Recombinant allergens for specific immunotherapy Cromwell, Oliver, PhD; Häfner, Dietrich, MD; Nandy, Andreas, PhD
Journal of allergy and clinical immunology,
04/2011, Letnik:
127, Številka:
4
Journal Article
Recenzirano
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Recombinant DNA technology provides the means for producing allergens that are equivalent to their natural counterparts and also genetically engineered variants with reduced IgE-binding activity. The ...proteins are produced as chemically defined molecules with consistent structural and immunologic properties. Several hundred allergens have been cloned and expressed as recombinant proteins, and these provide the means for making a very detailed diagnosis of a patient’s sensitization profile. Clinical development programs are now in progress to assess the suitability of recombinant allergens for both subcutaneous and sublingual immunotherapy. Recombinant hypoallergenic variants, which are developed with the aim of increasing the doses that can be administered while at the same time reducing the risks for therapy-associated side effects, are also in clinical trials for subcutaneous immunotherapy. Grass and birch pollen preparations have been shown to be clinically effective, and studies with various other allergens are in progress. Personalized or patient-tailored immunotherapy is still a very distant prospect, but the first recombinant products based on single allergens or defined mixtures could reach the market within the next 5 years.
Despite the efficacy of allergen-specific immunotherapy (AIT), the role of trained immunity and tolerance in this process has not been elucidated.
Here, we have performed a comprehensive longitudinal ...analysis of the systemic innate immune cell repertoire during the course of AIT.
Patients with allergy received standard preseasonal subcutaneous AIT with allergoids to birch and/or grass. Healthy controls were monitored without any intervention. Flow cytometry of innate lymphoid cell (ILC), natural killer cell, monocyte cell, and dendritic cell (DC) subsets was performed at baseline, 3 months (birch season), 6 months (grass seasons), and 12 months after the therapy in patients or at similar seasonal time points in controls. Additional analyses were performed in the third-year birch and grass season.
We observed a durable decrease in group 2 ILCs and an increase of group 1 ILCs after AIT, with dynamic changes in their composition. We found that an expansion of CD127+CD25++ clusters caused observed shifts in the heterogeneity of group 1 ILCs. In addition, we observed development of CD127+CD25++c-Kit+ group 3 ILC clusters. Moreover, we found an increase in the number of intermediate monocytes in parallel with a reduction in nonclassical monocytes during the first year after AIT. Classical and intermediate monocytes presented significant heterogeneity in patients with allergy, but AIT reduced the HLA-DR++ clusters. Finally, an increase in plasmacytoid DCs and CD141+ myeloid DCs was observed in individuals with allergy, whereas the number of CD1c+ myeloid DCs was reduced during the first year of AIT.
AIT induces changes in the composition and heterogeneity of circulating innate immune cells and brings them to the level observed in healthy individuals. Monitoring of ILCs, monocytes, and DCs during AIT might serve as a novel biomarker strategy.
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Background Phl p 4 is a major pollen allergen but exhibits lower allergenicity than other major allergens. The natural protein is glycosylated and shows cross-reactivity with related and structurally ...unrelated allergens. Objective We sought to determine the high-resolution crystal structure of Phl p 4 and to evaluate the immunologic properties of the recombinant allergen in comparison with natural Phl p 4. Methods Different isoallergens of Phl p 4 were expressed, and the nonglycosylated mutant was crystallized. The specific role of protein and carbohydrate epitopes for allergenicity was studied by using IgE inhibition and basophil release assays. Results The 3-dimensional structure was determined by using x-ray crystallography at a resolution of 1.9 Å. The allergen is a glucose dehydrogenase with a bicovalently attached flavin adenine dinucleotide. Glycosylated and nonglycosylated recombinant Phl p 4 showed identical inhibition of IgE binding, but compared with natural Phl p 4, all recombinant isoforms displayed a reduced IgE-binding inhibition. However, the recombinant protein exhibited an approximately 10-fold higher potency in basophil release assays than the natural protein. Conclusion The crystal structure reveals the compact globular nature of the protein, and the observed binding pocket implies the size of the natural substrate. Plant-derived cross-reactive carbohydrate determinants (CCDs) appear to reduce the allergenicity of the natural allergen, whereas the Pichia pastoris –derived glycosylation does not. Our results imply yet undescribed mechanism of how CCDs dampen the immune response, leading to a novel understanding of the role of CCDs.
Background The generation and maintenance of allergen-specific T-cell tolerance is a key step in healthy immune responses to allergens and successful allergen-specific immunotherapy. Breaking of ...peripheral T-cell tolerance to allergens can lead to the development of allergies, but the mechanisms are not completely understood. Objective We sought to identify molecular mechanisms that break allergen-specific T-cell tolerance in human subjects. Methods Proliferative responses of allergen-specific T cells from tonsils and peripheral blood were measured by using tritiated thymidine incorporation and carboxyfluorescein succinimidyl ester (CFSE) dilution experiments. Cytokine levels in cell-free supernatants were quantified by using the cytometric bead array, and mRNA expression of transcription factors and cytokines was determined by using quantitative PCR. Myeloid dendritic cells (DCs) were characterized by using flow cytometry. Results In allergic patients the immune profile of the tonsils represents the atopic status of patients, with low expression of the TH 1 cell-specific transcription factor T-bet and the cytokine IFN-γ, as well as IL-10. Human tonsils show very low levels of allergen-induced T-cell proliferation, thus representing a very suitable in vivo model to assess mechanisms of breaking allergen-specific T-cell tolerance. Triggering of Toll-like receptor (TLR) 4 or TLR8 and the proinflammatory cytokines IL-1β or IL-6 break allergen-specific T-cell tolerance in human tonsils and peripheral blood through a mechanism dependent on the adaptor molecule myeloid differentiation primary response gene (88) (MyD88). In particular, myeloid DCs and stimulations that activate them broke the tolerance of allergen-specific CD4+ T cells, whereas plasmacytoid DCs and stimulations that activate them, such as TLR7 and TLR9, did not have any effect. Tolerance-breaking conditions induced by different molecular mechanisms were associated with a mixed cytokine profile with a tendency toward increased levels of IL-13 and IL-17, which are TH 2 and TH 17 cytokines, respectively. Conclusion Certain innate immune response signals and proinflammatory cytokines break allergen-specific CD4+ T-cell tolerance in normally unresponsive subjects, which might lead to the development or exacerbation of allergic diseases after encountering microbes or inflammatory conditions.
Here we show that scavenger receptor class B type I is present in the small-intestine brush border membrane where it facilitates the uptake of dietary cholesterol from either bile salt micelles or ...phospholipid vesicles. This receptor can also function as a port for several additional classes of lipids, including cholesteryl esters, triacylglycerols, and phospholipids. It is the first receptor demonstrated to be involved in the absorption of dietary lipids in the intestine. In liver and steroidogenic tissues, the physiological ligand of this receptor is high-density lipoprotein. We show that binding of high-density lipoprotein and apolipoprotein A-I to the brush border membrane-resident receptor inhibits uptake of cholesterol (sterol) into the brush border membrane from lipid donor particles. This finding lends further support to the conclusion that scavenger receptor BI catalyzes intestinal cholesterol uptake. Our findings suggest new therapeutic approaches for limiting the absorption of dietary cholesterol and reducing hypercholesterolemia and the risk of atherosclerosis.
•Beginning and evolution of the official Allergen Nomenclature system 1980–2018.•Allergen Names abbreviated genus, species and number.•Expected data including characterization of protein amino acid ...sequence, cDNA, human serum donors and experimental data.•Challenges of identifying allergens including exposure and complex human exposure and immunity.•Complexity of new methods including “omics”.
A systematic nomenclature for allergens originated in the early 1980s, when few protein allergens had been described. A group of scientists led by Dr. David G. Marsh developed a nomenclature based on the Linnaean taxonomy, and further established the World Health Organization/International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-Committee in 1986. Its stated aim was to standardize the names given to the antigens (allergens) that caused IgE-mediated allergies in humans. The Sub-Committee first published a revised list of allergen names in 1986, which continued to grow with rare publications until 1994. Between 1994 and 2007 the database was a text table online, then converted to a more readily updated website. The allergen list became the Allergen Nomenclature database (www.allergen.org), which currently includes approximately 880 proteins from a wide variety of sources. The Sub-Committee includes experts on clinical and molecular allergology. They review submissions of allergen candidates, using evidence-based criteria developed by the Sub-Committee. The review process assesses the biochemical analysis and the proof of allergenicity submitted, and aims to assign allergen names prior to publication. The Sub-Committee maintains and revises the database, and addresses continuous challenges as new “omics” technologies provide increasing data about potential new allergens. Most journals publishing information on new allergens require an official allergen name, which involves submission of confidential data to the WHO/IUIS Allergen Nomenclature Sub-Committee, sufficient to demonstrate binding of IgE from allergic subjects to the purified protein.
...it seems advisable to select adjuvants for new AIT protocols1,E25,E26 for their ability to promote sialylated IgG(4) antibody responses. Peak identity was confirmed by analyzing the collected peak ...fractions by means of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, as previously described.E35,E38 Glycans with human or murine sialic acids (human N-acetylneuraminic acid or murine N-glycolylneuraminic acid) had different retention times. Mouse IgG antibodies rarely have a bisecting GlcNAc, whereas 10% to 15% of human IgG antibodies have a bisecting GlcNAc. Because more Fc glycans of total serum IgG from untreated C57BL/6 mice are sialylated than pooled serum IgG from healthy human donors (IVIG), percentages of sialylation of murine and human IgG antibodies cannot be directly compared. In all experiments normal distribution was assumed. 1 C.A. Akdis, M. Akdis, Advances in allergen immunotherapy: aiming for complete tolerance to allergens, Sci Transl Med, Vol. 7, 2015, 280ps6 2 F.D. Finkelman, M.V. Khodoun, R. Strait, Human IgE-independent systemic anaphylaxis, J Allergy Clin Immunol, Vol. 137, 2016, 1674-1680 3 R.T. Strait, S.C. Morris, F.D. Finkelman, IgG-blocking antibodies inhibit IgE-mediated anaphylaxis in vivo through both antigen interception and Fc gamma RIIb cross-linking, J Clin Invest, Vol. 116, 2006, 833-841 4 H. Beutier, C.M. Gillis, B. Iannascoli, O. Godon, P. England, R. Sibilano, IgG subclasses determine pathways of anaphylaxis in mice, J Allergy Clin Immunol, Vol. 139, 2017, 269-280.e7 5 C.M. Oefner, A. Winkler, C. Hess, A.K. Lorenz, V. Holecska, M. Huxdorf, Tolerance induction with T cell-dependent protein antigens induces regulatory sialylated IgGs, J Allergy Clin Immunol, Vol. 129, 2012, 1647-1655 6 C. Hess, A. Winkler, A.K. Lorenz, V. Holecska, V. Blanchard, S. Eiglmeier, T cell-independent B cell activation induces immunosuppressive sialylated IgG antibodies, J Clin Invest, Vol. 123, 2013, 3788-3796 7 M. Collin, M. Ehlers, The carbohydrate switch between pathogenic and immunosuppressive antigen-specific antibodies, Exp Dermatol, Vol. 22, 2013, 511-514 8 A. Pincetic, S. Bournazos, D.J. DiLillo, J. Maamary, T.T. Wang, R. Dahan, Type I and type II Fc receptors regulate innate and adaptive immunity, Nat Immunol, Vol. 15, 2014, 707-716 9 C. Möbs, H. Ipsen, L. Mayer, C. Slotosch, A. Petersen, P.A. Würtzen, Birch pollen immunotherapy results in long-term loss of Bet v 1-specific TH2 responses, transient TR1 activation, and synthesis of IgE-blocking antibodies, J Allergy Clin Immunol, Vol. 130, 2012, 1108-1116.e6
Allergens produced by recombinant DNA technology have the ability to improve allergy diagnosis and are used as reference standards for analytical methods. In addition, the use of recombinant ...allergens in specific immunotherapy has long been considered potentially superior compared with the use of conventional extracts. The advantages are clear: a complex natural substance that is difficult to characterize is replaced by only those components relevant for treatment, which furthermore can be reproduced in pharmaceutical quality. The challenges faced here include selecting the relevant allergen molecules and establishing a manufacturing that meets all the regulatory requirements for marketing authorization. In addition to unmodified recombinant allergens, hypoallergenic variants with lower IgE reactivity can also be made by genetic engineering. Proof of concept has been demonstrated for both these approaches in clinical trials.
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