Efficacy of Coxsackievirus A5 Vaccine Candidates in an Actively Immunized Mouse Model

Abstract

Coxsackievirus A5 (CV-A5) has recently emerged as a main hand, foot, and mouth disease (HFMD) pathogen. Following a large-scale vaccination campaign against enterovirus 71 (EV-71) in China, the number of HFMD-associated cases with EV-71 was reduced, especially severe and fatal cases. However, the total number of HFMD cases remains high, as HFMD is also caused by other enterovirus serotypes. A multivalent HFMD vaccine containing 4 or 6 antigens of enterovirus serotypes is urgently needed. A formaldehyde-inactivated CV-A5 vaccine derived from Vero cells was used to inoculate newborn Kunming mice on days 3 and 10. The mice were challenged on day 14 with a mouse-adapted CV-A5 strain at a dose that was lethal for 14-day-old suckling mice. Within 14 days postchallenge, groups of mice immunized with three formulations, empty particles (EPs), full particles (FPs), and a mixture of the EP and FP vaccine candidates, all survived, while 100% of the mock-immunized mice died. Neutralizing antibodies (NtAbs) were detected in the sera of immunized mice, and the NtAb levels were correlated with the survival rate of the challenged mice. The virus loads in organs were reduced, and pathological changes and viral protein expression were weak or not observed in the immunized mice compared with those in alum-inoculated control mice. Another interesting finding was the identification of CV-A5 dense particles (DPs), facilitating morphogenesis study. These results demonstrated that the Vero cell-adapted CV-A5 strain is a promising vaccine candidate and could be used as a multivalent HFMD vaccine component in the future.IMPORTANCE The vaccine candidate strain CV-A5 was produced with a high infectivity titer and a high viral particle yield. Three particle forms, empty particles (EPs), full particles (FPs), and dense particles (DPs), were obtained and characterized after purification. The immunogenicities of EP, FP, and the EP and FP mixture were evaluated in mice. Mouse-adapted CV-A5 was generated as a challenge strain to infect 14-day-old mice. An active immunization challenge mouse model was established to evaluate the efficacy of the inactivated vaccine candidate. This animal model mimics vaccination, similar to immune responses of the vaccinated. The animal model also tests protective efficacy in response to the vaccine against the disease. This work is important for the preparation of multivalent vaccines against HFMD caused by different emerging strains.

Keywords: CV-A5 vaccine; active immunization; efficacy; mouse model.

FIG 1

CV-A5 purification and identification. CV-A5 particles were harvested from infected Vero cells and purified by CsCl gradient ultracentrifugation. (A) The positions of empty, full, and dense particles (EPs, FPs, and DPs) are indicated. (B) The mock-infected Vero cell, EP, FP, and DP bands were subjected to SDS–4 to 20% PAGE and stained with Coomassie brilliant blue. (C) Proteins of the EPs, FPs, DPs, and mock-infected Vero cells were detected by Western blotting using the antibodies indicated at the top of each panel. VP0, VP1, VP2, VP3, and VP4 are indicated with arrows on the right, and molecular weight markers, in kilodaltons, are indicated with lines on the left. (D) The purified EPs, FPs, and DPs were inactivated with formaldehyde and examined by transmission electron microscopy. Scale bar,  200 nm.

FIG 2

Humoral immune response and persistence of the three formulations. Six-week-old BALB/c mice were primed and boosted intraperitoneally on days 0 and 14 with EPs, FPs, and EPs+FPs at doses of 0.5 μg, 1.5 μg, or 4.5 μg per injection. Bleeding was performed on days 14, 28, 49, and 70. For dose-dependent effects, NtAb titers were determined by using antisera collected on day 28. (A to C) The NtAb titers induced by EPs (A), FPs (B), and EPs+FPs (C) were determined. (D) NtAb levels induced by the three formulations at the three doses were compared. (E) For immune persistence, NtAb levels were analyzed at a dose of 1.5 μg/injection at 70 days postpriming. Two-way ANOVA was used for statistical significance analysis. Titers are presented as the means ± standard errors of the means (SEM). *, P < 0.05; ****, P < 0.0001; n.s., not significant. Ten mice were used in each group. Immunization doses are indicated in panels A, B, and C. NtAb titers below 8 were assigned to 2 for the convenience of presentation.

FIG 3

Virulence of CV-A5-M14 and establishment of the Kunming mouse model. CV-A5-M14 is a virulent, mouse-adapted strain that can infect 14-day-old (i.c. and i.p. routes) and 7-day-old (oral) Kunming mice. The criteria of clinical scores of infected Kunming mice are the following: 0, healthy; 1, fatigue or sleepiness; 2, weight loss and hunched back; 3, hindlimb weakness or jitter; 4, limb paralysis; 5, dying or death. The survival rates (percent) (A), mean clinical scores (B), and mean body weights (C) were compared in mice infected with CV-A5-M14 and mock-infected mice via the i.c., i.p. (14-day-old mice), and oral (7-day-old mice) routes at doses of 3 × 10⁷ CCID₅₀, 3 × 10⁸ CCID₅₀, and 3 × 10⁸ CCID₅₀, respectively. (D) The LD₅₀ of CV-A5-M14 was determined through the i.p. route at the doses indicated. The results are presented as the means ± SEM.

FIG 4

Efficacy of the vaccine in mice immunized with the three vaccine formulations. Kunming mice were primed and boosted on days 3 and 10 at a dose of 1.5 μg/injection and challenged at a dose of 10 LD₅₀ (2 × 10⁶ CCID₅₀/mouse) on day 14. (A) The survival rate (percent) was calculated 14 days postchallenge. Bleeding was performed on days 0, 14, and 28. NtAb titers of sera of mice vaccinated with EPs, FPs, EPs+FPs, or Al(OH)₃ (n = 5 for each group) were determined. NtAb titers on day 28 in mice infected with EPs, FPs, or EPs+FPs (n = 10 for each group) were determined. NtAb titers are presented as the means ± SEM. ****, P < 0.0001; n.s., not significant (by two-way ANOVA). (B) NtAb titers below 8 were assigned to 2 for the convenience of presentation. Mean clinical scores (C) and mean body weight (D) were recorded during the 14-day observation period. Black arrows indicate the date of vaccination. Orange arrows indicate the day of challenge.

FIG 5

Viral loads in organs in intraperitoneally immunized and challenged Kunming mice. Kunming mice immunized with EPs, FPs, or EPs+FPs or mock immunized were challenged with CV-A5-M14 at a dose of 10 LD₅₀ (2 × 10⁶ CCID₅₀/mouse). Heart, liver, spleen, lung, kidney, brain, and muscle tissues were retrieved for sample processing. Viral loads in organs were quantitated by qRT-PCR. The data were analyzed with two-way ANOVA. The results are presented as the means ± SEM. ****, P < 0.0001 (versus control treatment [by two-way ANOVA]).

FIG 6

Pathological changes and IHC in immunized-challenged Kunming mice. Histopathological changes (A) and expression of viral protein (B) in immunized-challenged Kunming mice were analyzed. Immunized Kunming mice were challenged via the i.p. route at 2 × 10⁶ CCID₅₀ with CV-A5-M14. The Al(OH)₃ groups were euthanized on day 6 postinfection, and mice in the vaccinated groups were euthanized at 14 days postinfection. Sections from lung, brain, muscle, and heart tissues were stained with hematoxylin to detect pathological changes. Viral proteins were detected in IHC assays with an anti-CV-A5 FP antibody, and sections were counterstained with hematoxylin. Original magnification, ×200.