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

but Reduced B Cell and T Cell Activation Capacity. PLoS ONE 6(9): e24558. doi:10.1371/journal.pone.0024558 In Vitro Evolution of Allergy Vaccine Candidates, with Maintained Structure, but Reduced B Cell and T Cell Activation Capacity Ola B. Nilsson 0 Justus Adedoyin 0 Claudio Rhyner 0 Theresa Neimert-Andersson 0 Jeanette Grundstro m 0 Kurt D. Berndt 0 Reto Crameri 0 Hans Gro nlund 0 Clive M. Gray, University of Cape Town, South Africa 0 1 Department of Medicine, Karolinska Institutet, Stockholm, Sweden, 2 Swiss Institute of Allergy and Asthma Research (SIAF), University of Zu rich, Davos, Switzerland, 3 Department Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden, 4 Center for Allergy Research, Karolinska Institutet , Stockholm , Sweden 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. - Funding: This work was supported in part by The Swedish Asthma and Allergy Associations Research Foundation, the Consul Th. C. Berghs Foundation, the Cancer and Allergy Foundation, the Centre for Allergy Research at Karolinska Institutet and the Bernard Osher Initiative for Research on Severe Asthma. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The domestic cat (Felis domesticus) is one of the most frequent pets and approximately 10% of the general population in industrialized countries is sensitized to cat allergens [1]. The symptoms deriving from cat allergy manifest mainly as rhinoconjunctivitis with a strong tendency to progress to asthma, especially in children [2]. Exposure to cat allergen is a result of the natural behavior of the cat to lick and groom. Proteins of the saliva is left to dry and spread as small airborne particles [3] where it can be detected in a variety of indoor environments [4,5]. The major allergen of the cat, formally termed Fel d 1, is a hetero-dimer member of the secretoglobin protein family [6,7] found in skin, lachrymal glands and in particular in the saliva. More than 95% of cat allergic patients show elevated serum IgE levels to Fel d 1 which is, therefore, the primary target for the development of immunotherapeutic vaccines for the treatment of cat allergy [8,9]. Allergen-specific IgE is the key molecule for the development of allergic symptoms. The synthesis of IgE requires a B cell to undergo class switch recombination in close contact with allergen-specific T helper 2 cells (Th2) [10]. The T cell receptor on primed CD4+ T cells recognize antigen presenting cells carrying MHC II molecules with tightly bound enzyme-digested linear polypeptides of 12 to 25 amino acids derived from the primary structure of the antigen [11]. B cells expressing allergen-specific IgE as a part of the B cell receptor (BCR) recognize the surface structures of the allergens. After allergen binding, the receptor-antigen complex is internalized, the antigen processed, and displayed as peptide-MHC II complex on the B cell surface [12]. These processes link the 3D structure of the allergen to the linear peptides presented to the T cell receptor by the MHC class II complex. Allergen-activated T cells are responsible for late-phase reactions (LPR), occurring 68 hours after allergen challenge eliciting symptoms ranging from oedema and itching to eczema, and also play a prominent and pivotal role in the pathogenesis of allergic asthma [13] as shown by the elevated numbers of CD4+ T cells found in bronchial mucosa, BAL fluid, and sputum in these patients. The most common treatment of allergic diseases is either alleviation of symptoms using drugs or advice the patient to avoid the allergen source [14,15]. However, allergen-specific immunotherapy (SIT), is the only treatment able to cure allergic diseases [16]. Successful SIT is thought to act through tolerance mechanisms induced by regulatory T cells and blocking IgG antibodies [17]. Induction of IgG antibodies may reduce clinical symptoms in several ways, by competition with IgE for binding epitopes on the allergens [18,19], by inhibition of IgE-facilitated antigen presentation to CD4+ T cells [20], but also by interference with mast cell degranulation by down-regulation of IgE receptors signaling via inhibitory motifs on the FccRIIb receptor [21]. Numerous studies have shown that crude allergen extracts currently used in SIT are clinically effective [22], a high allergen dose is more effective [23], although the potential risk of severe acute side effects is a limiting factor [24]. Attenuated allergenic molecules, i.e. hypoallergens or synthetic peptide fragments have been used as high dose and safer alternatives to conventional extract-based SIT [25,26]. However, such treatments have also been limited by recurring side effects, such as LPR [27,28]. T cell epitope (TCE) mappings of Fel d 1 have shown a scattered distribution of TCE located on both chains of Fel d 1 [29]. The relevance of T cells specific for Fel d 1 in cat allergic patients has been evaluated, where short overlapping peptides covering the major TCE of Fel d 1 elicited adverse effects, such as allergic rhinitis and late asthmatic reactions [28,30], despite the fact that the peptides were unable to bind IgE, and thus elicit immediate allergic react (...truncated)