The second intracellular loop of the calcitonin gene-related peptide receptor provides molecular determinants for signal transduction and cell surface expression

The calcitonin gene-related peptide (CGRP) receptor is a heterodimer of a family B G-protein-coupled receptor, calcitonin receptor-like receptor (CLR), and the accessory protein receptor activity modifying protein 1. It couples to Gs, but it is not known which intracellular loops mediate this. We have identified the boundaries of this loop based on the relative position and length of the juxtamembrane transmembrane regions 3 and 4. The loop has been analyzed by systematic mutagenesis of all residues to alanine, measuring cAMP accumulation, CGRP affinity, and receptor expression. Unlike rhodopsin, ICL2 of the CGRP receptor plays a part in the conformational switch after agonist interaction. His-216 and Lys-227 were essential for a functional CGRP-induced cAMP response. The effect of (H216A)CLR is due to a disruption to the cell surface transport or surface stability of the mutant receptor. In contrast, (K227A)CLR had wild-type expression and agonist affinity, suggesting a direct disruption to the downstream signal transduction mechanism of the CGRP receptor. Modeling suggests that the loop undergoes a significant shift in position during receptor activation, exposing a potential G-protein binding pocket. Lys-227 changes position to point into the pocket, potentially allowing it to interact with bound G-proteins. His-216 occupies a position similar to that of Tyr-136 in bovine rhodopsin, part of the DRY motif of the latter receptor. This is the first comprehensive analysis of an entire intracellular loop within the calcitonin family of G-protein-coupled receptor. These data help to define the structural and functional characteristics of the CGRP-receptor and of family B G-protein-coupled receptors in general. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.

Increased potential of a cationic liposome-based delivery system:enhancing stability and sustained immunological activity in pre-clinical development

The combination of dimethyl dioctadecyl ammonium bromide (DDA) and the synthetic cord factor trehalose dibehenate (TDB) with Ag85B-ESAT-6 (H1 fusion protein) has been found to promote strong protective immune responses against Mycobacterium tuberculosis. The development of a vaccine formulation that is able to facilitate the requirements of sterility, stability and generation of a vaccine product with acceptable composition, shelf-life and safety profile may necessitate selected alterations in vaccine formulation. This study describes the implementation of a sterilisation protocol and the use of selected lyoprotective agents in order to fulfil these requirements. Concomitantly, close analysis of any alteration in physico-chemical characteristics and parameters of immunogenicity have been examined for this promising DDA liposome-based tuberculosis vaccine. The study addresses the extensive guidelines on parameters for non-clinical assessment, suitable for liposomal vaccines and other vaccine delivery systems issued by the World Health Organisation (WHO) and the European Medicines Agency (EMEA). Physical and chemical stability was observed following alteration in formulations to include novel cryoprotectants and radiation sterilisation. Immunogenicity was maintained following these alterations and even improved by modification with lysine as the cryoprotective agent for sterilised formulations. Taken together, these results outline the successful alteration to a liposomal vaccine, representing improved formulations by rational modification, whilst maintaining biological activity.

Development of plasmid dna formulations for application as microspheric dna vaccines

The advent of DNA vaccines has heralded a new technology allowing the design and elicitation of immune responses more adequate for a wider range of pathogens. The formulation of these vaccines into the desired dosage forms extends their capability in terms of stability, routes of administration and efficacy. This thesis describes an investigation into the fabrication of plasmid DNA, the active principle of DNA vaccines, into microspheres, based on the tenet of an increased cellular uptake of microparticulate matter by phagocytic cells. The formulation of plasmid DNA into microspheres using two methods, is presented. Formulation of microspheric plasmid DNA using the double emulsion solvent evaporation method and a spray-drying method was explored. The former approach involves formation of a double emulsion, by homogenisation. This method produced microspheres of uniform size and smooth morphology, but had a detrimental effect on the formulated DNA. The spray-drying method resulted in microspheres with an improved preservation of DNA stability. The use of polyethylenimine (PEI) and stearylamine (SA) as agents in the microspheric formulation of plasmid DNA is a novel approach to DNA vaccine design. Using these molecules as model positively-charged agents, their influence on the characteristics of the microspheric formulations was investigated. PEI improved the entrapment efficiency of the plasmid DNA in microspheres, and has minimal effect on either the surface charge, morphology or size distribution of the formulations. Stearylamine effected an increase in the entrapment efficiency and stability of the plasmid DNA and its effect on the micropshere morphology was dependent on the method of preparation. The differences in the effects of the two molecules on microsphere formulations may be attributable to their dissimilar physico-chemical properties. PEI is water-soluble and highly-branched, while SA is hydrophobic and amphipathic. The positive charge of both molecules is imparted by amine functional groups. Preliminary data on the in vivo application of formulated DNA vaccine, using hepatitis B plasmid, showed superior humoral responses to the formulated antigen, compared with free (unformulated) antigen.

Formulation and characterisation of cationic microparticles for the delivery of DNA vaccines

The aim of this research was to formulate a novel biodegradable, biocompatible cationic microparticle vector for the delivery of DNA vaccines. The work builds upon previous research by Singh et al which described the adsorption of DNA to the surface of poly (D,L-lactide-co-glycolide) (PLG) microparticles stabilised with the surfactant cetyltrimethyl ammonium bromide (CT AB). This work demonstrated the induction of antibody and cellular immune responses to HIV proteins encoded on plasmid DNA adsorbed to the particle surface in mice, guinea pigs and non-human primates (Singh et aI, 2000; O'Hagan et aI, 2001). However, the use of surfactants in microparticle formulations for human vaccination is undesirable due to long term safety issues. Therefore, the present research aim was to develop an adsorbed DNA vaccine with enhanced potency and increased safety compared to CTAB stabilised PLG microparticles (PLG/CTAB) by replacement of the surfactant CTAB with an alternative cationic agent. The cationic polymers chitosan and poly (N- vinylpyrrolidone/2-dimethylaminoethyl methacrylate), dimethyl sulfate quaternary (PVP-PDAEMA) were investigated as alternative stabilisers to CTAB. From a variety of initial formulations, the most promising vector(s) for DNA vaccination were selected based on physicochemical data (chapter 3) and in vitro DNA loading and release characteristics (chapter 4). The chosen formulation(s) were analysed in greater depth (chapters 3 and 4), and gene expression was assessed by in vitro cell transfection studies using 293T kidney epithelial and C2C12 myoblast non-phagocytic cell lines (chapter 5). The cytotoxicity of the microparticles and their constituents were also evaluated in vitro (chapter 5). Stability and suitability of the formulation(s) for commercial production were assessed by cryopreparation and lyophilisation studies (chapters 3 and 4). Gene expression levels in cells of the immune response were evaluated by microparticle transfection of the dendritic cell (DC) line 2.4 and primary bone marrow derived DCs (chapter 6). In vivo, mice were injected i.m. with the formulations deemed most promising on the basis of in vitro studies and humoral and cellular immune responses were evaluated (chapter 6).

Characterisation of calcitonin generelated peptide (CGRP) and amylin receptors

The aims of this study were to examine the binding characteristics of the rat CGRP receptor and to further the classification of CGRP and amylin receptors in guinea-pig tissue preparations. Binding characteristics of CGRP were investigated on rat splenic, cerebellar and liver membrane preparations. Human-α-CGRP, rat-α-CGRP and the CGRP receptor analogues Tyrº -CGRPC28-37) and [Cys (ACM)2,7 ]-human CGRP and the CGRP receptor antagonist CGRPC8-37) were utilised in competitive radioligand binding experiments to identify possible CGRP receptor subtypes in these tissues. There appeared to be no significant differences between the rat CGRP receptors examined. A panel of monoclonal antibodies (Mabs) raised against CGRP were employed to investigate the structure-activity relationships of CGRP and its receptor. No differences between the tissue receptors were observed using this panel of Mabs. The effects of human-α, human-β, rat-α-CGRP, human and rat amylin and adrenomedullin(13-52) were examined on the spontaneously beating right atria and on electrically evoked twitch contractions of isolated guinea-pig ileum, vas deferens and left atria. All of the peptides caused concentration-dependent inhibition of twitch amplitude in the ileum and vas deferens. CGRP produced positive inotropic effects in the right and left atria and positive chronotropic effects in the right atria. A variety of CGRP receptor antagonists and putative amylin receptor antagonists were used to antagonise these effects. CGRP(8-37) is currently used as a basis for CGRP receptor classification (Dennis, et al., 1989). Based upon results obtained using CGRP(8-37) it has been shown that the guinea-pig ileum contains mainly CGRP 1 receptors and the vas deferens contain CGRP2 receptors. Amylin was shown to act at receptors distinct from those for CGRP and it is postulated that amylin has its own receptors in these preparations. Experiments using CGRP (19-37) and Tyrº -CGRP(28-37) indicate that human and rat CGRP act at distinct receptors in guinea-pig ileum and vas deferens. The amylin receptor antagonist amylin(8-37) and the putative antagonist AC187 provide evidence to suggest human and rat amylin also act at receptors able to distinguish between the two types of amylin.

Comparison of the depot effect and immunogenicity of liposomes based on dimethyldioctadecylammonium (DDA), 3β-[N-(N',N'-Dimethylaminoethane)carbomyl] cholesterol (DC-Chol), and 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP):prolonged liposome retention mediates stronger Th1 responses

The immunostimulatory capacities of cationic liposomes are well-documented and are attributed both to inherent immunogenicity of the cationic lipid and more physical capacities such as the formation of antigen depots and antigen delivery. Very few studies have however been conducted comparing the immunostimulatory capacities of different cationic lipids. In the present study we therefore chose to investigate three of the most well-known cationic liposome-forming lipids as potential adjuvants for protein subunit vaccines. The ability of 3ß-[N-(N',N'-dimethylaminoethane)carbomyl] cholesterol (DC-Chol), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), and dimethyldioctadecylammonium (DDA) liposomes incorporating immunomodulating trehalose dibehenate (TDB) to form an antigen depot at the site of injection (SOI) and to induce immunological recall responses against coadministered tuberculosis vaccine antigen Ag85B-ESAT-6 are reported. Furthermore, physical characterization of the liposomes is presented. Our results suggest that liposome composition plays an important role in vaccine retention at the SOI and the ability to enable the immune system to induce a vaccine specific recall response. While all three cationic liposomes facilitated increased antigen presentation by antigen presenting cells, the monocyte infiltration to the SOI and the production of IFN-? upon antigen recall was markedly higher for DDA and DC-Chol based liposomes which exhibited a longer retention profile at the SOI. A long-term retention and slow release of liposome and vaccine antigen from the injection site hence appears to favor a stronger Th1 immune response.

Surfactant vesicle-mediated delivery of DNA vaccines via the subcutaneous route

Compared to naked DNA immunisation, entrapment of plasmid-based DNA vaccines into liposomes by the dehydration-rehydration method has shown to enhance both humoural and cell-mediated immune responses to encoded antigens administered by a variety of routes. In this paper we have compared the potency of lipid-based and non-ionic surfactant based vesicle carrier systems for DNA vaccines after subcutaneous immunisation. Plasmid pI.18Sfi/NP containing the nucleoprotein (NP) gene of A/Sichuan/2/87 (H3N2) influenza virus in the pI.18 expression vector was incorporated by the dehydration-rehydration method into various vesicle formulations. The DRV method, entailing mixing of small unilamellar vesicles (SUV) with DNA, followed by dehydration and rehydration, yielded high DNA vaccine incorporation values (85-97% of the DNA used) in all formulations. Studies on vesicle size revealed lipid-based systems formed cationic submicron size vesicles whilst constructs containing a non-ionic surfactant had significantly large z-average diameters (>1500 nm). Subcutaneous vesicle-mediated DNA immunisation employing two DRV(DNA) formulations as well as naked DNA revealed that humoural responses (immunoglobulin total IgG, and subclasses IgG 1 and 1gG 2a) engendered by the plasmid encoded nucleoprotein were substantially higher after dosing twice, 28 days apart with 10 μg DRV-entrapped DNA compared to naked DNA. Comparison between the lipid and non-ionic based vesicle formulations revealed no significant difference in stimulated antibody production. These results suggest that, not only can DNA be effectively entrapped within a range of lipid and non-ionic based vesicle formulations using the DRV method but that such DRV vesicles containing DNA may be a useful system for subcutaneous delivery of DNA vaccines. © 2004 Elsevier B.V. All rights reserved.

Lyophilisation and sterilisation of liposomal vaccines to produce stable and sterile products

The advantages of liposomes as delivery systems for peptide, protein and DNA vaccines is well-recognised, unfortunately their application has been stinted by their instability during storage and their limited shelf-life. Further, sterilisation of these systems has been problematic, with degradation of the liposomes being reported after sterilisation using the various techniques available. Work form our laboratory has investigated techniques that can be applied to particulate liposomal vaccines such that they can be prepared in a freeze-dried and sterile format. In this article, we describe techniques for the lyophilisation, cryoprotection and sterilisation of liposomal vaccines. Applying these methods allows for the retention of both the chemical integrity of the lipids and the key physico-chemical characteristics of the liposomes (e.g., particle size, zeta potential, and dynamic viscosity), thus supporting the enhanced transition of liposomal vaccines from the bench to the clinic. © 2006 Elsevier Inc. All rights reserved.

Comparison of vesicle based antigen delivery systems for delivery of hepatitis B surface antigen

There is a clinical need for a more effective vaccine against hepatitis B, and in particular vaccines that may be suitable for therapeutic administration. This study assesses the potential of cationic surfactant vesicle based formulations using two agents; the cationic amine containing [N-(N′,N′-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) or dimethyl dioctadecylammonium bromide (DDA) with hepatitis B surface antigen (HBsAg). Synthetic mycobacterial cord factor, trehalose 6,6′-dibehenate (TDB) has been used as an adjuvant and the addition of 1-monopalmitoyl glycerol (C16:0) (MP) and cholesterol (Chol) to DDA-TDB is assessed for its potential to facilitate formation of dehydration-rehydration vesicles (DRV) at room temperature, and the effect of this on immune responses. A DRV formulation is directly compared to an adsorbed formulation of the same composition and preparation protocol (MP:dioleoyl phosphoethanolamine (DOPE):Chol:DC-Chol) and the direct substitution of MP with phosphatidylcholine (PC) is also compared in DRV antigen-entrapped formulations. MP and Chol were shown to facilitate the use of DDA-TDB in DRV formulations prepared at room temperature, whilst there was marginal alteration of immunogenicity (a reduction in HBsAg-specific IL-2). The HBsAg adsorbed DRV formulation was not significantly different from the HBsAg entrapped DRV formulation. Overall, DDA formulations incorporating TDB showed markedly increased antigen specific splenocyte proliferation and elicited cytokine production concomitant with a strong T cell driven response, delineating formulations that may be useful for further evaluation of their clinical potential. © 2007 Elsevier B.V. All rights reserved.

Reporter ion-based mass spectrometry approaches for the detection of non-enzymatic protein modifications in biological samples

Development of mass spectrometry techniques to detect protein oxidation, which contributes to signalling and inflammation, is important. Label-free approaches have the advantage of reduced sample manipulation, but are challenging in complex samples owing to undirected analysis of large data sets using statistical search engines. To identify oxidised proteins in biological samples, we previously developed a targeted approach involving precursor ion scanning for diagnostic MS3 ions from oxidised residues. Here, we tested this approach for other oxidations, and compared it with an alternative approach involving the use of extracted ion chromatograms (XICs) generated from high-resolution MSMS data using very narrow mass windows. This accurate mass XIC data methodology was effective at identifying nitrotyrosine, chlorotyrosine, and oxidative deamination of lysine, and for tyrosine oxidations highlighted more modified peptide species than precursor ion scanning or statistical database searches. Although some false positive peaks still occurred in the XICs, these could be identified by comparative assessment of the peak intensities. The method has the advantage that a number of different modifications can be analysed simultaneously in a single LC-MSMS run. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine. Biological significance: The use of accurate mass extracted product ion chromatograms to detect oxidised peptides could improve the identification of oxidatively damaged proteins in inflammatory conditions. © 2013 Elsevier B.V.

Investigating polymer-peptide conjugates and electrospinning for the production of advanced materials

This thesis describes the production of advanced materials comprising a wide array of polymer-based building blocks. These materials include bio-hybrid polymer-peptide conjugates, based on phenylalanine and poly(ethylene oxide), and polymers with intrinsic microporosity (PIMs). Polymer-peptides conjugates were previously synthesised using click chemistry. Due to the inherent disadvantages of the reported synthesis, a new, simpler, inexpensive protocol was sought. Three synthetic methods based on amidation chemistry were investigated for both oligopeptide and polymerpeptide coupling. The resulting conjugates produced were then assessed by various analytical techniques, and the new synthesis was compared with the established protocol. An investigation was also carried out focussing on polymer-peptide coupling via ester chemistry, involving deprotection of the carboxyl terminus of the peptide. Polymer-peptide conjugates were also assessed for their propensity to self-assemble into thixotropic gels in an array of solvent mixtures. Determination of the rules governing this particular self-assembly (gelation) was required. Initial work suggested that at least four phenylalanine peptide units were necessary for self-assembly, due to favourable hydrogen bond interactions. Quantitative analysis was carried out using three analytical techniques (namely rheology, FTIR, and confocal microscopy) to probe the microstructure of the material and provided further information on the conditions for self-assembly. Several polymers were electrospun in order to produce nanofibres. These included novel materials such as PIMs and the aforementioned bio-hybrid conjugates. An investigation of the parameters governing successful fibre production was carried out for PIMs, polymer-peptide conjugates, and for nanoparticle cages coupled to a polymer scaffold. SEM analysis was carried out on all material produced during these electrospinning experiments.

In silico prediction of peptide-MHC binding affinity using SVRMHC

The binding between peptide epitopes and major histocompatibility complex (MHC) proteins is a major event in the cellular immune response. Accurate prediction of the binding between short peptides and class I or class II MHC molecules is an important task in immunoinformatics. SVRMHC which is a novel method to model peptide-MHC binding affinities based on support rector machine regression (SVR) is described in this chapter. SVRMHC is among a small handful of quantitative modeling methods that make predictions about precise binding affinities between a peptide and an MHC molecule. As a kernel-based learning method, SVRMHC has rendered models with demonstrated appealing performance in the practice of modeling peptide-MHC binding.

VaxiJen:a server for prediction of protective antigens, tumour antigens and subunit vaccines

Background - Vaccine development in the post-genomic era often begins with the in silico screening of genome information, with the most probable protective antigens being predicted rather than requiring causative microorganisms to be grown. Despite the obvious advantages of this approach – such as speed and cost efficiency – its success remains dependent on the accuracy of antigen prediction. Most approaches use sequence alignment to identify antigens. This is problematic for several reasons. Some proteins lack obvious sequence similarity, although they may share similar structures and biological properties. The antigenicity of a sequence may be encoded in a subtle and recondite manner not amendable to direct identification by sequence alignment. The discovery of truly novel antigens will be frustrated by their lack of similarity to antigens of known provenance. To overcome the limitations of alignment-dependent methods, we propose a new alignment-free approach for antigen prediction, which is based on auto cross covariance (ACC) transformation of protein sequences into uniform vectors of principal amino acid properties. Results - Bacterial, viral and tumour protein datasets were used to derive models for prediction of whole protein antigenicity. Every set consisted of 100 known antigens and 100 non-antigens. The derived models were tested by internal leave-one-out cross-validation and external validation using test sets. An additional five training sets for each class of antigens were used to test the stability of the discrimination between antigens and non-antigens. The models performed well in both validations showing prediction accuracy of 70% to 89%. The models were implemented in a server, which we call VaxiJen. Conclusion - VaxiJen is the first server for alignment-independent prediction of protective antigens. It was developed to allow antigen classification solely based on the physicochemical properties of proteins without recourse to sequence alignment. The server can be used on its own or in combination with alignment-based prediction methods.

Synthesis and characterization of silica-supported L-Lysine-based dendritic branches

Two series of novel modified silicas have been prepared in which individual dendritic branches have been attached to aminopropylsilica using standard peptide coupling methodology. The dendritic branches are composed of enantiomerically pure l-lysine building blocks, and hence, the modified silicas have the potential to act as chiral stationary phases in chromatography. In one series of modified silicas, the surface of the dendritic branch consists of Boc carbamate groups, whereas the other has benzoyl amide surface groups. Different coupling reagents have been investigated in order to maximize the loading onto the solid phase. The new supported dendritic materials have been fully characterized with properties of the bulk material determined by elemental analysis, 13C NMR, and IR spectroscopy, whereas XPS provides important information about the surface of the modified silica exposed to the incident X-rays, the key region in which potential chromatographic performance of these materials will take place. Although the bulk analyses indicate that loading of the dendritic branch onto silica decreases with increasing dendritic generation (and consequently steric bulk), XPS indicates that the optimum surface coverage is actually obtained at the second generation of dendritic growth.

Peptide binding to the HLA-DRB1 supertype:a proteochemometrics analysis

A proteochemometrics approach was applied to a set of 2666 peptides binding to 12 HLA-DRB1 proteins. Sequences of both peptide and protein were described using three z-descriptors. Cross terms accounting for adjacent positions and for every second position in the peptides were included in the models, as well as cross terms for peptide/protein interactions. Models were derived based on combinations of different blocks of variables. These models had moderate goodness of fit, as expressed by r2, which ranged from 0.685 to 0.732; and good cross-validated predictive ability, as expressed by q2, which varied from 0.678 to 0.719. The external predictive ability was tested using a set of 356 HLA-DRB1 binders, which showed an r2(pred) in the range 0.364-0.530. Peptide and protein positions involved in the interactions were analyzed in terms of hydrophobicity, steric bulk and polarity.