This work set out to apply computational tools and biophysics principles to develop a systematic approach to the rational selection of synthetic antigens from appropriate DNA sequences. The wider potential to the rapid development of diagnostics will be demonstrated by application towards a chlamydia-specific immunodiagnostic, and the potential for vaccine and therapeutic development will be discussed.

The HPA-1a antigen is the most immunogenic and clinically important antigen of the human platelet antigen (HPA) system.  It is a major target in neonatal alloimmune thrombocytopenic purpura (NAITP).  As a first step towards peptide immunotherapy for NAITP, the aim was to map immunodominant helper epitopes by challenging peripheral blood T-helper cells <i>in vitro</i> with a panel of linear peptides derived from the GPIIIa protein sequence containing the HPA-1a polymorphic site.  A further aim was to determine whether the panel of HPA-1a/1b peptides can stimulate the production of cytokines from helper T-cells of women at risk of FMAIT. 15-mer peptides were synthesised corresponding to the sequences of the HPA-1 polymorphic region, with either Leu³³ or Pro³³ at each possible position.  Peripheral blood mononuclear cells (PBMC) were obtained from the blood of 21 HPA-1b1b women who had recently delivered an HPA-1a positive baby.  Fourteen women had developed anti-HPA-1a as a result of an incompatible pregnancy, and 13 of 14 were HLA-DRB3*01 positive.  Seven women had not produced anti-HPA-1a and 6 of 7 were HLA-DRB3*01 negative.  One unimmunised HPA-1b1b male donor and 11 HPA-1a positive blood donors acted as controls. PBMC from 9 of 14 alloimmunised women responded to particular HPA-1a Leu³³ peptides;  all 9 responders were HLA-DRB3*01 positive and they were negative for the HLA-DRB1*15 allele.  In contrast, only 2 of the 9 also responded to some of the HPA-1b Pro³³ peptides.  Two of 7 HPA-1b1b women who did not produce anti-HPA-1a also responded to some Leu³³ peptides;  neither woman was HLA-DRB3*01 positive.  Twelve of the 14 alloimmunised women were HLA-DQB1*02 positive and of those, 7 responded to peptides.  None of the control donors responded to either Leu³³ or Pro³³ peptides.  Certain peptides induced proliferation more commonly than others.  Peptides with Leu³³ near the C-terminus were immunodominant, with peptide L1_(19-33) eliciting a response in 50% of the alloimmunised women.  The immunodominant peptides (L1_(19-33), L2_(20-34), L3_(21-35), L4_(22-36) and L5_(23-37)) were examined for the presence of motifs that are bound by MHC class II molecules.  In addition to Leu³³ they were found to also require β3Trp²⁵.  The peptide panel was screened for the ability to stimulate PBMC proliferation in an assay designed to map T-cell epitopes, and the responding T-cells were examined by 3-colour flow cytometry for the presence of CD3+ and CD4+ T-cells and the surface expression of CD69 or CD71 activation markers.  The  majority of the responding cells were CD3+, CD4+ T-helper cells which carry the activation marker CD71 and CD69.  HLA class II blocking monoclonal antibodies were utilised to determine whether the HPA-1a response is restricted by MHC class II molecules.  The T helper cells responded to a single epitope, presented by peptide L1_(19-33), demonstrating that the Th-cell response is focused on one dominant epitope with one major restricting class II molecule.  This epitope could enable preparation of a short peptide, suitable for use for the prevention or reversal of the pathogenic alloimmune response to the HPA-1a alloantigen if presented by a tolerogenic route.

No description available.

Dendritic cells (DC) are unique antigen presenting cells which play a major role in antigen presentation and initiation of the immune response by regulating B- and T- cell activation. Antigen targeting to DC receptors is an effective, safe and specific method for vaccine development. The mannose receptor (MR) is an endocytic receptor expressed by subpopulations of DC and antigen targeting through MR leads to enhanced antigen uptake and presentation to T -cells. This makes MR a favourite receptor for the development of vaccines against diseases that require T-cell immunity such as cancer and viral infections. This project sought to develop tools to target antigens through MR and investigate their ability to induce T-cells activation in vitro and in vivo. We have used three approaches to deliver antigen through MR; (i) MRspecific mAbs: 503 and 6C3, have been chemically linked to the melanoma epitope TRP-2, (ii) MR-specific chimeric antibodies carrying several model antigens have been generated by using genetic engineering and (iii) Glycopolymers and the suitable antigens such as a shorter version of model antigen ovalbumin (OVA), with and without N-glycosylation sites have been generated and characterised. Glycopolymer-OVA conjugates were prepared by chemical coupling but it requires further optimization. The binding efficiency of anti-MR antibodies has been assessed using ELISA and BIACORE and the glycopolymers have been tested for their interaction with MR. Immunisations were performed with anti-MR mAb-TRP2 conjugates which induced TRP-2 specific COS+ T-cells activation and improved humoral response. Due to limitations in this approach in terms of chemical coupling being an inefficient method and the potential involvement of Fc recetors (FcRs), chimeric Abs fused to model antigens and bearing mutated Fc were generated. These chimeric Abs, have been tested for their ability to induce T-cell activation in vitro and in vivo. But the progress has been hampered due to the labile nature of these reagents. In future, anti-MR chimeric Abs will be used to generate anti-MR single chain antibodies carrying OVA (ScFv-OVA) and the glycopolymer project will be taken up Dr. Manovani Giuseppe (School of Pharmacy, University of Nottingham). It will involve further optimization of chemical coupling of glycopolymers to a-glycosylated OVA-mini protein, and the in vitro Ag presentation assay to investigate whether glycopolymers mediated Ag targeting of APe enhance T-cells activation. These further studies would greatly benefit the understanding of the mechanisms associated with the elicitation of immune resposes as a result of Ag targeting through MR. Anti-MR reagents generated in this study along with appropriate adjuvant could be exploited to target malarial, cancerous and viral Ags for robust T-cell activation against these infectious diseases. On the other hand, the role of MR in homeostasis and allergy has been already established, and the anti-MR reagents generated in this study can be used to target allergens and self-Ags to APes in an attempt to induce tolerance.

Errata inserted on back end paper. / Bibliography: leaves 226-254. / xviii, 261 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Describes the development of techniques to genotype and simultaneously assess allele dosage at the ABO locus using PCR and allele-specific restriction enzyme digestion. / Thesis (Ph.D.)--University of Adelaide, Dept. of Medicine, 1996