Sialidase fusion protein as a novel broad-spectrum inhibitor of influenza virus infection

Abstract

Influenza is a highly infectious disease characterized by recurrent annual epidemics and unpredictable major worldwide pandemics. Rapid spread of the highly pathogenic avian H5N1 strain and escalating human infections by the virus have set off the alarm for a global pandemic. To provide an urgently needed alternative treatment modality for influenza, we have generated a recombinant fusion protein composed of a sialidase catalytic domain derived from Actinomyces viscosus fused with a cell surface-anchoring sequence. The sialidase fusion protein is to be applied topically as an inhalant to remove the influenza viral receptors, sialic acids, from the airway epithelium. We demonstrate that a sialidase fusion construct, DAS181, effectively cleaves sialic acid receptors used by both human and avian influenza viruses. The treatment provides long-lasting effect and is nontoxic to the cells. DAS181 demonstrated potent antiviral and cell protective efficacies against a panel of laboratory strains and clinical isolates of IFV A and IFV B, with virus replication inhibition 50% effective concentrations in the range of 0.04 to 0.9 nM. Mouse and ferret studies confirmed significant in vivo efficacy of the sialidase fusion in both prophylactic and treatment modes.

FIG. 1.

Molecular model of DAS181. The catalytic domain of the sialidase (AvCD) is colored in green and the protruding anchoring domain (AR) on C terminus in blue. The model was built using the SWISS-MODEL software.

FIG. 2.

Sialic acid removal and turnover on MDCK cells. (A) Levels of α(2,6)- and α(2,3)-linked sialic acids and sialic acids in total on the surface of MDCK cells that were pretreated with vehicle (EDB-BSA) or various dilutions of DAS180 (His₆-AvCD) or DAS181 (AvCD-AR) in the vehicle for 1 h. (B) Level of cell surface sialic acid after a single treatment by DAS181. MDCK cells in confluent monolayers were treated with 100 mU of DAS181 for 2 h, washed, and chased for various times with fetal bovine serum-containing medium. The levels of sialic acids were detected with biotinylated lectins. The error bars indicate one standard deviation above or below the mean of three samples.

FIG. 3.

IFV binding to MDCK and fetuin-coated plates. Biotinylated A/PR/8/34 was allowed to bind to the DAS181-treated fetuin or MDCK monolayers for 30 min at 4°C. The bound virus was detected by using streptavidin-HRP and developed by using TMB. Virus binding to the untreated MDCK cells represented 100%. Wells without the virus were included for background streptavidin-HRP binding. The error bars indicate one standard deviation above or below the mean of three samples.

FIG. 4.

Effects of DAS181 on lung virus titer (A) and lung weight (B) in infected mice. The results are part of experiment 1 in Table 5. The data at each time point were derived from three mice. Statistically significant values are labeled with one (P < 0.05), two (P < 0.01), or three (P < 0.001) asterisks. Ribavirin was used at 75 mg/kg.

FIG. 5.

Total inflammatory cell counts (A) and protein concentrations (B) in nasal washes from ferrets. Infected ferrets were vehicle treated (▪) or were treated with DAS178 (▴). Uninfected animals were treated with DAS178 (⧫) only. Statistically significant values are labeled with one (P < 0.05) or two (P < 0.01) asterisks. No that only the DAS178-treated ferrets that showed positive virus shedding were included in the analysis.