Luca Santi and Hugh S. Mason
April, 2006

In the 20 years since the approval of recombinant insulin produced in Escherichia coli, biopharmaceuticals have experienced persistent growth, and demand is expected to climb. Classes of biologicals, especially recombinant proteins as antibodies, subunit vaccines, enzymes, hormones, immunomodulatory molecules, receptor agonists, and microbicides, promise to deliver a new wave of therapies. All these molecules are complex polypeptides that must be synthesized by living organisms. Currently most are produced in mammalian cells, which, together with yeast, insect cells, and E. coli, are the most traditional biological factories.

Plants as bio-factories
Transgenic plants comprise a convenient alternative system that has extensively demonstrated great potential in studies conducted during the last two decades.1 The production of subunit vaccines in particular has been widely validated using different plant heterologous gene expression approaches. Numerous candidate vaccines were proven to confer some degree of protection in animal challenge studies against toxins or viral or bacterial pathogens, and to stimulate humoral and mucosal immune responses in human subjects.

Plants are able to express a large variety of proteins and to perform the post-translational modifications required for proper biological function. Plant systems are much less likely to harbor microbes that are pathogenic to animals than are mammalian cells. Lastly, and significantly, plants offer the possibility of an easy scale-up, especially when compared to the previously mentioned expression systems that rely on fermentation technology.

The demand for hundreds of kilograms of recombinant proteins per annum requires utilization of bioreactors with capacities of up to 20,000 liters, with extraordinary associated costs for cleaning, revalidation procedures, and preparation of complex culture media. On the other hand, scaling up transgenic plant production would only involve increasing the acreage in open fields or greenhouse space. Furthermore, the use of edible plant tissues for direct oral delivery would eliminate most of the costs for downstream processing and purification. There are three main methods for production of recombinant proteins in plants: stable transformation of the nuclear or chloroplast genomes, and viral transient infection.

Plant viral expression systems
The use of plant viral vectors offers several advantages.2 Recombinant protein expression can reach very high levels in a relatively short time, ranging from 3 to 14 days post-infection, depending on the system used. The small genome size of most plant viruses facilitates molecular engineering, allowing the facile generation of large numbers of different constructs that can be quickly tested. Fully functional and systemic infectious vectors are easily transmissible by mechanical inoculation, making large-scale infections feasible. The major limitation is that the acquired trait is not genetically transmissible and a new infection must be performed on every new plant. Also the environmental containment of the modified virus causes some concerns.

Several expression vectors have been developed using different types of plant viruses; the most common are based on single stranded positive RNA viruses like the tobacco mosaic virus (TMV). The first vectors developed, the so called "full virus" vectors, consisted of the addition of a heterologous open reading frame, encoding for the protein of interest to the viral genome, and driven by an extra subgenomic promoter. The next generation systems were "gene replacement" vectors in which a viral gene, usually encoding the coat protein, was substituted with the gene of interest. The extreme evolution of this concept led to deconstructed viral vectors missing several components of the original virus and usually delivered to the plant by independent constructs.3

The etiologic agent of plague is the Gram-negative bacteria Yersinia pestis. It is generally accepted that in the course of human history plague has been the cause of three pandemic infections responsible for hundreds of millions of deaths worldwide. Nowadays plague is still endemic in Africa, Asia, regions of the former Soviet Union, and the Americas, where it persists mostly in rodent populations.

There are two major forms of the disease: bubonic and pneumonic. In bubonic plague, Y. pestis is transmitted to humans via the bite of infected fleas, often resulting in the formation of "bubos," which are enlarged lymph nodes typically localized in the axillary and femoral areas. Pneumonic plague occurs when the bacteria infect the lungs; it is considered fatal (mortality rates of almost 100%) and can be transmitted by aerosol from infected to naïve hosts.4 For these reasons pneumonic plague is of particular concern in light of biological warfare, and in fact, during the cold war the former Soviet Union produced enough aerosolized Y. pestis for use as a weapon. The United States, before dismantling its bio-weapon program in the late 1960s, also tested the deadly potential of aerosolized bacteria.

Although antibiotics for plague are available, their effectiveness relies on a prompt diagnosis of the disease, and moreover, strains with acquired resistance to antibiotics have been isolated. Killed whole cells (KWC) vaccines and live attenuated vaccines have been extensively investigated and used in different countries. In the U.S., until 1999, a KWC plague vaccine was used especially for subjects at risk. It is no longer in production due to the poor protection conferred against the pneumonic form, as well as the high incidence of side effects. A live attenuated vaccine was tested in the Soviet Union, but it also showed severe local and systemic side effects. Hence the development of a safe and effective vaccine was needed.

The protective efficacy of subunit vaccines against plague has been demonstrated for many years. Two antigens, F1 and V, and a fusion of both, F1-V, have been selected. The F1 antigen is highly expressed by Y. pestis and is exported to form an extra-cellular capsule that surrounds the bacteria. The V antigen is a secreted protein involved in the pathogenic process. In a collaborative effort that included the Biodesign Institute at Arizona State University, Icon Genetics (Halle, Germany), and the U.S. Army Medical Research Institute for Infectious Disease, we demonstrated that sequence-optimized genes and a robust transient expression system generated high levels of expression of all three antigens in leaves of N. benthamiana. The plant-derived antigens, administered subcutaneously in guinea pigs, generated systemic immune responses and provided protection against an aerosol challenge of virulent Y. pestis.5

Plant-derived subunit vaccine against Y. pestis
The first step was the creation of a synthetic version of the open reading frames of the antigens. The coding sequences were optimized for expression in dicotyledonous plants using preferred codons and eliminating spurious mRNA destabilizing signals, potential methylation sites, 5 intron splicing sites, and putative plant polyadenylation signals. The vector system used belongs to the last generation of deconstructed viral vectors. It was developed by Icon Genetics, recently acquired by Bayer Innovation GmbH; it is based on TMV and has been extensively modified to increase performance.3 The vectors are delivered to the plant cell nucleus by Agrobacterium tumefaciens (agroinfection) lines carrying two separated proviral cDNA modules: a 5 module containing the viral replicases and movement protein, and a 3 module harboring the gene of interest driven by the coat protein subgenomic promoter. Specific phage-derived recombination sequences are located on each module.

The co-delivery of the two proviral modules together with a third Agrobacterium line carrying a construct that directs constitutive expression of the phage PhiC31 integrase leads to the assembly of the complete viral vector in the plant cell nucleus. At this point the vector is transcribed, processed, and exported into the cytosol, where as a positive single stranded viral RNA molecule, it undergoes amplification and translation. This particular system has a deletion of the TMV coat protein, thus limiting its ability to spread systemically throughout the plant. Without the coat protein, the virus is still able to move from cell to cell but loses its ability for systemic infection, allowing stringent containment of the virus.

Distinct localization signals are located on different 5 modules. The pairwise combination of these modules with the 3 module allows the rapid generation of proteins that are targeted to different cellular compartments in order to evaluate the best localization for each target protein. In this specific case, the different 3 modules, each one containing one of the plant-optimized coding sequences for F1, V, and F1-V, were coupled with 5 modules for cytosolic, chloroplastic, and apoplastic accumulation. In all cases, the cytosolic accumulation gave the best results. F1 and V were expressed at levels of 2 mg/g of leaf fresh weight and the fusion F1-V at 1 mg/g. These amounts are at least an order of magnitude greater than any antigen expressed in stably nuclear-transformed plants. After purification, the proteins were assayed on SDS/PAGE with Coomassie staining. Antigenicity was evaluated using both ELISA and Western blot analyses.

For vaccine testing in animals, 25 μg of plant-derived antigens, mixed with alum as adjuvant, were administered subcutaneously. Groups of eight guinea pigs were dosed at days 0 (prime), 30 (boost 1) and 60 (boost 2). Serum analysis revealed that all proteins were highly immunogenic. The antibody titers specific for V in particular increased significantly just after the priming dose. Four weeks after the last dose was administered, the animals were challenged with an aerosol dose considered essentially 100% lethal to unvaccinated controls, and in fact, all sham-immunized mice were dead within 6 days after exposure.

Conversely, all antigen-vaccinated groups showed significant rates of survival at 21 days post-exposure. V-vaccinated animals showed the highest survival rate (six of eight), followed by F1-V (five of eight), and F1 (three of eight). Moreover, vaccinated animal mortality was significantly delayed beyond day 6. The majority of the animal challenge experiments reported in literature are carried out by injection of lethal doses of Y. pestis. The aerosol exposure used in our study offers a more reliable way to test protection for the pneumonic form of the disease. In addition, to our knowledge this is the first time that F1, V, and F1-V have been individually compared for protection in the same animal study. In conclusion, we have demonstrated that a rapid and robust plant based expression system could be used to produce an effective vaccine against plague.


1. Mason HS, Chikwamba R, Santi L, et al. (2004) Transgenic plants for mucosal vaccines. In Mucosal Immunity 3rd Ed, Mestecky J, Bienenstock J, Lamm ME, Mayer L, McGhee JR & Strober W (eds.), 1053-1060. Elsevier: San Diego, CA.

2. Canizares MC, Nicholson,L, Lomonossoff GP. (2005) Use of viral vectors for vaccine production in plants. Immunology and Cell Biology. 83(3), 263-270

3. Gleba Y, Klimyuk V, Marillonnet S. (2005) Magnifection-a new platform for expressing recombinant vaccines in plants. Vaccine 23(17-18), 2042-8

4. Plague manual: epidemiology, distribution surveillance and control. WHO Communicable Diseases Surveillance and Control. Available:

5. Santi L, Giritch A, Roy CJ, et al. (2006) Protection conferred by recombinant Yersinia pestis antigens produced by a rapid and highly scalable plant expression system. PNAS 103(4), 861-866

Hugh Mason, PhD
Associate Professor

Luca Santi, PhD
Research Assistant Professor

Center for Infectious Disease and Vaccinology
Biodesign Institute at Arizona State University