Selection of conserved epitopes from hepatitis C virus for pan-populational stimulation of T-cell responses

The hepatitis C virus (HCV) is able to persist as a chronic infection, which can lead to cirrhosis and liver cancer. There is evidence that clearance of HCV is linked to strong responses by CD8 cytotoxic T lymphocytes (CTLs), suggesting that eliciting CTL responses against HCV through an epitope-based vaccine could prove an effective means of immunization. However, HCV genomic plasticity as well as the polymorphisms of HLA I molecules restricting CD8 T-cell responses challenges the selection of epitopes for a widely protective vaccine. Here, we devised an approach to overcome these limitations. From available databases, we first collected a set of 245 HCV-specific CD8 T-cell epitopes, all known to be targeted in the course of a natural infection in humans. After a sequence variability analysis, we next identified 17 highly invariant epitopes. Subsequently, we predicted the epitope HLA I binding profiles that determine their potential presentation and recognition. Finally, using the relevant HLA I-genetic frequencies, we identified various epitope subsets encompassing 6 conserved HCV-specific CTL epitopes each predicted to elicit an effective T-cell response in any individual regardless of their HLA I background. We implemented this epitope selection approach for free public use at the EPISOPT web server. © 2013 Magdalena Molero-Abraham et al.

Structural characterization of CD81-Claudin-1 hepatitis C virus receptor complexes

Tetraspanins are thought to exert their biological function(s) by co-ordinating the lateral movement and trafficking of associated molecules into tetraspanin-enriched microdomains. A second four-TM (transmembrane) domain protein family, the Claudin superfamily, is the major structural component of cellular TJs (tight junctions). Although the Claudin family displays low sequence homology and appears to be evolutionarily distinct from the tetraspanins, CD81 and Claudin-1 are critical molecules defining HCV (hepatitis C virus) entry; we recently demonstrated that CD81-Claudin-1 complexes have an essential role in this process. To understand the molecular basis of CD81-Claudin-1 complex formation, we produced and purified milligram quantities of full-length CD81 and Claudin-1, alone and in complex, in both detergent and lipid contexts. Structural characterization of these purified proteins will allow us to define the mechanism(s) underlying virus-cell interactions and aid the design of therapeutic agents targeting early steps in the viral life cycle.

Design and development of cationic liposomes as DNA vaccine adjuvants

Cationic liposomes have been extensively explored for their efficacy in delivering nucleic acids, by offering the ability to protect plasmid DNA against degradation, promote gene expression and, in the case of DNA vaccines, induce both humoural and cellular immune responses. DNA vaccines may also offer advantages in terms of safety, but they are less effective and need an adjuvant to enhance their immunogenicity. Therefore, cationic liposomes can be utilised as delivery systems and/or adjuvants for DNA vaccines to stimulate stronger immune responses. To explore the role of liposomal systems within plasmid DNA delivery, parameters such as the effect of lipid composition, method of liposome preparation and presence of electrolytes in the formulation were investigated in characterisation studies, in vitro transfection studies and in vivo biodistribution and immunisation studies. Liposomes composed of 1,2-dioleoyl-sn-glycero 3-phosphoethanolamine (DOPE) in combination with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 1,2-stearoyl-3- trimethylammonium-propane (DSTAP) were prepared by the lipid hydration method and hydrated in aqueous media with or without presence of electrolytes. Whilst the in vitro transfection efficiency of all liposomes resulted to be higher than Lipofectin, DSTAP-based liposomes showed significantly higher transfection efficiency than DOTAP-based formulations. Furthermore, upon intramuscular injection of liposomal DNA vaccines, DSTAP-based liposomes showed a significantly stronger depot effect at the injection site. This could explain the result of heterologous immunisation studies, which revealed DSTAP-based liposomal vaccines induce stronger immune responses compared to DOTAP-based formulations. Previous studies have shown that having more liposomally associated antigen at the injection site would lead to more drainage of them into the local lymph nodes. Consequently, this would lead to more antigens being presented to antigen presenting cells, which are circulating in lymph nodes, and this would initiate a stronger immune response. Finally, in a comparative study, liposomes composed of dimethyldioctadecylammonium bromide (DDA) in combination with DOPE or immunostimulatory molecule of trehalose 6,6-dibehenate (TDB) were prepared and investigated in vitro and in vivo. Results showed that although DDA:TDB is not able to transfect the cells efficiently in vitro, this formulation induces stronger immunity compared to DDA:DOPE due to the immunostimulatory effects of TDB. This study demonstrated, while the presence of electrolytes did not improve immune responses, small unilamellar vesicle (SUV) liposomes induced stronger humoural immune responses compared to dehydration rehydration vesicle (DRV) liposomes. Moreover, lipid composition was shown to play a key role in in vitro and in vivo behaviour of the formulations, as saturated cationic lipids provided stronger immune responses compared to unsaturated lipids. Finally, heterologous prime/boost immunisation promoted significantly stronger immune responses compared to homologous vaccination of DNA vaccines, however, a single immunisation of subunit vaccine provoked comparable levels of immune response to the heterologous regimen, suggesting more immune efficiency for subunit vaccines compared to DNA vaccines.

Studying membrane trafficking in the worm C. elegans by RNA interference

A powerful approach to gain understanding of molecular machinery responsible for membrane trafficking is through inactivation of gene function by RNA interference (RNAi). RNAi-mediated gene silencing occurs when a double-stranded RNA is introduced into cells and targets a complementary mRNA for degradation. The subsequent lack of mRNA prevents the synthesis of the corresponding protein and ultimately causes depletion of a particular gene product from the cell. The effects of such depletion can then by analyzed by functional, morphological, and biochemical assays. RNAi-mediated knockdowns of numerous gene products in cultured cells of mammalian and other species origins have provided significant new insight into traffic regulation and represent standard approaches in current cell biology. However, RNAi in the multicellular nematode Caenorhabditis elegans model allows RNAi studies within the context of a whole organism, and thus provides an unprecedented opportunity to explore effects of specific trafficking regulators within the context of distinct developmental stages and diverse cell types. In addition, various transgenic C. elegans strains have been developed that express marker proteins tagged with fluorescent proteins to facilitate the analysis of trafficking within the secretory and endocytic pathways. This chapter provides a detailed description of a basic RNAi approach that can be used to analyze the function of any gene of interest in secretory and endosomal trafficking in C. elegans. © 2013 Elsevier Inc.

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.

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).

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.

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).

Immuno-potentiating activity of surfactant vesicles in DNA and subunit vaccination

Enhanced immune responses for DNA and subunit vaccines potentiated by surfactant vesicle based delivery systems outlined in the present study, provides proof of principle for the beneficial aspects of vesicle mediated vaccination. The dehydration-rehydration technique was used to entrap plasmid DNA or subunit antigens into lipid-based (liposomes) or non-ionic surfactant-based (niosomes) dehydration-rehydration vesicles (DRV). Using this procedure, it was shown that both these types of antigens can be effectively entrapped in DRV liposomes and DRV niosomes. The vesicle size of DRV niosomes was shown to be twice the diameter (~2µm) of that of their liposome counterparts. Incorporation of cryoprotectants such as sucrose in the DRV procedure resulted in reduced vesicle sizes while retaining high DNA incorporation efficiency (~95%). Transfection studies in COS 7 cells demonstrated that the choice of cationic lipid, the helper lipid, and the method of preparation, all influenced transfection efficiency indicating a strong interdependency of these factors. This phenomenon has been further reinforced when 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): cholesteryl 3b- [N-(N’ ,N’ -dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol)/DNA complexes were supplemented with non-ionic surfactants. Morphological analysis of these complexes using transmission electron microscopy and environmental scanning electron microscopy (ESEM) revealed the presence of heterogeneous structures which may be essential for an efficient transfection in addition to the fusogenic properties of DOPE. In vivo evaluation of these DNA incorporated vesicle systems in BALB/c mice showed weak antibody and cell-mediated immune (CMI) responses. Subsequent mock challenge with hepatitis B antigen demonstrated that, 1-monopalmitoyl glycerol (MP) based DRV, is a more promising DNA vaccine adjuvant. Studying these DRV systems as adjuvants for the Hepatitis B subunit antigen (HBsAg) revealed a balanced antibody/CMI response profile on the basis of the HBsAg specific antibody and cytokine responses which were higher than unadjuvated antigen. The effect of addition of MP, cholesterol and trehalose 6,6’-dibehenate (TDB) on the stability and immuno-efficacy of dimethyldioctadecylammonium bromide (DDA) vesicles was investigated. Differential scanning calorimetry showed a reduction in transition temperature of DDA vesicles by ~12°C when incorporated with surfactants. ESEM of MP based DRV system indicated an increased vesicle stability upon incorporation of antigen. Adjuvant activity of these systems tested in C57BL/6j mice against three subunit antigens i.e., mycobacterial fusion protein- Ag85B-ESAT-6, and two malarial antigens - merozoite surface protein-1, (MSP1), and glutamate rich protein, (GLURP) revealed that while MP and DDA based systems induced comparable antibody responses, DDA based systems induced powerful CMI responses.