Monoclonal antibody against EV71 3Dpol inhibits the polymerase activity of RdRp and virus replication

Fig. 1. Characteristics of IgG mAbs against EV71 3Dpol. a SDS-PAGE for mAbs 3A12, 2A10, 7A6G1. b Reactivity of indicated mAbs (3A12, 2A10, and 7A6G1) with EV71-expressed 3Dpol by IFA. c Western blot for mAbs IgG (lane 1, 3A12; lane 2, 2A10; lane 3, 7A6G1; lane 4, 5G10; and lane 5, positive antibody control). 3Dpol-immunized mice serum was used as positive antibody control. MAb 5G10, which was specific for Salmonella flagellin, served as negative antibody control

Fig. 2. Identification of regions recognized by 3Dpol-specific mAbs. a The intact 3Dpol protein and three C-terminal truncated proteins of 3Dpol were shown in the schematic diagram. b Truncated 3Dpol were used to identify mAb domains by using Western blot. c 3A12-recognized region covers 1–120 amino acid of 3Dpol, 2A10 in 120–250, and 7A6G1 in 360–462, respectively

Fig. 3. mAbs suppressed 3Dpol activities of RNA elongation. a Schematic diagram of RdRp-mediated RNA elongation. b Application of mAbs against 3Dpol suppressed the RNA elongation. Lane 1, no polymerase control; lane 2, positive control; lane 3, 3A12; lane 4, 2A10; lane 5, 7A6G1; lane 6, 5G10. Three independent experiments were performed, and the representative data were presented

Fig. 4. MAbs intracellularly inhibit EV71 replication. a Representative CPE on Vero-1008. b Indicated mAbs 3A12 and 2A10 inhibit EV71 replication when intracellularly delivered. c Indicated mAbs intracellularly inhibiting EV71 replication is dose-dependent. Three independent experiments were performed in duplicate, and the representative data were presented

Fig. 5. Kaplan-Meier analysis of antiviral efficacy of EV71 3Dpol-specific mAbs on ICR neonatal mice. a A neonatal mouse infected with EV71/MAV-VR exhibited limb paralysis (indicated by arrows). The virus inoculated mice were monitored daily for (b) clinical score and (c) survival for a period of 16 days. Clinical scores were graded as follows: 0, healthy; 1, reduced mobility; 2, limb weakness; 3, paralysis; 4, death. The number of mice in each group were indicated in one bracket