Newcastle disease virus-based live attenuated vaccine completely protects chickens and mice from lethal challenge of homologous and heterologous H5N1 avian influenza viruses

Abstract

H5N1 highly pathogenic avian influenza virus (HPAIV) has continued to spread and poses a significant threat to both animal and human health. Current influenza vaccine strategies have limitations that prevent their effective use for widespread inoculation of animals in the field. Vaccine strains of Newcastle disease virus (NDV), however, have been used successfully to easily vaccinate large numbers of animals. In this study, we used reverse genetics to construct a NDV that expressed an H5 subtype avian influenza virus (AIV) hemagglutinin (HA). Both a wild-type and a mutated HA open reading frame (ORF) from the HPAIV wild bird isolate, A/Bar-headed goose/Qinghai/3/2005 (H5N1), were inserted into the intergenic region between the P and M genes of the LaSota NDV vaccine strain. The recombinant viruses stably expressing the wild-type and mutant HA genes were found to be innocuous after intracerebral inoculation of 1-day-old chickens. A single dose of the recombinant viruses in chickens induced both NDV- and AIV H5-specific antibodies and completely protected chickens from challenge with a lethal dose of both velogenic NDV and homologous and heterologous H5N1 HPAIV. In addition, BALB/c mice immunized with the recombinant NDV-based vaccine produced H5 AIV-specific antibodies and were completely protected from homologous and heterologous lethal virus challenge. Our results indicate that recombinant NDV is suitable as a bivalent live attenuated vaccine against both NDV and AIV infection in poultry. The recombinant NDV vaccine may also have potential use in high-risk human individuals to control the pandemic spread of lethal avian influenza.

FIG. 1.

Generation and characterization of H5 HA gene recombinant NDV. (A) Schematic representation of the rLa genome with the PmeI site introduced between the P and M genes (I) and the wild-type H5 AIV HA gene (II) or mutated H5 AIV HA gene (III) that were inserted at the PmeI site. (B) Immunofluorescence analysis of H5 AIV HA protein expression. Confluent BHK-21 cells were infected with rLa (I), rLa-H5w (II), or rLa-H5m (III) at a MOI of 0.2. The infected cells were fixed and probed with chicken anti-NDV antiserum (a, d, g), chicken anti-H5 AIV antisera (b, e, h), or with the noninfected (NI) SPF chicken sera (c, f, i) followed by incubation with fluorescein isothiocyanate-conjugated rabbit anti-chicken IgG (Sigma). Cells were analyzed with a Leica DMIRE2 fluorescence microscope (Leica). (C) Western blot analyses of NDV recombinants expressing AIV H5. Lysates of cells infected with rLa, rLa-H5w, or rLa-H5m were incubated with chicken H5 AIV HA-specific antiserum that was generated with H5 HA gene DNA immunization (I) or chicken anti-NDV antiserum (II). Binding was visualized with 3,3′-diaminobenzidine reagent after incubation with peroxidase-conjugated secondary antibodies. Locations of marker proteins are indicated on the left, and the uncleaved (HA0) and processed forms (HA1 and HA2) of AIV hemagglutinin are indicated on the right.

FIG. 2.

Growth properties of recombinant viruses in embryonated eggs. The rescued NDV rLa (⋄), rLa-H5w (▵), or rLa-H5m (○) (0.1 ml of 100 EID₅₀) were inoculated into the allantoic cavities of 10-day-old embryonated eggs, and the allantoic fluid of six eggs from each group was harvested at the time points of 24, 48, 72, and 96 h postinoculation and pooled for the determination of EID₅₀ in eggs; data shown were acquired from three repeats.

FIG. 3.

Phylogenetic relationships of the hemagglutinin genes of H5N1 viruses. The tree includes avian influenza viruses that were isolated in countries from Southeast Asia during 2003 to 2005 and viruses isolated in the Middle East, Europe, and Africa during 2005 to 2006. The phylogenetic tree was generated with the PHYLIP program of the CLUSTALX software package (version 1.81) by using the neighbor-joining algorithm and based on bootstrap values of 1,000. The HA gene donor virus for the recombinant vaccine generation is underlined, and the challenge viruses used in this study are marked in italics. CK, chicken; DK, duck; GS, goose; MD, mallard; CO, Cygnus olor; CC, Cygnus cygnus; WS, whooper swan.

FIG. 4.

HI antibody duration induced by the recombinant virus rLa-H5w in SPF chickens. One-week-old white Leghorn SPF chickens were inoculated oculonasally with 10⁶ EID₅₀ of the rescued recombinant virus rLa-H5w in 50 μl. Sera were collected on a weekly basis for HI antibody detection to NDV (⋄) and H5N1 AIV (○). Data are represented as the means ± standard deviations.

FIG. 5.

Outcomes in vaccinated mice following lethal intranasal challenge with different H5N1 viruses. (A) Weight loss and (B) survival of the mice that were vaccinated with rLa (▴, ▪) or rLa-H5w (▵, □) and challenged by intranasal inoculation with 1,000 MLD₅₀ of influenza virus BHG/QH/05 (▵, ▴) or DK/FJ/02 (□, ▪) two weeks after the second immunization. Mean weight loss is expressed as a percentage of original weight.