High-Yield Expression of M2e Peptide of Avian Influenza Virus H5N1 in Transgenic Duckweed Plants

Abstract

Avian influenza is a major viral disease in poultry. Antigenic variation of this virus hinders vaccine development. However, the extracellular domain of the virus-encoded M2 protein (peptide M2e) is nearly invariant in all influenza A strains, enabling the development of a broad-range vaccine against them. Antigen expression in transgenic plants is becoming a popular alternative to classical expression methods. Here we expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005(H5N1) in nuclear-transformed duckweed plants for further development of avian influenza vaccine. The N-terminal fragment of M2, including M2e, was selected for expression. The M2e DNA sequence fused in-frame to the 5' end of β-glucuronidase was cloned into pBI121 under the control of CaMV 35S promoter. The resulting plasmid was successfully used for duckweed transformation, and western analysis with anti-β-glucuronidase and anti-M2e antibodies confirmed accumulation of the target protein (M130) in 17 independent transgenic lines. Quantitative ELISA of crude protein extracts from these lines showed M130-β-glucuronidase accumulation ranging from 0.09-0.97 mg/g FW (0.12-1.96 % of total soluble protein), equivalent to yields of up to 40 μg M2e/g plant FW. This relatively high yield holds promise for the development of a duckweed-based expression system to produce an edible vaccine against avian influenza.

Plant‐based oral vaccines against zoonotic and non‐zoonotic diseases

“…As this disease is usually spread in large farms, massive vaccinations are needed to control disease in birds and animals. Plant-based vaccines offer the best solution to control this disease in large animal farms (Firsov et al, 2015). Expressed HA in endoplasmic reticulum from avian influenza HPA1 resulted in high level expression; immunogenicity of transgenic Arabidopsis was confirmed from orally immunized mice with high-level HA-specific systematic IgG and mucosal IgA, strong Th1 responses together with IgG2b production and 72% of immunized mice were protected after viral challenge (Lee et al, 2015).…”

Platforms for Plant-Based Protein Production

“…Duckweed, the common name for Lemnaceae, is a monocot plant family consisting of four major genera: Lemna, Spirodela, Wolffia, and Wolffiella. Duckweed is propagated clonally without the need for pollen or seeds, which simplifies line management, propagation, and the process feed stream [32,117]. Duckweed is safe, fast growing in simple inorganic media (doubling time !…”

Production of vaccines for treatment of infectious diseases by transgenic plants

“…Subcutaneous immunization with purified antigen from both strains elicited hemagglutinin inhibition (HI) and virus neutralizing (VN) antibodies in mice, with HI titers > 1:40, which is the minimal titer consistent with protective immunity in humans. M2e has been expressed in tobacco (Tarasenko et al, 2013) and recently, in duckweed (Firsov et al, 2015) and also in a form of a hybrid protein M2eHBC, fused to hepatitis B core antigen (Ravin et al, 2012). Tarasenko et al (2013) expressed synthetic, codonoptimized 22-, 30-or 43-amino acids long terminal fragments of the M2 protein, encoding 3 different variants of M2e peptide (M122, M130 and M143, respectively, with M122 and M130 encoding truncated M2 protein variants), fused with Nterminus of β-glucuronidase, in Agrobacteriumtransformed N. tabacum.…”

“…90 % of the immunized mice was protected against the lethal influenza virus challenge. Recently, Firsov et al (2015) successfully expressed M2e peptide in the form of M2e-β-glucuronidase fusion protein in Agrobacterium-transformed duckweed. The team expressed 30 amino acids long N-terminal fragment of M2 protein, containing 24 amino acid long fragment encoding M2e, optimized for expression in duckweed and fused upstream of the β-gucuronidase gene, under the control of CaMV 35S promoter.…”

“…The team expressed 30 amino acids long N-terminal fragment of M2 protein, containing 24 amino acid long fragment encoding M2e, optimized for expression in duckweed and fused upstream of the β-gucuronidase gene, under the control of CaMV 35S promoter. Stably transformed plants were obtained (nuclear transformation) with the highest yield of M2e-β-glucuronidase fusion protein being 1.89 and 1.96 % TSP (0.82 and 0.97 mg/g fresh weight, respectively, Firsov et al, 2015), accounting for app. 40 µg/g of fresh weight of M2e alone.…”