Identification of chicken-derived antibodies targeting the Candida albicans Als3 protein

Abstract: Candida albicans is a major opportunistic pathogen, responsible for nearly half of clinical candidemia cases. The rising prevalence of azole-resistant Candida species represents a significant clinical challenge, underscoring the urgent need for alternative therapeutic strategies. Monoclonal antibody-based therapies have emerged as a promising and cost-effective approach to combating Candida infections. Agglutinin-like sequence protein 3 (Als3), a key cell surface protein of C. albicans, plays a pivotal role in adherence and biofilm formation, both of which are essential for its pathogenesis. In this study, recombinant Als3 protein was purified and utilized to immunize chickens, resulting in the production of Als3-specific immunoglobulin Y (IgY) antibodies. Two single-chain variable fragment (scFv) antibody libraries were subsequently constructed using phage display technology, yielding transformant counts of 5.3 × 10⁷ and 2.8 × 10⁷, respectively. Phage-based enzyme-linked immunosorbent assay (ELISA) revealed enhanced signals following bio-panning, enabling the identification and sequence validation of three scFv antibodies. These scFv antibodies exhibited strong binding activities to Als3, as confirmed through ELISA and western blot analyses. Binding affinities were determined to be ~ 10⁻⁸ M via serial titration ELISA and competitive ELISA. Additionally, the selected scFv antibodies specifically recognized endogenous Als3 protein in C. albicans, as demonstrated by western blot and cell-based ELISA assays. In conclusion, this study successfully generated and characterized high-affinity scFv antibodies targeting Als3, which exhibited exceptional specificity and binding activity. These findings highlight their potential as promising immunotherapeutic candidates for the treatment of C. albicans infections. Key points: • The Als3 protein of C. albicans is a critical biomarker and therapeutic target • Chicken-derived scFv antibodies against Als3 were developed via phage display • The scFv antibodies showed strong binding to endogenous Als3 in C.