RESULTS:
The inactivated vaccine provided
mild protection of the ferrets from
the SARS-CoV.
Results:
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Ferrets were inoculated with a mock
vaccine or with a formalin-inactivated
whole-virus SARS-CoV vaccine.
A booster immunization was given two
weeks later.
The ferrets were then inoculated
intratracheally and intranasally with live
SARS-CoV 4 weeks after the booster.
Results:
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Two days after inoculation
nasal and pharyngeal swabs
were taken for titration
analyses.
The viral titers were similar
in the pharynx and nose in
both the SARS-CoV vaccine
and the mock vaccine.
Ferrets given the SARS
vaccine cleared the virus
after 7 days, but 3 of the 4
ferrets given the mock virus
didn’t clear the virus until
day 14.
Figure 1. Viral titers in pharyngeal (A and B) and nasal (C and D) swabs
from ferrets vaccinated with formalin-inactivated severe acute respiratory
syndrome (SARS)–associated corona virus vaccine (FI-SARS) (A and C)
and mock-vaccinated ferrets (B and D). Virus was collected by use of
swabs in
Dulbecco’s modified Eagle medium and was analyzed by TCID50 assay.
The limit of viral detection for this assay was 1 log10 TCID50/mL.
What does this mean?
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The faster clearing of the virus from the
pharyngeal secretions indicated that the
SARS-CoV vaccine provided immune
protection to the ferrets.
• To examine the level of
immunity provided by the
SARS-CoV vaccine, serum
was used to perform
neutralizing assays. Ferrets
41267 and 50441 displayed
some neutralizing antibodies
after the booster shot which
tapered off after 3 weeks.
• The other two ferrets
showed little of no
neutralizing antibody.
• All 4 ferrets showed high
levels of neutralizing
antibody after being
inoculated with the virus.
• The mock virus showed no
Figure 2. Neutralizing antibody titers for ferrets vaccinated with
virus neutralizing antibody
formalin-inactivated severe acute respiratory syndrome (SARS)–
before and increasing
associated corona virus vaccine (FI-SARS) before (A) and after (B)
challenge and for mock-vaccinated ferrets after challenge (C). Data
neutralization after the
are expressed as the dilution of serum that prevented cytopathic
effect in 50% of the wells and were calculated by the method of
ferrets were inoculated with
Reed and Muench [17]. Results from individual ferrets are shown.
the virus but with lower titers.
Note the scale change between before and after challenge.
Characterization of the humoral
immune response to vaccination.
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To evaluate the potency of the humoral
immune response, virus specific IgG
levels in the serum were measured by
ELISA.
Results:
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No significant elevation of IgG levels due
to vaccination.
However, there was a marked elevation
in the levels of IgG antibodies after the
virus was administered.
The SARS-vaccinated ferrets produced
higher levels of spike-specific IgG after
inoculation and were detected 1 week
earlier than in the mock-vaccinated
ferrets, suggestive of a priming effect of
the vaccine.
To determine if this was caused by IgM
antibodies, IgG+IgA+IgM were
measured.
Results were similar to those of IgG
suggesting that although the vaccine only
generated a weak antigenic response to
the spike protein, it may have primed the
immune response, leading to higher
levels of other antibodies and faster
clearing of the virus.
Figure 3. Antibody titers measured by ELISA for IgG
alone (A and B) and for IgG+IgA+IgM (C and D) in
mock-vaccinated ferrets (A and C) and in ferrets
vaccinated with formalin-inactivated severe acute
respiratory syndrome (SARS)–associated corona
virus (SARS-CoV) vaccine (FI-SARS) (B and D).
Recombinant spike protein was used to measure
ferret antibodies to SARS-CoV.
Pathological finding in lung
tissue of vaccinated ferrets.
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Four SARS-vaccinated and 4 mockvaccinated ferrets were inoculated with
SARS-CoV and two were uninfected
controls.
The ferrets were euthanized 23 days
after inoculation of the virus.
Results:
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Focal bronchiolar hyperplasia was observed in
a mock vaccinated and SARS-vaccinated
ferrets.
Inflammatory cells were peribronchiolar.
There were perivascular cuffing around a few
small blood vessels.
The virus was not found in any of the lung
tissue.
Bronchial and bronchiolar hyperplasia was
evident in 7 of the 8 ferrets infected but not
in the control ferret.
This suggests that the SARS-CoV infection led
to the lesions, not the vaccine.
Figure 4. A, Normal bronchiole from a control ferret showing normal, uniformly layered epithelium (hematoxylin-eosin
[HE] stain; original magnification, 400). B, Bronchiole of ferret given mock vaccine and challenged with virus 23 days
earlier showing epithelial hyperplasia (arrow) and peribronchiolar inflammation primarily composed of lymphocytes (L)
(HE; original magnification, 400). C, Bronchiole of vaccinated, virus-challenged ferret showing focal bronchiolar
hyperplasia (arrow) and peribronchiolar inflammation primarily composed of lymphocytes (L) (HE; original
magnification, 400).
Hepatic Pathology:
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All the ferrets appeared normal upon
examination.
Except for a few foci of necrosis and
inflammation on the livers of the ferrets
exposed to the virus the livers appeared
normal.
A larger lesion was observed in a SARSvaccinated ferret than the mock vaccinated
ferret but no virus was present in the liver
suggesting the lesion was caused from
previous viral damage.
D, Liver of ferret given mock vaccine and challenged with virus showing a focal necrotizing inflammatory
lesion (arrow) (HE; original magnification,400). E, Liver of vaccinated, virus-challenged ferret showing that
the major portion of the section is normal (middle and right side) while a small inflammatory
focus is seen (arrow) (HE; original magnification, 100). F, Liver of vaccinated, virus-challenged ferret with a
focal necrotizing inflammatory lesion composed of a mixed population of cells, including lymphocytes and
macrophages (HE; original magnification, 400).
Ferret Health: Blood Chemistry
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To determine if exacerbated hepatitis is a
complication from the vaccine levels of alanine
aminotransferase (ALT) which is an indicator of
hepatitis were analyzed.
Elevated levels of alkaline phosphatase were
observed in 1 mock-vaccinated ferret.
A slightly elevated ALT level was observed in 1 SARSvaccinated ferret but levels returned to normal two
weeks later.
These results are consistent with the kinetics of the
SARS virus and not the result of an immune reaction
to the vaccine.
Therefore liver injury may not be caused the weak
formalin-inactivated whole vaccine, but may be
caused by the infection of the SARS-CoV.
Body weight and temperature:
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The ferret with the largest weight
loss also had the most severe lung
lesions.
Three of the 5 ferrets given the
mock vaccine lost some weight,
(Mean 15%) but made a full
recovery of body weight when they
cleared the virus except 1 ferret
who lost about 20% of its weight
after viral clearance.
Overall the SARS- vaccinated ferrets
maintained or gained weight after
infected with the virus.
One ferret from each group and a
spike in temperature on the day of
the booster indicating that the
increase was caused from stress.
Figure 5. Percent body weight and body temperature change in vaccinated
ferrets. Weights were recorded at time zero for mock-vaccinated ferrets
(A) and ferrets vaccinated with formalin-inactivated severe acute respiratory
syndrome (SARS)–associated corona virus vaccine (FI-SARS) (B). Rectal
temperatures were taken using a digital thermometer for mock-vaccinated
(C) and vaccinated (D) ferrets. The percentage of deviation from the starting
(100%) weight or temperature for each ferret is shown.
Discussion:
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The authors’ main goal: To provide an animal model for testing
of possible SARS-CoV vaccine.
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In one study 70% of the patients infected with SARS had
elevated ALT levels. Severe hepatitis has been shown to be
predictive of poor clinical outcome in patients with SARS.
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To date, animal models that mimic human SARS-CoV fatality rates
are lacking. No animal infected with SARS showed severe viral
pneumonia leading to death as seen in humans.
The authors did not observe elevated ALT levels in the ferrets.
Lung lesions were mild in the experiment and the lungs were
histologically normal. None of the lesions found would have
cause death or illness. The bronchiole and perivascular lesions
were related to a previous viral exposure.
Discussion:
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Antibody-dependent enhancement (ADE) may be part
of the pathogenesis in the SARS-CoV.
This enhanced disease should be marked by an
increase in viral titer after prior exposure to the visus.
The purposely designed weak vaccine was made to
elict a mild antibody response to test the possibility
that a low antibody titer is associated with enhanced
pulmonary disease.
There was no observations of an increase in titers in
the vaccinated ferrets suggesting that under the
conditions of an inactivated whole-virus vaccine,
some protection is provided without the risk of
enhanced liver or lung disease.
Problems and Questions:
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Why were sample collections from the
liver and lungs not collected in time to
recover the virus from these organs?
Why was there no data about the doses
of the vaccines?
Further Research:
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More experiments are needed to settle
the discrepancies of the author’s data
concerning elevated ALT levels and
other research data.
Experiments with the dose of the
vaccines
Primate studies of the SARS-vaccine
before human testing.