In view of addressing the global necessity of an effective vaccine in the SARS-CoV-2 pandemic, a plasmid DNA vaccine, expressing for the spike (S) protein and formulated in lipoplexes, was manufactured and tested for in vitro transfection and in vivo immunogenicity. Blank cationic liposomes of 130.9 ± 5.8 nm in size and with a zeta potential of +48 ± 12 mV were formulated using the thin-film layer rehydration method. Liposomes were complexed with pCMVkan-S at different N/P ratios. Ratios of 0.25:1 and 1:1 were selected according to their complex stability and controlled size compared to other ratios and tested in vitro for transfection studies and in vivo for immunogenicity. Both selected formulations showed enhanced neutralizing antibody responses compared to pCMVkan-S injected alone, as well as an increased T cell response. The titers observed were similar to those of intramuscular electroporation (IM-EP), which was set as an efficacy goal.
Previous investigations conducted on a liposomal formulation for a SARS-CoV-2 DNA vaccine manufactured using the thin-film layer rehydration method showed promising immunogenicity results in mice. The adaptation of the liposomal formulation to a scalable and reproducible method of manufacture is necessary to continue the investigation of this vaccine candidate. Microfluidics manufacture shows high potential in method translation. The physicochemical characterization of the blank liposomes produced by thin-film layer rehydration or microfluidics were shown to be comparable. However, a difference in lipid nanostructure in the bilayer resulted in a significant difference in the hydration of the thin-film liposomes, ultimately altering their complexation behavior. A study on the complexation of liposomes with the DNA vaccine at various N/P ratios showed different sizes and Zeta-potential values between the two formulations. This difference in the complexation behavior resulted in distinct immunogenicity profiles in mice. The thin-film layer rehydration-manufactured liposomes induced a significantly higher response compared to the microfluidics-manufactured samples. The nanostructural analysis of the two samples revealed the critical importance of understanding the differences between the two formulations that resulted in the different immunogenicity in mice.
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