Figure 2. Unconserved allergens are more allergenic and less related to human proteins. A, Euler diagrams showing groups of allergens without (Black circle) or with homologues (red circle) among helminth (brown), fungi (orange) and protozoa (green). B, Allergens displaying no homologues (black), any homologues (red) or that were highly conserved (blue) in the parasitic phyla were categorized as major allergens (among 357 allergens for which information was available) (Left Panel) or by the presence of a human homologue (Right Panel). doi:10.1371/journal.pone.0040552.g002 P = 0.0011) organisms suggesting that a conserved protein allergen in HPF is also conserved to a similar level in human. Further analysis found a highly significant negative correlation between the prevalence of anti-allergen IgE and the amino acid sequence identity level with the human homologues (r = 20.37, P,0.0001) (Figure 5A). As observed between allergens and helminth proteins, the prevalence of allergen-specific IgE in the population is higher for allergens without human homologues than for those allergens with human homologues but with lower amino acid identities. Because the prevalence of IgE and their classification as major or minor allergens is based on population prevalences to specific extracts, this type of classification may induce a biased analysis if some major allergens are derived from proteins that are rarely allergenic as would be the case for Gal d 2 a major egg-white allergen that is rarely allergenic [32,33,34]. To avoid this possible bias, we performed correlation analyses based on the prevalence of IgE to 75 allergens in a population of over 23,000 patients with allergic symptoms [35]. We found a similar negative relationship Tolerance to Self and Allergenicity using the prevalences reported (r = 20.37, P = 0.001). These data suggest that despite certain major allergens being of little importance in overall population, they contributed little to our analysis of 499 allergens. Of note, the absolute number of allergens and their predicted allergenicity decreases dramatically when the identity level with human proteins is .40% (Figure 5B). Interestingly, there is only one major allergen with an identity level with its human homologue above 63%, Asp f 27 (a fungal cyclophilin). Indeed, major allergens that exceed the 40% human identity level threshold were only 10.8% (23/213) of the major allergens for which IgE prevalence data were available. In contrast, 27.7% (59/ 213) of the major allergens showed homologues with human proteins with identity level up to 40%, while 61.5% (131/213) of the major allergens had no human homologues. This observation not only shows that molecular conservation decreases the allergenicity of an environmental protein dramatically, but also demonstrates that even moderate similarity with self-protein appears to limit allergenicity. Although including hundreds of allergens in our study strengthened the statistical analysis and the generalizability of our conclusions, it raised concern about the inclusion of several allergens from the same family. To avoid this bias, we performed correlation analysis using non-related allergens from specific extracts for which information could be obtained in Allergome and/or IUIS. We found similar negative correlation between conservation with human proteins and IgE prevalence for 13 non- duplicated allergens of Dermatophagoides pteronyssinus (Der p, r = 20.57, P = 0.040), 7 of Phleum pratense (Phl p, r = 20.80, P = 0.034), 16 allergens of Aspergillus fumigatus (Asp f, r = 20.64, P = 0.007) and a trend with the 9 non-duplicated allergens included of Bos domesticus (Bos d, r = 20.59, P = 0.097). Overall, for this specific group of allergens, the correlation was r = 20.58 (P,0.0001) (Table S3 and Figure S1). Discussion In the present study, we challenge one of the major hypotheses used to explain the intrinsic property of a protein that define allergenicity, i.e. homology. Our results show that rather than structural similarity it is the differences among allergenic proteins and their human (or microbial) counterparts that drive allerge- nicity. The development of allergies may be reflected not only by intrinsic host determinants, but also by factors related directly to the allergens themselves such as abundance within the allergenic extract, route of exposure to a given allergen, and the intrinsic, but yet undetermined, property that renders a protein allergenic. The most popular paradigm suggests that a protein’s similarity, either to proteins of microbial pathogens (i.e. Th2-inducing helminth parasites) or to humans, underlies their allergy-driving properties [26,28,29]. To address this hypothesis we compared 499 allergens with the whole deduced proteomes from the genomes of 15 microbes (including four helminths) and humans. We not only found an extraordinary level of conservation between allergens and helminth proteins, but we also found that the majority of allergens that had highly homologous proteins among the four helminth parasites also had homologues with protozoan, fungal and even human proteins. This finding indicates that similarity between allergens and helminth proteins does not underlie their allergenic properties, as intracellular protozoan parasites known to be major inducers of a Th1-dominated immune response [36] and fungi which are known to polarize toward the Th17-dominated response PLoS ONE | www.plosone.org 4 July 2012 | Volume 7 | Issue 7 | e40552