Optimization of SARS-CoV-2 Pseudovirion Production in Lentivirus Backbone With a Novel Liposomal System

• DOI: 10.3389/fphar.2022.840727
• Publication Date: 2022-03-29T16:38:13Z
• Authors: Gokulnath Mahalingam; Hari Krishnareddy...; Porkizhi Arjunan; Yogapriya Periyasami; Salma M; Saravanabhavan Th...; Kumarasamypet M. ...; Mahesh Moorthy; Shaji R. Velayudhan; Alok Srivastava; Srujan Marepally
• Keywords: liposomes; RM1-950; Coronavirus Disease 2019 Research; FOS: Health sciences; Diagnostic Methods for COVID-19 Detection; Transfection; Plasmid; Biochemistry; Gene; 03 medical and health sciences; lentivirus; Biochemistry, Genetics and Molecular Biology; Virology; Health Sciences; Genetics; pseudoviral neutralization; Biology; Vector (molecular biology); Pharmacology; 0303 health sciences; Recombinant DNA; Lentivirus; HEK 293 cells; Life Sciences; Virus; 3. Good health; Liposome; Chemistry; Infectious Diseases; Biosensors; Viral disease; transfection; Infectivity; FOS: Biological sciences; SARS-CoV-2 (COVID-19); Medicine; Lipofectamine; Therapeutics. Pharmacology; Gene Therapy Techniques and Applications

Due to the fast mutating nature of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the development of novel therapeutics, vaccines, and evaluating the efficacies of existing one’s against the mutated strains is critical for containing the virus. Pseudotyped SARS-CoV-2 viruses are proven to be instrumental in evaluating the efficiencies of therapeutics, owing to their ease in application and safety when compared to handling the live virus. However, a comprehensive protocol that includes selecting transfection reagents, validating different packaging systems for high-throughput screening of neutralizing antibodies, is still a requisite. To this end, we designed and synthesized amide linker-based cationic lipids with varying hydrophilic head groups from dimethyl (Lipo-DME) to methyl, ethylhydroxyl (Lipo-MeOH), and diethylhydroxyl (Lipo-DOH) keeping the hydrophobic tail, stearic acid, as constant. Among the liposomal formulations of these lipids, Lipo-DOH was found to be superior in delivering plasmids and demonstrated comparable transfection efficiencies with commercial standard Lipofectamine 3000. We further used Lipo-DOH for lentivirus and SARS-CoV-2 pseudovirion preparation. For comparing different lentivirus packaging systems, we optimized conditions using Addgene and BEI systems and found that the BEI lenti plasmid system was found to be efficient in making lentiviruses using Lipo-DOH. Using the optimized transfection reagent and the lentivirus system, we developed a robust protocol for the generation of SARS-CoV-2 pseudovirions and characterized their infectivity in human ACE2 expressing HEK-293T cells and neutralizing properties in IgG against spike protein of SARS-CoV-2 positive human sera from individuals recovered from COVID-19.