Gene therapy has shown potential in alleviating a wide range of diseases, ranging from viral infections to autosomal diseases. One of the obstacles to gene therapy reaching its full potential is the inadequacy of methods to deliver therapeutic nucleic sequences. Current delivery of gene therapy entails use of viral and non-viral vectors. Viral vectors are however associated with drawbacks such as potential toxicity, high cost and labour-intensive production. Thus non-viral delivery alternatives are being developed in an attempt to overcome difficulties associated with nucleic acid delivery for gene therapy. Virus-like particles are potentially very useful delivery vehicles as their production is simple and cost effective, the particle surface is amenable to modification and the capsid interior can be altered to accommodate a variety of cargoes. One such particle is the recombinant HBV capsid, which comprises a single species of protein and is tolerant of amino acid substitutions on the surface exposed immunodominant loops. This study aimed to enhance the vector-like properties of the HBV virus-like particle by including amino acid substitutions on the particle surface. These substituted residues in turn provided a conjugation site for tropic and immuno evasive moieties. It was found that lysine substitutions resulted in poor conjugation to the capsid surface, whereas substituted cysteine residues resulted in almost all protein-moiety conjugates forming. In order to introduce lysine and cysteine substitutions, a novel method of cloning into the HBV was generated. In doing so, complicated procedures associated with cloning into the immunodominant loop of the HBV capsid have been alleviated. Ligands containing galactose were utilised on the surface of both the HBV capsid and liposomes to confer hepatotropism. The presence of the galactose moieties on the surface of the HBV capsid prevented indiscriminate cellular uptake in cultured cells, however did not improve hepatotropism. Galactose ligands on the surface of liposomes did improve transfection efficiency, however they required a short linker distance between liposome surface and galactose group. The inclusion of galactose in liposome formulations also provided a means to deliver siRNA to the liver of transgenic HBV mice. It is believed that with alterations to the ligand structure, it is possible to provide HBV capsids with hepatotropism in future experimentation. This study demonstrated that the exposed external surface of the HBV capsid is amenable to convenient conjugation, which potentially facilitates immune evasion and conferring of defined tropism.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 4 uppsatser.

Orientador: Marjorie de Assis Golim / Resumo: A infecção crônica pelo vírus da hepatite B (VHB) é a principal causa de cronificação da hepatite, cirrose hepática e carcinoma hepatocelular em humanos. A vacinação contra hepatite B é essencial a saúde da população, sendo a medida de menor custo e maior eficiência para controlar o vírus. A vacina desencadeia resposta imune com produção de anticorpos contra o antígeno de superfície do VHB (anti-HBs), contudo, alguns indivíduos não desenvolvem imunidade efetiva, havendo necessidade de doses adicionais. Assim, estimular a resposta imune nos indivíduos já vacinados, porém pouco respondedores ou não respondedores previamente, poderia contribuir para o aumento da produção de anticorpos e persistência dos mesmos ao longo do tempo. Considerando o potencial imunomodulador de β-glucanas, inclusive no aumento da ativação de células T e B em resposta a antígenos, propôs-se neste estudo avaliar a influência do uso de β-glucanas como suplemento alimentar em indivíduos com imunidade não efetiva pós-vacina, que necessitassem de dose booster. Foram incluídos 46 doadores de sangue do Hemocentro de Botucatu, com idade entre 18 anos e 25 anos completos, do sexo masculino, vacinados com três doses para hepatite B na primeira infância. Aqueles que apresentaram anti-HBs<10UI/L foram considerados não imunes, sendo mantidos no estudo (n=31) e divididos em dois grupos de forma aleatória. Ambos os grupos receberam booster da vacina, sendo um grupo suplementado via oral com cápsulas de amido (n=11; pl... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Chronic hepatitis B virus (HBV) infection is the main cause of hepatitis chronification, liver cirrhosis and hepatocellular carcinoma in humans. Vaccination against hepatitis B is essential to the health of the population, being the least costly and most efficient measure to control the virus. The vaccine triggers an immune response with the production of antibodies against HBV surface antigen (Anti-HBs), however, some individuals do not develop effective immunity, requiring additional doses. Thus, stimulating the immune response in individuals who have already been vaccinated, but with little or no previous response, could contribute to increased antibody production and persistence over time. Considering the immunomodulatory potential of β-glucans, including the increased activation of T and B cells in response to antigens, it was proposed in this study to evaluate the influence of the use of βglucans as a food supplement in individuals with post-vaccine ineffective immunity , who needed a booster dose. 46 blood donors from the Hemocenter of Botucatu, aged between 18 and 25 years old, male, who received three doses of hepatitis B vaccine in childhood were included. Those who had anti-HBs <10 IU / L were considered non-immune, being maintained in the study (n = 31) and randomly divided into two groups. Both groups received a vaccine booster, one group supplemented orally with starch capsules (n = 11; placebo - 500mg / day), and the other with β-glucans (n = 20; 500mg / day) f... (Complete abstract click electronic access below) / Mestre

Zhu, Lin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 103-118). / Abstracts in English and Chinese. / Contents --- p.I / List of Abbreviations --- p.IV / List of Tables and Figures --- p.V / Chapter Chapter One: --- Introduction --- p.1 / Chapter 1.1 --- Viral Hepatitis --- p.2 / Chapter 1.2 --- Global Epidemiology of HBV --- p.3 / Chapter 1.3 --- Modes of Transmission --- p.4 / Chapter 1.4 --- Diagnostic Tests --- p.5 / Chapter 1.4.1 --- HBeAg and Anti-HBe --- p.7 / Chapter 1.4.2 --- Serum Enzymes --- p.8 / Chapter 1.4.3 --- HBV DNA Assays --- p.9 / Chapter 1.4.3.1 --- HBV DNA Assays --- p.9 / Chapter 1.4.3.2 --- Clinical Applications of DNA Assays --- p.10 / Chapter 1.4.4 --- Histology --- p.13 / Chapter 1.5 --- Natural Course of Chronic Hepatitis infection --- p.18 / Chapter 1.5.1 --- Phases of chronic hepatitis B --- p.18 / Chapter 1.5.2 --- HBeAg-negative chronic hepatitis B --- p.21 / Chapter 1.6 --- Molecular biology of HBV --- p.23 / Chapter 1.6.1 --- Overview --- p.23 / Chapter 1.6.2 --- Genomic structure and organization --- p.24 / Chapter 1.6.2.1 --- Surface ORF --- p.24 / Chapter 1.6.2.2 --- Precore/Core ORF --- p.25 / Chapter 1.6.2.3 --- Polymerase ORF --- p.25 / Chapter 1.6.2.4 --- X ORF --- p.26 / Chapter 1.7 --- Genetic Variation of HBV --- p.31 / Chapter 1.7.1 --- HBV genotypes --- p.31 / Chapter 1.7.2 --- Predominant genotypes and their subgroups in Asia --- p.33 / Chapter 1.7.3 --- HBV mutations --- p.36 / Chapter 1.7.3.1 --- Precore mutations --- p.37 / Chapter 1.7.3.2 --- Core promoter mutations --- p.38 / Chapter 1.7.3.3 --- Other Mutations associated with clinical outcome --- p.40 / Chapter Chapter Two: --- Methodology --- p.44 / Chapter 2.1 --- Aims and Hypothesis --- p.45 / Chapter 2.1.1 --- Aims --- p.46 / Chapter 2.1.2 --- Hypothesis --- p.47 / Chapter 2.2 --- Patient Recruitment --- p.48 / Chapter 2.3 --- Laboratory Assays --- p.49 / Chapter 2.3.1 --- Preparation of serum HBV DNA --- p.49 / Chapter 2.3.2 --- Quantification of serum HBV DNA --- p.51 / Chapter 2.4 --- Full-genome Amplification of HBV DNA --- p.53 / Chapter 2.5 --- Full-genome Sequencing of HBV DNA --- p.55 / Chapter 2.6 --- Assembly of HBV Full-genome Sequence --- p.58 / Chapter 2.7 --- Phylogenetic Analysis --- p.59 / Chapter 2.7.1 --- Construction of phylogenetic tree --- p.59 / Chapter 2.7.2 --- Genotype and subgenotype determination --- p.60 / Chapter 2.8 --- HBV Mutations --- p.62 / Chapter 2.9 --- Info-gain program --- p.64 / Chapter 2.10 --- Statistical Analysis --- p.65 / Chapter Chapter Three: --- Results --- p.67 / Chapter 3.1 --- Patient Information --- p.68 / Chapter 3.2 --- Phylogenetic Analysis --- p.69 / Chapter 3.3 --- HBV genotypes/subgenotypes --- p.76 / Chapter 3.4 --- “Hot-spo´tح HBV Mutants --- p.79 / Chapter 3.5 --- HBV Mutation Associated with Liver Fibrosis --- p.82 / Chapter 3.5.1 --- Mutant selection --- p.82 / Chapter 3.5.2 --- Clinical significance of novel mutants --- p.84 / Chapter Chapter Four: --- Discussion --- p.88 / Chapter 4.1 --- Full-genome Sequencing Strategy --- p.89 / Chapter 4.2 --- HBV genotypes/subgenotypes Distribution and Disease Activity --- p.90 / Chapter 4.2.1 --- HBV genotypes/subgenotypes distribution --- p.90 / Chapter 4.2.2 --- Clinical significance of genotypes/subgenotypes --- p.91 / Chapter 4.3 --- HBV Hotspot Mutants and Disease Activity --- p.93 / Chapter 4.4 --- HBV Novel Mutants --- p.96 / Chapter 4.5 --- Limitation of the Study and Future Work --- p.97 / Chapter 4.5.1 --- Limitation --- p.97 / Chapter 4.5.2 --- Future Direction --- p.98 / Chapter Chapter Five: --- Conclusions --- p.99 / References --- p.102