Inflammation in the female genital tract (FGT) is associated with increased HIV-1 viral replication, HIV-1 transmission and HIV-1 acquisition. The optimal commensal Lactobacillus bacterial species is associated with reduced inflammation in the FGT and dampened immune responses to non-optimal bacteria in vitro. Using a transcriptomics approach, this research aimed to investigate gene expression patterns in the FGT of HIV-infected women compared to peripheral blood. Furthermore, transcriptomics was used to investigate interactions between different vaginal Lactobacillus species and the host to elucidate its immunomodulatory mechanisms. Cervical cytobrushes and blood samples were collected from chronically HIV-infected South African women. Cervical and peripheral blood mononuclear cells (CMCs and PBMCs) were isolated and mRNA was extracted for microarray analysis using the Illumina HumanHT-12 v3 Expression BeadChip system. Eight Lactobacillus isolates, two of each L. jensenii, L. mucosae, L. crispatus and L. vaginalis species were included in this study. The effects of these lactobacilli on cytokine production by vaginal epithelial (VK2) cells stimulated with Gardnerella vaginalis (ATCC 14018) were tested in vitro, RNA was extracted and used for Affymetrix Genechip whole transcript microarray analysis. This study found that significantly over-expressed genes in CMCs compared to PBMCs were mapped to proinflammatory signaling pathways (including Nuclear factor kappa B (NFκB), Tumor necrosis factor (TNF), Toll-like receptor (TLR) and Nucleotide-binding and oligomerization domain (NOD)-like receptor). Concurrently, a signature of reduced potential for adaptive immunity was observed in CMCs compared to PBMCs, as evidenced by underrepresentation of the T cell receptor signaling and natural killer cell mediated cytotoxicity pathways. G. vaginalis induced a potent proinflammatory cytokine response by VK2 cells in vitro. Over-expressed genes in G. vaginalis-stimulated VK2 cells compared to unstimulated VK2 cells were mapped to inflammatory signalling pathways. In contrast, 3/8 Lactobacillus isolates, including two L. mucosae and one L. vaginalis species, reduced inflammatory cytokine production by VK2 cells in response to G. vaginalis and were thus termed “cytokinesuppressive”. Several genes, 7/8 of which are involved in inflammation, were downregulated in VK2 cells co-cultured with lactobacilli and G. vaginalis in combination compared to coculture with G. vaginalis only. Futhermore, when gene expression changes were investigated in cells cultured with cytokine-suppresive lactobacilli versus non-cytokine-suppressive lactobacilli, it was found that SAMD9L, DDX58, IFIT1 gene expression was downregulated exclusively in VK2 cells co-cultured with cytokine-suppressive lactobacilli and G. vaginalis compared to co-culture with G. vaginalis only. The findings of this study have identified distinct gene expression patterns in the FGT compared to peripheral blood. Furthermore, key genes that may play a critical role in the immunomodulatory effects of vaginal lactobacilli were identified, motivating for further confirmatory research.

Kaposi sarcoma herpes virus (KSHV) is a typical DNA virus that is associated with a number of proliferative diseases including Kaposi's sarcoma. The KSHV open reading frame (ORF) 4 encodes a complement regulatory protein (Kaposi complement-binding protein, KCP) that binds complement proteins and inhibits the complement-mediated lysis of cells infected by the virus, thus providing a strategy for evasion of the host complement system. Kaposi's sarcoma is an angiogenic skin lesion that has been recognized as one of the most abundant tumours found in many parts of Southern Africa and which can occasionally become highly invasive, aggressive and capable of causing death, particularly amongst AIDS patients. It is of major significance to understand how complement control proteins (CCPs) such as KCP perform their biological functions at the molecular and structural levels, because of their potentials as therapeutic agents, their implications in the pathology and importance in the etiology of many disease conditions. This study was therefore undertaken to characterise the structure-function relationship of KCP. Based on primary sequence analysis and comparison to other functionally and structurally similar proteins, oligonucleotide primers were designed to amplify by PCR, three regions of the predicted ORF 4 from human herpes virus-8 (llliV-8) DNA isolated from a primary effusion lymphoma cell line. The PCR products were inserted by ligation into the expression vector pPIC9 to generate three recombinant plasmids for heterologous expression in the yeast, Pichia pastoris and to produce separately, the 4 N-terminal Sushi domains (KCP-S, small), KCP protein lacking the putative transmembrane binding domain (KCP-M, medium) and the full-length protein (KCPF, full). Expression of the viral proteins was confirmed by SDS-PAGE and Western blot analyses using a rabbit polyclonal antibody directed against a selected peptide region that is common to all three recombinant KCPs. All the KCP proteins migrated electrophoretically as higher bands compared to their expected sizes. The lower mobilities of the proteins may be due to g1ycosy1ation since there are potential N-and O-glycosylation sites in the protein's primary sequence. Also, diffused bands were obtained in all the electrophoretic gels and Western blots carried out, which is characteristic of glycoproteins. Furthermore, the antibody recognized several larger and smaller bands that may represent aggregates and/or degradation products respectively. Both partially purified KCP-S and KCP-S directly from expression media were able to inhibit complement-mediated lysis of sensitized sheep erythrocytes by approximately 60% in a hemolysis assay. This result confirms previous reports that recombinant KCP is twice more efficient in inhibiting the classical pathway-mediated lysis of erythrocytes than the vaccinia virus complement control protein (VCP), which also contains 4 Sushi domains. The KCP-F and KCP-M proteins did not show any significant complement inhibitory activities. Preliminary immunohistochemical studies using the same antibody were carried out to determine the expression and distribution of KCP proteins in Kaposi's sarcoma.

Acinetobacter baumannii is a major pathogen causing nosocomial infections, particularly in critically ill patients. This organism has acquired the propensity to rapidly develop resistance to most antibiotics. At several hospitals within Cape Town, tobramycin and colistin remain frequently the only therapeutic options. The Vitek2 automated susceptibility testing (AST) is used in the clinical laboratory to determine selected susceptibility profiles. The suspicion of a possible AST-related technical error when testing for susceptibility to tobramycin in A. baumannii precipitated this study.

This study constitutes the first report on type-specific human papillomavirus (HPV) concordance and transmission in heterosexually active couples that are human immunodeficiency (HIV)-seronegative, HIV-seropositive or HIV-discordant and in which 71% of female participants have normal cervical cytology.

Includes bibliographical references (leaves 124-150). / The female genital tract serves as the major portal of entry for human immunodeficiency virus (HIV). Local immune factors unique to the mucosal micro-environment such as the genital tract cytokine milieu or the activation/differentiation status of T cells may play a significant role in heterosexual transmission of HIV and subsequent pathogenesis. Elucidation of the mechanisms underlying the persistent recruitment, activation and differentiation of mucosal T cells will give crucial insight into potential therapeutic targets to restore effective local immunity.

Bacterial vaginosis (BV) is an important predisposing factor for the acquisition of human immunodeficiency virus (HIV) and other sexually transmitted infections (STIs) in South African women. However, the microbial causes and the immunomodulatory effects of BV are not yet fully understood, and effective treatment strategies do not exist. BV is associated with upregulated inflammatory cytokine levels in the female genital tract (FGT), which in turn may increase HIV infection risk by recruiting and activating HIV target cells, reducing epithelial barrier function and directly promoting HIV replication. Lactobacillus species on the other hand are thought to protect against HIV by competitive exclusion, producing virucidal hydrogen peroxide (H2O2), maintaining an acidic pH by producing lactic acid and regulating immune responses in the FGT. This dissertation aimed to characterise the relative HIV inhibitory properties of clinical Lactobacillus isolates, to evaluate the immunoregulatory properties of lactobacilli, and determine the mechanisms underlying these relationships. Vaginal Lactobacillus isolates (n=103), including L. crispatus, L. jensenii, L. johnsonii, L. mucosae, L. plantarum, L. ruminis, L. salivarius and L. vaginalis, were isolated from young South African women who participated in the Women's Initiative in Sexual Health (WISH) study. The production of pro-inflammatory cytokines (IL-6, IL-1α, IL-1β), chemokines (IL-8, IP-10, MIP-3α, MIP-1α, MIP-1β) and regulatory IL-1RA by vaginal epithelial cells in response to lactobacilli in the presence or absence of Gardnerella vaginalis ATCC 14018 and Prevotella bivia ATCC 29303, was measured using Luminex. Growth rates, bacterial sizes, adhesion to cervical (Ca Ski) and vaginal epithelial cells (VK2), culture pH changes and D/L-lactate production by the lactobacilli were also measured in vitro. The properties of vaginal Lactobacillus isolates were also compared to those of commercial probiotics and ATCC reference strains. In order to evaluate differences between lactobacilli isolates that induced low (termed “non-inflammatory”) versus high (termed “inflammatory”) levels of inflammatory cytokine production, the proteomic profiles of 22 inflammatory and 22 non-inflammatory Lactobacillus isolates were analysed using liquid chromatography tandem mass spectrometry (LC-MS/MS) to investigate the underlying mechanisms leading to the different inflammatory profiles. Lastly, the influence of Lactobacillus culture supernatants (n=16) on HIV infectivity was evaluated using a Luciferase Reporter Gene Assay in TZM-BL cells. Lactobacilli isolated from women with non-optimal microbiota produced less lactic acid and induced greater inflammatory cytokine production than those from women with optimal microbiota, with IL-6, IL-8, IL-1a, IL-1b, MIP-1a and MIP-1b production significantly elevated. Proteomics analysis showed that 164 proteins were differentially abundant between inflammatory lactobacilli and non-inflammatory lactobacilli. Functional analysis revealed that isolates inducing low levels of inflammatory cytokine production had a significantly higher relative abundance of membrane-associated cellular components, metabolic biological processes and enzymatic molecular functions compared to isolates that induced higher levels of inflammation. A subset of sixteen lactobacilli significantly suppressed IL-6 (adjusted p<0.001) and IL-8 (adjusted p=0.0170) responses to G. vaginalis while L. crispatus isolates suppressed inflammatory cytokines responses to P. bivia. Culture supernatants from the same 16 isolates significantly suppressed HIV infectivity in TZM-BL cells (p=0.0078). Lactobacilli adhesion to VK2 cells correlated negatively with IL-6, IL-8, MIP-1a and IL-1RA production. Lactobacillus beneficial characteristics were highly strainspecific and vaginal isolates out-performed commercial probiotics and ATCC strains. Lactobacillus growth rates, bacterial sizes and adhesion to VK2 cells did not differ significantly between isolates from women with non-optimal microbiota versus those from women with optimal microbiota. These findings show that, while cervicovaginal lactobacilli suppressed overall inflammatory responses to G. vaginalis and P. bivia, isolates from women with non-optimal microbiota were more inflammatory, had lower relative protein abundance and produced less antimicrobial lactic acid than isolates from women with optimal microbiota. Additionally, vaginal Lactobacillus isolates performed better than existing commercial probiotics, suggesting room for improvement of current probiotic formulations available on the South African market to improve BV treatment outcomes and reduce inflammation in the FGT.

African swine fever (ASF) is a disease that affects domestic pigs and wild boars, resulting in up to 100% case fatality rate, and there is currently no effective treatment or vaccine. To date, there are 67 ASFV complete genome sequences available, but most of the sequences represent only genotypes I-V and VII-X of the 24 genotypes identified based on p72 sequencing, limiting inter and intra-genotype comparative studies. ASFVs encode several multigene families (MGFs) involved in virulence and host range which are found at the genomic termini and the majority of genomic differences between isolates are due to the composition of these MGFs. The comparison of the MGFs across ASFV isolates is of utmost importance in understanding genome variability and their contribution to virulence. The p72 gene has historically been used in phylogenetic analysis of ASFV. However, it lacks the capacity for higher resolution between isolates belonging to the same genotype. This study aimed to analyse and characterise six novel ASFV isolates of African origin from a domestic pig, warthog, wild boar and ticks in terms of genomic makeup, MGF composition and phylogenetic relationships, including identification of additional phylogenetic markers, specifically for use in discrimination between closely related isolates. Genomes of six novel isolates were sequenced and annotated by identifying open reading frames (ORFs) with a methionine START codon and performing BLASTx searches of each ORF against the NCBI data base. Differences between the genomes were analysed by generating dotplots and using Base-By-Base which showed them to be mostly collinear, but regions of difference were observed at the termini and the CCR. MGF analysis using sorting and clustering in Morpheus software, based on genotype, serogroup, country, host, virulence, and year, showed that genotype and serogroup play a role in the MGF arrangement patterns. Loci corresponding to regions of difference in the CCR were used for phylogenetic comparison to the previously identified marker p72. The tree topology of all of the alternative phylogenies differed from the current p72 classification. B117L and B169L provided slightly better resolution of genotypes I and II, respectively, and viruses from East Africa that are classified as belonging to genotype IX based on p72 were separated when using EP364R. This data adds to the pool of diverse ASFV isolates available for comparative genomics studies, and to the knowledge of ASFV in Africa. The sequencing of more diverse ASFV isolates of each genotype will help characterise the MGFs arrangement patterns among isolates. The novel alternative phylogenetic markers should further be investigated using more ASFV isolates representing the 24 genotypes described to date.

Includes bibliographical references. / Vaccinia virus is the most extensively studied, prototype vertebrate poxvirus, which was used as a vaccine in the eradication of smallpox. The genome of this virus has characteristic variable termini encoding open reading frames that are not essential for virus replication in cell culture. One such open reading frame, N1L situated at the left terminal region of the neurovirulent Western Reserve (WR) vaccinia virus strain, encodes a protein 13.8 kDa in size. In vivo studies in mouse brains revealed that a recombinant virus, vGK5, tacking the expression of the 13.8 kDa protein was rendered replication deficient in the brain. An essential requirement of poxviruses for their replication is the energy molecule adenosine triphosphate (ATP). The supply of this molecule in the brain to support replication of a virus is limited due to the high-energy requirements and small energy reserves of this organ. The specific function of the vaccinia virus 13.8 kDa protein in relation to viral replication in the brain was investigated. The South African (SA) Lister vaccinia virus strain was confirmed to encode an identical N1L gene to that of the WR vaccinia virus by amplification, cloning and sequencing of the Lister N1L open reading frame. The Lister vaccinia virus and a 13.8 kDa deletion strain (vGK5) were cultivated and used to intracranially infect mice. Using a luciferin/luciferase bioluminescence assay system the ATP levels in Lister and vGK5 vaccinia virus-infected mouse brains were measured and found to differ significantly after a 5-day infection period. The SA vaccine Lister vaccinia virus strain was found to be a slow growing virus in the brain. Subsequently, a possible role for the vaccinia virus 13.8 kDa protein in influencing ATP levels in the brain was postulated, yet a neurovirulent wild type strain is needed for further studies to consolidate this result. The 13.8 kDa protein was successfully expressed in the P. pastoris yeast expression system and positively identified by immunodetection studies.

Includes bibliographical references (leaves 176-221).

Includes bibliographical references (leaves 172-200).