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 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.

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, 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.

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 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.