Inhibition of respiratory syncytial virus infections with morpholino oligomers in cell cultures and in mice

Abstract

Respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in infants, young children, and high-risk adults. Currently, there is no vaccine to prevent RSV infection, and the available therapeutic agents are of limited utility. Peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) are a class of antisense agents that can enter cells readily and interfere with viral protein expression through steric blocking of complementary RNA. Two antisense PPMOs, designed to target sequence that includes the 5'-terminal region and translation start-site region of RSV L mRNA, were tested for anti-RSV activity in cultures of two human-airway cell lines. Both PPMOs showed minimal cytotoxicity and one of them, (AUG-2), reduced viral titers by >2.0 log(10). Intranasal (i.n.) treatment of BALB/c mice with AUG-2 PPMO before the RSV inoculation produced a reduction in viral titer of 1.2 log(10) in lung tissue at day 5 postinfection (p.i.), and attenuated pulmonary inflammation at day 7 postinfection. These data show that the AUG-2 PPMO possesses potent anti-RSV activity and is worthy of further investigation as a candidate for potential therapeutic application.

Figure 1. Structure, sequence, and cellular uptake of PPMO

(a) The deoxyribose ring and phosphodiester linkage of DNA are replaced by a morpholine ring and phosphorodiamidate linkage in PMO. The arginine-rich peptide (RXR)₄XB (see Materials and Methods), designated as P7, is covalently conjugated to the 5′ end of each PMO. “B” in the structure diagram represents the DNA bases A, C, G, or T. (b) PPMO (AUG-1 and AUG-2) were designed against a region of sequence that includes the AUG translation initiation codon of RSV L mRNA. DScr has a random PMO base sequence conjugated to a peptide identical to that of the other PPMO, to serve as a negative control. (c) HEp-2 cells were incubated with 20 μM of fluorescein-labeled DScr PPMO (Fl-PPMO) for 1h, 2h, and 4h. F1-PPMO entry was evaluated by confocal microscopy (top row) and flow cytometry (bottom row). Scale bar = 100 μm.

Figure 2. Evaluation of PPMO effect on cell viability

HEp-2 cells were treated with different concentrations of AUG-1, AUG-2 or DScr PPMO for 4 hours. The PPMO-containing media was then removed and the cells incubated in fresh media for an additional 24h before cell viability was evaluated by using the MTT assay. Data are expressed as percentage of viable cells compared to mock-treated cells (set at 100%). (See details in Materials and Methods).

Figure 3. Antiviral effect of PPMO in vitro

(a) HEp-2 and A549 cells treated with different concentrations of AUG-1, AUG-2 or DScr PPMO for 4h followed by infection with RSV A2 strain at an MOI of 0.01. Viral replication was determined 24h post-infection by HRP-staining plaque assay (see Methods). The activity of AUG-2 PPMO was also tested against the RSV B1 strain. hMPV-infected LL-MK2 cells were also challenged with PPMO, under the same conditions as above, as a control for RSV-specific PPMO activity. Vehicle-only treated cells produced titers that were almost identical to DScr-treated cells throughout these experiments. The limit of detection for this assay is 20 PFU/ml, indicated by the dotted line. *P< 0.05, **P< 0.01, ***P< 0.001 when comparing AUG-2- with DScr-treated cells. (b) HEp-2- cells were treated with 20 μM AUG-2 as described above and infected with rrRSV at different MOIs. Confocal images taken at 45 h p.i. show rrRSV-infected cells treated with DScr (top left) and AUG-2 (bottom left). Bar scale = 100 μm. RSV positive cells were determined by flow cytometry analysis. Numbers in each histogram represent percent of positive cells.

Figure 4. Pre- and post-infection treatment of cell cultures with PPMO

HEp-2 cells were infected with RSV at an MOI of 0.01. Different concentrations of PPMO were added at indicated time-points either before or after RSV infection. Viral replication was determined 24h after infection by HRP-staining plaque assay. The limit of detection for this assay was 20 PFU/ml, indicated by the dotted line.

Figure 5. Inhibition of RSV protein expression by PPMO treatment

HEp-2 cells were treated with PPMO for 4h followed by infection with RSV at an MOI of 3. Cells were lysed 24 h after infection and RSV proteins were detected by western blot using an anti-RSV polyclonal antibody. RSV proteins and molecular weight markers are denoted on the left and right sides respectively. The blots were also probed for β-actin as a control; shown below the viral proteins.

Figure 6. Biodistribution of Fl-PPMO in mouse lung

PPMO mixed 1:2 with trypan blue was administered i.n. to non-infected mice. Lungs were removed 2 hours later and opened along the sagittal plane. Panels a and b show lungs from a nontreated and PPMO-trypan blue-treated mouse, respectively. In a separate experiment, mice received DScr Fl-PPMO via i.n. administration, the lungs were removed 4h later and fixed. Lung section slides were examined by confocal microscopy as shown by (c) interference contrast, (d) fluorescent light, and (e) dual phase image.

Figure 7. PPMO treatment of RSV-infected mice

BALB/c mice were infected with 5 × 10⁶ PFU RSV and treated with indicated PPMO as follows: (a) one dose, at 4h before infection. (b) two doses, one at 4h before and one at 24h after infection. (c) three doses, at 4h before and at 24 h and 48 h after infection. (d) A single dose of 90 μg was administered 4 h before or 6 h after infection. Viral replication was quantified by HRP staining at day 5 after infection. Vehicle-only treated cells produced titers that were almost identical to DScr-treated cells throughout these experiments. The virus yields of AUG-2 PPMO were calculated by setting DScr PPMO values as 100%. Bar graphs represent mean ± SEM. n = 5 mice. *P< 0.05, **P< 0.01 when comparing AUG-2- with DScr-treated mice.

Figure 8. AUG-2 PPMO treatment reduces lung inflammation of RSV-infected mice

BALB/c mice were treated with 90 μg PPMO 4 h before RSV infection (5 × 10⁶ PFU). Lungs were harvested at day 7 after RSV infection, fixed for slide preparation and H&E stained. (a) Pathology score of prepared slides. Bar graphs represent mean ± SEM. n = 5 mice, with 6 slides/mouse. **P< 0.01. (b) Representative stained lung tissue sections from the indicated treatment. Arrows indicate cells infiltrating the perivascular and peribronchial spaces, (scored as described in Material and Methods). Scale bar = 200 μm.