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.

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)

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)

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)

Sheep polyclonal antibody to map Haemonchus contortus mimotopes using phage display library / Anticorpo policlonal de ovinos para mapear mimetopos de Haemonchus contortus usando a biblioteca de Phage display

The aim of this study was to evaluate phage display technology for mapping Haemonchus contortus mimotopes. We screened the PhD-7 Phage Display Peptide Library Kit with a sheep polyclonal antibody against H. contortus. After four rounds of selection, 50 phage peptide clones were selected by biopanning and sequenced. Two clones displaying peptide mimotopes of H. contortus proteins were chosen for sheep immunization: clone 6 - mimotope of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and clone 17 - mimotope of a disorganized muscle family member (Dim 1). Twelve sheep were allocated into 3 groups of 4 animals as follow: G1: control group; G2/GAPDH: immunized with clone 6; and G3/Dim1: immunized with clone 17. Four immunizations were performed at intervals of seven days (0, 7, 14, and 21 days). On day 28 post initial vaccination, all groups were orally challenged with 2500 H. contortus infective larvae. The mimotope peptides selected by phage display were recognized by IgG from sheep naturaly infected with H. contortus. The immunization protocol showed an increasein IgG anti-M13 phage titers, but no effect was observed in IgG-specific for the anti-mimotope peptides. This is the first report of successful use of a phage display library for the identification of mimotopes of H. contortus proteins.(AU)

O objetivo deste estudo foi avaliar a tecnologia de phage display no mapeamento de mimetopos de Haemonchus contortus. Anticorpo policlonal de ovinos anti-H. contortus foi usado para seleção a partir da biblioteca PhD-7 Phage Display Peptide Library Kit (New England BioLabs). Após quatro rodadas, 50 clones de fagos expressando peptídeos foram selecionados e sequenciados. Dois clones que exibiram mimetopos de H. contortus foram escolhidos para imunização de ovinos: clone 6 mimetopo de gliceraldeído-3-fosfato desidrogenase (GAPDH) e clone 17 - mimetopo da família do músculo desorganizado (Dim 1). Doze ovinos foram alocados em 3 grupos de 4 animais, da seguinte forma: G1: grupo controle, G2/GAPDH: imunizado com o clone 6 e G3/Dim1: imunizado com o clone 17. Quatro imunizações foram realizadas (0, 7, 14 e 21 dias). No dia 28 após a primeira imunização, todos os grupos foram desafiados oralmente com 2500 larvas infectantes de H. contortus . Os peptídeos mimetopos selecionados foram reconhecidos por IgG de ovinos naturalmente infectados por H. contortus. O ensaio de imunização revelou um aument dos títulos de IgG anti-fago M13, mas não ocorreu aumento de IgG anti-peptídeos mimetopos. Este é o primeiro relato de uso bem sucedido da biblioteca de Phage display para a identificação de mimetopos de H. contortus.(AU)

Characterization of Monoclonal Antibodies against Bovine herpesvirus type 1 selected by Phage Display Technology / Caracterização de Anticorpos Monoclonais reativos ao Herpesvírus bovino tipo 1 selecionados por Phage Display

The specificity of monoclonal antibodies (mAbs) to desired targets makes these molecules suitable for therapeutic and diagnostic uses against a wide range of pathogens. Phage display antibody libraries offer one method by which mAbs can be selected for, without the use of conventional hybridoma technology. In this work, phage display technology was used to construct, select and characterize a combinatorial single chain fragment variable (scFv) antibody library against bovine herpesvirus type 1 (BoHV-1) from the immune repertoire of chickens immunized with the virus. In silico analysis of the hypervariable domains of the antibody heavy chains revealed a high frequency of scFv fragments with low variability, suggesting that selection had probably been carried out and favored by a few im-munogenic viral antigens. The reactivity of the scFv fragments selected against BoHV-1 was demon-strated by Phage-ELISA. A significant increase in antibody reactivity to the target was observed after six rounds of library selection, showing its potential use as a molecule for BoHV-1 diagnosis. The strategy described here opens up a field for the use of phage display as a tool for selection of mono-clonal antibodies that could be used for theranostic applications against infectious and parasitic dis-eases of veterinary interest.

A especificidade dos anticorpos monoclonais (mAb) aos alvos desejados torna estas moléculas ade-quadas para uso em diagnóstico ou terapia de uma vasta gama de agentes patogênicos. Biblioteca de anticorpos apresentados em fagos filamentosos é uma metodologia para a produção de mAbs, poden-do ser utilizada como alternativa à tecnologia de hibridoma convencional, tradicionalmente empregada para este fim. Neste trabalho, a tecnologia de Phage display foi usada para construir, selecionar e ca-racterizar uma biblioteca combinatorial de fragmentos de anticorpos de cadeia única (scFv) contra o Herpesvírus bovino tipo 1 (BoHV-1) a partir do repertório imune de galinhas imunizadas com o vírus. A análise in silico dos domínios hipervariáveis das cadeias pesadas dos anticorpos revelou uma alta frequência de fragmentos scFv com baixa variabilidade, sugerindo que a seleção foi provavelmente conduzida e favorecida por poucos antígenos virais mais imunogênicos. A reatividade dos fragmentos scFv selecionados contra BoHV-1 foi demonstrada por Fago-ELISA. Observou-se um aumento signi-ficativo da reatividade dos anticorpos após seis ciclos de seleção, evidenciando sua utilização como molécula para o diagnóstico de BoHV-1. A estratégia aqui descrita abre a possibilidade do uso da tec-nologia de Phage display como ferramenta na seleção de anticorpos monoclonais com potencial uso tanto para o diagnóstico quanto para terapia de doenças infecciosas e/ou parasitárias de interesse veterinário.

Characterization of Monoclonal Antibodies against Bovine herpesvirus type 1 selected by Phage Display Technology / Caracterização de Anticorpos Monoclonais reativos ao Herpesvírus bovino tipo 1 selecionados por Phage Display

The specificity of monoclonal antibodies (mAbs) to desired targets makes these molecules suitable for therapeutic and diagnostic uses against a wide range of pathogens. Phage display antibody libraries offer one method by which mAbs can be selected for, without the use of conventional hybridoma technology. In this work, phage display technology was used to construct, select and characterize a combinatorial single chain fragment variable (scFv) antibody library against bovine herpesvirus type 1 (BoHV-1) from the immune repertoire of chickens immunized with the virus. In silico analysis of the hypervariable domains of the antibody heavy chains revealed a high frequency of scFv fragments with low variability, suggesting that selection had probably been carried out and favored by a few im-munogenic viral antigens. The reactivity of the scFv fragments selected against BoHV-1 was demon-strated by Phage-ELISA. A significant increase in antibody reactivity to the target was observed after six rounds of library selection, showing its potential use as a molecule for BoHV-1 diagnosis. The strategy described here opens up a field for the use of phage display as a tool for selection of mono-clonal antibodies that could be used for theranostic applications against infectious and parasitic dis-eases of veterinary interest.(AU)

A especificidade dos anticorpos monoclonais (mAb) aos alvos desejados torna estas moléculas ade-quadas para uso em diagnóstico ou terapia de uma vasta gama de agentes patogênicos. Biblioteca de anticorpos apresentados em fagos filamentosos é uma metodologia para a produção de mAbs, poden-do ser utilizada como alternativa à tecnologia de hibridoma convencional, tradicionalmente empregada para este fim. Neste trabalho, a tecnologia de Phage display foi usada para construir, selecionar e ca-racterizar uma biblioteca combinatorial de fragmentos de anticorpos de cadeia única (scFv) contra o Herpesvírus bovino tipo 1 (BoHV-1) a partir do repertório imune de galinhas imunizadas com o vírus. A análise in silico dos domínios hipervariáveis das cadeias pesadas dos anticorpos revelou uma alta frequência de fragmentos scFv com baixa variabilidade, sugerindo que a seleção foi provavelmente conduzida e favorecida por poucos antígenos virais mais imunogênicos. A reatividade dos fragmentos scFv selecionados contra BoHV-1 foi demonstrada por Fago-ELISA. Observou-se um aumento signi-ficativo da reatividade dos anticorpos após seis ciclos de seleção, evidenciando sua utilização como molécula para o diagnóstico de BoHV-1. A estratégia aqui descrita abre a possibilidade do uso da tec-nologia de Phage display como ferramenta na seleção de anticorpos monoclonais com potencial uso tanto para o diagnóstico quanto para terapia de doenças infecciosas e/ou parasitárias de interesse veterinário.(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)

Characterization of Leiurus abdullahbayrami (Scorpiones: Buthidae) venom: peptide profile, cytotoxicity and antimicrobial activity

Background Scorpion venoms are rich bioactive peptide libraries that offer promising molecules that may lead to the discovery and development of new drugs.Leiurus abdullahbayrami produces one of the most potent venoms among Turkish scorpions that provokes severe symptoms in envenomated victims.Methods In the present study, the peptide profile of the venom was investigated by electrophoretic methods, size-exclusion and reversed-phase chromatography and mass spectroscopy. Cytotoxic and antimicrobial effects were evaluated on a breast cancer cell line (MCF-7) and various bacterial and fungal species.Results Proteins make up approximately half of the dry weight of L. abdullahbayrami crude venom. Microfluidic capillary electrophoresis indicated the presence of 6 to 7 kDa peptides and proved to be a highly practical peptidomics tool with better resolution when compared to conventional polyacrylamide gel electrophoresis. Mass spectroscopy analysis helped us to identify 45 unique peptide masses between 1 to 7 kDa with a bimodal mass distribution peaking between molecular weights of 1 to 2 kDa (29%) and 3 to 4 kDa (31%). L. abdullahbayrami crude venom had a proliferative effect on MCF-7 cells, which may be explained by the high concentration of polyamines as well as potassium and calcium ions in the arachnid venoms. Antimicrobial effect was stronger on gram-negative bacteria.Conclusions This work represents the first peptidomic characterization of L. abdullahbayrami venom. Considering the molecular weight-function relationship of previously identified venom peptides, future bioactivity studies may lead to the discovery of novel potassium and chloride ion channel inhibitors as well as new antimicrobial peptides fromL. abdullahbayrami venom.(AU)

Characterization of Leiurus abdullahbayrami (Scorpiones: Buthidae) venom: peptide profile, cytotoxicity and antimicrobial activity

Background Scorpion venoms are rich bioactive peptide libraries that offer promising molecules that may lead to the discovery and development of new drugs.Leiurus abdullahbayrami produces one of the most potent venoms among Turkish scorpions that provokes severe symptoms in envenomated victims.Methods In the present study, the peptide profile of the venom was investigated by electrophoretic methods, size-exclusion and reversed-phase chromatography and mass spectroscopy. Cytotoxic and antimicrobial effects were evaluated on a breast cancer cell line (MCF-7) and various bacterial and fungal species.Results Proteins make up approximately half of the dry weight of L. abdullahbayrami crude venom. Microfluidic capillary electrophoresis indicated the presence of 6 to 7 kDa peptides and proved to be a highly practical peptidomics tool with better resolution when compared to conventional polyacrylamide gel electrophoresis. Mass spectroscopy analysis helped us to identify 45 unique peptide masses between 1 to 7 kDa with a bimodal mass distribution peaking between molecular weights of 1 to 2 kDa (29%) and 3 to 4 kDa (31%). L. abdullahbayrami crude venom had a proliferative effect on MCF-7 cells, which may be explained by the high concentration of polyamines as well as potassium and calcium ions in the arachnid venoms. Antimicrobial effect was stronger on gram-negative bacteria.Conclusions This work represents the first peptidomic characterization of L. abdullahbayrami venom. Considering the molecular weight-function relationship of previously identified venom peptides, future bioactivity studies may lead to the discovery of novel potassium and chloride ion channel inhibitors as well as new antimicrobial peptides fromL. abdullahbayrami venom.(AU)

Characterization of Leiurus abdullahbayrami (Scorpiones: Buthidae) venom: peptide profile, cytotoxicity and antimicrobial activity

Background Scorpion venoms are rich bioactive peptide libraries that offer promising molecules that may lead to the discovery and development of new drugs.Leiurus abdullahbayrami produces one of the most potent venoms among Turkish scorpions that provokes severe symptoms in envenomated victims.Methods In the present study, the peptide profile of the venom was investigated by electrophoretic methods, size-exclusion and reversed-phase chromatography and mass spectroscopy. Cytotoxic and antimicrobial effects were evaluated on a breast cancer cell line (MCF-7) and various bacterial and fungal species.Results Proteins make up approximately half of the dry weight of L. abdullahbayrami crude venom. Microfluidic capillary electrophoresis indicated the presence of 6 to 7 kDa peptides and proved to be a highly practical peptidomics tool with better resolution when compared to conventional polyacrylamide gel electrophoresis. Mass spectroscopy analysis helped us to identify 45 unique peptide masses between 1 to 7 kDa with a bimodal mass distribution peaking between molecular weights of 1 to 2 kDa (29%) and 3 to 4 kDa (31%). L. abdullahbayrami crude venom had a proliferative effect on MCF-7 cells, which may be explained by the high concentration of polyamines as well as potassium and calcium ions in the arachnid venoms. Antimicrobial effect was stronger on gram-negative bacteria.Conclusions This work represents the first peptidomic characterization of L. abdullahbayrami venom. Considering the molecular weight-function relationship of previously identified venom peptides, future bioactivity studies may lead to the discovery of novel potassium and chloride ion channel inhibitors as well as new antimicrobial peptides fromL. abdullahbayrami venom.

Tecnologia de Phage Display no mapeamento de Mimetopos de Haemonchus contortus e uso de RNA de interferência no silenciamento do Gene codificante da protéina Gliceraldeído-3-fosfato desidrogenase (GAPDH) do parasita

Haemonchus contortus é um nematoda hematófago de pequenos ruminantes, extremamente importante em escala mundial, sendo responsável por muitos danos à saúde dos animais, além de perdas produtivas e econômicas aos criadores. O panorama atual é de resistência do parasita à maioria das classes de anti-helmínticos disponíveis no mercado, o que demonstra necessidade urgente de métodos alternativos de controle. Portanto, os objetivos deste estudo foram selecionar mimetopos de H. contortus e avaliar o uso de RNA de interferência no silenciamento do gene codificante da proteína gliceraldeído-3-fosfato desidrogenase (GAPDH) do parasita. A presente tese está dividida em 3 capítulos apresentados na forma de introdução geral e 2 manuscritos. O manuscrito 1 consistiu no uso de anticorpo policlonal de ovinos para mapear mimetopos de H. contortus usando a biblioteca de phage display. O clone 6 mimetopo de GAPDH e clone 17 - mimetopo da família do músculo desorganizado (Dim 1) foram selecionados para ensaio de imunização de ovinos. Peptídeos mimetopos (sintéticos) foram avaliados como moléculas antigênicas por meio de ensaio imunoenzimático (ELISA). No ensaio de imunização constatou-se aumento dos títulos de IgG anti-fago M13, mas não ocorreu aumento de IgG anti-peptídeos mimetopos. Os peptídeos mimetopos sintéticos foram reconhecidos por IgG de ovinos naturalmente infectados por H. contortus. Este é o primeiro relato de uso bem sucedido da biblioteca de phage display para a identificação de mimetopos de H. contortus. O manuscrito 2 teve o objetivo de avaliar o uso de RNA de interferência (RNAi) para silenciamento do gene codificante da proteína GAPDH de H. contortus. Larvas infectantes foram incubadas com RNAi por 3; 6; 24 e 48h. Os resultados revelaram ausência de transcrição gênica em todas os períodos avaliados. Este estudo relata pela primeira vez o silenciamento de GAPDH em H. contortus, confirmando o gene como passível ao silenciamento por RNAi. Os dados expostos nesta tese apontam resultados promissores para uso em novas pesquisas voltadas ao desenvolvimento de terapias para o controle de H. contortus.

Haemonchus contortus is a hematophagous nematoda of small ruminants, extremely important on a worldwide scale, being responsible for severe impact to the animals health, besides productive and economic losses to the breeders. The current panorama is parasite resistance to the most anthelminthics classes commercially available for its control. This demonstrates the urgent need of alternative control methods. Therefore, the goals of this study were to select mimotopes from H. contortus and to evaluate the use of RNA interference (RNAi) to silence the gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of the parasite. This thesis is divided into 3 chapters presented as 1 general introduction and 2 manuscripts. The goal of the first manuscript was to evaluate polyclonal antibody to map H. contortus mimetopos using the phage display library. Clone 6 - GAPDH mimetope and clone 17 - mimetope from the disorganized muscle family (Dim 1) were selected for sheep an immunization assay. Peptide mimotopes (synthetic) were evaluated as antigenic molecules by enzyme-linked immunosorbent assay (ELISA). In the immunization assay, IgG titers anti-phage M13 showed increase, but IgG anti-peptide mimotopes did not increased. Peptide mimotopes (synthetic) were recognized by IgG from sheep naturally infected by H. contortus. This is the first report of successful use of the phage display library for identification of H. contortus mimotopes. The aim of the manuscript 2 was to evaluate the RNAi to silence the GAPDH gene of H. contortus. Infective larvae were incubated with RNAi for 3; 6; 24 and 48h. The results showed absence of gene transcription in all evaluated periods. This study reports for the first time the silencing of GAPDH gene in H. contortus, confirming the gene as susceptible to silencing by RNAi. The data presented in this thesis point out promising results for use in new researches to development H. contortus control therapies.

PROSPECÇÃO E CARACTERIZAÇÃO DE ANTÍGENOS MIMÉTICOS DE AFLATOXINA OBTIDOS POR PHAGE DISPLAY COM VALIDAÇÃO DO POTENCIAL IMUNOGÊNICO

A biotecnologia de phage display pode ser utilizada em uma grande variedade de aplicações, como a seleção de fagos que mimetizam a aflatoxina, um metabólito fúngico que compromete a saúde pública e a produção animal, causando doença hepática severa, neoplasias e perdas econômicas. Na técnica de phage display, bibliotecas de fagos podem ser rastreadas para seleção de peptídeos ligantes de anticorpos: a vacinação com estes fagos induz a produção de anticorpos anti-peptídeo que também podem reconhecer o antígeno original. Desta forma, os objetivos do presente trabalho foram selecionar clones altamente reativos miméticos de aflatoxina B1, buscando o desenvolvimento de imunógenos capazes de induzir a produção de anticorpos anti-aflatoxina. As adequações da técnica de phage display previamente estabelecida permitiram a prospecção dos clones miméticos de aflatoxina 3P8, 3P13, 3P16, 3P20, 3P23 e 3P30 que foram ligantes de anticorpo monoclonal anti-AFB1. Os resultados dos imunoensaios executados indicaram a alta especificidade de peptídeos expressos por estes fagos em mimetizar in vitro as características imunológicas correspondentes à AFB1 em anticorpo específico anti-AFB1. Desta forma, foi observado que a reatividade do fago com o anticorpo foi dependente das concentrações de fago estabelecidas, assim como não houve reatividade frente a moléculas irrelevantes. Fagos expressando mimotopos de baixa reatividade (como o fago 3P25 e o fago silvestre) não foram capazes de estabelecer esta condição, indicando que os mimotopos selecionados corresponderam complementarmente ao sítio de ligação ao antígeno em anticorpo anti-AFB1. A partir da sequência aminoacídica obtida dos mimotopos selecionados, QTDLDYLHPLINSWN, o peptídeo mimético de aflatoxina foi sintetizado e conjugado à molécula carreadora para validação do potencial imunogênico. O resultado das estratégias de imunização com mimotopos e peptídeo sintético indicou que os imunógenos foram eficientes na indução de anticorpo anti-peptídeo que também foi capaz de reconhecer o antígeno original. Portanto, o mimetismo molecular do peptídeo selecionado por phage display foi confirmado, permitindo o emprego do fago como imunógeno para o desenvolvimento de uma vacina de subunidades, através do encapsulamento em nanopartículas de quitosana. Este biopolímero permitiu a administração intranasal do imunógeno e o reconhecimento do antígeno pelo sistema imune de mucosa, configurando uma plataforma eficiente na indução de anticorpos específicos anti-aflatoxina. A proposta desse modelo vacinal para o controle de micotoxinas constitui uma nova linha de pesquisa na medicina veterinária e uma alternativa para o desenvolvimento da produção animal. Posteriormente, esta abordagem racional poderá ser aplicada a outras micotoxinas que afetam as populações humana e animal.

Phage display biotechnology is a widely applied technology, and has been proven as useful tool in selecting peptides epitopes that mimic antigens such as aflatoxin, a fungal metabolite that compromises animal production and human health, leading to severe liver intoxication, cancer and economic losses. By phage display technique, phage libraries could be screened for selection of mimotopes, small peptides that structurally mimic a given antibody-binding site of an epitope. Active immunization based on these mimotopes induced antibodies that recognized the mimicked epitope. In the study reported herein highly reactive clones that mimicry the immunological specificity of aflatoxin B1 were selected, seeking the development of immunogens capable to induce the production of anti-aflatoxin antibodies. Through adaptations of the previously established phage display technique the 3P8, 3P13, 3P16, 3P20, 3P23 and 3P30 clones were selected for being antigen-binding site specific ligands in anti-AFB1 monoclonal antibody. The immunoassays performed indicated high specificity of phage-displayed peptides in mimicking in vitro the immunological characteristics corresponding to AFB1 in specific antibody. The specificity was defined by the ability of a clone to be identified only by its cognate target anti-AFB1 monoclonal and polyclonal antibodies among different irrelevant ligands. Any clone showed specificity for BSA or irrelevant murine IgG. However, some clones 3P4, 3P5 and 3P16 exhibited recognition against the irrelevant monoclonal and rabbit polyclonal antibodies, which indicates that these clones were less specific than other selected clones. The affinity-selected phages exhibited a concentration-dependence profile: the reduction on phage concentration from 1011 to 108 pfu mL-1 causes a decreased reactivity towards the anti-AFB1 monoclonal antibody, while any interference was observed with the irrelevant phage 3P25 or WTP. These results also indicate that the phage-displayed peptides represented the binding site of the aforementioned antibody. Therefore, the peptide sequence of the selected mimotopes, QTDLDYLHPLINSWN, was synthesized and conjugated to protein carriers to validate the immunogenic potential. Immunization of mice with phage-displayed mimotopes and synthetic peptide suggested that the immunogens were efficiently specific in the induction of anti-AFB1 antibodies. Thus, the molecular mimicry of selected peptide was confirmed as an immunogen for development of epitope-based vaccine by encapsulation of bacteriophage into chitosan nanoparticles. This biopolymer allowed the intranasal administration of the immunogen and recognition of the antigen by the mucosal immune system, providing an efficient platform for the induction of specific anti-aflatoxin antibodies. The proposal of a vaccine model for the control of mycotoxins establishes a new research line in veterinary medicine and an alternative for the development of animal production. Hereafter, this rational approach may be applied to other mycotoxins that affect human and animal populations.

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.

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)

CARACTERIZAÇÃO DE ANTÍGENOS E IMUNOGENICIDADE DE Porcine circovirus 2 E Mycoplasma hyopneumoniae

Mycoplasma hyopneuminae e Porcine circovirus 2 (PCV2) são dois dos principais patógenos causadores de doenças em suínos, representando grandes prejuízos a suinocultura mundial. A vacinação é a principal medida de controle de ambas infecções. Considerando as vacinas de nova geração e a importância dos dois patógenos a suinocultura comercial, este trabalho apresenta dois estudos relacionados a importantes proteínas de Mycoplasma hyopneumoniae e do PCV2. No primeiro capítulo, escrito em formato de artigo, foi utilizada a técnica de phage display para seleção e identificação de regiões de epítopos do capsídeo do PCV2. Especificamente, o peptídeo denominando PC12, correspondente a região 164CKPVLDSTIDY-173 da cap-PCV2 foi capaz de estimular a produção de IgG total e IgG1. O peptídeo PC12 foi também capaz de discriminar soros de animais infectados com o PCV2 de soros de animais negativos para esse vírus. No segundo capitulo foram desenhadas três quimeras proteicas (Q1, Q2 e Q3) compostas de domínios potencialmente imunogênicos de proteínas de Mycoplasma hyopneumoniae. As proteínas foram expressas em sistema bacteriano e purificadas através da cauda de histidina. A avaliação da imunogenicidade foi realizada em modelo murino. Os animais inoculados com as quimeras apresentaram IgG e IgG1 contra as proteínas nativas de Mycoplasma hyopneumoniae cepa J. Variados padrões de produção de citocinas foram observados nos esplenócitos dos animais inoculados com as quimeras. Os resultados do presente estudo indicam que as quimeras podem ser utilizadas em futuros ensaios vacinais com suínos por apresentarem promissora capacidade imunogênica. Já os peptídeos de regiões antigênicas do PCV2 podem auxiliar no desenvolvimento de novas estratégias de vacinação e de novas moléculas de diagnósticos.

Mycoplasma hyopneuminae and Porcine circovirus 2 (PCV2) are the major pathogens that causes swine diseases and represents great losses to the pig farming in the world. The main control measure for both infections is vaccination. Considering the new generation vaccines and the importance of these two pathogens to the commercial pig farming, this work showed two studies related to important proteins of Mycoplasma hyopneumoniae and PCV2. In the first chapter the phage display technique was used to selected and identified regions of PCV2 capsid epitope. Specifically, PC12 peptide which correspond to region 164-CKPVLDSTIDY173 from PCV2 capsid that was capable of stimulate IgG total and IgG1 production. Also, PC12 peptide was capable to discriminate sera of animals infected with PCV2 from sera of animals negative to PCV2. In the second chapter were drawn three protein chimeras (Q1, Q2 and Q3) composed by potentially immunogenic domains of Mycoplasma hyopneumoniae proteins. The chimera proteins were expressed in bacterial system and purified using the histidine tail. The immunogenicity evaluation was performed in murine model. The animals inoculated with the chimeras presented IgG and IgG1 against Mycoplasma hyopneumoniae strain J native proteins. Several patterns of cytokine production were observed in the splenocytes of animals inoculated with chimeras. The results of the present study indicate that chimeras can be used in future vaccine trials with swine because they presented promising immunogenicity. And the PC2 peptides from antigenic regions can assist in the development of new vaccination strategies and in new diagnostic molecules.