Fig 1.
Phylogenetic tree of the EV-A71 isolates identified between 2018 and 2019 in Taiwan.
Phylogenetic analyses were based on viral protein VP1, VP2 and VP3 nucleotide sequences of the Taiwan EV-A71 isolates identified between 2018 and 2019 (black circles) and a representative set of EV-A71 and enterovirus isolates (891 bases for VP1, 762 bases for VP2, 726 bases for VP3). The 2018–2019 EV-A71 that were isolated from patients with severe neurological manifestations were labeled with open triangles (refer to S1 Table). Trees were constructed by using the neighbor-joining method with 1,000 replicates through MEGA 7.0.25 (http://www.megasoftware.net/). Coxsackievirus A16 strain Tainan/5079/98 (AF177911.1) was used as the outgroup. The distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. Genotype assignment, country, and year of isolation are provided in the virus names. TW, Taiwan.
Fig 2.
Neutralizing activities of human antibodies with the 2018–2019 genotype C1 EV-A71.
(A) Neutralizing titers of polyclonal convalescent sera to EV-A71. A total of 23 sera were collected from hospitalized children with genotype B5 and C4 EV-A71 infection, 2014–2017 (refer to S3 Table). Sera from genotype C1 EV-A71- and echovirus 11-infected children were included as controls. Post-genotype C1 EV-A71 infection sera EV-A71-25, -26, -27, and -28 were obtained from subject 19, 20, 21, and 4, respectively (refer to S1 Table). Two post-genotype C4 EV-A71 infection sera (EV-A71-3 and -6) were obtained after intravenous immunoglobulin administration and marked with a hash sign. Each serum was assayed in triplicate for each virus with equivalent results. The serum that had an 8-fold decline in titers against the 2018–2019 genotype C1 EV-A71 compared to titers against the B5 and C4 virus is marked with a star. (B) Neutralizing activities of human monoclonal antibodies to EV-A71. Twelve neutralizing monoclonal antibodies that recognize the 5-fold vertex, canyon, 2-fold plateau, and 3-fold plateau epitopes on the viral capsid were tested. An EV-A71 capsid-targeting non-neutralizing antibody 24-3-10A and an influenza H1-targeting antibody 2-12C were included as controls. Each symbol represents an independent measurement. Some measurements overlap. The geometric mean concentration of neutralization is shown as a middle bar. In the neutralization assay, the failure of antibody up to 100 μg/ml to prevent the cytopathic effect was determined as no virus neutralizing activity. Each antibody was assayed in duplicate for each virus with equivalent results and the assay was carried out three times. 16-2-11B, 17-1-12A, and 16-3-4D retained neutralizing activities against the majority of 2018–2019 genotype C1 EV-A71 and the analysis showed < 4-fold change in neutralization potencies for genotypes B5 and C1.
Fig 3.
Antigenic determinants on the viral capsid of genotype C1 EV-A71.
(A) Canyon epitopes, including VP1 D110, VP1 T232, VP1 K162, VP1 D164 and VP3 F186, recognized by human anti-EV-A71 MAbs 16-2-9D, 16-2-12D, 16-3-3C and 16-2-2D, were colored in cyan [24]. The surface residue 232 of VP3, located at the canyon region of capsid, was distinct in genotype C1 EV-A71 (S2 Fig) and interacted with VP3 D234 and L236 canyon residues (colored in orange). (B) Epitope mapping on the viral capsid by genotype C1 EV-A71-neutralizing MAbs. Escape variants were selected using MAbs 16-2-11B, 16-3-4D and 17-1-12A and revealed single amino acid substitutions at capsid residues VP1 S241 (colored in red), VP3 E81 (colored in blue) and VP2 D84 (colored in green), respectively. VP2 E88V and VP2 K149E were previously identified in the escape variant of genotype B5 and C4 EV-A71 with MAb 17-1-12A [24]. The surface view of pentamer shown with the 5-fold vertex at the center are created using the software program PyMOL (PDB 3VBS). The capsid VP1 protein is colored in black, VP2 colored in grey and VP3 colored in white. Abbreviations: 3, 3-fold axis; 2, 2-fold axis.
Fig 4.
The in vivo protection by genotype C1 EV-A71-neutralizing MAbs.
The hSCARB2-transgenic mice were administered with 16-2-11B, 17-1-12A, and 16-3-4D at 10 mg per Kg 24 hours prior to a lethal challenge of genotype C1 EV-A71 TW-5011-2018 (n = 3 per group). Weight change following infection and survival rate were measured. Non-neutralizing anti-EV-A71 canyon MAb 16-2-12D and anti-avian influenza H7 MAb 3A-44 were human IgG controls. The weight data are presented as the means ± standard error of the mean and the comparison of weight change among groups was analyzed using one-way ANOVA followed by Tukey’s post hoc analysis. *, p < 0.05; **, p < 0.01; ***, p < 0.001. d.p.i, day post infection.