The HLA complex is the most polymorphic genetic system known in man. The frequency of the HLA class II antigens have been well studied in Caucasoids but little data is available concerning HLA antigen frequencies in Negroes. In this thesis the class II antigens, excluding HLA-DP, were studied in South African (SA) Negroes (Xhosa), Cape Coloureds ( a group of mixed racial origin) and SA Caucasoids using serological, cellular ( HTC typing) and Southern blot techniques. The results obtained for the SA Negroes were compared with those previously found in Nigerians and American Negroes. Marked differences in HLA distribution occurred between these groups, which in part may be explained by Khoisan admixture in the SA Negroes. In addition, striking frequency differences were observed between the three SA populations. For example, in the Xhosa the HLA-DR1, DR4, DR7, DRw8, DQw2, DQw3, Dw1 and Dw3 specificities were found at a significantly lower frequency, whereas HLA-DR3, DRw6 and Dw' RSH' were found at a significantly higher frequency compared with the SA Caucasoids. The frequency in the Cape Coloureds was intermediate between those of the Xhosa and Caucasoids. In the SA Negroes and Cape Coloureds, several new specificities were detected such as HLA-DRw18, DR2 LUM(CT), DRwl2x6, DRw8x14, Dw' RSH', Dw' JOH' and Dw' BME'. The HLA-DR and DQ haplotypes in significant linkage disequilibrium were similar in the three groups. However, several haplotypes with unusual DR and DQ combinations such as HLA-DRw17,DQw7; DR9, DQw2 and DR4, DQw5 were present in the SA Negroes and Cape Coloured families. Al though some of these unusual haplotypes could be explained in terms of recombination between the common haplotypes, none could be typed using a panel of well defined homozygous typing cells, suggesting that the response observed in mixed lymphocyte culture arises from separate molecular determinants. The data on HLA class II antigen frequencies presented in this thesis is essential for future studies on HLA and disease associations and for establishing population relationships. Knowledge of new HLA class II antigens in the various population groups is also important in renal transplantation as matching for HLA-DR antigens is known to improve graft survival.

Brucella spp. are zoonotic infectious agents, primarily of livestock, that cause the disease brucellosis. Bovine brucellosis, caused by Brucella abortus, is of greatest concern due to the disease’s significant economical and public health impact. Canada fully eradicated bovine brucellosis from domesticated cattle herds in 1985, however, continued surveillance through screening for B. abortus exposure is paramount to the maintenance of bovine brucellosis eradication nationwide. The Canadian Food Inspection Agency (CFIA) is responsible for the surveillance of bovine brucellosis outbreaks in Canada and the maintenance of eradication. Current B. abortus serodiagnostics and serological screening is mostly based on the detection of antibodies against Brucella lipopolysaccharide (LPS), a highly immunogenic component of the outer cellular membrane. Such tests face difficulties with false positive results due to cross reactivity with other Gram-negative bacteria that produce LPS. The purpose of the research presented here was to address this issue through identifying new B. abortus protein antigens for the improvement of serological test specificity. In this study, 101 candidates were identified through predictive bioinformatic analyses and selected for immunogenic evaluation. While none of the expressed candidates displayed positive serological activity with in-house brucellosis positive bovine serum panels, the workflow presented here can be used for continued research and the assessment of more proteins from B. abortus and other bacterial pathogens.

Cancer immunotherapies focused on tumor-specific T cell responses are promising alternatives to chemotherapy/radiotherapy for various cancers as they can be engineered to specifically target tumours and establish long-term surveillance against relapsing tumours. The premise for the application of active immunotherapies is the recognition of tumor-specific and/or tumor-associated antigens by the immune system. Approaches that have been explored to this end include cancer vaccines and gene therapy/autologous cell transfer (T-cell receptor (TCR) or chimeric antigen receptor (CAR)-based). This study evaluated the use of oncolytic rhabdovirus-based vaccines (ORV) for the treatment of sarcoma with a focus on rhabdomyosarcoma (RMS). Sarcomas are amenable to to oncolytic virus (OV) infection and generate robust T-cell responses against tumour antigens. The ORV strategy undergoing clinical evaluation uses a prime-boost vaccine whereby a non-replicating adenovirus serotype 5 vector (Ad5) encoding an antigen is administered as a priming agent and boosted with a rhabdovirus encoding the same antigen (NCT02285816). However, the prevalence of pre-existing immunity to Ad5 in patients serves as an exclusion criterion and limits its effectiveness as a priming agent. To this end, we have shown that an alternative priming agent and antigen delivery vehicle, anti-DEC205 (aDEC205), targets antigens directly to dendritic cells (DCs), inducing robust immune responses. However, a lack of targetable antigens and methods to identify antigens is a limiting step for the application of ORVs for sarcoma. Thus, the identification of immunogenic sarcoma antigens is a critical step for the study ORVs. Current methodologies have important drawbacks in that they can be prohibitively time-consuming, complex or are ineffective in coupling antigen discovery and immunogenicity. Presented herein is the study of a novel methodology for the discovery of immunogenic antigens by probing for T cell activation marker CD107a and isolation by flow cytometry. In parallel, RMS antigen discovery was also performed via peptide elution and mass spectrometry resulting in the identification of 24 novel murine RMS antigens. Ultimately, therapeutic vaccination with a subset of these antigens encoded into DEC205 and ORV did not yield immune responses in a pre-clinical model; however, this research established immunization tools for further study of immunotherapy in RMS.

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, in fulfilment of the requirements for the degree of Doctor of Philosophy Johannesburg 2014 / Human receptor CD4 is a membrane-bound glycoprotein expressed on the surface of certain leukocytes where it plays a key role in the activation of immunostimulatory T cells. This function is diverted by the Human Immunodeficiency Virus (HIV) envelope glycoprotein (gp120), which uses CD4 as its primary receptor for cell entry. The requirement of CD4 for viral entry has rationalised the development of recombinant CD4-based proteins as competitive viral attachment inhibitors and immunotherapeutic agents. While growing evidence suggests that redox exchange reactions involving CD4 disulphides (potentially catalysed by cell surface-secreted oxidoreductases) play an essential role in regulating the activity of CD4, their mechanism(s), biological utility and structural consequences that may be applicable to the designs of novel antiviral therapies and vaccines remain incompletely understood. Herein, a novel recombinant CD4 protein designed to bind gp120 through a targeted disulphide-exchange mechanism is described. This molecule contains a conservative Ser60 to Cys mutation on the CD4 domain 1 α-helix which, according to theoretical crystal structure modelling, positions a thiol in close proximity of the gp120 V1/V2 loop disulphide (126Cys–Cys196) resulting in the formation of an interchain disulphide bond. Experimental evidence for this effect is provided by describing the expression, purification, refolding, receptor binding and antiviral activity analysis of a recombinant two-domain CD4 variant containing the S60C mutation (2dCD4-S60C). This 2dCD4-S60C binds HIV-1 gp120 with a significantly higher affinity than wild-type protein under conditions that facilitate disulphide exchange and this translates into a corresponding increase in the efficacy of CD4-mediated viral entry inhibition. To gain more insights into the importance of redox activity in the mechanism of HIV entry, a panel of recombinant 2-domain CD4 proteins (2dCD4), including wild-type and Cys/Ala variants, were used to show that Thioredoxin (Trx), an oxidoreductase found on the cell surface, reduces 2dCD4 highly efficiently, catalysing the formation of conformationally distinct monomeric 2dCD4 isomers, and a stable, disulphide-linked 2dCD4 dimer. HIV-1 gp120 was shown to be incapable of binding a fully oxidised, monomeric 2dCD4 in which both domain 1 and 2 disulphides are intact, but binds robustly to reduced equivalents that are the products of Trx-mediated isomerisation. This Trx-driven dimerisation of CD4, a process believed to be critical for the establishment of functional MHCII-TCR-CD4 antigen presentation complexes, is shown to be impaired when CD4 is bound to gp120. Finally, preliminary, low-resolution structural analysis of individual CD4 domains 1 and 2 are suggestive of intrinsic metastability in domain 2, and reduction of its resident allosteric disulphide bond likely underpins the structural rearrangements in CD4 that are required for efficient interaction with gp120. Overall, these findings emphasise the fundamental importance of redox pathways in the biochemical mechanism of HIV entry, and illustrate the feasibility of exploiting these for the development of novel antiviral ligands.

This investigation is concerned with cell surface antigens present on murine AKR/J mice spleen and thymus cells which have been extracted with papain. Isolation of individual proteins was accomplished by granulated gel electrofocusing. Similar patterns recorded by both electrofocusing procedures identified several proteins limited to the AKR/J and C3Heb/FeJ spleen and thymus samples, which represent Murine Leukemia Virus-associated surface proteins.

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