Severe acute respiratory syndrome (SARS) S protein
production in plants: Development of
recombinant vaccine
Natalia Pogrebnyak*, Maxim Golovkin*, Vyacheslav Andrianov, Sergei Spitsin, Yuriy Smirnov, Richard Egolf,
and Hilary Koprowski†
Biotechnology Foundation Laboratories, Thomas Jefferson University, Philadelphia, PA 19107-6799
Contributed by Hilary Koprowski, May 10, 2005
In view of a recent spread of severe acute respiratory syndrome
(SARS), there is a high demand for production of a vaccine to
prevent this disease. Recent studies indicate that SARS-coronavirus
(CoV) spike protein (S protein) and its truncated fragments are
considered the best candidates for generation of the recombinant
vaccine. Toward the development of a safe, effective, and inex-
pensive vaccine candidate, we have expressed the N-terminal
fragment of SARS-CoV S protein (S1) in tomato and low-nicotine
tobacco plants. Incorporation of the S1 fragment into plant ge-
nomes as well as its transcription was confirmed by PCR and RT-PCR
analyses. High levels of expression of recombinant S1 protein were
observed in several transgenic lines by Western blot analysis using
specific antibodies. Plant-derived antigen was evaluated to induce
the systemic and mucosal immune responses in mice. Mice showed
significantly increased levels of SARS-CoV-specific IgA after oral
ingestion of tomato fruits expressing S1 protein. Sera of mice
parenterally primed with tobacco-derived S1 protein revealed the
presence of SARS-CoV-specific IgG as detected by Western blot and
ELISA analysis.
immune response 兩 plant biotechnology 兩 severe acute respiratory
syndrome-coronavirus 兩 recombinant subunit vaccine
T he recent spread of severe acute respiratory syndrome (SARS)
has heightened demand for a SARS vaccine that is safe,
effective, economical, and easily administered. The production of
recombinant subunit vaccines has become a valuable modern
strategy for prevention of infectious diseases (1, 2). Development of
such vaccines relies on the identification of the best antigen and the
choice of expression system.
The causative agent of SARS has been identified as SARS-
coronavirus (CoV) (3–5), which is similar to other CoVs in both
virion structure and genomic organization (5, 6). Efforts to develop
a vaccine against SARS are necessarily based on only the limited
knowledge gained from studies of SARS-CoV, as well as on
anti-CoV strategies that have been devised over the years. Several
groups have shown that the spike glycoproteins are major inducers
of neutralizing antibodies, providing protective immunity against
many CoVs (7–11). Currently, the SARS-CoV S glycoprotein and
its truncated versions are considered the best candidates for gen-
eration of a recombinant vaccine against this disease (12–20). The
full-length SARS-CoV spike protein (S protein) is large (139 kDa)
and probably not cleaved into S1 and S2 subunits (21, 22). The
N-terminal part of the SARS-CoV S protein (S1) contains a
putative receptor-binding domain (RBD) that is responsible for cell
attachment (15, 23). Antibodies against this region were found to
be highly effective in blocking RBD–receptor interaction in other
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CoVs (24). Moreover, vaccinated animals developed an antibody
response against the S1 fragment of SARS S protein (25), and
SARS patient sera showed significant immunoreactivity with pep-
tide located within the S1 region (19).
Recent studies have shown that the plant system provides many
practical, economic, and safety advantages compared with conven-
9062–9067 兩 PNAS 兩 June 21, 2005 兩 vol. 102 兩 no. 25
tional systems for production and convenient delivery of the subunit
vaccines (26–30). Particularly, it is true for mucosal surfaces that
could provide a safe method for inducing protective immune
responses without injection-related hazards (31). Several groups
have reported development of S protein recombinant plant-based
vaccines against different CoVs for oral delivery that elicit protec-
tive immunity against virus challenge (9–11, 32). Recently, a S
protein plant-based vaccine candidate against swine transmissible
gastroenteritis CoV has advanced into early phase farming trials
(11). The neutralizing response in chickens immunized orally with
transgenic potato expressing S1 protein of another CoV (infectious
bronchitis virus) was comparable to that observed with the com-
mercial vaccine against this disease (9).
In this study, we report the successful expression of the S1
fragment of the SARS-CoV S protein in transgenic tomato and
low-nicotine tobacco plants and demonstrate its immunogenicity in
mice after parenteral or oral administration.
Materials and Methods
Cloning of the SARS-CoV Spike Gene into the Plant Transformation
Vector. The original gene encoding the human SARS-CoV spike
glycoprotein (strain TOR2, National Center for Biotechnology
Information no. NC 004718) is 3,768-bp long, encodes a protein of
1,255 aa with a molecular mass of 139 kDa, and was kindly provided
by I. Lipkin (Columbia University, New York). DNA encoding a
79-kDa S protein fragment, corresponding to amino acids 14–714,
was amplified by two consecutive rounds of PCR to generate XbaI
and SacI sites at the 5⬘ and 3⬘ ends, respectively, by using the
following primers: SP-F1-CCT TGC GCT TCT CAG CCA CGC
AAA CTC AAG AGG ATC GCA TCA CCA TCA CCA TCA
CAG TGA CCT TGA CCG GTG CAC, XbaI-F2-ATA ATC TAG
ATG ATC ATG GCT TCC TCC AAG TTA CTC TCC CTA GCC
CTC TTC CTT GCG CTT CTC AGC CAC G, and SacI-HDEL-
R-ATT CGA GCT CTT AAA GTT CAT CAT GAG CCA TAG
AAA CAG GCA TTA CT. The expression cassette of SARS-CoV
S1 protein comprised the plant-derived 23-aa {MIMASSKLLS-
LALFLALLSHANS}, signal peptide (SP), and a histidine tag
{RGSHHHHHH} at the N-terminal portion of the resulting
79-kDa polypeptide. After addition of the plant-specific endoplas-
mic reticulum retention signal {HDEL}, the cassette was subcloned
into the XbaI兾SacI site of the plant binary vector pE1801 (kindly
provided by S. Gelvin, Purdue University, West Lafayette, IN)
under synthetic (OCS)3MAS promoter (33), which is known as a
super promoter, followed by a tomato etch virus translation en-
hancer. Vector also contains the npt II gene for kanamycin selection
of transgenic plants. Plasmid pE1801-79SHDEL was electroporated
Abbreviations: SARS, severe acute respiratory syndrome; CoV, coronavirus; S protein, spike
protein; SP, signal peptide; g.i., gastric intubation; KmR, kanamycin-resistant.
*N.P. and M.G. contributed equally to this work.
†To whom correspondence should be addressed. E-mail: hilary.koprowski@jefferson.edu.
© 2005 by The National Academy of Sciences of the USA
www.pnas.org兾cgi兾doi兾10.1073兾pnas.0503760102
 into Agrobacterium tumefaciens strain LBA4404 and used for plant
transformations.
Generation of Transgenic Plants. Tomato plants (Lycopersicon escu-
lentum L. cv. Money Maker) were transformed by the Agrobacte-
rium-mediated method according to protocol described in ref. 34
with some modifications. Low-nicotine tobacco Nicotiana tabacum
cv. LAMD-609 (National Center for Genetic Resources Preserva-
tion accession no. PI 599689) was obtained from the Oxford
Tobacco Research Station (Oxford, NC). Tobacco plants (cvs.
LAMD-609 and Wisconsin) were transformed by the Agrobacte-
rium-mediated method according to Horsch et al. (35) with some
modifications. Independent kanamycin-resistant (KmR) tomato
and tobacco lines were used for molecular analyses.
Molecular Analysis of Transgenic Plant Material. The presence of the
spike gene in transgenic plants was confirmed by PCR using
genomic DNA isolated with the REDExtract-N-Amp Plant PCR
Kit (Sigma) and S protein gene-specific primers (ATG-GAC-TCA-
CTG-GTA-CTG-GTG-TGT-TAA-CTC-CTT-C and ACA-TGC-
TCA-GCT-CCT-ATA-AGA-CAG-CCT-GCT-TG) yielding the
product of 338 bp.
KmR transgenic plants with PCR-confirmed presence of the S
dilution and the corresponding horseradish peroxidase-conjugated
anti-rabbit or anti-mouse antibodies at 1兾10,000 dilution. Baculo-
virus expressed the full-length 139-kDa S protein (catalog no.
06450, Protein Science, Meriden, CT) and was used as a positive
control.
A freeze-dry lyophilizer system (VirTis, Gardner, NY) was used
to obtain lyophilized tobacco root and tomato fruit tissues. Tissues
were ground in liquid nitrogen and homogenized in extraction
buffer (50 mM Tris䡠Cl兾150 mM NaCl兾2% Nonidet P-40兾1%
desoxycholic acid兾0.5% SDS) at pH 8.0 with complete protease
inhibitor mixture (Roche Applied Science, Indianapolis) for SDS兾
PAGE and Western blot analysis at 50 ␮g per lane of total soluble
protein.
Immunological Assessment of Plant-Expressed S Protein in Mice.
Groups of 6- to 8-week-old female BALB兾c mice (five mice per
group) were used in all experiments. For oral immunization ex-
periments, mice were either fed lyophilized tissue material of
ripened tomato fruits or received powdered low-alkaloid tobacco
root material reconstituted in saline by gastric intubation (g.i.).
Each mouse consumed ⬇500 mg of dry tomato fruit tissue over a
period of 4–5 h. Intubated mice each received the equivalent of 50
mg of dry tobacco root material. Control groups of mice received

transgene were further analyzed for gene-specific mRNA expres-
sion by quantitative RT-PCR as described in ref. 36 by using the
Bio-Rad iCicler iQ Real Time Detection System with the primers
forward-GCT TCT CCA ATG TCT ATG CAG ATT C and
reverse-CAA GCA AGG ACA CAA CCC ATG AA and a
double-labeled probe 56-FAM兾AGC GCC AGG ACA AAC TGG
TGT TAT TGC T兾3BHQ-1 obtained from Integrated DNA Tech-
nologies (Coralville, IA). DNA of the S gene was used as a standard
to estimate the number of transcript copies per 50 ng of total cDNA.
Protein Expression Analysis. The XbaI兾SacI DNA fragment encod-
ing a 79-kDa truncated S protein was subcloned into a derivative of
the pGEX-3X vector for bacterial expression (Amersham Pharma-
cia Biotech). A 53-kDa version of the S protein was obtained by
additional HindIII兾SacI deletion of the same fragment. Both
plasmids were transformed into Escherichia coli Rosetta-2 (DE3)
strain (Novagen) for expression analysis.
Proteins were purified by using magnetic beads (following the
protocol of the manufacturer, Dynal, Oslo) mixed with Laemmli
buffer and heated and resolved on 4–20% gradient SDS兾PAGE.
Following the electroblotting procedure, proteins were detected
with SARS S protein-specific rabbit antibodies Sm and兾or Sn
(catalog nos. AP600b and AP6000a, respectively, Abgent, San
Diego) at 1兾1,000 dilution or with mouse sera (see below) at 1兾500
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PLANT BIOLOGY
the same amount of WT plant material. Blood and fecal pellets
were collected. Fecal pellets were extracted with 10 volumes of PBS
immediately after collection. Sera and fecal pellet extracts were
assayed for the presence of antigen-specific antibodies by Western
blot analysis and ELISA.
For parenteral immunization, mice were injected three times at
2-week intervals with an equivalent of 50 mg of dry tobacco root
material per mouse. Powdered plant material was reconstituted
with saline (1兾1 by weight) just before immunization. First and
second immunizations were given s.c. with complete and incom-
plete Freund’s adjuvant, respectively; the third dose was adminis-
tered i.p. in saline. Sera were collected retroorbitally from each
mouse before and 10 days after each immunization. Four weeks
after the last immunization, mice received an i.p. booster dose of 1
␮g of commercially obtained S peptide (Cell Sciences, Canton,
MA) without adjuvant. After 10 days, mice were killed and exan-
guinated by heart puncture, and sera were assayed by ELISA and
Western blot analysis.
Solid-phase ELISA was carried out as described in ref. 37 in
MaxiSorp 96-well plates (Nalge Nunc) coated overnight at 4°C with
the same S peptides obtained from Cell Sciences at a concentration
of 1 ␮g兾ml in PBS. Antigen-specific antibodies were detected by
using the following antibodies: rabbit anti-mouse IgG (total) and
anti-mouse IgG1 (both from BD Biosciences Pharmingen), anti-

Fig. 1. SARS-CoV S protein cloning strategy and expression analysis. (A) Schematic diagram of a full-length 139-kDa SARS-CoV S protein with two alleged regions
S1 and S2. SP cleaved from a mature protein is indicated by the dark gray box; transmembrane domain (TM) at the C terminus is represented by the light gray
box. Receptor-binding domain (RBD) is shown as a white box. The position of SARS-CoV S protein-specific antibody recognition sites are indicated as Sn and Sm.
The N-terminal S protein fragment (14 –714 aa, gray area) comprising all major antigenic epitopes was chosen for expression in planta by means of the pBI-based
vector that contains the (OCS)3MAS promoter (solid arrow) and nopaline synthase terminator (circle). (B) Western blot analysis of 79-kDa (lane 1) or 53-kDa (lane
2) S protein fragments expressed in E. coli to confirm the expected size and S protein specificity as detected by Sn and Sm antibodies. Control lane 3 contains
E. coli protein extract processed in the same manner as the S polypeptide fraction.

Pogrebnyak et al. PNAS 兩 June 21, 2005 兩 vol. 102 兩 no. 25 兩 9063
 mouse IgG2a, IgG2b, IgG3, IgM, and IgA (all from Organon
Teknika), and anti-mouse IgE (eBioscience, San Diego). A serum
dilution with an OD450 of 0.15 units above background was con-
sidered the ELISA titer.

Results
Construction of Expression Vectors. The construct for plant trans-
formation contained the 2.2-kb fragment of the SARS-CoV spike
gene encoding the truncated 79-kDa S1 region of the 139-kDa S
protein within the T-DNA of the pBI-based plant transformation
vector pE1801 under the strong (OCS)3MAS promoter. We sub-
stituted the viral SP (1–13 aa) of the original spike gene with the
plant-derived SP at the N terminus and added a plant-specific
HDEL endoplasmic reticulum retention signal at the C terminus of
the 14- to 714-aa S fragment (see Fig. 1A). The resulting plant
transformation construct pE1801-79SHDEL also contains the npt II
gene for selection of transgenic plants on kanamycin-supplemented
medium. The same 79-kDa version and the shorter 53-kDa version
of truncated SARS-CoV spike gene were also used for E. coli
expression and verification analysis with S-specific antibodies Sn
and Sm (Fig. 1B), which recognize two distinct parts of the S
protein.

Production of Transgenic Plants Expressing Truncated SARS-CoV S

Protein. Both tomato (cv. Money Maker) and tobacco (cvs. LAMD-
609 and Wisconsin) were transformed with pE1801-79SHDEL by
using the Agrobacterium-mediated transformation method. KmR
plants of tomato and tobacco (Figs. 2A Left and 3A Left) were tested
by PCR for the presence of the transgene in genomic DNA, yielding
the 338-bp expected product (Figs. 2 A Right and 3A Right) also
detected for the positive plasmid DNA controls. No products were
detected in the negative WT controls. Twelve tomato, 20 LAMD-
609, and 20 Wisconsin tobacco transgenic lines were chosen for
further evaluation. The transgenic tomato and tobacco plants did
not show any morphological differences compared with nontrans-
genic plants.
Transgene expression levels were analyzed by quantitative RT-
PCR. As expected, mRNA expression levels varied among inde-
pendent transgenic lines. Fig. 3B shows the detailed analysis of
transgenic tobacco plants. For some transgenic lines (lane 18 for
low-nicotine tobacco cv. LAMD-609 and lane 12 for control
tobacco cv. Wisconsin), expression levels reached 105-106 copies of
S protein transcript per 50 ng of total cDNA. mRNA levels in leaves
and roots of the same transgenic lines consistently revealed higher
levels of expression in the roots (see Fig. 3B Lower, for example).
Western blot analysis of transgenic lines with polyclonal SARS-
specific antibodies Sm and Sn confirmed the presence of SARS-
CoV S-specific 79-kDa protein and its derivatives at variable levels
(Figs. 2B and 3 C and D). The 79-kDa protein was barely detectable
in several green tomato fruits but was highly expressed in others
(compare lanes 2 and 4 in Fig. 2B Upper Right). In red, ripened
fruits, only lower molecular weight S-specific subproducts (50 kDa)
were detected in Fig. 2B Lower Right (lanes 1, 4, and 5). High
expression levels of the 79-kDa S protein were detected in several
KmR transgenic tobacco lines (Fig. 3C Right). Additional protein
bands were present in some extracts of transgenic and nontrans-
genic tobacco lines. The level of protein expression in tobacco roots
was significantly higher than that in leaves.
Several of the highest S1 protein-expressing tobacco and tomato
T0 transgenic lines were taken through a plant-breeding scheme to
generate homozygous lines to increase and genetically stabilize the
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Fig. 2. Analysis of transgenic tomato plants expressing SARS spike 79-kDa
peptide in fruit tissue. (A) KmR transgenic plants (example shown in Left)
revealed the presence of the SARS-S1 gene by PCR analysis using gene-specific
primers resulting in the 338-bp product (Right). No amplification product was
detected in the genomic DNA of the WT plant (Right). Lane ⫹ is a positive DNA
control for PCR. (B) Transgenic tomato fruits, green (Upper) and red, ripened
(Lower), were lyophilized and analyzed by Western blot (Right). S protein-
specific antibodies (Sn) detected protein with the expected molecular size (79
kDa) in some green fruit tissues (Upper Right); multiple lower molecular
weight fragments (⬇53 kDa) were detected in red tomato fruit tissue of
independent transgenic plants (Lower Right). (C) Increased IgA levels were
detected in S protein-specific ELISA assays of fecal pellet extracts from mice fed
with transgenic tomato fruit. Data are mean (⫾SD) values from each group of
five mice. Squares, mice fed with transgenic tomato fruits (⫹); diamonds, mice
fed with control tomato fruits (⫺); circles, preimmune control mice.
Immune Response of Mice to Plant-Derived SARS-CoV 79-kDa S
Protein. Each BALB兾c mouse was fed with 500 mg of lyophilized
tomato fruit containing the antigen and exhibited a significant
increase in IgA levels in the fecal pellet extracts (Fig. 2C). Samples
showed no increased IgA levels in mice fed with nontransgenic fruit
tissue and in the corresponding preimmune controls. No increase
in IgG levels was detected in fecal extracts of transgenic fruit-fed
mice or in corresponding sera of the same mice (data not shown).
This experiment was limited by the quantity of transgenic tomato
fruit material. In another experiment, mice were immunized orally
and parenterally with lyophilized transgenic tobacco root tissue
expressing the 79-kDa S1 protein. No increase in the level of S
protein reactive antibodies was detected during the course of three
immunizations with transgenic tobacco material given by either
route; however, after a booster dose of S peptide, a significant

high expression levels of S1 protein.
KmR T1 tobacco lines (cv. LAMD-609) were grown hydropon-
ically to obtain large amounts of root tissue (Fig. 3D Left) for
immunological experiments. Western blot analysis of T1 lines
revealed high levels of S protein expression comparable with the
original T0 transgenic lines (Fig. 3D Right).
increase of S protein-specific total IgG was detected in mice
immunized parenterally (Fig. 4A, group II ⫹), equal to a secondary
immune response obtained with commercially obtained S peptides
(Fig. 4A, group III). No increase in IgG levels was detected in
groups immunized parenterally with control plants or material
administered by g.i. (Fig. 4A, group II ⫺ and group I).

9064 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0503760102 Pogrebnyak et al.

 PLANT BIOLOGY
Fig. 3. Analysis of low-nicotine transgenic tobacco plants expressing SARS spike 79-kDa protein. (A) KmR transgenic tobacco plants (example shown in Left) were
analyzed by PCR using SARS-CoV S protein gene-specific primers that resulted in the expected 338-bp product (Right). No amplification product was detected in the
genomic DNA of a WT plant. Lane ⫹ is a positive DNA control for PCR. (B) Real-time quantitative RT-PCR expression analysis of transgenic low-nicotine cv. LAMD-609
(Left) and control cv. Wisconsin (Right) tobacco plants. The cDNA fragment containing the full-length S protein gene was used as a standard to estimate the number
of mRNA copies in the samples. (Upper) Absolute numbers of mRNA copies per 50 ng of total cDNA from individual transgenic KmR plants. (Lower) SARS spike-specific
mRNA expression in leaf and root tissues of selected KmR plants. (C) Tobacco transgenic plants (Left) showed variable expression levels of truncated SARS S protein in
Western blot analysis (Right) with S protein-specific Sm兾Sn antibodies. The expected 79-kDa protein was detected in several transgenic lines (1, 2, 3, 4, and 5), compared
with the corresponding bacterial extract (⫹). wt, WT tobacco control. (D) The T1 offspring (hydroponically grown roots shown in Left) from T0 plant lines 1 and 3
confirmed high levels of transgene expression. The expected 79-kDa protein was detected by Western blot analysis (Right) with Sm兾Sn antibodies.

Sera from group II mice were further tested by Western blot
analyses for the presence of SARS-CoV S protein-specific antibod-
ies. Results are shown for sera of mice primed i.p. with plant
material and boosted with S peptide (Fig. 4B Left). Sera from those
mice reacted with the 139-kDa commercial S protein (lane 1), the
E. coli-expressed 79-kDa form and its subfragments (lane 2), and
with commercial S peptide (lane 5). No reactivity with control
protein extracts from E. coli was detected (lanes 3 and 4), and no
reactivity was detected with any of the above proteins in sera of mice
immunized with control plants and boosted with S peptide (Fig. 4B
Left ⫺). Polyclonal rabbit antibodies were used to confirm S protein
specificity of the reactions (Fig. 4B Right). As expected, Sm anti-
bodies detected only the full-size S protein (lane 1) and the 79-kDa
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response equal to that produced after double immunization with
commercial S peptide in mice primed with plant material (Fig. 5A).
Levels of IgG2a and IgG2b in mice immunized with plant material
were about one-half of those obtained during secondary immune
response (Fig. 5 B and C). No changes in serum IgG3, IgM, or IgA
levels were detected, and no S protein-specific IgE antibodies were
detected in any sera at dilutions starting at 1:2 (data not shown).
Discussion
An enormous effort has been made by the scientific community to
develop a vaccine against SARS (12, 13, 18, 38, 39). With the goal
of generating a safe and inexpensive subunit vaccine that can be
delivered by an oral route, we have chosen to produce the recom-

truncated S protein (lane 2), whereas Sn antibodies reacted with the
79-kDa truncated S protein and with the booster S peptide (lane 5).
The expected reactivity with 53-kDa S protein was detectable but
after a longer exposure.
Detailed analysis of Ig subclasses in sera of mice from group II
and group III (Fig. 5) revealed an anti-S protein IgG1 immune
binant SARS antigen in transgenic plants. Recently, several groups
have presented key data regarding the immunogenicity of different
SARS proteins and have come to the conclusion that SARS-CoV
S protein is the best candidate for recombinant vaccine develop-
ment (12–19). Moreover, it was shown for other CoVs (swine
transmissible gastroenteritis virus, infectious bronchitis virus, and

Pogrebnyak et al. PNAS 兩 June 21, 2005 兩 vol. 102 兩 no. 25 兩 9065
 Fig. 4. Immune response to plant-derived 79-kDa S protein in mice. (A) ELISA titers in sera of mice immunized with lyophilized transgenic plant material. Each group
of five mice was immunized three times. Group I was immunized by means of g.i. with transgenic plant material (⫹) or negative control plant material (⫺); group II
mice were immunized parenterally with transgenic plant material (⫹) or with control WT plant material (⫺). Mice from both groups were subsequently boosted with
commercially available S peptide. Mice from positive control group III were immunized twice with the same peptide and complete Freund’s adjuvant. Data from each
group are mean ⫾ SD. (B) (Left)Western blot analysis of sera obtained from group II mice, immunized parenterally with transgenic (⫹) or negative control (⫺) plant
material. (Right) Immunodetection by SARS S protein-specific antibodies Sm and Sn. Identical blots with SARS S protein fragments were used in each experiment as
follows: lane 1, full-length 139-kDa SARS S protein commercially expressed in Baculovirus; lanes 2 and 3, 79- and 53-kDa SARS S proteins, respectively, expressed in E. coli;
lane 4, control protein extract from nontransformed E. coli; lane 5, commercial S peptide used for boosting.

porcine epidemic diarrhea virus) that plant-derived recombinant
antigen in the form of the S protein could also induce strong
protective immunity in animals (9–11, 32). Based on these studies
and others showing that the truncated version of the SARS-CoV S
protein (S1) contains important antigenic domains (14, 25), we have
selected this fragment for in planta expression. To ensure high
accumulation and proper posttranslational modification of the
glycoprotein, we assembled several expression cassettes with this
transgene. One of the cassettes that combines plant SP and the
endoplasmic reticulum retention signal (HDEL) (40) and is driven
by the super promoter (33) has proved to be the best in the
projected tissue-specific expression (fruits and roots) in transgenic
plants.
Two plant species were chosen to express the SARS-CoV
recombinant antigen: tomato and low-nicotine tobacco. Tomato is
an easy-to-transform crop plant with abundant edible fruit tissue
available for direct utilization and兾or downstream processing (41,
42). Tobacco is an extremely efficient plant transformation system
and is frequently used in plant biotechnology as the system of choice
(27–29). In this study, we explore the possibility of using a low-
nicotine tobacco variety that contains much less (0.06%) total
nicotine (compared with 2–4% in other tobacco varieties).
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After preliminary molecular characterization, the best transgenic
plants were tested for the presence of recombinant S protein by
Western blot analysis. This method makes possible a visual detec-
tion of recombinant protein in transgenic samples when it is
expressed兾accumulated at high levels (⬎0.1% total soluble pro-
tein). We have obtained plant-derived antigen of correct size in
several transgenic plants in adequate amounts to conduct immu-
nological experiments. Some variations in size of recombinant
protein were observed in mature and immature tissue of transgenic
tomato. Presumably, this variation is due to proteolytic cleavage of
the recombinant protein that occurs in the tissue of red, ripened
tomato fruits.
The demonstration that S proteins of several CoVs (swine
transmissible gastroenteritis virus, porcine epidemic diarrhea virus,
and infectious bronchitis virus) are immunogenic when adminis-
tered orally in animals (9–11, 32) led us to test plant-derived SARS
antigen by this route. Indeed, mice showed a significant increase in
mucosal IgA production after a single feeding, pointing to the
feasibility of developing a plant-based oral vaccine against SARS.
Using g.i. to deliver three doses of lyophilized tobacco root material
followed by a boost did not produce detectable immune response
in mice, in contrast with the results with direct tomato fruit feeding,
where IgA levels were increased after a single dose. The possible

Fig. 5. IgG subclass levels in sera of mice immunized parenterally. Sera from parenterally immunized mice of groups II and III (see Fig. 4) were analyzed by ELISA
for the presence of IgG1 (A), IgG2a (B), and IgG2b (C). Squares, sera from mice immunized with transgenic tobacco material; circles, sera from mice immunized
with negative control plant material; triangles, sera from mice immunized twice with commercial N-terminal S peptide. Data are mean ⫾ SD.

9066 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0503760102 Pogrebnyak et al.

 explanation for these results might be the smaller amount of antigen
present in tobacco root material and兾or the lower dose of plant
material that was administered to mice. Another explanation could
be a longer mucosal exposure to plant-derived antigen through the
process of chewing, swallowing, etc., associated with tomato feed-
ing, as compared with g.i. delivery of tobacco root material directly
to the stomach.
The mice parenterally primed with plant-derived antigen devel-
oped an immune response after a booster immunization with
N-terminal S protein peptide that by itself was not immunogenic
without adjuvant. The fact that plant-derived material was not
immunogenic by itself and required boosting immunization is not
surprising. Similar results were demonstrated in several other
studies. Boosting immunization was required for plant-expressed
rabies G protein (43), Tat protein from HIV-1 virus (44), and S
proteins from other CoVs (11). Lamphear et al. (11) reported swine
feeding studies of oral swine transmissible gastroenteritis virus
vaccine candidates to boost immunogenic responses in gilts previ-
ously sensitized with a commercially available, modified live viral
vaccine. For human vaccines, some data suggested that plant-
derived vaccines may be used as boosting vaccines, after primary
immunization with vaccines produced by more traditional technol-
ogies (45). At the same time, a number of successful attempts to
induce immune response with plant-expressed antigens without
boosting immunization with vaccines produced by traditional meth-
ods have been reported (28). Necessity of boosting immunization
may be due to the quantity and nature of expressed antigen as well
as usage of adjuvants.
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The type of immune response is known to be important in achieving
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about the requirements for an optimal immune response against
SARS-CoV.
In sum, our study demonstrates the successful expression of
SARS-CoV S protein in transgenic plants in amounts sufficient to
induce antibody response after feeding mice with transgenic ma-
terial. The mice parenterally primed with plant-derived antigen
developed an immune response after a booster immunization.
Progress toward the goal of establishing a safe and inexpensive
vaccination program awaits further study with other animals and
optimization of feeding protocols that might include, for example,
oral administration of subunit vaccine expressed in tomato fruit
followed by a boost with tobacco-derived antigen.
We thank Thomas Jefferson University兾Kimmel Cancer Center research
and animal facilities for their support, Dr. Deka and G. Golovin for help
with the greenhouse work, Dr. Borisjuk and Dr. Girard for helpful com-
PLANT BIOLOGY
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tion vector. This work was supported in part by grants from the United
States Department of Agriculture and from the Commonwealth of Penn-
sylvania to Biotechnology Foundation Laboratories (to H.K.).
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PNAS 兩 June 21, 2005 兩 vol. 102 兩 no. 25 兩 9067