Development of a SARS-Cov-2 Mimicking Model for Advancement of Antiviral Therapies

• Autor(a) principal: Jacinto, Manuel Costa Ferreira Lavrador
• Data de Publicação: 2023
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/160689
• topic: SARS-CoV-2; Lentiviral vectors; ACE2; TMPRSS2; Cathepsin L; Pseudotyping; Domínio/Área Científica::Engenharia e Tecnologia::Engenharia Química

Coronavirus Disease 2019 (COVID-19) is a highly contagious infection disease triggered by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that has impacted the world population immensely for the last 3 years. This work aims to establish a robust viral mimicking model of the infection process of SARS-CoV-2, focusing on the critical elements involved in viral entry. Such platform will allow for a better understanding of SARS-CoV-2 infection mechanism, providing a safe testing ground for potential antiviral drugs and therapeutics, as well to develop live cell biosensors for viral pathogen detection and quantification. Moreover, the developed model enables the manipulation of highly contagious and dangerous pathogens, like SARS-CoV-2, in a controlled laboratory environment. To develop such model, two infection-permissive cell lines expressing the ACE2 receptor and the TMPRSS2 protease were generated through lentiviral vector transduction. The expression of both proteins in the cell lines was confirmed via western blotting and flow cytometry techniques. Furthermore, to mimic the SARS-CoV-2 virus, lentiviral vectors pseudotyped with the spike proteins were generated, and their ability to transduce the developed cell lines was confirmed by flow cytometry. Transduction efficiency was optimized by testing different enhancers from which Polybrene and Vectofusin-1 emerged as the most efficient. Validation of the platform was achieved using Camostat Mesylate, Amantadine, and Dalbavancin, inhibitors well known to target ACE2, TMPRSS2 and Cathepsin-L, respectively. All inhibitors have shown to be effective in inhibiting SARS-CoV-2 spike pseudotyped lentiviral-vector transduction, confirming the reliability of the herein developed model for SARS-CoV-2 virus cell entry. This viral mimicking model demonstrated its effectiveness in replicating the viral entry mechanisms of SARS-CoV-2, offering a versatile and useful tool to develop effective antiviral drugs, novel viral detection methods, and overall advancing our comprehensive understanding of virus.