Evaluation of the immunomodulatory potential of chimera Bv1a-BLwt and its mutants on the co-culture model system

Allergen immunotherapy (AIT) is currently the only disease-modifying treatment for allergies. Pre-clinical models for the evaluation of novel therapeutics are crucial for ensuring their efficacy and safety. While cell culture models are cost-effective and efficient, they cannot fully replicate the cellular interactions in vivo. Therefore, it is essential to use more sophisticated model systems, such as co-cultures, to assess the potential of new therapeutics more accurately. Immunomodulatory protein banana lectin (BLwt) is an attractive candidate for adjuvant in AIT. Its mutant BLH84T was developed to reduce its potential mitogenicity. The aim of this study was the development of the coculture model system for testing the immunomodulatory effect of chimeras composed of the major birch pollen allergen (Bv1a) and BLwt (Bv1a-BLwt, Cwt), the hypoallergenic isoform of Bv1a (Bv1l) and BLH84T (Bv1l-BLH84T, C1 and BLH84T-Bv1l, C2). Chimeric structures were designed in silico, fully minimized, and relaxed without van der Waals atomic clashes. Afterward, proteins were successfully expressed in Escherichia coli and purified by IMAC yielding around 0.4 mg per 1L of expression medium. The IgE binding capacity was assessed using ELISA inhibition with birch pollen allergic patients’ sera. Caco-2 intestinal epithelial cells and THP-1 differentiated macrophages were used for the co-culture model system development. After protein application on the apical side of the co-culture, the integrity of the epithelial monolayer was not disturbed. The immunomodulatory potential of antigens was tested by measuring the gene expression levels for pro- and anti-inflammatory cytokines in both cell lines from co-culture. The obtained results indicate that the best anti-inflammatory response was favored after treatment with Cwt. Additionally, to further confirm the immunomodulatory effect of the recombinant chimeras, PBMCs obtained from individuals allergic to birch pollen were employed and treated