The goal of the work presented herein was to further our understanding of Bovine Leukocyte Antigen (BoLA) class I diversity of Holstein cattle and develop tools to measure class I restricted T cell responses to intracellular pathogens such as foot and mouth disease virus (FMDV) following vaccination. BoLA is a highly polymorphic gene region that allows the bovine immune system to differentiate pathogen-infected cells from healthy cells. Immune surveillance by CD8+ T cells plays an important role in clearing viral infections. These CD8+ T cells recognize BoLA class I molecules bearing epitopes (antigenic peptides) of intracellular origin in their peptide binding groove. Polymorphisms in the peptide binding region of class I molecules determine affinity of peptide binding and stability during antigen presentation. Different antigen peptide motifs are associated with specific genetic sequences of class I molecules. In order to better understand the adaptive immune response mediated by BoLA molecules, technologies from human medicine such as high-throughput sequencing, biochemical affinity and stability assays, tetramers and IFN-γ ELIspot assays could be applied. Therefore, it was hypothesized that we can translate these technologies from the study of human T cell responses to the study of cattle immunity. The first objective was to establish a comprehensive method for genotyping BoLA of Holstein cattle by using Illumina MiSeq, Sanger sequencing and polymerase chain reaction sequence-specific primers (PCR-SSP) (See Chapter 2). This is an important first step in order to study the BoLA restricted immune responses following FMDV vaccination. The second objective was to define the FMDV capsid protein peptide repertoire bound by BoLA class I molecules using bioinformatics and biochemical affinity and stability assays to facilitate the identification of T cell epitopes (See Chapter 3). The third objective was to demonstrate clonal T cell expansion for specific epitope polypeptides using ex-vivo multi-color flow cytometric MHC-epitope complexes (tetramers), followed by IFN-γ production measured by an ELIspot assay to quantify and define the antigen specific response of Holstein cattle to FMDV vaccination (see Chapter 4). In this, my dissertation studies aimed to improve our understanding of the BoLA class I restricted T-cell responses to candidate FMDV vaccines in Holstein cattle. In this manner, my research will improve animal health through the production of assays for characterizing the bovine immune response to intracellular pathogens and enhance vaccine design leading to improved biologicals to protect cattle from devastating infectious diseases.

Orientador: Paulo Lício de Geus / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Computação / Made available in DSpace on 2018-08-23T23:09:29Z (GMT). No. of bitstreams: 1 Pereira_AndreAugustodaSilva_M.pdf: 2078139 bytes, checksum: 3b321df6a81e4d3aaa8cf753b119f8a1 (MD5) Previous issue date: 2013 / Resumo: O resumo poderá ser visualizado no texto completo da tese digital / Abstract: The complete abstract is available with the full electronic document / Mestrado / Ciência da Computação / Mestre em Ciência da Computação

During acute and chronic infectious diseases hosts develop complex immune responses to cope with bacterial persistence. Depending on a variety of host and microbe factors, outcomes range from peaceful co-existence to detrimental disease. Mechanisms underlying immunity to bacterial stimuli span several spatiotemporal magnitudes and the summation of these hierarchical interactions plays a decisive role in pathogenic versus tolerogenic fate for the host. This dissertation integrates diverse data from immunoinformatics analyses, experimental validation and mathematical modeling to investigate a series of hypotheses driven by computational modeling to study mucosal immunity. Two contrasting microbes, enteroaggregative Escherichia coli and Helicobacter pylori, are used to perturb gut immunity in order to discover host-centric targets for modulating the host immune system. These findings have the potential to be broadly applicable to other infectious and immune-mediated diseases and could assist in the development of antibiotic-free and host-targeted treatments that modulate tolerance to prevent disease. / Ph. D.

Foot-and-mouth disease (FMD) is a highly contagious and economically important disease that affects even-toed hoofed mammals. The FMD virus (FMDV) is the causative agent of FMD, of which there are seven clinically indistinguishable serotypes. Three serotypes, namely, South African Territories (SAT)1, SAT2 and SAT3 are endemic to southern Africa and are the most antigenically diverse among the FMDV serotypes. A negative consequence of this antigenic variation is that infection or vaccination with one virus may not provide immune protection from other strains or it may only confer partial protection. The identification of B-cell epitopes is therefore key to rationally designing cross-reactive vaccines that recognize the immunologically distinct serotypes present within the population. Computational epitope prediction methods that exploit the inherent physicochemical properties of epitopes in their algorithms have been proposed as a cost and time-effective alternative to the classical experimental methods. The aim of this project is to employ in silico epitope prediction programmes to predict B-cell epitopes on the capsids of the SAT serotypes. Sequence data for 18 immunologically distinct SAT1, SAT2 and SAT3 strains from across southern Africa were collated. Since, only one SAT1 virus has had its structure elucidated by X-ray crystallography (PDB ID: 2WZR), homology models of the 18 virus capsids were built computationally using Modeller v9.12. They were then subjected to energy minimizations using the AMBER force field. The quality of the models was evaluated and validated stereochemically and energetically using the PROMOTIF and ANOLEA servers respectively. The homology models were subsequently used as input to two different epitope prediction servers, namely Discotope1.0 and Ellipro. Only those epitopes predicted by both programmes were defined as epitopes. Both previously characterised and novel epitopes were predicted on the SAT strains. Some of the novel epitopes are located on the same loops as experimentally derived epitopes, while others are located on a putative novel antigenic site, which is located close to the five-fold axis of symmetry. A consensus set of 11 epitopes that are common on at least 15 out of 18 SAT strains was collated. In future work, the epitopes predicted in this study will be experimentally validated using mutagenesis studies. Those found to be true epitopes may be used in the rational design of broadly reactive SAT vaccines / Life and Consumer Sciences / M. Sc. (Life Sciences)