Rachel J. Stephenson

Recent advances in self-assembled peptides: Implications for targeted drug delivery and vaccine engineering

Abstract Self‐assembled peptides have shown outstanding characteristics for vaccine delivery and drug targeting. Peptide molecules can be rationally designed to self‐assemble into specific nanoarchitectures in response to changes in their assembly environment including: pH, temperature, ionic strength, and interactions between host (drug) and guest molecules. The resulting supramolecular nanostructures include nanovesicles, nanofibers, nanotubes, nanoribbons, and hydrogels and have a diverse range of mechanical and physicochemical properties. These molecules can be designed for cell‐specific targeting by including adhesion ligands, receptor recognition ligands, or peptide‐based antigens in their design, often in a multivalent display. Depending on their design, self‐assembled peptide nanostructures have advantages in biocompatibility, stability against enzymatic degradation, encapsulation of hydrophobic drugs, sustained drug release, shear‐thinning viscoelastic properties, and/or adjuvanting properties. These molecules can also act as intracellular transporters and respond to changes in the physiological environment. Furthermore, this class of materials has shown sequence‐ and structure‐dependent impacts on the immune system that can be tailored to non‐immunogenic for drug targeting, and immunogenic for vaccine delivery. This review explores self‐assembled peptide nanostructures (beta sheets, alpha helices, peptide amphiphiles, amino acid pairing, elastin like polypeptides, cyclic peptides, short peptides, Fmoc peptides, and peptide hydrogels) and their application in vaccine delivery and drug targeting. Graphical abstract Figure. No Caption available.

Schistosome Vaccine Adjuvants in Preclinical and Clinical Research

There is currently no vaccine available for human use for any parasitic infections, including the helminth disease, schistosomiasis. Despite many researchers working towards this goal, one of the focuses has been on identifying new antigenic targets. The bar to achieve protective efficacy in humans was set at a consistent induction of 40% protection or better by the World Health Organisation (WHO), and although this is a modest goal, it is yet to be reached with the six most promising schistosomiasis vaccine candidates (Sm28GST, IrV5, Sm14, paramyosin, TPI, and Sm23). Adjuvant selection has a large impact on the effectiveness of the vaccine, and the use of adjuvants to aid in the stimulation of the immune system is a critical step and a major variable affecting vaccine development. In addition to a comprehensive understanding of the immune system, level of protection and the desired immune response required, there is also a need for a standardised and effective adjuvant formulation. This review summarises the status of adjuvants that have been or are being employed in schistosomiasis vaccine development focusing on immunisation outcomes at preclinical and clinical stages.

Revisiting glucose uptake and metabolism in schistosomes: new molecular insights for improved schistosomiasis therapies

A better understanding of the molecular mechanisms required for schistosomes to take up glucose, the major nutritional source exploited by these blood flukes from their mammalian hosts and the subsequent metabolism required to fuel growth and fecundity, can provide new avenues for developing novel interventions for the control of schistosomiasis. This aspect of parasitism is particularly important to paired adult schistosomes, due to their considerable requirements for the energy needed to produce the extensive numbers of eggs laid daily by the female worm. This review describes recent advances in characterizing glucose metabolism in adult schistosomes. Potential intervention targets are discussed within the insulin signaling and glycolysis pathways, both of which play critical roles in the carbohydrate and energy requirements of schistosomes.

Targeting the Mannose Receptor with Mannosylated Subunit Vaccines

The mannose receptor (MR) is an important component of the immune system and understanding the structural and conformational characteristics of this receptor is a key aspect of vaccine design. Improved understanding of the role of carbohydrate recognition domains 4-7 (CRDs 4-7) in recognising glycosylated ligands present on the surface of pathogens such as C.albicans, P. carinii, L. donovani, and M. tuberculosis has given new insight into MR vaccine development. Initial studies identified mannan and its derivatives to be important ligands in MR targeting, providing essential knowledge about the MR structural properties. The MR was found to be an early responder in immunogenic pathways. Many attempts have been made to mimic the structural properties of yeast mannan by attaching mannan or mannose to antigenic proteins or peptide epitopes. However, a more detailed understanding of the structural properties of the MR is necessary for the design of targeted vaccines. This review describes the structure of the MR and provides an overview of the use of mannosylated proteins and peptides for vaccine targeting.

An Overview of Structural Features of Antibacterial Glycoconjugate Vaccines That Influence Their Immunogenicity

Bacterial cell-surface-derived or mimicked carbohydrate moieties that act as protective antigens are used in the development of antibacterial glycoconjugate vaccines. The carbohydrate antigen must have a minimum length or size to maintain the conformational structure of the antigenic epitope(s). The presence or absence of O-acetate, phosphate, glycerol phosphate and pyruvate ketal plays a vital role in defining the immunogenicity of the carbohydrate antigen. The nature of the carrier protein, spacer and conjugation pattern used to develop the glycoconjugate vaccine also defines its overall spatial orientation which in turn affects its avidity and selectivity of interaction with the desired target(s). In addition, the ratio of carbohydrate to protein in glycoconjugate vaccines also makes an important contribution in determining the optimum immunological response. This Review article presents the importance of these variables in the development of antibacterial glycoconjugate vaccines and their effects on immune efficacy.

Synthesis and Characterisation of Self‐Assembled and Self‐Adjuvanting Asymmetric Multi‐Epitope Lipopeptides of Ovalbumin

Designing a lipopeptide (LP) vaccine with a specific asymmetric arrangement of epitopes may result in an improved display of antigens, increasing host-cell recognition and immunogenicity. This study aimed to synthesise and characterise the physicochemical properties of a library of asymmetric LP-based vaccine candidates that contained multiple CD4(+) and CD8(+) T-cell epitopes from the model protein antigen, ovalbumin. These fully synthetic vaccine candidates were prepared by microwave-assisted solid phase peptide synthesis. The C12 or C16 lipoamino acids were coupled to the N or C terminus of the OVA CD4 peptide epitope. The OVA CD4 LPs and OVA CD8 peptide constructs were then conjugated using azide-alkyne Huisgen cycloaddition to give multivalent synthetic vaccines. Physiochemical characterisation of these vaccines showed a tendency to self-assemble in aqueous media. Changes in lipid length and position induced self-assembly with significant changes to their morphology and secondary structure as shown by transmission electron microscopy and circular dichroism.

Systematic evaluation of self-adjuvanting lipopeptide nano-vaccine platforms for the induction of potent CD8+ T-cell responses

AIM Systematically evaluate lipid core peptide vaccine delivery platforms to identify core features promoting strong CD8(+) T-cell responses. MATERIALS & METHODS Three different self-adjuvanting lipid core peptide nanovaccines each comprising four copies of the dominant ovalbumin CD8(+) T-cell epitope and varying in the utilization of a polylysine or glucose core with 2-amino-hexadecanoic acid (C16) or 2-amino-dodecanoic acid (C12) lipids were synthesized. Vaccines were tested for ability to induce CD8(+) T-cell responses and inhibit tumor growth in vivo. RESULTS The construct utilizing C12 lipids and polylysine core induced very robust effector T cells shown to have in vivo effector capability as demonstrated by in vivo cytotoxicity and ability to inhibit tumor growth as well as modulation of dendritic cell activation. CONCLUSION The C12 polylysine platform was an effective configuration for induction of potent CD8(+) T-cell responses.

Synthesis of Mannosylated Lipopeptides with Receptor Targeting Properties

Present on the surface of antigen presenting cells (APCs), the mannose receptor (MR) has long been recognized as a front-line receptor in pathogen recognition. During the past decade many attempts have been made to target this receptor for applications including vaccine and drug development. In the present study, a library of vaccine constructs comprising fluorescently labeled mannosylated lipid-dendrimers that contained the ovalbumin CD4(+) epitope, OVA(323-339), as the model peptide antigen were synthesized using fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS). The vaccine constructs were designed with an alanine spacer between the O-linked mannose moieties to investigate the impact of distance between the mannose units on receptor-mediated uptake and/or binding in APCs. Uptake studies performed on F4/80(+) and CD11c(+) cells showed significant uptake and/or binding for lipopeptides containing mannose, and also the lipopeptide without mannose when compared to the control peptides (peptide with no lipid and peptide with no mannose and no lipid). Furthermore, mannan inhibition assays demonstrated that uptake of the mannosylated and lipidated peptides was receptor mediated. To address the specificity of receptor uptake, surface plasmon resonance studies were performed using biacore technology and confirmed high affinity of the mannosylated and lipidated vaccine constructs toward the MR. These studies confirm that both mannose and lipid moieties play significant roles in receptor-mediated uptake on APCs, potentially facilitating vaccine development.

Synthesis and Characterization of Bradykinin Derivatives Based on a β-Cyclodextrin Core

Mono-6A-fluorenylmethyloxycarbonylamino-mono-6X-succinyl-β-cyclodextrin (1), an amino acid-based bi-functionalized derivative of β-cyclodextrin (β-CD), has been functionalized with the bioactive peptide, bradykinin and/or sulfonamides using fluorenylmethyloxycarbonyl (Fmoc) solid phase peptide synthesis (SPPS). The all-in-one molecule contains a carrier (cyclodextrin), targeting agent (bradykinin), and/or model drug (sulfonamide). Varying combinations of these bradykinin-focussed molecules have been synthesized using Fmoc SPPS on Rink amide resin. The positioning of the sulfonamide group, the bradykinin peptide and the cyclodextrin carrier are essential for biological activity. The inclusion of spacers is also important. Structure–activity studies performed on three cancer cell lines in vitro support these conclusions.

Effect of lipidated gonadotropin-releasing hormone peptides on receptor mediated binding and uptake into prostate cancer cells in vitro

UNLABELLED Gonadotropin-releasing hormone (GnRH) receptors are overexpressed on many cancer cells but not on primary cell lines. This study was designed to investigate the targeting ability and uptake of dendritic lipidated [Gln(1)]-GnRH peptide analogues on receptor-positive prostate cancer PC-3 cells relative to receptor-negative ovarian carcinoma SKOV-3 cells for potential application in drug delivery. Direct antiproliferative effect of these was investigated on three GnRH-receptor positive cancer cells, PC-3, LNCaP and DU145. A significant dose dependent growth inhibitory effect was produced in DU145 cells by 5 dendrimers giving an IC50 value of 22-35 μM. All compounds were non-toxic to the normal peripheral blood mononuclear cells. FROM THE CLINICAL EDITOR This study demonstrates the use of specific dendritic lapidated GnRH analogues in growth inhibition of GnRH receptor positive prostate cancer cell lines, suggesting potential future clinical use of this or similar strategies to address GnRH receptor positive cancer cells.