Comparative Analysis of Chitosan, Lipid Nanoparticles, and Alum Adjuvants in Recombinant SARS-CoV-2 Vaccine: An Evaluation of Their Immunogenicity and Serological Efficacy

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  Figure 1. Humoral immune response induced by alum-, eLNP-, or chitosan-adjuvanted SARS-CoV-2 recombinant vaccines. (A) Experimental design of mice immunized with the adjuvanted recombinant SARS-CoV-2 vaccine. Serum levels of S1-specific IgG, IgG1, and IgG2c antibodies in vaccinated mice on day 14 post-boost are shown in (B), (C), and (D), respectively. Ratios of the indicated IgG subclasses in the vaccinated mice serum are shown in (E). Dissociation of low-affinity S1-specific IgGs was performed using 4 M urea, and the avidity index in the endpoint titer is presented in (F). Neutralizing titers were measured using a live virus neutralization assay (G). Results from two independent experiments. The group receiving eLNPs was administered at a dose equivalent to 3 µg of mRNA-LNP (this dose is equivalent to 90 µg of total lipid, or 45 µg of ionizable lipid). The control group received 5 µg of unadjuvanted recombinant SARS-CoV-2 protein. Data are presented as the mean ± SD (n = 5). p-values: * denotes p < 0.05, and ** p < 0.01.

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  Figure 2. Cellular immune responses induced by alum-, eLNP- or chitosan-adjuvanted SARS-CoV-2 recombinant vaccine. (A) Experimental design of mice immunized with the adjuvanted recombinant SARS-CoV-2 vaccine. S1-specific CD4+ and CD8+ T-cell responses were evaluated by flow cytometry (B,C). T cells were labeled with cellular markers and assessed for the intracellular cytokine expression of Th1 (IFN-γ, IL-2, and TNF-α), Th2 (IL-4), and Th17 (IL-17a). (D,E) Pie charts representing the percentage of CD4+ (D) and CD8+ (E) polyfunctional cytokine-producing cells positive for two or three cytokines. Results from two independent experiments. The group receiving eLNPs was administered a dose equivalent to 3 µg of mRNA-LNP (this dose is equivalent to 90 µg of total lipid, or 45 µg of ionizable lipid). The control group received 5 µg of unadjuvanted recombinant SARS-CoV-2 protein. Data are presented as the mean ± SD (n = 5). p-values: * denotes p < 0.05, ** p < 0.01, and *** p < 0.001.

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  Figure 3. Proinflammatory cytokine and chemokine production in muscle tissue after vaccination. (A) Experimental design of the muscle tissue extraction in samples 6, 24, and 48 h after vaccination with alum-, eLNP-, or chitosan-adjuvanted recombinant SARS-CoV-2 vaccine. (B) Production of proinflammatory cytokines: IFN-γ, IL-1α, IL-6, IL-10, IL-2, IL-7 and IL-9. (C) Production of proinflammatory chemokines: Eotaxin, IP-10 (CXCL10), MIP-1α, and RANTES. The eLNP group was administered a dose equivalent to 3 µg of mRNA-LNP (this dose is equivalent to 90 µg of total lipid, or 45 µg of ionizable lipid). Data are presented as the mean ± SD (n = 4) relative to the control group (PBS injection). p-values: * denotes p < 0.05, ** p < 0.01, and *** p < 0.001.

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  Figure 4. Proinflammatory cytokine and chemokine production in draining lymph nodes after vaccination. The samples were collected 6, 24 and 48 h after vaccination with alum- eLNP- or chitosan-adjuvanted recombinant SARS-CoV-2 vaccine. (A) Proinflammatory cytokines: IFN-γ, IL-1α, IL-10, IL-6 and IL-2 production. (B) Production of proinflammatory chemokines: IP-10 (CXCL10), MIG, and MIP-1α. The eLNP group was administered a dose equivalent to 3 µg of mRNA-LNP (this dose is equivalent to 90 µg of total lipid, or 45 µg of ionizable lipid). Data are presented as the mean ± SD (n = 4) relative to the control group (PBS injection). p-values: * denotes p < 0.05, ** p < 0.01, and *** p < 0.001.

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  Figure 5. Innate immune cell recruitment induced by adjuvanted SARS-CoV-2 recombinant vaccine at the injection site. Mice were injected intramuscularly (IM) with recombinant SARS-CoV-2 vaccine adjuvanted with alum, eLNPs, or chitosan. Innate immune cell recruitment was examined at 6, 24, and 72 h in the gastrocnemius muscle by flow cytometry. (A,B) Representative flow cytometry gating strategy for neutrophils (CD45+Ly6G+CD11b+) (A) and their percentages (B). (C,D) representative gating strategy for macrophages (CD45+Ly6G−CD11b+F4/80+CD64+) (C) and their percentages (D). (E–G) Representative gating strategy for dendritic cells (1: CD45+Ly6G−CD11b+CD11c+IA/IE+), monocytes, and natural killer cells (2: CD45+Ly6G−CD11b+CD11c+IA/IE−) (E) and their percentages ((F) and (G), respectively). The eLNP group was administered a dose equivalent to 3 µg of mRNA-LNP (this dose is equivalent to 90 µg of total lipid, or 45 µg of ionizable lipid). The control group received 5 µg of unadjuvanted recombinant SARS-CoV-2 protein. Data are presented as the mean ± SD (n = 4). p-values: * denotes p < 0.05, ** p < 0.01, and *** p < 0.001.