Molecular farming of antimicrobial peptides: available platforms and strategies for improving protein biosynthesis using modified virus vectors

LEITE, MICHEL L.; SAMPAIO, KAMILA B.; COSTA, FABRÍCIO F.; FRANCO, OCTÁVIO L.; DIAS, SIMONI C.; CUNHA, NICOLAU B.

doi:10.1590/0001-3765201820180124

MICHEL L. LEITE

Centro de Análises Proteômicas e Bioquímicas, Universidade Católica de Brasília/UCB, SGAN 916, Modulo B, Bloco C, 70790-160 Brasilia, DF, Brazil

Pós-Graduação em Ciências Genômicas e Biotecnologia, Centro de Análises Proteômicas e Bioquímicas, Universidade Católica de Brasília/UCB, SGAN 916, Modulo B, Bloco C, 70790-160 Brasilia, DF, Brazil

KAMILA B. SAMPAIO

Centro de Análises Proteômicas e Bioquímicas, Universidade Católica de Brasília/UCB, SGAN 916, Modulo B, Bloco C, 70790-160 Brasilia, DF, Brazil

Pós-Graduação em Ciências Genômicas e Biotecnologia, Centro de Análises Proteômicas e Bioquímicas, Universidade Católica de Brasília/UCB, SGAN 916, Modulo B, Bloco C, 70790-160 Brasilia, DF, Brazil

FABRÍCIO F. COSTA

Centro de Análises Proteômicas e Bioquímicas, Universidade Católica de Brasília/UCB, SGAN 916, Modulo B, Bloco C, 70790-160 Brasilia, DF, Brazil

Cancer Biology and Epigenomics Program, Northwestern University’s Feinberg School of Medicine, 60611, Chicago IL, USA

Genomic Enterprise, 2405 N. Sheffield Av., 14088, 60614, Chicago, IL, USA

MATTER Chicago, 222 W. Merchandise Mart Plaza, 12th Floor, 60654, Chicago, IL, USA

The Founder Institute, 3337 El Camino Real, 94306, Palo Alto, CA USA

OCTÁVIO L. FRANCO

Centro de Análises Proteômicas e Bioquímicas, Universidade Católica de Brasília/UCB, SGAN 916, Modulo B, Bloco C, 70790-160 Brasilia, DF, Brazil

Pós-Graduação em Ciências Genômicas e Biotecnologia, Centro de Análises Proteômicas e Bioquímicas, Universidade Católica de Brasília/UCB, SGAN 916, Modulo B, Bloco C, 70790-160 Brasilia, DF, Brazil

S-Inova Biotech, Pós-Graduação em Biotecnologia, Universidade Católica Dom Bosco, Av. Tamandaré, 6000, Jardim Seminário, 79117-010 Campo Grande, MS, Brazil

SIMONI C. DIAS

Centro de Análises Proteômicas e Bioquímicas, Universidade Católica de Brasília/UCB, SGAN 916, Modulo B, Bloco C, 70790-160 Brasilia, DF, Brazil

Pós-Graduação em Ciências Genômicas e Biotecnologia, Centro de Análises Proteômicas e Bioquímicas, Universidade Católica de Brasília/UCB, SGAN 916, Modulo B, Bloco C, 70790-160 Brasilia, DF, Brazil

NICOLAU B. CUNHA

Centro de Análises Proteômicas e Bioquímicas, Universidade Católica de Brasília/UCB, SGAN 916, Modulo B, Bloco C, 70790-160 Brasilia, DF, Brazil

Pós-Graduação em Ciências Genômicas e Biotecnologia, Centro de Análises Proteômicas e Bioquímicas, Universidade Católica de Brasília/UCB, SGAN 916, Modulo B, Bloco C, 70790-160 Brasilia, DF, Brazil

Correspondence to: Nicolau Brito da Cunha E-mail: nicolau.cunha@ucb.br / nicolaubrito@yahoo.com.br

Figures (3)
Tables (4)

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Figure 1
Agroinfiltration method for the large-scale biosynthesis of recombinant AMPs. After gene cloning using E. coli cells, pro-vectors carrying the 3`extremity of the expression cassette (green vector), the 5`extremity of the expression cassette (blue vector) and the gene that codifies the integrase for the extremities recombination (yellow vector) are inserted in A. tumefaciens cells, followed by cultivation in LB medium. The bacterial culture is then injected in the downside of N. benthamiana leaves. After 5 to 8 days, yields of recombinant AMPs can be obtained from harvested leaves after extraction and purification.

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Figure 2
Expression kinetics of foreign genes in N. benthamiana leaves. The gene delivery strategy mediated by A. tumefaciens allows the efficient spread of AMPs coding genes and integration to the host cells. Potent viral promoters induce high expression rates of the Green Fluorescent Protein (GFP) from Aequorea victoria gene, a frequent reporter gene used for experimental validation. The peak of expression is about the seventh or eighth day after inoculation.

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Figure 3
Pro-vector system for assembly of gene expression modules. Two viral vectors, donors respectively of the 5 ‘and 3’ ends, are specifically recognized by the enzyme Integrase C31 from Streptomyces sp. The enzyme catalyzes the recombination between the fragments by the pairing of homologous bases at the recombination sites. The final hybrid fragment presents all genetic elements for large-scale gene expression and for the addressing / purification of recombinant AMP.

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TABLE I
Comparison between different production systems of heterologous proteins of pharmaceutical interest.

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TABLE II
Available platforms for the biosynthesis of PMPs.

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TABLE III
Plant species used as bioreactors of PMPs.

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TABLE IV
Examples of transiently expressed antigens using the Magnifection^TM system.

Type	System	Average cost	Timescale	Scalability	Quality (folding, subunit assembly, glycosylation)	Post translational modifications	Contamination risks	Storage cost
Microorganisms	Bacteria	Low	Short	High	Low	None	Potential presence of endotoxins	Moderate
Microorganisms	Yeast	Moderate	Moderate	High	Moderate	Similar	Low	Moderate
Cell culture	Mammal cell culture	High	Long	Very low	Very high	Identical	Potential presence of virus, prions and oncogenes	High
Cell culture	Plant cell culture	Moderate	Moderate	Moderate	High	Similar	Low	Moderate
Transgenic multicellular organisms	Transgenic animals	High	Very long	Low	Very high	Identical	Potential presence of virus, prions and oncogenes	High
Transgenic multicellular organisms	Transgenic plants	Very low	Long	Very high	High	Similar	Low	Inexpressive
Transient platform	Plants infected with viral vectors	Very low	Short	Very high	High	Similar	Low	Inexpressive

Stable transformation		References
Type	Whole plants	Obembe et al. 2011 / Drake et al. 2017
Type	Stable nuclear transformation
Key Features	Stable incorporation of exogenous genes into the nuclear genome
	Stable inheritance of transgenes in successive generations
	Used to obtain the majority of transgenic plants until today
	Utilized commercially since 2014 in Japan by the company Hokusan for the production of interberry-alpha, a recombinant canine interferon-alpha produced in transgenic strawberry for the treatment of periodontal disease in dogs
Advantages	Transmission of new characters as traits inheritable to the progeny
Advantages	High scalability
Disadvantages	Possibility of undesirable crosses in some species
	Long cycle of production of some plant species
	Usually poor levels of transgene expression
Type	Whole plants	Meyers et al. 2010MEYERS B, ZALTSMAN A, LACROIX B, KOZLOVSKY SV and KRICHEVSKY A. 2010. Nuclear and plastid genetic engineering of plants: Comparison of opportunities and challenges. Biotechnol Adv 28: 747-756.
Type	Stable plastidial transformation
Key Features	Stable and simultaneous transformation of numerous copies of the plastidial genome
Key Features	Exclusively maternal inheritance in many species
Advantages	Natural biocontainment
	Minimizing gene flow by out-crossing
	High levels of expression (up to 70% TSP)
Disadvantages	Limited to few species: tomato, lettuce, soybeans and eggplant.
	Routine transformation of tobacco only
	Variable protein stability
Type	Plant cell-suspension cultures	Franconi et al. 2010FRANCONI R, DEMURTAS OC and MASSA S. 2010. Plant-derived vaccines and other therapeutics produced in contained systems. Expert Rev Vaccines 9: 877-892. / Drake et al. 2017
Key Features	Undifferentiated aggregates of plant cells dispersed and propagated in liquid medium
Key Features	System used for the production of the first PMP to achieve commercial production status by the FDA, in 2012: Elelyso, the replacement enzyme glucocerebrosidase from the Israeli company Protalix, in addition to the commercial chicken vaccine against Newcastle disease virus (NDV) from Dow Agroscience
Advantages	Fast, relatively inexpensive and high level of containment
	Usually high purity production and low downstream processing costs when the PMP is secreted into the culture medium
	Homogeneity of production
	Low N-glycans addition heterogeneity
Disadvantages	Need for sterile production conditions
	Decreased levels of protein biosynthesis in stationary phase, due to proteolytic activity
	Restricted to just a few crops such as tobacco, Arabidopsis, rice and carrots
Type	Transient expression systems	Regnard et al. 2010REGNARD GL, HALLEY-STOTT RP, TANZER FL, HITZEROTH II and RYBICKI EP. 2010. High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector. Plant Biotechnol J 8: 38-46./ Loh et al. 2017
Type	Agroinfiltration method
Key Features	Infiltration of tobacco leaves by suspension of Agrobacterium tumefaciens cells
Advantages	Transference of bacterial T-DNA to a high number of leaf cells
	Fast
	High expression. levels.
	Possibility of producing clinical grade pharmaceuticals
Disadvantages	Rapid decay of gene expression after peak expression.
Disadvantages	Inability to transfer transgene to progeny
Type	Virus infection method	McCormick et al. 2008MCCORMICK AA et al. 2008. Plant-produced idiotype vaccines for the treatment of non-Hodgkin’s lymphoma: Safety and immunogenicity in a phase I clinical study. Proc Natl Acad Sci U S A 105: 10131-10136. / Marsian and Lomonossoff 2016MARSIAN J and LOMONOSSOFF GP. 2016. Molecular pharming—VLPs made in plants. Curr Opin Biotechnol 37: 201-206.
Key Features	Non-integrative method
	Based on the use of plant viruses, such as Tobacco Mosaic Virus (TMV) and Potato Virus X (PVX), as infectious carriers of transgenes
	Used to infect tobacco
	Used by the Large Scale company to obtain vaccines against B-cell non-Hodgkin’s lymphoma
Advantages	Fast, scalable and capable of obtaining high levels of recombinant protein biosynthesis
Disadvantages	Restricted to tobacco
Disadvantages	Need for immediate processing due to protein instability
Type	Magnifection^TM	Gleba et al. 2014
Key Features	Platform that combines the characteristics of the methods Agroinfiltration and Virus infection
	Developed by Icon Genetics
	Use of deconstructed viral expression cassettes, lacking protein coat sequences and viral motility proteins sequences
	Systemic delivery of genes is mediated by Agrobacterium
Advantages	Improved infectivity
	Increased levels of gene expression and biosynthesis of recombinant proteins greater than 80% TSP
	Fast
	Capable of producing both small molecules, such as vaccine antigens, and large and complex IgGs
	Capable of co-expressing two or several polypeptides simultaneously
	Capable of assembling heterooligomeric proteins
	Ease of manipulation
Disadvantages	Restricted to tobacco
Disadvantages	Need for immediate processing due to protein instability

Species	Advantages	Disadvantages
Leafy crops
Tobacco	High biomass production	Low post-harvest protein stability
	Well established transformation and processing technologies High scalability	Presence of alkaloids
	Non-food and non-feed crop	Presence of alkaloids
Lettuce	High yield of biomass	Low post-harvest protein stability
	Edible
	Useful for human vaccination
Alfafa	High biomass production	Low post-harvest protein stability
	Useful for animal vaccination Clonal propagation	Presence of oxalic acid
	Addition of homogeneous N-glycans	Presence of oxalic acid
Clover	High biomass production	Low post-harvest protein stability
	Useful for animal vaccination Clonal propagation
	Addition of homogeneous N-glycans
Legumes
Soybeans	Abundant biomass, possibility of transgene expression in seed coat	Usual low levels of transgene expression
	High protein concentration in seeds
	High ratio of seed biomass /production cost
Pea	High protein concentration in seeds	Usual low levels of transgene expression
Pigeon pea	High protein concentration in seeds	Usual low levels of transgene expression
Cereals
Wheat	High protein stability during storage	Low yields
Wheat	High protein stability during storage	Difficulties in processing and handling
Barley	High protein stability during storage	Low yields
Barley	High protein stability during storage	Difficulties in processing and handling
Maize	High protein stability during storage	Usual low levels of transgene expression
	High biomass production
	Ease of processing and handling
Rice	High protein stability during storage	Usual low levels of transgene expression
	High biomass production
	Ease of processing and handling
Fruits
Tomato	Edible crop	Expensive cultivation
Tomato	Containment in greenhouse	Low stability after harvesting
Tubers
Carrot	Edible
	High protein stability in storage tissues
	High scalability
	Ease of purification and processing in cell suspension system
Potato	Edible	Usual low levels of transgene expression
Potato	High protein stability in storage tissues	Needs to be cooked before consumption
Oilcrops
Canola	Oleosin protein fusion platform	Low yields
Canola	Sprouting system developed	Low yields
Camelina sativa	Oleosin protein fusion platform	Low yields
Camelina sativa	Sprouting system developed	Low yields
Moss
Physcomitrella patens	Ease of culturing under containment	Low scaling
	Facilitated clone propagation
	Allows secretion in culture medium
	Exhibits homologous recombination
Green algae and aquatic plants
Chlamydomonas reinhardtii	Ease of culturing under containment	Low scaling
	Facilitated clone propagation
	Allows secretion in culture medium
Lemna	Ease of culturing under containment	Low scaling
	Facilitated clone propagation
	Allows secretion in culture medium
Model Plants
Arabidopsis thaliana	High availability of mutants	Low biomass
	High genetic accessibility
	Ease of transformation

Antigen	Disease/target	Status	Ref
Der p 1	Allergy	In vitro	Lienard et al. 2007LIENARD D et al. 2007. Suspension-cultured BY-2 tobacco cells produce and mature immunologically active house dust mite allergens. Plant Biotechnol J 5: 93-108.
Protective antigen Der p 2	Antherax	Animal pre-clinical trial	Koya et al. 2005KOYA V, MOAYERI M, LEPPLA SH and DANIELL H. 2005. Plant-Based Vaccine: Mice Immunized with Chloroplast-Derived Anthrax Protective Antigen Survive Anthrax Lethal Toxin Challenge. Infect Immun 73: 8266-8274.
L1 major capsid protein	Cervical cancer	Animal pre-clinical trial	Lenzi et al. 2008LENZI P et al. 2008. Translational fusion of chloroplast-expressed human papillomavirus type 16 L1 capsid protein enhances antigen accumulation in transplastomic tobacco. Transgenic Res 17: 1091-1102.
VCA antigen	Epstein–Barr virus	In vitro	Lee et al. 2006LEE MYT, ZHOU Y, LUNG RWM, CHYE M-L, YIP W-K, ZEE S-Y and LAM E. 2006. Expression of viral capsid protein antigen against Epstein-Barr virus in plastids of Nicotiana tabacum cv. SR1. Biotechnol Bioeng 94: 1129-1137.
HSP-A	Helicobacter pylori	Phase I/II clinical trial	Gu et al. 2005GU Q, HAN N, LIU J and ZHU M. 2005. Cloning of Helicobacter pylori urease subunit B gene and its expression in tobacco (Nicotiana tabacum L.). Plant Cell Rep 24: 532-539.
VP1	Foot and mouth disease	Animal pre-clinical trial	Wu et al. 2003WU L, JIANG L, ZHOU Z, FAN J, ZHANG Q, ZHU H, HAN Q and XU Z. 2003. Expression of foot-and-mouth disease virus epitopes in tobacco by a tobacco mosaic virus-based vector. Vaccine 21: 4390-4398.
Hepatitis B/C	HBsAg (Hep B)	Animal pre-clinical trial	Thanavala et al. 1995THANAVALA Y, YANG YF, LYONS P, MASON HS and ARNTZEN C. 1995. Immunogenicity of transgenic plant-derived hepatitis B surface antigen. Proc Natl Acad Sci U S A 92: 3358-3361.
F1-V	Plague	Animal pre-clinical trial	Del Prete et al. 2009DEL PRETE G, SANTI L, ANDRIANAIVOARIMANANA V, AMEDEI A, DOMARLE O, D’ ELIOS MM, ARNTZEN CJ, RAHALISON L and MASON HS. 2009. Plant-derived recombinant F1, V, and F1-V fusion antigens of Yersinia pestis activate human cells of the innate and adaptive immune system. Int J Immunopathol Pharmacol 22: 133-143.
SARS-CoV-S1	SARS	Animal pre-clinical trial	Pogrebnyak at al. 2005POGREBNYAK N, GOLOVKIN M, ANDRIANOV V, SPITSIN S, SMIRNOV Y, EGOLF R and KOPROWSKI H. 2005. Severe acute respiratory syndrome (SARS) S protein production in plants: Development of recombinant vaccine. Proc Natl Acad Sci U S A 102: 9062-9067.
Tet-C	Tetanus	Animal pre-clinical trial	Tregoning et al. 2005
Type 1 diabetes	GAD65	Animal pre-clinical trial	Ma et al. 2004MA S, HUANG Y, YIN Z, MENASSA R, BRANDLE JE and JEVNIKAR AM. 2004. Induction of oral tolerance to prevent diabetes with transgenic plants requires glutamic acid decarboxylase (GAD) and IL-4. Proc Natl Acad Sci U S A 101: 5680-5685.
HIV p24 capsid protein HIV	AIDS	In vitro	Zhang et al. 2002ZHANG GG, RODRIGUES L, ROVINSKI B and WHITE KA. 2002. Production of HIV-1 p24 protein in transgenic tobacco plants. Mol Biotechnol 20: 131-136.

[1] Correspondence to: Nicolau Brito da Cunha E-mail: nicolau.cunha@ucb.br / nicolaubrito@yahoo.com.br

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Molecular farming of antimicrobial peptides: available platforms and strategies for improving protein biosynthesis using modified virus vectors

Abstract