Abstract
Allergens from pollen, mites, and moulds often sensitise patients simultaneously, posing challenges for developing stable and effective combination vaccines. Alternaria alternata, a major source of indoor and outdoor allergens, is strongly linked to asthma development and contains proteolytic enzymes that can degrade other allergens, potentially reducing vaccine efficacy. This study aimed to evaluate the safety, efficacy, and stability of polymerised A. alternata extracts (allergoids) compared to native extracts and their compatibility with pollen extracts (Phleum pratense). Allergoids were prepared using glutaraldehyde and characterised through SDS-PAGE, LC-MS/MS, NMR, and gas chromatography. Their immunogenicity and IgE-binding properties were assessed via Western blot and ELISA competition assays, while enzymatic activity was analysed using ApiZym kits. Mice immunisation experiments were conducted to evaluate antibody responses. Polymerised extracts exhibited reduced IgE-binding capacity while maintaining IgG-binding and immunogenicity. Mice immunised with allergoids generated antibodies that efficiently blocked IgE binding in allergic patients. Proteolytic activity was significantly reduced in allergoids, and pollen extracts remained stable when combined with them. These findings demonstrate that A. alternata allergoids are a safer, more stable alternative for immunotherapy, offering enhanced efficacy and reduced injections for polysensitised patients. This study provides critical insights for designing optimised combination vaccines.