Antiviral activity of llama-inspired single domain antibody against Enterovirus A71

In the past few decades, Enterovirus A71 (EVA71) has caused devastating outbreaks in the Asia-Pacific region resulting in serious sequelae in infected young children. No preventive and therapeutic interventions are currently available for curing EVA71 infection, highlighting a great unmet medical need for this disease. Here, we showed that one novel single domain antibody (sdAb, F1) isolated from an immunized llama could alleviate EVA71 infection, both in vitro and in vivo We also confirmed that the sdAb clone F1 recognizes EVA71 through a novel conformational epitope comprising the highly conserved region of VP3 capsid protein by using competitive-binding and overlapping-peptide ELISA assays. Because of the virion's icosahedral structure, we reasoned that adjacent epitopes must be clustered within molecular ranges that may be simultaneously bound by an engineered antibody with multiple valency. Therefore, two single domain binding modules (F1) were fused to generate an sdAb-in-tandem design so that the capture of viral antigens can be further increased by valency effects. We showed that the tetravalent construct, F1xF1-hFc, containing two sdAb-in-tandem on an Fc scaffold, exhibits more potent neutralization activity against EVA71 than the bivalent sdAb, F1-hFc, for at least 5.8-fold. We also demonstrated that, using a human scavenger receptor class B member 2 (hSCARB2) transgenic mouse model, a half-dose of the F1xF1-hFc provided better protection against EVA71 infection than did the F1-hFc. Thus, our study furnishes important insights into multivalent sdAb engineering against viral infection and provides a novel strategic deployment approach for preparedness of emerging infectious diseases such as EVA71.