JOURNAL OF VIROLOGY, Aug. 2008, p. 7721–7724
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 82, No. 15
A Live Attenuated Severe Acute Respiratory Syndrome Coronavirus Is
Immunogenic and Efficacious in Golden Syrian Hamsters?
Elaine W. Lamirande,1Marta L. DeDiego,2Anjeanette Roberts,1Jadon P. Jackson,1Enrique Alvarez,2
Tim Sheahan,3Wun-Ju Shieh,4Sherif R. Zaki,4Ralph Baric,3Luis Enjuanes,2and Kanta Subbarao1*
Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, Maryland 208921; Department of Molecular and Cell Biology,
Centro Nacional de Biotecnologı ´a (CNB), Nacional de Biotecnologı ´a (CSIC), Campus Universidad Auto ´noma, Darwin 3,
Cantoblanco, 28049 Madrid, Spain2; Department of Epidemiology, University of North Carolina, Chapel Hill,
North Carolina 275993; and Infectious Diseases Pathology Activity, Centers for Disease Control and
Prevention, Atlanta, Georgia 303334
Received 11 February 2008/Accepted 29 April 2008
The immunogenicity and protective efficacy of a live attenuated vaccine consisting of a recombinant severe
acute respiratory syndrome (SARS) coronavirus lacking the E gene (rSARS-CoV-?E) were studied using
hamsters. Hamsters immunized with rSARS-CoV-?E developed high serum-neutralizing antibody titers and
were protected from replication of homologous (SARS-CoV Urbani) and heterologous (GD03) SARS-CoV in
the upper and lower respiratory tract. rSARS-CoV-?E-immunized hamsters remained active following wild-
type virus challenge, while mock-immunized hamsters displayed decreased activity. Despite being attenuated
in replication in the respiratory tract, rSARS-CoV-?E is an immunogenic and efficacious vaccine in hamsters.
Severe acute respiratory syndrome (SARS) is a respiratory
774 deaths (World Health Organization; http://www.who.int/csr
/sars/country/table2004_04_21/en/index.html). Since the initial
outbreak, there have been few sporadic cases of community-
acquired and laboratory-acquired infections. Masked palm civets
were identified as carriers of SARS-CoV (5) and horseshoe bats
carry a SARS-CoV-like virus (6, 7), suggesting that a future
outbreak would likely originate from an animal reservoir. The
threat of another SARS-CoV outbreak emphasizes the need
for vaccines and continued research on the prevention and
treatment of SARS-CoV. In the absence of ongoing human
infections, these experiments must be conducted with relevant
animal models. Several vaccine strategies are in development,
including inactivated virus, subunit, virus-like particles, DNA,
vectored, and reverse genetics-engineered vaccines (4, 8). In
this study, we examine the immunogenicity and efficacy of a
live-virus vaccine by use of an engineered SARS-CoV with the
structural E gene deleted in the Golden Syrian hamster model.
This model supports viral replication and associated pathology
in the lungs, and infected animals display reduced activity.
An infectious cDNA clone of SARS-CoV (Urbani) was as-
sembled as a bacterial artificial chromosome (1) and a virus
lacking the E gene (?E) was engineered as previously de-
scribed (2). Recombinant SARS-CoV-?E (rSARS-CoV-?E)
was restricted in replication in vitro and in vivo (2), prompting
us to evaluate the immunogenicity and efficacy of this virus as
a live attenuated vaccine in hamsters. The attenuated rSARS-
CoV-?E vaccine was compared with mock infection and
rSARS-CoV infection by use of 7-week-old male Golden Syr-
ian hamsters [LVG (SYR); Charles River Laboratories, Wil-
mington, MA] that were intranasally inoculated with 100 ?l of
103? the 50% tissue culture infectious dose (TCID50) of
rSARS-CoV or rSARS-CoV-?E or with medium only as pre-
viously described (2). Sera were collected from the hamsters
before immunization and on day 28 after immunization; two-
fold dilutions of heat-inactivated sera were tested for the pres-
ence of antibodies that neutralized the infectivity of 100
TCID50of SARS-CoV in Vero cell monolayers as described
previously (10). The immunogenicity and efficacy of the
rSARS-CoV-?E vaccine were evaluated using the homologous
virus, SARS-CoV Urbani, as well as a heterologous rSARS-
CoV bearing the spike (S) protein gene of the GD03 virus (3).
Similar titers of neutralizing antibodies were elicited by
rSARS-CoV and rSARS-CoV-?E against the homologous and
heterologous strains of SARS-CoV (Table 1). In both cases,
neutralizing antibody titers against the homologous virus were
higher (five- to eightfold) than those against the heterologous
virus, as reported earlier (3).
About 4 weeks after immunization, hamsters were chal-
lenged intranasally with 100 ?l of 103TCID50of the homolo-
gous SARS-CoV Urbani or the heterologous rSARS-CoV
GD03 strain. Four hamsters per group were sacrificed at two
time points after challenge (2 and 5 days) and their lungs and
nasal turbinates (NT) were harvested to determine the level of
virus replication and for histopathological examination. These
time points were selected because peak SARS-CoV titers in
the lungs of hamsters occur on day 2 postinfection and his-
topathological findings are most prominent on day 5 postin-
fection (9). Virus titers for 10% (wt/vol) tissue homogenates
were determined for Vero cell monolayers as described previ-
ously (10), and virus titers were expressed as TCID50/g of
tissue, with a lower limit of detection of 101.5TCID50/g.
Intranasal immunization with rSARS-CoV-?E and rSARS-
CoV provided complete protection from pulmonary replica-
tion of homologous challenge virus, while this virus replicated
* Corresponding author. Mailing address: Laboratory of Infectious
Diseases, NIAID, NIH, Bethesda, MD 20892. Phone: (301) 451-3839.
Fax: (301) 480-5719. E-mail: firstname.lastname@example.org.
?Published ahead of print on 7 May 2008.
to titers of 107.1and 107.2TCID50/g in the lungs of mock-
immunized hamsters on days 2 and 5 postchallenge, respec-
tively (Fig. 1). The challenge virus replicated to titers of 108.7
and 106.6TCID50/g on days 2 and 5 postchallenge, respectively,
in the NT of mock-immunized hamsters (Fig. 1A). In contrast,
virus titers of between 103and 105TCID50/g were observed for
the NT of the rSARS-CoV- and rSARS-CoV-?E-immunized
hamsters on day 2 postchallenge, a significant reduction
(10,000-fold) compared to the titers for mock-immunized
hamsters (P ? 0.05); moreover, challenge virus was not re-
covered from the NT of the rSARS-CoV- and rSARS-CoV-
?E-immunized hamsters on day 5 postchallenge, indicating
that the virus replicated to low titer and was cleared quickly
from the upper respiratory tract of immunized hamsters com-
pared to what was seen for mock-immunized hamsters.
The heterologous GD03 virus replicated to high titers of
107.3and 105.6TCID50/g in the NT and 105.8and 105.5
TCID50/g in the lungs of mock-immunized hamsters on days 2
and 5 postchallenge, respectively. In contrast, the lungs of
hamsters immunized with rSARS-CoV-?E and rSARS-CoV
were completely protected from replication of the GD03 virus,
and the challenge virus was cleared by day 5 postchallenge
from the NT (Fig. 1B).
The lungs of two hamsters per group harvested on days 2
and 5 postchallenge were fixed in 10% formalin and processed
for histopathological examination and immunohistochemical
analysis as described previously (10). Mock-immunized ham-
sters had focal antigen staining in the trachea (not shown) and
mild to moderate, focal to diffuse inflammatory infiltrates in
the lungs on day 2 and focal intense inflammatory infiltrates
(Fig. 2D) and scattered antigen staining on day 5 following
challenge with SARS-CoV Urbani (not shown). In contrast,
hamsters immunized with rSARS-CoV or rSARS-CoV-?E had
only focal mild inflammatory infiltrates on days 2 and 5 fol-
lowing challenge with homologous virus (Fig. 2), and viral
antigen was not detected in the lungs (not shown). On days 2
and 5 following challenge with the heterologous virus GD03,
focal to diffuse, mild to moderate infiltrates were noted in the
lungs of mock-immunized hamsters. Focal moderate infiltrates
were seen in the lungs of one hamster immunized with rSARS-
CoV-?E on day 2, but significant pulmonary inflammatory
infiltrates were not seen on day 5 postchallenge. Pulmonary
inflammation was not seen on day 2 or 5 when hamsters im-
munized with rSARS-CoV were challenged with the GD03
virus (not shown).
In order to determine whether hamsters became less active
following infection with SARS-CoV, a Nalgene activity wheel
(Nalge Nunc International, Rochester, NY) equipped with a
magnetic switch with an LCD counter that records revolutions
was placed in their cages overnight, with water and food avail-
able ad libitum. The time the hamsters spent in the cage with
the activity wheel and the number of revolutions were re-
corded, and the latter was reported as an average number of
revolutions per hour. Four hamsters were observed each night
for five consecutive nights before immunization (days ?7 to
?3) and after immunization. Activity was recorded again be-
fore and after challenge with SARS-CoV Urbani. The statis-
tical significance of the change in hamster activity following
immunization and challenge was compared as least-squares
means contrasts from a repeated-measures analysis of variance
with a Bonferroni correction.
The baseline activity level for hamsters prior to immuniza-
tion was between 700 and 1,000 revolutions/h (Fig. 3A). The
activity level of the rSARS-CoV-?E-immunized hamsters did
not change after immunization (P ? 0.32), but the activity level
of the hamsters immunized with rSARS-CoV decreased to
?300 revolutions/h (P ? 0.005). These data are consistent with
our previous observation that rSARS-CoV-?E was attenuated
compared to rSARS-CoV (2) and that activity was markedly
reduced following SARS-CoV infection (8). Three weeks after
immunization, the baseline activity level for all of the groups
FIG. 1. Replication of SARS-CoV Urbani (A) and GD03 (B) in
the NT and lungs of mock-immunized hamsters and hamsters immu-
nized with rSARS-CoV-?E or rSARS-CoV on days 2 and 5 postchal-
lenge. Virus titers represent the mean from four hamsters per day.
Error bars indicate standard errors.*, P ? 0.05 (Kruskal-Wallis,
Mann-Whitney U test). The lower limit of detection is 101.5TCID50/g
TABLE 1. Neutralizing antibody titers in sera of hamsters
immunized with rSARS-CoV or rSARS-CoV-?E against
homologous and heterologous strains of SARS-CoV
Mean (? SE) reciprocal neutralizing
antibody titer against:
?5.7 ? 0a
367 ? 97b
280 ? 73b
?5.7 ? 0a
45 ? 10b
52 ? 9b
aLower limit of detection is 5.7.
bP ? 0.05 compared to titers in mock-immunized animals.
returned to the preimmunization level (Fig. 3B). The activity
of the mock-immunized hamsters decreased to less than 100
revolutions/h (P ? 0.005) following challenge with SARS-CoV
Urbani, while the activities of the hamsters immunized with
rSARS-CoV (564 revolutions/h; P ? 0.005) and rSARS-
CoV-?E (608 revolutions/h; P ? 0.23) decreased only slightly.
The decreased activity of the rSARS-CoV-immunized ham-
sters following challenge does not appear to be biologically
significant, though it was statistically significant, presumably
because of the variance around the mean.
Our data indicate that an engineered rSARS-CoV-?E strain
did not cause clinical illness in hamsters, as measured by use of
an activity wheel. We have previously shown that rSARS-
CoV-?E is attenuated 20- to 200-fold in vitro and 100- to
1,000-fold in hamster lung or NT (2). The lower pulmonary
viral load was accompanied by less inflammation, consistent
with the difference we observed with the activity wheel (2).
rSARS-CoV-?E elicited serum-neutralizing antibodies against
both the homologous and heterologous viruses at levels com-
parable to those seen for rSARS-CoV. Neutralizing titers were
about eightfold higher against the homologous virus than
against the heterologous virus, reflecting the antigenic differ-
ences between the viruses (3).
Interestingly, immunization with rSARS-CoV-?E reduced
the replication of the wild-type challenge viruses in the upper
respiratory tract of hamsters and completely protected lungs
against homologous and heterologous challenge. The observa-
tion of complete protection in the lower respiratory tract and
FIG. 2. Lung histopathology of normal (uninfected) (A), rSARS-CoV-immunized (B), rSARS-CoV-?E-immunized (C), and mock-immunized
(D) hamsters 5 days after challenge with SARS-CoV Urbani. Hematoxylin and eosin staining. Magnification, ?20.
VOL. 82, 2008NOTES7723
partial protection in the upper respiratory tract is consistent Download full-text
with protection mediated by serum antibodies (10, 11). Wide-
spread eosinophilic pulmonary infiltrates described following
challenge to mice vaccinated with an alphavirus expressing the
SARS-CoV N protein (3) were not prominent in hamsters
vaccinated with rSARS-CoV-?E. This is reassuring, because
this live attenuated vaccine virus expresses the N protein in the
context of the other SARS-CoV proteins. Following challenge
with homologous or heterologous virus, the lungs of mock-
immunized hamsters had focal intense inflammatory infiltrates
and viral antigen was present. However, hamsters immunized
with rSARS-CoV-?E or rSARS-CoV showed only mild focal
infiltrates in the lungs and viral antigen was not detected.
These results correlated with the activity of the hamsters and
the quantitative virological data.
The ability of the rSARS-CoV-?E vaccine to protect against
challenge with the heterologous virus GD03 is significant, be-
cause this virus strain is antigenically one of the most divergent
strains from SARS-CoV Urbani; it clusters phylogenetically
with the animal SARS-CoV isolates (3), and was selected as a
representative of an animal SARS-CoV because if SARS were
to reemerge, it would likely come from an animal source. Our
data indicate that the rSARS-CoV-?E shows promise as a live
attenuated vaccine. In addition, it could be used to produce an
inactivated vaccine that may be safer to handle than virulent
wild-type viruses. Evaluation of this vaccine candidate in other
animal models is in progress.
We thank the staff of the Building 50 Shared Animal Facility,
NIAID, for assistance with the animal studies. We also thank Jeff
Skinner for assistance with the statistical analysis and Victor Barcelona
for computer graphics assistance.
This research was supported in part by the Intramural Research
Program of the NIH, NIAID; by NIH AID AI059136; and by the
European Community (projects DISSECT SP22-CT-2004-511060 and
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FIG. 3. Hamster activity wheel use. (A) Activity of hamsters re-
corded on indicated days before (squares) and after intranasal immu-
nization (arrow) for hamsters immunized with rSARS-CoV or rSARS-
CoV-?E. n ? 4 per group. Error bars indicate standard errors. The
difference in the activity of hamsters that received rSARS-CoV-?E
and the preimmunization activity of all hamsters was not significant
(P ? 0.32). The difference in the activity of hamsters that received
rSARS-CoV and the preimmunization activity of all hamsters was
significant (P ? 0.005). (B) Activity of hamsters immunized with
rSARS-CoV, rSARS-CoV-?E, or medium alone (L15; mock) re-
corded on indicated days before (squares) and after intranasal chal-
lenge with SARS-CoV Urbani (arrow). n ? 4 per group. Error bars
indicate standard error. The difference in the activity of hamsters that
received rSARS-CoV-?E and the prechallenge activity of all hamsters
was not significant (P ? 0.23). The difference in the activity of
hamsters that received rSARS-CoV and the prechallenge activity of
all hamsters was significant (P ? 0.005). The difference in the
activity of hamsters that were mock immunized and the prechal-
lenge activity of all hamsters was significant (P ? 0.005).
7724 NOTESJ. VIROL.