Generation and characterization of an anti ITGB4 monoclonal antibody that blocks Zika virus entry.

INTRODUCTION: Zika virus (ZIKV) causes congenital disease and neurological complications, yet no approved vaccines or antivirals are available. Integrin β4 (ITGB4) has been identified as an entry receptor for ZIKV. METHODS: Recombinant human ITGB4 ectodomain was expressed using a baculovirus-insect cell system and used to immunize BALB/c mice. Hybridoma screening identified monoclonal antibody 7C3. Binding affinity to ITGB4 was determined by biolayer interferometry. Epitope relationships between 7C3 and the previously reported anti-ITGB4 antibody 13H10 were assessed by competitive binding assay. Antiviral activity was evaluated in four cell lines by RT-qPCR quantification of cell-associated ZIKV RNA after antibody pretreatment and viral challenge. RESULTS: 7C3 bound ITGB4 with picomolar affinity (KD=30.9 pM) and high specificity. Competitive binding showed that 7C3 and 13H10 (KD=7.98 pM) recognize non-overlapping epitopes on ITGB4. Pretreatment with 7C3 significantly reduced cell-associated ZIKV RNA in all four cell lines compared with PBS and isotype controls. DISCUSSION: A second, independently generated anti-ITGB4 antibody with picomolar affinity can robustly block ZIKV entry across multiple cell types, including SY5Y neuroblastoma cells relevant to congenital neuropathology. The non-overlapping epitopes of 7C3 and 13H10 suggest potential for combination strategies to enhance ITGB4 blockade. High germline identity of the 7C3 variable regions (VH=94.46%, VL=95.82%) supports future humanization. These findings establish 7C3 as a host-directed antiviral candidate and provide a basis for ITGB4-targeting approaches against congenital ZIKV disease.