Complete protection against severe acute respiratory syndrome coronavirus-mediated lethal respiratory disease in aged mice by immunization with a mouse-adapted virus lacking E protein

Abstract

Zoonotic coronaviruses, including the one that caused severe acute respiratory syndrome (SARS), cause significant morbidity and mortality in humans. No specific therapy for any human coronavirus is available, making vaccine development critical for protection against these viruses. We previously showed that recombinant SARS coronavirus (SARS-CoV) (Urbani strain based) lacking envelope (E) protein expression (rU-ΔE) provided good but not perfect protection in young mice against challenge with virulent mouse-adapted SARS-CoV (MA15). To improve vaccine efficacy, we developed a second set of E-deleted vaccine candidates on an MA15 background (rMA15-ΔE). rMA15-ΔE is safe, causing no disease in 6-week-, 12-month-, or 18-month-old BALB/c mice. Immunization with this virus completely protected mice of three ages from lethal disease and effected more-rapid virus clearance. Compared to rU-ΔE, rMA15-ΔE immunization resulted in significantly greater neutralizing antibody and SARS-CoV-specific CD4 and CD8 T cell responses. After challenge, inflammatory cell infiltration, edema, and lung destruction were decreased in the lungs of rMA15-ΔE-immunized mice compared to those in rU-ΔE-immunized 12-month-old mice. Collectively, these results show that immunization with a species-adapted attenuated coronavirus lacking E protein expression is safe and provides optimal immunogenicity and long-term protection against challenge with lethal virus. This approach will be generally useful for development of vaccines protective against human coronaviruses as well as against coronaviruses that cause disease in domestic and companion animals.

Fig 1

Weights and survival in 6-week- and 12-month-old BALB/c mice after inoculation with 5 × 10⁴ PFU of rMA-ΔE. Six-week-old (A and B) or 12-month-old (C and D) BALB/c mice were inoculated with 5 × 10⁴ PFU of rMA-ΔE or MA15 (nonrecombinant) and monitored for weight loss and survival. Data are from 2 independent experiments with 6 mice per group (A and B) or with 9 (rMA-ΔE) or 6 (MA15) mice per group (C and D).

Fig 2

Immunization of 6-week-old BALB/c mice with rMA15-ΔE or rU-ΔE and challenge with MA15 at 21 days. Six-week-old mice were immunized with 6,000 PFU of rMA15-ΔE, rU-ΔE, or PBS. (A) Mice were sacrificed at days 2, 4, 6, and 8 postimmunization with rMA15-ΔE, and virus titers in the lungs were measured. (B) rMA15-ΔE-immunized mice were challenged with 10⁵ PFU of MA15 at day 21 postimmunization, and lung virus titers were measured. (C and D) Mice were immunized with rMA15-ΔE, rU-ΔE, or PBS and monitored for weight loss and survival. Data are representative of 2 experiments with 4 mice/group (A) or 2 experiments with 4 to 8 mice/group (B to D). (E) Serum neutralizing antibody titers were measured at day 21 as described in Materials and Methods. (F) Virus-specific CD4 and CD8 T cell responses in the lungs of immunized mice were analyzed by intracellular IFN-γ staining at day 7 as described in Materials and Methods (see Fig. 4F for representative fluorescence-activated cell sorter [FACS] plots). Average frequencies and numbers of N353-specific CD4 and S366-specific CD8 T cells are shown. Data are representative of one of two independent experiments with 4 mice per group. *, P < 0.05; **, P < 0.005; ****, P < 0.0001.

Fig 3

Histological changes observed after immunization with rMA-ΔE or rU-ΔE and challenge with MA15. Twelve-month-old BALB/c mice were immunized with 6,000 PFU of rMA15-ΔE (A to C), rU-ΔE (D to F), or PBS (G) and sacrificed at day 2, 4, or 6 postimmunization. Additional groups of mice were challenged with 10⁵ PFU of MA15 at days 21 (d21) (H to L) and 66 (d66) (M to Q) after immunization. Lungs were harvested and processed for histological examination as described in Materials and Methods. Representative images are shown. X, edema; ⊙, cellular debris, ↓, denuded epithelium. Original magnification, ×40.

Fig 4

Immunization of 12-month-old BALB/c mice with rMA15-ΔE or rU-ΔE and challenge with MA15 at 21 days. (A to D) Twelve-month-old mice were immunized with 6,000 PFU of rMA-ΔE, rU-ΔE, or PBS. (A) Virus titers at days 2, 4, 6, and 8 postimmunization. Data are combined results from 2 experiments (n = 4 to 9 mice/time point/group). (B to D) Mice were challenged at 21 days postimmunization with 10⁵ PFU of MA15. (B) Virus titers in the lungs were determined at days 1, 2, and 4 postchallenge (n = 4 to 6 mice/time point/group). (C and D) Mice were monitored for weight loss and survival. Data are representative of 1 of 2 independent experiments with 5 to 7 mice per group. (E and G) Serum neutralizing and ELISA antibody titers were measured at day 21 after immunization. (F and H) Virus-specific CD4 and CD8 T cell responses were analyzed by intracellular IFN-γ staining as described in Materials and Methods. (F) Representative flow cytometric plots of virus-specific CD4 and CD8 T cells in lungs of 12-month-old mice at day 7 after immunization. (H) Average frequency and number of N353-specific CD4 and S366-specific CD8 T cells in the lungs of 12-month-old mice. Data are representative of one of two independent experiments with 4 mice per group. *, P < 0.05; **, P < 0.005; ****, P < 0.0001.

Fig 5

Immunization of 18-month-old BALB/c mice with rMA15-ΔE and challenge with MA15 at 21 days. (A) Eighteen-month-old mice were immunized with 6,000 PFU of rMA15-ΔE or PBS and monitored for weight loss. (B and C) Twenty-one days postimmunization, mice were challenged with 10⁵ PFU of MA15 and monitored for weight loss and survival. Data are from 2 experiments with 5 to 6 mice per group.

Fig 6

Immunization of 6-week- and 12-month-old BALB/c mice with rMA15-ΔE or rU-ΔE and challenge with MA15 at 66 days. Six-week-old (A, C, and E) or 12-month-old (B, D, and F) mice were immunized with 6,000 PFU of rMA15-ΔE, rU-ΔE, or PBS and challenged 66 days later with 10⁵ PFU of MA15. (A to F) Mice were sacrificed for lung virus titers at the times shown postinfection (n = 3 to 6 mice/group/time) (A and B) or monitored for weight loss and survival (n = 5 mice/group) (C to F). (G to J) Neutralizing and ELISA antibody titers were measured at day 66 after immunization in 6-week-old (G and H) or 12-month-old (I and J) BALB/c mice. *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.