Characterization of a neuraminidase-deficient influenza a virus as a potential gene delivery vector and a live vaccine

Abstract

We recently identified a packaging signal in the neuraminidase (NA) viral RNA (vRNA) segment of an influenza A virus, allowing us to produce a mutant virus [GFP(NA)-Flu] that lacks most of the NA open reading frame but contains instead the gene encoding green fluorescent protein (GFP). To exploit the expanding knowledge of vRNA packaging signals to establish influenza virus vectors for the expression of foreign genes, we studied the replicative properties of this virus in cell culture and mice. Compared to wild-type virus, GFP(NA)-Flu was highly attenuated in normal cultured cells but was able to grow to a titer of >10(6) PFU/ml in a mutant cell line expressing reduced levels of sialic acid on the cell surface. GFP expression from this virus was stable even after five passages in the latter cells. In intranasally infected mice, GFP was detected in the epithelial cells of nasal mucosa, bronchioles, and alveoli for up to 4 days postinfection. We attribute the attenuated growth of GFP(NA)-Flu to virion aggregation at the surface of bronchiolar epithelia. In studies to test the potential of this mutant as a live attenuated influenza vaccine, all mice vaccinated with >/==" BORDER="0">10(5) PFU of GFP(NA)-Flu survived when challenged with lethal doses of the parent virus. These results suggest that influenza virus could be a useful vector for expressing foreign genes and that a sialidase-deficient virus may offer an alternative to the live influenza vaccines recently approved for human use.

FIG. 1.

Schematic diagram of the NA(183)GFP(157) vRNA in which most of the NA coding region has been replaced with the GFP gene. The regions shown in blue correspond to the noncoding regions of this segment, and regions in beige indicate the coding regions. This insertion yields a fusion protein consisting of 61 N-terminal residues of the NA protein and intact GFP. The lengths of the regions are not drawn to scale.

FIG. 2.

Growth kinetics of GFP(NA)-Flu and wild-type WSN virus in cell culture. MaKS (KS; broken line) or MDCK (CK; solid line) cells were infected with either GFP(NA)-Flu or wild-type WSN virus at an MOI of 10⁻⁴. At the indicated times after infection, virus titers in the supernatant were determined. The values are means of triplicate experiments. For GFP(NA)-Flu, the numbers of total plaques (open and closed black squares) and those of GFP-positive plaques (open and closed green triangles) were recorded separately.

FIG. 3.

GFP(NA)-Flu infection in respiratory organs of mice. GFP fluorescence was not detected immediately after virus inoculation (A) but was apparent in the nasal epithelium on day 1 postinfection (B). Intense fluorescence was apparent in bronchiolar epithelium on day 1 postinfection (C), shifting to the alveolus by day 2 (D) with diminishing intensity by day 3 (E) or 4 (F). (G and H) Aggregated virions were observed by electron microscopy on the surface of degenerating bronchiolar epithelium at 12 h postinfection. Bar, 500 nm.