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ISSN 0582-9879 Acta Biochim et Biophysica Sinica 2004, 36(1):37-41 CN 31-1300/Q

DNA Vaccine of SARS-Cov S Gene Induces Antibody Response
in Mice

Ping
ZHAO, Jin-Shan KE, Zhao-Lin QIN, Hao REN, Lan-Juan ZHAO, Jian-Guo YU1,

Jun
GAO, Shi-Ying ZHU, and Zhong-Tian QI*

( Department of Microbiology, Second Military Medical University,
Shanghai 200433, China;

1Department
of Infectious Diseases, PLA Hospital No.88, Tai’an 271000, China )

Abstract
The spike (S) protein,
a main surface antigen of SARS-coronavirus (SARS-CoV), is one of the most
important antigen candidates for vaccine design. In the present study, three
fragments of the truncated S protein were expressed in E.coli, and
analyzed with pooled sera of convalescence phase of SARS patients. The full
length S gene DNA vaccine was constructed and used to immunize BALB/c mice. The
mouse serum IgG antibody against SARS-CoV was measured by ELISA with E. coli
expressed truncated S protein or SARS-CoV lysate as diagnostic antigen. The
results showed that all the three fragments of S protein expressed by E.coli
was able to react with sera of SARS patients and the S gene DNA candidate
vaccine could induce the production of specific IgG antibody against SARS-CoV
efficiently in mice with seroconversion ratio of 75% after 3 times of
immunization. These findings lay some foundations for further understanding the
immunology of SARS-CoV and developing SARS vaccines.

Key
words severe acute
respiratory syndrome-coronavirus (SARS-CoV); spike protein; DNA vaccine;
antibody

The
severe acute respiratory syndrome (SARS), also named infectious atypical
pneumonia, is a newly described and highly contagious respiratory infection
that first occurred in late 2002 in Guangdong Province, China, and spread to
more than 30 countries in early 2003. It has been identified that the
etiological agent of SARS is a novel coronavirus, named as SARS-CoV [1]. The
spike (S) protein of coronavirus is the major envelope component, constituting
the spike projecting from the virion surface, which mediates many of the
biological properties of the virus, such as attachment to cell receptors,
penetration, and spread by virus-induced cell to cell fusion. The S protein
also plays an important role in the immune responses against the virus since
neutralizing antibody, passive antibody protection, and cellular immunity have
been related to the protein. Therefore, the spike protein may act as the most
potential antigen for SARS vaccine design [2]. The S gene of SARS-CoV encodes a
surface glycoprotein precursor predicted to be 1255 amino acids in length. It
belongs to type I membrance protein and has low level of similarity (20%–27%
amino acid identity) with other coronaviruses [3]. In the present study, the
truncated fragments of SARS-CoV S protein were expressed in E.coli and
theirs anti-genicity were analyzed, and furthermore, a candidate DNA vaccine
containing full-length S gene was constructed and its immunogenicity was
evaluated in mice.

Materials and Methods

Materials

The DNA
fragment SI which encodes nucleotide (nt) 1– 1650 of SARS-CoV S gene was
synthesized by Shanghai BioAsia Bio-tech Company. Two other DNA fragments, SII
encoding nt 1626–2934 and SIII encoding nt 2766– 3768 of SARS-CoV S gene, were
kindly provided by Prof. Zheng-Hong YUAN (Molecular Virology Lab, Fudan
University). The nucleotide sequences of the three DNA fragments are identical
to the published data (GenBank accession number: AY278554). Expand long
template PCR system was from Roche. Prokaryotic expression plasmid pPROEX HTa
was from LIFE TECHNOLOGIES. Prokaryotic expression plasmid pGEX-4T-1 and E.
coli strain BL21 (DE3) were the products of Amersham Bioscienences. Ni- NTA
affinity resin was from Qiagen. Sera of onvalescence phase of SARS patients
were collected from Beijing Xiaotangshan Hospital, and inactivated by heating
at 56 ℃ for
30 min. ELISA kits for IgG or IgM antibodies to SARS-CoV were from Beijing
BGI-GBI Biotech Co., Ltd.. Eukaryotic expression plasmid pCIDN, was constructed
from the vector pS2S/IL-2, in which the S2S gene of HBV and the murine IL-2
gene was replaced by synthetic rabbit beta-globin intron [4]. Six-week old
female BALB/c mice were purchased from Xiper-Bikai Experimental Animal Co.,
LTD, Shanghai. HRP conjugated goat-anti-human IgG and goat-anti-mouse IgG were
from Sigma.

Construction
of prokaryotic expression plasmid containing the fragments of S gene

DNA
fragment S301, encoding amino acid 14–314 of SARS-CoV S protein, was amplified
using the following primers: SAF: 5′-GAA TTC ATG AGT GAC CTT GACCGG TGC
ACC-3′;
SAR: 5′-GAA
TTC TTA ACC TCG AGT AAC ATC TCC TGA GGG AAC AAC C-3′. The fragment SI
was used as the template to amplify the S301, which was then inserted into
pMD18-T vector and subcloned into EcoRI site of pPROEX HTa. The
expression plasmid pHT-S301 was identified by EcoRI and PstI
digestion, respectively. DNA fragment S338, encoding aa 557–894 of SARS-CoV S
protein, was amplified by the following primers: SBF: 5′-GAA TTC ATG GAT TTC
ACT GAT TCC GTT CGA GAT CC-3′; and SBR: 5′– GCG GCC GCT TAG GTA ACT CCA ATG CCA
TTG AAC C-3′.
Fragment SII was used as the template for amplifying S338. The amplified
fragment was inserted into pMD18-T vector for the construction of the
expression plasmid pGEX-S338, which was obtained by subcloning the fragment
S338 into pGEX-4T-1 with EcoRI/NotI digestion. DNA fragment S324,
encoding a.a. 867–1190 of SARS-CoV S protein, was amplified by the primers:
SCF: 5′-GAA
TTC ATG GGA TGG ACA TTT GGT GCT GGC GCT G-3′ and SCR: 5′-GCG GCC GCT TAT TGC
TCA TAT TTT CCC AAT TCT TG-3′. Fragments SII and SIII were used as the
templates for amplifying the S324 by SOE-PCR methods [5]. The product was
cloned into pMD18-T vector and further inserted into the EcoRI/NotI
sites of pGEX-4T-1for the expression plasmid pGEXS324.

Expression
of truncated SARS-CoV S proteins in E.coli and identification of their
antigenicity

Plasmid
pHT-S301, pGEX-S338 or pGEX-S324, respectively. The positive colonies were
chosen and cultured overnight in LB medium containing 100 mg/L ampicillin. The
cultures were diluted 100 times with 2×YT medium and incubated at 37 ℃ until A600 reached 0.6. Then the IPTG was added to
a final concentration of 0.6 mmol/L for the induction of expression. The
bacteria were collected after 3 hours and lysed in 2×SDS loading buffer for
SDS-PAGE analysis. The antigenicity of these expressed proteins was determined
by Western blot, using 200 fold diluted pooled sera of convalescence phase of
two SARS patients as the primary antibody, and 100 fold diluted HRP conjugated
goat-anti-human IgG as the second antibody.

Construction
of SARS-CoV S gene DNA vaccine

A DNA
fragment encoding nt 1–2058 of S gene was amplified (with primers, NF: 5′-GAA TTC GCT AGC CAC
CAT GTT TAT TTT CTT ATT ATT TC-3′ and NR: 5′– GGA TCC TCT AGA TTA TGA ACT ATC AGC
ACC TAA AGA C-3′)
from the fragment SI and SII by SOE-PCR. The amplified fragment was inserted
into pMD18-T vector, and then subcloned into EcoRI/BamHI sites of
the eukaryotic expression plasmid pCIDN. The resulting plasmid was named
pCIDN-SA. Another DNA fragment, covering nt 1913–3768 of S gene was amplified
(with primers CF: 5′-GAG CTG AGC ATG TCG AC-3′ and CR: 5′– GGA TCC TCT AGA
TTA TGT GTA ATG TAA TTT GAC ACC-3′) using the template SII and SIII. After cloning
it into pMD18-T vector and sequenced, the amplified DNA fragment was fused with
pCIDN-SA at SalI/BamHI site. The eukaryotic expression plasmid
pCIDN-FS,containing the full-length S gene of SARS CoV was obtained.

Immunization
of mice [6]

Plasmid
pCIDN-FS and pCIDN were prepared with Mega plasmid preparation kit (Qiagen) and
dissolved in 0.01 mol/L PBS to a final concentration 2 g/L. Sixteen BALB/c mice
were randomly divided into two groups (eight mice in each group). The mice in
the experimental group were injected with 200 mg of pCIDN-FS in both tibialis anterior
muscles. Mice were boosted twice with the same dosage at 2 weeks intervals. The
mice in control group received the same amount of pCIDN vector with identical
route and frequency.

Detection
of anti-SARS-CoV IgG in mice

The
recombinant S301 protein was purified by Ni-NTA affinity resin according to the
manufacture’s directions. Microtiter plates were coated with the purified S301
protein (50 mg/L in carbonate buffer) to detect the antibody against SARS-CoV
in mouse sera. In addition, the microtiter plates coated with SARS-CoV lysate
was also used for the detection. Mouse sera were collected at 0, 2, 4, 6 and 8
week after the first immunization and diluted with PBS (pH 7.4) containing 12%
goat sera and 0.5% Tween-20. The detection antibody was HRP conjugated
goat-anti-mouse IgG (1:10,000 dilution). TMB/H2O2 was
used as the substrate. The absorbance at 450/630 nm was measured on an ELISA
reader (Bio-Rad). It was considered positive when the A450/A630 of the mice in the experimental group is larger
than or equal to 2.1-time of mean of the average value in the control group.

Statistical
analysis

All
data were analyzed using Student’s t-test for the significance of the
difference.

Results

Expression
of truncated S proteins in E. coli

The
plasmid pHT-S301 encodes a fusion protein about 37 kD with 6 histidines at its
N-terminal. The plasmid pGEX-S338 and pGEX-S324 encode two fragments of truncated
S protein fused with GST, and the molecular weights are 64 kD and 62 kD,
respectively. In SDS-PAGE, recombinant BL21(DE3) expressed proteins could be
visualized and the molecular weights were the same as predicted (Fig. 1).
protein is stronger than that of S324.

Fig. 1 SDS-PAGE analysis of expressed S protein from
recombinant E. coli BL21(DE3)

1, BL21(DE3)/pHT-S301 without IPTG induction; 2, BL21(DE3)/pHT-S301 with
IPTG induction; 3, BL21(DE3)/pGEX-S338 without IPTG induction; 4, BL21(DE3)/
pGEX-S338 with IPTG induction; 5, BL21(DE3)/pGEX-S324 without IPTG induction;
6, BL21(DE3)/pGEX-S324 with IPTG induction; M, molecular weight markers. Arrows
indicate the bands of target proteins.

Fig. 2 Western-blot analysis of expressed S protein from
recombinant E. coli BL21(DE3)

1, BL21(DE3)/ pHT-S301without IPTG induction; 2, BL21(DE3)/pHT-S301with
IPTG induction; 3, BL21(DE3)/ pGEX-S338 without IPTG induction; 4, BL21 (DE3)/
pGEX-S338 with IPTG induction; 5, BL21(DE3)/ pGEX-S324 without IPTG induction;
6, BL21(DE3)/ pGEX-S324 with IPTG induction.

Fig. 3 The structure of the DNA vaccine pCIDN-FS

Structure
and sequence analysis of DNA vaccine containing full-length S gene

The
structure of DNA vaccine pCIDN-FS is shown in Fig.3, in which “PCMV” is early promoter/ enhancer sequence
of CMV, “intron” is rabbit beta-globin intron, “S” represents the full-length S
gene of SARS-CoV and “BGH pA” is transcription terminal signal of bovine growth
hormone gene, “DHFR” is dihydrofolate reductase gene and “NEO” is neomycin
phosphotransferase gene. Three nucleotide mutations were found in the S gene of
pCIDN-FS compared with the parental sequence (GenBank accession number:
AY278554). The first one occurred at nt 822 (T → C), which is a nonsense mutation. The
second one at nt 1189 (A→G) resulting in amino acid Ile →Val mutation,

and the
last one at nt 2032 (A → T) leading to amino acid Thr → Ser mutation. There
is a restriction endonuclease PstI site at nt 750 and a SalI site
at nt 1924 in the S gene of SARS-CoV.

Antigenicity
analysis of S protein fragments

Antigenicity
of the S protein fragments expressed in E. coli were determined by
anti-SARS-CoV IgG positive sera of SARS patients in Western Blot. As shown in
Fig. 2, all of the three truncated S proteins could react with the pooled sera
of SARS patients, and the reactivity of S310 and S338 Antibody responses
induced by Full length S gene DNA vaccine

The
S301 protein was used as the diagnostic antigen in ELISA for the detection of
IgG antibodies against SARSCoV in mouse sera. Positive ratio was found to be
0%, 0%, 25%, 50% and 50% at various time courses of 0, 2, 4, 6 and 8 weeks
after the first immunization with pCIDN-FS. The positive ratio increased to 0%,
12.5%, 50%, 62.5% and 75% when serum samples were tested using the vi-rion
lysate of SARS-CoV as the diagnostic antigen.

All
sera positive in S301 protein based ELISA were also positive in virion lysate
based ELISA. Significant difference was observed by comparing the values of A450/A630 of ELISA detection between the pCIDN-FS immunized
group and pCIDN group. The difference developed at week 4 after the first
immunization, and increased gradually at the 6th and 8th weeks (Table 1, Table
2).

Discussion

The S
protein is important for the infectivity and pathogenicity of coronovirus.
Mutations in S gene have previously been correlated with altered pathogenesis,
virulence and tropism in other coronaviruses [7]. The S proteins of some
coronaviruses are cleaved by a host cell trypsin-like protease into S1 involved
in receptor binding and S2 involved in cell fusion. The S proteins are the
major targets of the neutralizing antibodies in some animal coronoviruses and
can also induce cytotoxic T lymphocytes. For example, the S proteins of mouse
hepatitis virus (MHV) and transmissible gastroenteritis virus (TGEV) could
elicit neutralizing antibodies which protect animals from virulent virus
challenge. In addition, the immune protection of S1 subunit at N-terminal of S
protein is more potent than that of S2 subunit at C-terminal [8,9]. However, it
is not the case in feline infectious peritonitis (FIPV), in the antibodies
against the S protein of FIPV are not protective. It has been found that the
antibodies against FIPV S protein enhance the disease progression, this is the
same as the antibody-dependent enhancement (ADE) of infection effects of Dengue
Virus, in which virus-antibody immune complexes bind to monocytes or
macrophages via Fc receptors for immunogobulin G or complement receptors on the
cell surface [10]. The target cells of SARS-CoV are not monocytes or
macrophages and convalescence sera of SARS patients were effective in clinical
treatment of SARS-CoV infection, therefore, the ADE effect may not occur in
SARS-CoV.

In the
present study, fragments of S proteins of SARSCoV were expressed in E.coli and
the antigenicity of these recombinant proteins were determined with the
convalescence sera of SARS patients. The antigenicity of S301 and S338 were
found to be stronger than that of S324, which is consistent with other reports
that major antigenic domains were located at the N-terminal of S protein and
the N-terminal of S2 subunits in some coronaviruses [11,12]. At present, the
above result may not reliably represent the distribution of epitopes in SARS-CoV
S protein, because only two serum samples were used, and the recombinant
proteins were non-glycosylated and denatured.

DNA
vaccine pCIDN-FS containing full-length S gene was used to immunize BALB/c mice
and significant difference of antibody response was developed between the DNA
vaccine group and the mock plasmid control group.

The
difference was further increased after boosts, indicating that the DNA vaccine
can induce specific humoral immune responses against S protein of SARS-CoV. In
the DNA vaccine group, one mouse generated specific antibodies right after the
first immunization. Using the virion lysate as diagnostic antigen for detecting
antibodies against SARS-CoV S protein, 62.5% of the DNA vaccine-immunized mice
was positive at the 2nd week after the last immunization and 75% was positive
at the 4th week after the last immunization. The reactivity of SARS-CoV virion
lysate is more sensitive than that of recombinant S301

Table 1 Detection of anti-SARS-CoV
IgG in sera of DNA immunized mice using recombinant S301 antigen coated
microplate (1∶50 diluted sera)

Table 2 Detection of anti-SARS-CoV IgG in sera of DNA
immunized mice using SARS-CoV viron lysate coated microplate (1∶50 diluted sera)

nature and covers only
partial epitopes of the complete S protein. In addition, the quantity and
structure of the coated antigen may also influence the sensitivity of ELISA.
The detective specificitiy of the recombinant S301 protein was the same as
virion lysate, indicating that the antibodies induced by the DNA vaccine are
specific for S protein. The S gene of SARS-CoV encodes type I membrance
glycoprotein, the elimination of the transmembrane domain at its carboxyl
terminus may make the DNA vaccine expressing secretive protein and inducing
stronger immune response.

In most
cases, DNA vaccines were less effective in larger species than in small
animals, so it isnot used very much on human. Antigens are produced in host
cells in DNA immunization, and the mechanisms are basically similar to
recombinant or inactivated vaccines, in addition to the immune stimulation of
vector DNA itself [13]. So, our results also uphold that recombinant S protein
may be a good candidate antigen for SARS vaccine design. The DNA vaccine
pCIDN-FS contains dihydrofolate reductase (DHFR) gene and neomycin
phosphotransferase (Neo) gene. These two markers can be used to select the
transfected cells andamplify the target gene, so this DNA construct can be used
to express S protein of SARS-CoV in CHO/dhfr– cell expression system to prepare recombinant SARS
vaccine.

Acknowledgments

We
thank Dr. Qiang XUE for his kindly help of preparing the manuscript.

References

1 Ksiazek TG, Erdman D, Goldsmith CS, Zaki SR, Peret T, Emery S, Tong S et
al. SARS Working Group. A novel coronavirus associated with severe acute
respiratory syndrome. N Engl J Med, 2003, 348(20):1953–1966

2 Popova R, Zhang X. The spike but not the hemagglutinin/esterase protein
of bovine coronavirus is necessary and sufficient for viral infection.Virology,
2002, 294(1): 222–236

3 Rota PA, Oberste MS, Monroe SS, Nix WA, Campagnoli R, Icenogle JP,
Penaranda S et al. Characterization of a novel coronavirus associated
with severe acute respiratory syndrome. Science, 2003, 300(5624): 1394–1399

4 Chow YH, Huang WL, Chi WK, Chu YD, Tao MH. Improvement of hepatitis B
virus DNA vaccines by plasmids coexpressing hepatitis B surface antigen and
interleukin-2. J Virol, 1997, 71(1): 169–178

5 Warrens AN, Jones MD, Lechler RI. Splicing by overlap extension by PCR
using asymmetric amplification: an improved technique for the generation of
hybrid proteins of immunological interest. Gene, 1997, 186(1): 29–35

6 Zhao P, Zhao LJ, Cao J, Hong HY, Qi ZT. Enhancement of immune responses
of hepatitis B virus core DNA vaccine by a signal peptide and a universal
helper T lymphocyte epitope. Acta Biochim Biophys Sin, 2002,34 (3): 341–346

7 Sanchez CM, Izeta A, Sanchez-Morgado JM, Alonso S, Sola I, Balasch M,
Plana-Duran J et al. Targeted recombination demonstrates that the spike
gene of transmissible gastroenteritis coronavirus is a determinant of its enteric
tropism and virulence. J Virol, 1999,73(9): 7607–7618

8 Daniel C, Talbot PJ. Protection from lethal coronavirus infection by
affinitypurified spike glycoprotein of murine hepatitis virus, strain A59.
Virology, 1990,174(1): 87–94

9 Yoo DW, Parker MD, Song J, Cox GJ, Deregt D, Babiuk LA. Structural
analysis of the conformational domains involved in neutralization of bovine
coronavirus using deletion mutants of the spike glycoprotein S1 subunit
expressed by recombinant baculoviruses. Virology, 1991, 183(1): 91–98

10 Corapi WV, Olsen CW, Scott FW. Monoclonal antibody analysis of
neutralization and antibody-dependent enhancement of feline infectious
peritonitis virus. J Virol, 1992, 66(11): 6695–6705

11 Kubo H, Yamada YK, Taguchi F. Localization of neutralizing epitopes and
the receptor-binding site within the amino-terminal 330 amino acids of the
murine coronavirus spike protein. J Virol, 1994, 68(9): 5403–5410

12 Delmas B, Rasschaert D, Godet M, Gelfi J, Laude H. Four major antigenic
sites of the coronavirus transmissible gastroenteritis virus are located on the
amino-terminal half of spike glycoprotein S. J Gen Virol, 1990, 71 ( Pt 6):
1313–1323

13 Whitton JL, Rodriguez F, Zhang J, Hassett DE. DNA immunization:
Mechanistic studies. Vaccine, 1999, 17(13–14): 1612–1619

Received: September 2,
2003 Accepted: September 24, 2003
This work was supported by grants from National Key Basic Research and
Development Program (No.2003CB514129) and SMMU Special Funds for SARS Prophylaxis
and Treatment (No.2003SARS03)

*Corresponding author: Tel, 86-21-25070312; Fax, 86-21-25070312; E-mail, [email protected]