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Everything you always wanted
to know about
… but were afraid to ask!
by Srilatha Bodla
Evaluation of modified vaccinia virus Ankara
based recombinant SARS vaccine in ferrets
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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.
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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.
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Transmission
Coronaviruses may be transmitted from person-to-person by
droplets, hand contamination, and small particle aerosols .
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Organization
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 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 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.
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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.
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Antiviral Drugs & Vaccine
Efforts are underway to assess potential anti-SARS-CoV agents in
vitro.
The availability of vaccines against animal coronaviruses, (avian
infectious bronchitis virus, feline infectious peritonitis virus) are
encouraging.
The S protein is generally thought to be a good target for vaccines
because it will elicit neutralizing antibody.
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Present study
Highly attenuated vaccinia virus ,modified vaccinia Ankara (MVA)
used as a vector.
Construction of rMVA expressing the S(rMVA-S) and N(rMVA-N)
2 major antigenic proteins responsible for inducing protective
immune responses against coronavirus.
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Animal model:
Ferrets (male castrated).
They are susceptible to SARS-CoV infection
To evaluate the efficacy and safety of rMVA based SARS
vaccines.
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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)
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Immunization of ferrets with rMVA-S & N
12 ferrets divided to 4 groups of 3 animals.
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 SARSCoV dissemination.
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Challenge of Immunized Ferrets
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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.
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No viral RNA was found in any tissue collected from the postmortem examination.
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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
21.
day
All other parameters were almost normal.
All the animals infected with SARS-CoV developed periportal and
pan-lobular hepatitis.
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ALT level following rMVA immunization
and SARS-CoV challenge.
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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.
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Pictures of Livers From Ferrets
Mild Hepatitis
MVA
PBS
Severe Hepatitis with Focal Necrosis
rMVA-SARS-N
rMVA-SARS-S
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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.
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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 SARSCoV challenge.is in line with reports of ADE of FIPV infection.
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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.
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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.
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SARS Panic & Humor
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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?
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THE END
Thank you!
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