Synthesis, Characterization and Immunological Evaluation of Self-Adjuvanting Group†A Streptococcal Vaccine Candidates Bearing Various Lipidic Adjuvanting Moieties.

Four group A streptococcal glycolipopeptide vaccine candidates with different lipidic adjuvanting moieties were prepared and characterized. The immunogenicity of the compounds was evaluated by macrophage and dendritic cell uptake studies and by in vivo quantification of systemic IgG antibody by ELISA. Three of the candidates showed significant induction of the IgG response.

Stability, permeability and growth-inhibitory properties of gonadotropin-releasing hormone liposaccharides.

PURPOSE: In this study we aimed to address the poor drug-like properties of Gonadotropin-Releasing Hormone (GnRH) peptide through modification with lipids and carbohydrates. METHODS: GnRH peptide was conjugated to 2-amino-D,L-octanoic acid (C8) and 2-amino-D,L-dodecanoic acid (C12) in monomer and dimer, along with (6-9) or without (2-5 and 11) a glucose moiety. Peptides were tested for their biological activity using different tumour cell lines. The toxicity of the constructs was evaluated in peripheral blood mononuclear cells (PBMC). RESULTS: All (glyco)lipopeptides showed improved metabolic stability in Caco-2 cell homogenates. Those with single lipid moiety (2, 4 and 8) exhibited prodrug-like properties. Permeability across Caco-2 cell monolayers was enhanced in the dimer C8-modified (glyco)lipopeptide (3) and the lipopeptide with C12 inserted mid-sequence (11). Most of the constructs showed moderate-to-high antiproliferative activity against GnRH-receptor positive DU145 and OVCAR-3 cells (up to 60%). Compound 11 was the most effective with IC50 = 26.4 ± 1.07 µg.ml(-1), which was comparable to triptorelin (25.1 ± 1.14 µg.mL(-1)). The sensitivity of OVCAR-3 cells to the effect of all analogues except for 11 decreased significantly in estrogen-reconstituted media. Only compounds 2, 4, 5 and 8 showed a steroid-dependent effect in DU145 cells. No compounds exhibited significant toxicity on PBMCs. CONCLUSION: These results indicated lipidation and glycosylation improves the druggability of GnRH and could lead to an increased direct antitumour activity in some hormone dependent and independent reproductive cancers.

Active immunisation of mice with GnRH lipopeptide vaccine candidates: Importance of T helper or multi-dimer GnRH epitope.

Active immunisation against gonadotropin releasing hormone (GnRH) is a potential alternative to surgical castration. This study focused on the development of a GnRH subunit lipopeptide vaccine. A library of vaccine candidates that contained one or more (up to eight) copies of monomeric or dimeric GnRH peptide antigen, an adjuvanting lipidic moiety based on lipoamino acids, and an additional T helper epitope, was synthesised by solid phase peptide synthesis. The candidates were evaluated in vivo in order to determine the minimal components of this vaccine necessary to induce a systemic immune response. BALB/c mice were immunised with GnRH lipopeptide conjugates, co-administered with or without Complete Freund's Adjuvant, followed by two additional immunisations. Significant GnRH-specific IgG titres were detected in sera obtained from mice immunised with four of the seven lipopeptides tested, with an increase in titres observed after successive immunisations. This study highlights the importance of for epitope optimisation and delivery system design when producing anti-hapten antibodies in vivo. The results of this study also contribute to the development of future clinical and veterinary immunocontraceptives.

Convergent synthetic methodology for the construction of self-adjuvanting lipopeptide vaccines using a novel carbohydrate scaffold.

A novel convergent synthetic strategy for the construction of multicomponent self-adjuvanting lipopeptide vaccines was developed. A tetraalkyne-functionalized glucose derivative and lipidated Fmoc-lysine were prepared by novel efficient and convenient syntheses. The carbohydrate building block was coupled to the self-adjuvanting lipidic moiety (three lipidated Fmoc-lysines) on solid support. Four copies of a group A streptococcal B cell epitope (J8) were then conjugated to the glyco-lipopeptide using a copper-catalyzed cycloaddition reaction. The approach was elaborated by the preparation of a second vaccine candidate which incorporated an additional promiscuous T-helper epitope.

Chemical methods for peptide and protein production.

Since the invention of solid phase synthetic methods by Merrifield in 1963, the number of research groups focusing on peptide synthesis has grown exponentially. However, the original step-by-step synthesis had limitations: the purity of the final product decreased with the number of coupling steps. After the development of Boc and Fmoc protecting groups, novel amino acid protecting groups and new techniques were introduced to provide high quality and quantity peptide products. Fragment condensation was a popular method for peptide production in the 1980s, but unfortunately the rate of racemization and reaction difficulties proved less than ideal. Kent and co-workers revolutionized peptide coupling by introducing the chemoselective reaction of unprotected peptides, called native chemical ligation. Subsequently, research has focused on the development of novel ligating techniques including the famous click reaction, ligation of peptide hydrazides, and the recently reported α-ketoacid-hydroxylamine ligations with 5-oxaproline. Several companies have been formed all over the world to prepare high quality Good Manufacturing Practice peptide products on a multi-kilogram scale. This review describes the advances in peptide chemistry including the variety of synthetic peptide methods currently available and the broad application of peptides in medicinal chemistry.

Characterisation of a cell wall-anchored protein of Staphylococcus saprophyticus associated with linoleic acid resistance.

BACKGROUND: The Gram-positive bacterium Staphylococcus saprophyticus is the second most frequent causative agent of community-acquired urinary tract infections (UTI), accounting for up to 20% of cases. A common feature of staphylococci is colonisation of the human skin. This involves survival against innate immune defenses including antibacterial unsaturated free fatty acids such as linoleic acid which act by disrupting bacterial cell membranes. Indeed, S. saprophyticus UTI is usually preceded by perineal skin colonisation. RESULTS: In this study we identified a previously undescribed 73.5 kDa cell wall-anchored protein of S. saprophyticus, encoded on plasmid pSSAP2 of strain MS1146, which we termed S. saprophyticus surface protein F (SssF). The sssF gene is highly prevalent in S. saprophyticus clinical isolates and we demonstrate that the SssF protein is expressed at the cell surface. However, unlike all other characterised cell wall-anchored proteins of S. saprophyticus, we were unable to demonstrate a role for SssF in adhesion. SssF shares moderate sequence identity to a surface protein of Staphylococcus aureus (SasF) recently shown to be an important mediator of linoleic acid resistance. Using a heterologous complementation approach in a S. aureus sasF null genetic background, we demonstrate that SssF is associated with resistance to linoleic acid. We also show that S. saprophyticus strains lacking sssF are more sensitive to linoleic acid than those that possess it. Every staphylococcal genome sequenced to date encodes SssF and SasF homologues. Proteins in this family share similar predicted secondary structures consisting almost exclusively of α-helices in a probable coiled-coil formation. CONCLUSIONS: Our data indicate that SssF is a newly described and highly prevalent surface-localised protein of S. saprophyticus that contributes to resistance against the antibacterial effects of linoleic acid. SssF is a member of a protein family widely disseminated throughout the staphylococci.

Modern lipid-, carbohydrate-, and peptide-based delivery systems for peptide, vaccine, and gene products.

Research related to peptide, vaccine, and gene delivery has grown exponentially over the last decade. In this review, we discuss the development of delivery systems for peptides, gene and vaccine products. Special focus is given to different lipidation and glycosylation strategies to improve the metabolic stability and membrane permeability of therapeutics, and their targeting to specific sites. The synthetic methods for preparation of the systems are also described.

Characterization of a serine protease homologous to house dust mite group 3 allergens from the scabies mite Sarcoptes scabiei.

The scabies mite, Sarcoptes scabiei var. hominis, infests human skin, causing allergic reactions and facilitating bacterial infection by Streptococcus sp., with serious consequences such as rheumatic fever and rheumatic heart disease. To identify a possible drug target or vaccine candidate protein, we searched for homologues of the group 3 allergen of house dust mites, which we subsequently identified in a cDNA library. The native protein, designated Sar s 3, was shown to be present in the mite gut and excreted in fecal pellets into mite burrows within the upper epidermis. The substrate specificity of proteolytically active recombinant rSar s 3 was elucidated by screening a bacteriophage library. A preference for substrates containing a RS(G/A) sequence at the P1-P2' positions was revealed. A series of peptides synthesized as internally quenched fluorescent substrates validated the phage display data and high performance liquid chromatography/mass spectrometry analysis of the preferred cleaved substrate and confirmed the predicted cleavage site. Searches of the human proteome using sequence data from the phage display allowed the in silico prediction of putative physiological substrates. Among these were numerous epidermal proteins, with filaggrin being a particularly likely candidate substrate. We showed that recombinant rSar s 3 cleaves human filaggrin in vitro and obtained immunohistological evidence that the filaggrin protein is ingested by the mite. This is the first report elucidating the substrate specificity of Sar s 3 and its potential role in scabies mite biology.

Pro-apoptotic activity of lipidic α-amino acids isolated from Protopalythoa variabilis.

Lipidic α-amino acids (LAAs) have been described as non-natural amino acids with long saturated or unsaturated aliphatic chains. In the continuing prospect to discover anticancer agents from marine sources, we have obtained a mixture of two cytotoxic LAAs (1a and 1b) from the zoanthid Protopalythoa variabilis. The anti-proliferative potential of 14 synthetic LAAs and 1a/1b were evaluated on four tumor cell lines (HCT-8, SF-295, MDA-MB-435, and HL-60). Five of the synthetic LAAs showed high percentage of tumor cell inhibition, while 1a/1b completely inhibited tumor cell growth. Additionally, apoptotic effects of 1a/1b were studied on HL-60 cell line. 1a/1b-treated cells showed apoptosis morphology, loss of mitochondrial potential, and DNA fragmentation.

Synthesis of a Streptococcus pyogenes vaccine candidate based on the M protein PL1 epitope.

Group A streptococcus is a Gram-positive bacteria that causes a range of infectious diseases. Targeting the bacteria, a new self-adjuvanting vaccine candidate, incorporating a carbohydrate carrier and an amino acid-based adjuvant, was synthesised utilising carbohydrate chemistry and solid-phase peptide synthesis procedures. Characterisation of the candidate was achieved using reverse-phase HPLC and electrospray ionisation mass spectrometry. The successful synthesis and characterisation of the vaccine candidate may contribute to the discovery of a therapeutically and clinically viable vaccine against group A streptococcus.

Carbohydrates in Vaccine Development.

Despite advances in the development of new vaccines, there are still some diseases with no vaccine solutions. Therefore, further efforts are required to more comprehensively discern the different antigenic components of these microorganisms on a molecular level. This review summarizes advancement in the development of new carbohydrate-based vaccines. Following traditional vaccine counterparts, the carbohydrate-based vaccines introduced a new approach in fighting infectious diseases. Carbohydrates have played various roles in the development of carbohydrate-based vaccines, which are described in this review, including carbohydrates acting as antigens, carriers or targeting moieties. Carbohydrate-based vaccines against infectious diseases, such as group A streptococcus, meningococcal meningitis and human immunodeficiency virus, are also discussed. A number of carbohydrate- based vaccines, such as Pneumovax 23, Menveo and Pentacel, have been successfully marketed in the past few years and there is a promising standpoint for many more to come in the near future.

Structure-activity relationship of a series of synthetic lipopeptide self-adjuvanting group a streptococcal vaccine candidates.

The development of 16 self-adjuvanting group A streptococcal vaccine candidates, composed of (i) a universal helper T-cell epitope (P25), (ii) a target GAS B-cell epitope (J14), and (iii) a lipid moiety, is described. Systemic J14-specific IgG antibodies were detected following subcutaneous immunization of BALB/c (H-2 (d)) mice with each construct without the need for an additional adjuvant. The effect of changing the order of P25, J14, and lipid moiety attachment or incorporation of P25 and J14 into a lipid-core peptide system on antibody titers was assessed. The point of lipid moiety attachment had the greatest influence on systemic J14-specific IgG antibody titers. Overall, the best vaccines featured a C-terminal lipid moiety, conjugated through a lysine residue to P25 at the N-terminus, and J14 on the lysine side chain.

Development of a liposaccharide-based delivery system and its application to the design of group A streptococcal vaccines.

Group A streptococcus (GAS) is associated with many human diseases, ranging in severity from benign to life-threatening. A promising strategy for developing vaccines against GAS involves the use of carbohydrates as carriers for peptide antigens. This study describes the optimized synthesis of d-glucose and d-galactose derived carriers, bearing an adipate linker and four tert-butoxycarbonyl protected aminopropyl groups. Prophylactic GAS vaccine candidates were synthesized by conjugating multiple copies of a single GAS M protein derived peptide antigen (either J8 or J14) onto the carbohydrate carriers. These antigens contain peptide sequences, which are highly conserved and offer the potential to prevent infections caused by up to 70% of GAS strains. Lipophilic amino acids were also conjugated to the d-glucose anomeric carbon to produce a self-adjuvanting liposaccharide vaccine. High serum IgG antibody titers against each of the incorporated peptide epitopes were detected following subcutaneous immunization of B10.BR (H-2 (k)) mice with the liposaccharide vaccine candidates.

Induction and characterization of an unusual alpha-D-galactosidase from Talaromyces flavus.

An extracellular alpha-d-galactosidase from Talaromyces flavus CCF 2686 with extremely broad and unusual acceptor specificity is produced exclusively in the presence of the specific inducer--6-deoxy-D-glucose (quinovose). The procedure for the preparation of this very expensive substance has been modified and optimized. Surprisingly, any of other common alpha-D-galactosidase inducers or substrates, e.g., D-galactose, melibiose and raffinose, did not stimulate its production. The crude alpha-D-galactosidase preparation was purified by anion-exchange chromatography and three isoenzymes with different substrate specificities were identified. The main isoenzyme (alphaGal1) was further purified by cation-exchange chromatography and fully characterized. When compared with other alpha-galactosidases and also with other isoenzymes produced by T. flavus, it showed a markedly different regioselectivity and also negligible hydrolytic activity towards melibiose. Moreover, it was active on polymeric substrates (locust bean gum, guar gum) and significantly inhibited by alpha-D-galactopyranosyl azide, D-galactose, D-xylose, melibiose, methyl alpha- and beta-D-galactopyranoside and lactose.

Evaluation of Lipopeptides as Toll-like Receptor 2 Ligands.

BACKGROUND: Peptide-based vaccines are considered to be the next generation of modern immunizations, as they are safe, easy to produce and well-defined. However, due to their weak immunogenic effect, it is important to first develop an appropriate adjuvant for peptide-based vaccines. OBJECTIVE: The aim of this work was to synthesize a series of four adjuvanting moieties as alkyne derivatives, incorporating dipalmitoyl serine (DPS), 1,3-diglyceride (DG), two hexadecane lipoamino acids (diLAA), and 2,3-dipalmitoyl-S-glycerylcysteine (Pam2Cys). Next aim was to synthesize and attach the azide derivative of biotinylated J14 peptide (model B-cell epitope) to the alkynes through copper- catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction. Final aim was to test the ability of the final biotin labeled conjugates to directly interact with in vitro expressed TLR2 and 8 using AlphaScreen proximity assay. METHOD: All of the peptides were synthesized by manual stepwise solid phase peptide synthesis (SPPS) on rink amide MBHA resin using HATU/DIPEA Fmoc-chemistry. The target compounds were synthesized in a solution phase using CuAAC reaction. RESULTS: Pam2Cys analogue bound to TLR2 as expected. Analogues of DPS and C16-LAA showed also affinity to TLR2, while it did not bind to the control protein (TLR8), demonstrating ability of the DPS and C16-LAA to be recognized by TLR2. CONCLUSION: Four alkyne derivatives of lipids were successfully synthesized and coupled to a biotinylated J14 peptide to give a series of self-adjuvanting ligands. These ligands showed different affinity to TLR2 upon testing by AlphaScreen assay. The DPS derivative showed the most promising affinity in comparison to the standard TLR2 agonist, Pam2Cys.

Glycosylation, an effective synthetic strategy to improve the bioavailability of therapeutic peptides.

Glycosylation of peptides is a promising strategy for modulating the physicochemical properties of peptide drugs and for improving their absorption through biological membranes. This review highlights various methods for the synthesis of glycoconjugates and recent progress in the development of glycosylated peptide therapeutics. Furthermore, the impacts of glycosylation in overcoming the existing barriers that restrict oral and brain delivery of peptides are described herein.

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, characterization and in vitro evaluation of amphiphilic ion pairs of erythromycin and kanamycin antibiotics with liposaccharides.

The hydrophilic ion paring strategy (HIP) is a method explored to improve the cell/tissue uptake of poorly adsorbed drugs and to optimize their physico-chemical characteristics. In this context, we here describe the synthesis of some ion pairs of two model cationic antibiotics, erythromycin (ERY) and kanamycin A (KAN), with liposaccharides having different levels of lipophilicity and charge. The formation of drug-liposaccharide complexes was confirmed by Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD) analysis. The effect of the amphiphilic liposaccharide moieties on the antimicrobial activity of ERY and KAN was assessed by measuring the minimal inhibitory concentration (MIC) of the compounds against a panel of bacterial strains that were susceptible or resistant to the parent antibiotics. The ion pairing did not depress the in vitro antibiotic activity, although no lowering of MIC values was registered. The experimental findings would motivate the future investigation of this ion pairing strategy in drug design, for instance allowing improvement of the encapsulation efficiency of hydrophilic antibiotics in lipid-based nanocarriers, or changing their in vivo biodistribution and pharmacokinetic profile.

Design, synthesis and evaluation of a gonadotropin releasing hormone-based subunit vaccine in rams (Ovis aries).

Immunocastration using gonadotropin-releasing hormone (GnRH)-based vaccines has been investigated in rams to reduce aggressive and sexual behaviour and to control meat quality. Despite considerable efforts, a practical GnRH vaccine has yet to be developed for rams. In the present study, a A GnRH-lipopeptide vaccine (GnRH-LP) including two copies of GnRH, 2-amino-d,l-hexadecanoic acid (C16), and a unique T helper epitope, was examined in rams. Rams received a primary and secondary vaccination of GnRH-LP without additional adjuvant (Group 1) or with the adjuvant AdjuVac™ (Group 2). In both Group 1 and 2 anti-GnRH antibody titres increased after secondary vaccination, however, the antibody titres were higher (p<0.01) for rams in Group 2. The latter rams showed a marked decrease in testicular size. The marked and sustained reduction in testicular size in rams treated with GnRH-LP+AdjuVac™ provides the basis for an effective immunocastration vaccine in rams.