Hybrid Adjuvant-Allergen H1sD2 Proteoforms Enhance Innate Immunity Activation via Distinct N-Glycosylation Profiles

Area of Science:

• Immunology
• Biotechnology
• Vaccine Development

Background:

• Allergen-specific immunotherapy (AIT) requires novel adjuvants for improved safety and efficacy in precision medicine.
• N-glycosylation of protein antigens influences innate immune responses via pattern recognition receptors.
• Therapeutic strategies for Type 1 hypersensitivity can be advanced by exploiting N-glycosylation for immune tolerance.

Purpose of the Study:

• To engineer and characterize N-glycosylated adjuvant configurations of hemagglutinin (H1s) and Der p 2 (D2) allergen.
• To investigate the impact of glycan profiles on innate immune responses.
• To evaluate the potential of these glycoengineered proteoforms in therapeutic strategies for Type 1 hypersensitivity.

Main Methods:

• Production of five distinct N-glycosylated H1sD2 proteoforms in *Pichia pastoris*.
• Structural verification using mass spectrometry.
• Assessment of DC-SIGN binding on THP-1-derived macrophages via confocal microscopy and flow cytometry.
• Stimulation of peripheral blood mononuclear cells (PBMCs) to measure cytokine levels (IL-10, IFN-γ).

Main Results:

• All engineered H1sD2 proteoforms demonstrated binding to DC-SIGN.
• Stimulation of PBMCs resulted in elevated IL-10 and IFN-γ levels, indicating a shift towards regulatory immune responses.
• The M2 glycovariant exhibited the most potent immunomodulatory signature.

Conclusions:

• Glycoengineering of allergen-adjuvant proteoforms can effectively modulate innate immunity.
• N-glycosylation offers a strategy to enhance the safety and efficacy of AIT.
• The M2 glycovariant is a promising therapeutic candidate for Type 1 hypersensitivity.