Previous research has provided evidence that CD8 T cells mediate immunity against infection with Theileria parva. However, the immunity induced by one parasite strain doesn‟t give complete protection against other strains and this is associated with parasite strain specificity of the CD8 T cell responses. There is evidence that such strain specificity is a consequence of the CD8 T cell responses of individual animals being focused on a limited number of immunodominant polymorphic peptide-MHC determinants. Dominant responses to the Tp2 antigen have been demonstrated in animals homozygous for the A10 MHC haplotype. Three Tp2 epitopes recognised by A10+ animals (Tp249-59, Tp250-59 and Tp298-106) have been defined. This project set out to investigate the dominance of these epitopes and to examine the T cell receptor (TCR) repertoires of the responding T cells. The specific objectives were to: (i) Determine the dominance hierarchies of the three defined Tp2 epitopes in both A10-homozygous and -heterozygous cattle. (ii) Examine the clonal repertoires of epitope-specific responses by analysis of TCR gene expression. (iii) Isolate full-length cDNAs encoding TCR α and β chain pairs from T cell clones of defined epitope specificity and use them to generate cells expressing the functional TCRs. Using MHC class I tetramers the relative dominance of CD8 T cell responses were found to differ between A10-homozygous and heterozygous cattle. All A10-homozygous cattle examined had detectable responses to all 3 Tp2 epitopes, the Tp249-59 epitope consistently being the most dominant. By contrast, only some A10-heterozygous cattle had detectable responses to Tp2 and when present the response was specific only for the Tp298-106 epitope. Analyses of the sequences of expressed TCR β chains showed that the responses in individual animals were clonotypically diverse, but often contained a few large expanded clonotypes. The TCRs of Tp298-106–specific T cells showed preferential usage of the Vβ13.5 gene and the frequent presence of a “LGG” motif within the CDR3 of the B chain. A conserved (public) TCRβ clonotype shared by the Tp250-59-specific CD8 T cells from all A10-homozygous cattle was identified. The TCRα chains co-expressed with this public TCRβ clonotype were identified for a number of T cell clones. Lentivirus transduction of Jurkat cells with three full-length TCR α and β chain pairs resulted in successful expression of one of the α/β chain pairs as a functional TCR, thus providing the basis for future work to generate bovine T cells expressing defined TCRs in vitro.

Equine infectious anemia virus (EIAV) has been used as a model to investigate protective mechanisms against lentiviruses. Unlike other lentiviruses, EIAV replication can be eventually controlled in most infected horses leading to an inapparent carrier state free of overt clinical signs which can last for many years. Maintenance of this carrier state is absolutely dependent on active immune responses as evidenced by the fact that immunosuppressive drugs can induce the recurrence of disease. However, the immune mechanisms that are responsible for this control of infection are not yet identified. As the resolution of the initial infection is correlated with the appearance of the virus-specific cytotoxic T lymphocytes (CTL), it appears that cellular immune responses play an important role. However, most studies into this protective mechanism have been limited to the identification of specific epitopes, usually at a single time point in the infection. Few studies have examined the cellular immune responses to the viral antigens throughout the infection period. Since the virus undergoes rapid mutation following infection, the adaptive immune response must also evolve to meet this challenge. Previously, the EIAV envelope (gp90) protein was shown to be the primary determinant of vaccine efficacy. Here, we hypothesized that the maturation of cellular immune responses is a lengthy process and involves envelope-specific T cell recognition shifting from immunodominant variable determinants to conserved immunorecessive determinants during the initial stages of the EIAV infection. The first part of this dissertation was to develop a new in vivo method to identify envelope-specific T cell responses. The second part of this dissertation was to investigate whether envelope-specific T cell recognition evolved in EIAV-infected ponies. Finally, the mechanisms for this T cell immunodominant shifting were also investigated from the point of both virus sequence mutation and T cell clone expansion and contraction. Also, a new EIAV attenuated vaccine which contained a consensus gp90 sequence was tested to see if it facilitated T cell recognition of the more conserved regions early in the infection. Our results indicated that envelope-specific T cell recognition patterns changed over time. Early after infection, dominant immune responses to the peptides in the carboxyl-terminus variable region were identified. By six months post infection, the recognized peptides spanned the entire envelope sequence, with a shift to the amino-terminus conserved region. The mechanisms responsible for this change remain unclear, but analysis of T cell receptor repertoire indicated that T cell clonal expansion and contraction might be one of the reasons. Our demonstration that envelope-specific peptide recognition shifts from the variable to the more conserved regions provides evidence that the maturation of cell mediated immune response is parallelled with long-term control of this infection.

Gene therapy has shown potential in alleviating a wide range of diseases, ranging from viral infections to autosomal diseases. One of the obstacles to gene therapy reaching its full potential is the inadequacy of methods to deliver therapeutic nucleic sequences. Current delivery of gene therapy entails use of viral and non-viral vectors. Viral vectors are however associated with drawbacks such as potential toxicity, high cost and labour-intensive production. Thus non-viral delivery alternatives are being developed in an attempt to overcome difficulties associated with nucleic acid delivery for gene therapy. Virus-like particles are potentially very useful delivery vehicles as their production is simple and cost effective, the particle surface is amenable to modification and the capsid interior can be altered to accommodate a variety of cargoes. One such particle is the recombinant HBV capsid, which comprises a single species of protein and is tolerant of amino acid substitutions on the surface exposed immunodominant loops. This study aimed to enhance the vector-like properties of the HBV virus-like particle by including amino acid substitutions on the particle surface. These substituted residues in turn provided a conjugation site for tropic and immuno evasive moieties. It was found that lysine substitutions resulted in poor conjugation to the capsid surface, whereas substituted cysteine residues resulted in almost all protein-moiety conjugates forming. In order to introduce lysine and cysteine substitutions, a novel method of cloning into the HBV was generated. In doing so, complicated procedures associated with cloning into the immunodominant loop of the HBV capsid have been alleviated. Ligands containing galactose were utilised on the surface of both the HBV capsid and liposomes to confer hepatotropism. The presence of the galactose moieties on the surface of the HBV capsid prevented indiscriminate cellular uptake in cultured cells, however did not improve hepatotropism. Galactose ligands on the surface of liposomes did improve transfection efficiency, however they required a short linker distance between liposome surface and galactose group. The inclusion of galactose in liposome formulations also provided a means to deliver siRNA to the liver of transgenic HBV mice. It is believed that with alterations to the ligand structure, it is possible to provide HBV capsids with hepatotropism in future experimentation. This study demonstrated that the exposed external surface of the HBV capsid is amenable to convenient conjugation, which potentially facilitates immune evasion and conferring of defined tropism.

Theileria parva is a complex protozoan parasite causing East Coast fever in Eastern and Central Africa. Vaccination using live parasites is an effective control measure and has been used in Zambia based on locally isolated and introduced T. parva stocks. Diversity among T. parva populations was investigated in parasites from two Zambian provinces with different disease epidemiologies and control histories. Isolates from the pre-vaccination era, local and exotic stocks used for vaccination, and one recent field isolate were cloned and passaged in vitro to study genomic stability over time. The results of the data from three genome-wide probes indicate a marked homogeneity and stability among the Zambian isolates in contrast to East African isolates. Results from Southern blot profiles and the polymorphic immunodominant molecule (PIM) sequence analysis suggest a common origin for the Zambian isolates from the pre-vaccination era, except for one isolate (Zam5) from Southern Province. This isolate showed characteristics suggesting a buffalo origin. Assays for genotype characterisation were developed using five allelic markers. Multilocus characterisation revealed identical profiles in a recent Zambian isolate from Southern Province and two components of an exotic cocktail vaccine, indicating the escape of one of the vaccine stocks in the field. Characterisation of T. parva field populations by RFLP-PCR assays after immunisation revealed the presence of dominant genotypes from those that had been used for vaccination. Circumstantial evidence for the involvement of one of the exotic vaccine parasites in epidemics in Southern Province is presented and a hypothesis formulated for the rapid spread of this genotype. Analysis of the characterisation data suggested the existence of two groups of T. parva parasites of different origin. The classic T. parva group, characterised by a dimorphism of the p150, p104 and p32 loci and the absence of a p67 insert and a buffalo-derived group which showed a polymorphism of p150, p104 and p32 and the presence of a p67 insert. There is evidence that recombination occurs, resulting in parasites that have characteristics of both groups. The relevance of these recombinant parasites in the epidemiology of the disease seems low. Characterisation of larger samples from areas of regular buffalo-cattle contact is necessary to clarify this. Sequence analysis of the most discriminative locus (PIM) was undertaken and gene conversion could be the main mechanism generating diversity. A more appropriate nomenclature for T. parva is proposed based on the growing evidence of molecular differences among isolates and stocks.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [93]-113).

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 64-80).

Oomycete Pythium insisiosum is the etiologic agent of pythiosis, chronic and granulomatous disease, which affects humans and animals in tropical and subtropical areas of the world. Due to ergosterol absence in the plasmatic membrane of this microorganism, treatments based on antifungal agents have been ineffective. Immunotherapy has emerged showing promising results. In this context, the improvement of the immunotherapy was the main goal of this study, whose first purpose was to characterize the immunodominant antigens from this species (P. insidosum ATCC 58637 and P. insidiosum 210-LAPEMI) through sodium dodecyl sulfate polyacrylamide gel eletrophoresis (SDS-PAGE). Some proteins showing 15 kDa to 120 kDa were detected, and bands were transferred to nitrocellulose membranes in order to develop the western blot tests. The second purpose was to characterize the humoral response to pythiosis in equine, in bovine and in experimental pythiosis in rabbits, considering the no treated disease, the disease cured by immunotherapy, the disease treated with immunotherapy but not cured, and the disease cured spontaneously. To encompass all these cases we have included sera from: a) horses cured by immunotherapy; b) horses with pythiosis but not responsive to immunotherapy; c) horses with pythiosis but not treated; d) bovines with pythiosis and showing spontaneous cure; e) rabbits with experimental pythiosis but not treated; and f) rabbits with experimental pythiosis treated with immunotherapy. All specimens recognized three immunodominant proteins: 74, 33 and 32 kDa. The sera from horses cured by immunotherapy (a) and bovine sera (d) recognized another immunodominant protein, 55 kDa, which was weakly positive or negative in the other groups. Thus, the 74, 33 and 32 kDa immunodominant proteins suggest a relevant function in the humoral response to pythiosis because they were recognized by horses, rabbits and bovines. In addition, the 55 kDa antigen, which is being reported here for the first time, is likely to be involved in the cure mechanisms stimulated by immunotherapy. / O oomiceto Pythium insidiosum é o agente etiológico da pitiose, doença granulomatosa crônica que afeta animais domésticos (eqüinos, bovinos, etc) e o homem, em regiões tropicais e subtropicais de todo mundo. A ausência de ergosterol na membrana plasmática deste microorganismo torna a antifungicoterapia muito limitada, donde a imunoterapia (a base de hifas rompidas) tem emergido com resultados promissores. A identificação de antígenos imunodominantes é fundamental para a consolidação dos imunoterápicos utilizados no tratamento da pitiose. Objetivando-se o refinamento destes imunoterápicos, o presente estudo teve como proposta inicial, caracterizar os antígenos imunodominantes desta espécie (P. insidosum ATCC 58637 e P. insidiosum 210-LAPEMI) através de eletroforese em gel de poliacrilamida (SDS-PAGE). Diversas proteínas de 120 a 15 kDa foram detectadas e, a seguir, eletrotransferidas para membranas de nitrocelulose para realização de western blot. O segundo objetivo foi caracterizar a resposta humoral à doença em eqüinos, bovinos e na infecção experimental em coelhos, considerando-se a doença curada pela imunoterapia, doença tratada pela imunoterapia e não curada, doença não tratada, e na doença evidenciando cura espontânea. Para tanto, utilizou-se o soro de: a) eqüinos doentes e curados pela imunoterapia; b) eqüinos doentes e sem resposta a imunoterapia; c) eqüinos doentes e não tratados; d) bovinos doentes e com cura espontânea; e) coelhos infectados e tratados pela imunoterapia; f) coelhos infectados e não tratados. Todos os soros reconheceram três proteínas imunodominantes: 74, 33 e 32 kDa. O soro dos eqüinos doentes curados pela imunoterapia (a) e o soro dos bovinos (d) reconheceram também outra proteína imunodominante de 55 kDa, a qual, foi fracamente reconhecida ou ausente nos demais grupos. Assim, comprova-se que as proteínas de 74, 33 e 32 kDa desempenham relevante papel na resposta imune à pitiose, pois foram imunogênicas nas três espécies avaliadas. Em adição, sugere-se que a proteína de 55 kDa possa estar envolvida no mecanismo de cura promovido pela imunoterapia; ressalta-se que esta proteína é aqui pela primeira vez relatada.

Porcine circovirus type 2 is a single stranded DNA virus that belongs to the genus Circovirus in the family Circoviridae. This virus is associated with many kinds of diseases in pigs and causes significant economic losses in swine-breeding. In this study, two approaches of vaccination were tested in order to develop an effective vaccine against PCV2. The first approach was to test DNA vaccines. For this purpose, eukaryotic expression plasmids encoding two form of PCV2 Cap protein were constructed. The expression plasmids encoding murine TNF-α and IFN-α1 were also prepared for co-immunization with antigen encoding plasmid to enhance the immune response. The second approach is based on the previous finding that chimeric pentamers of VP1 mouse polyomavirus capsid protein fused with PCV2 can induce protective immunity against PCV2. These chimeric pentamers were further modified by AA substitutions in PCV2 Cap immunodominant epitope in order to enhance protective antibody response directed against PCV2. The chimeric pentamers and DNA vaccines were tested for ability to induce antibody immune response against PCV2 in mice. The results showed that chimeric pentamers are more potent inducers of protective antibody immune response against PCV2 compared to DNA vaccines. However, the protective antibody...