SYNOPSIS SARS Vaccine Development

Developing effective and safe vaccines is urgently needed to prevent infection by severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV). The inactivated SARS-CoV vaccine may be the first one available for clinical use because it is easy to generate; however, safety is the main concern. The spike (S) protein of SARS-CoV is the major inducer of neutralizing antibodies, and the receptor-binding domain (RBD) in the S1 subunit of S protein contains multiple conformational neutralizing epitopes. This suggests that recombinant proteins containing RBD and vectors encoding the RBD sequence can be used to develop safe and effective SARS vaccines.

safe vaccines is urgently needed to prevent a new SARS epidemic and for biodefense preparedness. Currently, 3 major classes of SARS vaccines are under development: 1) inactivated SARS-CoV (Figure 1), 2) full-length S protein (Figure 2A), and 3) those based on fragments containing neutralizing epitopes ( Figure 2B).

Inactivated SARS-CoV-based Vaccines
SARS-CoV expresses several structural proteins, including nucleocapsid, membrane, envelope, and spike (S) proteins (1). All may serve as antigens to induce neutralizing antibodies and protective responses. In general, prior to identification of the protein that contains the major neutralizing epitopes, the inactivated virus may be used as the first-generation vaccine because it is easy to generate whole killed virus particles. However, once the neutralizing epitopes are identified, the inactivated virus vaccine should be replaced by vaccines based on fragments containing neutralizing epitopes since they are safer and more effective. Several reports have showed that SARS-CoV inactivated with formaldehyde, UV light, and β-propiolactone can induce virus-neutralizing antibodies in immunized animals (8)(9)(10)(11), and the first inactivated SARS-CoV vaccine is being tested in the clinical trials in China. However, safety of the inactivated vaccine is a serious concern; production workers are at risk for infection during handling of concentrated live SARS-CoV, incomplete virus inactivation may cause SARS outbreaks among the vaccinated populations, and some viral proteins may induce harmful immune or inflammatory responses, even causing SARS-like diseases (12,13).

S Protein-based Vaccines
The S protein of SARS-CoV, a type I transmembrane glycoprotein, is responsible for virus binding, fusion, and entry and is a major inducer of neutralizing antibodies (1,14). S protein consists of a signal peptide (SP: amino acids [aa] 1-12) and 3 domains: an extracellular domain (aa 13-1193), a transmembrane domain (aa 11194-1215), and an intracellular domain (aa 1216-1255). Its extracellular domain consists of 2 subunits, S1 and S2 (14), although the cleavage site between these subunits has not been clearly defined. The S1 subunit is responsible for virus binding to the receptor, angiotensin-converting enzyme 2 (ACE2) (15,16). A fragment located in the middle region of the S1 subunit (aa 318-510) is the receptor-binding domain (RBD) for ACE2 (17)(18)(19). SARS-CoV may also bind to cells through the alternative receptors DC-SIGN or L-SIGN (20,21), but the binding sites for these alternative receptors have not been defined. The S2 subunit, which contains a putative fusion peptide and 2 heptad repeats (HR1 and HR2), is responsible for fusion between the viral and target cell membranes. Infection by SARS-CoV is initiated by binding of RBD in the viral S protein S1 subunit to ACE2 on target cells. This forms a fusogenic core between the HR1 and HR2 regions in the S2 domain that brings the viral and target cell membranes into close proximity, which results in virus fusion and entry (22)(23)(24). This scenario indicates that the S protein may be used as a vaccine to induce antibodies for blocking virus binding and fusion.
Several recombinant vector-based vaccines expressing SARS-CoV S protein have been assessed in preclinical studies. Yang et al. (25) reported that a candidate DNA vaccine encoding the full-length S protein induced neutralizing antibodies (neutralizing titers ranging from 1:50 to 1:150) and protected mice from SARS-CoV challenge. Using DNA vaccines encoding the full-length and segments of S protein to immunize rabbits, Wang et al. have produced higher titers of neutralizing antibodies and demonstrated that major and minor neutralizing epitopes are located in the S1 and S2 subunits, respectively (26). Other groups also found neutralizing epitopes in the S2 subunit (27,28). Bisht et al. (29) have shown that intranasal or intramuscular inoculations of mice with highly attenuated modified vaccinia virus Ankara (MVA) vaccines encoding full-length SARS-CoV S protein also produce neutralizing antibodies with mean neutralizing titers of 1:284. Bukreyev et al. (30) reported that mucosal immunization of African green monkeys with an attenuated parainfluenza virus expressing S protein resulted in production of neutralizing antibodies and protected animals from infection by challenge with SARS-CoV. These data suggest that the S protein can induce neutralizing antibodies and protective responses in immunized animals.
Using convalescent-phase sera from SARS patients and a set of peptides spanning the entire sequence of the SARS-CoV S protein, we have identified 5 linear immunodominant sites (IDS) in the S protein ( Figure 2A). IDS I, II, III, and V reacted with >50% of the convalescent-phase sera from SARS patients, while IDS IV was reactive with >80% of SARS sera, suggesting that IDS IV is the major immunodominant epitope on the S protein (31). Synthetic peptides corresponding to IDS could induce high titers of S protein-specific antibodies, but none of these antibodies possesses neutralizing activity. These findings suggest that the IDS in S protein may not induce neutralizing antibodies. Whether these antibodies enhance infection by heterologous SARS-CoV strains or mediate harmful immune responses is unclear. The S protein of FIPV expressed by recombinant vaccinia can cause antibody-dependent enhancement of disease if vaccinated animals are subsequently infected with wild-type virus (32). Our previous studies on HIV-1 showed that antibodies against some immunodominant epitopes in the HIV-1 envelope glyco-

Vaccines Based on Fragments Containing Neutralizing Epitopes
RBD, a fragment (≈193 aa residues) in the middle of S1 subunit of S protein ( Figure 2B), is responsible for virus binding to the receptor on target cells. We have demonstrated that the antisera from SARS patients and from animals immunized with inactivated SARS-CoV reacted strongly with RBD (9,35). Absorption of antibodies by RBD from these antisera results in the removal of most of the neutralizing antibodies, and RBD-specific antibodies isolated from these antisera have potent neutralizing activity (35,36). We have also shown that rabbits and mice immunized with RBD produced high titers of neutralizing antibodies against SARS-CoV with 50% neutralizing titers at a >1:10,000 serum dilution (37). The immunized mice were protected from SARS-CoV challenge (unpub. data). The antibodies purified from the antisera against SARS-CoV significantly inhibited RBD binding to ACE2 (9,(36)(37)(38). Using spleen cells from mice immunized with RBD, we have generated a panel of 25 monoclonal antibodies (MAbs) that recognize different conformational epitopes on RBD and possess potent neutralizing activity (38). Our result is in agreement with the report by van den Brink et al. (39), who identified 3 human neutralizing anti-S MAbs from antibody phage display libraries by using inactivated SARS-CoV as the target. These researchers also found that all of these MAbs specifically bound to RBD and blocked interaction between RBD and ACE2. These findings suggest that RBD contains the major neutralizing epitopes in the S protein and is an ideal SARS vaccine candidate because RBD contains the receptor-binding site, which is critical for virus attachment to the target cell for infection (15,(17)(18)(19). Antibodies specific for RBD are expected to block binding of virus to the target cell. RBD induces higher titers of neutralizing antibodies than those vaccines expressing the full-length S protein (25,26,29,30,37,38). RBD sequences among the late SARS-CoV strains are highly conserved. When the early and late SARS-CoV strains are compared, only 3 to 5 aa residues are variable among the 193 residues in RBD and most of the isolates vary by only 1 residue (4). van den Brink et al. (39) showed that 1 human MAb (CR3014) specific for RBD of SARS-CoV strain FM1 can effectively bind to most RBDs of the early and late SARS-CoV strains. These data suggest that antibodies directed against RBD of a SARS-CoV isolate may neutralize infection by a broad spectrum of SARS-CoV strains. Therefore, recombinant proteins containing RBD or vectors encoding RBD may be used as vaccines for preventing infection by SARS-CoV with distinct genotypes.

Conclusions
An ideal SARS vaccine should 1) elicit highly potent neutralizing antibody responses against a broad spectrum of viral strains; 2) induce protection against infection and transmission; and 3) be safe by not inducing any infectionenhancing antibodies or harmful immune or inflammatory responses. Currently, an inactivated SARS-CoV vaccine is in clinical trials in China. Safety is the major concern for this type of vaccine (12). The S protein is the major inducer of neutralizing antibodies. Recombinant vector-based vaccines expressing full-length S protein of the late SARS-CoV are under development. These vaccines can induce potent neutralizing and protective responses in immunized animals but may induce antibodies that enhance infection by early human SARS-CoV and animal SARS-CoV-like viruses (6). Recent studies have demonstrated that recombinant RBD consists of multiple conformational neutralizing epitopes that induce highly potent neutralizing antibodies against SARS-CoV (9,26,(35)(36)(37)(38). Unlike fulllength S protein, RBD does not contain immunodominant sites that induce nonneutralizing antibodies. RBD sequences are relatively conserved. Thus, recombinant RBD or vectors encoding RBD may be used as safe and efficacious vaccines for preventing infection by SARS-CoV with distinct genotypes.
Dr. Jiang is associate member and head of the Viral Immunology Laboratory, Lindsley F. Kimball Research Institute, New York Blood Center. His primary research interests include development of vaccines and therapeutic agents against SARS-CoV and HIV.