Recently two healthcare workers infected with Ebola Virus (EBOV) were treated with a “mystery serum” which turned out to be a cocktail of monoclonal antibodies (Mabs) to EBOV, similar to receiving anti-venom for a snake bite. [see Mystery Serum Links below] What is unique about these Mabs is that they were produced in a plant, specifically, Nicotiana benthamiana, a relative of the tobacco plant. Antibodies produced in plants have been nicknamed “plantibodies”.
Why Make Proteins In Plants?
The ability to make mammalian, viral, and other proteins in plants has been around for about 25 years now. Early methods of expressing foreign proteins in plants were highly inefficient, but new methods have solved many of the early issues (ref). and produce high yields. Yields of 300mg/kg leaf fresh weight and 1-2 mg/g fresh weight (up to 10% of total soluble protein) have been reported (4,5). Some of the advantages of plant-based expression systems are (5):
- Relatively inexpensive, requiring no special culture equipment, bioreactors, media, components, or sterile environment.
- Fast: can grow high yields of the desired protein within a few days to a weekThe material retains biological activity and immunogenicity.
- Multi-vector systems consisting of replication-defective constructs can be used to minimize the chance of spurious replication.
- Unique storage possibilities in that proteins can be stored in the form of seeds or in freeze-dried leaves.
In addition to EBOV antigens, proteins of Foot & Mouth Disease Virus, HIV-1, malaria, rotovirus, Influenza virus, HPV, Yersinia pestis, Dengue Virus, and HbsAg, and monoclonal antibodies all have been produced in plants (5).
How Do They Do It?
There are two key genetic components to the technology for expressing exogenous proteins in plants. The first is a plant expression vector derived from a plant virus. The two most popular types of vectors are based on Tobacco Mosaic Virus (TMV) or Potato Virus X (PVX) (1-7, Wikipedia).
The second is the bacterium Agrobacterium tumefaciens (Agrobacterium), that infects certain plants and causes tumors. Agrobacterium cultures are used as the gene transfer and delivery system to induce protein expression in the plants.
Here’s how it works:
- The gene of interest is cloned into a plant expression vector plasmid.
- The plasmid is introduced into Agrobacterium by transformation/conjugation.
- The plant is infected with Agrobacterium by a process called agroinfiltration.
- Agrobacterium then transfers the vector and gene of interest into the plant where it is expressed at high copy number, producing large quantities of the protein of interest.
SIDE BAR: Transient Expression by Agroinfiltration (Wikipedia)
Agroinfiltration is a technique used to induce transient expression of genes in plants. A suspension of Agrobacterium tumefaciens is mechanically infused into the leaves of plants, where it transfers the desired gene to plant cells. The benefits of agroinfiltration compared to other plant transformation methods are speed and convenience. Once inside the leaf the Agrobacterium transfers the gene of interest in high copy numbers into the plant cells. The gene is then transiently expressed.
Plantibodies: Plant-Generated Monoclonal Antibodies
Monoclonal antibodies (Mab), such as the anti-EBOV antibodies, can also be produced in plants by agroinfiltration. Expressing complete antibody molecules is trickier because you need to express two different polypeptide chains and get them to assemble into a molecule consisting of two heavy chains (HC) and two light chains (LC), and retain antigen-binding specificity and capability (6,7).
Step 1: Generate monoclonal antibodies by immunizing mice and making hybridomas producing the Mab by the standard methods for making Mabs.
Step 2: “Humanize” the antibody by swapping gene and protein sequences common to mouse antibodies with those found in human antibodies, to prevent or decrease the chance of the recipient producing an immune reaction against the MAbs.
Step 3: Clone the Mab genes into plant expression vectors such as TMV- or PVX-based vectors; infect plants and let them grow (for about a week). Seems to work best if the HC and LC are expressed from non-competing vector systems such as HC in a TMV-based vector and LC in a PVX-based vector.
Step 4: Harvest the plant material, extract and purify the MAbs.
Although the production of proteins and antibodies in plants has not received much attention to date, the successful use of the anti-EBOV cocktail to treat people infected by EBOV will most likely propel this technology into the limelight in the near future.
Mystery Serum Links
References—Want To Read More?
- Gelvin SB. 2003. Agrobacterium-mediated plant transformation: the biology behind the “gene-jockeying” tool. Microbiol Mol Biol Rev. 67(1):16-37. ⊂⊃
- Marillonnet S, Giritch A, Gils M, Kandzia R, Klimyuk V, Gleba Y. 2004. In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proc Natl Acad Sci U S A. 101(18): 6852-6857. ⊂⊃
- Marillonnet S, Thoeringer C, Kandzia R, Klimyuk V, Gleba Y. 2005. Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants. Nat Biotechnol. 23(6): 718-723. ⊂⊃
- Giritch A, Marillonnet S, Engler C, van Eldik G, Botterman J, Klimyuk V, and Gleba Y. 2006. Rapid high-yield expression of full-size IgG antibodies in plants coinfected with non-competing viral vectors. Proc Natl Acad Sci U S A. 103(40): 14701–14706. ⊂⊃
- Hefferon KL. 2012. Plant virus expression vectors set the stage as production platforms for biopharmaceutical proteins. Virology 433: 1–6. ⊂⊃
- Olinger GG, Pettitt J, Kim D, Working C, Bohorov O, Bratcher B, Hiatt E, Hume SD, Ashley K. Johnson AK, Morton J, Pauly M, Whaley KJ, Lear CM, Biggins JE, Scully C, Hensley L, and Zeitlin L. 2012. Delayed treatment of Ebola virus infection with plant-derived monoclonal antibodies provides protection in rhesus macaques. Proc Natl Acad Sci U S A. 109(44): 18030-18035. ⊂⊃
- Pettitt J, Zeitlin L, Kim DH, Working C, Johnson JC, Bohorov O, Bratcher B, Hiatt E, Hume SD, Johnson AK, Morton J, Pauly MH, Whaley KJ, Ingram MF, Zovanyi A, Heinrich M, Piper A, Zelko J, and Olinger GG. 2013. Therapeutic Intervention of Ebola Virus Infection in Rhesus Macaques with the MB-003 Monoclonal Antibody Cocktail. Sci. Transl. Med. 5(199):199ra113, pp.1-6. ⊂⊃
Companies Involved In Plantibody Research