Prevention of measles virus infection by intranasal delivery of fusion inhibitor peptides

Abstract

Measles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV H and the fusion (F) envelope glycoprotein; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad-repeat (HR) regions of F can potently inhibit MV infection at the entry stage. We show here that specific features of H's interaction with its receptors modulate the susceptibility of MV F to peptide fusion inhibitors. A higher concentration of inhibitory peptides is required to inhibit F-mediated fusion when H is engaged to its nectin-4 receptor than when H is engaged to its CD150 receptor. Peptide inhibition of F may be subverted by continued engagement of receptor by H, a finding that highlights the ongoing role of H-receptor interaction after F has been activated and that helps guide the design of more potent inhibitory peptides. Intranasal administration of these peptides results in peptide accumulation in the airway epithelium with minimal systemic levels of peptide and efficiently prevents MV infection in vivo in animal models. The results suggest an antiviral strategy for prophylaxis in vulnerable and/or immunocompromised hosts.

Importance: Measles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that parenterally delivered fusion-inhibitory peptides protect mice from lethal CNS MV disease. Here we show, using established small-animal models of MV infection, that fusion-inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. Since the fusion inhibitors are stable at room temperature, this intranasal strategy is feasible even outside health care settings, could be used to protect individuals and communities in case of MV outbreaks, and could complement global efforts to control measles.

FIG 1

Inhibition of MV entry by HPIV3 F-derived HRC peptides. Vero-SLAM cell monolayers were infected with wild-type MV G954 in the presence of peptides VG (circles), VG_PEG4-chol (squares), [VG_PEG4]₂-chol (triangles), and VIKI_PEG4-chol (diamonds) at the indicated concentrations. Viral entry was assessed by plaque assay. Results are presented as percent reduction in plaque number (y axis) compared to the absence of treatment, as a function of compound concentration (x axis). Each point represents the mean (± standard error) of results from four experiments.

FIG 2

Inhibition of MV H/F-mediated fusion by HPIV3 F-derived HRC peptides. Fusion of MV H/F-coexpressing cells with SLAM-bearing cells in the presence of VG, VG_PEG4-chol, [VG_PEG4]₂-chol, and VIKI_PEG4-chol as indicated at 0.1 μM (A, D, and G), 1 μM (B, E, and H), and 10 μM (C, F, and I) was quantitated at 1 h (A, B, and C), 4 h (D, E, and F), or 6 h (G, H, and I), using a β-galactosidase complementation assay. Results are presented as percent reduction in luminescence (y axis) compared with no treatment. Each point is the mean (± standard error) of results from 3 experiments.

FIG 3

Protease sensitivity of MV- and HPIV3-derived peptides. The indicated peptides were incubated in the absence (−) or presence (+) of trypsin, at either 0.01 μg/μl (A) or 0.05 μg/μl (B) for 1 h at 0°C, 22°C, or 37°C. The products of the reaction were subjected to reducing SDS-PAGE gels and silver stained as described in Materials and Methods.

FIG 4

Inhibition of cell fusion in the presence of anti-MV H antibodies. Fusion inhibition (y axis) was assessed in the presence of HA55 neutralizing antibody added at the indicated times (x axis) in the absence (white bar) or presence (black bars) of 1 μM of VG_PEG4-chol peptide. Data represent averages (± standard deviation) of data from triplicate wells from a representative experiment.

FIG 5

Inhibition of cell fusion with cells bearing nectin 4 or SLAM. Fusion of MV H/F-coexpressing cells with cells transfected with nectin 4 (A) or SLAM (B) in the presence of MV HRC1 (circles), MV HRC2 (squares), or MV HRC4 (triangles) was quantitated after 6 h by a β-galactosidase complementation assay. Results are presented as percent reduction in luminescence (y axis) compared with no treatment, as a function of compound concentration (x axis). Each point is the mean (± standard error) of results from 3 experiments.

FIG 6

Peptides cross the human airway epithelium (HAE). (A) Drops containing peptides settle at the apical side (air interface) of the HAE. Medium (liquid interface) was collected at each time point for quantification of peptide by ELISA. PVDF, polyvinylidene difluoride. (B to D) Kinetic analysis (ELISA) of the peptide concentration in the liquid reservoir of the culture, reflecting minimal transit through the HAE (MV HRC2 in panel B, MV HRC4 in panel C, and VIKI_PEG4-chol in panel D).

FIG 7

Intranasal administration of MV-derived peptides protects cotton rats from MV infection. Cotton rats (n = 4) were infected i.n. with MV 24 h after the first peptide treatment and were euthanized 4 days postinfection. MV titration of lung homogenates showed that MV HRC2, MV HRC3, and MV HRC4 block infection in CR (***, P = 0.001 in Mann-Whitney U test). The limit of viral detection was 10² PFU/gram.

FIG 8

Intranasal administration of MV HRC4 protects mice from lethal MV encephalitis. (A to C) Immunofluorescent staining of MV HRC4 (in red) in the lungs of mice after i.n. administration. Nuclei were counterstained using DAPI. While peptide staining is absent from untreated animals (A), a high concentration of MV HRC4 (in red) is present in the lungs of treated mice (B), and magnification (C) reveals the presence of the fusion inhibitors in the bronchioles as well as in the parenchyma. One-week-old SLAM transgenic mice (n = 5) were challenged with intranasal MV (G954 strain) 24 h after the first MV-HRC peptide treatment and were monitored for 5 weeks. Control animals (mock) were injected with vehicle alone. (D) Peptide was administered subcutaneously daily from day −1 to day +7 after infection. (E) Mice received only 2 intranasal (i.n.) administrations of MV HRC peptides, 24 h before and 2 h after infection. Statistical significance of the difference between the MV HRC4 and untreated groups in E (*, P = 0.0334) was analyzed using the Mantel-Cox test. (F) Production of anti-NP antibodies in the sera of infected (as indicated on the x axis) SLAM transgenic mice. (G) Four- to 5-week-old SLAM transgenic/IFNAR knockout mice (n = 10) were challenged with intranasal MV (50 LD₅₀ of strain G954) 24 h after the first MV HRC peptide treatment and were monitored for 4 weeks. Control animals (mock; n = 10) were injected with vehicle alone. Mice received only two i.n. administrations of MV HRC peptides, 24 h and 6 h before infection. Statistical significance of the differences between the MV HRC4 and untreated groups (***, P < 0.0001) was analyzed using the Mantel-Cox test.