Supramolecular Peptide Self-Assemblies Facilitate Oral Immunization.

Oral immunization is a promising strategy for preventing and treating gastrointestinal (GI) infections and diseases, as it allows for direct access to the disease site. To elicit immune responses within the GI tract, however, there are many obstacles that oral vaccines must surmount, including proteolytic degradation and thick mucus barriers. Here, we employed a modular self-assembling peptide nanofiber platform to facilitate oral immunization against both peptide and small molecule epitopes. Synthesizing nanofibers with d-amino acids rendered them resistant to proteases in vitro, whereas l-amino acid nanofibers were rapidly degraded. Additionally, the inclusion of peptide sequences rich in proline, alanine, and serine (PAS), increased nanofiber muco-penetration, and accelerated nanofiber transport through the GI tract. Oral immunization with PASylated nanofibers and mucosal adjuvant generated local and systemic immune responses to a peptide epitope but only for l-amino acid nanofibers. Further, we were able to apply this design to also enable oral immunization against a small molecule epitope and illustrated the therapeutic and prophylactic effectiveness of these immunizations in mouse models of colitis. These findings demonstrate that supramolecular peptide self-assemblies have promise as oral vaccines and immunotherapies.

Active immunotherapy for C5a-mediated inflammation using adjuvant-free self-assembled peptide nanofibers.

The terminal protein in the complement cascade C5a is a potent inflammatory molecule and chemoattractant that is involved in the pathology of multiple inflammatory diseases including sepsis and arthritis, making it a promising protein to target with immunotherapies. Active immunotherapies, in which patients are immunized against problematic self-molecules and generate therapeutic antibodies as a result, have received increasing interest as an alternative to traditional monoclonal antibody treatments. In previous work, we have designed supramolecular self-assembling peptide nanofibers as active immunotherapies with defined combinations of B- and T-cell epitopes. Herein, the self-assembling peptide Q11 platform was employed to generate a C5a-targeting active immunotherapy. Two of three predicted B-cell epitope peptides from C5a were found to be immunogenic when displayed within Q11 nanofibers, and the nanofibers were capable of reducing C5a serum concentrations following immunization. Contrastingly, C5a's precursor protein C5 maintained its original concentration, promising to minimize side effects heretofore associated with C5-targeted therapies. Immunization protected mice against an LPS-challenge model of sepsis, and it reduced clinical severity in a model of collagen-antibody induced arthritis. Together, this work indicates the potential for targeting terminal complement proteins with active immunotherapies by leveraging the immunogenicity of self-assembled peptide nanomaterials. STATEMENT OF SIGNIFICANCE: Chronic inflammatory diseases such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease are currently treated primarily with monoclonal antibodies against key inflammatory mediators. While helpful for many patients, they have high non-response rates, are costly, and commonly fail as anti-drug antibodies are raised by the patient. The approach we describe here explores a fundamentally different treatment paradigm: raising therapeutic antibody responses with an active immunotherapy. We employ innovative supramolecular peptide nanomaterials to elicit neutralizing antibody responses against complement component C5a and demonstrate therapeutic efficacy in preclinical mouse models of sepsis and rheumatoid arthritis. The strategy reported may represent a potential alternative to monoclonal antibody therapies.

Engaging natural antibody responses for the treatment of inflammatory bowel disease via phosphorylcholine-presenting nanofibres.

Inflammatory bowel disease lacks a long-lasting and broadly effective therapy. Here, by taking advantage of the anti-infection and anti-inflammatory properties of natural antibodies against the small-molecule epitope phosphorylcholine (PC), we show in multiple mouse models of colitis that immunization of the animals with self-assembling supramolecular peptide nanofibres bearing PC epitopes induced sustained levels of anti-PC antibodies that were both protective and therapeutic. The strength and type of immune responses elicited by the nanofibres could be controlled through the relative valency of PC epitopes and exogenous T-cell epitopes on the nanofibres and via the addition of the adjuvant CpG. The nanomaterial-assisted induction of the production of therapeutic antibodies may represent a durable therapy for inflammatory bowel disease.

Randomized peptide assemblies for enhancing immune responses to nanomaterials.

Biomaterials capable of inducing immune responses with minimal associated inflammation are of interest in applications ranging from tissue repair to vaccines. Here we report the design of self-assembling randomized polypeptide nanomaterials inspired by glatiramoids, an immunomodulatory class of linear random copolymers. We hypothesized that peptide self-assemblies bearing similar randomized polypeptides would similarly raise responses skewed toward Type 2 immunity and TH2 T-cell responses, additionally strengthening responses to co-assembled peptide epitopes in the absence of adjuvant. We developed a method for synthesizing self-assembling peptides terminated with libraries of randomized polypeptides (termed KEYA) with good batch-to-batch reproducibility. These peptides formed regular nanofibers and raised strong antibody responses without adjuvants. 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, enhancing immune responses against epitopes relevant to influenza and chronic inflammation while 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, induced no measurable swelling in footpad injections, and decreased overall T cell expansion compared to unmodified nanofibers, further suggesting a TH2 T-cell response with minimal inflammation. Collectively, this work introduces a biomaterial capable of raising strong Type 2/TH2/IL-4 immune responses, with potential applications ranging from vaccination to tissue repair.

Advances in nanomaterial vaccine strategies to address infectious diseases impacting global health.

Despite the overwhelming success of vaccines in preventing infectious diseases, there remain numerous globally devastating diseases without fully protective vaccines, particularly human immunodeficiency virus (HIV), malaria and tuberculosis. Nanotechnology approaches are being developed both to design new vaccines against these diseases as well as to facilitate their global implementation. The reasons why a given pathogen may present difficulties for vaccine design are unique and tied to the co-evolutionary history of the pathogen and humans, but there are common challenges that nanotechnology is beginning to help address. In each case, a successful vaccine will need to raise immune responses that differ from the immune responses raised by normal infection. Nanomaterials, with their defined compositions, commonly modular construction, and length scales allowing the engagement of key immune pathways, collectively facilitate the iterative design process necessary to identify such protective immune responses and achieve them reliably. Nanomaterials also provide strategies for engineering the trafficking and delivery of vaccine components to key immune cells and lymphoid tissues, and they can be highly multivalent, improving their engagement with the immune system. This Review will discuss these aspects along with recent nanomaterial advances towards vaccines against infectious disease, with a particular emphasis on HIV/AIDS, malaria and tuberculosis.

Enabling sublingual peptide immunization with molecular self-assemblies.

Short peptides are poorly immunogenic when delivered sublingually - under the tongue. Nanomaterial delivery of peptides could be utilized to improve immunogenicity towards designed sublingual vaccines, but nanomaterials have not been widely successful in sublingual vaccines owing to the challenges of transport through the sublingual mucosa. Here, we report that the sublingual immunogenicity of peptides is negligible, even in the presence of sublingual adjuvants or when PEGylated, but can be dramatically enhanced by assembly into supramolecular polymer-peptide nanofibers bearing low-molecular weight PEG, optimally between 2000 and 3000 Da. Neither PEGylation nor a sublingual adjuvant were capable of rendering peptides immunogenic without assembly into nanofibers. We found that PEG decreased nanofiber interactions with mucin and promoted longer residence time at the sublingual immunization site. Parallel investigations with shortened nanofibers indicated that the size of the assemblies had a surprisingly negligible influence over sublingual immunogenicity. In mice, optimized formulations were capable of raising strong and highly durable systemic antibody responses, antibodies in the upper respiratory and reproductive tracts, and systemic antigen-specific T-cell responses. These nanofiber-based sublingual vaccines were effective with both protein and nucleotide adjuvants and raised responses against both a model peptide epitope and a peptide epitope from M. tuberculosis. Further, PASylation (modification of nanofibers with peptide sequences rich in Pro, Ala, and Ser) could be substituted for PEGylation to also achieve sublingual immunogenicity. These findings indicated that surface properties supersede nanomaterial size in modulating sublingual nanomaterial immunogenicity, having important implications for the design of synthetic sublingual vaccines.

Dual carrier-cargo hydrophobization and charge ratio optimization improve the systemic circulation and safety of zwitterionic nano-polyplexes.

While polymeric nano-formulations for RNAi therapeutics hold great promise for molecularly-targeted, personalized medicine, they possess significant systemic delivery challenges including rapid clearance from circulation and the potential for carrier-associated toxicity due to cationic polymer or lipid components. Herein, we evaluated the in vivo pharmacokinetic and safety impact of often-overlooked formulation parameters, including the ratio of carrier polymer to cargo siRNA and hydrophobic siRNA modification in combination with hydrophobic polymer components (dual hydrophobization). For these studies, we used nano-polyplexes (NPs) with well-shielded, zwitterionic coronas, resulting in various NP formulations of equivalent hydrodynamic size and neutral surface charge regardless of charge ratio. Doubling nano-polyplex charge ratio from 10 to 20 increased circulation half-life five-fold and pharmacokinetic area under the curve four-fold, but was also associated with increased liver enzymes, a marker of hepatic damage. Dual hydrophobization achieved by formulating NPs with palmitic acid-modified siRNA (siPA-NPs) both reduced the amount of carrier polymer required to achieve optimal pharmacokinetic profiles and abrogated liver toxicities. We also show that optimized zwitterionic siPA-NPs are well-tolerated upon long-term, repeated administration in mice and exhibit greater than two-fold increased uptake in orthotopic MDA-MB-231 xenografts compared to commercial transfection reagent, in vivo-jetPEI®. These data suggest that charge ratio optimization has important in vivo implications and that dual hydrophobization strategies can be used to maximize both NP circulation time and safety.

Zwitterionic Nanocarrier Surface Chemistry Improves siRNA Tumor Delivery and Silencing Activity Relative to Polyethylene Glycol.

Although siRNA-based nanomedicines hold promise for cancer treatment, conventional siRNA-polymer complex (polyplex) nanocarrier systems have poor pharmacokinetics following intravenous delivery, hindering tumor accumulation. Here, we determined the impact of surface chemistry on the in vivo pharmacokinetics and tumor delivery of siRNA polyplexes. A library of diblock polymers was synthesized, all containing the same pH-responsive, endosomolytic polyplex core-forming block but different corona blocks: 5 kDa (benchmark) and 20 kDa linear polyethylene glycol (PEG), 10 kDa and 20 kDa brush-like poly(oligo ethylene glycol), and 10 kDa and 20 kDa zwitterionic phosphorylcholine-based polymers (PMPC). In vitro, it was found that 20 kDa PEG and 20 kDa PMPC had the highest stability in the presence of salt or heparin and were the most effective at blocking protein adsorption. Following intravenous delivery, 20 kDa PEG and PMPC coronas both extended circulation half-lives 5-fold compared to 5 kDa PEG. However, in mouse orthotopic xenograft tumors, zwitterionic PMPC-based polyplexes showed highest in vivo luciferase silencing (>75% knockdown for 10 days with single IV 1 mg/kg dose) and 3-fold higher average tumor cell uptake than 5 kDa PEG polyplexes (20 kDa PEG polyplexes were only 2-fold higher than 5 kDa PEG). These results show that high molecular weight zwitterionic polyplex coronas significantly enhance siRNA polyplex pharmacokinetics without sacrificing polyplex uptake and bioactivity within tumors when compared to traditional PEG architectures.