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 with recombinant proteins. Only after treatment with Cwt, PBMCs
secreted the anti-inflammatory cytokine IL-10. Obtained results suggest that Cwt chimera
could have a therapeutic effect in AIT in birch pollen allergy
