Malaria is a major health problem and an effective vaccine is essential for the eradication of the disease. Despite extensive efforts, a malaria vaccine remains elusive due to the parasite's complex life cycle, diverse morphology, and immune system evasion mechanisms. Antibodies against C terminal domain of merozoite surface protein 1 (MSP1-19), a highly conserved protein and the main vaccine candidate for blood-stage malaria, can inhibit erythrocyte invasion by the parasite and alleviate the disease symptoms. However, MSP1-19 is poorly immunogenic and classic protein-in-adjuvant MSP1-19-based vaccine formulations failed to induce strong immune responses due to low immunogenicity and generation of ineffective antibodies. The aim of this study was to use hepatitis B virus core (HBc) particles to increase the immunogenicity of MSP1-19. HBc forms particles with protruding spikes and induces a strong and specific immune response against foreign epitopes inserted at the tips of the spikes. In addition, positioning of MSP1-19 on the particle can influence the accessibility of certain antibody binding sites, possibly altering elicited antibody fine specificity and vaccine efficiency. MSP1-19 domain was inserted into the middle of the HBc sequence so that it is displayed at the tips of the HBc particle. Two HBc-MSP1-19 constructs, having different insert flanking linkers, displayed soluble particle formation after bacterial expression and lysis optimization. The particles were purified and the suitability of these two constructs as malaria vaccine candidates was assessed. Firstly, binding of the conformational anti-MSP1-19 antibodies indicated that MSP1-19 domain in the chimeric proteins has the correct disulphide bond pattern which is crucial for the protective properties of an MSP1-19-based vaccine. Furthermore, electron microscopy imaging and determination of initial 3D structures confirmed that both HBc MSP1-19 constructs form particles resembling the wild-type HBc particles, meaning the insertion of MSP1-19 did not heavily distort the overall HBc particle structure. In addition, it was shown that MSP1-19 domains are displayed at the tips of the particle spikes. Particle formation and foreign epitope display are important for the epitope's immunogenicity improvement. The immunogenicity of the chimeric particles was then assessed in mice. Both constructs elicited similar high antibody titres without the use of additional adjuvants, but no difference was observed between the particulate constructs and a non-particulate control (an MSP1-19-based protein). Interestingly, although both HBc-MSP1-19 and non-particulate MSP1-19-elicited antibodies recognized native malarial parasite, only the particulate construct antibodies demonstrated a moderate parasite growth inhibition while the antibodies from the control group did not show parasite inhibition above the background levels. In conclusion, it was shown that MSP1-19 can be expressed in bacteria as a soluble correctly folded protein fused to HBc. More importantly, the fusion protein is capable of forming immunogenic particles which generate antibodies that recognize native MSP1 and inhibit parasite growth more effectively than the protein without the HBc. Therefore, this work lays grounds and supports further chimeric HBc-MSP1-19 research and development.

Plusieurs données expérimentales suggèrent que la protéine Core du virus de l’Hépatite B (HBV), en plus de ses fonctions structurales pour la formation des nucléocapsides dans le cytoplasme, pourrait avoir des fonctions régulatrices importantes dans le noyau des hépatocytes infectés. En effet, Core s’associe à l’ADNccc et aux promoteurs de certains gènes cellulaires dans le noyau des hépatocytes infectés et pourrait ainsi contrôler leur régulation transcriptionnelle. De plus, de par sa capacité à lier les ARN, elle pourrait également participer au métabolisme post-transcriptionnel de gènes viraux et/ou cellulaires. Pour caractériser ces fonctions, nous avons réalisé une analyse protéomique des facteurs cellulaires qui interagissent avec la protéine Core dans le noyau d’hépatocytes humains. Cet interactome a mis en évidence un grand nombre de protéines de liaison aux ARN (RBP), qui participent au métabolisme des ARN et en particulier aux mécanismes d’épissage. Deux interactants majeurs de Core ont été plus particulièrement étudiés, SRSF10 et RBMX, impliqués notamment dans l’épissage et la réparation de l’ADN. Une analyse fonctionnelle effectuée par une approche siRNA a montré que SRSF10 et RBMX affectent différemment le niveau des ARN viraux, vraisemblablement en agissant à des étapes différentes du cycle viral. De même, un composé ciblant l’activité de certaines RBP diminue fortement la réplication d’HBV en affectant l’accumulation des ARN viraux. Ainsi, ces résultats suggèrent que Core pourrait interagir avec certaines RBP pour contrôler le destin des ARN viraux et/ou cellulaires, une piste intéressante pour le développement de nouvelles stratégies antivirales ciblant l’hôte / Converging evidences suggest that the Hepatitis B virus (HBV) core protein, beside its well-known structural role to form nucleocapsids in the cytoplasm, could have important regulatory functions in the nucleus of infected hepatocytes. Indeed, nuclear Core was shown to associate with the cccDNA and to the promoters of some cellular genes, suggesting that Core may control viral and/or cellular gene expression. In addition, Core has the capacity to bind RNA, and may thus regulate HBV RNA metabolism. To elucidate these functions, we performed a proteomic analysis of the cellular factors interacting with nuclear Core in human hepatocytes. This interactome revealed a majority of highly interconnected RNA-binding proteins (RBPs), which participate in several steps of mRNA metabolism, including transcription, splicing and nuclear egress. We focused on two major Core-interacting factors, SRSF10 and RBMX that were previously involved in splicing and DNA repair. Functional analyses performed by a siRNA approach indicated that RBMX and SRSF10 were able to differentially regulate the levels of all viral RNAs most likely by acting at different steps of the viral life-cycle. Similarly, a small compound, affecting the activity of selected RBPs, severely impaired HBV replication by strongly reducing viral RNA accumulation. Altogether, these results strongly suggest that Core interacts with some selected RBPs to control the fate of viral and/or cellular RNAs and provide new critical information for the development of novel host-targeting antiviral agents (HTA)

Student Number : 9903144J - MSc (Med) dissertation - Faculty of Health Sciences / Since the introduction of vaccination against hepatitis B virus (HBV) infection in South Africa, at least one case of infection despite vaccination has occurred. The purpose of this study was to determine whether this infection was the result of mutations within the region of the surface (S) gene encoding the a determinant epitopes of the hepatitis B surface antigen, which permitted viral vaccine-escape. HBV DNA was extracted from the serum and liver tissue of the patient and amplified within the complete 3 215 bp genome and S gene, respectively. Following cloning, sequencing revealed a minor population displaying unique or uncommon S gene mutations that resulted in C138R, C139R, K141R, P142L, T143A, N146D, and T148A amino acid substitutions in the clones from the serum, and C139Y and D144N in the clones from the liver. Such isolates may represent South African HBV vaccine-escape mutants that caused chronic infection in the host prior to their reversion to wild-type.

Hepatitis B virus chronically infects 350-400 million people worldwide. It often leads to hepatocellular carcinoma, which causes >1 million deaths yearly. Current therapies prevent new viral genome formation but do not target pre-existing viral genomic DNA, thus curing only ~1/2 of patients. We targeted hepatitis B virus DNA for cleavage using zinc finger nucleases, which cleave as dimers. Co-transfection of our zinc finger nuclease pair with a target plasmid containing the hepatitis B virus genome resulted in specific cleavage. After three days in culture, 26% of the target remained linear, while ~10% was cleaved and mis-joined tail-to-tail. A portion of cleaved plasmids are repaired in cells, often with deletions and insertions. To track misrepair, we introduced an XbaI restriction site in the spacer between the zinc finger nuclease sites. Targeted cleavage and misrepair destroys the XbaI site. After three days in culture, ~6% of plasmids were XbaI resistant. 13 of 16 clones sequenced contained frameshift mutations that would lead to dramatic truncations of the viral core protein. These results demonstrate for the first time the feasibility of targeting episomal viral DNA genomes in cells using zinc finger nucleases. This strategy is broadly applicable toward inactivating other DNA viruses within cells. A major concern for the therapeutic use of zinc finger nucleases is off-target cleavage. To measure specificity, we employed in vitro assays and developed a bioinformatics method to find off-target cleavage sites in cultured cells. These sites can then be PCR amplified and tested using a mutation detection assay that we developed.

Background: The most recent estimate for the number of Australians living with chronic hepatitis B virus (HBV) infection is 150,000, with over one million ever having been infected. One in four people with chronic infection will die as a result. Worldwide, the burden of chronic HBV infection is great. As many as 400 million people are chronically infected, and the World Health Organisation estimates that as a result HBV infection is the tenth leading cause of death. / Aim: The aim of the research presented in this thesis is to improve the accuracy and relevance of our understanding of the epidemiology and control of HBV infection in Australia, through the development of new methodological approaches to the collection and analysis of relevant epidemiological data. / Methods: Three novel approaches were adopted. First, a serosurvey of a randomised, age-structured convenience sample of over 3200 specimens was performed spanning the period from 1995 to 2005 to estimate the prevalence of markers of infection with, and immunity to HBV. Secondly, a comparative analysis of the serosurvey results with national surveillance notifications since 1971 and migration records since 1945 was undertaken. Finally, a complex deterministic mathematical model of HBV infection in Australia was constructed simulating the entire population between 1951 and 2050. / Results: The serosurvey indicates that chronic infection with HBV is more common in the Victorian population than existing national serosurvey estimates suggest, and the coverage of immunisation programs (particularly of adolescents) is far from universal. Significant geographic, age, and gender disparities in the prevalence of chronic HBV infection were identified in the serosurvey, which appear in part to relate to historical migration patterns and which could be used to develop a targeted and effective public health response. The comparative analysis of the serosurvey results with notifications and migration data demonstrates coherence of these disparate sources of information, and suggest that knowledge of migration patterns can lead to robust predictions of future notifications. The novel regression model developed implies that at least 50,000 people with chronic HBV infection are undiagnosed. The mathematical model of HBV infection in the Australian population is unique in many respects, and has been validated against external data to provide reassurance regarding the accuracy of the simulated outcomes. Some of these outcomes include an estimated 160,000 Australians living with chronic HBV infection in 2009, increasing by several thousand people every year, and that less than 5 per cent of chronic infections entering the population are able to be addressed by domestic vaccination or other prevention programs. / Conclusion: The new insights into the epidemiology of HBV infection in Australia provided by the approaches described all suggest a large and increasing burden of chronic HBV infection. New approaches are needed to provide essential policy outcomes to assist and empower Australians living with chronic HBV infection. If this does not occur, the economic and human costs to our community are likely to become great.

No description available.

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Medicine / Master / Master of Philosophy

Woodchucks are the preferred animal model to study chronic viral hepatitis and the development of hepatocellular carcinoma (HCC), which occurs as a result of infection with woodchuck hepatitis virus. Significant elevations in the phosphomonoester peak in 31P-MRS spectrum correlated to the presence of HCC. Ex vivo 31P-NMR determined that HCC tissue had significantly elevated concentrations of PC compared to uninfected control tissues, confirming that PME is specific to the tumour’s growth. Finally, a recombinant vaccinia virus was constructed to stimulate the immune systems of infected woodchucks against cells expressing core antigens. Despite reductions in surface antigen expression and viral load, elevations in serum GGT and the PME in 31P-MRS indicated that there was tumour growth in treated woodchucks. In conclusion, the PME peak represents a potential biomarker of cancerous growth when used in conjunction with serological tests to detect HCC in the liver due to chronic hepatitis virus infection.

Hepatitis B virus (HBV) is a life-threatening pathogen with major economic significance. Acute infection in adults is common, albeit usually self-limiting. Importantly, infection in infants typically results in chronic infection and increased incidence of hepatocellular carcinoma (HCC). Furthermore, the infectious carrier state is perpetuated in chronically infected individuals. Successful immuno-therapeutic vaccination would reduce the incidence of chronic infection and of HCC as well as reduce transmission of the disease. Recovery from acute and chronic HBV infection typically occurs in the presence of robust antigen-specific humoral and cellular immune responses (CMI), whereas these responses are low or absent in chronically HBV-infected individuals. Therefore, it was hypothesised that effective stimulation of both humoral and CMI responses, in conjunction with currently available antiviral therapies, may contribute significantly to development of vaccines for treatment of chronic HBV infection. The duck hepatitis B virus (DHBV) model of HBV infection was used to test novel vaccine strategies that could complement existing antiviral therapeutic approaches to treat chronically HBV-infected humans. To this end, three separate vaccine studies were conducted to investigate potential therapeutic regimes. Methods to assess the efficacies of the vaccine strategies included immunoperoxidase detection of viral antigen and immune cell markers within the liver and development of sensitive assays to monitor levels of DHBV DNA, duck hepatitis B virus surface antigen (DHBsAg), antibodies to duck hepatitis B core (anti-DHBc) and surface antigens (anti-DHBs) in serum were developed and validated which allowed monitoring of the kinetics of the humoral immune response following vaccination and the course and outcome of experimental DHBV infection. The first vaccine study tested the protective efficacy of DNA vaccines encoding either the small form of DHBsAg (DHBs) protein or the larger antigen (DHBpre-S/S), These were administered to ducks at day 4 and 14 of age. On the same day as the second vaccination, ducks were challenged intravenously with DHBV. Immunoperoxidase staining of biopsy tissue collected at day 4 p.i. showed significant decreases in the number of DHBV infected hepatocytes in ducks receiving the DNA vaccines compared to the mock-vaccinated control ducks. Significant protection against development of chronic DHBV infection was observed in ducks vaccinated with DNA vaccines expressing either pre-S/S or S protein. Although anti-DHBs antibodies were not detected prior to DHBV challenge, the decrease in the percentages of DHBV-infected hepatocytes at day 4 p.i is suggestive that neutralisation of the inoculum by low-level anti-DHBs antibodies in cohort with CMI responses induced by vaccination were the most probable mechanisms of action. The second vaccine study examined the protective efficacy of a novel whole-cell vaccine that expressed the DHBV core antigen (DHBcAg). Ducks were vaccinated on day 4 and 14 of age and DHBV challenge was administered 4 days later. Detectable anti-DHBc antibodies were generated as soon as 4 days after the initial vaccination suggesting that this regimen elicited increased immunogenicity than vaccination with DNA vaccines alone. In contrast to the first vaccine study with DNA vaccines expressing DHBsAg, no significant differences in the percentage of DHBV-infected hepatocytes were observed in biopsy tissue collected at day 4 p.i.This finding is confirmation that anti-DHBc antibodies were not neutralising to the initial DHBV inoculum. However, significant protection against development of chronic DHBV infection was observed in the whole-cell vaccinated ducks suggesting that the mechanism of protection was consistent immune-mediated killing of DHBV-infected hepatocytes following CMI responses to determinants of DHBcAg. The final vaccine study involved a combination strategy of antiviral drug Entecavir (ETV) and prime-boost vaccination with DNA vaccines and recombinant fowlpoxvirus (rFPV) expressing DHBV antigens. Immediately following DHBV infection, ducks were dosed by oral gavage with the antiviral drug Entecavir (ETV) and at the same time received the priming DNA vaccines encoding DHBV antigens. Seven days later the boosting vaccination consisting of recombinant fowlpox viruses (rFPV) also expressing DHBV antigens was administered. Extraordinary protection was observed, with 100% of ducks given combination therapy rapidly resolved their DHBV infection while 100% of non-treated ducks developed chronic infection. It was concluded that protection resulted from a combination of at least three factors. First, reduction and control of DHBV levels with the aid of ETV; secondly, stimulation of surface antigen-specific humoral immune responses resulting in neutralisation of newly produced virions; and finally, the combined up-regulation of CMI responses against DHBV core and surface antigens, resulting in elimination of infected hepatocytes. The four manuscripts that comprise this thesis provide insights into the viral kinetics and immune responses that follow DHBV infection and/or vaccination of ducks. The results provide new directions for future vaccine studies aimed at developing effective treatments for chronic HBV infection. / Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, 2008