by Kok Dick Shun, Louis. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references. / Acknowledgements --- p.I / Table of Contents --- p.II / Abbreviations --- p.V / Aim and Scope of This Dissertation --- p.IX / Abstract --- p.X / Chapter Chapter One： --- General Introduction --- p.1 / Chapter 1.1 --- An Overview of the Immune System --- p.2 / Chapter 1.1.1 --- Innate Immunity --- p.2 / Chapter 1.1.2 --- Adaptive Immunity --- p.3 / Chapter 1.1.2.1 --- Humoral antibody immune response --- p.4 / Chapter 1.1.2.2 --- Cell- mediated immune response --- p.5 / Chapter 1.2 --- Immunomodulation --- p.6 / Chapter 1.3 --- An overview of the Host-mediated response against tumours --- p.9 / Chapter 1.3.1 --- T and B lymphocytes --- p.9 / Chapter 1.3.2 --- M acrophages --- p.14 / Chapter 1.3.3 --- Natural killer cells --- p.17 / Chapter 1.3.4 --- Lymphokines-activated killer cells --- p.20 / Chapter 1.3.5 --- Tumour infiltrating lymphocytes --- p.22 / Chapter 1.3.6 --- Cytokines --- p.23 / Chapter 1.4 --- Carbohydrates as Potential Immunostimulating agents --- p.33 / Chapter 1.5 --- General Properties of Bupleuri radix (B.R.) --- p.35 / Chapter Chapter Two： --- Materials and Methods --- p.36 / Chapter 2.1 --- Materials --- p.37 / Chapter 2.1.1 --- Animals --- p.37 / Chapter 2.1.2 --- Bupleuri radix --- p.37 / Chapter 2.1.3 --- "Buffers, culture media and chemicals" --- p.37 / Chapter 2.1.4 --- Cell lines --- p.48 / Chapter 2.2 --- Methods --- p.49 / Chapter 2.2.1 --- Extraction and fractionation of Bupleuri radix --- p.49 / Chapter 2.2.2 --- Purification of Bupleuri radix --- p.54 / Chapter 2.2.3 --- Characterization of Bupleuri radix --- p.55 / Chapter 2.2.4 --- In vivo Drug Treatment --- p.59 / Chapter 2.2.5 --- Isolation and preparation of cells --- p.59 / Chapter 2.2.6 --- Assays for the immunomodulatory activities of Bupleuri radix --- p.62 / Chapter 2.2.7 --- Assays for the immunorestorative properties of Bupleuri radix --- p.74 / Chapter 2.2.8 --- Assays for the anti-tumour activities of Bupleuri radix --- p.75 / Chapter 2.2.9 --- Statistical analysis --- p.83 / Chapter Chapter Three： --- "Fractionation, Purification and Characterization of Bioactive Compounds from Bupleuri radix" --- p.84 / Chapter 3.1 --- Results / Chapter 3.1.1 --- Extraction and Fractionation of Bupleuri radix --- p.85 / Chapter 3.1.2 --- Purification of Bupleuri radix --- p.85 / Chapter 3.1.3 --- Carbohydrate and Protein Contents of B.R. Fractions --- p.87 / Chapter 3.1.4 --- Lack of cytotoxicity of Bupleuri radix to Mouse Splenocytes --- p.91 / Chapter 3.1.5 --- LC50 of B.R. Fractions determined by Brine Shrimp Bioassay --- p.91 / Chapter 3.1.6 --- Heat stability of B.R. Fractions --- p.93 / Chapter 3.1.7 --- "Uronic Acid Content of BRIai, BRIaii, BRIbi and BRIbii" --- p.93 / Chapter 3.2 --- Discussion --- p.93 / Chapter Chapter Four： --- The Immunomodulatory Activities of Bupleuri radix --- p.96 / Chapter 4.1 --- Results / Chapter 4.1.1 --- Effect of Bupleuri radix on the Specific and Nonspecific Immunity --- p.97 / Chapter 4.1.1.1 --- Mitogenic effect of B.R. Fractions on Murine Splenocytes in vitro --- p.97 / Chapter 4.1.1.2 --- Mitogenic effect of B.R. Fractions on Murine Splenocytes ex vivo --- p.97 / Chapter 4.1.1.3 --- In vitro Mitogenic effect of B.R. Fractions treated with Periodate --- p.103 / Chapter 4.1.1.4 --- In vitro Mitogenic effect of B.R. Fractions treated with Acetic Acid --- p.103 / Chapter 4.1.1.5 --- In vitro Co -mitogenic effect of B.R. Fractions with Polymyxin B Sulphate --- p.107 / Chapter 4.1.1.6 --- Effect of B.R. Fractions on Lymphocyte sub-populations --- p.107 / Chapter 4.1.1.7 --- Primary Humoral Immune Response to SRBC in B.R.-treated mice --- p.107 / Chapter 4.1.1.8 --- Activity of cytotoxic T cells in B.R-treated mice --- p.111 / Chapter 4.1.1.9 --- Effect of B.R. Fractions on Interleukin-1 - like Factors Production --- p.111 / Chapter 4.1.1.10 --- Effect of B.R. Fractions on Interleukin-2 Production --- p.116 / Chapter 4.1.1.11 --- Effect of B.R. Fractions on Interleukin-2 Receptor Expression on Murine Splenocytes --- p.116 / Chapter 4.1.1.12 --- Effect of B.R. Fractions on GM-CSF Production --- p.119 / Chapter 4.1.1.13 --- Immunopotentiating effects of B.R. Fractions on Macrophages: --- p.119 / Chapter 4.1.1.13.1 --- In vivo Migration of Macrophages in B.R.-treated mice --- p.119 / Chapter 4.1.1.13.2 --- Effect of B.R. Fractions on the Fc Receptor Expression on Murine Resident Peritoneal Exudate Cells --- p.123 / Chapter 4.1.2 --- Immunorestorative Properties of Bupleuri radix --- p.123 / Chapter 4.1.2.1 --- Effect of B.R. Fractions on Lymphocyte Blastogenesis in Aged Mice --- p.123 / Chapter 4.1.2.2 --- Effect of B.R. Fractions on Lymphocyte Blastogenesis in Tumour-bearing Mice --- p.125 / Chapter 4.2 --- Discussion --- p.125 / Chapter Chapter Five： --- The Anti-tumour Activities of Bupleuri radix --- p.132 / Chapter 5.1 --- Results / Chapter 5.1.1 --- Cytostatic Effect of B.R. Fractions on Murine Tumour Cell Lines in vitro --- p.133 / Chapter 5.1.2 --- Effect of B.R. Fractions on the Growth of Tumour Ceils in vivo --- p.133 / Chapter 5.1.3 --- Effect of B.R. Fractions on the Survival of EAT-bearing mice --- p.140 / Chapter 5.1.4 --- Ex vivo Induction of Natural Killer Cell Activity by B.R. Fractions --- p.146 / Chapter 5.1.5 --- In vitro Induction of Lymphokine-activated Killer Cell Activity by B.R Fractions --- p.149 / Chapter 5.1.6 --- In vivo Induction of Tumour Infiltrating Lymphocytes by B.R. Fractions --- p.149 / Chapter 5.1.7 --- In vitro Induction of Macrophage-mediated Cytostatic Effect on Tumour Cells by B.R. Fractions --- p.151 / Chapter 5.1.8 --- In vitro Induction of Macrophage-mediated Cytostatic Eifect on Tumour Cells by B.R. Fractions --- p.153 / Chapter 5.1.9 --- Effect of B.R. Fractions on γ-interferon Production in vitro --- p.156 / Chapter 5.2 --- Discussion --- p.156 / Chapter Chapter Six： --- "General Discussion, Conclusion and Future Prospects" --- p.164 / Bibliography --- p.i

"Sin Nombre virus is a hantavirus first recognized in New Mexico in 1993. This virus is responsible for causing Hantavirus Pulmonary Syndrome, an acute, life threatening illness characterized by pulmonary edema, capillary leaking, and extreme respiratory distress. CD8+ cytotoxic T-cell lines specific for Sin Nombre virus were isolated from the peripheral blood mononuclear cells (PBMC) of a donor (NM3) who was naturally infected with the Sin Nombre virus, and has survived hantavirus pulmonary syndrome (HPS). Cytotoxic T lymphocyte (CTL) assays showed that one of these cell lines, 10K, specifically recognizes a nine amino acid epitope, TAHGVGIIP (amino acids 664-672 of the precursor GPC protein), which is located in the G2 protein after cleavage. Another cell line, 10c27, specifically recognized an eight amino acid epitope, AHGVGIIP (amino acids 665-672 of the precursor GPC protein), located in the G2 protein after cleavage. Using polymerase chain reaction (PCR) and CTL assays, the recognition of these epitopes was shown to be restricted by the B35.01 Class 1 human leukocyte associated antigen (HLA) allele. This information will be useful in creating a vaccine for use in immunizing people against the Sin Nombre hantavirus, as well as elucidating the pathogenesis of this disease. "

The most virulent malaria parasite, Plasmodium falciparum, makes use of extensive antigenic diversity to maximise its transmission potential. Parasite genomes contain several highly polymorphic gene families, whose products are the target of protective immune responses. The best studied of these are the PfEMP1 surface proteins, which are encoded by the var multi-gene family and are important virulence factors. During infection, the parasite switches expression between PfEMP1 variants in order to evade adaptive immune responses and prolong infection. On the population level, parasites appear to be structured with respect to their var genes into non-overlapping repertoires, which can lead to high reinfection rates. This non-random structuring of antigenic diversity can also be found at the level of individual var gene repertoires and var genes themselves. However, not much is known about the evolutionary determinants which select for and maintain this structure at different ecological scales. In this thesis I investigate the mechanisms by which multi-scale immune selection and other ecological factors influence the evolution of structured diversity. Using a suite of theoretical frameworks I show that treating diversity as a dynamic property, which emerges from the underlying infection and transmission processes, has a major effect on the relationship between the parasite’s transmis- sion potential and disease prevalence, with important implications for monitoring control efforts. Furthermore, I show that an evolutionary trade-off between within-host and between-host fitness together with functional constraints on diversification can explain the structured diversity found at both the repertoire and parasite population level and might also account for empirically observed exposure-dependent acquisition of immunity. Together, this work highlights the need to consider evolutionary factors acting at different ecological scales to gain a more comprehensive understanding of the complex immune-epidemiology of P. falciparum malaria.

vÃrus sincicial respiratÃrio humano (VSRh) à o agente viral mais freqÃentemente relacionado a infecÃÃes do trato respiratÃrio inferior em crianÃas menores de dois anos de idade. O VSRh à caracterizado antigenicamente em dois grupos: A e B, e cada grupo apresenta vÃrios subgrupos. A glicoproteÃna G à a principal responsÃvel pela a variaÃÃo antigÃnica inter e intragrupos desse vÃrus. Os objetivos desse estudo foram caracterizar os perÃodos epidÃmicos e a diversidade antigÃnica e genÃmica dos VSRh circulantes em Fortaleza, Cearà â Brasil, durante cinco perÃodos epidÃmicos consecutivos (2004-2008). A imunofluorescÃncia indireta (IFI) foi utilizada para a triagem de VSRh e de todos os vÃrus analisados e para a caracterizaÃÃo antigÃnica dos VSRh. A RT-nested-PCR seguida do seqÃenciamento parcial do gene G foi utilizada para a caracterizaÃÃo gÃnomica dos VSRh. O VSRh foi detectado em 456 das 2885 (15.8%) amostras. O pico dos perÃodos epidÃmicos de VSRh ocorreu nos meses de marÃo a maio relacionado à ocorrÃncia de chuvas. Um total de 282 VSRh (62,8%) foram caracterizados antigenicamente por IFI, sendo 170 VSRhA (60,3%) e 112 VSRhB (39,7%). Ambos os grupos circularam durante todo o perÃodo analisado sendo observado o predomÃnio de A em todos os anos. Um total de 250 VSRh (54,8%) foi submetido à RT-nested-PCR com amplificaÃÃo de 133 e seqÃenciamento de 86. A caracterizaÃÃo genÃmica dos VSRh identificou os subgrupos GA2 e GA5 para o VSRhA e os subgrupos GB3 e BA para o VSRhB. Esses quatro subgrupos co-circularam durante o ano de 2006. Nos anos de 2004, 2005 e 2007 verificou-se a presenÃa dos dois subgrupos de VSRhA. Em 2008 somente o GA2 circulou. Em 2004, 2007 e 2008 somente o subgrupo BA esteve presente. Em 2005 somente o GB3 circulou. Os VSRhA apresentaram uma maior variabilidade nas seqÃÃncias nucleotÃdicas, indicando uma possÃvel pressÃo seletiva positiva. Houve variaÃÃes no Ãnicio, fim e duraÃÃo de cada perÃodo epidÃmico de VSRh, assim como na circulaÃÃo de grupos e subgrupos. / The human respiratory syncytial virus (HRSV) is the major agent of lower respiratory tract in children under two years old. HRSV is characterized antigenically into two groups: A and B, and each group has several subgroups. Glycoprotein G is primarily responsible for the antigenic variation between and within groups of viruses. The aims of this study were to characterize the epidemic periods and the antigenic and genomic diversity of circulating HRSV in Fortaleza, Cearà - Brazil, for five consecutive epidemic periods (2004-2008). The screening of positive samples to HRSV and other viruses analyzed, as the antigenic characterization of HRSV was carried out by indirect immunofluorescence. RT-nested-PCR followed by partial sequencing of the gene G was used for genomic characterization of HRSV. The HRSV was detected in 456 of 2885 samples (15.8%). The peak of the epidemic periods of HRSV occurred from March to May related to rainfall. A total of 282 HRSV (62.8%) were characterized antigenically, with 170 HRSVA (60.3%) and 112 HRSVB (39.7%). Both groups circulated throughout the period analyzed with a predominance of HRSVA in all years of study. A total of 250 HRSV (54.8%) were submitted to RT-nested-PCR with amplification of 133 and sequencing of 86. The genomic characterization of HRSV identified subgroups GA2 and GA5 for HRSVA and subgroups GB3 and BA for HRSVB. In the years 2004, 2005 and 2007 both subgroups of HRSVA circulated. In 2008 only GA2 circulated. In 2004, 2007 and 2008 only the subgroup BA was present. In 2005 only the GB3 circulated. The HRSV A showed a higher variability in nucleotide sequences, indicating a possible positive selective pressure. There were variations in the beginning, end and duration of each epidemic period of HRSV, as well as in the occurrence of groups and subgroups.

Human respiratory syncytial virus (HRSV) is a negative sense, single strand RNA virus that causes respiratory tract infection with common cold-like symptoms, which can be severe in children, immunocompromised, and the elderly. Even with 60 years of research, the need for vaccine and effective treatment has not been met. In this work, recombinant viruses have been generated which will be valuable in gaining a better understanding of HRSV subtypes, glycoprotein evolution, and the polymerase localization, which would contribute to HRSV vaccine and therapeutics development. The differences in the fitness of A and B antigenic subtypes of HRSV and how it affects the regional circulation pattern is not well understood. To study and compare the two subtypes, it is important to use clinically relevant recombinant viruses and to use animal models that best represent human infection. Using a wild-type virus strain (A11 and B05) from each HRSV subtype, a wild-type like recombinant (r) virus, rHRSVA11, and recombinant viruses expressing fluorescent proteins, rHRSVA11EGFP(5) and rHRSVB05dTom(5), were generated. Characterization of rB05 viruses demonstrated that the differences in the fluorescent protein expressed did not affect virus growth kinetics. To prepare for an experiment in cotton rats, recombinant HRSVs generated were used to infect cotton rat lung cells in vitro. With confirmation of infection of cotton rat lung cells by rHRSV, cotton rat co-infection experiment was planned for the recombinant A11 and B05 viruses and a microneutralization assay was developed for post-infection processing of the in vivo samples. The BA genotype of HRSV B subtype is a strain of HRSV B subtype containing a 60 nucleotide duplication in the glycoprotein (G) gene. HRSV BA genotype was first isolated in 1998 and has quickly become the predominant genotype circulating globally. Although a role of immune evasion by the strains of BA genotype has been suggested to explain this phenomenon, few studies have supported this hypothesis. To compare the HRSV B subtype virus with and without the duplication, rB05 virus lacking the duplication, rHRSVB05EGFP(5)GΔ60b, and containing an epitope tag within the duplication, rHRSVB05EGFP(5)Gmycb, were generated. A serial passage experiment was set up using rHRSVB05EGFP(5) and rHRSVB05EGFP(5)GΔ60b to understand the mutations that accumulate in the G protein gene of each virus. This will be valuable in setting up a similar experiment in the presence of immune pressure to understand the advantage that is conferred to the virus containing the duplication. Expression of Gmyc was confirmed in rHRSVB05EGFP(5)Gmyc infection, which validated that this virus can be used to study the HRSVB05 G protein and modifications in the duplicated region. The HRSV large (L) protein is essential in HRSV transcription and replication, but is difficult to study due to lack of immunologic reagents and challenges with purification. Recombinant viruses expressing reporter and polymerase fusion proteins have been generated and used for studying various other viral polymerases. Expression plasmids for HRSV L protein containing a reporter protein in its variable region 2 have been published. However, the modification resulted in downregulation in the function of the protein and rHRSV expressing modified L protein have not yet been published. In this study, rHRSVB05LVenus was generated to study the effects of modification of HRSV L protein variable region and the localization of HRSV L protein. LVenus protein in rHRSVB05LVenus infected cells was visualized by confocal laser scanning microscopy and the expression levels were examined by immunoblotting. rHRSVB05LVenus was compared to rHRSVB05EGFP(5) with unmodified L protein to show that modification of HRSV L protein had no effect on virus replication. Viruses had equivalent growth kinetics and were equally sensitive to ribavirin, a known HRSV inhibitor. The recombinant viruses generated in this study are valuable tools in answering questions that are difficult to pursue without clinically relevant recombinant viruses. Characterization of the rHRSVs demonstrated that these viruses will have many applications. In this study, viruses were characterized for the basic growth kinetics, expression of proteins of interest, and assay development. With these validated tools, questions such as the cause of the epidemiological pattern observed for HRSV A and B subtypes, the role of host immune response in advantage conferred to HRSV BA genotype, and the effects of inhibitors to formation of HRSV polymerase complex can be addressed. / 2018-10-31T00:00:00Z

Abstract not available

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 89-111).

The diplomonads are a diverse group of eukaryotic flagellates found in microaerophilic and anaerobic environments. The most studied diplomonad is the intestinal parasite Giardia intestinalis, which infects a variety of mammals and cause diarrheal disease. Less is known about Spironucleus salmonicida, a parasite of salmonid fish, known to cause systemic infections with high mortality. We created a transfection system for S. salmonicida to study cellular functions and virulence in detail (Paper I). The system was applied to explore the mitochondrion-related organelle (MRO) in S. salmonicida. We showed that S. salmonicida possesses a hydrogenosome (Paper II) with a higher metabolic capacity than the corresponding MRO of Giardia, the mitosome. Evolutionary analysis of key hydrogenosomal proteins showed ancient origin, indicating their presence in the ancestral diplomonad and subsequent loss in Giardia. Annexins are of evolutionary interest since these proteins are found across all kingdoms. Annexin-like proteins are intriguingly expanded into multigene families in Giardia and Spironucleus. The annexins of S. salmonicida were characterized (Paper III) with distinct localizations to various cellular structures, including a putative adhesion structure anterior in the cell. The disease-causing Giardia trophozoites differentiate into infectious cysts, a process essential for transmission and virulence of the parasite. Cysts are often spread via contaminated water and exposed to environmental stressors, such as UV irradiation. We studied the survival and transcriptional response to this stress factor (Paper IV) and results showed the importance of active DNA replication machinery for parasite survival after DNA damage. In addition, we studied transcriptional changes along the trajectory of encystation (Paper V), which revealed a coordinated cascade of gene regulation. This was observed for the entire transcriptome as well as putative regulators. Large transcriptional changes appeared late in the process with the majority of differentially regulated genes encoding hypothetical proteins. We studied the localizations of several of these to gain information of their possible function. To conclude, the diplomonads are complex eukaryotic microbes with cellular processes adjusted to match their life styles. The work in this thesis has provided insight of their adaptations, differences and similarities, but also new interesting leads for future studies of diplomonad biology and virulence.

Trypanosomosis is a parasitic disease in man, domestic and wild animals and is of major economic importance in many parts of the world, particularly in Sub-Saharan Africa. Trypanosoma congolense, T vivax and T brucei brucei are the major pathogenic trypanosomes infecting cattle in sub-Saharan Africa. The parasite itself is not directly responsible for the disease, but rather causes illness through the release of pathogenic factors. One of the major pathogenic factors released by trypanosomes is proteinases. Trypanotolerant cattle produce antibodies against a trypanosomal proteinase, congopain, that inhibit congopain activity. Congopain thus has vaccine potential. This study describes the mapping of immunogenic epitopes of congopain to identify peptide regions of the protein that induce enzyme inhibitory antibodies for inclusion in a trypanosome vaccine. This vaccine approach targets the disease, rather than the parasite by focusing on a pathogenic factor. These peptides also have potential for use in diagnostic assays. Peptides from the catalytic domain of a trypanosomal cysteine proteinase, congopain, were selected using an epitope prediction program. Peptides selected were from the two forms of congopain called CP1 and CP2. Antibodies against peptide-carrier conjugates were produced in chickens. The antibodies recognised native congopain, recombinant CP2 and the recombinant catalytic domain (C2). This suggests that the peptides selected have promise for use in vaccines. The peptides were also used to determine whether they are natural immunogenic epitopes of CP2 and thus have potential for use in diagnostic assays. Antibodies in the sera from T. congolense infected cattle recognised all the peptides in an ELISA. Antibodies in the sera from C2-immunised, non-infected cattle recognised most of the peptides in an ELISA. In order to distinguish between T. congolense and T vivax infection, two different peptides from the C-terminal extensions of CP2 and vivapain were used in ELISA tests with sera from infected cattle. Although anti-peptide antibodies produced against the two C-terminal extension peptides were specific for their respective peptides, thereby indicating the discriminatory power of the peptides selected, there was cross-reactivity by the sera from T. congolense and T. vivax infected cattle. Optimal antibody binding peptide sequences of these two peptides need to be identified by testing modified sequences of these two peptides to improve the sensitivity of this assay. In addition to attempting to define the epitopes of congopain, preliminary studies to increase the immunogenicity of congopain were also undertaken. Alpha 2-macroglobulin is a natural host inhibitor of proteinases. Inhibition occurs by entrapment of an active proteinase within the alpha 2-macroglobulin cage. In addition, it has been demonstrated that antigen complexed with alpha 2-macroglobulin becomes more immunogenic, resulting in enhanced antigenic presentation of an entrapped antigen. This study reports the interaction between congopain and alpha 2-macroglobulin. The preliminary results of this study showing congopain-alpha 2-macroglobulin interaction could be used to explore the possibility of increasing the immunogenicity of congopain and congopain epitopes by complexing these to alpha 2-macroglobulin. Congopain epitopes complexed with alpha 2-macroglobulin could be used to form a peptide-based vaccine. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2003.

African animal trypanosomosis or nagana is a tsetse fly-transmitted disease, caused by Trypanosoma congolense, T. vivax and to a lesser extent T. brucei brucei. The disease causes major losses in revenue in many livestock-producing African countries. The available control methods, including chemotherapeutic drugs and insecticidal spraying, have become environmentally unacceptable. Antigenic variation displayed by the parasites has hindered vaccine development efforts. In this context, rather than focusing solely on the parasite itself, efforts in vaccine development have shifted towards targeting pathogenic factors released by the parasites during infection. Congopain, the major cysteine protease of T. congolense, has been shown to act as a pathogenic factor in the disease process. Analysis of the immune response of trypano-tolerant cattle revealed that these animals have the ability to control congopain activity in vivo. Therefore, congopain is an attractive vaccine candidate. To test the protective potential of congopain, immunisation studies had been conducted in cattle using the baculovirus-expressed catalytic domain of congopain (C2) in RWL, a saponin-based proprietary adjuvant from SmithKline-Beecham. Immunised animals were partially protected against a disease caused by an infection with T.congolense. Unfortunately, subsequent attempts to reproduce these results were disappointing. It was hypothesised that this failure could be due to the different expression system (P. pastoris) used to produce the antigen (C2), or the different adjuvant, ISA206 (Seppic), used, thus hinting towards an epitope presentation problem. Congopain had been shown to dimerise at physiological pH in vitro. Sera from trypano-tolerant cattle preferentially recognised the dimer conformation, advocating for protective epitopes to be dimer associated. For that reason, the present study aimed at improving the antigenicity of congopain through firstly, the elucidation of the protective epitopes associated with the dimer, secondly, the determination of the 3-D structure of the protease in order to map protective epitopes to later design mimotopes, and thirdly improve the delivery of congopain to the immune cells while maintaining the conformation of the protease by using a molecular adjuvant, BiP. A dimerisation model was proposed, identifying the amino acid residues forming the dimerisation motif of congopain. In the present study, particular amino acid residues located in the dimerisation motif were mutated by PCR-based site-directed mutagenesis to generate mutants with different dimerisation capabilities. The congopain mutants were expressed in yeast and their dimerisation capability was assessed by PhastGel® SDS-PAGE. The mutations altered both the electrophoretic mobility of the mutants and their enzymatic characteristics compared to wild-type congopain. This advocated for the involvement of these amino acid residues in the dimerisation process, although they seem not to be the only partakers. Wild-type C2 and mutant forms of C2 were heterologously expressed in P. pastoris and purified to crystallisation purity levels. Crystallisation of these proteins is currently underway, but the results are still unknown. While awaiting the crystallisation results, in silico homology modelling was employed to gain insight into the 3-D structure, using cruzipain crystal structure as a template. The modelled 3-D structure of congopain followed the common framework of cathepsin L-like cysteine proteases. Due to time constraints and awaiting the crystal-derived 3-D structure, the 3-D model of congopain was not exploited to design mimotopes with the potential to provide protection against the disease. As it was shown that protective epitopes are likely to be dimer-specific, maintaining the native conformation of congopain is essential for stimulating a protective immune response in animals. Chemically formulated adjuvants usually contain high salt concentration, at acidic or basic pH, thus might change the conformation of the protease. Adjuvants capable of efficiently delivering the antigen to immune cells while maintaining the conformation of the protease were sought. Proteins belonging to the HSP70 family are natural adjuvants in higher eukaryotes. A protein belonging to the HSP70 family was previously identified in T. congolense lysates and is homologous to mammalian BiP. Congopain was genetically fused with T. congolense BiP in order to improve antigen delivery and production of congopain activity-inhibiting antibodies. The chimeric proteins were successfully expressed in both bacteria and yeasts. The low yields of recombinantly expressed chimeras in yeast and problems associated with renaturation and purification of bacteria-expressed chimeras prevented immunisation studies in mice. However, the groundwork was laid for producing BiP-congopain chimeras for use in an anti-disease vaccine for African trypanosomosis. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2009.