Expression of Two Foreign Genes by a Newcastle Disease Virus Vector From the Optimal Insertion Sites through a Combination of the ITU and IRES-Dependent Expression Approaches

Abstract

Many Newcastle disease virus (NDV) strains have been developed as vectors to express a foreign gene (FG) for vaccine and cancer therapy purposes. The non-coding region between the phosphoprotein (P) and matrix protein (M) genes and the non-coding region behind the NP gene open reading frame (ORF) in the NDV genome have been identified as the optimal insertion sites for efficient FG expression through the independent transcription unit (ITU) and the internal ribosomal entry site (IRES) dependent expression approaches, respectively. To date, however, the majority of these NDV vectors express only a single or two FGs from suboptimal insertion sites in the NDV genome, obtaining various levels of FG expression. To improve the FG expression, we generated NDV LaSota vaccine strain-based recombinant viruses expressing two FGs, GFP, and RFP, from the identified optimal insertion sites through a combination of the ITU and IRESdependent approaches. Biological assessments of the recombinant viruses indicated that the recombinants expressing two FGs were slightly attenuated with approximately one order of magnitude lower in virus titers when compared to the viruses containing a single FG. The FG expression efficiencies from the two-FG viruses were also lower than those from the single-FG viruses. However, the expression of two FGs from the optimal insertion sites was significantly (p < 0.05) higher than those from the suboptimal insertion sites. The expressions of FGs as monocistronic ITU were approximately 4-fold more efficient than those expressed by the bicistronic IRES-dependent approach. These results suggest that the NDV LaSota vector could efficiently express two FGs from the identified optimal insertions sites. The ITU strategy could be used for "vectoring" FGs in circumstances where high expression of gene products (e.g., antigens) is warranted, whereas, the IRES-dependent tactic might be useful when lower amounts of IRES-directed FG products are needed.

Advances in the Study of Antitumour Immunotherapy for Newcastle Disease Virus

“…Therefore, this strategy ensures the expression of foreign proteins and the replication of viruses. He et al [65] also confirmed the above view by combining IRES and ITU to recombine NDV to express REF and EGFP in 2020, suggesting that the combination of two or more foreign proteins expressed in this way is a promising and feasible approach.…”

Rescue of an enterotropic Newcastle disease virus strain ZM10 from cloned cDNA and stable expressing an inserted foreign gene

“…A majority of recombinant NDVs expressing one foreign gene (FG) as vaccines or biological therapies express through either an independent transcription unit (ITU) or an internal ribosomal entry site (IRES) approach [ 29 , 32 , 33 ]. The efficacy of these two strategies has been investigated, showing that the ITU approach was more efficient than that through the IRES approach [ 32 ].…”

“…A majority of recombinant NDVs expressing one foreign gene (FG) as vaccines or biological therapies express through either an independent transcription unit (ITU) or an internal ribosomal entry site (IRES) approach [ 29 , 32 , 33 ]. The efficacy of these two strategies has been investigated, showing that the ITU approach was more efficient than that through the IRES approach [ 32 ]. The expression levels of FGs through ITU at different genomic locations were variable and the optimal insertion site in NDV genome for efficient expression has been identified as the non-coding region between the P and M genes [ 16 , 17 , 34 ].…”

Development and Scalable Production of Newcastle Disease Virus-Vectored Vaccines for Human and Veterinary Use

“…NDV constructs bearing genes encoding the SARS-CoV S protein [ 28 ] and the SARS-CoV-2 S protein [ 27 ], both around 3.8 kb in size, have been successfully generated, demonstrating this vector’s capacity for inserts in this size range. NDV has also been shown to tolerate multiple transgenes [ 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 ] using different co-expression strategies [ 59 ]. Each foreign gene must be flanked by untranslated regions (UTRs) of NDV genes called gene start (GS) and gene end (GE) sequences ( Figure 1 ).…”