Recombinant Allergens Need a Reality Check

During the last 6 decades we have seen an overwhelming expansion of our knowledge on the nature of allergens. Crucial information has resulted from the development of IgE-targeting reagents, mAbs to allergens, and DNA/RNA technology for analytic as well as preparative purposes. The general availability as recombinant proteins of major allergens from most allergen source materials and the extensive structural and functional information on these recombinant proteins might tempt us to discard the need for additional studies on the properties of allergens. This view is supported by the common view that allergens belong to a small number of protein families. The situation is not so simple: the number of allergens is still growing substantially. Seeing an article titled “IgE recognition of the house dust mite allergen Der p 37 is associated with asthma”1Huang H.-J. Resch-Marat Y. Casset A. Weghofer M. Zieglmayer P. Zieglmayer R. et al.IgE recognition of the house dust mite allergen Der p 37 is associated with asthma.J Allergy Clin Immunol. 2022; 149: 1031-1043Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar in this issue of the Journal of Allergy and Clinical Immunology may evoke the reflex reaction “Oh no, not again, not another mite allergen.” The second part of the title, which states that IgE to this allergen is associated with asthma, might be of more immediate interest. The same claim has been made for other minor mite allergens, as cited by the Huang et al.1Huang H.-J. Resch-Marat Y. Casset A. Weghofer M. Zieglmayer P. Zieglmayer R. et al.IgE recognition of the house dust mite allergen Der p 37 is associated with asthma.J Allergy Clin Immunol. 2022; 149: 1031-1043Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar One plausible explanation is that specific IgE to minor mite allergens is found more often in patients with a high level of specific IgE to mite extract because the level of specific IgE to mite extract is more likely to be higher in patients with severe asthma than in patients with mild asthma. Even more interesting are the results of the reality check obtained by Huang et al.1Huang H.-J. Resch-Marat Y. Casset A. Weghofer M. Zieglmayer P. Zieglmayer R. et al.IgE recognition of the house dust mite allergen Der p 37 is associated with asthma.J Allergy Clin Immunol. 2022; 149: 1031-1043Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar Are patients significantly exposed to this allergen? Are the structure and properties of recombinant Dermatophagoides pteronyssinus 37 (rDer p 37) essentially identical to those of the allergen that patients encounter in real life, namely, natural Der p 37 (nDer p 37)? To investigate this, Huang et al1Huang H.-J. Resch-Marat Y. Casset A. Weghofer M. Zieglmayer P. Zieglmayer R. et al.IgE recognition of the house dust mite allergen Der p 37 is associated with asthma.J Allergy Clin Immunol. 2022; 149: 1031-1043Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar purified antibodies from the serum of rabbits immunized with rDer p 37 and used these antibodies for affinity purification of Der p 37 from mite extract and for allergen immune assays on house dust extracts. The exposure data are preliminary, but they indicate that the levels of extractable Der p 37 are substantially lower than the levels of Der p 1. After immunoblotting of rDer p 37, the rabbit antibodies stained strongly at 30 kDa, corresponding to the expected molecular mass of 26.4 kDa. Unexpectedly, affinity-purified nDer p 37 stained most strongly at 15 kDa. Analysis of the nDer p 37 preparations by mass spectrometry showed peaks at 25.5, 15.8, 14.1, and 8.5 kDa. Neither the strong 25.5-kDa peak nor the minor 14.1-kDa peak reacted with anti–Der p 37, and they were suggested to correspond to Der p 1 and Der p 2, respectively. The 15.8- and 8.5-kDa proteins stained with anti–Der p 37 and are assumed to be cleavage products of nDer p 37. The 15-kDa nDer p 37 fragment showed IgE reactivity comparable to that of the complete recombinant Der p 37 allergen, suggesting that it contains a major IgE epitope. A dot blotting analysis of nDer p 37 with antibodies specific for other house dust mite allergens (ie, Der p 1, Der p 2, Der p 4, Der p 5, Der p 7, Der p 10, Der p 21, and Der p 23) revealed the presence of Der p 1 and, to a lesser extent, Der p 2 in the Der p 37–containing fractions. Huang et al1Huang H.-J. Resch-Marat Y. Casset A. Weghofer M. Zieglmayer P. Zieglmayer R. et al.IgE recognition of the house dust mite allergen Der p 37 is associated with asthma.J Allergy Clin Immunol. 2022; 149: 1031-1043Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar used ELISA plates coated with anti–Der p 1 followed by incubation with nDer p 37 and detection by enzyme-labeled rabbit anti–Der p 37. They concluded that the presence of complexes consisting of Der p 1 bound to Der p 37 was the likely explanation. As the Der p 2 results were less convincing, we will focus the discussion on Der p 1 as a potential partner of Der p 37. The preliminary conclusion of the reality check is that nDer p 37 differs from rDer p 37 in at least 2 ways: it is fragmented, and it forms a complex with Der p 1. The discovery that a natural allergen is a fragment of the protein expected from its DNA/RNA sequence does not come as a big surprise. Proteolytic processing is a very common posttranslational modification. For example, Der p 1 loses an N-terminal fragment (the 9.3-kDa propeptide) before it becomes the fully active 25-kDa allergen.2Meno K. Thorsted P.B. Ipsen H. Kristensen O. Larsen J.N. Spangfort M.D. et al.The crystal structure of recombinant proDer p 1, a major house dust mite proteolytic allergen.J Immunol. 2005; 175: 3835-3845Crossref PubMed Scopus (72) Google Scholar The Der p 1 propeptide is not known to be allergenic, but the 6.7-kDa propeptide of the peanut allergen Ara h 1, namely, Arah1Pro, is a relevant allergen.3Aalberse R.C. Mueller G.A. Derksen N.I.L. Aalberse J.A. Edwards L.L. Pomés A. et al.Identification of the amino-terminal fragment of Ara h 1 as a major target of the IgE-binding activity in the basic peanut protein fraction.Clin Exp Allergy. 2020; 50: 401-405Crossref PubMed Scopus (9) Google Scholar It is very different from the much larger “mature” allergen. The full-sized initial protein is called a polyprotein because it is the source of 2 independent proteins. As the peanut ripens inside the peanut plant vacuole, the initial protein is cleaved by a vacuolar enzyme. This results in the generation of 2 proteins with very different structures and functions: Arah1Pro is a small antimicrobial protein, whereas Ara h 1 is a big food storage protein. Both are allergens. The special features of Arah1Pro make it a strong candidate for initial percutaneous sensitization to peanut. This is an example of a single gene giving rise to 2 independent allergens (Fig 1, B2Meno K. Thorsted P.B. Ipsen H. Kristensen O. Larsen J.N. Spangfort M.D. et al.The crystal structure of recombinant proDer p 1, a major house dust mite proteolytic allergen.J Immunol. 2005; 175: 3835-3845Crossref PubMed Scopus (72) Google Scholar, 3Aalberse R.C. Mueller G.A. Derksen N.I.L. Aalberse J.A. Edwards L.L. Pomés A. et al.Identification of the amino-terminal fragment of Ara h 1 as a major target of the IgE-binding activity in the basic peanut protein fraction.Clin Exp Allergy. 2020; 50: 401-405Crossref PubMed Scopus (9) Google Scholar, 4Gajhede M. Osmark P. Poulsen F.M. Ipsen H. Larsen J.N. Joost van Neerven R.J. et al.X-ray and NMR structure of Bet v 1, the origin of birch pollen allergy.Nat Struct Biol. 1996; 3: 1040-1045Crossref PubMed Scopus (342) Google Scholar, 5Kaiser L. Velickovic T.C. Badia-Martinez D. Adedoyin J. Thunberg S. Hallén D. et al.Structural characterization of the tetrameric form of the major cat allergen Fel d 1.J Mol Biol. 2007; 370: 714-727Crossref PubMed Scopus (49) Google Scholar). Much more surprising is the finding of a complex allergen consisting of 2 proteins derived from 2 separate genes. However, it is not unprecedented. The best-known example is the cat allergen Fel d 1 (Fig 1, C). It is composed of 2 distantly related proteins (uteroglobins), Fel d 1A (7.9 kDa; 70 amino acids) and Fel d 1B (10.1 kDa; 92 amino acids).5Kaiser L. Velickovic T.C. Badia-Martinez D. Adedoyin J. Thunberg S. Hallén D. et al.Structural characterization of the tetrameric form of the major cat allergen Fel d 1.J Mol Biol. 2007; 370: 714-727Crossref PubMed Scopus (49) Google Scholar,6Griffith I.J. Craig S. Pollock J. Xu X.-B. Morgenstern J.P. Rogers B.L. Expression and genomic structure of the genes encoding Fel d 1, the major allergen of the domestic cat.Gene. 1992; 113: 263-268Crossref PubMed Scopus (70) Google Scholar These 2 proteins meet somewhere in the cat body and combine to form a covalent dimer of 2 antiparallel helical structures joined by 3 disulfide bridges. Two dimers combine to form a glycosylated tetramer of approximately 39 kD. As far as we now know, no cross-reacting allergen exists outside the cat family. This may reflect a technical problem: screening of cDNA-based expression libraries with IgE antibodies may be inefficient to detect the monomeric building blocks of such a relatively complex allergen. The DNA/mRNA sequence of Fel d 1 was largely obtained from the amino acid sequence of the purified protein.7De Groot H. Van Swieten P. Van Leeuwen J. Lind P. Aalberse R.C. Monoclonal antibodies to the major feline allergen Fel d 1, I. Serologic and biologic activity of affinity-purified Fel d 1 and of Fel d 1-depleted extract.J Allergy Clin lmmunol. 1988; 82: 772-786Google Scholar,8Morgenstern J.P. Griffith I.J. Brauer A.W. Rogers B.L. Bond J.F. Chapman M.D. et al.Determination of the amino acid sequence of Fel d I, the major allergen of the domestic cat: protein sequence analysis and cDNA cloning.Proc Natl Acad Sci U S A. 1991; 88: 9690-9694Crossref PubMed Scopus (201) Google Scholar Huang et al1Huang H.-J. Resch-Marat Y. Casset A. Weghofer M. Zieglmayer P. Zieglmayer R. et al.IgE recognition of the house dust mite allergen Der p 37 is associated with asthma.J Allergy Clin Immunol. 2022; 149: 1031-1043Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar are careful in pointing out that the information on the Der p 37/Der p 1 complex is still preliminary. It would be informative to know the size and composition of the fragments, as well as those of the complex, by using nondenaturing conditions (analyzed, for example, by size exclusion chromatography), particularly in combination with mass spectroscopy to establish the amino acid sequence of the 15.8- and 8.5-kDa proteins. The possibility of Der p 2 being another partner for Der p 37 is also still uncertain. At this moment, it seems most likely that most allergens are of the “Bet v 1 type” as illustrated in Fig 1, A: 1 gene giving rise to a single allergen. Bet v 1 is an ideal and spectacular textbook example of how a lot of a recombinant allergen can be obtained9Ferreira F. Hoffmann-Sommergruber K. Breiteneder H. Pettenburger K. Ebner C. Sommergruber W. et al.Purification and characterization of recombinant Bet v 1, the major birch pollen allergen. Immunological equivalence to natural Bet v 1.J Biol Chem. 1993; 268: 19574-19580Abstract Full Text PDF PubMed Google Scholar and what an allergen can look like.4Gajhede M. Osmark P. Poulsen F.M. Ipsen H. Larsen J.N. Joost van Neerven R.J. et al.X-ray and NMR structure of Bet v 1, the origin of birch pollen allergy.Nat Struct Biol. 1996; 3: 1040-1045Crossref PubMed Scopus (342) Google Scholar Part of the problem with the many mite allergens is their unpredictably complex fate after being secreted into the gut by the allergen-producing cell and ending up in floor dust or mattress dust. Subsequent extraction and storage conditions may also affect allergenicity, most likely diminishing their allergenic potency. However, some of the modifications potentially enhance allergenicity compared with that of the initial protein and that of the in vitro–produced clean and homogeneous recombinant protein. To resolve this, a reality check of recombinant allergens may be warranted. Some options are suggested in Table I. Clinical allergy remains a balancing act at the interface of sticky dust particles and crystal-clear proteins.Table ISuggestions for a reality check•Obtain or prepare antibodies (preferably monoclonal) to the recombinant protein, a pool of sera IgE-positive to the recombinant allergen, a labeled anti-IgE reagent, and a suitable environmental allergen source (eg, pollen extract, mite extract, or house dust extract)•Test these reagents in a 4-step ELISA as follows: (1) coat with the antibody to the recombinant allergen, (2) add titrated amounts of allergen (recombinant and natural), (3) add IgE antibody, and (4) detect bound IgE•To establish the native molecular size in a nondenaturing medium, use size exclusion chromatography (SEC) and analyze the fractions in the 4-step ELISA and/or RAST inhibition•Use these antibodies for SDS-PAGE immunoblotting of the recombinant allergen and the allergen extract (both nonreduced and reduced gels), and perform staining with animal antibodies and human IgE pool•Perform the same tests using affinity-purified, SEC-fractionated natural allergen and analyze the material by mass spectroscopyRAST, Radioallergosorbent test; SEC, size exclusion chromatography. Open table in a new tab RAST, Radioallergosorbent test; SEC, size exclusion chromatography. IgE recognition of the house dust mite allergen Der p 37 is associated with asthmaJournal of Allergy and Clinical ImmunologyVol. 149Issue 3PreviewHouse dust mite (HDM) allergens are major elicitors of allergic reactions worldwide. Full-Text PDF Open Access