HLA (Human Leukocyte Antigen) molecules provide a framework for T-cell recognition of antigenic peptides and thus play an important role in the immune system and defence against pathogens. HLA molecules are encoded for by genes on the short arm of chromosome six in the human genome, a region of over 4000 kilo bases (kb) known as the human major histocompatibility complex (MHC) or HLA complex. According to structural and functional characteristics, the genes of this region have been classified into three families, namely classes ƒ¹, ƒ¹ƒ¹ and ƒ¹ƒ¹ƒ¹. The HLA genes are highly polymorphic and because of this characteristic, it increases the functional range of recognition of different antigens and contributes to immunological specificity. Previous studies on population groups such as Indians and Cambodians, have identified an association between HLA polymorphisms and susceptibility to TB. A large number of different population groups with diverse gene pools reside in South Africa, of which the Cape Coloured population is one. This anthropologically distinct population group¡¦s diverse gene pool originated from founder individuals of the colonizing population, which came from various nations and cultural backgrounds, including Europe, Africa (such as Khoi, San Xhosa, Sotho and East African populations), Madagascar and the Far East. Unusual MHC allele frequencies and haplotypes have been identified in the Cape Coloured population. The Cape Coloureds are highly susceptible to TB and reside in the Western Cape, which has a TB incidence rate that is higher than that of any other province in South Africa. The recently admixed Coloured population is a valuable candidate population for the identification of genes/mutations underlying complex diseases. This study focussed on polymorphisms of class ƒ¹ƒ¹ genes, specifically HLA-DRB, and their possible contribution to disease susceptibility in populations of South Africa. Two questions have been formulated: 1) What knowledge can we gain from the current literature on HLA variants in the different population groups of South Africa and disease susceptibility? 2) Is there an association between alleles of the most polymorphic class ƒ¹ƒ¹ MHC gene, HLA-DRB, and susceptibility or resistance to TB in the Cape Coloured population? These two primary questions have been addressed by: 1) Reviewing the literature concerning HLA alleles in diverse population groups in South Africa and their contribution to disease susceptibility. Chapter 2 addresses this objective and is written in the format of a review article to facilitate its future publication, 2) Conducting a TB case-control study, typing HLA-DRB in Cape Coloured individuals residing in the Western Cape to investigate the possible association between TB susceptibility or resistance and specific DRB alleles. The HLA-DRB1, DRB3 and DRB4 typing by means of PCR-SSP (polymerase chain reaction ¡V sequence specific primers) was done on DNA isolated from 106 TB patients and 107 controls from the Cape Coloured population. The results obtained for this experimental investigation is presented (also in publication format) in Chapter 3. Summary of main findings: 1) The literature overview of publications describing HLA related disease association in the different population groups in South Africa (Chapter 2), revealed that unique alleles contributing to susceptibility of various diseases have been identified in some South African populations. The large number of ethnic groups in South Africa and unique populations such as the Cape Coloureds provide a genetic resource, which has the potential to be utilized for candidate gene hunting. 2) A weak association between susceptibility for TB and HLA-DRB1*0301-0302 (DR3) and HLA-DRB3*0101-0301 (DR52) exists in the Cape Coloured population (Chapter 3). Since the South African population consists of a large number of different population groups with diverse gene pools, a unique opportunity to study disease predispositions among specific population groups with diverse genetic make-up is presented. With these different populations, often residing in a common environment, the interplay of environmental and genetic factors in disease development could be studied. For example, HLA typing of these populations could clarify the extent to which inter-population HLA variation contributes towards disease susceptibility. The identification of certain HLA-DRB alleles potentially contributing to TB susceptibility, could lead to an understanding of the differential factors involved in disease susceptibility and in turn lead to the understanding of the fundamental mechanisms involved. This could result in therapeutic approaches and treatments that will be advantageous to the population concerned. / Prof. L. Bornman

African horsesickness virus (AHSV) , a member of the genus Orbivirus in the family Reo viridae, is the aetiological agent of African horsesickness, a highly infectious non-contagious disease of equines. The AHSV virion is composed of seven structural proteins organised into a double layered capsid, which encloses ten double-stranded RNA segments. The double stranded (ds) RNA genome of AHSV encodes, in addition to the seven structural proteins, at least three non-structural proteins (NS1 to NS3). The assembly of viral proteins in AHSV-infected cells results in at least three characteristic particulate structures. The first of these structures are the complete virions and viral cores. Empty virions or particles that simulate the virion surface can be produced synthetically by the co-expression of various combinations of AHSV structural genes in insect cells. Apart from the core particles and complete virions, there are two additional structures observed in AHSV-infected cells. Unique virus-specified tubular structures, composed of NS1, are observed in the cytoplasm of all orbivirus-infected cells. The second structure, distinctive hexagonal crystals, is unique to AHSV and is composed entirely of VP7, the major core protein. The assembly of all these particles can be produced synthetically when expressed individually in an insect cell expression system. The aim of this investigation was first of all to investigate the structure and assembly of these structures and secondly to evaluate their use as vehicles for foreign immunogens. The NS1 gene of AHSV-6 was cloned as a complete and full-length cDNA fragment from purified dsRNA genome segment 5 and the complete nucleotide sequence determined. The gene was found to be 1749 bp in length with one major open reading frame (ORF) of 1645 bp, encoding a protein comprising 548 amino acids. The 5' and 3' termini of the gene were found to contain the conserved terminal hexanucleotide sequences of AHSV RNA fragments, followed by inverted heptanucleotide repeats. The deduced amino acid sequence was analysed and found to define a hydrophobic protein of 63 kDa. Antigenic profile analysis indicated a hydrophilic domain with relative high antigenicity in the C-terminus of the protein. This represents a possible insertion site for immunogenic epitopes. The cloned NS1 gene of AHSV-6 was modified at the 5' and 3' terminal ends to facilitate expression of the gene. In vitro expression yielded a protein corresponding to the predicted size of NS1. The gene was also expressed in insect cells, using a recombinant baculovirus and yields of approximately 1.0mg NS1 protein/106 cells were obtained. Expression of NS1 in insect cells resulted in the intracellular formation of tubular structures with diameters of 23 ±2 nm. Biophysical analysis of the AHSV tubules suggests that they are more fragile and unstable than BTV NS1 tubules. To gain more insight into the structure, assembly and the biochemical characteristics of AHSV cores and virions, a number of baculovirus multigene expression vectors have been developed and utilised to co¬express various combinations of AHSV genes. Cells infected with a dual-recombinant baculovirus, expressing AHSV-9 VP3 and VP7 genes, contained high levels of VP7 and low levels of VP3. The simultaneous expression of the two proteins resulted in the spontaneous intracellular assembly of empty multimeric core-like particles (CLPs) with a diameter of approximately 72 nm. These particles structurally resembled authentic AHSV cores in size and appearance. The yield of CLP production was low as a result of the insolubility of VP7, which aggregates preferably into large hexagonal crystal as well as the low yield of VP3. The interaction of CLPs with either VP2 or VP5 was investigated by co-infection of the VP3 and VP7 dual recombinant baculovirus with a VP2 or VP5 single recombinant baculovirus. Each of the outer capsid proteins interacted separately with CLPs. Co-expression of all four major structural proteins of AHSV, using two dual recombinant baculoviruses one expressillJg VP2 and VP3, the other VP5 and VP7, resulted in the spontaneous assembly of empty virus-like particles with a diameter of 82 nm. Although co¬expression of the different combinations of AHSV proteins was obtained, the levels of expression were low. This low levels of the AHSV capsid proteins and the aggregation of VP7 down regulated the assembly process. In order to investigate the possibility of the use of CLPs and VP7 crystals as particulate delivery systems, insertion analysis of VP7 was used to identify certain sequences in the VP7 protein that are not essential for the assembly of CLPs or trimer-trimer interactions in the crystals. Two insertion mutants of VP7 (mt177 and mt200) were constructed. In each case three unique restriction enzyme sites were introduced that coded for six amino acids. In mt177 these amino acids were added to the hydrophilic RGD loop at position 177-178 and for mt200 to amino acid 200 - 201. Both regions were located in the top domain of VP7. Insertion mt177 increased the solubility of VP7, but did not abrogate trimerisation and CLP formation with VP3. The yield of mutant CLPs was significantly higher than the normal CLPs, possibly due to the increased solubility and availability of VP7 trimers. Evidence about the size of an insert that can be accommodated by VP7 was provided by the insertion of a 101 amino acid region of VP2, containing a previously identified immunodominant region of VP2. The two chimeric VP7/TrVP2 proteins were investigated for their ability to form crystal structures and CLPs. The chimeric proteins did not produce the typical hexagonal crystal structure, but rather small ball-like structures. This investigation yielded valuable information regarding the structure and assembly of AHSV tubules, CLPs and VLPs. These findings also have practical value, since the multimeric structures can be utilised as delivery systems for immunogens, like the AHSV VP2 immunodominant epitopes. / Thesis (PhD (Genetics))--University of Pretoria, 2007. / Veterinary Tropical Diseases / unrestricted

No description available.

In this study the effects of repeated antigen injections on total complement (C') and C'4 of guinea pig serum were investigated to determine if constant antigenic stimulation would show changes in the C' and C'4 titers. Attempts were also made to correlate any changes with variations in antibody titers during the repeated antigen injections.

Several cellular and subcellular fractions were prepared from Pseudomonas aeruginosa strain PA-7. Those found to be immunogenic in rabbits included a heat-stable lipopolysaccharide, a protein-lipopolysaccharide complex, a cell wall preparation arid a formalin-killed whole cell vaccine. However, a lipopolysaccharide preparation extracted with phenol and water was found to be a poor immunogen in rabbits. The cell wall fraction proved to be the most effective immunogen in terms of the amount of antibody evoked, and of the duration of the serum antibody response. Hyperimmune sera produced against all four antigens were found to contain a mixed population of 2-mercaptoethanol sensitive and 2-mercaptdethanol resistant antibodies. Gel filtration and ion exchange chromatography studies established the presence of both IgM and IgG immunoglobulins in all four types of hyperimmune serum. Whole immune serum, as well as the IgM and IgG serum fractions, afforded passive protection to mice challenged with twenty or more LD₅₀ of viable organisms. There was an indication that the IgG fraction of two of the four serum types provided better protection than did the IgM fraction, but precipitation studies indicated that this may have been due to greater numbers of IgG immunoglobulins. In addition serum containing a high proportion of 2-mercaptoethanol resistant antibody-was found to promote faster clearance of injected bacteria than did serum taken earlier in the response. Immunodiffusion studies indicated that all four antigenic preparations contained at least one common immunogen; moreover, all serum types were able to react with sheep red blood cells coated with the heat-stable lipopolysaccharide preparation in passive hemagglutination and hemolysin tests. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

It was established that antibodies could be evoked in rabbits against ferredoxin purified from cultures of Clostridium pasteurianum and against its performic acid oxidized derivative. The extent of cross-reaction was studied between the two antisera and four related antigens: native ferredoxin, iron-sulfide free ferredoxin, performic acid oxidized ferredoxin, arid S-carboxymethylated ferredoxin. All combinations demonstrated cross reactivity by complement fixation, and in the case of oxidized ferredoxin antiserum, three preparations, native ferredoxin, iron sulfide free ferredoxin, and performic acid oxidized ferredoxin precipitated antibody. The data obtained with these cross-reactivity studies Indicated that the cysteine-containing regions of the ferredoxin molecule were not critically involved as antigenic determinants. The C-terminal region of the protein was considered for further study. This octapeptide was synthesized and tested for its ability to combine with antibody directed against both native ferredoxin and its performic acid oxidized derivative. The peptide exhibited specific binding to both antisera as demonstrated by inhibition of complement fixation and precipitation, and by equilibrium dialysis experiments. It is suggested that C. pasteurianum ferredoxin is antigenic in rabbits, that cysteine residues are not involved in at least two of the antigenic regions of the protein, and that the C-terminal octapeptide is one of the antigenic determinants of this molecule. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The effect of continual antigenic stimulation on the immune system of mice was studied using two different experimental approaches. A GVHR was induced in Fi mice by the injection of parental spleen cells at weekly intervals. Several weeks later the spleen cells of mice undergoing a GVHR were shown to be immunosuppressed as their in vitro responses to the mitogens Con A and LPS were substancially lower than control animals. The serum from these treated mice was also immunosuppressive to normal spleen cells. The proliferative response to Con A and allogeneic cells of normal: syngeneic, allogeneic, and parental spleen cells was 90% suppressed when GVH serum was added in comparison to the addition of normal serum. Similarly, the in vitro antibody response to a T dependant antigen was impaired; however, the antibody response to a T independant antigen was not impaired. These results indicate that T cell functions are more sensitive than are B cell function to immunosuppressive factors in the serum of mice undergoing a GVHR. The serum was fractionated by gel filtration on a Bio-Gel P-200 column. The inhibitory material in GVH serum eluted in the immunoglobulin fraction of serum which indicate that it has a molecular weight of 150,000 or greater. The second approach studied involved continual allogeneic stimulation. Parental type mice were injected at five day intervals with Fi spleen cells in order to induce a HVG reaction. After several injections the spleen cells from these mice were tested in vitro. The spleen cells from HVG mice responded the same as normal spleen cells to the mitogens Con A and LPS. The spleen cells from HVG mice showed an enhanced in vitro antibody response as compared to normal spleen cells. This enhancement was attributed to the allogeneic effect. This series of experiments have shown that the induction of a GVHR in mice can later lead to immunosuppression and production of immunosuppressive factors in the serum of these mice. The induction of a short term HVG reaction has no adverse effects on the immune system except for enhancing an antibody response. It is possible that a more prolonged HVG reaction would parallel the immunosuppression observed in mice undergoing a GVHR. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Recent work has shown that in vitro, the cytotoxic immune response to cell surface antigens is enhanced if the antigen to which the immune response is directed, is on cells bearing major histocompatibility antigens identical to those of the responding cells. This 'H-2 restriction' has been demonstrated in the mouse using virally infected cells, haptenated cells, cells bearing the male Y antigen, and cells differing at the minor histocompatibility loci. Other investigations have shown that antigenic determinants coupled to tolerated antigens or isologous serum proteins, elicit a humoral response which is weaker than that to the same determinant coupled to a heterologous carrier. This and other evidence suggest an inverse relationship between humoral and cell mediated immunity. The purpose of this investigation was to explore the humoral response to antigens on cells which are syngeneic or allogeneic to the recipient, in order to determine the influence of a tolerated as opposed to allogeneic background. The approach used in this study was as follows: Mice were immunized with antigen (rabbit Fab' fragments) attached to syngeneic, allogeneic, or F₁ (semi syngeneic), irradiated spleen cells. Specific anti-rabbit Fab' plaque forming cell numbers were determined five days after the third, weekly injection of Fab' coated spleen cells. Some of the spleen cells taken from the responding animals, on the day of sacrifice, were incubated in vitro with soluble antigen (rabbit Fab' fragments not specific for mouse cells) for four days. The results showed that the humoral response to antigens attached to cells bearing 'self histocompatibility antigens (i.e. syngeneic or F₁ semi syngeneic cells) was significantly weaker than the humoral response to the same antigen on allogeneic cells. The effect of in vitro incubation of responder spleen cells for four days with soluble antigen was to reverse this difference. Those spleen cells exhibiting lowered plaque forming cell numbers initially (i.e. those cells from mice immunized with antigen on syngeneic or F₁ cell surfaces) showed, after incubation, a response equal to or greater than those cells which intially (before in vitro incubation) demonstrated a larger response (i.e. cells from those mice immunized with antigen on allogeneic cell surfaces). / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

DNA immunization, or the use of antigen-expressing DNAs to raise immune responses, represents a novel approach to the study and manipulation of immune responses. In this dissertation, we examine the role of antigen expression at the target site, the role of antigen presentation by bone marrow-derived cells, and the effect of secretion of antigen on DNA-raised responses in mice. Immunizations were conducted using either gene gun delivery of DNA to the epidermis or intramuscular (i.m.) saline injections. To examine the role of antigen expression at the target site, we excised target sites at different time points following immunization. We immunized with plasmid DNA expressing three different forms of antigens: influenza hemagglutinin H1, human growth hormone and influenza nucleoprotein NP (membrane-bound, secreted and intracellular, respectively). We hypothesized that antigen expression at the target site would be essential in initiating immune responses. We demonstrate here that the target site plays different roles in gene gun and i.m. immunizations. We found that the skin target site played an essential role in eliciting maximal antibody and cytotoxic T lymphocyte (CTL) responses by gene gun immunization, although low-level responses can be raised independent of the target site. In contrast, the muscle target site was not essential for eliciting maximal immune responses following i.m. immunization. We suggest that gene gun immunization results in transfection of keratinocytes and bone marrow-derived Langerhans cells at the target site, and these cells together initiate maximal responses. In i.m. immunizations, on the other hand, nonmuscle cells at distal sites, perhaps bone marrow-derived cells in lymphoid tissues, become transfected and are sufficient for initiation of maximal responses. We also examined the role of antigen presentation by bone marrow-derived cells in initiation of CTL responses to influenza NP following gene gun and i.m. immunization. We hypothesized that antigen presentation by bone marrow-derived cells would be involved in initiation of CTL responses. To test this hypothesis, irradiated F1 mice of MHC class I H-2bxd haplotype were reconstituted with bone marrow from either H-2b or H-2d donors, creating two sets of bone marrow chimeric mice (H-2b → H-2bxd and H-2d → H-2bxd, respectively). We immunized the two sets of bone marrow chimeric mice and determined the MHC haplotype restriction of the induced CTL responses using H-2b- or H-2d-restricted peptides of NP. We found that the CTL responses initiated following gene gun and i.m. immunization were restricted to the haplotype of the bone marrow donor. In H-2b→ H-2bxd chimeric mice, CTL responses were restricted to H-2b, while in H-2d→ H-2bxd chimeric mice, CTL responses were restricted to H-2d. Thus, antigen presentation by bone marrow-derived cells, and not by skin or muscle cells, initiates CTL responses following both gene gun and i.m. immunization. Finally, we examined the effect of secretion of a DNA-expressed antigen on antibody responses. We hypothesized that a secreted antigen would raise greater antibody responses than a membrane-bound antigen, due to easier access of a soluble antigen to lymphoid tissues and to uptake by professional antigen-presenting cells and by antigen-specific B cells. We immunized mice with plasmid DNA expressing either a secreted or the normal membrane-bound form of influenza hemagglutinin H1. We found that secretion of H1 (sH1) did not result in enhanced antibody responses, with sH1 appearing to be less effective than H1. We suggest that the effectiveness of DNA immunization with membrane-bound H1 in raising maximal antibody responses may be due to MHC class II presentation of H1 via an endogenous pathway, resulting from direct transfection of bone marrow-derived APCs. We also found that secretion of H1 influenced the predominant IgG subclass of antibody responses raised by i.m. immunization. Secreted H1 raised predominantly IgG1 responses and H1 raised predominantly IgG2a responses. The IgG1 response to sH1 following i.m. immunization was IL-4 dependent, suggesting that the response to sH1 had a T-helper type 2 phenotype. We propose a model for the mechanism of initiation of immune responses by DNA immunization based on our results and taking them within the context of results from other investigators in the field. We propose that DNA immunization may initiate immune responses primarily by the direct transfection of bone marrow-derived cells that then express and present the DNA vaccine-encoded antigen. However, antigen expression by nonhemopoietic cells, particularly in skin, may play a role in raising maximal responses.

The immunomodulatory effects of Trichinella spiralis or Trichinella pseudospiralis soluble antigen extracts were examined in an effort to characterize the differences in immune responses seen during these Trichinella infections. The newborn larvae extracts of either parasite exhibited similar potency for stimulating macrophage PGE production; however, the muscle larvae extracts of T. pseudospiralis stimulated greater levels of PGE than did the muscle larvae extracts of T. spiralis. These data clearly indicate that Trichinella antigens possess immunomodulatory capabilities.