Chimeric coronavirus-like particles carrying severe acute respiratory syndrome coronavirus (SCoV) S protein protect mice against challenge with SCoV

Abstract

We tested the efficacy of coronavirus-like particles (VLPs) for protecting mice against severe acute respiratory syndrome coronavirus (SCoV) infection. Coexpression of SCoV S protein and E, M and N proteins of mouse hepatitis virus in 293T or CHO cells resulted in the efficient production of chimeric VLPs carrying SCoV S protein. Balb/c mice inoculated with a mixture of chimeric VLPs and alum twice at an interval of four weeks were protected from SCoV challenge, as indicated by the absence of infectious virus in the lungs. The same groups of mice had high levels of SCoV-specific neutralizing antibodies, while mice in the negative control groups, which were not immunized with chimeric VLPs, failed to manifest neutralizing antibodies, suggesting that SCoV-specific neutralizing antibodies are important for the suppression of viral replication within the lungs. Despite some differences in the cellular composition of inflammatory infiltrates, we did not observe any overt lung pathology in the chimeric-VLP-treated mice, when compared to the negative control mice. Our results show that chimeric VLP can be an effective vaccine strategy against SCoV infection.

Figure 1

Characterization of VLPs. (A) SCoV VLPs, MHV VLPs and chimeric VLPs, all of which were produced from 293T cells, were independently purified by sucrose gradient centrifugation and 5 μg of purified VLPs were applied to each lane of SDS-PAGE. Colloidal Coomassie blue staining (CCB) and Western blot (WB) analysis of purified SCoV VLPs, MHV VLPs and chimeric VLPs are shown. (B) Negative staining of a chimeric VLP. Arrowheads indicate peplomers. Bars = 100 nm. Courtesy of Dr. Vsevolod L. Popov.

Figure 2

SCoV-specific neutralizing antibody titers of immunized mice at 28 days after immunization (A) and SCoV titers in lungs of mice 2 days after intranasal challenge with 1 × 10⁶ TCID₅₀ of SCoV at day 28 (B). (A) Mean SCoV-specific neutralizing antibody titers of the mice at 28 days after i.n. inoculation of SCoV or i.m. inoculation of placebo (medium from Vero E6 cells) or 2 μg chimeric VLP suspended in PBS or alum are shown. The lengths of the bars indicate mean virus-specific serum neutralizing antibody titers. The vertical dashed line demarks the minimal antibody detection level in this assay (i.e., 10). (B) Mice were independently inoculated i.m. with placebo (medium from Vero E6 cells), a mixture of 2 μg chimeric VLP and PBS or a mixture of 2 μg chimeric VLP and alum or i.n. with 1 × 10⁶ TCID₅₀ of infectious SCoV. After 28 days, these mice were challenged with 1 × 10⁶ TCID₅₀ of SCoV. Two days later, mice were sacrificed and the virus titers in the lungs were determined as shown in the graph. The lengths of the bars indicate mean pulmonary virus titers in each indicated group (log₁₀/lung). The vertical dashed line denotes the minimal virus detection level in this assay (i.e., 2.3 log₁₀/lung). Number of animals/group = 5.

Figure 3

SCoV-specific neutralizing serum antibody titers in the immunized mice and SCoV titers in lungs of mice 2 days after intranasal challenge with SCoV at day 56. On day 0, mice were either left untreated or were inoculated i.n. with 1 × 10⁶ TCID₅₀ of SCoV or injected i.m. with placebo (medium from Vero E6 cells), a mixture of 2 μg MHV VLPs and PBS, a mixture of 2 μg chimeric VLP and PBS, a mixture of 2 μg chimeric VLP and alum, a mixture of 1 μg chimeric VLP and alum, a mixture of 0.5 μg chimeric VLP and alum, a mixture of 1 μg influenza virus vaccine and alum, or alum alone. (A) The graph represents virus-specific serum neutralizing antibody titers at 28 days post inoculation. Number of animals/group = 5. (B) At 56 days blood samples were collected, and then the mice, excluding those inoculated with live SCoV and left untreated, were re-inoculated with the same material. The graph represents virus-specific, serum-neutralizing antibody titers at 56 days after initial inoculation. The vertical dashed lines in (A) and (B) denote the minimal antibody detection level in this assay (1/10 dilution). (C) Mice were inoculated with 1 × 10⁶ TCID₅₀ of SCoV at day 56. After 2 days, mice were euthanized, and the lung virus titers were determined. The vertical dashed line in (C) denotes the minimal virus detection level in this assay (2.3 log₁₀ TCID₅₀/g lung). The data from two independent experiments were combined and are represented in (B) and (C). The number of mice used for (B) and (C) were: 7 for placebo, SCoV, and 2 μg chimeric VLP with PBS; 6 for 2 μg chimeric VLP with alum; 5 for MHV VLP with alum and 1 μg chimeric VLP with alum; 4 for 0.5 μg chimeric VLP with alum; and 3 for all other groups.

Figure 4

Lung histopathology and immunohistochemistry of the control placebo and immunized mice at 2 days post SCoV challenge. Mice (n = 5 for all groups) were inoculated with VeroE6 cell culture fluids twice at days 0 and 28 (A–C), immunized with a mixture of chimeric VLPs and PBS at days 0 and 28 (D and G), chimeric VLPs and alum (E and H) at days 0 and 28, or live SCoV at day 0 only (F and I). These mice were challenged with SCoV at day 56 and sacrificed at day 58. (A) Bronchiolar epithelial cells showed swelling and blebbing of the luminal cytoplasm, and extensive cellular debris comprised of necrotic epithelium and inflammatory cells in the airway lumen. Moderate peribronchiolar mononuclear inflammatory cell infiltrates are also present (hematoxylin and eosin staining) (magnification, ×200). (B) Thickening of the bronchiolar interstitial tissues and alveolar walls with mononuclear cell infiltration (magnification, ×200). (C) SCoV N antigen was distributed in bronchiolar epithelial cells, as determined by immunohistochemistry (magnification, ×400). (D–F) Bronchiolar epithelial cells showed rare swelling and blebbing of the luminal cytoplasm, and the rare presence of cellular debris in airways (magnification, ×200). The peribronchiolar mononuclear, neutrophil and eosinophil infiltrates (D, inset) (magnification, ×400). (G–I) SCoV antigens were not detected by immunohistochemistry in the lungs of these mice (magnification, ×400).

Figure 5

Amino acid sequences of endodomains of SCoV, MHV and FIPV. A CLUSTALW alignment of the carboxy-terminus of S protein sequences of SCoV (strain Urbani, GenBank accession no. AY278741), MHV (strain A59, GenBank accession no. NC_001846), and FIPV (GenBank accession no. AY994055). The transmembrane domain, cytoplasmic domain, cystein-rich region, and charge-rich region are indicated. Charge-residues in the membrane proximal region are shaded. Twelve residues near C-terminal region are in boxes. Asterisks represent identical residues.