Vaccination is one of the transformative advances of the last century, allowing prevention of infection with a single dose. However, vaccines for diseases that continue to challenge public health must induce immune responses that are not only potent, but that exhibit tunable features such as polarizing responses toward cell- mediated or antibody-mediated immunity, promoting immunological memory over effector response, or directing immune cells to target tissue. Adjuvants could help deliver this control by activating specific immune pathways, or sets of pathways, that define how antigens are responded to. Toll-like receptor agonists (TLRas), for example, are a growing class of adjuvants that activate stimulatory pathogen-sensing pathways triggered by molecular patterns uncommon in humans, but common in pathogens. Many new studies confirm multifunctional or combination adjuvants able to activate several TLR pathways drive synergistic responses pre-clinically and clinically. However, adjuvant design has historically been dominated by empirical approaches. Thus, new strategies that simplify vaccine composition and create modular platforms for delivery of multiple adjuvants could generate insight into how adjuvants control the nature of immune function, individually or in concert. Biomaterials hold great potential along these lines because these materials offer the ability to deliver multiple cargos. However, many materials – polymer particles, for example – exhibit intrinsic features that can activate inflammatory pathways even in the absence of other immune cues. This feature can be harnessed in vaccination, but also hinders rational design because the role of each vaccine component is clouded by the intrinsic effects of the carrier. Materials that offer features of biomaterials – such as co-delivery – but that improve the modularity and definition of vaccines could provide new knowledge of how combination adjuvants polarize immunity and inform the design of a new generation of vaccines that elicit tunable responses. Toward this goal, we designed a new class of vaccine based on polyelectrolyte multilayers (PEMs) assembled entirely from immune signals. These immune-PEMs (iPEMs) are electrostatically self-assembled from peptide antigens and polyionic TLRas that serve as molecular adjuvants. In this project we will test the hypothesis that juxtaposition of antigens and TLRas in iPEMs can be used to program specific features of antigen-specific immunity. The specific aims are: 1) test if TLRa composition in iPEM correlates to in vitro TLR signaling & polarizes DC/T cell function, 2) test if iPEMs polarize T and B cell function depending on TLRas type and composition in iPEMs, 3) determine how iPEM composition drives local reorganization of LNs & changes in T cell migration, 4) use melanoma as a test bed to assess the efficacy of iPEMs as a function of TLRa combination. Importantly, we will benchmark these materials against potent biomaterial vaccines carriers, and against clinically-relevant adjuvants. Our work will generate new knowledge of how the juxtaposition and combination of antigens and adjuvants promote and polarize immunity, contributing new insight to support more rational vaccine design strategies.

PROJECT NARRATIVE Vaccines could play a transformative role in preventing and treating diseases ranging from infectious pathogens to cancer because these technologies harness the specificity of the immune system to clear pathogens without targeting the body's own cells. To realize these goals, new understanding of adjuvants – molecules added to vaccines to enhance function – is needed to support design of next-generation vaccines that elicit responses tailored for specific diseases. This project will combine natural biomaterials to create a simple nanotechnology platform that reveals how combination adjuvants work together, and how these powerful materials can be designed to elicit specific immune responses.