Adrian W. Zürcher

Peptide mimotopes displayed by phage inhibit antibody binding to Bet v 1, the major birch pollen allergen, and induce specific IgG response in mice

The major birch pollen allergen Bet v 1 is one of the most extensively characterized allergens both on the molecular and the immunological level. To define conformational B cell epitopes on Bet v 1, we screened filamentous phage libraries expressing circular or linear nonapeptides to select ligands specific for anti‐Bet v 1 murine monoclonal antibodies BIP1 and BIP4. The deduced amino acid sequence of the BIP1 ligand was CFPYCYPSESA, and of the BIP4‐ligand, CRQTRTMPGC. Both sequences derived from the circular phage library. Alignments to the sequence of Bet v 1 showed no similarities, indicating that the antibodies most likely recognize discontinuous epitopes. Phages displaying these mimotopes were capable of inhibiting interactions of the anti‐Bet v 1 monoclonals with Bet v 1 in a dose‐dependent manner in ELISA. In contrast, sequence‐identical synthetic peptides were ineffective in blocking the antibody‐allergen interactions. This is in agreement with the conformational inhomogeneity of the peptides in solution as observed by nuclear magnetic resonance spectroscopy. Intragastric administration of phages expressing the BIP1 mimotope induced a Bet v 1‐specific IgG response in Balb/c mice. We conclude that peptide mimotopes, when displayed on phages, may induce a protective IgG response preventing IgE‐mediated allergic reactions, suggesting a possible clinical application.—Jensen‐Jarolim, E., Leitner, A., Kalchhauser, H., Zürcher, A., Ganglberger, E., Bohle, B., Scheiner, O., Boltz‐Nitulescu, G., Breiteneder, H. Peptide mimotopes displayed by phage inhibit antibody binding to Bet v 1, the major birch pollen allergen, and induce specific IgG response in mice. FASEB J. 12, 1635–1642 (1998)

Allergen mimotopes in food enhance type I allergic reactions in mice

BIP1 is a murine IgG antibody capable of enhancing the IgE binding to Bet v 1, the major birch pollen allergen. We have previously generated a mimotope of BIP1, designated Bet mim 1, from a constrained phage display peptide library. We demonstrated that oral immunization of BALB/c mice with the Bet mim 1 mimotope resulted in the induction of Bet v 1‐specific IgG. The aim of this study was to test the influence of such an oral immunization with Bet mim 1 on a subsequent type I allergic response to Bet v 1. Phages displaying Bet mim 1 or control mimotopes, or PBS alone, were delivered to BALB/c mice by intragastric gavages prior to systemic sensitization with recombinant Bet v 1 and Al(OH)3, an adjuvant inducing preferentially IgE antibody responses. Only mice fed with Bet mim 1‐phages displayed substantially enhanced type I allergic skin reactivity to Bet v 1, as compared to mice pretreated with control mimotopes or PBS. A gastric digestion assay indicated that Bet v 1 and its homologue from apple, Mal d 1, were degraded within seconds under physiological conditions. In contrast, phage‐displayed mimotopes were resistant to digestion. Our data indicate that allergen mimics in the diet that resist digestion, can induce allergen specific IgG able to enhance an allergic response. We therefore conclude that sensitization via the oral route may represent a mechanism for aggravating type I allergic reactions, probably leading to an earlier onset of symptoms even at lower allergen dosage.—Jensen‐Jarolim, E., Wiedermann, U., Ganglberger, E., Zürcher, A., Stadler, B. M., Boltz‐Nitulescu, G., Scheiner, O., Breiteneder, H. Allergen mimotopes in food enhance type I allergic reactions in mice. FASEB J. 13, 1586–1592 (1999)

Culture and IgE Synthesis of Nasal B Cells

The interplay of inflammatory cells, mediators and cytokines during type I allergic reactions in the nose is well described. But even though allergen-specific IgE is known to play a central role for the induction of these events, little is known about nasal B cells and their role in the local allergic reaction. It was the aim of the present study to examine the possibility to isolate and culture B cells from the nose. For this purpose allergic and nonallergic volunteers were challenged by nasal provocation with allergen. Cells were collected sequentially after nasal provocation by nasal lavage, B cells grown in the CD40 system and IgE production assessed by enzyme-linked filter spot assay and radioimmunoassay. IgE-producing B cells were detected after culture of nasal lavage cells in the CD40 system. Elevated levels of IgE were measured in culture supernatants from cells collected during the late-phase allergic reaction. We conclude that B cells can be isolated from the nose and may serve as an interesting source of B cells after in vivo contact with antigen.

IgE-producing hybridomas established after B-cell culture in the CD40 system.

While total IgE synthesis can be easily induced in human PBL or B cells by different stimuli, no systems are known for the induction of allergen-specific IgE in vitro. In this study we investigated whether a specific Ig response could be induced using the CD40 culture system with the final intention to generate B-cell hybridomas secreting IgE of defined specificity. B cells derived from immunized donors normally give rise to many specific hybridomas after cell fusion. However, if cultured in the CD40 system and then immortalized and screened for anti-tetanus specificity, no tetanus-specific clones were found but a large number of IgE-secreting hybridomas had been generated. Also allergen-specific B cells could not be expanded in the CD40 system but long-term cultures yielded again B cells that were efficiently immortalized by cell fusion resulting in stable IgE-secreting hybridomas but of undefined specificity. One of these IgE-producing clones was further characterized and had an IgE production rate of 4.5 micrograms/10(6) cells/24 h. This paper provides two findings. (1) Our cell lines represent a valuable new source of human IgE. (2) Most importantly, our data indicate that the CD40 system is not suitable to expand specific B cells, suggesting that other systems have to be developed for the induction of a significant antigen-specific Ig response.

Mimicry of human IgE epitopes by anti-idiotypic antibodies

Abstract 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 hyperimmunisation, and confirm Jerne’s hypothesis that both foreign antigens and self structures can be mimicked by our own immunoglobulins.

Mimotope and Anti–Idiotypic Vaccines to Induce an Anti–IgE Response

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 Cε3 and Cε4 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 Cε3 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.

Anti-IgE in allergic sensitization

Anti‐IgE autoantibodies exist predominantly in the sera of patients with atopic disease. For some time such anti‐IgE autoantibodies have been considered a phenomenon that may not be of clinical importance. The cloning of such anti‐IgE autoantibodies has eliminated doubts of whether these antibodies exist, but it is still unclear whether such autoantibodies play a pathophysiological role. However, there are ongoing clinical trials that use humanized anti‐IgE antibodies for passive immunization of atopic individuals. While this approach may not definitely clarify the role of anti‐IgE autoantibodies, it will nevertheless clarify the role of IgE.

Neuropeptides accentuate interleukin-4 induced human immunoglobuline E synthesis in vitro

Abstract Corticotropin releasing factor, adrenocorticotropic hormone (ACTH) and alpha‐melanocyte stimulating hormone either inhibit or enhance in a dose‐dependent fashion an interleukin‐4 (IL‐4) driven human IgE synthesis in vitro. Here, we show that culture conditions strongly influence the earlier observed dose‐ and donor‐dependent effects of adrenocorticotropic hormone. The effect of ACTH on IgE synthesis became only apparent late during culture periods, suggesting an indirect effect via the cellular microenvironment rather than by acting directly at the level of B‐cell isotype switching. Thus, we studied other proopiomelanocortin (POMC) derived peptides and neuropeptides known to influence the cellular microenvironment. Indeed, similar modulatory effects on IgE synthesis were also observed by the addition of other proopiomelanocortin‐derived peptides such as α‐, β‐, and γ‐endorphins as well as by the opioid binding pentapeptide Leu‐enkephalin. Furthermore the neuropeptide substance P accentuated an IL‐4 or an IL‐4 and anti‐CD40 antibody driven class switch to IgE. In contrast to ACTH, substance P interfered not only with IgE synthesis but also with the synthesis of the other immunoglobulin isotypes. Thus, systemically acting neuroendocrine peptides such as ACTH and locally acting neuropeptides such as the enkephalins and substance P can modulate the magnitude of an IL‐4 induced IgE response.

Can Active Immunization Redirect an Anti-IgE Immune Response?

We used as a template a mouse monoclonal antibody against IgE to isolate peptides from random peptide phage display libraries. Thereby, two types of peptides were isolated that corresponded to two different epitopes on the human IgE molecule. These peptides, also called mimotopes, seem to be a suitable tool in conjunction with carriers to induce an autoimmune response with a beneficial effect in humans, because the originally used template antibody is capable of neutralizing IgE, is nonanaphylactogenic, and inhibits IgE synthesis. The vaccination approach is further supported by the fact that we were capable of isolating anti-idiotypic antibodies from antibody phage display libraries against the template antibody. These anti-idiotypic antibodies were inhibited by both of the isolated IgE mimotopes. Thus, active vaccination with defined IgE mimotopes may represent a follow-up drug for the presently used anti-IgE antibodies.

Natural And Recombinant ANTI-IgE Autoantibodies

Since the first description of anti-IgE autoantibodies by Williams et al. in 19721, these antibodies have remained a much disputed subject. Most immunologists considered such natural autoantibodies as low affinity antibodies of no importance. Despite the fact that Manning et al. described only 4 years later that repeated injections of a rabbit anti-Ig antiserum to neonatal mice suppressed not only IgM but also IgG, IgA and IgE synthesis2, it was still not generally accepted that anti-IgE autoantibodies may have the same profound effect as heterologous anti-IgE antisera. Subsequent animal experiments, showing that anti-IgE antibodies are strongly immunoregulatory and lead to the suppression of IgE synthesis in vivo have not been associated with the possibility to use such antibodies for therapy3,4, or to postulate that such antibodies may represent a normal physiological control. Only recently, both possibilities have become feasible. Here we demonstrate that anti-IgE antibodies, isolated from the human genome, as recombinant antibodies, may possess the necessary fine specificity for future therapeutic application.