Everything you always wanted to know about … but were afraid to ask! by Srilatha Bodla

1. Everything you always wanted to know about … but were afraid to ask! by Srilatha Bodla SARS Evaluation of modified vaccinia virus Ankara based recombinant SARS vaccine in ferrets

2. Introduction Severe Acute Respiratory Syndrome (SARS) is an acute respiratory illness caused by Corona virus infection . Fever followed by a respiratory compromise are the signs and symptoms, which also include chills, muscular aches, headache and loss of appetite. The first world-wide SARS epidemic occurred between late 2002 and the first half of 2003 caused severe stress in global society. Much has been learned about SARS, including its causation by a new coronavirus (SARS-CoV); however, our knowledge about the ecology of SARS coronavirus infection remains limited. At present, the most efficacious treatment regimen for SARS is still subject to debate.

3. Introduction (cont.) The coronaviruses ( family Coronaviridae) are members of a family of large, enveloped, positive-sense single-stranded RNA viruses . The genomes of coronaviruses range in length from 27 to 32 kb and about 100 and 140 nanometers in diameter. Human coronaviruses (HCoVs) were previously only associated with mild diseases. Several coronaviruses can cause fatal systemic diseases in animals, including feline infectious peritonitis virus (FIPV), hemagglutinating encephalomyelitis virus (HEV) of swine, etc.

4. Transmission Coronaviruses may be transmitted from person-to-person by droplets, hand contamination, and small particle aerosols .

5. Organization The main function of the S protein is to bind to species-specific host cell receptors and to trigger a fusion event between the viral envelope and a cellular membrane. The SARS-CoV genome contains five major open reading frames (ORFs) that encode the replicase polyprotein; the spike (S), envelope (E), and membrane (M) glycoproteins; and the nucleocapsid protein (N). The spike protein has been shown to be a virulence factor in many different coronaviruses. The S protein is the principal viral antigen that elicits neutralizing antibody on behalf of the host. The M protein is the major component of the virion envelope.

6. Detection SARS Co-V has been detected from extracts of lung and kidney ,sputum or upper respiratory tract swab, by virus isolation,electron microscopy and PCR.

7. Efforts are underway to assess potential anti-SARS-CoV agents in vitro. Antiviral Drugs & Vaccine The S protein is generally thought to be a good target for vaccines because it will elicit neutralizing antibody. The availability of vaccines against animal coronaviruses, (avian infectious bronchitis virus, feline infectious peritonitis virus) are encouraging.

8. Highly attenuated vaccinia virus ,modified vaccinia Ankara (MVA) used as a vector. Present study Construction of rMVA expressing the S(rMVA-S) and N(rMVA-N) 2 major antigenic proteins responsible for inducing protective immune responses against coronavirus.

9. Animal model: Ferrets (male castrated). They are susceptible to SARS-CoV infection To evaluate the efficacy and safety of rMVA based SARS vaccines.

10. Results and Discussion Expression of SARS-CoV S and N proteins by rMVA –N and S recombinant viruses: Confirmed by Western Blot. S-specific mouse monoclonal antibody (detection of SARS-CoV S protein) SARS patient serum (detection of SARS-CoV N protein)

11. 12 ferrets divided to 4 groups of 3 animals. Immunization of ferrets with rMVA-S & N Immunized with PBS ,Parental MVA, rMVA-S, or rMVA-N. ELISA and Micro plaque reduction neutralizing assay Antibody was detected in ferrets immunized with rMVA-S. Ferrets immunized with rMVA-S showed peek antibody titre between 7 and 9 days. Other ferrets showed comparable levels of antibodies between 19 and 21 days. Rapid memory immune response occurred in ferrets immunized with rMVA-S. The presence of anribody did not lead to the prevention of SARS-CoV dissemination.

12. Challenge of Immunized Ferrets

13. SARS-CoV Challenge of Immunized Ferrets Challenged the vaccinated and control animals with SARS-CoV No clinical signs were found up to 29 days . Detected viral RNA in feces, pharyngeal swabs and blood samples by RTPCR. Viral RNA detected in pharyngeal and feces within 7 days, but not in the blood. The viral RNA persisted in blood longer than in pharyngeal excretion and feces .SARS-CoV replicates in ferrets.

14. No viral RNA was found in any tissue collected from the post-mortem examination.

15. Blood Chemistry & Histopathology Performed to investigate any pathological effects as consequence of rMVA vaccination and SARS-CoV challenge. Vet Test dry chemistry analyzer was used. Blood samples taken were examined for levels of alkaline phosphatase, alanine amino transferase, albumin, creatinine, total bilirubin, total keratin, and urea. Ferrets vaccinated with rMVA-N or rMVA-S showed higher levels of ALT. Elevated level of ALT was evidenced on 5th dpi and lasted until day 21. All other parameters were almost normal. All the animals infected with SARS-CoV developed periportal and pan-lobular hepatitis.

16. ALT level following rMVA immunization and SARS-CoV challenge.

17. Ferrets immunized with rMVA-S developed more severe lesions than other animals. Ferret #9,(immunized with rMVA-S) which developed rapid antibody response had most severe hepatitis. Mild hepatitis was observed in control animals. Ferrets immunized with rMVA-N also showed elevated level of ALT, only ferret #4 developed severe hepatitis.

18. Pictures of Livers From Ferrets Mild Hepatitis MVA PBS Severe Hepatitis with Focal Necrosis rMVA-SARS-N rMVA-SARS-S

19. Perivascular mononuclear infiltrates were present in all livers. The tissue samples for pathological sectioning for collected postmortem. (ie 27-29days after challenge) ALT level had already declines to the normal range (27-29 days after the challenge) The liver inflammation may not truly reflect the severity of the hepatitis associated with rMVA-S or N. Other organs were mildly affected.

20. Antibody-Dependant Enhancement of Virus Infectivity ADE is invitro serological phenomemon, in which viral infection of susceptible cells is modified by the addition of virus –reactive abs. Neutralizing antibody induced by the spike protein of feline infectious peritonitis virus failed to protect cats from the virus challenge Antibodies acquired either through a passive transfer of immune serum against the spike protein or Immunization with a recombinant vaccinia virus expressing the spike protein often lead to accelerated infection. SARS–CoV infect hepatocytes and cause hepatitis in human. Immunization with rMVA-S induced hepatitis in ferrets after SARS-CoV challenge.is in line with reports of ADE of FIPV infection.

21. FUTURE STUDIES Detailed pathological examination must be perform when the ALT level is high. To improve the immune responses by the use of different immunization regimen. More ferrets which would allow post-mortem examination at various time points after vaccination and challenge. More detailed analysis of the immune responses and immuno histological studies should be done. Other vaccination strategies should be examined.

22. CONCLUSION rMVA-S can induce rapid and vigorous neutralizing antibody response in ferrets. Neutralizing antibody did not prevent virus infection and spreading. Vaccination with SARS-CoV S/N protein may lead to enhanced pathology during infection and may cause damage of the liver. SARS-CoV does not cause clinical disease in ferrets. Ferrets are useful model in evaluating the safety of the vaccination strategy. Extra caution must be taken in future human trials of SARS vaccination.

23. SARS Panic & Humor

25. Unanswered Questions… What is the origin of SARS-CoV? What is the animal reservoir, if any? If SARS-CoV was present in an unknown animal species, did it jump to humans because of a unique combination of random mutations? Can SARS-CoV now infect both its original host and humans? Why are children less likely to develop SARS ? Do they have a lower clinical manifestation index, or do they possess a (relative) (cross-?) immunity against SARS-CoV? Are there environmental sources of SARS-CoV infection, such as foodstuff, water, sewage? How important is genetic diversity among SARS-CoV strains? Which factors determine the period of time between infection and the onset of infectiousness?

26. THE ENDThank you!