Molecular Mechanisms of Adjuvant Triplet Combinations

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Abstract: Molecular Mechanisms of Adjuvant Triplet Combinations The immune system makes decisions in response to complex combinations of microbial inputs. Live vaccines that are empirically attenuated from pathogens have been a powerful means to yield life-long immunity against many deadly pathogens because they mimic immune responses to combinations of microbial signals. However, the rational design of non-live vaccines using immunomodulatory agents such as adjuvants has remained an elusive task in many cases where live vaccination is not efficacious or feasible, in part because identifying potent adjuvant combinations and associated molecular mechanisms that explain cross talk remains a major challenge. Based on our recent findings and extensive preliminary data, we propose to define the molecular mechanisms through which two adjuvant triplets – containing agonists for Toll-like receptor (TLR), C-type lection receptor (CLR), RIG-I-like receptor (RLR), and cytosolic dsDNA sensor (CDS) pathways – induce protective CD4+ and CD8+ T cell responses in mice. We will use an innovative approach which is (i) comparative – by contrasting the quantitative effects of adjuvant triplets and matching singles and pairs as means to accurately pinpoint molecular mechanisms explaining cross talk; and (ii) multiscale – by studying molecular mechanisms at play in cells, tissues, and the whole body. First, we will determine the molecular mechanisms of intra-cellular signaling cross talk by adjuvant triplets by testing hypotheses at the level of protein complexes proximal to adjuvant receptors, phosphorylation cascades and kinase-substrate relationships, and gene regulatory networks. Second, we will identify the molecular mechanisms through which adjuvant combinations impact inter-cellular signaling between dendritic cells (DCs) and T cells by testing hypotheses on the regulatory mechanisms shaping the cellular, surface, and secreted proteome of DCs. Third, we will test hypotheses on the effects of adjuvant triplets on the organism-wide spreading and seeding of effector and memory T cells, and the underlying cell circuits of the skin (vaccination site) and draining lymph node that explain the induction of protective, long-term T cell immunity. Results from this work will produce critical insights at the forefront of adjuvant combination research by characterizing higher-order combinations of adjuvants that can mimic the effects of well-established, potent live attenuated vaccines and inform future vaccine designs against infection.