Acetalated Dextran Microparticles for Codelivery of STING and TLR7/8 Agonists

Abstract

Vaccines are the most effective tool for preventing infectious diseases; however, subunit vaccines, considered the safest type, suffer from poor immunogenicity and require adjuvants to create a strong and sustained immune response. As adjuvants, pathogen-associated molecular patterns (PAMPs) offer potent immunostimulatory properties and defined mechanisms of action through their cognate pattern recognition receptors (PRRs). Their activity can be further enhanced through combining two or more PAMPs, particularly those that activate multiple immune signaling pathways. However, the cytosolic localization of many PRRs requires intracellular delivery of PAMPs for optimal biological activity, which is particularly true of the stimulator of interferon genes (STING) PRR. Using acetalated dextran (Ace-DEX) microparticles (MPs) encapsulating STING agonist 3'3'-cyclic GMP-AMP (cGAMP) combined with soluble PAMPS, we screened the effect of codelivery of adjuvants using primary mouse bone marrow derived dendritic cells (BMDCs). We identified that codelivery of cGAMP MPs and soluble Toll-like receptor 7/8 (TLR7/8) agonist resiquimod (R848) elicited the broadest cytokine response. cGAMP and R848 were then coencapsulated within Ace-DEX MPs via electrospray. Using the model antigen ovalbumin, we observed that Ace-DEX MPs coencapsulating cGAMP and R848 (cGAMP/R848 Ace-DEX MPs) induced antigen-specific cellular immunity, and a balanced Th1/Th2 humoral response that was greater than cGAMP Ace-DEX MPs alone and PAMPs delivered in separate MPs. These data indicate that polymeric Ace-DEX MPs loaded with STING and TLR7/8 agonists represent a potent cellular and humoral vaccine adjuvant.

Keywords: STING; acetalated dextran; cGAMP adjuvant; microparticles; vaccine adjuvants.

Figure 1.

Combined treatment of cGAMP microparticles (MPs) with various soluble pathogen-associated molecular patterns (PAMPs). Bone marrow derived dendritic cells (BMDCs) from C57BL/6 mice were treated with soluble (Sol.) murabutide (10 μg/mL), MPL (1 μg/mL), poly(dA:dT) (10 μg/mL), CpG (1 μg/mL), poly(I:C) (10 μg/mL), or R848 (0.01 μg/mL) alone, or in combination with 1 μg/mL cGAMP MPs or an equivalent amount of blank MPs. The left-most data group displayed in each panel includes a PBS control (checkered bar), an equivalent amount of blank MPs to the cGAMP MPs (black bar), and 1 μg/mL cGAMP MPs only (gray bar). Cell supernatants were collected after 22 h and were analyzed for (A) IL-6, (B) TNF, (C) IL-1β, (D) IL-12p70, and (E) IFN-β (n = 4 ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p <0.0001).

Figure 2.

Morphology and release kinetics of cGAMP and R848 individually encapsulated in acetalated dextran microparticles (Ace-DEX MPs) made by electrospray. Scanning electron micrographs of (A) cGAMP Ace-DEX MPs and (B) R848 Ace-DEX MPs. Scale bars = 4 μm. (C) Release profiles of cGAMP and R848 individually encapsulated into acetalated dextran MPs were conducted at pH 7.4. Data are presented as mean ± SEM (n = 3).

Figure 3.

Optimal ratio determination for cGAMP and resiquimod (R848) microparticles. Bone marrow derived dendritic cells from C57BL/6 mice were treated for 22 h with indicated concentrations of resiquimod (R848) and cGAMP, both of which were individually encapsulated within acetalated dextran (Ace-DEX) microparticles. Supernatants were analyzed for (A) IL-6, (B) TNF, (C) IL-1β, (D) IL-12p70, and (E) IFN-β. Data are presented as mean ± SEM (n = 4).

Figure 4.

Morphology and release kinetics of combination microparticles made by electrospray. (A) Scanning electron micrographs of acetalated dextran microparticles encapsulating a combination of cGAMP and resiquimod (cGAMP/R848 Ace-DEX MPs). Scale bar indicates 4 μm. (B) Release profiles of cGAMP and R848 within combination MPs conducted at pH 7.4. (C) Drug loading of cGAMP and R848 (μg/mg). Data are presented as mean ± SEM (n = 3).

Figure 5.

Coencapsulation of cGAMP and R848 in acetalated dextran microparticles (Ace-DEX MPs) compared to individual encapsulation. Bone marrow derived dendritic cells from C57BL/6 mice were treated with indicated concentrations of cGAMP and R848, respectively, delivered as soluble (Sol.) drugs, encapsulated in separate Ace-DEX MPs (cGAMP MPs + R848 MPs) or coencapsulated within the same Ace-DEX MPs (cGAMP/R848 MPs). After 22 h, supernatants were harvested and analyzed for (A) IL-6, (B) TNF, (C) IL-1β, (D) IL-12p70, and (E) IFN-β (n = 4 ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 6.

Cytokine profiles of combination cGAMP/R848 acetalated dextran (Ace-DEX) or poly(lactic-co-glycolic acid) (PLGA) MPs. Bone marrow derived dendritic cells from C57BL/6 mice were treated with indicated concentrations of cGAMP and R848, respectively, coencapsulated within Ace-DEX or PLGA MPs. After 22 h, supernatants were harvested and analyzed for (A) IL-6, (B) TNF, (C) IL-1β, (D) IL-12p70, and (E) IFN-β (n = 4 ± SEM *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Figure 7.

Comparison of antibody responses induced by cGAMP and R848 delivered via acetalated dextran (Ace-DEX) or poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs). C57BL/6 mice were immunized intramuscularly on days 0 and 21. All groups received 10 μg of OVA, except the PBS group. OVA was given alone or in combination with cGAMP (200 ng) and R848 (18 ng) delivered in single-loaded or dual-loaded Ace-DEX or PLGA MPs, blank Ace-DEX or PLGA MPs, or alum. On day 28, serum was collected and analyzed for ovalbumin (OVA)-specific (A) total IgG, (B) IgG1, and (C) IgG2c as well as (D) the ratio between IgG2c and IgG1 isotypes (n = 4–5 ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Data are presented as n = 5 ± SD.

Figure 8.

Comparison of T cell responses induced by cGAMP and R848 delivered via acetalated dextran (Ace-DEX) or poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs). C57BL/6 mice were immunized intramuscularly on days 0, 21, and 35. All groups received 10 μg of OVA, except the PBS group. OVA was given alone or in combination with cGAMP (200 ng) and R848 (18 ng) delivered in single-loaded or dual-loaded Ace-DEX or PLGA MPs, blank Ace-DEX or PLGA MPs, or alum. On day 42, mice were sacrificed and (A) IFN-γ and (C) IL-2 ELISpots were performed on splenocytes restimulated with SIINFEKL peptide (10 μg/mL) for 36 h. Alternatively, splenocytes were stimulated with 10 μg/mL ovalbumin (OVA) protein for 36 h. Supernatants were analyzed for (B) IFN-γ and (D) IL-2 by ELISA (n = 4–5 ± SD, *p < 0.05, **p ← 0.01, ***p < 0.001, ****p < 0.0001).