Expression and Immunogenicity of M2e Peptide of Avian Influenza Virus H5N1 Fused to Ricin Toxin B Chain Produced in Duckweed Plants

Abstract

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.

Efficient genetic transformation and CRISPR/Cas9‐mediated genome editing in Lemna aequinoctialis

“…In comparison to previous Agrobacterium ‐mediated transformation protocols in L. minor (Cantó‐Pastor et al ., ; Chhabra et al ., ; Firsov et al ., , ; Ko et al ., ; Sun et al ., ; Yamamoto et al ., ; Yang et al ., , , ), the increased efficiency of our protocol is likely resulted from adopting sonication coupled with vacuum infiltration infection method. In several previous studies, such as banana (Subramanyam et al ., ), chickpea (Indurker et al ., ) and cowpea (Bakshi et al ., ), it has been shown that the combination of vacuum infiltration and sonication can greatly improve the efficiency of Agrobacterium infection.…”

“…The development of genetic manipulation tools requires both an efficient transformation protocol and an effective genome editing system. Among the 37 duckweed species that belong to five genera: Spirodela , Landoltia , Lemna , Wolffiella and Wolffia (Landolt, ; Sree et al ., ), the Agrobacterium‐ mediated transformation system has been developed in at least seven of them, including Lemna gibba (Cantó‐Pastor et al ., ; Yamamoto et al ., ), Lemna minor (Chhabra et al ., ; Firsov et al ., , ; Ko et al ., ; Sun et al ., ; Yamamoto et al ., ; Yang et al ., , ), Lemna turionifera (Yang et al ., ), Landoltia punctata (formerly Spirodela oligorrhiza ) (Rival et al ., ; Vunsh et al .,), Spirodela polyrhiza (Yang et al ., ), Wolffia arrhiza (Khvatkov et al ., , ) and Wolffia globosa (Heenatigala et al ., ). However, the efficiency of the transformation varied dramatically among species (from 0.14% to 82.5%).…”

“…In addition, duckweeds are capable of rapidly accumulating heavy metals, nutrients and pollutants, which can be used for bio‐remediation of wastewater (Cheng and Stomp, ; Khellaf and Zerdaoui, ; Megateli et al ., ; Stout et al ., ; Xu et al ., ). The extremely fast population doubling rate (3–5 days per asexual doubling under ideal conditions) also makes duckweeds a good candidate for the production of recombinant proteins including antigens, monoclonal antibodies and exogenous enzymes (Cox et al ., ; Firsov et al ., , ; Khvatkov et al ., ; Ko et al ., ; Naik et al ., ; Stomp, ; Sun et al ., ), as well as dietary supplements for humans and animals because of its high nutritional value (Appenroth et al ., ; Beukelaar et al ., ; Bhanthumnavin and Mcgarry, ). Despite the potential for both basic research and industrial applications that established duckweeds as one of the most promising alternative future crops (Appenroth et al ., ; Lam et al ., ), current research and development in duckweeds are constrained by the lack of efficient genetic manipulation tools that allow to knockout or knock‐in a gene of interest in the duckweed genome within a short time.…”

Enhancing the antivirus activity of chimeric canine interferon with ricin subunit B by using nanoparticle formulations

“…[15][16][17] Studies have shown that RTB is non-toxic and could be a superior drug carrier when fused with other molecules. [18][19][20][21][22] Furthermore, our present study investigated the immune-modulatory effects of RTB on the murine macrophage cell line RAW264.7, nding that RTB could activate macrophages and release downstream cascades of JAK-STAT. Phosphorylation levels were increased at 20 sites in the RTB-treated macrophage cell line, 23 including JAK1/TYK2 and STAT1/2, which are the docking sites and specic adapter protein of the type 1 interferon signaling pathway.…”

Development of Wolffia arrhiza as a Producer for Recombinant Human Granulocyte Colony-Stimulating Factor

“…To date, there are already examples of successful expression of commercially important proteins in duckweed plants: the industrial enzyme endoglucanase E1 from Acidothermus cellulolyticus (Sun et al, 2007 ), anti-CD20, anti-CD30, and anti-interferon a2b monoclonal antibodies (Cox et al, 2006 ; Biolex Therapeutics website), and the protective antigens of influenza virus - peptide M2e (Firsov et al, 2015 , 2018 ), and hemagglutinin (Bertran et al, 2015 ). Despite that Wolffia is very promising plant for biotechnology, yet there is no reports of its use for recombinant proteins expression.…”