SMART-lipid nanoparticles enabled mRNA vaccine elicits cross-reactive humoral responses against the omicron sub-variants.

The continual emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has necessitated the development of broad cross-reactive vaccines. Recent findings suggest that enhanced antigen presentation could lead to cross-reactive humoral responses against the emerging variants. Toward enhancing the antigen presentation to dendritic cells (DCs), we developed a novel shikimoylated mannose receptor targeting lipid nanoparticle (SMART-LNP) system that could effectively deliver mRNAs into DCs. To improve the translation of mRNA, we developed spike domain-based trimeric S1 (TS1) mRNA with optimized codon sequence, base modification, and engineered 5' and 3' UTRs. In a mouse model, SMART-LNP-TS1 vaccine could elicit robust broad cross-reactive IgGs against Omicron sub-variants, and induced interferon-γ-producing T cells against SARS-CoV-2 virus compared with non-targeted LNP-TS1 vaccine. Further, T cells analysis revealed that SMART-LNP-TS1 vaccine induced long-lived memory T cell subsets, T helper 1 (Th1)-dominant and cytotoxic T cells immune responses against the SARS-CoV-2 virus. Importantly, SMART-LNP-TS1 vaccine produced strong Th1-predominant humoral and cellular immune responses. Overall, SMART-LNPs can be explored for precise antigenic mRNA delivery and robust immune responses. This platform technology can be explored further as a next-generation delivery system for mRNA-based immune therapies.

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

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.