Hepatitis B virus (HBV) is hyperendemic to southern Africa and parts of Asia where it is a major cause of serious liver disease. Licensed antivirals for chronically infected individuals are only partially effective and approximately one million deaths occur annually as a result ofpersistent infection with the virus. Although RNA interference (RNAi) based gene silencingof HBV has been successfully demonstrated, difficulties with delivery of anti-HBV RNAieffectors remains an obstacle to their clinical use. Recombinant adenoviruses (Ads), amongst the most efficient hepatotropic gene vectors following systemic administration, have been successfully used to deliver expressed anti-HBV RNAi sequences. However, a drawback of Ad vectors is diminished efficacy and toxicity that results from stimulation of innate andadaptive immunity.To attenuate these effects we used polyethylene glycol (PEG) to modify first generation recombinant Ad (FG Ad) vectors that express an anti-HBV short hairpin (shRNA) sequence. Efficient hepatocyte transduction occurred and expressed shRNAs were processed to generate intended HBV-targeting guides. Inhibition of HBV replication was achieved after intravenous administration of PEGylated or native recombinant first generation Ads (FG Ads) to HBV transgenic mice. Circulating HBV viral particle equivalents (VPEs) remained low for 3 weeks and began to increase after 5 weeks. A second dose of PEGylated anti-HBV Ad caused a less sustained decrease in circulating VPEs, but no silencing after a second dose was observed in animals treated with unmodified vector. Release of inflammatory cytokines was elevated in animals receiving unmodified vectors and only a modest increase in monocyte chemotactic protein-1 (MCP-1) was observed in mice that received a second dose of PEG Abstract Ads. Also, polymer-conjugated vectors induced a weaker adaptive immune response and were less hepatotoxic than their unmodified counterparts. To address concerns about the transient nature of transgene expression by FG Ads resulting from immunostimulation, third generation helper-dependent (HD Ad) were utilised to delivered anti-HBV RNAi effectors. Seven days after intravenous administration of infectious HD Ads to HBV transgenic mice, 80-90% of hepatocytes were transduced and markers of HBV replication were decreased by approximately 95% which was sustained for 8 weeks. HD Ad-induced release of proinflammatory cytokines was minimal in preparations that were enriched with infectious particles. PEGylated HD Ad vectors caused similar anti- HBV effects and may be useful to evade interaction with vector-sequestrating receptors and further attenuate immunostimulation. Collectively these observations indicate that PEG modification of Ads and the use of HD Ads may have utility for delivery of therapeutic HBVsilencing sequences. Future work will focus on improving strategies to avoid immune detection and utilisation of HD Ad vectors for other HBV targeting sequences.

M.Tech. (Homoeopathy) / According to the World Health Organisation (2008), an estimated two billion individuals globally, are infected with Hepatitis B (HBV). South Africa reported 864 notified new cases between 2001 and 2004 (Department Of Health, 2005), with an estimated 3-4 million chronic HBV infected black South Africans (Kew, 2008). Kwa-Zulu Natal and Free State were the most affected provinces; while 20-39 years was the most affected age group as of 2005 (Department Of Health, 2005). Workers in the health industry, intravenous drug users and children of women who have Hepatitis B are at the most risk for contracting this disease from blood products and body fluids (Immunization Action Coalition, 2007; Boon et al., 2006), where contraction of the disease can lead to liver cirrhosis, fibrosis and hepato-cellular carcinoma (Highleyman, 2008). The Expanded Program on Immunization (EPI) of the South African Department of Health (2009) suggests vaccination for Hepatitis B should be administered at six, ten and fourteen weeks, or a dose every month for 3 months. Adverse reactions associated with the vaccine include “Guillain-Barre Syndrome, arthritis, demyelinating nervous system disease” (Pratt, 2008) and anaphylaxis (Danis & Halm, 1997). Alternatives that may assist in avoiding such symptoms include: waiting until adolescence to vaccinate (Slonim et al., 2005); only vaccinating high risk groups (Francois et al., 2002); or researching an alternative (Romm, 2001). Homeoprophylaxis is the use of homeopathy to prevent the contraction or development of disease (Zoltan, 2000) and its successful use has been recorded in various disease types and locations. There have been very few studies to show the effect of individual homeopathic nosodes used as prophylactic treatment in their related diseases, with almost none of these utilising any means of serological testing (Bevan-Jones, 2009; Frost et al., 2003; Sheffield, 2006). The aim of this study was to determine the production of the antibody to the surface antigen of Hepatitis B (anti-Hbs) due to Hepatitis B 12cH nosode administration.   Forty-three participants ranging, in ages 18 to 65 years, who tested negative for the presence of anti-HBs, took part in this four week long, double-blind, placebo controlled study. Participants were randomly placed into either the Verum or Placebo group, each group receiving four lactose powders to be taken weekly for four weeks. The Active group received lactose powders medicated with Hepatitis B 12cH, whereas the Placebo group received lactose powders medicated with 96% alcohol. Participants underwent a repeat of the serum/plasma antibody testing at the conclusion of the study to determine if there were anti-HBs present in their blood. The results were then statistically analysed using nonparametric testing: Chi-squared independent test, Mann-Whitney test and Sign test. These showed that there was no change measurable effect on the surface antigen of hepatitis B (anti-HBs) of either the Verum (active medication) or Placebo group. Primary preventative medicine is becoming increasingly popular (Kuehlein et al., 2010). Both vaccination and homeoprophylaxis are examples of primary preventative medicine, where the aim is to prevent future disease. Vaccinations encourage the production of antibodies via the activation of T-helper cells and B-lymphocytes, thus providing a template for immunity against future infections (Miller, 2000; Janeway et al., 2001). While the mechanisms of vaccination are well understood, those of homeoprophylaxis are still being investigated. One theory is that nosodes enable the body to overcome diseases. Several studies have been conducted on the effects of nosodes (Bracho et al., Prophylactic vaccination against human papilloma virus infection and disease in women: a systemic review of randomized control trial.; Gosavi et al., 2012; Shuller, 2010) and have shown favourable results in the prevention of diseases associated with those homeopathic nosodes. However, only two studies have investigated the effects that nosodes have on the antibodies of the immune system (Hoover, 2006; Neustaedter, 2002) showing the need for further studies conducted in this area. The study showed that homeopathically prepared Hepatitis B 12cH nosode is not capable of eliciting an immune response that would result in the production of the antibody to the surface antigen of Hepatitis B, and thus not able to provide immunity against Hepatitis B.

Hepatitis B virus (HBV) is a major global public health burden, with over 350 million people chronically infected. This results in approximately 600,000 liver cancer-related deaths annually. Chronic HBV infections are normally managed with long-term anti-HBV therapeutics, such as reverse transcription inhibitors, which target post-transcriptional viral processes without affecting the cccDNA. Treatment failure however is largely as a result of the stability of this episomal viral DNA. The cccDNA minichromosome serves as a reservoir of HBV DNA and is capable of re-establishing viral replication following withdrawal of treatment. Designer nucleases, like transcription activator-like effector nucleases (TALENs), have recently been used to create double stranded breaks (DSBs) at target-specific endogenous DNA loci. These nucleases are designed as pairs, which upon dimerisation cleave double-stranded DNA. Subsequent activation of the cellular non-homologous end-joining (NHEJ) pathway often results in targeted mutagenesis at the DSB site. As TALENs may be designed to bind to any DNA sequence, they are commonly used as genetic engineering agents. Inactivation of HBV cccDNA, using these engineered TALENs, presents a unique approach to disabling viral replication permanently. To investigate this, a panel of TALENs targeting the core (C), surface (S) and two different polymerase (P1 and P2) regions of HBV cccDNA were generated using a Golden gate modular assembly approach. TALENs were initially tested in two liver-derived cell lines. Firstly as transient co-transfections in Huh7 cells using a HBV replication competent plasmid, followed by long term investigations in HepG2.2.15 cells which model HBV replication in vitro. Inactivation of HBV was determined by measuring markers of viral replication, whilst TALEN-mediated targeted disruption was verified by T7 endonuclease 1 (T7E1) or CELI endonuclease assays and sequencing. In vitro, the S TALEN inhibited HBsAg secretion by 80% in Huh7 cells and 60% in HepG2.2.15 cells. Furthermore, S TALEN-mediated targeted disruption occurred in 35-47% of cccDNA copies, whilst the C TALEN resulted in 11% targeted disruption of cccDNA in without inhibition of HBsAg expression. The P2 TALEN showed no anti-HBV efficacy, however the P1 TALEN inhibited HBsAg expression by up to 60% without any evidence of site-directed cleavage. As this TALEN binding site spans the HBV Enhancer I sequence, knock-down of HBsAg expression is most likely to occur as a result of transient transcriptional repression. To confirm whether permanent repression of HBV transcription could be achieved, a KRAB-based transcription activator-like repressor (rTALE) targeting the HBV pre-S2 promoter was generated. Using an in vitro reporter gene assay, the pre-S2 rTALE inhibited luciferase expression by up to 90%. However this was only achieved using high molar concentrations of the repressor, suggesting multiple rTALEs may improve HBV transcriptional repression. As the S and C TALENs displayed significant anti-HBV efficiency in vitro, they were tested in a murine hydrodynamic injection model of HBV replication. In vivo, the S TALEN inhibited HBsAg secretion by 95% and induced disruption in 77–87% of HBV DNA targets. In addition the C TALEN inhibited HBcAg expression and induced disruption in 78-93% of HBV DNA targets. Additionally, serological analysis showed a reduction in circulating virions and no apparent liver toxicity, as determined by real-time PCR (qPCR) and aspartate transaminase (AST)/ alanine aminotransferase (ALT) liver function tests respectively. Deep sequencing at the S and C TALEN binding sites showed targeted mutagenesis of HBV DNA in samples extracted from murine hepatocytes transfected with TALENs, however wild-type sequences were exclusively detected in mice that had not been treated with anti-HBV TALENs. Furthermore, frameshift deletions were predominantly detected indicating major disruptions in the HBV surface and core sequences. These results indicate that TALENs designed to disable and silence HBV cccDNA are effective both in vitro and in vivo and as such provide a promising therapeutic approach to treat this serious infection.

Master of Dental Surgery / This work was digitised and made available on open access by the University of Sydney, Faculty of Dentistry and Sydney eScholarship . It may only be used for the purposes of research and study. Where possible, the Faculty will try to notify the author of this work. If you have any inquiries or issues regarding this work being made available please contact the Sydney eScholarship Repository Coordinator - ses@library.usyd.edu.au

Master of Dental Surgery / This work was digitised and made available on open access by the University of Sydney, Faculty of Dentistry and Sydney eScholarship . It may only be used for the purposes of research and study. Where possible, the Faculty will try to notify the author of this work. If you have any inquiries or issues regarding this work being made available please contact the Sydney eScholarship Repository Coordinator - ses@library.usyd.edu.au