RESUMEN
BACKGROUND: Grass pollen allergens are the most important and widespread elicitors of pollen allergy. One of the major plant allergens which millions of people worldwide are sensitized to is Phl p 2, a small protein from timothy grass pollen. Phl p 2 is representative of the large family of cross-reacting plant allergens classified as group 2/3. Recombinant Phl p 2 has been demonstrated by immunological cross-reactivity studies to be immunologically equivalent to the natural protein. RESULTS: We have solved the solution structure of recombinant Phl p 2 by means of nuclear magnetic resonance techniques. The three-dimensional structure of Phl p 2 consists of an all-beta fold with nine antiparallel beta strands that form a beta sandwich. The topology is that of an immunoglobulin-like fold with the addition of a C-terminal strand, as found in the C2 domain superfamily. Lack of functional and sequence similarity with these two families, however, suggests an independent evolution of Phl p 2 and other homologous plant allergens. CONCLUSIONS: Because of the high homology with other plant allergens of groups 1 and 2/3, the structure of Phl p 2 can be used to rationalize some of the immunological properties of the whole family. On the basis of the structure, we suggest possible sites of interaction with IgE antibodies. Knowledge of the Phl p 2 structure may assist the rational structure-based design of synthetic vaccines against grass pollen allergy.
Asunto(s)
Alérgenos/química , Inmunoglobulina E/química , Proteínas de Plantas/química , Polen/química , Secuencia de Aminoácidos , Dicroismo Circular , Mapeo Epitopo , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Poaceae/inmunología , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Homología de Secuencia de AminoácidoRESUMEN
The interaction of immunoglobulin E and otherwise harmless antigens (allergens) leads in sensitized individuals through effector cell activation to the immediate induction of a cascade of inflammatory reactions, the hallmark of type I allergy. Recently, the molecular and structural characterization of allergens, specific IgE antibodies and their epitopes has made rapid progress. Here we discuss active and passive strategies for therapy of type I allergy, which are based on interfering with the IgE-allergen interaction.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad/inmunología , Hipersensibilidad/terapia , Inmunoglobulina E/inmunología , Alérgenos/química , Animales , Reacciones Antígeno-Anticuerpo , Epítopos de Linfocito B/inmunología , Humanos , VacunaciónRESUMEN
Type I allergy represents a hypersensitivity occurring in almost 20% of the population that is based on the recognition of innocuous airborn antigens (pollen, mite, mould and pet allergens) by specific immunoglobulin E. Allergic symptoms (e.g. allergic rhinitis, conjunctivitis, asthma) are caused by the release of biological mediators from effector-cells after allergen-induced crosslink of receptor-bound IgE. Here we discuss strategies to obtain recombinant allergen-specific antibody fragments (Fabs) from mouse and human cell lines as well as directly from allergic patients lymphocytes via the combinatorial library technology. It is suggested to use recombinant allergen-specific Fabs for the standardization of allergen extracts currently used for diagnosis and treatment, to determine allergen contents in allergen sources and the environment to allow preventive measures and to use allergen-specific Fabs as therapeutic tools to interfere with the allergen-IgE interaction. The latter appears possible because IgE represents the least abundant class of immunoglobulins and there is increasing evidence for a limited diversity among allergens and their B-cell epitopes. Moreover, allergic effector reactions are mostly confined to accessible target organs so that a local application of competing Fabs prior to allergen exposure might represent a feasible therapeutic approach.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Animales , Humanos , Hipersensibilidad/diagnóstico , Hipersensibilidad/prevención & control , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/uso terapéutico , Ratones , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunologíaRESUMEN
BACKGROUND: Bet v 1 and homologous proteins represent major allergens for almost 95% of patients allergic to tree pollen and approximately 70% of those allergic to fruits and vegetables. As yet, no continuous (sequential) IgE epitopes have been determined for Bet v 1, and evidence has accumulated that Bet v 1 IgE epitopes belong to the conformational (discontinuous) type. OBJECTIVE: A panel of 85 mouse monoclonal anti-Bet v 1 antibodies was raised as a tool with which to study the interaction of human IgE antibodies with Bet v 1. METHODS: The epitopes of selected monoclonal antibodies (mAbs) were characterized by mapping with synthetic overlapping peptides and by cross-competition experiments. Cross-reactivity of Bet v 1-specific mAbs with tree and plant food allergens was investigated by Western blotting. The influence of Bet v 1-specific mAbs on the IgE-Bet v 1 interaction was studied by competition assays with immobilized purified recombinant Bet v 1 and by basophil histamine release experiments. RESULTS: Antibodies that increased the IgE binding to Bet v 1 up to fivefold could be defined, whereas others inhibited IgE binding to Bet v 1 up to 99% and competed with the Bet v 1-induced histamine release from patients' basophils. CONCLUSION: The activity of the enhancing antibodies is interpreted as a stabilization of Bet v 1 states/IgE epitopes, which are either more accessible for certain IgE antibodies or are recognized with higher affinity. Those mAbs that competed with the Bet v 1-IgE interaction, if humanized or produced as recombinant antibody fragments, might be considered as potential tools for local allergy therapy.
Asunto(s)
Alérgenos , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Reacciones Antígeno-Anticuerpo , Epítopos/análisis , Inmunoglobulina E/inmunología , Proteínas de Plantas/inmunología , Animales , Anticuerpos Bloqueadores/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Antígenos de Plantas , Basófilos/inmunología , Basófilos/metabolismo , Western Blotting , Cromatografía de Afinidad , Reacciones Cruzadas/inmunología , Ensayo de Inmunoadsorción Enzimática , Liberación de Histamina , Humanos , Ratones , Ratones Endogámicos BALB C , Mapeo Peptídico , Proteínas de Plantas/genética , Pruebas de Precipitina , Proteínas Recombinantes/inmunologíaRESUMEN
Recombinant allergens have made it possible to dissect the mechanisms of allergen-antibody interactions at a molecular level. It becomes clear that monoclonal human IgG antibodies as well as animal derived antibodies can block the interaction of specific IgE antibodies as well as the allergen induced allergic effector reaction. Using PCR technology and combinatorial plasmid vectors, recombinant antibody fragments can be produced and it has even become possible to isolate allergen-specific IgE Fabs out of combinatorial IgE libraries constructed from allergic patients lymphocytes. Recombinant Fabs will represent useful tools to study the IgE-allergen interaction as well as for the standardization of allergen extracts and quantitative allergen measurements. Moreover, allergen-specific recombinant Fabs which block the allergen-IgE interaction have to be considered as tools for local therapy in effector organs of allergic patients.
Asunto(s)
Alérgenos/inmunología , Fragmentos Fab de Inmunoglobulinas/genética , Animales , Anticuerpos Bloqueadores/genética , Anticuerpos Bloqueadores/uso terapéutico , Clonación Molecular , Humanos , Hipersensibilidad Inmediata/terapia , Inmunización Pasiva , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Estándares de ReferenciaRESUMEN
The interaction of a mouse monoclonal antibody (4A6) and birch profilin, a structurally well conserved actin- and phosphoinositide-binding protein and cross-reactive allergen, was characterized. In contrast to serum IgE from allergic patients, which shows cross-reactivity with most plants, monoclonal antibody 4A6 selectively reacted with tree pollen profilins. Using synthetic overlapping peptides, a continuous hexapeptide epitope was identified. The exchange of a single amino acid (Gln-47 --> Glu) within the epitope was found to abolish the binding of monoclonal antibody 4A6 to other plant profilins. The NMR analyses of the birch and the nonreactive timothy grass profilin peptides showed that the loss of binding was not due to major structural differences. Both peptides adopted extended conformations similar to that observed for the epitope in the x-ray crystal structure of the native birch profilin. Binding studies with peptides and birch profilin mutants generated by in vitro mutagenesis demonstrated that the change of Gln-47 to acidic amino acids (e.g. Glu or Asp) led to electrostatic repulsion of monoclonal antibody 4A6. In conclusion the molecular and structural analyses of the interaction of a monoclonal antibody with a continuous peptide epitope, recognized in a conformation similar to that displayed on the native protein, are presented.
Asunto(s)
Alérgenos/inmunología , Anticuerpos Monoclonales/inmunología , Proteínas Contráctiles , Epítopos/química , Proteínas de Microfilamentos/inmunología , Secuencia de Aminoácidos , Animales , Reacciones Cruzadas , Epítopos/inmunología , Femenino , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Profilinas , Proteínas Recombinantes/inmunología , ÁrbolesRESUMEN
BACKGROUND: Type I allergy represents a severe health problem in industrialized countries where up to 20% of the population suffers from allergic rhinitis, conjunctivitis and allergic asthma bronchiale and in severe cases from anaphylaxis, leading to death. OBJECTIVE: The aim of this study was to evaluate recombinant Bet v 1, the major birch pollen allergen for in vivo and in vitro diagnosis of birch pollen allergy. METHODS: A group of 51 birch pollen allergic patients and eight non-allergic control individuals were tested for birch pollen allergy by skin-prick and intradermal testing, comparing commercial birch pollen extracts with recombinant Bet v 1. Quantitative and qualitative serological testing was done with natural and recombinant allergens by radioallergosorbent test (RAST), enzyme-linked immunosorbent assay (ELISA) and immunoblotting. RESULTS: Recombinant Bet v 1 allowed accurate in vivo and in vitro diagnosis of tree pollen allergy in 49/51 patients tested. No false positive results were obtained in any in vitro assay system (ELISA, Westernblot) or by skin testing (skin-prick, intradermal test) with recombinant Bet v 1. CONCLUSION: Our results document that recombinant Bet v 1 produced in bacterial expression systems allows accurate in vitro and in vivo diagnosis of birch pollen allergy in > 95% of birch pollen allergic patients.
Asunto(s)
Alérgenos , Asma/diagnóstico , Hipersensibilidad Inmediata/diagnóstico , Pruebas Intradérmicas , Proteínas de Plantas , Polen/inmunología , Hipersensibilidad Respiratoria/diagnóstico , Adulto , Anciano , Especificidad de Anticuerpos/inmunología , Antígenos de Plantas , Asma/inmunología , Femenino , Humanos , Hipersensibilidad Inmediata/inmunología , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Masculino , Persona de Mediana Edad , Proteínas de Plantas/inmunología , Proteínas Recombinantes/inmunología , Hipersensibilidad Respiratoria/inmunología , ÁrbolesRESUMEN
Birch pollen allergy is a very frequent pathology in Europe and North America. More than 95% of the tree pollen allergic patients display IgE reactivity against Bet v 1, the major birch pollen allergen. Starting with PBL from a patient desensitized by immunotherapy, we have generated five B cell lines (BAB1 to BAB5) that secrete human IgG mAbs of high affinity for Bet v 1. Although competition studies indicated that these human IgG mAb recognized different epitopes, broad cross-reactivity was found with Bet v 1 homologous allergens present in tree pollens and plant-derived foods. When tested for interference with allergic patients' IgE, BAB1 inhibited (by 80-100%) the binding of IgE to nitrocellulose-blotted Bet v 1, while BAB2 enhanced it. The biologic significance of the ability of BAB1 to interfere with patients' IgE binding is indicated by the finding that BAB1 completely inhibited Bet v 1-induced histamine release from allergic patients' basophils. Allergen-specific human IgG mAbs such as BAB1, which presents high blocking activity in both immunochemical and cellular IgE competition experiments, might have therapeutical application.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Alérgenos/metabolismo , Anticuerpos Monoclonales/farmacología , Sitios de Unión de Anticuerpos/efectos de los fármacos , Inmunoglobulina E/metabolismo , Inmunoglobulina G/farmacología , Proteínas de Plantas/metabolismo , Polen/inmunología , Adyuvantes Inmunológicos/biosíntesis , Adyuvantes Inmunológicos/química , Alérgenos/efectos de los fármacos , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/química , Especificidad de Anticuerpos , Antígenos de Plantas , Basófilos/inmunología , Unión Competitiva , Reacciones Cruzadas , Mapeo Epitopo , Liberación de Histamina/efectos de los fármacos , Humanos , Inmunoglobulina E/efectos de los fármacos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/química , Proteínas de Plantas/efectos de los fármacosRESUMEN
BACKGROUND: Grass pollen allergens belong to the potent elicitors of type I allergy. Approximately 40% of allergic individuals display IgE reactivity with grass pollen allergens. In previous studies we have reported the complementary DNA cloning and expression in Escherichia coli of three of the most relevant timothy grass pollen allergens: Phl p 1, Phl p 2, and Phl p 5. OBJECTIVE: To achieve high level expression of immunologically active timothy grass pollen allergens in E. coli, the cDNAs were inserted into expression plasmids. METHODS: The three recombinant grass pollen allergens were expressed at high levels in E. coli as recombinant nonfusion proteins, purified by conventional protein chemical methods and tested for their IgE-binding capacity by immunoblot and ELISA, as well as in histamine release assays. RESULTS: Milligram amounts of pure recombinant allergens were obtained from cultured E. coli. IgE binding to purified recombinant Phl p 1, Phl p 2, and Phl p 5 could be demonstrated by immunoblot and ELISA. With ELISAs the percentage of grass pollen-specific IgE directed against the individual recombinant allergens could be estimated. In addition, the purified recombinant timothy grass pollen allergens induced dose-dependent and specific histamine release from patients' blood basophils. CONCLUSION: Purified recombinant timothy grass pollen allergens represent useful tools for diagnosis and therapy of grass pollen allergy.
Asunto(s)
Alérgenos/inmunología , Polen/química , Polen/inmunología , Alérgenos/química , Alérgenos/aislamiento & purificación , Relación Dosis-Respuesta Inmunológica , Epítopos/farmacología , Escherichia coli/genética , Escherichia coli/inmunología , Vectores Genéticos/inmunología , Liberación de Histamina , Humanos , Inmunoglobulina E/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/inmunología , Proteínas de Plantas/aislamiento & purificación , Poaceae/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Rinitis Alérgica Estacional/inmunología , Rinitis Alérgica Estacional/metabolismoRESUMEN
BACKGROUND: Type I allergy represents a severe health problem in industrialized countries where up to 20% of the population suffer from allergic rhinitis, conjunctivitis and allergic asthma bronchiale and in severe cases from anaphylaxis, leading to death. OBJECTIVE: The aim of this study was to evaluate recombinant Bet v I, the major birch pollen allergen for in vivo and in vitro diagnosis of birch pollen allergy. METHODS: A group of 51 birch pollen allergic patients and eight non-allergic control individuals were tested for birch pollen allergy by skin-prick and intradermal testing, comparing commercial birch pollen extracts with recombinant Bet v I. Quantitative and qualitative serological testing was done with natural and recombinant allergens by radioallergosorbent test (RAST), enzyme-linked immunosorbent assay (ELISA) and immunoblotting. RESULTS: Recombinant Bet v I allowed accurate in vivo and in vitro diagnosis of tree pollen allergy in 49/51 patients tested. No false positive results were obtained in any in vitro assay system (ELISA, Western blot) or by skin testing (skin-prick, intradermal test) with recombinant Bet v I. CONCLUSION: Our results document that recombinant Bet v I produced in bacterial expression systems allows accurate in vitro and in vivo diagnosis of birch pollen allergy in > 95% of birch pollen allergic patients.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad Inmediata/diagnóstico , Proteínas de Plantas/inmunología , Polen/inmunología , Proteínas Recombinantes/inmunología , Pruebas Cutáneas , Adulto , Anciano , Alérgenos/genética , Reacciones Antígeno-Anticuerpo , Antígenos de Plantas , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Hipersensibilidad Inmediata/inmunología , Immunoblotting , Inmunoglobulina E/análisis , Inmunoglobulina G/análisis , Masculino , Persona de Mediana Edad , Proteínas de Plantas/genética , Polen/genética , Prueba de Radioalergoadsorción , Árboles/inmunologíaRESUMEN
Up to 20% of the population in industrialized countries suffer from type I allergic symptoms (rhinitis, conjunctivitis, and bronchial asthma). The cDNA coding for birch pollen profilin, a highly conserved cross-reactive allergen and actin-binding protein was expressed in Escherichia coli. Upon induction with IPTG up to 30 mg recombinant profilin per liter culture could be obtained. A single step purification protocol based on the high affinity of profilin to poly-(L-proline) Sepharose was used to obtain large amounts of soluble and pure recombinant birch profilin. Recombinant birch pollen profilin specifically bound IgE, elicited dose dependent histamine release from patients basophils and could be used for skin prick testing without toxic effects. The results indicate that by using purified recombinant profilin, specific diagnosis of type I allergy might be improved.
Asunto(s)
Alérgenos/genética , Proteínas Contráctiles , Escherichia coli/genética , Expresión Génica , Proteínas de Microfilamentos/genética , Polen/química , Árboles , Adsorción , Alérgenos/inmunología , Alérgenos/aislamiento & purificación , Secuencia de Bases , Basófilos/inmunología , Liberación de Histamina , Humanos , Hipersensibilidad/inmunología , Immunoblotting , Inmunoglobulina E/sangre , Proteínas de Microfilamentos/inmunología , Proteínas de Microfilamentos/aislamiento & purificación , Datos de Secuencia Molecular , Plásmidos , Profilinas , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Pruebas CutáneasRESUMEN
BACKGROUND: Interleukin (IL)-4 and IL-13 have been shown to be potent switch factors for IgE synthesis in human B cells. OBJECTIVE: In this study we investigated the effects of recombinant human IL-4 and IL-13 on total and allergen specific IgE synthesis by peripheral blood mononuclear cells (PBMC) from pollen allergic patients and healthy control individuals. METHODS: Peripheral blood mononuclear cells (PBMC) from allergic patients were investigated for their capacity to produce allergen specific IgE in vitro. Total protein extracts from birch pollen and timothy grass pollen as well as purified recombinant birch pollen allergens, Bet v I, birch profilin (Bet v II) and recombinant timothy grass pollen allergens, Phl p I, Phl p II, and Phl p V were used to measure specific IgE-antibody synthesis in cell culture supernatants by IgE-immunoblot and ELISA. RESULTS: PBMC obtained from allergic patients spontaneously secreted allergen specific IgE in the culture supernatants. Addition of Interleukin 4, Interleukin 13 and anti-CD40 antibody to the cultures alone or in combinations significantly induced total IgE production whereas allergen specific IgE production was not affected. CONCLUSION: Our results indicate that the peripheral blood of allergic individuals contains long lived allergen specific B cells which have already switched to IgE production and which are not sensitive to IL-4 and IL-13 treatment. These results may have implications on attempts to use cytokines or cytokine antagonists in therapy of Type I allergy.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad/sangre , Inmunoglobulina E/biosíntesis , Interleucina-12/farmacología , Interleucina-4/farmacología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Polen/inmunología , Alérgenos/farmacología , Animales , Especificidad de Anticuerpos , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Células CHO/metabolismo , Células Cultivadas , Cricetinae , Humanos , Inmunoglobulina E/inmunología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
We used the vapor phase of acrolein as an anhydrous fixative for timothy grass pollen in an immunogold double-labeling localization study of two different major allergens, Phl p I and Phl p V. More than 48 hr of fixation were needed for the subcellular pollen structures to be satisfactorily stabilized. The immunoreactivity of acrolein-fixed pollen allergens was not destroyed even after prolonged acrolein fixation. By immunoblotting, the two allergens differ in their immunological and structural characteristics. Electron microscopic localization traced the allergens at least partially to different subcellular pollen compartments.
Asunto(s)
Acroleína , Alérgenos/ultraestructura , Inmunohistoquímica , Microscopía Inmunoelectrónica/métodos , Polen/ultraestructura , Fijación del Tejido , Animales , Anticuerpos Monoclonales , Fijadores , Gases , Immunoblotting , Ratones , Poaceae , ConejosRESUMEN
Grass pollen allergens belong to the most important and widespread elicitors of pollen allergy. Using serum IgE from a grass pollen allergic patient, a complete cDNA encoding a group II allergen was isolated from a timothy grass (Phleum pratense) pollen expression library. The deduced amino acid sequence of the Phl p II allergen shows an average sequence identity of 61% with the protein sequences determined for group II/III allergens from rye grass (Lolium perenne) and a sequence identity of 43% with the C-terminal portion of group I grass pollen allergens from different species. A hydrophobic leader peptide similar to leader peptides found in other major grass pollen allergens heads the deduced amino acid sequence, indicating that group II/III grass pollen allergens belong to a family of secreted proteins. Serum IgE specific for Phl p II, detected the protein exclusively in pollen and not in other plant tissues. The recombinant Phl p II was expressed in Escherichia coli and showed similar IgE-binding capacity as the natural allergen.