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

Construction of an sIgE:FLAG-mIgE:GFP Reporter Mouse Strain

Like all other immunoglobulins, IgE can be secreted into the blood or expressed as a membrane receptor on the surface of B lymphocytes. Secreted immunoglobulins trace the antigen and contribute to its destruction. Membrane immunoglobulins accompany the B cell along its differentiation pathway, regulating processes like the induction and maintenance of immunological memory and differentiation of plasma cells. The regulation of the expression of IgE is very complex. A lot of positive and negative regulators influence the synthesis of IgE. In previous publications, we were able to show that the membrane IgE (mIgE) antigen receptor itself controls the quantity and quality of serum IgE produced. However, the knowledge about the regulatory function of the antigen receptor on these processes is at best limited. In the present paper, we present the construction of a reporter mouse strain, which will help us to follow an mIgE-bearing B cell during the immune response more precisely