A New Generation of Modified Live-Attenuated Avian Influenza Viruses Using a Two-Strategy Combination as Potential Vaccine Candidates

Abstract

In light of the recurrent outbreaks of low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI), there is a pressing need for the development of vaccines that allow rapid mass vaccination. In this study, we introduced by reverse genetics temperature-sensitive mutations in the PB1 and PB2 genes of an avian influenza virus, A/Guinea Fowl/Hong Kong/WF10/99 (H9N2) (WF10). Further genetic modifications were introduced into the PB1 gene to enhance the attenuated (att) phenotype of the virus in vivo. Using the att WF10 as a backbone, we substituted neuraminidase (NA) for hemagglutinin (HA) for vaccine purposes. In chickens, a vaccination scheme consisting of a single dose of an att H7N2 vaccine virus at 2 weeks of age and subsequent challenge with the wild-type H7N2 LPAI virus resulted in complete protection. We further extended our vaccination strategy against the HPAI H5N1. In this case, we reconstituted an att H5N1 vaccine virus, whose HA and NA genes were derived from an Asian H5N1 virus. A single-dose immunization in ovo with the att H5N1 vaccine virus in 18-day-old chicken embryos resulted in more than 60% protection for 4-week-old chickens and 100% protection for 9-to 12-week-old chickens. Boosting at 2 weeks posthatching provided 100% protection against challenge with the HPAI H5N1 virus for chickens as young as 4 weeks old, with undetectable virus shedding postchallenge. Our results highlight the potential of live att avian influenza vaccines for mass vaccination in poultry.

Influenza Viruses with Rearranged Genomes as Live-Attenuated Vaccines

“…The H9N2 wild-type (wt) virus and the H9:pH1N1 reassortant have been previously described (12,13). The HPAIV A/Vietnam/1203/04 (H5N1) (here, H5N1 wt) was a kind gift from Ruben Donis, CDC, Atlanta, GA. Recombinant viruses used in this study were generated from cloned cDNAs and are described below and in Table 1.…”

“…The eight-plasmid reverse genetic system for H9N2 wt has been previously described, and it is based in the bidirectional plasmid vector pDP2002 (13). The HA and NA genes from the H5N1 wt strain were cloned into the pDP2002 vector.…”

Differential regulation of antiviral and proinflammatory cytokines and suppression of Fas-mediated apoptosis by NS1 of H9N2 avian influenza virus in chicken macrophages

“…Recombinant viruses were generated with an eight-plasmid reverse genetics system (Hoffmann et al, 2001(Hoffmann et al, , 2002Song et al, 2007). Briefly, viruses were rescued in 293T/MDCK co-cultures transfected with pDP2000 (Song et al, 2007;Wan & Perez, 2007) in the TransIT-LT1 reagent (Mirus), each harbouring one of the eight viral segment cDNAs. Recombinant viruses were grown in 9-day-old embryos from specific pathogen-free (SPF) hens (Charles River) and allantoic fluids (VAF) were harvested 48 h after inoculation.…”

TLR2 Engagement on Dendritic Cells Promotes High Frequency Effector and Memory CD4 T Cell Responses

“…Influenza virus (A/PR/8/34) was grown in the allantoic fluid of 10-day-old embryonated chicken eggs as previously described (43). Determination of influenza viral titers in lung homogenates was accomplished by the tissue culture infectious dose 50 assay (TCID 50 ) as described previously (44), with titers expressed as the reciprocal of the dilution of lung extract that corresponds to 50% virus growth in Madin-Darby canine kidney cells or calculated by the Reed Muench method.…”