Resolving the Nanoscale Structure of ß-Sheet Peptide Self-Assemblies Using Single-Molecule Orientation-Localization Microscopy.

Synthetic peptides that self-assemble into cross-ß fibrils are versatile building blocks for engineered biomaterials due to their modularity and biocompatibility, but their structural and morphological similarities to amyloid species have been a long-standing concern for their translation. Further, their polymorphs are difficult to characterize by using spectroscopic and imaging techniques that rely on ensemble averaging to achieve high resolution. Here, we utilize Nile red (NR), an amyloidophilic fluorogenic probe, and single-molecule orientation-localization microscopy (SMOLM) to characterize fibrils formed by the designed amphipathic enantiomers KFE8L and KFE8D and the pathological amyloid-beta peptide Aß42. Importantly, NR SMOLM reveals the helical (bilayer) ribbon structure of both KFE8 and Aß42 and quantifies the precise tilt of the fibrils' inner and outer backbones in relevant buffer conditions without the need for covalent labeling or sequence mutations. SMOLM also distinguishes polymorphic branched and curved morphologies of KFE8, whose backbones exhibit much more heterogeneity than those of typical straight fibrils. Thus, SMOLM is a powerful tool to interrogate the structural differences and polymorphism between engineered and pathological cross-ß-rich fibrils.

Resolving the nanoscale structure of ß-sheet assemblies using single-molecule orientation-localization microscopy.

Synthetic peptides that self-assemble into cross-ß fibrils have remarkable utility as engineered biomaterials due to their modularity and biocompatibility, but their structural and morphological similarity to amyloid species has been a long-standing concern for their translation. Further, their polymorphs are difficult to characterize using spectroscopic and imaging techniques that rely on ensemble averaging to achieve high resolution. Here, we utilize single-molecule orientation-localization microscopy (SMOLM) to characterize fibrils formed by the designed amphipathic enantiomers, KFE8L and KFE8D, and the pathological amyloid-beta peptide Aß42. SMOLM reveals that the orientations of Nile red, as it transiently binds to both KFE8 and Aß42, are consistent with a helical (bilayer) ribbon structure and convey the precise tilt of the fibrils' inner and outer backbones. SMOLM also finds polymorphic branched and curved morphologies of KFE8 whose backbones exhibit much more heterogeneity than those of more typical straight fibrils. Thus, SMOLM is a powerful tool to interrogate the structural differences and polymorphism between engineered and pathological cross ß-rich fibrils.

Amyloidogenic propensity of self-assembling peptides and their adjuvant potential for use as DNA vaccines.

De novo designed peptides that self-assemble into cross-ß rich fibrillar biomaterials have been pursued as an innovative platform for the development of adjuvant- and inflammation-free vaccines. However, they share structural and morphological properties similar to amyloid species implicated in neurodegenerative diseases, which has been a long-standing concern for their successful translation. Here, we comprehensively characterize the amyloidogenic character of the amphipathic self-assembling cross-ß peptide KFE8, compared to pathological amyloid and amyloid-like proteins α-synuclein (α-syn) and TDP-43. Further, we developed plasmid-based DNA vaccines with the KFE8 backbone serving as a scaffold for delivery of a GFP model antigen. We find that expression of tandem repeats of KFE8 is non-toxic and efficiently cleared by autophagy. We also demonstrate that preformed KFE8 fibrils do not cross-seed amyloid formation of α-syn in mammalian cells compared to α-syn preformed fibrils. In mice, vaccination with plasmids encoding the KFE32-GFP fusion protein elicited robust immune responses, inducing production of significantly higher levels of anti-GFP antibodies compared to soluble GFP. Antigen-specific CD8+T cells were also detected in the spleens of vaccinated mice and cytokine profiles from antigen recall assays indicate a balanced Th1/Th2 response. These findings illustrate that cross-ß-rich peptide nanofibers have distinct physicochemical properties from those of pathological amyloidogenic proteins, and are an attractive platform for the development of DNA vaccines with self-adjuvanting properties and improved safety profiles. STATEMENT OF SIGNIFICANCE: Biomaterials comprised of self-assembling peptides hold great promise for the development of new vaccines that do not require use of adjuvants. However, these materials have safety concerns, as they self-assemble into cross-ß rich fibrils that are structurally similar to amyloid species implicated in disease. Here, we comprehensively study the properties of these biomaterials. We demonstrate that they have distinct properties from pathological proteins. They are non-toxic and do not trigger amyloidogenesis. Vaccination of these materials in mice elicited a robust immune response. Most excitingly, our work suggests that this platform could be used to develop DNA-based vaccines, which have few storage requirements. Further, due to their genetic encoding, longer sequences can be generated and the vaccines will be amenable to modification.

Nanomaterials-based vaccines to target intracellular bacterial pathogens.

Development of novel immunization approaches to combat a growing list of emerging and ancient infectious agents is a global health priority. Intensive efforts over the last several decades have identified alternative approaches to improve upon traditional vaccines that are based on live, attenuated agents, or formulations of inactivated agents with adjuvants. Rapid advances in RNA-based and other delivery systems for immunization have recently revolutionized the potential to protect populations from viral pathogens, such as SARS-CoV-2. Similar efforts to combat bacterial pathogens, especially species with an intracellular niche, have lagged significantly. In the past decade, advances in nanotechnology have yielded a variety of new antigen/adjuvant carrier systems for use in vaccine development against infectious viruses and bacteria. The tunable properties of nanomaterial-based vaccines allow for balancing immunogenicity and safety which is a key hurdle in traditional antigen and adjuvant formulations. In this review, we discuss several novel nanoparticle-based vaccine platforms that show promise for use against intracellular bacteria as demonstrated by the feasibility of construction, enhanced antigen presentation, induction of cell mediated and humoral immune responses, and improved survival outcomes in in vivo models.

Self-adjuvanting nanovaccines boost lung-resident CD4+ T cell immune responses in BCG-primed mice.

Heterologous vaccine regimens could extend waning protection in the global population immunized with Mycobacterium bovis Bacille Calmette-Guerin (BCG). We demonstrate that pulmonary delivery of peptide nanofibers (PNFs) bearing an Ag85B CD4+ T cell epitope increased the frequency of antigen-specific T cells in BCG-primed mice, including heterogenous populations with tissue resident memory (Trm) and effector memory (Tem) phenotype, and functional cytokine recall. Adoptive transfer of dendritic cells pulsed with Ag85B-bearing PNFs further expanded the frequency and functional repertoire of memory CD4+ T cells. Transcriptomic analysis suggested that the adjuvanticity of peptide nanofibers is, in part, due to the release of damage-associated molecular patterns. A single boost with monovalent Ag85B PNF in BCG-primed mice did not reduce lung bacterial burden compared to BCG alone following aerosol Mtb challenge. These findings support the need for novel BCG booster strategies that activate pools of Trm cells with potentially diverse localization, trafficking, and immune function.

Coiled Coil Crosslinked Alginate Hydrogels Dampen Macrophage-Driven Inflammation.

Alginate hydrogels are widely used for tissue engineering and regenerative medicine due to their excellent biocompatibility. A facile and commonly used strategy to crosslink alginate is the addition of Ca2+ that leads to hydrogelation. However, extracellular Ca2+ is a secondary messenger in activating inflammasome pathways following physical injury or pathogenic insult, which carries the risk of persistent inflammation and scaffold rejection. Here, we present graft copolymers of charge complementary heterodimeric coiled coil (CC) peptides and alginate that undergo supramolecular self-assembly to form Ca2+ free alginate hydrogels. The formation of heterodimeric CCs was confirmed using circular dichroism spectroscopy, and scanning electron microscopy revealed a significant difference in crosslink density and homogeneity between Ca2+ and CC crosslinked gels. The resulting hydrogels were self-supporting and display shear-thinning and shear-recovery properties. In response to lipopolysaccharide (LPS) stimulation, peritoneal macrophages and bone marrow-derived dendritic cells cultured in the CC crosslinked gels exhibited a 10-fold reduction in secretion of the proinflammatory cytokine IL-1ß compared to Ca2+ crosslinked gels. A similar response was also observed in vivo upon peritoneal delivery of Ca2+ or CC crosslinked gels. Analysis of peritoneal lavage showed that macrophages in mice injected with Ca2+ crosslinked gels display a more inflammatory phenotype compared to macrophages from mice injected with CC crosslinked gels. These results suggest that CC peptides by virtue of their tunable sequence-structure-function relationship and mild gelation conditions are promising alternative crosslinkers for alginate and other biopolymer scaffolds used in tissue engineering.

Peptide-based supramolecular vaccine systems.

Currently approved replication-competent and inactivated vaccines are limited by excessive reactogenicity and poor safety profiles, while subunit vaccines are often insufficiently immunogenic without co-administering exogenous adjuvants. Self-assembling peptide-, peptidomimetic-, and protein-based biomaterials offer a means to overcome these challenges through their inherent modularity, multivalency, and biocompatibility. As these scaffolds are biologically derived and present antigenic arrays reminiscent of natural viruses, they are prone to immune recognition and are uniquely capable of functioning as self-adjuvanting vaccine delivery vehicles that improve humoral and cellular responses. Beyond this intrinsic immunological advantage, the wide range of available amino acids allows for facile de novo design or straightforward modifications to existing sequences. This has permitted the development of vaccines and immunotherapies tailored to specific disease models, as well as generalizable platforms that have been successfully applied to prevent or treat numerous infectious and non-infectious diseases. In this review, we briefly introduce the immune system, discuss the structural determinants of coiled coils, ß-sheets, peptide amphiphiles, and protein subunit nanoparticles, and highlight the utility of these materials using notable examples of their innate and adaptive immunomodulatory capacity. STATEMENT OF SIGNIFICANCE: Subunit vaccines have recently gained considerable attention due to their favorable safety profiles relative to traditional whole-cell vaccines; however, their reduced efficacy requires co-administration of reactogenic adjuvants to boost immune responses. This has led to collaborative efforts between engineers and immunologists to develop nanomaterial-based vaccination platforms that can elicit protection without deleterious side effects. Self-assembling peptidic biomaterials are a particularly attractive approach to this problem, as their structure and function can be controlled through primary sequence design and their capacity for multivalent presentation of antigens grants them intrinsic self-adjuvanticity. This review introduces the various architectures adopted by self-assembling peptides and discusses their application as modulators of innate and adaptive immunity.

Microneedle patch for the ultrasensitive quantification of protein biomarkers in interstitial fluid.

The detection and quantification of protein biomarkers in interstitial fluid is hampered by challenges in its sampling and analysis. Here we report the use of a microneedle patch for fast in vivo sampling and on-needle quantification of target protein biomarkers in interstitial fluid. We used plasmonic fluor-an ultrabright fluorescent label-to improve the limit of detection of various interstitial fluid protein biomarkers by nearly 800-fold compared with conventional fluorophores, and a magnetic backing layer to implement conventional immunoassay procedures on the patch and thus improve measurement consistency. We used the microneedle patch in mice for minimally invasive evaluation of the efficiency of a cocaine vaccine, for longitudinal monitoring of the levels of inflammatory biomarkers, and for efficient sampling of the calvarial periosteum-a challenging site for biomarker detection-and the quantification of its levels of the matricellular protein periostin, which cannot be accurately inferred from blood or other systemic biofluids. Microneedle patches for the minimally invasive collection and analysis of biomarkers in interstitial fluid might facilitate point-of-care diagnostics and longitudinal monitoring.

Small Animal Model of Post-chemotherapy Tuberculosis Relapse in the Setting of HIV Co-infection.

Tuberculosis relapse following drug treatment of active disease is an important global public health problem due to the poorer clinical outcomes and increased risk of drug resistance development. Concurrent infection with HIV, including in those receiving anti-retroviral therapy (ART), is an important risk factor for relapse and expansion of drug resistant Mycobacterium tuberculosis (Mtb) isolates. A greater understanding of the HIV-associated factors driving TB relapse is important for development of interventions that support immune containment and complement drug therapy. We employed the humanized mouse to develop a new model of post-chemotherapy TB relapse in the setting of HIV infection. Paucibacillary TB infection was observed following treatment with Rifampin and Isoniazid and subsequent infection with HIV-1 was associated with increased Mtb burden in the post-drug phase. Organized granulomas were observed during development of acute TB and appeared to resolve following TB drug therapy. At relapse, granulomatous pathology in the lung was infrequent and mycobacteria were most often observed in the interstitium and at sites of diffuse inflammation. Compared to animals with HIV mono-infection, higher viral replication was observed in the lung and liver, but not in the periphery, of animals with post-drug TB relapse. The results demonstrate a potential role for the humanized mouse as an experimental model of TB relapse in the setting of HIV. Long term, the model could facilitate discovery of disease mechanisms and development of clinical interventions.

Self-Assembly of Block Heterochiral Peptides into Helical Tapes.

Patterned substitution of d-amino acids into the primary sequences of self-assembling peptides influences molecular-level packing and supramolecular morphology. We report that block heterochiral analogs of the model amphipathic peptide KFE8 (Ac-FKFEFKFE-NH2), composed of two FKFE repeat motifs with opposite chirality, assemble into helical tapes with dimensions greatly exceeding those of their fibrillar homochiral counterparts. At sufficient concentrations, these tapes form hydrogels with reduced storage moduli but retain the shear-thinning behavior and consistent mechanical recovery of the homochiral analogs. Varying the identity of charged residues (FRFEFRFE and FRFDFRFD) produced similarly sized nonhelical tapes, while a peptide with nonenantiomeric l- and d-blocks (FKFEFRFD) formed helical tapes closely resembling those of the heterochiral KFE8 analogs. A proposed energy-minimized model suggests that a kink at the interface between l- and d-blocks leads to the assembly of flat monolayers with nonidentical surfaces that display alternating stacks of hydrophobic and charged groups.

Intratumoral injection of the seasonal flu shot converts immunologically cold tumors to hot and serves as an immunotherapy for cancer.

Reprogramming the tumor microenvironment to increase immune-mediated responses is currently of intense interest. Patients with immune-infiltrated "hot" tumors demonstrate higher treatment response rates and improved survival. However, only the minority of tumors are hot, and a limited proportion of patients benefit from immunotherapies. Innovative approaches that make tumors hot can have immediate impact particularly if they repurpose drugs with additional cancer-unrelated benefits. The seasonal influenza vaccine is recommended for all persons over 6 mo without prohibitive contraindications, including most cancer patients. Here, we report that unadjuvanted seasonal influenza vaccination via intratumoral, but not intramuscular, injection converts "cold" tumors to hot, generates systemic CD8+ T cell-mediated antitumor immunity, and sensitizes resistant tumors to checkpoint blockade. Importantly, intratumoral vaccination also provides protection against subsequent active influenza virus lung infection. Surprisingly, a squalene-based adjuvanted vaccine maintains intratumoral regulatory B cells and fails to improve antitumor responses, even while protecting against active influenza virus lung infection. Adjuvant removal, B cell depletion, or IL-10 blockade recovers its antitumor effectiveness. Our findings propose that antipathogen vaccines may be utilized for both infection prevention and repurposing as a cancer immunotherapy.

Modulating Mechanical Properties of Collagen-Lignin Composites.

Three-dimensional matrices of collagen type I (Col I) are widely used in tissue engineering applications for its abundance in many tissues, bioactivity with many cell types, and excellent biocompatibility. Inspired by the structural role of lignin in a plant tissue, we found that sodium lignosulfonate (SLS) and an alkali-extracted lignin from switchgrass (SG) increased the stiffness of Col I gels. SLS and SG enhanced the stiffness of Col I gels from 52 to 670 Pa and 52 to 320 Pa, respectively, and attenuated shear-thinning properties, with the formulation of 1.8 mg/mL Col I and 5.0 mg/mL SLS or SG. In 2D cultures, the cytotoxicity of collagen-SLS to adipose-derived stromal cells was not observed and the cell viability was maintained over 7 days in 3D cultures. Collagen-SLS composites did not elicit immunogenicity when compared to SLS-only groups. Our collagen-SLS composites present a case that exploits lignins as an enhancer of mechanical properties of Col I without adverse cytotoxicity and immunogenicity for in vitro scaffolds or in vivo tissue repairs.

Nanoscale Peptide Self-assemblies Boost BCG-primed Cellular Immunity Against Mycobacterium tuberculosis.

Bacillus Calmette-Guerin (BCG) is the only vaccine against TB and has limited protection efficacy, which wanes past adolescence. Multifunctional CD8+ T cells (IFN-γ+/TNF-α+/IL-2+) are associated with lower reactivation risk and enhanced control of active Mtb infection. Since boosting with BCG is contraindicated, booster vaccines that augment T cell immunity in the lungs of BCG-vaccinated individuals are urgently needed. We developed a vaccination strategy based on self-assembling peptide nanofibers presenting Mtb-specific CD8+ or CD4+ T cell epitopes that induce high frequency and antigen-specific effector memory T cells producing IFN-γ and IL-2. Intranasal immunization with peptide nanofibers was well tolerated in mice leading to increased antigen-specific CD8+ T cell population in the lungs. Co-assembled nanofibers of CD8+ T cell epitopes and toll-like receptor 2 (TLR2) agonists induced a 8-fold expansion in multifunctional CD8+ T cell populations in the lungs of vaccinated mice. Aerosol challenge with Mtb in BCG-primed and nanofiber-boosted mice provided an additional 0.5-log CFU reduction in lung bacterial load and indicating enhanced protection compared to BCG alone. Together, these data suggest that heterologous prime-boost with BCG and peptide nanofiber vaccines induces cell mediated immunity in the lung, reduces bacterial burden, and is a potentially safer alternative for boosting BCG-primed immunity.

Microwave-Assisted Synthesis and Immunological Evaluation of Self-Assembling Peptide Vaccines.

Self-assembling peptides spontaneously associate into functional supramolecular scaffolds, which have found numerous biomedical applications. These molecular assemblies have applications in nerve regeneration, wound healing, and both prophylactic and therapeutic vaccination. They can also be useful tools for proliferation assays, sustained culture of difficult cell lines, or activation of cell lines for immunoassays. This protocol will describe the basic peptide synthesis and purification of model self-assembling peptide immunogen and methods for vaccinating mice, collecting lymph nodes, and stimulating cells ex vivo.

Environmental Enrichment and Social Isolation Mediate Neuroplasticity of Medium Spiny Neurons through the GSK3 Pathway.

Resilience and vulnerability to neuropsychiatric disorders are linked to molecular changes underlying excitability that are still poorly understood. Here, we identify glycogen-synthase kinase 3ß (GSK3ß) and voltage-gated Na+ channel Nav1.6 as regulators of neuroplasticity induced by environmentally enriched (EC) or isolated (IC) conditions-models for resilience and vulnerability. Transcriptomic studies in the nucleus accumbens from EC and IC rats predicted low levels of GSK3ß and SCN8A mRNA as a protective phenotype associated with reduced excitability in medium spiny neurons (MSNs). In vivo genetic manipulations demonstrate that GSK3ß and Nav1.6 are molecular determinants of MSN excitability and that silencing of GSK3ß prevents maladaptive plasticity of IC MSNs. In vitro studies reveal direct interaction of GSK3ß with Nav1.6 and phosphorylation at Nav1.6T1936 by GSK3ß. A GSK3ß-Nav1.6T1936 competing peptide reduces MSNs excitability in IC, but not EC rats. These results identify GSK3ß regulation of Nav1.6 as a biosignature of MSNs maladaptive plasticity.

A combined carrier-adjuvant system of peptide nanofibers and toll-like receptor agonists potentiates robust CD8+ T cell responses.

Improving CD8+ T cell responses activated by subunit vaccination is crucial for improving vaccine efficacy and safety. Here we report a carrier-adjuvant system composed of self-assembling peptide nanofibers presenting an immunodominant antigen from herpes simplex virus (HSV) and toll-like receptor (TLR) agonists that induces robust effector and memory CD8+ T cell responses in mice. The effector function of vaccine-induced CD8+ T cells was influenced by the type of TLR agonist. The use of CpG (TLR9 agonist) resulted in significantly greater specific in vivo cytotoxicity and trended towards more cells producing both IFN-γ and TNF-α compared to gardiquimod (TLR7 agonist). Prime-boost immunization with peptide nanofibers combined with either adjuvant resulted in development of HSV-specific CD8+ memory T cells further demonstrating the capability of the carrier-adjuvant system to induce strong HSV-specific CD8+ T cell responses. Inclusion of peptide epitope-nanofibers in protein-based subunit vaccines should increase the functional spectrum of the vaccine-elicited immune response and protection.

Biomaterials-Based Vaccination Strategies for the Induction of CD8+T Cell Responses.

Natural and synthetic biomaterials are increasingly being used for the development of vaccines and immunotherapies as alternatives to traditional live-attenuated formulations due to their improved safety profiles and no risk of reversion to virulence. Polymeric materials in particular enjoy attention due to the ease of fabrication, control over physicochemical properties, and their wide range of immunogenicity. While the majority of studies focus on inducing protective antibody responses, in recent years, materials-based strategies for the delivery of antigens and immunomodulators to improve CD8+T cell immunity against infectious and non-infectious diseases have gained momentum. Notably, platforms based on polymeric nanoparticles, liposomes, micelles, virus-like particles, self-assembling peptides and peptidomimetics, and multilayer thin films show considerable promise in preclinical studies. In this Review, we first introduce the concepts of CD8+T cell activation, effector and memory functions, and cytotoxic activity, followed by vaccine design for eliciting robust and protective long-lived CD8+T cell immunity. We then discuss different materials-based vaccines developed in the past decade to elicit CD8+T cell responses based on molecular composition or fabrication methods and conclude with a summary and glimpse at the future trends in this area.

Supramolecular Peptide Nanofibers Engage Mechanisms of Autophagy in Antigen-Presenting Cells.

Supramolecular peptide nanofibers are attractive for applications in vaccine development due to their ability to induce strong immune responses without added adjuvants or associated inflammation. Here, we report that self-assembling peptide nanofibers bearing CD4+ or CD8+ T cell epitopes are processed through mechanisms of autophagy in antigen-presenting cells (APCs). Using standard in vitro antigen presentation assays, we confirmed loss and gain of the adjuvant function using pharmacological modulators of autophagy and APCs deficient in multiple autophagy proteins. The incorporation of microtubule-associated protein 1A/1B-light chain-3 (LC3-II) into the autophagosomal membrane, a key biological marker for autophagy, was confirmed using microscopy. Our findings indicate that autophagy in APCs plays an essential role in the mechanism of adjuvant action of supramolecular peptide nanofibers.

Supramolecular peptide hydrogel adjuvanted subunit vaccine elicits protective antibody responses against West Nile virus.

A crucial issue in vaccine development is to balance safety with immunogenicity. The low immunogenicity of most subunit antigens warrants a search for adjuvants able to stimulate both cell-mediated and humoral immunity. In recent years, successful applications of nanotechnology and bioengineering in the field of vaccine development have enabled the production of novel adjuvant technologies. In this work, we investigated totally synthetic and supramolecular peptide hydrogels as novel vaccine adjuvants in conjunction with the immunoprotective envelope protein domain III (EIII) of West Nile virus as an immunogen in a mouse model. Our results indicate that, compared to the clinically approved adjuvant alum, peptide hydrogel adjuvanted antigen elicited stronger antibody responses and conferred significant protection against mortality after virus challenge. The high chemical definition and biocompatibility of self-assembling peptide hydrogels makes them attractive as immune adjuvants for the production of subunit vaccines against viral and bacterial infections where antibody-mediated protection is desirable.