Preparation of a Nanobody Specific to Dectin 1 and Its Anti-inflammatory Effects on Fungal Keratitis.

OBJECTIVE

To prepare a nanobody specific to dectin 1 and verify its specificity and anti-inflammatory effects on Aspergillus fumigatus keratitis.

METHODS

The nanobody was selected from a high-quality shark-antibody library constructed with phage-display technology. The nanobody was developed in the expression systems of Escherichia coli. Indirect ELISA was used to determine the specificity of the nanobody to recombinant dectin 1 protein. The potential of the nanobody to be recognized and expressed on the surfaces of cells and corneas was detected by immunofluorescence, and its anti-inflammatory effect on A. fumigatus keratitis was further verified. After infection with A. fumigatus, eyes of C57B L/6 mice were treated with nanobodies. Human corneal epithelial cells (HCECs) were pretreated with nanobodies and then incubated with A. fumigatus. Clinical scores and slit-lamp photography were used to assess disease response in mouse corneas. RT-PCR and ELISA were used to evaluate mRNA and protein expression of IL1β and IL6 in both mouse corneas and HCECs.

RESULTS

The nanobody was successfully expressed through microbial system and showed specific high-affinity binding to recombinant dectin 1. Furthermore, it exhibited specific binding to dectin 1 expressed on the surfaces of cells and recognized dectin 1 in mouse corneas. Importantly, it reduced clinical scores of A. fumigatus keratitis in mice compared with a PBS-treatment group. In addition, it decreased mRNA and protein expression of IL1β and IL6 in infected corneas and HCECs stimulated with A. fumigatus.

CONCLUSION

These results suggest that this nanobody can bring about anti-inflammatory effects. This highlights the potential of these nanobodies as innovative therapeutic agents in A. fumigatus.