To be optimally effective, peptide-based vaccines need to be administered with adjuvants. Many currently available adjuvants are toxic, not biodegradable; they invariably invoke adverse reactions, including allergic responses and excessive inflammation. A nontoxic, biodegradable, biocompatible, self-adjuvanting vaccine delivery system is urgently needed. Herein, we report a potent vaccine delivery system fulfilling the above requirements. A peptide antigen was coupled with poly-hydrophobic amino acid sequences serving as self-adjuvanting moieties using solid-phase synthesis, to produce fully defined single molecular entities. Under aqueous conditions, these molecules self-assembled into distinct nanoparticles and chain-like aggregates. Following subcutaneous immunization in mice, these particles successfully induced opsonic epitope-specific antibodies without the need of external adjuvant. Mice immunized with entities bearing 15 leucine residues were able to clear bacterial load from target organs without triggering the release of soluble inflammatory mediators. Thus, we have developed a well-defined and effective self-adjuvanting delivery system for peptide antigens.

Please cite this article as: Zhao, G., Chandrudu, S., Skwarczynski, M., Toth, I., The application of self-assembled nanostructures in peptide-based subunit vaccine development, European Polymer Journal (2017), doi: http:// dx.doi.org/10. 1016/j.eurpolymj.2017.02.014 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 1The application of self-assembled nanostructures in

Necator americanus (hookworm) infects over half a billion people worldwide. Anthelminthic drugs are commonly used to treat the infection; however, vaccination is a more favorable strategy to combat this parasite. We designed new B‐cell peptide epitopes based on the aspartic protease of N. americanus (Na‐APR‐1). The peptides were conjugated to self‐adjuvanting lipid core peptide (LCP) systems via stepwise solid‐phase peptide synthesis (SPPS) and copper catalyst azide–alkyne cycloaddition (CuAAC) reactions. The LCP vaccine candidates were able to self‐assemble into nanoparticles, were administered to mice without the use of additional adjuvant, and generated antibodies that recognized the parent epitope. However, only one LCP derivative was able to produce a high titer of antibodies specific to Na‐APR‐1; circular dichroism analyses of this compound showed a β‐sheet conformation for the incorporated epitope. This study provides important insight in epitope and delivery system design for the development of a vaccine against hookworm infections.

Hybanthus enneaspermus is an Indian folk medicinal herb that has been widely used as a libido enhancer. This plant belongs to the Violaceae plant family, which ubiquitously contains disulfide-rich cyclic peptides named cyclotides. Cyclotides are an expanding plant-derived peptide family with numerous interesting bioactivities, and their unusual stability against proteolysis has attracted much attention in drug design applications. Recently, H. enneaspermus has been reported to be a rich source of cyclotides, and hence, it was of interest to investigate whether cyclotides contribute to its aphrodisiac activity. In this study, we evaluated the in vivo aphrodisiac activity of the herbal powder, extract, and the most abundant cyclotide, hyen D, extracted from H. enneaspermus on rats in a single dose regimen. After dosing, the sexual behaviors of male rats were observed, recorded, analyzed, and compared with those of the vehicle group. The results show that the extract and hyen D significantly decreased the intromission latency of sexually naïve male rats and the extract improved a range of other measured sexual parameters. The results suggest that the extract could enhance libido as well as facilitate erectile function in male rats and that the cyclotide hyen D could contribute to the libido-enhancing activity of this ethnomedicinal herb.

Most vaccines require co-delivery of an adjuvant in order to generate the desired immune responses. However, many currently available adjuvants are non-biodegradable, have limited efficacy, and/or poor safety profile. Thus, new adjuvants, or self-adjuvanting vaccine delivery systems, are required. Here, we proposed a self-adjuvanting delivery system that is fully defined, biodegradable, and non-toxic. The system is produced by conjugation of polyleucine to peptide antigen, followed by selfassembly of the conjugate into nanoparticles. The protocol includes solid-phase peptide synthesis of the vaccine conjugate, purification, self-assembly and physicochemical characterization of the product.Overall, this protocol describes, in detail, the production of a well-defined and effective self-adjuvanting delivery system for peptide antigens, along with tips for troubleshooting.

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A decline in the prevalence of parasites such as hookworms appears to be correlated with the rise in non-communicable inflammatory conditions in people from high-and middle-income countries. This correlation has led to studies that have identified proteins produced by hookworms that can suppress inflammatory bowel disease (IBD) and asthma in animal models. Hookworms secrete a family of abundant netrin-domain containing proteins referred to as AIPs (Anti-Inflammatory Proteins), but there is no information on the structure-function relationships. Here we have applied a downsizing approach to the hookworm AIPs to derive peptides of 20 residues or less, some of which display anti-inflammatory effects when co-cultured with human peripheral blood mononuclear cells and oral therapeutic activity in a chemically induced mouse model of acute colitis. Our results indicate that a conserved helical region is responsible, at least in part, for the antiinflammatory effects. This helical region has potential in the design of improved leads for treating IBD and possibly other inflammatory conditions.

The back cover picture shows the action of a vaccine candidate against hookworm. B‐cell peptide epitopes derived from hookworm protease Na‐APR‐1 were incorporated into lipid core peptides (structure shown on the right). Proper epitope conformation (β‐sheet and not α‐helix) was found to be crucial for pathogen protein recognition by the antibodies produced. More details can be found in the Full Paper by Mariusz Skwarczynski et al. on page 1647 in Issue 10, 2015. (DOI: ).