Pseudotyped Influenza A Virus as a Vaccine for the Induction of Heterotypic Immunity

Abstract

cThere is a need for vaccines that can protect broadly across all influenza A strains. We have produced a pseudotyped influenza virus based on suppression of the A/PR/8/34 hemagglutinin signal sequence (S-FLU) that can infect cells and express the viral core proteins and neuraminidase but cannot replicate. We show that when given by inhalation to mice, S-FLU is nonpathogenic but generates a vigorous T cell response in the lung associated with markedly reduced viral titers and weight loss after challenge with H1 and H3 influenza viruses. These properties of S-FLU suggest that it may have potential as a broadly protective A virus vaccine, particularly in the setting of a threatened pandemic before matched subunit vaccines become available.

Focused antibody response to influenza linked to antigenic drift

“…T1-3B and control serum had similar neutralization titers to all the viruses. In our MN assays, the OD 450 readout was dependent on NP expression following viral entry (65). The MN assays were performed in duplicate.…”

“…The MN assay detects inhibition of virus entry into MDCK-SIAT-1 cells in the absence of trypsin (67), with antibody present throughout the assay (23). MN assays with clade 6B viruses incorporated a replication step in the presence of trypsin for 48 hours, which gave lower background staining with the developing antibody to NP AA5H (65). Serum and plasma samples were heat inactivated at 56°C without RDE treatment, and monoclonal antibodies were untreated.…”

“…Assays were performed using standard procedures as described previously (65). The ELISA or MN titers were expressed as half maximal effective concentrations (EC 50 : midpoint between negative and plateau positive controls) derived by linear interpolation from neighboring points in the titration curve.…”

“…transfer of 500 μl antibody at a dose of 10 mg/kg into anesthetized animals. Mice were weighed and scored according to previously described criteria (65), and mice with 20% weight loss or morbid clinical scores were humanely killed. (14,37).…”

“…cDNA corresponding to codons 1 to 520 of the ectodomain of HA from influenza A/England/195/2009 virus was amplified and purified as previously described (39,65). Briefly, the construct incorporated at the C-terminus included a thrombin cleavage site, a trimerization sequence, a histidine-tag region for protein purification, and the BirA recognition sequence LNDIFEAQKIEW for site-specific biotinylation.…”