Single-shot dendritic cell targeting SARS-CoV-2 vaccine candidate induces broad, durable and protective systemic and mucosal immunity in mice

Abstract

Current coronavirus disease 2019 vaccines face limitations including waning immunity, immune escape by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, limited cellular response, and poor mucosal immunity. We engineered a Clec9A-receptor binding domain (RBD) antibody construct that delivers the SARS-CoV-2 RBD to conventional type 1 dendritic cells. Compared with non-targeting approaches, single dose immunization in mice with Clec9A-RBD induced far higher RBD-specific antibody titers that were sustained for up to 21 months after vaccination. Uniquely, increasing neutralizing and antibody-dependent cytotoxicity activities across the sarbecovirus family was observed, suggesting antibody affinity maturation over time. Consistently and remarkably, RBD-specific follicular T helper cells and germinal center B cells persisted up to 12 months after immunization. Furthermore, Clec9A-RBD immunization induced a durable mono- and poly-functional T-helper 1-biased cellular response that was strongly cross-reactive against SARS-CoV-2 variants of concern, including Omicron subvariants, and with a robust CD8⁺ T cell signature. Uniquely, Clec9A-RBD single-shot systemic immunization effectively primed RBD-specific cellular and humoral immunity in lung and resulted in significant protection against homologous SARS-CoV-2 challenge as evidenced by limited body weight loss and approximately 2 log₁₀ decrease in lung viral loads compared with non-immunized controls. Therefore, Clec9A-RBD immunization has the potential to trigger robust and sustained, systemic and mucosal protective immunity against rapidly evolving SARS-CoV2 variants.

Keywords: COVID-19; Clec9A; antibody-dependent cytotoxicity; dendritic cell targeting; mucosal immunity; receptor binding domain; sarbecoviruses.

Graphical abstract

Figure 1

RBD-specific antibody responses induced upon single dose immunization with Clec9A-RBD (A) Experimental design schematic to characterize the antigen-specific antibody response elicited in 5- to 6-week-old BALB/c mice (n = 5–6 per group) s.c. immunized once with 2 μg Clec9A-RBD adjuvanted with 50 μg poly I:C. (B) Longitudinal monitoring of serum SARS-CoV-2 RBD-specific IgG, IgG1, and IgG2a titers were determined by ELISA. The dashed line represents the limit of detection (2.3 log₁₀). The ratio of anti-RBD IgG2a:IgG1 was also calculated. (C) Longitudinal monitoring of serum neutralizing activity against ancestral SARS-CoV-2 was determined by c-Pass sVNT. The dashed line at 30% represents the cut-off value, above which samples are considered positive for neutralizing activity. (D and E) ADCC activity and (E) IgG avidity index of immune sera against ancestral SARS-CoV-2 at 1, 6, 12, and 21months after immunization were determined by ADCC Reporter Bioassay, and urea wash ELISA, respectively. The dashed line represents the baseline ADCC fold induction (1.0). (B–E) Symbols represent individual animals and bars represent (B) geometric mean or (C–E) mean. Statistical analysis: (B–E) Non-parametric two-tailed Mann-Whitney test (between Clec9A-RBD vs. naive) or (D) Friedman test with Dunnett’s correction for multiple comparisons (between different time points of Clec9A-RBD immunized group). ∗p < 0.05 or ∗∗p < 0.01.

Figure 2

Functional analysis of Clec9A-RBD immune sera against SARS-CoV-2 variants and other sarbecoviruses Adult (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.

Figure 3

RBD-specific T_FH and B cell subsets induced upon single dose immunization with Clec9A-RBD (A) Experimental design schematic to characterize the antigen-specific humoral 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 and C) Percentage of spleen RBD-specific (B) GC B cells (B220⁺ IgD⁻ GL-7⁺ CD95⁺ RBD⁺⁺) and (C) MBC (B220⁺ IgD⁻ CD27⁺ RBD⁺⁺) measured at 7 days and 1, 6, 9, and 12 months (circle = 6 months, triangle = 9 months, diamond = 12 months) after immunization were determined by flow cytometry. (D) Frequency of bone marrow ASC reactive to ancestral and variant SARS-CoV-2 RBD measured at 1, 6, 9, and 12 months (circle = 6 months, triangle = 9 months, diamond = 12 months), and 21 months after immunization were determined by B cell ELISPOT. Bone marrow cells from naive mice were assessed against ancestral SARS-CoV-2 RBD as a negative control. (E) Percentage of Activation-induced markers positive (AIM⁺) T_FH cells CD3⁺ CD4⁺ CXCR5⁺ PD-1⁺ CD25⁺ CD69⁺) in response to re-stimulation with ancestral and variant SARS-CoV-2 RBD peptides in spleens at 7 days, and 6, 9, and 12 months (circle = 6 months, triangle = 9 months, diamond = 12 months) after immunization were determined by flow cytometry. Splenocytes from naive mice were restimulated with ancestral SARS-CoV-2 RBD peptides as a negative control. (B–E) Symbols represent individual animals and bars represent the mean. Statistical analysis: (B and C) Non-parametric two-tailed Mann-Whitney test or (D and E) Kruskal-Wallis test with Dunnett’s correction for multiple comparisons. ∗p < 0.05 or ∗∗p < 0.01.

Figure 4

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.

Figure 5

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.

Figure 6

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.