Figure 4. Allergens with homologues among microbes display similar levels of conservation with human proteins. The percent identity levels between human proteins (x-axis) a representative helminth (B. malayi, A), protozoan (P. falciparum, B) and fungus (C. albicans, C) protein (y-axis). Correlations were evaluated by Spearman rank test. Each dot represents one allergen and the squared-dot at the origin of the axis represents allergens that had neither homologues in humans nor in the microbes: (A) n = 222, (B) n = 247 and (C) n = 210 allergens. doi:10.1371/journal.pone.0040552.g004 or by failing to induce a strong Th1 response [29]. Therefore, it is reasonable to speculate that allergenicity favors the distance from the self while adjuvanticity may be associated with molecular mimicry. There may also be a ‘‘breakpoint’’, so that an allergen may have characteristics of both strong antigenicity and strong adjuvanticity in which case 30–40% identity with self-proteins may be optimal. Another important implication of the present study is related to cross-reactivity between allergens and parasite proteins. Having pre-formed allergen-specific IgE can have important implications Tolerance to Self and Allergenicity for vaccine development and can lead potentially to serious allergic adverse events [39] following vaccination. If vaccinating individuals prior to the acquisition of the parasite (hookworm in this case [39]) could circumvent this problem, there would still be the concern of cross-reactive IgE to a homologous (non-helminth) allergen. For example, it has been demonstrated that Bla g 5 (a cockroach glutathione-S –transferase (GST) allergen) and hel- minth GST are cross-reactive in humans. Moreover, experimental helminth infection in mice can induce cross-sensitization to Bla g 5, a major cockroach allergen [25]. Although this phenomenon of cross-sensitization has yet to be formally demonstrated in humans, we presume that a significant proportion of new vaccines against infectious organisms might induce IgE-mediated responses because of pre-existing cross-reactive IgE to vaccine antigens. Indeed, epidemiological studies estimate the skin reactivity to cockroach extract to be ,26% in North Americans and in people living in helminth-endemic areas [40]. Since the prevalence of anti-Bla g 5 IgE ranges between 30–90% of the cockroach allergic population, it suggests that at least 7% of a given population might be at risk for a cross-reactive allergy to a GST vaccine, two of which (schistosome-GST and hookworm GST) are being suggest- ed as potential parasite candidates in humans [39,41]. Finally, our results show that molecular uniqueness rather than molecular similarity between allergens and microbial/helminth or human proteins, underlies allergic responsiveness to environmen- tal proteins, as close to a third of the allergens and allergen families, including most of the major allergens, fail to have homologous proteins among microbial genera. These results associate allergenicity with immunogenicity rather than with similarity. It is reasonable to assume that, for some allergens, similarity and functional mimicry play important roles in Th2 polarization [27] giving them properties of adjuvanticity. For the majority of allergens, however, similarity or relatedness to microbial and/or human proteins, leads to allergenic tolerance. Materials and Methods Allergen Lists and Genomes A list of 1600 allergens were downloaded from the Allergome database (www. Allergome.org) and filtered for molecularly and immunologically defined allergens present in the Allergen Families (AllFam) [3] database (http://www.meduniwien.ac.at/allergens/ allfam). After selection, a comprehensive list of 499 allergens, covering 145 of 180 AllFams listed, was used to build a fasta file containing the amino acid sequence of the allergens. This fasta file was used to perform in silico searches for homologues across 15 genomes of microbes and the human genome. Fasta files with translated CDS annotated protein sequences for Brugia malayi, Loa loa and Wuchereria bancrofti (all version 1) were downloaded from the Broad Institute (http://www.broadinstitute. org/annotation/genome/filarial_worms/MultiDownloads.html), Schistosoma mansoni fasta file (version GeneDB v 4.0 h) was downloaded from Sanger Institute (ftp://ftp.sanger.ac.uk/pub/ pathogens/), Plasmodium falciparum (PlasmoDB 6.4) from Plasmodb (http://plasmodb.org/common/downloads/), Trypanosoma cruzi CL Brener (TriTrypDB-2.2) and Leishmania major Friedlin (TriTrypDB-2.2) from Tritrypdb (http://tritrypdb.org/common/ downloads/), Toxoplasma gondii ME-49 (ToxoDB 6.0) from Toxobd (http://toxodb.org/common/downloads/). Genome-predicted protein fasta file for Escherichia coli b088, Listeria monocytogenes EGD, Staphyloccocus aureus 55/2053, Mycobacterium tuberculosis C, Aspergillus fumigatus, Candida albicans WO-1 and Histoplasma capsulatum Nam1 were also downloaded from the Broad Institute, (http://www.broadinstitute.org//scientific-community/data). The PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e40552