In Vitro Evolution of Allergy Vaccine Candidates, with Maintained Structure, but Reduced B Cell and T Cell Activation Capacity

Allergy and asthma to cat (Felis domesticus) affects about 10% of the population in affluent countries. Immediate allergic symptoms are primarily mediated via IgE antibodies binding to B cell epitopes, whereas late phase inflammatory reactions are mediated via activated T cell recognition of allergen-specific T cell epitopes. Allergen-specific immunotherapy relieves symptoms and is the only treatment inducing a long-lasting protection by induction of protective immune responses. The aim of this study was to produce an allergy vaccine designed with the combined features of attenuated T cell activation, reduced anaphylactic properties, retained molecular integrity and induction of efficient IgE blocking IgG antibodies for safer and efficacious treatment of patients with allergy and asthma to cat. The template gene coding for rFel d 1 was used to introduce random mutations, which was subsequently expressed in large phage libraries. Despite accumulated mutations by up to 7 rounds of iterative error-prone PCR and biopanning, surface topology and structure was essentially maintained using IgE-antibodies from cat allergic patients for phage enrichment. Four candidates were isolated, displaying similar or lower IgE binding, reduced anaphylactic activity as measured by their capacity to induce basophil degranulation and, importantly, a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. In addition, all mutants showed ability to induce blocking antibodies in immunized mice.The approach presented here provides a straightforward procedure to generate a novel type of allergy vaccines for safer and efficacious treatment of allergic patients

Identification of Critical Amino Acids in an Immunodominant IgE Epitope of Pen c 13, a Major Allergen from Penicillium citrinum

Background: Pen c 13, identified as a 33-kDa alkaline serine protease, is a major allergen secreted by Penicillium citrinum. Detailed knowledge about the epitopes responsible for IgE binding would help inform the diagnosis/prognosis of fungal allergy and facilitate the rational design of hypoallergenic candidate vaccines. The goal of the present study was to characterize the IgE epitopes of Pen c 13. Methodology/Principal Findings: Serum samples were collected from 10 patients with mold allergy and positive Pen c 13 skin test results. IgE-binding epitopes on rPen c 13 were mapped using an enzymatic digestion and chemical cleavage method, followed by dot-blotting and mass spectrometry. A B-cell epitope-predicting server and molecular modeling were used to predict the residues most likely involved in IgE binding. Theoretically predicted IgE-binding regions were further confirmed by site-directed mutagenesis assays. At least twelve different IgE-binding epitopes located throughout Pen c 13 were identified. Of these, peptides S16 (A 148 –E 166) and S22 (A 243 –K 274) were recognized by sera from 90 % and 100 % of the patients tested, and were further confirmed by inhibition assays. Peptide S22 was selected for further analysis of IgE-binding ability. The results of serum screening showed that the majority of IgE-binding ability resided in the C-terminus. One Pen c 13 mutant, G270A (T 261 –K 274), exhibited clearly enhanced IgE reactivity, whereas another, K274A, exhibited dramatically reduced IgE reactivity

An update on molecular cat allergens: Fel d 1 and what else? Chapter 1: Fel d 1, the major cat allergen

Background: Cats are the major source of indoor inhalant allergens after house dust mites. The global incidence of cat allergies is rising sharply, posing a major public health problem. Ten cat allergens have been identified. The major allergen responsible for symptoms is Fel d 1, a secretoglobin and not a lipocalin, making the cat a special case among mammals. Main body: Given its clinical predominance, it is essential to have a good knowledge of this allergenic fraction, including its basic structure, to understand the new exciting diagnostic and therapeutic applications currently in development. The recent arrival of the component-resolved diagnosis, which uses molecular allergens, represents a unique opportunity to improve our understanding of the disease. Recombinant Fel d 1 is now available for in vitro diagnosis by the anti-Fel d 1 specific IgE assay. The first part of the review will seek to describe the recent advances related to Fel d 1 in terms of positive diagnosis and assessment of disease severity. In daily practice, anti-Fel d 1 IgE tend to replace those directed against the overall extract but is this attitude justified? We will look at the most recent arguments to try to answer this question. In parallel, a second revolution is taking place thanks to molecular engineering, which has allowed the development of various forms of recombinant Fel d 1 and which seeks to modify the immunomodulatory properties of the molecule and thus the clinical history of the disease via various modalities of anti-Fel d 1-specific immunotherapy. We will endeavor to give a clear and practical overview of all these trends

Crystal Structure of the Dog Lipocalin Allergen Can f 2: Implications for Cross-reactivity to the Cat Allergen Fel d 4

Allergic diseases are a major health problem, affecting up to 30% of the population in affluent countries. Domestic pets such as cats, dogs and horses are among the most common agents of IgE-mediated allergies. Conventional diagnostics and allergen-specific immunotherapy (SIT) still rely on crude allergen extracts, even though several commercial extracts have demonstrated poor quality with regard to the included allergen content. Particularly in dog allergy, SIT may show a low clinical effectiveness upon treatment with allergenic extracts. As an alternative, recombinant DNA technologies allow for the construction of recombinant (r) allergens for improved allergy diagnostics and induction of a long-lasting protection by SIT. However, this therapy is still limited by unwanted side-effects such as IgE-mediated acute reactions, or late phase reactions (LPR). To combat these limitations, allergens have been modified for safer use in SIT, i.e. hypoallergens, or several allergenic molecules have been combined in one construct for improved diagnostics and treatment of allergic patients. The aim of this thesis was to demonstrate how development in molecular allergology may increase the understanding of the allergenicity and cross-reactivity of dog allergens. We also set out to demonstrate how this knowledge can be used to engineer safer and more efficacious allergenic constructs for improved diagnostics and therapy. In paper I, we described the crystal structure and the structural characteristics of the dog lipocalin allergen Can f 2. The recombinant protein was shown to mimick the IgEbinding characteristics of the natural allergen. Furthermore, we demonstrated an IgEmediated cross-reactivity between Can f 2 and the cat lipocalin allergen Fel d 4, and suggested a potential conserved epitope as a cross-reactive site. In paper II, the primary structures for Fel d 4 and the major horse allergen Equ c 1 were used to identify a novel dog lipocalin allergen, Can f 6. The recombinant allergen elicited a positive IgE-reaction in ~40% of dog-sensitized subjects, and most individuals also had a positive reaction to the homologous allergens Fel d 4 and Equ c 1. The biological activity of rCan f 6 and the homologous allergens was demonstrated by basophil activation test (BAT). Finally, we identified IgE-mediated cross-reactivity between these three allergens, which may account for cross-species sensitization between dog, cat and horse. In paper III, the method phage display was for the first time used to construct hypoallergenic mutants of the major cat allergen, Fel d 1. The four vaccine candidates were produced as folded proteins in E. coli and showed a lower IgE-binding than the wild type protein in ELISA. By T cell proliferation and BAT assays, we demonstrated that two of the four candidates had a significantly lower T cell activation capacity. Moreover all mutants had a lower allergenic activity than rFel d 1, which could indicate a safer profile for SIT of allergic patients. The effectiveness of the mutants was demonstrated in a mouse model for cat allergy, by the induction of blocking IgG antibodies. In paper IV, a different vaccine concept was described, where four dog lipocalin allergens were assembled in one molecule. The corresponding fusion protein comprised the biochemical and immunological properties of the original allergens, investigated by CD-spectra and ELISA. Importantly, the linked construct bound more IgE than the individual allergens, demonstrating the usefulness for diagnostics. Finally, the linked molecule induced comparable IgG levels to all included allergens compared with an equimolar mix, while showing a lower T cell reactivity which could implicate a lower risk of LPR. To summarize, this thesis demonstrates how molecular approaches may be used to improve our understanding of allergenicity, to characterize new allergens and define their relationship with other allergens. This detailed knowledge was used to formulate modified allergen constructs for improved diagnosis and vaccination of allergic patients