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

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.

New therapeutic strategies are needed to prevent and treat RSV infection, which causes significant morbidity and mortality worldwide each year (1). RSV can produce acute respiratory illness in patients of all ages, but strikes the age extremes, infants and the elderly, with highest frequency. RSV is a major, global cause of severe lower respiratory tract viral illness in infants and children, responsible for 70,000–126,000 infant hospitalizations for pneumonia or bronchiolitis and 400 infant deaths every year in the US alone (2, 3). The elderly are also at risk for severe disease (4, 5), with up to 62,000 RSV-associated hospitalizations reported annually in the US (6). RSV attack rates in US nursing homes are approximately 5%–10% per year, with a 2%–8% case fatality rate, amounting to approximately 10,000 deaths yearly among individuals older than 64 years (7).

A humanized monoclonal antibody against RSV-F, palivizumab, is the current mainstay for prophylaxis of children younger than 24 months who are at high risk of severe RSV infection, but is only moderately effective at preventing infection (8, 9). Although ribavirin, a nucleoside analog with modest in vitro activity against RSV, is FDA approved for the treatment of RSV infection, its routine use in infants and children is not recommended due to lack of proven efficacy (10). The morbidity and mortality associated with RSV infection have continued to invigorate efforts to develop peptide (11–13), small-molecule (14–18), DNA (19, 20), siRNA-based (21), and vaccine strategies (22–24) to combat RSV infection. For example, a RhoA-derived peptide, termed C3, was shown to inhibit the interaction between the RSV-F fusion protein and RhoA GTPase, thereby blocking syncytium formation. The small molecules RFI-641 (17) and VP-14637 (15) were reported to inhibit viral entry by targeting RSV-F. Despite a broad range of anti-RSV efforts, new FDA-approved drugs have yet to emerge. However, a recent breakthrough in the design of RSV-F glycoprotein stabilized to preserve a highly antigenic site in its prefusion state successfully yielded RSV-specific neutralizing antibodies in mice and macaques, providing new hope for an RSV vaccine (23, 25).

RSV is a member of the Paramyxoviridae family of viruses, which contain 3 glycoproteins on the viral envelope. The RSV-F surface protein mediates fusion of the RSV envelope with the host cell membrane (26). Like HIV-1, influenza, Ebola, and SARS viruses, the RSV fusion process requires formation of a 6-helix bundle (6-HB) that enables the virus to penetrate the host membrane (27). Initially, RSV-F exists as a core trimer composed of the N-terminal heptad repeat domain (NHR), upon which the C-terminal heptad repeat domain (CHR) folds back to form a hairpin turn, yielding an antiparallel trimer of dimers (14). The crystal structure of hRSV-F fragments confirmed the structural homology between the hRSV-F1 protein core and several other viral fusion proteins, including HIV-1 gp41, which suggests that therapeutic strategies designed to target HIV-1 viral fusion may be adapted to subvert RSV (26).

The peptidic drug enfuvirtide (T-20) contains the CHR sequence of HIV-1 gp41 and effectively targets gp41 in vivo, potently inhibiting HIV-1 fusion and entry (28). CHR peptides derived from RSV such as T118, which contains residues 488–522 of RSV-F, exhibited nanomolar inhibitory activity in RSV infectivity and syncytia formation assays (11). The mechanism of action mandates that T118 form an α-helical complex upon binding to the NHR trimeric core. However, T118 and other CHR derivatives exist primarily as random coils in solution, as evidenced by their less than 10% α-helical content (29). Thus, T118 and its analogs must pay the entropic cost of folding into the active conformation upon target binding, which translates into decreased binding affinity and biological activity. Furthermore, peptide unfolding renders the amide backbone highly susceptible to proteolytic degradation in vivo. Such liabilities have stifled the development of peptide-based RSV fusion inhibitors.

The poor pharmacologic properties of peptides have long motivated chemists to develop new synthetic strategies for stabilizing secondary structure so that the inherent potency and selectivity of peptides can be harnessed for targeting protein interactions. An important breakthrough in peptide α-helix stabilization derived from installing an all-hydrocarbon “staple” into native peptide sequence. Non-natural α,α-disubstituted amino acids bearing olefin tethers are substituted for natural amino acids at positions spanning 1 (i,i+4) or 2 (i,i+7) turns of the α-helix, followed by ruthenium-catalyzed olefin metathesis to effectively cross-link the inserted residues (30). The all-hydrocarbon constraint can confer α-helical structure, proteolytic stability, and improved biological activity by recapitulating bioactive structure (30, 31). Here we applied hydrocarbon stapling to develop stabilized α-helices of RSV-F (referred to herein as SAH-RSVF peptides) for evaluation as next-generation inhibitors of RSV infection (Figure 1A).

Figure 1

Design, α-helicity, and 5-HB binding activity of i,i+7–stapled SAH-RSVF peptides. (A) RSV fusion mechanism and its disruption by a decoy HR2 helix. (B) Sequence compositions and staple placement of i,i+7–stapled SAH-RSVF peptides. (C) Circular dichroism analysis of i,i+7–stapled SAH-RSVF peptides, demonstrating the observed range of α-helical stabilization by hydrocarbon stapling. (D) Competitive binding activity of i,i+7–stapled SAH-RSVF peptides against the FITC-RSVF/5-HB interaction. Data represent mean ± SEM for experiments performed in triplicate.

Synthesis and binding activity of SAH-RSVF peptides. We previously explored the utility of inserting i,i+4 staples, which span 1 turn of an α-helix, into HR2 peptides derived from gp41, yielding a lead i,i+4 double-stapled peptide with optimized α-helical structure, proteolytic resistance, and anti–HIV-1 activity (32). By analogy, we installed single i,i+4 staples within the N- and C-terminal portions of an RSV HR2 peptide spanning residues 488–522, generating peptides SAH-RSVF_a–SAH-RSVF_f (Supplemental Figure 1A; supplemental material available online with this article; doi: 10.1172/JCI71856DS1). Circular dichroism analysis revealed that insertion of a single i,i+4 staple enhanced α-helical structure up to 2.3-fold over the unmodified SAH-RSVF peptide (Supplemental Figure 1B). To test for the capacity of the single i,i+4–stapled SAH-RSVF peptides to target the RSV fusion apparatus, we developed a recombinant 5-HB assay, as previously described for HIV-1 gp41 (33). The RSV 5-HB is composed of alternating HR1 and HR2 helices separated by short glycine/serine linkers. The corresponding recombinant DNA was synthesized as short overlapping oligonucleotides, assembled by PCR, and then subcloned into a vector containing a C-terminal hexahistidine tag. Recombinant 5-HB protein was expressed as inclusion bodies, and, after denaturing lysis, nickel affinity chromatography, dialysis, and protein refolding, yields of 10 mg/l were achieved. The fluorescently labeled RSVF template peptide directly bound to the RSV 5-HB with a K_D of 63 nM, as measured by fluorescence polarization (FP) assay (Supplemental Figure 1C), completing the formation of an RSV 6-HB (Supplemental Figure 1D), which was then deployed in competition FP binding assays. Comparative binding analyses revealed that single i,i+4–stapled SAH-RSVF peptides either matched (SAH-RSVF_a–SAH-RSVF_c and SAH-RSVF_e) or exhibited weaker (SAH-RSVF_d and SAH-RSVF_f) competitive binding activity than that of the unlabeled RSVF template peptide (Supplemental Figure 1E). To determine whether an equipotent binder could target native RSV virus, we conducted colocalization imaging analyses of A549 cells treated with FITC–SAH-RSVF_b and RSV-A2 virus. Both live confocal microscopy and immunoelectron microscopy documented striking colocalization of peptide and virus, visualized as punctate structures at the plasma membrane (Supplemental Figure 2, A and B). Thus, SAH-RSVF_b, which bound to recombinant RSV 5-HB in vitro, likewise engaged native RSV in the setting of viral infection.

In an effort to improve upon the binding affinity of the unmodified SAH-RSVF peptide, we explored the effect of structural stabilization by use of i,i+7 staples, which cross-link a 2-turn span of α-helical peptide. A panel of N- and C-terminally i,i+7–stapled RSV peptides, designated SAH-RSVF_A–SAH-RSVF_F, were successfully synthesized (Figure 1B) and exhibited up to 3-fold stabilization of α-helical structure, as determined by circular dichroism (Figure 1C). In contrast to the i,i+4 series, the majority of i,i+7 single-stapled peptides showed notably enhanced competitive binding activity for RSV 5-HB compared with the unmodified peptide (Figure 1D). Interestingly, for those constructs with impaired binding activity, the degree of impairment correlated with progressive encroachment of the staple on the critical hydrophobic binding interface. Thus, for the RSVF template, 2-turn helix stabilization by i,i+7 stapling markedly enhances binding affinity for the RSV 5-HB.

Enhanced α-helicity and protease resistance of i,i+7 double-stapled SAH-RSVF peptides. We combined the best N- and C-terminal i,i+7 single-stapled peptides, in addition to carrying forward the nonbinding C-terminal F staple position as a negative control, to generate a series of i,i+7 double-stapled constructs (Figure 2A). In each case, the i,i+7 double-stapled peptides exhibited additive enhancement in α-helical content compared with the corresponding single-stapled analogs (Figure 2B), which suggests that double stapling in this context optimally reinforces α-helical structure throughout the length of the SAH-RSVF peptide. Of the i,i+7 double-stapled constructs, SAH-RSVF_BD and SAH-RSVF_CD retained the most potent competitive binding activity, followed by SAH-RSVF_BE and SAH-RSVF_CE, and the predictably poor SAH-RSVF_BF and SAH-RSVF_CF analogs that contained the disruptive F staple (Figure 2C).

Figure 2

Design, α-helicity, 5-HB binding activity, and protease resistance of i,i+7 double-stapled SAH-RSVF peptides. (A) Sequence compositions and staple placement of i,i+7 double-stapled SAH-RSVF peptides. (B) Circular dichroism analysis of i,i+7 double-stapled SAH-RSVF peptides, demonstrating enhanced α-helical stabilization compared with the corresponding i,i+7 single-stapled peptides. ^, exceeds the calculated ideal α-helicity of an undecapeptide standard. (C) Competitive binding activity of i,i+7 double-stapled SAH-RSVF peptides against the FITC-RSVF/5-HB interaction. SAH-RSVF_BD and SAH-RSVF_CD were among the most effective competitive binders, whereas the negative control constructs SAH-RSVF_BF and SAH-RSVF_CF showed substantially impaired 5-HB binding activity. Data represent mean ± SEM for experiments performed in triplicate. (D) Trypsin resistance profile of i,i+7 single- and double-stapled SAH-RSVF peptides. (E) Proteinase K resistance profile of an exemplary subpanel of i,i+7 single- and double-stapled SAH-RSVF peptides. Data (mean ± SEM) represent fraction intact for experiments performed in triplicate.

An important optimization criterion for peptide therapeutics is protease resistance. To measure the comparative protease susceptibility of unmodified and i,i+7 single- and double-stapled peptides, we subjected the lead SAH-RSVF constructs and their negative controls to trypsin digestion, followed by liquid chromatography/mass spectrometry (LC/MS) analysis of the reaction products over time. Whereas i,i+7 single stapling conferred up to 4-fold enhancement in peptide half-life compared with the unmodified peptide, i,i+7 double-stapled analogs manifested 3- to 33-fold improvement in proteolytic resistance (Figure 2D). In each case, the double-stapled peptides displayed at least additive proteolytic resistance of the corresponding single-stapled peptides, with synergistic benefit typically observed. We further subjected one of our lead RSV 5-HB competitive binders (SAH-RSVF_BD), its corresponding single-stapled analogs (SAH-RSVF_B and SAH-RSVF_D), and a control panel (SAH-RSVF_F and SAH-RSVF_BF) to even more aggressive proteolytic conditions using proteinase K, and again found striking proteolytic resistance of both double-stapled peptides compared with the corresponding single-stapled peptides and unmodified RSVF template (Figure 2E). Taken together, these data demonstrated that i,i+7 double stapling optimizes α-helicity and protease resistance, yielding structurally stable and high-affinity competitive inhibitors of RSV 6-HB formation.

SAH-RSVF peptides inhibit RSV infectivity in vitro. To determine whether the 5-HB–targeting capability of i,i+7 single- and double-stapled peptides correlated with functional blockade of RSV infection, we pretreated A549 cells with vehicle or 5-μM concentrations of unmodified or i,i+7–stapled peptide, followed by infection with a recombinant GFP-expressing RSV (rgRSV). After a 1-hour infection period, the medium was replaced with fresh peptide-containing medium, and cells were incubated for an additional 24 hours, followed by harvesting for determination of percent GFP⁺ cells by flow cytometry. Consistent with 5-HB binding avidity, SAH-RSVF_B, SAH-RSVF_C, SAH-RSVF_D, SAH-RSVF_BD, and SAH-RSVF_CD demonstrated potent suppression of rgRSV infection, whereas all constructs containing the negative control F staple were even less effective at inhibiting infectivity than the unmodified SAH-RSVF peptide (Figure 3A and Supplemental Figure 3A). Correspondingly, the 3 best inhibitors of rgRSV infection, SAH-RSVF_B, SAH-RSVF_C, and SAH-RSVF_BD, were most effective at blocking viral syncytia formation, with those analogs containing the F staple again showing no inhibitory activity (Figure 3B). Next, the lead double-stapled peptides, SAH-RSVF_BD and SAH-RSVF_CD, along with their negative controls, SAH-RSVF_BF and SAH-RSVF_CF, were subjected to RSV-A2 plaque assay and viral titer measurement. A549 cells were treated with vehicle or 5 μM stapled peptide as above, except that cells were infected with RSV-A2 for a 2-hour period. The cells and media were harvested after 24 hours of incubation in fresh peptide-containing medium. Cells were analyzed for RSV-N mRNA levels, and the collected supernatant was applied to HEp-2 cells for plaque assay and measurement of viral titers after 5 days. Whereas SAH-RSVF_BF and SAH-RSVF_CF treatment had no suppressive effect on RSV-A2 viral titers or RSV-N mRNA levels compared with the untreated control, SAH-RSVF_BD and SAH-RSVF_CD peptides significantly reduced both viral titers and RSV-N transcript levels, with SAH-RSVF_BD manifesting the most potent inhibitory activity (Figure 3C and Supplemental Figure 4). To confirm the efficacy of SAH-RSVF_BD, plaque assay and viral titer measurements were repeated using 30 minutes of peptide pretreatment followed by infection with both lab and clinical RSV isolates, including RSV-A2, RSV 2-20, and the recombinant RSV-A2 strain expressing fusion protein from clinical isolate Line 19 (RSV-rA2Line19F) (34). In each case, SAH-RSVF_BD showed marked suppression of viral titers compared with the vehicle control (Figure 3D). Finally, we applied the method of Saika et al. (35) to confirm that SAH-RSVF_BD–mediated inhibition of RSV infectivity indeed derived from blockade of viral fusion with the cell membrane. RSV-A2 virus was labeled with the fluorescent lipids R18 (red) and DiOC18 (green) and applied to A549 cells. Labeled virus is initially red due to the quenching of the DiOC18 fluorescent signal by R18; upon fusion with cellular membrane, the lipids are diluted, and dequenching of DiOC18 is reflected by the shift in red coloration to orange-yellow or green, as imaged by confocal microscopy. We found that SAH-RSVF_BD treatment (5 μM) reduced the number of RSV-A2 fusion events by 60% (Supplemental Figure 5), highlighting the mechanistic basis for peptide activity.

Figure 3

Inhibition of RSV infection and syncytia formation by i,i+7–stapled SAH-RSVF peptides. (A) The capacity of SAH-RSVF peptides to suppress infection of A549 cells by GFP-expressing rgRSV correlated with their competitive binding potency in the 5-HB FP assay (see Figure 1D and Figure 2C). SAH-RSVF_B, SAH-RSVF_C, SAH-RSVF_D, SAH-RSVF_BD, and SAH-RSVF_CD demonstrated the most robust viral inhibitory activity, whereas controls SAH-RSVF_F, SAH-RSVF_BF, and SAH-RSVF_CF were markedly impaired. Data represent mean ± SEM for experiments performed in triplicate. (B) Comparative activity of SAH-RSVF peptides in inhibiting syncytia formation after rgRSV infection of A549 cells, as counted in 3 different wells at 4 discrete locations per well at 48 hours after infection. Data are mean ± SEM. (C) Inhibition of RSV-A2 infection by SAH-RSVF peptides, as assessed by HEp-2 cell plaque assay and measurement of viral titers (pfu/ml). Data are mean ± SEM for 2 independent experiments performed in technical triplicates. (D) SAH-RSVF_BD prevented viral infection of A549 cells by RSV-A2, RSV 2-20, and RSV-rA2Line19F, as assessed by HEp-2 cell plaque assay and measurement of viral titers (pfu/ml). Data are mean ± SEM for 2 independent experiments performed in technical triplicates. *P < 0.001, **P < 0.0001.

Intranasal pretreatment with SAH-RSVF_BD blocks nasal and pulmonary RSV infection. To determine whether the in vitro anti-RSV activity of SAH-RSVF_BD translated to an in vivo context, we administered vehicle, SAH-RSVF_BD, or SAH-RSVF_BF by nasal drop (50 μl of 125 μM solution in 1.2% DMSO) to 10-week-old BALB/c mice (n = 4 per cohort) followed by intranasal inoculation with a single dose of rgRSV (1 × 10⁶ pfu) 1 hour later. Mice were sacrificed 24 hours after infection, and noses were harvested, sectioned, stained with DAPI, and subjected to fluorescence imaging. The microscopic images demonstrated a marked reduction in GFP⁺ nasal tissue in SAH-RSVF_BD–treated mice, whereas SAH-RSVF_BF showed no such effect (Figure 4A). Quantitation of GFP positivity by ImageJ analysis demonstrated a statistically significant reduction in nasal tissue fluorescence of greater than 50% compared with that from mice treated with vehicle or SAH-RSVF_BF (Figure 4B), demonstrative of significantly reduced RSV infection. H&E staining of the nose sections revealed thickened mucosa and inflammation in rgRSV-infected mice treated with vehicle or SAH-RSVF_BF (Figure 4C). In contrast, rgRSV-infected mice treated with SAH-RSVF_BD showed little to no inflammation, with nasal histology appearing similar to that of mock-infected animals (Figure 4C). Thus, pretreatment of rgRSV-infected mice with intranasal SAH-RSVF_BD markedly decreases nasal infection in a sequence-dependent manner.

Figure 4

Inhibition of nasal RSV infection by intranasal pretreatment with SAH-RSVF_BD. (A) 10-week-old BALB/c mice were treated with vehicle (1.2% DMSO) or the indicated SAH-RSVF peptides (50 μl of 125 μM solution in 1.2% DMSO) by nasal drop, followed by inoculation with rgRSV (1 × 10⁶ pfu/mouse). Tissues were harvested 24 hours after infection and processed for fluorescence imaging, which revealed marked suppression of GFP positivity by SAH-RSVF_BD, but not the control SAH-RSVF_BF. Original magnification, ×20. (B) Average GFP positivity, quantified by ImageJ analysis of 8 images (4 sections) per mouse. Data (mean ± SEM) represent percent GFP⁺ of total DAPI⁺ cells. (C) H&E staining of the nasal sections demonstrated airway constriction from mucous production (a disease hallmark in mice; yellow arrow) and inflammation (mononuclear cell infiltrates; white arrow) in rgRSV-infected mice treated with vehicle and SAH-RSVF_BF, but notably less airway constriction and immune cell infiltration in SAH-RSVF_BD–treated mice. Sections from mock-infected mice served as a negative control for nasal mucosal inflammation. Insets show magnified views of yellow boxed regions. Original magnification, ×20; ×40 (insets). *P < 0.001.

As nasal RSV infection can spread to the lungs and cause bronchiolitis and pneumonia, we sought to determine whether intranasal SAH-RSVF_BD could likewise suppress the development of pulmonary RSV infection in mice using established models (21, 36–38). Vehicle, SAH-RSVF_BD, or SAH-RSVF_BF were administered by nasal drop (50 μl of 125 μM solution in 1.2% DMSO) to 10-week-old BALB/c mice (n = 4 per cohort), followed by intranasal inoculation with RSV-A2 (1 × 10⁶ pfu) 1 hour later. 4 days later, mice were sacrificed, the lungs were subjected to 4% paraformaldehyde perfusion, and left lung lobes were harvested for cryopreservation, sectioning, and anti-RSV immunofluorescence microscopy. The immunofluorescence images demonstrated a marked reduction in RSV antigen in the lung tissue of SAH-RSVF_BD–treated mice, whereas the level of RSV detection in SAH-RSVF_BF–treated mice was as robust as that of vehicle-treated mice (Figure 5A). ImageJ analysis documented that SAH-RSVF_BD treatment reduced RSV immunofluorescence more than 70% compared with that observed for mice treated with vehicle or SAH-RSVF_BF (Figure 5B). We further generated homogenates from the cranial lobe of the right lungs of mice and subjected the supernatants to viral titer determination by HEp-2 cell plaque assay. Again, only the lung homogenates harvested from SAH-RSVF_BD–treated mice exhibited reduced viral titers (Figure 5C). Finally, we extracted total RNA from the caudal lobe of the right lungs and performed quantitative RT-PCR for the RSV-N transcript. Compared with tissue from mock-infected mice, RSV-A2 inoculation led to a robust increase in RSV-N expression. Whereas RSV-N expression levels in lung tissue from vehicle- and SAH-RSVF_BF–treated mice were similarly elevated, SAH-RSVF_BD treatment reduced expression levels by approximately 65% (Figure 5D). The experiment was also performed with peptide dosing and viral reinoculation 16 hours after the first infection (n=3 per cohort), and similar sequence-dependent inhibitory effects of SAH-RSVF_BD were observed (Supplemental Figure 6). Thus, by each measure, prophylactic intranasal treatment with SAH-RSVF_BD effectively reduced both upper and lower respiratory infection by RSV.

Figure 5

Intranasal prophylaxis with SAH-RSVF_BD inhibits spread of RSV infection to the lung. (A) Anti-RSV immunofluorescence microscopy of lung sections from 10 week-old BALB/c mice treated with vehicle and SAH-RSVF peptides (50 μl of 125 μM solution in 1.2% DMSO) followed by intranasal RSV-A2 inoculation (1 × 10⁶ pfu/mouse) after 1 hour and sacrifice 4 days later. Original magnification, ×20. (B) Average RSV positivity (red) by ImageJ analysis of 8 images (4 sections) per mouse. RSV⁺ cells are expressed as a percentage of total DAPI⁺ cells (mean ± SEM). (C) Viral titers (log₁₀ pfu/ml) were measured in the supernatants of lung homogenates by HEp-2 plaque assay. Data are mean ± SEM. (D) Total RNA was isolated from the lungs (right caudal lobe) of treated mice, and RSV-N expression was quantitated by RT-PCR. Data are mean ± SEM. *P < 0.0001.

Prophylactic intratracheal treatment with a nanoparticle preparation of SAH-RSVF_BD blocks pulmonary RSV infection. Although local pretreatment with SAH-RSVF_BD at the intranasal site of RSV infection reduced nasal infection and subsequent spread to the lungs, we next sought to determine whether peptide delivery to the lung via the intratracheal route could prevent the natural progression of RSV infection after intranasal inoculation. To assess and potentially augment lung tissue exposure, we generated a chitosan-based nanoparticle (NP) (21) preparation of Cy5-labeled SAH-RSVF_BD (100 μM peptide, 1:2.5 peptide/NP ratio, 50 μl volume) and compared lung tissue fluorescence 24 hours after intratracheal administration of vehicle, Cy5-labeled SAH-RSVF_BD peptide alone, or the corresponding NP preparation. With both peptide treatments, lung tissue sections demonstrated robust fluorescence, but specimens from mice treated with the NP preparation exhibited more uniform and higher-level delivery, as assessed by confocal microscopy and ImageJ analysis (Figure 6, A and B). These data suggest that pharmacologic approaches to concentrating and retaining SAH-RSVF_BD at the pulmonary mucosal surface may be an effective means of maximizing its therapeutic potential.

Figure 6

Intratracheal SAH-RSVF_BD as a NP preparation inhibits pulmonary RSV infection. (A) 10-week-old BALB/c mice were treated intratracheally with vehicle or Cy5-labeled SAH-RSVF_BD 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-RSVF_BD (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-RSVF_BD/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.

To measure the capacity of SAH-RSVF_BD to confer anti-RSV prophylaxis for a prolonged period when administered by the intratracheal route, we pretreated mice (n = 3 per cohort) with vehicle, SAH-RSVF_BD, or the corresponding NP preparation, followed by a single intranasal inoculation with RSV-rA2Line19F (1 × 10⁶ pfu/mouse) or mock infection 48 hours later. Anti-RSV immunofluorescence confocal microscopy of the lung revealed a striking near absence of lung tissue fluorescence in infected mice pretreated with the NP preparation of SAH-RSVF_BD, whereas no statistically significant effects were observed by ImageJ analysis for mice treated with SAH-RSVF_BD or NPs alone (Figure 6, C and D). Consistent with these imaging data, only the lung homogenates from mice treated with the NP preparation of SAH-RSVF_BD demonstrated marked reduction of viral titers, by HEp-2 cell plaque assay, and RSV-N expression, by quantitative RT-PCR (Figure 6, E and F). We repeated the experiment (n = 3 per cohort) with an extended 72-hour prophylactic window and obtained nearly identical results, with striking protection against RSV infection by the NP preparation of SAH-RSVF_BD (Supplemental Figure 7). Thus, for intratracheal administration of SAH-RSVF_BD in the context of 48- and 72-hour intervals between peptide prophylaxis and intranasal viral inoculation, the higher-level tissue exposure conferred by the NP preparation of SAH-RSVF_BD effectively suppressed the spread of RSV infection to the lung.

New technologies that fortify lengthy peptides, such as hydrocarbon double stapling, are reinvigorating efforts to optimize natural peptides for therapeutic application. Indeed, the fusion domain helices of a variety of pathogenic viruses, including RSV, Ebola, SARS, and influenza, may hold the key to a series of next-generation inhibitors of viral infections. The pioneering agent, enfuvirtide, demonstrated bona fide therapeutic activity against HIV-1 with a compelling mechanism of action (28). Although still prescribed in the context of multidrug-resistant HIV-1 infection, its utility has been far outpaced by the extensive arsenal of alternative and highly effective anti–HIV-1 agents with oral bioavailability. The large doses (and attendant costs) of enfuvirtide required to achieve inhibitory activity, along with the twice-daily subcutaneous mode of administration, are a direct consequence of the structural instability and proteolytic susceptibility of lengthy peptides. However, with recent advances in conformational control of peptides, including the advancement of peptide double stapling to address traditional peptide liabilities (32), the antiviral activity of natural α-helical peptide interaction domains implicated in viral fusion warrant a second look, especially when treatment options for specific viruses are scarce.

Here, we found that the insertion of 2 i,i+7 staples (each spanning 2 turns of an α-helix) into the peptide sequence of the HR2 domain of RSV yielded a lead peptide with enhanced structure in solution, striking protease resistance, and the capacity to block infection by laboratory and clinical RSV isolates in vitro and in vivo. Following our previously reported design principles for the development of stapled peptides for biomedical research (39), we explored (a) differential hydrocarbon staple compositions, including i,i+4 and i,i+7 staples; (b) the comparative benefits of single versus double stapling in the context of a lengthy peptide template; and, importantly, (c) the biophysical and biological effect of alternative placement of staples within the peptide sequence. Disruption of the binding interface by staple F exemplifies the importance of preserving key peptide/target interactions. In addition, disruptive staple positions provide mutant controls for verifying the specificity and sequence dependence of lead construct bioactivity. By vetting SAH-RSVF_BD in a comprehensive battery of assays, spanning biochemical, cellular, and in vivo analyses, we demonstrate proof of concept for the potential therapeutic utility of SAH-RSVF peptides in blocking both the intranasal establishment and the pulmonary spread of RSV infection.

Having applied all-hydrocarbon stapling to 2 viral fusion peptides with distinct amino acid sequences, but common mechanisms of action (32), we noted the emergence of several general trends that may inform the broader applicability of the approach. First, we found that scanning with i,i+4 and i,i+7 staples was a key first step in determining the optimal type and placement of hydrocarbon staples for a given peptide template. Second, double stapling was markedly superior to single stapling in enforcing the structure and protease resistance of such lengthy peptides. Third, a battery of correlative in vitro and cellular structure-function assays was essential to selecting lead constructs for in vivo testing. Finally, the sequence-dependent results observed in cells and in vivo, facilitated by the use of negative control constructs with point mutants or disruptive staples, was critical for validating on-mechanism biological activity. The capacity to generate both anti–HIV-1 and anti-RSV double-stapled peptides that blocked viral infection by fusion inhibition suggests that all-hydrocarbon stapling may be a general approach for developing antivirals for the Paramyxoviridae family.

We combined peptide stapling and NP delivery technologies, for the first time to our knowledge, in an effort to maximize tissue delivery and persistence. In the context of antiviral prophylaxis, the capacity of NPs to concentrate the therapeutic agent at the site of viral infection, coupled with the structural stability and protease resistance conferred by peptide stapling, yielded a dramatic suppression of viral infection not achieved under the same experimental conditions by the stapled peptide alone. The observed prevention of pulmonary spread of nasal RSV infection by intratracheally administered SAH-RSVF_BD–coated polysaccharide NPs correlated with higher-level and more uniform distribution of Cy5-labeled peptide, and with suppression of RSV infection, by 3 measures: viral imaging, viral titers, and RSV-N expression in lung specimens. Thus, hydrocarbon double-stapled SAH-RSVF peptides may provide a new pharmacologic opportunity to combat RSV infection, while also serving as a paradigm for extending the utility of peptide stapling technology to other undrugged viruses that share this fusion mechanism. Key next steps toward translation include expanding preclinical in vivo testing to define and optimize (a) the maximal duration of prophylaxis and (b) the efficacy in mitigating established infection. Given the documented neutralizing immunogenicity of RSV-F epitopes (23, 25), the capacity of SAH-RSVF exposure to elicit neutralizing antibodies for potential vaccine development is also worthy of exploration.

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We thank E. Smith for assistance with figure preparation and M. Moore for providing the engineered RSV strains and clinical isolate 2-20. This work was supported in part by NIH grant 5R01 AI084102 and a Burroughs Wellcome Fund Career Award in the Biomedical Sciences to L.D. Walensky, a Research Career Scientist Award and VA Merit Review Award 5I01BX000954-02 by the Department of Veteran Affairs and NIH grant 1P30HL101265-01 to S.S. Mohapatra, and a University of South Florida Signature Research Program Fellowship to T. Wong. This study is based on work supported in part by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development; the contents of this report do not represent the views of the Department of Veterans Affairs or the United States Government.

Address correspondence to: Loren D. Walensky, Dana-Farber Cancer Institute, 450 Brookline Avenue, Mayer 664, Boston, Massachusetts 02215, USA. Phone: 617.632.6307; Fax: 617.582.8240; E-mail: loren_walensky@dfci.harvard.edu. Or to: Shyam S. Mohapatra, James A. Haley VA Hospital, 13000 Bruce B. Downs Boulevard, Tampa, Florida 33612, USA. Phone: 813.974.8568; Fax: 813.974.8575; E-mail: shyam.mohapatra@va.gov.

Conflict of interest: Loren D. Walensky is a scientific advisory board member and consultant for Aileron Therapeutics. Shyam S. Mohapatra is a scientific advisory board member for TransGenex Nanobiotech Inc.

Reference information: J Clin Invest. 2014;124(5):2113–2124. doi:10.1172/JCI71856.

See the related article at Repurposing staples for viruses: applying peptide design to RSV prophylaxis.

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