Subunit Vaccines Against MERS-CoV
Subunit vaccines against MERS-CoV have been developed extensively, almost all of which are based on the S protein, including full-length S timer, NTD, S1, and S2, particularly RBD. These subunit vaccines, including their antigenicity, functionality, immunogenicity, and protective efficacy in different animal models, are summarized in Table 2.
TABLE 2  Subunit Vaccines against MERS-CoVa.
Name  Functionality and antigenicity  Adjuvant  Route  Animal models  Antibody response  Cellular immune response  Protective efficacy  References
Subunit vaccines based on MERS-CoV full-length S protein
MERS S-2P protein  Binds to DPP4 receptor and MERS-CoV S-NTD, RBD, and S2-specific neutralizing mAbs (G2, D12, and G4, respectively)  SAS  I.M.  BALB/c mice  Elicits neutralizing Abs in mice, neutralizing 7 pseudotyped MERS-CoV  N/A  N/A  Pallesen et al., 2017
Subunit vaccines based on MERS-CoV RBD protein
rRBD (S367-606) protein  N/A  Alum Hydro + CpG or poly(I:C); IFA + CpG (mouse); Alum (NHPs)  I.M. or S.C.  BALB/c mice; NHPs  Elicits MERS-CoV RBD-specific Abs in mice (IgG, IgG1, IgG2a, and IgG2b) and NHPs (IgG), neutralizing pseudotyped (mouse: < 1:5 × 102) and live (NHPs: < 1:5 × 102) MERS-CoV (EMC2012 strain)  Induces MERS-CoV RBD-specific cellular immune responses (IFN-γ, TNF-α, IL-2, IL-4, IL-6, and IL-10) in mice and/or monkeys  Partially protects vaccinated NHPs from challenge of MERS-CoV (EMC2012 strain, 6.5 × 107 TCID50) with alleviated pneumonia and decreased viral load  Lan et al., 2014, 2015
RBD (S377-662)-Fc protein  Binds to DPP4 receptor  Poly(I:C); Montanide  I.N. or S.C.  BALB/c mice  Elicits MERS-CoV S1- and RBD-specific Abs (IgA, IgG (> 1:104), IgG1, IgG2a, and IgG3) in mice, neutralizing (≥ 1:2.4 × 102) live MERS-CoV (EMC2012 strain)  Induces MERS-CoV S1-specific cellular immune responses (IFN-γ and IL-2) in mice  N/A  Du et al., 2013c; Ma et al., 2014b
RBD (S377-588)-Fc protein  Binds to DPP4 receptor and MERS-CoV RBD specific neutralizing mAbs (Mersmab1, m336, m337, and m338)  Montanide; MF59; AddaVax  I.M. or S.C.  BALB/c mice; hDPP4-Tg mice; Rabbits  Elicits MERS-CoV S1 and RBD-specific Abs in mice (IgG (> 1:105), IgG1, and IgG2a) and rabbits (IgG), neutralizing 17 pseudotyped (≥ 1:104) and 2 live (≥ 1:103) MERS-CoV (EMC2012 and London1-2012 strains)  Induces MERS-CoV S1-specific cellular immune responses (IFN-γ and IL-2) in mice  Protects vaccinated Ad5/hDPP4-transduced BALB/c mice and majority (4/6) of vaccinated hDPP4-Tg mice from MERS-CoV (EMC2012 strain, 105 PFU for BALB/c; 103–4 TCID50 for Tg) challenge, without immunological toxicity or eosinophilic immune enhancement  Du et al., 2013a; Ma et al., 2014b; Tang et al., 2015; Zhang et al., 2016; Nyon et al., 2018
RBD-Fd protein  Binds to DPP4 receptor and MERS-CoV RBD-specific neutralizing mAbs (Mersmab1, m336, m337, and m338)  MF59; Alum  I.M. or S.C.  BALB/c mice; hDPP4-Tg mice  Elicits MERS-CoV S1-specific Abs (IgG (> 1:105), IgG1, and IgG2a) in mice, neutralizing at least 9 pseudotyped (> 1:104) and live (> 1:103) MERS-CoV (EMC2012 strain)  N/A  Protects majority (5/6) of vaccinated hDPP4-Tg mice from challenge of MERS-CoV (EMC2012 strain, 104 TCID50)  Tai et al., 2016
RBD (T579N) protein  Binds to receptor DPP4 and MERS-CoV RBD-specific neutralizing mAbs (hMS-1, m336, m337, and m338)  Montanide; Alum  I.M. or S.C.  BALB/c mice; hDPP4-Tg mice  Elicits neutralizing Abs (> 1:3 × 103) in mice against live MERS-CoV (EMC2012 strain)  N/A  Protects all vaccinated hDPP4-Tg mice from challenge of MERS-CoV (EMC2012 strain, 104 TCID50)  Du et al., 2016a
Subunit vaccines based on non-RBD MERS-CoV S protein fragments
S1 protein  N/A  Ribi; Alum pho  I.M.  BALB/c mice; NHPs  Elicits MERS-CoV S1-specific Abs in mice (IgG and IgG1) and NHPs (IgG), neutralizing 8 pseudotyped and live MERS-CoV (JordanN3 strain)  N/A  Protects vaccinated NHPs from challenge of MERS-CoV (JordanN3 strain, 5 × 106 PFU) with reduced abnormalities on chest CT  Wang et al., 2015
S1 protein  N/A  Advax + SAS  I.M.  Dromedary camels; Alpacas  Elicits neutralizing Abs in dromedary camels (≥ 1:80) and alpacas (≥ 1:6.4 × 102) against live MERS-CoV (EMC2012 strain)  N/A  Protects vaccinated dromedary camels and alpacas from challenge of MERS-CoV (EMC2012 strain, 107 TCID50) with reduced and delayed viral shedding in the upper airways (in camels) or complete protection (in alpacas)  Adney et al., 2019
rNTD protein  N/A  Alum pho + CpG  I.M.  BALB/c mice; Ad5-hDPP4 mice  Elicits MERS-CoV S-NTD-specific Abs (IgG, ≥ 1:104) in mice, neutralizing pseudotyped and live (1:40) MERS-CoV (EMC2012 strain)  Induces MERS-CoV S-NTD-specific cellular immune responses (IFN-γ, IL-2, IL-6, IL-10, and IL-17A) in mice  Protects vaccinated Ad5-hDPP4-transduced mice from challenge of MERS-CoV (EMC2012 strain, 105 PFU) with reduced lung abnormalities and respiratory tract pathology  Jiaming et al., 2017
SP3 peptide (aa736-761)  N/A  Freund’s  N/A  BALB/c mice; NZW rabbits  Elicits MERS-CoV S-specific Abs (IgG, 1:104) in rabbits, neutralizing pseudotyped MERS-CoV  N/A  N/A  Yang et al., 2014a
aaa, 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.

MERS-CoV Subunit Vaccines Based on Full-Length S Protein
Subunit vaccines based on the full-length S protein cover both RBD and non-RBD neutralizing epitopes, some of which may be located in the conserved S2 subunit; thus this type of subunit vaccines are expected to induce high-titer neutralizing antibodies. Although several MERS-CoV full-length S protein-based vaccines have been reported in other vaccine types, including viral vectors and DNAs (Wang et al., 2015; Wang C. et al., 2017; Haagmans et al., 2016; Zhou et al., 2018), only a few subunit vaccines have been developed that rely on the full-length S protein. For example, a recombinant MERS-CoV S protein trimer (MERS S-2P) in prefusion conformation binds to the DPP4 receptor, as well as to the MERS-CoV NTD, RBD, and S2-specific neutralizing mAbs (Pallesen et al., 2017). Whereas this protein induces neutralizing antibodies in mice against divergent pseudotyped MERS-CoV in vitro, its in vivo protective activity against MERS-CoV infection is unknown (Pallesen et al., 2017). Therefore, more studies are needed to elucidate the potential for the development of MERS-CoV full-length S-based subunit vaccines, including understanding their protective efficacy and identifying possible harmful immune responses.

MERS-CoV Subunit Vaccines Based on RBD
Numerous MERS-CoV RBD-based subunit vaccines have been developed and extensively evaluated in available animal models since the emergence of MERS-CoV (Table 2) (Du et al., 2013c; Tai et al., 2017; Zhou et al., 2018). In general, these subunit vaccines have strong immunogenicity and are capable of inducing high neutralizing antibodies and/or protection against MERS-CoV infection (Ma et al., 2014b; Zhang et al., 2016; Tai et al., 2017; Wang Y. et al., 2017). Most subunit vaccines based on the MERS-CoV RBD have been described in detail in a previous review article (Zhou et al., 2019). In this section, we will briefly introduce these RBD-targeting MERS vaccines, and compare their functionality, antigenicity, immunogenicity, and protection against MERS-CoV infection.
Co-crystallographic analyses of MERS-CoV RBD and/or RBD/DPP4 complexes have confirmed that the RBD is attributed to residues 367–588 (Chen et al., 2013) or 367–606 (Lu et al., 2013) in the MERS-CoV S1 subunit. Indeed, a recombinant MERS-CoV RBD (rRBD) fragment (residues 367–606) elicits RBD-specific antibody and cellular immune responses and neutralizing antibodies in mice and/or non-human primates (NHPs) (Lan et al., 2014, 2015). However, it only partially protects NHPs from MERS-CoV infection by alleviating pneumonia and clinical manifestations, as well as decreasing viral load (Lan et al., 2015). In addition, an RBD protein fragment containing MERS-CoV S residues 377–622 fused with the Fc tag of human IgG can induce MERS-CoV S1- and/or RBD-specific humoral and cellular immune responses in the immunized mice with neutralizing activity against MERS-CoV infection (Du et al., 2013c; Jiang et al., 2013). However, after comparing several versions of MERS-CoV RBD fragments with different lengths, it was found that a truncated RBD (residues 377–588) had the highest DPP4-binding affinity and induced the highest-titer IgG antibodies and neutralizing antibodies against MERS-CoV, identifying its role as a critical neutralizing domain (Ma et al., 2014b).
Subsequently, several MERS-CoV subunit vaccines have been designed based on the identified critical neutralizing domain of RBD fragment, including those expressed in a stable CHO cell line (S377-588-Fc), fusing with a trimeric motif foldon (RBD-Fd), or containing single or multiple mutations in the RBD of representative human and camel strains from the 2012–2015 MERS outbreaks (Tai et al., 2016, 2017; Nyon et al., 2018). These RBD proteins maintain good conformation, functionality, antigenicity, and immunogenicity, with ability to bind the DPP4 receptor and RBD-specific neutralizing mAbs and to elicit robust neutralizing antibodies cross-neutralizing multiple strains of MERS pseudoviruses and live MERS-CoV (Tai et al., 2016, 2017; Nyon et al., 2018). It is noted that the wild-type MERS-CoV RBD proteins consisting of the identified critical neutralizing domain confer partial protection of hDPP4-transgenic (hDPP4-Tg) mice from MERS-CoV infection without causing immunological toxicity or eosinophilic immune enhancement (Tai et al., 2016; Wang Y. et al., 2017; Nyon et al., 2018); nevertheless, a structurally designed mutant version of such RBD protein with a non-neutralizing epitope masked (T579N) preserves intact conformation and significantly improves overall neutralizing activity and protective efficacy, resulting in the full protection of hDPP4-Tg mice against high-dose MERS-CoV challenge (Du et al., 2016a).
The above studies indicate that protein lengths to be chosen as MERS-CoV subunit vaccines and/or structure-based vaccine design can impact on the immunogenicity and/or protection of RBD-based subunit vaccines.

MERS-CoV Subunit Vaccines Based on Non-RBD S Protein Fragments
MERS vaccines targeting non-RBD regions of S protein have been developed and investigated in mice and NHPs. It has been shown that a MERS-CoV S1 protein formulated with Ribi (for mice) or aluminum phosphate (for NHPs) adjuvant elicited robust neutralizing antibodies in mice and NHPs against divergent strains of pseudotyped and live MERS-CoV, protecting NHPs from MERS-CoV infection (Wang et al., 2015). In addition, MERS-CoV S1 protein adjuvanted with Advax and Sigma Adjuvant System induced low-titer neutralizing antibodies in dromedary camels with reduced and delayed viral shedding after MERS-CoV challenge, but high-titer neutralizing antibodies in alpacas with complete protection of viral shedding from viral infection, indicating that protection of MERS-CoV infection is positively correlated with serum neutralizing antibody titers (Adney et al., 2019). Moreover, immunization with a recombinant MERS-CoV NTD protein (rNTD) can induce neutralizing antibodies and cell-mediated responses, protecting Ad-hDPP4-transduced mice against MERS-CoV challenge (Jiaming et al., 2017). Notably, specific antibodies with neutralizing activity have been elicited by a S2 peptide sequence (residues 736–761) of MERS-CoV in rabbits (Yang et al., 2014a), but the protective efficacy of this peptide vaccine is unknown. The above reports demonstrate the potential for the development of MERS subunit vaccines based on the non-RBD fragments of MERS-CoV S protein.

MERS-CoV Subunit Vaccines Based on Non-S Structural Proteins
Unlike SARS subunit vaccines which have been designed based on viral N and M proteins, it appears that very few subunit vaccines have been developed based on the non-S structural protein(s) of MERS-CoV. One study reports the induction of specific antibodies by MERS-CoV N peptides (Yang et al., 2014a), and another report shows that N protein is used for development of vaccines based on viral vector Vaccinia virus, modified Vaccinia Ankara (MVA) (Veit et al., 2018). This may be potentially a consequence of the weak immunogenicity and/or protective efficacy of non-S structural proteins, further confirming the role of MERS-CoV S protein as the key target for the development of MERS vaccines, including subunit vaccines.

Potential Factors Affecting MERS-CoV Subunit Vaccines
Similar to SARS-CoV subunit vaccines, the immunogenicity and/or protection of MERS-CoV subunit vaccines may also be affected by a number of factors, such as antigen sequences, fragment lengths, adjuvants, vaccination pathways, antigen doses, immunization doses and intervals used.
As described above, MERS-CoV subunit vaccines containing different antigens or fragment lengths, particularly those based on the RBD, have apparently variable immunogenicity and/or protective efficacy, and a critical neutralizing domain that contains an RBD fragment corresponding to residues 377–588 of S protein elicits the highest neutralizing antibodies among several fragments tested (Ma et al., 2014b; Zhang et al., 2015).
Adjuvants play an essential role in enhancing host immune responses to MERS-CoV subunit vaccines, including those based on the RBD, and different adjuvants can promote host immune responses to variant levels (Lan et al., 2014; Zhang et al., 2016). For example, while a MERS-CoV RBD subunit vaccine (S377-588 protein fused with Fc) alone induced detectable neutralizing antibody and T-cell responses in immunized mice, inclusion of an adjuvant enhanced its immunogenicity. Particularly, among the adjuvants (Freund’s, aluminum, Monophosphoryl lipid A, Montanide ISA51 and MF59) conjugated with this RBD protein, MF59 could best potentiate the protein to induce the highest-titer anti-S antibodies and neutralizing antibodies, protecting mice against MERS-CoV infection (Zhang et al., 2016). Moreover, a recombinant RBD (rRBD) protein plus alum and CpG adjuvants elicited the highest neutralizing antibodies against pseudotyped MERS-CoV infection, whereas the strongest T-cell responses were induced by this protein plus Freund’s and CpG adjuvants (Lan et al., 2014).
Vaccination pathways are important in inducing efficient immune responses, and different immunization routes may elicit different immune responses to the same protein antigens. For example, immunization of mice with a MERS-CoV subunit vaccine (RBD-Fc) via the intranasal route induced higher levels of cellular immune responses and stronger local mucosal neutralizing antibody responses against MERS-CoV infection than those induced by the same vaccine via the S.C. pathway (Ma et al., 2014a). In addition, while Freund’s and CpG-adjuvanted rRBD protein elicited higher-level systematic and local IFN-γ-producing T cells via the S.C. route, this protein adjuvanted with Alum and CpG induced higher-level tumor necrosis factor-alpha (TNF-α) and interleukin 4 (IL-4)-secreting T cells via the I.M. route (Lan et al., 2014).
Antigen dosage, immunization doses, and intervals may significantly affect the immunogenicity of MERS-CoV subunit vaccines. Notably, a MERS-CoV RBD (S377-588-Fc) subunit vaccine immunized at 1 μg elicited strong humoral and cellular immune responses and neutralizing antibodies in mice although the one immunized at 5 and 20 μg elicited a higher level of 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 in the highest antibodies and neutralizing antibodies against MERS-CoV infection (Wang Y. et al., 2017).