RESULTS:

1
RESULTS

• The inactivated vaccine provided mild protection
  of the ferrets from the SARS-CoV.

2
Results

• 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.

3
Results

• 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 didnt 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 Dulbeccos modified
Eagle medium and was analyzed by TCID50 assay.
The limit of viral detection for this assay was 1
log10 TCID50/mL.
4
What does this mean?

• The faster clearing of the virus from the
  pharyngeal secretions indicated that the SARS-CoV
  vaccine provided immune protection to the ferrets.

5

• 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 virus neutralizing
  antibody before and increasing neutralization
  after the ferrets were inoculated with the virus
  but with lower titers.

Figure 2. Neutralizing antibody titers for
ferrets vaccinated with formalin-inactivated
severe acute respiratory syndrome
(SARS)associated corona virus vaccine (FI-SARS)
before (A) and after (B) challenge and for
mock-vaccinated ferrets after challenge (C). Data
are expressed as the dilution of serum that
prevented cytopathic effect in 50 of the wells
and were calculated by the method of Reed and
Muench 17. Results from individual ferrets are
shown. Note the scale change between before and
after challenge.
6
Characterization of the humoral immune response
to vaccination.

• To evaluate the potency of the humoral immune
  response, virus specific IgG levels in the serum
  were measured by ELISA.

7
Results

• 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, IgGIgAIgM 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 IgGIgAIgM (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.

8
Pathological finding in lung tissue of vaccinated
ferrets.

• Four SARS-vaccinated and 4 mock-vaccinated
  ferrets were inoculated with SARS-CoV and two
  were uninfected controls.
• The ferrets were euthanized 23 days after
  inoculation of the virus.

9
Results

• 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).
10
Hepatic Pathology

• 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 SARS-vaccinated
  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).
11
Ferret Health Blood Chemistry

• 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
  SARS-vaccinated 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.

12
Body weight and temperature

• 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.
13
Discussion

• The authors main goal To provide an animal
  model for testing of possible SARS-CoV vaccine.
• 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.
• 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.
• 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.

14
Discussion

• 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.

15
Problems and Questions

• 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?

16
Further Research

• More experiments are needed to settle the
  discrepancies of the authors 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.