Combinatorial Delivery of Dual and Triple TLR Agonists via Polymeric Pathogen-like Particles Synergistically Enhances Innate and Adaptive Immune Responses

Despite decades of research very few vaccine-adjuvants have received FDA approval. Two fundamental challenges plague clinical translation of vaccine-adjuvants: reducing acute toxicities that result from systemic diffusion of many soluble adjuvants, and delivering multiple adjuvants at the same time to mimic the synergistic immune-stimulation of pathogens, while being safe. In order to address these barriers, we evaluated combinations of four clinically relevant immune-agonists, specifically Toll-like receptor (TLR) ligands, using biodegradable, polymer microparticles. We tested them alone and in combinations of 2 or 3, for a total of 10 unique conditions. We evaluated primary bone-marrow-derived Dendritic Cell phenotypes and functionality, and identified several synergistic combinations. We picked a dual and a triple adjuvant combination, TLR4/TLR9 and TLR4/TLR7/TLR9, for further evaluation and found that both combinations promoted antigen cross-presentation in vitro. Studies in mice using the model antigen Ovalbumin, showed that both combinations enhanced lymph node germinal center and T follicular helper cell responses. The triple adjuvant combination showed increased antigen-specific antibody titer with an overall balanced Th1/Th2 response, while the dual combination promoted Th1-polarized IgG responses. Our results show how polymeric particulate-carriers can be adopted to safely deliver combinatorial adjuvants and selectively synergize specific types of immune responses for vaccine applications.