Antiviral Activity of a Llama-Derived Single-Domain Antibody against Enterovirus A71

Abstract

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 or 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 enzyme-linked immunosorbent assays (ELISAs). 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 could be further increased by valency effects. We showed that the tetravalent construct F1×F1-hFc, containing two sdAb-in-tandem on a fragment crystallizable (Fc) scaffold, exhibits more potent neutralization activity against EVA71 than does the bivalent sdAb F1-hFc by 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 F1×F1-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.

The Bottlenecks of Preparing Virus Particles by Size Exclusion for Antibody Generation

“…The neutralization assay was used for measuring the ability of the antibody to inhibit the cytopathic effects induced by viruses. Briefly, 96-well tissue culture plates were seeded with 3 × 10 4 cells/well in DMEM with 10% FBS and incubated at 37 °C for 24 h. The various concentration of antibodies were incubated with the virus at an MOI of 0.005 PFU/cell, which was the 100 TCID 50 s (a hundred time of 50% tissue culture infective doses), at 37 °C for 1 h [ 62 ]. The RD cells were infected by the mixture of antibodies and virus.…”

Therapeutic nanobodies against SARS-CoV-2 and other pathogenic human coronaviruses

“…Because of their unique properties and a variety of advantages over conventional antibodies, nanobodies have been developed rapidly in recent years to prevent and treat viral infections, including those caused by influenza virus, Ebola virus, HIV-1, respiratory syncytial virus, and enterovirus 71 [75][76][77][78]. Several nanobodies have proceeded to clinical trials or have been officially approved for treatment of human diseases, such as acquired thrombotic thrombocytopenic purpura, rheumatoid arthritis, plaque psoriasis, solid tumors, and cancer [79][80][81][82].…”

Research Progress and Applications of Multivalent, Multispecific and Modified Nanobodies for Disease Treatment

“…(2020) isolated a nanobody (F1), which inhibited EVA71 infection both in vitro and in vivo . The neutralizing activity was improved when multivalent formats were used and the most effective constructs were the ones in which the structure geometry enabled to maximally exploit the avidity effect ( 141 ).…”