Abstract
Recombinant viruses based on the cDNA copy of the tobacco mosaic virus (TMV) genome carrying different versions of the conserved M2e epitope from influenza virus A cloned into the coat protein (CP) gene were obtained and partially characterized by our group previously; cysteines in the human consensus M2e sequence were changed to serine residues. This work intends to show some biological properties of these viruses following plant infections. Agroinfiltration experiments on Nicotiana benthamiana confirmed the efficient systemic expression of M2e peptides, and two point amino acid substitutions in recombinant CPs significantly influenced the symptoms and development of viral infections. Joint expression of RNA interference suppressor protein p19 from tomato bushy stunt virus (TBSV) did not affect the accumulation of CP-M2e-ser recombinant protein in non-inoculated leaves. RT-PCR analysis of RNA isolated from either infected leaves or purified TMV-M2e particles proved the genetic stability of TMV‑based viral vectors. Immunoelectron microscopy of crude plant extracts demonstrated that foreign epitopes are located on the surface of chimeric virions. The rod‑shaped geometry of plant-produced M2e epitopes is different from the icosahedral or helical filamentous arrangement of M2e antigens on the carrier virus-like particles (VLP) described earlier. Thereby, we created a simple and efficient system that employs agrobacteria and plant viral vectors in order to produce a candidate broad-spectrum flu vaccine.
Highlights
Influenza A is one of the widespread human and animal viruses
Cloning of different versions of the human consensus [14] influenza M2e epitope into the open reading frame (ORF) of the coat protein (CP) gene of tobacco mosaic virus (TMV) strain U1 [12] between 155 and 156 codons was described in detail previously [13]
This article describes the first successful example of the efficient systemic expression of the conserved influenza M2e epitope in plants that provided the high-level accumulation of recombinant coat proteins and the efficient assembly of stable rod-shaped M2e-containing chimeric particles in plant leaves
Summary
Influenza A is one of the widespread human and animal viruses. Most of the commercial vaccines contain hemagglutinin and neuraminidase as the protective antigens. The immunogenic and protective properties of the influenza M2e epitope have been studied for more than 10 years [2,3,4] This antigen was previously expressed in E. coli as a fusion with HBcAg from human hepatitis virus B [5], TLR5 ligand flagellin [6], coat proteins of woodchuck hepatitis virus [7], papaya mosaic virus (PapMV) [8] and phage Qbeta [9]. Genomes, respectively, but expression levels were low and plant infections limited to inoculated leaves This investigation was aimed at studying biological properties of two recombinant viruses created previously for the presentation of the M2e epitope using a cDNA copy of the tobacco mosaic virus (TMV-U1) [12] genome. Chimeric particles can be isolated from infected plants, and improved immunogenicity of the epitope that is repeated in many copies (up to 2000 times per one particle) was shown [13]
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