title: Generation and characterization of a protective mouse monoclonal antibody against human enterovirus 71.
abstract: Human enterovirus 71 (EV71) infection has emerged as a major threat to children; however, no effective antiviral treatment or vaccine is currently available. Antibody-based treatment shows promises to control this growing public health problem of EV71 infection, and a few potent monoclonal antibodies (mAbs) targeting viral capsid protein have been well described. Here, we generated an EV71-specific mouse mAb 2G8 that conferred full protection against lethal EV71 challenge in a suckling mouse model. 2G8 belonged to IgM isotype and neutralized EV71 at the attachment stage. Biochemical assays mapped the binding epitope of 2G8 to the SP70 peptide, which spanning amino acid residues 208-222 on the VP1 protein. Alanine scanning mutagenesis defined the essential roles of multiple residues, including Y208, T210, G212, K215, K218, L220, E221, and Y222, for 2G8 binding. Then, a panel of single mutation was individually introduced into the EV71 infectious clone by reverse genetics, and three mutant viruses, K215A, K218A, and L220A, were successfully recovered and characterized. Biochemical and neutralization assays revealed that K218A mutant partially escaped 2G8 neutralization, while L220A completely abolished 2G8 binding and neutralization. In particular, neutralization assays with human sera demonstrated that K218A and L220A substitutions are also critical for antibody neutralization in natural infection population. These findings not only generate a protective mAb candidate with therapeutic potential but also provide insights into antibody-mediated EV71 neutralization mechanism.
authors.affiliation: Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, 100071, China.
authors.forename: Yong-Qiang
authors.initials: YQ
authors.lastname: Deng
authors.forename: Jie
authors.initials: J
authors.lastname: Ma
authors.forename: Li-Juan
authors.initials: LJ
authors.lastname: Xu
authors.forename: Yue-Xiang
authors.initials: YX
authors.lastname: Li
authors.forename: Hui
authors.initials: H
authors.lastname: Zhao
authors.forename: Jian-Feng
authors.initials: JF
authors.lastname: Han
authors.forename: Jiang
authors.initials: J
authors.lastname: Tao
authors.forename: Xiao-Feng
authors.initials: XF
authors.lastname: Li
authors.forename: Shun-Ya
authors.initials: SY
authors.lastname: Zhu
authors.forename: E-De
authors.initials: ED
authors.lastname: Qin
authors.forename: Cheng-Feng
authors.initials: CF
authors.lastname: Qin
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000831:Animals, Newborn; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000302:isolation & purification / Q000627:therapeutic use*; D057134:Antibodies, Neutralizing / Q000276:immunology / Q000302:isolation & purification / Q000627:therapeutic use; D000914:Antibodies, Viral / Q000276:immunology / Q000302:isolation & purification / Q000627:therapeutic use*; D004252:DNA Mutational Analysis; D004195:Disease Models, Animal; D029821:Enterovirus A, Human / Q000235:genetics / Q000276:immunology*; D004769:Enterovirus Infections / Q000628:therapy*; D057131:Immune Evasion; D007116:Immunization, Passive; D007075:Immunoglobulin M / Q000276:immunology / Q000302:isolation & purification / Q000627:therapeutic use; D051379:Mice; D009500:Neutralization Tests; D011485:Protein Binding; D059386:Reverse Genetics; D016019:Survival Analysis; D015678:Viral Structural Proteins / Q000235:genetics / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D057134:Antibodies, Neutralizing; D000914:Antibodies, Viral; D007075:Immunoglobulin M; D015678:Viral Structural Proteins