| Summary: | Newcastle Disease (ND) is an economically important viral disease in poultry. Although commercial vaccines are available, ND continues to cause disease outbreaks, particularly in low- and middle-income countries where strict biosecurity measures are often not feasible. Therefore, there is an urgent need for a cost-effective approach to develop improved ND vaccines. This study used Agrobacterium tumefaciens-mediated transient expression in Nicotiana benthamiana via pEAQ-HT expression system, with the aim of developing improved plant expressed NDV vaccines. Bioinformatics analysis was also used to support ND vaccine development.
We successfully expressed NDV structural proteins (Fusion [F], Haemagglutinin-Neuraminidase [HN], and Matrix [M]) in N. benthamiana, with the highest yield observed at 9 days post-infiltration. The use of the A.tumefaciens NMX021 strain, co-expression of heterologous chaperone proteins (calnexin and/or calreticulin), and the pH of the extraction buffer were important factors that influenced protein expression. Co-expression of NDV F, HN, and M proteins together with chaperone proteins led to the observation of particulate structures under electron microscopy that resembled virus-like particles (VLPs). However, due to the abundance of plant-derived vesicular structures and protein aggregates, definitive identification of these as VLPs could not be confirmed, and further characterisation is required.
Bioinformatics analysis of the F and HN glycoproteins included multiple sequence alignment across representative NDV genotypes and identified amino acid substitutions relevant to vaccine design. E347G and G362R in the HN protein are located in the receptor-binding domain, while D170S and S/T543A in the F protein involve antigenic and glycosylation-associated regions. These substitutions may influence immune recognition and support homologous vaccine design to reduce viral shedding. Further functional validation using site-directed mutagenesis and biological assays is recommended.
Keywords: Newcastle disease virus; plant-based vaccines; Agrobacterium; pEAQ-HT; homologous vaccines, viral shedding.
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