capsid proteins (Scotti and Rybicki 2013; Rybicki 2014) and non-Hodgkin lymphoma vaccines, which proceeded to a phase I clinical trial (see also Tables 4 and 5)
(McCormick et al. 2008).
Fraunhofer USA Center for Molecular Biotechnology (CMB) has developed
a “launch vector”, an advanced, hybrid, gene infiltration system that combines
the elements of TMV vector and A. tumefaciens binary plasmids (Musiychuk et al.
2007). Among others developed, the launch vector pBID4 contains the 35S promoter from cauliflower mosaic virus (35S CaMV) that drives transcription of the
viral genome, the nopaline synthase terminator, genes for virus replication and cellto-cell movement and the target gene cloned under the transcriptional control of
the coat protein subgenomic mRNA promoter. Following infiltration, primary transcripts produced in the nucleus are transported into the cytoplasm, resulting in robust
protein production (such as 1 mg/g of fresh weight) (Musiychuk et al. 2007; Massa
et al. 2007).
The pEAQ system is based on full-length or truncated versions of CPMV
RNA-2 for efficient and rapid protein production without viral replication (Sainsbury
et al. 2009). These vectors contain the 35S CaMV promoter, nos terminator, the
p19 sequence encoding a suppressor of silencing and 5
0 - and 3
0 -UTRs from
CPMV RNA-2. The gene of interest is inserted between the UTRs. The “HT”
(hypertranslatable) variants of these family of vectors provide extremely high
translational efficiency and, eventually, high level of the recombinant protein
accumulation in plant biomass (Peyret and Lomonossoff 2015).
Transient expression of target proteins in plants is, therefore, considered a more
feasible approach when compared to stable transformation, due to its rapid production capabilities often leading to the expression of large amounts of recombinant
protein in a short time. The process is scalable just by virus-infecting or agroinfiltrating more plants.
Agro-infiltration has demonstrated the highest efficiency and highest levels of
target protein expression with the potential for cost-effective production (Loh et al.
2017). Nicotiana benthamiana is particularly amenable to infiltration methods.
The maximum of protein expression is generally observed within 7 days postinfiltration which is faster if compared to the mere virus strategy that requires at
least 2 weeks to generate a systemic spreading and expression. As mentioned, yields
using this approach can reach 1 g of product per kilogram of leaves, though the
levels are obviously protein-dependent. Successful clinical trial has indicated safety
and efficacy of the protein therapeutics and biologics made by agro-infiltrated plants
(see Table 5). The main example is represented by the production in Nicotiana
benthamiana of a vaccine candidate against H1N1 influenza pandemic that occurred
in 2009: the Canadian company Medicago was able to produce a ready to be
administered hemagglutinin-based vaccine by transient expression in 19 days
(D'Aoust et al. 2010).
Engineering Plants for the Future: Farming with Value-Added Harvest
75
(McCormick et al. 2008).
Fraunhofer USA Center for Molecular Biotechnology (CMB) has developed
a “launch vector”, an advanced, hybrid, gene infiltration system that combines
the elements of TMV vector and A. tumefaciens binary plasmids (Musiychuk et al.
2007). Among others developed, the launch vector pBID4 contains the 35S promoter from cauliflower mosaic virus (35S CaMV) that drives transcription of the
viral genome, the nopaline synthase terminator, genes for virus replication and cellto-cell movement and the target gene cloned under the transcriptional control of
the coat protein subgenomic mRNA promoter. Following infiltration, primary transcripts produced in the nucleus are transported into the cytoplasm, resulting in robust
protein production (such as 1 mg/g of fresh weight) (Musiychuk et al. 2007; Massa
et al. 2007).
The pEAQ system is based on full-length or truncated versions of CPMV
RNA-2 for efficient and rapid protein production without viral replication (Sainsbury
et al. 2009). These vectors contain the 35S CaMV promoter, nos terminator, the
p19 sequence encoding a suppressor of silencing and 5
0 - and 3
0 -UTRs from
CPMV RNA-2. The gene of interest is inserted between the UTRs. The “HT”
(hypertranslatable) variants of these family of vectors provide extremely high
translational efficiency and, eventually, high level of the recombinant protein
accumulation in plant biomass (Peyret and Lomonossoff 2015).
Transient expression of target proteins in plants is, therefore, considered a more
feasible approach when compared to stable transformation, due to its rapid production capabilities often leading to the expression of large amounts of recombinant
protein in a short time. The process is scalable just by virus-infecting or agroinfiltrating more plants.
Agro-infiltration has demonstrated the highest efficiency and highest levels of
target protein expression with the potential for cost-effective production (Loh et al.
2017). Nicotiana benthamiana is particularly amenable to infiltration methods.
The maximum of protein expression is generally observed within 7 days postinfiltration which is faster if compared to the mere virus strategy that requires at
least 2 weeks to generate a systemic spreading and expression. As mentioned, yields
using this approach can reach 1 g of product per kilogram of leaves, though the
levels are obviously protein-dependent. Successful clinical trial has indicated safety
and efficacy of the protein therapeutics and biologics made by agro-infiltrated plants
(see Table 5). The main example is represented by the production in Nicotiana
benthamiana of a vaccine candidate against H1N1 influenza pandemic that occurred
in 2009: the Canadian company Medicago was able to produce a ready to be
administered hemagglutinin-based vaccine by transient expression in 19 days
(D'Aoust et al. 2010).
Engineering Plants for the Future: Farming with Value-Added Harvest
75