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).

Characterisation of propionibacterium acnes

Propionibacterium acnes forms part of the normal flora of the skin, oral cavity, large intestine and the external ear. Historically, P. acnes is considered to be of low virulence; however, in recent years it has been found as the aetiological agent in various pathologies including acne vulgaris, endophthalmitis, endocarditis, osteomyelitis, sarcoidosis, prosthetic hip infections and sciatica. It currently remains unclear why this normally harmless commensal can cause infection and contribute to a number of clinically significant conditions. This thesis has sought to investigate the phenotypic, genetic and antigenic properties of P.acnes strains isolated from sciatica patients undergoing microdiscectomy, normal skin, blood cultures, prosthetic hips and acne lesions. Isolates' phenotype was examined by determining their biotype by analytical profile index, antimicrobial susceptibility, virulence factor expression and serotype. A molecular typing method for P.acnes was developed using random amplification of polymorphic DNA (RAPD). Patient serum was used to screen P.acnes strains for antigens expressed in vivo and the chemical composition determined. The serodiagnostic potential and inflammatory properties of identified antigens were assessed. The optimised and reproducible RAPD protocol classified strains into three major clusters and was found to distinguish between the serotypes I and II for a large number of clinical isolates. Molecular typing by RAPD also enabled the identification of a genotype that did not react with the type I or II monoclonal antibodies and these strains may therefore constitute a previously undiscovered subspecies of P.acnes with a genetic background different from the type I and II serotypes. A major cell associated antigen produced by all strains was identified and characterised. A serological assay based on the antigen was used to measure IgG and IgM levels in serum from patients with acne, sciatica and controls. No difference in levels of antibodies was detected. Inflammatory properties of the antigen were measured by exposing murine macrophage-like cells and measuring the release of nitric oxide and tumour necrosis factor-alpha (TNF-α). Only TNF-α was elicited in response to the antigen. The phenotypic, genotypic and antigenic properties of this organism may provide a basis for future studies on P.acnes virulence and provide an insight into its mechanisms of pathogenesis.

Characterisation of coagulase-negative staphylococci associated with endocarditis

Coagulase-negative staphylococci are major aetiological agents of prosthetic valve endocarditis and an occasional cause of native valve disease. It is currently unclear how this group of usually avirulent microorganisms produces an infection associated with high rates of morbidity and mortality. The aim of this thesis was to investigate whether there are specific genotypes and/or phenotypes of coagulase-negative staphylococci with a propensity to cause infective endocarditis and to investigate any identified virulence factors as markers of infection. In this study, strains of endocarditis-related coagulase-negative staphylococci were genotyped by determining their macrorestriction genomic profile using pulsed-field gel electrophoresis. The strains were also investigated for phenotypic characteristics that predisposed the microorganisms to infect heart valves. By comparing coagulase-negative staphylococcal strains recovered from endocarditis patients with isolates from other significant infections (prosthetic device-related osteomyelitis and catheter-associated sepsis), no specific genotype or phenotype with a predilection to cause endocarditis was identified. However, the majority of the endocarditis-associated and other infection strains expressed the potential virulence factors lipase and esterase. Another approach to the investigation of virulence determinants used patient's serum to screen a Staphylococcus epidermidis NCTC 11047 genomic DNA library for cellular and secreted staphylococcal products that were expressed in vivo. The characterisation of two clones, which reacted with serum collected from a S. epidermidis-related endocarditis patient identified a staphylococcal pyruvate dehydrogenase complex E2 subunit and a novel secreted protein with homology to a Staphylococcus aureus staphyloxanthin biosynthesis protein and a secreted protein of unknown function described in Staphylococcus carnosus. Investigation of the secreted protein previously undetected in S. epidermidis, termed staphylococcal secretory antigen (SsaA), identified a potential marker of S. epidermidis-related endocarditis.

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.

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.

Non-curliation of Escherichia coli O78:K80 isolates associated with IS1 insertion in csgB and reduced persistence in poultry infection

The elaboration of curli fimbriae by Escherichia coli is associated with the development of a lacy colony morphology when grown on colonisation factor antigen agar at 25 degrees C. Avian colisepticaemia E. coli isolates screened for curliation by this culture technique showed lacy and smooth colonial morphologies and the genetic basis of the non-curliated smooth colonial phenotype was analysed. Two smooth E. coli O78:K80 isolates possessed about 40 copies of the IS1 element within their respective genomes of which one copy insertionally inactivated the csgB gene, the nucleator gene for curli fibril formation. One of these two isolates also possessed a defective rpoS gene which is a known regulator of curli expression. In the day-old chick model, both smooth isolates were as invasive as a known virulent O78:K80 isolate as determined by extent of liver and spleen colonisation post oral inoculation but were less persistent in terms of caecal colonisation.