Nature, while slow to change, has had the benefit of billions of years competitive pressure to hone approaches to interesting problems. In this work, we take advantage of this preexisting work, and make progress towards replicating the benefits found in nature in artificial systems. Taking inspiration from dynamic, living structures such as bone, and muscle, we demonstrate partial synthetic recreations of the conceptual pieces required to mimic such materials. Diffusion controlled etching is used to demonstrate functional optimization of preexisting structures. Incorporating growth into the system allows dynamic, reversible changes in structure to take place. We show the migration of a channel through a solid matrix. We also took inspiration from deep sea fish to develop a device that concentrates gas more efficiently than a simple countercurrent flow separator. Existing methods including hollow fiber membrane contactors, vacuum swing adsorption, and cryogenic distillation represent a significant portion of the worlds energy needs. Countercurrent amplifiers found in the swimbladders of teleost fish, can efficiently concentrate gases at least 15,000 times above the ambient concentrations. Incorporating the architecture found in the swim bladders of deep sea fish with modern CO2 and O2 capture fluids enables a feedback loop of concentration within our artificial device to drive an increase in the release rates of both gases.

Toll-like receptors (TLRs) on innate immune cells are important in the early detection of pathogens and initiation of immune responses. Spatial and temporal aspects of signaling via these receptors are critical in defining unique and effective immune responses. These factors, however, are not well-characterized and there are currently no methods yet to probe them. Herein, I describe approaches developed by the Esser-Kahn lab to address spatial and temporal activation of immune pathways. We have designed photo-labile immunostimulants for controlled activation of cells in space and time. Variants of pyrimido indoles (TLR4 agonist), imidazoquinolines (TLR7 and 8 agonists), and palmitylated-peptides (TLR1, 2, and 6 agonist) were generated with photo-labile protecting groups installed at moieties necessary for productive receptor interaction. We demonstrate controlled activation using light of the NF-κB pathway in RAW macrophages, bone marrow-derived dendritic cells, and TLR-bearing fibroblasts.

Although one of the greatest achievements of modern medicine, traditional vaccination strategies have failed to generate effective vaccines for many infections including global diseases, like tuberculosis, and regional diseases, like Q-Fever. 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 TLR agonists 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. Secondly, no multi-TLR systems to date provide systematic analysis of the combinatorial space of three TLR agonists. This work describes the effects of conjugation between combinations of three TLR agonists and between TLR agonists and antigens on immunological activity. 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-B, ISGs), cytokine profiles (IL12-p70, TNF-, IL-6, IL-10, CCL2, IFN-, IFN-, IFN-) and in vivo cytokine serum levels (IL-6, TNF-, IL12-p40, IFN-, IFN-). 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 TH2 biasing profiles despite the presence of a TLR9 agonist. To assess their efficacy in a vaccine, we formulated antigens from C. burnetii, the causative agent of Q-Fever, with TLR tri-agonists and evaluated the outcomes of vaccination in vivo. We found that the Tri-agonists elicited unique, antigen-specific immune responses in vivo which matched our initial in vitro analysis. We evaluated our top candidates in a live C. burnetii aerosol challenge model in mice and found two of our TLR tri-agonist containing formulations conferred partial protection to the challenged animals. Our findings characterize a novel adjuvant platform and offer an alternative approach to generating protective and effective vaccine candidates against C. burnetii. 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. In addition, numerous studies have shown that conjugation of TLR agonists to antigens can beneficially influence their potency, toxicity, pharmacokinetics, and function. Here, we present our work in developing site-specific conjugation approaches of TLR agonists to antigens.