DAS181 inhibits H5N1 influenza virus infection of human lung tissues

Abstract

DAS181 is a novel candidate therapeutic agent against influenza virus which functions via the mechanism of removing the virus receptor, sialic acid (Sia), from the adjacent glycan structures. DAS181 and its analogues have previously been shown to be potently active against multiple strains of seasonal and avian influenza virus strains in several experimental models, including cell lines, mice, and ferrets. Here we demonstrate that DAS181 treatment leads to desialylation of both alpha2-6-linked and alpha2-3-linked Sia in ex vivo human lung tissue culture and primary pneumocytes. DAS181 treatment also effectively protects human lung tissue and pneumocytes against the highly pathogenic avian influenza virus H5N1 (A/Vietnam/3046/2004). Two doses of DAS181 treatment given 12 h apart were sufficient to block H5N1 infection in the ex vivo lung tissue culture. These findings support the potential value of DAS181 as a broad-spectrum therapeutic agent against influenza viruses, especially H5N1.

FIG. 1.

Lectin binding to lung tissues with and without DAS181 treatment. Lectin binding of SNA (A, D, and G), MAA (B, E, and H), and PNA (C, F, and I) on normal lung tissue with no incubation with DAS181 (A to C), after 2 h of incubation with 500 U/ml DAS181 (D to F), and after 2 h of incubation with 500 U/ml, followed by removal of DAS181 and then incubation in medium for 48 h (G to I). Neo-fuchsin staining with hematoxylin counterstaining was used. Magnification, ×200.

FIG. 2.

Sia expression on type I-like pneumocytes in vitro. Primary human type I-like pneumocytes were incubated with control SAGM (left panels) or 500 U/ml of DAS181 (right panels) for 2 h at 37°C and fixed. Sia in α2-3 linkages was stained reddish brown by biotinylated MAL-I (A and B) and MAL-II (C and D) lectins, while Sia in α2-6 linkages was stained red by horseradish peroxidase-conjugated SNA (E and F). Magnification, ×400.

FIG. 3.

MALDI-TOF mass spectra of permethylated N-glycans from human ex vivo lung biopsy material. Results obtained without DAS181 treatment (A) and after 2 h of DAS181 (500 U/ml) treatment (B) are shown. The red peaks represent the abundance of a specific sialylated glycan present in the lung tissue, and the green peaks represent the desialylated glycans. All molecular ions are [M+Na]+. Structural assignments are based on monosaccharide composition, MALDI-TOF/TOF MS/MS analysis, and knowledge of biosynthetic pathways.

FIG. 4.

Effective inhibition of influenza virus infection of primary human pneumocytes by DAS181. Cultures were incubated with medium (vehicle) (A and C) or 500 U/ml DAS181 (B and D) for 2 h, followed by infection with influenza virus A/HK/54/98 (H1N1) (A and B) or A/VN/3046/04 (H5N1) (C and D). After being washed, the cultures were incubated for 18 h at 37°C before fixation and staining with an antibody against influenza virus antigen. Brown staining represents sites of antibody binding.

FIG. 5.

Immunohistochemical analysis of lung tissue for influenza virus NP. The number of positive cells detected in infected lung biopsy samples after a single 2-h incubation with 500 U/ml DAS181 before A/VN/3046/04 (H5N1) infection (open circles) and with continuous exposure to DAS181 (closed circles) compared to that in the control treatment (closed squares). Error bars represent standard deviations of three experiments, and an asterisk indicates a P value of <0.05 with the Student one-tailed t test. hpf is high-power fields.

FIG. 6.

Influenza virus M gene analysis of normal human lung tissue with and without DAS181 treatment. Lung tissue was infected with A/VN/3046/04 (H5N1) and sampled 24 h (A) and 48 h (B) after treatment with DAS181 (100 U/ml) at different dosage regimens. Error bars indicate the standard deviations of three experiments. The indicated P values were calculated with the post-hoc Bonferroni test. M gene/10⁵ ß-actin represents the number of influenza virus M gene copies/10⁵ copies of the gene for ß-actin.