Selection and characterization of peptides mimetic to Campylobacter fetus subsp. venerealis using phage display / Seleção e caracterização de peptídeos miméticos a Campylobacter fetus subsp. venerealis usando phage display

Bovine genital campylobacteriosis (BGC) is a venereal and subclinical disease that affects the fertility of cattle herds, and it is caused by Campylobacter fetus subsp. venerealis . This study selected peptides mimetic to the BGC-causing agent from a phage library. Phage display is a technique that applies bacteriophage libraries that reveal peptides fused to the viral capsid in biological selections against target proteins. Biopannings were performed for biological selection in the phage library using rabbit hyperimmune serum and C. fetus subsp. venerealis protein extract. Five selected heptapeptides were considered mimetic to Cfv-NCTC 10354 based on the results of bioinformatics analysis and assays with hyperimmune serum and cervicovaginal mucus obtained from heifers. ALASLPL and LSYLFPP were the most reactive peptides and considered promising as possible mimetic immunogens for C. fetus subsp. venerealis.

Campilobacteriose Genital Bovina (CGB) é uma doença venérea e subclínica que causa problemas reprodutivos em rebanhos, causada por Campylobacter fetus subsp. venerealis. Este trabalho teve como objetivo selecionar peptídeos miméticos ao agente da CGB de uma biblioteca de fagos. Phage display é uma técnica que aplica bibliotecas de bacteriófagos que expõem peptídeos fusionados ao capsídeo viral em seleções biológicas contra proteínas alvo. Biopannings foram realizados para seleção biológica na biblioteca de fagos por meio de soro hiperimune de coelho e extrato proteico de C. fetus subsp. venerealis. Cinco heptapeptídeos selecionados foram considerados miméticos para Cfv-NCTC 10354 a partir de análises de bioinformática e ensaios com soro hiperimune e muco cérvico-vaginal de novilhas. ALASLPL e LSYLFPP foram os peptídeos mais reativos e considerados promissores como possíveis imunógenos miméticos para C. fetus subsp. venerealis.

Selection and characterization of peptides mimetic to Campylobacter fetus subsp. venerealis using phage display / Seleção e caracterização de peptídeos miméticos a Campylobacter fetus subsp. venerealis usando phage display

ABSTRACT: Bovine genital campylobacteriosis (BGC) is a venereal and subclinical disease that affects the fertility of cattle herds, and it is caused by Campylobacter fetus subsp. venerealis . This study selected peptides mimetic to the BGC-causing agent from a phage library. Phage display is a technique that applies bacteriophage libraries that reveal peptides fused to the viral capsid in biological selections against target proteins. Biopannings were performed for biological selection in the phage library using rabbit hyperimmune serum and C. fetus subsp. venerealis protein extract. Five selected heptapeptides were considered mimetic to Cfv-NCTC 10354 based on the results of bioinformatics analysis and assays with hyperimmune serum and cervicovaginal mucus obtained from heifers. ALASLPL and LSYLFPP were the most reactive peptides and considered promising as possible mimetic immunogens for C. fetus subsp. venerealis.

RESUMO: Campilobacteriose Genital Bovina (CGB) é uma doença venérea e subclínica que causa problemas reprodutivos em rebanhos, causada por Campylobacter fetus subsp. venerealis. Este trabalho teve como objetivo selecionar peptídeos miméticos ao agente da CGB de uma biblioteca de fagos. Phage display é uma técnica que aplica bibliotecas de bacteriófagos que expõem peptídeos fusionados ao capsídeo viral em seleções biológicas contra proteínas alvo. Biopannings foram realizados para seleção biológica na biblioteca de fagos por meio de soro hiperimune de coelho e extrato proteico de C. fetus subsp. venerealis. Cinco heptapeptídeos selecionados foram considerados miméticos para Cfv-NCTC 10354 a partir de análises de bioinformática e ensaios com soro hiperimune e muco cérvico-vaginal de novilhas. ALASLPL e LSYLFPP foram os peptídeos mais reativos e considerados promissores como possíveis imunógenos miméticos para C. fetus subsp. venerealis.

Selection and characterization of peptides mimetic to Campylobacter fetus subsp. venerealis using phage display / Seleção e caracterização de peptídeos miméticos a Campylobacter fetus subsp. venerealis usando phage display

Bovine genital campylobacteriosis (BGC) is a venereal and subclinical disease that affects the fertility of cattle herds, and it is caused by Campylobacter fetus subsp. venerealis . This study selected peptides mimetic to the BGC-causing agent from a phage library. Phage display is a technique that applies bacteriophage libraries that reveal peptides fused to the viral capsid in biological selections against target proteins. Biopannings were performed for biological selection in the phage library using rabbit hyperimmune serum and C. fetus subsp. venerealis protein extract. Five selected heptapeptides were considered mimetic to Cfv-NCTC 10354 based on the results of bioinformatics analysis and assays with hyperimmune serum and cervicovaginal mucus obtained from heifers. ALASLPL and LSYLFPP were the most reactive peptides and considered promising as possible mimetic immunogens for C. fetus subsp. venerealis.(AU)

Campilobacteriose Genital Bovina (CGB) é uma doença venérea e subclínica que causa problemas reprodutivos em rebanhos, causada por Campylobacter fetus subsp. venerealis. Este trabalho teve como objetivo selecionar peptídeos miméticos ao agente da CGB de uma biblioteca de fagos. Phage display é uma técnica que aplica bibliotecas de bacteriófagos que expõem peptídeos fusionados ao capsídeo viral em seleções biológicas contra proteínas alvo. Biopannings foram realizados para seleção biológica na biblioteca de fagos por meio de soro hiperimune de coelho e extrato proteico de C. fetus subsp. venerealis. Cinco heptapeptídeos selecionados foram considerados miméticos para Cfv-NCTC 10354 a partir de análises de bioinformática e ensaios com soro hiperimune e muco cérvico-vaginal de novilhas. ALASLPL e LSYLFPP foram os peptídeos mais reativos e considerados promissores como possíveis imunógenos miméticos para C. fetus subsp. venerealis.(AU)

Recombinant antibodies against Iranian cobra venom as a new emerging therapy by phage display technology

The production of antivenom from immunized animals is an established treatment for snakebites; however, antibody phage display technology may have the capacity to delivery results more quickly and with a better match to local need. Naja oxiana, the Iranian cobra, is a medically important species, responsible for a significant number of deaths annually. This study was designed as proof of principle to determine whether recombinant antibodies with the capacity to neutralize cobra venom could be isolated by phage display. Methods: Toxic fractions from cobra venom were prepared by chromatography and used as targets in phage display to isolate recombinant antibodies from a human scFv library. Candidate antibodies were expressed in E. coli HB2151 and purified by IMAC chromatography. The selected clones were analyzed in in vivo and in vitro experiments. Results: Venom toxicity was contained in two fractions. Around a hundred phage clones were isolated against each fraction, those showing the best promise were G12F3 and G1F4. While all chosen clones showed low but detectable neutralizing effect against Naja oxiana venom, clone G12F3 could inhibit PLA2 activity. Conclusion: Therefore, phage display is believed to have a good potential as an approach to the development of snake antivenom.(AU)

Recombinant antibodies against Iranian cobra venom as a new emerging therapy by phage display technology

Background:The production of antivenom from immunized animals is an established treatment for snakebites; however, antibody phage display technology may have the capacity to delivery results more quickly and with a better match to local need. Naja oxiana, the Iranian cobra, is a medically important species, responsible for a significant number of deaths annually. This study was designed as proof of principle to determine whether recombinant antibodies with the capacity to neutralize cobra venom could be isolated by phage display.Methods:Toxic fractions from cobra venom were prepared by chromatography and used as targets in phage display to isolate recombinant antibodies from a human scFv library. Candidate antibodies were expressed in E. coli HB2151 and purified by IMAC chromatography. The selected clones were analyzed in in vivo and in vitro experiments.Results:Venom toxicity was contained in two fractions. Around a hundred phage clones were isolated against each fraction, those showing the best promise were G12F3 and G1F4. While all chosen clones showed low but detectable neutralizing effect against Naja oxiana venom, clone G12F3 could inhibit PLA2 activity.Conclusion:Therefore, phage display is believed to have a good potential as an approach to the development of snake antivenom.(AU)

Chicken antibodies against venom proteins of Trimeresurus stejnegeri in Taiwan

The venom of bamboo vipers (Trimeresurus stejnegeri - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived antivenom is a specific treatment against snakebites, but its production costs are high and there are some inevitable side effects. The aim of the present work is to help in the development of an affordable and more endurable therapeutic strategy for snakebites. Methods: T. stejnegeri venom proteins were inactivated by glutaraldehyde in order to immunize hens for polyclonal immunoglobulin (IgY) antibodies production. After IgY binding assays, two antibody libraries were constructed expressing single-chain variable fragment (scFv) antibodies joined by the short or long linker for use in phage display antibody technology. Four rounds of biopanning were carried out. The selected scFv antibodies were then further tested for their binding activities and neutralization assays to TS proteins. Results: Purified IgY from egg yolk showed the specific binding ability to TS proteins. The dimensions of these two libraries contain 2.4 × 107 and 6.8 × 107 antibody clones, respectively. An increase in the titers of eluted phage indicated anti-TS clones remarkably enriched after 2nd panning. The analysis based on the nucleotide sequences of selected scFv clones indicated that seven groups of short linkers and four groups of long linkers were identified. The recombinant scFvs showed significant reactivity to TS venom proteins and a cross-reaction to Trimeresurus mucrosquamatus venom proteins. In in vivo studies, the data demonstrated that anti-TS IgY provided 100% protective effects while combined scFvs augmented partial survival time of mice injected with a lethal amount of TS proteins. Conclusion: Chickens were excellent hosts for the production of neutralization antibodies at low cost. Phage display technology is available for generation of monoclonal antibodies against snake venom proteins. These antibodies could be applied in the development of diagnostic kits or as an alternative for snakebite envenomation treatment in the near future.(AU)

Chicken antibodies against venom proteins of Trimeresurus stejnegeri in Taiwan

The venom of bamboo vipers (Trimeresurus stejnegeri - TS), commonly found in Taiwan, contains deadly hemotoxins that cause severe envenomation. Equine-derived antivenom is a specific treatment against snakebites, but its production costs are high and there are some inevitable side effects. The aim of the present work is to help in the development of an affordable and more endurable therapeutic strategy for snakebites. Methods: T. stejnegeri venom proteins were inactivated by glutaraldehyde in order to immunize hens for polyclonal immunoglobulin (IgY) antibodies production. After IgY binding assays, two antibody libraries were constructed expressing single-chain variable fragment (scFv) antibodies joined by the short or long linker for use in phage display antibody technology. Four rounds of biopanning were carried out. The selected scFv antibodies were then further tested for their binding activities and neutralization assays to TS proteins. Results: Purified IgY from egg yolk showed the specific binding ability to TS proteins. The dimensions of these two libraries contain 2.4 × 107 and 6.8 × 107 antibody clones, respectively. An increase in the titers of eluted phage indicated anti-TS clones remarkably enriched after 2nd panning. The analysis based on the nucleotide sequences of selected scFv clones indicated that seven groups of short linkers and four groups of long linkers were identified. The recombinant scFvs showed significant reactivity to TS venom proteins and a cross-reaction to Trimeresurus mucrosquamatus venom proteins. In in vivo studies, the data demonstrated that anti-TS IgY provided 100% protective effects while combined scFvs augmented partial survival time of mice injected with a lethal amount of TS proteins. Conclusion: Chickens were excellent hosts for the production of neutralization antibodies at low cost. Phage display technology is available for generation of monoclonal antibodies against snake venom proteins. These antibodies could be applied in the development of diagnostic kits or as an alternative for snakebite envenomation treatment in the near future.(AU)

Isolation of recombinant antibodies directed against surface proteins of Clostridium difficile

Clostridium difficile has emerged as an increasingly important nosocomial pathogen and the prime causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis in humans. In addition to toxins A and B, immunological studies using antisera from patients infected with C. difficile have shown that a number of other bacterial factors contribute to the pathogenesis, including surface proteins, which are responsible for adhesion, motility and other interactions with the human host. In this study, various clostridial targets, including FliC, FliD and cell wall protein 66, were expressed and purified. Phage antibody display yielded a large panel of specific recombinant antibodies, which were expressed, purified and characterised. Reactions of the recombinant antibodies with their targets were detected by enzyme-linked immunosorbent assay; and Western blotting suggested that linear rather than conformational epitopes were recognised. Binding of the recombinant antibodies to surface-layer proteins and their components showed strain specificity, with good recognition of proteins from C. difficile 630. However, no reaction was observed for strain R20291a representative of the 027 ribotype. Binding of the recombinant antibodies to C. difficile M120 extracts indicated that a component of a surface-layer protein of this strain might possess immunoglobulin-binding activities. The recombinant antibodies against FliC and FliD proteins were able to inhibit bacterial motility.(AU)

Seleção de peptídeos específicos para anticorpos anti Leptospira Interrogans

Leptospira interrogans sorovar Copenhageni é o principal sorovar envolvido na Leptospirose humana, nas Regiões Sudeste e Nordeste do Brasil, sendo ainda escassas as informações nas demais regiões do país. É caracterizada por ser uma doença de caráter populacional e ambiental, seu controle está intimamente ligado a medidas de prevenção, aplicadas aos animais e ao ambiente no qual os mesmos são mantidos. A pesquisa de anticorpos constitui a principal prova de diagnóstico da leptospirose, incluindo teste sorogrupos – específicos, dos quais a prova de soro aglutinação microscópica (SAM) é a mais importante e amplamente utilizada. O objetivo nesse estudo foi verificar a viabilidade do uso da técnica Phage Display para selecionar peptídeos similares aos epítopos de anti Leptospira sorovar Copenhageni para subsidiar futuramente, um novo teste de diagnóstico. Após 3 ciclos de seleção, 92 clones foram selecionados e submetidos ao seqüenciamento. Assim foram seqüenciados 57 clones que demonstraram 54 seqüências distintas. Testes ELISA foram realizados para validação dos clones reativos e 3 deles foram reativos com altas absorbâncias. A tecnologia de Phage Display foi capaz de demonstrar reatividade, sinalizando a mimetização de possíveis peptídeos aos epítopos da Leptospira interrogans sorovar Copenhageni.

Leptospira interrogans serovar Copenhageni is the main sorovar involved in the Leptospirosis human, in the Southeast Regions and Northeast of Brazil, being still scarce the information in the too regions of the country. It is characterized by be an illness of his, environmental and population character control is intimate linked in proportion to prevention, applied to the animals and to the environment in which they are maintained. To research of antibodies I constituted to main test of diagnosis of the leptospirosis, including test serogroups – specific, of the which the microscopic amalgamation serum test (BE) is to more important and broadly utilized. The objective in that study was verify the feasability of the use of the technical one Phage Display for select similar peptides to the epítopos of anti Leptospira serovar Copenhageni for subsidize future, a new test of diagnosis. After 3 cycles of selection, 92 clones were selected and submitted to the sequencing. So were sequenced 57 clones that showed 54 distinct sequences. ELISA tests were performed to validate the reactive clones and three of them were reactive with high absorbance. Phage display technology was able to demonstrate reactivity, signaling the possible peptides mimicking the epitopes of Leptospira interrogans serovar Copenhageni.

Seleção de peptídeos específicos para anticorpos anti Leptospira Interrogans

Leptospira interrogans sorovar Copenhageni é o principal sorovar envolvido na Leptospirose humana, nas Regiões Sudeste e Nordeste do Brasil, sendo ainda escassas as informações nas demais regiões do país. É caracterizada por ser uma doença de caráter populacional e ambiental, seu controle está intimamente ligado a medidas de prevenção, aplicadas aos animais e ao ambiente no qual os mesmos são mantidos. A pesquisa de anticorpos constitui a principal prova de diagnóstico da leptospirose, incluindo teste sorogrupos específicos, dos quais a prova de soro aglutinação microscópica (SAM) é a mais importante e amplamente utilizada. O objetivo nesse estudo foi verificar a viabilidade do uso da técnica Phage Display para selecionar peptídeos similares aos epítopos de anti Leptospira sorovar Copenhageni para subsidiar futuramente, um novo teste de diagnóstico. Após 3 ciclos de seleção, 92 clones foram selecionados e submetidos ao seqüenciamento. Assim foram seqüenciados 57 clones que demonstraram 54 seqüências distintas. Testes ELISA foram realizados para validação dos clones reativos e 3 deles foram reativos com altas absorbâncias. A tecnologia de Phage Display foi capaz de demonstrar reatividade, sinalizando a mimetização de possíveis peptídeos aos epítopos da Leptospira interrogans sorovar Copenhageni.(AU)

Leptospira interrogans serovar Copenhageni is the main sorovar involved in the Leptospirosis human, in the Southeast Regions and Northeast of Brazil, being still scarce the information in the too regions of the country. It is characterized by be an illness of his, environmental and population character control is intimate linked in proportion to prevention, applied to the animals and to the environment in which they are maintained. To research of antibodies I constituted to main test of diagnosis of the leptospirosis, including test serogroups specific, of the which the microscopic amalgamation serum test (BE) is to more important and broadly utilized. The objective in that study was verify the feasability of the use of the technical one Phage Display for select similar peptides to the epítopos of anti Leptospira serovar Copenhageni for subsidize future, a new test of diagnosis. After 3 cycles of selection, 92 clones were selected and submitted to the sequencing. So were sequenced 57 clones that showed 54 distinct sequences. ELISA tests were performed to validate the reactive clones and three of them were reactive with high absorbance. Phage display technology was able to demonstrate reactivity, signaling the possible peptides mimicking the epitopes of Leptospira interrogans serovar Copenhageni.(AU)

DESENVOLVIMENTO DE BIBLIOTECAS DE PHAGE DISPLAY PARA A OBTENÇÃO DE MARCADORES E/OU INIBIDORES BIOLÓGICOS CONTRA Trypanosoma evansi

MORAES, Julio Cesar. Desenvolvimento de bibliotecas de phage display para a obtenção de marcadores e/ou inibidores biológicos contra Trypanosoma evansi. 2017. 115 f. Tese (Doutorado em Ciência Animal). Universidade do Estado de Santa Catarina. Programa de Pós-graduação em Ciência Animal, Lages, 2017. Trypanosoma evansi é um hemoprotozoário parasito, causador da doença conhecida como Surra ou Mal das Cadeiras, tendo sido descrito em várias espécies, como cães, capivaras, quatis, bovinos, búfalos, sendo os equinos a espécie mais acometida. É responsável por prejuízos diretos no setor de equinocultura e, indiretos no setor pecuário que utilizam essa espécie no manejo de outros animais de produção. O T. evansi é transmitido de maneira acíclica por insetos (Tabanidae e Stomoxydae) e morcegos hematófagos e, de maneira iatrogênica (vacinações e coleta de sangue). Com o objetivo de identificar novos alvos para o controle e ou diagnóstico do T. evansi, utilizamos a tecnologia de phage display associada à técnica Kunkel de mutagênese direcionada para construir uma biblioteca de proteínas com diversidade superior a um milhão de proteínas distintas e mesmo após a diversificação, a biblioteca manteve como base estrutural o esqueleto protéico de uma toxina atenuada com Domínio Kunitz presente no veneno do escorpião Mesobuthus tamulus As mutações foram direcionadas, com auxílio da estrutura cristalográfica da toxina, para resíduos específicos na porção C-terminal da -hélice e no loop menor da proteína. O DNA mutante foi purificado e amplificado pela técnica de Círculo Rolante Seletivo. Clones da biblioteca foram sequenciados, onde foi evidenciado uma taxa próxima de 100% de inserções de mutações resíduo específicas. Em seguida a biblioteca foi utilizada em seleções de afinidade contra o T. evansi para busca de ligantes específicos ao parasito. Após um round in vivo e dois in vitro, seis clones foram selecionados para estudos individuais de potência, especificidade de ligação e toxicidade. Entre todos os clones, em especial, os clones 5 e 1 apresentaram um maior potencial de ligação ao T. evansi e ausência de ligação contra outros tripanosomatídeos (T. cruzi e T. rangeli). Os clones 1, 2 e 5 demostraram ser tóxicos para o T. evansi causando uma mortalidade de 13%, 31,5% e 19,7%, respectivamente. O DNA dos clones 1, 2 e 5 foram sequenciados, demonstrando que houve a inserção de mutações. A biblioteca derivada de toxinas, bem como os clones 1 e 5 identificados demonstraram possuir potencial para o desenvolvimento de novos testes para o diagnóstico direto de T. evansi. Os clones 1, 2 e 5 que demonstraram atividade tóxica, poderão servir como base para outros estudos, visando o desenvolvimento de novas drogas com atividade tripanocida.

MORAES, Julio Cesar. Development of phage display libraries for obtaining markers and/or biological inhibitors against Trypanosoma evansi. 2017. 115 f. Thesis (DSc in Animal Science). Universidade do Estado de Santa Catarina. Programa de Pós-graduação em Ciência Animal, Lages, 2017. Trypanosoma evansi is a parasitic hemoprotozoan, which causes the disease known as "surra" or "mal das cadeiras" and has been described in several species, such as dogs, capybaras, quatis, cattle, buffaloes, and horses are the most affected species. It is responsible for direct losses in the equinoculture sector and indirect in the livestock sector that use this species in the handling of other production animals. T. evansi is transmitted acyclically by insects (Tabanidae and Stomoxydae) and hematophagous bats and iatrogenically (vaccinations, collection of blood). With the objective of identify new compounds for the control and / or diagnosis of T. evansi, We used phage display technology associated with Kunkel's mutagenesis technique aimed at constructing a protein library with diversity of more than one million distinct proteins and even after diversification, the library retained as a structural basis the protein skeleton of an attenuated toxin with Kunitz Domain present in the venom of Mesobuthus tamulus scorpion The mutations were directed, with the support of the crystallographic structure of the toxin, to specific residues in the C-terminal portion of the -helix and in the smaller loop of the protein. The mutant DNA was purified and amplified by the Selective Rolling Circle technique. Clones from the library were sequenced, where a close to 100% rate of insertions of specific residue mutations was evidenced. Then the library was used in affinity selections against T. evansi to search for specific ligands to the parasite. After one round in vivo and two in vitro, six clones were selected for individual studies of potency, binding specificity and toxicity. Among all clones, in particular, clones 5 and 1 showed a greater potential for binding to T. evansi and lack of binding against other trypanosomatids (T. cruzi and T. rangeli). Clones 1, 2 and 5 proved to be toxic to T. evansi causing a mortality of 13%, 31.5% and 19.7%, respectively. DNA from clones 1, 2 and 5 were sequenced, demonstrating that mutations were inserted. The toxin-derived library as well as clones 1 and 5 identified has potential for the development of new tests for the direct diagnosis of T. evansi. Clones 1, 2 and 5 that demonstrated toxic activity may serve as the basis for other studies aimed at the development of new drugs with trypanocidal activity.

In silico analysis for identification of tick phagotopes selected by phage-displayed libraries

Phage display techniques have been widely employed to map epitope structures which have served as the basis for developing molecular vaccines. We have applied this technique to map specific epitopes of Rhipicephalus (Boophilus) microplus. In the present study, we have identified the potential immunogens using a process in which the selected phage clones were analyzed through bioinformatics, prior to final field tests. The present study demonstrates the feasibility of identifying important R. (B.) microplus phagotopes for vaccine development through screening of phage-displayed random peptide libraries and bioinformatics tools.

Técnicas de phage display têm sido amplamente empregadas para o mapeamento de epítopos os quais tem servido como base para o desenvolvimento de vacinas moleculares. Esta técnica foi aplicada no mapeamento de epítopos do Rhipicephalus (Boophilus) microplus. Neste estudo, potenciais imunógenos foram identificados pela adoção de um processo em que os clones de fagos foram analisados por bioinformática, previamente à realização dos testes. Os resultados demonstraram a possibilidade da identificação de importantes mimetopos do R. (B.) microplus para o desenvolvimento de vacinas através da seleção de bibliotecas de phage display associada à análise de bioinformática.

In silico analysis for identification of tick phagotopes selected by phage-displayed libraries

Phage display techniques have been widely employed to map epitope structures which have served as the basis for developing molecular vaccines. We have applied this technique to map specific epitopes of Rhipicephalus (Boophilus) microplus. In the present study, we have identified the potential immunogens using a process in which the selected phage clones were analyzed through bioinformatics, prior to final field tests. The present study demonstrates the feasibility of identifying important R. (B.) microplus phagotopes for vaccine development through screening of phage-displayed random peptide libraries and bioinformatics tools.

Técnicas de phage display têm sido amplamente empregadas para o mapeamento de epítopos os quais tem servido como base para o desenvolvimento de vacinas moleculares. Esta técnica foi aplicada no mapeamento de epítopos do Rhipicephalus (Boophilus) microplus. Neste estudo, potenciais imunógenos foram identificados pela adoção de um processo em que os clones de fagos foram analisados por bioinformática, previamente à realização dos testes. Os resultados demonstraram a possibilidade da identificação de importantes mimetopos do R. (B.) microplus para o desenvolvimento de vacinas através da seleção de bibliotecas de phage display associada à análise de bioinformática.

Produção e aplicação de imunoglobulinas Y anti-leptospira

A dissertação foi dividida em dois estudos. No primeiro estudo objetivou-se verificar se galinhas imunizadas com uma solução de Leptospiras inativadas e proteínas de membrana externa do sorovar Hardjo, poderiam produzir anticorpos policlonais específicos anti-Leptospiras, detectáveis em testes ELISA. Foram imunizados oito galinhas com 25 semanas de idade, da raça White Leghorn, sendo três imunizadas com uma suspensão de Leptospiras inativadas, três com uma solução de proteínas de membrana externa extraída do sorovar Hardjo e duas controle. Coletas de sangue foram realizadas quinzenalmente e de ovos diariamente. A IgY foi purificada a partir da gema dos ovos utilizando para a delipidação o método de diluição em água ácida e a precipitação com sulfato de amônio. Realizou-se testes ELISA para verificar a especificidade da IgY, nestes foi possível observar a produção de anticorpos anti-Leptospira, tanto no soro quanto nas gemas purificadas. O pico de produção de anticorpos específicos ocorreu na 5º semana após a primeira imunização. Foi possível induzir a produção de anticorpos específicos em galinhas, por meio de imunizações destas com Leptospira inativada e proteínas de membrana externa do sorovar Hardjo. Galinhas imunizadas com uma suspensão de Leptospiras inativadas e com PME de Leptospira interrogans do sorovar Hardjo produziram anticorpos reativos a PME Hardjo detectáves por teste ELISA. No segundo estudo objetivou-se com identificar epítopos ou mimetopos (sequências que imitam epítopos verdadeiros), por meio da técnica de phage display tendo como alvo anticorpos IgY anti-Leptospira e anti-PME Hardjo. Os anticorpos IgY utilizados como alvo foram produzidos em galinhas imunizadas com proteínas de membrana externa de Leptospira interrogans sorovar sorovar Hardjo (anti-PME Hardjo), suspensão de Leptospiras inativada (anti-Leptospira) e IgY controle. No biopanning uma biblioteca comercial de peptídeos randômicos Ph.D. 12 mer, New England BioLabs®Inc foi incubada primeiramente com a IgY controle, realizando um processo denominado seleção negativa e depois com a IgY anti-Leptospira e anti-PME Hardjo, a eluição dos fagos selecionados foi então realizada de forma competitiva e ácida. Após três ciclos de seleção, 288 clones foram isolados e sequenciados, gerando 132 sequências válidas. Os clones foram submetidos a testes ELISA com o anticorpo usado como alvo no biopanning para verificar a especificidade destes. Nos resultados dos testes ELISAs verificou-se que nove fagos (PMEcomp17, PMEac10, PMEac12, PMEac19, PMEac29, PMEac35, Lep6, Lep7 e Lep20) apresentaram melhores resultados. Estes foram alinhados com a sequência primária de proteínas de Leptospira spp e Leptospira interrogans sorovar Hardjo, e as análises in Silico mostraram que os nove peptídeos têm similaridade com proteínas imunogênicas de Leptospiras. Foram selecionados nove peptídeos com similaridade a proteínas de Leptospira interrogans consideradas imunogênicas. Os peptídeos foram similares a proteínas de membrana externa, lipoproteína, LipL32, LipL41, LigA, SecY, LenE