Modulation of Innate Immune Responses via Covalently Linked TLR Agonists.

We present the synthesis of novel adjuvants for vaccine development using multivalent scaffolds and bioconjugation chemistry to spatially manipulate Toll-like receptor (TLR) agonists. TLRs are primary receptors for activation of the innate immune system during vaccination. Vaccines that contain a combination of small and macromolecule TLR agonists elicit more directed immune responses and prolong responses against foreign pathogens. In addition, immune activation is enhanced upon stimulation of two distinct TLRs. Here, we synthesized combinations of TLR agonists as spatially defined tri- and di-agonists to understand how specific TLR agonist combinations contribute to the overall immune response. We covalently conjugated three TLR agonists (TLR4, 7, and 9) to a small molecule core to probe the spatial arrangement of the agonists. Treating immune cells with the linked agonists increased activation of the transcription factor NF-κB and enhanced and directed immune related cytokine production and gene expression beyond cells treated with an unconjugated mixture of the same three agonists. The use of TLR signaling inhibitors and knockout studies confirmed that the tri-agonist molecule activated multiple signaling pathways leading to the observed higher activity. To validate that the TLR4, 7, and 9 agonist combination would activate the immune response to a greater extent, we performed in vivo studies using a vaccinia vaccination model. Mice vaccinated with the linked TLR agonists showed an increase in antibody depth and breadth compared to mice vaccinated with the unconjugated mixture. These studies demonstrate how activation of multiple TLRs through chemically and spatially defined organization assists in guiding immune responses, providing the potential to use chemical tools to design and develop more effective vaccines.

In vivo characterization of the physicochemical properties of polymer-linked TLR agonists that enhance vaccine immunogenicity.

The efficacy of vaccine adjuvants such as Toll-like receptor agonists (TLRa) can be improved through formulation and delivery approaches. Here, we attached small molecule TLR-7/8a to polymer scaffolds (polymer-TLR-7/8a) and evaluated how different physicochemical properties of the TLR-7/8a and polymer carrier influenced the location, magnitude and duration of innate immune activation in vivo. Particle formation by polymer-TLR-7/8a was the most important factor for restricting adjuvant distribution and prolonging activity in draining lymph nodes. The improved pharmacokinetic profile by particulate polymer-TLR-7/8a was also associated with reduced morbidity and enhanced vaccine immunogenicity for inducing antibodies and T cell immunity. We extended these findings to the development of a modular approach in which protein antigens are site-specifically linked to temperature-responsive polymer-TLR-7/8a adjuvants that self-assemble into immunogenic particles at physiologic temperatures in vivo. Our findings provide a chemical and structural basis for optimizing adjuvant design to elicit broad-based antibody and T cell responses with protein antigens.

A Light-Controlled TLR4 Agonist and Selectable Activation of Cell Subpopulations.

The spatial and temporal aspects of immune cell signaling are key parameters in defining the magnitude of an immune response. Toll-like receptors (TLRs) on innate immune cells are important in the early detection of pathogens and initiation of an immune response. Controlling the spatial and temporal signaling of TLRs would enable further study of immune synergies and assist in the development of new vaccines. Here, we show a light-based method for the spatial control of TLR4 signaling. A TLR4 agonist, pyrimido[5,4-b]indole, was protected with a cage at a position critical for receptor binding. This afforded a photocontrollable agonist that was inactive while caged, yet effected NF-κB activity in cells following UV photocontrolled deprotection. We demonstrated spatial control of NF-κB activation within a population of cells by treating all cells with the caged TLR4 agonist and constraining light exposure and consequent activation to a region of interest.

Controlling the origins of inflammation with a photoactive lipopeptide immunopotentiator.

Inflammatory immune responses are mediated by signaling molecules that are both produced by and recognized across highly heterogeneous cell populations. As such, the study of inflammation using traditional immunostimulants is complicated by paracrine and autocrine signaling, which obscures the origin of a propagating response. To address this challenge, we developed a small-molecule probe that can photosensitize immune cells, thus allowing light-mediated inflammation. This probe was used to control the origin of inflammation using light. Following this motif, inflammation was initiated from fibroblasts or dendritic cells. The contributions of fibroblasts and dendritic cells in initiating inflammation in heterogeneous co-culture are reported, thus providing insights into the future development of vaccines and treatment of inflammation.

Stimulation of innate immune cells by light-activated TLR7/8 agonists.

The innate immune response is controlled, in part, by the synergistic interaction of multiple Toll-like receptors (TLRs). This multi-receptor cooperation is responsible for the potent activity of many vaccines, but few tools have been developed to understand the spatio-temporal elements of TLR synergies. In this Communication, we present photo-controlled agonists of TLR7/8. By strategically protecting the active agonist moiety based on an agonist-bound crystal structure, TLR activity is suppressed and then regained upon exposure to light. We confirmed NF-κB production upon light exposure in a model macrophage cell line. Primary cell activity was confirmed by examining cytokine and cell surface marker production in bone-marrow-derived dendritic cells. Finally, we used light to activate dendritic cell sub-populations within a larger population.

Directing the immune system with chemical compounds.

Agonists of immune cell receptors direct innate and adaptive immunity. These agonists range in size and complexity from small molecules to large macromolecules. Here, agonists of a class of immune cell receptors known as the Toll-like receptors (TLRs) are highlighted focusing on the distinctive molecular moieties that pertain to receptor binding and activation. How the structure and combined chemical signals translate into a variety of immune responses remain major questions in the field. In this structure-focused review, we outline potential areas where the tools of chemical biology could help decipher the emerging molecular codes that direct immune stimulation.