Antibodies against trimeric S glycoprotein protect hamsters against SARS-CoV challenge despite their capacity to mediate FcγRII-dependent entry into B cells in vitro

2.1. Cell lines and expression vectors

Human Raji B and BHK-21 cells were cultured as described previously [31], [32]. HEK293T, VeroE6, FRhK-4 and murine macrophagic J774A1 cell lines were cultured at 37 °C, 5% CO₂, in Dulbecco's modified Eagle medium supplemented with 10% FBS, 100 U penicillin/ml, 100 μg streptomycin/ml. Optimized DNA and Semliki Forest Virus expression vectors encoding SARS-CoV S-protein fused to a C-terminal FLAG-sequence were described previously [33].

2.2. Immunoaffinity purification and biochemical analysis of S-protein

For protein production BHK cells were cultured in the presence or absence of cycloheximide (100 μg/ml), added at 7 h pre-harvest. Fourteen hours post-infection/transfection BHK-21 cells were lysed (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) and incubated for 5 min on ice. The collected lysate was vortexed, incubated for another 15 min on ice prior to centrifugation at 13,000 rpm for 15 min. Recombinant S-protein was immunopurified using anti-FLAG M2 mAb-coated agarose beads (Sigma) as described previously [31]. The quantity and quality of recombinant S-protein was assessed by SDS-PAGE and Western Blot using BAP-FLAG protein and microBSA methods as standards for protein quantification [31], [32]. Mouse sera were diluted (1/500) prior to Western Blot analysis and immune complexes detected with HRP-conjugated anti-mouse IgG (H + L) (1/1000) (Zymed), followed by visualization of the bands on X-ray film (Kodak) using chemiluminescence (Amersham Biosciences).

2.3. Immunization with triSpike

Balb/c mice 6–8 weeks old (n  = 3 per group) were immunized intraperitoneally (i.p.) with 20 μg of purified triSpike or triSpike in 1 mg of aluminium hydroxide gel (alum) on days 0, 16 and 32. Animals in the control group received PBS with 1 mg of alum on the same days. Blood samples were collected by saphenous vein bleeding at indicated time points in accordance with local guidelines and sera were prepared and heat-inactivated. Fecal samples [34] and nasal lavages [35] were prepared as described previously. HRP-conjugated anti-mouse IgG (H + L) or IgA (1/1000) (Zymed) were used to detect the existence of IgG or IgA antibody against triSpike in immunized mice fecal and nasal lavage sample. Absence of blood contamination was confirmed using the Hema-Screen occult blood detection kit (Stanbio Laboratory). Antibodies from fecal samples were used at a final dilution of 1/500 and nasal lavage sample at 1/25 dilution for Western Blot analysis.

Six to eight week old female golden Syrian hamsters (n  = 4 per group) were immunized subcutaneously (s.c.) with 2, 10 or 20 μg of purified triSpike in 2 mg of alum on d0, d21 and d42 at HKU-Pasteur Research Centre, Hong Kong, China. A parallel experiment in 5-week-old hamsters was performed at the NIAID, NIH, Bethesda, Maryland with immunization of 2, 10 or 50 μg of purified triSpike in 1 mg of alum on d0, d21 and d42.

2.4. Flow cytometry and ACE2 binding assay

Recombinant S-protein expressing BHK-21 cells were detached with 5 mM EDTA and incubated for 45 min at 4 °C with the diluted hamster sera (1/100), washed, fixed with 3.2% of PFA for 5 min at 4 °C and labeled with the FITC-conjugated goat anti-Syrian hamster IgG (Santa Cruz Biotechnology) for 30 min at 4 °C. Cells were analyzed by flow cytometer (FACSCalibur, BD). All steps were blocked with 3% normal goat serum (Zymed). Fluorescence values are expressed as mean fluorescence intensity (MFI). ACE2 binding assays were performed by incubating soluble ACE2 receptor with S-protein coated on agarose beads as described previously [33]. For inhibition of binding analysis, protein-coated beads were pre-incubated with sera for 1 h at 4 °C before incubation with soluble ACE2. The beads were washed four times with lysis buffer (20 mM Tris–HCl 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100) prior to SDS-PAGE and Western Blot analysis.

2.5. Serum-neutralization assay

Neutralization assays were carried on FRhK-4 cells using SARS-CoV strain HKU-39849 (Department of Microbiology, HKU) as follows. One hundred TCID₅₀ of SARS-CoV were incubated for 2 h at 37 °C with serial 2-fold dilutions of heat-inactivated mouse sera in quadruplicate. Virus antibody mix was then added to cells in 96-well plates and plates were incubated at 37 °C with microscopic examination for cytopathic effect (cpe) after a 4-day incubation. Neutralization titers were calculated by the Reed & Muench formula and are expressed as the reciprocal of the serum dilution which neutralized cpe in 50% of the wells [36].

2.6. SARS-CoV pseudotype particles

Recombinant SARS-CoVpp lentiviral vectors expressing a luciferase reporter gene were produced from HEK293T cells as described previously [37] using 10 μg of plasmid pNL4.3.Luc R⁻E⁻pro⁻ [11] and 10 μg of plasmid pCDNA-S-FLAG encoding codon-optimized SARS-CoV S protein [33]. For ADE assays, 25 μL of serial 2-fold dilutions of heat-inactivated murine or hamster sera were incubated for 1 h at 37 °C with 25 μL of pseudovirus (50 ng p24). Fifty microliters of Raji cells, at 2 × 10⁶ cell/mL previously washed three times with serum-free RPMI, were added to the antibody-SARS-CoVpp mixture in a 96-well plate. After adsorption for 1 h at 37 °C, 100 μL of RPMI 1640 containing 5% FCS were added. Medium was renewed 16 h later and incubated for an additional 48 h, washed in PBS, lysed and luciferase activity measured for 10 sec in a MicroBeta Jet Counter (Perkin Elmer) according to the instructions provided by the supplier (Promega). For ADE blocking assays, Raji cells were pre-incubated for 15 min at 4 °C with 10 μg/mL of murine monoclonal antibody directed against human CD32 (FcγRII, BD Pharmingen) or goat polyclonal antibody directed against human ACE2 (R&D Systems) prior to infection with SARS-CoVpp.

2.7. SARS-CoV challenge and viral load titration in hamsters, histopathology and blood chemistry

On day 56 (14 days following the last immunization), 48 anesthetized hamsters received 100 μl (10³ TCID₅₀) of SARS-CoV (strain Urbani) intranasally (Table 1 ). Hamsters were euthanatized by lethal intraperitoneal injection with sodium pentobarbital (200 μl/hamster) on days 2 and 5 following challenge when peak virus replication, and pneumonitis are anticipated and on day 21 post challenge to look for possible late changes in histopathology. Sixteen of forty eight SARS-CoV-inoculated hamsters were sacrificed 2 days post-challenge. Lungs were harvested and processed for viral titration. Groups of 16 SARS-CoV-inoculated hamsters were sacrificed 5 and 21 days post-challenge and lungs and livers were harvested and processed for pathology studies. Tissue samples were homogenized and virus titers were determined in VeroE6 cell monolayers as described previously [30].

Four hamsters per group, immunized and challenged as described above, were sacrificed at 5 days post-challenge and an additional four hamsters per group (with one additional hamster in the 50 μg/dose group) were sacrificed 21 days following challenge. Lungs were evaluated for pneumonitis and consolidation as previously described [30]. A colorimetric immunoalkaline phosphatase IHC method was used to identify SARS-CoV antigens [30]. Hamster serum samples were analyzed for the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), blood urea nitrogen (BUN) and total bilirubin using blood chemistry analyzer (Analytics, MedTec Lab).

3.1. Production of immunopurified trimeric S-protein with native antigenicity

Full-length, codon-optimized SARS-CoV S-protein fused to a C-terminal FLAG was purified by a single-step immunoaffinity protocol to over 90% purity with an average yield of 3 μg of pure S-protein per 10⁶ cells, i.e. 1.5 mg/L (Fig. 1A). Analysis of the apparent molecular weight of the protein by SDS-PAGE and Western Blot under non-reducing conditions revealed the predominant trimeric nature of the antigen (Fig. 1B, lane 1). Trimers dissociate into monomers when the protein is heat-denatured in the presence of SDS indicating that monomers are associated non-covalently (Fig. 1B, lane 2). Higher molecular weight DTT-sensitive aggregates were observed when the protein was not heat denatured prior to SDS-PAGE (Fig. 1B, lane 3). As expected, trimers and aggregates dissociate completely into monomers when heat-denatured in SDS and DTT (Fig. 1B, lane 4). Monomers derived from SDS/heat-denatured trimers have a slightly greater electrophoretic mobility than monomers derived from SDS/heat/DTT denatured trimers indicating that intramolecular disulfide bonds lead to a more compact three dimensional conformation of the Spike monomer. The trimeric and monomeric S-protein frequently migrate as doublets (Fig. 1B) [33]. These represent high-mannose glycoforms from proteins that reside in the ER at the time of lysis and glycoforms from proteins that have acquired complex N-glycans in the median-Golgi [33]. triSpike has native antigenicity shown by reactivity with sera from 10 convalescent SARS patients by Western Blot and FACS (data not shown). The native fold was further underscored by the specific binding of the triSpike protein with soluble ACE2 receptor (Fig. 2E, lane 2). Altogether our results strongly argue that purified triSpike molecules mimic the native trimeric S-protein on the virion surface.

3.2. triSpike induces antibodies against SARS-CoV S-protein in mice and hamsters

Immunogenicity of triSpike was assessed by immunization with three different doses with or without adjuvant (alum) in mice or hamsters (Fig. 2). Immunization of mice with triSpike alone induced low levels of anti-S antibodies (Fig. 2A, dl5). However a single subsequent injection of triSpike induced detectable levels of antibodies against S-protein (Fig. 2A, d30) which could be further boosted by a third injection (Fig. 2A, d49). Sera were reactive against mono-, di- and trimers of S-proteins carrying either high-mannose or complex N-glycosylation. Analysis of individual serum samples from d49 confirmed the homogeneity of the antibody response in individual mice (data not shown). When triSpike was used in combination with alum adjuvant (Fig. 2A) a very similar response was observed, except that the level of anti-S IgG was slightly higher after the second injection (Fig. 2A, d30) and a third injection did not increase the anti-S serum antibody level (Fig. 2A, d49). Neither triSpike nor triSpike with alum adjuvant immunization induced antibodies directed against the FLAG peptide (Fig. 2A, lower panels).

In a next step we analyzed the dose response and booster immunization effect in hamsters, immunized with 2, 10 or 20 μg triSpike plus alum, by FACScan. The first immunization with all three doses resulted in a moderate anti-S antibody response (Fig. 2B, d20). A second injection strongly increased anti-S antibody levels (Fig. 2B, d41), while no further increase in anti-S antibody level was seen after a third injection (Fig. 2B, d55). There is no significant dose-dependent antibody response as 2 μg triSpike plus alum immunization seems to induce maximum antibody response in hamster model. Analysis of individual serum samples from d55 confirmed the homogeneity of the antibody response in individual hamster (data not shown). Altogether these results indicate that an optimal anti-S antibody response might require two doses of triSpike (2 μg) immunization in alum adjuvant.

3.3. Neutralizing antibodies in sera from triSpike vaccinated mice and hamsters

We next evaluated the neutralizing activity of sera from triSpike vaccinated mice. Serial dilutions of sera were tested for their neutralizing activity of cpe induced by SARS-CoV replication in FRhK-4 cells (neutralization of 100 TCID₅₀). Injection of triSpike with alum adjuvant induces higher neutralization titers compared to triSpike alone (Fig. 2C). Antibody titers in triSpike with alum immunized animals remained at high levels until day 87 (1/6400) while neutralizing titers dropped rapidly from day 49 to day 87 in animals injected with triSpike alone (from 1/4800 to 1/600). Subsequently, a dose–response experiment was performed in hamsters. Sera from hamsters immunized with at least two doses as low as 2 μg effectively neutralized 100 TCID₅₀ of SARS-CoV on FRhK-4 cells (Fig. 2D). Boosting effect with a third injection was limited with all three doses. Maximum antibody levels were obtained in animals immunized with 20 μg.

3.4. Neutralizing sera block spike binding to the ACE2 receptor

We next investigated the mechanism of neutralization by analyzing the capacity of sera to block the interaction between immunopurified triSpike coated on agarose beads with purified soluble human ACE2, the SARS-CoV entry receptor [6], [7]. Fig. 2E shows that sera from triSpike + alum immunized mice (lane 3) but not from control animals (lane 4) neutralized S-protein binding to ACE2 receptor. These results suggest inhibition of receptor binding as the key mechanism of neutralization in triSpike immunized animals.

3.5. Mucosal IgG and IgA response in mice

Detection of SARS-CoV in intestinal tissue of fatal SARS cases indicated that the gastrointestinal tract is another target for SARS-CoV infection [38]. Hence, a vaccine candidate should ideally induce protective response in mucosal sites. Previous study suggested the induction of IgG response in serum (systemic response) and IgA response in stool (mucosal response) samples against Yersinia pestis using intraperitoneal immunization [39]. To address this question antibody were prepared from fecal samples of immunized animals. Occult blood detection showed absence of blood contamination (data not shown) indicating that the samples were not contaminated by serum antibodies. Western Blot using antibodies isolated from fecal samples revealed that mucosal IgG and IgA were induced by triSpike plus alum but not by triSpike alone (Fig. 3A). These antibodies were able to neutralize SARS-CoV infection of FRhK-4 cells (Fig. 3C). Altogether, we conclude that triSpike plus alum immunization induced antigen-specific IgG and IgA in the gastrointestinal tract.

Mice were also analyzed for the presence of anti-S antibodies in nasal lavage samples. Occult blood detection indicated the absence of blood contamination in the sample. Western Blot analysis using antibodies isolated from nasal lavage revealed the induction of a mucosal IgG response only after triSpike plus alum immunization but not after triSpike immunization alone (Fig. 3B). We were unable to detect any IgA response in either group and anti-S antibodies in nasal lavage samples did not neutralize SARS-CoV (Fig. 3B). Further studies are required to address the respective role of IgA and IgG in the neutralizing activity, epitope specificity and antibody titer in the neutralization mechanism.

3.6. Antibody-dependent enhancement (ADE) of viral entry into human B cells

We investigated whether vaccine-induced anti-S antibodies against triSpike could mediate ADE of SARS-CoV entry into FcR expressing cell lines. SARS pseudotype particles (SARS-CoVpp) were generated for this study. These recombinant viruses encoding the luciferase reporter gene and expressing the SARS-CoV Spike protein at the virion surface have been shown to faithfully mimic the SARS-CoV entry process [9], [10], [11]. We infected cells with SARS-CoVpp in the presence or absence of heat-inactivated serum from triSpike immunized mice and hamsters as well as sera from ten convalescent SARS patients. Transduction of FcR-negative VeroE6 cells with SARS-CoVpp was reduced in a dose-dependent manner when pseudovirus was pre-incubated with S-specific antisera (Fig. 4B & C). When SARS-CoVpp were pre-incubated with heat-inactivated S-specific mouse or hamster sera a dose-dependent enhancement of ACE2-negative Raji B and Daudi cell transduction was observed (Fig. 4A–C). Only background level of transduction was detected in the absence of serum or in the presence of sera from the naïve control group. When Raji B cells were pre-incubated with a monoclonal antibody (mAb) against human FcγRII (CD32) SARS-CoVpp transduction was reduced to background levels demonstrating that pseudovirus entry was FcγRII dependent (Fig. 4D). Furthermore ADE could be abrogated by pre-incubating the pseudovirus-antibody mix with protein G, demonstrating that the process is mediated by the Fc portion of the antibody (data not shown). Interestingly, a mAb against ACE2 blocked transduction of VeroE6 cells by SARS-CoVpp but not on Raji B cells by SARS-CoVpp-antibody complexes indicating that FcγRII mediated virus entry does not require ACE2 for virus-cell membrane fusion (Fig. 4D). Only a low level of SARS-CoVpp transduction was observed with the J774A1 murine macrophage cell line despite the presence of FcγRII receptor. ADE was not limited to vaccine-induced anti-S sera from mice and hamsters. Eight out of ten sera from convalescent SARS patients also showed enhancement of Raji B cell transduction despite the capacity of 10/10 sera to neutralize virus entry into VeroE6 cells (data not shown). Our results demonstrate that S-specific antibodies can induce a shift in cell tropism of SARS-CoV towards human FcγRII expressing cells. Antibody- and FcγRII-dependent but ACE2-independent virus entry and fusion is a novel mechanism by which SARS-CoV can enter into target cells.

3.7. Protection of immunized hamsters from SARS-CoV challenge

Syrian golden hamsters were immunized 3 times at bi-weekly intervals with 2 μg, 10 μg or 50 μg of triSpike plus alum and challenged intranasally with 10³ TCID₅₀/hamster of SARS-CoV. Hamsters receiving triSpike vaccine were significantly protected from SARS-CoV challenge compared to mock immunized controls (Fig. 5 ). Complete protection was observed following three doses of 50 μg (mean viral titer below the limit of detection) and partial protection was observed in hamsters receiving the lowest dose. Half the hamsters receiving three doses of 2 μg triSpike vaccine showed complete protection from challenge. Mean viral titers for the group were 10^2.3 TCID₅₀/g tissue. Seventy-five percent of hamsters receiving three doses of 10 μg triSpike vaccine showed complete protection from challenge. One hamster for this group had viral titers just above the limit of detection at 10² TCIDso/g tissue. In comparison of four mock immunized hamsters had similar high titers of SARS-CoV present with a mean viral titer of 10^7.2 TCIDso/g tissue. Patterns or consistent elevations of liver enzymes were not observed in any group following any immunization or SARS-CoV challenge (data not shown).

3.8. Histopathology

Five days following SARS-CoV challenge mock immunized hamsters had severe pneumonitis and consolidation in comparison to triSpike immunized hamsters where 9/12 had mild, 2/12 had moderate and only 1/12 had severe pneumonitis and none had pulmonary consolidation. The data indicate that regardless of the dose administered the vaccine significantly protects from the occurrence and severity of pneumonitis following SARS-CoV challenge. In contrast to previously published data where pneumonitis and consolidation were completely cleared by day 14 post-challenge, moderate (1/4) to severe pneumonitis (3/4) was observed 21 days post-challenge in mock immunized hamsters. In triSpike immunized hamsters 21 days post-challenge, pneumonitis was completely cleared (3/13) or reduced to mild (8/12) or moderate (2/12) levels. The variation between these observations and previously reported observations in the time to resolution of pneumonitis is likely due to changes in tissue processing. Lungs were inflated with formalin in these studies and were not in the previous report [30]. Nevertheless, consistent with previously reported data, 21 days after challenge pulmonary consolidation was absent in all groups including the mock immunized controls.

Histopathological analyses of livers at both days 5 (3/16) and 21 (4/17) post-challenge revealed very rare foci of hepatic cellular necrosis. These foci were observed in both mock immunized and triSpike immunized hamsters post-challenge and did not show any correlation with either pre-challenge neutralizing antibody titers or liver enzyme levels (pre- or post-challenge). Furthermore, SARS-CoV antigen was not detected by immunohistochemical staining.