RESUMO
Monoclonal antibodies have a wide range of applications in basic and applied research as well as in the medical and pharmaceutical industries. Phage display antibody libraries offer an alternative to hybridoma technology for the generation of monoclonal antibodies and can be applied to high-throughput screening and facilitate the generation of novel antibodies. Despite their utility in several fields of research there has been limited application of antibody libraries in the study of trematode parasites. Fasciola hepatica causes considerable loss to the agriculture sector and is also a human pathogen. The parasite's excretory/secretory material contains numerous molecules that facilitate its invasion and survival within the mammalian host, including cathepsin B and L proteases. F. hepatica cathepsin B2 is expressed during the initial weeks of infection and has suspected roles in immune evasion and as a digestive enzyme in the parasite's gut; it is considered a good target for vaccination or therapeutic inhibitors. In this study, we produced a single-chain variable fragment (scFv) phage display library from naïve mice. The library was used to identify several scFv that can bind to antigens from adult F. hepatica homogenate, and a scFv that can bind to F. hepatica cathepsin B2. The results highlight the potential applicability of such a library to facilitate the study of F. hepatica and other parasites. This is the first report of the application of a naïve phage display antibody library to the study of F. hepatica.
Assuntos
Anticorpos Anti-Helmínticos/metabolismo , Antígenos de Helmintos/imunologia , Fasciola hepatica/imunologia , Anticorpos de Cadeia Única/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Western Blotting , Catepsinas/imunologia , Técnicas de Visualização da Superfície Celular , Eletroforese em Gel de Ágar , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Camundongos , Modelos Moleculares , Reação em Cadeia da PolimeraseRESUMO
Phage display technology to produce recombinant monoclonal antibodies or antibody fragments permits the identification of sought after antibodies in short time frames at low cost along with direct and rapid selection for antibody characteristics. Monoclonal antibodies can facilitate the identification and characterisation of parasite molecules that function at the host-parasite interface to help understand at the molecular level the biology of the parasite and disease progression, which often leads to new drug targets, diagnostic antigens or vaccine candidates. The trematode Fasciola hepatica is an important veterinary and human parasite. In this work, we infected rats with F. hepatica and amplified the generated antibody repertoire to produce a single-chain variable fragment (scFv) phage display library. The library was used to identify a scFv that recognises cathepsin L1, a major component of the adult parasites excretory/secretory material and an important vaccine candidate. This is the first report of the construction of a phage display antibody library from a F. hepatica infected host, and also the first instance such a library has been used to identify an affinity-matured monoclonal antibody fragment that binds to a F. hepatica antigen. The scFv library and methods detailed should facilitate future research characterising F. hepatica antigens.
Assuntos
Anticorpos Anti-Helmínticos/imunologia , Catepsinas/imunologia , Fasciola hepatica/imunologia , Biblioteca de Peptídeos , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Antígenos de Helmintos/imunologia , Técnicas de Visualização da Superfície Celular , Clonagem Molecular , Eletroforese em Gel de Ágar , Biblioteca Gênica , Immunoblotting , Região Variável de Imunoglobulina/química , Reação em Cadeia da Polimerase , Ratos , Ratos Endogâmicos Lew , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologiaRESUMO
Fasciola hepatica infection continues to be a major problem in the agriculture sector, particularly in sheep and cattle. Cathepsin L and B proteases are major components of the excretory/secretory material of the parasite, and their roles in several important aspects of parasite invasion and survival has led to their use as targets in rational vaccine design. Previous studies in rats demonstrated that the use of stage-specific antigens, cathepsin B2 and cathepsin L5, as part of a multivalent vaccine, was able to confer significant protection against challenge. In the present study, recombinant versions of cathepsin L5 and cathepsin B2 produced in yeast were used in combination to vaccinate sheep. Intramuscular and intranasal forms of administration were applied, and sheep were subsequently challenged with 150 F. hepatica metacercariae. Intramuscular vaccination was able to induce a strong systemic antibody response against both antigens, but failed to confer significant protection. Conversely, no elevated antibody response was detected against the vaccine antigens following nasal vaccination; however, a reduction in parasite egg viability (>92%) and a statistically significant (pâ¯=â¯0.006), predominantly adjuvant-mediated reduction in worm burdens was observed.
Assuntos
Adjuvantes Imunológicos/administração & dosagem , Catepsinas/imunologia , Fasciola hepatica/imunologia , Fertilidade , Proteínas Recombinantes/imunologia , Vacinação/veterinária , Administração Intranasal/métodos , Animais , Anticorpos Anti-Helmínticos/sangue , Antígenos de Helmintos/imunologia , Catepsinas/administração & dosagem , Catepsinas/genética , Bovinos , Cisteína Endopeptidases/administração & dosagem , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , Fasciola hepatica/química , Fasciola hepatica/genética , Injeções Intramusculares , Contagem de Ovos de Parasitas , Carga Parasitária , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/genética , Ovinos/imunologia , Doenças dos Ovinos/imunologia , Doenças dos Ovinos/parasitologia , Doenças dos Ovinos/prevenção & controle , Vacinas/administração & dosagem , Vacinas/genética , Vacinas/imunologia , Leveduras/genéticaRESUMO
Lymphocyte responses in the blood, peritoneal fluid and both mesenteric and hepatic lymph nodes of cDNA-FhPGK/pCMV vaccinated and/or Fasciola hepatica infected rats of both sexes were investigated to provide an insight into the immune responses that develop in different body compartments. The immune response that developed in cDNA-FhPGK/pCMV vaccinated females contributed to partial protection against F. hepatica infection (54% reduction in fluke recovery), while more liver flukes were found in the livers and bile ducts of cDNA-FhPGK/pCMV vaccinated male rats than in unvaccinated animals (increase of 13%). Rat sex not only affected the ultimate effectiveness of vaccination but also lymphocyte responses following vaccination and/or infection. Different CD4+ and CD8+ T cell profiles were noted in peritoneal fluid and lymph nodes, but not in blood, during acute and chronic fasciolosis. Moreover, independent lymphocyte responses developed in distinct body compartments. Immune responses of rats were polarized towards Th2/Treg with lymphocytes isolated from male rats showing higher IL-4 and IL-10 production than females. Lymphocyte proliferative capacities in response to mitogen (PHA) or vaccine antigen (FhPGK) were impaired in both sexes with a considerably higher reduction observed for males and restored lymphocyte proliferative capacities reported for females vaccinated with cDNA-FhPGK/pCMV during chronic fasciolosis.
Assuntos
Proteínas de Helminto/metabolismo , Linfócitos/imunologia , Fosfoglicerato Quinase/metabolismo , Vacinação , Animais , DNA Complementar/metabolismo , Fasciola hepatica/imunologia , Fasciolíase/prevenção & controle , Feminino , Fígado/imunologia , Linfonodos/imunologia , Masculino , Ratos , Linfócitos T/imunologiaRESUMO
Immune responses of rats and sheep following vaccination with cDNA encoding phosphoglycerate kinase of Fasciola hepatica (cDNA-FhPGK/pCMV) and F. hepatica infection were investigated in the present study. cDNA-FhPGK/pCMV vaccinated female Sprague-Dawley rats were better protected by vaccination than their male counterparts - 48% reduction in fluke burden for females and no protection for males when compared with appropriate infection control groups. Moreover, male rats developed marked leukocytosis during the study with higher neutrophil, eosinophil and monocyte responses than females. Additionally, dynamics of eosinophil and monocyte responses varied between sexes. Increased titres of anti-FhPGK IgG1 and IgG2a correlated with the protective effect of vaccination that was observed among female rats. In the case of male sheep, no differences in worm burdens and in the course of the immune response were observed following vaccination. Titres of specific antibodies detected were low, and cellular responses were not significant. Apparently, sheep immune responses induced by cDNA-FhPGK/pCMV vaccination are not effective at controlling F. hepatica infection. Poor immunogenicity of DNA vaccines in large animals is still a major obstacle of this technology that has to be overcome.
Assuntos
Fasciola hepatica/enzimologia , Fasciolíase/prevenção & controle , Fosfoglicerato Quinase/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Animais , Anticorpos Anti-Helmínticos/sangue , Modelos Animais de Doenças , Eosinófilos/imunologia , Fasciola hepatica/genética , Fasciolíase/imunologia , Fasciolíase/patologia , Feminino , Imunoglobulina G/sangue , Leucocitose , Masculino , Monócitos/imunologia , Fosfoglicerato Quinase/genética , Ratos Sprague-Dawley , Ovinos , Resultado do Tratamento , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologiaRESUMO
Fasciola hepatica infections cause large economic losses and are a serious veterinary medicine problem in many regions of the world. Recent studies examining fascioliasis in the bison population from Bialowieza National Park have shown that the prevalence of infection with this parasite is up to 100%. Liver flukes isolated from bison from Bialowieza National Park in Poland were compared with a fluke strain originally obtained from the Central Veterinary Laboratory, Weybridge, UK, to determine variations in cercarial production and establish the ability of their metacercariae to activate in vitro. Some small differences in cercarial production between the two isolates are shown, while significant differences in the ability of their metacercariae to activate in vitro were observed.