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. 2007 Jan 8;25(4):729-40.
doi: 10.1016/j.vaccine.2006.08.011. Epub 2006 Aug 22.

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

Yiu Wing Kam  1 , François Kien, Anjeanette Roberts, Yan Chung Cheung, Elaine W Lamirande, Leatrice Vogel, Shui Ling Chu, Jane Tse, Jeannette Guarner, Sherif R Zaki, Kanta Subbarao, Malik Peiris, Béatrice Nal, Ralf Altmeyer
Affiliations

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

Yiu Wing Kam et al. Vaccine. .

Abstract

Vaccine-induced antibodies can prevent or, in the case of feline infectious peritonitis virus, aggravate infections by coronaviruses. We investigated whether a recombinant native full-length S-protein trimer (triSpike) of severe acute respiratory syndrome coronavirus (SARS-CoV) was able to elicit a neutralizing and protective immune response in animals and analyzed the capacity of anti-S antibodies to mediate antibody-dependent enhancement (ADE) of virus entry in vitro and enhancement of replication in vivo. SARS-CoV-specific serum and mucosal immunoglobulins were readily detected in immunized animals. Serum IgG blocked binding of the S-protein to the ACE2 receptor and neutralized SARS-CoV infection in vitro. Entry into human B cell lines occurred in a FcgammaRII-dependent and ACE2-independent fashion indicating that ADE of virus entry is a novel cell entry mechanism of SARS-CoV. Vaccinated animals showed no signs of enhanced lung pathology or hepatitis and viral load was undetectable or greatly reduced in lungs following challenge with SARS-CoV. Altogether our results indicate that a recombinant trimeric S protein was able to elicit an efficacious protective immune response in vivo and warrant concern in the safety evaluation of a human vaccine against SARS-CoV.

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Figures

Fig. 1
Fig. 1
Biochemical characterization of purified trimeric SARS-CoV S-protein (triSpike). (A) Silver staining of immunopurified triSpike, lane M contains a pre-stained protein marker and lane 1 had 720 ng of purified triSpike. (B) triSpike was expressed in BHK-21 cells in the presence of cycloheximide. Recombinant triSpike purification was performed by immunoaffinity as described in Section 2. Eluted protein was treated as indicated and analyzed by 4–8% Tris-Acetate SDS-PAGE gel and Western Blot using M2 monoclonal antibody against the FLAG peptide. Sizes of molecular weight markers are indicated on the right (HiMark™ Pre-stained HMW Protein Standard, Invitrogen).
Fig. 2
Fig. 2
Immunogenicity of triSpike. Sera collected at indicated time points from vaccinated mice or hamsters were analyzed for reactivity with triSpike. (A) Western Blot analysis of pooled sera from immunized mice with or without alum adjuvant. Sera were collected and used at 1/1000 dilution against S-FLAG that was separated under conditions that allowed simultaneous detection of mono-, di-, and trimers of S-protein. A SARS patient serum (SARS), a rabbit serum against S1 and M2 monoclonal antibody against the FLAG peptide was used as control. FLAG-tagged bacterial alkaline phosphatase (BAP-FLAG) was used to assess the presence of antibodies against the FLAG tag. Immune complexes were detected with HRP-conjugated goat anti-mouse, -human or -rabbit IgG polyclonal antibody. Sizes of molecular weight markers are indicated on the right. (B) Reactivity of immune sera from pooled immunized hamster with live BHK-21 cells expressing S-protein at the plasma membrane using FACS analysis. Values were expressed as mean ± standard deviations. (C) Effect of alum adjuvant on longevity of neutralizing response in mouse sera against SARS-CoV (determined on FRhK-4 cells). Values were expressed as mean ± standard deviations. (D) Dose effect of triSpike immunization with alum adjuvant on neutralizing response against SARS-CoV in hamsters (determined on FRhK-4 cells). Values were expressed as mean ± standard deviations. (E) Inhibition of S-protein binding to ACE2 by sera from immunized mice. S-protein coated beads were pre-incubated with sera prior to incubation with soluble ACE2 (sACE2) and detection of the receptor was performed with a polyclonal goat-anti human ACE2 antibody. BAP-FLAG coated beads were used as control.
Fig. 3
Fig. 3
Induction of mucosal immune response in triSpike plus alum vaccinated mice. (A) Antibodies prepared from fecal samples collected at day 44 post-immunization were reacted against S-protein as described in Fig. 2A. Immune complexes were detected with HRP-conjugated goat anti-mouse IgG or IgA polyclonal antibody. (B) Similar as (A) except that Western Blot analysis was performed with pooled nasal lavage samples collected at day 65. (C). Antibodies prepared from fecal samples of vaccinated mice were analyzed for neutralizing activity against SARS-CoV infection on FRhK-4 cells. Weak neutralizing activity was detected after the third immunization only. Asterisk (**) indicates the value of p < 0.01 in two-tailed t tests. Values were expressed as mean ± standard deviations.
Fig. 4
Fig. 4
Analysis of antibody-dependent enhancement of SARS-CoVpp entry. (A) Analysis of various cell lines for ADE of Spike-mediated viral entry. SARS-CoVpp were incubated with a 1/1000 dilution of sera from mice immunized with triSpike and transduction was measured as SARS-CoVpp-expressed luciferase activity. (B) Serial dilutions of sera from mice immunized with triSpike neutralize SARS-CoVpp transduction of VeroE6 cells and enhance transduction of Raji B cells. (C) Same as (B) except that sera from hamsters immunized with triSpike were used. (D) FcγRII (CD32) involvement in antibody-dependent enhancement of SARS-CoVpp entry. Same as (C), SARS-CoVpp were incubated with a 1/1000 dilution of sera and Raji cells pre-incubated with 10 mg/mL of a mAb against ACE2 or CD32 (left panel) prior to infection. Inhibition of SARS-CoVpp entry by the mAb against ACE2 was controlled on VeroE6 cells (right panel). All experiments were performed in triplicates and data are presented as mean ± standard deviations.
Fig. 5
Fig. 5
Protection of triSpike-immunized hamsters from SARS-CoV challenge. Hamsters were inoculated intranasally with 103 TCID50 of SARS-CoV (Urbani strain) and virus titers in lung homogenates were determined. Error bars indicated standard errors and asterisk (***) indicate the value of p < 0.001 in two-tailed t tests. Values were expressed as TCID50 per g of lung tissue.
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