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The spike protein of SARS-CoV - A target for vaccine and therapeutic development

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Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease caused by a novel coronavirus, SARS-coronavirus (SARS-CoV). The SARS-CoV spike (S) protein is composed of two subunits; the S1 subunit contains a receptor-binding domain that engages with the host cell receptor angiotensin-converting enzyme 2 and the S2 subunit mediates fusion between the viral and host cell membranes. The S protein plays key parts in the induction of neutralizing-antibody and T-cell responses, as well as protective immunity, during infection with SARS-CoV. In this Review, we highlight recent advances in the development of vaccines and therapeutics based on the S protein.
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The spike protein of SARS-CoV — a target for vaccine and
therapeutic development
Lanying Du*, Yuxian He*, Yusen Zhou, Shuwen Liu§, Bo-Jian Zheng, and Shibo Jiang*
*Lindsley F. Kimball Research Institute, New York Blood Center, 310 East 67th Street, New York,
New York 10065, USA.
State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and
Epidemiology, Beijing 100071, China.
§School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515, China.
Department of Microbiology, University of Hong Kong, Pokfulam, Hong Kong SAR, China.
Abstract
Severe acute respiratory syndrome (SARS) is a newly emerging infectious disease caused by a novel
coronavirus, SARS-coronavirus (SARS-CoV). The SARS-CoV spike (S) protein is composed of two
subunits; the S1 subunit contains a receptor-binding domain that engages with the host cell receptor
angiotensin-converting enzyme 2 and the S2 subunit mediates fusion between the viral and host cell
membranes. The S protein plays key parts in the induction of neutralizing-antibody and T-cell
responses, as well as protective immunity, during infection with SARS-CoV. In this Review, we
highlight recent advances in the development of vaccines and therapeutics based on the S protein.
Severe acute respiratory syndrome (SARS) was the first new infectious disease identified in
the twenty-first century. This acute, and often severe, respiratory illness originated in the
Guangdong province of China in November 2002 (REF. 1). A global effort coordinated by
WHO led to the identification, in April 2003, of a new coronavirus, SARS-coronavirus (SARS-
CoV), as the agent that caused the outbreak2.
SARS-CoV is an enveloped, single and positive-stranded RNA virus2. Its genome RNA
encodes a non-structural replicase polyprotein and structural proteins, including spike (S),
envelope (E), membrane (M) and nucleocapsid (N) proteins3-5. SARS-CoV, a zoonotic virus,
resides in hosts that form its natural reservoir, such as bats, but can also infect intermediate
hosts, such as small animals (for example, palm civets), before being transmitted to
humans6-8. SARS-CoV can infect and replicate in several cell types in the human body and
causes serious pathological changes (BOX 1, FIG 1). A further understanding of the life cycle
© 2009 Macmillan Publishers Limited. All rights reserved
Correspondence to S.J. e-mail: sjiang@nybloodcenter.org.
DATABASES
Entrez Genome: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=genome
HIV-1 | MHV | SARS-CoV
UniProtKB: http://www.uniprot.org
ACE2 | E | M | N | S
FURTHER INFORMATION
Shibo Jiang's homepage: http://www.nybc.org/research/research/index.do?sid0=7&sid1=32&page_id=31&content_id=91
WHO update 7: http://www.who.int/csr/don/2004_05_18a/en/index.html
WHO update 49: http://www.who.int/csr/sars/archive/2003_05_07a/en/
NIH Public Access
Author Manuscript
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Published in final edited form as:
Nat Rev Microbiol. 2009 March ; 7(3): 226–236. doi:10.1038/nrmicro2090.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
and pathogenesis of SARS-CoV will help us to develop vaccines and therapeutics to prevent
and treat SARS-CoV and SARS-like coronavirus (SL-CoV) infections in the future.
After its first occurrence, SARS rapidly spread around the world along international air-travel
routes, reaching all five continents and 29 countries, resulting in 8,098 cases and 774 deaths
by 23 September 2003 (REF. 9). The overall fatality of SARS is about 10% in the general
population, but >50% in patients aged 65 years and older (WHO update 49; see Further
information). The global outbreak of SARS was brought under control in July 2003 by effective
quarantine, patient-isolation and travel restrictions. Four sporadic SARS cases caused by
different SARS-CoV isolates than those that predominated in the 2002-2003 outbreak were
reported in late 2003 and early 2004 (REFS 10-12). The most recent epidemic of SARS
occurred in Beijing and Anhui in China in April 2004 and originated from laboratory
contamination (WHO update 7; see Further information). Since then, no new case of SARS
has been reported, possibly because of continued global vigilance and surveillance and
laboratory bio-safety practices, as well as the euthanizing or quarantining of animals that may
have been exposed to SARS-CoV13,14. Although the outbreaks of SARS seem to be over,
SARS is still a safety concern because of the possible reintroduction of a SL-CoV into humans
and the risk of an escape of SARS-CoV from laboratories15,16.
Infection with SARS-CoV can trigger a series of humoral and cellular immune responses.
Specific antibodies against SARS-CoV (immunoglobulin G (IgG) and IgM) were detectable
approximately 2 weeks post-infection, reaching a peak 60 days post-infection and remaining
at high levels until 180 days post-infection (REF. 17). High titres of neutralizing antibodies
and SARS-CoV-specific cytotoxic T lymphocyte responses were detected in patients who had
recovered from SARS18,19, and the levels of the responses correlated well with the disease
outcome20. This suggests that both humoral and cellular immune responses are crucial for the
clearance of infection by SARS-CoV.
Zoonotic virus
A virus that normally exists in vertebrate animals, but can also be transmitted
to humans and can cause disease in both animals and humans.
Box 1
Pathology of SARS and the life cycle of SARS-CoV infection
Severe acute respiratory syndrome-coronavirus (SARS-CoV) spreads primarily through
droplets (respiratory secretions) and close person-to-person contact. After the virus enters
into the body, it binds to primary target cells that express abundant virus receptor, the
angiotensin-converting enzyme 2 (ACE2), including pneumocytes and enterocytes in the
respiratory system. The virus enters and replicates in these cells. The matured virions are
then released to infect new target cells121 (FIG. 1). SARS-CoV can also infect mucosal
cells of intestines, tubular epithelial cells of kidneys, epithelial cells of renal tubules,
cerebral neurons and immune cells122,123. Infectious viral particles in patients with SARS
can be excreted through respiratory secretions, stool, urine and sweat. SARS-CoV infection
damages lung tissues owing to elevated levels of production and activation of
proinflammatory chemokines and cytokines124, resulting in atypical pneumonia with rapid
respiratory deterioration and failure.
Neutralizing antibodies and/or T-cell immune responses can be raised directly against several
SARS-CoV proteins21-23, but mainly target the S protein20,24-26, suggesting that S protein-
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induced specific immune responses play important parts in the fight against SARS-CoV
infection18. SARS-CoV S protein also has a key role in the ability of SARS-CoV to overcome
the species barrier, as adaptive evolution of S protein can contribute to the animal-to-human
transmission of SARS-CoV27. Because the S protein of SARS-CoV is involved in receptor
recognition, as well as virus attachment and entry, it represents one of the most important
targets for the development of SARS vaccines and therapeutics.
Structure of the SARS-CoV S protein
The spikes of SARS-CoV are composed of trimers of S protein, which belongs to a group of
class I viral fusion glycoproteins that also includes HIV glyco-protein 160 (Env), influenza
haemagglutinin (HA), paramyxovirus F and Ebola virus glycoprotein28. The SARS-CoV S
protein encodes a surface glycoprotein precursor that is predicted to be 1,255 amino acids in
length, and the amino terminus and most of the protein is predicted to be on the outside of the
cell surface or the virus particles3. The predicted S protein consists of a signal peptide (amino
acids 1–12) located at the N terminus, an extracellular domain (amino acids 13-1,195), a
transmembrane domain (amino acids 1,196–1,215) and an intracellular domain (amino acids
1,216–-1,255)29-32 (FIG. 2a). Similarly to other coronaviruses, the S protein of SARS-CoV
can be cleaved into the S1 and S2 subunits by proteases, such as trypsin33, factor Xa34 and
cathepsin L35. The trypsin cleavage site occurs at R667–S668 (REF. 36), whereas cathepsin
L cleavage is mapped to T678-M679 in the S protein35. Cathepsin L cleaves the S protein of
SARS-CoV upstream of, rather than adjacent to, the fusion peptide, and the cleavage is required
for activation of the membrane fusion domain of the S protein following entry into target
cells35.
Angiotensin-converting enzyme 2 (ACE2) has been identified as the receptor of SARS-
CoV37. A fragment that is located in the S1 subunit and spans amino acids 318–510 is the
minimal receptor-binding domain (RBD)30,38,39. Crystallographic studies have shown the
structure of RBD complexed with its receptor ACE2 (REFS 29,40). During the interaction of
RBD with the receptor, RBD presents a concave surface for the N terminus of the receptor
peptidase, on which amino acids 445–460 anchor the entire receptor-binding loop of the RBD
core (FIG. 2b). This loop (amino acids 424– 494 of the RBD), which makes complete contact
with the receptor ACE2, was referred to as receptor-binding motif (RBM) (FIG. 2a). The RBM
region is tyrosine rich. Among the 14 residues of RBM that are in direct contact with ACE2,
six are tyrosine, representing both the hydroxyl group and hydrophobic ring. The RBD region
also contains multiple cysteine residues that are linked by disulphide bonds29 (FIG. 2c). Two
residues in particular, those at positions 479 and 487, determine SARS disease progression and
SARS-CoV tropism (host range)41,42. Any residue changes in these two positions might
therefore enhance animal-to-human or human-to-human transmission29.
Human and animal SARS-CoVs depend on ACE2 for cell entry. Animal SARS-CoV could
evolve to infect humans by a series of transmission events between animals and humans. For
example, a chimeric recombinant SARS-CoV that bears the S protein of civet SARS-CoV
(icSZ16-S) can adapt to human airway epithelial cells and displays enhanced affinity for human
ACE2 (REF. 43). Changes of only a few residues in the RBD of the civet SARS-CoV S protein,
which is responsible for binding with the peptidase domain of ACE2, result in enhanced human
ACE2-binding affinity of SARS-CoVs from animals, including civets, mice and rats,
facilitating efficient cross-species infections29. However, the SL-CoV from bats does not
infect ACE2-expressing cells7,8, suggesting that, unlike SARS-CoVs from human and civets,
the SL-CoV from bats does not use ACE2 as a cellular receptor. Thus, the SL-CoV from bats
might be the precursor of animal SARS-CoVs, which may act as the intermediates for animal-
to-human transmission.
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Functions of the SARS-CoV S protein
SARS-CoV S protein has pivotal roles in viral infection and pathogenesis44,45. S1 recognizes
and binds to host receptors, and subsequent conformational changes in S2 facilitate fusion
between the viral envelope and the host cell membrane30,33.
Receptor binding
The RBD in S1 is responsible for virus binding to host cell receptors30,37,39. ACE2 from
SARS-CoV-permissive Vero E6 cells efficiently binds S1, and its soluble form blocks S1 from
associating with Vero E6 cells. In addition, SARS-CoV replicates efficiently in ACE2-
transfected cells, and anti-ACE2 antibodies block virus entry and replication in Vero E6 cells.
This shows that ACE2 is a functional receptor for SARS-CoV37,46,47. A total of 18 residues
of ACE2 keep contact with 14 amino acids in the RBD of SARS-CoV S protein29. K341 of
ACE2 and R453 of the RBD are important for the complex formation48. N479 and T487 of
the RBD are important for the high-affinity association of S protein with ACE2 (REF. 42). A
point mutation at R441 or D454 of the RBD disrupts the antigenic structure and binding activity
of RBD to ACE2 (REFS 30,49).
Adaptive evolution
A process that enables living organisms to cope with environmental stresses
and pressures for survival in a new host. for example, under positive selective
pressure, civet SArS-CoV can evolve and subsequently adapt to the human
host.
SARS-CoV can also bind to host cells through alternative receptors, such as DC-SIGN
(dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin) and/or L-
SIGN (liver/lymph node-SIGN)50,51. Seven asparagine-linked glycosylation sites in the S
protein, including residues at positions 109, 118, 119, 158, 227, 589 and 699, are crucial for
DC-SIGN-or L-SIGN-mediated virus entry. These residues differ from those of the ACE2-
binding domain located at amino acids 318–510 (REF. 52). This would suggest that S protein
can also use DC-SIGN or L-SIGN as a receptor, independently of ACE2. However, the actual
function of DC-SIGN and L-SIGN needs to be further verified.
Viral fusion
The fusion process that is mediated by S protein of SARS-CoV is similar to that mediated by
class I viral fusion proteins of other viruses, such as HIV-1 and murine hepatitis virus
(MHV)53,54, but may occur in the acidic environment of the endosomes, rather than on the
cell surface. S2 contains heptad repeat 1 (HR1) and HR2 domains, which play an important
part in SARS-CoV fusion with target cells. Binding of the RBD of S1 to the receptor ACE2
triggers a conformational change of the S2 from a pre-fusion form to a post-fusion form,
resulting in insertion of the putative fusion peptide (amino acids 770–788)31 into the target
cell membrane and association of HR1 and HR2 domains to form a six-helix bundle fusion
core structure. This brings the viral envelope and target cell membrane into close proximity
for fusion. The crystal structure of the SARS-CoV fusion core is described in detail in REF.
55 (FIG. 3). Similarly to the S protein of MHV, but not gp41 of HIV-1, SARS-CoV S protein
has a longer HR1 region than HR2 region. The six-helix bundle fusion core has a rod-shaped
structure with a length of 70 Å and a diameter of 28 Å. Three HR1 helices form a parallel
trimeric coiled-coil that is surrounded by three HR2 helices in an oblique, antiparallel
manner55 (FIG. 3). A synthetic peptide derived from the HR2 region could interact with an
HR1 peptide to form a stable six-helix bundle and inhibit SARS-CoV infection in a dose-
dependent manner53. Consequently, both the HR1 and HR2 regions in the S2 domains are
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expected to participate in the viral fusion and entry processes and will serve as attractive targets
for the development of anti-SARS-CoV therapeutics and vaccines.
Vaccines based on the SARS-CoV S protein
The roles of S protein in receptor binding and membrane fusion indicate that vaccines based
on the S protein could induce antibodies to block virus binding and fusion or neutralize virus
infection. Among all structural proteins of SARS-CoV, S protein is the main antigenic
component that is responsible for inducing host immune responses, neutralizing antibodies
and/or protective immunity against virus infection. S protein has therefore been selected as an
important target for vaccine and anti-viral development. A comparison of these approaches is
provided in TABLE 1.
It has been reported that antibodies raised to amino acids 485–625 in S1 or 1,029–1,192 in S2
neutralize infection by SARS-CoV strains (for example, Tor2 and Sin2774) in Vero E6
cells56,57. Vaccination of African green monkeys with an attenuated parainfluenza virus that
encodes the full-length S protein of SARS-CoV urbani strain resulted in the production of S
protein-specific neutralizing antibodies, which protected vaccinated monkeys from subsequent
homologous SARS-CoV challenge58, suggesting that immunization with the S protein of
SARS-CoV is highly effective in the prevention of SARS.
Vaccines based on the full-length S protein
Several vaccines that are based on the full-length S protein of SARS-CoV have been reported.
Yang et al.59 showed that a DNA vaccine encoding the full-length S protein SARS-CoV urbani
strain could induce both T-cell and neutralizing-antibody responses, as well as protective
immunity, in a mouse model. Other groups have also shown that vaccination of mice or
monkeys with highly attenuated modified vaccinia virus Ankara (MVA), which encodes the
full-length S protein of the SARS-CoV urbani strain or HKU39849 strain, elicited S-specific
neutralizing antibodies and protective immunity, as evidenced by decreased virus titres in the
respiratory tracts of animals after homologous SARS-CoV challenge60,61. Passive transfer of
murine serum to naive mice also protected these mice from the challenge of homologous
SARS-CoV60,61. In addition, vaccination of mice or hamsters with a full-length S protein
trimer protected these animals from infection by homologous SARS-CoV (HKU39849 strain)
62. Furthermore, a recombinant baculovirus-expressed full-length S protein of the urbani strain
and its trimer could induce sufficient neutralizing antibodies against human and palm civet
SARS pseudoviruses that bore S proteins of homologous and heterologous SARS-CoV variants
(for example, Tor2, GD03T13 and SZ3 strains) in vaccinated mice63. These reports suggest
that the full-length S protein is highly immunogenic and induces protection against SARS-
CoV challenge and that neutralizing antibodies alone may be able to suppress virus
proliferation, further justifying the rationale that vaccines can be developed based on the S
protein.
Although full-length S protein-based SARS vaccines can induce neutralizing antibody
responses against SARS-CoV infection, they may also induce harmful immune responses that
cause liver damage of the vaccinated animals or enhanced infection after challenge with
homologous SARS-CoV64,65, raising concerns about the safety and ultimate protective
efficacy of vaccines that contain the full-length SARS-CoV S protein.
SARS pseudovirus
A synthetic virus that bears the SArS-CoV S protein and contains an Env-
defective, luciferase-expressing genome of a retrovirus (for example, HiV),
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and can infect but does not replicate in cells that express receptors for SArS-
CoV.
Vaccines based on the RBD
Previous studies have shown that the RBDs of the S proteins of the coronaviruses MHV and
HCoV-229E contain major antigenic determinants that can induce neutralizing antibodies66,
67. We have discovered that the recombinant RBD (rRBD) antigen of SARS-CoV is highly
reactive with the neutralizing antibodies against SARS pseudoviruses that bear S proteins of
SARS-CoV (Tor2 strain) in the antisera of mice and rabbits immunized with inactivated SARS-
CoV68. The RBD strongly reacts with the antisera from patients with SARS in the convalescent
phase, and depletion of the RBD-specific antibodies from patients with SARS results in
significant elimination of the neu ralizing activity69. Chen et al.61 have also shown that most
neutralizing antibodies of antisera of mice, rabbits and monkeys induced by a live-attenuated
MVA virus that expressed the full-length S protein could be absorbed and removed by rRBD.
using a fusion protein that contained the RBD linked to human IgG1 Fc fragment (designated
RBD-Fc) as an immunogen, we have successfully induced highly potent neutralizing
antibodies against SARS-CoV BJ01 strain in immunized rabbits with neutralizing titres greater
than 1:10,000 (REF. 70). The antibodies effectively cross-neutralize infection by SARS
pseudoviruses that bear S proteins of both homologous and heterologous SARS-CoV isolates,
including the representative strains of human 2002–2003 and 2003–2004 SARS-CoV (Tor2
and GD03, respectively) and palm civet SARS-CoV (SZ3)71. Immunization of mice with
RBD-Fc induces long-term protective immunity against challenge with homologous SARS-
CoV BJ01 strain70,72. Administration of an adeno-associated virus (AAV)-based vaccine that
contains RBD (RBD-rAAV) by intramuscular and mucosal pathways elicits sufficient
neutralizing antibodies to inhibit homologous SARS-CoV (GZ50) challenge in the established
mouse model, and the immune responses can be enhanced by priming with RBD-rAAV and
boosting with RBD-specific peptides73-75.
The SARS-CoV S protein can also induce CD8+ T-cell responses. One H-2(b)- and one H-2
(d)-restricted T-cell epitope are mapped to RBD (S436–S443 and S366–S374, respectively)
24. Immunization of mice with a RBD-based subunit vaccine (S318–S510) elicits both
antibody and cellular immune responses against SARS-CoV26. The RBD of S protein contains
multiple conformation-dependent epitopes and is the main domain that induces neutralizing
antibody and T-cell immune responses against SARS-CoV infection76,77, making it an
important target for vaccine development. The approaches for developing RBD-based vaccines
against SARS-CoV have provided useful information for designing vaccines against other
viruses with class I fusion proteins, as these proteins also contain RBDs in their S proteins.
It should be noted that the efficacy of these vaccine candidates is mainly tested in young-mouse
and primate animal models. These models are usually less robust, providing virus replication
but lacking clinical symptoms and diseases. It is necessary, therefore, to develop more-robust
animal models of human diseases for evaluation of vaccine efficacy. Baric and colleagues78,
79 have recently reported several lethal SARS-CoV challenge models in BALB/c mice that
recapitulated the age-related SARS disease by using recombinant SARS-CoV that bore the S
protein of early human and zoonotic strains (GZ02 and HC/SZ/61/03, respectively). They also
developed another pathogenic model for young mice after 15 passages of the urbani isolate in
BALB/c mice, which resulted in a lethal virus, MA15, that replicates to high titres in the lungs
of mice, causing clinical disease of SARS78,79. Other reports80,81 list examples for the use
of senescent mouse models for vaccine evaluation. One candidate vaccine, Venezuelan equine
encephalitis virus replicon particles, that expressed the urbani SARS-CoV S protein partially
protected the aged mice from challenge with a recombinant heterologous SARS-CoV that bore
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epidemic and zoonotic S proteins (icGDO3-S), providing a model to mimic the age-related
susceptibility observed in the elder population80. The animal models discussed above can be
used as valuable tools to evaluate the efficacy of SARS vaccines.
S protein-based therapeutics
Peptides that interrupt the RBD–ACE2 interaction
It has been shown that rRBD blocks S protein-mediated entry of lentivirus pseudotypes into
ACE2-expressing 293T cells with a half maximal inhibitory concentration (IC50) of less than
10 nM30. Similarly, a peptide that overlaps the RBD sequence (amino acids 471–503) blocks
the RBD–ACE2 interaction, inhibiting SARS-CoV entry into Vero cells with an IC50 of
approximately 40 μM82. A polypeptide that contains two RBD-binding motifs of ACE2 (amino
acids 22–44 and 351–357) linked by a glycine exhibits high potent inhibitory activity on SARS
pseudovirus infection in ACE2-expressing HeLa cells with an IC50 of 100 nM83. These
findings suggest that peptides derived from both RBD and ACE2 that block RBD–ACE2
binding could be developed as novel therapeutics against SARS-CoV infection. However, the
in vivo inhibitory activity of these peptides should be evaluated in animal models before
considering further development.
Peptides that interfere with the cleavage of S protein
Cleavage of the S protein trimer is an important event in infection, making the potential
cleavage site between S1 and S2 domains another target for development of anti-SARS-CoV
agents. Synthetic peptides, including P6 (amino acids 598–617) and P8 (amino acids 737–756),
both of which are close to the S1–S2 connection and cleavage site, exhibit potent inhibitory
activity against the GZ50 strain of SARS-CoV infection in fetal rhesus kidney (FRhK4) cells,
and have IC90 values of approximately 100 and 25 μM84. This suggests that binding of the
peptides to the S protein interferes with the cleavage of S1 and S2, inhibiting the production
of functional S1 and S2 subunits and subsequent fusion of the viral envelope and the host cell
membrane. Again, the in vivo antiviral efficacy of these peptides should be tested in animal
models.
Peptides that block the HR1–HR2 interaction from forming a fusion-active core
In the early 1990s, Jiang et al.85 and Wild et al.86 discovered the highly potent anti-HIV
peptides derived from the HIV-1 gp41 HR2 region. One of the HR2 peptides, T20 (enfuvirtide),
was approved by the uS Food and Drug Administration for the treatment of patients with HIV
or AIDS, especially those who have failed to respond to the current antiretroviral drugs. These
HR2 peptides could interact with the viral gp41 HR1 region at fusion-intermediate
conformation and block six-helix bundle formation, resulting in the inhibition of HIV fusion
at the nanomolar level87,88. Because the SARS-CoV S protein S2 domain also contains HR1
and HR2 sequences, we anticipated that peptides derived from the HR2 region of the SARS-
CoV S protein S2 domain would also have antiviral activity against SARS-CoV. We designed
and synthesized several peptides that overlapped the HR2 sequence and found that one of these,
designated CP-1, could interact with an HR1 peptide to form a stable six-helix bundle and
inhibited infection by SARS-CoV WHu strain in Vero E6 cells with an IC50 of approximately
20 μM53. Later, several other research groups also identified anti-SARS-CoV peptides from
the S2 domain HR2 region that had viral fusion inhibitory activity at the micromolar level84,
89,90. An NMR study has shown that in the pre-fusion intermediate state, the HR2 region
forms a symmetric coiled-coil trimer, which has not been observed for other class I viral fusion
proteins. The poor antiviral activity of anti-SARS-CoV peptides, compared with the anti-HIV
peptides, could be attributed to the tendency of the SARS-CoV S protein HR2 region to form
the trimeric coiled-coil. Replacement of the key residues in the HR2 peptide to reduce its ability
to form the trimer, but increase its affinity of binding with the HR1 region, to form the six-
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helix bundle could lead to improvement of its antiviral efficacy. The peptidic antiviral drugs
for SARS and other emerging infectious diseases with short incubation periods could have
more advantages than the anti-HIV drug enfuvirtide, as enfuvirtide must be injected twice per
day for the patient's lifetime. This results in an intolerable injection-site reaction and a high
cost to patients, whereas a few injections of the peptidic drugs against SARS-CoV in the early
stage of the acute phase could be enough to save patients' lives. One of the disadvantages of
using HR2-based peptide inhibitors is the potential selection of escape mutants with altered
host-range phenotypes91.
mAbs that target the S protein
Neutralizing mouse mAbs
Using rRBD and inactivated SARS-CoV as immunogens, we have successfully generated a
panel of highly potent neutralizing mouse monoclonal antibodies (mAbs) that could block
receptor binding and cross-neutralize infection by pseudoviruses that bore S proteins of the
representative human SARS-CoV strains that caused the 2002–2003 and 2003–2004 outbreaks
(Tor2 and GD03T13) and palm civet SARS-CoV (SZ3)63,69,71,92. Mouse mAbs that target
other fragments of the SARS-CoV S protein (for example, amino acids 1,143–1,157) could
also effectively inhibit SARS-CoV infection56,93. These neutralizing mouse mAbs can be
administered to patients with SARS for early and urgent treatment of SARS-CoV infection9,
but cannot be repeatedly used owing to the risk of a human–anti-mouse antibody response.
Such a response could rapidly clear the murine antibody from the blood, thus preventing the
mouse antibodies from producing the desired therapeutic effect and causing the patient to have
an allergic reaction94. Some antibodies against trimeric S protein have the potential to mediate
FcγRII-dependent entry into B cells in vitro and thereby cause antibody-dependent
enhancement62.
Neutralizing human mAbs
A range of neutralizing human mAbs have been generated from B cells of patients infected
with SARS-CoV95,96 or from human immunoglobulin transgenic mice immunized with full-
length SARS-CoV S protein97-99. These S-specific mAbs, such as 80R and CR3014, could
block SARS-CoV S protein binding with the ACE2 receptor and neutralize infection by human
SARS-CoV strains Tor2 and HKu39849 and/or palm civet SARS-CoV strain SZ3 (REFS 32,
100,101). mAbs m396 and S230.15 neutralize human SARS-CoV and/or pseudoviruses that
bear S proteins of human SARS-CoV strains (urbani, Tor2 and GD03) and palm civet SARS-
CoV strains (SZ3 and SZ16)97. Human anti-S mAbs S109.8, S215.17, S227.14 and S230.15
cross-neutralize infection by a panel of recombinant SARS-CoV strains bearing variant S
proteins that are representative of human strains (GZ02, CuHK-W1 and urbani) and zoonotic
strains found in palm civet (HC/SZ/61/03) and raccoon dog (A031G)99. Some human mAbs,
such as 80R32, m396 (REF. 97), 201 and 68 (REF. 102), exhibit potent antiviral effects against
homologous SARS-CoV challenge in young-mouse replication models. However, others, such
as S109.8, S227.14 and S230.15 (REF. 99), could induce broad protection against lethal
homologous and heterologous SARS-CoV challenge in both young- and aged-mouse models,
providing a strategy to minimize the emergence of mAb escape mutants.
Antiviral compounds and small molecules
Inhibitors of cathepsin L
Cathepsin L activates S protein-mediated membrane fusion by facilitating receptor-dependent
and acid-dependent conformational changes in the S2 domain. This occurs in endosomes in
which a low pH allows for optimal proteolytic activity35,103,104. Thus, cathepsin L inhibitors,
such as E63c, E64d and MDL28170, can block viral entry or inhibit in vitro infection of SARS-
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CoV or SARS pseudoviruses103,105,106. These findings suggest that compounds which
inhibit the activity of cathepsin L protease could be developed as therapeutics for the inhibition
of SARS-CoV infection, but their in vivo antiviral activity should be further tested in animal
models.
Other compounds and small molecules that target the S protein
Several other compounds and small molecules that target the S protein have been reported. For
example, amiodarone blocks the in vitro spread of SARS-CoV by inhibiting virus infection at
a post-endosomal level107. Yi et al.108 identified two small molecules, tetra-o-galloyl-beta-
d-glucose (TGG) and luteolin, which have inhibitory activity, that blocked SARS-CoV or
SARS pseudovirus entry into Vero E6 cells. Kao et al.109 identified 18 small molecules that
targeted S protein–ACE2-mediated viral entry. One of these, VE607, exhibits potent inhibitory
activity on SARS pseudovirus entry into ACE2-expressing 293T cells. These reports suggest
that the small molecules discussed above can function as effective antiviral inhibitors against
S protein-mediated viral entry. However, further studies are needed to determine the in vivo
efficacy of these antiviral agents in animal models and select optimal formulations to deliver
effective concentrations of the drugs to the target tissues.
Gene targeting with small interfering RNA
RNA interference induced by a small interfering RNA (siRNA) has been successfully used
recently as a specific and efficient method for silencing specific viral genes, interrupting protein
synthesis and suppressing virus replication110,111. It has been demonstrated that siRNAs
directed against S sequences of SARS-CoV inhibited SARS-CoV replication in virus-infected
Vero E6 cells112. Several research groups113-117 reported that S-specific siRNAs could
reduce S protein expression by blocking S mRNA accumulation or reducing the number of
copies of the viral genome in FRhK4 cells, indicating that S gene expression in SARS-CoV-
infected cells can be effectively silenced by S-specific siRNAs. The in vivo study used a rhesus
macaque model to indicate that siRNA duplexes (siSC2–5) that targeted the S protein and
ORF1b of SARS-CoV could suppress SARS-like symptoms, inhibit virus replication in the
monkey respiratory tract and protect lungs from acute damage118. The findings discussed
above reveal the function of siRNA in the inhibition of SARS-CoV infection, replication and/
or interruption of S gene expression, raising hopes for the development of effective, novel
antiviral agents against SARS-CoV.
Conclusions and prospects
In summary, the S protein of SARS-CoV possesses some unique features that are different
from other type I glycoproteins. Many class I fusion proteins, such as HIV Env, influenza HA
and MHV S, are post-translationally cleaved at the N-proximal region of the fusion peptide by
specific proteases into the surface and transmembrane subunits. By contrast, cleavage of the
SARS-CoV S protein may occur far upstream of the predicted fusion peptide (FIG. 2a). unlike
the S proteins of coronaviruses cleaved by furin-like proteases, the S protein of SARS-CoV
can be cleaved by cathepsin L at position T678 or by trypsin at R667. In contrast to the entrance
mechanism of HIV, SARS-CoV can enter cells from an acidic environment of the
endosome119. Nevertheless, SARS-CoV can also enter the target cell surface, which is
mediated by proteases on the cell surface through a non-endosomal-dependent pathway120.
The interaction between the SARS-CoV S protein and ACE2 is essential for SARS-CoV entry.
The natural evolution of the epidemic SARS-CoV strains probably occurred over a long period,
through the repeated transmission of viruses from animals to humans and from humans to
animals, resulting in mutations in both the SARS-CoV S protein and ACE2, so that human and
animal SARS-CoVs could enter cells that bore human or animal ACE2. Further understanding
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of the tropism of the virus and the mechanism of the SARS-CoV S protein in receptor binding
and entry is therefore important for the development of anti-SARS-CoV therapeutics and
vaccines.
As the major component for the development of vaccines against SARS, S protein, and
especially the RBD, has been shown to induce highly potent neutralizing antibodies to block
virus binding and membrane fusion and/or protective immunity against virus infection. Owing
to the absence of human SARS cases in recent years, future SARS epidemics will probably
originate from zoonotic transmission. SARS vaccines should therefore protect against not only
human SARS-CoV strains, including those from early, middle and late phases of the epidemic,
but also those of zoonotic origin. Although current vaccine candidates effectively neutralize
SARS-CoV in young-animal replication models without clinical symptoms, they may not
protect an elderly population against SARS-CoV infection. Thus, it is essential to test the
vaccine candidates in robust lethal-challenge models using aged animals. Future vaccines
should effectively protect both the young and the elderly populations from infection by either
human or animal SARS-CoV strains that may cause future SARS epidemics.
Peptides and non-peptidic small molecules that target the functional domain of the SARS-CoV
S protein, particularly the RBD in the S1 subunit and the HR2 region in the S2 subunit, are
mainly virus entry inhibitors and can be further developed as anti-SARS-CoV therapeutics. To
develop these molecules as effective and safe antiviral drugs for the treatment of SARS, the
urgent task is to improve their potency. Mouse and human mAbs that target the S protein of
SARS-CoV have shown potent inhibition and/or neutralization to homologous and
heterologous SARS-CoV isolates and can be further developed as immunotherapeutics or
passive immunization agents for therapy and prophylaxis of SARS-CoV infection. Future
studies are needed to test the in vivo efficacy of these antiviral agents in animal models.
Amiodarone
A medication commonly used to treat patients with irregular heart beats or
cardiac arrhythmias, including ventricular tachycardia and ventricular
fibrillation.
Luteolin
A flavonoid extracted from Chinese herbs, including Prunella vulgaris and
Saussurea lappa Clarks.
Overall, the feasibility of using peptides and small molecules as anti-SARS therapeutics is
partially limited by their low antiviral potency. Furthermore, the possibility of enhancing viral
entry might restrict mAbs as immunotherapeutics for long-term use. It is likely, however, that
S protein-based vaccines will bear fruit in the near future, as they have been proven to induce
long-term and potent neutralizing antibodies and/or protective immunity against SARS-CoV.
But the in vivo efficacy of these vaccine candidates in elderly and lethal-challenge models, and
their protection against zoonotic virus infection, should be determined before a clinical study
is initiated. To take these factors into full consideration, a combination of different strategies
with multiple vaccines and antiviral therapeutics may be needed to induce broad and cross
protection against various virus strains, especially isolates that have mutated quickly. Early
clinical studies that were based on such strategies have been carried out, but it is difficult to
push the clinical trials of these candidate vaccines and therapeutics forwards owing to a lack
of SARS-CoV-infected subjects and insufficient financial support. Thus, most big
pharmaceutical companies have no interest in developing SARS vaccines and therapeutics
because of the concern of profitability. However, studies on SARS will provide important
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information for designing novel strategies for prophylaxis and therapies of other newly
emerging infections caused by enveloped viruses with class I fusion proteins.
Acknowledgements
We thank all three anonymous reviewers for their constructive comments and informative suggestions. Our research
was supported by the National Institutes of Health (NIH) of the United States (RO1 AI68002), by the Research Fund
for the Control of Infectious Diseases, the Food and Health Bureau of the Hong Kong SAR Government, and by the
National 973 Basic Research Program of China (2005CB523001).
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Figure 1. The life cycle of SARS-CoV in host cells
Severe acute respiratory syndrome-coronavirus (SARS-CoV) enters target cells through an
endosomal pathway113,121,125-127. S protein first binds to the cellular receptor angiotensin-
converting enzyme 2 (ACE2)129, and the ACE2–virus complex is then translocated to
endosomes, where S protein is cleaved by the endosomal acid proteases (cathepsin L)105 to
activate its fusion activity. The viral genome is released and translated into viral replicase
polyproteins pp1a and 1ab, which are then cleaved into small products by viral proteinases.
Subgenomic negative-strand templates are synthesized from discontinuous transcription on the
plus-strand genome and serve as templates for mRNA synthesis. The full-length negative-
strand template is made as a template for genomic RNA. Viral nucleocapsids are assembled
from genomic RNA and N protein in the cytoplasm, followed by budding into the lumen of
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the ERGIC (endoplasmic reticulum (ER)–Golgi intermediate compartment)128. Virions are
then released from the cell through exocytosis.
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Figure 2. SARS-CoV S protein structure and its complex with the receptor AcE2
a | Schematic of the S protein 29–32. The residue numbers of each region represent their
positions in the S protein of severe acute respiratory syndrome-coronavirus (SARS-CoV). b|
Crystal structures of the RBD complexed with the receptor. RBD (the core structure is cyan
and the loop RBM is red) interacts with the receptor angiotensin-converting enzyme 2 (ACE2;
green). A five-stranded anti-parallel β-sheet (β1–β4 and β7) that connects with three short α-
helices (αA-αC) constitutes the core, whereas a two-stranded β-sheet (β5 and β6) forms the
loop. N* and C* represent the amino and carboxyl termini of the RBD, respectively. c | The
RBD tyrosine (magenta) and cysteine (yellow) residue distribution29. The asterisks represent
six ACE2-contacting tyrosines on the RBD, and two disulphide bonds are shown to link C323
to C348 and C467 to C474. CP, cytoplasm domain; FP, fusion peptide; HR, heptad repeat;
RBD, receptor-binding domain; RBM, receptor-binding motif; SP, signal peptide; TM,
transmembrane domain. Parts b and c are adapted, with permission, from REF. 29 © (2005)
American Association for the Advancement of Science.
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Figure 3. The fusion core structure
The fusion core is a six-helix bundle with three HR2 α-helices packed in an oblique antiparallel
manner against the hydrophobic grooves on the surface of the central HR1 trimer55,130. A
top (a) and side (b) view is shown of the severe acute respiratory syndrome-coronavirus
(SARS-CoV) S protein six-helix bundle fusion core structure formed by the HR1 and HR2
domains in the S2 subunit. C, carboxyl; N, amino. Figure adapted, with permission, from REF.
55 © (2004) American Society for Biochemistry and Molecular Biology.
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Table 1
S protein-based vaccines and antiviral therapies against SARS-CoV
category Advantages Disadvantages Refs
Vaccines*
Full-length S protein Induces effective neutralizing-antibody and
T-cell responses, as
well as protective immunity
Might induce harmful immune
responses
that cause liver damage or
enhanced
infection
64,65
DNA-based Easier to design; induces immunoglobulin G,
neutralizing-
antibody and T-cell responses and/or
protective immunity
Might have low efficacy in
humans;
repeated doses may cause
toxicity
59,131
Viral vector-based Induces neutralizing-antibody responses,
protective immunity
and/or T-cell responses
Might induce ADE effect;
possibly
present pre-existing immunity
60,61,65
Recombinant S
protein-based Induces high neutralizing-antibody
responses and protective
immunity
Mainly humoral responses; need
repeated doses and adjuvants 62
RBD Induces highly potent neutralizing-antibody
and T-cell responses
and protective immunity
Not identified 70-73
DNA-based Induces neutralizing-antibody and T-cell
responses and/or
protective immunity
Induces low responses; might not
neutralize mutants 132-134
Viral vector-based Induces neutralizing-antibody responses,
protective immunity
and/or T-cell responses
Possible genomic integration of
foreign
DNA; viral vector instability
75,135
Recombinant RBD
protein-based Safer and more effective than other RBD
vaccines; induces
neutralizing-antibody and T-cell responses,
protective immunity
and cross protection
Needs repeated doses and
adjuvants 26,70-72
Therapeutics*
Peptides Inhibits virus infection by preventing S
protein-mediated
receptor binding and blocking viral fusion
and entry
Low antiviral potency 53,82-84,
136-138
RBD–ACE2 blockers Blocks RBD–ACE2 binding and S protein-
mediated infection Not identified 82,83
S cleavage inhibitors Might interfere with S cleavage Not identified 84,136,137
Fusion core blockers Easy to design; inhibits virus infection with
high specificity Not identified 53,89,90,138
Neutralizing
antibodies Highly potent virus inhibition and/or
neutralization activity
against homologous and heterologous
SARS-CoV isolates
Might enhance SARS-CoV
entry; further
studies needed
139
Neutralizing mouse
antibodies Easier to generate than human neutralizing
antibodies; neutralizes SARS-CoV in vitro
and prevents virus
replication
Repeated use can cause HAMA
response;
might not recognize mutants with
key
substitutions in S protein
65,94,
140,141
Neutralizing human
antibodies Inhibits virus entry, neutralizes virus
infection, induces cross
protection and reduces disease severity and
viral burden; more
suitable to development as human
immunotherapeutics
Not identified 97,142,143
Small compounds Oral bioavailability Low antiviral potency 103-105,
107-109
Nat Rev Microbiol. Author manuscript; available in PMC 2009 September 24.
NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Du et al. Page 24
category Advantages Disadvantages Refs
Protease inhibitors Blocks virus entry and/or inhibits protease
(cathepsin L)
proteolysis
Not identified 103-105
S protein inhibitors Specifically inhibits S protein-mediated
SARS-CoV fusion and
entry into the host cell
Not identified 107-109
Small interfering RNAs Reduces virus replication and/or silences S
gene expression Low antiviral potency; limited
usefulness 113-117
*All candidates are at the preclinical study stage. ACE2, angiotensin-converting enzyme 2; ADE, antibody-dependent enhancement; HAMA, human–
anti-mouse antibody, RBD, receptor-binding domain, SARS-CoV; severe acute respiratory syndrome-coronavirus.
Nat Rev Microbiol. Author manuscript; available in PMC 2009 September 24.
... The S protein consists of two subunits, S1 and S2. The S1 subunit binds the target cells expressing viral receptor through the receptor-binding domain (RBD), whereas the S2 subunit promotes virus-cell membrane fusion [6,7]. According to the vital roles of S protein, current US FDA-approved vaccines focus on challenging immune responses against the S1 subunit of SARS-CoV-2 spike. ...
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... The 'S' protein is known to induce both humoral, and cellular immune responses, and remains the target of vaccines that are based on full-length S protein, and its receptor-binding domain, including DNA, viral vector, and subunit-based vaccines. In addition, the peptides, antibodies, organic compounds, and short interfering RNAs (siRNAs) are additional therapeutics under development [5,6]. Interestingly, the COVID-19 mRNA vaccines that are in use currently have been shown to induce neutralizing antibody response against the SARS-CoV-2 [7]. ...
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Nature is the international weekly journal of science: a magazine style journal that publishes full-length research papers in all disciplines of science, as well as News and Views, reviews, news, features, commentaries, web focuses and more, covering all branches of science and how science impacts upon all aspects of society and life.
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