Allergen-specific anergisation with pseutotyped vesicles

It is well established that T lymphocytes play a central role in the pathogenesis of allergic disease and that modulation of T lymphocyte function can ameliorate symptoms. In order to elicit a curative response in type I allergy we aim at exclusively targeting allergen specific T lymphocytes by delivering to them preformed `packets of information` with an anergizing potential. Antigen-presenting cells, e.g. dendritic cells, constitutively produce subcellular particles called exosomes, which can be used to elicit an immune response in vivo. In this project we plan to bypass antigen-presenting cells and we will instead explore the usefulness of pseudotyped microvesicles as surrogate antigen presenting particles to induce, modulate or abrogate (i.e. `anergosomes`) allergen-specific immune responses. We have shown recently that assembly and budding of enveloped viruses takes place in special, hydrophobic compartments of the plasma membrane of producer cells, called lipid rafts. For the generation of `anergosomes` we will take advantage of these principles. Microvesicle formation in producer cells will be induced by the expression of viral core-proteins derived from enveloped viruses, e.g. Moloney murine leukemia virus, HIV or Influenza. Relevant receptor molecules will be targeted to the microvesicle surface by introduction of posttranslational lipid modification sites into their amino acid sequence. Such anergy-inducing microvesicles would lack classical co- stimulators for T cell activation. Additionally, we will make anergosomes to express ligands for inhibotory receptors on T lymphocytes. To achieve allergen-specificity within the system, `anergosomes` will have to express a histocompatible MHC Class II molecule and an allergen-derived peptide capable of binding to the appropriate restriction element. The relevant MHC molecules will be over-expressed in the producer cell line and will be modified with membrane microdomain (lipid raft) targeting sequences (from e.g. glycosyl phosphatidyl inositol (GPI) - anchored molecules, CD4, or the phosphoprotein associated with glycosphingolipid-enriched microdomains (PAG)). The allergenic peptides will be expressed as fusion-genes (e.g. with the invariant chain), which should guarantee their access to the HLA Class II peptide loading compartment - the MIIC - and lead to homogeneous loading of HLA Class II heterodimers. The presence of invariant chains in the producer cells will also target the HLA Class II molecules to the MIIC. Allergen specific peripheral blood T cells or T cell clones derived from patients with defined allergies (e.g. Bet v 1) will serve as the read-out system. The proposed approach will avoid the administration of soluble whole allergenic proteins or of soluble allergen-derived T cell peptide epitopes. Both forms can cause adverse reactions in vivo, such as anaphylactic shock or asthmatic reactions. The project aims at evaluating the feasibility of the proposed anergosome-approach in an allergen-specific model in vitro. In the case that promising results can be obtained we will explore the possibility to modulate allergen- specific immune responses in vivo in further studies.

During the last decades the prevalence of Type I allergies has markedly increased. Today, diseases like atopic asthma, atopic dermatitis and allergic rhinoconjunctivitis account for an important cause of morbidity and impaired quality of life. The sharp increase of allergic disorders demands for efficient therapeutic interventions. Allergen- specific T lymphocytes have a central role in the pathogenesis of allergic diseases. In allergic patients, an overwhelming Th2 response results in the production of allergen-specific IgE antibodies. Furthermore, allergen- specific T cells induce late phase reactions and allergic inflammation in target organs, e.g. the lung and skin. In this project we have bypassed classical antigen presenting cells and we have instead explored the usefulness of pseudotyped virus-like particles (VLP) as surrogate antigen presenting platforms to influence allergen-specific immune responses. For this purpose we have developed a system for the generation of VLP with immunomodulatory capacity, i.e. immunosomes. Immunosomes are lipid raft enriched, cellular plasma membrane derived microvesicles, which bud from producer cells by virtue of introduced core proteins from enveloped viruses. The replacement of transmembrane/intracellular domains with glycosylphosphatidylinositol (GPI) anchor attachment sequences helps to target relevant immune receptors, such as MHC and immunomodulatory molecules, to lipid rafts and consequently to immunosomes. Within this project we have succeeded in creating cell free, modular antigen presenting platforms that fulfill the tasks of professional antigen presenting cells. Such immunosomes have been equipped and functionally evaluated with a considerable number of immunostimulatory molecules or combinations thereof in T lymphocyte activation assays as well as phenotypically by Western blots, ELISA and electron microscopy. Immunosomes appear as 100 nm-sized vesicles in the particulate fraction of cell culture supernatants, and consist mainly of lipid raft resident molecules. They show an impressive in vitro T cell stimulatory capacity, both in polyclonal and antigen-specific T cell stimulation models. T cell stimulation is accessory-cell independent it depends, however, on secondary signals such as co-stimulation and adhesion and can even be augmented by membrane-bound cytokines. To obtain HLA- Class II specific VLP we have successfully cloned and expressed the required restriction elements - able to present allergen-specific model antigens - first on artificial antigen presenting cells and then on VLP. Furthermore, we have learned how to express such antigens in an immunogenic way, with the help of invariant-chain based constructs. The research within this project has laid the ground to influence the allergen specific immune response at the level of antigen presentation with the help of a novel modular antigen-presenting platform.