<p>The family 1 cellulose-binding modules (CBM1) form a groupof small, stable carbohydrate-binding proteins. These modulesare essential for fungal cellulosedegradation. This thesisdescribes both functional studies of the CBM1s as well asprotein engineering of the modules for several objectives.</p><p>The characteristics and specificity of CBM1s from the<i>Trichoderma reesei</i>Cel7A and Cel6A, along with severalother wild type and mutated CBMs, were studied using bindingexperiments and transmission electron microscopy (TEM). Datafrom the binding studies confirmed that the presence of onetryptophan residue on the CBM1 binding face enhances itsbinding to crystalline cellulose. The two<i>T. reesei</i>CBM1s as well as the CBM3 from the<i>Clostridium thermocellum</i>CipA were investigated by TEMexperiments. All three CBMs were found to bind in lineararrangements along the sides of the fibrils. Further analysesof the bound CBMs indicated that the CBMs bind to the exposedhydrophobic surfaces, the so called (200) crystalline face of<i>Valonia</i>cellulose crystals.</p><p>The function and specificity of CBM1s as a part of an intactenzyme were studied by replacing the CBM from the exo-actingCel7A by the CBM1 from the endoglucanase Cel7B. Apart fromslightly improved affinity of the hybrid enzyme, the moduleexchange did not significantly influence the function of theCel7A. This indicates that the two CBM1s are analogous in theirbinding properties and function during cellulosehydrolysis.</p><p>The CBM1 was also used for immobilization studies. Toimprove heterologous expression of a CBM1-lipase fusionprotein, a linker stability study was carried out in<i>Pichia pastoris</i>. A proline/threonine rich linker peptidewas found to be stable for protein production in this host. Forwhole bacterial cell immobilization, the<i>T. reesei</i>Cel6A CBM1 was expressed on the surface of thegram-positive bacteria,<i>Staphylococcus carnosus</i>. The engineered<i>S. carnosus</i>cells were shown to bind cellulosefibers.</p><p>To exploit the stable CBM1 fold as a starting point forgenerating novel binders, a phage display library wasconstructed. Binding proteins against an amylase as well asagainst a metal ion were selected from the library. Theamylase-binding proteins were found to bind and inhibit thetarget enzyme. The metal binding proteins selected from thelibrary were cloned on the surface of the<i>S. carnosus</i>and clearly enhanced the metal bindingability of the engineered bacteria.</p><p><b>Keywords</b>: cellulose-binding, family 1carbohydrate-binding module, phage display, bacterial surfacedisplay, combinatorial protein library, metal binding, proteinengineering,<i>Trichoderma reesei, Staphyloccus carnosus</i>.</p>

Three epitope-mapping procedures were used to identify B-cell epitopes on Betanodaviruses: neutralisation escape mutant sequence analysis, phage display, and pepscan. Betanodaviruses have emerged as major pathogens of marine fish. These viruses are the aetiological agents of a disease referred to as viral nervous necrosis (VNN), which affects many species of fish that are economically valuable to the aquaculture industry. The identification of betanodavirus B-cell epitopes will facilitate the rational development of vaccines to counter VNN. A panel of mouse monoclonal antibodies (MAbs) was produced using hybridoma methodology for use in each of the epitope mapping procedures. These antibodies were characterised in Western blotting, ELISA, and virus neutralisation tests. Rabbit polyclonal sera, and serum samples from nodavirus-infected fish were also used for pepscan analyses. Attempts to produce betanodavirus neutralisation escape mutants, using plaque assay or limiting dilution based methods, were not successful. Two phage libraries expressing random peptides of seven (Ph.D.7™) or twelve (Ph.D.12™) amino acids in length as fusions to the coat protein were used to identify the ligands recognised by MAbs directed against betanodavirus. Neither of these phage libraries yielded conclusive results. Phage clones containing tandem inserts were obtained after MAb selection from library Ph.D.7™. Extensive screening and nucleotide sequence analysis of MAb-selected clones from library Ph.D.12™) failed to yield a consensus sequence. Pepscan analyses were performed using the recently developed suspension array technology (SAT). This was used to map the recognition sites of MAbs and serum samples onto a panel of overlapping synthetic peptides (12mers) that mimicked the betanodavirus coat protein. The results of pepscan analyses required careful interpretation due to the binding of antibodies and serum samples to multiple peptides. However, three regions of the nodavirus coat protein were identified as containing B-cell epitopes: amino acids 1-50, 141-162, and 181-212. These results are discussed in relation to previous studies of immune responses to betanodaviruses, and to the future development of betanodavirus vaccines and diagnostic reagents.

Les matériaux inorganiques ont des propriétés uniques à l'échelle nanométrique. Ces propriétés ont généré beaucoup d'intérêt pour fabriquer des nouveaux matériaux utilisant des nano-objets comme unité de construction. Nous avons suivi une approche biomimétique pour la fabrication de dispositifs à base de nanoparticules afin d'améliorer les méthodes actuelles de fabrication top-down et bottom-up. Certaines protéines naturelles se lient en effet spécifiquement à des matériaux inorganiques, et déclenchent notamment la croissance de cristaux inorganiques. Une première étape dans cette approche biomimétique est de comprendre comment des protéines se lient spécifiquement à des nanomatériaux inorganiques. Nous avons exploré ce mécanisme de reconnaissance en sélectionnant des anticorps (les protéines de notre système immunitaire spécialisées dans les interactions avec de nombreuses cibles) contre des matériaux inorganiques par la méthode combinatoire biotechnologique appelée "phage display". Cette technique permet d'obtenir la séquence génétique codante des anticorps sélectionnés se liant à leur cible à partir d'une banque aléatoire d'anticorps. L'analyse statistique des séquences des anticorps sélectionnés fournit de nouvelles informations sur les interactions protéines/matériaux inorganiques. Notre principale conclusion est l'identification de l'acide aminé arginine en tant que contributeur majeur dans les interactions protéine/or. L'ingénierie génétique des anticorps permet de fonctionnaliser ces nouvelles sondes de matériaux inorganiques en vue de leur utilisation pour des applications dans le domaine des nanomatériaux. Les anticorps recombinants sélectionnés et leurs dérivés fonctionnalisés peuvent être exprimés par sécrétion à l'aide d'un hôte eucaryote (Dictyostelium discoideum) mis au point au cours de cette thèse.

Notre laboratoire a développé une stratégie d'isolement de fragments d'anticorps recombinants à partir de primates non humains (Macaca fascicularis) immunisés, en utilisant la technologie des phages. Dans le cadre de cette thèse, une comparaison des séquences d'anticorps de macaques (Macaca Mulatta) et d'anticorps humains a toutefois montré que les anticorps des deux espèces présentent des différences qui rendent souhaitable une étape d'humanisation des anticorps de macaques. Cette stratégie a été utilisée dans le cadre du projet Européen AntiBotABE (www.antibotabe.com) et l'étape de criblage a été adaptée pour isoler des scFv neutralisant de façon croisée les toxines botuliques BoNT/B des sous-types B1 et B2, en utilisant séquentiellement l'holotoxine BoNT/B1 et un fragment recombinant représentant la région C-terminale de la chaîne lourde de BoNT/B2. Le meilleur scFv ciblant les régions C-terminales des chaînes lourdes de BoNT/B1 et BoNT/B2, B2-7, a montré une bonne capacité de neutralisation de BoNT/B1 et BoNT/B2 dans le test ex vivo de paralysie hémidiaphragmatique. Les régions charpentes du scFv B2-7 ont un pourcentage d'identité élevé (80 %) avec leurs homologues humains. Des scFv neutralisant BoNT/A1 en ciblant sa chaîne légère ont aussi été isolés, dont le scFv le plus efficace, 2H8, induit une diminution de 50% de l'activité endopeptidasique à une concentration correspondant à un rapport molaire 2H8/BoNT/A1 de 64000. Les régions charpentes de 2H8 ont également un pourcentage d'identité élevée (88%) avec leurs homologues humains. La versatilité de cette stratégie en fait un outil permettant l'isolement de nombreux autres fragments d'anticorps à visée thérapeutique.

Combinatorial protein engineering, taking advantage of large libraries of protein variants and powerful selection technology, is a useful strategy for developing affinity proteins for applications in biotechnology and medicine. In this thesis, two small affinity proteins have been subjected to combinatorial protein engineering to improve or redirect the binding. In two of the projects, a three-helix protein domain based on staphylococcal protein A has been used as scaffold to generate so called Affibody molecules capable of binding to key proteins related to two diseases common among elderly people. In the first project, Affibody molecules were selected using phage display technology for binding to Ab-peptides, believed to play a crucial role in Alzheimer’s disease, in that they can oligomerize and contribute to the formation of neural plaques in the brain. The selected Affibody molecules were found to efficiently capture Ab from spiked human plasma when coupled to an affinity resin. The structure of the complex was determined by nuclear magnetic resonance (NMR) and demonstrated that the original helix 1 in the two Affibody molecules was unfolded upon binding, forming intermolecular b-sheets that stabilized the Ab peptide as buried in a tunnel-like cavity. Interestingly, the complex structure also revealed that the Affibody molecules were found to homo-dimerize via a disulfide bridge and bind monomeric Ab-peptide with a 2:1 stoichiometry. Furthermore, Affibody molecule-mediated inhibition of Ab fibrillation in vitro, suggested a potential of selected binders for future therapeutic applications. In the second project, two different selection systems were used to isolate Affibody molecules binding to tumor necrosis factor alpha (TNF), which is involved in inflammatory diseases such as rheumatoid arthritis. Both selection systems, phage display and Gram-positive bacterial display, could successfully generate TNF-binding molecules, with equilibrium dissociation constants (KD) in the picomolar to nanomolar range. Initial characterization of the binding to TNF was evaluated by competitive binding studies between the Affibody molecules and clinically approved TNF antagonists (adaliumumab, infliximab and etanercept) and demonstrated overlapping binding sites with both adaliumumab and etanercept. Furthermore, linkers of different lengths were introduced between Affibody moieties, in dimeric and trimeric constructs that were evaluated for their ability to block the binding between TNF and a recombinant form of its receptor. In the dimeric constructs, a linker length of 20-40 amino acids seemed to have an advantage compared to shorter and longer linkers, and the tested trimeric construct could block the TNF binding at even lower concentration. The results provided valuable information for the design of future Affibody-based molecules that could be investigated in therapeutic or medical imaging applications. In the third project aiming to generate a protein domain with capacity to influence the pharmacokinetics of protein therapeutics, a natural serum albumin-binding domain (ABD) was subjected to an engineering effort aiming at improving the affinity to human serum albumin (HSA), a protein with an exceptional long half-life in serum (19 days). First-generation affinity improved ABD variants were selected using phage display technology from a constructed ABD library. After additional rational engineering of such first generation variants, one variant with a 10,000-fold improved affinity to HSA (KD ≈ 120 fM) was obtained. Furthermore, characterization of this molecule also demonstrated improved affinity to several other serum albumins. When used as a gene fusion partner, this affinity-maturated variant denoted ABD035, should have the potential to extend the half-life of biopharmaceuticals in humans, and several other animal species. / QC 20100722

Made available in DSpace on 2014-06-11T19:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-12-22Bitstream added on 2014-06-13T18:56:05Z : No. of bitstreams: 1 fernandes_cc_me_jabo.pdf: 1349174 bytes, checksum: b0d752648346a29041d0fbd5f330fbf6 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Anticorpos monoclonais se constituem na base de vários testes usados na detecção e na identificação de antígenos. Nesse contexto, tais imuno-reagentes têm sido extensivamente empregados na identificação de estirpes virais envolvidas na etiologia de surtos de bronquite infecciosa a campo, permitindo o aperfeiçoamento das técnicas de detecção e caracterização antigênica do vírus da bronquite infecciosa das galinhas (VBI). No presente estudo, uma biblioteca de fragmentos de anticorpos de galinha originalmente preparada por “phage display” contra a estirpe vacinal (H120) do VBI, foi usada para a seleção de fragmentos de anticorpos recombinantes com reatividade cruzada para as estirpes heterólogas IBVPR01, IBVPR05, isoladas de surtos a campo no Brasil e SE-17, isolada nos Estados Unidos. Após três ciclos de “panning”, foi identificado pelo ELISA um conjunto de 15 anticorpos scFv expressos em fagos e com reatividade cruzada para essas mesmas estirpes do VBI. A análise por Western-blotting revelou que três desses clones apresentavam fagos expressando fragmentos de anticorpos monoclonais com reatividade cruzada para a nucleoproteína N das três estirpes do VBI e também para a forma recombinante dessa nucleoproteína derivada da estirpe M41. Concluindo, os fragmentos de anticorpos monoclonais recombinantes scFv-N produzido em fagos interagem com um epítopo mais conservado da proteína N do VBI e apresentam um grande potencial para utilização na detecção e no diagnóstico direto desse vírus e no estudo de evolução de variantes desse vírus. / Monoclonal antibodies are the basis of various techniques used for antigen detection or characterization, and their use is specially recommended for the identification of viral strains involved in the etiology of outbreaks of infectious bronchitis, because these antibodies are homogeneous, highly specific and fully characterized, allowing the improvement of detection of immunological techniques and antigenic characterization of avian infectious bronchitis virus strains (IBV). We used a phage display library prepared previously against the IBV vaccine strain (H120) for the selection of new scFv antibody fragments reacting with heterologous IBV strains isolated from outbreaks in Brazil (IBVPR01, IBVPR05) and USA (SE-17). After three cycles of panning a set of 15 scFv antibodies was expressed in phages and exhibited crossreaction in ELISA with these three viral strains. Western-blotting analysis showed that three of this clone set were expressing scFv specific for the nucleoprotein of these IBV strains, as well as to the recombinant form of this protein derived from M41 strain of IBV. In conclusion, the recombinant fragments of monoclonal antibodies expressed by phage-display technique have a great potential for future use in immunodiagnostic techniques and study the evolution of variant strains of this virus.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Human strongyloidiasis is an important intestinal parasitic infection worldwide, 50% of infected individuals are asymptomatic, however it can cause hyper infection and dissemination in immunocompromised hosts leading to death. Early detection of the disease prevents the development of hyper infection and dissemination syndromes, so the use of an efficient diagnostic tool has great importance to identify and control this parasitic disease. Due to the lack of efficiency in parasitological and serological tests currently available to detect human strongyloidiasis it is necessary to improve immunodiagnostic tests using recombinant and synthetic antigens once there are limitations to obtain and use homologous antigens produced from the parasite. The aim of this study was to select using phage display technology Strongyloides stercoralis mimetic peptides ligands to immunoglobulin G from patients with strongyloidiasis. The PhDTM-C7C library was used in the selection process and the DNA of the selected clones was extracted, sequenced and analyzed using bioinformatics tools. ELISA tests were done by using five distinct phage clones, which presented significant similarity with proteins from S. stercoralis, and the two synthetic peptides corresponding to the sequences displayed on two phage clones. Binding specificity of each phage clone to the pool of sera from patients with strongyloidiasis was analyzed by competitive ELISA. Sensitivity, specificity, diagnostic efficiency, area under curve and likelihood ratio were calculated for each antigen. All phage clones presented high diagnostic potential achieving area under curves higher than 0.8, the C9 clone presented reasonable sensitivity (87.5%), specificity (80%) and diagnostic efficiency (82.5%).Synthetic peptides C10 and D3 showed superior diagnostic performance, with areas under the curve greater than 0.9 and excellent sensitivity (95%, 95%), specificity (86.3%, 92.5%) and diagnostic efficiency (89 2%, 93.3%) respectively. It was concluded that the selected peptides by Phage display can mimic S. stercoralis epitopes and represent promising alternative to the currently available antigens for human strongyloidiasis diagnosis. / Estrongiloidiase humana é uma importante parasitose intestinal em todo mundo, 50% das pessoas infectadas são assintomáticas, entretanto pode haver hiperinfecção e disseminação em pacientes imunocomprometidos, levando à morte. A detecção precoce da doença previne o desenvolvimento das síndromes de hiperinfecção e disseminação, assim, o uso de uma ferramenta diagnóstica eficiente é de grande importância para identificar e controlar esta parasitose. Devido à falta de eficiência nos testes parasitológicos e sorológicos disponíveis atualmente para detectar estrongiloidiase humana é necessário aprimorar os testes imunodiagnósticos utilizando antígenos recombinantes e sintéticos, uma vez que há limitações para obter e utilizar antígenos homólogos, produzidos a partir do parasito. O objetivo deste trabalho foi selecionar por Phage display peptídeos miméticos a Strongyloides stercoralis ligantes a imunoglobulinas G de pacientes com estrongiloidiase. Uma biblioteca PhDTM-C7C foi utilizada no processo de seleção e o DNA dos clones selecionados foi extraído, sequenciado e analisado por ferramentas de bioinformática. Os testes ELISA foram feitos utilizando cinco clones de fagos distintos, os quais tiveram similaridades significantes com proteínas de S. stercoralis, e dois peptídeos sintéticos correspondentes às sequências expressas por dois clones selecionados. A especificidade de ligação de cada clone de fago ao pool de soros de pacientes com estrongiloidiase foi analisada por ELISA de competição. Sensibilidade, especificidade, eficiência diagnóstica, áreas sob a curva e razão de verossimilhança foram calculados para cada antígeno. Todos os clones de fagos analisados apresentaram alto potencial diagnóstico alcançando área sob a curva maiores que 0,8, se destacando o clone C9 com razoável sensibilidade (87,5%), especificidade (80%) e eficiência de diagnóstica (82,5%). Os peptídeos sintéticos C10 e D3 apresentaram desempenho diagnóstico superior, com áreas sob a curva maiores que 0,9 e excelente sensibilidade (95%, 95%), especificidade (86,3%, 92,5%) e eficiência de diagnóstica (89,2%, 93,3%) respectivamente. Concluiu-se que os peptídeos selecionados por Phage display podem mimetizar epitopos de S. stercoralis e representam alternativa promissora aos antígenos atualmente disponíveis para utilização no diagnóstico da estrongiloidiase humana. / Doutor em Imunologia e Parasitologia Aplicadas

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Introduction: HPV infection is the most common sexually transmitted disease in humans, which can cause benign diseases (warty lesion) or malignant as the anogenital cancer, oral cancer and, with the highest incidence, cervical cancer. Overall, the main HPV detection method is based on cytological evaluation of Papanicolaou; however, false-negative results and unsatisfactory sampling complicate the disease diagnosis and prevention. In this context, the investigation of new diagnosis platforms and prophylaxys has great interest. Objectives: The aim of this study was to conduct an epidemiological study of HPV infection in the women population of southeastern Brazil and then propose new diagnostic and vaccine approaches. Methodology: Genital samples of 5,223 women were evaluated by a new molecular tool based on PCR (Nested Multiplex PCR - NMPCR), using a cocktail of primers for simultaneous detection of 38 different HPV types, in single tube. Another molecular approach was proposed by Phage Display technique to select the IgA binding peptides from cervical samples of patients with HPV infection. In silico (Linear and 3D bioinformatics) and in vitro (ELISA, Slot blot and Bioelectrode) analysis were performed to validate the selected phages and synthetic peptides in cervical (n=91) and salivary specimens (n=66). The sensitivity and specificity parameters of HPV diagnosis were determined by ROC curve. To test the vaccine potential of synthetic peptides, in silico (3D bioinformatic), in vivo (female BALB/c mice immunization) or in vitro analyzes (MTT, ELISA, splenocytes culture, CBA and Neutralization assay) were carried out. Results: Among the 5,223 women evaluated, there was a prevalence of 58.9% of HPV infection, especially the types 53, 52 and 06, showing the relevance to propose new alternatives for HPV detection and prevention. In molecular approach of Phage Display, thirty-two distinct phage clones were selected against IgA from positive HPV cervical samples. Phage C.B1 showed higher reactivity against HPV samples compared to the negative control group by ELISA. This peptide is a putative epitope of HPV major capsid protein (L1) and has distinguished efficiently infection from HPV controls in cervical and saliva samples (p <0.0001), with high sensitivity (above 95%) and specificity (above 71%) in both fluids. The C.B1 peptide was also successfully incorporated (p<0.05) onto a graphite bioelectrode for HPV direct diagnosis in saliva. In the vaccine approach, three peptides (PEP1, PEP3 and PEP4) showed no toxicity in murine macrophages. Immunizations showed potent induction of serum IgG antibodies production by PEP3, and increase in IgA titer in the vaginal mucosa under PEP1 and PEP3 stimuli. The evaluation of cellular immune response indicated that the PEP1 and, particularly, the PEP3 chimeric peptide were able to polarize the immune response towards to Th1 profile and sensitize the cells to a pro-inflammatory \"status\", suitable for antiviral activity. The HPV-16 PsVs neutralization assay showed that PEP3 was able to reduce the infection by 49%. Conclusions: Overall, the studies presented here showed a high prevalence of HPV among Brazilian women by viral types still little explored in the literature. We selected mimotopes by Phage Display tool capable of detecting HPV in both saliva samples as in the cervical secretion, providing a low cost, simple and non-invasive diagnostics for population screening. In addition, the mimotopes selected have attractive applicability in vaccine formulations, but further studies are still needed. / Introdução: A infecção por HPV é a doença sexualmente transmissível mais comum em humanos, que pode causar doenças benignas (lesões verrucosas) ou malignas como o câncer anogenital, câncer oral e, com maior incidência, o câncer do colo uterino. Globalmente, o principal método de detecção do HPV baseia-se na avaliação citopatológica de Papanicolaou, entretanto, resultados falsonegativos e obtenção de amostras insatisfatórias dificultam o diagnóstico e a prevenção da doença. Neste contexto, a busca por novas plataformas diagnósticas e profiláticas para o HPV é de grande interesse. Objetivos: O objetivo do presente estudo foi realizar um levantamento epidemiológico da infecção por HPV na populacão feminina da região Sudeste do Brasil e, a partir de então, propor novas abordagens diagnósticas e vacinais. Metodologia: Amostras genitais de 5.223 mulheres foram avaliadas por uma nova ferramenta molecular baseada em PCR (Nested Multiplex PCR - NMPCR), utilizando-se um coquetel de primers para detecção simultânea de 38 diferentes tipos virais de HPV, em um único tubo. Outra abordagem molecular foi proposta através da técnica de Phage Display, a fim de selecionar peptídeos ligantes a anticorpos IgA oriundos de amostras cervicas de pacientes com infecção por HPV. Análises in silico (Bioinformática linear e 3D) e in vitro (ELISA, Slot blot e Bioeletrodo) foram realizadas para a validação dos fagos selecionados e dos peptídeos sintetizados em espécimes cervicais (n=91) e salivares (n=66). Os parâmetros de sensibilidade e especificidade do diagnóstico do HPV foram determinados pela curva ROC. Para testar o potencial vacinal dos peptídeos sintéticos, análises in silico (Bioinformática 3D), in vivo (imunização em camundongos fêmeas BALB/c) e in vitro (MTT, ELISA, Cultura de esplenócitos, CBA e Ensaio de Neutralização) foram conduzidas. Resultados: Dentre as 5.223 mulheres avaliadas, houve uma prevalência de 58,9% de infecção por HPV, com predominância dos tipos 53, 52 e 06, evidenciando a relevância de se propor novas alternativas de detecção e profilaxia do HPV. Na abordagem molecular de Phage Display trinta e dois clones de fagos distintos foram selecionados contra a IgA de amostras cervicais positivas para o HPV. O fago C.B1 apresentou maior reatividade contra amostras de HPV em comparação ao grupo controle negativo, por ELISA. Este peptídeo é um epítopo putativo da proteína principal do capsídeo do HPV (L1) e tem discriminado eficientemente amostras HPV de controles, em amostras cervicais e salivares (p<0,0001); com alta sensibilidade (acima de 95%) e especificidade (acima de 71%) em ambos fluidos. O peptídeo C.B1 também foi incorporado com sucesso (p<0,05) em um bioeletrodo de grafite para o diagnóstico do HPV direto na saliva. Na abordagem vacinal, três peptídeos (PEP1, PEP3 e PEP4) não apresentaram toxicidade em macrófagos murinos. As imunizações revelaram potente indução na produção de anticorpos séricos IgG pelo PEP3, e aumento nos títulos de IgA na mucosa vaginal sob estímulo dos PEP1 e PEP3. A avaliação da resposta imune celular indicou que o PEP1 e, sobretudo o peptídeo quimérico PEP3, foram capazes de polarizar a resposta imune para o perfil Th1 e sensibilizar as células a um status pró-inflamatório, propício para atuação antiviral. O ensaio de neutralização de PsVs do HPV-16 mostrou que o PEP3 foi capaz de reduzir a infecção em 49%. Conclusões: De maneira geral, os estudos aqui apresentados evidenciaram uma alta prevalência de HPV em mulheres brasileiras por tipos virais ainda pouco explorados na literatura mundial. Nós selecionamos mimotopos pela ferramenta de Phage Display capazes de detectar o HPV tanto em amostras de saliva quanto em secreção cervical, possibilitando um diagnóstico simples, de baixo custo, e não invasivo para rastreios populacionais. Além disso, os mimotopos aqui selecionados possuem atraente aplicabilidade em formulações vacinais, porém estudos adicionais ainda são necessários. / Doutor em Genética e Bioquímica

Ticks are implicated in serious economic losses to animal production worldwide in the order of billions of dollars. Although many antigens have been in vaccine tests, none has been effective in the control of ticks, which justifies the development of new antigens as well as new vaccine strategies. Phage Display techniques have been widely employed to map epitope structures, which have served as the basis for developing molecular vaccines. In the present study, we have applied this technique to map specific epitopes of Rhipichephalus (Boophilus) microplus and to validate the peptides as potential immunogens, we have adopted a process of selection prior to final field tests. This strategy was established in order to reduce the number of clones to be tested in the field. Six Phage -displayed random peptide libraries were selected with seven different strategies against the purified hyperimmune serum of chickens (IgY) that were immunized with total proteins of larvae and adults of Rhipicephalus (Boophilus) microplus. The selected Phage clones were sequenced, translated and analyzed through bioinformatics. To evaluate the potential of these phagotopes as effective candidate vaccines, ELISA assays, dot-blot, mice immunization (MI), humoral immune response (HIR) of cattle against the clones and a cutaneous hypersensitivity tests in cattle were performed. The selected Phage clones were analyzed through bioinformatics, generating 107 different peptides in a total of 281 sequences. Some selected phagotopes showed excellent matches with linear sequences of known proteins of the Rhipicephalus (Boophilus) microplus. Peptides that did not present significant matches with known proteins, but shared extensive homology among each other, were clustered and classified as conformational epitopes of Rhipicephalus (Boophilus) microplus, or considered as mimotopes of unknown proteic antigens. Among all clones tested by dot-blots, the 40 most reactive ones were further screened by HIR and MI. The mice sera raised against the Phage clones clearly recognized tick proteins indicating that the phagotope-induced immune responses were antigen-specific, but not all could be identified by the sera of naturally infested cattle (Bos taurus and Bos indicus). Four clones were then selected to go through the cutaneous hypersensitivity tests, and apparently all of them were effectives at diferent degrees. The stringent selection processes coupled with this schemes of validation assays allowed us to narrow down the number of peptides that should be tested in the field. One of the advantages of using whole recombinant M13 virus, carrying the recombinant peptide in fusion with the pIII protein of the virus, is that its capsid acts as adjuvant and could be tested directly without any further mixture. Additionally, we have finally used anergy skin tests (cutaneous hypersensitivity tests) once they are sometimes used to guide clinical decisions. We have assumed that cutaneous hypersensitivity tests measure a common property of cellular immune function relevant to the outcome, which together with the humoral response in naturally infested animals may provide us with sequences of peptides that may be relevant as vaccines. Finally, we have used those sequences to produce multiple antigen peptide system (MAPS), which will be used in future field tests. The present work demonstrates that the whole epitope profile can obtained through screening the Phage Displayed peptide libraries with the hyperimmune serum and reveals the potential of using epitopedisplaying Phage s as peptide vaccines against ticks. / Os carrapatos tem provocado significativas perdas econômicas na produção animal mundial na ordem de bilhões de dólares anuais. Embora vários antígenos específicos tenham sido empregados em testes vacinais, nenhum tem propiciado controle eficaz, sendo necessária a identificação de novos antígenos, bem como de novas estratégias vacinais. A tecnologia de Phage Display tem sido amplamente empregada no mapeamento de estruturas antigênicas as quais tem servido como base para o desenvolvimento de vacinas moleculares e representa uma alternativa as metodologias tradicionais. No presente trabalho, utilizou-se a tecnologia para o mapeamento de epítopos do Rhipicephalus (Boophilus) microplus e, para validar os peptídeos selecionados como potencial imunógenos, foi adotado um processo com múltiplas etapas de caracterização dos clones previamente aos ensaios clínicos. Esta estratégia foi estabelecida com o objetivo de reduzir o número de clones a ser testados em bovinos como imunógenos candidatos. Foram utilizadas para a seleção seis bibliotecas distintas de peptídeos randômicos expressados em bacteriófagos filamentosos e sete estratégias de seleção contra as imunoglobulinas (IgY) purificadas de soro hiperimune de galinhas imunizadas com proteínas totais de larvas e adultos de Rhipicephalus (Boophilus) microplus. As seqüências do DNA correspondente aos insertos dos fagos selecionados foram seqüenciados, traduzidos e analisados por análises in silico. Foram realizados testes de ELISA, Dot-Blot, imunização em camundongos, determinação da resposta imune humoral de bovinos naturalmente infestados, e a medição da hipersensibilidade cutânea com o objetivo de evoluir o potencial destes fagotopos como candidatos efetivos a novos imunógenos. Foram gerados 107 peptídeos distintos em um total de 281 seqüências, sendo alguns destes similares às seqüências protéicas lineares do Rhipicephalus (Boophilus) microplus. Os peptídeos sem similaridades significativas com proteínas conhecidas, mas que apresentaram seqüências consenso entre os peptídeos obtidos, foram agrupados e classificados como epítopos conformacionais ou considerados como mimetopos de antígenos protéicos desconhecidos do Rhipicephalus (Boophilus) microplus. Entre os clones testados por Dot-Blot, os 40 mais reativos foram posteriormente testados em ensaios de imunização em camundongos e também submetidos ao reconhecimento humoral de bovinos naturalmente infestados com carrapatos. Adicionalmente, utilizou-se o teste cutâneo de hipersensibilidade para a análise da imunidade celular. Os soros de camundongos imunizados com os clones de fagos foram reativos as proteínas de carrapato indicando que os fagotopos foram capazes de gerar resposta imune antígeno específica, embora nem todos os fagos fossem reconhecidos pelo soro de animais naturalmente infestados (Bos taurus e Bos indicus). Após a caracterização e validação, quatro fagotopos foram escolhidos para a realização dos testes cutâneos, os quais apresentaram graus variados de reatividade. Uma das vantagens da utilização do vírus M13 recombinante, contendo o peptídeo recombinante fusionado na proteína PIII do vírus, é que o próprio capsídio atua como adjuvante e pode ser testado diretamente após a seleção sem procedimentos adicionais de preparo do antígeno. Finalmente, as seqüências determinadas foram utilizadas para a produção e expressão de um sistema recombinante de múltiplos antígenos peptídicos (MAPS). Foram escolhidos 10 peptideos para a expressão na forma de multi-epitopos, os quais serão futuramente utilizados em ensaios clínicos. O presente trabalho demonstrou que um perfil geral dos epítopos pode ser obtido através da utilização de bibliotecas de peptídeos randômicos expressados em bacteriófagos filamentosos contra anticorpos obtidos de soro hiperimune e revela o potencial da seleção e utilização peptídeos epítopos específicos como potenciais vacinas recombinantes multiantigênicas para o controle do carrapato bovino. / Doutor em Genética e Bioquímica

Anaplasmosis, a persistent intraerythrocytic infection of cattle by Anaplasma marginale, causes severe anemia and a higher rate of abortion, resulting in significant loss to both dairy and beef industries. Clinical diagnosis is based on symptoms and confirmatory laboratory tests are required. Currently, all the diagnostic assays have been developed with whole antigens with indirect ELISA based on multiple epitopes. In a pioneer investigation we demonstrated the use of critical motifs of an epitope as biomarkers for immunosensor applications. Mimotopes of the MSP1a protein functional epitope were obtained through Phage Display after three cycles of selection of a 12-mer random peptide library against the neutralizing monoclonal antibody 15D2. Thirty-nine clones were randomly selected, sequenced, translated and aligned with the native sequence. The consensus sequences SxSSQSEASTSSQLGA was obtained, which is located in C-terminal end of the 28-aa repetitive motif of the MSP1a protein, but the alignment and sequences variation among mimotopes allowed us to map the critical motif STSSxL within the consensus sequence. Based on these results, two peptides were chemically synthesized; one based on the critical motif (STSSQL, Am1) and the other based on the consensus sequence aligned with the native epitope (SEASTSSQLGA, Am2). Sera from 24 infected and 52 healthy animals were tested by ELISA for reactivity against Am1 and Am2, which presented sensitivities of 96% and 100%, respectively. The Am1 peptide was incorporated onto a biolectrode (graphite modified with poly-3-hydroxyphenylacetic acid) and direct serum detection was demonstrated by impedance, differential pulse voltammetry, and atomic force microscopy. The electrochemical sensor system proved to be highly effective in discriminating sera from positive and negative animals. These immunosensors were highly sensitive and selective for positive IgG, contaminants did not affect measurements, and were based on a simple, fast and reproducible electrochemical system. / Anaplasmose, uma infecção intraeritrocitária obrigatória de bovinos, causada pela Anaplasma marginale, causa severa anemia e alta taxa de aborto, resultando em perda significativa para a indústria de laticínios e carne. O diagnóstico clínico é baseado nos sintomas e exames laboratorias confirmatórios são necessários. Atualmente, todos os ensaios de diagnósticos têm sido desenvolvidos com ELISA indireto de antígenos totais baseado em epítopos múltiplos. Em uma investigação pioneira demonstramos o uso de motivos críticos de um epítopo como biomarcador para a aplicação em imunossensores. Mimotopos do epítopo funcional da proteína MSP1a foram obtidos por meio de Phage Display, após três ciclos de seleção, de uma biblioteca randômica de peptídeos 12-mer contra o anticorpo monoclonal 15D2. Trinta e nove clones foram selecionados aleatoriamente, sequenciados, traduzidos e alinhados com a sequência nativa. Foi obtida a sequência consenso SxSSQSEASTSSQLGA, que está localizada na extremidade C-terminal do motivo repetitivo de 28-aa da proteína MSP1a, mas o alinhamento e a variação das sequências entre os mimotopos nos permitiu mapear o motivo crítico STSSxL dentro da sequência consenso. Com base nesses resultados, dois peptídeos foram quimicamente sintetizados; um baseado no motivo crítico (STSSQL, Am1) e o outro baseado na sequência consenso alinhada com o epítopo nativo (SEASTSSQLGA, Am2). Soro de 24 animais infectados e 52 animais saudáveis foram testados por ELISA quanto à reatividade contra Am1 e Am2, que apresentou sensibilidades de 96% e 100%, respectivamente. O peptide Am1 foi incorporado a um bioeletrodo (grafite modificado com ácido poli-3-hidroxyfenilacético) e a detecção direta de soro foi demonstrada por impedância, voltametria de pulso diferencial e microscopia de força atômica. O sistema de sensor eletroquímico provou ser altamente eficaz em soro de animais positivos de negativos. Este imunossensor foi altamente sensível e seletivo para IgG positiva, contaminantes não afetaram as medições, e foram baseados e um sistema simples, rápido e reprodutível. / Doutor em Genética e Bioquímica