Rhesus theta-defensin prevents death in a mouse model of severe acute respiratory syndrome coronavirus pulmonary disease

Abstract

We evaluated the efficacy of rhesus theta-defensin 1 (RTD-1), a novel cyclic antimicrobial peptide, as a prophylactic antiviral in a mouse model of severe acute respiratory syndrome (SARS) coronavirus (CoV) lung disease. BALB/c mice exposed to a mouse-adapted strain of SARS-CoV demonstrated 100% survival and modest reductions in lung pathology without reductions in virus titer when treated with two intranasal doses of RTD-1, while mortality in untreated mice was approximately 75%. RTD-1-treated, SARS-CoV-infected mice displayed altered lung tissue cytokine responses 2 and 4 days postinfection compared to those of untreated animals, suggesting that one possible mechanism of action for RTD-1 is immunomodulatory.

FIG. 1.

Treatment with RTD-1 protects mice against lethality of SARS-CoV infection. (A and B) BALB/c mice 6 to 8 weeks old were treated with sham control (40 μl phosphate-buffered saline, no virus), RTD-1 alone (125 μg [∼5 mg/kg] intranasal RTD-1 15 min prior to infection, followed by an identical dose 18 h later), SARS-CoV alone (3 × 10⁵ PFU MA15 intranasally in 40 μl phosphate-buffered saline), or RTD-1 followed with SARS-CoV infection. Mice were monitored daily for weight loss (A) and survival (B) (n = 6 or 7/group). SARS-CoV-infected mice without RTD-1 treatment had a 30% survival rate and 25% decrease in weight in those that survived (*, P ≤ 0.05 by Student's t test for SARS alone versus all other groups). Data presented in panels A and B are representative of two independent experiments. (C) Lung tissue was harvested from mice, and viral titer levels were determined. (D) RTD-1 has no direct antiviral effect on SARS-CoV. Viral titers were determined using Vero cells and samples of 1 × 10⁵ PFU SARS-CoV (Urbani) that were preincubated for 30 min at 37°C with RTD-1 in serum-free phosphate-buffered saline at the indicated concentrations prior to plaque assay in Vero cells. Results in panels A, C, and D are presented as means ± standard errors (A and B, n = 6 or 7; C and D, n = 3). All experiments were performed under biosafety level 3 containment. This study was approved by the University of Iowa Animal Care and Use Committee.

FIG. 2.

(A to L) Pulmonary tissue histopathology in SARS-CoV (3 × 10⁵ PFU MA15)-infected mice with or without RTD-1 (5 mg/kg, two doses) treatment. Hematoxylin- and eosin-stained (4 μM) tissue sections were examined 2, 4, and 10 days postinfection. See the text for additional details. Asterisks indicate alveolar edema; arrows indicate necrotizing bronchiolitis. n = 4 at each time point. Scale bar, 100 μm. (M to O) Pulmonary histopathology scores in SARS-CoV-infected mice treated with or without RTD-1. Tissues were harvested at 2 and 4 days and scored by a veterinary pathologist (D.K.M.) blinded to the treatment protocol, using a severity scale from 1 (absent/rare) to 3 (severe/multifocal). Data are presented for alveolar edema (M), perivascular cellular infiltrates (N), and necrotizing bronchiolitis (O). Results are presented as means ± standard errors (n = 4; *, P ≤ 0.05).

FIG. 3.

RTD-1 treatment alters pulmonary cytokine responses to SARS-CoV. Lung tissues were harvested at days 2 (A) and 4 (B) postinfection, and cytokine responses were evaluated using the Bio-Plex cytokine assay (Bio-Plex Cytokine 23-Plex kit; Bio-Rad Laboratories) to identify changes in cytokine protein expression in lung homogenates from baseline and days 2 and 4 postinfection. The results are shown for sham treatment (normal saline alone), RTD-1 alone (5 mg/kg, two doses), SARS-CoV (3 × 10⁵ PFU MA15) with RTD-1 treatment, and SARS-CoV alone. Note differences in y-axis scales between left and right panels. See the text for interpretation. Results are presented as means ± standard errors (n = 3; *, P ≤ 0.05 by Student's t test for SARS-CoV alone versus SARS-CoV plus RTD-1).