Dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) is a cellular receptor for delta inulin adjuvant

Abstract

Delta inulin, or Advax, is a polysaccharide vaccine adjuvant that significantly enhances vaccine-mediated immune responses against multiple pathogens and was recently licensed for use in the coronavirus disease 2019 (COVID-19) vaccine SpikoGen. Although Advax has proven effective as an immune adjuvant, its specific binding targets have not been characterized. In this report, we identify a cellular receptor for Advax recognition. In vitro uptake of Advax particles by macrophage cell lines was substantially greater than that of latex beads of comparable size, suggesting an active uptake mechanism by phagocytic cells. Using a lectin array, Advax particles were recognized by lectins specific for various carbohydrate structures including mannosyl, N-acetylgalactosamine and galactose moieties. Expression in nonphagocytic cells of dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), a C-type lectin receptor, resulted in enhanced uptake of fluorescent Advax particles compared with mock-transfected cells. Advax uptake was reduced with the addition of ethylenediaminetetraacetic acid and mannan to cells, which are known inhibitors of DC-SIGN function. Finally, a specific blockade of DC-SIGN using a neutralizing antibody abrogated Advax uptake in DC-SIGN-expressing cells. Together, these results identify DC-SIGN as a putative receptor for Advax. Given the known immunomodulatory role of DC-SIGN, the findings described here have implications for the use of Advax adjuvants in humans and inform future mechanistic studies.

Keywords: Adjuvant; C‐type lectin; DC‐SIGN; vaccine.

Figure 1.. Advax particle uptake in comparison to latex beads or zymosan bioparticles.

Differentiated THP-1 cells were treated with fluorescent Advax particles or latex beads at the same particle per cell (PPC) rate, and incubated at 37°C. At 15 min or 60 min, cells were dissociated for flow cytometric analysis. Representative dot plots of THP-1 cells after 60 min incubation are shown in (a) with mean proportion of cells shown in (b). Differentiated Thp-1 cells were also treated with the same particle number of Advax or zymosan bioparticles (10 particles per cell) and measured for uptake using flow cytometry (c). Cells were also incubated for 60 min with 10 PPC Advax particles or latex beads, transferred to a glass slide and stained with CellMask Deep Red and DAPI prior to fluorescent microscopy (d). Flow cytometric data are representative of 2 independent experiments with n = 3 technical replicates each experiment, and imaging is representative of a single experiment with n = 3 technical replicates. Data in b and c were analysed using a 2-way ANOVA with post-hoc Fisher’s LSD test or Sidak test respectively, where P < 0.0332 (*), P < 0.0021 (**), P < 0.0002 (***). Data show mean ± SEM.

Figure 2.. Advax particles are recognised by lectins of known specificity and various mammalian glycans.

(a) Fluorescent Advax particles were added to an array of 88 lectins, imaged using an Innopsys InnoScan 1100AL then analysed using Mapix software (Innopsys). Binding was determined by positive replicate spots in four replicate experiments. A result was considered positive if the relative fluorescence units of spots was greater than one-fold above the mean background (average background of negative control spots plus 3 standard deviations) and if significance was reached (determined by the two-tailed t-test, where P < 0.005 was considered significant). Data show mean of technical triplicates. N.D. means lectin classification is not determined. (b) In a separate experiment, fluorescent Advax particles were added to a glycan array, imaged using an Innopsys InnoScan 1100AL then analysed using Mapix software (Innopsys). Binding was determined by positive replicate spots in three replicate experiments. Positive binding was determined by the average relative fluorescence units of a specific structure with a value above mean background and had P < 0.005 (determined by the two-tailed Student’s t-test). Data show mean ± SEM.

Figure 3.. DC-SIGN expression enhances cellular association with Advax particles.

HEK293T cells were transfected with a plasmid encoding DC-SIGN-OFP-Spark plasmid or with RFP as a control. Transfection efficiencies are displayed in (a) or (b) for DC-SIGN-OFP or RFP respectively. Forty-eight h after transfection, cells were incubated with fluorescent Advax particles at particles per cell (PPC) values of 10 or 50 for 1 hour, then analysed using flow cytometry for association/uptake by percentage of cells fluorescein positive (c) or median fluorescence intensity (MFI) of fluorescein (d). Significance of difference were calculated using a 2-way ANOVA with post-hoc Sidak test, where P < 0.0332 (*), P < 0.0021 (**), P < 0.0002 (***). Data show mean ± SEM.

Figure 4.. Advax particles localise near the lysosome in DC-SIGN-transfected COS-7 cells.

COS-7 cells were mock transfected (a), transfected with RFP and LAMP1-BFP as a control (b) or with DC-SIGN-OFP and LAMP1-BFP (c). Forty-eight h after transfection, fluorescent Advax particles were added at a particles per cell (PPC) of 1 and incubated for 3 h. Cells were then washed and stained with CellMask Deep Red, then fixed for confocal imaging.

Figure 5.. Advax cellular uptake is abrogated by inhibitors of DC-SIGN function.

HEK293T cells were transfected with DC-SIGN-OFP-Spark plasmid or with RFP as a control. Forty-eight h after transfection, cells were pre-incubated with 5 mM EDTA (a–c) or 200 μg mL⁻¹ Saccharomyces cerevisiae mannan (d–f) for 1 h at 37°C. Advax particles were then added at a concentration of 38 μg/well and cells incubated for another hour at 37°C before washing, followed by analysis by flow cytometry. Proportion of cells fluorescein positive (a), median fluorescence intensity (MFI) (b) and representative histograms (c) after EDTA pre-treatment are shown. Proportion of cells fluorescein positive (d), MFI (e) and representative histograms after mannan pre-treatment are also shown. Cells were also treated with 25 μg mL⁻¹ mAb clone 120612, a neutralising antibody for DC-SIGN, or isotype control antibody for 30 min at 4°C prior to the addition of Advax particles. Proportion of cells fluorescein positive (g), MFI (h) and representative histograms (i) are shown. Data depict n = 3 replicates per experiment and is pooled from two independent experiments. Statistics were calculated using 1-way ANOVA with the post-hoc Tukey test, where P < 0.0332 (*), P < 0.0021 (**), P < 0.0002 (***). Data show mean ± SEM.