PROJECT ABSTRACT Infectious diseases are a major cause of morbidity and mortality in the expanding older population. While vaccines are efficient measures to prevent infections, a critical problem is that aging-associated changes in the immune system lead to decreased immunogenicity and clinical efficacy of most currently used vaccines. Novel strategies that increase vaccination efficacy and specifically target the aged immune system are imperative. An essential facet of these efforts is the use of combinatory adjuvants that synergistically potentiate more effective vaccine-induced immune responses against various pathogens. In this project we will define the mechanism of action (MOA) of a combination of a unique parasite protein adjuvant, rASP-1 with each of three well- characterized innate adjuvants: STING, TLR7/8 and RIG-I agonists, known to activate cytoplasmic pattern recognition receptors and Type I IFN signaling, which are specifically dampened in the aged population. We have shown that rASP-1 is a powerful adjuvant as it activates human and mouse dendritic cells to potentiate the differentiation of naïve CD4+ T cells into Th1, Th17 and Tfh-like cells. Transcriptomic analyses of rASP-1- activated hMoDCs revealed upregulation of the MyD88-independent activation pathway and interferon related genes. In mice, rASP-1 elicits a balanced Th1/Th2 antibody response, Th1-biased cellular immunity, and enhanced responses when co-administered with vaccines leading to increased survival following microbial challenge. Importantly, our preliminary data indicate that rASP-1 in combination with 2’3’-cGAMP (STING), R848 (TLR7/8) or 5’3p-hpRNA (RIG-I) in activated adult and aged mouse BMDCs can dramatically synergize beyond merely additive effects; it enhanced secretion of IL-12p40, IP-10 and/or IL-10. The synergy is exceptionally evident when it activates aged mouse BMDCs with the STING ligand. Therefore, we hypothesize that combining rASP-1 with STING, TLR7/8 or RIG-I agonists will synergistically activate early innate immune signaling that critically contributes to the establishment and nature of immune responses, and the duration and intensity of immune activation. We posit that this will help restore the aging-associated deficits in the critical regulatory pathways of the antiviral responses. Through comprehensive immunologic and transcriptomic analyses, the MOA through which rASP-1 in combination with these three PRR agonists leads to optimal activation of helper T cells driving humoral and cellular antiviral responses will be established in both mouse (Aim 1) and human (Aim 2) DCs (adult and aged), to establish that they are conserved, and then test their efficacy in vivo in adult and aged mice using the trivalent inactivated influenza vaccine (IIV3) as a model antigen (Aim 3). This project brings together a dynamic team with a history of collaboration and complementary expertise in vaccinology, immunology, biochemistry, innate cell receptor biology, ...