The Combination of TLR4 and TLR9 Agonists with Self-Amplifying RNA Lipid Nanoparticles Leads to a More Powerful Immune Response Against SARS-CoV-2

The aim of this study was to compare immune response against SARS-CoV-2 in Balb/C mice when self-amplifying RNA lipid nanoparticles (saRNA LNPs) combined with TLR4 Agonist (monophosphoryl lipid A) as the adjuvant 1 and TLR9 Agonist (CpG) as the adjuvant 2. Here, we found that the size distribution of saRNA LNPs was 88-165 nm with a mean size of 126 nm. Although TLR4 Agonist (adjuvant 1) and TLR9 Agonist (adjuvant 2) could increase the expression of S-protein in HEK293T/17 cells compared with saRNA LNPs alone, the combination of both adjuvants had a significant effect on the expression of the S-protein. Moreover, combining TLR4 Agonist (adjuvant 1) and TLR9 Agonist (adjuvant 2) increased the antibody (IgG and IgA) titer. Here, the ratio of IgG2a/IgG1 showed a T helper type 1-biased response. ELISpot test showed the mice vaccinated with saRNA LNPs+ TLR4 Agonist and TLR9 Agonist had significantly more secreting cells compared with other vaccinated mice (<i>p</i> < 0.05). The secretion of interleukin (IL)-4 and interferons (IFN)-γ by re-stimulated splenocytes confirmed these data. Significant differences in concentration of IL-4 and IFN-γ produced by activated splenocytes were also seen in the mice vaccinated with saRNA LNPs+ TLR4 Agonist and microparticles compared with other groups (<i>p</i> < 0.05). The highest quantity of S-protein was detected in the blood, followed by the small intestine and spleen. The interesting thing was that no significant difference was seen between the amount of S-protein induced by different formulations and the type of adjuvant did not affect the biodistribution.