Size dependent enhancement of helium ion irradiation tolerance in sputtered Cu/V nanolaminates

We have investigated the evolution of radiation damage and changes in hardness of sputter-deposited Cu/V nanolaminates upon room temperature helium ion irradiation. As the individual layer thickness decreases from 200 to 5 nm, helium bubble density and radiation hardening both decrease. The magnitude of radiation hardening becomes negligible for individual layer thickness of 2.5 nm or less. These observations indicate that nearly immiscible Cu/V interface can effectively absorb radiation-induced point defects and reduce their concentrations.

Small cationic DDA:TDB liposomes as protein vaccine adjuvants obviate the need for TLR agonists in inducing cellular and humoral responses

Most subunit vaccines require adjuvants in order to induce protective immune responses to the targeted pathogen. However, many of the potent immunogenic adjuvants display unacceptable local or systemic reactogenicity. Liposomes are spherical vesicles consisting of single (unilamellar) or multiple (multilamellar) phospholipid bi-layers. The lipid membranes are interleaved with an aqueous buffer, which can be utilised to deliver hydrophilic vaccine components, such as protein antigens or ligands for immune receptors. Liposomes, in particular cationic DDA:TDB vesicles, have been shown in animal models to induce strong humoral responses to the associated antigen without increased reactogenicity, and are currently being tested in Phase I human clinical trials. We explored several modifications of DDA:TDB liposomes--including size, antigen association and addition of TLR agonists--to assess their immunogenic capacity as vaccine adjuvants, using Ovalbumin (OVA) protein as a model protein vaccine. Following triple homologous immunisation, small unilamellar vesicles (SUVs) with no TLR agonists showed a significantly higher capacity for inducing spleen CD8 IFN? responses against OVA in comparison with the larger multilamellar vesicles (MLVs). Antigen-specific antibody reponses were also higher with SUVs. Addition of the TLR3 and TLR9 agonists significantly increased the adjuvanting capacity of MLVs and OVA-encapsulating dehydration-rehydration vesicles (DRVs), but not of SUVs. Our findings lend further support to the use of liposomes as protein vaccine adjuvants. Importantly, the ability of DDA:TDB SUVs to induce potent CD8 T cell responses without the need for adding immunostimulators would avoid the potential safety risks associated with the clinical use of TLR agonists in vaccines adjuvanted with liposomes.

The production, harvest and adsorptive recovery of an infectious herpes simplex virus vaccine

At present there is not a reliable vaccine against herpes virus. Viral protein vaccines as yet have proved unsuccessful to meet the challenge of raising an appropriate immune response. Cantab Pharmaceuticals has produced a virus vaccine that can undergo one round of replication in the recipient in order to produce a more specific immune reaction. This virus is called Disabled Infectious Single Cycle Herpes Simplex Virus (DISC HSV) which has been derived by deleting the essential gH gene from a type 2 herpes virus. This vaccine has been proven to be effective in animal studies. Existing methods for the purification of viruses rely on laboratory techniques and for vaccine production would be on a far too small a scale. There is therefore a need for new virus purification methods to be developed in order to meet these large scale needs. An integrated process for the manufacture of a purified recombinant DISC HSV is described. The process involves culture of complementing Vero (CR2) cells, virus infection and manufacture, virus harvesting and subsequent downstream processing. The identification of suitable growth parameters for the complementing cell line and optimal limes for both infection and harvest are addressed. Various traditional harvest methods were investigated and found not to be suitable for a scaled up process. A method of harvesting, that exploits the elution of cell associated viruses by the competitive binding of exogenous heparin to virus envelope gC proteins, is described and is shown to yield significantly less contaminated process streams than sonication or osmotic approaches that involve cell rupture (with> 10-fold less complementing cell protein). High concentrations of salt (>0.8M NaCl) exhibit the same effect, although the high osmotic strength ruptures cells and increase the contamination of the process stream. This same heparin-gC protein affinity interaction is also shown to provide an efficient adsorptive purification procedure for herpes viruses which avoids the need to pre-treat the harvest material, apart from clarification, prior to chromatography. Subsequent column eluates provide product fractions with a 100-fold increase in virus titre and low levels of complementing cell protein and DNA (0.05 pg protein/pfu and 1.2 x 104 pg DNA/pfu respectively).

Influence of microbial antigen formulation and delivery route on the immune response

Recent technological advances have resulted in the production of safe subunit and synthetic small peptide vaccines. Unfortunately, these vaccines are weakly or non-immunogenic in the absence of an immunological adjuvant (agents that can induce strong immunity to antigens). In addition, in order to prevent and/or control infection at the mucosal surface, stimulation of the mucosal immune system is essential. This may be achieved via the common mucosal immune system by exposure to antigen at a mucosal surface remote from the area of infection. Initial studies investigated the potential of multiple emulsions in effecting oral absorption and the subsequent immune responses to a lipopolysaccharide vaccine (LPS) after immunisation. Nasal delivery of LPS was carried out in parallel work using either aqueous solution or gel formulations. Tetanus toxoid vaccine in simple solution was delivered to guinea pigs as free antigen or entrapped in DSPC liposomes. In addition, adsorbed tetanus toxoid vaccine was delivered nasally free or in an aerosil gel formulation. This work was extended to investigate guinea pigs immunised by various mucosal routes with a herpes simplex virus subunit vaccine prepared from virus infected cells and delivered in gels, multiple emulsions and liposomes. Comparable serum antibody responses resulted but failed to produce enhanced protection against vaginal challenge when compared to subcutaneous immunisation with alhydrogel adjuvanted vaccine. Thus, immunisation of the mucosal surface by these methods may have been inadequate. These studies were extended in an attempt to protect against HSV genital challenge by construction of an attenuated Salmonella typhimurium HWSH aroA mutant expressing a cloned glycoprotein D-l gene fused to the Es-cherichia coli lac z promoter. Preliminary work on the colonisation of guinea pigs with S. typhimurium HWSH aroA mutants were carried out, with the aim of using the guinea pig HSV vaginal model to investigate protection.

Immunological studies in multiple low dose streptozotocin induced diabetes in mice

1. Multiple low doses of streptozotocin (MSZ) treatment successfully induced diabetes in male TO, MFI and HO lean mice. In contrast however, BALB/c mice failed to develop persistent hyperglycaemia. Single streptozotocin (SSZ) treatment also produced diabetes in TO mice. SSZ treatment however, produced severe weight loss and atrophy of the lymphoid organs. MSZ treatment on the other hand, was not cytotoxic towards lymphoid organs and, whilst there was no loss of body weight, growth rates were reduced in MSZ treated mice. 2. Following sheep red blood cell (SRBC) immunisation of MSZ-treated mice, haemagglutination titres, and numbers of antigen reactive cells and plaque forming cells were all significantly lower than control values. 3. In vitro proliferation of spleen cells in response to phytohaemagglutinin (PHA) and conconavalin A (ConA) was found to be significantly depressed in MSZ treated mice. However, T-lymphocyte responses were intact when the mice were not overtly hyperglycaemic. In contrast, however, T cell independent responses to lipopolysaccharide (LPS) were generally intact throughout the study period. 4. Cell mediated immunity, as assessed by measurements of delayed (Type IV) hypersensitivity, was also depressed in MSZ treated mice. This suppression could be reversed by insulin therapy. 5. Both natural killer cell activity and antibody dependent cell mediated cytotoxicity were found to be significantly increased in MSZ treated mice. 6. Histological examination of the pancreas showed the presence of insulitis, in MSZ treated mice, and cytotoxic effector cells against obese mice islet cells (as assessed by 51Cr release) and HIT-T15 cells (as assessed by insulin secretion) were found to be significantly increased. Furthermore, these effector cells were also found to show increased proliferation in the presence of homogenates prepared from HIT-T15 cells. Examination of the Sera from MSZ treated mice showed that islet cell surface antibodies were present.

Exploring oxidative modifications of tyrosine:an update on mechanisms of formation, advances in analysis and biological consequences

Protein oxidation is increasingly recognised as an important modulator of biochemical pathways controlling both physiological and pathological processes. While much attention has focused on cysteine modifications in reversible redox signalling, there is increasing evidence that other protein residues are oxidised in vivo with impact on cellular homeostasis and redox signalling pathways. A notable example is tyrosine, which can undergo a number of oxidative post-translational modifications to form 3-hydroxy-tyrosine, tyrosine crosslinks, 3-nitrotyrosine and halogenated tyrosine, with different effects on cellular functions. Tyrosine oxidation has been studied extensively in vitro, and this has generated detailed information about the molecular mechanisms that may occur in vivo. An important aspect of studying tyrosine oxidation both in vitro and in biological systems is the ability to monitor the formation of oxidised derivatives, which depends on a variety of analytical techniques. While antibody-dependent techniques such as ELISAs are commonly used, these have limitations, and more specific assays based on spectroscopic or spectrometric techniques are required to provide information on the exact residues modified and the nature of the modification. These approaches have helped understanding of the consequences of tyrosine oxidation in biological systems, especially its effects on cell signalling and cell dysfunction, linking to roles in disease. There is mounting evidence that tyrosine oxidation processes are important in vivo and can contribute to cellular pathology.

Design and synthesis of potential antimicrobial agents

Tuberculosis is one of the most devastating diseases in the world primarily due to several decades of neglect and an emergence of multidrug-resitance strains (MDR) of M. tuberculosis together with the increased incidence of disseminated infections produced by other mycobacterium in AIDS patients. This has prompted the search for new antimycobacterial drugs. A series of pyridine-2-, pyridine-3-, pyridine-4-, pyrazine and quinoline-2-carboxamidrazone derivatives and new classes of carboxamidrazone were prepared in an automated fashion and by traditional synthesis. Over nine hundred synthesized compounds were screened for their anti mycobacterial activity against M. fortutium (NGTG 10394) as a surrogate for M. tuberculosis. The new classes of amidrazones were also screened against tuberculosis H37 Rv and antimicrobial activities against various bacteria. Fifteen tested compounds were found to provide 90-100% inhibition of mycobacterium growth of M. tuberculosis H37 Rv in the primary screen at 6.25 μg mL-1. The most active compound in the carboxamidrazone amide series had an MIG value of 0.1-2 μg mL-1 against M. fortutium. The enzyme dihydrofolate reductase (DHFR) has been a drug-design target for decades. Blocking of the enzymatic activity of DHFR is a key element in the treatment of many diseases, including cancer, bacterial and protozoal infection. The x-ray structure of DHFR from M. tuberculosis and human DHFR were found to have differences in substrate binding site. The presence of glycerol molecule in the Xray structure from M. tuberculosis DHFR provided opportunity to design new antifolates. The new antifolates described herein were designed to retain the pharmcophore of pyrimethamine (2,4- diamino-5(4-chlorophenyl)-6-ethylpyrimidine), but encompassing a range of polar groups that might interact with the M. tuberculosis DHFR glycerol binding pockets. Finally, the research described in this thesis contributes to the preparation of molecularly imprinted polymers for the recognition of 2,4-diaminopyrimidine for the binding the target. The formation of hydrogen bonding between the model functional monomer 5-(4-tert-butyl-benzylidene)-pyrimidine-2,4,6-trione and 2,4-diaminopyrimidine in the pre-polymerisation stage was verified by 1H-NMR studies. Having proven that 2,4-diaminopyrimidine interacts strongly with the model 5-(4-tert-butylbenzylidene)- pyrimidine-2,4,6-trione, 2,4-diaminopyrimidine-imprinted polymers were prepared using a novel cyclobarbital derived functional monomer, acrylic acid 4-(2,4,6-trioxo-tetrahydro-pyrimidin-5- ylidenemethyl)phenyl ester, capable of multiple hydrogen bond formation with the 2,4- diaminopyrimidine. The recognition property of the respective polymers toward the template and other test compounds was evaluated by fluorescence. The results demonstrate that the polymers showed dose dependent enhancement of fluorescence emissions. In addition, the results also indicate that synthesized MIPs have higher 2,4-diaminopyrimidine binding ability as compared with corresponding non-imprinting polymers.

Pegylation of DDA:TDB liposomal adjuvants reduces the vaccine depot effect and alters the Th1/Th2 immune responses

The adjuvant efficacy of cationic liposomes composed of dimethyldioctadecylammonium bromide and trehalose dibehenate (DDA:TDB) is well established. Whilst the mechanism behind its immunostimulatory action is not fully understood, the ability of the formulation to promote a 'depot effect' is a consideration. The depot effect has been suggested to be primarily due to their cationic nature which results in electrostatic adsorption of the antigen and aggregation of the vesicles at the site of injection. The aim of the study was to further test this hypothesis by investigating whether sterically stabilising DDA:TDB with polyethylene glycol (PEG) reduces aggregation, and subsequently influences the formation of a depot at the site of injection. Results reported demonstrate that high (25%) levels of PEG was able to significantly inhibit the formation of a liposome depot and also severely limit the retention of antigen at the site, resulting in a faster drainage of the liposomes from the site of injection. This change in biodistribution profile was reflected in the immunisation response, where lower levels of IgG2b antibody and IFN-? and higher level of IL-5 cytokine were found. Furthermore entrapping antigen within DDA:TDB liposomes did not improve antigen retention at the injection site compared surface adsorbed antigen. © 2011 Elsevier B.V. All rights reserved.

The vesicle size of DDA:TDB liposomal adjuvants plays a role in the cell-mediated immune response but has no significant effect on antibody production

The use of cationic liposomes as experimental adjuvants for subunit peptide of protein vaccines is well documented. Recently the cationic liposome CAF01, composed of dimethyldioctadecylammonium (DDA) and trehalose dibehenate (TDB), has entered Phase I clinical trials for use in a tuberculosis (TB) vaccine. CAF01 liposomes are a heterogeneous population with a mean vesicle size of 500 nm; a strong retention of antigen at the injection site and a Th1-biassed immune response are noted. The purpose of this study was to investigate whether CAF01 liposomes of significantly different vesicle sizes exhibited altered pharmacokinetics in vivo and cellular uptake with activation in vitro. Furthermore, the immune response against the TB antigen Ag85B-ESAT-6 was followed when various sized CAF01 liposomes were used as vaccine adjuvants. The results showed no differences in vaccine (liposome or antigen) draining from the injection site, however, significant differences in the movement of liposomes to the popliteal lymph node were noted. Liposome uptake by THP-1 vitamin D3 stimulated macrophage-like cells did not show a liposome size-dependent pattern of uptake. Finally, whilst there were no significant differences in the IgG1/2 regardless of the liposome size used as a delivery vehicle for Ag85B-ESAT-6, vesicle size has a size dependent effect on cell proliferation and IL-10 production with larger liposomes (in excess of 2 µm) promoting the highest proliferation and lowest IL-10 responses, yet vesicles of ~500 nm promoting higher IFN-? cytokine production from splenocytes and higher IL-1ß at the site of injection.

Formulation and the characterisation of cationic liposomal adjuvants for the delivery of a promising subunit tuberculosis vaccine

Cationic liposomes of dimethyldioctadecylammonium bromide (DDA) incorporating the glycolipid trehalose 6,6-dibehenate (TDB) forms a promising liposomal vaccine adjuvant. To be exploited as effective subunit vaccine delivery systems, the physicochemical characteristics of liposomes were studied in detail and correlated with their effectiveness in vivo, in an attempt to elucidate key aspects controlling their efficacy. This research took the previously optimised DDA-TDB system as a foundation for a range of formulations incorporating additional lipids of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), by incrementally replacing the cationic content within DDA-TDB or reducing the total DDA-TDB dose upon its substitution, to ascertain the role of DDA and the effect of DDA-TDB concentration in influencing the resultant immunological performance upon delivery of the novel subunit TB vaccine, Ag85B–ESAT-6-Rv2660c (H56 vaccine). With the aim of using the DPPC based systems for pulmonary vaccine delivery and the DSPC systems for application via the intramuscular route, initial work focused on physicochemical characterisation of the systems with incorporation of DPPC or DSPC displaying comparable physical stability, morphological structure and levels of antigen retention to that of DDA-TDB. Thermodynamic analysis was also conducted to detect main phase transition temperatures and subsequent in vitro cell culture studies demonstrated a favourable reduction in cytotoxicity, stimulation of phagocytic activity and macrophage activation in response to the proposed liposomal immunoadjuvants. Immunisation of mice with H56 vaccine via the proposed liposomal adjuvants showed that DDA was an important factor in mediating resultant immune responses, with partial replacement or substitution of DDA-TDB stimulating Th1 type cellular immunity characterised by elevated levels of IgG2b antibodies and IFN-? and IL-2 cytokines, essential for providing protective efficacy against TB. Upon increased DSPC content within the formulation, either by DDA replacement or reduction of DDA and TDB, responses were skewed towards Th2 type immunity with reduced IgG2b antibody levels and elevated IL-5 and IL-10 cytokine production, as resultant immunological responses were independent of liposomal zeta potential. The role of the cationic DDA lipid and the effect of DDA-TDB concentration were appreciated as the proposed liposomal formulations elicited antigen specific antibody and cellular immune responses, demonstrating the potential of cationic liposomes to be utilised as adjuvants for subunit vaccine delivery. Furthermore, the promising capability of the novel H56 vaccine candidate in eliciting protection against TB was apparent in a mouse model.

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.

A comparative study of cationic liposome and niosome-based adjuvant systems for protein subunit vaccines:characterisation, environmental scanning electron microscopy and immunisation studies in mice

Vesicular adjuvant systems composing dimethyldioctadecylammonium (DDA) can promote both cell-mediated and humoral immune responses to the tuberculosis vaccine fusion protein in mice. However, these DDA preparations were found to be physically unstable, forming aggregates under ambient storage conditions. Therefore there is a need to improve the stability of such systems without undermining their potent adjuvanticity. To this end, the effect of incorporating non-ionic surfactants, such as 1-monopalmitoyl glycerol (MP), in addition to cholesterol (Chol) and trehalose 6,6′-dibehenate (TDB), on the stability and efficacy of these vaccine delivery systems was investigated. Differential scanning calorimetry revealed a reduction in the phase transition temperature (T c) of DDA-based vesicles by ∼12°C when MP and cholesterol (1:1 molar ratio) were incorporated into the DDA system. Transmission electron microscopy (TEM) revealed the addition of MP to DDA vesicles resulted in the formation of multi-lamellar vesicles. Environmental scanning electron microscopy (ESEM) of MP-Chol-DDA-TDB (16:16:4:0.5 μmol) indicated that incorporation of antigen led to increased stability of the vesicles, perhaps as a result of the antigen embedding within the vesicle bilayers. At 4°C DDA liposomes showed significant vesicle aggregation after 28 days, although addition of MP-Chol or TDB was shown to inhibit this instability. Alternatively, at 25°C only the MP-based systems retained their original size. The presence of MP within the vesicle formulation was also shown to promote a sustained release of antigen in-vitro. The adjuvant activity of various systems was tested in mice against three subunit antigens, including mycobacterial fusion protein Ag85b-ESAT-6, and two malarial antigens (Merozoite surface protein 1, MSP1, and the glutamate rich protein, GLURP). The MP- and DDA-based systems induced antibody responses at comparable levels whereas the DDA-based systems induced more powerful cell-mediated immune responses. © 2006 The Authors.

Hepatitis C virus infection reduces hepatocellular polarity in a vascular endothelial growth factor-dependent manner

Background & Aims - Hepatitis C virus (HCV) infection leads to progressive liver disease, frequently culminating in fibrosis and hepatocellular carcinoma. The mechanisms underlying liver injury in chronic hepatitis C are poorly understood. This study evaluated the role of vascular endothelial growth factor (VEGF) in hepatocyte polarity and HCV infection. Methods - We used polarized hepatoma cell lines and the recently described infectious HCV Japanese fulminant hepatitis (JFH)-1 cell culture system to study the role of VEGF in regulating hepatoma permeability and HCV infection. Results - VEGF negatively regulates hepatocellular tight junction integrity and cell polarity by a novel VEGF receptor 2–dependent pathway. VEGF reduced hepatoma tight junction integrity, induced a re-organization of occludin, and promoted HCV entry. Conversely, inhibition of hepatoma expressed VEGF with the receptor kinase inhibitor sorafenib or with neutralizing anti-VEGF antibodies promoted polarization and inhibited HCV entry, showing an autocrine pathway. HCV infection of primary hepatocytes or hepatoma cell lines promoted VEGF expression and reduced their polarity. Importantly, treatment of HCV-infected cells with VEGF inhibitors restored their ability to polarize, showing a VEGF-dependent pathway. Conclusions - Hepatic polarity is critical to normal liver physiology. HCV infection promotes VEGF expression that depolarizes hepatoma cells, promoting viral transmission and lymphocyte migration into the parenchyma that may promote hepatocyte injury.