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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.
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:
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ACE2 | E | M | N | S
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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
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
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
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-
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
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
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
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
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.
A medication commonly used to treat patients with irregular heart beats or
cardiac arrhythmias, including ventricular tachycardia and ventricular
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.
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
Full-length S protein Induces effective neutralizing-antibody and
T-cell responses, as
well as protective immunity
Might induce harmful immune
that cause liver damage or
DNA-based Easier to design; induces immunoglobulin G,
antibody and T-cell responses and/or
protective immunity
Might have low efficacy in
repeated doses may cause
Viral vector-based Induces neutralizing-antibody responses,
protective immunity
and/or T-cell responses
Might induce ADE effect;
present pre-existing immunity
Recombinant S
protein-based Induces high neutralizing-antibody
responses and protective
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
DNA; viral vector instability
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
Peptides Inhibits virus infection by preventing S
receptor binding and blocking viral fusion
and entry
Low antiviral potency 53,82-84,
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
antibodies Highly potent virus inhibition and/or
neutralization activity
against homologous and heterologous
SARS-CoV isolates
Might enhance SARS-CoV
entry; further
studies needed
Neutralizing mouse
antibodies Easier to generate than human neutralizing
antibodies; neutralizes SARS-CoV in vitro
and prevents virus
Repeated use can cause HAMA
might not recognize mutants with
substitutions in S protein
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
Not identified 97,142,143
Small compounds Oral bioavailability Low antiviral potency 103-105,
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category Advantages Disadvantages Refs
Protease inhibitors Blocks virus entry and/or inhibits protease
(cathepsin L)
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. ...
Full-text available
Concerns over vaccine efficacy after the emergence of the SARS-CoV-2 Delta variant prompted revisiting the vaccine design concepts. Monoclonal antibodies (mAbs) have been developed to identify the neutralizing epitopes on spike protein. It has been confirmed that the key amino acid residues in epitopes that induce the formation of neutralizing antibodies do not have to be on the receptor-binding domain (RBD)- angiotensin-converting enzyme 2 (ACE2) contact surface, and may be conformationally hidden. In addition, this epitope is tolerant to amino acid mutations of the Delta variant. The antibody titers against RBD in health care workers in Thailand receiving two doses of CoronaVac, followed by a booster dose of BNT162b2, were significantly increased. The neutralizing antibodies against the Delta variant suggest that the overall neutralizing antibody level against the Wuhan strain, using the NeutraLISA, was consistent with the levels of anti-RBD antibodies. However, individuals with moderate anti-RBD antibody responses have different levels of a unique antibody population competing with a cross-neutralizing mAb clone, 40591-MM43, determined by in-house competitive ELISA. Since 40591-MM43 mAb indicates cross-neutralizing activity against the Delta variant, this evidence implies that the efficiency of the vaccination regimen should be improved to facilitate cross-protective antibodies against Delta variant infections. The RBD epitope recognized by 40591-MM43 mAb is hidden in the close state.
... It is known that the virus enters the host cell by binding of the viral spike glycoprotein to the host receptor, angiotensinconverting enzyme 2 (ACE2) [35] therefore (6M0J) seems to be a biologically meaningful receptor. ...
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In this study, FDA-approved HCV antiviral drugs and their structural analogues—several of them in clinical trials—were tested for their inhibitory properties toward the SARS-CoV-2 spike protein bound to angiotensin-converting enzyme 2 (6M0J) using a virtual screening approach and computational chemistry methods. The most stable structures and the corresponding binding affinities of thirteen such antiviral compounds were obtained. Frontier molecular orbital theory, global reactivity descriptors, molecular docking calculations and electrostatic potential analysis were used to hypothesize the bioactivity of these drugs against 6M0J. It is found that an increased affinity for the protein is shown by inhibitors with large compound volume, relatively higher electrophilicity index, aromatic rings and heteroatoms that participate in hydrogen bonding. Among the tested drugs, four compounds 10–13 showed excellent results—binding affinities − 11.2 to − 11.5 kcal mol⁻¹. These four top scoring compounds may act as lead compounds for further experimental validation, clinical trials and even for the development of more potent antiviral agents against the SARS-CoV-2. Graphical abstract Approved HCV drugs and analogues were tested for their bioactivity towards the SARS-CoV-2 (6M0J) using virtual screening, ESP and MD analysis.
... 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]. ...
Full-text available
The ongoing pandemic (also known as coronavirus disease-19; COVID-19) by a constantly emerging viral agent commonly referred as the severe acute respiratory syndrome corona virus 2 or SARS-CoV-2 has revealed unique pathological findings from infected human beings, and the postmortem observations. The list of disease symptoms, and postmortem observations is too long to mention; however, SARS-CoV-2 has brought with it a whole new clinical syndrome in “long haulers” including dyspnea, chest pain, tachycardia, brain fog, exercise intolerance, and extreme fatigue. We opine that further improvement in delivering effective treatment, and preventive strategies would be benefited from validated animal disease models. In this context, we designed a study, and show that a genetically engineered mouse expressing the human angiotensin converting enzyme 2; ACE-2 (the receptor used by SARS-CoV-2 agent to enter host cells) represents an excellent investigative resource in simulating important clinical features of the COVID-19. The ACE-2 mouse model (which is susceptible to SARS-CoV-2) when administered with a recombinant SARS-CoV-2 spike protein (SP) intranasally exhibited a profound cytokine storm capable of altering the physiological parameters including significant changes in cardiac function along with multi-organ damage that was further confirmed via histological findings. More importantly, visceral organs from SP treated mice revealed thrombotic blood clots as seen during postmortem examination. Thus, the ACE-2 engineered mouse appears to be a suitable model for studying intimate viral pathogenesis thus paving the way for identification, and characterization of appropriate prophylactics as well as therapeutics for COVID-19 management.
... Among them, the S protein-based vaccines include full-length S protein vaccines, viral vectorbased vaccine, DNA-based vaccine, recombinant S protein based and recombinant RBD protein-based vaccines. On the other hand, S protein-based antiviral therapies include RBD-ACE2 blockers, S cleavage inhibitors, fusion core blockers, neutralizing antibodies, protease inhibitors, S protein inhibitors and small interfering RNAs [80]. Even though such therapeutic options have proven efficacy in in-vitro studies, most of them did not undergo randomized animal or human trials and, hence, are of limited use in our present COVID-19 scenario. ...
Full-text available
A new class of coronavirus, known as the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has been discovered, which is responsible for the occurrence of the disease, COVID-19. A comparative study with SARS, MERS and other human viruses was conductedand concluded that SARS-CoV-2 spread more rapidly due to increased globalization and adaptation of the virus in every environment. According to recent WHO reports, by 16 May 2021, the current outbreak of COVID-19 had affected over 174,054,314 people and killed more than 3,744,116 people in more than 222 countries acrossthe world. Finding a solution against the deadly COVID-19 has become an enormous challenge for researchers and virologists. A ring vaccination trial, which recruits subjects connected to a known case either socially or geographically, is a solution to evaluate vaccine efficacy and control the spread of the disease simultaneously, although its implementation is challenging. This review aims to summarize the noteworthy features of the world-intimidating SARS-CoV-2 global pandemic along with its evaluation, problems and challenges in the treatment strategies, clinical efficiency and detection methods proposed so far. This paper describes the impact of the lockdown in response to the COVID-19 pandemic on social, economic, health, and National Health Programs in India; possible ways to control the disease are also discussed.
Full-text available
The coronavirus disease‑19 (COVID‑19) pandemic has already claimed millions of lives and remains one of the major catastrophes in the recorded history. While mitigation and control strategies provide short term solutions, vaccines play critical roles in long term control of the disease. Recent emergence of potentially vaccine‑resistant and novel variants necessitated testing and deployment of novel technologies that are safe, effective, stable, easy to administer, and inexpensive to produce. Here we developed three recombinant Newcastle disease virus (rNDV) vectored vaccines and assessed their immunogenicity, safety, and protective efficacy against severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) in mice and hamsters. Intranasal administration of rNDV‑based vaccine candidates elicited high levels of neutralizing antibodies. Importantly, the nasally administrated vaccine prevented lung damage, and significantly reduced viral load in the respiratory tract of vaccinated animal which was compounded by profound humoral immune responses. Taken together, the presented NDV‑based vaccine candidates fully protected animals against SARS‑CoV‑2 challenge and warrants evaluation in a Phase I human clinical trial as a promising tool in the fight against COVID‑19.
The SARS-CoV-2 pandemia had stimulated the numerous publications emergence on the α1-proteinase inhibitor (α1-PI, α1-antitrypsin), primarily when it was found that high mortality in some regions corresponded to the regions with deficient α1-PI alleles. By analogy with the last century's data, when the root cause of the α1-antitrypsin, genetic deficiency leading to the elastase activation in pulmonary emphysema, was proven. It is evident that proteolysis hyperactivation in COVID-19 may be associated with α1-PI impaired functions. The purpose of this review is to systematize scientific data, critical directions for translational studies on the role of α1-PI in SARS-CoV-2-induced proteolysis hyperactivation as a diagnostic marker and a target in therapy. This review describes the proteinase-dependent stages of a viral infection: the reception and virus penetration into the cell, the plasma aldosterone-angiotensin-renin, kinins, blood clotting systems imbalance. The ACE2, TMPRSS, ADAM17, furin, cathepsins, trypsin- and elastase-like serine proteinases role in the virus tropism, proteolytic cascades activation in blood, and the COVID-19-dependent complications is presented. The analysis of scientific reports on the α1-PI implementation in the SARS-CoV-2-induced inflammation, the links with the infection severity, and comorbidities were carried out. Particular attention is paid to the acquired α1-PI deficiency in assessing the patients with the proteolysis overactivation and chronic non-inflammatory diseases that are accompanied by the risk factors for the comorbidities progression, and the long-term consequences of COVID-19 initiation. Analyzed data on the search and proteases inhibitory drugs usage in the bronchopulmonary cardiovascular pathologies therapy are essential. It becomes evident the antiviral, anti-inflammatory, anticoagulant, anti-apoptotic effect of α1-PI. The prominent data and prospects for its application as a targeted drug in the SARS-CoV-2 acquired pneumonia and related disorders are presented.
Drug repurposing involves the process of investigating already existing drugs with an aim to use them for different therapeutic purposes than the intended one. This approach is relatively faster, less costly, and reliable in terms of safety as the drug under study is already derisked and known for its other chemistry and pharmacokinetic properties. With these benefits in mind, it is a very reliable way to undertake drug development for emerging diseases such as COVID-19 which demand immediate interventions to slow or completely stop its havoc on mankind. One of the biggest challenges that drug repurposing has is the possibility of the occurrence of new mechanisms of action between the drug ligand and some proteins in the human physiology. Drug repurposing appears to have settled in the meantime in drug development, though more studies in the future will be warranted particularly in regards to resistance.
Purpose: To evaluate IgG production in a group of vaccinated and unvaccinated subjects previously infected, or not, with SARS-CoV-2. Methods: A total of 316 subjects were enrolled at different times after vaccination and/or infection. IgG against target S1 subunit of the spike protein of SARS-COV-2 was assessed by a chemiluminescent microparticle immunoassay. Participant data was collected using a clinical-epidemiological survey. Results: A total of 56.2% (n = 146) of our cohort was vaccinated, with 27.5% (n = 36) reporting a previous infection. Of these, all were IgG positive at the time of the study, regardless of gender, age category, vaccine type, and elapsed time since vaccination. The vaccinated group without a previous infection (72.5%, n = 95) showed a slightly lower IgG seropositivity and median values, overall, although significantly higher in females and lower with the ChAdOx1 nCoV-19 (AstraZeneca) vaccine. Vaccinated subjects above the age of 65 showed a trend towards higher median IgG values (13,911.0 AU/mL), when previously infected with SARS-CoV-2, but comparatively lower IgG median value (5158.7 AU/mL) in its absence. In all vaccinated groups, IgG antibody production increased at 1-2 weeks, peaking at 4-6 weeks. Afterward, IgG decreased progressively but almost all subjects (97.7%, n = 128) were seropositive for the remainder of our study. Fully vaccinated individuals with a past infection showed a lower IgG rate of decrease versus their uninfected counterparts (17.9 vs 22.6%, respectively). Conclusion: Our findings suggest a higher effect of vaccination on the production IgG antibodies, as opposed to natural infection. Nonetheless, in general, antibody titers waned rapidly.
Although the amino acid sequences of SARS-CoV-1 and SARS-CoV-2 fusion peptides (FPs) are highly conserved, the cryo-electron microscopy structures of the SARS-CoV-1 and SARS-CoV-2 spike proteins show that the helix length of SARS-CoV-1 FP is longer than that of SARS-CoV-2 FP. In this work, we simulated the membrane-binding models of SARS-CoV-1 and SARS-CoV-2 FPs and compared the binding modes of the FPs with the POPC/POPE/cholesterol bilayer membrane. Our simulation results show that the SARS-CoV-2 FP binds to the bilayer membrane more effectively than the SARS-CoV-1 FP. It is seen that the short N-terminal helix of SARS-CoV-2 FP is more favorable to insert into the target membrane than the long N-terminal helix of SARS-CoV-1 FP. Meanwhile, the potential of mean force calculations showed that the SARS-CoV-2 FP would prefer only one binding mode (N-terminal binding), whereas the SARS-CoV-1 FP has two favorable membrane-binding modes (C-terminal and N-terminal binding modes). Moreover, in the case of SARS-CoV-1 FP binding to the target membrane, the transition between the two binding modes is relatively fast. Finally, we discovered that the membrane-binding mode would influence the helix length of SARS-CoV-1 FP, while the helix length of SARS-CoV-2 FP could be stably maintained in the membrane-bound configurations. This work suggests that the short helix might endow the FP with high membrane-anchoring strength. In particular, the membrane-penetrating residues (Phe, Ile, and Leu) of short α-helix interact with the cell membrane more strongly than those of long α-helix.
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The severe acute respiratory syndrome coronavirus (SARS-CoV) caused a worldwide epidemic in late 2002/early 2003 and a second outbreak in the winter of 2003/2004 by an independent animal-to-human transmission. The GD03 strain, which was isolated from an index patient of the second outbreak, was reported to resist neutralization by the human monoclonal antibodies (hmAbs) 80R and S3.1, which can potently neutralize isolates from the first outbreak. Here we report that two hmAbs, m396 and S230.15, potently neutralized GD03 and representative isolates from the first SARS outbreak (Urbani, Tor2) and from palm civets (SZ3, SZ16). These antibodies also protected mice challenged with the Urbani or recombinant viruses bearing the GD03 and SZ16 spike (S) glycoproteins. Both antibodies competed with the SARS-CoV receptor, ACE2, for binding to the receptor-binding domain (RBD), suggesting a mechanism of neutralization that involves interference with the SARS-CoV–ACE2 interaction. Two putative hot-spot residues in the RBD (Ile-489 and Tyr-491) were identified within the SARS-CoV spike that likely contribute to most of the m396-binding energy. Residues Ile-489 and Tyr-491 are highly conserved within the SARS-CoV spike, indicating a possible mechanism of the m396 cross-reactivity. Sequence analysis and mutagenesis data show that m396 might neutralize all zoonotic and epidemic SARS-CoV isolates with known sequences, except strains derived from bats. These antibodies exhibit cross-reactivity against isolates from the two SARS outbreaks and palm civets and could have potential applications for diagnosis, prophylaxis, and treatment of SARS-CoV infections. • epitope • paratope • vaccine • therapeutic
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Skowronski, DA Astell, C Brunham, RC Low, DE Petric, M Roper, RL Talbot, PJ Tam, T Babiuk, L
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Unlike other class I viral fusion proteins, spike proteins on severe acute respiratory sydrome coronavirus virions are uncleaved. As we and others have demonstrated, infection by this virus depends on cathepsin proteases present in endosomal compartments of the target cell, suggesting that the spike protein acquires its fusion competence by cleavage during cell entry rather than during virion biogenesis. Here we demonstrate that cathepsin L indeed activates the membrane fusion function of the spike protein. Moreover, cleavage was mapped to the same region where, in coronaviruses carrying furin-activated spikes, the receptor binding subunit of the protein is separated from the membrane-anchored fusion subunit.
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Although the 2003 severe acute respiratory syndrome (SARS) outbreak was controlled, repeated transmission of SARS coronavirus (CoV) over several years makes the development of a SARS vaccine desirable. We performed a comparative evaluation of two SARS vaccines for their ability to protect against live SARS-CoV intranasal challenge in ferrets. Both the whole killed SARS-CoV vaccine (with and without alum) and adenovirus-based vectors encoding the nucleocapsid (N) and spike (S) protein induced neutralizing antibody responses and reduced viral replication and shedding in the upper respiratory tract and progression of virus to the lower respiratory tract. The vaccines also diminished haemorrhage in the thymus and reduced the severity and extent of pneumonia and damage to lung epithelium. However, despite high neutralizing antibody titres, protection was incomplete for all vaccine preparations and administration routes. Our data suggest that a combination of vaccine strategies may be required for effective protection from this pathogen. The ferret may be a good model for SARS-CoV infection because it is the only model that replicates the fever seen in human patients, as well as replicating other SARS disease features including infection by the respiratory route, clinical signs, viral replication in upper and lower respiratory tract and lung damage.
Heptad repeat regions (HR1 and HR2) are highly conserved sequences located in the glycoproteins of enveloped viruses. They form a six-helix bundle structure and are important in the process of virus fusion. Peptides derived from the HR regions of some viruses have been shown to inhibit the entry of these viruses. SARS-CoV was also predicted to have HR1 and HR2 regions in the S2 protein. Based on this prediction, we designed 25 peptides and screened them using a HIV-luc/SARS pseudotyped virus assay. Two peptides, HR1-1 and HR2-18, were identified as potential inhibitors, with EC50 values of 0.14 and 1.19 μM, respectively. The inhibitory effects of these peptides were validated by the wild-type SARS-CoV assay. HR1-1 and HR2-18 can serve as functional probes for dissecting the fusion mechanism of SARS-CoV and also provide the potential of further identifying potent inhibitors for SARS-CoV entry.
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Cellular entry of enveloped viruses is often dependent on attachment proteins expressed on the host cell surface. Viral envelope proteins bind these receptors, and, in an incompletely understood process, facilitate fusion of the cellular and viral membranes so as to introduce the viral core into the cytoplasm. Only a small fraction of viral receptors have been identified so far. Recently, a novel coronavirus was identified as the etiological agent of severe acute respiratory syndrome (SARS). The fusion protein gene of SARS coronavirus (SARS-CoV) was cloned and characterized, and shortly thereafter, angiotensin-converting enzyme 2 (ACE2) was shown to be its functional receptor. Identification of ACE2 as a receptor for SARS-CoV will likely contribute to the development of antivirals and vaccines. It may also contribute to the development of additional animal models for studying SARS pathogenesis, and could help identify the animal reservoir of SARS-CoV.
We have cloned, expressed, and characterized the full-length and various soluble fragments of the SARS-CoV (Tor2 isolate) S glycoprotein. Cells expressing S fused with receptor-expressing cells at neutral pH suggesting that the recombinant glycoprotein is functional, its membrane fusogenic activity does not require other viral proteins, and that low pH is not required for triggering membrane fusion; fusion was not observed at low receptor concentrations. S and its soluble ectodomain, Se, were not cleaved to any significant degree. They ran at about 180–200 kDa in SDS gels suggesting post-translational modifications as predicted by previous computer analysis and observed for other coronaviruses. Fragments containing the N-terminal amino acid residues 17–537 and 272–537 but not 17–276 bound specifically to Vero E6 cells and purified soluble receptor, ACE2, recently identified by M. Farzan and co-workers [Nature 426 (2003) 450–454]. Together with data for inhibition of binding by antibodies developed against peptides from S, these findings suggest that the receptor-binding domain is located between amino acid residues 303 and 537. These results also confirm that ACE2 is a functional receptor for the SARS virus and may help in the elucidation of the mechanisms of SARS-CoV entry and in the development of vaccine immunogens and entry inhibitors.
Severe acute respiratory syndrome (SARS) is an emerging infectious viral disease characterized by severe clinical manifestations of the lower respiratory tract. The pathogenesis of SARS is highly complex, with multiple factors leading to severe injury in the lungs and dissemination of the virus to several other organs. The SARS coronavirus targets the epithelial cells of the respiratory tract, resulting in diffuse alveolar damage. Several organs/cell types may be infected in the course of the illness, including mucosal cells of the intestines, tubular epithelial cells of the kidneys, neurons of the brain, and several types of immune cells, and certain organs may suffer from indirect injury. Extensive studies have provided a basic understanding of the pathogenesis of this disease. In this review we describe the most significant pathological features of SARS, explore the etiological factors causing these pathological changes, and discuss the major pathogenetic mechanisms. The latter include dysregulation of cytokines/chemokines, deficiencies in the innate immune response, direct infection of immune cells, direct viral cytopathic effects, down-regulation of lung protective angiotensin converting enzyme 2, autoimmunity, and genetic factors. It seems that both abnormal immune responses and injury to immune cells may be key factors in the pathogenesis of this new disease.
The only severe acute respiratory syndrome (SARS) vaccine currently being tested in clinical trial consists of inactivated severe acute respiratory syndrome-associate coronavirus (SARS-CoV). However, limited information is available about host immune responses induced by the inactivated SARS vaccine. In this study, we demonstrated that SARS-CoV inactivated by beta-propiolactone elicited high titers of antibodies in the immunized mice and rabbits that recognize the spike (S) protein, especially the receptor-binding domain (RBD) in the S1 region. The antisera from the immunized animals efficiently bound to the RBD and blocked binding of RBD to angiotensin-converting enzyme 2, the functional receptor on the susceptible cells for SARS-CoV. With a sensitive and quantitative single-cycle infection assay using pseudovirus bearing the SARS-CoV S protein, we demonstrated that mouse and rabbit antisera significantly inhibited S protein-mediated virus entry with mean 50% inhibitory titers of 1:7393 and 1:2060, respectively. These data suggest that the RBD of S protein is a major neutralization determinant in the inactivated SARS vaccine which can induce potent neutralizing antibodies to block SARS-CoV entry. However, caution should be taken in using the inactivated SARS-CoV as a vaccine since it may also cause harmful immune and/or inflammatory responses.