A Dual-Adjuvanted Parenteral-Intranasal Subunit Nanovaccine generates Robust Systemic and Mucosal Immunity Against SARS-CoV-2 in Mice

Abstract

Existing parenteral SARS-CoV-2 vaccines produce only limited mucosal responses, essential for reducing transmission and achieving sterilizing immunity. Appropriately designed mucosal boosters can overcome the shortcomings of parenteral vaccines and enhance pre-existing systemic immunity. Here, a new protein subunit nanovaccine is developed by utilizing dual-adjuvanted (RIG-I: PUUC RNA and TLR-9: CpG DNA) polysaccharide-amino acid-lipid nanoparticles (PAL-NPs) along with SARS-CoV-2 S1 trimer protein, that can be delivered both intramuscularly (IM) and intranasally (IN) to generate balanced mucosal-systemic SARS-CoV-2 immunity. Mice receiving IM-Prime PUUC+CpG PAL subunit nanovaccine, followed by an IN-Boost, developed high levels of IgA, IgG, and cellular immunity in the lungs and showed robust systemic humoral immunity. Interestingly, as a purely intranasal subunit vaccine (IN-Prime/IN-Boost), PUUC+CpG PAL-NPs induced stronger lung-specific T cell immunity than IM-Prime/IN-Boost, and a comparable IgA and neutralizing antibodies, although with a lower systemic antibody response, indicating that a fully mucosal delivery route for SARS-CoV-2 vaccination may also be feasible. The data suggest that PUUC+CpG PAL subunit nanovaccine is a promising candidate for generating SARS-CoV-2 specific mucosal immunity.

Keywords: SARS‐CoV‐2 subunit nanovaccine; antiviral immunity; combination adjuvants; mucosal immunity; parenteral and intranasal vaccination; polymer nanoparticles.

Figure 1

Synthesis and characterization of adjuvant loaded PAL‐NPs. A) Multistep synthetic scheme of polysaccharide‐amino acid‐lipid (PAL) amphiphilic polymer. B) Schematic of PAL‐NPs fabrication and adjuvants loading (encapsulated: R848 and surface‐loaded nucleic acids: PUUC RNA, CpG DNA). C) Physiochemical characterization of PAL‐NPs: hydrodynamic diameter and zeta potential (inset: TEM image, scale bar: 500 nm). D) Murine GM‐CSF differentiated Bone Marrow Dendritic Cells (mBMDCs) were treated with single/dual/triple adjuvanted PAL‐NP formulations and controls for 24 h. E–G) Analysis of proinflammatory cytokines: IL‐1β E), IFN‐β F), and IL12p70 G) after treatment of adjuvanted PAL‐NPs (n = 6) with differentiated mBMDCs. H) Designed in vivo studies for the assessment of adaptive immune responses (both lung‐specific and systemic) using adjuvant nanovaccine formulations via two strategies: a) adjuvant‐mediated and b) different prime‐boost vaccination routes. Error bars represent SEM (standard error of the mean). Statistical significance was determined by one‐way ANOVA followed by Tukey's post‐hoc test for multiple comparisons. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 for all graphs.

Figure 2

A dual‐adjuvanated PAL‐NPs (PUUC+CpG) subunit nanovaccine elicits robust SARS‐CoV‐2 mucosal and systemic humoral immunity, when delivered IM‐Prime/IN‐Boost. A) Vaccination study design: Female BALB/c mice (n=3 for PBS and n=6 for other PAL‐NP formulations) were immunized IM at day 0 (1st dose) with nanovaccine formulation of adjuvanted PAL‐NPs (NPs: 250 µg, PUUC: 20 µg, CpG: 40 µg and R848: 20 µg) and stabilized spike (Sp) S1 trimer protein at a dose of 1 µg respectively. On day 21, mice received the 2nd dose of vaccine formulation IN using similar doses of adjuvants, PAL‐NPs, and spike protein, except for the CpG dose, which was reduced to 20 µg. Mice were euthanized after 2 weeks on day 35 to collect BAL fluid and serum. BAL fluid and serum from vaccinated mice were assayed with ELISA assay. B–E) BAL fluid from vaccinated mice was assayed for anti‐spike IgG B), IgA C), IgG1 D), and IgG2a E) with ELISA at 1:5 dilution. F) Quantification of BAL IgG2a/IgG1. G,H) Anti‐spike total IgG in serum at various dilutions measured by absorbance (A450‐A630 nm) and comparison of area under the curve (AUC). I) ACE‐2 signal measured by absorbance (A450‐A630 nm) in spike protein neutralization assay with ELISA. Lower absorbance values indicate higher spike‐neutralizing antibody levels in serum. J,K) Quantification of serum anti‐spike IgG1 and comparison of AUC. L,M) Quantification of serum anti‐spike IgG2a and comparison of AUC. N) Quantification of serum IgG2a/IgG1 ratio. Error bars represent the SEM. Normality was assessed with the Kolmogorov–Smirnov test. Statistical significance was determined with the Kruskal–Wallis test and Dunn's post‐hoc test for multiple comparisons. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 for all graphs.

Figure 3

PUUC+CpG dual‐adjuvanted PAL‐NPs subunit nanovaccine elicits robust SARS‐CoV‐2 mucosal cellular immunity when delivered IM‐Prime/IN‐Boost. A) Vaccination study design: Female BALB/c mice were immunized on days 0 (IM prime) and 21 (IN boost) with adjuvanted PAL‐NP vaccine formulation combined with S1 spike protein (see Table S1, Supporting Information for doses). Harvested lung cells on day 35 were restimulated with spike peptide pools for 6 h and stained for analysis by flow cytometry. B–D) CD4⁺ T_RM flow cytometry (FCM) plots B), percentage of CD4⁺CD69⁺CD103⁻ C) and CD4⁺CD69⁺CD103⁺ cell populations D). E–G) CD8⁺ T_RM FCM plots E), percentage of CD8⁺CD69⁺CD103⁻ F) and CD8⁺CD69⁺CD103⁺ cell populations G). H–J) CD4⁺CD44⁺ T_RM FCM plots H), percentage of CD4⁺CD44⁺CD69⁺CD103⁻ I) and CD4⁺CD44⁺CD69⁺CD103⁺ J) cell populations. K,L) Percentage of CD8⁺CD44⁺CD69⁺CD103⁻ K) and CD8⁺CD44⁺CD69⁺CD103⁺ L) cell populations. M–O) Percentage of monofunctional CD4⁺ T_RM M), CD8⁺ T_RM N), and CD4⁺CD44⁺ T_RM O) cells expressing TNF‐α. P) Percentage of monofunctional CD4⁺T_RM cell population expressing IFN‐γ. Lung cells were stained for B cell markers and analyzed by flow cytometry. Q,R) Representative FCM plots and percentage of RBD tetramer⁺ B220 cells. Outliers were identified by the ROUT method and removed. Error bars represent the SEM. Statistical significance was calculated using one‐way ANOVA followed by Bonferroni's post‐hoc test for the figures (C–N) and Tukey's post‐hoc test for figures O) and R) for multiple comparisons. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 for all graphs. ns represents the non‐significant values.

Figure 4

PUUC+CpG dual‐adjuvanted PAL‐NPs protein subunit vaccine formulation elicit robust SARS‐CoV‐2 mucosal and systemic humoral immunity with IM‐Prime/IN‐Boost group and induces a significant level of mucosal humoral responses with IN‐Prime/IN‐Boost group. A) Vaccination study design: Female BALB/c mice (n = 8 for all groups) were immunized using PUUC+CpG PAL‐NP vaccine formulation combined with S1 spike protein (see Table S1, Supporting Information for doses) through three different prime‐boost strategies (day 0: prime and day 21: boost): IM‐Prime/IN‐Boost, IN‐Prime/IM‐Boost, and IN‐Prime/IN‐Boost. On Day 35, BAL fluid and serum samples were collected from vaccinated mice, and assayed using ELISA. B–F) BAL fluid from vaccinated mice was assayed for anti‐spike IgA B), IgG C), spike nAbs D), IgG1 E), and IgG2a F) with ELISA at 1:5 dilution except for IgA and neutralization assay which was performed at 1:2 dilution. G,H) Anti‐spike total IgG in serum at various dilutions measured by absorbance (A450‐A630 nm) and comparison of AUC. I) ACE‐2 signal measured by absorbance (A450‐A630 nm)  in spike protein neutralization assay with ELISA. J,K) Quantification of serum anti‐spike IgG1 at various dilutions and comparison of AUC. L,M) Quantification of serum anti‐spike IgG2a at various dilutions and comparison of AUC. Error bars represent the SEM. Normality was assessed with the Kolmogorov–Smirnov test. Statistical significance was determined with the Kruskal–Wallis test and Dunn's post‐hoc test for multiple comparisons. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 for all graphs.

Figure 5

PUUC+CpG dual‐adjuvanted PAL‐NPs protein subunit vaccine formulation elicits robust SARS‐CoV‐2 T cell (T_RM) immunity with IN‐Prime/IN‐Boost and B cell responses with IM‐Prime/IN‐Boost. A) Vaccination study design: Female BALB/c mice (n = 8 for all groups) were immunized using PUUC+CpG PAL‐NP vaccine formulation combined with S1 spike protein (see Table S1, Supporting Information for doses) through three different prime‐boost strategies (day 0: prime and day 21: boost): IM‐Prime/IN‐Boost, IN‐Prime/IM‐Boost, and IN‐Prime/IN‐Boost. On Day 35, mice were sacrificed, and lung cells were collected and restimulated with spike peptide for 6 h. B) Gating strategies for analysis of CD4⁺ and CD8⁺ T_RM responses. C,D) Representative FCM plots of CD4⁺ T_RM and CD8⁺ T_RM populations in groups: PBS, IM‐Prime/IN‐Boost. E,F) Percentage of CD4⁺CD69⁺CD103^‐ and CD4⁺CD69⁺CD103⁺ T cells were represented through graphs along with their respective values (n = 8 for all groups) in a table format. G,H) Percentage of CD4⁺CD44⁺CD69⁺CD103^‐ and CD4⁺CD44⁺ T_RM cells were represented through graphs along with their respective values (n = 8 for all groups) in table format. I–L) Percentage of CD8⁺CD44⁺CD69⁺CD103^‐ I), CD8⁺CD44⁺ T_RM J), CD8⁺CD44⁺CD69⁺CD103^‐ K), and CD8⁺CD44⁺ T_RM L) T cell populations. M,N) Representative FCM plots and percentage of RBD tetramer⁺ B220⁺ cells. Error bars represent the SEM. Statistical significance was calculated using one‐way ANOVA followed by Tukey's post‐hoc test for the Figures (E,G, and I–L) and Bonferroni's post‐hoc test for Figures F) and H) for multiple comparisons. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 for all graphs.

Figure 6

PUUC+CpG dual‐adjuvanted PAL‐NPs subunit vaccine formulation enhances T_H1‐type immunity with IN‐Prime/IN‐Boost group. A) Female BALB/c mice (n = 8 for all groups) were immunized using PUUC+CpG PAL‐NP vaccine formulation combined with S1 spike protein (see Table S1, Supporting Information for doses) through three different prime‐boost strategies (day 0: prime and day 21: boost): IM‐Prime/IN‐Boost, IN‐Prime/IM‐Boost, and IN‐Prime/IN‐Boost. Mice were sacrificed on Day 35, and the collected lung cells were restimulated with overlapping spike peptides for 6 h and analyzed using flow cytometry to detect antigen‐specific T cell cytokine production. B–D) Representative FCM plots (groups: PBS, IM‐Prime/IN‐Boost, and IN‐Prime/IN‐Boost) of monofunctional CD4⁺ T_RM cells expressing TNF‐α B), IFN‐γ C), and GrzB D). E) Percentages of monofunctional CD4⁺ T_RM cells expressing TNF‐α, IFN‐γ, and GrzB. F–I) Percentages of polyfunctional CD4⁺ T_RM cells expressing TNF‐α+GrzB F), IFN‐γ+GrzB G), TNF‐α+GrzB H), and TNF‐α+IFN‐γ+GrzB I). J) Comparative summary (bar graph) of a percentage of monofunctional and polyfunctional CD4⁺ T_RM cells expressing TNF‐α, IFN‐γ, and GrzB in IM‐Prime/IN‐Boost and IN‐Prime/IN‐Boost (donut graph showing the percentage of each group as a fraction of total response). K–M) Representative FCM plots (groups: PBS, IM‐Prime/IN‐Boost, and IN‐Prime/IN‐Boost) of monofunctional CD8⁺ T_RM cells expressing TNF‐α K), IFN‐γ L), and GrzB M). N) Percentages of monofunctional CD8⁺ T_RM cells expressing TNF‐α, IFN‐γ, and GrzB. O–R) Percentages of polyfunctional CD8⁺ T_RM cells expressing TNF‐α+GrzB O), IFN‐γ+GrzB P), TNF‐α+GrzB Q), and TNF‐α+IFN‐γ+GrzB R). S) Comparative summary (bar graph) of percentage of monofunctional and polyfunctional CD8⁺ T_RM cells expressing TNF‐α, IFN‐γ, and GrzB in IM‐Prime/IN‐Boost and IN‐Prime/IN‐Boost (donut graph showing the percentage of each group as a fraction of total response). Error bars represent the SEM. Statistical significance was calculated using One‐Way ANOVA and Tukey post‐hoc test for multiple comparisons. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 for all graphs.

Figure 7

Summary of the adaptive immune responses, including lung‐specific (both humoral and cellular) and systemic (humoral) immune responses, generated in mice following treatment with adjuvanted PAL‐NP nanovaccine formulations and evaluated through two in vivo SARS‐CoV‐2 vaccination studies. A) Investigation of adjuvant‐mediated adaptive immune responses with various adjuvant combinations on PAL‐NPs, delivered with S1 spike trimer antigen via IM‐prime and IN‐boost strategy. Identification of best combination adjuvant PAL nanovaccine that generates robust and balanced mucosal‐systemic responses, thereby enhancing existing IM immunity and triggering potent mucosal SARS‐CoV‐2 immunity. B) Comparison study of vaccination route‐specific immune responses generated in mice after administration of PUUC+CpG PAL nanovaccine through different prime‐boost routes (IM‐P/IN‐B and IN‐P/IN‐B). The purely intranasal route (IN‐P/IN‐B) induced higher T cell responses with T_H1 type immunity and a comparable humoral response, suggesting its potential use for mucosal vaccine delivery.