RESUMEN
BACKGROUND AND OBJECTIVES: To evaluate standard intravenous immunoglobulin (IVIG) as an alternative to intravenous cytomegalovirus hyperimmune immunoglobulin (CMVIG) for prophylaxis and therapy of cytomegalovirus (CMV) disease, we measured the ELISA and neutralizing titres of CMV-specific antibodies in CMVIG and IVIG preparations. MATERIALS AND METHODS: Anti-CMV-IgG ELISA and neutralizing titres (fibroblast-based test) in CMVIG CG (Cytogam®, n = 20), CMVIG CT (Cytotect® CP, n = 3), IVIG P (Privigen®, n = 32) and IVIG K/G (Kiovig®/Gammagard®, n = 5) were compared, and IgG subclasses 1-4 were determined by nephelometry. RESULTS: Cytomegalovirus hyperimmune immunoglobulins contained more than fourfold higher CMV ELISA and CMV-neutralizing activity per gram of IgG than the standard IVIGs. Pooled data for all four products showed a significant correlation between anti-CMV-IgG ELISA and neutralizing titres (r = 0·93, P < 0·001). There was a good correlation between the IgG3 content and CMV-neutralizing antibodies amongst lots of CMVIGs (r = 0·91, P = 0·01), but this did not extend to the IVIGs. CMVIG CG contained the highest CMV-neutralizing activity (3497 ± 395 PEIU/g IgG) of any product tested. CONCLUSION: The higher anti-CMV neutralization capacity of CMVIG per gram of IgG vs. standard IVIG suggests that standard IVIGs are not equivalent to or interchangeable with CMVIG.
Asunto(s)
Anticuerpos Antivirales/análisis , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina G/análisis , Inmunoglobulinas Intravenosas/inmunología , Inmunoglobulinas/inmunología , Anticuerpos Antivirales/inmunología , Citomegalovirus/inmunología , Infecciones por Citomegalovirus/tratamiento farmacológico , Infecciones por Citomegalovirus/virología , Humanos , Inmunoglobulinas/uso terapéutico , Inmunoglobulinas Intravenosas/uso terapéuticoRESUMEN
BACKGROUND: Intravenous immunoglobulin (IVIG) preparations are increasingly used for the treatment of autoimmune and chronic inflammatory diseases. Naturally occurring autoantibodies against Siglec-9 and Fas are thought to contribute to the anti-inflammatory effects of IVIG via cell death regulation of leukocytes and tissue cells. Dimeric IVIG fractions are suspected to contain idiotypic (Id)-anti-idiotypic complexes of antibodies, which might also include anti-Siglec-9 and anti-Fas autoantibodies. METHODS: Dimeric IVIG fractions were separated from monomeric IVIG by size-exclusion chromatography and remonomerized by low pH treatment. Binding studies of total, monomeric, and dimeric IVIG were performed using surface plasmon resonance and flow cytometry on primary human neutrophils. RESULTS: Anti-Siglec-9 and anti-Fas autoantibodies were contained in both monomeric and dimeric IVIG fractions, but anti-Siglec-9 antibodies were highly enriched in dimeric IVIG. The propensity to engage in dimer formation was paratope dependent. IVIG binding to Siglec-9 was specific and sialylation independent. Interestingly, we detected anti-idiotypic antibodies (anti-Ids) against anti-Siglec-9 autoantibodies in dimeric, but not in monomeric fractions of IVIG. CONCLUSIONS: Our study supports the concept that idiotype-anti-idiotype (Id-anti-Id) interactions contribute to the dimer formation in IVIG preparations. To our knowledge, this is the first description of Id-anti-Id dimers of death receptor-specific antibodies in IVIG. Such Id-anti-Id interactions might determine the activity of immunomodulatory antibodies present both in IVIG and the patient.
Asunto(s)
Antígenos CD/inmunología , Autoanticuerpos/análisis , Idiotipos de Inmunoglobulinas/análisis , Inmunoglobulinas Intravenosas/análisis , Lectinas/inmunología , Humanos , Inmunoglobulinas Intravenosas/inmunología , Neutrófilos , Multimerización de Proteína , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico , Receptor fas/inmunologíaRESUMEN
Polyvalent Ig preparations, derived from the pooled plasma of thousands of healthy donors, contain a complex mix of both 'acquired' and natural antibodies directed against pathogens as well as foreign and self/auto antigens (Ag). Depending on their formulation, donor pool size, etc., liquid Ig preparations contain monomeric and dimeric IgG. The dimeric IgG fraction is thought to represent mainly idiotype-antiidiotype Ab pairs. Treatment of all IgG fractions at pH 4 effectively monomerizes the IgG dimers resulting in separated idiotype-antiidiotype Ab pairs and thus in a comparable F(ab')(2) binding site availability of the different IgG fractions. Previously, we identified an increased anti-self-reactivity within the monomerized dimer fraction. This study addressed if, among the different IgG fractions, an analogous preferential reactivity was evident in the response against different pathogen-derived protein and carbohydrate antigens. Therefore, we assessed the activity of total unseparated IgG, the monomeric and dimeric IgG fractions against antigenic structures of bacterial and viral antigens/virulence factors. All fractions showed similar reactivity to protein antigens except for exotoxin A of Pseudomonas aeruginosa, where the dimeric fraction, especially when monomerized, showed a marked increase in reactivity. This suggests that the production of antiidiotypic IgG antibodies contributes to controlling the immune response to certain categories of pathogens. In contrast, the monomeric IgG fractions showed increased reactivity towards pathogen-associated polysaccharides, classically regarded as T-independent antigens. Taken together, the differential reactivity of the IgG fractions seems to indicate a preferential segregation of antibody reactivities according to the nature of the antigen.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Anticuerpos Antivirales/inmunología , Reacciones Antígeno-Anticuerpo , Antígenos Bacterianos/inmunología , Antígenos Virales/inmunología , Inmunoglobulina G/inmunología , Anticuerpos Antibacterianos/química , Anticuerpos Antibacterianos/aislamiento & purificación , Anticuerpos Antivirales/química , Anticuerpos Antivirales/aislamiento & purificación , Toxinas Bacterianas/inmunología , Línea Celular , Humanos , Inmunoglobulina G/química , Inmunoglobulina G/aislamiento & purificación , Pruebas de Neutralización , Polisacáridos Bacterianos/inmunología , Multimerización de Proteína , Toxoides/inmunologíaRESUMEN
High-dose intravenous immunoglobulin (IVIg) preparations are used currently for the treatment of autoimmune or inflammatory diseases. Despite numerous studies demonstrating efficacy, the precise mode of action of IVIg remains unclear. Paradoxically, IgG can exert both pro- and anti-inflammatory activities, depending on its concentration. The proinflammatory activity of low-dose IVIg requires complement activation or binding of the Fc fragment of IgG to IgG-specific receptors (FcgammaR) on innate immune effector cells. In contrast, when administered in high concentrations, IVIg has anti-inflammatory properties. How this anti-inflammatory effect is mediated has not yet been elucidated fully, and several mutually non-exclusive mechanisms have been proposed. This paper represents the proceedings of a session entitled 'IVIg--Understanding properties and mechanisms' at the 6th International Immunoglobulin Symposium that was held in Interlaken on 26-28 March 2009. The presentations addressed how IgG may affect the cellular compartment, evidence for IVIg-mediated scavenging of complement fragments, the role of the dimeric fraction of IVIg, the anti-inflammatory properties of the minor fraction of sialylated IgG molecules, and the genetic organization and variation in FcgammaRs. These findings demonstrate the considerable progress that has been made in understanding the mechanisms of action of IVIgs, and may influence future perspectives in the field of Ig therapy.
Asunto(s)
Inmunoglobulinas Intravenosas/uso terapéutico , Animales , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina G/uso terapéutico , Inmunoglobulinas Intravenosas/inmunología , Inmunomodulación/inmunología , Inflamación/terapia , Ratones , Polimorfismo de Nucleótido Simple , Receptores de IgG/genética , Receptores de IgG/inmunologíaRESUMEN
BACKGROUND AND OBJECTIVES: Complement inhibition is considered important in the mechanism of action of intravenous immunoglobulin (IVIG) in a number of inflammatory and autoimmune disorders. The capacity of different IVIG preparations to 'scavenge' activated C3 and thereby inhibit complement activation was assessed by a new in vitro assay. MATERIALS AND METHODS: Diluted human serum as a complement source, with or without addition of different concentrations of IVIG, was incubated in microtitre plates coated with heat-aggregated human IgG. Complement scavenging was measured by detecting reduced C3 binding and determining fluid phase C3b-IgG complex formation. Complement activation induced by the IVIG preparations was measured as C5a formation. RESULTS: All IVIG preparations exhibited a dose-dependent inhibition of C3b deposition, correlating strongly with binding of C3b to fluid-phase IgG, but the extent of complement scavenging varied considerably between different IVIG preparations. At an IVIG concentration of 0.9 mg/ml, the inhibition of C3b deposition ranged from 72 +/- 16% to 22 +/- 4.1%. The reduction of C3b deposition on the complement-activating surface was not due to IVIG-induced complement activation in the fluid phase, as shown by the low C5a formation in the presence of serum. CONCLUSION: In vitro analysis allows comparison of the complement-inhibitory properties of IVIG preparations. The extent of complement scavenging varies between the products.
Asunto(s)
Activación de Complemento , Complemento C3a/química , Complemento C5a/análisis , Inmunoglobulinas Intravenosas/análisis , Complemento C3a/inmunología , Complemento C5a/inmunología , Pruebas de Fijación del Complemento/métodos , Relación Dosis-Respuesta Inmunológica , Humanos , Inmunoglobulinas Intravenosas/inmunologíaRESUMEN
In this first-in-man study, we assessed the pharmacokinetics, safety and tolerability of MonoRho, a human recombinant monoclonal anti-RhD immunoglobulin G1 (IgG1) antibody. Eighteen RhD-negative healthy male volunteers were randomized in two groups to receive a single administration of 300 micro g of MonoRho either intravenously or intramuscularly. There were no symptoms of allergic or anaphylactic type reaction in any subject, and there was no evidence of any MonoRho-related changes in laboratory safety parameters. None of the subjects mounted a detectable immune response to MonoRho. Serum samples were obtained up to 91 days after injection to measure anti-D IgG concentrations by flow cytometry. After intramuscular administration of MonoRho, anti-D IgG concentrations gradually increased reaching peak levels after a mean of 3.4 days. After 3 weeks, the mean anti-D IgG concentrations after intravenous and intramuscular administration became virtually equal to each other and remained so thereafter. In both the treatment groups, the mean elimination half-life was about 18 days and thus similar to that described for plasma-derived anti-D IgG. The bioavailability of MonoRho after intramuscular administration was estimated as 46%. The excellent tolerability and safety of MonoRho as well as its expected elimination half-life supports the continued clinical development of this compound.
Asunto(s)
Anticuerpos Monoclonales/farmacocinética , Fragmentos de Inmunoglobulinas/administración & dosificación , Isoanticuerpos/sangre , Sistema del Grupo Sanguíneo Rh-Hr/inmunología , Adulto , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/efectos adversos , Disponibilidad Biológica , Semivida , Humanos , Fragmentos de Inmunoglobulinas/efectos adversos , Inmunoglobulina G , Masculino , Farmacocinética , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/farmacocinéticaRESUMEN
Anti-idiotypic antibodies (anti-Id) have been described against idiotypes expressed on various autoantibodies. Since an immunoregulatory effect has been postulated for anti-Id, modulation of the anti-Id response in autoimmune disease may be of interest. In chronic immune thrombocytopenic purpura (AITP), autoantibodies directed mainly against platelet membrane glycoprotein (GP) IIb/IIIa cause platelet destruction by Fc-mediated phagocytosis or by complement lysis. We have previously reported on the generation of two recombinant anti-GPIIb/IIIa autoantibody fragments (PDG-X, PDG-B), that are specific for conformationally intact GPIIb/IIIa and inhibit binding of autoantibodies from patients with AITP. In the present study, we show that anti-GPIIb/IIIa specificities are not limited to a single individual by isolating five additional anti-GPIIb/IIIa autoantibody fragments from a second phagemid Fab library of an unrelated healthy donor. Using soluble Fab of PDG-X and PDG-B as antigens for panning Fab phagemid libraries from healthy human individuals, we isolated anti-Id phage clones specific for PDG-X or PDG-B. In addition they inhibited the binding of PDG-X or PDG-B to GPIIb/IIIa. Amino acid sequence comparison between these specific antiId and GPIIb/IIIa was performed. Generation of these anti-Id directed against pathologically relevant anti-GPIIb/IIIa autoantibodies may represent a new suitable and specific therapeutic option for the treatment of antibody-mediated AITP.
Asunto(s)
Anticuerpos Antiidiotipos/metabolismo , Autoanticuerpos/metabolismo , Sitios de Unión de Anticuerpos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/inmunología , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Anticuerpos Antiidiotipos/uso terapéutico , Afinidad de Anticuerpos/inmunología , Reacciones Antígeno-Anticuerpo/inmunología , Sitios de Unión de Anticuerpos/inmunología , Unión Competitiva/inmunología , Clonación Molecular , Mapeo Epitopo/métodos , Humanos , Fragmentos de Inmunoglobulinas/uso terapéutico , Inmunoterapia , Datos de Secuencia Molecular , Biblioteca de Péptidos , Púrpura Trombocitopénica Idiopática/inmunología , Púrpura Trombocitopénica Idiopática/terapia , Proteínas Recombinantes/uso terapéuticoRESUMEN
Natural antibodies provide an early defense mechanism against pathogens, show a frequent self-reactivity, and are present throughout life. Two questions concern the physiological control of self-reactivity and the pathogenetic link to autoimmune disease. Here we propose a concept of conditional autoimmunity involving natural antibodies against the alpha chain of the high-affinity receptor for IgE (Fc(epsilon)RIalpha ). Like other natural antibodies, anti-Fc(epsilon)RIalpha antibodies are found in sera of healthy donors. We now report the first human recombinant anti-Fc(epsilon)RIalpha autoantibodies isolated by repertoire cloning from a human tonsillar IgM library. These high-affinity antibodies recognize Fc(epsilon)RIalpha on cells and trigger histamine release from freshly isolated blood basophils. However, the latter effect requires IgE removal from the Fc(epsilon)RI. The same conditional histamine release is seen when using sera from individual normal donors and affinity-purified anti-Fc(epsilon)RIalpha antibodies isolated from multidonor therapeutic IgG preparations. We propose that such anti-Fc(epsilon)RIalpha antibodies can become pathogenic and that this is dependent on the state of occupancy of the Fc(epsilon)RIalpha by its natural ligand IgE. We suggest that an imbalance between Fc(epsilon)RIalpha occupancy and natural anti-Fc(epsilon)RIalpha antibodies may be implicated in the pathogenesis of autoimmune urticaria.
Asunto(s)
Autoanticuerpos/inmunología , Autoinmunidad , Receptores de IgE/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Autoanticuerpos/genética , Basófilos/inmunología , Células Cultivadas , Liberación de Histamina , Humanos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Región Variable de Inmunoglobulina/genética , Modelos Inmunológicos , Datos de Secuencia Molecular , Biblioteca de Péptidos , Proteínas Recombinantes/inmunologíaRESUMEN
By means of repertoire cloning we have isolated human anti-IgE antibodies as well as human anti-FcepsilonRI antibodies. Whether the naturally occurring anti-IgE autoantibodies play a pathophysiological role may be disputed, but the beneficial role of recombinant anti-IgE antibodies as a therapeutic agent has been shown. On the other hand, the natural antibodies isolated from an antibody library of a nonallergic individual against the FcepsilonRI alpha-chain are anaphylactogenic, if FcepsilonRI was not occupied. Thus, anti-FcepsilonRI autoantibodies may be part of a conditional autoimmune reaction, leading to urticaria if local IgE is consumed, e.g. after an immediate reaction. Thus, anti-FcepsilonRI antibodies may represent an amplification arm of the late reaction. The normal occurrence of anti-IgE and anti-IgE receptor autoantibodies may suggest that it might also be feasible to induce such autoantibodies by vaccination. In a monkey model using a mimotope of human IgE it was possible to induce a beneficial anti- IgE autoimmune response. The actual epitope of the IgE molecule was then also molecularly reconstructed by generating recombinant anti-idiotypic antibodies. These antibodies also induced effectively an anti-IgE response in monkeys, suggesting that not only mimotopes but also anti-idiotypic antibodies may be used to generate an autoimmune response. Both of our projects suggest that active immunization may be a new form of immunomodulation for the treatment of allergic disease.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Autoanticuerpos/inmunología , Autoinmunidad , Hipersensibilidad Inmediata/inmunología , Receptores de IgE/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Autoanticuerpos/genética , Haplorrinos , Humanos , Biblioteca de Péptidos , Urticaria/inmunologíaRESUMEN
The role of autoantibodies against the alpha-subunit of the human high-affinity IgE receptor (FcepsilonRIalpha) in the pathogenesis of chronic idiopathic urticaria (CIU) is controversial. We have shown that these antibodies are widespread, apparently non-pathogenic and belong to the natural antibody repertoire. To clarify this controversy, we constructed antibody libraries from both healthy donors and CIU patients with active disease. Here we describe the first three high affinity IgM anti-FcepsilonRIalphaautoantibodies isolated from normal and urticaria libraries. Sequence analysis revealed germline VH in both cases paired with a slightly mutated VL, thus supporting their classification as natural antibodies. Strikingly, one major IgM clone was present in both CIU patients and normal donors. The anti-FcepsilonRIalpha autoantibodies recognize FcepsilonRIalpha on cells, but are non-anaphylactogenic on blood basophils, except when IgE is removed from the receptor. Based on their functional activities we propose a concept of "conditional autoimmunity" where natural anti-FcepsilonRIalphaautoantibodies can become pathogenic dependent on the state of occupancy of the FcepsilonRIalpha by its natural ligand IgE.
Asunto(s)
Autoanticuerpos/inmunología , Basófilos/inmunología , Receptores de IgE/inmunología , Urticaria/inmunología , Animales , Afinidad de Anticuerpos , Autoanticuerpos/sangre , Células CHO , Enfermedad Crónica , Cricetinae , Humanos , Inmunoglobulina E/fisiología , Proteínas Recombinantes/inmunologíaRESUMEN
Chronic urticaria may be characterized by conditional autoantibodies against the alpha-chain of the high-affinity receptor for IgE (FcepsilonRI). These autoantibodies are termed conditional as they only recognize unoccupied FcepsilonRI. The same conditional reactivity pattern has also been found in sera of atopic and normal healthy donors. Any condition resulting in accessibility of FcepsilonRI will render these autoantibodies anaphylactogenic. This finding offers a unifying hypothesis for the manifestation of different forms of urticaria. Non-immunologic triggers may thereby influence directly or indirectly the number of accessible FcepsilonRI allowing the conditional autoantibodies to induce urticaria symptoms.
Asunto(s)
Autoanticuerpos/inmunología , Urticaria/inmunología , Liberación de Histamina/inmunología , Humanos , Inmunoglobulina E/inmunología , Receptores de IgE/inmunologíaRESUMEN
Replacement of the hyperimmune anti-Rhesus (Rh) D immunoglobulin, currently used to prevent haemolytic disease of the newborn, by fully recombinant human anti-RhD antibodies would solve the current logistic problems associated with supply and demand. The combination of phage display repertoire cloning with precise selection procedures enables isolation of specific genes that can then be inserted into mammalian expression systems allowing production of large quantities of recombinant human proteins. With the aim of selecting high-affinity anti-RhD antibodies, two human Fab libraries were constructed from a hyperimmune donor. Use of a new phage panning procedure involving bromelin-treated red blood cells enabled the isolation of two high-affinity Fab-expressing phage clones. LD-6-3 and LD-6-33, specific for RhD. These showed a novel reaction pattern by recognizing the D variants D(III), D(IVa), D(IVb), D(Va), D(VI) types I and II. D(VII), Rh33 and DFR. Full-length immunoglobulin molecules were constructed by cloning the variable regions into expression vectors containing genomic DNA encoding the immunoglobulin constant regions. We describe the first, stable, suspension growth-adapted Chinese hamster ovary (CHO) cell line producing a high affinity recombinant human IgG1 anti-RhD antibody adapted to pilot-scale production. Evaluation of the Fc region of this recombinant antibody by either chemiluminescence or antibody-dependent cell cytotoxicity (ADCC) assays demonstrated macrophage activation and lysis of red blood cells by human lymphocytes. A consistent source of recombinant human anti-RhD immunoglobulin produced by CHO cells is expected to meet the stringent safety and regulatory requirements for prophylactic application.
Asunto(s)
Biotecnología/métodos , Fragmentos Fab de Inmunoglobulinas/genética , Inmunoglobulina G/genética , Globulina Inmune rho(D)/metabolismo , Animales , Bacteriófagos , Secuencia de Bases , Bromelaínas/farmacología , Células CHO , Clonación Molecular , Cricetinae , Eritrocitos , Humanos , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Isoinmunización Rh/prevención & controlRESUMEN
We purified a bacteriocin from the cell-free supernatant of Propionibacterium jensenii DF1 isolated from Swiss raw milk, and named it propionicin SM1. The heat-stable protein was strongly bactericidal against P. jensenii DSM20274. On the basis of the N-terminal amino acid sequence of the purified protein, a degenerate oligonucleotide probe was designed to locate and clone the corresponding gene of P. jensenii DF1. It hybridized exclusively with the DF1l-resident plasmid pLME106, but not with chromosomal DNA. Sequencing of the 6.9-kb plasmid revealed the targeted amino acid sequence within an open reading frame (ORF4) of 207 amino acids (molecular mass, 22,865 Da). The corresponding gene was named ppnA. It encodes the prepeptide PpnA that is processed to the mature protein (19,942 Da) propionicin SM1. No sequence homology is detectable with known proteins. However, the proposed leader peptide sequence containing 27 amino acids has typical signal peptide features and shows good homology to the leader peptide of Usp45, a protein excreted from Lactococcus lactis (VAN ASSELDONK et al., 1993). Plasmid pLME106 contains at least 9 ORFs, some exhibiting significant homologies to plasmid-encoded functions from other bacteria. The highest identity values were found for ORF1 with the theta replicase (acc. no. U39878) of Brevibacterium linens (58.8%) and ORF6 with the recombinase/invertase (acc. no. AF060871) found in Rhodococcus rhodochrous (46.4%).
Asunto(s)
Bacteriocinas/genética , Genes Bacterianos , Propionibacterium/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Bacteriocinas/farmacología , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plásmidos/genética , Señales de Clasificación de Proteína/genética , Mapeo Restrictivo , Análisis de Secuencia de ADNRESUMEN
IgE Abs mediate allergic responses by binding to specific high affinity receptors (FcepsilonRI) on mast cells and basophils. Therefore, the IgE/FcepsilonRI interaction is a target for clinical intervention in allergic disease. An anti-IgE mAb, termed BSW17, is nonanaphylactogenic, although recognizing IgE bound to FcepsilonRI, and interferes with binding of IgE to FcepsilonRI. Thus, BSW17 represents a candidate Ab for treatment of IgE-mediated disorders. By panning BSW17 against random peptide libraries displayed on phages, we defined mimotopes that mimic the conformational epitope recognized on human IgE. Two types of mimotopes, one within the Cepsilon3 and one within the Cepsilon4 domain, were identified, indicating that this mAb may recognize either a large conformational epitope or eventually two distinct epitopes on IgE. On the basis of alignments of the two mimotopes with the human IgE sequence, we postulate that binding of BSW17 to the Cepsilon3 region predominantly blocks binding of IgE to FcepsilonRI, leading to neutralization of IgE. Moreover, binding of BSW17 to the Cepsilon4 region may explain how BSW17 recognizes FcepsilonRI-bound IgE, and binding to this region may also interfere with degranulation of IgE sensitized cells (basophils and mast cells). As a practical application of these findings, mimotope peptides coupled to a carrier protein may be used for the development of a peptide-based anti-allergy vaccine by induction of anti-IgE Abs similar to the current approach of using humanized nonanaphylactogenic anti-IgE Abs as a passive vaccine.
Asunto(s)
Anafilaxia/inmunología , Anticuerpos Antiidiotipos/química , Anticuerpos Antiidiotipos/metabolismo , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/metabolismo , Inmunoglobulina E/inmunología , Secuencia de Aminoácidos , Anafilaxia/metabolismo , Anticuerpos Antiidiotipos/farmacología , Anticuerpos Monoclonales/farmacología , Bacteriófagos/química , Bacteriófagos/inmunología , Sitios de Unión de Anticuerpos , Unión Competitiva/inmunología , Epítopos/inmunología , Epítopos/metabolismo , Humanos , Regiones Constantes de Inmunoglobulina/química , Regiones Constantes de Inmunoglobulina/metabolismo , Inmunoglobulina E/farmacología , Cadenas epsilon de Inmunoglobulina/química , Cadenas epsilon de Inmunoglobulina/metabolismo , Imitación Molecular , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Biblioteca de Péptidos , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
According to Jerne's network hypothesis, the binding site of an anti-idiotypic antibody also represents the internal image of an epitope present on a foreign, or even a self antigen. In recent years, antigen mimicry has been defined at the molecular level for some xeno-antigens. However, until now there has been no demonstration of structural mimicry between a human anti-idiotypic antibody and a self structure. To address this question, we used human IgE as the self structure and a well-defined anti-human IgE mAb (BSW17). We describe the isolation of two anti- idiotypic antibodies specific for the anti-IgE antibody BSW17 from a non-immune human Fab phage display library. Interestingly, these two anti-idiotypic antibodies mimic the same molecular surface region as a previously described IgE peptide mimotope isolated by panning on BSW17, but they cover a much larger epitope on the IgE molecule. Accordingly, immunisation of rabbits with the two anti-idiotypic antibodies induced high-affinity antibodies with the same characteristics as BSW17. Thus, our data demonstrate that it is possible to isolate anti-idiotypic antibodies derived from the human genome without the need for hyperimmunization, and confirm Jerne's hypothesis that both foreign antigens and self structures can be mimicked by our own immunoglobulins.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Epítopos/inmunología , Inmunoglobulina E/inmunología , Imitación Molecular , Secuencia de Aminoácidos , Animales , Anticuerpos Antiidiotipos/química , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos , Especificidad de Anticuerpos , Autoanticuerpos/química , Células CHO , Cricetinae , Humanos , Inmunización , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/inmunología , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/inmunología , Modelos Inmunológicos , Modelos Moleculares , Datos de Secuencia Molecular , Biblioteca de Péptidos , Conejos , Alineación de SecuenciaRESUMEN
An essential requirement for oral vaccines is the ability to survive the harsh environment of the stomach in an antigenically intact form. As bacteriophages are adapted to this environment we used epitope-displaying M13 bacteriophages as carriers for an experimental oral anti-IgE vaccine. The feasibility of this approach was tested in a simulated gastric fluid using two different mimotopes as well as an anti-idiotypic Fab of the non-anaphylactogenic monoclonal anti-IgE antibody BSW17. All phage clones remained infective after this treatment. However, only epitopes displayed on the pVIII protein were still recognized by BSW17 whereas pIII-expressed epitopes were rapidly inactivated. Surprisingly, when used for oral immunization of mice all phage clones induced anti-IgE antibodies. In contrast, oral immunization with the purified, pVIII protein displaying the mimotope induced anti-phage but no anti-IgE antibodies. After feeding a single dose of mimotope-displaying bacteriophage, phage DNA could be detected in mouse feces for 10 days. Our results show that epitope-displaying bacteriophages can be used to induce an epitope-specific antibody response via the oral route.
Asunto(s)
Bacteriófagos/genética , Inmunoglobulina E/inmunología , Vacunas/administración & dosificación , Administración Oral , Animales , Anticuerpos Antiidiotipos/sangre , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Epítopos , Femenino , Inmunización , Fragmentos Fab de Inmunoglobulinas/inmunología , Ratones , Ratones Endogámicos BALB C , Reacción en Cadena de la PolimerasaRESUMEN
Antigen-specific human IgE is in short supply. Thus, we sought to determine the yet unknown specificity of a widely available human IgE, namely the myeloma cell line U266-derived IgE-ND. For this purpose highly specific peptides able to mimic the putative antigen recognized by IgE-ND were isolated from phage-display random peptide libraries. Interestingly, we found linear sequence homologies of the IgE-ND-binding peptides with self antigens and a xenoantigen from Thiobacillus ferrooxidans. However, none of these antigens was recognized by IgE-ND. Nevertheless, our approach may be applied to identify antigen specificities of myeloma antibodies. Importantly, the mimotopes were anaphylactogenic in a histamine release assay using human basophils sensitized with IgE-ND. Thus, our mimotopes represent functional albeit synthetic antigens and may be used to study human antigen-specific IgE responses.
Asunto(s)
Antígenos de Neoplasias/inmunología , Inmunoglobulina E/química , Imitación Molecular/inmunología , Mieloma Múltiple/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antineoplásicos/química , Especificidad de Anticuerpos , Bacteriófagos/inmunología , Unión Competitiva , Células CHO , Células Cultivadas , Cricetinae , Epítopos/aislamiento & purificación , Humanos , Inmunoglobulina E/metabolismo , Datos de Secuencia Molecular , Biblioteca de Péptidos , Receptores de IgE/metabolismoRESUMEN
We have defined epitopes on human IgE by screening different phage display random peptide libraries with a monoclonal anti-IgE antibody termed BSW17. The selected mimotopes and epitopes within the Cepsilon3 and Cepsilon4 region of IgE induced antibodies that were nonanaphylactogenic and had biological activity similar to BSW17. The chemically synthesized and KLH-coupled IgE epitopes or mimotopes were used to induce an anti-IgE response in rhesus monkeys. The immunized rhesus monkeys were subsequently protected in a PCA test when sensitized with human IgE and triggered with the corresponding allergen. Furthermore, using the same monoclonal anti-IgE antibody, we also generated an anti-idiotypic antibody that showed sequence homology with the IgE epitope in the Cepsilon3 domain. This anti-idiotypic antibody as well as the mimotopes were then used in a mouse model to induce orally an anti-IgE immune response. For this purpose mice were fed by intragastric gavages with bacteriophages displaying the small IgE-homologous structures. Orally immunized mice produced serum anti-IgE antibodies that were inhibited by BSW17 suggesting that it may be possible to induce a systemic anti-IgE response orally.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Hipersensibilidad/inmunología , Inmunoglobulina E/inmunología , Vacunas Sintéticas/inmunología , Animales , Anticuerpos Antiidiotipos/genética , Anticuerpos Antiidiotipos/uso terapéutico , Mapeo Epitopo , Humanos , Hipersensibilidad/prevención & control , Ratones , Biblioteca de Péptidos , Proteínas Recombinantes/inmunología , Vacunas Sintéticas/genéticaRESUMEN
Natural antibodies (Ab) reacting with self antigens have been shown to be present in all individuals. These autoantibodies (auto-Ab) can be either pathogenic or non-pathogenic. Auto-Ab reacting with the alpha-subunit of the high-affinity receptor for IgE (FcepsilonRIalpha) have been implicated in the pathogenesis of a subset of patients with chronic idiopathic urticaria (CIU). Intravenous immunoglobulin (IVIg) preparations have been used with variable clinical benefit in the treatment of these patients. Here we show that anti-FcepsilonRIalpha auto-Ab are present in a therapeutic IVIg preparation as well as in atopic and chronic urticaria patients and healthy individuals. We affinity-purified the anti-FcepsilonRIalpha Ab from an IVIg preparation using recombinant FcepsilonRIalpha. Interestingly, these anti-FcepsilonRIalpha auto-Ab showed no evidence of histamine release but strongly cross-reacted with an external antigen, tetanus toxoid (TTd) with a higher affinity for TTd than for the FcepsilonRIalpha. Since the cross-reacting Ab are non-anaphylactogenic, there is no evidence that TTd immunization may contribute to the pathogenesis of CIU. However, our results may indicate that the anti-FcepsilonRIalpha auto-Ab belong to the natural Ab and serve as the parental Ab for some anti-TTd Ab.