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.

The amino acid sequence of the extracellular domain of the virus-encoded M2 matrix protein (peptide M2e) is conserved among all subtypes of influenza A strains, enabling the development of a broad-range vaccine against them. We expressed M2e from avian influenza virus A/chicken/Kurgan/5/2005 (H5N1) in nuclear-transformed duckweed plants for further development of an avian influenza vaccine. The 30-amino acid N-terminal fragment of M2, including M2e (denoted M130), was selected for expression. The M2e DNA sequence fused in-frame to the 3′ end of ricin toxin B chain (RTB) was cloned under control of the CaMV 35S promoter into pBI121. The resulting plasmid was used for duckweed transformation, and 23 independent transgenic duckweed lines were obtained. Asialofetuin-binding ELISA of protein samples from the transgenic plants using polyclonal anti-RTB antibodies confirmed the expression of the RTB–M130 fusion protein in 20 lines. Quantitative ELISA of crude protein extracts from these lines showed RTB–M130 accumulation ranging from 0.25–2.5 μg/g fresh weight (0.0006–0.01% of total soluble protein). Affinity chromatography with immobilized asialofetuin and western blot analysis of protein samples from the transgenic plants showed expression of fusion protein RTB–M130 in the aggregate form with a molecular mass of about 70 kDa. Mice were immunized orally with a preparation of total soluble protein from transgenic plants, receiving four doses of 7 μg duckweed-derived RTB–M130 each, with no additional adjuvant. Specific IgG against M2e was detected in immunized mice, and the endpoint titer of nti-M2e IgG was 1,024. It was confirmed that oral immunization with RTB-M130 induces production of specific antibodies against peptide M2e, one of the most conserved antigens of the influenza virus. These results may provide further information for the development of a duckweed-based expression system to produce a broad-range edible vaccine against avian influenza.

To date, the expression of recombinant proteins in transgenic plants is becoming a powerful alternative to classical expression methods. Special efforts are directed to the development of contained cultivation systems based on cell culture or rhyzosecretion, which reliably prevents the heterologous DNA releasing into the environment. A promising object for the development of such systems is the tiny aquatic plant of Wolffia arrhiza, which can be used as a dipped culture in bioreactors. Herein we have expressed the human granulocyte colony-stimulating factor (hG-CSF) in nuclear-transformed Wolffia. The nucleotide sequence of hG-CSF was optimized for expression in Wolffia and cloned into the vector pCamGCSF downstream of double CaMV 35S promoter. Wolffia plants were successfully transformed and 34 independent transgenic lines with hG-CSF gene were obtained, PCR and Southern blot analysis confirmed the transgenic origin of these lines. Western blot analysis revealed accumulation of the target protein in 33 transgenic lines. Quantitative ELISA of protein extracts from these lines showed hG-CSF accumulation up to 35.5 mg/kg of Wolffia fresh weight (0.194% of total soluble protein). This relatively high yield holds promise for the development of Wolffia-based expression system in strictly controlled format to produce various recombinant proteins.

The M2e peptide of the H5N1 A/Chicken/Kurgan/05/2005 avian influenza virus was successfully synthesized in transgenic tobacco plants. The amino terminal segment of the M2 protein, comprised of 22, 30, or 43 amino acids, including the M2e peptide (M122, M130, and M143 variants, respectively), was trans lationally fused with the N terminus of β glucuronidase in the pBI121 plant expression vector. The nucle otide sequence of the target fragment was synthesized by ligation from synthetic oligonucleotides; its codon composition was adapted for expression in plants. Tobacco plants were successfully transformed with the obtained vectors (pBIM122, pBIM130, and pBIM143, respectively). In the plants transformed with pBIM143, the Ml43-β glucuronidase fusion protein was not produced, probably due to the presence of the M2 protein transmembrane domain (25-43 aa of M2) in this construct. In the pBIM122 and pBIMl30 transformed plants, the target M2e peptide was expressed as a component of the Ml22-β glucuronidase and M130-β glucuronidase fusion proteins, respectively, as was detected by Western blot analysis. These proteins were detected as bands of the expected size without apparent degradation. As a result, the M2e peptide of the H5N1 avian influenza virus was successfully synthesized for the first time in nuclear transformed transgenic plants. The results obtained in this study will be used for developing a transgenic plant based edible anti influenza vaccine.

No abstract