Recombinant Newcastle disease virus as a vaccine vector

Abstract

A complete cDNA clone of the Newcastle disease virus (NDV) vaccine strain Hitchner B1 was constructed, and infectious recombinant virus expressing an influenza virus hemagglutinin was generated by reverse genetics. The rescued virus induces a strong humoral antibody response against influenza virus and provides complete protection against a lethal dose of influenza virus challenge in mice, demonstrating the potential of recombinant NDV as a vaccine vector.

FIG. 1

Schematic representation of pNDV/B1, pNDV/B1-CAT, and pNDV/B1-HA cDNA constructs. (A) pNDV/B1 was generated by seven PCR fragments spanning the following nucleotide positions: F1, T7 promoter, nt 1755 (SacII); F2, nt 1 to 3321; F3, nt 1755 (SacII) to 6580; F4, nt 6151 to 10,210; F5, nt 7381 to 11,351; F6, nt 11,351 to 14,995; and F7, nt 14,701 to 15,186. These sequences were followed by the hepatitis delta virus (HDV) ribozyme and the T7 terminator. The cDNA fragments were joined at shared restriction sites and assembled in plasmid pSL1180 (Amersham Pharmacia Biotech). SacII and XbaI are shown in italics to indicate that they are genomic tag sequences. (B) The pNDV/B1-CAT and pNDV/B1-HA constructs were made by inserting the CAT and influenza virus A/WSN/33 HA open reading frames (ORF), respectively, into the unique XbaI cloning site (nt 3163) located between the P and M genes of the pNDV/B1 clone. The inserted gene contains the gene end (GE; 5′-TTAGAAAAAA-3′), intercistronic nucleotide (T), and the gene start sequence (GS; 5′-ACGGGTAGAA-3′). In addition, seven nucleotides (5′-CGCCACC-3′) were inserted upstream of the initiation site to introduce an optimal Kozak sequence (13). In the case of pNDV/B1-HA, the gene start sequence is followed by the 5′ untranslated region (26 nt) of the HA gene.

FIG. 2

Detection of the HA protein on infected cells and in purified virions. (A) rNDV/B1- or rNDV/B1-HA-infected cells were fixed with 1% paraformaldehyde at day 2 postinfection, and cells were used for immunostaining analysis. NDV protein expression on the surfaces of cells infected with rNDV/B1 (a) or rNDV/B1-HA passaged in eggs at a low multiplicity of infection three times (3^rdP) (b) or 10 times (10^thP) (c) was analyzed by using mouse anti-NDV serum. HA expression on the cell surfaces of rNDV/B1 (e)- or rNDV/B1-HA (f and g)-infected cells was analyzed by using anti-HA monoclonal antibody (2G9). Mock-infected cells were also analyzed as a control (d and h). (B) rNDV/B1 and rNDV/B1-HA were purified from allantoic fluids of infected embryonated chicken eggs. Influenza A/WSN/33 virus was purified from MDBK cell culture supernatants as a control. Serial twofold dilutions of A/WSN/33 viral proteins (1.5 to 0.19 μg) and 3 μg of rNDV/B1-HA or rNDV/B1 viral proteins were separated on a sodium dodecyl sulfate–10% polyacrylamide gel. The gel was transferred to a nitrocellulose membrane, and the HA protein was detected by chemiluminescence using a mixture of anti-HA monoclonal antibodies and an anti-mouse IgG peroxidase-labeled antibody (DAKO). HA₀ and HA₁ indicate uncleaved and cleaved HA protein, respectively.

FIG. 2

Detection of the HA protein on infected cells and in purified virions. (A) rNDV/B1- or rNDV/B1-HA-infected cells were fixed with 1% paraformaldehyde at day 2 postinfection, and cells were used for immunostaining analysis. NDV protein expression on the surfaces of cells infected with rNDV/B1 (a) or rNDV/B1-HA passaged in eggs at a low multiplicity of infection three times (3^rdP) (b) or 10 times (10^thP) (c) was analyzed by using mouse anti-NDV serum. HA expression on the cell surfaces of rNDV/B1 (e)- or rNDV/B1-HA (f and g)-infected cells was analyzed by using anti-HA monoclonal antibody (2G9). Mock-infected cells were also analyzed as a control (d and h). (B) rNDV/B1 and rNDV/B1-HA were purified from allantoic fluids of infected embryonated chicken eggs. Influenza A/WSN/33 virus was purified from MDBK cell culture supernatants as a control. Serial twofold dilutions of A/WSN/33 viral proteins (1.5 to 0.19 μg) and 3 μg of rNDV/B1-HA or rNDV/B1 viral proteins were separated on a sodium dodecyl sulfate–10% polyacrylamide gel. The gel was transferred to a nitrocellulose membrane, and the HA protein was detected by chemiluminescence using a mixture of anti-HA monoclonal antibodies and an anti-mouse IgG peroxidase-labeled antibody (DAKO). HA₀ and HA₁ indicate uncleaved and cleaved HA protein, respectively.

FIG. 3

Growth curves of wtNDV/B1, rNDV/B1, rNDV/B1-CAT, and rNDV/B1-HA viruses in embryonated chicken eggs. Embryonated eggs were inoculated with 100 PFU of each virus, and allantoic fluids were harvested at different time points (24, 48, and 72 h postinoculation). Viral titers (TCID₅₀) in CEF were determined by immunofluorescence assay using anti-NDV rabbit serum and an anti-rabbit IgG fluorescein isothiocyanate-labeled antibody (DAKO).

FIG. 4

Average body weights of vaccinated mice after influenza virus challenge. Vaccinated mice were challenged with a lethal dose (100 LD₅₀) of influenza A/WSN/33 virus on day 35 (2 weeks after the booster injection). Relative average daily body weights (percentages) of mice vaccinated with rNDV/B1-HA intravenously (i.v.) (▪) or intraperitoneally (i.p.) (▴), with rNDV/B1 intravenously (♦), or with PBS (●) are shown. The vaccinating dose was 3 × 10⁷ PFU/ml in all cases. The error bars indicate ±0.5 times the standard deviation.