TRAF6-IRF5 kinetics, TRIF, and biophysical factors drive synergistic innate responses to particle-mediated MPLA-CpG co-presentation

Abstract

Innate immune responses to pathogens are driven by co-presentation of multiple pathogen-associated molecular patterns (PAMPs). PAMPs and PAMP-analogs are also used as immune-adjuvants to enhance vaccine efficacy by activating various Pattern Recognition Receptors (PRRs), like Toll-like receptors (TLRs). Various combinations of PAMP adjuvants can trigger synergistic immune responses, but the underlying molecular mechanisms driving that synergy are poorly understood. Here, we used synthetic particulate carriers co-loaded with MPLA (TLR4-adjuvant) and CpG (TLR9-adjuvant) as pathogen-like particles (PLPs) to dissect the signaling pathways responsible for the integrated, dual-adjuvant immune response. PLP-based co-presentation of MPLA and CpG to mouse bone marrow derived antigen-presenting cells (BM-APCs) elicited synergistic Type-I Interferon (IFN-{beta}) and IL-12p70 responses, which were strongly influenced by the biophysical properties of PLPs. Mechanistically, we found that the adapter protein MyD88 and the Interferon-Regulatory-Factor-5 (IRF-5), but not the canonical factors IRF-3 or IRF-7, were necessary for production of both IFN-{beta} and IL12p70. TRIF signaling was required to elicit the synergistic response; the absence of TRIF abolished synergy. Importantly, both the kinetics and magnitude of downstream TRAF6 and IRF5 signaling (TRIF-TRAF-IRF5 pathway kinetics) drove the observed synergy. These results identify not only the key signaling mechanism that cooperates to generate a combinatorial response to MPLA-CpG dual engagement in BM-APCs, but they also underscore the critical role that signaling kinetics and biophysical presentation plays in integrated responses to combination adjuvants.