D1.224 - Rational Design of a T-Cell Epitope-Based Chimeric Allergen from Dermatophagoides pteronyssinus Toward Precision Immunotherapy

Allergen-specific immunotherapy remains the only disease-modifying treatment for allergic diseases, yet conventional allergen extracts present variability, incomplete allergen representation, and risk of IgE-mediated adverse reactions. Epitope based recombinant constructs offer a precision approach by selectively preserving T-cell immunogenicity while minimizing IgE reactivity to improve safety. We designed a recombinant chimeric molecule containing selected T-cell epitopes from Der p 1, Der p 2, and Der p 23 from Dermatophagoides pteronyssinus, deliberately excluding predicted IgE-binding regions. 

T-cell epitopes were selected using in silico prediction tools, prioritizing immunodominant regions while avoiding IgE-reactive sequences. The construct incorporated GPGPG and KK linkers to preserve epitope independence and included cysteine-to-serine substitutions to enhance structural stability. Codon optimization for bacterial expression was performed, and a C-terminal His-tag was added for purification. Three-dimensional modeling was performed using an ab initio trRosetta algorithm and validated by Ramachandran plot, Procheck, Watcheck, and ProSA-Web analyses. The synthetic gene was cloned into pET45b(+), confirmed by NcoI/XhoI restriction digestion, and expressed in Escherichia coli BL21 (DE3) upon IPTG induction.

Restriction analysis confirmed correct gene insertion. Structural validation demonstrated favorable stereochemical parameters and predicted conformational stability. Recombinant expression was successfully induced with IPTG (2–6 h). SDS PAGE analysis of soluble fractions revealed distinct protein bands within the expected molecular range (~31–33 kDa), consistent with the designed construct. Western blot using anti-His antibody confirmed specific recombinant expression. Both constructs demonstrated detectable soluble expression under initial experimental conditions. 

We successfully achieved the design, structural validation, and preliminary soluble expression of a T-cell epitope-based chimeric allergen from D. pteronyssinus. Ongoing work focuses on systematic optimization of recombinant expression and purification, followed by detailed structural and biochemical characterization. Subsequent steps will assess immunological functionality, including the induction of regulatory and blocking antibody responses, advancing this construct toward a safer, precision-based HDM immunotherapy platform.