An enterovirus A71 virus-like particle with replaced loops confers partial cross-protection in mice

Fig. 1. Structural modeling and the spatial locations of loops. Cartoon representations of superpositions with EV-A71 and CV-A16 VP1(A) or EV-A71 and CV-A10 VP1 (B). The VP1 of EV-A71, CV-A16, and CV-A10 are colored in yellow, red, and blue, respectively. Loop regions are magnified on the right side of the pictures. The pentamer spatial models of EV-A71(C), EV-A71CHI3 (D), CV-A10 (E), and CV-A16 (F) capsid was constructed with the amino acid sequence of EV-A71 P1, EV-A71-P1CHI3, CV-A10 P1, CV-A16 P1, respectively. G-H, E-F, and B-C loops of EV-A71 VP1 are colored in light blue, light green, and light red, respectively. The chimeric G-H loop of CV-A16 VP1, E-F and B-C loops of CV-A10 VP1 are colored in blue, green, and red, respectively.

Fig. 2. The expression and characterization of EV-A71-VLPCHI3. The Coomassie stained gel (A) and the western blotting detection (B) of protein in supernatant: lane M, protein marker; lane PC, positive control; lane D1 to D6 were Sf9 cell culture supernatants harvested on days 1 to 6 after infection with rBV-EV-A71-P1CHI3–3CD. The Coomassie stained gel (C) and the western blotting detection (D) of purified supernatant: M, protein marker; lane PC, positive control; lane 1, purified supernatant harvested on day six after infection with rBV-EV-A71-P1CHI3–3CD. Electron microscopy of purified inactivated EV-A71 virus (E) and EV-A71-VLPCHI3 (F). The purified VLPs suspension was added to a 200-mesh ultrathin carbon supporting film, which was subsequently negatively stained for 5 min with 2 % phosphotungstic acid. The morphology of VLPs was observed with a Hitachi HT-7700 transmission electron microscope at 100 kV.

Fig. 3. The specific and cross IgG levels in sera from immunized mice. Four groups of 6-week-old female BALB/c mice were immunized with Sf9 culture supernatant, PBS, inactivated EV-A71 and EV-A71-VLPCHI3, respectively. Serum from immunized mice was collected at weeks two, four, and six and analyzed for total IgG. (A), (B) and (C) represented ELISA endpoint titers of immune sera against inactivated EV-A71, CV-A16 or CV-A10, respectively. Data are presented as the mean ± standard deviation (S.D.) of the independent immunization group, and the means of total IgG levels elicited by EV-A71-VLPCHI3 and inactivated EV-A71 were compared using a student's two-tailed t-test. ** P < 0.01.

Fig. 4. The specific and cross-neutralizing antibody titration in sera from immunized mice. Sera were harvested two weeks after the second booster and tested by microneutralization assay against EV-A71 (A), CV-A16 (B) or CV-A10 (C), respectively. The data were analyzed by student's two-tailed t-test. ** P < 0.01.

Fig. 5. Passive protection conferred by EV-A71-VLPCHI3. Each immunized ICR female mouse gave birth to about ten neonatal mice. Groups of neonatal mice were challenged within 24 h after birth, respectively. (A-C) Survival curves of the neonatal mice challenged with 50 × LD50 of EV-A71, CV-A16 or CV-A10; (D-F) Health scores of the neonatal mice. The survival rates were evaluated by the Mantel-Cox log-rank test. The health scores were compared using Dunn's multiple-comparison test. *P < 0.05.