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Letter
SARS Vaccine Protective in
Mice
Konrad Stadler,*1 Anjeanette Roberts,†1
Stephan Becker,‡1 Leatrice Vogel,† Markus
Eickmann,‡ Larissa Kolesnikova,‡ Hans-Dieter Klenk,‡ Brian Murphy,† Rino
Rappuoli,* Sergio Abrignani,*
and Kanta Subbarao†
*Chiron Vaccines, Siena, Italy; †National Institutes of Health, Bethesda,
Maryland, USA; and ‡Institut für Virologie, Marburg, Germany
Suggested
citation for this article
To the Editor: Less than a year after the identification of the
severe acute respiratory syndrome coronavirus (SARS-CoV) (1),
3 independent laboratories reported protection from SARS-CoV challenge
in animal models using a DNA vaccine or recombinant forms of the modified
vaccinia Ankara or a parainfluenza virus, encoding the spike gene (2–4).
Their protective efficacies are encouraging because they provide proof
that a SARS-CoV vaccine is feasible. However, vaccines based on those
technologies are not licensed for human use, and recommendation and licensing
will likely take many years. We have developed an inactivated virus vaccine
that induces neutralizing antibodies and protects against SARS-CoV challenge.
The vaccine was produced as described elsewhere (5).
Briefly, the SARS-CoV (strain FRA, GenBank accession no. AY310120) was
grown in Vero cells, inactivated with β-propiolactone (BPL), and
complete inactivation was confirmed by 2 consecutive passages on Vero
cells. Inactivated virus was purified by column chromatography followed
by sucrose gradient centrifugation. The fraction containing virus was
dialyzed against phosphate-buffered saline pH 7.2, and total protein content
was determined by using the Micro BCA Protein Assay Kit (Pierce Biotechnology,
Rockford, IL, USA). Immunogenicity of the vaccine was tested by immunizing
BALB/c mice at 0, 2, and 4 weeks with 5 μg of inactivated virus combined
with the adjuvant MF59, an oil squalene-in-water emulsion (6)
approved for human use in Europe for an influenza vaccine. Ten days after
the third immunization, serum samples were tested for the presence of
SARS-CoV spike protein–specific antibodies by using enzyme-linked immunosorbent
assay, and high titers (1–3 × 104) of anti-SARS-CoV spike
immunoglobulin (Ig) G antibodies were detected. IgG subclass determination
indicated a predominant Th2-type immune response similar to that observed
in BALB/c mice vaccinated with a UV-inactivated SARS-CoV plus alum (7).
Efficacy of the inactivated virus vaccine was also assessed in a BALB/c
mouse model of SARS-CoV infection (8). Animal studies
were approved by the National Institutes of Health Animal Care and Use
Committee and were conducted in an animal biosafety level 3 facility.
SARS-CoV replicates in the respiratory tract of BALB/c mice following
intranasal infection with 104 50% tissue culture infectious
doses (TCID50) of virus. Generally, virus titers peak within
2 days after infection and are cleared within 7 days (8).
BALB/c mice were immunized at 0, 2, and 4 weeks with 5 μg of BPL-inactivated
SARS virus with or without the MF59 adjuvant. Mice were also immunized
with 4 different control preparations: phosphate-buffered saline, adjuvant
MF59 alone, and 5 μg of BPL inactivated influenza A virus vaccine with
or without MF59. Serum samples were collected 2 weeks after each dose,
and assayed for their ability to neutralize SARS-CoV (8).
After 2 vaccine doses, SARS-CoV neutralizing antibodies were detected
only in the group of mice immunized with the BPL-inactivated SARS virus
vaccine plus MF59 adjuvant (1:91). Two weeks after the third dose, the
BPL-inactivated SARS virus vaccine without MF59 induced neutralizing titers
of 1:64, while the adjuvanted vaccine elicited neutralizing titers >1:600
(Table).
Mice were challenged intranasally at this point with 104 TCID50
SARS-CoV (Urbani strain, GenBank accession no. AY278741). Nasal turbinates
and lung tissues were analyzed for infectious virus 2 days later (Table).
SARS-CoV titers in mice from the control groups were ≈106
TCID50 virus/g of lung tissue and ≈103 TCID50
virus/g of nasal turbinate tissue. Complete protection from virus replication
was observed in mice that received the MF59 adjuvanted SARS-CoV vaccine.
Immunization with the nonadjuvanted vaccine resulted in complete protection
of the upper respiratory tract and a significant reduction (30,000-fold)
of viral titers in the lower respiratory tract compared to the control
groups. The incomplete protection of this group was attributed to a single
animal that contained detectable infectious virus in the lung.
Accelerated or enhanced virus replication or disease in immunized persons
is a concern in developing any vaccine. This may be particularly true
for SARS-CoV vaccines since adverse effects have been reported for one
animal coronavirus vaccine, feline infectious peritonitis virus (9).
Additionally, some in vitro experiments were performed with pseudotyped
lentiviruses that expressed the spike glycoprotein derived from SARS-like
virus isolated from civets. In these experiments, the presence of antibodies
that neutralized most human isolates of SARS-CoV demonstrated enhanced
entry into renal epithelial cells (10). In our studies,
we did not find enhanced virus replication in the respiratory tract of
vaccinated mice upon SARS-CoV challenge. However, since mice are a model
of SARS-CoV infection but not disease, the issue of disease enhancement
will have to be carefully evaluated if and when an appropriate animal
model in which this phenomenon can be demonstrated becomes available.
In summary, an inactivated SARS-CoV vaccine, produced with a technology
that has a safety record established by immunizing hundreds of millions
of persons, protects mice from challenge with SARS-CoV. The vaccine adjuvanted
with MF59 elicits neutralizing antibodies (titer 1:91) after only 2 doses.
We conclude that the vaccine described here has desirable properties,
and our data support further development and plans for clinical trials.
Acknowledgments
This study was supported
by the Fonds der Chemischen Industrie, the 6th Framework Program of
the European Commission (FP6-511065), and Chiron Vaccines.
References
- Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret
T, Emery S, et al. A
novel coronavirus associated with severe acute respiratory syndrome.
N Engl J Med. 2003;348:1953–66.
- Yang ZY, Kong WP, Huang Y, Roberts A, Murphy BR, Subbarao K, et al.
DNA
vaccine induces SARS coronavirus neutralization and protective immunity
in mice. Nature. 2004;428:561–4.
- Bisht H, Roberts A, Vogel L, Bukreyev A, Collins PL, Murphy BR, et
al. Severe
acute respiratory syndrome coronavirus spike protein expressed by attenuated
vaccinia virus protectively immunizes mice. Proc Natl Acad Sci U
S A. 2004;101:6641–6.
- Bukreyev A, Lamirande EW, Buchholz UJ, Vogel LN, Elkins WR, St Claire
M, et al. Mucosal
immunisation of African green monkeys (Cercopithecus aethiops)
with an attenuated parainfluenza virus expressing the SARS coronavirus
spike protein for the prevention of SARS. Lancet. 2004;363:2122–7.
- Song HC, Seo M-Y, Stadler K, Yoo BJ, Choo Q-L, Coates S, et al. Synthesis
and characterization of a native, oligomeric form of recombinant severe
acute respiratory syndrome coronavirus spike glycoprotein. J Virol.
2004;78:10328–35.
- Podda A, Del Giudice G. MF59-adjuvanted
vaccines: increased immunogenicity with an optimal safety profile.
Expert Rev Vaccines. 2003;2:197–203.
- Takasuka N, Fujii H, Takahashi Y, Kasai M, Morikawa S, Itamura S,
et al. A
subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits
systemic humoral immunity in mice. Int Immunol. 2004;16:1423–30.
- Subbarao K, McAuliffe J, Vogel L, Fahle G, Fischer S, Tatti K, et
al. Prior
infection and passive transfer of neutralizing antibody prevent replication
of severe acute respiratory syndrome coronavirus in the respiratory
tract of mice. J Virol. 2004;78:3572–7.
- Vennema H, de Groot RJ, Harbour DA, Dalderup M, Gruffydd-Jones T,
Horzinek MC, et al. Early
death after feline infectious peritonitis virus challenge due to recombinant
vaccinia virus immunization. J Virol. 1990;64:1407–9.
- Yang ZY, Werner HC, Kong WP, Leung K, Traggiai E, Lanzavecchia A,
et al. Evasion
of antibody neutralization in emerging severe acute respiratory syndrome
coronaviruses. Proc Natl Acad Sci U S A. 2005;102:797–801.
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Table. Immunogenicity
and efficacy of β-propiolactone (BPL)–inactivated severe acute
respiratory syndrome coronavirus (SARS-CoV) vaccine in mice against
subsequent challenge with live SARS-CoV
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Immunogen*
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Neutralization titer†
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Virus replication upon challenge‡
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Lungs
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Nasal turbinates
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2 wk post 1st dose
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2 wk post 2nd dose
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2 wk post 3rd dose
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No. infected/ no. tested
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Mean (± SE) virus titer§
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No. infected/ no. tested
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Mean (± SE) virus titer§
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PBS
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<1:8
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<1:8
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<1:8
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4/4
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6.3 ± 0.3
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3/4
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2.8 ± 0.35
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MF59
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<1:8
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<1:8
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<1:8
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4/4
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6.1 ± 0.13
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3/4
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3.0 ± 0.58
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Influenza A (5 μg)
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<1:8
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<1:8
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<1:8
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4/4
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6.3 ± 0.07
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3/4
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2.9 ± 0.36
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Influenza A (5 μg) + MF59
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<1:8
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<1:8
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<1:8
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4/4
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6.0 ± 0.19
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4/4
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3.0 ± 0.11
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BPL-SARS-CoV (5 μg)
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<1:8
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<1:8
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1:64
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1/4
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2.0 ± 0.0¶#
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0/4
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≤1.8 ± 0**††
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BPL-SARS-CoV (5 μg) + MF59
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<1:8
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1:91
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1:645
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0/4
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≤1.5 ± 0¶**
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0/4
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≤1.8 ± 0**††
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*The indicated immunogens or control preparations
were administered to mice by subcutaneous injection on 3 occasions
2 weeks apart; PBS, phosphate-buffered saline.
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†Neutralization titers were determined as described
(8).
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‡Mice were challenged with 104 50% tissue
culture infectious doses (TCID50) SARS-CoV intranasally.
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§Virus titers are expressed as log10
TCID50/g of tissue.
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¶p<0.00001 in a 2-tailed Student t test,
compared to titers seen in mice that were immunized with PBS.
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#Indicates the titer of a single animal. The remaining
3 mice had no detectable levels of virus.
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**Virus not detected; the lower limit of detection
of infectious virus was 1.5 log10 TCID50/g
in a 10% wt/vol suspension of lung homogenate and 1.8 log10
TCID50/g in a 5% wt/vol suspension of nasal turbinates.
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††p = 0.025 in a 2-tailed Student t test,
compared to titers seen in mice that were immunized with PBS.
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1These authors contributed equally to this
work.
Suggested citation
for this article:
Stadler K, Roberts
A, Becker S, Vogel L, Eickmann M, Kolesnikova L, et al. SARS vaccine protective
in mice. Emerg Infect Dis [serial on the Internet]. 2005 Aug [date
cited]. Available from http://www.cdc.gov/ncidod/EID/vol11no08/04-1003.htm
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