published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

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ABSTRACT Since its discovery as the etiological agent of AIDS in 1983, HIV-1 has been the focus of unrelenting research into an effective vaccine to control viral infection. Neutralizing antibodies constitute a correlate of immune protection for most available vaccines, but the induction of these antibodies against HIV-1 has become a major challenge. The HIV-1 envelope glycoprotein has evolved to evade neutralizing antibodies in an extraordinary way, yet a vaccine that can stimulate such antibodies remains the best hope to provide sterilizing immunity. The existence of a group of monoclonal antibodies, such as IgG1b12, 2G12, 2F5 and 4E10, capable of neutralizing a broad range of primary isolates signals vulnerable areas on the envelope glycoprotein. Furthermore, passive transfer of these antibodies can completely protect against viral challenge in animal models. The epitopes recognized by these antibodies are being intensely pursued as vaccine targets, in the hope of inducing such specificities. This thesis encompasses a series of studies on characterizing the epitopes recognized by these broadly cross-reactive monoclonal antibodies in the context of subtype C viruses. HIV-1 subtype C is responsible for the vast majority of infections worldwide, however, until recently, little research has been done on these viruses in contrast to the well characterized subtype B strains. Chapter Two describes the characterization of paediatric subtype C viruses for their sensitivity to IgG1b12, 2G12, 2F5 and 4E10. This study was done because of a planned clinical trial of some of these antibodies as post-exposure prophylaxis to prevent mother-to-child HIV-1 subtype C transmission. Only the MAb 4E10 was able to neutralize all the viruses tested, while IgG1b12 was only partially effective. 2F5 and 2G12 did not neutralize any of the viruses. The conclusion was that only 4E10 and IgG1b12 would be suitable for use as prophylactic agents in a population where HIV-1 subtype C is prevalent. Given that subtype C viruses were found to be largely insensitive to 2G12 neutralization, the commonly absent glycan at iv position 295 was introduced into envelope glycoproteins from this clade. The The work presented in Chapter Three explores the requirements of the 2G12 epitope on the envelopes of subtype C viruses. However, this antibody binding site was not readily reconstituted, suggesting structural differences from other HIV-1 subtypes in which the 2G12 epitope is naturally expressed. Chapter Four describes the study of 4E10 resistant virus quasispecies isolated from a seven year old perinatally HIV-1 infected child, in whom anti-MPER antibodies were found. Determinants of 4E10 neutralization were mapped to the epitope of this antibody in the MPER, as well as to the cytoplasmic tail, in particular, to four amino acids in the LLP-2 region. The role of neutralizing antibodies in natural HIV-1 subtype C infection was examined in Chapter Five by following the development of autologous and heterologous neutralizing antibodies in 14 patients during the first year of infection. Potent but relatively strain-specific neutralizing antibody responses were detected within 3-12 months of infection. The magnitude of the responses was associated with shorter V1-to-V5 envelope length and fewer glycosylation sites, in particular in the V1-V2 region. Furthermore, anti-MPER and anti-CD4i neutralizing antibodies were detected in some individuals; however, they were not associated with neutralization breadth. Finally, in Chapter Six these results are analyzed collectively, in the context of the latest findings in the field, and suggestions for further research are discussed.

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The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily. The result of RAGE-ligand interactions augments the proinflammatory mechanisms acting in chronic inflammatory diseases. RAGE recognises a wide range of ligands that have no apparent structural similarities. It is unclear what controls this promiscuity of RAGE. The extracellular domain of RAGE has two potential glycosylation sites. It is speculated that N-linked glycosylation may have significant impact on ligand recognition, especially of S100 calcium binding protein ligands. Two objectives of this thesis were to establish whether S100A9 acts as a ligand for RAGE and to investigate whether glycosylation of RAGE has any influence on ligand recognition. These were achieved by generating two forms of RAGE. HEK 293 cells were transfected to express full-length, membrane-bound RAGE or a secreted form comprising the extracellular domain of RAGE. Site-directed mutagenesis of RAGE showed that asparagine at position 25 is the pre-dominant N-linked glycosylation site. The carbohydrate added to asparagine 25 was further modified to a non-sialylated carboxylated N-linked glycan, specifically recognised by monoclonal antibody GB 3.1. Binding studies showed that different RAGE ligands have individual requirements for glycosylation of the receptor. Binding of AGE-modified AGE-BSA or of S100B to RAGE occured independent of N-linked glycosylation of the receptor. RAGE also binds the S100 protein, MRP-14 (S100A9). In contrast to AGE-BSA or S100B, the non-sialylated carboxylated N-glycan expressed on RAGE is crucial for binding to MRP-14. However, RAGE produced in tunicamycin containing medium and thus lacking N-linked glycosylation, shows strong binding to MRP-14. It was concluded that two forms of binding are involved: the first mechanism relies on the non-sialylated carboxylated N-glycan attached to RAGE and acts in a "tethering" fashion. The second mechanism involves a conformational change of RAGE, which results in exposure of a binding site(s) and a more conventional receptor-ligand interaction. Another objective for this thesis is to study the expression of RAGE and its alternatively spliced variants. PCR analysis has revealed several variants of RAGE that result from alternative splicing mechanisms. The variant proteins are soluble due to a lack of membrane localising sequence. PCR results confirmed the presence of transcripts encoding for spliced variants of RAGE in several tumour cell lines. Among these were transcripts that should encode a soluble form of sRAGE 2. Furthermore, it was shown that sRAGE 2 transcript can be present in forms that contain the ligand-binding V-domain of RAGE or that are N-truncated and lack the V-domain. This is the first report of a soluble, N-truncated sRAGE 2 variant. The results in this thesis add to our knowledge of RAGE biology. MRP-14 (S100A9) is identified as a new ligand. The control of MRP-14/RAGE interaction relies on N-linked glycosylation of the receptor and further modification of the carbohydrate. "Tethering" or stronger receptor-ligand interactions are suggested as mechanisms for controlling RAGE recognition of multiple ligands. Soluble RAGE variants that lack or contain V-domain binding regions, and hence sites for glycosylation were produced. These have the capacity to compete with membrane-bound receptor for available ligand. The control of the expression of soluble RAGE variants, in concert with the control of various modification to carbohydrate expressed on the receptor, adds a level of complexity to ligand specificity. This may ultimately result in different paradigms of the inflammatory process.

Addendum pasted inside the back end-paper. / x, 179 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis presents a hypothesis that there are abnormalities of N-glycosylation in classical galactosaemia and that these abnormalities could contribute to the long-term complications. The aim of the thesis is to characterise and model N-glycosylation in skin fibroblasts from patients with galactosaemia. The study identifies a disturbance in the synthesis and processing of dolichol-linked oligosaccharides. It is anticipated that the serum glycoproteins in untreated galactosaemia may contain N-glycans that are partly absent or truncated. / Thesis (M.D.)--University of Adelaide, Dept. of Paediatrics, 1997

Recent advances in DNA vaccine technology has brought about a promising strategy for the control of viruses that contain surface membrane glycoproteins. This type of vaccine involves the intramuscular injection of a bacterial plasmid containing a gene encoding a viral protein. The strategy uses eukaryotic processing of the protein as would naturally occur during a viral infection. In this study, plasmid DNA encoding the glycoproteins of infectious hematopoietic necrosis virus (pcDNA3-IHNV-g), snakehead rhabdovirus (pcDNA3-SHRV-g), or spring viremia of carp virus (pcDNA3-SVCV-g) was injected into the skeletal muscle of rainbow trout fry. At 30 days post-vaccination, fish were challenged with IHNV. Protection against IHNV was observed among all DNA vaccinated groups. Fish injected with plasmid pcDNA3-IHNV-g, pcDNA3-SHRV-g, or pcDNA3-SVCV-g had relative survival rates of 93.2%, 98.3% and 94.9%, respectively. The mechanisms for the viral mediated resistance induced by these glycoprotein based DNA vaccines is unknown. A parallel study conducted by Dr. Carol Kim on the production of Mx proteins in these fish indicates that the observed protection might be a consequence of the stimulation of interferon. / Graduation date: 1999