Development and evaluation of an inactivated coxsackievirus A16 vaccine in gerbils

Figures

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  Figure 1.. Phylogenetic tree based on the entire VP1 genome of 38 representative strains. The neighbor-joining method was used to construct the phylogenetic tree, and the bootstrap values (%) for 1000 replicates were calculated and shown at the nodes of major clades. The CVA16-393 sequence was marked with a circle (●), while the five sequences (11203SD, 10246, TJ-224, 40812TXT and CA16-194) used in this study were marked with a triangle (▴). The other 32 reference sequences were available in the GenBank database. MEGA 5.1 software was used in this study.

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  Figure 2.. Replication kinetics of CVA16-393-p7 and growth curves of CVA16-393 at various passages in MRC-5 cells. (A) The viral titres of CVA16-393-p7 were determined by a TCID50 assay at 24, 36, 48, 60, 72 and 84 h post-infection at MOIs of 0.1, 0.5 and 1.0. (B) Cells were infected with 7th, 12th, 17th, and 22nd passages (CVA16-393-p7, CVA16-393-p12, CVA16-393-p17, CVA16-393-p22) of CVA16-393 at an MOI of 0.5. The culture supernatants were collected at 24, 36, 48, 60, 72 and 84 h post-infection, and the virus titres were determined and shown as TCID50/ml.

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  Figure 3.. Survival rate and severity of disease in the 7th and 22nd passages of CVA16-393-infected gerbils. (A) Survival curves for groups of 21-day-old gerbils (n = 5-9) when infected with the 7th and 22nd passages (p7 and p22) of CVA16-393 at a TCID50 of 101 to 103. (B) Mean clinical scores for 21-day-old gerbils infected with the 7th and 22nd passages of CVA16-393 at a TCID50 of 101 to 103.

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  Figure 4.. Dynamic profile of the neutralizing antibody response elicited by three CVA16 vaccine doses at different immunization intervals and the IFN-γ response in BALB/c mice. Gerbils were immunized with 50, 100 U/0.1 ml or 400 U/0.1 ml of the inactivated CVA16 vaccine, and received a booster vaccination at one- (A), two- (B), and four-week (C) intervals. Blood samples were collected at 1, 3, 5 and 8 weeks after the booster immunization. The dotted lines indicated the detection limit of this assay. (D) Eight-week-old BALB/c mice (n = 5 each group) were immunized with 100 U of CVA16 vaccine and boosted one week later. PBS containing 0.5 mg/ml of aluminum hydroxide was used as the control. Four weeks after immunization, the ELISPOT assay was performed to measure the IFN-γ response of the splenocytes. *Significant difference between the two groups (p < 0.05).

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  Figure 5.. Neutralizing antibody titres against four CVA16 strains. The 30-day-old gerbils were immunized with 100 U/0.1 ml of the CVA16 vaccine and boosted one week later. Seven days after the second dose, the NATs of sera were determined by 100× TCID50 of five CVA16 strains (10246, 40812TXT, 11203SD, TJ-224 and CA16-194). Each group included 10 gerbils. PBS containing 0.5 mg/ml of aluminum hydroxide was used as the negative control. The dotted lines indicated the detection limit of this assay. *Significant difference between the two groups (p < 0.05).

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  Figure 6.. Evaluation of the cross-protection elicited by CVA16 vaccines in gerbils. Gerbils (aged 7 days) were immunized intraperitoneally with 0.1 ml of the CVA16 vaccine at four-fold serial dilutions (100, 25, 6.25, or 1.56 U/gerbil) and boosted one week later. One week after the booster immunization, gerbils were challenged intraperitoneally with 100 × LD50 of strains 11203SD, 10246, TJ-224, 40812TXT and CA16-194. (A) Survival curves for the challenged gerbils (n = 5). (B) Mean clinical scores of the challenged gerbils (n = 5). PBS containing the aluminum hydroxide adjuvant (0.5 mg/ml) was used as the negative control. The gerbils were monitored for clinical signs for 20 days. Seven days post-infection, muscle and brainstem tissues were collected from both the healthy and diseased gerbils belonging to the 1.56 U vaccine-immunized sub-groups. (C) Representative images of hematoxylin and eosin stained muscle tissues from both the healthy and diseased gerbils. The arrows indicated the inflammatory cell infiltration, and the degeneration of muscle fibres in the muscle tissue of the diseased gerbils. Bar: 50 μm. (D) Seven days post-infection, representative images of Nissl stained brainstem…