Production of H5N1 Influenza Virus Matrix Protein 2 Ectodomain Protein Bodies in Tobacco Plants and in Insect Cells as a Candidate Universal Influenza Vaccine

Abstract

The spread of influenza A viruses is partially controlled and prevented by vaccination. The matrix protein 2 ectodomain (M2e) is the most conserved sequence in influenza A viruses, and is therefore a good potential target for a vaccine to protect against multiple virus subtypes. We explored the feasibility of an M2e-based universal influenza A vaccine candidate based on the highly pathogenic avian influenza A virus, H5N1. A synthetic M2e gene was human- and plant-codon optimized and fused in-frame with a sequence encoding the N-terminal proline-rich domain (Zera®) of the γ-zein protein of maize. Zera®M2e was expressed transiently in Nicotiana benthamiana and Sf21 baculovirus/insect cell expression systems, and Zera®M2e protein bodies (PBs) were successfully produced in both expression systems. The plant-produced Zera®M2e PBs were purified and injected into Balb/c mice. Western blot analysis using insect cell-produced Zera®M2e PBs and multiple tandem M2e sequences (5xM2e) fused with the avian influenza H5N1 transmembrane and cytosolic tail (5xM2e_tHA) confirmed the presence of M2e-specific antibodies in immunized mice sera. The immunogenicity of the Zera®M2e indicates that our plant-produced protein has potential as an inexpensive universal influenza A vaccine.

Genetic engineering approaches to improve posttranslational modification of biopharmaceuticals in different production platforms

“…In terms of N-glycosylation, biopharmaceuticals generated from the plant-based system N. tabacum show plant-specific glycan structures and lack mammalian-specific sialic acids, which can result in immunogenicity and poor pharmacokinetics, respectively (Mercx et al, 2017;Raven et al, 2015). With BICS, the turnover of producing and approving new vaccines is streamlined, thereby supporting the annual need of vaccines due to the seasonal and epidemic character of viruses (Mbewana, Mortimer, Pêra, Hitzeroth, & Rybicki, 2015). With BICS, the turnover of producing and approving new vaccines is streamlined, thereby supporting the annual need of vaccines due to the seasonal and epidemic character of viruses (Mbewana, Mortimer, Pêra, Hitzeroth, & Rybicki, 2015).…”

“…Even the simultaneous production of several protein subunits is realizable (Roldão, Vicente, Peixoto, Carrondo, & Alves, 2011 (Meghrous et al, 2015). With BICS, the turnover of producing and approving new vaccines is streamlined, thereby supporting the annual need of vaccines due to the seasonal and epidemic character of viruses (Mbewana, Mortimer, Pêra, Hitzeroth, & Rybicki, 2015). As for most eukaryotic expression platforms, insect cells are capable to provide different PTM, whereas protein processing, folding, and secretion of secreted and membrane-bound products are limited and need to be initiated (Ailor & Betenbaugh, 1999;Mandal et al, 2016).…”

Plant‐derived protein bodies as delivery vehicles for recombinant proteins into mammalian cells

“…Furthermore, it was discovered that the N‐terminal 93 amino acids of 27 kDa γ‐zein (abbreviated gz93 from here on) are sufficient to produce PBs in other plants, and even in heterologous expression systems such as fungal, insect, and mammalian cells (Llop‐Tous et al, 2010; Torrent et al, 2009). Various proteins with different properties in terms of molecular mass and function, including growth factors (Torrent et al, 2009), viral vaccine candidate proteins (Hofbauer et al, 2016; Mbewana, Mortimer, Pêra, Hitzeroth, & Rybicki, 2015; Whitehead et al, 2014), and enzymes (Llop‐Tous, Ortiz, Torrent, & Ludevid, 2011), have been successfully incorporated into newly induced PBs in plants like Nicotiana benthamiana when fused to gz93. N. benthamiana is frequently used for the production of biopharmaceuticals because it is well suited for the transient expression of recombinant proteins, and this method offers advantages over other expression systems in terms of speed, safety, scalability, and reduced upstream production costs.…”

“…Furthermore, it was discovered that the N-terminal 93 amino acids of 27 kDa γ-zein (abbreviated gz93 from here on) are sufficient to produce PBs in other plants, and even in heterologous expression systems such as fungal, insect, and mammalian cells (Llop-Tous et al, 2010;Torrent et al, 2009). Various proteins with different properties in terms of molecular mass and function, including growth factors (Torrent et al, 2009), viral vaccine candidate proteins (Hofbauer et al, 2016;Mbewana, Mortimer, Pêra, Hitzeroth, & Rybicki, 2015;Whitehead et al, 2014), and enzymes (Llop-Tous, Ortiz, Torrent, & Ludevid, 2011), have been successfully incorporated into newly induced PBs in plants like…”

Characterization and Immunogenicity of HIV Envelope gp140 Zera® Tagged Antigens

“…Genetically fusing antigens to protein body (PB)-producing fusion tags such as elastin-like polypeptides (derived from animal proteins), hydrophobin (fungal protein) and zein (plant protein) is another strategy to improve their immunogenicity, while also improving their accumulation and recovery (Conley et al, 2011). The use of a zein-derived tag for PB formation is especially promising, and it has been used as a genetic fusion to various antigens including HIV Nef (de Virgilio et al, 2008), a HPV type 16 E7 protein (Whitehead et al, 2014), an influenza virus A matrix 2 protein ectodomain (M2e) (Mbewana et al, 2015), the ectodomain of influenza A haemagglutinin (HA) subtype 5 (Hofbauer et al, 2016), bluetongue virus VP2 serotype-specific antigen (van Zyl et al, 2017), Yersinia pestis F1-V (Alvarez et al, 2010), xylanase (Llop-Tous et al, 2010) and various fluorescent proteins (Torrent et al, 2009b;Joseph et al, 2012;Hofbauer et al, 2014;Saberianfar et al, 2015). Zera R (γ -zein ER-accumulating domain) is a 112 amino acid domain derived from the N-terminus of γ -zein which comprises the CGC motif downstream of the signal peptide, a central proline-rich domain containing a hexapeptide repeat (PPPVHL) 8 , as well as a C-terminal Pro-X domain with 4 cysteine residues (Geli et al, 1994).…”