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Taura syndrome (TS) is one of the most devastating diseases affecting the shrimp farming industry worldwide. The causative virus, Taura syndrome virus (TSV), has been identified. My work is centred on the development of monoclonal antibodies against TSV. I have also characterized a novel variant of the virus from Venezuela and evaluated the effect of hyperthermia on TSV infection. This work has resulted in 3 manuscripts, which constitute the core of this dissertation. The taxonomy throughout this dissertation is done according to Holthuis (1980).The first manuscript describes the production of a monoclonal antibody reacting with the Belize strain of TSV. The antibody, MAb 2C4, exhibits good sensitivity and specificity for TSV in immunohistochemistry (IHC) and dot blot immunoassay. MAb 2C4 reacted with the TSV-HI94, TSV-SI98 and TSV-BZ02 variants, but not with the TSV-VE05 and TSV-TH05 variants. This antibody adds and improves tools to those available for TSV diagnosis.Chapter three describes a relatively novel variant of TSV from Venezuela, which was characterized by our laboratory. By genetic sequencing, this new isolate exhibits a 94% similarity with TSV-HI94. IHC, dot blot immunoassay and bioassays were also performed. While processed samples reacted only weakly with the TSV monoclonal antibody MAb 1A1, the virus in its native state reacted strongly with the antibody. In bioassays, TSV-VE05 presented mortality comparable to TSV-HI94 in Penaeus vannamei. These data confirm the presence of TSV in Venezuela and that a new variant of the virus was responsible for the outbreak of TS.In chapter four, the behavior of TSV infection under hyperthermic conditions was examined. I compared the susceptibility of Kona stock P. vannamei to the infection by two variants of TSV under hyperthermic conditions (32oC). Shrimp, infected with TSV-HI94, were resistant to infection at high temperature. However, under the same hyperthermic conditions, the challenged shrimp were fully susceptible to the infection by TSV-BZ02. This susceptibility to TSV-BZ02 at higher temperatures was independent both of the route of infection and of the salinity of water. I conjecture that TSV-BZ02 might be a temperature permissible mutant of TSV.

Monoclonal antibodies (mAbs) recognizing different regions of PrP are potential tools in the study of prion diseases and immunotherapy. We used shuffled recombinant prion protein containing elk and mouse PrP as antigen to produce monoclonal antibodies in mice. We found that mAb 5C6 mapped to a discontinuous epitope comprised of amino acid 132 and 158 (mouse numbering). Monoclonal anibody 9E9 which maps to a unique N-terminal epitope at amino acid preferentially recognized cervid PrP. In contrast, the epitope of mAb 9H9 is located in the C-terminus and only reacted with mouse and hamster. The epitope for mAb 7H11 appears to be affected by the glycosylation of PrP and by the presence or absence of the disulfide bond. To confirm the epitopes of these mAbs, we constructed elk and mouse mutants both with and without reactivity to 5C6 and 9E9. We then used these mutants to investigate the effect of each epitope on the conversion of PrPC to PrPsc. In one approach to map the epitopes of newly-generated monoclonal antibodies (mAbs), we generated a series of contiguous ten amino acids deletion constructs spanning amino acids 107 to 230 and expressed these recombinant proteins in mammalian cells (RK13) or bacteria. Using Western blotting, all deletion constructs could be recognized with antibodies to the extreme C-terminus of PrP, or the N-terminal region upstream of the structured globular domain of PrP. However, mAb 5C6 failed to react with all internally deleted PrP constructs expressed in mammalian cells, and to a lesser extent bacterially produced mutant recombinant proteins. We confirmed the surprising result using the well-defined antibodies 6H4 and D18, which recognize epitopes in the same internal region as 5C6. Our results suggest the formation of an ultra-stable, SDS-resistant conformation in PrP harboring deletions mutations in the globular domain of PrP. We hypothesize that epitope burying within this stable conformation(s) precludes mAb recognition by 5C6, 6H4 and D18. It will be of extreme interest to determine the relationship of this previously undefined PrP conformation to the pathogenic process of PrP conformational change.

Therapeutic monoclonal antibodies (MAb) are produced as secreted complex glycoproteins from mammalian cell systems and represent one of the most important classes of therapeutic medicines for the treatment of a variety of human diseases. Their benefit in health care and high economic impact provide the driving force for the development of improved production levels with the focus of optimizing clinical efficacy. One important issue is the optimization of monoclonal antibody production. A frequent approach used to address this challenge is the engineering of mammalian cell lines to increase antibody production levels through genetic manipulation. Valuable information can then be obtained by monitoring the effects of genetic changes on the biochemistry of the cell associated with MAb production. Global protein expression profiling of mammalian cells used for the production of biopharmaceuticals may reveal key biochemical characteristics associated with MAb-producing cell lines. A better understanding of these characteristics can in turn lead to more rational strategies for cell line and process development. The proposed research relates to a larger NSERC Strategic Network (MAbNet) Grant to develop and establish a novel platform for the large-scale manufacture of specific glycoforms of therapeutic monoclonal antibodies. The efficacy of these recombinant MAbs will be enhanced by the control of their glycosylation profiles. The work presented in this thesis will assist MAbNet in meeting their objectives. Specifically, we use 2D-Differential In-Gel Electrophoresis (2D-DIGE) to quantify protein expression differences between EG2-hFc1-producing Chinese Hamster Ovary cells (CHO-1A7) with its parental cell line (CHO-BRI). Here, we identified 34 unique differentially expressed proteins associated with EG2-hFc1 production that relate to various biological processes including protein processing, carbohydrate metabolism, amino acid metabolism, energy metabolism, apoptosis, and cell proliferation pathways. The majority of identified significant protein expression changes and their associated metabolic processes seem to prioritize energy production in CHO-1A7 cells. Due to the metabolic load of recombinant antibody production, the CHO-1A7 cell line attempts to meet the energy requirements needed for recombinant protein biosynthesis while maintaining cell viability and efficient protein folding mechanisms. A 2-D proteome reference map was also constructed for the CHO-BRI host cell line containing 131 identified protein spots. The map provides information that will further expand our understanding of this particular cell line. It will be a useful tool for studies investigating physiological responses and protein expression patterns of CHO-BRI to genetic and environmental perturbations. The set of identified differentially expressed proteins provides data on the downstream changes in protein expression due to genetic manipulation, and furthermore can provide targets for cell-line specific optimization of antibody production. The work described in this thesis furthers our understanding of antibody production in a specific CHO cell line.

Diss. (sammanfattning) Umeå : Univ., 2006. / Härtill 4 uppsatser.

These studies examined the in vivo pharmacokinetics and efficacy of five anti-methamphetamine monoclonal antibodies (mAbs, KD values from 11 to 250 nM) in rats. While no substantive differences in mAb systemic clearance (t1/2 = 6.1-6.9 days) were found, in vivo function was significantly reduced within 1-3 days for four of the five mAbs. Only mAb4G9 was capable of prolonged efficacy, as judged by prolonged high methamphetamine serum concentrations. MAb4G9 also maintained high amphetamine serum concentrations, along with reductions in methamphetamine and amphetamine brain concentrations, indicating neuroprotection. The combination of broad specificity for methamphetamine-like drugs, high affinity, and prolonged action in vivo suggests mAb4G9 is a potentially efficacious medication for treating human methamphetamine-related medical diseases.

Master of Science / Department of Diagnostic Medicine/Pathobiology / Weiping Zhang / Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea to pigs at all ages, resulting in high mortality rate of 80-100% in piglets less than one week old. Within one year after the outbreak in April 2013, PEDV has rapidly spread in the US and causes the loss of over 10% of the US pig population. Monoclonal antibody (mAb) is a key reagent for rapid diagnosis of PEDV infection. In this study, we produced a panel of mAbs against nonstructural protein 8 (nsp8), spike(S) protein, and nucleocapsid (N) protein of PEDV. Four mAbs were selected, which can be used in various diagnostic assays, including indirect immunofluorescence assay (IFA), enzyme-linked immunoabsorbent assay (ELISA), Western Blot, immunoprecipitation (IP), immunohistochemistry (IHC) test and fluorescence in situ hybridization (FISH). The mAb 51-79 recognizes amino acid (aa) 33-60 of nsp8, mAb 70-100 recognizes aa1371-1377 of S2 protein, and mAb 66-155 recognizes aa 241-360 of N protein, while mAb 13-519 is conformational. Using the mAb70-100, the immunoprecipitated S2 fragment was examined by protein N-terminal sequencing, and cleavage sites between S1 and S2 was identified. In addition, this panel of mAbs was further applied to determine the infection site of PEDV in the pig intestine. IHC test result showed that PEDV mainly located at the mid jejunum, distal jejunum and ileum. Results from this study demonstrated that this panel of mAbs provides a useful tool for PEDV diagnostics and pathogenesis studies.

Master of Science / Department of Diagnostic Medicine and Pathobiology / Weiping Zhang / Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea to pigs at all ages, resulting in high mortality rate of 80-100% in piglets less than one week old. Within one year after the outbreak in April 2013, PEDV has rapidly spread in the US and causes the loss of over 10% of the US pig population. Monoclonal antibody (mAb) is a key reagent for rapid diagnosis of PEDV infection. In this study, we produced a panel of mAbs against nonstructural protein 8 (nsp8), spike(S) protein, and nucleocapsid (N) protein of PEDV. Four mAbs were selected, which can be used in various diagnostic assays, including indirect immunofluorescence assay (IFA), enzyme-linked immunoabsorbent assay (ELISA), Western Blot, immunoprecipitation (IP), immunohistochemistry (IHC) test and fluorescence in situ hybridization (FISH). The mAb 51-79 recognizes amino acid (aa) 33-60 of nsp8, mAb 70-100 recognizes aa1371-1377 of S2 protein, and mAb 66-155 recognizes aa 241-360 of N protein, while mAb 13-519 is conformational. Using the mAb70-100, the immunoprecipitated S2 fragment was examined by protein N-terminal sequencing, and cleavage sites between S1 and S2 was identified. In addition, this panel of mAbs was further applied to determine the infection site of PEDV in the pig intestine. IHC test result showed that PEDV mainly located at the mid jejunum, distal jejunum and ileum. Results from this study demonstrated that this panel of mAbs provides a useful tool for PEDV diagnostics and pathogenesis studies.

No útero gestante dos animais com placentação do tipo hemocorial, ocorre uma migração e acúmulo transitório de linfócitos natural killer (NK) , cuja atuação na gestação não está totalmente elucidada. Estas células NK do ambiente uterino (uNK) apresentam comportamento distinto daquelas encontradas no sangue circulante (cNK), constituindo uma sub-população das células NK com expressão gênica específica ditada pelo ambiente uterino gestante. De fato, se estas células isoladas do útero de camundongos prenhes forem inoculadas em machos da mesma espécie eram capazes de induzir a resposta imunológica com produção de anticorpos que reagem especificamente com as células uNK. No presente trabalho foi utilizado um destes anticorpos monoclonais denominados de \"mouse anti-mouse uterine natural killer cell clone 1 (mam-uNK1)\" obtidos anteriormente em nosso laboratório para avaliar a especificidade deste anticorpo e a localização da molécula antigênica correspondente. Para tanto, foram utilizados cortes histológicos do útero no 9º dia de gestação, dos órgãos linfóides (baço, timo e linfonodo), do cérebro, do fígado e do coração submetidos à reações imunocitoquímicas com o anticorpo mam-uNK1 em nível da microscopia de luz e, pela imunomicroscopia eletrônica nas células uNK do útero gestante e células estriadas cardíacas do miocárdio. Foram obtidos homogenados teciduais dos mesmos órgãos avaliados pela imunocitoquímica para realização do SDS-PAGE e Western-blot com o intuído de identificar as frações protéicas reativas e homologia entre os diversos órgãos. Os padrões de imunomarcação com o mam-uNK1 foram comparadas com o padrão de reatividade da lectina DBA (Dolichos biflorus) tanto nos cortes histológicos quanto nos homogenados submetidos ao Western-blot. Os resultados demonstraram reação positiva distribuída difusamente no citoplasma, com maior intensidade no perímetro das células uNK, e marcação difusa no citoplasma das células deciduais e das células musculares lisas do miométrio. Reações positivas foram encontradas também no citoplasma das células musculares cardíacas, no citoplasma das células reticulares dos órgãos linfóides, nos feixes de nervos do sistema nervoso central e no citoplasma dos hepatócitos. Pela imunomicroscopia eletrônica foram observadas partículas de ouro coloidal em maior número no citoplasma que preenchem os prolongamentos citoplasmáticos tipo microvilosidades e no citoplasma marginal abaixo da membrana plasmática nas células uNK. Nas células musculares cardíacas as marcações mais intensas foram constatadas no citoplasma da extremidade destas células onde as miofibrilas eram menos organizadas e se ancoravam à membrana plasmática. Pelo Western-blot, foram identificadas duas bandas reativas ao mam-uNK1 com peso molecular de 52 e 54 kDa comuns a todos os órgãos analisados. Estes dados demonstram que a molécula reconhecida pelo anticorpo mam-uNK1 tem ampla distribuição em diversos tipos celulares não sendo específica para as células uNK, porém apresentam uma localização peculiar nestas células e nas células musculares cardíacas. Pelo padrão de localização identificado em imunomicroscopia eletrônica, presume-se que estas moléculas estejam associadas com a modulação do citoesqueleto nas diversas atividades que estes componentes estruturais desempenham nas células, sendo particularmente interessante a relação com a motilidade celular. / In the pregnant uterus of animals developing hemochorial type placentation occurs a transient migration and accumulation of natural killer lymphocytes (NK) which activity in the pregnancy has not been fully elucidated. These NK cells from uterine environment (uNK) present a distinct behavior from those found in the peripheral circulating blood (cNK), composing a subset of NK cells with specific gene expression that is regulated by pregnant uterus. Actually, these cells isolated from pregnant mice uteri were inoculated in the male mice of same strains, they induced immune response with production of antibodies reactivity specifically to uNK cells. In the present work it was used one of these mouse anti-mouse uterine natural killer cells clone 1 (mam-uNK1) monoclonal antibody that was obtained previously in our laboratory, in the aim to evaluate the specificity of this antibody and localization of corresponding antigenic molecule. It was used histological sections of uteri on 9º gestational day, lymphoid organs (spleen, thymus and lymph node), brain, liver and heart processed for immunocytochemistry with mam-uNK antibody at light microscopy and immunoelectron microscopy for uNK cells in pregnant uterus and cardiac muscle cells. Tissue homogenates from the same organs that were evaluated by immunocytochemistry were obtained to perform SDS-PAGE and Western-blot primary to identify the proteins fractions reactive with mam-uNK antibody and possible homology among the tissues. The immunolabeling pattern using mam-uNK both, in histological sections and Western-blot were compared with pattern of Dolichos biflorus (DBA) lectin reactions. The results showed diffuse positive reaction distributed in the cytoplasm with higher intensity on perimeter of uNK cells and diffuse labeling in the cytoplasm of decidual cells and, in smooth muscle cells of miometrium. Positive reaction was also found in the cytoplasm of cardiac muscle cells, reticular cells of lymphoid organs, hepatocytes and in the axon bundles of the brain. By immunelectron microscopy, were observed higher number of gold particles in the cytoplasm of microvillous-like cell processes and in the marginal cytoplasm near the plasma membrane of uNK cells. In the cardiac muscle cells the most conspicuous labeling was seen in the cytoplasm of muscle cells at the end portions, that is, where the myofibrills were less organized and anchoring to plasma membrane. The Western-blot identified two bands with 52 and 54 kDa reactive do mam-uNK constantly found in all organs analyzed. These data show that the molecule that is recognized by mam-uNK1 antibody is widely distributed in several cell types, not specific for mouse uNK cells, but has a very peculiar localization in these cells and in the cardiac muscle cells. To the localization pattern that was identified by immunelectron microscopy it was suggested that, these molecules were associated to the modulation of the cytoskeleton in many activities that these structural component potentially could carry out in the cells, being particularly attractive those related to cell motility.

Trois bactéries du genre Yersinia sont pathogènes pour l’homme : Yersinia pestis (bacille de la peste), et les bactéries entéropathogènes Yersinia pseudotuberculosis et Yersinia enterocolitica. Yersinia pestis est responsable de plus de 20 000 cas humains de peste déclarés à l’Organisation Mondiale de la Santé (OMS) ces dix dernières années dans différents foyers en Afrique, Asie et Amériques. Considérée aujourd’hui à tort comme une maladie du passé, elle est au contraire classée parmi les maladies réémergentes Même si elle ne se présente plus sous la forme d’épidémies massives, elle pose encore au monde actuel d’importants défis de par son extrême gravité, sa rapidité de dissémination, une apparition de résistances aux antibiotiques et une éventuelle utilisation terroriste du bacille. Dans ce contexte, l’immunothérapie contre Y. pestis pourrait être une bonne alternative pour traiter la peste bubonique et pulmonaire. Un des objectifs de cette thèse était de produire des anticorps monoclonaux murins contre trois protéines de l’injectisome (YscF, YscC et LcrV), un facteur de virulence clé des Yersinia. Les anticorps obtenus ont été caractérisés et pour certains leurs épitopes identifiés. Par la suite, en collaboration avec Elisabeth Carniel à l’Institut Pasteur, leur pouvoir neutralisant a été évalué in vivo dans un modèle murin de peste bubonique. Ces mêmes anticorps monoclonaux, produits contre les protéines de l’injectisome sont en cours d’évaluation pour la mise au point d’un test de diagnostic rapide de Y. pestis dans différents fluides et échantillons biologiques. Yersinia pseudotuberculosis et Yersinia enterocolitica sont présentes dans le monde entier et sont transmises par contamination à partir de viande de porc mal cuite, de lait ou produits laitiers et de végétaux, ou par contact avec des animaux porteurs sauvages ou domestiques. Une transmission interhumaine par voie fécale-orale est également possible. Ces bactéries sont responsables très fréquemment d’infections entériques. Cependant leur recherche dans les coprocultures n’est pas réalisée de façon systématique en laboratoires d’analyses médicales du fait de leur croissance lente et difficile sur les milieux usuels, ce qui rend leur isolement à partir de fèces difficile. De plus, les procédures de routine sont coûteuses et longues. Cela entraine probablement une sous-estimation de l’incidence des infections à Yersinia entéropathogènes, la prescription de traitements non adaptés et la réalisation d’appendicectomies non nécessaires, d’où la nécessité de développer des tests de diagnostic rapides, spécifiques, sensibles et faciles à utiliser. Un des objectifs de cette thèse était de produire un panel d‘anticorps monoclonaux murins contre les principaux biotypes et sérotypes pathogènes de Y. pseudotuberculosis et Y. enterocolitica pour le développement de tests de diagnostic immunologiques (ELISA et tests bandelettes) répondant aux caractéristiques recherchées et utilisables directement avec des échantillons biologiques humains. / Three bacteria of the genus Yersinia are pathogenic for the human: Yersinia pestis (the plague bacillus) and the enteropathogenic bacteria: Yersinia pseudotuberculosis and Yersinia enterocolitica. Yersinia pestis is responsible for more than 20,000 human cases of plague declared to the World Health Organization (WHO) during the ten last years in different areas from Africa, Asia and America. Mistakenly considered today as a disease from the past, on the contrary, the plague is re-emerging. Even if it doesn’t occur as a massive epidemic, it still lays down a challenge to the world for its important severity, its quick spreading, the appearance of antimicrobial resistance and a potential use for terrorism. Under the circumstances, the immunotherapy against Y. pestis could be a good option to treat bubonic and pneumonic plague. One the aims of this thesis was to produce murine monoclonal antibodies against the three proteins of the injectisom (YscF, YscC, LcrV), a key virulence factor of Yersinia. The obtained antibodies were characterized and for certain, the epitopes were identified. Then, in collaboration with Elisabeth Carniel from Institut Pasteur, their therapeutic effect was evaluated in vivo with a bubonic plague model in mice. The antibodies generated against the proteins from the injectisom are now evaluated in a diagnosis test for a fast detection of Y. pestis in different biological samples. Yersinia enterocolitica and Yersinia pseudotuberculosis, the two enteropathogenic Yersinia species for humans, have a worldwide distribution and are among the most frequent agents of human diarrhea in temperate and cold countries. However, research of enteropathogenic Yersinia is not consistently performed in medical laboratories because of their specific growth characteristics, which makes their isolation from the stool samples difficult. Moreover, current procedures for isolation are expensive and time consuming, which leads to underestimation of the incidence of yersiniosis and prescriptions of inappropriate antibiotic treatments. One the aims of this thesis was to produce different murine monoclonal antibodies against the main pathogenic biotypes and serotypes of Y. pseudotuberculosis and Y. enterocolitica for the development of fast, sensitive, specific and easy-to-use immunoassays (ELISA and dipsticks), useful for both human and veterinary diagnosis.