(671f) Design of Self-Assembled Vaccines to Program Activation of Multiple Adjuvant Pathways

Biomaterials have the potential to better control immune function through co-delivery of signals, targeting, and controlled release. However, addition of polymers increases the complexity of vaccine composition. Further, many polymeric carriers trigger stimulatory pathways even without other immune cues. While potentially a trait to be harnessed, these effects can also hinder rational design since carriers can alter the types of immune response that develop. Development of materials that juxtapose multiple antigens and adjuvants at high density while eliminating carrier effects could simplify vaccine composition and support tunable control of immune responses to infectious disease or cancer. Along these lines, recent studies have explored vaccines using combinations of molecular adjuvants based on toll-like receptor (TLR) ligands. These signaling pathways have evolved to detect patterns common in pathogens but uncommon in humans. Thus new vaccines are seeking synergistic benefits using combinations of TLR agonists (TLRas) that simultaneously activate several TLR pathways.

Polyelectrolyte multilayers (PEMs) offer fundamental features to provide a unique level of control over the delivery of multiple TLRAs. These nanoscale structures are prepared by electrostatic, layer-by-layer assembly of oppositely charged polymers. Recently, PEMs has been studied as vaccines using biocompatible polymers to encapsulate or adsorb antigens or adjuvants. We sought to design PEMs composed entirely of peptide antigens and combinations of TLRas (i.e., TLRa3, TLRa7, and TLRa9). These assemblies are termed immune-PEMs (iPEMs). iPEMs were assembled from a model antigen (SIINFEKL) and combinations of TLR3a polyinosinic-polycytidylic acid (polyIC), TLR7a polyuridylic acid (polyU), TLR9a Class B CpG oligonucleotide (CpG). Each of these TLRas is polyanionic, thus SIINFEKL was modified with arginine residues (SIIN*) to support electrostatic assembly. We initially assembled iPEMs containing a single TLRa, observing linear growth of SIIN*/polyIC, SIIN*/polyU, and SIIN*/CpG iPEMs on silicon substrates. We observed that iPEMs grow proportional to the number of layers deposited on the silicon substrates. Similarly, iPEMs containing two types of TLRas, or all three types of TLRas could be deposited with tunable control over the loading of these components. We next tested if SIIN*/polyIC IPEMs activate primary dendritic cells (DCs) and expand antigen-specific T cells in culture and in mice. We observed that SIIN and polyIC in iPEMs maintained the specificity and functionality of each component by promoting upregulation of DC maturation markers (i.e., CD40, CD80, and CD86) and inflammatory cytokine secretions (i.e, IFN-γ, IL-6 and IL-1β). These structures also drove expansion of SIINFEKL-specific CD8⁺ T cells. Direct measurements of TLR3 signaling also revealed iPEMs containing polyIC activated TLR3, while iPEMs containing other TLRas or irrelevant nucleic-acid sequences did not.

To test whether SIIN*/polyIC iPEMs generate functional, antigen-specific T cell responses incorporated in iPEMs, mice were primed at day 0 and boosted on day 15. iPEMs significantly expanded antigen-specific CD8⁺ T cells at day 7 (pâ?¤0.05) and 22 (pâ?¤0.001) compared to both naïve mice and admixed vaccines (i.e., free) containing the same dose of antigens and adjuvants. In tumor study, mice were primed and boosted (day 15 and day 28) with SIIN*/polyIC iPEMs, then challenged with 1x10⁶ B16-OVA cells on Day 36 (N=6). In these studies, SIIN*/polyIC iPEMs conferred a 56% increase in median survival compared to admixed vaccines. Ongoing studies are assessing these same ideas using iPEMs assembled from multiple TLRas. This platform could thus offer modularity to selectively program the activation of TLR pathways in development of new vaccines and tumor therapies.