Semiconductive and Biocompatible Nanofibrils from the Self-Assembly of Amyloid π-Conjugated Peptides.

Owing to their capacity to self-assemble into organized nanostructures, amyloid polypeptides can serve as scaffolds for the design of biocompatible semiconductive materials. Herein, symmetric and asymmetric amyloid π-conjugated peptides were prepared through condensation of perylene diimide (PDI) with a natural amyloidogenic sequence derived from the islet amyloid polypeptide. These PDI-bioconjugates assembled into long and linear nanofilaments in aqueous solution, which were characterized by a cross-ß-sheet quaternary organization. Current-voltage curves exhibited a clear signature of semiconductors, whereas the cellular assays revealed cytocompatibility and potential application in fluorescence microscopy. Although the incorporation of a single amyloid peptide appeared sufficient to drive the self-assembly into organized fibrils, the incorporation of two peptide sequences at the PDI's imide positions significantly enhanced the conductivity of nanofibril-based films. Overall, this study exposes a novel strategy based on amyloidogenic peptide to guide the self-assembly of π-conjugated systems into robust, biocompatible, and optoelectronic nanofilaments.

Site-Specific Alkylation of the Islet Amyloid Polypeptide Accelerates Self-Assembly and Potentiates Perturbation of Lipid Membranes.

The accumulation of insoluble amyloids in the pancreatic islets is a pathological hallmark of type II diabetes and correlates closely with the loss of ß-cell mass. The predominant component of these amyloid deposits is the islet amyloid polypeptide (IAPP). The factors contributing to the conversion of IAPP from a monomeric bioactive peptide hormone into insoluble amyloid fibrils remain partially elusive. In this study, we investigated the effect of the oxidative non-enzymatic post-translational modification induced by the reactive metabolite 4-hydroxynonenal (HNE) on IAPP aggregation and cytotoxicity. Incubation of IAPP with exogenous HNE accelerated its self-assembly into ß-sheet fibrils and led to the formation of a Michael adduct on the His-18 side chain. To model this covalent modification, the imidazole N(π) position of histidine was alkylated using a close analogue of HNE, the octyl chain. IAPP lipidated at His-18 showed a hastened random coil-to-ß-sheet conformational conversion into fibrillar assemblies with a distinct morphology, a low level of binding to thioflavin T, and a high surface hydrophobicity. Introducing an octyl chain on His-18 enhanced the ability of the peptide to perturb synthetic lipid vesicles, to permeabilize the plasma membrane, and to induce the death of pancreatic ß-cells. Alkylated IAPP triggered the self-assembly of unmodified IAPP by prompting primary nucleation and increased its capacity to perturb the plasma membrane, indicating that only a small proportion of the modified peptide is necessary to shift the balance toward the formation of proteotoxic species. This study underlines the importance of studying IAPP post-translational modifications induced by oxidative metabolites in the context of pancreatic amyloids.

Synthetic Multicomponent Nanovaccines Based on the Molecular Co-assembly of ß-Peptides Protect against Influenza A Virus.

Peptides with the ability to self-assemble into nanoparticles have emerged as an attractive strategy to design antigen delivery platforms for subunit vaccines. While toll-like receptor (TLR) agonists are promising immunostimulants, their use as soluble agents is limited by their rapid clearance and off-target inflammation. Herein, we harnessed molecular co-assembly to prepare multicomponent cross-ß-sheet peptide nanofilaments exposing an antigenic epitope derived from the influenza A virus and a TLR agonist. The TLR7 agonist imiquimod and the TLR9 agonist CpG were respectively functionalized on the assemblies by means of an orthogonal pre- or post-assembly conjugation strategy. The nanofilaments were readily uptaken by dendritic cells, and the TLR agonists retained their activity. Multicomponent nanovaccines induced a robust epitope-specific immune response and completely protected immunized mice from a lethal influenza A virus inoculation. This versatile bottom-up approach is promising for the preparation of synthetic vaccines with customized magnitude and polarization of the immune responses.

Engineered Curli Nanofilaments as a Self-Adjuvanted Antigen Delivery Platform.

Proteinaceous nanoparticles constitute efficient antigen delivery systems in vaccine formulations due to their size and repetitive nature that mimic most invading pathogens and promote immune activation. Nonetheless, the coadministration of an adjuvant with subunit nanovaccines is usually required to induce a robust, long-lasting, and protective immune response. Herein, the protein Curli-specific gene A (CsgA), which is known to self-assemble into nanofilaments contributing to bacterial biofilm, is exploited to engineer an intrinsically immunostimulatory antigen delivery platform. Three repeats of the M2e antigenic sequence from the influenza A virus matrix 2 protein are merged to the N-terminal domain of engineered CsgA proteins. These chimeric 3M2e-CsgA spontaneously self-assemble into antigen-displaying cross-ß-sheet nanofilaments that activate the heterodimeric toll-like receptors 2 and 1. The resulting nanofilaments are avidly internalized by antigen-presenting cells and stimulate the maturation of dendritic cells. Without the need of any additional adjuvants, both assemblies show robust humoral and cellular immune responses, which translate into complete protection against a lethal experimental infection with the H1N1 influenza virus. Notably, these CsgA-based nanovaccines induce neither overt systemic inflammation, nor reactogenicity, upon mice inoculation. These results highlight the potential of engineered CsgA nanostructures as self-adjuvanted, safe, and versatile antigen delivery systems to fight infectious diseases.

Recombinant Bacillus subtilis flagellin Hag is a potent immunostimulant with reduced proinflammatory properties compared to Salmonella enterica serovar Typhimurium FljB.

Flagellin constitutes a potential adjuvant for vaccines owing to its robust immunostimulatory properties. However, clinical trials have revealed that flagellin derived from Salmonella enterica serovar Typhimurium induces high levels of proinflammatory markers and substantial adverse effects. The flagellin from Bacillus subtilis, Hag, shares high sequence homology with Salmonella FljB within the D0 and D1 domains responsible for TLR5 engagement, while the D2 and D3 domains associated with an off-target immune response are absent. Accordingly, we compared the immunostimulatory and proinflammatory properties of Hag with FljB by harnessing an epitope from the matrix 2 protein (M2e) of the influenza virus. Both flagellins engaged TLR5, with FljB showing a 2.5-fold higher potency than Hag. Mice inoculation showed a robust FljB- or Hag-induced M2e-specific antibody response, with Hag demonstrating a decreased secretion of proinflammatory markers and reduced weight loss. This study revealed that flagellin Hag is a potent immunoadjuvant with reduced proinflammatory properties.

Self-Assembly of Flagellin into Immunostimulatory Ring-like Nanostructures as an Antigen Delivery System.

Proteinaceous nanoparticles represent attractive antigen carriers for vaccination as their size and repetitive antigen displays that mimic most viral particles enable efficient immune processing. However, these nanocarriers are often unable to stimulate efficiently the innate immune system, requiring coadministration with adjuvants to promote long-lasting protective immunity. The protein flagellin, which constitutes the primary constituent of the bacterial flagellum, has been widely evaluated as an antigen carrier due to its intrinsic adjuvant properties involving activation of the innate immune receptor Toll-like receptor 5 (TLR5). Although flagellin is known for its ability to self-assemble into micron-scale length nanotubes, few studies have evaluated the potential usage of flagellin-based nanostructures as immunostimulatory antigen carriers. In this study, we reported for the first time a strategy to guide the self-assembly of a flagellin protein from Bacillus subtilis, Hag, into lower aspect ratio nanoparticles by hindering non-covalent interactions responsible for its elongation into nanotubes. We observed that addition of an antigenic sequence derived from the influenza A virus (3M2e) at the C-terminus of this flagellin, as opposed to positioning the epitope into mid-sequence, precluded filament elongation and resulted in low aspect ratio ring-like nanostructures upon salting-out-induced self-assembly. These nanostructures displayed the antigen at their surface and shared morphological and structural characteristics with flagellin nanotubes, with a diameter of approximately 12 nm, and an α-helix-rich secondary structure. Flagellin ring-like nanostructures were efficiently internalized by antigen-presenting cells, and avidly activated the TLR5 in vitro as well as the innate and adaptive immune responses. Intranasal immunization of mice with these nanostructures resulted in the potentiation of the antigen-specific antibody response and protection against a lethal infection with the influenza A virus, illustrating the potential of these intrinsically immunostimulatory nanostructures as antigen carriers.

Contribution of the 12-17 hydrophobic region of islet amyloid polypeptide in self-assembly and cytotoxicity.

The islet amyloid polypeptide (IAPP) is a 37-residue aggregation-prone peptide hormone whose deposition as insoluble fibrils in the islets of Langerhans is associated with type II diabetes. Therapeutic interventions targeting IAPP amyloidogenesis, which contributes to pancreatic ß-cell degeneration, remain elusive owing to the lack of understanding of the self-assembly mechanisms and of the quaternary proteospecies mediating toxicity. While countless studies have investigated the contributions of the 20-29 amyloidogenic core in self-assembly, IAPP central region, i.e. positions 11 to 19, has been less studied, notwithstanding its potential key role in oligomerization. In this context, the present study aimed at investigating the physicochemical and conformational properties driving IAPP self-assembly and associated cytotoxicity. Computational tools and all-atom molecular dynamics simulation suggested that the hydrophobic 12-17 segment promotes IAPP self-recognition and aggregation. Alanine scanning revealed that the hydrophobic side chains of Leu12, Phe15 and Val17 are critical for amyloid fibril formation. Destabilization of the α-helical folding by Pro substitution enhanced self-assembly when the pyrrolidine ring was successively introduced at positions Ala13, Asn14 and Phe15, in comparison to respective Ala-substituted counterparts. Modulating the peptide backbone flexibility at position Leu16 through successive incorporation of Pro, Gly and α-methylalanine, inhibited amyloid formation and reduced cytotoxicity, while the isobutyl side chain of Leu16 was not critical for self-assembly and IAPP-mediated toxicity. These results highlight the importance of the 12-17 hydrophobic region of IAPP for self-recognition, ultimately supporting the development of therapeutic approaches to prevent oligomerization and/or fibrillization.

Self-assembled peptide nanorod vaccine confers protection against influenza A virus.

Proteinaceous nanostructures have emerged as a promising strategy to develop safe and efficient subunit vaccines. The ability of synthetic ß-sheet self-assembling peptides to stabilize antigenic determinants and to potentiate the epitope-specific immune responses have highlighted their potential as an immunostimulating platform for antigen delivery. Nonetheless, the intrinsic polymorphism of the resulting cross-ß fibrils, their length in the microscale and their close structural similarity with pathological amyloids could limit their usage in vaccinology. In this study, we harnessed electrostatic capping motifs to control the self-assembly of a chimeric peptide comprising a 10-mer ß-sheet sequence and a highly conserved epitope derived from the influenza A virus (M2e). Self-assembly led to the formation of 100-200 nm long uniform nanorods (NRs) displaying the M2e epitope on their surface. These cross-ß assemblies differed from prototypical amyloid fibrils owing to low polydispersity, short length, non-binding to thioflavin T and Congo Red dyes, and incapacity to seed homologous amyloid assembly. M2e-NRs were efficiently uptaken by antigen presenting cells and the cross-ß quaternary architecture activated the Toll-like receptor 2 and stimulated dendritic cells. Mice subcutaneous immunization revealed a robust M2e-specific IgG response, which was dependent on self-assembly into NRs. Upon intranasal immunization in combination with the polymeric adjuvant montanide gel, M2e-NRs conferred complete protection with absence of clinical signs against a lethal experimental infection with the H1N1 influenza A virus. These findings indicate that by acting as an immunostimulator and delivery system, synthetic peptide-based NRs constitute a versatile self-adjuvanted nanoplatform for the delivery of subunit vaccines.

Protein Supramolecular Structures: From Self-Assembly to Nanovaccine Design.

Life-inspired protein supramolecular assemblies have recently attracted considerable attention for the development of next-generation vaccines to fight against infectious diseases, as well as autoimmune diseases and cancer. Protein self-assembly enables atomic scale precision over the final architecture, with a remarkable diversity of structures and functionalities. Self-assembling protein nanovaccines are associated with numerous advantages, including biocompatibility, stability, molecular specificity and multivalency. Owing to their nanoscale size, proteinaceous nature, symmetrical organization and repetitive antigen display, protein assemblies closely mimic most invading pathogens, serving as danger signals for the immune system. Elucidating how the structural and physicochemical properties of the assemblies modulate the potency and the polarization of the immune responses is critical for bottom-up design of vaccines. In this context, this review briefly covers the fundamentals of supramolecular interactions involved in protein self-assembly and presents the strategies to design and functionalize these assemblies. Examples of advanced nanovaccines are presented, and properties of protein supramolecular structures enabling modulation of the immune responses are discussed. Combining the understanding of the self-assembly process at the molecular level with knowledge regarding the activation of the innate and adaptive immune responses will support the design of safe and effective nanovaccines.

Harnessing the Activation of Toll-Like Receptor 2/6 by Self-Assembled Cross-ß Fibrils to Design Adjuvanted Nanovaccines.

Protein fibrils characterized with a cross-ß-sheet quaternary structure have gained interest as nanomaterials in biomedicine, including in the design of subunit vaccines. Recent studies have shown that by conjugating an antigenic determinant to a self-assembling ß-peptide, the resulting supramolecular assemblies act as an antigen delivery system that potentiates the epitope-specific immune response. In this study, we used a ten-mer self-assembling sequence (I10) derived from an amyloidogenic peptide to biophysically and immunologically characterize a nanofibril-based vaccine against the influenza virus. The highly conserved epitope from the ectodomain of the matrix protein 2 (M2e) was elongated at the N-terminus of I10 by solid phase peptide synthesis. The chimeric M2e-I10 peptide readily self-assembled into unbranched, long, and twisted fibrils with a diameter between five and eight nm. These cross-ß nanoassemblies were cytocompatible and activated the heterodimeric Toll-like receptor (TLR) 2/6. Upon mice subcutaneous immunization, M2e-fibrils triggered a robust anti-M2e specific immune response, which was dependent on self-assembly and did not require the use of an adjuvant. Overall, this study describes the efficacy of cross-ß fibrils to activate the TLR 2/6 and to stimulate the epitope-specific immune response, supporting usage of these proteinaceous assemblies as a self-adjuvanted delivery system for antigens.

Roles of Near and Far Fields in Plasmon-Enhanced Singlet Oxygen Production.

In plasmon-enhanced singlet oxygen (1O2) production, irradiation of a hybrid photosensitizer-metal nanoparticle leads to a significant alteration of the photosensitizer's 1O2 yield. The quest for a more rational design of these nanomaterials calls for a better understanding of the enhancement mechanism that, to this day, remains largely unexplored. Herein, we introduce a new methodology to distinguish the near- and far-field contributions to the plasmon-enhanced 1O2 production using a tunable model nanoplatform, Rose Bengal-decorated silica-coated metal nanoparticles. By correlating 1O2 production to the experimental and simulated optical properties of our nanoparticles, we effectively discriminate how the near- and far-field effects contribute to the plasmonic interactions. We show that these effects work in synergy; i.e., for nanoparticles with a similar local field, the production of 1O2 correlates with maximized scattering yields. Our results expound the critical plasmonic aspects in terms of near and far fields for the design of an efficient hybrid plasmonic nanoparticle photosensitizer.