Template-based coiled-coil antigens elicit neutralizing antibodies to the SARS-coronavirus

Abstract

The Spike (S) glycoprotein of coronaviruses (CoV) mediates viral entry into host cells. It contains two hydrophobic heptad repeat (HR) regions, denoted HRN and HRC, which oligomerize the S glycoprotein into a trimer in the native state and when activated collapse into a six-helix bundle structure driving fusion of the host and viral membranes. Previous studies have shown that peptides of the HR regions can inhibit viral infectivity. These studies imply that the HR regions are accessible and that agents which can interact with them may prevent viral entry. In the present study, we have investigated an approach to generate antibodies that specifically recognize the HRN and HRC regions of the SARS-CoV spike (S) glycoprotein in order to evaluate whether these antibodies can inhibit viral infectivity and thus neutralize the SARS-CoV. In this regard, we incorporated HRN and HRC coiled-coil surface residues into a de novo designed two-stranded alpha-helical coiled-coil template for generating conformation-specific antibodies that recognize alpha-helices in proteins (Lu, S.M., Hodges, R.S., 2002. J. Biol. Chem. 277, 23515-23524). Eighteen surface residues from two regions of HRN and HRC were incorporated into the template and used to generate four anti-sera, HRN1, HRN2, HRC1, and HRC2. Our results show that all of the elicited anti-sera can specifically recognize HRN or HRC peptides and the native SARS-CoV S protein in an ELISA format. Flow cytometry (FACS) analysis, however, showed only HRC1 and HRC2 anti-sera could bind to native S protein expressed on the cell surface of Chinese hamster ovary cells, i.e., the cell surface structure of the S glycoprotein precluded the ability of the HRN1 or HRN2 anti-sera to see their respective epitope sites. In in vitro viral infectivity assays, no inhibition was observed for either HRN1 or HRN2 anti-serum, whereas both HRC1 and HRC2 anti-sera could inhibit SARS-CoV infection in a dose-dependent manner. Interestingly, the HRC1 anti-serum, which was a more effective inhibitor of viral infectivity compared to HRC2 anti-serum, could only bind the pre-fusogenic state of HRC, i.e., the HRC1 anti-serum did not recognize the six-helix bundle conformation (fusion state) whereas HRC2 anti-serum did. These results suggest that antibodies that are more specific for the pre-fusogenic state of HRC may be better neutralizing antibodies. Overall, these results clearly demonstrate that the two-stranded coiled-coil template acts as an excellent presentation system for eliciting helix-specific antibodies against highly conserved viral antigens and HRC1 and HRC2 peptides may represent potential candidates for use in a peptide vaccine against the SARS-CoV.

Fig. 1

Top panel: the two-stranded α-helical coiled-coil template sequence. The 18 residue positions which can be substituted with native S protein residues are indicated with an asterisk (*). Residues which form the 4–3 hydrophobic repeat of the coiled-coil structure are underlined. The relative position of the * residues when in a two-stranded coiled structure are shown in an end cross-sectional view (below left) and cartoon (below right). In the cross-sectional view, the direction of the helices is into the page from NH₂ to COOH terminus with the polypeptide chains parallel and in-register. Heptad positions are labeled a–g, with the prime indicating corresponding positions on the opposing helix. Arrows depict the hydrophobic interactions that occur between residues in the “a” and “d” positions. In the cartoon model, the shaded circles denote the substituted positions on the front helix of the dimer. Middle and Bottom panels show the amino acid sequence of the HRN and HRC peptides used in this study. Peptide names are indicated on the left. Numbers in parentheses indicate the amino acid sequence region of the native S protein which the peptide spans. The location of the disulfide bridge between cysteine residues is denoted by a solid line. Core a and d residues in template are underlined. Ac, denotes N^α-acetyl; -amide, denotes C^α-amide; BB, denotes N^α-benzoylbenzoyl; nL, denotes norleucine; and X, denotes either pAbz-Cys or Ac-Cys depending on the use of the peptide.

Fig. 2

Molecular graphics representation of the SARS-CoV S HRN (residues 896–972, blue) and molecular modeling representation of HRC (residues 1150–1185, red) trimeric coiled-coil domains. Residues chosen to be incorporated into the coiled-coil template sequence are colored in yellow (A, C, E, and G). Accessibility of the chosen surface residues when in the six-helix bundle conformation of HRN/HRC is also shown (B, D, F, and H). The molecular graphics of the HRN trimeric coiled and six-helix bundle structure are based on the X-ray crystallographic structure (pdb Accession No. 1WNC ) shown in ribbon representation (center). The HRC trimeric coiled-coil model was based on the X-ray structure of a trimeric GCN4 coiled-coil (pdb Accession No. 1GCM ). Models were prepared using the molecular graphics program VMD, version 1.8.3 (Humphrey et al., 1996). In the six-helix bundle structures (B, D, F, and H) the HRC helices are antiparallel to the HRN helices as shown (center).

Fig. 3

(A) Far UV CD spectra of the SARS-CoV S immunogen peptides. Spectra were recorded in a 0.1 M KCl, 0.05 M K₂HPO₄, pH 7 buffer. Peptide concentrations were 100 μM. (B) Temperature denaturation profiles of the helical template immunogen peptides. Denaturations were monitored by CD at 222 nm in a 0.1 M KCl, 0.05 M K₂HPO₄, pH 7 buffer. For analysis of reduced peptides, the above buffer also contained 2 mM dithiothreitol (DTT). Peptide concentrations were 100 μM.

Fig. 4

(A) ELISA reactivity of anti-HR sera with a panel of HR synthetic peptides. HRN monomer denotes single stranded HRN(916–950) peptide conjugated to BSA. HRN trimer denotes covalently cross-linked trimeric HRN(902–950) peptide conjugated to BSA. HRC monomer denotes HRC(1150–1185) peptide conjugated to BSA. HRC trimer denotes covalently cross-linked trimeric HRC(1150–1185) peptide conjugated to BSA. Sera were diluted 1:5000 and 0.2 μg/well of peptide conjugate plated. (B) HRN1 and HRN2 sera binding to covalently cross-linked trimeric HRN(902–950) peptide. (C) HRC1 and HRC2 sera binding to covalently cross-linked trimeric HRC(1150–1185) peptide. For (B and C), serial (fourfold) dilutions of the sera were applied to the peptide (0.2 μg/well) and the amount of bound antibodies measured by an ELISA assay as described in Section 2. The background was estimated by the amount of antibody bound to BSA and subtracted.

Fig. 5

Kinetic analysis of HRN1 and HRC1 purified sera binding to immobilized HRN(902–950)-trimer peptide (A) and immobilized HRC(1150–1185)-trimer (B) at various IgG concentrations, respectively. The binding reactions are expressed as response units (RU) as a function of time. Total IgG concentration is given to the right of each trace in nM. Each analysis consisted of a 110 μl injection association phase in PBS buffer at a flow rate of 70 μl/min, followed by a 240 s dissociation phase in PBS buffer. The fit lines (smooth lines) obtained from global analysis of the data set are shown overlaid with each curve.

Fig. 6

ELISA reactivity of anti-HR sera against plated HRN(902–950)/HRC(1150–1185) complex (six-helix bundle state). The average of duplicate runs is reported.

Fig. 7

ELISA reactivity of anti-HR sera against plated soluble S protein 1180K. The average of triplicate runs is reported.

Fig. 8

Flow cytometric analysis of cell surface S glycoprotein. CHO cells were stained using HRN1, HRN2, HRC1 and HRC2 sera and a secondary phycoerythrin (PE)-conjugated anti-rabbit antibody. Mock transfected CHO cells are shown on the left (first four columns) and CHO cells transiently expressing the wild type S protein (SΔ19) are shown on the right (columns five to eight). Cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA, USA), and propidium iodide-staining (dead) cells were excluded. Five thousand cells were counted per analysis. The percentage of cells with an increase in fluorescence relative to mock cells stained with secondary antibody only (representing background) is shown. The average of triplicate analysis with error bars indicating the standard deviation is reported.

Fig. 9

Neutralization activity of anti-HR sera on in vitro SARS-CoV infection of Vero E6 cells. Each point represent the mean of triplicate determinant, error bars represent variance of data. HRC1 and HRC2 significantly inhibited the infection of SARS-CoV into Vero E6 cells and the inhibition was effected in a dose-dependent manner, whereas the control sera and HRN1 and HRN2 anti-sera did not cause any effect under the same conditions.