two partners (SVA and IT) and whose role is, to act as the interface between the consortium and the IMI office, to hold the accounting of the project, to ensure that project deliverables and reports are timely submitted to the IMI offices, to organize the General Assembly (GA) and Executive Committee (ExCom) meetings, drafting the agenda and minutes of these meetings, and liaise with the project advisory board.
Within WP2 we have verified the antiviral activity of recombinant soluble human Angiotensin-converting enzyme 2 (srhACE-2) against different Variants of Concorn (VOC). In addition, we demonstrated the antiviral activity of srhACE-2 in Vivo and also investigated the role of ACE2 in molecular pathogenesis of COVID-19 in diabetic condition, using 3D-organoids.
Within WP3, we performed unbiased forward genetic screening to identify host cell factors required for SARS-CoV-2 infection and replication. Using varying screening setups and SARS-CoV-2 variants of concern, several candidate host cell factors were identified, with their validation being explored. We continuously optimized the screening systems and implemented new modalities. In addition, libraries of host cell sugar-binding proteins, so called lectins, have been investigated for their role in the SARS-CoV-2 tethering process. A newly developed human lectin library was subjected to SPIKE protein interaction screens, which confirmed candidates from the mouse library (reporting periods 1 and 2) and revealed additional SPIKE interactors. Interestingly, the lectin binding capacities to the SPIKE protein strongly depend on the variant of concern. During the fourth reporting period (RP4), a range of approaches was undertaken to identify and characterize the host cell factors essential for SARS-CoV-2 infection and replication, as outlined in Work Package 3 (WP3). A significant development was the exploration of a novel homogeneous murine-based screening setup for chemical mutagenesis, which successfully confirmed the previously identified host cell factor, Ccz1. Additionally, the team engineered and characterized various cellular systems designed for both gene trap mutagenesis (Task 3.2.1) and chemical mutagenesis (Task 3.2.2) screens, expanding the toolkit available for studying viral interactions with host cells.
Within WP4, we have continued the work on the host cell factor SPEN/SHARP. SPEN/SHARP is a transcription repressor that was published to be an important SARS-CoV-2 host cell factor (Daniloski et al., Cell 2021, DOI: 10.1016/j.cell.2020.10.030). To further pursue this, JLU equipped wild-type (wt) and SPEN/SHARP CRISPR/Cas9 KO HeLa cells with ACE2 to make them permissible for SARS-CoV-2. Subsequent infection experiments however, did not reveal a particular dependency of SARS-CoV-2 replication on SPEN/SHARP, indicating it is not an essential host factor. This subproject was therefore discontinued. Partner JLU investigated the sensitivity of different SARS-CoV-2 variants to the antiviral type I interferons (IFNs),and found that the now dominant variant of concern Omicron (BA.1) has evolved towards an increased IFN resistance (Shalamova et al., PNAS Nexus 2022, DOI: 10.1101/2022.02.17.480904). In addition, we used results from a previous genome-wide screen and discovered the membrane-anchored cell protein LRP1 as an important cofactor of infection by a series of RNA viruses including SARS-CoV-2. However, the replication cycle of SARS-CoV-2 was found to be not LRP1-dependent enough to pursue this further for the development of antivirals. The results were published (Devignot et al., Life Sci Alliance. 2023, DOI: 10.26508/lsa.202302005. In RP4, the focus of WP4 shifted to validating the pro-viral host factors identified through the genome-wide mutational screens conducted in WP3. The teams successfully identified several compounds and host targets that demonstrated potent inhibitory effects against SARS-CoV-2 and other coronaviruses, based in part on the screening results. These findings highlight potential therapeutic avenues for combating viral infections.
Within WP5, IT has prepared the project website, and the project communication pack in period 1. Furthermore, partner IBEC has been in charge to supervise MAD-CoV 2 Virtual Reality video and mobile app. Both dissemination tools were delivered by the expected time (31/07/2021). MAD-CoV 2 activity has been disseminated by the partners to the scientific community and the general public in various conferences, courses and workshops.
Within WP2 we have verified the antiviral activity of recombinant soluble human Angiotensin-converting enzyme 2 (srhACE-2) against different Variants of Concorn (VOC). In addition, we demonstrated the antiviral activity of srhACE-2 in Vivo and also investigated the role of ACE2 in molecular pathogenesis of COVID-19 in diabetic condition, using 3D-organoids.
Within WP3, we performed unbiased forward genetic screening to identify host cell factors required for SARS-CoV-2 infection and replication. Using varying screening setups and SARS-CoV-2 variants of concern, several candidate host cell factors were identified, with their validation being explored. We continuously optimized the screening systems and implemented new modalities. In addition, libraries of host cell sugar-binding proteins, so called lectins, have been investigated for their role in the SARS-CoV-2 tethering process. A newly developed human lectin library was subjected to SPIKE protein interaction screens, which confirmed candidates from the mouse library (reporting periods 1 and 2) and revealed additional SPIKE interactors. Interestingly, the lectin binding capacities to the SPIKE protein strongly depend on the variant of concern. During the fourth reporting period (RP4), a range of approaches was undertaken to identify and characterize the host cell factors essential for SARS-CoV-2 infection and replication, as outlined in Work Package 3 (WP3). A significant development was the exploration of a novel homogeneous murine-based screening setup for chemical mutagenesis, which successfully confirmed the previously identified host cell factor, Ccz1. Additionally, the team engineered and characterized various cellular systems designed for both gene trap mutagenesis (Task 3.2.1) and chemical mutagenesis (Task 3.2.2) screens, expanding the toolkit available for studying viral interactions with host cells.
Within WP4, we have continued the work on the host cell factor SPEN/SHARP. SPEN/SHARP is a transcription repressor that was published to be an important SARS-CoV-2 host cell factor (Daniloski et al., Cell 2021, DOI: 10.1016/j.cell.2020.10.030). To further pursue this, JLU equipped wild-type (wt) and SPEN/SHARP CRISPR/Cas9 KO HeLa cells with ACE2 to make them permissible for SARS-CoV-2. Subsequent infection experiments however, did not reveal a particular dependency of SARS-CoV-2 replication on SPEN/SHARP, indicating it is not an essential host factor. This subproject was therefore discontinued. Partner JLU investigated the sensitivity of different SARS-CoV-2 variants to the antiviral type I interferons (IFNs),and found that the now dominant variant of concern Omicron (BA.1) has evolved towards an increased IFN resistance (Shalamova et al., PNAS Nexus 2022, DOI: 10.1101/2022.02.17.480904). In addition, we used results from a previous genome-wide screen and discovered the membrane-anchored cell protein LRP1 as an important cofactor of infection by a series of RNA viruses including SARS-CoV-2. However, the replication cycle of SARS-CoV-2 was found to be not LRP1-dependent enough to pursue this further for the development of antivirals. The results were published (Devignot et al., Life Sci Alliance. 2023, DOI: 10.26508/lsa.202302005. In RP4, the focus of WP4 shifted to validating the pro-viral host factors identified through the genome-wide mutational screens conducted in WP3. The teams successfully identified several compounds and host targets that demonstrated potent inhibitory effects against SARS-CoV-2 and other coronaviruses, based in part on the screening results. These findings highlight potential therapeutic avenues for combating viral infections.
Within WP5, IT has prepared the project website, and the project communication pack in period 1. Furthermore, partner IBEC has been in charge to supervise MAD-CoV 2 Virtual Reality video and mobile app. Both dissemination tools were delivered by the expected time (31/07/2021). MAD-CoV 2 activity has been disseminated by the partners to the scientific community and the general public in various conferences, courses and workshops.
MAD-CoV 2 succeeded to bring evidence that srhACE can act as as a universal antiviral against all SARS-COV-2 variants. By using 3D organoid model system MAD-CoV 2 has contributed to increase the knowledge on molecular pathogenesis of SARS-CoV-2 in diabetic patients. MAD-CoV 2 has also demonstrated that these 3D organoid infection models will give us, and other scientists, tools for investigating the molecular pathogenesis of the pathogens. In addition, we have also established a unique human lectin library, which can be used to identify lectins with antiviral activities, not only against SARS-CoV-2 but also against other emerging virus.