Immunization with an attenuated severe acute respiratory syndrome coronavirus deleted in E protein protects against lethal respiratory disease

Abstract

The severe acute respiratory syndrome coronavirus (SARS-CoV) caused substantial morbidity and mortality in 2002-2003. Deletion of the envelope (E) protein modestly diminished virus growth in tissue culture but abrogated virulence in animals. Here, we show that immunization with rSARS-CoV-DeltaE or SARS-CoV-Delta[E,6-9b] (deleted in accessory proteins (6, 7a, 7b, 8a, 8b, 9b) in addition to E) nearly completely protected BALB/c mice from fatal respiratory disease caused by mouse-adapted SARS-CoV and partly protected hACE2 Tg mice from lethal disease. hACE2 Tg mice, which express the human SARS-CoV receptor, are extremely susceptible to infection. We also show that rSARS-CoV-DeltaE and rSARS-CoV-Delta[E,6-9b] induced anti-virus T cell and antibody responses. Further, the E-deleted viruses were stable after 16 blind passages through tissue culture cells, with only a single mutation in the surface glycoprotein detected. The passaged virus remained avirulent in mice. These results suggest that rSARS-CoV-DeltaE is an efficacious vaccine candidate that might be useful if SARS recurred.

Fig. 1

Clinical effects and vaccine efficacy of rSARS-CoV-ΔE and rSARS-CoV-Δ[E,6-9b]. (A, B). Six-week-old BALB/c mice were infected with 12,000 PFU of rSARS-CoV-ΔE (circles, n = 10) or rSARS-CoV-Δ[E,6-9b] (squares, n = 10) and monitored daily for survival (A) and weight loss (B). (C, D). Six-week-old BALB/c mice were immunized with 12,000 PFU of rSARS-CoV-ΔE (circles, n = 10) or rSARS-CoV-Δ[E,6-9b] (squares, n = 10) or phosphate-buffered saline (PBS) (triangles, n = 11) and challenged at day 21 post-infection with 1 × 10⁵ PFU of MA15 virus. Survival (C) and weight loss (D) were recorded daily. Differences in survival were statistically significant (p < 0.002).

Fig. 2

Virus titers and pulmonary histopathological changes in MA15 virus-infected mice. Six-week-old BALB/c mice were immunized with 12,000 PFU of rSARS-CoV-ΔE or PBS and challenged at day 21 p.i. with 1 × 10⁵ PFU of MA15 virus. Mice were sacrificed at days 3 and 5 post-challenge and analyzed for infectious virus (A) and histopathology (B–E) as described in Materials and Methods.

Fig. 3

Vaccine efficacy in SARS-CoV-infected hACE2 Tg mice. Six-week-old hACE2 Tg mice were immunized with 12,000 PFU of either rSARS-CoV-ΔE (circles, n = 6) or rSARS-CoV-Δ[E,6-9b] (squares, n = 10) or PBS (triangles, n = 6) and challenged at day 21 post-immunization with 12,000 PFU of the Urbani strain of SARS-CoV. Mice were monitored daily for survival.

Fig. 4

Virus-specific CD8 T cell responses in hACE2 Tg mice after immunization with rSARS-CoV-ΔE and rSARS-CoV-Δ[E,6-9b]. Six-week-old hACE2 Tg mice were immunized by intranasal inoculation with rSARS-CoV-ΔE or rSARS-CoV-Δ[E,6-9b]. (A) Blood was obtained 7 days p.i. and the virus-specific CD8 T cell response was analyzed by intracellular IFN-γ staining as described in Materials and Methods. Representative flow cytometry plots of antigen-specific CD8 T cells in the blood of Tg mice at 7 days post-immunization with rSARS-CoV-ΔE. (B) Average frequency of S436- and S525-specific CD8 T cells in the blood of hACE2 Tg mice at day 7 post-immunization with either rSARS-CoV-ΔE (open bars, n = 6) or rSARS-CoV-Δ[E,6-9b] (filled bars, n = 6). (C) Average frequency of virus-specific CD8 T cells in the spleen of hACE2 Tg mice at day 21 post-immunization with rSARS-CoV-ΔE or rSARS-CoV-Δ[E,6-9b] (n = 3 for both viruses and both peptides). The number of epitope S436-specific cells were greater in the blood (p = 0.07) and spleen (p = 0.08) in rSARS-CoV-ΔE compared to rSARS-CoV-Δ[E,6-9b]-immunized mice.

Fig. 5

Virus-specific CD8 T cell responses in BALB/c mice after immunization with rSARS-CoV-ΔE and rSARS-CoV-Δ[E,6-9b]. Six-week-old BALB/c mice were immunized by intranasal inoculation with rSARS-CoV-ΔE or rSARS-CoV-Δ[E,6-9b]. Blood was obtained 7 days later and the virus-specific CD8 T cell response was analyzed by intracellular IFN-γ staining. (A) Representative flow cytometry plots of antigen-specific CD8 T cells in the blood of rSARS-CoV-ΔE or rSARS-CoV-Δ[E,6-9b]-immunized BALB/c mice at 7 days post-immunization. (B) Average frequency of S366-specific CD8 T cells in the blood of BALB/c mice at day 7 post-immunization with either rSARS-CoV-ΔE (open bars) or rSARS-CoV-Δ[E,6-9b] (filled bars) (n = 8 for both viruses ). Numbers indicate fraction of mice with detectable virus-specific CD8 T cell responses. Samples with undetectable responses were not included in the figure.

Fig. 6

Enhanced replication of rSARS-CoV-ΔE and rSARS-CoV-Δ(E,6-9b) after serial passage in Vero E6 cells. rSARS-CoV-ΔE and rSARS-CoV-Δ[E,6-9b] were serially passaged 16 times in Vero E6 cells. Virus was harvested and analyzed in one step growth curves by infecting Vero cells at a multiplicity of infection of 0.05. Titers were measured at the indicated times p.i. p1 and p16 indicate the original virus stock and virus passaged 16 times, respectively. Differences in titers at each time point were statistically significant (p < 0.04).

Fig. 7

Clinical effects and vaccine efficacy of rSARS-CoV-ΔE and rSARS-CoV-Δ[E,6-9b] after 16 passages through Vero E6 cells. (A, B). Six-week-old hACE2 Tg mice were inoculated with 12,000 PFU of rSARS-CoV-ΔE-p16 (circles, n = 14) or rSARS-CoV-Δ[E,6-9b]-p16 (squares, n = 18) or wild-type rSARS-CoV (triangles, n = 4) and monitored daily for survival (A) and weight loss (B). Differences in survival were statistically significant (p < 0.002). (C, D). Six-week-old hACE2 Tg mice were immunized with 12,000 PFU of rSARS-CoV-ΔE-p16 (circles, n = 8) or rSARS-CoV-Δ(E,6-9b)-p16 (squares, n = 10) or PBS (triangles, n = 4) and challenged 21 days post-immunization with 12,000 PFU of SARS-CoV Urbani. Survival (C) and weight loss (D) were recorded daily.