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Oomycetes are a class of organisms within which there are a range of devastating pathogens with a variety of different hosts including plants and animals. Phytophthora infestans, the causal agent of late blight in potato, is one of the most important plant pathogens in economical terms. Saprolegnia parasitica is a fish pathogenic oomycete capable of causing disease in freshwater fish species. This study uses both pathogens to work towards understanding the fundamental biology of oomycetes and the development of effective control strategies.

A research report submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in partial fulfilment of the requirements for the degree Master of Medicine in Paediatrics (MMed) Johannesburg 2013 / Introduction Invasive pneumococcal disease, including meningitis, caused by Streptococcus pneumoniae is a major cause of morbidity and mortality world-wide. The introduction of pneumococcal polysaccharide-protein conjugate vaccine (PCV) in the United States has resulted in a reduction in incidence of pneumococcal meningitis. PCV was introduced into the South African expanded programme on immunization (EPI) in 2009. Objective We evaluated the temporal association which the introduction of PCV into the South African EPI had on the incidence of pneumococcal meningitis in children. Methods The study was undertaken in Soweto. All children admitted to Chris Hani Baragwanath Academic Hospital (CHBAH) <14 years of age with meningitis from January 2006 to November 2011 were identified through an electronic database and their microbiological records reviewed to identify the causative bacteria. The results were time framed into two groups: prior to introduction of PCV 1 January 2006 to 31 March 2009 (pre-vaccine era) and post PCV-introduction 1 April 2009 to 30 November 2011 (post-vaccine era). Results 783 patients were admitted with suspected meningitis during the study period, of these 243 (31.0%) met the criteria for bacterial meningitis. The incidence of pneumococcal meningitis was decreasing in the CHBAH in-patient paediatric population by 4.7% per annum prior to the introduction of the vaccine in April 2009. The decline in incidence after PCV introduction accelerated to 18% per annum post-vaccine introduction (P=0.391). In the population most at risk for pneumococcal meningitis, children <1 year of age, the annual reduction in incidence of pneumococcal meningitis accelerated from 1.1% in the pre-vaccine era to 43.4% following PCV introduction (P= 0.011). Conclusions The introduction of PCV resulted in a decline in the incidence of pneumococcal meningitis in all age groups. This decline was most dramatic in the <1 year age group.

Infants born to HIV-1 positive mothers are at risk of acquiring the infection through pro-longed breastfeeding due to the presence of HIV-1 cell-free RNA and cell-associated DNA in breast milk. However, there is limited focus on vaccines to prevent mother-to-child transmission (MTCT) via breastfeeding. Mycobacterium tuberculosis (M. tuberculosis) the causative agent of Tuberculosis (TB) is the most common cause of AIDS-related deaths. Most infants in Africa receive Bacillus Calmette-Guérin (BCG) at birth or soon after birth and it is the only licensed vaccine for TB. The development of a dual platform vaccine against HIV-1 and TB would be a logical effort to combat these two deadly diseases. Thus, rBCG expressing an HIV-1 derived immunogen may induce HIV-1 responses at birth and these responses can be boosted at adolescence, by a heterologous vector such as modified vaccinia Ankara (MVA) or Chimpanzee adenovirus serotype 63 (ChAdV63). In the first study, I assessed BCG-based vaccines derived from BCG Danish SSI-1331 (BCG₁₃₃₁), expressing an HIV-1 immunogen HIVconsv either by an episomal plasmid (BCG.HIVconsv401^epi) or integrated into the BCG chromosome (BCG.HIVconsv401^int) in a prime-boost regimen. BALB/c mice were immunised with the different prime-boost regimens. rBCG alone was unable to induce detectable HIV-1-specific T-cell responses, however, when used in a prime-boost strategy, elevated HIV-1-specific T-cell responses were observed. In the second study, I aimed to construct marker-less mycobacterium-vectored HIV-1 vaccines using the operator-repressor titration (ORTA®) system as an alternative system for antibiotic resistance gene free vaccines. This rBCG vaccine would express the HIVconsv immunogen. I first constructed plasmids carrying the lac operator lacO and tetracycline operator tetO to enable use in ORT Escherichia coli (E.coli) and Mycobacterium strains, respectively. The ORT system was successful in E. coli and not in mycobacterium. I also constructed plasmids carrying mycobacterium essential genes that would allow for genetic manipulation in Mycobacterium and the use of ORT in mycobacterium. Although the plasmid construction was successful, in the end, genetic manipulations in Mycobacterium and the production of an ORT based BCG (ORT-VAC) was not successful. Finally, I evaluated the immunogenicity of conventional DNA plasmid pTH.HIVconsv compared to Semliki Forest virus replicon DREP.HIVconsv in rhesus macaques. Immunisations were done in a prime-boost strategy with heterologous vectors MVA or ChAdV63 delivering the same immunogen, HIVconsv. It was found that DREP.HIVconsv which was at least 20-fold lower dose than pTH.HIVconsv was capable of inducing comparable T-cell responses and in some experiments, the responses were superior to the conventional DNA plasmid pTH.HIVconsv.

Despite the progress in elucidating antigens for both therapeutic and prophylactic vaccines, safety concerns over current vaccine delivery vehicles and adjuvants has limited the development of new vaccines. In particular, there is an urgent need for effective vaccines capable of stimulating cytotoxic T lymphocyte (CTL) responses against intracellular pathogens or tumor cells. Liposomes are under investigation as a particulate vaccine delivery system with the required safety profile and demonstrated ability to target antigens to dendritic cells (DC), the cells of the immune system responsible for initiating effective and long lasting CTL immune responses. Unmodified liposomes however, are inherently non-immunogenic and thus not capable of stimulating activation of DC, which is a necessary step in immune activation. In this thesis the use of modified liposomes to more efficiently target vaccine antigens to DC and then activate the DC sufficiently to initiate down-stream immune responses was investigated. In the first approach to liposome modification, mannosylated phospholipids were incorporated within the liposome bilayer to target C-type lectins on DC. Incorporation of mono- or tri-mannosylated phospholipids within liposomes was found to be an effective means of attaching mannose-containing ligands to the liposome surface without compromising the integrity of the liposome structure. The uptake of tri-mannose-containing liposomes was enhanced in human monocyte derived DC (MoDC) compared to both unmodified liposomes and mono-mannose-containing liposomes. In contrast, neither mono- nor tri-mannose-containing liposomes were taken up by murine bone marrow derived DC (BMDC) to a greater extent than unmodified liposomes. This finding may reflect the differences in ligand specificity for C-type lectins on DC derived from different mammalian species. It was also found in these studies that increased uptake of liposomal antigens by DC does not necessarily result in increased DC activation, as evidenced by a lack of up-regulation of DC surface activation markers and ability to stimulate T cell proliferation. The second approach to liposome modification involved the incorporation of lipid core peptides (LCPs) into the liposome structure. LCPs alone were demonstrated to be able to stimulate DC and subsequent CD8+ T cell activation in vitro. LCP-based vaccines were also able to stimulate effective cytotoxic immune responses in vivo, and protect against tumor challenge, but only if administered in alum with CD4 help. Liposomes containing LCPs were able to stimulate greater DC activation and subsequent CD8+ T cell proliferation in vitro compared with unmodified liposomes. In the in vivo studies however, LCP-containing liposomes were not able to stimulate a cytotoxic immune response or protect against tumor challenge as effectively as LCP administered in alum. In the final approach to liposome modification, inclusion of the adjuvant Quil A was investigated for its ability to increase the immunogenicity of LCP-containing liposomes. It was found that small amounts of Quil A could be incorporated into liposomes without compromising the liposome bilayer. The inclusion of as little as 2% Quil A was able to stimulate DC activation and subsequent T cell proliferation in in vitro studies. In addition, immunisation of mice with LCP-containing liposomes with incorporated Quil A was found to stimulate an in vivo CTL immune response comparable to LCPs administered under optimal vaccine conditions. In conclusion, the work presented in this thesis demonstrates that modified liposomes are a useful vaccine delivery system for the initiation of in vivo cytotoxic and prophylactic immune responses.

Copies of author's previously published article inserted onto back cover. Bibliography: leaves 164-187. Confirms the value of DHBV infection in ducks as a model to evaluate the protective and therapeutic efficacy of DNA vaccines against hepadnavirus infection. The possibility that this model could be explored further to evaluate various combinations of antigens and cytokines 'cocktail' DNA vaccines that elicit the most effective humoral and effective CMI responses for prevention and treatment of HBV infection is discussed.

Graduation date: 1995

Background: Rotavirus disease is the leading global cause of severe diarrhea in children under 5 years. We examined the association between different rotavirus vaccines doses and severity of diarrhea. Methods: A secondary analysis of surveillance of children with acute gastroenteritis (AGE) symptoms during two seasons (January-June) in 2010 and 2011 from three pediatric hospitals in Atlanta, Georgia was conducted. Enrolled children were tested for rotavirus, using EIA (Rotaclone) and vaccination records were collected from the state immunization registry and healthcare providers. Cases were defined as any enrolled child who tested positive for rotavirus. Each enrolled child was assigned a Vesikari score to assess AGE severity. Results: 63.9% of participants had severe AGE. Cases were more likely to have severe AGE than controls (OR 3.8, 95% CI: 2.2-6.5). Receiving a mixed vaccine regimen had similar protection against severe disease to receiving only RotaTeq® or Rotarix® (Mixed: OR 0.1, 95% CI: 0.02-0.5; RotaTeq®: OR 0.1, 95% CI: 0.02-0.5; Rotarix®: OR 0.1; 95% CI 0.01-0.3). When controlling for vaccine type and demographic covariates, three doses of vaccine offered significant protection against severe disease (OR 0.3, 95% CI: 0.2-0.6). Conclusions: Receiving a mixed regimen of rotavirus vaccine is effective in preventing severe AGE. Mixed rotavirus vaccine regimens were equally efficacious to receiving a single type of vaccine in preventing severe disease. Three doses of vaccine, regardless of type, were effective in preventing severe disease but one or two doses were not.