(17a) Determining the Role of Peptide Nanocluster Characteristics on Dendritic Cell Antigen Processing in Peptide Vaccines

Peptide subunit vaccines are desirable because they increase control over the induced immune response and safety of the vaccine by reducing the risk of off-target responses to molecules other than the target antigen. The immunogenicity of most peptides, however, is often insufficient to produce robust and lasting immunity. Peptide nanoclusters (PNCs) are proposed as a subunit peptide vaccine delivery system made completely of crosslinked peptide antigen that could improve the immunogenicity of a peptide vaccine. PNCs provide similar benefits to other delivery systems in that they increase the size of the antigen material to be internalized, potentially increasing in vivo retention and trafficking to areas of high immune cell activity, as well as increasing antigen presenting cell (APC) recognition of larger particles that mimic the size of whole pathogens. PNCs have been successfully synthesized from several therapeutically relevant peptides as small as 8 amino acids in length, meaning the peptide antigen is already small enough to be presented on APC surfaces without proteolysis. Furthermore, since PNCs are crosslinked using functional groups on residues that may be contained within the epitope sequence, this could also affect how APCs can process and present them. To allow PNCs to be a useful delivery platform for small peptides, it is important to determine the optimal length, sequence, and method of crosslinking that will provide the best processing and presentation of antigens by APCs like dendritic cells (DCs).

To address these questions, PNCs were synthesized by desolvation from the model protein ovalbumin epitope SIINFEKL. The peptide was also altered several ways, including flanking with 3 extra residues on each end from either the original ovalbumin sequence or strategically chosen amino acids. The length was chosen to be just larger than most MHC I presented antigens to require proteolysis for antigen presentation, and distinct flanking residues were chosen to determine if these residues play a role in the efficient processing or presentation of the SIINFEKL sequence. Additionally, several types of crosslinkers were used to stabilize the PNCs to represent various types of crosslinking including completely reversible, reversible with leftover linker arms, irreversible, etc. For each PNC variation, optimization of the desolvation conditions was performed to yield PNCs that would be comparable in size and dispersity. Six variations of PNCs were successfully synthesized in a range of sizes all within 150-350 nm in diameter and PDI .160-.450. These PNCs will be administered to DCs in vitro to evaluate how these differences in PNCs affect several aspects of DC antigen processing including levels of internalization, MHC-mediated presentation, DC maturation levels, and ultimately SIINFEKL-specific T cell activation. This knowledge will guide future PNC synthesis methods to maximize PNC efficiency in increasing immunogenicity of peptide vaccines.