Thesis (M Med (Virology))-- University of Limpopo (Medunsa Campus), 2013 / Introduction: The hepatitis B virus (HBV) causes a serious type of liver disease referred to as hepatitis B, which is associated with various fatal sequelae following the onset of chronic infection. As a result of the global burden of chronic HBV infection, HBV-related mortality is currently estimated at 620 000 annual deaths worldwide (Hwang and Cheung, 2011). The World Health Assembly (WHA) recommended in 1992 that the hepatitis B vaccine be incorporated into national immunization programmes universally, especially in the hyperendemic regions of the world, in order to curb the global burden of hepatitis B (WHO, 1992). Accordingly, South Africa introduced the hepatitis B vaccine into the national Expanded Programme on Immunization (EPI-SA) in April 1995. Almost 17 years later, South Africa has not conducted any nationwide serosurveys to monitor the population impact of the hepatitis B vaccine. Instead, a number of field and laboratory studies have been conducted only in vaccinated children within the first 5 years of life and as such reports on the short¬term impact made by the hepatitis B vaccine in the country have largely relied on these studies (Tsebe et al., 2001; Schoub et al., 2002; Simani et al., 2008). The aim of the current study therefore was to assess the population impact made by the hepatitis B vaccine post its introduction into EPI-SA using an age stratified, cross-sectional study. The objectives were to compare the prevalence of HBV exposure between the post- and pre-vaccination populations of South Africa, determine the influence of HIV infection on the prevalence of HBV exposure between the post- and pre-vaccination populations of South Africa by performing a subset analyses, and lastly, to perform molecular characterization of hepatitis B surface and polymerase genes in HBV DNA positive individuals. Materials and Methods: This was an explorative and descriptive retrospective, cross¬ sectional study based on recently tested and stored blood samples from the NHLS fi Diagnostic Laboratory in the Department of Virology. For the purpose of this study, these samples were obtained after Ethics approval, and two target populations identified based on the year (Le. 1995) the hepatitis B vaccine was introduced into EPI-SA; a post-vaccination population consisting of 605 blood samples from individuals aged 1-15 years and a pre¬vaccination population consisting of 601 blood samples from individuals aged 16-25 years. The post-vaccination population was further stratified by age as follows; 1-5,6-10 and 11-15 years, in order to assess immunity and chronic carriage of HBV across the different age groups. All samples were tested for the following primary serological markers; HBsAg, anti¬HBc and anti-HBs, to determine the prevalence of HBV chronic carriage, past HBV exposure and immunity to HBV infection respectively. Samples were further assessed for the vi incidence of acute HBV infection by testing for IgM anti-HBc. All serological testing was performed using the Elecsys@ 2010 Immunoassay System. Samples with serological evidence of infection or exposure to HBV were selected and screened for HBV DNA using a real time PCR assay to determine the prevalence of active HBV infection within this group. Study subjects with records of their HIV status, either positive or negative, were also pooled for subset analyses in order to determine the influence of HIV infection on immunity and chronic carriage of HBV. Finally, samples positive for HBV DNA were subjected to molecular characterization of the hepatitis B surface (S) and polymerase (pol) genes. Results: Following serological screening, immunity to HBV infection was found to be significantly (p<0.001) higher (56.7%) in the post-vaccination population than in the pre-vaccination population (15.5%). Within the post-vaccination population alone, immunity was found to wane with increasing age from 76.1 % in those 1-5 years of age to 50.0% in those 6-10 years and 44.2% in those 11-15 years of age. Chronic carriage on the other hand was significantly (p=0.008) reduced in the post-vaccination population with 1.5% HBsAg prevalence as compared to 4.0% in the pre-vaccination population. Within the different age strata, chronic carriage increased with increasing age (0.5% in 1-5 years; 1.3% in 6-10 years; 2.5% in 11-15 years). Overall, no acute HBV infection was detected within the post-vaccination population, while a 14.6% prevalence rate of acute HBV infection was found for the pre-vaccination population. From the subset analyses, immunity was found to be significantly (p<0.001) higher in the HIV uninfected population as compared to the HIV infected population; 82.5% versus 22.0% in the post-vaccination population and 26.7% versus 0% in the pre-vaccination population, while chronic carriage was found to be higher in the HIV infected population than in the HIV uninfected population. Following molecular characterization of the HBV S gene, it was revealed that the majority of the viral isolates were genotype A, with only 1 genotype D isolate found. A number of notable amino acid i$ variations were also detected within the antigenic region of the HBsAg of viral isolates, inCluding the K122R, N131T, T143S, and E164D mutations. Conclusion: Introduction of the hepatitis B vaccine into EPI-SA has shown remarkable success in children under the age of 5 years. Overall, immunity and chronic carriage of HBV within the post-vaccination population has been greatly impacted by hepatitis B immunization. Within the HIV infected population, susceptibility to HBV infection remains a cause for concern. Finally, although amino acid variations within the viral HBsAg are present, vaccine escape-related mutants appear to be rare or even absent within the South African population. vii

Sub-Saharan Africa has a high prevalence of hepatitis B virus (HBV) infections. Health care workers (HCWs) are at high risk of contracting HBV infection through their occupation. Vaccination of HCWs against HBV is standard practice in many countries, but is often not implemented in resource-poor settings. We aimed with this cross-sectional study to determine HBV prevalence, HCW vaccination status, and the risk factors for HCWs contracting HBV infection in Tanzania. We enrolled 600 HCWs from a tertiary Tanzanian hospital. Their demographics, medical histories, HBV vaccination details and risk factors for contracting blood-borne infections were collected using a standardized questionnaire. Serum samples were tested for HBV and hepatitis C virus (HCV) markers by ELISA techniques, PCR and an anti-HBs rapid test. HCWs were divided in two subgroups: those at risk of contracting HBV (rHCW 79.2 %) via exposure to potentially infectious materials, and those considered not at risk of contracting HBV (nrHCW, 20.8 %). The overall prevalence of chronic HBV infection (HBsAg+, anti-HBc+, anti-HBs-) was 7.0 % (42/598). Chronic HBV infection was found in 7.4 % of rHCW versus 5.6 % of nrHCW (p-value = 0.484). HCWs susceptible to HBV (HBsAg-, anti-HBc-, anti-HBs-) comprised 31.3 %. HBV immunity achieved either by healed HBV infection (HBsAg-, anti-HBc+, anti-HBs+) or by vaccination (HBsAg-, anti-HBc-, anti-HBs+) comprised 36.5 % and 20.2 %, respectively. 4.8 % of participants had indeterminate results (HBsAg-, anti-HBc+, anti-HBc-IgM-, anti-HBs-). Only 77.1 % of HCWs who received a full vaccination course had an anti-HBs titer >10 ml/U. An anti-HBs point-of-care test was 80.7 % sensitive and 96.9 % specific. There was a significantly higher risk for contracting HBV (anti-HBc+) among those HCW at occupational risk (rHCW) of older age (odds ratios (OR) in rHCW 3.297, p < 0.0001 vs. nrHCW 1.385, p = 0.606) and among those HCW being employed more than 11 years (OR 2.51, p < 0.0001***). HCV prevalence was low (HCV antibodies 1.2 % and HCV-RNA 0.3 %). Chronic HBV infection is common among Tanzanian HCWs. One third of HCWs were susceptible to HBV infection, highlighting the need for vaccination. Due to high prevalence of naturally acquired immunity against HBV pre-testing might be a useful tool to identify susceptible individuals. / Die Studie konnte in einem Krankenhaus der Maximalversorgung im Norden Tansanias eine hohe Prävalenz chronischer Hepatitis B-Infektionen beim Gesundheitspersonal zeigen. Weiterhin hatten die Teilnehmer ein hohes Risiko, eine HBV-Infektion im Laufe ihres Berufslebens zu erwerben. Bezüglich der Hepatitis C-Infektion zeigte sich insgesamt eine sehr niedrige Prävalenz. Ein besonders hohes Risiko, mit Hepatitis B infiziert zu werden, hatte Personal mit direktem Kontakt zu Blut oder Nadelmaterial. Ein Drittel des Krankenhauspersonals wies keine Immunität gegen Hepatitis B auf und war somit weiterhin einem Infektionsrisiko ausgesetzt. Etwas mehr als ein Drittel des Kollektivs wies die Antigen-/Antikörperkonstellation einer ausgeheilten Infektion auf. Der Infektionszeitpunkt ist aufgrund der häufig inapparenten klinischen Verläufe retrospektiv nicht zu eruieren. Ein weiterer Teil konnte mittels Immunisierung durch eine bereits im Vorfeld durch Hilfsgelder finanzierte Impfaktion eine Immunität erwerben. Der Impferfolg wurde nach dieser Impfaktion jedoch nicht serologisch verifiziert. Bei 40 Personen, die angaben an der Impfaktion teilgenommen zu haben, konnten keinerlei Antikörper nachgewiesen werden. Retrospektiv zeigte die Impfaktion mit 76% eine niedrige Erfolgsrate, was unter anderem auf einen hohen Teil an bereits HBV-Infizieren Teilnehmer zurückzuführen ist, bei denen eine Impfung nutzlos ist.

Master of Science / Periodontal disease is one of the most widespread diseases in humans and is characterised by chronic gingival inflammation and B cell accumulation and resorption of the crest of alveolar bone with subsequent loss of teeth. Porphyromonas gingivalis has been identified as a putative aetiological agent for periodontitis. The aim of the research presented in this thesis was to investigate, using in vitro systems, the responses of autoreactive B-1 and B-2 cells to enterobacterial and nonenterobacterial lipopolysaccharide (LPS) to shed light on the pathogenesis of chronic periodontitis and other diseases involving B cell accumulation and autoantibody production. The hypotheses tested were: (1) B cells respond differently to enterobacterial and non-enterobacterial LPS. (2) B-1 cells are activated by a lower concentration of LPS than B-2 cells. (3) LPS stimulation results in preferential accumulation of B-1 cells. Findings consistent with these hypotheses would provide new evidence for different roles for B-1 and B-2 cells in immune responses and that LPS stimulation could lead to B-1 cell accumulation in diseases thus characterised. Initial experiments investigated the responses of representative B-1 (CH12) and B-2 (WEHI-279) cell lines to preparations of P. gingivalis and Salmonella enteritidis LPS utilising flow cytometric and quantitative molecular methods. The cell lines responded differently to the two LPS preparations. There were significant but limited effects on viability and proliferation in the WEHI-279 cell line, but no significant changes in mRNA expression levels for genes including Toll-like receptors (TLR2, TLR4, RP105), immunoglobulin (IgM), cytokines (IL-6, IL-10), co-stimulatory molecules (CD80, CD86), and regulators of apoptosis (Bcl-2, Bax). In the CH12 cell line however, LPS stimulation had greater effect. Addition of S. enteritidis LPS from a threshold level of 100ng/mL was found to rescue the cells from death, reflected by the percentage viability and proliferation. Stimulation of CH12 cells with S. enteritidis LPS also led to a decrease in expression of RP105 mRNA, which may be part of a negative feedback loop. Interestingly, stimulation with low concentrations P. gingivalis LPS appeared to inhibit proliferation but high LPS concentrations stimulated proliferation of CH12 cells, although no further significant effects were noted in other analyses. Evidence was found that CH12 cells have a high basal level of activation. This suggests that this line is constitutively activated. Stimulation with P. gingivalis or S. enteritidis LPS did not affect the level of CD80 mRNA expression. It is possible that the CH12 line constitutively expresses a maximal level of CD80 (and possibly CD86) and further stimulation will not cause any increase. Since S. enteritidis LPS appeared to have more pronounced effects on both B cell populations, this LPS was used to further investigate B cell subset responses in a mixed splenocyte culture system. Experiments examining percentage viability and number of viable cells indicated that B-1 and B-2 B cells responded differently to LPS stimulation. A threshold level for B-2 cell response (significant increase in cell number) was found to be 100ng/mL LPS, in contrast to the B-1 B cell subset which were only significantly different to the unstimulated cells when stimulated with 50μg/mL LPS. By examining the expression of CD80, the majority of murine splenic B-1 cells were found to activated prior to any LPS stimulation in vitro. In contrast, the B-2 subset showed significant increase in CD80 expression only at high (≥10μg/mL) LPS concentrations. Studies of the division index of B-1 and B-2 cells showed a significant response in both subsets following stimulation with 1μg/mL and 10μg/mL LPS. However, overall, the results are inconsistent with LPS driving the preferential accumulation of B-1 cells in disease states. These experiments provided useful evidence that supported the idea that B-1 and B-2 cells respond differently to LPS. However, these studies were unable to directly address the role of P. gingivalis LPS in periodontitis. It may be that P. gingivalis LPS could have different effects to S. enteritidis LPS on primary B cells. It is still possible that B-1 cells may be more sensitive to P. gingivalis, as opposed to S. enteritidis LPS. Studies by other groups have suggested that the TH1/TH2 profile is skewed towards TH2 in chronic periodontitis and that P. gingivalis may drive this shift via its ability to signal through TLR2 (and modulate TLR4 signalling). Further, recent studies in our laboratories have found that P. gingivalis gingipains are able to polyclonally activate B cells and to break down both IFNγ and IL-12. Future studies should further examine the effects of B-1 and B-2 interactions in the mixed lymphocyte system together with subsequent studies utilising human periodontitis biopsies. The results presented in this thesis, together with work undertaken by other investigators, suggests that LPS could perturb the normal homeostatic mechanisms of the B-1 B cell-subset and increase polyclonal activation therefore contributing to the genesis of pathologies such as chronic periodontitis.

The human hepatitis B virus (HBV) is a small hepatotropic virus, which affects approximately 350 million chronic sufferers worldwide. It has a compact 3.2 kbp dsDNA genome encoding four major overlapping genes namely core, polymerase, surface and X required for its replication. The virus synthesises a pregenomic RNA (pgRNA) which functions both as an RNA intermediate for reverse transcription into the DNA genome and as the mRNA for the translation of the core (C) and polymerase (P) proteins. The core overlaps the polymerase gene and is translated at a 10 to 1 ratio. The polymerase gene translated from the P AUG codon is preceded by at least 4 upstream AUG codons (uAUGs), namely C AUG, C1 AUG, J AUG and C2 AUG. Various mechanisms have been implicated in the synthesis of the polymerase protein. This led to the currently accepted model which involves leaky scanning and a reinitiation mechanism in polymerase synthesis. However, multiple sequence alignment of the pgRNA revealed a short upstream open reading frame (uORF) highly conserved at the nucleotide level in all HBV subtypes and mammalian hepadnaviruses. This previously unreported uORF, designated as C0 ORF in this study is also conserved in its position and length. Past studies have either omitted this uORF in their test constructs or ignored its potential role. The C0 ORF has a conserved weak initiation context and is located within the epsilon structure within the 5' leader of the pgRNA, required for viral encapsidation. Importantly, the C0 ORF precedes and overlaps the core ORF, which may suggest an alternative model in which the core and polymerase may be translated and coordinately regulated. Fusion of the C0 ORF to luciferase showed for the first time that this uORF is translated through the detection of reporter activity (~20% of C) and also visualisation of the fusion protein via western analysis using anti-C0 and anti-luciferase antibodies. Subsequent removal of the C0 ORF implicated a role in repressing downstream core fusion protein synthesis in HepG2 cells. A similar repression was observed on J expression. To study the effect of C0 on downstream polymerase translation, a pgRNA-like DNA construct was made and subsequent mutations introduced. Mutation of the C0 AUG led to an increase in initiation at the downstream P AUG. Alteration of the existing weak initiation context to an optimal context which favours stronger initiation consistently showed a potential role for C0 ORF in facilitating reinitiation at certain downstream initiation codons including P AUG. Mutations of other uAUGs preceding the P AUG were also done to better understand their roles in regulating polymerase synthesis. The removal of the C AUG markedly increased expression from the P AUG. This study revealed other internal uAUGs in-frame to the C AUG, namely the C1 and C2 AUGs are also effectively translated, further reducing availability of translating ribosomes to downstream P AUG. Indeed the removal of the C1 and C2 AUGs led to a corresponding increase in initiation from the P AUG. Initiation at the internal J AUG was also reported and its removal showed a significant decrease in expression from the P AUG, consistent with the previous model implicating reinitiation at the P initiation site after translation of the short J ORF. The inhibitory role of the 5 uAUGs prior to the P AUG were confirmed when all were removed, giving rise to translation almost equal to that at C AUG. Taken together, these results suggest a new model in which the HBV C0 ORF plays a key role in controlling core and polymerase synthesis by repressing core translation and making available more ribosomes to downstream AUGs possibly facilitating translation reinitiation. In addition, the translation of the C0 ORF across the [epsilon] region may also preclude encapsidation, potentially acting as a switch discriminating the pgRNA template between encapsidation and translation. Therefore, the highly conserved [epsilon] region and C0 ORF present an excellent target for molecular based antiviral drugs (antisense oligonucleotides, aptamers, ribozymes) potentially providing new anti HBV drugs.

The human hepatitis B virus (HBV) is a small hepatotropic virus, which affects approximately 350 million chronic sufferers worldwide. It has a compact 3.2 kbp dsDNA genome encoding four major overlapping genes namely core, polymerase, surface and X required for its replication. The virus synthesises a pregenomic RNA (pgRNA) which functions both as an RNA intermediate for reverse transcription into the DNA genome and as the mRNA for the translation of the core (C) and polymerase (P) proteins. The core overlaps the polymerase gene and is translated at a 10 to 1 ratio. The polymerase gene translated from the P AUG codon is preceded by at least 4 upstream AUG codons (uAUGs), namely C AUG, C1 AUG, J AUG and C2 AUG. Various mechanisms have been implicated in the synthesis of the polymerase protein. This led to the currently accepted model which involves leaky scanning and a reinitiation mechanism in polymerase synthesis. However, multiple sequence alignment of the pgRNA revealed a short upstream open reading frame (uORF) highly conserved at the nucleotide level in all HBV subtypes and mammalian hepadnaviruses. This previously unreported uORF, designated as C0 ORF in this study is also conserved in its position and length. Past studies have either omitted this uORF in their test constructs or ignored its potential role. The C0 ORF has a conserved weak initiation context and is located within the epsilon structure within the 5� leader of the pgRNA, required for viral encapsidation. Importantly, the C0 ORF precedes and overlaps the core ORF, which may suggest an alternative model in which the core and polymerase may be translated and coordinately regulated. Fusion of the C0 ORF to luciferase showed for the first time that this uORF is translated through the detection of reporter activity (~20% of C) and also visualisation of the fusion protein via western analysis using anti-C0 and anti-luciferase antibodies. Subsequent removal of the C0 ORF implicated a role in repressing downstream core fusion protein synthesis in HepG2 cells. A similar repression was observed on J expression. To study the effect of C0 on downstream polymerase translation, a pgRNA-like DNA construct was made and subsequent mutations introduced. Mutation of the C0 AUG led to an increase in initiation at the downstream P AUG. Alteration of the existing weak initiation context to an optimal context which favours stronger initiation consistently showed a potential role for C0 ORF in facilitating reinitiation at certain downstream initiation codons including P AUG. Mutations of other uAUGs preceding the P AUG were also done to better understand their roles in regulating polymerase synthesis. The removal of the C AUG markedly increased expression from the P AUG. This study revealed other internal uAUGs in-frame to the C AUG, namely the C1 and C2 AUGs are also effectively translated, further reducing availability of translating ribosomes to downstream P AUG. Indeed the removal of the C1 and C2 AUGs led to a corresponding increase in initiation from the P AUG. Initiation at the internal J AUG was also reported and its removal showed a significant decrease in expression from the P AUG, consistent with the previous model implicating reinitiation at the P initiation site after translation of the short J ORF. The inhibitory role of the 5 uAUGs prior to the P AUG were confirmed when all were removed, giving rise to translation almost equal to that at C AUG. Taken together, these results suggest a new model in which the HBV C0 ORF plays a key role in controlling core and polymerase synthesis by repressing core translation and making available more ribosomes to downstream AUGs possibly facilitating translation reinitiation. In addition, the translation of the C0 ORF across the [epsilon] region may also preclude encapsidation, potentially acting as a switch discriminating the pgRNA template between encapsidation and translation. Therefore, the highly conserved [epsilon] region and C0 ORF present an excellent target for molecular based antiviral drugs (antisense oligonucleotides, aptamers, ribozymes) potentially providing new anti HBV drugs.

Thesis (D. Min.)--Harding Graduate School of Religion, 1987. / Includes bibliographical references (leaves 240-254).

The homeodomain-interacting protein kinase (HIPK) family is comprised of four evolutionarily conserved and highly related serine/threonine kinases originally identified as co-repressors for homeodomain-containing transcription factors. While the HIPKs are most noted for regulation of apoptosis, proliferation and differentiation, I report a pleiotropic function of HIPK1 within the B cell lineage. Although lymphocyte development was normal within the thymus and bone marrow of HIPK1-deficient (HIPK1-/-) mice, the spleen exhibited a reduced number of transitional and follicular (FO) B cells, but with an increase in the marginal zone (MZ) B cell population. HIPK1-/- B cells exhibited impaired proliferation in response to B cell receptor (BCR) cross-linking in vitro; and immunization of HIPK1-/- mice with T-independent type 2 (TI-2) antigen resulted in a significantly impaired humoral response despite the expanded MZ B cell population. Immunization with T-dependent (TD) antigen resulted in a kinetically delayed response, with impaired affinity maturation. Identification of a kinase-substrate interaction between HIPK1 and the B cell adaptor 3BP2 suggests a potential context for HIPK1 function in BCR signaling. HIPK1-/- B cells were uniquely resistant to reactive oxygen species (ROS)-induced apoptosis, but equally susceptible to UV- and γ-irradiation compared to controls. In vitro class-switch recombination (CSR) assays revealed that HIPK1 is required for the negative regulation of CSR. HIPK1-/- B cell cultures harbored more viable cells, more switched cells, and elevated AID mRNA levels. The findings presented in this thesis demonstrate that HIPK1 is required for splenic B cell homeostasis and optimal BCR-responsiveness. In contrast, HIPK1 is also required for the negative regulation of CSR, possibly by mediating CSR-induced apoptosis.

Graduation date: 1994

Thesis (M.A.)--Ohio State University. / Includes bibliographical references (leaves 119-128). Available online via OhioLINK's ETD Center

Thesis (M. Med. Sc.)--University of Hong Kong, 2006.