RATIONAL CONTROL OF TOLL-LIKE RECEPTOR SIGNALING USING MICRONEEDLES FOR ENHANCED VACCINATION

Abstract

Vaccines have successfully prevented and eradicated a number of diseases; however, there are many diseases for which vaccines do not exist or for which current vaccines are ineffective. A multitude of strategies are being explored to improve vaccines; specifically, directly targeting antigen-presenting cells (APCs), which are involved in potentiating immune responses. APC targeting can be accomplished in multiple ways; one strategy is by tuning the composition of vaccines to target pathogen sensors in and on APCs. Vaccines contain antigens, which are identifying pieces of pathogens. Vaccines that do not incite a strong enough or desired immune response alone also contain adjuvants, which dictate the types of costimulatory markers and signals APCs will present to lymphocytes or other effector cells. Studies have shown that different types of adjuvants can bind receptors on APCs, such as toll-like receptors (TLRs), and activate discrete pathways. This differential activation dictates the activation and polarization of numerous subsets of T cells and B cells. For example, cytotoxic CD8+ T cells may be activated by one type of TLR agonist (TLRa), while another TLRa may promote the activation of helper Th2 CD4+ T cells. Importantly, recent preclinical studies have shown that utilizing multiple TLRas can produce synergistic immune responses by activating a broader set of immune stimulatory pathways; however, the tunability of these immune responses remains understudied. To direct this combinatorial signaling, it would be advantageous to colocalize antigen with defined compositions of multiple classes of TLRas. Toward this goal, biomaterials provide an excellent alternative to soluble delivery of cues because they enable processes such as self-assembly. This allows colocalization of multiple cargos and targeted delivery.Another consideration in targeting APCs is their preferential distribution across APC-lean and APC-rich tissues. One of these APC-rich tissues is the skin. While conventional vaccine delivery relies on passive drainage into the bloodstream from immune cell-poor tissues such as the subcutaneous space or muscle, the skin contains many times the number of APCs compared to blood. The skin is also easily accessible, making it an attractive option for delivery. An emerging strategy to target the skin uses microneedle arrays (MNAs), which are patches displaying hundreds of micron-scale polymer needles that can penetrate skin. MNAs can be made of many different types of materials; for example, a non-dissolvable polymer coated with immune signals, or a dissolving polymer loaded with vaccine cargo. In both cases, TLRas and antigen can be combined into MNAs utilizing electrostatics to achieve the benefits of biomaterials while directly targeting the skin immune niche. This dissertation outlines innovative methods to coat and load combinations of TLR agonists (TLRas) onto and into MNAs, then evaluates these multi-TLRa MNAs in cellular and pre-clinical animal models. With coated MNAs, I show that splenic dendritic cells (DCs), a specialized type of APC, exhibit tunable behavior as a function of both the total coating mass and relative TLRa loading. Nearly 100% of T cells co-cultured with DCs and MNAs proliferate, and the ratio of TLRa changes their cytokine profiles. Next, I developed a novel type of immune complex to precisely load multiple TLRas with an antigen into dissolvable MNAs. I found that these MNAs enabled localized, sustained cargo release over time. Among the cells in the skin, APCs were primarily responsible for taking up this cargo. I also examined how gene expression in the skin changed as a function of MNA application and demonstrated the dependence of transcriptional signaling on MNA application. Motivated by the localized effects of MNAs on the skin, I next tested the geographic effects of MNA placement on antigen-specific effector cell proliferation and localized disease outcomes. To do this, I defined the site of disease and changed the side of MNA application or application distance (that is, draining to a non-disease draining lymph node) from the site of disease. I found that matching MNA application side to that of the disease draining lymph node was most important in improving disease outcomes, but the distance from this lymph node did not have an effect. Next, I used the multi-TLRa MNA library to probe how the TLRa ratio changed immune responses. I tested how genes differentially changed in the lymph node due to TLRa ratio and found that each type of TLRa showed a gene signature indicative of a microenvironment conducive to a distinct effector cell polarization relevant to vaccination. Interestingly, this polarization could be directly correlated with antigen-specific cell expansion and disease outcomes. Together, this body of work describes two novel vaccine delivery platforms that can be used to improve disease outcomes. Findings from these discoveries could hold relevance for geographically restricted diseases, such as melanomas. These results could also inform improved tunable vaccines for a variety of diseases.