Subunit Vaccines Against Sars-CoV and Mers-CoV
Subunit vaccines are vaccines developed based on the synthetic peptides or recombinant proteins. Unlike inactivated or live-attenuated virus and some viral vectored vaccines, this vaccine type mainly contains specific viral antigenic fragments, but without including any components of infectious viruses, eliminating the concerns of incomplete inactivation, virulence recovery, or pre-existing immunity (Du et al., 2008; Deng et al., 2012). Similar to DNA or VLP-based vaccines, subunit vaccines are generally safe without causing potential harmful immune responses, making them promising vaccine candidates. Moreover, subunit vaccines may target specific, well-defined neutralizing epitopes with improved immunogenicity and/or efficacy (Du et al., 2008; Zhang et al., 2014).
A number of subunit vaccines against SARS-CoV and MERS-CoV have been developed, and these are described in detail in the next paragraphs. The targets used for the development of SARS-CoV and MERS-CoV subunit vaccines are also be discussed.

Potential Targets for Development of SARS-CoV and MERS-CoV Subunit Vaccines
The S protein of SARS-CoV and MERS-CoV plays a vital role in receptor binding and membrane fusion. Thus, the S protein, but not other structural proteins, is the major antigen to induce protective neutralizing antibodies to block viruses from binding their respective receptor and thus inhibit viral infection (Bisht et al., 2004; Buchholz et al., 2004; Bukreyev et al., 2004; Yang et al., 2004). As a result, the S protein is also a major target for the development of subunit vaccines against SARS-CoV and MERS-CoV. Both full-length S protein and its antigenic fragments, including S1 subunit, NTD, RBD, and S2 subunit, can serve as important targets for the development of subunit vaccines (Guo et al., 2005; Mou et al., 2013; Wang et al., 2015; Jiaming et al., 2017; Zhou et al., 2018).
Although subunit vaccines based on the full-length S protein may elicit potent immune responses and/or protection, studies have found that antibodies induced by some of these vaccines mediate enhancement of viral infection in vitro, as in the case of SARS-CoV (Kam et al., 2007; Jaume et al., 2012), raising safety concerns for the development of full-length S protein-based subunit vaccines against SARS-CoV and MERS-CoV. In contrast, RBD-based subunit vaccines comprise the major critical neutralizing domain (Du and Jiang, 2015; Zhou et al., 2019). Therefore, these vaccines may generate potent neutralizing antibodies with strong protective immunity against viral infection. S1 subunit, for example, is much shorter than the full-length S protein, but it is no less able to induce strong immune responses and/or protection against viral infection (Li et al., 2013; Adney et al., 2019). Thus, this fragment can be used 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, cellular immune responses, and/or protection than the other regions, such as full-length, S1, and RBD (Guo et al., 2005; Jiaming et al., 2017). Therefore, in terms of safety and efficacy, the RBD and/or S1 of S protein could be applied as critical targets for the development of subunit vaccine candidates against SARS-CoV, MERS-CoV, SARSr-CoV, and MERSr-CoV. Because of its conserved amino acid sequences and high homology among different virus strains (Elshabrawy et al., 2012; Zhou et al., 2018), the S2 subunit has potential to be used as a target for the development of universal vaccines against divergent virus strains.
In addition to the S protein, the N protein of SARS-CoV and MERS-CoV may serve as an additional target for the development of subunit vaccines. Unlike S protein, the N protein has no ability to elicit neutralizing antibodies to block virus-receptor interaction and neutralize viral infection, but it may induce specific antibody and cellular immune responses (Liu et al., 2006; Zheng et al., 2009). Several immunodominant B-cell and T-cell epitopes have been identified in the N protein of SARS-CoV and MERS-CoV, some of which are conserved in mice, non-human primates, and humans (Liu et al., 2006; Chan et al., 2011; Veit et al., 2018). Other proteins, such as M protein, can be used as potential targets of SARS-CoV and MERS-CoV subunit vaccines. Notably, SARS-CoV M protein-derived peptides have immunogenicity to induce high-titer antibody responses in the immunized animals (He et al., 2005b), suggesting the potential for utilizing this protein to develop subunit vaccines.

Subunit Vaccines Against SARS-CoV
Numerous subunit vaccines against SARS-CoV have been developed since the outbreak of SARS, the majority of which use the S protein and/or its antigenic fragments, in particular, RBD, as the vaccine target (Table 1).
TABLE 1  Subunit Vaccines against SARS-CoVa.
Name  Antigenicity and functionality  Adjuvant  Route  Animal models  Antibody response  Cellular immune response  Protection  References
Subunit vaccines based on SARS-CoV full-length or trimeric S protein
FL-S and EC-S proteins  Bind to SARS-CoV S1, NTD, RBD, and S2-specific mAbs  MPL + TDM  S.C.  BALB/c mice  Elicit SARS-CoV S-specific Abs (IgG, > 1: 2 × 105), neutralizing (> 1:2.4 × 104) pseudotyped SARS-CoV (Tor2, GD03, and SZ3 strains)  N/A  N/A  He et al., 2006a
S andS-foldon proteins  N/A  TiterMax Gold; Alum Hydro+MPL  S.C. or I.M.  BALB/c mice  Elicit SARS-CoV S-specific Abs (IgG, > 1:104) in mice, neutralizing (∼2.4 × 102 for S; ∼1:7 × 102 for S-foldon) live SARS-CoV (Urbani strain)  N/A  Protect vaccinated mice from challenge of SARS-CoV (Urbani strain, 105 TCID50) with undetectable viral load in lungs  Li et al., 2013
triSpike protein  N/A  Alum hydro  I.P. or S.C.  BALB/c mice; Hamsters  Elicits SARS-CoV S-specific mucosal and serum Abs (IgA and IgG) in mice and hamsters, blocking S-ACE2 receptor binding and neutralizing live SARS-CoV (HKU-39849 strain); induces ADE  N/A  Protects vaccinated hamsters from challenge of SARS-CoV (Urbani strain, 103 TCID50) with undetectable or reduced viral load in lungs  Kam et al., 2007; Jaume et al., 2012
Subunit vaccines based on SARS-CoV RBD protein
RBD-Fc protein  N/A  Freund’s  I.D. or I.M.  BALB/c mice; Rabbits  Elicits SARS-CoV S/RBD-specific Abs (IgG) in mice and rabbits, neutralizing pseudotyped (rabbits: ≥ 7.3 × 104) and live (mice: 1:4 × 103; rabbits: > 1:1.5 × 104) SARS-CoV (BJ01 strain)  N/A  Protects majority (4/5) of vaccinated mice from challenge of SARS-CoV (BJ01 strain, 106 TCID50), with one mouse showing mild alveolar damage in lungs  He et al., 2004; Du et al., 2007
RBD193-CHO; RBD219-CHO proteins  Binds to SARS-CoV RBD-specific mAbs (neutralizing 24H8, 31H12, 35B5, 33G4, 19B2; non-neutralizing 17H9)  Freund’s  S.C.  BALB/c mice  Elicit SARS-CoV RBD-specific Abs, neutralizing pseudotyped (< 1:104 for RBD193-CHO; 1:5.8 × 104 for RBD219-CHO) and live (< 1:103 for RBD193-CHO; 1:103 for RBD219-CHO) SARS-CoV (GZ50 strain)  Induce SARS-CoV RBD-specific cellular immune responses (IFN-γ, IL-2, IL-4, IL-10) in mice  Protect all (for RBD219-CHO) or majority (3/5, for RBD219-CHO) of vaccinated mice from challenge of SARS-CoV (GZ50 strain, 100 TCID50 for RBD193-CHO; 5 × 105 TCID50 for RBD219-CHO) with undetectable viral RNA or no, to reduced, viral load in lungs  Du et al., 2009c, 2010
RBD-293T protein  Binds to SARS-CoV RBD-specific mAbs (neutralizing 24H8, 31H12, 35B5, 33G4, 19B2; non-neutralizing 17H9)  SAS  S.C.  BALB/c mice  Elicits SARS-CoV RBD-specific Abs (IgG), neutralizing pseudotyped (1:6.9 × 105) and live (1:1.6 × 103) SARS-CoV (GZ50 strain)  N/A  Protects all vaccinated mice from challenge of SARS-CoV (GZ50 strain, 100 TCID50) with undetectable viral RNA and viral load in lungs  Du et al., 2009b
S318-510 protein  N/A  Alum; Alum + CpG  S.C.  129S6/SvEv mice  Elicits SARS-CoV-specific Abs (IgG, IgG1, and IgG2a) in mice. Reduces neutralization after removing glycosylation  Induces SARS-CoV S-specific cellular immune responses (IFN-γ) in mice  N/A  Zakhartchouk et al., 2007
Subunit vaccines based on non-RBD SARS-CoV S protein fragments
S1 and S1-foldon proteins  N/A  TiterMax Gold; Alum Hydro + MPL  S.C. or I.M.  BALB/c mice  Elicit SARS-CoV S-specific Abs (IgG, > 1:104) in mice, neutralizing (1:1.7 × 102 for S1; 1:90 for S1-foldon) live SARS-CoV (Urbani strain)  N/A  Protect vaccinated mice from challenge of SARS-CoV (Urbani strain, 105 TCID50) with undetectable viral load in lungs  Li et al., 2013
S2 protein  N/A  Freund’s  S.C.  BALB/c mice  Elicits SARS-CoV S2-specific Abs (IgG, 1:1.6 × 103) in mice with no neutralizing activity  Induces SARS-CoV S2-specific cellular immune responses (IFN-γ and IL-4) in mice  N/A  Guo et al., 2005
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 Abs (IgG (1:1.8 × 103), IgG1, and IgG2a) in mice  Induces cellular immune responses with up-regulated IFN-γ and IL-10 cytokines in mice  N/A  Zheng et al., 2009
rN protein  N/A  Montanide + CpG; Freund’s  S.C.  BALB/c mice  Elicits SARS-CoV N-specific Abs (IgG) in mice  Induces SARS-CoV N-specific cellular immune responses (IFN- γ) in mice  N/A  Liu et al., 2006
M1-31 and M132-161 peptides  Bind to sera from SARS patients or immunized mice and rabbits  Freund’s  I.D.  BALB/c mice; NZW rabbits  Induce SARS-CoV M-specific Abs (IgG) in rabbits  N/A  N/A  He et al., 2005b
aAbs, 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.

SARS-CoV Subunit Vaccines Based on Full-Length S Protein
Subunit vaccines based on SARS-CoV S protein, including full-length or trimeric S protein, are immunogenic with protection against SARS-CoV infection (He et al., 2006a; Kam et al., 2007; Li et al., 2013). Either insect cell-expressed full-length (FL-S) or extracellular domain (EC-S) SARS-CoV S protein developed high-titer S-specific antibodies with neutralizing activity against pseudotyped SARS-CoV expressing S protein of representative SARS-CoV human and palm civet strains (Tor2, GD03, and SZ3) isolated during the 2002 and 2003 or 2003 and 2004 outbreaks (He et al., 2006a). In addition, full-length S-ectodomain proteins fused with or without a foldon trimeric motif (S or S-foldon) could elicit specific antibody responses and neutralizing antibodies, protecting immunized mice against SARS-CoV challenge with undetectable virus titers in the lungs (Li et al., 2013). Moreover, a subunit vaccine (triSpike) based on a full-length S protein trimer induced specific serum and mucosal antibody responses and efficient neutralizing antibodies against SARS-CoV infection (Kam et al., 2007). Nevertheless, this vaccine also resulted in Fcγ receptor II (FcγRII)-dependent and ACE2-independent ADE, particularly in human monocytic or lymphoblastic cell lines infected with pseudotyped SARS-CoV expressing viral S protein, or in Raji B cells (B-cell lymphoma line) infected with live SARS-CoV (Kam et al., 2007; Jaume et al., 2012), raising significant concerns over the use of full-length S protein as a SARS vaccine target.

SARS-CoV Subunit Vaccines Based on RBD
SARS-CoV RBD contains multiple conformation-dependent epitopes capable of eliciting high-titer neutralizing antibodies; thus, it is a major target for the development of SARS vaccines (He et al., 2004, 2005a; Jiang et al., 2012; Zhu et al., 2013). Subunit vaccines based on the SARS-CoV RBD have been extensively explored. Studies have found that a fusion protein containing RBD and the fragment crystallizable (Fc) region of human IgG1 (RBD-Fc) elicited highly potent neutralizing antibodies against SARS-CoV in the immunized rabbits and mice, which strongly blocked the binding between S1 protein and SARS-CoV receptor ACE2 (He et al., 2004). This RBD protein induced long-term, high-level SARS-CoV S-specific antibodies and neutralizing antibodies that could be maintained for 12 months after immunization, protecting most of the vaccinated mice against SARS-CoV infection (Du et al., 2007). In addition, recombinant RBDs (residues 318–510 or 318–536) stably or transiently expressed in Chinese hamster ovary (CHO) cells bound strongly to RBD-specific monoclonal antibodies (mAbs), elicited high-titer anti-SARS-CoV neutralizing antibodies, and protected most, or all, of the SARS-CoV-challenged mice, with undetectable viral RNA and undetectable or significantly reduced viral load (Du et al., 2009c, 2010). Significantly, a 293T cell-expressed RBD protein maintains excellent conformation and good antigenicity to bind SARS-CoV RBD-specific neutralizing mAbs. It elicited highly potent neutralizing antibodies that completely protected immunized mice against SARS-CoV challenge (Du et al., 2009b). Particularly, RBDs from the S proteins of Tor2, GD03, and SZ3, representative strains of SARS-CoV isolated from human 2002–2003, 2003–2004, and palm civet strains, can induce high-titer cross-neutralizing antibodies against pseudotyped SARS-CoV expressing respective S proteins (He et al., 2006c). Different from the full-length S protein-based SARS subunit vaccines, no obvious pathogenic effects have been identified in the RBD-based SARS subunit vaccines (Kam et al., 2007; Jaume et al., 2012).

SARS-CoV Subunit Vaccines Based on Non-RBD S Protein Fragments
SARS subunit vaccines based on S protein fragments (S1 and S2), other than the RBD, have shown immunogenicity and/or protective efficacy against SARS-CoV infection (Guo et al., 2005; Li et al., 2013). For example, recombinant S1 proteins fused with or without foldon elicited specific antibodies with neutralizing activity that protected immunized mice against high-dose SARS-CoV challenge (Li et al., 2013). Although some studies have demonstrated that recombinant SARS-CoV S2 (residues 681–980) protein elicits specific non-neutralizing antibody response in mice (Guo et al., 2005), others have indicated that mAbs targeting highly conserved heptad repeat 1 (HR1) and HR2 domains of SARS-CoV S protein have broad neutralizing activity against pseudotyped SARS-CoV expressing S protein of divergent strains (Elshabrawy et al., 2012), indicating the potential of utilizing the S2 region as a broad-spectrum anti-SARS-CoV vaccine target (Zheng et al., 2009).

SARS-CoV Subunit Vaccines Based on Non-S Structural Proteins
Subunit vaccines based on the N and M proteins of SARS-CoV have shown immunogenicity in vaccinated animals (Liu et al., 2006; Zheng et al., 2009). Studies have revealed that a plant-expressed SARS-CoV N protein conjugated with Freund’s adjuvant elicited specific IgG antibodies, including IgG1 and IgG2a subtypes, and cellular immune responses in mice, whereas another E. coli-expressed N protein conjugated with Montanide ISA-51 and cysteine-phosphate-guanine (CpG) adjuvants induced specific IgG antibodies toward a Th1 (IgG2a)-type response in mice (Liu et al., 2006; Zheng et al., 2009). Although N-specific antibodies have been detected in convalescent-phase SARS patient and immunized rabbit sera, they have no neutralizing activity against SARS-CoV infection (Qiu et al., 2005). In addition, immunodominant M protein peptides (M1-31 and M132-161) identified using convalescent-phase sera of SARS patients and immunized mouse and rabbit sera have immunogenicity to elicit specific IgG antibodies in rabbits (He et al., 2005b). In spite of their immunogenicity, it appears that these N- and M-based SARS subunit vaccines have not been investigated for their protective efficacy against SARS-CoV infection. Thus, it is unclear whether these non-S structural protein-based SARS subunit vaccines can prevent SARS-CoV infection.

Potential Factors Affecting SARS-CoV Subunit Vaccines
A number of factors may affect the expression of proteins to be used as SARS subunit vaccines; apart from their immunogenicity and/or protective efficacy. Understanding of these factors is important to generate subunit vaccines with good quality, high immunogenicity, and excellent protection against SARS-CoV infection.
The expression of recombinant protein-based SARS subunit vaccines may be changed by the following factors. First, addition of an intron splicing enhancer to the truncated SARS-CoV S protein fragments results in better enhancement of protein expression in mammalian cells than the exon splicing enhancers, and different cells may result in different fold increase of protein expression (Chang et al., 2006). Second, inclusion of a post-transcriptional gene silencing suppressor p19 protein from tomato bushy stunt virus to a SARS-CoV N protein may significantly increase its transient expression in tobacco (Zheng et al., 2009).
The following factors may affect the immunogenicity and protective efficacy of protein-based SARS subunit vaccines, including same proteins expressed in different expression systems, and same proteins with various lengths, amino acid mutations, or deletions (He et al., 2006b; Du et al., 2009b). For example, RBD proteins containing different lengths (193-mer: RBD193-CHO or 219-mer: RBD219-CHO) elicited different immune responses and protective efficacy against SARS-CoV challenge (Du et al., 2009c, 2010). A recombinant SARS-CoV RBD (RBD-293T) protein expressed in mammalian cell system was able to induce stronger neutralizing antibody response than those expressed in insect cells (RBD-Sf9) and E. coli (RBD-Ec) (Du et al., 2009b), suggesting that RBD purified from mammalian cells has preference for further development due to its ability to maintain native conformation. Notably, a single mutation (R441A) in the RBD of SARS-CoV disrupted its major neutralizing epitopes and affinity to bind viral receptor ACE2, thus abolishing the vaccine’s immunogenicity, and hence, its ability to induce neutralizing antibodies in immunized animals (He et al., 2006b). Additionally, deletion of a particular amino acid by changing a glycosylation site in the SARS-CoV RBD (RBD219-N1) also resulted in the alteration of subunit vaccine’s immunogenicity (Chen et al., 2014).
Other factors that potentially affect the immunogenicity of SARS subunit vaccines include immunization routes and adjuvants (Zakhartchouk et al., 2007; Li et al., 2013). Significantly high-titer antibodies were induced by monomeric or trimeric SARS-CoV S and S1 proteins through the intramuscular (I.M.) route compared to the subcutaneous (S.C.) route (Li et al., 2013). Moreover, a SARS-CoV RBD subunit vaccine conjugated with Alum plus CpG adjuvants elicited a higher level of IgG2a antibody and interferon gamma (IFN-γ) secretion than the RBD with Alum alone (Zakhartchouk et al., 2007).

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).