Assessment of Th1/Th2 Bias of STING Agonists Coated on Microneedles for Possible Use in Skin Allergen Immunotherapy

Abstract

Microneedle-based skin allergen-specific immunotherapy (AIT) can benefit from adjuvants that can stimulate a stronger Th1 response against the allergen. We evaluated two stimulator of interferon genes (STING) agonists, namely, cyclic diguanylate monophosphate (c-di-GMP) and cyclic diadenylate monophosphate (c-di-AMP), as skin adjuvants using coated microneedles (MNs). For comparison, the approved subcutaneous (SC) hypodermic injection containing alum was used. Ovalbumin (Ova) was used as a model allergen. Ova-specific IgG2a antibody in serum, which is a surrogate marker for Th1 type immune response was significantly higher when STING agonists were used with coated MNs as compared to SC injection of Ova+alum in mice. In contrast, IgG1 antibody, a surrogate marker for Th2 type immune response, was at comparable levels in the MN and SC groups. Restimulation of splenocytes with Ova produced higher levels of Th1 cytokines (IFN-γ and IL-2) in the STING agonists MN groups as compared to the SC group. In conclusion, delivery of STING agonists into the skin using coated MNs activated the Th1 pathway better than SC- and MN-based delivery of alum. Thus, STING agonists could fulfill the role of adjuvants for skin AIT and even for infectious disease vaccines, where stimulation of the Th1 pathway is of interest.

Keywords: STING adjuvants; allergy adjuvant; cutaneous-allergen immunotherapy; microneedles; skin immunotherapy.

Figure 1.

Allergen-coated microneedles. (A) Schematic showing the concept of allergen-coated microneedles for skin allergen-specific immunotherapy. (B) Digital photograph of an uncoated microneedle patch containing micron-sized needles. (C) Stereomicroscope image of Ova-coated MN patch. (D) Zoomed stereomicroscope image of a single microneedle coated with Ova. (E) In vivo delivery efficiency of MNs coated with Ova+STING adjuvant in mice skin. Gene expression analysis of (F) IRF-3, and (G) IFN-γ in skin of mice immunized with Ova and STING using coated MNs. Error bars denote ± SEM. * p < 0.05, ** p < 0.01, and **** p < 0.0001

Figure 2.

Immunization schedule and treatment groups. (A) Immunization and sample collection schedule. (B) The different treatment groups, and vaccination doses.

Figure 3.

Anti-Ova response in serum at different time points. (A) anti-Ova IgG response at 1:100 serum dilution, (B) anti-Ova IgG1 response at 1:100 serum dilution, (C) anti-Ova IgG2a response at 1:100 serum dilution, and (D) IgE response at 1:20 serum dilution. ELISA was used to measure anti-Ova antibody responses in the form of optical density at 492 nm. Individual mice serum was used in analysis. Error bars denote ± SEM. * p < 0.05, *** p < 0.001, **** p < 0.0001, and ns: not significant.

Figure 4.

Splenocyte culture supernatant analysis. Spleens were collected at end of the experiment and cultured for 72 h with medium alone as a negative control, 200 μg/ml Ova, or 5 μg/ml of concanavalin A as a positive control. Supernatant of cultured cells were collected after 14 h for IL-2, 72 h for IFN-, IL-4 and IL-5 analysis. Expression levels of (A) IFN-γ, (B) IL-2, (C) IL-4, and (D) IL-5 cytokines. Error bars denote ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001, and ns: not significant. Values are presented after subtraction of media alone cytokine levels. Data for concanavalin A is not plotted.