Combinatorial control of TLR signaling using self-assembly of immune signals on microneedle arrays to access the skin niche - Project Summary. Vaccines have been crucial technologies to prevent and stop the spread of global diseases, such as smallpox and COVID. Despite these accomplishments, more robust vaccines are needed to combat existing and emerging pathogens. To be effective, vaccines must contain at least two classes of signals: i) an antigen – the specific identifying molecular sequence on a pathogen, and ii) an adjuvant – a signal to the cell that the antigen is dangerous. Specialized antigen presenting cells (APCs) take up antigen and adjuvants in the periphery, then migrate to specialized immune organs known as lymph nodes (LNs) to present antigen and additional markers to naive lymphocytes, such as T cells. T cells then return to the periphery, destroying cells or pathogens displaying that same antigen. Specialized subsets of these APCs, dendritic cells (DCs) and Langerhans cells (LCs), are concentrated in skin and dermal layers; thus, targeting vaccines to these cells has potential to increase vaccine potency. DCs and LCs contain toll-like receptors (TLRs) in and on their surface, which recognize toll-like receptor agonists (TLRas) - molecular patterns commonly found on pathogens but not mammalian cells. Because activating TLRs induces inflammatory responses from these cells, TLRas are clinically used as adjuvants to enhance vaccine responses against a variety of pathogens. Recent studies show that activating multiple TLRs can improve disease outcomes compared to single TLRa delivery. One way to deliver multiple TLRas is by using biomaterials, which can achieve controlled and targeted engagement of multiple TLRs on immune cells. Our lab has developed immune polyelectrolyte multilayers, or iPEMs, comprised entirely of antigen and a TLRa. Injectable iPEM particles activate DCs more potently than soluble signals. However, these materials do not specifically access the skin niche, where APCs are concentrated. Microneedle arrays (MNAs) are emerging technologies being explored clinically that penetrate the skin via projections long enough to reach concentrated APCs in dermal layers, but too short to trigger pain receptors. Motivated by improved vaccine outcomes from combinatorial adjuvants, I have shown that constant amounts of antigen and varying combinations of TLRas can be assembled on MNAs to develop a library of compositions. DCs treated with this library in vitro presented antigen and costimulatory markers in a compositionally-dependent format. Funding of this proposal will allow me to test the hypothesis that inclusion of two classes TLRas on MNAs maximizes APC activation at the site of application. I will also link the relative TLRa compositions to APC migration to the LNs, resulting effector cell response, and improvements in a melanoma model. These goals will be accomplished in three aims: 1) Connect defined ratios of TLRas on MNAs to TLR engagement on skin-resident APCs in vitro, 2) Test how TLRa composition on MNAs polarizes APC localization and T cell response in vivo. and 3) Use melanoma as a test bed to link TLRa composition on MNAs to overall disease outcomes. These aims will provide understanding regarding the delivery of multiple TLRas via MNAs to improve disease outcomes. Combined with my holistic training plan, the research planned in this F31 award period will advance my ultimate goal of leading an independent research lab.
