A Toll-like Receptor-Activating, Self-Adjuvant Glycan Nanocarrier

Abstract

The global pandemic of COVID-19 highlights the importance of vaccination, which remains the most efficient measure against many diseases. Despite the progress in vaccine design, concerns with suboptimal antigen immunogenicity and delivery efficiency prevail. Self-adjuvant carriers-vehicles that can simultaneously deliver antigens and act as adjuvants-may improve efficacies in these aspects. Here, we developed a self-adjuvant carrier based on an acetyl glucomannan (acGM), which can activate toll-like receptor 2 (TLR2) and encapsulate the model antigen ovalbumin (OVA) via a double-emulsion process. In vitro tests showed that these OVA@acGM-8k nanoparticles (NPs) enhanced cellular uptake and activated TLR2 on the surface of dendritic cells (DCs), with increased expression of co-stimulatory molecules (e.g. CD80 and CD86) and pro-inflammatory cytokines (e.g. TNF-α and IL12p70). In vivo experiments in mice demonstrated that OVA@acGM-8k NPs accumulated in the lymph nodes and promoted DCs' maturation. The immunization also boosted the humoral and cellular immune responses. Our findings suggest that this self-adjuvant polysaccharide carrier could be a promising approach for vaccine development.

Keywords: glucomannan; polysaccharides; self-adjuvant; toll-like receptors; vaccination.

FIGURE 1

Schematic illustration of designing a self-adjuvant carrier for antigen delivery. (1) Acetylation of glucomannans (GM) with different molecular weights (100 and 8 kDa). (2) Encapsulation of ovalbumin (OVA) into acetyl GM (acGM) through a double-emulsion method. (3) Evaluation of the OVA-loaded acGM-8k NPs (OVA@acGM-8k NPs) in promoting the humoral and cellular immunity in mice.

FIGURE 2

Screening acGM for antigen encapsulation. (A) Scheme of different acetylation methods to synthesize acGM. (B) Turbidity of the mixture of the reaction solution and dd-H₂O (V/V = 1:1) at designed time points. (C) The mixture of the reaction solution and dd-H₂O (V/V = 1:1) was precipitated overnight. (a) TFAA/acetic acid; (b) Acetic anhydride/pyridine; (c) Acetic chloride/pyridine. (D) Effect of reaction time on the measurement of the degree of substitution (DS). (E) Effect of molecular weight on the solubility of acGM with the DS of 1.8 in volatile solvents. (F) Effect of molecular weight on OVA loading capacity and encapsulation efficiency.

FIGURE 3

Characterization of acGM-8k and acGM-8k nanoparticles (NPs). (A) 600 MHz 1D ¹H-NMR spectrum of GM-8k and acGM-8k. (B) The infrared spectra of GM-8k and acGM-8k. (C) Particle size distribution and morphology of acGM-8k NPs (inserted picture, scale bar: 200 nm). (D) Zeta potential of acGM-8k NPs.

FIGURE 4

acGM-8k NPs activate BMDCs through TLR2 in vitro. (A) Cell viability of acGM-8k NPs at different concentrations. (B) Detection of secreted embryonic alkaline phosphatase (SEAP) activity in TLR2 reporter cell (murine TLR2-expressing HEK 293 cells, Invivogen). HEK-Blue™ Null2 Cells were used as a negative control. (C–D) Determination of cytokines (C) TNF-α and (D) IL-12p70 secreted by BMDCs isolated from TLR2^−/− mice or WT mice. (E–F) Percentage of (E) CD80⁺ and (F) CD86⁺ in CD11c⁺ cells isolated from TLR2^−/− mice or WT mice. TLR2^−/−: TLR2 knockout. WT: wild type. *p < 0.05; **p < 0.01; ***p < 0.001 vs the PBS treatment; ns: no significance; n = 3: For (A) and (B), data were obtained from three replicates of one representative experiment out of three; for (C)–(F), data were obtained from three mice in each group. All numerical values are given as average values ± standard deviation. Statistical analysis was performed using Prism Software (GraphPad, United States), followed by one-way ANOVA analysis with Dunnett’s post hoc evaluation.

FIGURE 5

acGM-8k NPs enhanced cellular uptake of OVA. (A) Cellular uptake efficiency of OVA@acGM-8k NPs. (B) Screening of potential mechanisms of OVA@acGM-8k NPs internalization by DC2.4 cells. (C) Confocal microscopy imaging of OVA@acGM-8k NPs in DC2.4 cells (scale bar: 10 μm). Green: FITC-OVA; Red: lysotracker red; Blue: DAPI. ***p < 0.001 vs the control group; ns: no significance; n = 3: data were obtained from three replicates of one representative experiment out of three. All numerical values are given as average values ± standard deviation. Statistical analysis was performed using Prism Software (GraphPad, United States), followed by one-way ANOVA analysis with Dunnett’s post hoc evaluation.

FIGURE 6

Distribution of OVA@acGM-8k NPs and maturation of lymph node-resident DC in vivo. (A) Migration of OVA@acGM-8k NPs from the injection site to popliteal lymph nodes (LNs) in vivo. The pink arrow indicates the popliteal LNs. (B) Popliteal LNs were isolated and visualized at 36 h after footpad injection. (C) Average radiant efficiency of popliteal LNs at 36 h after footpad injection. (D) The percentages of Cy5-positive in all LNs and CD11c⁺ DCs 36 h after footpad injection. (E) The percentage CD11c⁺ DCs in LNs 72 h after immunization. (F) The percentage of CD86⁺ in CD11c⁺ DCs in LNs 72 h after immunization. *p < 0.05, **p < 0.01; ***p < 0.001 vs the OVA group; ns: no significance; n = 3: For (B)–(F), data were obtained from three mice in each group. All numerical values are given as average values ± standard deviation. Statistical analysis was performed using Prism Software (GraphPad, United States), followed by one-way ANOVA analysis with Dunnett’s post hoc evaluation.

FIGURE 7

OVA@acGM-8k NPs enhanced the humoral and cellular immune response. (A) Schematic of the experiment design. (B–D) Levels of anti-OVA IgG, IgG1, and IgG2a antibodies in serum were determined. (E–F) Splenocytes were restimulated with OVA (100 μg/ml) or SIINFEKL (2 μg/ml) for 6 h at 37°C; then, flow cytometry was used to determine percentages of OVA-specific (E) IFN-γ–producing CD4⁺ T cells and (F) IFN-γ-producing CD8⁺ T cells. *p < 0.05, **p < 0.01, ***p < 0.001 vs the OVA group; ns: no significance. For (B)–(D), data were obtained from five mice in each group; For (E)–(F), data were obtained from three mice in each group. All numerical values are given as average values ± standard deviation. Statistical analysis was performed using Prism Software (GraphPad, United States), followed by one-way ANOVA analysis with Dunnett’s post hoc evaluation.

FIGURE 8

Systemic toxicities of OVA@acGM-8k NPs. Histological evaluation of heart, liver, spleen, lung, and kidney. Mice were subcutaneously injected with OVA, OVA@acGM-8k NPs and OVA@acDEX NPs at an interval of 2 weeks. 14 days after the last injection, mice were sacrificed and the heart, liver, spleen, lung, and kidney were separated for paraffin section and H&E staining. Scale bar: 100 μm.