Subunit Vaccines Against Emerging Pathogenic Human Coronaviruses

REVIEW
published: 28 February 2020

doi: 10.3389/fmicb.2020.00298
Subunit Vaccines Against Emerging

Pathogenic Human Coronaviruses
Ning Wang 1 , Jian Shang 2 , Shibo Jiang 1,3* and Lanying Du 1*
1
Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY, United States, 2 Department of Veterinary
and Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, Saint Paul, MN, United States, 3 Key
Laboratory of Medical Molecular Virology (MOE/NHC/CAMS), School of Basic Medical Sciences, Shanghai Medical College,
Fudan University, Shanghai, China
Seven coronaviruses (CoVs) have been isolated from humans so far. Among them, three
emerging pathogenic CoVs, including severe acute respiratory syndrome coronavirus
(SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and a newly
identified CoV (2019-nCoV), once caused or continue to cause severe infections in
humans, posing significant threats to global public health. SARS-CoV infection in
humans (with about 10% case fatality rate) was first reported from China in 2002, while
MERS-CoV infection in humans (with about 34.4% case fatality rate) was first reported
from Saudi Arabia in June 2012. 2019-nCoV was first reported from China in December
2019, and is currently infecting more than 70000 people (with about 2.7% case
Edited by: fatality rate). Both SARS-CoV and MERS-CoV are zoonotic viruses, using bats as their
Lijun Rong,
The University of Illinois at Chicago,
natural reservoirs, and then transmitting through intermediate hosts, leading to human
United States infections. Nevertheless, the intermediate host for 2019-nCoV is still under investigation
Reviewed by: and the vaccines against this new CoV have not been available. Although a variety of
Ahmed Mohamed Kandeil, vaccines have been developed against infections of SARS-CoV and MERS-CoV, none
National Research Centre, Egypt
Liang Qiao, of them has been approved for use in humans. In this review, we have described the
Loyola University Chicago, structure and function of key proteins of emerging human CoVs, overviewed the current
United States
vaccine types to be developed against SARS-CoV and MERS-CoV, and summarized
*Correspondence:
Shibo Jiang
recent advances in subunit vaccines against these two pathogenic human CoVs. These
[email protected] subunit vaccines are introduced on the basis of full-length spike (S) protein, receptor-
Lanying Du binding domain (RBD), non-RBD S protein fragments, and non-S structural proteins,
[email protected]
and the potential factors affecting these subunit vaccines are also illustrated. Overall,
Specialty section: this review will be helpful for rapid design and development of vaccines against the new
This article was submitted to
2019-nCoV and any future CoVs with pandemic potential. This review was written for
Virology,
a section of the journal the topic of Antivirals for Emerging Viruses: Vaccines and Therapeutics in the Virology
Frontiers in Microbiology section of Frontiers in Microbiology.
Received: 28 November 2019
Keywords: human coronaviruses, pathogenesis, SARS-CoV, MERS-CoV, 2019-nCoV, subunit vaccines
Accepted: 10 February 2020
Published: 28 February 2020
Citation:
Wang N, Shang J, Jiang S and
INTRODUCTION
Du L (2020) Subunit Vaccines Against
Emerging Pathogenic Human
Coronaviruses (CoVs) belong to the subfamily Othocoronavirinae, in the family Coronaviridae of
Coronaviruses. the order Nidovirales. According to the 10th Report on Virus Taxonomy from the International
Front. Microbiol. 11:298. Committee on Taxonomy of Viruses (ICTV), the Othocoronavirinae is comprised of four
doi: 10.3389/fmicb.2020.00298 genera, including alphacoronavirus (alpha-CoV), betacoronavirus (beta-CoV), gammacoronavirus
Frontiers in Microbiology | www.frontiersin.org 1 February 2020 | Volume 11 | Article 298

Wang et al. Subunit Vaccines Against Coronaviruses
(gamma-CoV), and deltacoronavirus (delta-CoV) (King et al., Fouchier et al., 2004; Woo et al., 2005; Gerna et al., 2006). HCoV-
2018). Alpha- and beta-CoVs can infect mammals, including 229E and HCoV-OC43 may lead to central nervous system
but not limited to bats, pigs, cats, mice, and humans (Kusanagi infection since viral RNAs are detected in the brain of some
et al., 1992; Li et al., 2005b; Poon et al., 2005; Drexler et al., patients (Arbour et al., 2000; Desforges et al., 2014).
2014; Pedersen, 2014; Kudelova et al., 2015; Cui et al., 2019). Unlike the above four human CoVs, SARS-CoV, MERS-
Gamma- and delta-CoVs usually infect birds, while some of CoV, and 2019-nCoV have caused severe pneumonia and/or
them could infect mammals (Woo et al., 2009a, 2012, 2014; Ma failure of other organs, even death, among infected populations
et al., 2015). Since the late sixties, CoVs have been recognized (Nicholls et al., 2003; Zhong et al., 2003; Zaki et al., 2012;
as one of the viral sources responsible for the common cold. Zhu et al., 2020). The epidemic outbreak of SARS-CoV began
Among all CoVs identified so far, seven have the ability to in the Guangdong Province of China in November 2002, and
infect humans, including human coronavirus 229E (HCoV-229E) spread through human-to-human transmission to other parts
and human coronavirus NL63 (HCoV-NL63), which belong to of the world within a few months (Ksiazek et al., 2003). From
alpha-CoVs (Hamre and Procknow, 1966; Chiu et al., 2005), November 2002 to August 2003, SARS-CoV infected more than
as well as human coronavirus OC43 (HCoV-OC43), human 8,098 people in 29 counties, resulting in over 774 deaths with
coronavirus HKU1 (HCoV-HKU1), severe acute respiratory ∼10% fatality rate (Du et al., 2009a). Palm civets serving as a
syndrome coronavirus (SARS-CoV), Middle East respiratory potential intermediate host of this virus were traced immediately
syndrome coronavirus (MERS-CoV), and the newly emerged (Tu et al., 2004). Chinese horseshoe bats (Rhinolophus sinicus)
coronavirus (2019-nCoV), which are known to be beta-CoVs are the natural reservoir of SARS-CoV (Li et al., 2005b). Various
(Drosten et al., 2003; Ksiazek et al., 2003; Vabret et al., 2003; Woo bat SARS-related CoVs (SARSr-CoV) have been identified in
et al., 2005; Zaki et al., 2012; Du et al., 2016b; Zhang et al., 2020; Yunnan, China, several of which can infect human cells, and
Zhu et al., 2020) (Figure 1). have been further characterized (Ge et al., 2013; Hu et al., 2017).
Four human CoVs, including HCoV-229E, HCoV-NL63, These discoveries indicate the threat of re-emergence of SARS-
HCoV-OC43, and HCoV-HKU1, have been identified in humans, CoV or SARSr-CoV.
but without causing severe infections. HCoV-229E was isolated A decade later, another highly pathogenic human CoV,
from nasal secretions of medical students with minor upper MERS-CoV, emerged, and the first patient with MERS-CoV
respiratory disease. This virus was an original isolate, and was first infection was reported in Saudi Arabia in June 2012 (Zaki
reported in the 1960s (Hamre and Procknow, 1966). In addition et al., 2012). By December 26, 2019, a total of 2,494 laboratory-
to HCoV-229E, several studies have reported the recovery of confirmed cases of MERS, including 858 associated deaths in
HCoV-OC43 from patients with upper respiratory tract illness 27 countries (fatality rate 34.4%), were reported to the WHO1 .
(Tyrrell and Bynoe, 1965; Hamre et al., 1967; McIntosh et al., Globally, the majority (about 80%) of human cases have been
1967; Kapikian et al., 1969). In 2004, HCoV-NL63 was isolated reported in Saudi Arabia, where people get infected through
from clinical species of infants suffering from pneumonia or direct contact with infected dromedary camels or persons2
bronchiolitis, and characterized for its ability to infect human (Zaki et al., 2012). Isolation of MERS-CoV and detection of
respiratory tract (Fouchier et al., 2004; van der Hoek et al., neutralizing antibodies from dromedary camels suggest that
2004). The subsequent study in 2005 identified a new member these camels are potentially an important intermediate host
of CoVs, named HCoV-HKU1, from a 71-year-old man with (Reusken et al., 2013; Azhar et al., 2014). Similar to SARS-
pneumonia (Woo et al., 2005). Generally, these four viruses are CoV, MERS-CoV is also an emerging zoonotic virus (Li and
the most common pathogens causing mild upper respiratory Du, 2019). Bats habituate several CoVs phylogenetically related
infection or asymptomatic infection, and count for about 30% to MERS-CoV, and some of them are identical to MERS-CoVs,
of all colds (Myint, 1994; Lau et al., 2006; Kim et al., 2017). suggesting that MERS-CoV may originate from bats (Annan
In the serological surveillance on healthy adults, HCoV-229E, et al., 2013; Lelli et al., 2013; Lau et al., 2018; Luo et al., 2018a).
HCoV-NL63, and HCoV-OC43 demonstrated more than 90% Different from SARS-CoV, which has not caused infections
seropositive with the immunological assay. It appears common in humans since 2004 (Du et al., 2009a), the transmission
for these CoVs to infect children (Mourez et al., 2007; Shao of MERS-CoV has not been interrupted, and the infected
et al., 2007; Severance et al., 2008). In contrast to the above human cases continue increasing1 (Mobaraki and Ahmadzadeh,
three human CoVs, HCoV-HKU1 has around 50% seropositive 2019). Currently, human-to-human transmission of MERS-
in healthy individuals and a relatively low exposure rate in CoV is limited.
children (Lehmann et al., 2008; Severance et al., 2008). Although A new CoV, 2019-nCoV, has caught worldwide attention
the prevalence of various CoVs is different, the incidence (Liu and Saif, 2020; Zhang et al., 2020). It was first identified
among these viruses shows no significant difference (Woo et al., in Wuhan, China in December 2019, from patients with
2009b). The afore-mentioned four CoVs have been detected pneumonia (Zhu et al., 2020), and has infected more than
in 2.1–17.9% of clinical specimens (Esper et al., 2006; Lau 70000 people globally, including 2,009 deaths (∼2.7% fatality
et al., 2006; Gerna et al., 2007; Regamey et al., 2008; Matoba
et al., 2015; Killerby et al., 2018). These viruses have also 1
https://www.who.int/emergencies/mers-cov/en/
been associated with lower respiratory tract illness in children, 2
https://www.who.int/docs/default-source/coronaviruse/situation-reports/
elders, and immunodeficient individuals (Falsey et al., 2002; 20200219-sitrep-30-covid-19.pdf?sfvrsn=6e50645_2

FIGURE 1 | Phylogenetic tree of coronaviruses (CoVs) based on the nucleotide sequences of RNA dependent RNA polymerase (RdRp). The Tree, with 1,000
bootstrap values, was constructed by the maximum likelihood method using MEGA 6. The four main phylogenetic clusters correspond to genera alpha-CoV,
beta-CoV, gamma-CoV, and delta-CoV. Each CoV genus contains different subgenera. The letters in blue indicate human CoVs.
rate), as of February 19, 2020, particularly in China, and the GENOME OF EMERGING HUMAN
other parts of the world, including Australia, Japan, Malaysia, CORONAVIRUSES, AS WELL AS
Singapore, South Korea, Viet Nam, Cambodia, Philippines,
Thailand, Nepal, Sri Lanka, India, United States, Canada, France, STRUCTURE AND FUNCTION OF THEIR
Finland, Germany, Italy, Russian Federation, Spain, Sweden, KEY PROTEINS
United Kingdom, Belgium, Egypt, and United Arab Emirates3 .
Different from MERS-CoV but similar to SARS-CoV, 2019-nCoV The human CoVs are enveloped viruses with a positive-sense,
can cause human-to-human transmission, and its intermediate single-stranded RNA genome. They are 80–160 nm in diameter.
host that leads to the current human infection and outbreak is Like other CoVs, human CoVs contain the largest viral genome
still under investigation. [27–32 kilobase pairs (kb)] among the RNA viruses, and they
share similar genome organization (Fehr and Perlman, 2015).
3
https://www.who.int/docs/default-source/coronaviruse/situation-reports/ Two large overlapping open reading frames (ORFs), ORF 1a and
20200204-sitrep-15-ncov.pdf?sfvrsn=88fe8ad6_2 ORF 1b, occupy two-thirds of the genome at the 50 -terminus, and

a third of the genome at the 30 -terminus encodes four common relies on the interaction between viral and cellular membrane
structural proteins in the gene order of spike (S), envelope proteins. Recognition of S1 subunit with a receptor and/or
(E), membrane (M), and nucleocapsid (N) (50 –30 ) (Fehr and sugar on the cell surface initiates the infection (Li, 2015). After
Perlman, 2015). The large ORF 1ab is a replicase gene encoding the initial recognition and binding, the S protein undergoes
polyproteins 1a (pp1a) and pp1b/1ab, which can be cleaved into conformational changes, followed by membrane fusion through
15–16 non-structural proteins (nsp2-nsp16 or nsp1-nsp16) by the S2 region (Li, 2015, 2016; Du et al., 2017). Consequently, the
3C-like proteinase (3CLpro , nsp5) and papain-like proteinase viral genetic materials are delivered into the host cell through the
(PLpro , nsp3) (Bailey-Elkin et al., 2014; Fehr and Perlman, 2015; fusion core (Du et al., 2009a).
Tomar et al., 2015; Snijder et al., 2016). In addition to the Similar to HCoV-NL63 (Hofmann et al., 2005; Li, 2015),
genes encoding the above structural proteins, the genes encoding SARS-CoV recognizes, through the RBD in the CTD region of
accessory proteins have also been detected in the 30 region its S1 subunit, angiotensin-converting enzyme 2 (ACE2) as the
between S–E–M–N (Fehr and Perlman, 2015). Some beta-CoVs, receptor on the target cell (Li et al., 2003). The RBD (CTD) in
such as HCoV-OC43 and HCoV-HKU1, contain hemagglutinin- two states (standing or lying) has been observed in the trimeric S
esterase (HE) gene located between ORF 1ab and S gene encoding protein. ACE2 binds to standing RBD, specifically in the receptor-
an additional structural protein, HE (De Groot et al., 2011; binding motif (RBM), keeping the RBD in the “standing” state
Desforges et al., 2013; Huang et al., 2015). Similar to other human (Yuan et al., 2017). In addition to human ACE2, SARS-CoV S
CoVs, SARS-CoV possesses a ∼29-kb genome, which encodes protein could also bind to palm civet and mouse ACE2s (Li et al.,
pp1a and pp1ab, four main structural proteins (S, E, M, and 2004; Li, 2008). Mutations in the RBD of S1 subunit are required
N), and eight accessory proteins, such as 3a, 3b, 6, 7a, 7b, 8a, for cross-species transmission of SARS-CoV (Li et al., 2005c; Li,
8b, and 9b (Figure 2A) (Marra et al., 2003; Snijder et al., 2003). 2008, 2016). Several bat SARSr-CoVs have been identified, and
The MERS-CoV genome is about 30 kb in length and encodes these CoVs can utilize human ACE2 as their receptor to bind
pp1a, pp1ab, four structural proteins (S, E, M, and N), and the target cells (Ge et al., 2013; Hu et al., 2017). The structure
five accessory proteins (3, 4a, 4b, 5, and 8b) (Figure 2A) (van of SARS-CoV S trimer and RBD binding to the ACE2 receptor is
Boheemen et al., 2012). The genomic RNA of SARS-CoV and shown in Supplementary Figures S1A,B.
MERS-CoV is packed inside capsid formed by the N protein, MERS-CoV RBD shares a structure similar to that of the
while the M, E, and S proteins form the envelope surrounding the homology domain of SARS-CoV (Wang et al., 2013). However,
capsid (Figure 2B). Accessory genes may incorporate into virions antibodies induced by SARS-CoV RBD have no cross-reactivity
at low levels (Liu et al., 2014). Nevertheless, neither SARS-CoV and/or cross-neutralizing activity to MERS-CoV (Du et al.,
nor MERS-CoV appears to contain the HE gene (Rota et al., 2003; 2013b). Moreover, MERS-CoV utilizes dipeptidyl peptidase 4
Zaki et al., 2012). (DPP4) as a receptor through the RBD (CTD) region (Raj et al.,
Several proteins of human CoVs are important for viral 2013), which is distinct from the SARS-CoV receptor ACE2.
infection and/or pathogenesis. For example, most nsps Although the core regions of SARS-CoV and MERS-CoV RBDs
participate in viral RNA replication and/or transcription are similar, their RBM regions are significantly different, which
(Snijder et al., 2016). The accessory proteins interact with host explains why they recognize different receptors (Li, 2015). MERS-
cells, potentially helping the viruses to evade the immune system CoV S trimer also maintains a structure similar to that of SARS-
and increase their virulence (Menachery et al., 2017). The HE CoV S trimer. Both standing and lying states can be detected in
protein assists in the attachment of virus–host cells, thus playing the MERS-CoV RBDs, whereas DPP4 only binds to the standing
a key role in the production of infectious virions, as in the case RBD (Pallesen et al., 2017; Yuan et al., 2017). MERS-CoV is
of HCoV-OC43 (Desforges et al., 2013). The M and E proteins clustered with Ty-BatCoV HKU4 and Pi-BatCoV HKU5 in the
are responsible for virus assembly or promote virulence (Scobey subgenus Merbecovirus. Ty-BatCoV HKU4, but not Pi-BatCoV
et al., 2013; DeDiego et al., 2014). Different from the above HKU5, could use human DPP4 as a receptor (Yang et al., 2014b).
proteins, the S protein of human CoVs mediates viral entry Recently, some MERS-related CoVs (MERSr-CoVs) have been
into host cells and subsequent membrane fusion, enabling viral discovered from bats that can enter human DPP4-expressing cells
infection (Du et al., 2009a; Lu et al., 2014). The S protein is a (Lau et al., 2018; Luo et al., 2018a). These findings suggest that
class I viral protein, which can be cleaved into two functional the emergence of MERSr-CoV may threaten human health owing
subunits, an amino-terminal S1 subunit and a carboxyl-terminal to their potential for cross-species transmission. The MERS-CoV
S2 subunit. The S1 subunit is responsible for virus–host cell S protein and RBD and their complexes, along with the DPP4
receptor binding, whereas the S2 subunit is involved in virus– receptor, are shown in Supplementary Figures S1C,D.
host membrane fusion (Li et al., 2005a; Lu et al., 2014). The S1 Recent studies have found that the new human CoV, 2019-
contains two major domains, an N-terminal domain (NTD) and nCoV, which belongs to the species of SARSr-CoV, shares high
a C-terminal domain (CTD). In general, NTDs mediate sugar sequence identify (about 79.5%) to SARS-CoV (Zhou et al., 2020).
binding (Li, 2016; Li et al., 2017; Ou et al., 2017; Hulswit et al., The genome of 2019-nCoV encodes pp1ab (translated from ORF
2019; Tortorici et al., 2019), whereas CTDs facilitate protein 1ab), four structural proteins (S, E, M, and N), and six accessory
receptor recognition (Wong et al., 2004; Hofmann et al., 2006; proteins (3a, 6, 7a, 7b, 8, and 10) (Figure 2A). Same as SARS-
Lu et al., 2013). The NTDs and CTDs of the S1 subunit can bind CoV and MERS-CoV, 2019-nCoV appears to have no HE gene.
host receptors or function as receptor-binding domains (RBDs) The virion of 2019-nCoV consists of similar structure as SARS-
(Lu et al., 2013; Hulswit et al., 2019). The entry of human CoVs CoV and MERS-CoV (Figure 2B). Like SARS-CoV, 2019-nCoV

FIGURE 2 | Schematic structure of SARS-CoV, MERS-CoV, and 2019-nCoV. (A) Schematic diagram of genomic organization of SARS-CoV, MERS-CoV, and
2019-nCoV. The genomic regions or open-reading frames (ORFs) are compared. Structural proteins, including spike (S), envelope (E), membrane (M) and
nucleocapsid (N) proteins, as well as non-structural proteins translated from ORF 1a and ORF 1b and accessory proteins, including 3a, 3b, 6, 7a, 7b, 8a, 8b, and 9b
(for SARS-CoV), 3, 4a, 4b, 5, and 8b (for MERS-CoV), and 3a, 6, 7a, 7b, 8, and 10 (for 2019-nCoV) are indicated. 50 -UTR and 30 -UTR, untranslated regions at the N-
and C-terminal regions, respectively. Kb, kilobase pair. (B) Schematic structure of virion of SARS-CoV, MERS-CoV, and 2019-nCoV and its major structural proteins.
also uses ACE2 as its cellular receptor to enter host cells (Zhou Zhu et al., 2020). With the increasing numbers of 2019-nCoV and
et al., 2020). Currently, the structure of 2019-nCoV RBD and/or MERS-CoV infections and continuous threat of re-emergence of
its binding with the viral receptor has not yet been available. SARS-CoV, as well as the potential of SARS- and MERS-related
CoVs to cause human infections, it is critical to develop vaccines
with strong efficacy and safety targeting these viruses to prevent
OVERVIEW OF VACCINES AGAINST their infections in humans. Since the vaccines against 2019-nCoV
have not been available, the rest of the review will focus on the
EMERGING PATHOGENIC HUMAN vaccines against SARS-CoV and MERS-CoV.
CORONAVIRUSES Although a variety of vaccines have been developed against
SARS-CoV and MERS-CoV, most of them are in the preclinical
Unlike the four low pathogenic human CoVs, including studies, and only several have been tested in clinical trials4 , 5
HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1, (Du et al., 2016b; Cho et al., 2018). Nevertheless, no vaccines
which cause mild to no pathogenesis in humans, SARS-CoV,
MERS-CoV, and 2019-CoV are three highly pathogenic human 4
https://clinicaltrials.gov/ct2/show/NCT03615911
CoVs (Channappanavar and Perlman, 2017; Cui et al., 2019; 5
https://clinicaltrials.gov/ct2/show/NCT03399578

have been approved for the prevention of SARS and MERS Potential Targets for Development of
in humans, demonstrating the need to develop effective and SARS-CoV and MERS-CoV Subunit
safe vaccines to control current MERS-CoV infection, or to
be stockpiled for potential use against re-emerged SARS-CoV Vaccines
or SARSr-CoV. Particularly, effective and safe vaccines are The S protein of SARS-CoV and MERS-CoV plays a vital role
urgently needed to prevent and control the current outbreak in receptor binding and membrane fusion. Thus, the S protein,
of 2019-nCoV. but not other structural proteins, is the major antigen to induce
Most SARS-CoV and MERS-CoV vaccines developed thus protective neutralizing antibodies to block viruses from binding
far are based on the inactivated or live attenuated viruses, their respective receptor and thus inhibit viral infection (Bisht
DNAs, proteins, nanoparticles, viral vectors, including virus- et al., 2004; Buchholz et al., 2004; Bukreyev et al., 2004; Yang
like particles (VLPs) (Zeng et al., 2004; Jiang et al., 2005; et al., 2004). As a result, the S protein is also a major target for the
Liu et al., 2005; Du et al., 2009a, 2016b; Pimentel et al., development of subunit vaccines against SARS-CoV and MERS-
2009; Al-Amri et al., 2017). Each vaccine type has different CoV. Both full-length S protein and its antigenic fragments,
advantages and disadvantages. For instance, inactivated and live- including S1 subunit, NTD, RBD, and S2 subunit, can serve as
attenuated virus-based vaccines are vaccine types developed important targets for the development of subunit vaccines (Guo
using the most traditional approaches. Although they generally et al., 2005; Mou et al., 2013; Wang et al., 2015; Jiaming et al.,
induce highly potent immune responses and/or protection, the 2017; Zhou et al., 2018).
possibility for incomplete inactivation of viruses or recovering Although subunit vaccines based on the full-length S protein
virulence exists, resulting in significant safety concerns (Zhang may elicit potent immune responses and/or protection, studies
et al., 2014). Also, these traditional vaccines may induce the have found that antibodies induced by some of these vaccines
antibody-dependent enhancement (ADE) effect, as in the case mediate enhancement of viral infection in vitro, as in the
of SARS-CoV infection (Luo et al., 2018b). Similarly, some case of SARS-CoV (Kam et al., 2007; Jaume et al., 2012),
viral-vectored vaccines can elicit specific antibody and cellular raising safety concerns for the development of full-length S
immune responses with neutralizing activity and protection, but protein-based subunit vaccines against SARS-CoV and MERS-
they might also induce anti-vector immunity or present pre- CoV. In contrast, RBD-based subunit vaccines comprise the
existing immunity, causing some harmful immune responses. major critical neutralizing domain (Du and Jiang, 2015; Zhou
Instead, DNA and nanoparticle vaccines maintain strong et al., 2019). Therefore, these vaccines may generate potent
safety profile; however, the immunogenicity of these vaccines neutralizing antibodies with strong protective immunity against
is usually lower than that of virus- or viral vector-based viral infection. S1 subunit, for example, is much shorter than
vaccines, often requiring optimization of sequences, components, the full-length S protein, but it is no less able to induce strong
or immunization routes, inclusion of appropriate adjuvants, immune responses and/or protection against viral infection (Li
or application of combinational immunization approaches et al., 2013; Adney et al., 2019). Thus, this fragment can be used
(Zhang et al., 2014). as an alternative target for subunit vaccine development. Despite
their ability to induce immune responses and/or neutralizing
antibodies, NTD and S2 as the targets of subunit vaccines are
less immunogenic, eliciting significantly lower antibody titers,
SUBUNIT VACCINES AGAINST cellular immune responses, and/or protection than the other
SARS-CoV AND MERS-CoV regions, such as full-length, S1, and RBD (Guo et al., 2005;
Jiaming et al., 2017). Therefore, in terms of safety and efficacy,
Subunit vaccines are vaccines developed based on the synthetic the RBD and/or S1 of S protein could be applied as critical
peptides or recombinant proteins. Unlike inactivated or live- targets for the development of subunit vaccine candidates against
attenuated virus and some viral vectored vaccines, this vaccine SARS-CoV, MERS-CoV, SARSr-CoV, and MERSr-CoV. Because
type mainly contains specific viral antigenic fragments, but of its conserved amino acid sequences and high homology among
without including any components of infectious viruses, different virus strains (Elshabrawy et al., 2012; Zhou et al., 2018),
eliminating the concerns of incomplete inactivation, virulence the S2 subunit has potential to be used as a target for the
recovery, or pre-existing immunity (Du et al., 2008; Deng development of universal vaccines against divergent virus strains.
et al., 2012). Similar to DNA or VLP-based vaccines, subunit In addition to the S protein, the N protein of SARS-CoV
vaccines are generally safe without causing potential harmful and MERS-CoV may serve as an additional target for the
immune responses, making them promising vaccine candidates. development of subunit vaccines. Unlike S protein, the N protein
Moreover, subunit vaccines may target specific, well-defined has no ability to elicit neutralizing antibodies to block virus-
neutralizing epitopes with improved immunogenicity and/or receptor interaction and neutralize viral infection, but it may
efficacy (Du et al., 2008; Zhang et al., 2014). induce specific antibody and cellular immune responses (Liu
A number of subunit vaccines against SARS-CoV and et al., 2006; Zheng et al., 2009). Several immunodominant B-cell
MERS-CoV have been developed, and these are described and T-cell epitopes have been identified in the N protein of SARS-
in detail in the next paragraphs. The targets used for the CoV and MERS-CoV, some of which are conserved in mice,
development of SARS-CoV and MERS-CoV subunit vaccines are non-human primates, and humans (Liu et al., 2006; Chan et al.,
also be discussed. 2011; Veit et al., 2018). Other proteins, such as M protein, can be

used as potential targets of SARS-CoV and MERS-CoV subunit expressed in Chinese hamster ovary (CHO) cells bound strongly
vaccines. Notably, SARS-CoV M protein-derived peptides have to RBD-specific monoclonal antibodies (mAbs), elicited high-
immunogenicity to induce high-titer antibody responses in the titer anti-SARS-CoV neutralizing antibodies, and protected most,
immunized animals (He et al., 2005b), suggesting the potential or all, of the SARS-CoV-challenged mice, with undetectable viral
for utilizing this protein to develop subunit vaccines. RNA and undetectable or significantly reduced viral load (Du
et al., 2009c, 2010). Significantly, a 293T cell-expressed RBD
Subunit Vaccines Against SARS-CoV protein maintains excellent conformation and good antigenicity
Numerous subunit vaccines against SARS-CoV have been to bind SARS-CoV RBD-specific neutralizing mAbs. It elicited
developed since the outbreak of SARS, the majority of which use highly potent neutralizing antibodies that completely protected
the S protein and/or its antigenic fragments, in particular, RBD, immunized mice against SARS-CoV challenge (Du et al., 2009b).
as the vaccine target (Table 1). Particularly, RBDs from the S proteins of Tor2, GD03, and SZ3,
representative strains of SARS-CoV isolated from human 2002–
SARS-CoV Subunit Vaccines Based on Full-Length S 2003, 2003–2004, and palm civet strains, can induce high-titer
Protein cross-neutralizing antibodies against pseudotyped SARS-CoV
Subunit vaccines based on SARS-CoV S protein, including full- expressing respective S proteins (He et al., 2006c). Different
length or trimeric S protein, are immunogenic with protection from the full-length S protein-based SARS subunit vaccines, no
against SARS-CoV infection (He et al., 2006a; Kam et al., obvious pathogenic effects have been identified in the RBD-based
2007; Li et al., 2013). Either insect cell-expressed full-length SARS subunit vaccines (Kam et al., 2007; Jaume et al., 2012).
(FL-S) or extracellular domain (EC-S) SARS-CoV S protein
developed high-titer S-specific antibodies with neutralizing SARS-CoV Subunit Vaccines Based on Non-RBD S
activity against pseudotyped SARS-CoV expressing S protein of Protein Fragments
representative SARS-CoV human and palm civet strains (Tor2, SARS subunit vaccines based on S protein fragments (S1 and
GD03, and SZ3) isolated during the 2002 and 2003 or 2003 S2), other than the RBD, have shown immunogenicity and/or
and 2004 outbreaks (He et al., 2006a). In addition, full-length protective efficacy against SARS-CoV infection (Guo et al., 2005;
S-ectodomain proteins fused with or without a foldon trimeric Li et al., 2013). For example, recombinant S1 proteins fused with
motif (S or S-foldon) could elicit specific antibody responses or without foldon elicited specific antibodies with neutralizing
and neutralizing antibodies, protecting immunized mice against activity that protected immunized mice against high-dose SARS-
SARS-CoV challenge with undetectable virus titers in the lungs CoV challenge (Li et al., 2013). Although some studies have
(Li et al., 2013). Moreover, a subunit vaccine (triSpike) based demonstrated that recombinant SARS-CoV S2 (residues 681–
on a full-length S protein trimer induced specific serum and 980) protein elicits specific non-neutralizing antibody response in
mucosal antibody responses and efficient neutralizing antibodies mice (Guo et al., 2005), others have indicated that mAbs targeting
against SARS-CoV infection (Kam et al., 2007). Nevertheless, this highly conserved heptad repeat 1 (HR1) and HR2 domains of
vaccine also resulted in Fcγ receptor II (FcγRII)-dependent and SARS-CoV S protein have broad neutralizing activity against
ACE2-independent ADE, particularly in human monocytic or pseudotyped SARS-CoV expressing S protein of divergent strains
lymphoblastic cell lines infected with pseudotyped SARS-CoV (Elshabrawy et al., 2012), indicating the potential of utilizing the
expressing viral S protein, or in Raji B cells (B-cell lymphoma S2 region as a broad-spectrum anti-SARS-CoV vaccine target
line) infected with live SARS-CoV (Kam et al., 2007; Jaume et al., (Zheng et al., 2009).
2012), raising significant concerns over the use of full-length S
protein as a SARS vaccine target. SARS-CoV Subunit Vaccines Based on Non-S
Structural Proteins
SARS-CoV Subunit Vaccines Based on RBD Subunit vaccines based on the N and M proteins of SARS-
SARS-CoV RBD contains multiple conformation-dependent CoV have shown immunogenicity in vaccinated animals (Liu
epitopes capable of eliciting high-titer neutralizing antibodies; et al., 2006; Zheng et al., 2009). Studies have revealed
thus, it is a major target for the development of SARS vaccines that a plant-expressed SARS-CoV N protein conjugated with
(He et al., 2004, 2005a; Jiang et al., 2012; Zhu et al., 2013). Subunit Freund’s adjuvant elicited specific IgG antibodies, including
vaccines based on the SARS-CoV RBD have been extensively IgG1 and IgG2a subtypes, and cellular immune responses in
explored. Studies have found that a fusion protein containing mice, whereas another E. coli-expressed N protein conjugated
RBD and the fragment crystallizable (Fc) region of human IgG1 with Montanide ISA-51 and cysteine-phosphate-guanine (CpG)
(RBD-Fc) elicited highly potent neutralizing antibodies against adjuvants induced specific IgG antibodies toward a Th1
SARS-CoV in the immunized rabbits and mice, which strongly (IgG2a)-type response in mice (Liu et al., 2006; Zheng et al.,
blocked the binding between S1 protein and SARS-CoV receptor 2009). Although N-specific antibodies have been detected in
ACE2 (He et al., 2004). This RBD protein induced long-term, convalescent-phase SARS patient and immunized rabbit sera,
high-level SARS-CoV S-specific antibodies and neutralizing they have no neutralizing activity against SARS-CoV infection
antibodies that could be maintained for 12 months after (Qiu et al., 2005). In addition, immunodominant M protein
immunization, protecting most of the vaccinated mice against peptides (M1-31 and M132-161) identified using convalescent-
SARS-CoV infection (Du et al., 2007). In addition, recombinant phase sera of SARS patients and immunized mouse and rabbit
RBDs (residues 318–510 or 318–536) stably or transiently sera have immunogenicity to elicit specific IgG antibodies in

Frontiers in Microbiology | www.frontiersin.org
Wang et al.
TABLE 1 | Subunit Vaccines against SARS-CoVa .
Name Antigenicity and Adjuvant Route Animal Antibody response Cellular immune Protection References
functionality models response
Subunit vaccines based on SARS-CoV full-length or trimeric S protein

FL-S and EC-S Bind to SARS-CoV MPL + TDM S.C. BALB/c mice Elicit SARS-CoV S-specific Abs N/A N/A He et al., 2006a
proteins S1, NTD, RBD, and (IgG, > 1: 2 × 105 ), neutralizing
S2-specific mAbs (> 1:2.4 × 104 ) pseudotyped
SARS-CoV (Tor2, GD03, and
SZ3 strains)
S andS-foldon N/A TiterMax Gold; S.C. or I.M. BALB/c mice Elicit SARS-CoV S-specific Abs N/A Protect vaccinated Li et al., 2013
proteins Alum Hydro (IgG, > 1:104 ) in mice, mice from challenge of
+ MPL neutralizing (∼2.4 × 102 for S; SARS-CoV (Urbani
∼1:7 × 102 for S-foldon) live strain, 105 TCID50 ) with
SARS-CoV (Urbani strain) undetectable viral load
in lungs
triSpike protein N/A Alum hydro I.P. or S.C. BALB/c mice; Elicits SARS-CoV S-specific N/A Protects vaccinated Kam et al., 2007;
Hamsters mucosal and serum Abs (IgA hamsters from Jaume et al., 2012
and IgG) in mice and hamsters, challenge of SARS-CoV
blocking S-ACE2 receptor (Urbani strain, 103
binding and neutralizing live TCID50 ) with
SARS-CoV (HKU-39849 strain); undetectable or
induces ADE reduced viral load in
lungs
8
Subunit vaccines based on SARS-CoV RBD protein

RBD-Fc protein N/A Freund’s I.D. orI.M. BALB/c mice; Elicits SARS-CoV N/A Protects majority (4/5) He et al., 2004; Du
Rabbits S/RBD-specific Abs (IgG) in of vaccinated mice et al., 2007
mice and rabbits, neutralizing from challenge of
pseudotyped SARS-CoV (BJ01
(rabbits: ≥ 7.3 × 104 ) and live strain, 106 TCID50 ),
(mice: 1:4 × 103 ; with one mouse
rabbits: > 1:1.5 × 104 ) showing mild alveolar
SARS-CoV (BJ01 strain) damage in lungs
RBD193-CHO; Binds to Freund’s S.C. BALB/c mice Elicit SARS-CoV RBD-specific Induce SARS-CoV Protect all (for Du et al., 2009c,
RBD219-CHO SARS-CoV Abs, neutralizing pseudotyped RBD-specific RBD219-CHO) or 2010
proteins RBD-specific mAbs (< 1:104 for RBD193-CHO; cellular immune majority (3/5, for
(neutralizing 24H8, 1:5.8 × 104 for RBD219-CHO) responses (IFN-γ, RBD219-CHO) of
31H12, 35B5, and live (< 1:103 for IL-2, IL-4, IL-10) in vaccinated mice from
Subunit Vaccines Against Coronaviruses

February 2020 | Volume 11 | Article 298
33G4, 19B2; RBD193-CHO; 1:103 for mice challenge of SARS-CoV

non-neutralizing RBD219-CHO) SARS-CoV (GZ50 strain, 100
17H9) (GZ50 strain) TCID50 for
RBD193-CHO; 5 × 105
TCID50 for
RBD219-CHO) with
undetectable viral RNA
or no, to reduced, viral
load in lungs
(Continued)
Wang et al.
TABLE 1 | Continued
Name Antigenicity and Adjuvant Route Animal Antibody response Cellular immune Protection References
functionality models response
RBD-293T Binds to SAS S.C. BALB/c mice Elicits SARS-CoV RBD-specific N/A Protects all vaccinated Du et al., 2009b
protein SARS-CoV Abs (IgG), neutralizing mice from challenge of
RBD-specific mAbs pseudotyped (1:6.9 × 105 ) and SARS-CoV (GZ50
(neutralizing 24H8, live (1:1.6 × 103 ) SARS-CoV strain, 100 TCID50 ) with
31H12, 35B5, (GZ50 strain) undetectable viral RNA
33G4, 19B2; and viral load in lungs
non-neutralizing
17H9)
S318-510 N/A Alum; Alum + CpG S.C. 129S6/SvEv Elicits SARS-CoV-specific Abs Induces SARS-CoV N/A Zakhartchouk et al.,
protein mice (IgG, IgG1, and IgG2a) in mice. S-specific cellular 2007
Reduces neutralization after immune responses
removing glycosylation (IFN-γ) in mice
Subunit vaccines based on non-RBD SARS-CoV S protein fragments
S1 and N/A TiterMax Gold; S.C. or I.M. BALB/c mice Elicit SARS-CoV S-specific Abs N/A Protect vaccinated Li et al., 2013
S1-foldon Alum Hydro + MPL (IgG, > 1:104 ) in mice, mice from challenge of
proteins neutralizing (1:1.7 × 102 for S1; SARS-CoV (Urbani
1:90 for S1-foldon) live strain, 105 TCID50 ) with
SARS-CoV (Urbani strain) undetectable viral load
in lungs
9
S2 protein N/A Freund’s S.C. BALB/c mice Elicits SARS-CoV S2-specific Induces SARS-CoV N/A Guo et al., 2005
Abs (IgG, 1:1.6 × 103 ) in mice S2-specific cellular
with no neutralizing activity immune responses
(IFN-γ and IL-4) in
mice
Subunit vaccines based on SARS-CoV non-S structural proteins (i.e. N and M)
rN protein N/A Freund’s I.P. BALB/c mice Elicits SARS-CoV N-specific Induces cellular N/A Zheng et al., 2009
Abs (IgG (1:1.8 × 103 ), IgG1, immune responses
and IgG2a) in mice with up-regulated
IFN-γ and IL-10
cytokines in mice
rN protein N/A Montanide + CpG; S.C. BALB/c mice Elicits SARS-CoV N-specific Induces SARS-CoV N/A Liu et al., 2006
Freund’s Abs (IgG) in mice N-specific cellular

immune responses
(IFN- γ) in mice
M1-31 and Bind to sera from Freund’s I.D. BALB/c mice; Induce SARS-CoV M-specific N/A N/A He et al., 2005b
M132-161 SARS patients or NZW rabbits Abs (IgG) in rabbits
peptides immunized mice
and rabbits
a Abs,antibodies; ADE, antibody-dependent enhancement; Alum hydro, aluminum hydroxide; CHO, Chinese hamster ovary; CpG, cysteine-phosphate-guanine; I.D., intradermal; I.M., intramuscular; IFN-γ , interferon
gamma; IL-2, interleukin 2; IL-4, interleukin 4; IL-10, Interleukin 10; I.P., intraperitoneal; mAbs, monoclonal antibodies; Montanide, Montanide ISA-51; MPL + TDM, monophosphoryl lipid A and trehalose dicorynomycolate;
N/A, not reported; NTD, N-terminal domain; NZW rabbits, New Zealand White rabbits; RBD, receptor-binding domain; SAS, Sigma adjuvant system; S.C., subcutaneous; TCID50 , median tissue culture infectious dose.
rabbits (He et al., 2005b). In spite of their immunogenicity, antibody and interferon gamma (IFN-γ) secretion than the RBD
it appears that these N- and M-based SARS subunit vaccines with Alum alone (Zakhartchouk et al., 2007).
have not been investigated for their protective efficacy against
SARS-CoV infection. Thus, it is unclear whether these non- Subunit Vaccines Against MERS-CoV
S structural protein-based SARS subunit vaccines can prevent Subunit vaccines against MERS-CoV have been developed
SARS-CoV infection. extensively, almost all of which are based on the S protein,
including full-length S timer, NTD, S1, and S2, particularly
Potential Factors Affecting SARS-CoV Subunit RBD. These subunit vaccines, including their antigenicity,
Vaccines functionality, immunogenicity, and protective efficacy in
A number of factors may affect the expression of proteins different animal models, are summarized in Table 2.
to be used as SARS subunit vaccines; apart from their
immunogenicity and/or protective efficacy. Understanding of MERS-CoV Subunit Vaccines Based on Full-Length S
these factors is important to generate subunit vaccines with good Protein
quality, high immunogenicity, and excellent protection against Subunit vaccines based on the full-length S protein cover both
SARS-CoV infection. RBD and non-RBD neutralizing epitopes, some of which may
The expression of recombinant protein-based SARS subunit be located in the conserved S2 subunit; thus this type of
vaccines may be changed by the following factors. First, addition subunit vaccines are expected to induce high-titer neutralizing
of an intron splicing enhancer to the truncated SARS-CoV antibodies. Although several MERS-CoV full-length S protein-
S protein fragments results in better enhancement of protein based vaccines have been reported in other vaccine types,
expression in mammalian cells than the exon splicing enhancers, including viral vectors and DNAs (Wang et al., 2015; Wang C.
and different cells may result in different fold increase of et al., 2017; Haagmans et al., 2016; Zhou et al., 2018), only
protein expression (Chang et al., 2006). Second, inclusion of a few subunit vaccines have been developed that rely on
a post-transcriptional gene silencing suppressor p19 protein the full-length S protein. For example, a recombinant MERS-
from tomato bushy stunt virus to a SARS-CoV N protein CoV S protein trimer (MERS S-2P) in prefusion conformation
may significantly increase its transient expression in tobacco binds to the DPP4 receptor, as well as to the MERS-CoV
(Zheng et al., 2009). NTD, RBD, and S2-specific neutralizing mAbs (Pallesen et al.,
The following factors may affect the immunogenicity and 2017). Whereas this protein induces neutralizing antibodies
protective efficacy of protein-based SARS subunit vaccines, in mice against divergent pseudotyped MERS-CoV in vitro,
including same proteins expressed in different expression its in vivo protective activity against MERS-CoV infection
systems, and same proteins with various lengths, amino acid is unknown (Pallesen et al., 2017). Therefore, more studies
mutations, or deletions (He et al., 2006b; Du et al., 2009b). are needed to elucidate the potential for the development
For example, RBD proteins containing different lengths (193- of MERS-CoV full-length S-based subunit vaccines, including
mer: RBD193-CHO or 219-mer: RBD219-CHO) elicited different understanding their protective efficacy and identifying possible
immune responses and protective efficacy against SARS-CoV harmful immune responses.
challenge (Du et al., 2009c, 2010). A recombinant SARS-CoV
RBD (RBD-293T) protein expressed in mammalian cell system MERS-CoV Subunit Vaccines Based on RBD
was able to induce stronger neutralizing antibody response than Numerous MERS-CoV RBD-based subunit vaccines have been
those expressed in insect cells (RBD-Sf9) and E. coli (RBD- developed and extensively evaluated in available animal models
Ec) (Du et al., 2009b), suggesting that RBD purified from since the emergence of MERS-CoV (Table 2) (Du et al., 2013c;
mammalian cells has preference for further development due Tai et al., 2017; Zhou et al., 2018). In general, these subunit
to its ability to maintain native conformation. Notably, a single vaccines have strong immunogenicity and are capable of inducing
mutation (R441A) in the RBD of SARS-CoV disrupted its major high neutralizing antibodies and/or protection against MERS-
neutralizing epitopes and affinity to bind viral receptor ACE2, CoV infection (Ma et al., 2014b; Zhang et al., 2016; Tai et al.,
thus abolishing the vaccine’s immunogenicity, and hence, its 2017; Wang Y. et al., 2017). Most subunit vaccines based on the
ability to induce neutralizing antibodies in immunized animals MERS-CoV RBD have been described in detail in a previous
(He et al., 2006b). Additionally, deletion of a particular amino review article (Zhou et al., 2019). In this section, we will briefly
acid by changing a glycosylation site in the SARS-CoV RBD introduce these RBD-targeting MERS vaccines, and compare
(RBD219-N1) also resulted in the alteration of subunit vaccine’s their functionality, antigenicity, immunogenicity, and protection
immunogenicity (Chen et al., 2014). against MERS-CoV infection.
Other factors that potentially affect the immunogenicity Co-crystallographic analyses of MERS-CoV RBD and/or
of SARS subunit vaccines include immunization routes and RBD/DPP4 complexes have confirmed that the RBD is attributed
adjuvants (Zakhartchouk et al., 2007; Li et al., 2013). Significantly to residues 367–588 (Chen et al., 2013) or 367–606 (Lu et al.,
high-titer antibodies were induced by monomeric or trimeric 2013) in the MERS-CoV S1 subunit. Indeed, a recombinant
SARS-CoV S and S1 proteins through the intramuscular (I.M.) MERS-CoV RBD (rRBD) fragment (residues 367–606) elicits
route compared to the subcutaneous (S.C.) route (Li et al., RBD-specific antibody and cellular immune responses and
2013). Moreover, a SARS-CoV RBD subunit vaccine conjugated neutralizing antibodies in mice and/or non-human primates
with Alum plus CpG adjuvants elicited a higher level of IgG2a (NHPs) (Lan et al., 2014, 2015). However, it only partially protects

Wang et al.
TABLE 2 | Subunit Vaccines against MERS-CoVa .
Name Functionality and Adjuvant Route Animal Antibody response Cellular immune Protective efficacy References
antigenicity models response
Subunit vaccines based on MERS-CoV full-length S protein

MERS S-2P Binds to DPP4 SAS I.M. BALB/c mice Elicits neutralizing Abs in mice, N/A N/A Pallesen et al.,
protein receptor and neutralizing 7 pseudotyped 2017
MERS-CoV S-NTD, MERS-CoV
RBD, and
S2-specific
neutralizing mAbs
(G2, D12, and G4,
respectively)
Subunit vaccines based on MERS-CoV RBD protein
rRBD N/A Alum I.M. or S.C. BALB/c mice; Elicits MERS-CoV RBD-specific Induces Partially protects Lan et al., 2014,
(S367-606) Hydro + CpG NHPs Abs in mice (IgG, IgG1, IgG2a, MERS-CoV vaccinated NHPs from 2015
protein or poly(I:C); and IgG2b) and NHPs (IgG), RBD-specific challenge of
IFA + CpG neutralizing pseudotyped cellular immune MERS-CoV (EMC2012
(mouse); (mouse: < 1:5 × 102 ) and live responses (IFN-γ, strain, 6.5 × 107
Alum (NHPs) (NHPs: < 1:5 × 102 ) TNF-α, IL-2, IL-4, TCID50 ) with alleviated
MERS-CoV (EMC2012 strain) IL-6, and IL-10) in pneumonia and
mice and/or decreased viral load
monkeys
RBD Binds to DPP4 Poly(I:C); I.N. or S.C. BALB/c mice Elicits MERS-CoV S1- and Induces N/A Du et al., 2013c;
(S377-662)-Fc receptor Montanide RBD-specific Abs (IgA, IgG MERS-CoV Ma et al., 2014b
11
protein (> 1:104 ), IgG1, IgG2a, and S1-specific cellular

IgG3) in mice, neutralizing immune responses
(≥ 1:2.4 × 102 ) live MERS-CoV (IFN-γ and IL-2) in
(EMC2012 strain) mice
RBD Binds to DPP4 Montanide; I.M. or S.C. BALB/c mice; Elicits MERS-CoV S1 and Induces Protects vaccinated Du et al., 2013a;
(S377-588)-Fc receptor and MF59; hDPP4-Tg RBD-specific Abs in mice (IgG MERS-CoV Ad5/hDPP4- Ma et al., 2014b;
protein MERS-CoV RBD AddaVax mice; (> 1:105 ), IgG1, and IgG2a) S1-specific cellular transduced BALB/c Tang et al., 2015;
specific neutralizing Rabbits and rabbits (IgG), neutralizing immune responses mice and majority (4/6) Zhang et al., 2016;
mAbs (Mersmab1, 17 pseudotyped (≥ 1:104 ) and (IFN-γ and IL-2) in of vaccinated Nyon et al., 2018
m336, m337, and 2 live (≥ 1:103 ) MERS-CoV mice hDPP4-Tg mice from
m338) (EMC2012 and London1-2012 MERS-CoV (EMC2012
strains) strain, 105 PFU for
BALB/c; 103−4 TCID50
for Tg) challenge,

without immunological
toxicity or eosinophilic
immune enhancement
RBD-Fd protein Binds to DPP4 MF59; I.M. or S.C. BALB/c mice; Elicits MERS-CoV S1-specific N/A Protects majority (5/6) Tai et al., 2016
receptor and Alum hDPP4-Tg mice Abs (IgG (> 1:105 ), IgG1, and of vaccinated
MERS-CoV IgG2a) in mice, neutralizing at hDPP4-Tg mice from
RBD-specific least 9 pseudotyped (> 1:104 ) challenge of
neutralizing mAbs and live (> 1:103 ) MERS-CoV MERS-CoV (EMC2012
(Mersmab1, m336, (EMC2012 strain) strain, 104 TCID50 )
m337, and m338)
(Continued)
Wang et al.
TABLE 2 | Continued
Name Functionality and Adjuvant Route Animal Antibody response Cellular immune Protective efficacy References
antigenicity models response
RBD (T579N) Binds to receptor Montanide; I.M. or S.C. BALB/c mice; Elicits neutralizing Abs N/A Protects all vaccinated Du et al., 2016a
protein DPP4 and Alum hDPP4-Tg mice (> 1:3 × 103 ) in mice against hDPP4-Tg mice from
MERS-CoV live MERS-CoV (EMC2012 challenge of
RBD-specific strain) MERS-CoV (EMC2012
neutralizing mAbs strain, 104 TCID50 )
(hMS-1, m336,
m337, and m338)
Subunit vaccines based on non-RBD MERS-CoV S protein fragments
S1 protein N/A Ribi; I.M. BALB/c mice; Elicits MERS-CoV S1-specific N/A Protects vaccinated Wang et al., 2015
Alum pho NHPs Abs in mice (IgG and IgG1) and NHPs from challenge of
NHPs (IgG), neutralizing 8 MERS-CoV (JordanN3
pseudotyped and live strain, 5 × 106 PFU)
MERS-CoV (JordanN3 strain) with reduced
abnormalities on chest
CT
S1 protein N/A Advax + SAS I.M. Dromedary Elicits neutralizing Abs in N/A Protects vaccinated Adney et al., 2019
camels; dromedary camels (≥ 1:80) and dromedary camels and
Alpacas alpacas (≥ 1:6.4 × 102 ) against alpacas from challenge
live MERS-CoV (EMC2012 of MERS-CoV
strain) (EMC2012 strain, 107
12
TCID50 ) with reduced

and delayed viral
shedding in the upper
airways (in camels) or
complete protection (in
alpacas)
rNTD protein N/A Alum I.M. BALB/c mice; Elicits MERS-CoV Induces Protects vaccinated Jiaming et al., 2017
pho + CpG Ad5-hDPP4 S-NTD-specific Abs MERS-CoV Ad5-hDPP4-
mice (IgG, ≥ 1:104 ) in mice, S-NTD-specific transduced mice from
neutralizing pseudotyped and cellular immune challenge of
live (1:40) MERS-CoV responses (IFN-γ, MERS-CoV (EMC2012
(EMC2012 strain) IL-2, IL-6, IL-10, strain, 105 PFU) with
and IL-17A) in mice reduced lung
abnormalities and

respiratory tract
pathology
SP3 peptide N/A Freund’s N/A BALB/c mice; Elicits MERS-CoV S-specific N/A N/A Yang et al., 2014a
(aa736-761) NZW rabbits Abs (IgG, 1:104 ) in rabbits,
neutralizing pseudotyped
MERS-CoV
a aa,amino acid; Abs, antibodies; Ad5, adenovirus serotype 5; Ad5-hDPP4 mice, Ad5-hDPP4-transuced mice; Alum hydro, aluminum hydroxide; Alum pho, Aluminum phosphate; hDPP4, human dipeptidyl peptidase
4; hDPP4-Tg mice, transgenic mice expressing MERS-CoV receptor human DPP4; IFA, incomplete Freund’s adjuvant; I.M., intramuscular; I.N., intranasal; mAbs, monoclonal antibodies; Montanide, Montanide ISA51;
N/A, not reported; NHPs, non-human primates; NZW, rabbits, New Zealand White rabbits; PFU, plaque-forming unit; rRBD, recombinant RBD; SAS, Sigma Adjuvant System; S.C., subcutaneous; TCID50 , median tissue
culture infectious dose; TNF-α, tumor necrosis factor (TNF)-alpha.
NHPs from MERS-CoV infection by alleviating pneumonia and that protection of MERS-CoV infection is positively correlated
clinical manifestations, as well as decreasing viral load (Lan et al., with serum neutralizing antibody titers (Adney et al., 2019).
2015). In addition, an RBD protein fragment containing MERS- Moreover, immunization with a recombinant MERS-CoV NTD
CoV S residues 377–622 fused with the Fc tag of human IgG can protein (rNTD) can induce neutralizing antibodies and cell-
induce MERS-CoV S1- and/or RBD-specific humoral and cellular mediated responses, protecting Ad-hDPP4-transduced mice
immune responses in the immunized mice with neutralizing against MERS-CoV challenge (Jiaming et al., 2017). Notably,
activity against MERS-CoV infection (Du et al., 2013c; Jiang specific antibodies with neutralizing activity have been elicited
et al., 2013). However, after comparing several versions of MERS- by a S2 peptide sequence (residues 736–761) of MERS-CoV
CoV RBD fragments with different lengths, it was found that in rabbits (Yang et al., 2014a), but the protective efficacy
a truncated RBD (residues 377–588) had the highest DPP4- of this peptide vaccine is unknown. The above reports
binding affinity and induced the highest-titer IgG antibodies and demonstrate the potential for the development of MERS
neutralizing antibodies against MERS-CoV, identifying its role as subunit vaccines based on the non-RBD fragments of MERS-
a critical neutralizing domain (Ma et al., 2014b). CoV S protein.
Subsequently, several MERS-CoV subunit vaccines have been
designed based on the identified critical neutralizing domain of MERS-CoV Subunit Vaccines Based on Non-S
RBD fragment, including those expressed in a stable CHO cell Structural Proteins
line (S377-588-Fc), fusing with a trimeric motif foldon (RBD- Unlike SARS subunit vaccines which have been designed based
Fd), or containing single or multiple mutations in the RBD of on viral N and M proteins, it appears that very few subunit
representative human and camel strains from the 2012–2015 vaccines have been developed based on the non-S structural
MERS outbreaks (Tai et al., 2016, 2017; Nyon et al., 2018). protein(s) of MERS-CoV. One study reports the induction
These RBD proteins maintain good conformation, functionality, of specific antibodies by MERS-CoV N peptides (Yang et al.,
antigenicity, and immunogenicity, with ability to bind the DPP4 2014a), and another report shows that N protein is used for
receptor and RBD-specific neutralizing mAbs and to elicit robust development of vaccines based on viral vector Vaccinia virus,
neutralizing antibodies cross-neutralizing multiple strains of modified Vaccinia Ankara (MVA) (Veit et al., 2018). This
MERS pseudoviruses and live MERS-CoV (Tai et al., 2016, 2017; may be potentially a consequence of the weak immunogenicity
Nyon et al., 2018). It is noted that the wild-type MERS-CoV RBD and/or protective efficacy of non-S structural proteins, further
proteins consisting of the identified critical neutralizing domain confirming the role of MERS-CoV S protein as the key target for
confer partial protection of hDPP4-transgenic (hDPP4-Tg) mice the development of MERS vaccines, including subunit vaccines.
from MERS-CoV infection without causing immunological
toxicity or eosinophilic immune enhancement (Tai et al., 2016; Potential Factors Affecting MERS-CoV Subunit
Wang Y. et al., 2017; Nyon et al., 2018); nevertheless, a Vaccines
structurally designed mutant version of such RBD protein with Similar to SARS-CoV subunit vaccines, the immunogenicity
a non-neutralizing epitope masked (T579N) preserves intact and/or protection of MERS-CoV subunit vaccines may also be
conformation and significantly improves overall neutralizing affected by a number of factors, such as antigen sequences,
activity and protective efficacy, resulting in the full protection fragment lengths, adjuvants, vaccination pathways, antigen doses,
of hDPP4-Tg mice against high-dose MERS-CoV challenge immunization doses and intervals used.
(Du et al., 2016a). As described above, MERS-CoV subunit vaccines containing
The above studies indicate that protein lengths to be chosen different antigens or fragment lengths, particularly those based
as MERS-CoV subunit vaccines and/or structure-based vaccine on the RBD, have apparently variable immunogenicity and/or
design can impact on the immunogenicity and/or protection of protective efficacy, and a critical neutralizing domain that
RBD-based subunit vaccines. contains an RBD fragment corresponding to residues 377–588 of
S protein elicits the highest neutralizing antibodies among several
MERS-CoV Subunit Vaccines Based on Non-RBD S fragments tested (Ma et al., 2014b; Zhang et al., 2015).
Protein Fragments Adjuvants play an essential role in enhancing host immune
MERS vaccines targeting non-RBD regions of S protein have responses to MERS-CoV subunit vaccines, including those
been developed and investigated in mice and NHPs. It has based on the RBD, and different adjuvants can promote
been shown that a MERS-CoV S1 protein formulated with host immune responses to variant levels (Lan et al., 2014;
Ribi (for mice) or aluminum phosphate (for NHPs) adjuvant Zhang et al., 2016). For example, while a MERS-CoV RBD
elicited robust neutralizing antibodies in mice and NHPs subunit vaccine (S377-588 protein fused with Fc) alone
against divergent strains of pseudotyped and live MERS- induced detectable neutralizing antibody and T-cell responses
CoV, protecting NHPs from MERS-CoV infection (Wang in immunized mice, inclusion of an adjuvant enhanced its
et al., 2015). In addition, MERS-CoV S1 protein adjuvanted immunogenicity. Particularly, among the adjuvants (Freund’s,
with Advax and Sigma Adjuvant System induced low-titer aluminum, Monophosphoryl lipid A, Montanide ISA51 and
neutralizing antibodies in dromedary camels with reduced MF59) conjugated with this RBD protein, MF59 could best
and delayed viral shedding after MERS-CoV challenge, but potentiate the protein to induce the highest-titer anti-S
high-titer neutralizing antibodies in alpacas with complete antibodies and neutralizing antibodies, protecting mice against
protection of viral shedding from viral infection, indicating MERS-CoV infection (Zhang et al., 2016). Moreover, a

recombinant RBD (rRBD) protein plus alum and CpG adjuvants expected that one or several of these promising subunit vaccines
elicited the highest neutralizing antibodies against pseudotyped can be further processed into clinical trials to confirm their
MERS-CoV infection, whereas the strongest T-cell responses immunogenicity against viral infections in humans.
were induced by this protein plus Freund’s and CpG adjuvants
(Lan et al., 2014).
Vaccination pathways are important in inducing efficient RAPID DEVELOPMENT OF SUBUNIT
immune responses, and different immunization routes may elicit VACCINES AGAINST THE NEW
different immune responses to the same protein antigens. For
example, immunization of mice with a MERS-CoV subunit
PATHOGENIC HUMAN CORONAVIRUS
vaccine (RBD-Fc) via the intranasal route induced higher Currently, the newly identified 2019-nCoV is spreading to infect
levels of cellular immune responses and stronger local mucosal people, resulting in significant global concerns. It is critical to
neutralizing antibody responses against MERS-CoV infection rapidly design and develop effective vaccines to prevent infection
than those induced by the same vaccine via the S.C. pathway (Ma of this new coronavirus. Since S protein and its fragments, such
et al., 2014a). In addition, while Freund’s and CpG-adjuvanted as RBD, of SARS-CoV, and MERS-CoV are prime targets for
rRBD protein elicited higher-level systematic and local IFN- developing subunit vaccines against these two highly pathogenic
γ-producing T cells via the S.C. route, this protein adjuvanted human CoVs, it is expected that similar regions of 2019-nCoV
with Alum and CpG induced higher-level tumor necrosis factor- can also be used as key targets for developing vaccines against
alpha (TNF-α) and interleukin 4 (IL-4)-secreting T cells via the this new coronavirus (Jiang et al., 2020). Similarly, other regions
I.M. route (Lan et al., 2014). of 2019-nCoV, including S1 and S2 subunits of S protein and
Antigen dosage, immunization doses, and intervals may N protein, can be applied as alternative targets for vaccine
significantly affect the immunogenicity of MERS-CoV subunit development. Taken together, the approaches and strategies in
vaccines. Notably, a MERS-CoV RBD (S377-588-Fc) subunit the development of subunit vaccines against SARS and MERS
vaccine immunized at 1 µg elicited strong humoral and cellular described in this review will provide important information for
immune responses and neutralizing antibodies in mice although the rapid design and development of safe and effective subunit
the one immunized at 5 and 20 µg elicited a higher level of vaccines against 2019-nCoV infection.
S1-specific antibodies (Tang et al., 2015). In addition, among
the regimens at one dose and two doses at 1-, 2-, and 3-week
intervals, 2 doses of this protein boosted at 4 weeks resulted AUTHOR CONTRIBUTIONS
in the highest antibodies and neutralizing antibodies against
MERS-CoV infection (Wang Y. et al., 2017). SJ and LD conceived the idea and revised and edited the
manuscript. NW and LD collected information and drafted the
manuscript. JS performed the structural analysis. All authors read
POTENTIAL CHALLENGES AND FUTURE and made final approval of the manuscript.
PERSPECTIVES FOR SARS-CoV AND
MERS-CoV SUBUNIT VACCINES
FUNDING
Compared with other vaccine types such as inactivated virus
and viral-vectored vaccines, SARS and MERS subunit vaccines This work was supported by the NIH grants R01AI137472
are much safer and do not cause obvious side effects. However, and R01AI139092.
these subunit vaccines may face some important challenges,
mostly arising from their relatively low immunogenicity, which
must be combined with appropriate adjuvants or optimized for SUPPLEMENTARY MATERIAL
suitable protein sequences, fragment lengths, and immunization
schedules. In addition, structure and epitope-based vaccine The Supplementary Material for this article can be found
design has become a promising strategy to improve the efficacy online at: https://www.frontiersin.org/articles/10.3389/fmicb.
of subunit vaccines. This is evidenced by a structurally designed 2020.00298/full#supplementary-material
MERS-CoV RBD-based protein which has significantly improved
neutralizing activity and protection against MERS-CoV infection FIGURE S1 | Structures of SARS-CoV and MERS-CoV S proteins, RBDs, and
their complexes with respective receptor. Trimeric S proteins of SARS-CoV (PDB:
(Du et al., 2016a). It is prospected that more structure- 5×5b) (A) and MERS-CoV (PDB: 5×5f) (C) are colored differently for each
guided novel strategies will be developed to improve the monomer. Two conformations of the RBD in each trimeric S protein are labeled as
overall immunogenicity and efficacy of subunit vaccines against standing and lying states. The RBDs of each S protein are shown as light blue on
emerging pathogenic human coronaviruses, including those the right panel. ACE2 and DPP4 receptors are respectively modeled to the trimeric
targeting SARS-CoV and MERS-CoV. Although a large number S proteins by match-alignment of SARS-CoV RBD-ACE2 complex (PDB: 2AJF) to
SARS-CoV S trimer (B) or MERS-CoV RBD-DPP4 complex (PDB: 4kr0) to
of SARS and MERS subunit vaccines have been developed with MERS-CoV S trimer (D). Each of the RBD-receptor complexes is shown on the
potent immunogenicity and/or protection in available animal right panel. ACE2, angiotensin-converting enzyme 2; DPP4, dipeptidyl peptidase
models, virtually all remain in the preclinical stage. It is thus 4; RBD, receptor-binding domain; S, spike.

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761–774. doi: 10.1586/14760584.2014.912134 use, distribution or reproduction is permitted which does not comply with these terms.

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Subunit Vaccines Against Emerging

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Subunit vaccines based on SARS-CoV full-length or trimeric S protein

Subunit vaccines based on SARS-CoV RBD protein

Subunit Vaccines Against Coronaviruses

33G4, 19B2; RBD193-CHO; 1:103 for mice challenge of SARS-CoV

Subunit Vaccines Against Coronaviruses

Frontiers in Microbiology | www.frontiersin.org 10 February 2020 | Volume 11 | Article 298

Subunit vaccines based on MERS-CoV full-length S protein

protein (> 1:104 ), IgG1, IgG2a, and S1-specific cellular

Subunit Vaccines Against Coronaviruses

TCID50 ) with reduced

Subunit Vaccines Against Coronaviruses

Frontiers in Microbiology | www.frontiersin.org 13 February 2020 | Volume 11 | Article 298

Frontiers in Microbiology | www.frontiersin.org 14 February 2020 | Volume 11 | Article 298

Frontiers in Microbiology | www.frontiersin.org 15 February 2020 | Volume 11 | Article 298

Frontiers in Microbiology | www.frontiersin.org 16 February 2020 | Volume 11 | Article 298

Frontiers in Microbiology | www.frontiersin.org 17 February 2020 | Volume 11 | Article 298

Frontiers in Microbiology | www.frontiersin.org 18 February 2020 | Volume 11 | Article 298

Frontiers in Microbiology | www.frontiersin.org 19 February 2020 | Volume 11 | Article 298

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