Viruses are comprised of several structural proteins which make up the outer shell of the virus particle. Genetic material (either RNA or DNA) is contained within the core of these virus particles. When virus particles are produced without genetic material in their core they are unable to replicate and are referred to as virus like particles (VLP). These VLP’s still retain the ability to elicit an immune response as they maintain the critical structural proteins and have been shown to be of use in biomedical applications. VLPs have, for example been used as vaccines (such as Engerix®, for hepatitis B virus and Cervarix®, for papillomavirus), and have several advantages over conventional attenuated viral vaccines since there is no risk of them becoming infectious genetic material.
The development and modification of the plant derived virus Cowpea mosaic virus (CPMV) has provided a simple and effective route into the use of VLP’s in medical applications. CPMV derived VLP’s offer high degrees of stability when formed, deliver good yields in their preparation and can be structurally modified. CPMV VLPs have been used, for example to express parts of other viruses or proteins to elicit an immune response1, used as drug carriers which provide a protective shell for the delivery of drugs and chemicals2 and modified with dyes for bio-imaging applications3.
The challenges of conventional purification techniques
CPMV VLPs can be produced by either natural infection or by Agrobacterium-mediated transient expression. However, the small-scale preparation of these VLP’s using plants has several challenges when increased quantities of material are required for further development studies. For example, the use of ultracentrifugation is a common technique used in the laboratory to aid separation of impurities, but in a commercial process this is labour intensive, not easily scaleable and presents challenges of cleaning equipment for the production of pharmaceutical products.
Leaf Expression Systems’ solution
CPMV VLPs can be produced in plants through transient co-expression of the VP60 coat protein precursor and a 24K proteinase4. This is performed by infiltrating Agrobacterium into Nicotiana benthamiana leaves, resulting in the production of empty VLPs. This method is applied on a large scale at Leaf Expression Systems, using our unique industrial scale vacuum infiltration equipment.
To address the challenges of ultracentrifugation, Leaf Expression Systems has developed a purification procedure which allows for the scaleable production of VLPs using chromatography to replace ultracentrifugation. The separation of VLPs by standard chromatography media is challenging, due to the size of the VLPs. We have successfully combined media with novel bead technology and monolithic columns, which allows for the efficient purification of large proteins, such as VLPs. Using these technologies, we can purify CPMV VLPs to a protein purity of >95% with yields of around 100mg/kg of infiltrated leaf material. The ability to process infiltrated leaf material resulting in the levels of purity and yield demonstrated, makes plant-based expression a cost effective option in comparison to traditional production methods.
1. Usha R, Rohll JB, Spall VE, Shanks M, Maule AJ, Johnson JE, Lomonossoff GP. Expression of an animal virus antigenic site on the surface of a plant virus particle. Virology. 197(1):366-74 (1993)
2. Czapar AE, Steinmetz NF. Plant viruses and bacteriophages for drug delivery in medicine and biotechnology. Curr Opin Chem Biol. 38:108-116 (2017)
3. Leong HS, Steinmetz NF, Ablack A, Destito G, Zijlstra A, Stuhlmann H,Manchester M, Lewis JD. Intravital imaging of embryonic and tumor neovasculature using viral nanoparticles. Nat Protoc. 5(8):1406-17 (2010)
4. Montague NP, Thuenemann EC, Saxena P, Saunders K, Lenzi P, Lomonossoff GP. Recent advances of cowpea mosaic virus-based particle technology. Hum Vaccin. 7(3):383-90 (2011).
Figure: Negative stained TEM of CPMV VLPs.