Encoding of a transgene in-frame with a Newcastle disease virus protein increases transgene expression and stability

Abstract

Newcastle disease virus (NDV) has been extensively explored as a vector for vaccine and oncolytic therapeutic development. In conventional NDV-based vectors, the transgene is arranged as a separate transcription unit in the NDV genome. Here, we expressed haemagglutinin protein (HA) of an avian influenza virus using an NDV vector design in which the transgene ORF is encoded in-frame with the ORF of an NDV gene. This arrangement does not increase the number of transcription units in the NDV genome, and imposes a selection pressure against mutations interrupting the transgene ORF. We placed the HA ORF upstream or downstream of N, M, F and HN ORFs of NDV so that both proteins are encoded in-frame and are separated by either a self-cleaving 2A peptide, furin cleavage site or both. Only constructs in which HA was placed downstream of the NDV HN were viable. These constructs expressed the transgene at a higher level compared to the vector encoding the same transgene in the same position in the NDV genome but as a separate transcription unit. Furthermore, the transgene expressed in one ORF with the NDV protein proved to be more stable over multiple passages. Thus, this design may be useful for applications where the stability of the transgene expression is highly important for a recombinant NDV vector.

Keywords: Newcastle disease virus vector; avian influenza vaccine; transgene expression; vectored vaccine.

Fig. 1.

Construction and recovery of recombinant NDVs expressing AIV HA. (a) Schemes of in-frame arrangements of ORFs linked by the 2A and furin cleavage site sequences used in this study. Green and red arrows indicate 2A and furin cleavage positions, respectively. (b) Schemes of recombinant NDV genomes with the AIV HA inserts (blue) constructed. The rescued constructs are indicated.

Fig. 2.

Characterization of recombinant NDVs. (a) Multicycle growth kinetics. DF-1 cells were infected at an m.o.i. of 0.01 of each virus and the virus titre in the medium was detected at 12 h intervals in three independent experiments except for the control HN/L-HA which was repeated twice. (b) Fluorescent foci phenotypes of recombinant NDVs in DF-1 cells. Areas of individual fluorescent foci were measured using Carl Zeiss ZEN software, and the data were normalized to the average foci area of the wt virus. Bar, 200 µm.

Fig. 3.

Characterization of expression of AIV HA by recombinant NDVs. (a) DF-1 cells were infected with an m.o.i. of 1 of each construct, incubated for 48 h and processed for western blot analysis. The same membrane was sequentially probed with the indicated antibodies. The graph shows the quantification of HA expression relative to N protein normalized to the control construct HN/L-HA from three independent experiments. (b) HeLa cells infected at a low m.o.i. were incubated without exogenous proteases to prevent virus spread from the originally infected cells. Cells were fixed and stained at 24 h p.i. for 24 h. All images were taken under the same conditions. (c) Purified NDV virions were analysed in western blots for the NDV structural protein and AIV HA content (left) and for total protein composition using Coomassie staining (right).

Fig. 4.

Analysis of the stability of expression of AIV HA by recombinant NDVs. All recombinant NDVs were passaged 10 times in embryonated chicken eggs and the percentage of infectious viruses expressing AIV HA was analysed by immunofluorescence. (a) Images of HeLa cells infected at a low m.o.i. with the viruses from passage 4 and incubated without exogenous proteases to prevent virus spread from the originally infected cells. The cells were fixed and stained at 24 h p.i. with anti-NDV polyclonal chicken antibodies and anti-HA polyclonal rabbit antibodies. The images are intentionally overexposed to make the differences in the transgene expression easily detectable. Arrows indicate cells positive for both NDV and AIV HA antigens (i.e. infected with a recombinant virus expressing the transgene), while arrowheads indicate cells positive only for NDV antigens (i.e. infected with a virus that lost the transgene expression). (b) Scatter plots showing quantification of AIV HA expression by recombinant NDVs upon serial passages using the immunofluorescence assay as in (a). Each dot represents data from a randomly chosen field of view; at least 300 total infected cells were counted for each construct for each passage.