Fig 2: Protective efficacy of single dose immunization with Clec9A-RBD(A) Experimental design schematic to compare the antigen-specific immune responses elicited in 5- to 6-week-old BALB/c mice (n = 5 per group) s.c. immunized once with 2 μg Clec9A-RBD adjuvanted with 50 μg poly I:C, relative to mice immunized once or twice with 0.05 μg Comirnaty mRNA vaccine. (B) SARS-CoV-2 RBD-specific IgG titers in immune sera and BAL samples harvested at 1 month after immunization were determined by ELISA. The dashed and dotted line represents the limit of detection in serum (2.3 log10) and BAL (1 log10) samples, respectively. (C and D) Neutralizing and (D) ADCC activity of immune sera and BAL samples against ancestral SARS-CoV-2 at 1 month after immunization were determined by c-Pass sVNT and ADCC reporter bioassay, respectively. The dashed line represents the cut-off value above which samples are considered positive for neutralizing activity (30%) and baseline ADCC fold induction (1.0), respectively. (E) Frequency of IFN-γ+ splenocytes and lung cells upon re-stimulation with ancestral SARS-CoV-2 RBD peptides at 7 days after immunization were determined by IFN-γ ELISPOT. (F) Percentage of CD4+ and CD8+ T cells expressing IFN-γ, IL-2, and/or TNF-α in spleen upon re-stimulation with ancestral SARS-CoV-2 RBD peptides at 7 days after immunization were determined by flow cytometry. (G) Experimental design schematic of the SARS-CoV-2 MA10 challenge experiment in 5- to 6-week-old BALB/c mice (n = 8 for body weight loss and clinical scores; n = 5 for lung viral load) immunized according to regimen in (A). (H) Percentage of body weight loss compared with body weight at the start of the experiment. (I) Average clinical scores of infected mice over 10 dpi. Clinical scores were based on five criteria: appearance of mouse coat, level of consciousness, activity level, eye condition, and respiratory quality scored on a scale of 1–5). (H and I) Symbols represent mean percentages (H)/clinical score (I) ± SD. (J) Lung viral titers in infected mice at 3 dpi were quantified via plaque assay. The dashed line represents the LOD (1.92 log10) and undetected samples were given an arbitrary value corresponding to half the LOD value (1.62 log10). (B–E and J) Symbols represent individual animals and bars represent (C-F, J) mean or (B) geometric mean ± (F) SD. Statistical analysis: (B–F, H–J) non-parametric two-tailed Kruskal-Wallis test with Dunnett’s correction for multiple comparisons. ∗p < 0.05, ∗∗p < 0.01 or ∗∗∗p < 0.001. Asterisk colors represent statistical significance between corresponding groups.

Fig 3: RBD-specific cellular responses induced upon single dose immunization with Clec9A-RBD(A) Experimental design schematic to characterize the antigen-specific cellular response elicited in 5- to 6-week-old BALB/c mice (n = 5 per group) s.c. immunized once with 2 μg Clec9A-RBD adjuvanted with 50 μg poly I:C. (B) Frequency of total IFN-γ+ and IL-5+ splenocytes upon re-stimulation with ancestral and variant SARS-CoV-2 RBD peptides at 7 days after immunization were determined by IFN-γ/IL-5 FluoroSPOT. (C) Frequency of splenocytes producing IFN-γ, IL-2, and/or TNF-α upon re-stimulation with ancestral and variant SARS-CoV-2 RBD peptides at 7 days after immunization was determined by IFN-γ/IL-2/TNF-α FluoroSPOT (B, C). (D) Percentage of spleen CD4+ and CD8+ T cells expressing IFN-γ, IL-2, and/or TNF-α upon re-stimulation with ancestral and variant SARS-CoV-2 RBD peptides at 7 days after immunization was determined by flow cytometry. (B–D) Splenocytes from naive mice were restimulated with ancestral SARS-CoV-2 RBD peptides as a negative control. Bars represent the mean ± SD. Statistical analysis: (B–D) Non-parametric two-tailed Kruskal-Wallis test with Dunnett’s correction for multiple comparisons. ∗p < 0.05.

Fig 4: Induction of RBD-specific lung mucosal immune responses upon single dose systemic immunization with Clec9A-RBD(A) Experimental design schematic to characterize the antigen-specific immune response elicited in lung tissues of 5- to 6-week-old BALB/c mice (n = 5 per group) s.c. immunized once with 2 μg Clec9A-RBD adjuvanted with 50 μg poly I:C. (B) Presence of Clec9A-RBD construct in lung homogenate at 4 days after immunization was detected by ELISA. (C) Percentage of RBD-specific GC B cells in perfused lung at 7 days after immunization was determined by flow cytometry. (D) SARS-CoV-2 RBD-specific IgG, IgG1, and IgG2a titers in BAL samples at 2 weeks after immunization were determined by ELISA. The dashed line represents the limit of detection (1 log10). The ratio of anti-RBD IgG2a:IgG1 was also calculated. (E and F) Neutralizing and (F) ADCC activity of immune sera and BAL samples against ancestral SARS-CoV-2 at 2 weeks after immunization were determined by c-Pass sVNT and ADCC Reporter Bioassay, respectively. The dashed line represents the cut-off value above which samples are considered positive for neutralizing activity (30%) and baseline ADCC fold induction (1.0), respectively. (G) Percentage of CD4+ and CD8+ T cells expressing IFN-γ, IL-2, and/or TNF-α in perfused lung upon re-stimulation with ancestral SARS-CoV-2 RBD peptides at 7 days after immunization were determined by flow cytometry. (B–F) Symbols represent individual animals and bars represent (B, C, and E–G) mean ± (G) SD and (D) geometric mean. Statistical analysis: (B–G) Non-parametric two-tailed Mann-Whitney test. ∗p < 0.05.

Fig 5: Functional analysis of Clec9A-RBD immune sera against SARS-CoV-2 variants and other sarbecovirusesAdult (5- to 6-week-old) BALB/c mice (n = 5 per group) were s.c. immunized once with 2 μg Clec9A-RBD adjuvanted with 50 μg poly I:C. (A) Experimental design schematic to characterize the antigen-specific antibody response against SARS-CoV-2 variants and other sarbecoviruses. (B) Serum neutralizing activity against a panel of 20 sarbecoviruses at 1, 6, 12, and 21 months after immunization was determined by Multiplex sVNT. The dashed line at 30% represents the cut-off value, above which samples are considered positive for neutralizing activity. (C) IgG avidity index and (D) ADCC activity of immune sera against ancestral and variant SARS-CoV-2 at 1, 6, 12, and 21 months after immunization were determined by urea wash ELISA, and ADCC Reporter Bioassay, respectively. The dashed line represents the baseline ADCC fold induction (1.0). (E) Serum RBD-specific IgG titers against ancestral and variant SARS-CoV-2 RBD were measured via ELISA by pooling serum harvested from each respective individual mouse at 6 and 12 months after immunization. The dashed line represents the ELISA limit of detection (2.3 log10). (B–E) Symbols represent individual animals and bars represent (B–D) mean or (E) geometric mean. Statistical analysis: (B and D) Non-parametric two tailed Friedman test with Dunnett’s correction for multiple comparisons. ∗p < 0.05 or ∗∗p < 0.01.