Linked Toll-Like Receptor Triagonists Stimulate Distinct, Combination-Dependent Innate Immune Responses

Abstract

Traditional vaccination strategies have failed to generate effective vaccines for many infections like tuberculosis and HIV. New approaches are needed for each type of disease. The protective immunity and distinct responses of many successful vaccines come from activating multiple Toll-like receptors (TLRs). Vaccines with multiple TLRs as adjuvants have proven effective in preclinical studies, but current research has not explored two important elements. First, few multi-TLR systems explore spatial organization-a critical feature of whole-cell vaccines. Second, no multi-TLR systems to date provide systematic analysis of the combinatorial space of three TLR agonists. Here, we present the first examination of the combinatorial space of several spatially defined triple-TLR adjuvants, by synthesizing a series of five triple-TLR agonists and testing their innate activity both in vitro and in vivo. The combinations were evaluated by measuring activation of immune stimulatory genes (Nf-κB, ISGs), cytokine profiles (IL12-p70, TNF-α, IL-6, IL-10, CCL2, IFN-α, IFN-β, IFN-γ), and in vivo cytokine serum levels (IL-6, TNF-α, IL12-p40, IFN-α, IFN-β). We demonstrate that linking TLR agonists substantially alters the resulting immune response compared to their unlinked counterparts and that each combination results in a distinct immune response, particularly between linked combinations. We show that combinations containing a TLR9 agonist produce more Th1 biasing immune response profiles, and that the effect is amplified upon conjugation. However, combinations containing TLR2/6 agonist are skewed toward T_H2 biasing profiles despite the presence of TLR9. These results demonstrate the profound effects that conjugation and combinatorial administration of TLR agonists can have on immune responses, a critical element of vaccine development.

Figure 1

(A) TLR agonists are naturally present in pathogens. To recapitulate TLR activation by a pathogen, they can be added as a mixture or conjugated to mimic the spatial confinement in pathogens. (B) Conjugating the agonists ensures that all of the agonists are taken up by immune cells into the same endosome, leading to differing immune responses compared to the analogous unlinked combination.

Figure 2

(A) Chemical structures of individual TLR agonists with covalent linkage sites shown. (B) General synthetic strategy to create five distinct TLR triagonists. Mal. = Maleimide.

Figure 3

Innate immune activation as measured by (A) NF-κB and (B) ISG activity in RAW-Blue 264.7 macrophage cell assay treated with TLR triagonists or a 1:1:1 mixture of the analogous unlinked agonists. Cells were incubated with each compound (100 nM) for 18 h at 37 °C. Supernatant was removed and incubated with QUANTI-Blue for 1 h (NF-κB) or 4 h (ISG), and the absorbance at 620 nm was measured. Error bars represent SD. Samples were run in triplicate, where ** p < 0.01; *** p < 0.001; ns, not significant as determined by a two-tailed student t test. All noted statistical analyses represent the asterisked compound compared to the analogous unlinked compounds. See the SI for a full data set. AU = absorbance units.

Figure 4

In vitro cytokine expression from BMDCs as measured by CBA (TNF-α, IL-10, and CCL2) and ELISA (IL-12p70, IL-6, and IFN-β). Cells were incubated with each TLR triagonist (100 nM) or a 1:1:1 (molar ratio) mixture of the analogous unlinked TLR agonists (100 nM each) for 6 h at 37 °C. Error bars represent standard deviation of the mean. Samples were run in triplicate, where * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant. Statistical analysis is between the linked vs unlinked agonists, performed by the two-tailed Student t test. TLR1/2_4_7a and TLR2/6_4_7a were tested separately from TLR1/2_4_9a, TLR2/6_4_9a, and TLR4_7_9a due to assay size constraints. No measurable levels of IFN-γ or IFN-α were detected.

Figure 5

(A) In vivo IL-6, (B) TNF-α, (C) IL-12p40, (D) IFN-α, and (E) IFN-β serum levels in C57BL/6 mice 3 h (IL-6 and IL-12p40) and 1 h (TNF-α, IFN-α, and IFN-β) postinjection as measured by ELISA. Mice were injected via IM with TLR triagonists (1 nmol) or a 1:1:1 molar ratio mixture of the analogous unlinked TLR agonists (1 nmol each). (F) Percent weight change 24 h post-triagonist-injection mixed with 0.5 nmol CBU_1910 antigen. Samples were run in triplicate for ELISA experiments, and in n = 8 for the percent weight experiment (except PBS and TLR2/6_4_7a where n = 5), where * p < 0.05; ** p < 0.01; *** p < 0.001; ns, not significant. Statistical analysis is between the linked versus unlinked agonist for the indicated compound, performed using a two-tailed Student t test.