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Immunization with SARS Coronavirus Vaccines Leads to
Pulmonary Immunopathology on Challenge with the
SARS Virus
Chien-Te Tseng
1,2
, Elena Sbrana
1
, Naoko Iwata-Yoshikawa
1,2
, Patrick C. Newman
1
, Tania Garron
1
,
Robert L. Atmar
3,4
, Clarence J. Peters
1,2
, Robert B. Couch
3,4
*
1Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, Texas, United States of America, 2Center for Biodefense and
Emerging Disease, The University of Texas Medical Branch, Galveston, Texas, United States of America, 3Department of Medicine, Baylor College of Medicine, Houston,
Texas, United States of America, 4Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas, United States of America
Abstract
Background:
Severe acute respiratory syndrome (SARS) emerged in China in 2002 and spread to other countries before
brought under control. Because of a concern for reemergence or a deliberate release of the SARS coronavirus, vaccine
development was initiated. Evaluations of an inactivated whole virus vaccine in ferrets and nonhuman primates and a virus-
like-particle vaccine in mice induced protection against infection but challenged animals exhibited an immunopathologic-
type lung disease.
Design:
Four candidate vaccines for humans with or without alum adjuvant were evaluated in a mouse model of SARS, a
VLP vaccine, the vaccine given to ferrets and NHP, another whole virus vaccine and an rDNA-produced S protein. Balb/c or
C57BL/6 mice were vaccinated IM on day 0 and 28 and sacrificed for serum antibody measurements or challenged with live
virus on day 56. On day 58, challenged mice were sacrificed and lungs obtained for virus and histopathology.
Results:
All vaccines induced serum neutralizing antibody with increasing dosages and/or alum significantly increasing
responses. Significant reductions of SARS-CoV two days after challenge was seen for all vaccines and prior live SARS-CoV. All
mice exhibited histopathologic changes in lungs two days after challenge including all animals vaccinated (Balb/C and
C57BL/6) or given live virus, influenza vaccine, or PBS suggesting infection occurred in all. Histopathology seen in animals
given one of the SARS-CoV vaccines was uniformly a Th2-type immunopathology with prominent eosinophil infiltration,
confirmed with special eosinophil stains. The pathologic changes seen in all control groups lacked the eosinophil
prominence.
Conclusions:
These SARS-CoV vaccines all induced antibody and protection against infection with SARS-CoV. However,
challenge of mice given any of the vaccines led to occurrence of Th2-type immunopathology suggesting hypersensitivity to
SARS-CoV components was induced. Caution in proceeding to application of a SARS-CoV vaccine in humans is indicated.
Citation: Tseng C-T, Sbrana E, Iwata-Yoshikawa N, Newman PC, Garron T, et al. (2012) Immunization with SARS Coronavirus Vaccines Leads to Pulmonary
Immunopathology on Challenge with the SARS Virus. PLoS ONE 7(4): e35421. doi:10.1371/journal.pone.0035421
Editor: Stefan Poehlmann, German Primate Center, Germany
Received January 31, 2012; Accepted March 15, 2012; Published April 20, 2012
Copyright: ß2012 Tseng et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Research performed by the authors and summarized in this report was supported by Public Health Service Contract NO1 AI 30039 from the National
Institute of Allergy and Infectious Diseases. The content of this publication does not necessarily reflect the views or policies of the Department of Health and
Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. The funders had no role in
study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: rcouch@bcm.edu
Introduction
Severe acute respiratory syndrome (SARS) emerged in
Guangdong, People’s Republic of China, in late 2002, and spread
to other countries in Asia and to Canada in the ensuing months
[1–3]. Infection control efforts brought the infection under control
by mid-2003 [4]. More than 8000 cases, including almost 800
deaths, were reported during the outbreak period [4]. Increasing
age and comorbidity were risk factors for severe disease and death
[5,6,7]. Since 2003, only sporadic cases have been reported;
however, the possibility that SARS outbreaks could reemerge
naturally or be deliberately released is a public health concern.
SARS is caused by a Coronavirus (SARS-CoV) [8,9]. Limited
data are available about the ecology of SARS-CoV, but bats are
thought to be the animal reservoir for the virus which may be
transmitted to small mammals with exposure to these small
animals as the source of human infections [10]. The clinical
disease is similar to other severe acute respiratory infections,
including influenza; the SARS case definition includes clinical,
epidemiologic, and laboratory criteria [11,12]. A number of
therapeutic efforts were employed for the disease in Asia and in
Canada; however, no treatment of clear value was identified.
Animal models were developed using mice, hamsters, ferrets and
PLoS ONE | www.plosone.org 1 April 2012 | Volume 7 | Issue 4 | e35421
nonhuman primates, and efforts to identify useful treatments and
effective vaccines are ongoing.
Vaccine candidates for preventing SARS have been developed
by various groups and include inactivated whole virus, spike (S)
protein preparations, virus-like particles (VLPs), plasmid DNA and
a number of vectors containing genes for SARS-CoV proteins
[13–28]. Phase I studies in humans have been conducted with a
whole virus vaccine and a DNA vaccine [29–30].
An early concern for application of a SARS-CoV vaccine was
the experience with other coronavirus infections which induced
enhanced disease and immunopathology in animals when
challenged with infectious virus [31], a concern reinforced by
the report that animals given an alum adjuvanted SARS vaccine
and subsequently challenged with SARS-CoV exhibited an
immunopathologic lung reaction reminiscent of that described
for respiratory syncytial virus (RSV) in infants and in animal
models given RSV vaccine and challenged naturally (infants) or
artificially (animals) with RSV [32,33]. We and others described a
similar immunopathologic reaction in mice vaccinated with a
SARS-CoV vaccine and subsequently challenged with SARS-CoV
[18,20,21,28]. It has been proposed that the nucleocapsid protein
of SARS-CoV is the antigen to which the immunopathologic
reaction is directed [18,21]. Thus, concern for proceeding to
humans with candidate SARS-CoV vaccines emerged from these
various observations.
The studies reported here were conducted to evaluate the safety,
immunogenicity, and efficacy of different SARS-CoV vaccines in a
murine model of SARS.
Materials and Methods
Tissue Cultures and Virus
Vero E6 tissue cultures [obtained from The American Type
Culture Collection (ATCC), CRL:1586] were grown in Dulbec-
co’s modified minimum essential medium (DMEM) supplemented
with penicillin (100 units/ml), streptomycin (100 mg/ml), 0.2%
sodium bicarbonate and 10% fetal bovine serum (FBS). The
Urbani strain of SARS-CoV was obtained from T.G. Ksiazek at
the Centers for Disease Control and Prevention (Atlanta, GA), and
a working stock of this virus was prepared by serially passaging a
portion of the seed virus three times (p3) in Vero E6 cultures. The
culture fluid from infected cells was clarified by low-speed
centrifugation, filtered through a 0.45 mm filter, aliquoted, and
stored at 280uC.
Vaccines
Four different SARS-CoV vaccines were evaluated in these
studies (Table 1). Two whole virus vaccines were evaluated; one
was prepared in Vero tissue cultures, zonal centrifuged for
purification, and double-inactivated with formalin and UV
irradiation, the DI vaccine (DIV); it was tested with and without
alum adjuvant [16]. The other whole virus vaccine was prepared
in Vero cells, concentrated, purified, inactivated with beta
propiolactone and packaged with alum adjuvant (BPV) [13]. A
recombinant DNA spike (S) protein vaccine (SV) was produced in
insect cells and purified by column chromatography was tested
with and without alum adjuvant [17]. The fourth vaccine (the
VLP vaccine) was a virus-like particle vaccine prepared by us as
described previously; it contained the SARS-CoV spike protein (S)
and the Nucleocapsid (N), envelope (E) and membrane (M)
proteins from mouse hepatitis coronavirus (MHV) [20].
Animals
Six- to eight-week-old, female Balb/c and C57BL/6 mice
(Charles River Laboratory, Wilmington, MA), were housed in
cages covered with barrier filters in an approved biosafety level 3
animal facility maintained by the University of Texas Medical
Branch (UTMB) at Galveston, Texas. All of the experiments were
performed using experimental protocols approved by the Office of
Research Project Protections, Institutional Animal Care and Use
Committee (IACUC), University of Texas Medical Branch and
followed National Institutes of Health and United States
Department of Agriculture guidelines.
Study Design
Three different experiments, performed for comparing different
vaccines, are reported here. Adjuvanted (alum) and non-
adjuvanted (PBS) vaccines were obtained from the NIH/BEI
resource. Groups of mice (N = 12–13 per group) were adminis-
tered various dosages of each vaccine intramuscularly (IM) on days
0 and 28; mice given only PBS, alum, trivalent inactivated
influenza vaccine or live SARS-CoV were included as controls in
various experiments. On day 56, five mice from each group were
sacrificed for assessing serum neutralizing antibody titers and lung
histopathology; the remaining seven or eight mice in each group
were challenged with 10
6
TCID
50
/60 ml of SARS-CoV intrana-
sally (IN). Challenged mice were euthanized on day 58 for
determining virus quantity and preparing lung tissue sections for
histopathologic examination.
Neutralizing Antibody Assays
Mice were anesthetized with isoflurane and then bled from the
retro-orbital sinus plexus. After heat inactivation at 56uC for
30 minutes, sera were stored at 280uC until tested. Assays for
virus-specific neutralizing antibodies were performed on serial 2-
fold diluted samples of each serum using 2% FBS-DMEM as the
diluent in 96-well tissue culture plates (Falcon 3072); the final
volume of the serially diluted samples in each well was 60 ml after
addition of 120 TCID
50
of SARS-CoV in 60 ml into each well.
The beginning dilution of serum was 1:20. The dilutions were
incubated for 45–60 minutes at room temperature; then 100 mlof
each mixture was transferred into duplicate wells of confluent
Vero E6 cells in 96-well microtiter plates. After 72 hours of
incubation, when the virus control wells exhibited advanced virus-
induced CPE, the neutralizing capacity of individual serum
samples were assessed by determining the presence or absence of
cytopathic effect (CPE). Neutralizing antibody titers were
expressed as the reciprocal of the last dilution of serum that
completely inhibited virus-induced CPE.
Collection and Processing of Lungs for Histology and
Virus Quantity
Two days post SARS-CoV challenge, mice were euthanized
and their lungs were removed. Lung lobes were placed in 10%
neutral buffered formalin for histological examination and
immunohistochemistry (IHC), as described previously [34,35].
For virus quantitation, the remaining tissue specimen was weighed
and frozen to 280uC. Thawed lung was homogenized in PBS/
10% FBS solution using the TissueLyser (Qiagen; Retsch, Haan,
Germany). The homogenates were centrifuged and SARS-CoV
titers in the clarified fluids were determined by serial dilution in
quadruplicate wells of Vero E6 cells in 96-well plates. Titers of
virus in lung homogenates were expressed as TCID
50
/g of lung
(log
10
); the minimal detectable level of virus was 1.6 to 2.6 log
10
TCID
50
as determined by lung size.
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Histopathology
Evaluations for histopathology were done by pathologists
masked as to the vaccine/dosage of each specimen source;
numeric scores were assigned to assess the extent of pathologic
damage and the eosinophilic component of the inflammatory
infiltrates.
Statistical Analysis
Neutralizing antibody titers, lung virus titers, histopathologic
lesion score and eosinophilic infiltration scores were averaged for
each group of mice. Comparisons were conducted using
parametric and nonparametric statistics as indicated.
Results
Experiments
The three experiments performed, vaccines and dosages used
and controls for each experiment are shown in Table 1. The
vaccines were evaluated for immunogenicity and efficacy;
however, because of the previous report of immunopathology on
challenge of ferrets and nonhuman primates that had been
vaccinated with a whole virus adjuvanted vaccine and mice that
had been vaccinated with a VLP vaccine, the primary orientation
was to assess for immunopathology among animals in relation to
type of vaccine, dosage, serum antibody responses, and virus
infection. The vaccine preparations were made for human trials so
identifying a preparation that was likely to be both safe and
protective in humans was desired. The rationale for each
experiment is described.
Comparison of Vaccines (Experiment 1). To differentiate
between vaccines, three vaccine preparations were simultaneously
evaluated, the double-inactivated (formalin and UV) whole virus
vaccine (DIV), the rDNA-expressed S protein vaccine (SV), and
the previously evaluated chimeric viral-like particle vaccine (VLP)
that had led to immunopathology with virus challenge [16,17,20].
Geometric mean serum neutralizing antibody titers for each
group on day 56 are shown in figure 1A. Geometric mean titers for
those given a nonadjuvanted or alum adjuvanted vaccine were not
different for the double-inactivated whole virus vaccine (DIV), and
the VLP vaccine, (p.0.05, student’s t-test), but were different for
the S protein vaccine (SV) (p = 0.001, student’s t test). Geometric
mean titers for the different dosage groups given the DI vaccine
(DIV) with alum and those for the groups given the S protein
vaccine (SV) with or without alum were significantly different
(p = 0.007, p = 0.028, and p = 0.01, respectively, Kruskall-Wallis)
while the geometric means for those dosage groups given the DI
vaccine (DIV) without alum were not (p.0.05, Kruskall-Wallis).
In a multiple regression analysis, postvaccination titers for the DI
vaccine (DIV) were significantly increased by both alum and
higher dosage (for alum, p = 0.012, for dosage, p ,0.001); for the
S protein vaccine (SV), only alum increased responses (p = 0.001).
Table 1. Experimental Groups for Evaluation of SARS Coronavirus Vaccines.
Group
Exp 1
1
Vaccine Comparisons
Exp 2
1
Higher SV Dosage plus DIV and BPV Comparisons
Exp 3
1,3
Mouse and Vaccine Specificity
1 DIV/1 mg
2
PBS PBS-PBS
2 DIV/0.5 mg Live virus PBS
3 DIV/0.25 mgSV/9mg Live virus
4 DIV/0.125 mgSV/3mg Flu vaccine
5 DIV/1 mg+alum SV/1 mg DIV/1 mg
6 DIV/0.5 mg+alum SV/9 mg+alum DIV/1 mg+alum
7 DIV/0.25 mg+alum SV/3 mg+alum BPV/undil +alum
8 DIV/0.125 mg+alum SV/1 mg+alum PBS-PBS
9SV/2
mg
2
DIV/1 mgPBS
10 SV/1 mg DIV/0.25 mg (50 ml) Live virus
11 SV/0.5 mg DIV/1 mg+alum Flu vaccine
12 SV/0.25 mg DIV/0.25 mg+alum (50 ml) DIV/1 mg
13 SV/2 mg+alum BPV/undil +alum
2
DIV/1 mg+alum
14 SV/1 mg+alum BPV/undil +alum (25 ml) BPV/undil +alum
15 SV/0.5 mg+alum
16 SV/0.25 mg+alum
17 VLP/2 mg
2
18 VLP/2 mg+alum
19 Alum
20 PBS
1
Design =All experiments in Balb/c mice except as noted in Exp 3. Each group contained 12–13 mice; all were given 100 ml of vaccine IM at dosages with or without
alum as indicated on days 0 and 28 except as noted. Five mice in each group were sacrificed on day 56 for serum antibody; remaining mice were given 10
6
TCID
50
of
SARS-CoV intranasal on day 56 and sacrificed on day 58 for virus and lung histology.
2
DIV/dosage = Vaccine DIV = Zonal centrifuge purified doubly inactivated (formalin and UV) whole virus SV/dosage = Vaccine SV = Recombinant baculovirus expressed S
glycoprotein of SARS-CoV VLP/dosage = Vaccine VLP= Virus-like particles containing SARS-CoV S glycoprotein and E, M, and N proteins from mouse hepatitis
coronavirus BPV/dosage = Vaccine BPV = Purified beta propiolactone inactivated whole virus plus alum.
3
Experiment 3 = Groups 1 to 7 were Balb/c mice; groups 8 to 14 were C57BL/6 mice. Flu vaccine was licensed trivalent 2009-10 formulation of high dosage vaccine
(60 mg of HA of each strain). Groups 1 and 8 were given PBS (placebo) and challenged with PBS; all others were challenged with live SARS-CoV.
doi:10.1371/journal.pone.0035421.t001
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Two days after challenge, lungs were obtained from all animals
for virus quantitation and histology. CoV titers are shown in
figure 1B. Geometric mean lung titers in the alum and PBS control
groups were 10
7.3
and 10
6.3
TCID
50
/g, respectively. All vaccine
groups exhibited lower titers or no detectable virus on day two
after challenge. None of the animals given any of the alum-
adjuvanted DI vaccine (DIV) dosages and only an occasional
animal in the lower dosages of nonadjuvanted vaccine yielded
virus (Kruskall-Wallis and Mann Whitney U tests, p.0.05 for all
comparisons). All groups given the S protein vaccine (SV) yielded
virus after challenge and the differences between groups were
significant (p = 0.002 for all groups, p = 0.023 for alum and
p = 0.008 for no adjuvant, Kruskall-Wallis); also, geometric mean
titers were higher for the groups given lower vaccine dosages.
Geometric mean titers for the VLP vaccine groups were similar
(p.0.05).
In the vaccine comparison experiment, lung lesion scores for
histopathology were graded for individual animals on a scale of 0
to 4 where 0–2 represented degree of cellular infiltration and 3–4
represented the degree of bronchiolar epithelial cell necrosis and
airway cellular debris (figure 2A). As shown, all animals exhibited
pathologic changes after challenge including those animals with no
measurable virus on day two suggesting virus infection had
occurred but was not detectable on day two because of a short
duration of infection or neutralization of virus by antibody in the
lung during processing. The higher scores (.3) in some groups
related primarily to the fact that virus infection had induced
inflammatory infiltrates and epithelial cell necrosis with desqua-
mation of the epithelium and collection of cellular debris in
airways of these animals. Mean score differences were noted
among the various vaccines (p = ,0.001, Anova). Those groups
given the DI vaccine (DIV) without alum had higher mean scores
than did those given DI vaccine (DIV) with alum (p = 0.001,
Mann-Whitney U); similarly, the group given the VLP vaccine
without alum had a higher mean score than for those given VLP
vaccine with alum (p = 0.008, Mann-Whitney U). Post hoc
comparisons for the three different vaccines indicated that the
DI vaccine (DIV) group overall had lower lesion scores than either
the S protein vaccine (SV) group or the alum and PBS control
groups (p = 0.001 comparing the DI and S protein vaccines (DIV
and SV) and p,0.001 for DIV vs. control groups, Tukey HSD
and Dunnett t, respectively), but not the VLP vaccine group
(p.0.05, Tukey HSD). The S protein vaccine group (SV) was also
lower overall than the control groups (p = 0.048, Dunnett t).
When the characteristics of the infiltrates were compared,
animals given alum or PBS exhibited epithelial cell necrosis and
peribronchiolar and perivascular mononuclear cell infiltrates
consistent with epithelial cell infection and an inflammatory
response seen in viral infections. In addition to mononuclear cells,
however, infiltrates among vaccinated animals contained neutro-
phils and eosinophils that were not seen in the lesions of the
animals that had been previously given PBS or alum only
(figure 2B) suggesting a T helper cell type 2 hypersensitivity
reaction; increased eosinophils are a marker for a Th2-type
hypersensitivity reaction. Percent eosinophils was lower in these
vaccinated animals (mean 1–3.2%) than had been seen in animals
given VLP vaccines in the earlier study (mean 13.269.6% and
2269.9% of cells for VLP with PBS or alum, respectively in that
study) but no (0%) eosinophils were seen in the lung infiltrates of
control animals in this experiment. This pattern of excess
eosinophils in cellular infiltrates seen in lung sections from animals
given vaccine and not in control animals was as seen in the earlier
study with VLP vaccine and those later with other vaccines
although the percent eosinophils was lower in this study.
The mean percent eosinophils differed between groups
(p,0.001, Anova). Overall, the percent was lower for the groups
given the DI and S protein alum adjuvanted vaccines than for the
corresponding nonadjuvanted group (p = 0.049 for DIV and 0.001
for SV, Mann-Whitney U). For the vaccines, the eosinophil mean
percentages were lower for the S protein vaccine (SV) than for
either the DI vaccine (DIV) or VLP vaccine (DIV vs. SV,
p = 0.002; VLP vs. SV, p = ,0.001, Tukey HSD). Additionally,
eosinophil percentages for all three vaccines, including the S
protein vaccine, were significantly greater than the controls (SV,
DIV and VLP vaccine, p,0.001 for each, Tukey HSD).
Higher Dosages of the S Protein Vaccine Plus the bp
Inactivated Whole Virus Vaccine, Experiment 2. This
experiment was conducted to verify the findings in the initial
Figure 1. Vaccine Comparisons of Three SARS-CoV Vaccines,
Experiment 1. Serum neutralizing (neut) antibody and lung virus titers
for each vaccine dosage group. A. Geometric mean serum antibody titer
as log
2
and standard error of the mean (S.E.) on day 56 for each vaccine
dosage group. Seven to eight mice per group. Vaccines: double
inactivated whole virus (DIV), recombinant S protein (SV), viral-like
particle vaccine (VLP), with alum (+A). Five mice per group were given
0.1 ml of vaccine intramuscularly on days 0 and 28. B. Geometric mean
virus titer (log
10
TCID
50
/g) and standard error of the mean (S.E.) in lungs
on day 58 (two days after SARS-CoV challenge) for each vaccine dosage
group. Analyses: A. GMT with compared to without alum: DIV p..05,
VLP p..05, SV p = .001. GMT for different vaccine dosage: DIV with alum
p = .007, DIV without alum p..05, SV with alum p = .028, SV without
alum p = .01. Multiple regression: GMT increased for alum p = .012 and
dosage p,.001, for SV alum only p =.001. B. GMT for all DIV groups not
different p..05, GMT for SV group without alum p .008 and with alum p
.023. GMT for VLP group is not different p..05.
doi:10.1371/journal.pone.0035421.g001
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PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e35421
experiment of a hypersensitivity immunopathologic-like reaction
after SARS-CoV challenge of vaccinated animals, to determine if
a higher dosage of the S protein vaccine (SV) would suppress
infection and still exhibit a similar reaction, and whether the
original bpropiolactone inactivated whole virus vaccine (BPV)
that had shown an immunopathologic-like reaction after challenge
of vaccinated ferrets and nonhuman primates exhibited a similar
immunopathologic reaction in the mouse model [13,14].
Additionally, a live virus ‘‘vaccination’’ group was added in this
experiment for comparison of challenge results following
vaccinations with inactivated vaccines to those following earlier
infection.
Serum neutralizing antibody responses are shown in figure 3A.
The bp inactivated vaccine (BPV), was only available at one
dosage with alum so a smaller volume (25 ml) was given to one
group for a dosage comparison. Geometric mean titers for the
groups given the alum adjuvanted version of the DI and the S
protein vaccines were greater than for the unadjuvanted vaccine
(DIV P = 0.014, SV p,0.001, student’s t test). In multiple
regression analysis, titers were also significantly increased after
both the DI and S protein vaccines with use of alum (p#0.01); no
dosage effect was noted. The geometric mean neutralizing
antibody titers of the two bp inactivated vaccine groups (BPV)
were different (p = 0.039, Mann-Whitney U).
Figure 2. Vaccine Comparisons of Three SARS-CoV Vaccines,
Experiment 1. Mean lung cellular infiltration/lesion pathology and
percent eosinophils in infiltrates for each vaccine dosage group two
days after challenge with SARS-CoV. A. Mean lesion score and standard
error of the mean (S.E.) for each vaccine dosage group. All mice
exhibited lung histopathology. Scores are mean of scores for seven to
eight mice per group. Scoring. 0 – no pathology, 1 and 2 – (1) minimal
(2) moderate peribronchiole and perivascular cellular infiltration, 3 and
4 – 1 and/or 2 plus minimal (3) or moderate (4) epithelial cell necrosis of
bronchioles with cell debris in the lumen. B. Mean percent eosinophils
on histologic evaluation for seven to eight mice in each vaccine dosage
group. Mean for each mouse is the mean percent eosinophils on five
separate microscopy fields of lung sections. Analyses: A. Mean lesion
scores were different p,.001. DIV without alum greater than with alum
p = .001, VLP without alum greater than with alum p = .008. Posthoc
comparisons: DIV lower than SV p = .001 and controls p,.001 but not
VLP p..05. SV lower than controls p .048. B. Mean percent eosinophils
were different p,.001. Mean percent eosinophils lower for DIV with
alum than without alum p = .049 and lower for SV with alum than
without alum p = .001. Mean percent eosinophils lower for SV than DIV
p = .002 or VLP. P = ,.001. Mean percent eosinophils greater than
controls for DIV, SV and VLP, all three vaccines p,.001.
doi:10.1371/journal.pone.0035421.g002
Figure 3. Higher Dosages of SV Vaccine plus DIV and BPV
Vaccine Comparisons, Experiment 2. Serum neutralizing (neut)
antibody and lung virus titers for each vaccine dosage group. A.
Geometric mean serum antibody titer and standard error of the mean
(S.E.) on day 56 for each vaccine dosage group. Five mice per group
given 0.1 ml of vaccine intramuscularly on days 0 and 28. B. Geometric
mean virus titer (log
10
TCID
50
/g) and standard error of the mean (S.E.) in
lungs on day 58 (two days after SARS-CoV challenge) for each vaccine
dosage group. Seven to eight mice per group. Vaccines: double
inactivated whole virus (DIV), recombinant S protein (SV), bpropiolac-
tone inactivated whole virus (BPV) with alum (+A). Analyses: A. GMT
with alum greater than without alum: SV p,.001, DIV p = .014. GMT for
the two BPV groups are different p = .039. Multiple regression: DIV and
SV increased with alum p#.01, no dosage effect p..05.
doi:10.1371/journal.pone.0035421.g003
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Two days after challenge with 10
6
TCID
50
of SARS-CoV, titers
in mice given PBS varied between 10
7.0
and 10
8.0
TCID
50
per g of
tissue; one vaccinated animal in the group given the S protein
vaccine (SV) at the 3 mg and the 1 mg dosage without alum yielded
virus but all other animals in all other groups were culture negative
for virus (figure 3B).
Shown in figure 4A are the mean lesion scores on histologic
evaluations. The scoring system for experiments two and three
were developed by a replacement pathologist who preferred a
scale of 0 to 3 which corresponded to a judgment of mild,
moderate or severe (figure 4A). Mean lesion scores for this grading
system overall were significantly different from each other
(p,0.001, Anova) and scores were lower for the S protein vaccine
than for either of the whole virus vaccines (SV versus DIV and
BPV, p,0.001 and p = 0.006, respectively, Tukey HSD). Of
interest is that those given live virus and then challenged with live
virus two months later exhibited an infiltrative disease severity
comparable to the PBS and vaccinated groups despite no
detectable virus on day two, again suggesting some degree of
infection may have occurred earlier.
The mean eosinophil scores for the lung infiltrations were lower
for the S protein vaccine groups [SV vs. DIV p,0.001; SV vs.
BPV, p,0.001, Tukey HSD]; however, they were clearly greater
than seen in those given PBS or live virus earlier (p,0.001, Tukey
HSD) (figure 4B).
Representative photo micrographs of lung sections from mice in
this experiment two days after challenge with SARS-CoV are
shown in figure 5. The pathologic changes were extensive and
similar in all challenged groups (H & E stains). Perivascular and
peribronchial inflammatory infiltrates were observed in most fields
along with desquamation of the bronchial epithelium, collections
of edema fluid, sloughed epithelial cells, inflammatory cells and
cellular debris in the bronchial lumen. Large macrophages and
swollen epithelial cells were seen near lobar and segmental
bronchi, small bronchioles and alveolar ducts. Necrotizing
vasculitis was prominent in medium and large blood vessels,
involving vascular endothelial cells as well as the tunica media, and
included lymphocytes, neutrophils, and eosinophils in cellular
collections. Occasional multinucleated giant cells were also seen.
The eosinophil component of infiltrates was very prominent in
animals vaccinated with the experimental vaccine preparations
when compared to animals mock-vaccinated using PBS, or those
exposed earlier to live virus (figure 6); few to no eosinophils were
seen in those lung sections. Thus, while pathology was seen in
sections from the control mice, the hypersensitivity-type pathologic
reaction with eosinophils was not seen. The morphological
identification of eosinophils in H&E stains was supported by using
Giemsa stain to highlight intracytoplasmic granules in selected
lung sections (not shown), and confirmed by immunostaining with
antibodies against mouse eosinophil major basic protein (provided
by the Lee Laboratory, Mayo Clinic, Arizona) [36].
The different groups of vaccinated animals showed similar
trends in severity of pathology and of eosinophils in inflammatory
infiltrates; however, the DIV and BPV preparations at high dosage
tended to produce a greater infiltration with eosinophils.
Mouse and Vaccine Specificity (Experiment
3). Experiment 3 was performed to evaluate vaccine and
mouse strain specificity. SARS-CoV vaccines used were the DI
vaccine (DIV) with and without alum and the bp inactivated
vaccine (BPV), which contains alum, at the highest dosage. For
mouse strain specificity, Balb/c mice were included for consistency
between experiments; C57BL/6 mice were given the same
vaccines and dosages as Balb/c mice for comparison as C57BL/
6 mice do not exhibit a bias for Th2 immunologic responses as do
Balb/c mice [37–39]. PBS and live virus controls were again
included and trivalent 2010-11 formulation influenza vaccine at a
dosage of 12 mg per component was given to assess vaccine
specificity.
Neutralizing antibody titers are shown in figure 7A. Geometric
mean titers for the highest dose of the DI vaccine were higher for
those vaccine groups in the Balb/c mice than the C57BL/6 mice
but only the nonadjuvanted DI vaccine group was significantly
higher (p = 0.008, Mann Whitney U). The serum antibody
responses after BPV and live virus administration were similar
for the two mouse strains. After challenge, mean lung virus titers
Figure 4. Higher Dosages of SV Vaccine plus DIV and BPV
Vaccine Comparisons, Experiment 2. Mean lung cellular infiltra-
tion/lesion pathology and mean percent eosinophils in infiltrates for
each vaccine dosage group two days after challenge with SARS-CoV. A.
Mean lesion score and standard error of the mean (S.E.) for each vaccine
dosage group. Scores are mean of scores for seven to eight mice per
group. Scoring - 0 - no definite pathology, 1 - mild peribronchiole and
perivascular cellular infiltration, 2 - moderate peribronchiole and
perivascular cellular infiltration, 3 - severe peribronchiolar and
perivascular cellular infiltration with thickening of alveolar walls,
alveolar infiltration and bronchiole epithelial cell necrosis and debris
in the lumen. Ten to 20 microscopy fields were scored for each mouse
lung. B. Mean score and standard error of the mean (S.E.) for eosinophils
as percent of infiltrating cells for each vaccine dosage group. Scores are
mean of scores for seven to eight mice per group. Scoring: 0 - ,5% of
cells, 1 - 5–10% of cells, 2 - 10–20% of cells, 3 - .20% of cells. Ten to 20
microscopy fields were scored for each mouse lung. Analyses: A. Mean
lesion scores were different p,.001. Mean scores were lower for SV
than DIV p,.001 and less than BPV p = .006. B. Mean eosinophil scores
were lower for SV than DIV p,.001 and less than BPV p,.001.
Eosinophil scores greater for SV than PBS or live virus p,.001.
doi:10.1371/journal.pone.0035421.g004
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were similar for the PBS control challenged mice of both mouse
strains (10
6.7–7.3
TCID
50
/g lung) (figure 7B). None of the Balb/c
mouse groups given either vaccine or live virus earlier yielded virus
after challenge but some virus was detected in C57BL/6 mice
given the DIV without alum and the BPV with alum (C57BL/6
versus Balb/c, p = 0.004, Mann Whitney U).
Mean lung lesion scores two days after challenge were similar
for all groups and indicated a moderate to severe degree of cellular
infiltration (p.0.05 for each, Anova) (figure 8A). However,
eosinophil scores were significantly different between groups
(p,0.001, Anova) with significantly lower scores for nonvaccine
groups than for vaccine groups of both mouse strains (p,0.001 for
all comparable group comparisons, Tukey’s HSD). Eosinophil
scores for the vaccine groups were not different between the two
mouse strains (p.0.05, t test) (figure 8B). Photomicrographs of the
different vaccine and mouse strain groups are shown in figure 9.
Both vaccines in both mouse strains exhibited significant cellular
infiltrations that included numerous eosinophils as shown in the
MBP stained sections, a finding consistent with a hypersensitivity
component of the pathology. Prior influenza vaccine did not lead
to an eosinophil infiltration in the lung lesions after challenge.
Discussion
The emergence of the disease SARS and the rapid identification
of its severity and high risk for death prompted a rapid
mobilization for control at the major sites of occurrence and at
the international level. Part of this response was for development
of vaccines for potential use in control, a potential facilitated by the
rapid identification of the causative agent, a new coronavirus [8–
9]. Applying the principles of infection control brought the
epidemic under control but a concern for reemergence naturally
or a deliberate release supported continuation of a vaccine
development effort so as to have the knowledge and capability
necessary for preparing and using an effective vaccine should a
need arise. For this purpose, the National Institute of Allergy and
Infectious Diseases supported preparation of vaccines for evalu-
ation for potential use in humans. This effort was hampered by the
Figure 5. Photographs of Lung Tissue. Representative photomicrographs of lung tissue two days after challenge of Balb/c mice with SARS-CoV
that had previously been given a SARS-CoV vaccine. Lung sections were separately stained with hematoxylin and eosin (H&E) and an
immunohistochemical protocol using an eosinophil-specific staining procedure with a monoclonal antibody to a major basic protein of eosinophils.
DAB chromogen provided the brown eosinophil identity stain. The procedure and antibody were kindly provided by the Lee Laboratory, Mayo Clinic,
Arizona. The H&E stain column is on the left and eosinophil-specific major basic protein (EOS MBP) stain column is on the right. Vaccines: double
inactivated whole virus (DIV), bpropiolactone inactivated whole virus vaccine (BPV). As shown in the images, eosinophils are prominent (brown DAB
staining) in all sections examined. Exposure to SARS-CoV is associated with prominent inflammatory infiltrates characterized by a predominant
eosinophilic component.
doi:10.1371/journal.pone.0035421.g005
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occurrence in the initial preclinical trial of an immunopathogenic-
type lung disease among ferrets and Cynomolgus monkeys given a
whole virus vaccine adjuvanted with alum and challenged with
infectious SARS-CoV [14]. That lung disease exhibited the
characteristics of a Th2-type immunopathology with eosinophils in
the lung sections suggesting hypersensitivity that was reminiscent
of the descriptions of the Th2-type immunopathologic reaction in
young children given an inactivated RSV vaccine and subse-
quently infected with naturally-occurring RSV [32–33]. Most of
these children experienced severe disease with infection that led to
a high frequency of hospitalizations; two children died from the
infection [33,40,41]. The conclusion from that experience was
clear; RSV lung disease was enhanced by the prior vaccination.
Subsequent studies in animal models that are thought to mimic the
human experience indicate RSV inactivated vaccine induces an
increased CD4
+
T lymphocyte response, primarily of Th2 cells
and the occurrence of immune complex depositions in lung tissues
[32,42,43]. This type of tissue response is associated with an
increase in type 2 cytokines including IL4, IL5, and IL13 and an
influx of eosinophils into the infected lung; [32,33,42,44].
Histologic sections of tissues exhibiting this type of response have
a notable eosinophilic component in the cellular infiltrates. Recent
studies indicate that the Th2-type immune response has both
innate and adaptive immune response components [33,43].
In addition to the RSV experience, concern for an inappropri-
ate response among persons vaccinated with a SARS-CoV vaccine
emanated from experiences with coronavirus infections and
disease in animals that included enhanced disease among infected
animals vaccinated earlier with a coronavirus vaccine [31]. Feline
infectious peritonitis coronavirus (FIPV) is a well-known example
of antibody-mediated enhanced uptake of virus in macrophages
that disseminate and increase virus quantities that lead to
enhanced disease [31,45]. Antigen-antibody complex formation
with complement activation can also occur in that infection and
some other coronavirus infections in animals. Thus, concern for
safety of administering SARS-CoV vaccines to humans became an
early concern in vaccine development.
As a site proposed for testing vaccines in humans, we requested
and were given approval for evaluating different vaccine
candidates for safety and effectiveness. Two whole coronavirus
Figure 6. Photomicrographs of Lung Tissue. Representative photomicrographs of lung tissue from unvaccinated unchallenged mice (normal)
and from Balb/c mice two days after challenge with SARS-CoV that had previously been given PBS only (no vaccine) or live virus. H&E and
immunohistochemical stains for eosinophil major basic protein were performed as described for figure 5. The H&E column is on the left and the Eos
MBP column is on the right. Shown are sections from normal mice (no vaccine or live virus) and mice given PBS (no vaccine) or live SARS-CoV and
then challenged with SARS-CoV. As shown in the middle and bottom row images, although exposure to SARS-CoV elicits inflammatory infiltrates and
accumulation of debris in the bronchial lumen, eosinophils in all groups remain within normal limits.
doi:10.1371/journal.pone.0035421.g006
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vaccines, one rDNA-expressed S protein vaccine and a VLP
vaccine prepared by us were evaluated in a Balb/c mouse model,
initially described by others, of SARS-CoV [46,47]. The concern
for an occurrence of lung immunopathology on challenge of mice
vaccinated with an inactivated virus vaccine, as reported by
Haagmans, et al. for ferrets and nonhuman primates, was seen by
us after challenge of mice vaccinated with a SARS VLP vaccine
[20]. This finding was duplicated in an experiment reported here
and was also seen in mice vaccinated with a range of dosages of a
double-inactivated whole virus vaccine (DIV) and an rDNA S
protein vaccine (SV) although the immunopathologic reaction
appeared reduced among animals given the S protein vaccine
when compared to those given the whole virus vaccine. In later
experiments, these findings were confirmed and the vaccine
utilized by Haagmans, et al. was also shown to induce the
immunopathology in mice. Thus, all four vaccines evaluated
induced the immunopathology; however, all four also induced
neutralizing antibody and protection against infection when
compared to control challenged animals.
The immunopathology in all experiments in the present study
occurred in the absence of detectable virus in lungs of mice two days
after challenge with infectious virus. In two experiments, a live virus
group subsequently challenged with live virus was included. These
challenged animals also exhibited similar histopathologic changes
after challenge although no infectious virus was detected in lungs on
day two; however, in the latter case, the infiltrates were nearly 100%
Figure 7. Mouse and Vaccine Specificity, Experiment 3. Serum
neutralizing (neut) antibody and lung virus titers for each vaccine
dosage group. A. Geometric mean serum antibody titer and standard
error of the mean (S.E.) on day 56 for each vaccine dosage group for
each mouse strain (Balb/c or C57BL/6). Five mice per group given 0.1 ml
of vaccine intramuscularly on days 0 and 28. B. Geometric mean virus
titer (log
10
TCID
50
/g) and standard error of the mean (S.E.) in lungs on
day 58 (two days after SARS-CoV challenge for each vaccine dosage
group for each mouse strain. Seven to eight mice per group. Vaccines:
Double inactivated whole virus, (DIV), bpropiolactone inactivated
whole virus (BPV), with alum (+A). Analyses: A. GMT for highest DIV
dosage without alum greater for Balb/c than C57BL/6 p = .008 but not
for alum p..05. GMT for the BPV vaccine and live virus were not
different for the two strains p..05. B. GMT for PBS control mice were
not different p..05. GMT for DIV without alum and BPV with alum
greater for C57BL/6 than Balb/c p = .004.
doi:10.1371/journal.pone.0035421.g007
Figure 8. Mouse and Vaccine Specificity, Experiment 3. Mean
lung cellular infiltration/lesion pathology and percent eosinophils in
infiltrates for each vaccine dosage group for each mouse strain (Balb/c
or C57BL/6) two days after challenge with SARS-CoV. A. Mean lesion
score and standard error of the mean (S.E.) for each vaccine dosage
group. Scores are mean of scores for seven to eight mice per group.
Scoring 0 - no definite pathology, 1 - mild peribronchiole and
perivascular cellular infiltration, 2 - moderate peribronchiole and
perivascular cellular infiltration, 3 - severe peribronchiole and perivas-
cular cellular infiltration with thickening of alveolar walls, alveolar
infiltration and bronchiole epithelial cell necrosis and debris in the
lumen. Ten to 20 microscopy fields were scored for each mouse lung. B.
Mean score and standard error of the mean (S.E.) for eosinophils as
percent of infiltrating cells for each vaccine dosage group. Scores are
mean of scores for seven to eight mice per group. Scoring: 0 - ,5% of
cells, 1 - 5–10% of cells, 2 - 10–20% of cells, 3 - .20% of cells. Ten to 20
microscopy fields were scored for each mouse lung. Analyses: A. Mean
lesion scores were not different p..05. B. Mean eosinophil scores were
different p,.001. Mean scores for vaccine groups greater than non-
vaccine groups for Balb/c and C57BL/6 p,.001 for all comparisons.
Mean eosinophil scores for the same groups not different for Balb/c and
C57BL/6 p..05.
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monocytes and lymphocytes without the eosinophil component seen
in the vaccinated challenged animals. In a separate test to assess the
effects of the challenge inoculum, mice were given an IN challenge
with 10
8
TCID
50
of inactivated whole SARS-CoV. Lungs of these
animals revealed minimal or no histopathologic damage (data not
shown). These findings suggest that virus replication probably
occurred early after challenge, including in animals given live CoV
earlier, and is required for development of pathology, including for
the immunopathology. Infection would have been transient, below
the limit of detection two days after challenge, or neutralized in lung
homogenates before testing for virus.. Nevertheless, the Th2-type
immunopathology pattern was seen only in animals given an
inactivated vaccine earlier.
During the course of these experiments, a report appeared
describing a similar immunopathologic-type reaction with prom-
inent eosinophils in SARS-CoV challenged Balb/c mice that had
been given Venezuelan equine encephalitis (VEE) vector contain-
ing the SARS nucleocapsid protein gene [18]. Those challenged
animals exhibited infection similar to unvaccinated animals as well
as Th2-type immunopathology. A similar experiment with a VEE
vector containing only the S gene exhibited protection against
infection and no immunopathology. More recently, this group has
reported immunopathology with prominent eosinophil infiltration
after SARS-CoV challenge in Balb/c mice vaccinated with the
same double-inactivated whole virus vaccine used in our
experiments [28]. They attribute the immunopathologic reaction
following these SARS-CoV vaccinations to presence of the
nucleocapsid protein (N) in the vaccine.
In another report, vaccinia was used as a vector vaccine for
immunizing Balb/c mice with each of the SARS-CoV structural
proteins (N, S, membrane, and envelope) and then challenged with
SARS-CoV [21]. Virus infection was present in all groups after
challenge but reduced in the S vector vaccine group. Histopa-
thology scores were high for the N containing vector group and
low for the S containing group and for the vehicle control group.
Eosinophilic infiltrates and IL-5 were increased in the N vaccine
group but only IL-5 was increased in the S vaccine group.
To be certain the Th2 type immunopathology was elicited by
the S protein vaccine in our studies and in hopes a greater immune
response would result from higher dosages of the vaccine and
induce greater protection against infection as well as reduce or
prevent the immunopathology, our experiment 2 used up to 9 mg
of the S protein for immunization. While increased titers of serum
antibody were induced and no virus was detected day two after
challenge in most animals, the Th2-type immunopathology
occurred after challenge, and the immunopathology seen earlier
after vaccination with the DI whole virus vaccine was seen again.
This experiment also included the whole virus vaccine tested
earlier in ferrets and nonhuman primates where the Th2-type
immunopathology was initially seen. That vaccine, the BPV in this
report, exhibited a pattern of antibody response, protection against
infection and occurrence of immunopathology after challenge
similar to the DI whole virus vaccine (DIV).
A final experiment was conducted to evaluate specificity. The
Balb/c mouse was compared to C57BL/6 mice which do not
exhibit the Th2 response bias known to occur in Balb/c mice.
C57BL/6 mice in that same experiment exhibited results on
challenge similar to those seen in Balb/c mice. Challenge of
animals given prior influenza vaccine were infected and exhibited
histopathologic damage similar to animals given PBS earlier;
neither group exhibited the eosinophil infiltrations seen in animals
given a SARS-CoV vaccine.
In these various experiments alum was used as an adjuvant and
this adjuvant is known to promote a Th2 type bias to immune
responses [48]. However, the immunopathology seen in vaccinated-
challenged animals also occurred in animals given vaccine without
alum. In an effort to determine whether an adjuvant that induced a
bias for a Th1-type response would protect and prevent the
immunopathology, we initiated an experiment where the DI PBS
suspended vaccine was adjuvanted with Freund’s complete
adjuvant, a Th1-type adjuvant. However, this experiment was
aborted by the September, 2008, Hurricane Ike induced flood of
Galveston, Texas. An experiment with a SARS-CoV whole virus
vaccine with and without GlaxoSmithKline (GSK) adjuvant ASO1
in hamsters has been reported [25]. This adjuvant is thought to
induce Th1-type immune responses [49]. The authors indicate no
lung immunopathology was seen among animals after challenge,
including the group given vaccine without adjuvant; however,
whether the hamster model could develop a Th2-type immunopa-
thology is uncertain. Finally, a number of other studies of vaccines in
animal model systems have been reported but presence or absence
of immunopathology after challenge was not reported.
Figure 9. Photomicrographs of Lung Tissue. Representative
photomicrographs of lung tissue two days after challenge of Balb/c
and C57BL/6 mice that had previously been given a SARS-CoV vaccine.
Lung sections were separately stained with H&E (pink and blue
micrographs) or the immunohistochemical stain for eosinophil major
basic protein (blue and brown micrographs). Balb/c mice lung sections
are in the left column and C57BL/6 are in the right column; doubly
inactivated whole virus vaccine is in the upper four panels and those
from mice given the bpropiolactone inactivated whole virus vaccine
are in the lower four panels. Pathologic changes observed (inflamma-
tory infiltrates) are similar in Balb/c and C57BL/6 and eosinophils are
prominent in both groups.
doi:10.1371/journal.pone.0035421.g009
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A summary of the SARS-CoV vaccine evaluations in animal
models (including the current report) that indicated an evaluation
for immunopathology after challenge is presented in Table 2. As
noted all vaccines containing S protein induced protection against
infection while the studies with VEE and vaccinia vector
containing the N protein gene only did not. Also shown is that a
Th2-type immunopathology was seen after challenge of all
vaccinated animals when evaluation for immunopathology was
reported except the study in hamsters with a GSK whole virus
vaccine. Thus, inactivated whole virus vaccines whether inacti-
vated with formalin or beta propiolactone and whether given with
our without alum adjuvant exhibited a Th2-type immunopatho-
logic in lungs after challenge. As indicated, two reports attributed
the immunopathology to presence of the N protein in the vaccine;
however, we found the same immunopathologic reaction in
animals given S protein vaccine only, although it appeared to be of
lesser intensity. Thus, a Th2-type immunopathologic reaction on
challenge of vaccinated animals has occurred in three of four
animal models (not in hamsters) including two different inbred
mouse strains with four different types of SARS-CoV vaccines
with and without alum adjuvant. An inactivated vaccine
preparation that does not induce this result in mice, ferrets and
nonhuman primates has not been reported.
This combined experience provides concern for trials with
SARS-CoV vaccines in humans. Clinical trials with SARS
coronavirus vaccines have been conducted and reported to induce
antibody responses and to be ‘‘safe’’ [29,30]. However, the
evidence for safety is for a short period of observation. The
concern arising from the present report is for an immunopatho-
logic reaction occurring among vaccinated individuals on exposure
to infectious SARS-CoV, the basis for developing a vaccine for
SARS. Additional safety concerns relate to effectiveness and safety
against antigenic variants of SARS-CoV and for safety of
vaccinated persons exposed to other coronaviruses, particularly
those of the type 2 group. Our study with a VLP SARS vaccine
contained the N protein of mouse hepatitis virus and Bolles, et al.,
reported the immunopathology in mice occurs for heterologous
Gp2b CoV vaccines after challenge [25]. This concern emanates
from the proposal that the N protein may be the dominant antigen
provoking the immunopathologic reaction.
Because of well documented severity of the respiratory disease
among infants given an inactivated RSV vaccine and subsequently
infected with RSV that is considered to be attributable to a Th2-
type immunopathologic reaction and a large number of studies in
the Balb/c mouse model that have described and elucidated many
components of the immunopathologic reaction to RSV vaccines,
the similarity to the SARS-CoV vaccine evaluations in Balb/c
mice supports caution for clinical vaccine trials with SARS-CoV
vaccines in humans. Of interest are the similar occurrences in
C57BL/6 mice and in ferrets and nonhuman primates that
provide alternative models for elucidating vaccine-induced
mechanisms for occurrences of Th2 immunopathologic reactions
after infection. As indicated, strong animal model evidence
indicates expression of the N protein by SARS-CoV vector
vaccines can induce sensitization leading to a Th2–type immuno-
pathology with infection. In contrast to our results, those studies
did not find clear evidence of the Th2 type immunopathology on
challenge of mice given a vector vaccine for the S protein. The
finding of a Th-2-type pathology in our studies in animals
immunized with an rDNA-produced S protein is unequivocal. In
this regard, animal model studies with FIPV in cats and RSV in
mice have indicated that viral surface proteins may be the
sensitizing protein of inactivated vaccines for immunopathology
with infection [32,45]. This suggests that presentation of the S
protein in a vector format may direct immune responses in a
different way so that sensitization does not occur.
Limitations of the present studies include their performance in
mice only and uncertainty of the relevance of rodent models to
SARS-CoV vaccines in humans. Additionally, a more intense
study for virus replication including quantitative RT-PCR assays
might have confirmed the probability that virus replication is
required for induction of the immunopathology after vaccination.
Evaluations of mechanisms for the immunopathology, including
immunoglobulin and cytokine responses to vaccines and tests for
antigen-antibody complexes in tissues exhibiting the reaction,
could have strengthened the Th2-type immunopathology finding.
Finally, a successful study with a Th1-type adjuvant that did not
exhibit the Th2 pathology after challenge would have confirmed a
Th2 bias to immune responses as well as provide a potential safe
vaccination approach for SARS.
Acknowledgments
We thank I. Darlene Kirk, CCRP, for aid in coordinating the study and
preparing the manuscript. MBP antibodies were kindly provided by the
laboratory of Drs. Jamie and Nancy Lee, Mayo Clinic Arizona; e-mail
address: jjlee@mayo.edu
Table 2. Summary of Reported Protection and
Immunopathology in Animal Model Studies with SARS
Coronavirus Vaccines.
Animal Model Vaccine
1
Protection
2
Immunopathology
3
Mice Whole virus
tr
w alum Yes Yes
Whole virus
25,tr
w alum Yes Yes
wo alum Yes Yes
VLP
17,tr
w alum Yes Yes
wo alum Yes Yes
S Protein
tr
w alum Yes Yes
wo alum Yes Yes
VEE Vector
15
for N protein No Yes
for S protein Yes No
Vaccinia vector
18
for N protein No Yes
for S protein Yes ?No
Ferrets Whole virus
11
w alum Yes Yes
Nonhuman Primate
4
Whole virus
11
w alum Yes Yes
Hamsters Whole virus
22
w ASO1 Yes No
1
Reference for each indicated; tr = this report; w = with, wo = without.
2
Protection against infection (reduced lung virus after challenge).
3
Th2-type immunopathology as indicated by cellular infiltrates with
prominence of eosinophils.
4
Cynomolgus monkeys.
doi:10.1371/journal.pone.0035421.t002
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Author Contributions
Conceived and designed the experiments: RBC CJP C-TT. Performed the
experiments: C-TT ES NI-Y PCN TG. Analyzed the data: RLA RBC C-
TT. Contributed reagents/materials/analysis tools: RBC C-TT RLA ES.
Wrote the paper: RBC C-TT ES.
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