The highly hydrophobic viral protein (VP) 7 of African horse sickness virus (AHSV) folds into a trimeric structure that aggregates to form flat, hexagonal crystals (Chuma et al., 1992). These crystals are composed of flat sheets of hexameric rings, similar to the rings of trimers seen in the outer core surface layer. The crystals have been shown to be highly immunogenic when used as a subunit vaccine and are able to elicit a strong immune response against subsequent viral infections (Wade-Evans et al., 1997). The aim of this study is to investigate the structural constraints of using these structures as a particulate, multiple peptide vaccine delivery system. Three hydrophilic regions at amino acid position 144, 177°and 200 on the VP7 surface of this trimeric structure were targeted for insertion of peptides and a new vector was constructed in this study with a multiple cloning site at each one of the three top domain sites. The newly constructed three-site VP7 mutant gene was expressed in the Bac- To-Bac expression system and the recombinant proteins were investigated for its solubility and crystal formation by sucrose density gradient centrifugation. The structure and stability of the modified, trimeric VP7 was confirmed and further analyzed. Scanning electron microscopy showed the formation of large structures by the trimeric modified VP7 protein units. These' structures differed from the hexagonal crystals formed by unmodified VP7, resulting in rough-looking, flat circular structures attached by protein cables. The high yield of protein expression and the ease, with which these particles can be purified, makes this vector ideal for vaccine use. These protein structures also seemed to remain stable after being stored under different conditions. Studies were also conducted on the stability of these structures after sonication, enabling a range of different size particles to be presented to the immune system. The purpose for the creation of multiple cloning sites was for the vaccine to be able to accommodate and efficiently present multiple epitopes to the immune system. An investigation was launched into the effect of peptide insertion at one or more of the multiple cloning sites. The initial study included the insertion of two small peptides from AHSV VP2 at amino acid sites 144 and 177 respectively. The size of the peptides that can be inserted is also very important in the use of virus-like particles as antigen carriers. In order to utilize the full potential of the VP7 particles as an antigen presentation system, it must be possible to accommodate large epitope-containing insertions. At the extreme, a stretch of 250 amino acids from AHSV VP2 was inserted into the 177 amino acid multiple cloning site of the three-site VP7. Structural evaluation of all these expressed proteins indicated that the structure of the VP7 subunit vaccine is stable and still retains the ability to form large aggregated structures from the trimeric units. Scanning electron microscope revealed that all these peptide-containing constructs retain approximately the same structural shape as the structures formed by the three-site VP7 mutant. / Dissertation (MSc(Genetics))--University of Pretoria, 2005. / Genetics / unrestricted

Commercial (high-grade) BaS04 selectively adsorbs two proteins from crude 0. savignyi salivary gland extracts. They co-purify during reversed-phase HPLC, but can be separated by hydrophobic-interaction chromatography. Both proteins have been characterized in terms of their molecular mass, amino acid composition and one partial internal amino acid sequence was determined. Their molecular masses were established through electro-spray mass spectrometry as 9333 Da and 9173 Da, respectively. The 9.3 kDa protein was designated BSAP1 and the 9.1 kDa protein BSAP2. Their amino acid compositions shows significant differences, in particular the presence of 6-7 and 8 cysteine residues in BSAP1 and BSAP2, respectively. It is therefore unlikely that these proteins are isoforms. All of the cysteine residues are involved in the formation of disulphide bonds, the only possible exception being one residue in BSAP1. Both proteins appear to be N-terminally blocked. An internal amino acid sequence Asp/Ser-Gly-Gly-Xxx-Xxx-Ile-Leu-Gly was obtained by sequencing a fragment of the cyanogen bromide cleaved BSAP2. It was suspected that these proteins might exhibit anticoagulant activity. The prothrombin time (PT) and activated partial thromboplastin time (aPPT) in the presence of the presumptive inhibitors were therefore evaluated. The aPPT was not significantly prolonged. The PT however did indicate a slight delay in the clotting time. This delay is not due to inhibition of factor VII, one of only two unique coagulation factors in the extrinsic pathway. The other factor is thromboplastin, also known as tissue factor. The nature of the protein adsorption to BaS04 was examined. From literature it is known that ϒ-carboxyglutamic acid-containing proteins, as well as some hydroxyproline and hydroxylysine-rich glycoproteins adsorb selectively to BaS04. The BSAPs were analysed for the presence of these modified amino acids, but all tests proved negative. The absence of Gla residues was determined using a Gla-specific stain on a polyacrylamide gel and was confirmed by performing mass spectrometry on native and decarboxylated protein samples. The absence of hydroxyproline and hydroxylysine was demonstrated by amino acid analysis. Both BSAPI and BSAP2 bind to neutral and negative membranes. BSAPI binds neutral and negative membranes more strongly than BSAP2. Its affinity for negative membranes is however much lower than its affinity for neutral membranes. In contrast, BSAP2 binds both membranes equally strongly. The binding of the proteins to the membranes was significantly lowered upon pre-incubation with Ca2+. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2006. / Biochemistry / unrestricted

In this study, the genomes of both the attenuated South African lumpy skin disease virus (LSDV) Neethling vaccine strain (LW) and a virulent field isolate from a recent outbreak namely the South African lumpy skin disease virus (LSDV) Neethling Warmbaths isolate (LD) have been cloned, sequenced and analysed. The genomic sequences of the South African LSDV Neethling Warmbaths isolate (LD) and the South African LSDV Neethling vaccine strain (LW), were compared to each other. The virulent South African isolate, LD was also compared to the previously sequenced virulent LSDV Neethling strain 2490 (LK), to determine molecular differences. The LSDV genome is approximately 150 kbp in size and consists of 156 putative genes. Of the 156 potential encoded proteins of the virulent LSDV field isolates, the South African LSDV Neethling Warmbaths isolate (LD) and the LSDV Kenyan Neethling strain 2490 (LK), 120 were identical, 21 showed differences of a single amino acid, 7 showed two amino acid differences, while only one showed three amino acid differences. These were mostly found in the variable terminal regions. The LSDV Kenyan Neethling strain 2490 (LK) was isolated in Kenya in 1958 and than re-isolated in 1987 from lesions of an experimentally infected cow (Tulmanet al. 2001). The South African LSDV Neethling Warmbaths isolate (LD) was isolated from lesions of a severely infected calf in the Northern Province of the Republic of South Africa, on the farm Bothasvlei in 2001 (David Wallace, Biotechnology Division, Onderstepoort Veterinary Institute, Republic of South Africa; Personal communication, 2001). Considering the geographically distant African regions of the isolates, namely South Africa (LD) and Kenya (LK) as well as the time when these viruses were isolated, minimal genetic variation was observed thereby suggesting that lumpy skin disease virus is genetically stable. When the attenuated vaccine strain (L W) was compared with the South African field isolate LD, a total of 480 amino acid differences were observed in 121 of the 156 potential encoded proteins. These were again mainly in genes of the terminal regions and a number of these led to frameshifts that caused truncated open reading frames (ORFs) as well as deletions of up to nine amino acids and insertions of up to 42 amino acids. These modified open reading frames (ORFs) encode proteins that are involved in various aspects, such as the regulation of host immune responses [a soluble interferon (IFN)-gamma receptor, and an interleukin-l (IL-l) receptor-like protein], gene expression (mutT motif proteins), DNA repair (superoxide dismutase), host-range specificity (ankyrin-repeat protein, kelch-like proteins) including proteins with unassigned functions. These differences could lead to a reduction in immuno evasive mechanisms and virulence factors present in attenuated LSDV strains. At this stage, it is not possible to define which amino acid differences in particular are responsible for dramatic alterations in viral virulence. A good indication, however are differences occurring in functional domains. A mutation in a trans-membrane region, for example, could alter the levels of secretion of a protein involved in the regulation of the host immune response. We conclude that the attenuated effect is likely to be the sum of the altered phenotypes of the expressed proteins, although it is also likely that a few specific proteins carry more weight. Further studies to determine the functions of the relevant encoded gene products will hopefully confirm this. The molecular design of an effective vaccine is likely to be based on the strategic manipulation of such genes. / Dissertation (MSc (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Please read the abstract in the section 00front of this document / Dissertation (MSc (Agric))--University of Pretoria, 2005. / Genetics / unrestricted

Submitted by Maria Creuza Silva (mariakreuza@yahoo.com.br) on 2015-04-15T14:46:57Z No. of bitstreams: 1 Tese Bethânia de Araújo Almeida. 2014.pdf: 800350 bytes, checksum: 1d096cce2c9cc1288163d0a75f44d3c5 (MD5) / Approved for entry into archive by Maria Creuza Silva (mariakreuza@yahoo.com.br) on 2015-04-15T14:47:12Z (GMT) No. of bitstreams: 1 Tese Bethânia de Araújo Almeida. 2014.pdf: 800350 bytes, checksum: 1d096cce2c9cc1288163d0a75f44d3c5 (MD5) / Made available in DSpace on 2015-04-15T14:47:12Z (GMT). No. of bitstreams: 1 Tese Bethânia de Araújo Almeida. 2014.pdf: 800350 bytes, checksum: 1d096cce2c9cc1288163d0a75f44d3c5 (MD5) / A vacinação é a principal estratégia de saúde pública para prevenir, controlar e erradicar uma série de doenças. Com o intuito de aportar contribuições e suscitar discussões acerca do desenvolvimento e provimento de vacinas, o estudo caracteriza a dinâmica de inovação no setor a partir de atores envolvidos nas etapas de desenvolvimento e produção, cujos investimentos e estratégias são condicionados por aspectos regulatórios e de apropriação dos benefícios oriundos das inovações. Por ser um setor dominado por poucas empresas multinacionais, que possuem alta taxa de inovação e amplo acesso ao mercado mundial, a direção da dinâmica de inovação no setor tende a acirrar ainda mais as desigualdades relacionadas ao desenvolvimento e provimento de vacinas para responder às necessidades de saúde das populações. No que tange aos aspectos empíricos, optou-se por analisar as instituições envolvidas no desenvolvimento inicial e intermediário de candidatas à vacina contra dengue, considerada uma tecnologia inovadora por não existir vacina licenciada contra a doença.