Peptide nanoparticles as novel immunogens: design and analysis of a prototypic severe acute respiratory syndrome vaccine

Abstract

Severe acute respiratory syndrome (SARS) is an infectious disease caused by a novel coronavirus that cost nearly 800 lives. While there have been no recent outbreaks of the disease, the threat remains as SARS coronavirus (SARS-CoV) like strains still exist in animal reservoirs. Therefore, the development of a vaccine against SARS is in grave need. Here, we have designed and produced a prototypic SARS vaccine: a self-assembling polypeptide nanoparticle that repetitively displays a SARS B-cell epitope from the C-terminal heptad repeat of the virus' spike protein. Biophysical analyses with circular dichroism, transmission electron microscopy and dynamic light scattering confirmed the computational design showing alpha-helcial nanoparticles with sizes of about 25 nm. Immunization experiments with no adjuvants were performed with BALB/c mice. An investigation of the binding properties of the elicited antibodies showed that they were highly conformation specific for the coiled-coil epitope because they specifically recognized the native trimeric conformation of C-terminal heptad repeat region. Consequently, the antisera exhibited neutralization activity in an in vitro infection inhibition assay. We conclude that these peptide nanoparticles represent a promising platform for vaccine design, in particular for diseases that are characterized by neutralizing epitopes with coiled-coil conformation such as SARS-CoV or other enveloped viruses.

Figure 1

(A) Schematic of full length SARS‐CoV S protein, residues 1–1255, which is divided into S1 (1–770) and S2 (771–1185) domains. The S1 domain contains the receptor‐binding domain (RBD). The S2 domain contains the predicted fusion peptide (FP), the N‐terminal heptad repeat region (HRN), interhelical domain (IHD), the C‐terminal heptad repeat region (HRC) and the transmembrane domain (TM). (B) Nanoparticle sequence (top) and HRC1 nanoparticle sequence (bottom): in black, the signaling sequence and the His‐tag used for purification, in green the pentameric coiled‐coil sequence, in blue the trimeric coiled‐coil sequence and in red the HRC1 epitope sequence. Alanines (shown in black) in the f position of the heptad repeat of the coiled‐coils are used to optimize interhelical contacts. (C) From top to bottom: HRC1 epitope sequence used in the nanoparticle immunogen (21); native HRC sequence (1150–1185) and schematic of maltose‐binding protein (MBP) fusion construct (MBP‐HRC‐GCN4). MBP was used as expression tag and purification tag, modified GCN4 sequence to stabilize the HRC sequence as a trimeric coiled‐coil and maintain the construct as a trimer (36). The GCN4 sequence is shown in purple.

Figure 2

(A) 3D monomeric building block of P6HRC1 composed of a modified pentameric coiled‐coil domain from COMP (green) and trimeric de novo designed coiled‐coil domain (blue) which is extended by the coiled‐coil sequence of SARS HRC1 (red). (B) Computer models of the complete peptide nanoparticle P6HRC1 with T = 1 (top) and T = 3 (bottom) icosahedral symmetry. The calculated diameters of these particles are about 23 and 28 nm and the molecular weight 757 and 2271 kDa, respectively.

Figure 3

(A) Far UV circular dichroism (CD) spectra of P6HRC1. Spectra were recorded in 20 mm sodium phosphate pH 7.5, 150 mm NaCl, 10% glycerol. Peptide concentration was 0.36 mg/mL. (B) Temperature denaturation profile of the helical peptide nanoparticle P6HRC1. Denaturation was monitored by CD at 222 nm in 20 mm sodium phosphate pH 7.5, 150 mm NaCl, 10% glycerol.

Figure 4

(A) Transmission electron microscopy image of P6HRC1 nanoparticles at 242 000×. The sample was negatively stained with 1% uranyl acetate. Sample concentration was 0.076 mg/mL and the buffer was 20 mm Tris pH 7.5, 150 mm NaCl, 10% glycerol. Nanoparticle size ranges from 25 to 30 nm. (B) Dynamic light scattering data of P6HRC1. Size distribution by volume shows a peak corresponding to a size of 26 nm. Sample concentration was 0.072 mg/mL and the buffer was 20 mm Tris pH 7.5, 150 mm NaCl, 10% glycerol.

Figure 5

(A) Sedimentation velocity ultracentrifugation of P6HRC1. The overlay shows the normalized sedimentation coefficient distribution g(s*) plots obtained from program DcDt+ for three different concentrations of P6HRC1. The distributions peak near 29S, which under the conditions used correspond to a molecular weight of 1.4 MDa or 110 monomers per nanoparticle. The buffer used was 20 mm Tris pH 7.5, 150 mm NaCl, 5% glycerol. (B) Sedimentation velocity ultracentrifugation of P6HRC1 showing a model of a continuous distribution of molecular masses, i.e. a c(M) analysis obtained using program Sedfit. The buffer used was 20 mm Tris pH 7.5, 150 mm NaCl, 5% glycerol. Molecular weight for P6HRC1 ranges from 700 kDa to 3 MDa.

Figure 6

(A) ELISA reactivity of nanoparticle mice antisera with nanoparticles (1 μg/well) coated on the plate. (B) ELISA reactivity of P6HRC1 nanoparticle mice antisera with P6HRC1 nanoparticles (1 μg/well) coated on the plate. (C) ELISA reactivity of P6HRC1 nanoparticle mice antisera with SARS‐CoV S protein (1–1180) (0.2 μg/well) coated on the plate. (D) ELISA reactivity of P6HRC1 nanoparticle mice antisera with MBP‐HRC‐GCN4 trimer (0.5 μg/well) coated on the plate. (E) ELISA reactivity of P6HRC1 nanoparticle mice antisera with native monomeric HRC peptide (0.5 μg/well) coated on the plate.

Figure 7

In vitro SARS‐CoV neutralizing activities of antibodies to P6HRC1 nanoparticle. Neutralization is shown as percentage of virus plus buffer alone. HRC1 nanoparticle sera significantly inhibited SARS‐CoV infection of Vero E6 cells, whereas antisera to nanoparticles alone did not neutralize virus infectivity.