Mucosal delivery of a double-stapled RSV peptide prevents nasopulmonary infection
Gregory H. Bird, Sandhya Boyapalle, Terianne Wong, Kwadwo Opoku-Nsiah, Raminder Bedi, W. Christian Crannell, Alisa F. Perry, Huy Nguyen, Viviana Sampayo, Ankita Devareddy, Subhra Mohapatra, Shyam S. Mohapatra, Loren D. Walensky
Gregory H. Bird, Sandhya Boyapalle, Terianne Wong, Kwadwo Opoku-Nsiah, Raminder Bedi, W. Christian Crannell, Alisa F. Perry, Huy Nguyen, Viviana Sampayo, Ankita Devareddy, Subhra Mohapatra, Shyam S. Mohapatra, Loren D. Walensky
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Research Article

Mucosal delivery of a double-stapled RSV peptide prevents nasopulmonary infection

Abstract

Respiratory syncytial virus (RSV) infection accounts for approximately 64 million cases of respiratory disease and 200,000 deaths worldwide each year, yet no broadly effective prophylactic or treatment regimen is available. RSV deploys paired, self-associating, heptad repeat domains of its fusion protein, RSV-F, to form a fusogenic 6-helix bundle that enables the virus to penetrate the host cell membrane. Here, we developed hydrocarbon double-stapled RSV fusion peptides that exhibit stabilized α-helical structure and striking proteolytic resistance. Pretreatment with double-stapled RSV peptides that specifically bound to the RSV fusion bundle inhibited infection by both laboratory and clinical RSV isolates in cells and murine infection models. Intranasal delivery of a lead double-stapled RSV peptide effectively prevented viral infection of the nares. A chitosan-based nanoparticle preparation markedly enhanced pulmonary delivery, further preventing progression of RSV infection to the lung. Thus, our results provide a strategy for inhibiting RSV infection by mucosal and endotracheal delivery of double-stapled RSV fusion peptides.

Authors

Gregory H. Bird, Sandhya Boyapalle, Terianne Wong, Kwadwo Opoku-Nsiah, Raminder Bedi, W. Christian Crannell, Alisa F. Perry, Huy Nguyen, Viviana Sampayo, Ankita Devareddy, Subhra Mohapatra, Shyam S. Mohapatra, Loren D. Walensky

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Figure 6

Intratracheal SAH-RSVFBD as a NP preparation inhibits pulmonary RSV infection.

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Intratracheal SAH-RSVFBD as a NP preparation inhibits pulmonary RSV infe...
(A) 10-week-old BALB/c mice were treated intratracheally with vehicle or Cy5-labeled SAH-RSVFBD administered alone (100 μM) or in combination with NPs (1:2.5 peptide/NP ratio) in a 50-μl volume. Mice were sacrificed 24 hours after treatment, and lungs were harvested after 1% paraformaldehyde perfusion, followed by cryopreservation in OCT. Sections (5 μm) were mounted in DAPI-containing medium and imaged with an Olympus fluorescent microscope. Original magnification, ×20. (B) ImageJ analysis was used to quantify Cy5+ cells, expressed as a percentage of total DAPI+ cells. Data represent mean ± SEM. (C) 10-week-old BALB/c mice were treated intratracheally with 50 μl NP, SAH-RSVFBD (250 μM peptide in 1.2% DMSO), the combination (1:2.5 peptide/NP ratio), or vehicle. 48 hours after treatment, mice were inoculated intranasally with single dose recombinant RSV-rA2Line19F (1 × 106 pfu/mouse). Control mice received 50 μl vehicle intratracheally followed by mock inoculation 48 hours later. Mice were sacrificed 4 days after infection, and 5-μm sections from 1% paraformaldehyde-perfused and fixed lungs were subjected to anti-RSV immunofluorescence (IF) analysis and H&E staining. Lung tissue from mice treated with the SAH-RSVFBD/NP formulation demonstrated decreased inflammatory cell infiltrates. Original magnification, ×20. (D) Average RSV positivity (red), quantified by ImageJ analysis of 8 images (4 sections) per mouse. GFP+ cells are expressed as a percentage of total DAPI+ cells. (E) Viral titers (log10 pfu/ml) in the supernatants of lung homogenates, measured by HEp-2 plaque assay. (F) Total RNA was isolated from the lungs of treated mice, and RSV-N expression was quantitated by RT-PCR. All data are mean ± SEM. *P < 0.01, **P < 0.001, ***P < 0.0001.