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

Displaying Fel d1 on virus-like particles prevents reactogenicity despite greatly enhanced immunogenicity: a novel therapy for cat allergy

Allergen-specific desensitization is the only disease-modifying therapy currently available for the treatment of allergies. These therapies require application of allergen over several years and some may induce life-threatening anaphylactic reactions. An ideal vaccine for desensitization should be highly immunogenic and should alleviate allergic symptoms upon few injections while being nonreactogenic. We describe such a vaccine for the treatment of cat allergy, consisting of the major cat allergen Fel d1 coupled to bacteriophage Qβ-derived virus-like particles (Qβ–Fel d1). Qβ–Fel d1 was highly immunogenic, and a single vaccination was sufficient to induce protection against type I allergic reactions. Allergen-specific immunoglobulin G antibodies were shown to be the critical effector molecules and alleviated symptoms by two distinct mechanisms. Although allergen-induced systemic basophil degranulation was inhibited in an FcγRIIb-dependent manner, inhibition of local mast cell degranulation in tissues occurred independently of FcγRIIb. In addition, treatment with Qβ–Fel d1 abolished IgE memory responses upon antigen recall. Despite high immunogenicity, the vaccine was essentially nonreactogenic and vaccination induced neither local nor systemic anaphylactic reactions in sensitized mice. Moreover, Qβ–Fel d1 did not induce degranulation of basophils derived from human volunteers with cat allergies. These data suggest that vaccination with Qβ–Fel d1 may be a safe and effective treatment for cat allergy

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

Recombinant cat and mite allergens as tools in the study of diagnosis and treatment of allergy

Allergic diseases, such as allergic asthma, allergic rhinitis and atopic eczema, are a major health problem world-wide, affecting up to 30% of the population. A large proportion of the patients are allergic to indoor allergens, such as mite and cat allergens, which are airborne and thus cause symptoms in the airways upon inhalation. Hundreds of single allergens from different sources have been identified, cloned and expressed as highly pure and well-defined recombinant allergens. They can be produced in large amounts and are gradually replacing allergen extracts in allergy diagnosis and allergen-specific immunotherapy (SIT), the only treatment that may affect the natural course of allergic diseases. SIT may be further improved by genetically modifying allergens into hypoallergens, which have a reduced allergenic capacity, but a retained T-cell reactivity. The aims of this thesis were to clone and characterise new allergens from the storage mite Lepidoglyphus destructor, and to apply the major recombinant cat allergen Fel d 1 in allergy diagnosis and in the construction and evaluation of hypoallergens for use in therapy of cat allergy. In paper I, PCR and screening with sera were used to identify and isolate new allergen clones from a phage display cDNA library that was constructed previously from L. destructor. Two new clones, showing homology to tropomyosin and α-tubulin, were obtained and subsequently expressed in Escherichia coli. The recombinant proteins bound to IgE antibodies in sera of mite-allergic patients and were characterised as putative minor allergens Lep d 10 and L. destructor α-tubulin. Lep d 10 was found to be cross-reactive with tropomyosins from other mite species and crustaceans. In paper II, the previously well-characterised major recombinant cat allergen rFel d 1 was compared to cat dander extract (CDE) in the diagnosis of cat allergy in children of a large prospective birth cohort. The rFel d 1 was at least as good as CDE in detecting allergen-specific IgE and may be a better marker for early cat sensitisation. The study showed that children with allergen-specific IgE, but without symptoms to cat at 4 years of age all developed allergic symptoms at 8 years of age. In paper III, the three-dimensional structure of rFel d 1 was genetically altered in a rational approach comprising duplication of known T-cell epitopes and disruption of disulphide bonds through introduction of point mutations. Three out of seven Fel d 1 derivatives generated were identified as hypoallergens with a strongly reduced IgE-binding capacity, a reduced allergenicity and a retained T-cell reactivity. The most promising candidate for SIT, rFel d 1 (DTE III) was further evaluated in vivo in paper IV. Therapeutic treatment of cat-allergic mice with the hypoallergen resulted in decreased airway hyperreactivity and induction of allergen-specific IgG with blocking capacity. In contrast to unmodified rFel d 1, the hypoallergen was tolerated at a high treatment dose without any observed side effects. In addition, rFel d 1 (DTE III) induced less skin prick test-reactivity compared to rFel d 1 in cat-allergic patients. In summary, the results of this thesis show the wide usage of recombinant allergens. They are important in the characterisation of single allergens of allergen sources, as shown for the mite L. destructor. Furthermore, they reveal sensitisation to a specific allergen component, e.g. the major cat allergen Fel d 1, which in respect to diagnosis of cat allergy seems to be at least as good as CDE. In addition, recombinant allergens can be used in the development of safer and more efficient tools for SIT, as demonstrated with Fel d 1 (DTE III)

The cat lipocalin Fel d 7 and its cross-reactivity with the dog lipocalin Can f 1

We investigated the prevalence of sensitization to the cat lipocalin Fel d 7 among 140 cat-sensitized Swedish patients and elucidated its allergenic activity and cross-reactivity with the dog lipocalin Can f 1. Sixty-five of 140 patients had IgE to rFel d 7 whereof 60 also had IgE to rCan f 1. A moderate correlation between IgE levels to rFel d 7 and rCan f 1 was found. rFel d 7 activated basophils in vitro and inhibited IgE binding to rCan f 1 in 4 of 13 patients, whereas rCan f 1 inhibited IgE binding to rFel d 7 in 7 of 13 patients. Fel d 7 and Can f 1 showed high similarities in protein structure and epitopes in common were found using cross-reactive antisera. Fel d 7 is a common allergen in a Swedish cat-sensitized population that cross-reacts with Can f 1, and may contribute to symptoms in cat-but also in dog-allergic patients