Newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens

Abstract

The international outbreak of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) in 2002-2003 highlighted the need to develop pretested human vaccine vectors that can be used in a rapid response against newly emerging pathogens. We evaluated Newcastle disease virus (NDV), an avian paramyxovirus that is highly attenuated in primates, as a topical respiratory vaccine vector with SARS-CoV as a test pathogen. Complete recombinant NDV was engineered to express the SARS-CoV spike S glycoprotein, the viral neutralization and major protective antigen, from an added transcriptional unit. African green monkeys immunized through the respiratory tract with two doses of the vaccine developed a titer of SARS-CoV-neutralizing antibodies comparable with the robust secondary response observed in animals that have been immunized with a different experimental SARS-CoV vaccine and challenged with SARS-CoV. When animals immunized with NDV expressing S were challenged with a high dose of SARS-CoV, direct viral assay of lung tissues taken by necropsy at the peak of viral replication demonstrated a 236- or 1,102-fold (depending on the NDV vector construct) mean reduction in pulmonary SARS-CoV titer compared with control animals. NDV has the potential for further development as a pretested, highly attenuated, intranasal vector to be available for expedited vaccine development for humans, who generally lack preexisting immunity against NDV.

Fig. 1.

NDV vaccine constructs. The SARS-CoV S or S1 ORF was cloned by using XbaI sites (italicized) into NDV-BC or NDV-VF antigenomic cDNA under the control of a set of NDV gene-start (GS) and gene-end (GE) transcription signals that direct its expression as a separate mRNA. NDV genes are shown as gray boxes and the SARS-CoV S gene as black boxes. The nucleotide sequence spanning the inserted expression cassette was identical in both NDV vectors and is shown at the top of the figure in the DNA-positive sense. The intergenic nucleotide between each gene is indicated by an arrow.

Fig. 2.

Quantitation of SARS-CoV S-specific CD8+ T cells in the peripheral blood of AGM immunized with the NDV vaccine constructs. Peripheral blood mononuclear cells (PBMC) were stimulated with peptides specific to the S protein, stained, and analyzed by flow cytometry. Cells positive for either IFN-γ or TNF-α were plotted as a percentage of total CD8+ cells. The individual values and the mean values (horizontal bars) were plotted for each group of animals; each group contained four animals except that the empty NDV-BC vector control group contained two animals. The arrows indicate the timing of the first and second doses (days 0 and 28).

Fig. 3.

SARS-CoV replication in the upper and lower respiratory tract of AGM after challenge. Animals were immunized by the i.n. and i.t. routes (10⁷ pfu per site) with the indicated NDV recombinant on days 0 and 28, and they were challenged on day 56 by the i.n. and i.t. sites with 10⁶ TCID₅₀ of SARS-CoV per site. Two days later, the animals were killed, and duplicate samples were taken from the nasal turbinates, the trachea, and the indicated lung regions. The replicate tissue specimens were each titrated in quadruplicate on separate days to measure infectious SARS-CoV. The reported value for each organ is the average of 8 log-transformed titers for each group (i.e., two samples per organ taken from each of four animals). The lower limit of detection was 10 TCID₅₀ per ml. The average ± SE of the log-transformed TCID₅₀ per ml values are shown. P values were calculated by using a two-tailed Student's t test. ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.