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Background: Current s.c. allergen-specific immunotherapy (SIT) leads to amelioration of IgE-mediated allergy, but it requires numerous allergen injections over several years and is frequently associated with severe side-effects. The aim of this study was to test whether modified recombinant allergens can improve therapeutic efficacy in SIT while reducing allergic side-effects.
Methods: The major cat allergen Fel d 1 was fused to a TAT-derived protein translocation domain and to a truncated invariant chain for targeting the MHC class II pathway (MAT-Fel d 1). The immunogenicity was evaluated in mice, while potential safety issues were assessed by cellular antigen stimulation test (CAST) using basophils from cat-dander-allergic patients.
Results: MAT-Fel d 1 enhanced induction of Fel d 1-specific IgG2a antibody responses as well as the secretion of IFN-γ and IL-2 from T cells. Subcutaneous allergen-specific immunotherapy of mice using the modified Fel d 1 provided stronger protection against anaphylaxis than SIT with unmodified Fel d 1, and MAT-Fel d 1 caused less degranulation of human basophils than native Fel d 1.
Conclusion: MAT-Fel d 1 allergen enhanced protective antibody and Th1 responses in mice, while reducing human basophil degranulation. Immunotherapy using MAT-Fel d 1 allergen therefore has the potential to enhance SIT efficacy and safety, thus, shortening SIT. This should increase patient compliance and lower treatment costs.
Subcutaneous allergen-specific immunotherapy (SIT) is the only causal treatment of allergies, has a long-lasting effect and can stop progression of the allergy to multiple sensitizations or to asthma. Subcutaneous SIT typically requires 50–70 injections during 3–5 years (1) and bears a significant risk of allergic side-effects including anaphylaxis. Hence, the number of injections and also the risk of allergic side-effects should be reduced. Current efforts to enhance SIT focus on optimizing the allergen molecules (2–4), the adjuvants (5, 6), the route of administration (7–9), and also their dosage form, including use of particulate delivery systems, such as virus-like (10) or poly(lactide-co-glycolide) particles (11).
Approaches to increase the safety of SIT include pretreatment with anti-histamines or anti-IgE antibodies (12). Safety can also be improved by disrupting IgE-binding epitopes by heat denaturation (13), by chemical modifications to generate allergoids (14), using genetically engineered allergens with reduced IgE-binding capacity (2, 3), or using synthetic peptides that do not bind IgE but stimulate T-cell responses (4). However, reduced IgE-binding has often been followed by reduced immunogenicity of the allergens (15).
We have recently proposed a new concept for allergy vaccines based on targeting the MHC class II antigen presentation pathway (16). These so-called modular antigen transporter (MAT) recombinant allergens consist of an allergen fused to a TAT-derived translocation peptide and to the first 110 amino acids of the human invariant chain (Ii). The TAT peptide mediates cytoplasmic uptake of extracellular proteins (17, 18). Small molecules are believed to enter cells via electrostatic interactions in an energy-independent manner, whereas large molecules are taken up by energy-dependent macropinocytosis (19). Early in biosynthesis, MHC class II αβ heterodimers assemble in the endoplasmic reticulum with an Ii trimer to form a nonameric complex (20). Ii binds to the MHC class II molecule and blocks the class II peptide-binding groove until the MHC II–Ii complexes are transported to the endosomes where Ii is removed by proteolysis, thus permitting loading of the groove with endosomal peptides (21). Fusing the allergen to Ii therefore directly links the allergen with the class II pathway of antigen presentation.
In this study, we used Fel d 1 as a model allergen and tested the influence of the above described modifications on immune responses in vivo. To avoid potential differences in distribution and pharmacokinetics of the different proteins, the allergens were administered directly into the inguinal lymph nodes of mice (22).
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Although SIT offers medical advantages over symptomatic treatments of IgE-mediated allergies, merely 3–4% of allergic patients choose to undergo SIT, mainly because of the long treatment duration and the frequently associated allergic side-effects. For use in SIT it would therefore be highly desirable to have allergens that enhance efficacy and at the same time reduce allergic side-effects, i.e. to render SIT faster and safer. In the present study, different modified recombinant Fel d 1 allergens were tested for their therapeutic potential in SIT. The allergens were in part modified by fusing them to a protein translocation sequence to improve cellular uptake and a truncated invariant chain for targeting to the MHC class II pathway of antigen presentation (16).
Subcutaneous allergen-specific immunotherapy aims at regulating allergen-specific immune responses, i.e. it aims to trigger Th1 T cells and neutralizing antibody responses. The presence of Th1 cytokines such as IFN-γ inhibits the production of IL-4 and thereby prevents the IL-4-mediated IgE-switch of B cells. IgE enhances the expression of high affinity IgE receptors (FcεRI) (23), hence, allergy is self-promoting. Our study revealed that mice immunized with MAT-Fel d 1 had higher levels of IFN-γ and lower levels of IL-4 than mice immunized with the unmodified Fel d 1 allergen. In line with this, immunotherapy of sensitized mice using MAT-Fel d 1 induced higher IgG2a levels and conferred better protection against a challenge with a high dose of the cat-fur allergen extract; enhanced production of Th1-assisted antibodies, IgG2a in mice and IgG1 and IgG4 antibodies in humans (24), have been reported after SIT (13).
In addition to being more efficient in stimulating Th1 immune responses, our study revealed that MAT-Fel d 1 also reduced basophil degranulation and leukotriene release by 100-fold when compared with unmodified Fel d 1 or the cat-fur allergen extract. This potentially increased safety profile of MAT-Fel d 1 was confirmed in sensitized mice that showed no anaphylaxis after a high-dose challenge with MAT-Fel d 1. This increased safety of the MAT-Fel d 1 can be explained by its reduced IgE-binding capacity (data not shown) and the more rapid cellular uptake of TAT-containing proteins (18, 25). These properties prevent the allergen from binding to FcεRI on mast cells and basophils. In conclusion, the results demonstrated that MAT-Fel d 1 not only increased SIT efficacy but also safety.
Over the last 10 years, several studies have reported enhanced presentation of antigens through the MHC class-II pathway by different strategies that involved the Ii (26–28). The so called Ii-Key peptides, consist of a short fragment of the human Ii (hIi aa 77–92 or aa 77–80) fused to an antigenic peptide (28). The Ii-Key binds to an allosteric site of the MHC-II molecules on the cell surface and the antigenic peptide binds to the epitope-binding groove, increasing the loading of MHC class II molecules and, therefore, enhancing the immunogenicity of the loaded antigen. The MAT allergens used in our study had a longer sequence of the hIi (aa 1–110). This targets the MHC class-II molecules to the endoplasmic reticulum (16) where it occupies the peptide binding groove, and thereby avoids interaction with other antigens. The complex formed by the MAT-allergen and the MHC class-II molecule is directed through the Golgi to the endo/lysosomal compartment where the Ii is degraded together with the fused allergen. Now, the allergenic peptides can be loaded to the MHC class-II molecules and be transported to the cell surface (29). As a result, MAT allergens increase the efficiency of allergen presentation, which furthermore result in an increase in the effective allergen dose available for stimulation of protective immune response. Indeed, high allergen doses suppress IgE production while low doses are supportive of its production (30), and human immunotherapy with high allergen doses is more efficient than using lower allergen doses (31). While this in theory could be advantageously used for SIT, the risk of allergic adverse events limits the possibility of increasing the therapeutic allergen dose and explains the need for a controlled dose-escalation during the early phase of SIT. However, the safety profile as well as the cell-penetration and MHC-targeting properties of MAT allergens may allow SIT with high allergen doses, making such allergen highly attractive in human therapy.
This study, as well as other studies in mice (22) and humans (manuscript in preparation), also demonstrated that i.l. allergen administration may strongly improve SIT efficacy and allow lower allergen doses to be used, which again would improve safety as compared with s.c. SIT. Alternative routes of allergen administration, e.g. sublingual (SLIT), oral and intranasal applications (7–9) have been introduced as a means to improve SIT. However, as most proteins have short half-lives, most of such administered allergens are degraded before exerting their immunotherapeutic effects. For this reason, i.l. injections, in which the allergen is administered directly into a s.c. lymph node, i.e. to the site of action on the immune system, represent an advantage over other administration routes.
In conclusion, our data demonstrated that the two main problems of current SIT, its relatively low efficiency requiring a high number of injections, as well as the risk of allergic reactions, could both be improved by using MAT allergens.