Many studies conducted over the last two decades have demonstrated that transgenic plants are a convenient antigen expression system, and that plant tissue can be used directly as a delivery system for orally administered subunit vaccines [25
]. Prior studies focused on oral vaccine delivery of unprocessed (raw potato [6
]) and minimally processed (freeze-dried [7
]) plant material. As was previously discussed [26
], the use of unpurified plant-delivered antigens as vaccines may meet regulatory approval in animal health markets, but will likely face daunting challenges for commercialization as human vaccines, due to regulatory factors including quality assurance for antigen quantity and uniformity in perishable or freeze-dried plant tissues. The current study was designed to explore the feasibility of developing cost-effective antigen purification from plant tissue as a step toward formulation of a standardized subunit vaccine. We focused our efforts on: 1) finding a plant-based expression system that drives high level antigen accumulation to increase cost-effectiveness of the purification process, and 2) validating the oral immunogenic properties of processed plant-derived antigen.
We previously showed that rNV accumulates in transgenic tobacco, tomato and potato [6
], and that the VLPs resemble those produced in insect cells driven by a baculovirus expression system [4
]. Furthermore, the oral immunogenicity of plant made rNV were similar to that obtained from insect cells in both preclinical and human clinical trials [6
]. However, we noted that a major limitation of the system was a modest level of antigen accumulation in the transgenic plant tissue, which required that volunteers eat a very large bolus of plant sample [18
]. Subsequent studies by our group (unpublished) have failed to identify a strategy to overcome constraints in NVCP accumulation in stably nuclear transgenic plants. We therefore evaluated transient approaches to NVCP expression, and we here describe our success in adopting a plant viral vector system to achieve rNV accumulation at a level that is suitable for commercial development of a subunit vaccine.
In 2004 a novel new strategy was introduced in which the viral genome was “deconstructed” and modified to utilize the bacterium Agrobacterium tumefaciens
for delivery of DNA vectors into fully expanded plant leaves [20
]. Vectors delivered by Agrobacterium
into leaf cells trigger replication and transient expression of RNA. For small scale production, the bacteria can be infiltrated into leaves using a syringe; while for large scale production, whole plants can be vacuum infiltrated [22
]. This “magnICON” system produced very high levels of protein expression [16
] compared with stably transgenic lines and other viral transient systems that are often hampered by post-transcriptional RNAi-mediated gene silencing. It is likely that the robust performance of magnICON vectors is at least in part due to the gene silencing suppressor function of one of the TMV replicase subunits [28
In plant tissues, the dominant soluble protein is Rubisco [29
]; it comprises up to 50% of the total leaf protein and must, therefore, be removed if a target protein is to be utilized for demanding vaccine applications. We employed protocols that can be utilized in cost-effective plant extract manipulation, asking how wine and food industry technology can be applied to antigen purification. Our protocol is a low pH extraction of leaves followed by centrifugation/filtration, similar to cell disruption in a wine press followed by filtration. As shown in , we removed all easily detectable levels of Rubisco from our sample, and enriched the NVCP to produce an “antigen concentrate”.
There is extensive prior evidence that VLPs can assemble in plant cells [30
]. We therefore asked if rNV were formed when the antigen was expressed using the magnICON vector and purified; by both EM and sucrose gradient sedimentation we gained affirmative results (, , and ). The results discussed above allow us to reach a conclusion – very “low technology” procedures (and therefore cost-effective) can be used to concentrate and partially purify rNV from leaves after use of the magnICON expression system. The expression level (0.86 mg antigen/g leaf FW) is ~30-fold greater than we have previously achieved in nuclear stably transformed N. tabacum
], ~35-fold higher than transient expression in N. benthamiana
leaf with a nonreplicating vector (Mason et al., unpublished data), and ~60-fold greater than in stably transformed tomato fruit [17
It was essential to validate the mucosal immunogenicity of the partially purified rNV. We observed both humoral and mucosal responses, which is consistent with previous data using both insect cell-derived and plant-derived rNV [6
]. All animals responded to antigen presentation with mucosal antibody responses (enteric and vaginal IgA), and CT adjuvant enhanced these responses. We conclude that the magnICON expression system coupled with a “low technology” purification protocol will yield immunogenically active rNV.
There is no commercial vaccine to prevent norovirus infections. Introduction of a vaccine must take into account the variability of capsid protein composition in different viral serotypes [31
]. Additional research is needed: 1) to determine the extent of cross-protection among VLPs from different serotypes, 2) to derive a correlate of protection to define a successful immunization regime, and 3) to establish the most effective formulation and delivery strategy (including adjuvants). The data in this paper have validated a robust plant-based expression technology that is highly cost-effective and rapid (2-3 weeks after vector construction) for the production of candidate subunit vaccines for noroviruses. Ongoing research by our group is establishing protocols for Good Manufacturing Practices for production of rNV for human testing to meet stringent federal guidelines. We anticipate that plant-based antigen production technology will speed research on norovirus vaccine candidates and provide the capacity to allow cost efficiencies that will make the manufacture of a multivalent subunit vaccine a feasible venture.