Lack of innate interferon responses during SARS coronavirus infection in a vaccination and reinfection ferret model

Abstract

In terms of its highly pathogenic nature, there remains a significant need to further define the immune pathology of SARS-coronavirus (SARS-CoV) infection, as well as identify correlates of immunity to help develop vaccines for severe coronaviral infections. Here we use a SARS-CoV infection-reinfection ferret model and a functional genomics approach to gain insight into SARS immunopathogenesis and to identify correlates of immune protection during SARS-CoV-challenge in ferrets previously infected with SARS-CoV or immunized with a SARS virus vaccine. We identified gene expression signatures in the lungs of ferrets associated with primary immune responses to SARS-CoV infection and in ferrets that received an identical second inoculum. Acute SARS-CoV infection prompted coordinated innate immune responses that were dominated by antiviral IFN response gene (IRG) expression. Reinfected ferrets, however, lacked the integrated expression of IRGs that was prevalent during acute infection. The expression of specific IRGs was also absent upon challenge in ferrets immunized with an inactivated, Al(OH)(3)-adjuvanted whole virus SARS vaccine candidate that protected them against SARS-CoV infection in the lungs. Lack of IFN-mediated immune enhancement in infected ferrets that were previously inoculated with, or vaccinated against, SARS-CoV revealed 9 IRG correlates of protective immunity. This data provides insight into the molecular pathogenesis of SARS-CoV and SARS-like-CoV infections and is an important resource for the development of CoV antiviral therapeutics and vaccines.

Figure 1. Mean levels of serum neutralizing antibody to SARS-CoV.

Neutralizing antibody levels in serum were determined as outlined in the materials and methods section. Inverse neutralization titre is represented on the y-axis vs. day of study on the x-axis. Note that ferrets were infected on study day 1 and reinfected ferrets were also innoculated on study day 30. Mock infected animals received an intranasal instillation of serum-free media on study day 1 and had undetectable titres. Values shown represent group mean of 3–4 ferrets per group, and error bars show standard deviation.

Figure 2. Viral burden in lung tissue.

Viral burdens in sections of lung were determined by the TCID₅₀ method, as outlined in the materials and methods section. Log10 virus titres are shown on the vertical axis vs. study day on the horizontal axis. Note that ferrets were infected on study day 1, and reinfected ferrets were also inoculated on study day 30. Mock infected animals received an intranasal instillation of serum-free media on study day 1 and had undetectable virus. Values shown represent group means of 3–4 ferrets per group, and error bars show standard deviation.

Figure 3. Lung histopathology to SARS-CoV challenge following reinfection.

Histological lung sections (5 µm) were obtained from multiple lung lobes at 7 d postchallenge and stained by hematoxylin and eosin. Representative micrographs from uninfected (A), SARS infected alone (B), or SARS infected and re-infected (C and D) are shown. Primary SARS-CoV infection produced inflammation and the appearance of lung immune cells primarily surrounding small-to-medium bronchial airways at 7 days following challenge (B). Ferrets that received infection-reinfection were largely protected from lung histopathology (C).

Figure 4. Microarray analysis of gene expression in lung tissue from ferrets infected and reinfected with SARS-CoV.

EDGE analysis across all time points identified 3454 genes as significantly differently expressed (≥2-fold change in at least one time point, p≤0.05, and q≤0.1) as described in the Methods. Genes were then one-way hierarchically clustered by gene using Pearson correlation and average distance metrics (red, upregulated; blue, downregulated). The most significant canonical signaling pathways according to IPA for the resulting clusters are noted. Full gene lists are publically available on GEO (see Methods). DPI, days post-infection. DPR, days post-reinfection.

Figure 5. Complement and IL-6 signaling in SARS-CoV infected-reinfected ferret lungs.

(A) Complement and IL-6 signaling (C) genes selected by pathway analysis are shown in a one-way hierarchical cluster (red = upregulated, blue = downregulated). (B) IPA canonical complement system pathway analysis at 2 DPI. (D) IPA canonical IL-6 signaling pathway analysis at 5 DPI. All genes are significantly differently expressed (EDGE analysis: ≥2-fold change in at least one time point, p≤0.05, and q≤0.1).

Figure 6. IFN responses in SARS-CoV infected-reinfected ferret lungs.

(A) Fifty IRGs selected by pathway analysis are shown in a one-way hierarchical cluster. (B) IPA canonical IFN-signaling pathway analysis at 3 DPI and 3 DPR. All genes are significantly differently expressed (EDGE analysis: ≥2-fold change in at least one time point, p≤0.05, and q≤0.1).