Self-Assembling Peptide Biomaterials Enable Vaccines Against Global Infectious Diseases

Abstract

Vaccines are widely accepted to be one of the most important aspects of public health which have prevented disease and decreased mortality for infectious diseases in the last century. Further improving vaccine technology to support development of new and improved vaccines against a variety of targets is paramount where the global infectious disease landscape changes rapidly. Delivery platforms using biomaterials-based strategies show promise for meeting the unique challenges presented in developing vaccines against a wide variety of targets. Mucosal delivery of vaccines is additionally of particular relevance, both to enable protective responses at mucosal sites, where most pathogens enter the body, and to enable self-delivery and decrease resources needed for vaccine delivery, including cold-chain requirements and trained medical personnel. Here, we address a variety of aspects of vaccine development using self-assembling peptide nanofiber platforms to increase antigen multivalency. Firstly, we leverage the Coil29 nanofiber platform for sublingual (under the tongue) administration for a variety of peptide antigens. We additionally use a Design of Experiments (DOE) approach to examine the sublingual delivery of Coil29 in a multi-epitope combinatorial context, using antigens from gonorrhea as models for this application. Next, we use the self-assembling Q11 platform to increase antigen multivalency of hemagglutinin (HA) for influenza vaccines, and incorporate nanoscale, non-reactogenic adjuvants which increase breadth of antibody binding for vaccines targeting H3N2 influenza. In Chapter 3, we describe the application of Coil29, a self-assembling peptide nanofiber platform based on stacked α-helices, to sublingual immunization. We have previously shown that sublingual peptide immunization is enabled with -sheet-forming peptide nanofibers, but the translatability of this delivery route to other biomaterial platforms has not been investigated. Surprisingly, we found that the addition of mucus-inert peptides, namely combinations of proline, alanine, and serine (PASylation), to sublingual formulations was only required in an epitope-dependent fashion, unlike with Q11, where mucus inert peptides or polymers were required in all cases. Upon further investigation, we revealed that PASylation decreases viscosity and mucus complexation and increases nanofiber transport of hydrophobic Coil29 formulations through the oral epithelium. We correlated these biophysical findings with the hydrophobicity of Coil29 nanofiber constructs, where hydrophobic formulations required PASylation while more hydrophilic ones did not. We additionally demonstrate the ability of Coil29 nanofibers to be formed into “tabletized” solid-state formulations, further enabling self-administration. In this chapter, we demonstrate the applicability of Coil29 to sublingual immunization and the ability to tune this modular platform to antigens of varying physicochemical characteristics.To further develop Coil29 as a platform for sublingual immunization (discussed in Chapter 4), we next investigated combining multiple peptide epitopes into one formulation. Using antigens from gonorrhea as a delivery route relevant model, we screened several sequences for sublingual immunogenicity before using a mixture DOE approach to combine two epitopes – 2C7 and MtrE – with PAS-Coil29, which revealed the formulation which maximized serum IgG and IgA responses to both epitopes. We further examined the role of peptide hydrophobicity, immunogenicity, and immunodominance in the epitope dependency of sublingual immunization with Coil29, as well as the role of PASylation in this combinatorial context. We validated the results of the DOE with several mucosal adjuvants with differing T-helper biasing including c-di-AMP and CTB. We examined the heat stability of the final combination formulation, with no loss of efficacy after storage at 40 °C for 7 days. This work further highlighted the epitope-dependent nature of mucosal vaccine delivery and the careful considerations which must be made to maximize immune responses for each vaccine target.In addition to advancing mucosal delivery technologies for peptide antigens, we also leveraged self-assembling nanofibers to increase multivalency and efficacy for protein antigens, here using HA from influenza A. In Chapter 5, we investigated two alternative bioconjugation strategies to construct HA-Q11. In the first, B-tail co-assembly, HA with a B-sheet forming tag self-inserts into Q11 nanofibers. The second, SortaseA (SrtA) mediated ligation, utilizes enzymatic coupling of pre-formed tagged Q11 and tagged HA protein. In addition to increased multivalency achieved via bioconjugation, we also included a nanoscale, non-reactogenic adjuvant KEYA-Q11, a randomized peptide based on glatiramer acetate which engages T cells and augments immune responses. We found that the B-tail co-assembly scheme increases anti-HA humoral and cellular responses in a one- and two-dose vaccine format, even with low fractions of KEYA-Q11 in the nanofiber formulation. However, this scheme did not result in significantly higher measures of protection against influenza virus and demonstrated some decrease in HA antigenicity upon conjugation. The SrtA mediated conjugation scheme also demonstrated increased humoral and cellular immune responses compared to unconjugated protein, but also a marked increase in breadth of antibody binding to heterologous HA proteins and an increase in correlates of protection to influenza infection. In particular, the increase in antibody breadth achieved here demonstrates a significant advancement towards a flu vaccine with increased durability to antigenic drift, potentially helping to eliminate the need for yearly vaccination.This dissertation represents critical advancements in the use of self-assembling nanofiber platforms to deliver vaccines targeting globally relevant infectious diseases. We demonstrate the applicability of the α-helical nanofiber platform Coil29 to sublingual immunization with peptide antigens of varying physicochemical properties, both alone and in a combinatorial format. We also use the B-sheet self-assembling nanofiber Q11 to increase the multivalency of HA for influenza vaccines, increasing antibody binding breadth with the nanoscale, non-reactogenic, polysequence adjuvant KEYA-Q11. This interdisciplinary research synergizes knowledge from a variety of fields including materials science, immunology, vaccinology, chemistry, and microbiology and presents original and impactful contributions to these fields.