A neonatal mouse model of coxsackievirus A16 for vaccine evaluation - PubMed (original) (raw)

A neonatal mouse model of coxsackievirus A16 for vaccine evaluation

Qunying Mao et al. J Virol. 2012 Nov.

Abstract

To evaluate vaccine efficacy in protecting against coxsackievirus A16 (CA16), which causes human hand, foot, and mouth disease (HFMD), we established the first neonatal mouse model. In this article, we report data concerning CA16-induced pathological changes, and we demonstrate that anti-CA16 antibody can protect mice against lethal challenge and that the neonatal mouse model could be used to evaluate vaccine efficacy. To establish a mouse model, a BJCA08/CA16 strain (at 260 50% lethal doses [LD(50)]) was isolated from a patient and used to intracerebrally (i.c.) inoculate neonatal mice. The infection resulted in wasting, hind-limb paralysis, and even death. Pathological examination and immunohistochemistry (IHC) staining indicated that BJCA08 had a strong tropism to muscle and caused severe necrosis in skeletal and cardiac muscles. We then found that BJCA08 pretreated with goat anti-G10/CA16 serum could significantly lose its lethal effect in neonatal mice. When the anti-G10 serum was intraperitoneally (i.p.) injected into the neonatal mice and, within 1 h, the same mice were intracerebrally inoculated with BJCA08, there was significant passive immunization protection. In a separate experiment, female mice were immunized with formaldehyde-inactivated G10/CA16 and BJCA08/CA16 and then allowed to mate 1 h after the first immunization. We found that there was significant protection against BJCA08 for neonatal mice born to the immunized dams. These data demonstrated that anti-CA16 antibody may block virus invasion and protect mice against lethal challenge, and that the neonatal mouse model was a viable tool for evaluating vaccine efficacy.

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Figures

Fig 1

Intracerebral inoculation of BJCA08/CA16 results in age-related disease and mortality. ICR mice (n = 8 to 10 per age) were intracerebrally inoculated with BJCA08 (1.9 × 104 PFU/mouse) at 1, 3, 5, 7, or 14 days of age. BJCA08-induced mean score of clinical disease and BJCA08-induced mortality were monitored and recorded daily after infection. Representative results from two similar experiments are shown. The Mantel-Cox log-rank test was used to compare the survival rates of pups between each age group and the 1-day-old group at 21 days postinfection. ***, P < 0.0001.

Fig 2

Intracerebral inoculation of BJCA08/CA16 results in dose-related disease and mortality. One-day-old ICR mice (n = 8 to 10 per group) were intracerebrally inoculated with increasing doses of BJCA08 (0.3, 2.4, 1.9 × 10, 3.0 × 102, and 1.9 × 104 PFU/mouse). Control animals were given medium instead of a virus. Mortality and clinical disease were monitored and recorded daily after infection. One representative from two independent experiments is shown. The LD50 was calculated as 1.2 PFU/mouse, and 3.0 × 102 PFU/mouse (260 LD50) was chosen as the challenge dose.

Fig 3

Histological examination of infected mice after intracerebral challenge with lethal doses of BJCA08/CA16. One-day-old ICR mice were intracerebrally inoculated with medium (mock control) or BJCA08 (260 LD50). (a) A representative picture of wasting caused by BJCA08 at day 8 of postinfection is shown (arrow). (b) A representative picture of right-hind-limb paralysis caused by BJCA08 at day 8 of postinfection is shown (arrow). Mice on the right side of panels a and b labeled in yellow were naive age-matched controls. Representative sections are shown. (c) Infected mice (grades 4 to 5) exhibited severe necrotizing myositis in hind-limb skeletal muscles (arrow). (d) Severe necrotizing myositis in skeletal muscles near the spine (arrow) was also detected in infected mice (grades 4 to 5). (e) A moribund mouse due to BJCA08 exhibited focal myocarditis (arrow). (f) In contrast, no histological change was observed in the heart of the mock-infected control mice. No histological change in the brain was observed in the brain of the infected mice (grades 3 to 5) (g) or the mock control mice (h). Samples in panels c to h were stained with hematoxylin and eosin stain; magnification, ×400.

Fig 4

Immunohistochemical results for infected mice after intracerebral challenge with lethal doses of BJCA08/CA16. One-day-old ICR mice were intracerebrally inoculated with medium (mock control) or BJCA08 (260 LD50). Representative sections are shown. (a) Infected mice (grades 4 to 5) exhibited numerous viral antigen-positive fibers in hind-limb skeletal muscles (arrow). (b) In contrast, no viral antigen was observed in hind-limb skeletal muscles of the mock control mice. Numerous viral antigen-positive fibers (c, arrow) were also detected in skeletal muscles near the spine, but no viral antigen was observed in intramedullary nerve cells (d) of infected mice (grades 4 to 5). (e) A moribund mouse due to BJCA08 exhibited numerous viral antigen-positive fibers in the heart (arrow). (f) In contrast, no viral antigens were observed in the heart of the mock-infected control mice. Viral antigens were detected in ependymal cells and choroid plexus epithelium (g, arrow), but no viral antigen was found in other parts of the brain of the infected mice (grades 3 to 5) (h). Samples in panels a to h were subjected to IHC analysis with DAB (3,3′-diaminobenzidine) chromogen and hematoxylin counterstain; magnification, ×400.

Fig 5

Mean tissue viral loads in BJCA08/CA16-infected mice. One-day-old ICR mice were intracerebrally inoculated with BJCA08 (260 LD50). Virus loads were assessed by real-time quantitative reverse transcriptase PCR in samples of the intestine, lung, liver, muscle, brain, spleen, kidney, heart, and blood from the infected mice. Samples were collected at the times indicated. Results represent the mean virus load (log10 copies) per milligram of tissue or per milliliter of blood ± SD (three mice per group).

Fig 6

Passive immunization with anti-G10/CA16 serum protected pups against CA16 challenge in vivo. One-day-old ICR mice (n = 8 to 10 per group) were intraperitoneally inoculated with 50 μl of 10-fold serially diluted anti-G10 serum (NTAb titer, 1,280) or medium. Within 1 h after inoculation, each mouse was intracerebrally challenged with 260 LD50 of BJCA08. Mortality and clinical disease were monitored and recorded daily after infection. The Mantel-Cox log-rank test was used to compare the survival of pups between each antiserum group and the medium control group at 21 days postinfection. ***, P < 0.0001.

Fig 7

BJCA08/CA16 pretreated in vitro with anti-G10/CA16 serum could significantly lose its lethal effect in neonatal mice. Serially diluted anti-G10 serum (NTAb titer, 1,280) or medium was incubated with an equal volume of 300 PFU BJCA08 at 37°C for 1 h. One-day-old ICR mice (n = 8 to 10 per group) were intracerebrally inoculated with the mixture described above. Mortality and clinical disease were monitored and recorded daily after infection. The Mantel-Cox log-rank test was used to compare the survival of pups between each antiserum group and the medium control group at 21 days postinfection. ***, P < 0.0001; **, P < 0.001.

Fig 8

Maternal immunization with inactivated whole-virus CA16 antigen protected newborn mice against lethal challenge. Adult female ICR mice (n = 2 per group) were intraperitoneally injected with formaldehyde-inactivated G10 antigen (1.5 × 106 PFU/mouse), BJCA08 antigen (4.8 × 105 PFU/mouse), or medium twice at a 2-week interval and allowed to mate at 1 h after the first injection. After delivery, pups were challenged with BJCA08 (300 PFU/mouse) on postnatal day 1. Mortality and clinical disease were monitored and recorded daily after infection. The Mantel-Cox log-rank test was used to compare the survival of pups between each maternal immunization group and the medium control group at 21 days postinfection. Dunn's multiple-comparison test was used to compare the weight of pups between each maternal immunization group and the medium control group at 21 days postinfection. ***, P < 0.0001.

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A neonatal mouse model of coxsackievirus A16 for vaccine evaluation - PubMed (original) (raw)