Rapid high-yield expression of a candidate influenza vaccine based on the ectodomain of M2 protein linked to flagellin in plants using viral vectors

Eugenia S. Mardanova, Roman Y. Kotlyarov, Victor V. Kuprianov, Liudmila A. Stepanova, Liudmila M. Tsybalova, George P. Lomonosoff, Nikolai V. Ravin

The extracellular domain of matrix protein 2 (M2e) of influenza A virus is a promising target for the development of a universal vaccine against influenza because M2e sequences are highly conserved among human influenza A strains. However, native M2e is poorly immunogenic, but its immunogenicity can be increased by delivery in combination with adjuvants or carrier particles. It was previously shown that fusion of M2e to bacterial flagellin, the ligand for Toll-like receptor (TLR) 5 and powerful mucosal adjuvant, significantly increases the immunogenicity and protective capacity of M2e. In this study, we report for the first time the transient expression in plants of a recombinant protein Flg-4M comprising flagellin of Salmonella typhimurium fused to four tandem copies of the M2e peptide. The chimeric construct was expressed in Nicotiana benthamiana plants using either the self-replicating potato virus X (PVX) based vector, pA7248AMV-GFP, or the cowpea mosaic virus (CPMV)-derived expression vector, pEAQ-HT. The highest expression level up to 30 % of total soluble protein (about 1 mg/g of fresh leaf tissue) was achieved with the PVX-based expression system. Intranasal immunization of mice with purified Flg-4M protein induced high levels of M2e-specific serum antibodies and provided protection against lethal challenge with influenza virus. This study confirms the usefulness of flagellin as a carrier of M2e and its relevance for the production of M2e-based candidate influenza vaccines in plants.