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Background: Human IgE (hIgE) mAbs against major mite allergen Der p 2 developed using human hybridoma technology were used for IgE epitope mapping and analysis of epitopes associated with the hIgE repertoire., Objective: We sought to elucidate the new hIgE mAb 4C8 epitope on Der p 2 and compare it to the hIgE mAb 2F10 epitope in the context of the allergenic structure of Der p 2., Methods: X-ray crystallography was used to determine the epitope of anti-Der p 2 hIgE mAb 4C8. Epitope mutants created by targeted mutagenesis were analyzed by immunoassays and in vivo using a human high-affinity IgE receptor (FcεRIα)-transgenic mouse model of passive systemic anaphylaxis., Results: The structure of recombinant Der p 2 with hIgE mAb 4C8 Fab was determined at 3.05 Å. The newly identified epitope region does not overlap with the hIgE mAb 2F10 epitope or the region recognized by 3 overlapping hIgE mAbs (1B8, 5D10, and 2G1). Compared with wild-type Der p 2, single or double 4C8 and 2F10 epitope mutants bound less IgE antibodies from allergic patients by as much as 93%. Human FcεRIα-transgenic mice sensitized by hIgE mAbs, which were susceptible to anaphylaxis when challenged with wild-type Der p 2, could no longer cross-link FcεRI to induce anaphylaxis when challenged with the epitope mutants., Conclusions: These data establish the structural basis of allergenicity of 2 hIgE mAb nonoverlapping epitopes on Der p 2, which appear to make important contributions to the hIgE repertoire against Der p 2 and provide molecular targets for future design of allergy therapeutics., Competing Interests: Disclosure statement Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) (award nos. R01AI077653-13 [to A.P., M.D.C., and M.C.] and R01AI155668 and R21AI123307 [to S.A.S.]). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The research was supported in part by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences (Z01-ES102906 [to G.A.M.]). The structural results shown in this report are derived from data collected at Southeast Regional Collaborative Access Team (SER-CAT; 22ID) beamline at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions may be found at www.ser-cat.org/members.html. Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences (contract nos. DE-AC02-06CH11357 and W-31-109-Eng-38). This work was partially supported by an ASPIRE III grant from the Office of the Vice President of Research at the University of South Carolina. This research is funded by the above mentioned NIH/NIAID award to InBio. Disclosure of potential conflict of interest: A. Pomés is an employee of InBio; and is the contact principal investigator of the NIH R01 award that provided funding for the study. M.D. Chapman is an employee of InBio; has a financial interest in InBio; and is a co-investigator on the NIH R01 award. A. Ball is an employee of InBio. The hIgE mAbs and some of the allergens described herein were produced by InBio. InBio has a license agreement with the VUMC for commercialization of hIgE mAbs for research and diagnostic purposes. The hIgE mAbs covered by this agreement are available from InBio (www.inbio.com). S.A. Smith is an inventor on US patent 10908168-B2 for generation of hIgE mAbs; has received patent royalties; and has related patents pending. The rest of the authors declare that they have no relevant conflicts of interest., (Copyright © 2024 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Background: Sensitization to house dust mite allergens is strongly correlated with asthma. Der p 7 elicits strong IgE antibody and T-cell responses in patients with mite allergy. However, the structure and biological function of this important allergen are unknown. Allergen function might contribute to allergenicity, as shown for the protease activity of group 1 mite allergens and the interaction with the innate immune system by group 2 mite allergens., Objective: We sought to determine the crystal structure of Der p 7 and to investigate its biological function., Methods: X-ray crystallography was used to determine the Der p 7 structure. Nuclear magnetic resonance analysis and biochemical assays were used to examine the binding of Der p 7 to predicted ligands., Results: Der p 7 has an elongated structure, with two 4-stranded antiparallel beta-sheets that wrap around a long C-terminal helix. The fold of Der p 7 is similar to that of LPS-binding protein (LBP), which interacts with Toll-like receptors after binding LPS and other bacterially derived lipid ligands. Nuclear magnetic resonance and biochemical assays indicate that Der p 7 does not bind LPS but binds with weak affinity to the bacterial lipopeptide polymyxin B in the predicted binding site of Der p 7., Conclusions: Der p 7 binds a bacterially derived lipid product, a common feature of some allergens. The finding that the group 7, as well as the group 2, mite allergens are structurally similar to different proteins in the Toll-like receptor pathway further strengthens the connections between dust mites, innate immunity, and allergy., (Copyright (c) 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.)

Background: The major house dust mite group 1 allergens Der p 1 and Der f 1, which belong to the papain-like cysteine protease family, are the most potent of indoor allergens. However, little information is available on the location of IgE epitopes., Objective: We investigated the allergenicities of recombinant proforms and mature forms of Der p 1 and Der f 1 to compare them with natural Der p 1 and Der f 1 and to obtain information on the conformational IgE-binding epitopes., Methods: Secreted pro-Der p 1 and pro-Der f 1 and their mutants without hyperglycosylation expressed in yeast were converted to mature forms. We purified the proforms and mature forms and analyzed their apparent molecular sizes and secondary structures by means of gel-filtration and circular dichroism analysis and their allergenicities by means of assays for IgE binding, IgE-binding inhibition, and basophil histamine release. The tertiary structure of pro-Der f 1 was predicted by molecular modeling., Results: The recombinant mature forms exhibited similar molecular sizes, secondary structures, and allergenicities as their natural types. On the other hand, their proforms exhibited different secondary structures and less allergenicities than the mature forms in all sera and volunteers tested. Molecular modeling revealed that the prosegment is anchored at the prosegment-binding loop and the substrate-binding cleft on the surface of the mature portion., Conclusions: Our studies indicate that the prodomains of Der p 1 and Der f 1 reduce allergenicity and that the major conformational IgE epitopes commonly found in a broad population of patients exist within the 2 regions blocked by the prosegments. Recombinant Der p 1 and Der f 1 and the findings in the present study will be the basis for allergen standardization and the design of safer and more effective allergen vaccines.

Background: Local nasal immunotherapy (LNIT) is an effective immunotherapy. Peptides derived from the group 2 allergen of Dermatophagoides pteronyssinus, Dp2 28-40 and Dp2 28-40A, and fungal immunomodulatory peptide (FIP) have been shown to act as T(H)1 potential and response-inducing adjuvant. LNIT by the use of Dp2 peptides in conjunction with FIP were investigated., Objective: We sought to determine whether Dp2-induced airway inflammation in mice could be downregulated by Dp2 peptides or a mixture of Dp2 peptides with FIP., Method: Mice were sensitized with rDp2 followed by LNIT with Dp2 peptides, FIP, or FIP and a mixture of Dp2 peptides. After intratracheal challenge with rDp2, the airway inflammation and hyperresponsiveness were determined by bronchoalveolar lavage fluid (BALF) analysis and methacholine challenge., Results: Both Dp2 peptides and FIP were able to inhibit rDp2-induced airway inflammation and airway hyperresponsiveness. An increase in IFN-gamma and a decrease in IL-5 in BALF and sera were found after LNIT with Dp2 peptides, FIP, and mixtures of both. Serum levels of TGF-beta were reduced after LNIT with FIP and Dp2 28-40. Penh values were significantly decreased after methacholine challenge in both the early and late phase., Conclusions: LNIT with allergen-derived peptides and FIP can produce an anti-inflammatory effect on allergen-induced airway inflammation. LNIT with selected peptides and FIP might be a good alternative therapy for allergic airway disease.