key: cord-290088-g9559ux3
authors: Loh, Hwei-San; Green, Brian J; Yusibov, Vidadi
title: Using transgenic plants and modified plant viruses for the development of treatments for human diseases
date: 2017-08-08
journal: Curr Opin Virol
DOI: 10.1016/j.coviro.2017.07.019
sha: 
doc_id: 290088
cord_uid: g9559ux3

Production of proteins in plants for human health applications has become an attractive strategy attributed by their potentials for low-cost production, increased safety due to the lack of human or animal pathogens, scalability and ability to produce complex proteins. A major milestone for plant-based protein production for use in human health was achieved when Protalix BioTherapeutics produced taliglucerase alfa (Elelyso(®)) in suspension cultures of a transgenic carrot cell line for the treatment of patients with Gaucher's disease, was approved by the USA Food and Drug Administration in 2012. In this review, we are highlighting various approaches for plant-based production of proteins and recent progress in the development of plant-made therapeutics and biologics for the prevention and treatment of human diseases.

Hwei-San Loh 1,2 , Brian J Green 3 

Production of proteins in plants for human health applications has become an attractive strategy attributed by their potentials for low-cost production, increased safety due to the lack of human or animal pathogens, scalability and ability to produce complex proteins. A major milestone for plant-based protein production for use in human health was achieved when Protalix BioTherapeutics produced taliglucerase alfa (Elelyso 1 ) in suspension cultures of a transgenic carrot cell line for the treatment of patients with Gaucher's disease, was approved by the USA Food and Drug Administration in 2012. In this review, we are highlighting various approaches for plant-based production of proteins and recent progress in the development of plant-made therapeutics and biologics for the prevention and treatment of human diseases.

Infectious diseases remain as one of the leading causes of mortality and morbidity in developing countries and are exacerbated by the lack of resources and infrastructure to prevent, treat and control diseases. Therefore, emerging and re-emerging pathogens have frequently resulted in epidemics in these countries. Over the past several decades, production of proteins in plants has been shown to be a promising approach for the manufacture of targets for human health applications. Plants, when compared to other production systems, offer some advantages, including ease of scaling and lack of human and animal pathogens [1] [2] [3] (Table 1) .

This review focuses on several approaches that have been used to produce proteins in plants for prophylactic and therapeutic applications to combat human disease conditions. The various approaches for plant-based production of proteins are illustrated in Figure 1 .

Stable nuclear and chloroplast transformations are the two approaches utilized to express heterologous recombinant proteins in plants. Agrobacterium-mediated stable transformation has a long history in plant genetic manipulation, and is achieved by stable integration of T-DNA into plant nuclear genome [4] . However, the approach is time consuming, with a lead time ranging from 12 to 18 months and typically has low levels of the target protein expressed [5] . Stable introduction of target genes into chloroplast genome, that is, chloroplast transformation or transplastomics, however, allows for higher levels of target expression as compared to nuclear transformation, largely due to the lack of gene silencing and high gene copy number [6] , but it is technically difficult, lacks most post-translational modifications and has only been successful in a limited number of plant species.

Transient expression of target proteins in plants using modified plant viruses or viral vectors integrated into binary vectors delivered via Agrobacterium [7, 8 ] is often considered a more robust approach when compared to stable transformation, due to its rapid production capabilities and relatively high protein expression [8 ] . The majority of plant viral vectors used to date are based on single-stranded RNA viruses, such as tobacco mosaic virus, potato virus X and cowpea mosaic virus (CPMV), which encode for at least three proteins with functions in viral replication (replicase), encapsidation (coat protein) and movement from cell-to-cell (movement protein) [9]. The initial strategy involved production of recombinant proteins using plant viruses by exploiting their natural ability to infect (full virus) plants. However, this approach generally failed due to instability of viral genome modified by the introduction of large target genes [7] . This issue was largely resolved by using Agrobacterium-mediated gene delivery or agroinfiltration. The target gene can either be directly cloned into an Agrobacterium vector or through a modified plant viral vector which has been integrated into an Agrobacterium binary plasmid, and delivered into the plant tissues by infiltration with the transformed Agrobacterium [7, 8 ] . Agroinfiltration allows for high levels of target protein expression with the Table 1 General comparison of expression hosts for the production of heterologous proteins for medical and pharmaceutical applications potential for cost-effective production [5, 10] . The peak protein expression is typically observed in less than 7 days postinfiltration which is significantly faster when compared to the full virus strategy which requires more than 2 weeks in order to generate a systemic infection for expression. The promise of this platform has been evidenced in numerous successful clinical trials, which demonstrated safety and efficacy of plant-made protein therapeutics and biologics [11 ] . For example, in responding to the H1N1 influenza virus pandemic that occurred in 2009, Medicago, a Canadian company, reported producing the vaccine candidate, hemagglutinin in 19 days in Nicotiana benthamiana [10] . As such, agroinfiltration provides a rapid response capability and is currently the preferred approach for the production of proteins in plants.

Modified plant viruses for treatment of human diseases Loh, Green and Yusibov 83 Detection of serum IgG in immunized mice (SC) and fecal IgA in immunized mice via oral administration. [33 ] Hepatitis B virus (HBV) small surface antigen (S-HBsAg) eVLP vaccine against HBV Lactuca sativa (lettuce)/transgenic (nuclear)

Detection of serum IgG in immunized mice via oral administration. [34] HBV surface antigen (HBsAg) SUV against HBV Solanum tuberosum (potato)/transgenic (nuclear)

Induction of serum antibodies and stable immunological memory in immunized mice fed with transgenic potato tubers. [35] Human immunodeficiency virus (HIV) gp120 multi-epitopic envelope protein (C4(V3)6)

Lettuce/transgenic (nuclear)

Detection of cell-mediated and humoral immunities in immunized mice via oral administration. [36] HIV gp120 and gp41 multi-epitopic envelope proteins (Multi-HIV)

Tobacco/transgenic (chloroplast)

Detection of antibody and cellular responses as well as specific IFN-g production in immunized mice via oral administration.

[37]

HIV-1 envelope proteins (Gag/Dgp41) eVLPs vaccine against HIV-1 Purified CTB-EX4 increased level of insulin secretion from pancreatic cells.

Oral feeding of lyophilized CTB-EX4 lowered blood glucose level in mice. [55] Keys for abbreviations: ADE, antibody-dependent enhancement; C H , constant domains of immunoglobulin heavy chain; C L , constant domain of immunoglobulin light chain; CTB, cholera toxin B; cVLP, chimeric virus-like particle; DIII, domain III; DPP, dipeptidyl peptidase; E, envelope; eVLP, enveloped virus-like particle; EX, exendin; F, coagulation factor; GAA, acid alpha glucosidase; GLP, glucagon like peptide; GP, glycoprotein; HA, hemagglutinin; HI, hemagglutination-inhibition; HC, heavy chain; Ig, immunoglobulin; LTB, heat-labile enterotoxin B subunit; IM, intramuscular; IN, intranasal; IP, intraperitoneal; IV, intravenous; MAb, monoclonal antibody; N, nucleocapsid; PA, protective antigen; RTB, ricin toxin B; sAg, surface antigen; SC, subcutaneous; scFv, single-chain variable fragment of immunoglobulin; SUV, subunit vaccine; VLP, virus-like particle. 

Numerous examples of plant-produced proteins targeting prophylactic and therapeutic applications (subsectioned as vaccines, antibodies and other biopharmaceuticals) in preclinical development are shown in Table 2 . Several lead candidates have gone through clinical trials (Table 3) and have been comprehensively reviewed [12,13 ].

Vaccines are highly effective tools for the prevention of infections. Over the last three decades, plant-produced antigens targeting various pathogens have been shown to be effective in animal models ( Table 2) . Several of these candidates have progressed into early stage clinical development and were evaluated in Phase 1-2 human clinical trials ( (Table 3) .

Planet Biotechnology (Hayward, CA) produced the world's first plant-derived clinically tested secretory IgA monoclonal antibody which recognizes the surface antigen I/II of Streptococcus mutans (CaroRx TM ) that predominantly causes dental caries. Following the successful demonstration of safety and efficacy in a Phase 2 clinical trial, CaroRx TM has been licensed in Europe in a medical device category [17, 18] and applied as an oral topical solution to prevent tooth decay.

In 1986, the recombinant human growth hormone was the first plant-based biopharmaceutical protein produced in plants [19] . Then over two decades later, the FDA in May 2012 approved ELELYSO 1 (human recombinant taliglucerase alfa or glucocerebrosidase), an enzyme produced in genetically engineered carrot cells for treating type 1 Gaucher's disease (GD) by Protalix BioTherapeutics and its partner, Pfizer [20] . GD is a lysosomal storage disorder caused by a hereditary deficiency of the enzyme, glucocerebrosidase (GCD). GD is currently treated by enzyme replacement therapy using this recombinant GCD that is administered intravenously every 2 weeks [21] .

In addition to offering a versatile production platform for numerous plant-made proteins, plant viruses have been engineered to provide medical applications in other ways [22] . VLPs offer advantages over recombinant protein vaccines as they tend to elicit a higher immune response [23] . Virus nanoparticles have also been developed for the targeted delivery for disease treatment and diagnostic purposes. For example, CPMV represents an icosahedral nanoparticle with its capsid surface displaying 300 accessible lysine residues; each of these can be conjugated to various chemical moieties like fluorescent dyes/arrays, polyethylene glycol polymers and subcellular targeting molecules [24, 25] . The use of this technology includes 86 Engineering for viral resistance the construction of CPMV nanoparticles displaying gastrin-releasing peptide receptors that are overexpressed in human prostate cancers [26] . Another example, cowpea chlorotic mottle virus can stably assemble in vitro and package the RNA derived from sindbis virus, a mammalian virus. These hybrid cowpea chlorotic mottle virusbased VLPs were shown to protect against RNA degradation by cellular nucleases and were able to deliver and release their RNA contents within the cytoplasm of mammalian cells. Moreover, these hybrid VLPs with the fusion of subcellular targeting moieties could be directed toward distinct sites within the cell [27] and potentially applied as a medical targeted delivery tool. Plant viruses have also been engineered to act as adjuvants to elicit an immune response that is more potent and effective. The rodshaped papaya mosaic virus nanoparticles have been engineered to express an influenza epitope on their surface, and mice and ferrets immunized with these recombinant nanoparticles exhibited an increase in robust humoral response to influenza virus infection [28] .

There is growing evidence that plants are capable of making proteins with desired quality to address a range of human health-related issues. Plant production platforms for protein therapeutics and biologics, in particular the transient agroinfiltration approach, have demonstrated the ability to be used for broad research and development, as well as commercial needs. It has been extensively discussed that the transient agroinfiltration approach is the ideal platform for fast and scalable production in response to new outbreaks of highly infectious diseases and has been demonstrated under various programs. The success of Protalix Biotherapeutics in gaining FDA approval for the therapeutic enzyme, ELELYSO 1 for human use was a significant milestone for the plant molecular pharming field. More importantly, the primary benefits of plant-made protein therapeutics and biologics in terms of product safety and potential cost-effectiveness will further contribute to global public health in both developed and developing nations. 

Yao J, Weng YQ, Dickey A, Wang YJ: Plants as factories for human pharmaceuticals: applications and challenges. Int J Mol Sci 2015, 16:28549-28565. This paper illustrates the plant molecular farming or pharming concept in relation to human pharmaceutical applications. Several types of plantbased production platforms are described. The challenges such as planttype glycosylations, downstream bioprocesses and biosafety concerns are also discussed. Besides, some of the preclinical and clinical studies that have been enlisted in our review paper are elaborated here.

The production of recombinant pharmaceutical proteins in plants

Production of vaccines and therapeutic antibodies for veterinary applications in transgenic plants: an overview

Comparative evaluation of recombinant protein production in different biofactories: the green perspective

The integration of T-DNA into plant genome

Magnifection -a new platform for expressing recombinant vaccines in plants

Engineering the chloroplast genome for hyperexpression of human therapeutic proteins and vaccine antigens

A launch vector for the production of vaccine antigens in plants. Influenza Other Respir Viruses

Production of secretory IgA antibodies in plants

Production of antibodies in plants: status after twenty years

The expression of a nopaline synthase -human growth hormone chimaeric gene in transformed tobacco and sunflower callus tissue

First plant-made biologic approved

Plant-based oral delivery of b-glucocerebrosidase as an enzyme replacement therapy for Gaucher's disease

Plant virus expression vector development: new perspectives

Virus-like particles as a highly efficient vaccine platform: diversity of targets and production systems and advances in clinical development

Plant viral capsids as nanobuilding blocks: construction of arrays on solid supports

Cowpea mosaic virus nanoparticles target surface vimentin on cancer cells

Intravital imaging of human prostate cancer using viral nanoparticles targeted to gastrinreleasing peptide receptors

Reconstituted plant viral capsids can release genes to mammalian cells

Improvement of the trivalent inactivated flu vaccine using PapMV nanoparticles

A plantproduced protective antigen vaccine confers protection in rabbits against a lethal aerosolized challenge with Bacillus anthracis Ames spores. Hum Vaccines Immunother

Generation of protective immune response against anthrax by oral immunization with protective antigen plant-based vaccine

Novel vaccination approach for dengue infection based on recombinant immune complex universal platform

Expression of an immunogenic Ebola immune complex in Nicotiana benthamiana

Expression of an immunogenic LTB-based chimeric protein targeting Zaire ebolavirus epitopes from GP1 in plant cells

Brief background about Ebola virus is mentioned in this paper. Development of plant-based vaccine against Ebola virus is in fact scarcely reported till date. The authors have demonstrated the successful expression of the chimeric protein, LTB-EBOV in plant cells and its immunogenicity in BALB/c mice via subcutaneous and oral administrations

Freeze-drying of plant tissue containing HBV surface antigen for the oral vaccine against hepatitis B

Study of the immunogenicity of hepatitis B surface antigen synthesized in transgenic potato plants with increased biosafety

Immunogenic properties of a lettuce-derived C4 (V3)6 multiepitopic HIV protein

A plantderived multi-HIV antigen induces broad immune responses in orally immunized mice

Immunological characterization of plant-based HIV-1 Gag/Dgp41 virus-like particles

Transgenic tobacco expressed HPV16-L1 and LT-B combined immunization induces strong mucosal and systemic immune responses in mice

Immunization with an HPV-16 L1-based chimeric virus-like particle containing HPV-16 E6 and E7 epitopes elicits long-lasting prophylactic and therapeutic efficacy in an HPV-16 tumor mice model

A plant produced H1N1 trimeric hemagglutinin protects mice from a lethal influenza virus challenge

Immunogenicity of H1N1 influenza virus-like particles produced in Nicotiana benthamiana. Hum Vaccines Immunother

The authors have developed the recombinant hemagglutinin from the A/ California/04/09 strain of H1N1 influenza A virus in the form of enveloped VLPs (HAC-VLPs) in plants. HAC-VLPs resemble the influenza A virus by morphology

Expression of H3N2 nucleoprotein in maize seeds and immunogenicity in mice

Purification and immunogenicity of hemagglutinin from highly pathogenic avian influenza virus H5N1 expressed in Nicotiana benthamiana

Expression of rabies glycoprotein and ricin toxin B chain (RGP-RTB) fusion protein in tomato hairy roots: a step towards oral vaccination for rabies

Antigen production in plant to tackle infectious diseases flare up: the case of SARS

A non-glycosylated, plant-produced human monoclonal antibody against anthrax protective antigen protects mice and non-human primates from B. anthracis spore challenge. Hum Vaccines

Therapeutic intervention of Ebola virus infection in rhesus macaques with the MB-003 monoclonal antibody cocktail

Structural and functional characterization of an anti-West Nile virus monoclonal antibody and its single-chain variant produced in glycoengineered plants

Generation and analysis of novel plantderived antibody based therapeutic molecules against West Nile virus

A plant produced antigen elicits potent immune responses against West Nile virus in mice

Suppression of inhibitor formation against FVIII in a murine model of hemophilia A by oral delivery of antigens bioencapsulated in plant cells

Low cost industrial production of coagulation factor IX bioencapsulated in lettuce cells for oral tolerance induction in hemophilia B

Oral delivery of acid alpha glucosidase epitopes expressed in plant chloroplasts suppresses antibody formation in treatment of Pompe mice

The authors have demonstrated the successful expression of acid alpha glucosidase (GAA) in fusion with a transmucosal carrier, Cholera toxin B in chloroplasts. By using oral administration of GAA bioencapsulated in plant cells, an induction of oral tolerance and significant suppression of GAA-specific inhibitory antibody (which will jeopardize the enzyme replacement therapy) were evidenced in Pompe mice

Oral delivery of bioencapsulated exendin-4 expressed in chloroplasts lowers blood glucose level in mice and stimulates insulin secretion in beta-TC6 cells

The authors would like to thank Dr Stephen Streatfield (FhCMB) for editorial assistance.

The authors declare no conflict of interest.Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest