Application of a small molecule calcium influx inducer as a vaccine adjuvant: enhancing Th2-biased immune responses

Abstract

Introduction: Vaccines are highly effective in preventing the spread of communicable diseases and are critical to overall public health. As immune stimulants vaccine adjuvants augment the level and longevity of these protective responses. Seeking novel adjuvants using parallel high throughput screens and subsequent systematic structure-activity relationship studies we identified an analogue of a hit compound, 2G272, that in screening assays retained in vitro induction of calcium (Ca²⁺) influx, tetraspanin CD63 EV reporter activity and cell viability. Here, we further our analyses of the biological activity of 2G272 related its potential use as a vaccine adjuvant.

Methods: 2G272 was tested for activation of murine bone marrow-derived dendritic cells (mBMDC) by flow cytometry for Ca²⁺ entry, levels of CD80 and CD86 expression, and in vitro stimulation of antigen-specific T cell proliferation. Cytokines and IgG responses from BALB/c mice injected with 2G272 as a single agent or as an adjuvant with ovalbumen were measured by ELISA.

Results: 2G272 triggered store-operated Ca²⁺ entry in mBMDC as well as increases in CD80 and CD86 surface expression. In co-culture experiments, this compound amplified the stimulation of cognate T cell proliferation. Intramuscular injection of 2G272 elicited minimal systemic cytokine and chemokine release. When used as an adjuvant with ovalbumen, 2G272 generated a significant antigen-specific IgG1 response with a higher splenocyte T helper 2 (Th2) cytokine response.

Discussion: 2G272 activated mBMDCs associated with EV release and a store-operated calcium entry response. Enhanced cognate T cell proliferation was mediated either through direct engagement with compound-stimulated mBMDCs or indirectly via immunostimulatory extracellular vesicles released by 2G272-activated mBMDCs. 2G272 elicited minimal systemic cytokine and chemokine release, demonstrating a promising safety profile. When used as an adjuvant in a murine vaccination model, 2G272 enhanced the IgG1 response with an associated T helper 2 cytokine profile. Hence this compound shows promise as an adjuvant if a Th2 response is beneficial or in combination with other agents to provide a balanced immune response in vaccines.

Keywords: Ca2+ influx; T helper 2; adjuvant; dendritic cell; extracellular vesicle; vaccine.

Figure 1

Chemical structures. Shown are the structures for compounds 634; ethyl 2-(benzo[c][1,2,5]thiadiazole-4-sulfonamido)-4,5-dimethylthiophene-3-carboxylate, 2G272; ethyl 2-(b3enzo[c][1,2,5]selenadiazole-4-sulfonamido)-4,5-dimethylthiophene-3-carboxylate, and 2E281; ethyl 4,5-dimethyl-2-(phenylsulfonamido)thiophene-3-carboxylate (8).

Figure 2

2G272 increases intracellular Ca²⁺ levels associated with APC functions. (A, B) Calcium mobilization in mBMDCs treated with 2G272 (A), or 2E281 (B). mBMDCs were loaded with Fura-8 and stimulated with 1, 2, 5 and 10 µM of the indicated compound. The time-course of intracellular Ca²⁺ levels was recorded for 25 minutes after stimulation. The dashed line indicates the addition of compound. Representative data from two independent experiments with similar results are shown. (C) Ca²⁺ restoration assay to assess SOCE in mBMDCs. Fura-8-loaded mBMDCs were treated with ION (1 µM), 2G272 (10 µM) alone or in combination with 10 µM BTP2 (SOCE inhibitor) for 10 minutes in Ca²⁺ free medium. Subsequently, Ca²⁺ was added (final concentration: 1.8 mM), and the resulting Ca²⁺ influx was measured. Representative data from two independent experiments with similar results are shown. (D, E) Expression of costimulatory molecules, CD80 (D) and CD86 (E) were measured by flow cytometry 24 hours after treatment with 2G272 (10 µM), 2E281 (10 µM), or MPLA (1 µg/ml) in the presence or absence of BTP2 (10 µM). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, by two-way ANOVA with Tukey’s post-hoc test. Representative data from two independent experiments with similar results are shown.

Figure 3

Immunomodulatory effects of 2G272 on dendritic cells and T cell proliferation. (A, B) BMDCs from BALB/c mice were treated with vehicle (0.5% DMSO), 10 µM 2G272, 10 µM 2E281, or 1 µM MPLA overnight. Following a 4-hour incubation with OVA protein, CFSE-labeled CD4⁺ T cells from DO11.10 mice were added to mBMDCs and incubated for 5 days. (A) Dot plots of CD4⁺ T cell division by CSFE generational dilution. (B) Percentage of divided CD4⁺ T cells. ****p < 0.0001, by ordinary one-way ANOVA with Dunnett’s post-hoc test. Representative data from two independent experiments with similar results are shown.

Figure 4

Compound 2G272 promotes the release of immune-stimulatory EVs from mBMDCs. (A) Characterization of EVs by immunoblotting. mBMDCs were treated with vehicle (Veh, 0.25% DMSO), 2G272 (10 μM), 2E281 (10 μM), or ION (1 μM) overnight. EVs from the supernatant were harvested, lysed and proteins (2 µg/well for CD80 and CD86, 4 μg/well for CD81 and Alix) were separated by electrophoresis and immunoblotted as shown. Full blots are shown in Supplementary Figure S2. (B) EV_2G272 enhances T cell proliferation in the presence of antigenic peptides. CFSE-labeled CD4⁺ T cells from transgenic DO11.10 mice were treated with 20µg in equal volumes of EV_2G272, EV_2E281, EV_ION, or EV_Veh in the presence or absence of OVA_323–339 peptide for 5 days. T cell proliferation was determined by CFSE dilution using flow cytometry, shown as dot plots and (C) percentages of divided T cells. (D) IL-2 concentrations in the culture supernatants at day 5 were measured by ELISA. Data are means ± SDs of triplicates, representative of two independent experiments. *p < 0.05, ****p < 0.0001 by ordinary one-way ANOVA and Dunnett’s post-hoc test compared to EV_Veh.

Figure 5

Cytokine, chemokine and inflammatory responses in mice administered with 2G272. BALB/c mice (n=5/group) were i.m. injected with 2G272 (200 nmol/animal) or vehicle (0.5% DMSO). MF59 (25 µL/animal) was used as a positive control. Sera samples were collected 8, or 48, hours after injection. The levels of (A) IL-6 (8 hours), (B) CXCL1 (8 hours), (C) CCL2 (8 hours), and (D) alpha 1 acid glycoprotein (48 hours) were measured in serum samples using ELISA and Luminex bead assays. Serum concentrations of IL-12, TNF, CCL5 and CXCL10 were below detectable levels. Statistical significance was determined by Kruskal-Wallis test followed by Dunn’s post-hoc test compared to the MF59 control: ***p < 0.001, **p < 0.01, and *p < 0.05.

Figure 6

2G272 elicits Th2 biased immune responses in a murine model. (A, B) OVA-specific IgG1 and IgG2a antibody responses. Female BALB/c mice (n=4-9) were immunized with OVA (5 µg/animal) adjuvanted with 2G272 (200 nmol/animal), 2E281 (200 nmol/animal), MPLA (1 µg/animal), or ION (200 nmol/animal) on days 0 and 21. Serum samples were collected on day 28 and OVA-specific IgG1 and IgG2a levels were determined by ELISA. (C, D) On day 42, splenocytes were cultured with OVA for 5 days, and IL-5 (C) and IFN-γ (D) in the supernatant were measured by ELISA. Data are presented as individual values and as means ± SEM. Data shown are pooled from two experiments. Statistical significance was determined by Kruskal-Wallis test followed by Dunn’s post-hoc test compared to the vehicle control: **p < 0.01, *p < 0.05.