Protection from lethal challenge in a neonatal mouse model by circulating recombinant form coxsackievirus A16 vaccine candidates

Abstract

Circulating coxsackievirus A16 (CA16) is a major cause of hand, foot and mouth disease (HFMD) in South-east Asia. At present, there is no vaccine against CA16. Pathogenic animal models that are sensitive to diverse circulating CA16 viruses would be desirable for vaccine development and evaluation. In this study, we isolated and characterized several circulating CA16 viruses from recent HFMD patients. These CA16 viruses currently circulating in humans were highly pathogenic in a newly developed neonatal mouse model; we also observed and analysed the pathogenesis of representative circulating recombinant form CA16 viruses. An inactivated CA16 vaccine candidate, formulated with alum adjuvant and containing submicrogram quantities of viral proteins, protected neonatal mice born to immunized female mice from lethal-dose challenge with a series of CA16 viruses. Further analysis of humoral immunity showed that antibody elicited from both the immunized dams and their pups could neutralize various lethal viruses by a cytopathic effect in vitro. Moreover, viral titres and loads in the tissues of challenged pups in the vaccine group were far lower than those in the control group, and some were undetectable. This lethal-challenge model using pathogenic CA16 viruses and the vaccine candidates that mediated protection in this model could be useful tools for the future development and evaluation of CA16 vaccines.

Fig. 1.

Circulating CA16CC024 virus resulted in dose-related disease and mortality in newborn mice. One-day-old ICR mice were intracerebrally inoculated with increasing doses of CA16CC024, from 10^2.5 to 10^6.5 CCID₅₀ ml⁻¹ (10 µl). Survival rates and clinical scores were then monitored and recorded daily after infection. Control mice were given medium instead of virus suspension. Each group contained 8–10 mice. The results are from three independent experiments. Varying grades of clinical disease are identified as: 0, healthy; 1, lethargy and inactivity; 2, wasting; 3, limb-shake weakness; 4, hind-limb paralysis; 5, moribund or dead.

Fig. 2.

Pathological analysis of CA16-infected neonatal mice. One-day-old ICR mice were intracerebrally inoculated with medium or CA16CC024. No histological change was observed in the hind-limb muscle (a) or spine skeletal muscle (e) of the non-infected control mice. Infected mice (grades 4–5) exhibited severe necrosis, including muscle bundle fracture, dissolution of muscle fibre cells, nuclear shrinkage and swelling (b), and inflammatory cell infiltration (arrow) (c, d). Infected mice (grades 4–5) showed loose muscle fibres in the juxtaspinal skeletal muscle (f). No histological change was observed in the brains of the non-infected control mice (g) or CA16-infected mice (h). No histological changes were observed in the lungs of the non-infected control mice (i). Mice infected with CA16CC024 exhibited severe alveolar shrinkage (j), a few areas of scattered pulmonary fibrosis (k), a large number of red cells (j, arrow), and vascular dilatation and congestion (j) in the lung tissue. Magnification: a, b, e–j: ×400 (scale bars, 20 μm); c, d, k, l: ×2000. The results are representative of three independent experiments.

Fig. 3.

Viral antigen expression in tissues from CA16CC024-infected neonatal mice. The tissues were obtained on day 5 after intracerebral inoculation with medium or CA16CC024 at 10^3.5 CCID₅₀ ml⁻¹. Samples were embedded and frozen in liquid nitrogen, then fixed in 3.7 % paraformaldehyde and treated with hydrogen peroxide (2.5 %). Rabbit anti-CA16 antibody and HRP-conjugated anti-rabbit IgG antibody were used for immunohistochemical staining. Magnification:×400 (scale bars, 20 μm). The viral antigen was exhibited in the heart (b), intestine (d), liver (f), spinal skeletal muscle (h), lung (j), hind-limb muscle (l) and cortex of the brain (p) of infected mice, but not in the spleen (m) or brain medulla (o). In contrast, no viral antigen was detected in the heart (a), intestine (c), liver (e), spine skeletal muscle (g), lung (i), hind-limb muscle (k) or brain (n) of non-infected mice. The results are representative of three independent experiments.

Fig. 4.

Viral load variations in various tissues of CA16CC024-infected mice at different time points. One-day-old ICR mice were inoculated intracerebrally with 10 µl CA16CC024 at 10^3.5 CCID₅₀ ml⁻¹. Virus loads were assessed by real-time quantitative reverse transcriptase-PCR in samples of the intestine, lung, liver, hind-limb muscle, brain, spleen, kidney, heart, spine and blood from the infected mice. Samples were collected at the times indicated. The results represent the mean virus loads [log₁₀ copies (mg tissue)⁻¹ or log₁₀ copies (ml blood)⁻¹]±sd (three mice per group, repeated three times).

Fig. 5.

Maternal immunization with inactivated CA16CC024 protected newborn mice from CC024 lethal challenge. Adult female ICR mice were immunized twice at 2 week intervals and allowed to mate with male mice after the first injection. After delivery, pups (n = 8–10) were challenged with the lethal CA16CC024 strain at (a) 10^3.5, (b) 10^4.5 or (c) 10^5.5 CCID₅₀ ml⁻¹. Survival rates and clinical scores were monitored for 21 days post-challenge.

Fig. 6.

Maternal immunization with inactivated CA16CC024 virus protected newborn mice from lethal challenge with (a) CC045, (b) CC090, (c) CC097 and (d) CC163 (d). Adult female ICR mice were immunized twice at 2 week intervals and allowed to mate with male mice after the first injection. After delivery, pups (n = 8–10) were challenged with the lethal strain. Clinical scores and survival rates were monitored for 21 days post-challenge.

Fig. 7.

Neutralization titres of anti-CA16 sera in immunized dams and pups. (a) Diagram of the experimental procedure. (b, c) Adult female ICR mice were immunized and examined for NTAb titres as described in Methods. The sera were collected from three dams and their pups (8–10 mice per group, mixed) after delivery. The mean neutralization titres against various CA16 strains were measured as described in Methods. The values for the negative control groups were all <2.

Fig. 8.

Protective efficacy of mating female mice after the first or second injection. (a) Diagram of the experimental procedure. (b) Adult female mice were immunized intraperitoneally twice at 2 week intervals with inactivated CA16CC024 viruses or PBS formulated with alum. One group containing three female mice immunized with inactivated CC024 vaccine was allowed to mate with the male mice after the second injection and sera were collected for measurement of NTAb titres as shown in diagram (a) after delivery. The mean neutralization titres against various CA16 strains were measured and compared with those generated using a different procedure, as described in Fig. 7. (c) After delivery, pups (n = 8–10) born to immunized female mice mated with male mice after the first or second injection were challenged with the lethal CA16CC024 strain at 10^3.5 CCID₅₀ ml⁻¹ (equal to 100 LD₅₀). Survival rates and clinical scores were monitored for 21 days post-challenge.