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Access to abstract restricted until May 2014 / Access to thesis restricted until May 2014 / Department of Biology

Data from ¹H nuclear magnetic resonance studies on the Fv fragment of protein 315, a Dnp-binding BALB/c mouse lgA(λ₂) myeloma protein, have been used to refine a predicted structure of the combining site of the protein. The Dnp-binding subsite in the modified structure is composed of the side chains of three aromatic amino acids Trp 93_L, Tyr 3⁴_L and 3⁴_H. A fourth aromatic amino acid residue is close to the side chain-NH-CH 2 -group, this is Tyr 33 H - The antibody-hapten binding is a simple encounter process, which causes no extensive conformation change in the Fv fragment. A method for paramagnetic structural studies has been devised using Dnp derivatives with cllgophosphate side chains, which create a specific manganese binding site on the Fv fragment-hapten complex. The distances from the bound metal ion to the imidazole side chains of two of the three hlstldine residues of protein 315 have been determined. ¹H nuclear magnetic resonance has been used to study the histidine residues of the Fv fragment of protein 315. It has been shown that one of the three histidine residues (102_H) is close to the combining site, but that this residue does not participate directly in binding haptens. ³¹ P nuclear magnetic resonance studies have shown the presence of a positively charged amino acid side chain near the entrance of the combining site of the Fv fragment. This residue has been identified as Arg 95_L. The mode of binding of trini trophenyl derivatives to the Fv fragment has been studied by ¹H nuclear magnetic resonance. It is concluded that these haptens, when bound to the Kv fragment, make contacts with the same amino acid side chains as Dnp derivatives.

Human cytotoxic T lymphocytes (CTL) recognize antigen in the form of short peptide fragments presented by Human Leukocyte Antigen (HLA) class 1 molecules. The HLA B27-restricted CTL response to Influenza A virus is directed against a single nine amino acid epitope within the viral nucleoprotein NP383-391. Influenza A-specific CTL lines and clones, generated from the peripheral blood of three unrelated individuals, exhibited remarkable fine specificity for NP383-391, failing to recognize synthetic variants containing single amino acid substitutions at positions 1, 4, 7 or 8. The sequences of rearranged T cell receptor (TCR) alpha and beta chain genes showed remarkable conservation of variable and joining gene segment usage, and also of non germline-encoded B chain residues proposed to interact with peptide, even between clones from unrelated individuals. Correlation of fine specificity for peptide with TCR alpha chain usage suggested a possible orientation for the TCR/HLA/peptide ternary complex in this immune response. The finding of restricted TCR usage in the HLA B27-restricted CTL response to influenza A virus has implications for the study and potential therapy of HLA B27 associated autoimmune disease. The TCR heterodimer from one clone was expressed in a rat basophil cell line and shown to confer specificity for HLA B27 + NP383-391. Influenza A-specific CTL were also shown to recognize bacterial superantigens in an MHC class II dependent but unrestricted manner. A rapid screening assay for candidate superantigens was developed and used to identify a novel superantigen produced by Clostridium perfringens. This superantigen was found to have a unique specificity for human TCRβ chains.

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Bibliography : leaves 147-185. / xi, 185, [6] leaves [16] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1990

"February 1977." / Includes bibliographical references (leaves [105]-[120]) / xiv, 104, [16] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The malignant transformation, and the relevance of developmental antigens to the cancer process, is broadly reviewed. The two developmental antigens - foetal and placental - are then examined in experimental mouse and rat tumour systems. / Thesis (Ph.D.)--University of Adelaide, Dept. of Animal Physiology, 1978

There is an abundance of information available on the molecular mechanisms of antigenic variation in Plasmodium falciparum. The variant antigen PfEMP1, which mediates antigenic variation as well as cytoadherence and rosetting, has been extensively characterised. Genes coding for the antigen belong to the gene family var, and several var genes have been cloned and characterised. The rodent malaria parasite P. chabaudi is a widely studied in vivo model for P. falciparum. The P. c. chabaudi AS parasite strain has been shown to exhibit antigenic variation and the variant antigen has been detected by surface fluorescence. As with P. falciparum, there is a link between antigenic variation and cytoadherence, however genes coding for the variant antigen in P. chabaudi have not been cloned to date. Therefore, potentially useful in vivo experiments on antigenic variation are restricted. In this thesis it is shown for the first time that the P. c. adami DS parasite strain also exhibits antigenic variation. / Chapter 3 describes efforts to locate genes coding for variant antigens in P. c. adami DS. The main strategy involved a genome survey, by sequencing and analysing randomly selected clones from a P. c. adami DS genomic library. DNA sequences were compared to Plasmodium spp. sequence databases to look for similarity to var genes or other genes encoding variant antigens. Of the 297 clones analysed none had significant sequence similarity to genes coding for variant antigens. However, in a small proportion of sequences some similarity to var genes was noted. Several genes of potential interest were identified, most importantly the gene coding for the vaccine candidate rhoptry associated protein 1 (RAP1), which was subsequently cloned and characterised. Further attempts to locate var gene homologues in P. c. adami involved amplification of P. c. adami genomic DNA using degenerate oligonucleotide primers corresponding to conserved regions of var genes. This strategy proved to be unsuccessful, most likely due to lack of sequence similarity between P. falciparum and P. c. adami genes. In several vaccination studies with the apical membrane antigen 1 (AMA1) of P. c. adami DS, mice were significantly protected against homologous parasite challenge. However, some mice developed late, low-level breakthrough parasitaemias. In Chapter 4, the characterisation of two such breakthrough parasitaemias is described. The ama1 genes of the breakthrough parasites were found to be identical to the ama1 gene of the parental parasites. Similarly, no alteration in AMA1 expression was observed. However, the breakthrough parasites were found to be more resistant than the parental parasites to the effects of passive immunisation with rabbit antisera to AMA1, RAP1 and possibly also MSP119. P. chabaudi infections in mice have been previously shown to consist of a primary parasitaemia followed by a short period of subpatency, and a recrudescent parasitaemia. In surface immunofluorescence studies with P. c. chabaudi, parasites of the recrudescence were shown to be distinct from parasites of the primary parasitaemia, with respect to antigens expressed on the surface of late trophozoite- and schizont-infected erythrocytes. / Chapter 4 describes similar surface immunofluorescence assays carried out with P. c. adami infected erythrocytes, and quantitation of fluorescence by flow cytometry. As with P. c. chabaudi, the recrudescent parasites were found to be antigenically distinct from the primary parasitaemia, indicating that antigenic variation had taken place. Because breakthrough parasites from the AMA1 vaccination trial were similar to recrudescences in peak and duration, we hypothesised that breakthrough parasitaemias, like recrudescent parasitaemias, occur as a result of antigenic variation. In Chapter 4 it was shown by surface immunofluorescence and flow cytometry using hyperimmune sera raised against different parasite populations, that breakthrough parasites express antigens on the surface of late trophozoite- and schizont infected erythrocytes that differ from those expressed by the parental and recrudescent parasites. These results support the hypothesis that switching of the variant antigen on the infected erythrocyte surface enables parasites to evade protective antibody responses directed against merozoite antigens. / Chapter 5 describes the cloning and characterisation of P. c. adami RAP1 which was identified in the process of the genomic survey described in Chapter 3, as well as P. berghei RAP1. Both rodent parasite orthologues of RAP1 were found to have 30% sequence similarity to P. falciparum RAP1, and 6 of 8 cysteines were conserved in the rodent parasite orthologues. However the three polypeptides vary significantly in size. P. c. adami RAP1 and P. berghei RAP1 consist of 691 aa and 604 aa respectively, whereas P. falciparum RAP1 consists of 783 aa residues. These size differences reflect very different N-terminal sequences prior to the first cysteine, whereas the cysteine-rich C-terminal regions are more conserved. Both P. falciparum RAP1 and P. c. adami RAP1 contain N-terminal repeats, however they bear no sequence similarity to each other. P. berghei RAP1 lacks N-terminal sequence repeats that are characteristic of P. falciparum and P. c. adami RAP1. The large cysteine-rich C-terminal region P. c. adami RAP1 (PcRAP1 C3) was expressed in E. coli as a hexa-his fusion protein. Rabbit antiserum to recombinant PcRAP1 C3 was used to characterise the expression and sub-cellular localisation of the RAP1 antigen. P. c. adami RAP1 was found to have a Mr of approximately 80,000 and was shown by immunofluorescence to localise to the merozoite rhoptries. Passive immunisation of mice with rabbit anti-RAP1 serum was shown to protect against fulminant parasitaemia and mortality. In a mouse vaccination trial using the recombinant PcRAP1 C3 polypeptide partial protection was conferred against homologous parasite challenge.

Thesis (MScMed)--University of Stellenbosch, 2006. / Bibliography. Also available via the Internet.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser. I publ. felaktig serietitel: Umeå University medical dissertation.