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Prior Immunization with Severe Acute Respiratory Syndrome (SARS)-Associated Coronavirus (SARS-CoV) Nucleocapsid Protein Causes Severe Pneumonia in Mice Infected with SARS-CoV

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The details of the mechanism by which severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneumonia are unclear. We investigated the immune responses and pathologies of SARS-CoV-infected BALB/c mice that were immunized intradermally with recombinant vaccinia virus (VV) that expressed either the SARS-CoV spike (S) protein (LC16m8rVV-S) or simultaneously all the structural proteins, including the nucleocapsid (N), membrane (M), envelope (E), and S proteins (LC16m8rVV-NMES) 7-8 wk before intranasal SARS-CoV infection. The LC16m8rVV-NMES-immunized group exhibited as severe pneumonia as the control groups, although LC16m8rVV-NMES significantly decreased the pulmonary SARS-CoV titer to the same extent as LC16m8rVV-S. To identify the cause of the exacerbated pneumonia, BALB/c mice were immunized with recombinant VV that expressed the individual structural proteins of SARS-CoV (LC16mOrVV-N, -M, -E, -S) with or without LC16mOrVV-S (i.e., LC16mOrVV-N, LC16mOrVV-M, LC16mOrVV-E, or LC16mOrVV-S alone or LC16mOrVV-N + LC16mOrVV-S, LC16mOrVV-M + LC16mOrVV-S, or LC16mOrVV-E + LC16mOrVV-S), and infected with SARS-CoV more than 4 wk later. Both LC16mOrVV-N-immunized mice and LC16mOrVV-N + LC16mOrVV-S-immunized mice exhibited severe pneumonia. Furthermore, LC16mOrVV-N-immunized mice upon infection exhibited significant up-regulation of both Th1 (IFN-gamma, IL-2) and Th2 (IL-4, IL-5) cytokines and down-regulation of anti-inflammatory cytokines (IL-10, TGF-beta), resulting in robust infiltration of neutrophils, eosinophils, and lymphocytes into the lung, as well as thickening of the alveolar epithelium. These results suggest that an excessive host immune response against the nucleocapsid protein of SARS-CoV is involved in severe pneumonia caused by SARS-CoV infection. These findings increase our understanding of the pathogenesis of SARS.
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of October 29, 2015.
This information is current as Infected with SARS-CoV
Protein Causes Severe Pneumonia in Mice
Coronavirus (SARS-CoV) Nucleocapsid
Respiratory Syndrome (SARS)-Associated
Prior Immunization with Severe Acute
Matsushima and Michinori Kohara
Hisatoshi Shida, Minoru Kidokoro, Kyosuke Mizuno, Kouji
Hidenori Suzuki, Katsuo Karamatsu, Yasuhiro Yasutomi,
Satoshi Sekiguchi, Kouichi Morita, Tsunekazu Hishima,
Inoue, Misako Yoneda, Shoji Yokochi, Ryoichi Kase,
Fumihiko Yasui, Chieko Kai, Masahiro Kitabatake, Shingo
http://www.jimmunol.org/content/181/9/6337
doi: 10.4049/jimmunol.181.9.6337
2008; 181:6337-6348; ;J Immunol
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Prior Immunization with Severe Acute Respiratory Syndrome
(SARS)-Associated Coronavirus (SARS-CoV) Nucleocapsid Protein
Causes Severe Pneumonia in Mice Infected with SARS-CoV
1
Fumihiko Yasui,* Chieko Kai,
Masahiro Kitabatake,
2
* Shingo Inoue,
Misako Yoneda,
Shoji Yokochi,*
§
Ryoichi Kase,* Satoshi Sekiguchi,* Kouichi Morita,
Tsunekazu Hishima,
Hidenori Suzuki,
Katsuo Karamatsu,
#
Yasuhiro Yasutomi,
#
Hisatoshi Shida,**
Minoru Kidokoro,
††
Kyosuke Mizuno,
‡‡
Kouji Matsushima,
§
and Michinori Kohara
3
*
The details of the mechanism by which severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneu-
monia are unclear. We investigated the immune responses and pathologies of SARS-CoV-infected BALB/c mice that were immunized
intradermally with recombinant vaccinia virus (VV) that expressed either the SARS-CoV spike (S) protein (LC16m8rVV-S) or simul-
taneously all the structural proteins, including the nucleocapsid (N), membrane (M), envelope (E), and S proteins (LC16m8rVV-NMES)
7– 8 wk before intranasal SARS-CoV infection. The LC16m8rVV-NMES-immunized group exhibited as severe pneumonia as the
control groups, although LC16m8rVV-NMES significantly decreased the pulmonary SARS-CoV titer to the same extent as
LC16m8rVV-S. To identify the cause of the exacerbated pneumonia, BALB/c mice were immunized with recombinant VV that ex-
pressed the individual structural proteins of SARS-CoV (LC16mOrVV-N, -M, -E, -S) with or without LC16mOrVV-S (i.e.,
LC16mOrVV-N, LC16mOrVV-M, LC16mOrVV-E, or LC16mOrVV-S alone or LC16mOrVV-N LC16mOrVV-S, LC16mOrVV-M
LC16mOrVV-S, or LC16mOrVV-E LC16mOrVV-S), and infected with SARS-CoV more than 4 wk later. Both LC16mOrVV-N-
immunized mice and LC16mOrVV-N LC16mOrVV-S-immunized mice exhibited severe pneumonia. Furthermore, LC16mOrVV-
N-immunized mice upon infection exhibited significant up-regulation of both Th1 (IFN-
, IL-2) and Th2 (IL-4, IL-5) cytokines and
down-regulation of anti-inflammatory cytokines (IL-10, TGF-
), resulting in robust infiltration of neutrophils, eosinophils, and lym-
phocytes into the lung, as well as thickening of the alveolar epithelium. These results suggest that an excessive host immune response
against the nucleocapsid protein of SARS-CoV is involved in severe pneumonia caused by SARS-CoV infection. These findings increase
our understanding of the pathogenesis of SARS. The Journal of Immunology, 2008, 181: 6337– 6348.
From November 2002 to July 2003, an outbreak of severe
acute respiratory syndrome (SARS),
4
which originated in
China, spread worldwide, resulting in 8098 cases with 774
deaths (http://www.who.int/csr/sars/country/en/index.html). Pa-
tients with SARS usually develop high fever followed by severe
clinical symptoms, which include acute respiratory distress syn-
drome with diffuse alveolar damage, and ultimately death. A novel
type of coronavirus (CoV), termed SARS-associated CoV (SARS-
CoV), was identified as the etiologic agent of SARS (1–3). The
genome of SARS-CoV is a single strand of positive-sense RNA of
30 kb in length with 14 putative open reading frames, which
encode nonstructural replicase polyproteins and several structural
proteins, including spike (S), envelope (E), membrane (M), and
nucleocapsid (N) proteins (4). The S protein of SARS-CoV, like
the S proteins of other CoVs, plays an important role in the first
step of viral infection by binding to a host cell receptor. Angio-
tensin-converting enzyme 2 was identified as the host receptor for
SARS-CoV (5). Angiotensin-converting enzyme 2 is abundantly
expressed in the epithelia of the lung and small intestine and may
mediate SARS-CoV entry in humans (6). Although intensive in-
vestigations rapidly unraveled the sequence of the SARS-CoV ge-
nome and its receptor in humans, the precise molecular mechanism
underlying the development of SARS is not fully understood.
The possible roles of host anti-SARS-CoV immune responses
have been suggested in severe clinical cases. The uncontrolled
release of immune mediators, called a “cytokine storm,” has been
*Department of Microbiology and Cell Biology,
Laboratory of Electron Microscopy,
The Tokyo Metropolitan Institute of Medical Science,
Laboratory Animal Research Cen-
ter, The Institute of Medical Science,
§
Department of Molecular Preventive Medicine,
School of Medicine, The University of Tokyo, and
Department of Pathology, Tokyo
Metropolitan Komagome Hospital, Tokyo, Japan;
Department of Virology, Institute of
Tropical Medicine, Nagasaki University, Nagasaki, Japan;
#
Laboratory of Immunoregu-
lation and Vaccine Research, Tsukuba Primate Research Center, National Institute of
Biomedical Innovation, Ibaraki, Japan; **Division of Molecular Virology, Institute for
Genetic Medicine, Hokkaido University, Sapporo, Japan;
††
Third Department of Virol-
ogy, National Institute of Infectious Diseases, Musashimurayama, Japan; and
‡‡
The
Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan
Received for publication January 23, 2008. Accepted for publication August 23, 2008.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This study was supported in part by a Grant for Research on Emerging and Re-emerging
Infectious Diseases from the Ministry of Health, Labor and Welfare, Japan, by the 21st Cen-
tury Centers of Excellence program on Global Strategies for Control of Tropical and Emerging
Infectious Diseases at Nagasaki University, and by the Ministry of Education, Culture, Sports,
Science and Technology of Japan. Strategic cooperation to control emerging and re-emerging
infections is funded by the Special Co-ordination Fund for Promoting Science and Technology
of the Ministry of Education, Culture, Sports, Science and Technology.
2
Current address: Department of Immunology, Graduate School of Medicine, Kum-
amoto University, 1-1-1 Honjo, Kumamoto, 860-8556, Japan.
3
Address correspondence and reprint requests to Dr. Michinori Kohara, Department
of Microbiology and Cell Biology, The Tokyo Metropolitan Institute of Medical
Science, 3-18-22 Honkomagome, Bunkyo-ku, Tokyo 113-8613, Japan. E-mail ad-
dress: kohara-mc@igakuken.or.jp
4
Abbreviations used in this paper: SARS, severe acute respiratory syndrome; CoV,
coronavirus; VV, vaccinia virus; HA, hemagglutinin; MOI, multiplicity of infection;
VLP, virus-like particle; TCID
50
, tissue culture ID
50
.
Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00
The Journal of Immunology
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implicated in the pathogenesis of SARS. However, the cytokine
profiles of SARS patient sera do not correlate with the severity of
pneumonia because of their diversity. For example, Jones et al. (7)
have reported a decreased number of IL-2-, IL-4-, IL-10-, and
IL-12-producing cells in SARS-CoV-infected patients. In contrast,
Wong et al. (8) have demonstrated increased production of IFN-
,
IL-1, IL-6, and IL-12 p70, but not of IL-2, IL-4, IL-10 or TNF-
,
which is consistent with a Th1 response. The data from these adult
patients with SARS show no clear trend toward either a Th1 or
Th2 bias. These results might be related to patient anamnesis.
Therefore, the development of animal models for SARS is needed
to understand the pathogenesis of SARS. Non-human primates,
mice, ferrets, and hamsters have been found to support the repli-
cation of SARS-CoV (9 –14). However, an animal model that
mimics the clinical symptoms and pathology observed in SARS
patients has not been reported to date. Recently, Roberts et al. (15)
reported that aged BALB/c mice (older than 12 mo) exhibited high
and prolonged levels of viral replication, signs of clinical symp-
toms, and histopathologic changes in the lung. Aged BALB/c mice
represent a conventional animal model that mimics the findings in
elderly SARS patients, many of whom exhibit severe disease re-
quiring intensive care and ventilation support, as well as increased
mortality.
In the present study, we investigated the pulmonary immune
responses and pathologies of intranasally SARS-CoV-infected
BALB/c mice older than 6 mo of age that were previously immu-
nized with SARS-CoV structural proteins using vaccinia virus
(VV) vectors, by measuring various cytokine mRNAs and his-
topathologies of the lungs.
Materials and Methods
Cells and viruses
RK13 cells (CCL-37) from the American Type Culture Collection (ATCC)
and Vero E6 cells (CRL-1586) from ATCC were cultured in MEM (Nissui
Pharmaceutical) that contained 5% FCS. To generate recombinant VV
LC16m8, which expresses the structural proteins of SARS-CoV, primary
rabbit kidney cell cultures were prepared by overnight digestion with 100
PU/ml dispase (Sanko Jun-yaku) of kidneys extirpated from 7-day-old in-
bred JW rabbits (Kitayama Labs). The cells were grown in T175 flasks in
lactalbumin medium with Hank’s salts (LH) that contained 5% FCS, 100
U/ml penicillin, and 100
g/ml streptomycin. When the cell confluency
was 50%, the culture medium was replaced with lactalbumin medium
with Eagle’s salts (LE) that contained 5% FCS, 100 U/ml penicillin, and
100
g/ml streptomycin. SARS-CoV Vietnam/NB-04/2003 strain, which
was isolated from the throat wash fluid of one patient (16), was provided
by Dr. M. Quynh Le. VVs LC16m8 (m8) and LC16mO (mO) were pro-
vided by the Chemo-Sero-Therapeutic Research Institute (Kumamoto, Ja-
pan). All work using SARS-CoV was performed in BioSafety Level 3
facilities by personnel wearing powered air-purifying respirators (Shige-
matsu Works).
Generation of recombinant VV
To generate a pBR322-based plasmid vector (pBMSF) for homologous
recombination into the hemagglutinin (HA) locus of m8, we cloned the HA
gene, which contained the ATI/p7.5 synthetic hybrid promoter, from the
pSFJ1–10 plasmid and inserted it into the pBM vector, which was recon-
structed in our laboratory. Full-length cDNAs for the SARS-CoV nucleo-
capsid (N), membrane (M), and envelope (E) proteins were cloned from the
Vietnam/NB-04/2003 strain of SARS-CoV by RT-PCR (16). Full-length
SARS-CoV spike (S) protein gene was prepared from pSFJ1-10-SARS-S,
which is described in our previous report (17). Next, the genes that encode
the SARS-CoV structural proteins were ligated by inserting internal ribo-
somal entry site sequence of hepatitis C virus (genotypes 2a and 1b/2b)
fused with the 2A sequence of foot and mouth disease virus and Thosea
asigna virus or encephalomyocarditis virus by PCR (see Fig. 1A). The
generated DNA fragment was digested with EcoRI and inserted down-
stream of the ATI/p7.5 hybrid promoter of pBR322-based plasmid vector
pBMSF, thereby generating pBMSF-SARS-NMES. The pBMSF-SARS-
NMES plasmid was linearized with PvuI, and transfected into primary
rabbit kidney cells that had been infected with m8 at a multiplicity of
infection (MOI) of 10. After 36 h, the virus-cell mixture were harvested by
scraping, and frozen at 80°C until use. The resulting HA-negative re-
combinant viruses were purified as previously described (17), and named
m8rVV-NMES. Furthermore, recombinant mO that expressed the SARS-
CoV N, M, or E protein with a six histidine tag at the C terminus was
generated (mOrVV-NHis, mOrVV-MHis, and mOrVV-EHis), as was mO
that expressed six histidine-tagged S protein (mOrVV-SHis), as previously
described (17).
Western blot analysis
Vero E6 cells were infected with m8rVV-NMES at an MOI of 5. After
18 h, the cells were lysed with lysis buffer (10 mM Tris (pH 7.4), 150 mM
NaCl, 1% SDS, 0.5% Nonidet P-40, protease inhibitor cocktail). The cell
lysates (30
g) were resolved by SDS-PAGE and transferred to a polyvi-
nylidene difluoride membrane (Immobilon-P; Millipore). After blocking
the membranes with 5% skim milk solution at room temperature for 1 h,
the membrane was incubated with polyclonal Abs against the N, M, E, or
S protein. Vero E6 cell lysates infected with mOrVV-NHis, mOrVV-MHis,
mOrVV-EHis, or mOrVV-SHis was used as positive controls. We used the
anti-S polyclonal Abs described in our previous study (17). Polyclonal Abs
against N and E proteins were prepared from rabbit sera immunized with
KLH-conjugated N peptide (residues aa 250 –263) and E peptide (residues
aa 61–73). Polyclonal Abs against the M protein were provided by Dr.
Mizutani (National Institute of Infectious Diseases, Musashimurayama,
Tokyo). We purified the IgG fractions of these antisera using the protein A
Ampure PA kit (Amersham Biosciences). After washing with TBS that
contained 0.1% Tween 20 (TBST), the membranes were reacted with HRP-
conjugated F(ab)
2
of anti-rabbit IgG (GE Healthcare). Each specific pro-
tein band was visualized using the ECL system (GE Healthcare).
Indirect immunofluorescence analysis
Vero E6 cells were infected with m8rVV-NMES at an MOI of 5 at 30°C
for 4 h. The cells were washed with PBS and fixed with cold acetone/
methanol (1/1) mixture for 10 min. After blocking with TNB blocking
buffer (NEN Life Science Products) at room temperature for 1 h, the fixed
cells were incubated with polyclonal Abs against the N, M, or E protein or
mAb against the S protein (designated as anti-S-His protein, clone no.
13B8), which was originally prepared in our laboratory, at 4°C overnight.
After washing, the cells were incubated with Alexa Fluor 488-conjugated
anti-rabbit IgG or mouse IgG Ab at room temperature for 1 h. Nuclei were
stained with DAPI (4,6-diamidino-2-phenylindole). Fluorescence images
were acquired using a confocal microscope (LSM510 META; Carl Zeiss).
Confirmation of SARS-CoV-like particle formation
RK13 cells were cultured in 150-mm dishes, and then infected with
m8rVV-NMES at an MOI of 5. After 48 h of incubation, the culture su-
pernatants were collected and centrifuged to remove cell debris at 3000
rpm for 30 min at 4°C. The supernatants were concentrated 100-fold
using the Pellicon XL (cut off molecular weight 3 10
5
; Millipore). The
isolation of virus-like particles (VLP) was performed as previously de-
scribed, with a slight modification (18). Briefly, the concentrated superna-
tant was placed on 60% (w/w) sucrose cushion and centrifuged at 4.0
10
4
rpm for 5 h. The opalescent band was collected and centrifuged in a
20 60% (w/w) sucrose gradient at 2.7 10
4
rpm for 4 h, and then divided
into 20 fractions. The protein content of each fraction was determined with
the DC protein assay kit (Bio-Rad). The 20
l of each fraction were sep-
arated by SDS-PAGE (7.5%, 10%, or 15% polyacrylamide gel), and trans-
ferred onto a polyvinylidene difluoride membrane. The membrane was
incubated with mAb against S protein (13B8), mAb against N protein
(IMG-654; Imgenex) or polyclonal Abs against the M or E protein. After
washing, the membranes were reacted and visualized as described. The
VLPs in the concentrated culture supernatant were visualized using trans-
mission electron microscopy. For immunogold staining, VLPs were loaded
onto a collodion-coated electron microscopy grid for 5 min. After the re-
moval of excess sample solution, polyclonal Ab against S protein was
added onto the grid and incubated at room temperature for 1 h. The grids
were washed six times with Sorensen’s phosphate buffer at room temperature
and incubated with 5-nm gold-conjugated anti-rabbit IgG for 1 h. After wash-
ing with Sorensen’s phosphate buffer for 10 s, the samples were stained with
2% phosphotungstic acid for 1 min. After draining off the excess phospho-
tungstic acid, the samples were observed under the electron microscopy.
Immunization of rabbits with m8rVV-NMES
Groups of three New Zealand White rabbits (SLC) were immunized intra-
dermally with 1 10
8
PFU/body of m8rVV-NMES or with 1 10
8
PFU/body of m8, at 0 and 6 wk. Sera were collected at the indicated time
6338 N PROTEIN OF SARS-CoV AS A CAUSE OF PNEUMONIA EXACERBATION
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points (see Fig. 2A), and used in ELISA analysis and in the in vitro neu-
tralization assay described below. All animal experiments using rabbits
were approved by The Tokyo Metropolitan Institute of Medical Science
Animal Experiment Committee and were performed in accordance with the
animal experimentation guidelines of The Tokyo Metropolitan Institute of
Medical Science.
ELISA
Recombinant SARS-CoV N, M, E, and S proteins tagged with six histi-
dines at the C terminus were expressed in RK13 cells by infecting with
mOrVV-N-His, mOrVV-E-His, mOrVV-M-His, or mOrVV-S-His at an
MOI of 5. These proteins were purified using nickel Sepharose (6 Fast
Flow; GE Healthcare). His-tagged E and M proteins were further purified
by SDS-PAGE. These full-length structural proteins (0.2
g/ml, 50
l/
well) were coated onto 96-well plates at 4°C overnight. The plates were
blocked with 1% BSA in PBS() that contained 0.5% Tween 20 and 2.5
mM EDTA, and then incubated with serial 2-fold dilutions of sera from the
rabbits immunized with m8rVV-NMES or m8. After extensive washing,
the plates were assayed as previously described, except that o-phenylene-
diamine was used as the substrate (17). The individual SARS-CoV struc-
tural protein-specific IgG titers are presented as the end point dilution Ab
titers. The end point titer was defined as the reciprocal of the highest di-
lution of serum at which the absorbance at 490 nm (A
490
) ratio (A
490
of
m8rVV-NMES-immunized serum/A
490
of m8-immunized serum (negative
control)) was greater than 2.0, as previously described (19).
In vitro neutralization assay for SARS-CoV
The neutralizing Ab titers of the sera of rabbits immunized with m8rVV-
NMES or m8 were determined as previously described (17). Briefly, serial
2-fold dilutions of heat-inactivated sera were mixed with equal volumes of
200 tissue culture ID
50
(TCID
50
) of SARS-CoV and incubated at 37°C for
1 h. Vero E6 cells were then infected with 100
l of the virus-serum
mixtures in 96-well plates. After 5 days (or 6 days in the SARS-CoV
challenge experiment) of infection, the neutralization titer was determined
as the end point dilution of the serum at which there was 50% inhibition of
the SARS-CoV-induced cytopathic effect. The method used for end point
calculation was that described by Reed and Muench (20).
SARS-CoV challenge experiment
Female BALB/c mice older than the 6 mo of age (SLC) were used in this
study. Four groups of eight BALB/c mice (seven mice in the vehicle-
immunized group) were inoculated intradermally with either 1 10
7
PFU/
body of m8, m8rVV-S, or m8rVV-NMES or 70
l of vehicle (MEM with-
out FCS). At 7– 8 wk postimmunization, the mice were infected
intranasally with 1 10
5
TCID
50
/body of SARS-CoV (20
l/mouse), as
previously described (11). Four mice from each group were sacrificed 2
and 9 days later, except for the three mice of the vehicle-immunized group,
which were sacrificed 2 days later. The mice were sacrificed under anes-
thesia and the lung, liver, small intestine, and spleen were extirpated. Ali-
quots of these tissues were frozen immediately at 80°C or fixed with 10%
formalin. The collected blood was used for the in vitro neutralization assay.
In addition, BALB/c mice were injected intradermally with 1 10
7
PFU/
body of recombinant VV that expressed each structural protein of SARS-
CoV (mOrVV-NHis, mOrVV-MHis, mOrVV-EHis, mOrVV-SHis) with or
without LC16mOrVV-SHis (i.e., LC16mOrVV-N, -M, -E, -S alone or
LC16mOrVV-N LC16mOrVV-S, -M LC16mOrVV-S, or -E
LC16mOrVV-S), and infected with 1 10
5
TCID
50
/body of SARS-CoV
more than 4 wk later. After 2 and 9 days, mice (n3–5 per group) were
sacrificed following blood collection under anesthesia, and their lungs were
extirpated. All animal experiments using mice were approved by the An-
imal Experiment Committee at The Institute of Medical Science, Univer-
sity of Tokyo, and were performed in accordance with the animal exper-
imentation guidelines of The Institute of Medical Science, University of
Tokyo.
Determination of viral titers in the organs
The SARS-CoV titers in the mouse organs were determined as previously
described (11). Briefly, tissue samples (i.e., lung, liver, small intestine, and
spleen) were homogenized in a 10-fold volume of Leibovitz 15 medium
(Invitrogen). The homogenates were centrifuged at 2000 rpm for 10 min at
4°C. Serial 10-fold dilutions of the supernatants of these homogenates were
added to Vero E6 cells seeded on 96-well plates. After 6 days of incuba-
tion, the cells were fixed with 10% formalin. Viral titer was determined as
the 50% end point dilution of the homogenate that induced the cytopathic
effect. The method used for end point calculation was that described by
Reed and Muench (20).
Lung histopathology and inflammation scores
In accordance with a previous report (11), 10% formalin-fixed lung tissues
of the SARS-CoV-infected mice were embedded in paraffin. Paraffin block
sections (4-
m thickness) were stained with H&E staining. The peribron-
chial and perivascular scores were recorded in a blinded fashion by a pa-
thologist. We evaluated pulmonary pathology using the histopathologic
scoring systems developed by Cimolai et al. (21), in which the scoring
system is weighted heavily for bronchial lesions. This scoring system al-
lowed us to differentiate the severity of pulmonary pathology in small
groups of animals. The pathology grading system consisted of a numerical
score ranging from 0 to 26. In brief, each section was scored based upon
a cumulative total from five categories that incorporated evaluations of the
following: A) number of bronchiolar and bronchial sites affected by the
periluminal infiltrate (range, 0 to 3); B) severity of the periluminal infiltrate
(range, 0 to 3); C) luminal exudate severity (range, 0 to 2); D) frequency
of perivascular infiltrate (range, 0 to 3); and E) severity of parenchymal
pneumonia (range, 0 to 5). The accumulated numeric score was derived
from the sum of the subscores: A3(BC)DE. Eosinophils were de-
tected in tissue sections by method of Luna (22).
Extraction of total RNA and quantitative RT-PCR of cytokine
or chemokine mRNA
To measure the levels of cytokine or chemokine mRNA, total RNA sam-
ples were extracted from the lungs using the RNeasy Mini kit (Qiagen).
Quantitative RT-PCR was conducted with TaqMan Gene Expression as-
says (Applied Biosystems) using the ABI Prism 7700 and Sequence De-
tection System software v.1.7. The fold change in copy number of each
cytokine/chemokine mRNA was revealed using the 2
⫺⌬⌬Ct
method using
18 S rRNA as an endogenous calibrator.
Statistical analysis
Data are presented as mean SD. Statistical analysis was performed by
one-way ANOVA, followed by the Dunnett or Bonferroni test. A value of
p0.05 was considered to be statistically significant.
Results
Generation of recombinant VV that expresses the structural
proteins of SARS-CoV
A multicistronic transgene that expresses simultaneously four
structural proteins (N, M, E, and S proteins) of SARS-CoV was
constructed and inserted into the HA locus of LC16m8 (m8) by
homologous recombination (Fig. 1A). Expression of the transgene
was placed under the control of the powerful ATI/p7.5 hybrid pro-
moter. We screened for m8rVV-NMES using the erythrocyte ag-
glutination assay (17), and confirmed the insertion of the transgene
by PCR. Expression of the N, M, E, and S proteins in Vero E6 cells
infected with m8rVV-NMES was detected by Western blot anal-
ysis. Recombinant LC16mO (mO) expressing the C-terminal his-
tidine-tagged N, M, E or S protein (mOrVV-NHis, -MHis, -EHis,
and -SHis) was generated as previously described, and used as a
positive control for each protein. We also used m8rVV-S (17). As
shown in Fig. 1B, the expression levels of the N and S proteins in
the m8rVV-NMES-infected cells were high and moderate, respec-
tively. In contrast, the expression levels of the M and E proteins
in m8rVV-NMES-infected cells were weaker than those in mOrVV-
MHis- and mOrVV-EHis-infected cells. The M protein in the
m8rVV-NMES-infected cells was 20 kDa, whereas that in the
mOrVV-MHis-infected cells was observed as forms of 20 kDa
(nonglycosylated form) and 25 kDa (glycosylated form) (23). Fur-
thermore, we investigated the cellular localizations of these struc-
tural proteins by indirect immunofluorescence (Fig. 1C). In
m8rVV-NMES-infected cells, all of the SARS-CoV proteins were
localized in the perinuclear regions. In particular, the localization of
the N protein in m8rVV-NMES-infected cells was different from that
in mOrVV-NHis-infected cells, in which the N-His protein was found
diffusely in the cytoplasm. VLPs are formed by the assembly of struc-
tural proteins in the cytoplasm, followed by release into the culture
medium. By infecting m8rVV-NMES into RK13 cells, we confirmed
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FIGURE 1. Construction of recombinant VV that express four structural proteins of SARS-CoV (m8rVV-NMES). A, DNA fragments that encode the
SARS-CoV N, M, E, and S proteins were ligated with the internal ribosomal entry site sequence of hepatitis C virus (2a and 1b/2b) and fused with the 2A
sequences of foot and mouth disease virus (FMDV) and Thosea asigna virus (TaV) or encephalomyocarditis (EMCV). After digestion with EcoRI, the DNA
fragment was inserted into the pBMSF vector, and the resultant plasmid was designated as pBMSF-NMES. PvuI-linearized pBMSF-NMES was used for
homologous recombination into the HA locus of the LC16m8 genome. Recombinant mO that expressed the SARS-CoV N, M, E, or S protein was generated
(mOrVV-NHis, -MHis, -EHis, and -SHis) as described in Materials and Methods.B, Vero E6 cells were infected with m8rVV-NMES or m8. Uninfected
Vero E6 cells were used as a negative control (NC). Structural proteins mOrVV-NHis, mOrVV-MHis, mOrVV-EHis, and mOrVV-SHis were used as
positive controls. SARS-CoV structural proteins were detected using rabbit polyclonal Abs and donkey anti-rabbit IgG polyclonal Abs, which were
conjugated with HRP. The lane between m8rVV-NMES and the mOrVV-N, mOrVV-M, mOrVV-E, and mOrVV-S samples was left empty, to exclude the
possibility of leakage of sample solution between lanes. C, Vero E6 cells were infected with m8rVV-NMES at an MOI of 5 at 30°C for 4 h. The SARS-CoV
proteins in the fixed cells were visualized with the polyclonal Abs against the N, M, or E protein or mAb against the S protein (designated as 13B8). Nuclei
were stained with DAPI). Structural proteins mOrVV-NHis, mOrVV-MHis, mOrVV-EHis, and mOrVV-SHis were used as positive controls (PC). D, The
VLPs were isolated from the culture supernatants of RK13 cells infected with m8rVV-NMES at an MOI of 5 for 48 h at 30°C. After sucrose gradient
centrifugation, 20 fractions were collected. E, Equal amounts of the gradient fractions (nos. 3–16) were examined by Western blot analyses. m8, m8-infected
RK13 cell lysate; ppt, m8rVV-NMES-infected RK13cell lysate; PC, RK13 cell lysates infected with mOrVV-NHis, mOrVV-MHis, mOrVV-EHis, or
mOrVV-SHis. F, A concentrated culture supernatant was subjected to transmission electron microscopy. VLPs were probed with polyclonal Ab against the
S protein and incubated with 5-nm gold-conjugated anti-rabbit IgG.
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the formation of VLPs in the culture medium. After sucrose gradient
centrifugation, 20 fractions (500
l each) were collected (Fig. 1D).
The four SARS-CoV structural proteins were monitored by Western
blot analysis. As shown in Fig. 1E, fraction number 10 contained all
the SARS-CoV proteins, and the buoyant density of this fraction was
1.15 g/ml, a value that is consistent with previous reports (18, 24,
25). Moreover, we confirmed the formation of VLPs in the concen-
trated culture supernatant using scanning electron microscopy and im-
munogold-labeling with the anti-S protein polyclonal Ab. The parti-
cles were 70 –100 nm in diameter, which is consistent with the sizes
as reported previously (18, 24, 25). The particles were positively
stained with immunogold (Fig. 1F).
Induction of Abs specific for SARS-CoV structural proteins in
rabbits immunized with m8rVV-NMES
To investigate the immunogenicity of m8rVV-NMES, 1 10
8
PFU/
body of either m8rVV-NMES or m8, its parental strain, was inocu-
lated intradermally on the backs of New Zealand White rabbits at 0
and 6 wk (Fig. 2A). Rabbit antisera specific for the full-length struc-
tural proteins of SARS-CoV were detected by ELISA (Fig. 2B). In
agreement with previous reports (26 –28), the N and S proteins both
exhibited strong immunogenicity in rabbits. IgG-specific for the N or
S protein was induced as early as 1 wk after m8rVV-NMES immu-
nization, and the titer exceeded 1:10000 2 wk later. The titers of Abs
against the N and S proteins were dramatically increased by booster
immunization with m8rVV-NMES. It was also observed that the Ab
titer of the N protein, but not that of the S protein, decreased after
reaching the peak titer. Immunization with m8rVV-NMES did not
induce Abs specific for the E and M proteins, even after booster im-
munization (Fig. 2B). The antigenicity of the purified E and M pro-
teins coated onto the ELISA plates was confirmed using each rabbit
anti-E or anti-M peptide Ab (data not shown). Therefore, we believe
that the lack of induction of Abs specific for the E and M proteins in
the rabbit sera results from the poor immunogenicity and lower ex-
pression levels of these proteins.
Induction of SARS-CoV-neutralizing serum Abs in rabbits by
immunizing with m8rVV-NMES
We determined the neutralization titers against SARS-CoV us-
ing the same rabbit antisera. The neutralization titer was 1:30
FIGURE 2. Immunogenicity of m8rVV-NMES in rabbits. A, New Zea-
land White rabbits (n3) were inoculated intradermally with 10
8
PFU/
body of m8rVV-NMES or m8 at 0 and 6 wk. Blood samples were collected
at the indicated time points. B, Induction of serum IgG specific for the four
structural proteins of SARS-CoV. The individual SARS-CoV structural
protein-specific IgG titers are presented as the end point dilution Ab titers.
The end point titer was defined as the reciprocal of the highest dilution of
serum at which the absorbance at 490 nm (A
490
) ratio (A
490
of m8rVV-
NMES-immunized serum/A
490
of m8-immunized serum (negative con-
trol)) was greater than 2.0. C, Induction of neutralizing Abs against SARS-
CoV. The neutralization titer of m8rVV-NMES-immunized rabbit sera was
defined as the end point dilution of the serum at which there was 50%
inhibition (NT
50
) of the SARS-CoV-induced cytopathic effect. Immuniza-
tion with m8rVVs or m8 was conducted using the schedule described in
Fig. 3A. ND, Not detectable.
FIGURE 3. SARS-CoV challenge to BALB/c mice immunized with
m8rVV-NMES or m8rVV-S. A, Four groups of eight BALB/c mice (seven
mice in the vehicle-immunized group) were inoculated intradermally with
m8rVV-NMES, m8rVV-S, m8, or vehicle and challenged 7– 8 wk later
with 1 10
5
TCID
50
/body of SARS-CoV delivered via the intranasal
route. Blood and lung tissues samples were collected at the indicated time
points. B, After 2 and 9 days, the titers of SARS-CoV in the lungs of four
mice in each group (except for three mice of the vehicle-immunized group,
which were examined 2 days later) were determined. Virus titers are ex-
pressed as log
10
TCID
50
/g of tissue. C, At 2 and 9 days after SARS-CoV
infection, the serum neutralization titers of all groups were measured as
described in Materials and Methods.,p0.05; ⴱⴱ,p0.01, as com-
pared with both the vehicle- and m8-immunized groups.
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(range, 1:25 to 1:36) after 2 wk, and was sustained for 6 wk
(Fig. 2C). Booster immunization with m8rVV-NMES further
increased the neutralization titer more than 10-fold 2 wk later.
These values are somewhat lower than those induced by
m8rVV-S in our previous report (17). In contrast, the antisera
from rabbits immunized with m8 did not exhibit any neutraliz-
ing activity against SARS-CoV (Fig. 2C).
SARS-CoV challenge of BALB/c mice having prior
immunization with m8rVV-NMES or m8rVV-S
As m8rVV-NMES and m8rVV-S could induce high levels of neu-
tralizing Abs against SARS-CoV (Fig. 2C), we investigated the
influences of m8rVV-NMES and m8rVV-S on SARS-CoV chal-
lenge of BALB/c mice (Fig. 3A). The m8rVV-NMES and
m8rVV-S constructs were inoculated intradermally on the backs of
BALB/c mice at 1 10
7
PFU/body. At 7– 8 wk after this single
immunization, the mice were infected intranasally with SARS-
CoV at 1 10
5
TCID
50
/body. After 2 and 9 days, the lung, liver,
small intestine, and spleen were extirpated from the mice under
anesthesia, and the SARS-CoV titers were measured. As shown in
Fig. 3B, 200- and 100-fold reductions in pulmonary virus titers
were observed in the m8rVV-NMES-immunized and m8rVV-S-
immunized groups 2 days after infection. The virus titers in the
lungs of the m8rVV-NMES-immunized and m8rVV-S-immunized
groups were 5.40 10
5
and 1.52 10
6
TCID
50
/g of lung, re-
spectively. In contrast, the vehicle-immunized and LC16m8-im-
munized groups exhibited virus titers of 1.07 10
8
and 1.18
10
8
TCID
50
/g of lung, respectively. The virus was not detected in
the lungs of any group 9 days later, as reported previously (11, 15).
In contrast, virus titers in other organs, including liver, small in-
testine, and spleen, were lower than that of the detection limit 2
and 9 days after infection (data not shown).
We also measured the neutralization titers in these mice sera 2
and 9 days after SARS-CoV infection (Fig. 3C). Two days postin-
fection, the neutralization titers of the m8rVV-NMES-immunized
and m8rVV-S-immunized groups were 1:11.1 1.01 and 1:14
3.94, respectively, whereas those of the negative control groups
were below the limit of detection. At 9 days postinfection, the
serum neutralization titers of m8rVV-NMES-immunized and
m8rVV-S-immunized groups had increased to 1:838.0 681.0
and 1:367.9 132.1, respectively. In contrast, the serum neutral-
izing titers of the vehicle-immunized and m8-immunized groups
were 1:59.7 35.4 and 1:67.8 18.6, respectively. These results
suggest that both the m8rVV-NMES- and m8rVV-S-immunized
groups could elicit neutralizing Abs against SARS-CoV and alle-
viate SARS-CoV infection.
FIGURE 4. Pulmonary histopathology of m8rVV-S-
preimmunized BALB/c mice after SARS-CoV chal-
lenge. At 7– 8 wk after immunization with m8rVV-
NMES, m8rVV-S, m8, or vehicle, the mice were
infected intranasally with 1 10
5
TCID
50
/body of
SARS-CoV. A, Four mice from each group (three mice
from the vehicle-immunized group were killed 2 days
later) were sacrificed 2 and 9 days later. Extirpated lung
tissues were fixed with 10% formalin and embedded in
paraffin. Paraffin block sections (4-
m thickness) were
stained with H&E staining. Histopathologic sections
were prepared for vehicle-immunized mice at 2 days
postinfection (dpi) (a) and 9 dpi (e), m8-immunized
mice at 2 dpi (b) and 9 dpi (f), m8rVV-NMES-immu-
nized mice at 2 dpi (c) and 9 dpi (g), m8rVV-S-immu-
nized mice at 2 dpi (d) and 9 dpi (h), and uninfected
mice (i). B, The degree of pulmonary inflammation was
determined in a blinded fashion on a subjective 27-
point scale (0, minimal inflammation; 26, massive in-
flammation) as described in Materials and Methods.
Each symbol represents an individual mouse. ,p
0.05. C, Representative lung sections from m8-immu-
nized mice (a) and m8rVV-NMES-immunized mice (b)
after staining with Luna method (for eosinophils and
neutrophils) and H&E (for plasma cells). Arrows indi-
cate neutrophils (yellow), eosinophils (red), and plas-
ma-like cells (green). D, The numbers of neutrophils,
eosinophils, and plasma-like cells that infiltrated the
lung were counted using Luna method and H&E stain-
ing. Data are mean SD for n5 mice. Fields viewed
at a magnification of 400. ,p0.05; ⴱⴱ,p0.01,
for significant differences evaluated using the Bonfer-
roni test.
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Histopathologic findings in the lungs of m8rVVs-immunized
BALB/c mice after SARS-CoV infection
We performed histopathologic analyses of lung tissues. Two
days after SARS-CoV infection, the vehicle-, m8-, and m8rVV-
S-immunized groups showed only slight pulmonary inflamma-
tion (Fig. 4A, a, b, and d), whereas the m8rVV-NMES-immu-
nized group showed infiltration of lymphocytes into the areas
surrounding the bronchi and slight thickening of the alveolar
epithelium (Fig. 4A, c). We scored pulmonary inflammation in
all the groups 2 days after SARS-CoV infection as follows (Fig.
4B): in the m8rVV-NMES-immunized group, 5.00 2.71; in
the vehicle-immunized group, 2.00 2.00; in the m8-immu-
nized group, 1.33 0.82; and in the m8rVV-S-immunized
group, 2.50 1.00. At 9 days postinfection, the vehicle-, m8-,
and m8rVV-NMES-immunized groups exhibited severe pulmo-
nary inflammation, i.e., infiltration of inflammatory cells and
thickening of alveolar epithelia (Fig. 4A, e, f, and g). In contrast,
the m8rVV-S-immunized group showed only slight pulmonary in-
flammation (Fig. 4A, h). As shown in Fig. 4B, the pulmonary in-
flammation score for the m8rVV-NMES-immunized group
(12.75 2.87) 9 days after SARS-CoV infection was significantly
higher than that for the m8rVV-S-immunized group (3.50 3.00).
In contrast, this score was comparable to those obtained for the
vehicle-immunized and m8-immunized groups (9.75 2.87 and
FIGURE 5. Identification of SARS-CoV structural protein implicated in severe pulmonary inflammation. A, Five groups of six BALB/c mice were
inoculated intradermally with mOrVV-NHis, mOrVV-MHis, mOrVV-EHis, mOrVV-SHis, or mO and challenged 4 wk later with 1 10
5
TCID
50
/body
of SARS-CoV via the intranasal route. B, After 2 days, the titers of SARS-CoV in the lungs of three mice in each group were determined. Virus titers are
expressed as log
10
TCID
50
/g of tissue. ,p0.05, as compared with the mO-immunized group using the Dunnett test. C, Histopathologic findings for all
the groups 9 days after SARS-CoV infection. Extirpated lung tissues were fixed with 10% formalin and embedded in paraffin. Paraffin block sections (4-
m
thickness) were subjected to H&E staining. D, The degree of pulmonary inflammation was determined in a blinded fashion on a subjective 27-point scale
(0, minimal inflammation; 26, massive inflammation). Each symbol represents an individual mouse. ,p0.05. E, Representative lung sections from
mO-immunized mice (a) and mOrVV-N-immunized mice (b) after staining with Luna method (for eosinophils and neutrophils). Arrows indicate neutrophils
(yellow) and eosinophils (red).
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8.33 2.31, respectively). The m8rVV-NMES-immunized group
exhibited as severe inflammation as the control groups, although
m8rVV-NMES contains the S protein and protects as well as
m8rVV-S against SARS-CoV infection. In addition, marked infil-
tration of neutrophils, eosinophils, plasma-like cells, and lympho-
cytes was observed in the m8rVV-NMES-immunized group, as
compared with the control groups, after SARS-CoV infection (Fig.
4C, b and D).
These results suggest that the severe pulmonary inflammation
seen in m8rVV-NMES-immunized mice after SARS-CoV infec-
tion results from host immune responses rather than a direct cy-
topathic effect of SARS-CoV, because the virus titers for all the
group were negligible 9 days after SARS-CoV infection and the
virus titer of the m8rVV-NMES-immunized group was signifi-
cantly decreased 2 days postinfection.
Identification of the factor that results in the exacerbation of
pulmonary inflammation in m8rVV-NMES-immunized BALB/c
mice after SARS-CoV infection
We hypothesized that the severe pulmonary inflammation seen in
the m8rVV-NMES-immunized mice resulted from the host im-
mune responses to SARS-CoV components expressed by m8rVV-
NMES. This notion was supported by the observation of negligible
virus titers 9 days after SARS-CoV infection. Therefore, we in-
vestigated the influence of recombinant VV expressing each struc-
tural protein of SARS-CoV (mOrVV-NHis, mOrVV-MHis,
mOrVV-EHis, and mOrVV-SHis) on subsequent intranasal infec-
tion with SARS-CoV. BALB/c mice were immunized with
mOrVV-NHis, -MHis, -EHis, and -SHis at 1 10
7
PFU/body, and
4 wk later infected intradermally with 1 10
5
TCID
50
of SARS-
CoV (Fig. 5A). After 2 and 9 days, three mice from each group
were sacrificed following blood collection under anesthesia, and
their lungs were extirpated. Consistent with earlier results, a sig-
nificant reduction of pulmonary virus titer was observed after 2
days in only the mOrVV-SHis-immunized group (Fig. 5B). In con-
trast, immunization with the other SARS-CoV structural proteins,
including the N, M, and E proteins, did not confer protection
against the subsequent SARS-CoV infection. As shown in Fig. 5C,
the alleviation of pulmonary inflammation was also observed in
the mOrVV-SHis-immunized group. Severe infiltration of lym-
phocytes and thickening of the alveolar epithelia were observed in
the lung tissues of the mOrVV-NHis-immunized mice 9 days after
SARS-CoV infection (Fig. 5C). The pulmonary damage in the
mOrVV-NHis-immunized mice (15.00 5.56) was significantly
more severe than that in the mOrVV-SHis-immunized mice
(5.67 2.52) (Fig. 5D). However, there were no significant dif-
ferences among the other groups. Furthermore, infiltration of neu-
trophils, eosinophils, and lymphocytes was observed in the
FIGURE 6. Cytokine profiles of the
lungs of BALB/c mice preimmunized with
each SARS-CoV structural protein and chal-
lenged with SARS-CoV. Three mice from
each group were sacrificed 2 and 9 days
postinfection. The total RNA of the lung
was extracted. Quantitative RT-PCR was
conducted as described in Materials and
Methods. The fold change in copy number
of each cytokine or chemokine mRNA was
calculated by the 2
⫺⌬⌬Ct
method using 18 S
rRNA as an endogenous calibrator. ,p
0.05; ⴱⴱ,p0.01, as compared with the
uninfected control group using the Bonfer-
roni test. A, The levels of mRNA for proin-
flammatory cytokines and chemokines 2
days after SARS-CoV infection. B, The
mRNA expression levels of cytokines re-
lated to T cell activation 2 days after SARS-
CoV infection. C, The mRNA expression
levels of anti-inflammatory cytokines 9 days
after SARS-CoV infection.
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mOrVV-NHis-immunized mice after SARS-CoV infection (Fig.
5E, b), although the extent of infiltration of these cells into the
lungs of these mice was somewhat lower than that observed in
the m8rVV-NMES-immunized mice after SARS-CoV infection
(Fig. 4D). This may explain the observed differences in the
histopathologic findings for the mOrVV-NHis-immunized mice
and m8rVV-NMES-immunized mice.
Pulmonary cytokine responses of SARS-CoV-infected BALB/c
mice previously immunized with recombinant VV expressing
each structural protein of SARS-CoV
To elucidate the reason for the severe pulmonary inflammation
observed in the mOrVV-NHis-immunized mice after SARS-CoV
infection, we measured by quantitative RT-PCR the mRNA levels
for various cytokines and chemokines in the lungs of BALB/c
mice preimmunized with mOrVV-NHis, -MHis, -EHis, -SHis, or
mO. Several proinflammatory cytokine and chemokine mRNAs,
including those for IL-6, CXCL10, CCL2, and CCL3, were in-
creased in all the groups, with the exception of the mOrVV-SHis
group, 2 days after SARS-CoV infection (Fig. 6A). In contrast, the
mOrVV-SHis-immunized group showed low levels of mRNA ex-
pression for these proinflammatory cytokines or chemokines, es-
pecially IL-6, resulting in reduced lung pathology after immuni-
zation. The mRNA levels for IFN-
, IL-2, IL-4, and IL-5 were
highest in the mOrVV-NHis-immunized group (Fig. 6, Aand B).
None of the other groups showed up-regulation of these cytokines,
with the exception of the IL-5 mRNA level in the mOrVV-SHis-
immunized group. Furthermore, the mRNA expression levels of
anti-inflammatory cytokines (IL-10 and TGF-
) in the mOrVV-
NHis-immunized group were markedly lower than expression lev-
els in any of the other groups, which exhibited high virus titers,
and were comparable to those of the mOrVV-SHis group, in which
pulmonary inflammation was alleviated (Fig. 6C).
Verification of exacerbating effect of prior immunization with
N protein in SARS-CoV-infected Balb/c mice
To verify the exacerbating effect of N protein immunization, we
investigated the pulmonary virus titers and histopathology in
BALB/c mice that were previously immunized with the
combination of mOrVV-N and mOrVV-S (mOrVV-NS-immu-
nized group) 2 and 9 days after SARS-CoV infection, and com-
pared them to those of all other groups, including the mO-,
mOrVV-MS-, mOrVV-ES-, and mOrVV-S-immunized groups.
The mOrVV-NS-immunized group showed significantly decreased
pulmonary virus titers compared with the mO-immunized group (Fig.
7A). However, the mOrVV-NS-immunized group exhibited as
FIGURE 7. Severe pneumonia in BALB/c mice that were previously immunized with the combination of N protein and S protein of SARS-CoV. A, Five
groups of BALB/c mice (n8 –10 per group) were inoculated intradermally with the combinations of mOrVV-NHis and mOrVV-SHis (mOrVV-NS),
mOrVV-MHis and mOrVV-SHis (mOrVV-MS), mOrVV-EHis and mOrVV-SHis (mOrVV-ES), mOrVV-SHis, and mO, and challenged 7 wk later
with 1 10
5
TCID
50
/body of SARS-CoV via the intranasal route. After 2 days, the titers of SARS-CoV in the lungs of n3–5 mice from each group
were determined. Virus titers are expressed as log
10
TCID
50
/g of tissue. ,p0.05, ⴱⴱ,p0.01, as compared with the mO-immunized group using the
Bonferroni test. B, Histopathologic findings for all the groups 9 days after SARS-CoV infection. Extirpated lung tissues were fixed with 10% formalin and
embedded in paraffin. Paraffin block sections (4-
m thickness) were subjected to H&E staining. C, The degree of pulmonary inflammation was determined
in a blinded fashion on a subjective 27-point scale (0, minimal inflammation; 26, massive inflammation). Each symbol represents an individual mouse. †,
p0.05; ‡, p0.01, as compared with the mO-immunized group using the Bonferroni test. ,p0.05; ⴱⴱ,p0.01, as compared with the mOrVV-N
S-immunized group using the Bonferroni test.
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severe pneumonia as the mO-immunized group (Fig. 7, Band C). In
contrast, both the mOrVV-MS-immunized group and the mOrVV-
ES-immunized group were protected against SARS-CoV infection
to the same extent as the mOrVV-S-immunized group (Fig. 7, A–C).
Discussion
SARS-CoV is newly identified as an agent of SARS. However, the
detailed mechanism by which SARS-CoV causes severe pneumo-
nia remains unclear. The uncontrolled release of immune media-
tors has been implicated in the pathogenesis of SARS, whereas the
cytokine profiles of SARS patients have not elucidated the
cause the pneumonia owing to their diversity. It seems likely
that the diverse cytokine profiles noted among adult SARS pa-
tients are related to patient anamnesis.
In the present study, we observed severe pulmonary inflamma-
tion in m8rVV-NMES-immunized BALB/c mice 9 days after
SARS-CoV infection (Fig. 4A, g), even though the initial virus titer
was significantly lower than those of the control groups, which
included vehicle- and m8-immunized mice (Fig. 3B). The severity
of pulmonary inflammation did not correlate with the virus titer in
the m8rVV-NMES-immunized mice, in contrast to the correlations
observed for the vehicle-, m8-, and m8rVV-S-immunized groups.
We identified the N protein of SARS-CoV as the cause of the
severe pneumonia observed during SARS-CoV infection (Fig. 5, C
and D, and 7, Band C). To date, no studies have been reported to
our knowledge regarding SARS patients with severe pneumonia
who were previously immunized with either SARS-CoV or a
highly related species. In contrast, there are several reports of an-
tisera against human CoV (229E and OC43) and host factor IL-11
cross-reacting with the SARS-CoV Ag (29, 30). Furthermore, the
N protein of SARS-CoV has been shown to induce both cellular
and humoral immune responses (31–33). Taken together, these
results raise the possibility that a percentage of SARS patients
already possess the adaptive immune response elements that can
interact with SARS-CoV components, including the N protein, and
that their adaptive immune response may be involved in the ex-
acerbation of pneumonia. The temporal changes in immune re-
sponse and the pathogenesis after SARS-CoV infection of an an-
imal model that had previously been immunized with SARS-CoV
components are not well understood, as almost all the previous
studies reported only protection within a few days of SARS-CoV
infection (34 –39). In the present study, we demonstrate that
mOrVV-NHis-immunized mice after SARS-CoV infection exhibit
an imbalance between T cell activation (high expression levels of
IFN-
, IL-2, IL-4, and IL-5) and subsequent suppression (low ex-
pression levels of IL-10 and TGF-
), as well as high-level pro-
duction of proinflammatory cytokines (IL-6 and TNF-
) and che-
mokines (CCL2, CCL3, and CXCL10). Jiang et al. (40) reported
elevation of CXCL10 or IP-10 production in the pneumocytes,
CD3
T cells, and monocytes and macrophages of the lungs of
patients with SARS. CXCL10 may be responsible for the infiltra-
tion of activated T cells and monocytes or macrophages, which is
a pathologic finding in SARS patients (41– 43). It has been re-
ported that elevated expression of monocyte or macrophage acti-
vation factors (CCL2 and CCL3) was observed in SARS patients
(8, 44). Furthermore, the highest expression of IL-6 in mOrVV-
NHis-immunized mice is reasonable (Fig. 6A), as the elevation of
IL-6 levels is considered one of the causes in the severe pneu-
monia of SARS patients. Zhang et al. (45) reported recently the
molecular mechanism of IL-6 expression induction by the N
protein of SARS-CoV. In contrast, both IL-10 and TGF-
play
important roles in suppressing inflammatory responses (46).
Thus, the reduced production of both anti-inflammatory cyto-
kines in the mOrVV-NHis-immunized mice after SARS-CoV
infection may be related to the severity of the pulmonary in-
flammation in these mice. Weingartl et al. (47) and Czub et al.
(48) reported that immunization with S protein expressing-recom-
binant modified VV Ankara (rMVA-S) induced stronger inflam-
matory responses and focal necrosis in liver tissues after SARS-
CoV challenge than in control animals. However, the precise
mechanism underlying this liver inflammation has not been clari-
fied. Feline infectious peritonitis virus, which is another member
of the coronavirus family, exhibits enhanced infection into mono-
cytes or macrophages through virus-specific Ab binding to the Fc
receptors of these cells and causes enhanced inflammation (49). It
has also been reported for dengue virus that secondary infection
with a different genotype results in more severe symptoms, includ-
ing dengue hemorrhagic fever and dengue shock syndrome. The
exacerbation of this symptom is also positively associated with
pre-existing Abs with specificity for dengue virus (50). In the case
of SARS-CoV, Ab-dependent enhancement of infection has not
been reported previously. We hypothesized that the severe pneu-
monia observed in mOrVV-NHis-immunized mice after SARS-
CoV infection does not result from Ab-dependent enhancement
because the virus titers in the mouse lungs 9 days later were below
the detection limit. Deming et al. (51) reported recently the inten-
sive infiltration of eosinophils as well as lymphocytes after SARS-
CoV infection of aged BALB/c mice previously immunized with
the N protein of SARS-CoV. It has also been reported that immu-
nization with formalin-inactivated respiratory syncytial virus vac-
cine and VV that expresses the G glycoprotein of respiratory syn-
cytial virus correlates with the augmentation of Th2-type immune
responses and enhanced pulmonary disease (52, 53). Therefore, the
authors speculated that the Th2-biased responses of vaccinated
hosts after SARS-CoV infection might aggravate pulmonary in-
flammation, although the main host response remains unknown. In
contrast, our current data suggest that N protein-immunized mice
exhibit activation of both Th1 and Th2 responses after SARS-CoV
infection. In agreement with our data, Jin et al. (54) have demon-
strated that prior immunization with N protein generates stronger
Ag-specific Th1 and Th2 responses than immunization with M or
E protein. In addition, we demonstrate the suppression of anti-
inflammatory cytokine responses in N protein-immunized mice.
Interestingly, Shi et al. (55) demonstrated that coinjection of M
protein with N protein not only enhanced the production of Th1
cytokines (IFN-
and IL-2), but also reduced the rates of mortality
and pathologic change in SARS-CoV-infected voles. These results
suggest that further studies, including epitope analysis, are re-
quired to reveal the precise mechanism underlying the severe pul-
monary inflammation that results from SARS-CoV infection of
BALB/c mice immunized with the N protein of SARS-CoV.
In contrast, intradermal immunization of aged BALB/c mice
with m8rVV-S at 1 10
7
PFU/body significantly reduced the
pulmonary virus titer 2 days after SARS-CoV infection (Fig. 3B).
Furthermore, the m8rVV-S-immunized group exhibited alleviation
of the pulmonary histopathology, as compared with both control
groups after 9 days. To date, various types of SARS vaccine, in-
cluding recombinant vaccines, inactivated vaccines, and DNA vac-
cine, have been reported (34 –39). There are only a few reports on
the effect of a single immunization with recombinant SARS vac-
cines, namely SARS-CoV S protein-expressing vaccines based on
rabies virus (56), vesicular stomatitis virus (39), and adeno-asso-
ciated virus (57). It is noteworthy that a single i.m. immunization
with recombinant adeno-associated virus that expresses the recep-
tor-binding domain of S protein conferred long-term protection
against SARS-CoV infection (57). In the present study, we also
show that a single immunization with m8rVV-S reduces viral load
and improves the histopathologic findings in the lungs of BALB/c
6346 N PROTEIN OF SARS-CoV AS A CAUSE OF PNEUMONIA EXACERBATION
by guest on October 29, 2015http://www.jimmunol.org/Downloaded from
mice infected with high-titer (1 10
5
TCID
50
/body) SARS-CoV,
although a relatively low titer of SARS-CoV was used in the pre-
vious study conducted by Du et al. (57). These results suggest that
the systemic immune responses induced by a single immunization
with SARS vaccine successfully protect the animal model against
intranasal SARS-CoV infection.
In summary, we demonstrate that the immunization of BALB/c
mice with the N protein of SARS-CoV causes severe pulmonary
inflammation upon subsequent SARS-CoV infection, probably via
the imbalance created between T cell activation and suppression,
as well as by massive proinflammatory cytokine production. These
results provide new insights into the mechanisms involved in the
pathogenesis of SARS and help in the development of safe
vaccines.
Acknowledgments
We are grateful to Dr. Ryuichi Miura (University of Tokyo) for arranging
the SARS-CoV challenge experiment. We are also grateful to Iyo Kataoka
(Institute of Medical Science, University of Tokyo). We thank Dr. Masa-
hiro Shuda of the University of Pittsburgh for helpful discussions. We also
thank Dr. Tetsuya Mizutani and Dr. Shigeru Morikawa (Department of
Virology I, National Institute of Infectious Diseases) for providing antisera
from rabbits immunized with the M protein peptide and inactivated SARS-
CoV particles.
Disclosures
The authors have no financial conflict of interest.
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... The type of response that vaccines should trigger must always be Th1 cell response or balanced T-cell responses, because when the response is Th2 cell, it has been associated with enhanced respiratory disease (266)(267)(268)(269)(270). Moreover, we have looked at how T cells also play a critical role in B-cell maturation and therefore the induction of a strong and durable antibody response (150). ...
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Two years after the appearance of the SARS-CoV-2 virus, the causal agent of the current global pandemic, it is time to analyze the evolution of the immune protection that infection and vaccination provide. Cellular immunity plays an important role in limiting disease severity and the resolution of infection. The early appearance, breadth and magnitude of SARS-CoV-2 specific T cell response has been correlated with disease severity and it has been thought that T cell responses may be sufficient to clear infection with minimal disease in COVID-19 patients with X-linked or autosomal recessive agammaglobulinemia. However, our knowledge of the phenotypic and functional diversity of CD8+ cytotoxic lymphocytes, CD4+ T helper cells, mucosal-associated invariant T (MAIT) cells and CD4+ T follicular helper (Tfh), which play a critical role in infection control as well as long-term protection, is still evolving. It has been described how CD8+ cytotoxic lymphocytes interrupt viral replication by secreting antiviral cytokines (IFN-γ and TNF-α) and directly killing infected cells, negatively correlating with stages of disease progression. In addition, CD4+ T helper cells have been reported to be key pieces, leading, coordinating and ultimately regulating antiviral immunity. For instance, in some more severe COVID-19 cases a dysregulated CD4+ T cell signature may contribute to the greater production of pro-inflammatory cytokines responsible for pathogenic inflammation. Here we discuss how cellular immunity is the axis around which the rest of the immune system components revolve, since it orchestrates and leads antiviral response by regulating the inflammatory cascade and, as a consequence, the innate immune system, as well as promoting a correct humoral response through CD4+ Tfh cells. This review also analyses the critical role of cellular immunity in modulating the development of high-affinity neutralizing antibodies and germinal center B cell differentiation in memory and long-lived antibody secreting cells. Finally, since there is currently a high percentage of vaccinated population and, in some cases, vaccine booster doses are even being administered in certain countries, we have also summarized newer approaches to long-lasting protective immunity and the cross-protection of cellular immune response against SARS-CoV-2.
... Apart from some Flavivirus like Dengue virus, ADE has been described for few other human viruses, like Human Immunodeficiency Virus-1 (HIV1) [9], West Nile virus [10], Zika virus [11] or Ebola virus [12]. ADE has also been described for some respiratory infections like Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), Middle East Respiratory Syndrome Coronavirus (MERS-CoV) or Human Influenza virus [13][14][15][16][17][18][19] and it has been reported both in vivo and in vitro [20]. In these respiratory infections, nNabs enhance viral entry into lung phagocytic cells, disseminating the infection and initiating a powerful immune cascade that results in lung pathology. ...
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... In view of this, it is necessary to evaluate the functional avidity degree among the vaccine-elicited T cells. 2) In SARS-CoV, Th2 bias CD4 + T-cell immune responses have been demonstrated in a mice trial, and host immune responses to SARS-CoV N protein resulted in severe pneumonia (Yasui et al., 2008). Although the current SARS-CoV-2-targeted CD4 + T-cell response is Th1 bias (Aleebrahim-Dehkordi et al., 2022), it still requires more clinical data and rigorous assessment for the N-protein-targeted candidate vaccine. ...
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... 12 Data from the study of SARS-CoV and other respiratory viruses suggest that anti-SARS-CoV-2 antibodies could exacerbate COVID-19 through ADE. 13 This ADE was shown by animal trials of SARS-CoV-1 vaccines from 2003. 14- 16 Recently, studies on the COVID-19 virus have shown a non-canonical ADE mechanism independent of Fc receptors. Antibodies directed against a specific site on the NTD (N-Terminal Domain) of the SARS-CoV-2 spike protein were found to directly increase the binding of ACE2 (Angiotensin Converting Enzyme2, cellular virus receptor) to the spike, thereby increasing the infectivity of SARS-CoV-2. ...
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... Another study found pulmonary eosinophilic infiltrates in a variety of vaccines in Balb/c mice, however these mice were also ultimately protected against challenge (89). Tseng et al. demonstrated that Th2 immunopathology is induced by a variety of vaccines and hypothesised that non-neutralising antibody responses against nucleocapsid protein might explain much of the observed immunopathology, as shown previously (90,91). This is because although the recombinant spike protein vaccine (SV) induced immunopathology, it was to a lesser extent than that observed in the other vaccines evaluated (89). ...
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Vaccine-associated enhanced disease (VAED) is a difficult phenomenon to define and can be confused with vaccine failure. Using studies on respiratory syncytial virus (RSV) vaccination and dengue virus infection, we highlight known and theoretical mechanisms of VAED, including antibody-dependent enhancement (ADE), antibody-enhanced disease (AED) and Th2-mediated pathology. We also critically review the literature surrounding this phenomenon in pathogenic human coronaviruses, including MERS-CoV, SARS-CoV-1 and SARS-CoV-2. Poor quality histopathological data and a lack of consistency in defining severe pathology and VAED in preclinical studies of MERS-CoV and SARS-CoV-1 vaccines in particular make it difficult to interrogate potential cases of VAED. Fortuitously, there have been only few reports of mild VAED in SARS-CoV-2 vaccination in preclinical models and no observations in their clinical use. We describe the problem areas and discuss methods to improve the characterisation of VAED in the future.
... There were a large number of Th2 CD4 T cells and eosinophils in lung tissue (Polack et al. 2003;Ruckwardt et al. 2019). In preclinical studies of Middle East respiratory syndrome (MERS)-CoV and SARS-CoV-1 vaccines from different vaccine platforms, immunopathological evidence of Th2 type hypersensitivity similar to VAERD was observed in the lungs of animal models (Agrawal et al. 2016;Arvin et al. 2020;Yasui et al. 2008). In the development of COVID-19 vaccine, previous work on RSV vaccine should be considered to avoid or reduce the potential risk of VAERD. ...
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