Hemagglutinin-pseudotyped green fluorescent protein-expressing influenza viruses for the detection of influenza virus neutralizing antibodies

Abstract

Influenza virus is a highly contagious virus that causes yearly epidemics and occasional pandemics of great consequence. Influenza virus neutralizing antibodies (NAbs) are promising prophylactic and therapeutic reagents. Detection of NAbs in serum samples is critical to evaluate the prevalence and spread of new virus strains. Here we describe the development of a simple, sensitive, specific, and safe screening assay for the rapid detection of NAbs against highly pathogenic influenza viruses under biosafety level 2 (BSL-2) conditions. This assay is based on the use of influenza viruses in which the hemagglutinin (HA) gene is replaced by a gene expressing green fluorescent protein (GFP). These GFP-expressing influenza viruses replicate to high titers in HA-expressing cell lines, but in non-HA-expressing cells, their replication is restricted to a single cycle.

FIG. 1.

Generation of HA-pseudotyped GFP-expressing influenza viruses. (A) Schematic representation of the GFP viral RNA (vRNA) segment. The GFP segment contains the GFP open reading frame flanked by the terminal untranslated regions (black), along with 45 nucleotides (nt) from the coding region of the influenza HA vRNA (gray), which are required for efficient incorporation of the GFP vRNA into the virus particle. (B) Viral rescue. A 293T/WSN HA MDCK coculture was cotransfected with the polymerase (PA, PB1, and PB2) and nucleoprotein (NP) pCAGGs expression plasmids required for viral replication and transcription, together with the pCAGGs WSN HA expression plasmid (to facilitate viral rescue) and the pPOLI vRNA plasmids (NS, M, NA, NP, PA, PB1, and PB2) plus the pPOLI HA(45)GFP(80) vRNA. Forty-eight hours posttransfection, tissue culture supernatants were passaged in fresh HA-WSN MDCK-expressing cells, and virus rescue was confirmed by GFP expression. (C) Characterization of the HA-MDCK cell lines. HA-expressing MDCK cells were tested for HA protein expression by immunofluorescence with the MAbs 2G9 (WSN), 5C2 (A/New Caledonia/1/99 [NC]), 39E4 (1918), and 4G4 (A/Vietnam/1203/04 [Viet]). Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole). (D) Propagation of HA-pseudotyped GFP-expressing influenza viruses. WSN HA-pseudotyped virus was used to infect MDCK cells constitutively expressing the indicated influenza HAs. HA-pseudotyped viruses carrying the influenza viral hemagglutinin in the virus surface are represented.

FIG. 2.

Characterization of the HA-pseudotyped GFP-expressing influenza viruses. (A) Viral growth kinetics. Kinetics analyses of wild-type A/WSN/33 and HA-pseudotyped GFP-expressing viruses were performed in parental and HA-expressing MDCK cells. At indicated times postinfection, representative pictures were taken using fluorescent microscopy. At 72 h postinfection, light microscopy pictures were taken to show the cytopathic effect on the MDCK-HA-expressing cell lines. (B) Tissue culture supernatants shown in panel A were titrated by indirect immunofluorescence (wild-type [wt] WSN virus [top]) with a WSN polyclonal antibody or by GFP expression (HA-pseudotyped viruses [bottom]) on WSN-HA-expressing MDCK cells. hpi, hours postinfection. (C) Detection of NP and GFP. At 72 h postinfection, cell extracts were prepared and subjected to Western blotting for viral nucleoprotein (NP) and green fluorescent protein (GFP). The top two panels show Western blots of wild-type virus (WSN), while the bottom two panels show Western blots of WSN HA-pseudotyped GFP-expressing influenza virus (pWSN) in wild-type and HA-expressing MDCK cells. (D) Parental and HA-expressing MDCK cells were infected with the WSN-HA-pseudotyped GFP-expressing virus (MOI of 2). Twenty-four hours postinfection, representative pictures were taken using fluorescent microscopy. (E) Plaque assay. Confluent monolayers of the indicated MDCK cell lines were infected with the recombinant WSN-HA-pseudotyped GFP virus in the presence of agar media. Forty-eight hours postinfection, cells were stained with crystal violet.

FIG. 3.

GFP neutralization assay with A/BM/1/18 MAbs. (A) Indicated 1918 MAbs (left) were tested for their neutralizing ability with the 1918-HA-pseudotyped GFP-expressing influenza virus. The absence of neutralizing activity (GFP-positive signal) and presence of NAbs (no GFP signal) were monitored with fluorescence microscopy. Purified MAbs were diluted 1:2 with phosphate-buffered saline (PBS; starting concentration of 1 μg of MAbs in 50 μl) in 96-well plates and incubated with the indicated HA-pseudotyped viruses. The amount of HA-pseudotyped GFP-influenza virus used for these studies was previously determined, to give an approximately 50 to 75% positive GFP signal in the HA-expressing MDCK cells. After 30 to 60 min of incubation at room temperature, the antibody-virus mix was used to infect a 96-well plate of MDCK-HA-expressing cells. After 1 h of absorption at room temperature, the antibody-virus mix was removed, and cells were maintained in infectious media. Representative pictures of each field are indicated. HA-WSN-pseudotyped virus (pWSN) was included as a control. (B) Parental, WSN HA, and 1918 HA MDCK cells were fixed, permeabilized, and immunostained with 1 μg/ml of the indicated MAbs. (C, D) GFP expression levels produced by the p1918 (C)- and pWSN (D)-pseudotyped viruses with the indicated monoclonal antibodies were determined under a fluorescent plate reader. The y axis indicates the green fluorescence produced by viral infection. The x axis indicates viral inhibition in the presence of larger to smaller amounts of the monoclonal antibodies (starting concentration of 1 μg total, as previously described). The assays were conducted in triplicate. Error bars indicate standard deviations. +, indicates GFP expression levels in the absence of monoclonal antibodies.