Viruses. 2014 Apr 21;6(4):1789-800. doi: 10.3390/v6041789.

High-level systemic expression of conserved influenza epitope in plants on the surface of rod-shaped chimeric particles.


Petukhova NV1, Gasanova TV2, Ivanov PA3*, Atabekov JG4.
  • 1Department of Virology, Lomonosov Moscow State University, Moscow 119991, Russia.
  • 2Department of Virology, Lomonosov Moscow State University, Moscow 119991, Russia.
  • 3Department of Virology, Lomonosov Moscow State University, Moscow 119991, Russia. *Corresponding author
  • 4Department of Virology, Lomonosov Moscow State University, Moscow 119991, Russia.



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.

PMID: 24755563



A new approach for super-expression of conserved M2e epitope in plants has been developed on the basis of a recombinant tobacco mosaic virus (TMV-U1) genome designed for Agrobacterium-mediated delivery into the plant cell nucleus. The TMV coat protein (CP) served as a carrier and three versions of human consensus M2e sequence were inserted into the C-terminal surface loop between amino acid residues 155 and 156. Cysteine residues in the heterologous peptide were thought likely to impede efficient assembly of chimeric particles. Therefore, in addition to the initial sequence, we introduced mutations by the substitution of cysteines at the positions 17 and 19 with either serine (Ser) or alanine (Ala) residues. Agroinfiltration experiments proved that all the recombinant viruses were capable of spreading via vascular system of Nicotiana benthamiana. The chimeric particles were stable in plant extracts and during preparation procedures; foreign epitopes were exposed on their surface as shown by immunoelectron microscopy. Reverse transcription of genomic RNA and total RNA from systemic leaves proved genetic stability of TMV-M2e recombinant viruses. Nicotiana benthamiana plants produced as much as 5 g of TMV-M2e-ala or 1 g of TMV-M2e-ser CPs per 1 kg of fresh weight of upper non-inoculated leaves in 2 weeks. Following the purification, particles contained up to 90% of CP-M2e fusion protein. During vaccination, experimental mice did not lose weight comparing with the control animals. Antisera to chimeric virions contain far more antibodies specific to influenza virus than those specific to TMV particle itself (up to 5/1 ratio). IgG1/IgG2a ratio varies from 0.7 (Ser) to 3.2 (Ala). The longevity of M2e-specific immune response declined insignificantly during the 7 month period. A whole cell ELISA assay confirmed that the antisera to TMV-M2e-ala and TMV-M2e-ser are capable of recognizing M2 protein exposed on the surface of epithelial cells infected with influenza A/PR/8/34 (H1N1). Mice immunized with the chimeric virions were resistant to five lethal doses (LD50) of the homologous influenza virus strain, A/PR/8/34 (H1N1) and TMV-M2e-ala conferred protection (5LD50, 70% of survival rate) against a heterologous strain (4 amino acid changes in M2e plus cysteine substitutions) A/California/07/2009 (H1N1). Similarly, we constructed another vector designed for expression of fusion peptide (fp, GLFGAIAGFIEGGW) from hemagglutinin. Infection of N. benthamiana with TMV-fp vector caused severe symptoms and genetic stability of TMV-fp vector was demonstrated as well. Electron microscopy of TMV-fp preparation represented the rod-shaped particles similar in morphology to TMV-wt virions.



Figure. Immunogold labeling of purified TMV-based chimeric particles carrying conserved influenza epitope M2e. On the left: TMV-M2e-ser. On the right: TMV-M2e-ala. Primary antibodies were specific to M2e antigen, secondary anti-mouse antibodies were conjugated with 6 nm gold particles. Magnifications – 200.000. Scale bars are indicated.



  1. Petukhova N.V., Gasanova T.V., Stepanova L.A., Rusova O.A., Potapchuk M.V., Korotkov A.V., Skurat E.V., Tsybalova L.M., Kiselev O.I., Ivanov P.A., Atabekov J.G. Immunogenicity and protective efficacy of candidate universal influenza A nanovaccines produced in plants by Tobacco mosaic virus-based vectors. Curr. Pharm. Des. 2013. 19: 5587-5600.
  2. Petukhova N.V., Ivanov P.A., Migunov A.I. Virus-like particles – a new strategy for production of vaccines against influenza virus. Vopr. Virusol. 2013. 58(2): 10-14



This work was partly supported by the Russian Scientific Foundation (research project № 14-24-00007).



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