CPMV inactivation by UV light was previously reported and only required a dose of 2.5 J.cm-2 to prevent infection.This difference highlights one of the hurdles to standardizing UV inactivation across systems; we employed a UVP cross linker delivering 7 m watts.cm-2 , and samples were prepared using a 1 mg mL-1 solution placed 20 cm away from the UV source. Rae et al relied on a Stratalinker 1800 UV Cross linker delivering 3 mWatts cm-2 , and their samples were irradiated 15 cm from the UV source at a concentration of 2 mg mL-1 . In addition, the volume to surface area ratio of the prepared samples could have also influenced the results. Single-stranded RNA containing mammalian viruses have also been inactivated to produce vaccines. Among them hepatitis A,HIV,and influenza reported inactivation doses of 0.3, 1, and 1 J cm-2 , respectively. The need for use of higher dosage to inactivate the plant virus may reflect the higher stability of the plant viral nanoparticle and its requirement to remain stable in the environment when exposed to UV light. As previously stated, βPL and formalin are commonly used to produce inactivated mammalian virus vaccines.The dose required to inactivate CPMV using βPL and formalin is very similar to those reported in the literature for mammalian viruses. For example, the eastern equine encephalitis and poliomyelititis type II, equine herpesvirus type I, HIV, and the influenza virus have been successfully inactivated with 5-60 mM βPL; similarly, Hepatitis A, HIV , influenza A virus, Japanese encephalitis virus, and rabies were successfully inactivated using 5-120 mM of formalin.
CPMV viral capsids contain 300 solvent-exposed lysine residues that can been chemically modified to impart new functionalities through isothiocyanate and N-hydroxysuccinimide – ester coupling. Examples include the conjugation of targeting ligands , therapeutics , and fluorescent dyes . To verify that CPMV retained its chemical reactivity, CPMV, UV-CPMV, βPL-CPMV, and Form-CPMV were incubated with 5 molar excess of sulfo-Cyanine 5-NHS per coat protein overnight,dutch buckets system followed by centrifugation and desalting column techniques to remove the excess dyes. The level of Cy5 labeling of the CPMVCy5, UV-CPMVCy5, βPL-CPMVCy5, and Form-CPMVCy5 formulation was quantified using UV-visible spectroscopy; all samples produced dyes per particle values similar to CPMV . SDS-PAGE analysis further confirmed the chemical conjugation of C55 to both the L and S coat protein subunits . Thus, UV, βPL and formalin inactivated CPMV particles retain similar surface chemical reactivity. To determine whether inactivated CPMV retained its strong immunogenicity, we first assayed immunogenicity using the RAW-BlueTM assay . RAW-BlueTM cells are derived from the murine RAW264.7 macrophages and express numerous pattern-recognition receptors such as toll-like receptors, nucleotide-binding oligomerization domain like receptors, retinoic acid-inducible gene-I like receptors, and C-type lectin receptors. Upon activation of the receptors, RAW-BlueTM cells secrete alkaline phosphatase , which serves as the read-out. While this assay does not give any information about which innate pathways the CPMV particle activate, the assay provides a quantitative read out to compare whether all formulations activate innate signaling pathways to a similar or varying degree. 5 μg of CPMV, UV-CPMV, βPL-CPMV, and Form-CPMV were incubated with RAW-BlueTM cells for 18 h before the cell’s SEAP secretion levels were quantified . A positive and a negative control were included.
CPMV, UV-CPMV, and Form-CPMV showed significant activation in the RAW-BlueTM assay resulting in the highest and comparable signaling: while CPMV resulted in a 1.9-fold increase of SEAP level, both UV-CPMV and Form-CPMV resulted in 2.2-fold increase of SEAP levels. Yet, the difference was not statistically significant. Of note is that βPLCPMV appeared to be less effective in activating RAW-BlueTM and SEAP levels were increased only by 1.3-fold – therefore CPMV, UV-CPMV and Form-CPMV were almost 2-fold more effective . These differences in SEAP secretion levels may be attributed to differences in rate of cellular internalization; flow cytometry indicate that all particle formulations showed similar internalization rates ; 100% internalization was achieved within 8 h of incubation – however βPL-CPMV were internalized to a lesser degree . We hypothesize that the diminished immunogenicity of βPL treated CPMV can be explained by a combination of reduced cell uptake and more severe RNA damage including RNA breakage . Based on the aforementioned results, UV treatment remains the cheapest, fastest, and safest treatment modality and could be easily integrated in the CPMV production process. While UV treatment led to particle aggregation at the required inactivation dose of 7.5 J cm-2 , the resulting UV-CPMV remained strongly immunogenic. In contrast, βPL treatment resulted in monodisperse particles but with severely damaged RNA which resulted in reduced immunogenicity as per RAWBlueTM assay. Formalin could also effectively inactivate CPMV while maintaining its immunogenicity, but formalin treatment requires a long incubation step followed by purification steps that lower the final recovery of CPMV to 40-60 % of the original stock.
In the end, selecting the most suitable inactivation modality will demand further in vivo testing; future work should focus on whether in situ vaccination with inactivated CPMV particles can still increase the tumor burden using a mice model. In a pilot study, we injected C57BL/6J mice transdermally with the murine B16F10 melanoma cell line . Treatment started on day 7 post tumor challenge, and was given 4 times on day 7, 12, 17, and 22. Tumor volumes was recorded every other day, and mice were sacrificed once the tumor volume reached 1000 mm3 . The PBS control group reached the terminal tumor volume within 20 days. On the other hand, native CPMV and inactivated CPMV formulations demonstrated slower tumor growth and tumor shrinkage. In addition to the previously collected data, an additional animal study with a larger animal group is currently underway. We disagree with Leifeld fundamentally on the purpose of our agricultural system—our goal should be to produce nutritious, affordable and accessible food in a socially and environmentally sustainable manner and not just ‘keeping prices low’. So-called low-cost food produced by industrialized, conventional agriculture comes at a great price to our soils, water, biodiversity, atmosphere and worker health. In turn, low calories also contribute to a rise in obesity, which is associated with increased risks of diabetes and heart diseases, and paradoxically is often accompanied by various types of malnutrition. Thus, the price of food does not reflect these costs. Arguably, the people that pay the highest price for this ‘low-cost food’ are the farm workers who sacrifice their health, living conditions and sometimes even lives, although they may not be able to afford food for their own tables. Accessible, affordable,dutch buckets nutritious food is not synonymous with ‘low-cost food’, and low-income families should not have to choose between whether to eat, versus accepting harms to their own families, as well as the environmental consequences and exploitation of farm workers. Needing food to be ‘low-cost’ to feed people results from low wages and the inequitable distribution of wealth, issues that need to be tackled in order to attain a sustainable food system. Further, the negative consequences of large-scale, industrialized conventional agriculture undermine the earth’s capacity to continue producing food.In deciding the future trajectory of agriculture, we must balance the known harm caused by our current, high-input agriculture system with the potential costs and benefits associated with a transition to alternatives. Yield is only one factor within a set of complex socio-economic forces that determine what management practices growers adopt, how much land is dedicated to agricultural production, and how much food is available and accessible for the hungry. Yield, however, has continued to be a focus in the debate surrounding the adoption of alternative, less environmentally and socially damaging agriculture practices. A focus solely on increasing yields will not solve the problem of world hunger; this is clearly the case, because two billion people today are either chronically hungry or malnourished despite the fact that we produce more than enough food to feed the current population. Instead, all aspects of a farming system, including ecosystem services, production and livelihoods, must be evaluated in considering the future of agriculture. Leifeld argues for larger-scale studies to examine conventional and organic agriculture, but simply increasing the scale does not equate to a systems approach, although it may be an important component. We must instead invest in research aimed at understanding the full social–ecological consequences of transitioning or not transitioning to more sustainable food systems. Responding to Leifeld’s four specific points, we note that Leifeld, in focusing on how consideration of large scales might reveal negative consequences of transitioning to alternative agriculture practices, overlooks the potential positive implications of this transition at large scales. 1a.
Leifeld states that broad-scale adoption of organic practices could result in decreased yields in organic system because of reduced nitrogen deposition from conventional farms. This is an interesting hypothetical; however, there is absolutely no evidence in the published literature. Further, it is difficult to argue that hypothetical yield decreases in organic farms caused by the loss of nutrient spillover from conventional systems could offset the well-established negative impacts of nitrogen runoff, which include dead zones in the ocean and soil acidification.There is, however, growing evidence from landscape-scale studies that greater proportions of land devoted to organic and diversified techniques enhance ecosystem services such as pest control and pollination on farms. Across hundreds of cereal fields and nine regions in Europe, the potential for pest control was positively associated with the proportion of the landscape using organic and diversified farming techniques and negatively related to the amount of pesticide application. Similarly, in wheat fields across three regions in Germany, increasing the proportion of organic fields in the landscape from 5% to 20% more than doubled the richness and abundance of pollinators, on both organic and conventional farms. Increasing the use of diversified and organic agriculture in a landscape thus has the potential to improve yields, both through reducing pest damage and increasing pollination services . This would also contribute to the profitability of these systems by reducing the costs of inputs such as pesticides or managed honeybees. Expanding organic and diversified practices may also enhance food security by reducing reliance on fossil fuels owing to higher energy efficiency and managed honeybees through promoting wild pollinator populations. At the same time, organic soils also have higher water storage and infiltration, leading to higher resilience to severe weather conditions than in conventional systems. The next step is to understand when the enhanced provision of ecosystem services in diversified systems translates to improvements in human health and livelihoods. 1b. As landscapes transition to organic, it will also be interesting to study how the supply chain for supplemental external inputs evolves. Because organic agriculture is currently only a small proportion of the land in production, it relies on inputs like manure from conventional systems, but as the landscape changes, so will the source of inputs. Alternative, under exploited sources of nutrient inputs include municipal composting, and livestock integration may help eliminate the need for importing manure . 2. Leifeld points out that we do not know whether pest control services promoted by diversification practices will be sufficient for stable production at a large scale. We also rarely study the effects of pesticides at a landscape scale. We agree with Leifeld that integrated pest management where organic or synthetic pesticides are used only as a last resort, is a key component of sustainable agricultural systems. Only in organic systems, however, is IPM encoded into the best management practices. In conventional systems, no such guidelines exist. IPM is mentioned in the FAO’s International Code of Conduct on the Distribution and Use of Pesticides, but this is simply a recommendation with a purely voluntary agreement and only 15% of countries have signed onto it. In fact, worldwide the use of IPM is diminishing as systemic pesticides, such as neonicotinoids, are increasingly applied prophylactically as seed treatments ,protecting against pest pressures that may never materialize. Pesticide use is associated with extreme negative impacts on human health and wildlife. Simplified landscapes and high pesticide use are strongly associated with increased pest pressure, not pest control. Instead, we need to promote pest control through diversification, using methods of conservation biological control. Leifeld argues that alternative forms of agriculture such as organic should not be considered more sustainable because they may require more land for production.