Polysequence Nanomaterials for Immunomodulation
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Peptide-based vaccines have received growing interest due to their specificity and ability to limit off-target effects, and they are currently being explored toward a variety of infectious diseases and therapeutic targets. However, the efficacy and applicability of such epitope-based vaccines are currently limited by difficulties in predicting immunogenic epitopes in outbred populations and a reliance on carrier proteins and adjuvants that can cause pain and swelling. Current vaccine platforms are further limited in their ability to combine multiple different epitopes, making it difficult to adjust humoral and cellular responses systematically. A vaccine platform containing broadly reactive T-cell epitopes that boosts responses to co-delivered antigens with minimal inflammation could address these limitations. To that end, the focus of this dissertation was to create peptide epitopes that can be incorporated within a supramolecular nanomaterial platform, together acting as a nano-adjuvant, a term that we will use here to describe materials whose adjuvanting properties depend on their nanoscale structure. To achieve this, we took inspiration from a class of materials termed glatiramoids, which promote anti-inflammatory and TH2 immune responses. We created an immunomodulatory supramolecular nanomaterial system inspired by the randomized nature of glatiramoids termed KEYA-Q11. By creating a glatiramoid-like peptide library integrated within self-assembling Q11 nanofibers, numerous epitopes can be presented simultaneously along the nanofibers for maximum antigen presenting cell uptake and activation. The first half of this document (Chapters 3 and 4) describes how this nanomaterial increased immunogenicity of co-assembled epitopes while also creating a KEYA-specific non-inflammatory response to the randomized component. Additionally, capitalizing on the potential for KEYA-Q11 to amplify immune responses to co-assembled epitopes, this technology is applied in the second half of this document (Chapters 5 and 6) to an epitope-based influenza vaccine. Initially we designed and synthesized a self-assembling nanomaterial inspired by glatiramoids and evaluated its TH2 T-cell polarizing properties (Chapter 3). Glatiramoids raise strong, protective immune responses in patients and have been examined in a variety of contexts from Multiple Sclerosis to HIV. However, due to their randomized polysequence structure, it remains challenging to incorporate glatiramoids into other materials and strategies to optimize them for specific therapeutics. Therefore, we designed a polysequence peptide sequence and synthesized it onto the chemically defined, supramolecular Q11 nanofiber platform to straightforwardly titrate it into other nanomaterial formulations. This polysequence nanomaterial was termed KEYA-Q11 for the four amino acids, lysine, glutamic acid, tyrosine, and alanine, that comprise its structure. Due to the extensive number of possible KEYA sequences, multiple batches of KEYA-Q11 were first examined with an array of biophysical characterization techniques to confirm reproducible synthesis and assembly. The optimal number of polysequence amino acid additions was determined to be 20 amino acids as (KEYA)20Q11 could reliably be synthesized and raise strong Type 2/TH2/IL-4 immune responses. Moreover, by modulating the concentration of KEYA-Q11 in a Q11 immunization, the strength of KEYA-specific B-cell responses were similarly altered. KEYA modifications dramatically improved uptake of peptide nanofibers in vitro by antigen presenting cells and served as strong B-cell and T-cell epitopes in vivo, inducing a KEYA-specific Type 2/TH2/IL-4 phenotype. KEYA modifications also increased IL-4 production by T cells, extended the residence time of nanofibers, and decreased overall T cell expansion compared to unmodified nanofibers, further suggesting a TH2 T-cell response with minimal inflammation. Subsequently, we exploited the modularity of the self-assembling system to maximize application of KEYA-Q11 as a nanoscale adjuvant without inflammation (Chapter 4). Adjuvants are commonly required to raise strong immune responses to peptide therapeutics, but often induce swelling and pain at the injection site and typically drive immune phenotype. Relative to common adjuvants, KEYA-Q11 had no detectable injection site swelling and was more effective at raising humoral responses despite a genetically diverse in vivo population. Furthermore, when combined with peptide epitopes KEYA-Q11 augmented antibody production against co-assembled B-cell epitopes for cytokine TNF, D-chiral MMP cross linker, and a conserved segment of the M2 influenza protein, and increased T-cell stimulation specific to co-assembled T-cell epitopes PADRE and a conserved segment of the nucleoprotein of influenza. Likewise, when combined with the influenza surface protein hemagglutinin, KEYA modifications strengthened the resulting influenza-specific cellular immune responses. Augmented immune responses typically followed native epitope polarization, as in a co-assembly of KEYA-Q11 and the nucleoprotein epitope raised Type 2/TH2/IL4 producing KEYA-specific responses and magnified the Type 1/IFN producing nucleoprotein-specific responses that epitope would produce without an adjuvant, and thus using KEYA-Q11 as the adjuvant allowed for finer control over immune phenotype. Building on the success of KEYA-Q11 as a nano-scale adjuvant without inflammation, we utilized these properties to decrease the severity of influenza infection and provide broad protection via immunization with peptide epitopes (Chapters 5 and 6). Much of the current focus on influenza vaccines revolves around partial or whole proteins to induce broadly protective antibodies, while other have demonstrated cross-reactive T-cell responses are vital for heterologous protection. Conserved peptide epitopes have been discovered but typically are included with larger proteins and adjuvants to increase immunogenicity. Supramolecular assemblies based on the Q11 peptide system containing KEYA, a B-cell epitope from a conserved surface protein on influenza, and CD4+ and CD8+ T-cell epitopes from influenza nucleoprotein and polymerase acidic protein, respectively, raised strong immune responses against all three epitopes. Inclusion of the KEYA component in prophylactic immunizations with these materials significantly improved protection following a lethal influenza challenge. It has been established that while peptide-based immunotherapies can have finely directed specificity for chosen epitopes, they generally lack sufficient immunogenicity to provoke suitable immune responses. This new strategy for augmenting immune responses to peptide-based therapeutics, especially those employing nanomaterials, and especially for applications where non-inflammatory responses are prioritized, can be employed for a variety of potential applications in vaccine development, towards infectious diseases and towards non-infectious applications such as inflammatory autoimmune diseases, wound healing, or graft rejection. KEYA-Q11 is a unique fusion of two materials, a highly ordered system with a highly disordered system, and examination of this nanomaterial has provided valuable insight into both randomly polymerized structures and non-inflammatory nano-scale adjuvants.
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