Toward Consensus Epitopes B and T of Tropomyosin Involved in Cross-Reactivity across Diverse Allergens: An In Silico Study

Abstract

Tropomyosin (TM) is a pan-allergen with cross-reactivity to arthropods, insects, and nematodes in tropical regions. While IgE epitopes of TM contribute to sensitization, T-cell (MHC-II) epitopes polarize the Th2 immune response. This study aimed to identify linear B and T consensus epitopes among house dust mites, cockroaches, Ascaris lumbricoides, shrimp, and mosquitoes, exploring the molecular basis of cross-reactivity in allergic diseases. Amino acid sequences of Der p 10, Der f 10, Blo t 10, Lit v 1, Pen a 1, Pen m 1, rAsc l 3, Per a 7, Bla g 7, and Aed a 10 were collected from Allergen Nomenclature and UniProt. B epitopes were predicted using AlgPred 2.0 and BepiPred 3.0. T epitopes were predicted with NetMHCIIpan 4.1 against 10 HLA-II alleles. Consensus epitopes were obtained through analysis and Epitope Cluster Analysis in the Immune Epitope Database. We found 7 B-cell epitopes and 28 linear T-cell epitopes binding to MHC II. A unique peptide (residues 160-174) exhibited overlap between linear B-cell and T-cell epitopes, highly conserved across tropomyosin sequences. These findings shed light on IgE cross-reactivity among the tested species. The described immuno-informatics pipeline and epitopes can inform in vitro research and guide synthetic multi-epitope proteins' design for potential allergology immunotherapies. Further in silico studies are warranted to confirm epitope accuracy and guide future experimental protocols.

Keywords: B-cell epitope; IgE epitope; T-cell epitope; allergen; cross-reactivity; tropomyosin.

Figure 1

The flow chart of epitope prediction analysis applied to predict B-cell and T-cell epitopes.

Figure 2

Comparison of TM’s amino acid sequence identities (%) from different species. (a) Multiple sequence alignment between tropomyosins of HDM, cockroaches, crustaceans, helminths, and mosquito species; identical [*], conserved [:], and semiconserved [.]. (b) Sequence identities and similarities were calculated with Sequence Identities and Similarities (SIAS). For the similarity calculation, the default parameters offered by SIAS were chosen. The color scale (red to white to blue) represents the percent sequence identity from greatest to least. Molecular phylogenetic tree based on published amino acid sequences of tropomyosin from the edible crustacean, HDM, cockroach species, Aedes aegypti, and nematodes A. lumbricoides. The phylogenetic tree inferred from 1000 replicates taken to represent the evolutionary history of the taxa was analyzed and constructed using the evolutionary minimum method in MEGA11. Dermatophagoides pteronyssinus (Der p 10); Dermatophagoides farinae (Der f 10); Blomia tropicalis (Blo t 10); Ascaris lumbricoides (Asc l 3); Blattella germanica (Bla g 7); Periplaneta americana (Per a 7); Aedes aegypti (Aed a 10); Litopenaeus vannamei (Lit v 1); Penaeus monodon (Pen m 1); Penaeus aztecus (Pen a 1).

Figure 3

Heatmap representing the number of matches in specific residues of tropomyosin amino acid composition in different species. The Heatmap was generated using Heatmapper and clustered using the Euclidean distance metric method. The color grading, as indicated in the top left, represents the number of amino acid matches found in the specific residues of tropomyosin. Yellow indicates matches, and blue indicates maximum mismatches.

Figure 4

Conservation analysis of individual amino acids in tropomyosin (TM) was determined using the Consurf server. The conservation grades were subsequently superimposed onto the query sequence and structure, employing the ConSurf color-coding scheme, where the range from turquoise through maroon signifies positions from variable (grade 1) to conserved (grade 9). The conservation coloring scale is shown below.

Figure 5

B-cell and T-cell consensus epitopes superimposition on the surface of tropomyosin allergen structure. Regions of final single B-cell and T-cell consensus epitope sequences are shown in red. Images rendered using PyMOL.