RESUMEN
Numerous allergens have been cloned and produced by the use of recombinant DNA technology. In several cases recombinant variants with reduced IgE-reactivity have also been developed as candidates for allergen specific immunotherapy. Only very few of these proteins have as yet been tested in the clinic, and the major focus has been on birch and grass pollen, two of the most common causes of IgE-mediated allergic disease. This article serves to justify the rational for using recombinant products and reviews the progress that has been made to date with their clinical assessment.
Asunto(s)
Desensibilización Inmunológica/métodos , Hipersensibilidad/inmunología , Hipersensibilidad/terapia , Tolerancia Inmunológica , Alérgenos/administración & dosificación , Alérgenos/inmunología , Alérgenos/uso terapéutico , Animales , Antígenos de Plantas/administración & dosificación , Antígenos de Plantas/inmunología , Antígenos de Plantas/uso terapéutico , Gatos , Humanos , Inmunoglobulina E/inmunología , Polen/inmunología , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Rinitis Alérgica Estacional/inmunología , Rinitis Alérgica Estacional/terapiaRESUMEN
BACKGROUND: Phl p 5 is a major allergen of Timothy grass (Phleum pratense). A recombinant native Phl p 5 has already been used in clinical trials of allergen-specific immunotherapy as a component of a cocktail of allergens. Recombinant hypoallergenic allergens should further improve the treatment by reducing the risk of anaphylactic reactions at an increased therapeutic dosage. Native Phl p 5 is formed by α-helical regions separated by regions containing prolines. In order to generate hypoallergenic mutants, we studied the effect of proline mutations in single and multiple regions. METHODS: All mutants were analyzed by IgE inhibition assays and size exclusion chromatography with on-line mass determination. Selected mutants were additionally analyzed by field-flow fractionation, dynamic light scattering, circular dichroism spectroscopy, basophil activation and T-cell proliferation assays. RESULTS: Variants lacking prolines in a single region were obtained as soluble monomers. Six of eight molecules showed a slightly reduced IgE-binding capacity. Mutants carrying proline deletions in multiple regions formed monomers, dimers or insoluble aggregates. The mutant MPV.7 with five proline deletions and a substitution of proline 211 to leucine is monomeric, shows a strongly diminished IgE binding and maintains T-cell reactivity. The hydrodynamic radius and the content of the α-helical structure of MPV.7 are well comparable with the wild-type allergen. CONCLUSIONS: The hypoallergenic Phl p 5 variant MPV.7 combines multiple proline deletions with a substitution of proline 211 to leucine and meets basic demands for a pharmaceutical application. MPV.7 is a promising candidate for grass pollen immunotherapy with a cocktail of recombinant hypoallergens.
Asunto(s)
Alérgenos/genética , Alérgenos/inmunología , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Polen/genética , Polen/inmunología , Adulto , Anciano , Sustitución de Aminoácidos , Basófilos/inmunología , Desensibilización Inmunológica/métodos , Femenino , Humanos , Inmunoglobulina E/metabolismo , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/inmunología , Phleum/genética , Phleum/inmunología , Proteínas de Plantas/química , Prolina/genética , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Rinitis Alérgica Estacional/inmunología , Rinitis Alérgica Estacional/terapia , Eliminación de Secuencia , Solubilidad , Linfocitos T/inmunologíaRESUMEN
The mechanisms by which immune responses to nonpathogenic environmental antigens lead to either allergy or nonharmful immunity are unknown. Single allergen-specific T cells constitute a very small fraction of the whole CD4+ T cell repertoire and can be isolated from the peripheral blood of humans according to their cytokine profile. Freshly purified interferon-gamma-, interleukin (IL)-4-, and IL-10-producing allergen-specific CD4+ T cells display characteristics of T helper cell (Th)1-, Th2-, and T regulatory (Tr)1-like cells, respectively. Tr1 cells consistently represent the dominant subset specific for common environmental allergens in healthy individuals; in contrast, there is a high frequency of allergen-specific IL-4-secreting T cells in allergic individuals. Tr1 cells use multiple suppressive mechanisms, IL-10 and TGF-beta as secreted cytokines, and cytotoxic T lymphocyte antigen 4 and programmed death 1 as surface molecules. Healthy and allergic individuals exhibit all three allergen-specific subsets in different proportions, indicating that a change in the dominant subset may lead to allergy development or recovery. Accordingly, blocking the suppressor activity of Tr1 cells or increasing Th2 cell frequency enhances allergen-specific Th2 cell activation ex vivo. These results indicate that the balance between allergen-specific Tr1 cells and Th2 cells may be decisive in the development of allergy.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad/inmunología , Linfocitos T/inmunología , Células Th2/inmunología , Adulto , Antígenos CD , Antígenos de Diferenciación/fisiología , Antígeno CTLA-4 , Humanos , Hipersensibilidad/etiología , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Interleucina-4/biosíntesis , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
Recombinant production in bacteria of soluble and monomeric Phl p 1, a major allergen of Timothy grass pollen, has proved to be very problematic. In order to facilitate expression and purification of this allergen, a recombinant variant was designed with a single amino acid substitution. Several comparative analyses with natural counterparts using electrophoretic and HPLC separations, together with immunological assays, demonstrated high equivalence. This is the first description of an approach aiming at an improvement of a natural like recombinant allergen.
Asunto(s)
Alérgenos/metabolismo , Proteínas de Plantas/metabolismo , Alérgenos/química , Alérgenos/genética , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Electroforesis en Gel de Poliacrilamida , Inmunohistoquímica , Datos de Secuencia Molecular , Peso Molecular , Mapeo Peptídico , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Recombinant DNA technology has delivered the prospect of a new generation of preparations for allergen-specific immunotherapy. The first clinical studies with recombinant allergens have yielded encouraging results, suggesting that there is a good chance that such preparations will become available for use in the routine management of allergic disease.
Asunto(s)
Alérgenos/inmunología , Desensibilización Inmunológica/métodos , Proteínas Recombinantes/inmunología , Vacunas/inmunología , Ensayos Clínicos como Asunto , HumanosRESUMEN
Approximately 400 million allergic patients are sensitized against group 1 grass pollen allergens, a family of highly cross-reactive allergens present in all grass species. We report the eukaryotic expression of the group 1 allergen from Timothy grass, Phl p 1, in baculovirus-infected insect cells. Domain elucidation by limited proteolysis and mass spectrometry of the purified recombinant glycoprotein indicates that the C-terminal 40% of Phl p 1, a major IgE-reactive segment, represents a stable domain. This domain also exhibits a significant sequence identity of 43% with the family of immunoglobulin domain-like group 2/3 grass pollen allergens. Circular dichroism analysis demonstrates that insect cell-expressed rPhl p 1 is a folded species with significant secondary structure. This material is well behaved and is adequate for the growth of crystals that diffract to 2.9 A resolution. The importance of conformational epitopes for IgE recognition of Phl p 1 is demonstrated by the superior IgE recognition of insect-cell expressed Phl p 1 compared to Escherichia coli-expressed Phl p 1. Moreover, insect cell-expressed Phl p 1 induces potent histamine release and leads to strong up-regulation of CD203c in basophils from grass pollen allergic patients. Deglycosylated Phl p 1 frequently exhibits higher IgE binding capacity than the recombinant glycoprotein suggesting that rather the intact protein structure than carbohydrate moieties themselves are important for IgE recognition of Phl p 1. This study emphasizes the important contribution of conformational epitopes for the IgE recognition of respiratory allergens and provides a paradigmatic tool for the structural analysis of the IgE allergen interaction.
Asunto(s)
Alérgenos/inmunología , Epítopos/química , Inmunoglobulina E/inmunología , Proteínas de Plantas/inmunología , Alérgenos/genética , Animales , Línea Celular , Humanos , Espectrometría de Masas , Filogenia , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , SpodopteraRESUMEN
Type I allergy is an immunoglobulin E (IgE)-mediated hypersensitivity disease affecting more than 25% of the population. Currently, diagnosis of allergy is performed by provocation testing and IgE serology using allergen extracts. This process defines allergen-containing sources but cannot identify the disease-eliciting allergenic molecules. We have applied microarray technology to develop a miniaturized allergy test containing 94 purified allergen molecules that represent the most common allergen sources. The allergen microarray allows the determination and monitoring of allergic patients' IgE reactivity profiles to large numbers of disease-causing allergens by using single measurements and minute amounts of serum. This method may change established practice in allergy diagnosis, prevention, and therapy. In addition, microarrayed antigens may be applied to the diagnosis of autoimmune and infectious diseases.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad Inmediata/diagnóstico , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Alérgenos/genética , Alérgenos/aislamiento & purificación , Humanos , Hipersensibilidad Inmediata/inmunología , Hipersensibilidad Inmediata/terapia , Inmunoglobulina E/inmunología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
BACKGROUND: Grass pollen, in particular from Lolium multiflorum is a major allergen source in temperate climate zones of Southern Brazil. The IgE sensitization profile of Brazilian grass pollen allergic patients to individual allergen molecules has not been analyzed yet. OBJECTIVE: To analyze the IgE sensitization profile of a Brazilian grass pollen allergic population using individual allergen molecules. METHODS: We analyzed sera from 78 grass pollen allergic patients for the presence of IgE antibodies specific for 103 purified micro-arrayed natural and recombinant allergens by chip technology. IgE-ELISA inhibition experiments with Lolium multiflorum, Phleum pratense extracts and a recombinant fusion protein consisting of Phl p 1, Phl p 2, Phl p 5 and Phl p 6 were performed to investigate cross-reactivities. RESULTS: Within the Brazilian grass pollen allergic patients, the most frequently recognized allergens were Phl p 1 (95%), Phl p 5 (82%), Phl p 2 (76%) followed by Phl p 4 (64%), Phl p 6 (45%), Phl p 11 (18%) and Phl p 12 (18%). Most patients were sensitized only to grass pollen allergens but not to allergens from other sources. A high degree of IgE cross-reactivity between Phleum pratense, Lolium multiflorum and the recombinant timothy grass fusion protein was found. CONCLUSIONS: Component-resolved analysis of sera from Brazilian grass pollen allergic patients reveals an IgE recognition profile compatible with a typical Pooideae sensitization. The high degree of cross-reactivity between Phleum pratense and Lolium multiflorum allergens suggests that diagnosis and immunotherapy can be achieved with timothy grass pollen allergens in the studied population.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad/patología , Lolium/metabolismo , Polen/inmunología , Adulto , Brasil , Reacciones Cruzadas/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Hipersensibilidad/inmunología , Inmunoglobulina E/inmunología , Inmunoglobulina E/metabolismo , Masculino , Phleum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Proteínas de Plantas/metabolismo , Análisis por Matrices de Proteínas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Pruebas CutáneasRESUMEN
The recombinant major grass pollen allergen Phl p 6 has been expressed with a N-terminal 6 x His-tag sequence and subsequently purified using nickel-chelating Sepharose. After cleavage of the tag-sequence, a second pass over the affinity chromatography revealed that even untagged rPhl p 6 bound tightly. In order to determine if that property is typical for Phl p 6, the natural allergen was purified in the same way starting with a grass pollen extract. Indeed, nPhl p 6 could be highly enriched in one step using nickel-chelating Sepharose. In addition to this new powerful purification method, the results provide further information in that the recombinant and natural allergens share a lot of properties, since biochemical characteristics are reflected in the purification strategies. The preparations of natural and recombinant Phl p 6 were used for comparative electrophoretic, chromatographic and immunological analysis which demonstrated high similarity.
Asunto(s)
Alérgenos/aislamiento & purificación , Proteínas de Plantas/aislamiento & purificación , Alérgenos/química , Alérgenos/inmunología , Secuencia de Aminoácidos , Electroforesis en Gel de Poliacrilamida , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
A process for bacterial expression and purification of the recombinant major wasp allergen Antigen 5 (Ves v 5) was developed to produce protein for diagnostic and therapeutic applications for type 1 allergic diseases. Special attention was focused on medium selection, fermentation conditions, and efficient refolding procedures. A soy based medium was used for fermentation to avoid peptone from animal origin. Animal-derived peptone required the use of isopropyl-beta-D-thiogalactopyranoside (IPTG) for the induction of expression. In the case of soy peptone, a constitutive expression was observed, suggesting the presence of a component that mimics IPTG. Batch cultivation at reduced stirrer speed caused a reduced biomass due to oxygen limitation. However, subsequent purification and processing of inclusion bodies yielded significantly higher amount of product. Furthermore, the protein composition of the inclusion bodies differed. Inclusion bodies were denatured and subjected to diafiltration. Detailed monitoring of diafiltration enabled the determination of the transition point. Final purification was conducted using cation-exchange and size-exclusion chromatography. Purified recombinant Ves v 5 was analyzed by RP-HPLC, CD-spectroscopy, SDS-PAGE, and quantification ELISA. Up to 15 mg highly purified Ves v 5 per litre bioreactor volume were obtained, with endotoxin concentrations less than 20 EU mg(-1) protein and high comparability to the natural counterpart. Analytical results confirm the suitability of the recombinant protein for diagnostic and clinical applications. The results clearly demonstrate that not only biomass, but especially growth conditions play a key role in the production of recombinant Ves v 5. This has an influence on inclusion body formation, which in turn influences the renaturation rate and absolute product yield. This might also be true for other recombinant proteins that accumulate as inclusion bodies in Escherichia coli.
Asunto(s)
Alérgenos/biosíntesis , Reactores Biológicos , Escherichia coli/crecimiento & desarrollo , Proteínas de Insectos/biosíntesis , Proteínas Recombinantes/biosíntesis , Venenos de Avispas/biosíntesis , Alérgenos/química , Alérgenos/genética , Biomasa , Reactores Biológicos/microbiología , Escherichia coli/genética , Cuerpos de Inclusión/química , Cuerpos de Inclusión/metabolismo , Proteínas de Insectos/química , Proteínas de Insectos/genética , Oxígeno/metabolismo , Consumo de Oxígeno/fisiología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Venenos de Avispas/química , Venenos de Avispas/genéticaRESUMEN
BACKGROUND: Allergen-specific immunotherapy uses aqueous extracts of natural source materials as a basis for preparations to down regulate the allergic response. Recombinant DNA technology has enabled the cloning of many allergens, thus facilitating investigations aimed at improving efficacy and safety of immunotherapy. OBJECTIVE: To determine the effectiveness of a mixture of 5 recombinant grass pollen allergens in reducing symptoms and need for symptomatic medication in patients allergic to grass pollen. METHODS: A randomized, double-blind, placebo-controlled study of subcutaneous injection immunotherapy was performed in subjects with allergic rhinoconjunctivitis, with or without asthma. Primary endpoint was a symptom medication score compiled from separate symptom and medication scores. Secondary endpoints included a rhinitis quality of life questionnaire, conjunctival provocation, and specific antibody responses. RESULTS: The symptom medication score showed significant improvements in subjects receiving recombinant allergens as opposed to placebo, with reductions in both symptoms and medication usage. The rhinitis quality of life questionnaire revealed clinically relevant significant improvements in overall assessment and in 5 of 7 separate domains, and conjunctival provocation showed a clear trend in favor of active treatment. All treated subjects developed strong allergen-specific IgG(1) and IgG(4) antibody responses. Some patients were not sensitized to Ph l p 5 but nevertheless developed strong IgG antibody responses to that allergen. CONCLUSION: A recombinant allergen vaccine can be a effective and safe treatment to ameliorate symptoms of allergic rhinitis. The clinical benefit is associated with modification of the specific immune response with promotion of IgG(4) and reduction of IgE antibodies consistent with the induction of IL-10-producing regulatory T cells.
Asunto(s)
Conjuntivitis Alérgica/terapia , Desensibilización Inmunológica , Proteínas Recombinantes/uso terapéutico , Rinitis Alérgica Estacional/tratamiento farmacológico , Adulto , Alérgenos/inmunología , Femenino , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Masculino , Poaceae/inmunología , Polen/inmunologíaRESUMEN
Grass pollen allergy is one of the most important allergic diseases world-wide. Several meadow grasses, like timothy grass and rye grass, contribute to allergic sensitizations, but also allergens from extensively cultivated cereals, especially rye, make a profound contribution. The group 4 allergens are well known as important major allergens of grasses. We have cloned for the first time group 4 sequences from Phleum pratense, Lolium perenne, Secale cereale, Triticum aestivum, and Hordeum vulgare, and investigated the IgE-reactivity of recombinant Phl p 4 as a candidate for allergy diagnostic and therapeutic applications.
Asunto(s)
Alérgenos/metabolismo , Phleum/química , Proteínas de Plantas/metabolismo , Alérgenos/química , Alérgenos/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Expresión Génica , Datos de Secuencia Molecular , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismoRESUMEN
BACKGROUND: Recently, recombinant hypoallergenic derivatives of the major birch pollen allergen, Bet v 1, were used to treat birch-pollen-allergic patients in a double-blind, placebo-controlled, multi-centre immunotherapy study. The aim of this study was to evaluate the effects of vaccination with aluminium-hydroxide-adsorbed recombinant Bet v 1 derivatives versus placebo on T-cell, cytokine and antibody responses in a subgroup of patients. METHODS: Blood was drawn from patients of the Swedish centre (n = 27; rBet v 1 fragments: n = 10; rBet v 1 trimer: n = 8, and placebo-aluminium hydroxide: n = 9) before the start and after completion of the treatment. PBMC were stimulated with rBet v 1 and analysed for cytokine (IL-4, IL-5, IL-10, IL-12, IL-13 and IFN-gamma)-secreting cells by ELISpot. Bet v 1-specific antibody levels in serum (IgG(1-4), IgE and IgA) were measured by ELISA. Skin prick tests with defined Bet v 1 concentrations were performed before and 10-11 months after the beginning of the study. RESULTS: Bet v 1-specific IgG levels, consisting of IgG(1), IgG(2) and IgG(4), were significantly increased after treatment with recombinant allergen derivatives. Treatment with rBet v 1 trimer led to a significant (p < 0.05) reduction of Bet v 1-reactive IL-5- and IL-13-producing cells, reflecting a reduced Th2 response. In addition, a decreased number of Bet v 1-reactive IL-4 producing (p = 0.07) and an increase of IL-12-producing (p = 0.06) cells was noted in the trimer-treated patients. In contrast to placebo, active treatment resulted in significantly reduced immediate-type skin reactions to Bet v 1 even 10-11 months after treatment. CONCLUSION: Vaccination with recombinant hypoallergenic Bet v 1 derivatives induces a Bet v 1-specific IgG response and leads to reduced skin reactivity in allergic patients. A reduction of Bet v 1-specific Th2 responses was observed in trimer-treated patients, which may reflect the intrinsic property of this allergen derivative.
Asunto(s)
Anticuerpos/efectos de los fármacos , Citocinas/efectos de los fármacos , Desensibilización Inmunológica/métodos , Hipersensibilidad Inmediata/tratamiento farmacológico , Proteínas de Plantas/uso terapéutico , Proteínas Recombinantes/uso terapéutico , Adulto , Alérgenos/genética , Alérgenos/inmunología , Alérgenos/uso terapéutico , Anticuerpos/sangre , Anticuerpos/inmunología , Betula/inmunología , Células Cultivadas , Citocinas/sangre , Citocinas/inmunología , Método Doble Ciego , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Hipersensibilidad Inmediata/inmunología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Masculino , Persona de Mediana Edad , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Polen/inmunología , Proteínas Recombinantes/inmunología , Pruebas CutáneasRESUMEN
The cloning and production of an increasing number of allergens through the use of DNA technology has provided the opportunity to use these proteins instead of natural allergen extracts for the diagnosis and therapy of IgE-mediated allergic disease. For diagnostic purposes, it is essential that the molecules exhibit IgE-reactivity comparable with that of the natural wild-type molecules, whereas T cell reactivity and immunogenic activity may be more important for allergen-specific immunotherapy. In relation to the latter, the development of hypoallergenic recombinant allergen variants is an approach which shows great promise. Clinical application of the proteins requires that they must be produced under conditions of Good Manufacturing Practice and meet the specifications set down in the appropriate Regulatory Guidelines, principally the ICH-Guidelines. Special consideration has to be given to the choice of expression system, the design of the expression vectors, and the purification strategy to obtain a pure product free from toxins and contamination. The availability of the pure recombinant molecules provides the opportunity to formulate preparations that are free from the non-allergenic ballast proteins present in natural allergen extracts and which contain relative concentrations of the allergens in clinically appropriate proportions.
Asunto(s)
Alérgenos/inmunología , Proteínas Recombinantes/inmunología , Alérgenos/biosíntesis , Alérgenos/aislamiento & purificación , Animales , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Clonación Molecular/métodos , Humanos , Inmunoensayo/métodos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificaciónAsunto(s)
Alérgenos/inmunología , Extractos Vegetales/inmunología , Extractos Vegetales/normas , Alérgenos/análisis , Alergoides , Animales , Anticuerpos , Anticuerpos Monoclonales , Antígenos de Plantas/análisis , Antígenos de Plantas/inmunología , Desensibilización Inmunológica/normas , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/normas , Humanos , Inmunoensayo/métodos , Inmunoensayo/normas , Extractos Vegetales/análisis , Proteínas Recombinantes/análisis , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/normas , Estándares de ReferenciaRESUMEN
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot are amongst the most popular methods for allergen characterization, such as comparison of recombinant allergens with their natural counterparts. Native PAGE was evaluated as a possible robust and simple method offering high-resolution capacity for characterization of the major grass pollen allergen Phl p 2. Analytical separation of recombinant Phl p 2 provided a superior quality control in terms of homogeneity and, after Western blotting, immunoglobulin E (IgE) reactivity. Separation of natural Phl p 2 identified two major isoforms which were shown to have different N-terminal sequences and IgE-binding properties. After isolation using preparative native PAGE in combination with electrodialysis, both isoforms were investigated by specific proteolysis and reversed-phase high-performance liquid chromatography (RP-HPLC). The results demonstrate differences in the primary structures and that the recombinant counterpart corresponds exactly to one isoform. Analytical and preparative native PAGE thus proved to be powerful tools for the investigation of allergen isoforms and quality control of recombinant counterparts.
Asunto(s)
Alérgenos/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida/métodos , Proteínas de Plantas/aislamiento & purificación , Polen/inmunología , Alérgenos/análisis , Alérgenos/química , Secuencia de Aminoácidos , Afinidad de Anticuerpos , Cromatografía Líquida de Alta Presión , Escherichia coli/genética , Inmunoglobulina E , Datos de Secuencia Molecular , Proteínas de Plantas/análisis , Proteínas de Plantas/química , Isoformas de Proteínas/química , Isoformas de Proteínas/aislamiento & purificación , Proteínas Recombinantes , Alineación de SecuenciaRESUMEN
Grass pollen belong to the most important allergen sources involved in the elicitation of allergic asthma. We have isolated cDNAs coding for Bermuda grass (Cynodon dactylon) and timothy grass (Phleum pratense) pollen allergens, belonging to a family of pectin-degrading enzymes (i.e., polygalacturonases). The corresponding allergens, termed Cyn d 13 and Phl p 13, represent glycoproteins of approximately 42 kDa and isoelectric points of 7.5. rPhl p 13 was expressed in Escherichia coli and purified to homogeneity. Immunogold electron microscopy using rabbit anti-rPhl p 13 Abs demonstrated that in dry pollen group 13, allergens represent primarily intracellular proteins, whereas exposure of pollen to rainwater caused a massive release of cytoplasmic material containing submicronic particles of respirable size, which were coated with group 13 allergens. The latter may explain respiratory sensitization to group 13 allergens and represents a possible pathomechanism in the induction of asthma attacks after heavy rainfalls. rPhl p 13 was recognized by 36% of grass pollen allergic patients, showed IgE binding capacity comparable to natural Phl p 13, and induced specific and dose-dependent basophil histamine release. Epitope mapping studies localized major IgE epitopes to the C terminus of the molecule outside the highly conserved functional polygalacturonase domains. The latter result explains why rPhl p 13 contains grass pollen-specific IgE epitopes and may be used to diagnose genuine sensitization to grass pollen. Our finding that rabbit anti-rPhl p 13 Abs blocked patients' IgE binding to the allergen suggests that rPhl p 13 may be used for immunotherapy of sensitized patients.
Asunto(s)
Alérgenos/inmunología , Artemisia/inmunología , Phleum/inmunología , Polen/enzimología , Polen/inmunología , Poligalacturonasa/inmunología , Hipersensibilidad Respiratoria/enzimología , Hipersensibilidad Respiratoria/inmunología , Alérgenos/biosíntesis , Alérgenos/química , Alérgenos/aislamiento & purificación , Alérgenos/ultraestructura , Secuencia de Aminoácidos , Anticuerpos Bloqueadores/biosíntesis , Anticuerpos Bloqueadores/metabolismo , Artemisia/enzimología , Artemisia/ultraestructura , Basófilos/inmunología , Basófilos/metabolismo , Unión Competitiva/inmunología , Biomarcadores/análisis , Secuencia Conservada , Desensibilización Inmunológica/métodos , Liberación de Histamina/inmunología , Inmunoglobulina E/metabolismo , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/metabolismo , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Tamaño de la Partícula , Pectinas/metabolismo , Phleum/enzimología , Phleum/ultraestructura , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Polen/ultraestructura , Poligalacturonasa/química , Poligalacturonasa/ultraestructura , Unión Proteica/inmunología , Estructura Terciaria de Proteína , Hipersensibilidad Respiratoria/diagnóstico , Análisis de Secuencia de ProteínaRESUMEN
More than 400 million individuals are sensitized to grass pollen allergens. Group 5 allergens represent the most potent grass pollen allergens recognized by more than 80 % of grass pollen allergic patients. The aim of our study was to reduce the allergenic activity of group 5 allergens for specific immunotherapy of grass pollen allergy. Based on B- and T-cell epitope mapping studies and on sequence comparison of group 5 allergens from different grasses, point mutations were introduced by site-directed mutagenesis in highly conserved sequence domains of Lol p 5, the group 5 allergen from ryegrass. We obtained Lol p 5 mutants with low IgE-binding capacity and reduced allergenic activity as determined by basophil histamine release and by skin prick testing in allergic patients. Circular dichroism analysis showed that these mutants exhibited an overall structural fold similar to the recombinant Lol p 5 wild-type allergen. In addition, Lol p 5 mutants retained the ability to induce proliferation of group 5 allergen-specific T cell lines and clones. Our results demonstrate that a few point mutations in the Lol p 5 sequence yield mutants with reduced allergenic activity that represent potential vaccine candidates for immunotherapy of grass pollen allergy.