Human scFv antibodies selected by phage display technology exhibit distinct Zika virus binding profiles and minimal cross-reactivity with dengue virus serotype 2

Zika virus (ZIKV) is a global public health concern, mainly due to the congenital and neurological syndromes associated with the disease. The similarity of clinical symptoms to those of other flavivirus infections makes clinical diagnosis particularly challenging. Additionally, developing specific and sensitive diagnostic assays remains difficult because of the high antigenic similarity among these arboviruses, and effective immunotherapeutic treatments are still limited. In this study, four human anti-ZIKV single-chain variable fragment (scFv) antibodies were isolated by phage display through multiple rounds of biopanning against ZIKV. After panning, the scFvs were selected by pairing the most enriched VH and VL domains, using NGS and bioinformatic approaches. The selected scFvs were expressed in bacteria, purified, and characterized for their interactions against ZIKV antigens. All four scFvs showed a medium to high affinity for recombinant Envelope protein (rEZIKV) and ZIKV particles, as shown by ELISA and Surface Plasmon Resonance. One scFv also showed low cross-reactivity against Dengue Virus Serotype 2 (DENV2). Epitope prediction suggested that this low cross-reactive scFv likely interacts with a region within domain III of the EZIKV. Thus, this work provides new insights that advance the development of more sensitive and specific diagnostic reagents for ZIKV infections by identifying scFvs with high affinity, low cross-reactivity to DENV2, and targeted recognition of epitopes within the DIII domain.