Identification and evaluation of a highly effective fusion inhibitor for human metapneumovirus

Abstract

Human metapneumovirus (hMPV) can cause acute upper and lower respiratory tract infections that are particularly severe in young children, elderly subjects, and immunocompromised patients. To date, no treatments or vaccines are available for hMPV infections. Our objective was to assess the inhibitory potential of several peptides derived from the heptad repeat A and B (HRA and HRB) domains of the hMPV fusion protein. Nine candidate peptides were expressed in Escherichia coli or obtained synthetically and tested in vitro and in an animal model. Excellent in vitro inhibition of an hMPV strain of the A1 subgroup was obtained with five peptides, with 50% inhibitory concentrations ranging from 1.4 nM to 3.3 microM. One peptide, HRA2, displayed very potent activity against all four hMPV subgroups. It was also moderately active against human respiratory syncytial virus (strain A2) but displayed no activity against human parainfluenza virus type 3. BALB/c mice that received the HRA2 peptide and a lethal hMPV intranasal challenge simultaneously were completely protected from clinical symptoms and mortality. On day 5 postinfection, HRA2-treated mice had undetectable lung viral loads which were significantly less than those of untreated mice (3 x 10(4) 50% tissue culture infective doses/lung). Pulmonary inflammation, levels of proinflammatory cytokines/chemokines (RANTES, gamma interferon, and monocyte chemoattractant protein 1) and airway obstruction were also significantly decreased in HRA2-treated mice. The results of this study demonstrate that potent antivirals can be derived from the hMPV fusion protein HR domains. Moreover, hMPV, compared to other paramyxoviruses and to the human immunodeficiency virus, seems to be more susceptible to HRA- than HRB-derived peptides.

FIG. 1.

(A) Body weight loss in groups of treated and untreated mice (6 mice/group). The weight of mice was evaluated on a daily basis until day 5 postinfection. (B) Mean lung viral titers on day 5 postinfection. *, Results are statistically different (P < 0.05) from those for infected, untreated mice (hMPV only). The error bars indicate SEM.

FIG. 2.

Airway obstruction in groups of treated and untreated mice (6 mice/group). The degree of airway obstruction as measured by the P_enh value was evaluated on day 5 postinfection. *, Results are statistically different (P < 0.05) from those for infected, untreated mice (hMPV only). The error bars indicate SEM.

FIG. 3.

Pulmonary levels of the cytokines and chemokines RANTES, MCP-1, and IFN-γ in mice (6 mice/group). The levels of the different cytokines/chemokines in lungs of treated and untreated mice were determined by enzyme-linked immunosorbent assay. *, Results are statistically different (P < 0.05) from those for infected, untreated mice (hMPV only). The error bars indicate SEM.

FIG. 4.

Lung inflammation on day 5 postinfection in groups of treated and untreated mice (6 mice/group). (A) Lungs were removed and fixed with 10% buffered formalin. Histopathological scores were determined based on peribronchiolar, perivascular, interstitial, and alveolar inflammation. (B) Five-micrometer sections of paraffin-embedded lung tissues stained with hematoxylin/eosin. A representative section (10× enlarged) is shown for each group.

FIG. 5.

Alignment of the HRA and HRB amino acid sequences from hMPV groups A1, A2, B1, and B2, as well as from hRSV (strain A2) and hPIV-3 fusion proteins. The sequences were aligned using ClustalW with BioEdit software (version 7.0.5.2). (A) Alignment of hMPV, hRSV, and hPIV-3 HRA sequences. Gray letters are amino acids added to the HRA sequence to form the HRA2 peptide. (B) Alignment of hMPV, hRSV, and hPIV-3 HRB sequences. (C) Amino acid identity for HRA, HRA2, and HRB sequences of hMPV, hRSV, and hPIV-3 fusion protein sequences. hMPV clinical strains C-85473 and NL-001 (GenBank accession no. AF371337) were used as representatives of the hMPV A1 group; hMPV Can97-83 (GenBank AY145296) was used as representative of the hMPV A2 group; hMPV Can97-82 (GenBank AY145295) was used as representative of the hMPV B1 group; hMPV Can98-75 (GenBank AY145289) was used as representative of the hMPV B2 group; hRSV strain A2 (GenBank M11486) and hPIV-3 (GenBank M14892) were also used in this alignment.