Development of a Chimeric Molecule for Sublingual Allergen-Specific Immunotherapy to Treat House Dust Mite Allergy

• Grant number: 25/07392-9
• Support Opportunities: Regular Research Grants
• Start date: October 01, 2025
• End date: September 30, 2028
• Field of knowledge: Health Sciences - Medicine - Medical Clinics
• Principal Investigator: Keity Souza Santos
• Grantee: Keity Souza Santos
• Host Institution: Faculdade de Medicina (FM). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated researchers: Ana Iochabel Soares Moretti ; CESAR MANUEL REMUZGO RUIZ ; Marco Antonio Stephano
• Associated scholarship(s): 25/20355-5 - Development of a hypoallergenic chimeric molecule against house dust mite, BP.DR

Abstract

Allergic diseases represent a serious public health concern, affecting over 20% of the global population with projections reaching 50% by 2030. Allergen-specific immunotherapy, the only treatment capable of modifying the disease course, faces significant technological challenges. Traditional allergen extracts, while containing multiple allergens in a single preparation, often include proteins to which patients are not sensitized, potentially leading to new allergic sensitizations. Moreover, these extracts frequently lack adequate amounts of clinically relevant allergens, such as Der p 23, a small-sized protein often lost during the extraction process despite its recognized clinical importance due to high sensitization prevalence.In contrast, the approach using individual recombinant molecules, despite its precision, would require combining multiple proteins per patient, significantly increasing treatment costs and complexity. Additionally, this approach maintains the risk of adverse reactions as it preserves IgE epitopes in their native conformation.This project proposes an innovative solution through the development of a single chimeric molecule containing selected T-cell epitopes from major mite allergens, including Der p 1, Der p 2, Der p 10 and Der p 23. This strategy simultaneously overcomes the limitations of traditional extracts and the challenges of individual recombinant molecules.The methodology employs advanced bioinformatics tools for epitope selection and prediction, combined with protein engineering techniques for chimeric molecule construction. The project utilizes optimized linkers GPGPG and KK, and implements strategic cysteine residue substitutions. Protein expression will be performed in specialized Escherichia coli strains BL21-DE3 and ClearColi, followed by rigorous purification chromatography processes.Physicochemical characterization will include mass spectrometry, circular dichroism, and specific endolysosomal degradation assays. Functional evaluation will comprise comprehensive allergenicity tests using ELISA with allergic patients' sera. Immunogenicity studies will involve BALB/c mouse immunization with detailed analysis of blocking IgG and cellular response through ELISPOT. Therapeutic efficacy will be assessed in a murine allergic rhinitis model followed by specific immunotherapy with the chimeric molecule, monitoring parameters such as airway inflammation through bronchoalveolar lavage, specific IgE levels, and cytokine profiles.This innovative approach combines the advantages of molecular standardization, avoiding extract variability, with the safety provided by IgE epitope removal, all integrated into a single protein construct. Development will be conducted at the FMUSP Immunology Laboratory, equipped with state-of-the-art infrastructure and a highly qualified multidisciplinary team ensuring proper execution of all project stages.Beyond its immediate impact on mite allergy treatment, this strategy opens promising perspectives for future applications in other allergy types, including food allergies and insect venom hypersensitivity. The expected outcome is a therapeutic alternative that surpasses current limitations, offering greater precision than conventional extracts and improved safety and cost-effectiveness compared to multiple recombinant molecule approaches, with potential to transform respiratory allergy treatment and establish new frontiers in allergen immunotherapy. (AU)