Lack of Innate Interferon Responses during SARS Coronavirus Infection in a Vaccination and Reinfection Ferret Model

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

Lack of Innate Interferon Responses during SARS Coronavirus Infection in a Vaccination and Reinfection Ferret Model

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

Number of regulated genes in different functional categories following primary infection and reinfection.

<p>Number of regulated genes in different functional categories with at least 1.5-fold change and a significant t-test of p<0.05 (↑ upregulated, ↓ downregulated).</p

Mean levels of serum neutralizing antibody to SARS-CoV.

<p>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.</p

Expression of selected significantly changed genes over time in SARS-CoV infected–reinfected ferret lungs.

a<p>Mean gene expression ratios (log2) for selected genes is relative to the mock-infected ferret gene expression at 2 DPI.</p>b<p>Statistical significance of gene expression differences over time is determined by EDGE analysis as described in the Methods.</p>c<p><i>Canis familiaris</i> UniGene Build #11 (April, 2005) identifiers as per the Affymetrix GeneChip Canine Genome 2.0 Array probe library.</p

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

<p>(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).</p

Lung histopathology to SARS-CoV challenge following reinfection.

<p>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).</p

Viral burden in lung tissue.

<p>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.</p