Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine

Materials and methods

Expression of recombinant RBD-Fc and S1-C9 proteins. Plasmid encoding a 193-amino-acid fragment of SARS-CoV S protein (residues 318–510), corresponding to the receptor-binding domain, fused with the Fc domain of human IgG1 (RBD-Fc) and plasmid encoding S1 protein (residues 12–672) tagged with C9 at the C-terminus (S1-C9) have been described previously [20], [24]. The RBD-Fc and S1-C9 proteins were expressed by transfecting 293T cells with the plasmids using Fugene 6 reagents (Boehringer–Mannheim, Indianapolis, IN) according to the manufacturer’s protocol. Supernatants were harvested 72 h post-transfection. Recombinant RBD-Fc fusion proteins were purified by Protein A–Sepharose 4 Fast Flow (Amersham Biosciences, Piscataway, NJ), and S1-C9 proteins were purified by affinity chromatography with anti-C9 mouse monoclonal antibody (mAb) 1D4 (National Cell Culture Center, Minneapolis, MN).

Immunization of rabbits. Two NZW rabbits were immunized intradermally with 150 μg purified RBD-Fc resuspended in phosphate-buffered solution (PBS, pH 7.2) in the presence of Freund’s complete adjuvant (FCA), and boosted three times with freshly prepared emulsion of 150 μg immunogen and Freund’s incomplete adjuvant (FIA) at 3-week intervals. Pre-immune sera were collected before starting the immunization and antisera were collected 10 days after each boost. Sera were kept at 4 °C before use.

Enzyme-linked immunosorbent assay. The reactivity of rabbit sera with various antigens was determined by enzyme-linked immunosorbent assay (ELISA). Briefly, 1 μg/ml recombinant proteins (RBD-Fc or S1-C9) or purified human IgG (Zymed, South San Francisco, CA) were used, respectively, to coat 96-well microtiter plates (Corning Costar, Acton, MA) in 0.1 M carbonate buffer (pH 9.6) at 4 °C overnight. After blocking with 2% non-fat milk, serially diluted rabbit sera were added and incubated at 37 °C for 1 h, followed by four washes with PBS containing 0.1% Tween 20. Bound antibodies were detected with HRP-conjugated goat anti-rabbit IgG (Zymed) at 37 °C for 1 h, followed by washes. The reaction was visualized by addition of the substrate 3,3′,5,5′-tetramethylbenzidine (TMB) and absorbance at 450 nm was measured by an ELISA plate reader (Tecan US, Research Triangle Park, NC).

Immunoabsorption assay. The anti-human IgG-Fc in the rabbit antisera were depleted by immunoabsorption. Briefly, the immunoaffinity resins were prepared by coupling the human IgG (Zymed) to cyanogens bromide-activated Sepharose beads (Pharmacia, Piscataway, NJ) according to the manufacturer’s instructions. Immunoabsorption was performed by diluting rabbit antisera 10-fold with PBS and incubating with the human IgG resin overnight at 4 °C with constant rotation. Resins were then packed into columns and flowthroughs (anti-Fc depleted antisera) were collected.

Neutralization of SARS-CoV infection. Neutralization of SARS-CoV infection was assessed as previously described [29]. Briefly, Vero E6 cells were plated at 5 × 10⁴  cells/well in 96-well tissue culture plates and grown overnight. One hundred TCID₅₀ (50% tissue-culture infectious dose) of SARS-CoV BJ01 strain (GenBank Accession No. AY278488) was mixed with an equal volume of diluted rabbit sera and incubated at 37 °C for 1 h. The mixture was added to monolayers of Vero E6 cells. Cytopathic effect (CPE) was recorded on days 3 post-infection as previously described [29]. The neutralizing titers represented the dilutions of rabbit antisera that completely prevented CPE in 50% of the wells [34] as calculated by Reed–Meunch method [40].

Neutralization of pseudovirus infection. A sensitive, quantitative, and safe neutralization assay based on reported SARS-CoV pseudovirus system [34], [41], [42], [43] was developed. HIV pseudotyped with SARS-CoV S protein was prepared as previously described [23], [24], [43]. In brief, 293T cells were co-transfected with a plasmid encoding codon-optimized SARS-CoV S protein and a plasmid encoding Env-defective, luciferase-expressing HIV-1 genome (pNL4-3.luc.RE) using Fugene 6 reagents (Boehringer–Mannheim). Supernatants containing HIV/SARS-CoV S protein were harvested 48 h post-transfection and used for single-cycle infection of ACE2-transfected 293T cells. Briefly, ACE2-expressed 293T cells were plated at 10⁴  cells/well in 96-well tissue-culture plates and grown overnight. The pseudovirus was preincubated with serially diluted rabbit sera at 37 °C for 1 h before addition to cells. The culture was re-fed with fresh medium 24 h later and incubated for an additional 48 h. Cells were washed with PBS and lysed using lysis reagent included in a luciferase kit (Promega, Madison, WI). Aliquots of cell lysates were transferred to 96-well Costar flat-bottomed luminometer plates (Corning Costar, Corning, NY), followed by addition of luciferase substrate (Promega). Relative light units were determined immediately on the Ultra 384 luminometer (Tecan US).

Inhibition of S1-protein binding to soluble ACE2. Recombinant soluble ACE2 (R&D systems, Minneapolis, MN) at 2 μg/ml was coated to 96-well ELISA plates (Corning Costar) in 0.1 M carbonate buffer (pH 9.6) at 4 °C overnight. After blocking with 2% non-fat milk, 2 μg/ml S1-C9 was added to the wells in the presence or absence of serially diluted rabbit sera. After incubation at 37 °C for 1 h, the anti-C9 mAb 1D4 was added and incubated at 37 °C for an additional 1 h. After washing, the HRP-conjugated goat anti-mouse IgG (Zymed) and the substrate TMB were used for detection.

Measurement of inhibition of S1 binding to cell-expressed ACE2 by flow cytometry. 10⁶ stable 293T/ACE2 cells were detached and washed with Hanks’ balanced salt solution (HBSS) (Sigma, St. Louis, MO). S1-C9 was added to the cells to a final concentration of 1 μg/ml in the presence or absence of rabbit sera at indicated dilutions, followed by incubation at room temperature for 30 min. After thorough washes, the mAb 1D4 was added to the cells to a final concentration of 10 μg/ml and incubated at room temperature for 30 min. Cells were washed with HBSS and incubated with anti-mouse IgG-FITC conjugate (Sigma) at 1:50 dilution at room temperature for an additional 30 min. After washing, cells were fixed with 1% formaldehyde in PBS and analyzed in a Becton FACSCalibur flow cytometer (Mountain View, CA) using CellQuest software.

Results

Discussion

During the SARS pandemic of 2002/2003, despite the lack of effective and specific therapy, most SARS patients recovered from the acute illness and few were re-infected by SARS-CoV (http://www.who.int/csr/sars/en). Neutralizing antibodies were detectable in the convalescent sera of SARS patients [43], [44]. Inoculation of hyperimmune sera from mice infected by SARS-CoV [45] or immunized with MVA/S [35] in mice, or administration of neutralizing monoclonal antibodies into ferrets [46] reduced the titers of SARS-CoV in the respiratory tracts of these animals after challenge. These data suggest that protective humoral immunity is achievable and vaccines can be developed for prevention of SARS.

A number of vaccine candidates are of preclinical study, including inactivated vaccines, DNA vaccines, and attenuated viruses encoding SARS-CoV S protein [9], [10], [34], [35]. These agents are effective in inducing a protective neutralizing-antibody response in animals [34], [35], [36]. In the present study, we used a recombinant fusion protein (RBD-Fc) as an immunogen to immunize rabbits since RBD is a key functional domain in the S protein responsible for viral binding to receptor on the target cell [24], [25], [26] and contains neutralizing epitopes [47]. Antibodies against RBD on the S proteins of other coronaviruses, such as MHV, transmissible gastroenteritis virus (TGEV), and human coronavirus (HCoV-229E) [48], [49], [50], and those against receptors for the coronaviruses [51], [52] are highly effective in blocking RBD-receptor interaction and neutralizing infection by the corresponding coronaviruses. Here we found that the RBD-Fc fusion protein elicited robust antibody responses in the immunized rabbits and the rabbit antisera could completely block SARS-CoV infection at the serum dilution of 1:10,240 (Fig. 3).

Since full-length S protein contains RBD and other viral functional domains and multiple neutralizing epitopes, it is expected to induce more potent neutralizing antibodies than RBD alone. One possible reason why RBD actually elicited much higher titers of neutralizing antibodies than full-length S protein is that the latter contains non-neutralizing epitopes that may elicit enhancing antibodies, like those induced by antigenic sites on the envelope glycoproteins of HIV and Ebola virus [39], [53], [54], [55]. The S proteins from some coronaviruses could also induce enhancing antibodies. For example, immunization of felines with a vaccinia virus vector encoding the S protein of feline infectious peritonitis virus (FIPV) resulted in enhancement of virus replication after virus challenge [37], [38] and the epitopes that elicit enhancing antibodies were localized in the S protein [56]. Although no enhanced virus replication may be observed [34], [35], this may not exclude the possibility that the enhancing antibody titers are lower than neutralizing antibody titers. In such a case, enhancing antibodies may suppress or “neutralize” the neutralizing-antibody activity, resulting in reduced neutralizing-antibody titers.

The rabbit antisera directed against RBD are effective in binding to RBD on the S1 domain of SARS-CoV S protein (Fig. 2) and blocking RBD binding to soluble and cell-expressed ACE2 (Fig. 6). These confirm that the rabbit antisera contain antibodies specifically targeted to RBD. Although we have not tested the protective activity of the rabbit anti-RBD antibodies in animal models against SARS-CoV challenge, the high neutralizing titers of these antisera tested in vitro suggest that RBD-Fc may induce strong protective immunity in animals and humans, considering that the effective protection against SARS-CoV infection can be achieved by the convalescent sera from SARS patients with neutralizing antibody titers ranging from 1:20 to 1:1280 [44] and by antisera from mice and monkeys immunized by DNA vaccines and attenuated viruses encoding S protein, respectively, with low neutralizing antibody titers (1:16–1:284) [34], [35].

The sequences of S proteins, especially the S1 domains, of most coronaviruses are highly variable [14], which is a major concern in developing effective vaccines against virus strains with distinct genotypes and phenotypes. However, recent studies have shown that SARS-CoV strains are quite stable and do not change as much as that was originally predicted [10]. At the early phase of SARS endemic in 2002/2003, 5 out of the 193 amino acid residues in the RBD of SARS-CoV S protein are variable due to the positive selection pressure in the process of transition from animal (e.g., palm civet) SARS-like-CoV to human SARS-CoV. However, at the middle and late phases (most virus strains were isolated from SARS patients during these two phases), there is no mutation in the RBD sequence [6]. Furthermore, the conformation of RBD is relatively conserved to ensure the binding of virus with different subtypes to a specific receptor on the target cells, even though the linear sequence of RBD may be variable. One example is B12 mAb which recognizes the neutralizing epitopes on the CD4-binding domain on HIV-1 gp120 and neutralizes a broad range of HIV-1 primary isolates, although the linear sequences of CD4-binding regions in gp120 from the corresponding strains are highly variable [57], [58]. These suggest that anti-antibodies against conserved RBD of the S protein may have neutralizing activity against a broad spectrum of SARS-CoV strains.

In summary, the RBD of S protein has potential to be developed as a subunit vaccine since it induces highly potent antibodies to block S protein binding with receptor and to neutralize SARS-CoV infection and has low level of risk compared with inactivated viruses or live attenuated virus vectors.