Jai S. Rudra

A self-assembling peptide acting as an immune adjuvant

The development of vaccines and other immunotherapies has been complicated by heterogeneous antigen display and the use of incompletely defined immune adjuvants with complex mechanisms of action. We have observed strong antibody responses in mice without the coadministration of any additional adjuvant by noncovalently assembling a T and B cell epitope peptide into nanofibers using a short C-terminal peptide extension. Self-assembling peptides have been explored recently as scaffolds for tissue engineering and regenerative medicine, but our results indicate that these materials may also be useful as chemically defined adjuvants. In physiological conditions, these peptides self-assembled into long, unbranched fibrils that displayed the epitope on their surfaces. IgG1, IgG2a, and IgG3 were raised against epitope-bearing fibrils in levels similar to the epitope peptide delivered in complete Freund’s adjuvant (CFA), and IgM production was even greater for the self-assembled epitope. This response was dependent on self-assembly, and the self-assembling sequence was not immunogenic by itself, even when delivered in CFA. Undetectable levels of interferon-gamma, IL-2, and IL-4 in cultures of peptide-challenged splenocytes from immunized mice suggested that the antibody responses did not involve significant T cell help.

Multi-component extracellular matrices based on peptide self-assembly

Extracellular matrices (ECMs) are challenging design targets for materials synthesis because they serve multiple biological roles, and they are composed of multiple molecular constituents. In addition, their composition and activities are dynamic and variable between tissues, and they are difficult to study mechanistically in physiological contexts. Nevertheless, the design of synthetic ECMs is a central consideration in applications such as regenerative medicine and 3D cell culture. In order to produce synthetic matrices having both multi-component construction and high levels of compositional definition, strategies based on molecular self-assembly are receiving increasing interest. These approaches are described in this tutorial review and compared with the structures and processes in native ECMs that serve as their inspiration.

Modulating adaptive immune responses to peptide self-assemblies.

Self-assembling peptides and peptide derivatives have received significant interest for several biomedical applications, including tissue engineering, wound healing, cell delivery, drug delivery, and vaccines. This class of materials has exhibited significant variability in immunogenicity, with many peptides eliciting no detectable antibody responses but others eliciting very strong responses without any supplemental adjuvants. Presently, strategies for either avoiding strong antibody responses or specifically inducing them are not well-developed, even though they are critical for the use of these materials both within tissue engineering and within immunotherapies. Here, we investigated the molecular determinants and immunological mechanisms leading to the significant immunogenicity of the self-assembling peptide OVA-Q11, which has been shown previously to elicit strong antibody responses in mice. We show that these responses can last for at least a year. Using adoptive transfer experiments and T cell knockout models, we found that these strong antibody responses were T cell-dependent, suggesting a route for avoiding or ensuring immunogenicity. Indeed, by deleting amino acid regions in the peptide recognized by T cells, immunogenicity could be significantly diminished. Immunogenicity could also be attenuated by mutating key residues in the self-assembling domain, thus preventing fibrillization. A second self-assembling peptide, KFE8, was also nonimmunogenic, but nanofibers of OVA-KFE8 elicited strong antibody responses similar to OVA-Q11, indicating that the adjuvant action was not dependent on the specific self-assembling peptide sequence. These findings will facilitate the design of self-assembled peptide biomaterials, both for applications where immunogenicity is undesirable and where it is advantageous.

Self-assembled peptide nanofibers raising durable antibody responses against a malaria epitope.

Biomaterials that modulate innate and adaptive immune responses are receiving increasing interest as adjuvants for eliciting protective immunity against a variety of diseases. Previous results have indicated that self-assembling β-sheet peptides, when fused with short peptide epitopes, can act as effective adjuvants and elicit robust and long-lived antibody responses. Here we investigated the mechanism of immunogenicity and the quality of antibody responses raised by a peptide epitope from Plasmodium falciparum circumsporozoite (CS) protein, (NANP)(3),conjugated to the self-assembling peptide domain Q11. The mechanism of adjuvant action was investigated in knockout mice with impaired MyD88, NALP3, TLR-2, or TLR-5 function, and the quality of antibodies raised against (NANP)(3)-Q11 was assessed using a transgenic sporozoite neutralizing (TSN) assay for malaria infection. (NANP)(3)-Q11 self-assembled into nanofibers, and antibody responses lasted up to 40 weeks in C57BL/6 mice. The antibody responses were T cell- and MyD88-dependent. Sera from mice primed with either irradiated sporozoites or a synthetic peptide, (T1BT*)(4)-P3C, and boosted with (NANP)(3)-Q11 showed significant increases in antibody titers and significant inhibition of sporozoite infection in TSN assays. In addition, two different epitopes could be self-assembled together without compromising the strength or duration of the antibody responses raised against either of them, making these materials promising platforms for self-adjuvanting multi-antigenic immunotherapies.

Self-Assembling Peptide-Polymer Hydrogels Designed From the Coiled Coil Region of Fibrin

Biomaterials constructed from self-assembling peptides, peptide derivatives, and peptide-polymer conjugates are receiving increasing attention as defined matrices for tissue engineering, controlled therapeutic release, and in vitro cell expansion, but many are constructed from peptide structures not typically found in the human extracellular matrix. Here we report a self-assembling biomaterial constructed from a designed peptide inspired by the coiled coil domain of human fibrin, the major protein constituent of blood clots and the provisional scaffold of wound healing. Targeted substitutions were made in the residues forming the interface between coiled coil strands for a 37-amino acid peptide from human fibrinogen to stabilize the coiled coil peptide bundle, while the solvent-exposed residues were left unchanged to provide a surface similar to that of the native protein. This peptide, which self-assembled into coiled coil dimers and tetramers, was then used to produce triblock peptide-PEG-peptide bioconjugates that self-assembled into viscoelastic hydrogel biomaterials.

Antimicrobial polypeptide multilayer nanocoatings

A multilayer coating (or film) of nanometer-thick layers can be made by sequential adsorption of oppositely charged polyelectrolytes on a solid support. The method is known as layer-by-layer assembly (LBL). No special apparatus is required for LBL and nanofilms can be prepared under mild, physiological conditions. A multilayer nanofilm in which at least one of the constituent species is a polypeptide is a polypeptide multilayer nanofilm. The present work was aimed at assessing whether polypeptide multilayer nanofilms with specific antimicrobial properties could be prepared by incorporation of a known antimicrobial agent in the film structure, in this case the edible protein hen egg white lysozyme (HEWL). The chicken enzyme is widely employed as a human food preservative. An advantage of LBL in this context is that the nanofilm is fabricated directly on the surface of interest, eliminating the need to incorporate the antimicrobial in other packaging materials. Here, nanofilms were made of poly(L-glutamic acid) (PLGA), which is highly negatively charged in the mildly acidic pH range, and HEWL, which has a high net positive charge at acidic pH. We show that PLGA/HEWL nanofilms inhibit growth of the model microbe Microccocus luteus in the surrounding liquid medium. The amount of HEWL released from PLGA/HEWL films depends on the number of HEWL layers and therefore on the total quantity of HEWL in the films. This initial study provides a sketch of the scope for further development of LBL in the area of antimicrobial polypeptide multilayer films. Potential applications of such films include strategies for food preservation and coatings for implant devices.

Antigenic peptide nanofibers elicit adjuvant-free CD8 + T cell responses

Vaccines that elicit robust CD8⁺ T cell responses are desirable for protection against infectious diseases and cancers. However, most vaccine adjuvants fail to elicit robust CD8⁺ T cell responses without inflammation and associated toxicity. We recently reported that self-assembling peptides that form nanofibers in physiological buffers elicited strong adjuvant-free and antigen-specific antibody responses in mice. However, whether or not such nanofibers likewise can elicit strong CD8⁺ T cell responses is unknown. Here, we demonstrate that the self-assembling peptide Q11 conjugated to a CD8⁺ T cell epitope of ovalbumin (Q11-OVA), elicits strong antigen-specific primary and recall responses, and in a vaccination regimen protects against subsequent infection. Importantly, we show that these antigenic peptide nanofibers do not persist as an inflammatory antigen depot at the injection site. Our results demonstrate for the first time that self-assembling peptides may be useful as carriers for vaccines where CD8⁺ T cell-mediated protection is needed.

A Self-Adjuvanting Supramolecular Vaccine Carrying a Folded Protein Antigen

This work illustrates a strategy for the design of molecularly defined immunotherapies, using a blend of supramolecular peptide self-assembly and active site-directed protein capture.

Suppression of Cocaine-Evoked Hyperactivity by Self-Adjuvanting and Multivalent Peptide Nanofiber Vaccines

The development of anti-cocaine vaccines that counteract the rewarding effects of the drug are currently being investigated as adjunct therapies for prevention of relapse in abstinent users. However, cocaine is weakly immunogenic and requires conjugation to carrier proteins and coadministration with strong adjuvants, which carry the risk of local reactogenicity and systemic toxicity. Here we report synthetic and multivalent self-assembling peptide nanofibers as adjuvant-free carriers for cocaine vaccines. A novel cocaine hapten modified at the P3 site was conjugated to the N-terminus of an amphipathic self-assembling domain KFE8. In aqueous buffers the cocaine-KFE8 conjugate assembled into β-sheet rich nanofibers, which raised anti-cocaine antibodies without the need for added adjuvants in mice. Vaccinated mice were treated with cocaine and a significant negative correlation was observed between antibody levels and cocaine-evoked hyperactivity. These totally synthetic and multivalent nanofibers with well-defined chemical composition represent the first generation of adjuvant-free cocaine vaccines.

Quantitative Proteomics Reveals a Role for Epigenetic Reprogramming During Human Monocyte Differentiation

The differentiation of monocytes into macrophages and dendritic cells involves mechanisms for activation of the innate immune system in response to inflammatory stimuli, such as pathogen infection and environmental cues. Epigenetic reprogramming is thought to play an important role during monocyte differentiation. Complementary to cell surface markers, the characterization of monocytic cell lineages by mass spectrometry based protein/histone expression profiling opens a new avenue for studying immune cell differentiation. Here, we report the application of mass spectrometry and bioinformatics to identify changes in human monocytes during their differentiation into macrophages and dendritic cells. Our data show that linker histone H1 proteins are significantly down-regulated during monocyte differentiation. Although highly enriched H3K9-methyl/S10-phos/K14-acetyl tri-modification forms of histone H3 were identified in monocytes and macrophages, they were dramatically reduced in dendritic cells. In contrast, histone H4 K16 acetylation was found to be markedly higher in dendritic cells than in monocytes and macrophages. We also found that global hyperacetylation generated by the nonspecific histone deacetylase HDAC inhibitor Apicidin induces monocyte differentiation. Together, our data suggest that specific regulation of inter- and intra-histone modifications including H3 K9 methylation, H3 S10 phosphorylation, H3 K14 acetylation, and H4 K16 acetylation must occur in concert with chromatin remodeling by linker histones for cell cycle progression and differentiation of human myeloid cells into macrophages and dendritic cells.