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).