Microcrystalline Tyrosine and Aluminum as Adjuvants in Allergen-Specific Immunotherapy Protect from IgE-Mediated Reactivity in Mouse Models and Act Independently of Inflammasome and TLR Signaling

Abstract

Allergen immunotherapy (AIT) is the only modality that can modify immune responses to allergen exposure, but therapeutic coverage is low. One strategy to improve AIT safety and efficacy is the use of new or improved adjuvants. This study investigates immune responses produced by microcrystalline tyrosine (MCT)-based vaccines as compared with conventional aluminum hydroxide (alum). Wild-type, immune-signaling-deficient, and TCR-transgenic mice were treated with different Ags (e.g., OVA and cat dander Fel d 1), plus MCT or alum as depot adjuvants. Specific Ab responses in serum were measured by ELISA, whereas cytokine secretion was measured both in culture supernatants by ELISA or by flow cytometry of spleen cells. Upon initiation of AIT in allergic mice, body temperature and further clinical signs were used as indicators for anaphylaxis. Overall, MCT and alum induced comparable B and T cell responses, which were independent of TLR signaling. Alum induced stronger IgE and IL-4 secretion than MCT. MCT and alum induced caspase-dependent IL-1β secretion in human monocytes in vitro, but inflammasome activation had no functional effect on inflammatory and Ab responses measured in vivo. In sensitized mice, AIT with MCT-adjuvanted allergens caused fewer anaphylactic reactions compared with alum-adjuvanted allergens. As depot adjuvants, MCT and alum are comparably effective in strength and mechanism of Ag-specific IgG induction and induction of T cell responses. The biocompatible and biodegradable MCT seems therefore a suitable alternative adjuvant to alum-based vaccines and AIT.

Copyright © 2018 The Authors.

Figures

FIGURE 1.

Immunogenicity testing of tyrosine- and alum-based vaccines in BALB/c mice. (A) Mice (n = 3–4) were injected with 0.01 or 0.1 μg OVA on alum (black symbols), MCT (open symbols), or in PBS (dashed lines) on days 0 and 28, and Ab titers were measured on days 28 and 42. Titration curves for individual mice are illustrated. (B) After a third injection with OVA 0.1 μg on day 56, splenocytes were harvested on day 62 and restimulated with OVA for analysis of cytokine secretion (mean + SD). (C) Mice (n = 5) were immunized with 1 (left) or 100 (right) μg OVA on days, 0, 14, and 28; total IgG, IgG1, IgG2a, IgG2b, IgG3, and IgE Abs were determined (mean ± SEM). The latter experiment is representative of three independent experiments with comparable results.

FIGURE 2.

T cell response after MCT- or alum-adjuvanted immunization. C57BL/6 mice (n = 5) were adoptively transferred with OT-I and OT-II cells and immunized with 100 μg OVA on MCT (gray bars) or alum (black bars) 1 and 8 d later (individual mice are indicated). On day 15, spleen cells were analyzed by flow cytometry. (A) Dot blots and histograms show SIINFEKL-specific CD8 T cell activation (CD44) and proliferation (H2Kb pentamer). Frequencies of IFN-γ–producing and IFN-γ– and TNF-α–double-producing CD8 T cells (B) and CD4 T cells (C). (D) Splenocytes were also restimulated in vitro with MHC class I–restricted OVA aa 257–264 (SIINFEKL), MHC class II–restricted OVA aa 323–339, or OVA protein, and IFN-γ in culture supernatants was measured by ELISA. Mean ± SD of means are illustrated. The experiment is representative of two independent experiments with comparable results. *p < 0.05, **p < 0.01.

FIGURE 3.

Immunotherapy of cat dander allergy. BALB/C mice (n = 5) were sensitized by weekly injections of cat fur allergen extract and received AIT with recombinant Fel d 1 allergen on alum (red) or MCT (blue) thrice s.c., as indicated (A). Finally, the mice were challenged with cat fur allergen extract to test tolerance to anaphylaxis. (B) Cat fur allergen-specific IgE (left) and Fel d 1–specific IgG2a (right) Ab titers were measured in sera before and after sensitization and AIT. (C) After challenge, changes in the body temperature were measured as an indicator for anaphylaxis. Left, Rectal body temperature as function of time after challenge. Right, Integrated AUC with baseline 38°C for the body temperature curves. (D) Splenocytes were restimulated with rFel d 1 for 20 h (IL-2) or 96 h (IFN-γ and IL-10), and cytokines were measured in the supernatants by ELISA. Abs are illustrated as mean ± SEM, and other results are illustrated as mean ± SD.

FIGURE 4.

Safety testing of MCT- and alum-based vaccines in mice. BALB/C mice (n = 5) were sensitized by four weekly i.p. injections of OVA adsorbed on alum. Four weeks later, the mice received a single s.c. AIT with OVA on MCT (open circles and bars) or on alum (closed circles and bars). Body temperature changes are illustrated as a function of time after AIT (A) and integrated as AUC (B). The experiment is representative of two independent experiments with comparable results.

FIGURE 5.

Immunogenicity testing in mice deficient in TLR signaling. (A) C57BL/6 wild-type mice as well as syngeneic MyD88- and TLR4-deficient mice were immunized with 40 μg OVA-MCT (gray circles) or 40 μg OVA-alum (black circles) on days 0 and 14. Abs in blood were measured by ELISA. (B) C57BL/6 mice as well as syngeneic TRIF-deficient mice were immunized with 10 μg OVA-MCT on days 0 and 14. Abs in blood were measured by ELISA. The experiment is representative of two independent experiments with comparable results and with four to six mice per group.

FIGURE 6.

Assessment of inflammasome activation and other inflammatory reactions in vivo and in vitro. (A–C) THP-1 human monocytes were incubated with various doses of MCT in the presence or absence of zVAD. IL-1β secretion (A) and viability (B) were measured by cytokine and lactate dehydrogenase cytotoxicity ELISA, respectively, in supernatants. (C) Pro–IL-1β and cleaved IL-1β were separated by Western blot using nigericin as a positive control. For the sake of presentation, the blot was cropped and spliced as indicated with the vertical white line, excess sample replicates being omitted. (D) MCT, alum, or PBS were injected i.p. in mice, and cell populations in the peritoneal lavage were analyzed by flow cytometry as indicated. Infiltration of CD11b– and Ly6G–double-positive neutrophils (E), CD11b– and SiglecF–double-positive eosinophils (F), F4/80-positive macrophages (G), MHC class II– and CD11b–double-positive cells (H), and CD11c–CD11b double-positive DCs (I) were measured 4 h (open bars) and 24 h (closed bars) postinjection. The results with wild-type mice are representative of three independent experiments. (J) Percentage of neutrophils, macrophages, and DCs in wild-type versus NALP3 inflammasome knockout mice.

FIGURE 7.

Analysis of the role of inflammasome activation on adjuvant mechanism of action of MCT and alum with regard to stimulation of B and T cell responses. Wild-type C57BL/6 and ASC knockout mice (n = 5) were immunized on days 0, 14, and 26 with 0.1 μg OVA on MCT or alum. (A) Mice were bled on day 42, and OVA-specific IgG1, IgG2b, IgG2c, IgG3, and IgE Abs were measured by ELISA. The Ab results (mean + SD) are illustrated as OD measured at a serum dilution of 1:3000. (B) Mice were euthanized on day 48, and splenocytes were restimulated with OVA (+) or were not restimulated (−) for analysis of cytokine secretion by ELISA (mean + SD). The experiment was not repeated.