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BY DANIEL NORERO
\nCornell Alliance For Alliance<\/p>\n
While large companies and public sector consortiums in the United States, Canada, China and Europe are running at full speed to develop a vaccine grown in genetically modified (GM) tobacco plants, a research group at a Mexican university is working toward the same objective, but with a different and innovative strategy. They are using bioinformatics and computational genetic engineering to identify candidate antigens for a vaccine that can be expressed in tomato plants. Eating the fruit from these plants would then confer immunity against COVID-19.<\/em><\/p>\n At the time I write these lines,\u00a0there are already<\/a>\u00a0more than 3.6 million people reportedly infected by the COVID19 pandemic and some 252,000 deaths globally. In the US, which has the world\u2019s highest rate of infection, COVID-19 deaths\u00a0have surpassed<\/a>\u00a0deaths from cancer, coronary heart disease and even influenza\/pneumonia in just the few \u00a0months since the novel coronavirus arrived.<\/p>\n This critical situation has led the entire world to embark on a real race to develop a vaccine that immunizes the population against this new strain of coronavirus, which\u00a0apparently emerged<\/a>\u00a0in the autumn of 2019 in China. So far,\u00a0more than 100 vaccines<\/a>\u00a0are being\u00a0investigated for COVID-19<\/a>\u00a0by universities, public research centers and especially private companies. Some are already under clinical trial.<\/p>\n The\u00a0approaches<\/a>\u00a0used for their production don\u2019t differ much from the ones classically used in vaccines, where the antigens \u2014 a compound of the pathogen used to generate immunity in the patient \u2014 can be the inactivated virus, as well as the genetic material or a virus protein, which is grown on a large scale in chicken eggs, mammalian\/insect cell tissue or genetically modified microorganisms.<\/p>\n A lesser-known approach to produce antigens and vaccines on a large scale is the use of plants as biofactories. The plants are genetically modified (Figure 1) to produce, for example, virus-like-particles (VLPs<\/a>), which are structural proteins of the virus, or \u201cmulti-epitope\u201d proteins, where different sequences of an antigen allow us to generate an immunizing and protective response in humans.<\/p>\n The most widely used plant is\u00a0Nicotiana benthamiana<\/em>, a close relative of tobacco, due to its biomass, easy laboratory management and rapid growth. But scientists have also worked with other crops, such as lettuce, carrots, potatoes, rice, tomatoes and corn, among others.<\/p>\n At the beginning of 2020,\u00a097 experimental vaccines<\/a>\u00a0had been obtained with this methodology, including plant-grown antigens for HIV, polio, hepatitis B, rabies, HPV, cholera and tuberculosis, among other pathogens. Work even has been carried out on the cultivation of compounds against cancer and autoimmune diseases.<\/p>\n Some of the plant-based vaccines that have made it to advanced clinical trials include a\u00a0flu vaccine<\/a>\u00a0developed by Medicago, a Fraunhofer\u00a0malaria vaccine<\/a>\u00a0and\u00a0ZMapp<\/a>, a three-monoclonal antibody serum developed by Kentucky Bioprocessing, which has already been used with patients in outbreaks of Ebola from 2014-2015 and 2018-2019 in Africa. All these vaccines were obtained through cultivation of GM tobacco.<\/p>\n Currently, plant-based drugs are already a reality and at least one has entered the market: taliglucerase alfa, an\u00a0enzyme grown in GM carrots<\/a>\u00a0and obtained in bioreactors, which is prescribed as replacement therapy for Gaucher disease.<\/p>\n The advantages of vaccines cultivated in plants include the facilitation of their transport and storage without the need for a cold chain, which lowers costs. In addition, there is no need to worry about contamination of toxins and pathogens for humans, a risk that can occur in the production of vaccines in microorganisms or mammalian cultures.<\/p>\n Within the COVID-19 vaccine race, the strategy of plant cultivation \u2014 also know as biopharming or molecular farming \u2014 hasn\u2019t been left out. Two companies already mentioned are working on COVID-19 plant-based antigens by expressing VLPs in GM tobacco. One of them is Medicago, whose CEO claimed the Canadian company would be able to manufacture \u201c10 million doses per month<\/a>\u201d if its innovative production method and clinical trials obtain US Food and Drug Administration (FDA) approval. On the other hand, the American company Kentucky Bioprocessing is using a fast-growing GM tobacco of its own and\u00a0publicly stated<\/a>\u00a0that it is already conducting preclinical tests and possesses the ability to manufacture up to three million doses per week.<\/p>\n The third private sector research group is an alliance between the US company iBio and China\u2019s Beijing CC-Pharming, which are\u00a0combining the cultivation<\/a>\u00a0of VLPs from COVID-19 and a lichenase carrier immunostimulatory adjuvant in GM tobacco.<\/p>\n Meanwhile, the public sector is not far behind. The University of California, San Diego, is working on an\u00a0innovative collaborative<\/a>\u00a0project between internal research groups to develop a microneedle patch-vaccine that uses proteins grown in GM plants.<\/p>\n On the other hand, the Center for Research in Agricultural Genomics (CRAG) of Spain, will develop antigens for COVID 19 in\u00a0GM lettuce and tobacco<\/a>, and the international project NEWCOTIANA, which works on the development of medicines and vaccines in plants with funding from the European Union, has\u00a0released the complete genetic sequence<\/a>\u00a0of\u00a0Nicotiana benthamiana<\/em>\u00a0in order to accelerate the development of a plant-based vaccine. This last work\u00a0was led<\/a>\u00a0by IBMCP (Spain) and Queensland University of Technology (Australia).<\/p>\n Although the plant-based vaccines mentioned above have certain advantages over conventional vaccines, their route of administration continues to be through parenteral injection, the \u201cjab\u201d that can cause so much pain to children. But what if, instead of using GM tobacco and purifying antigens to make an injectable vaccine, we could eat a GM fruit that directly confers immunity?<\/p>\n Although something like this is not yet in clinical use, it is not a novelty in experimental terms.\u00a0Since the 1990s,<\/a>\u00a0several research groups have worked on the modification of edible plants and fruits that generate an immune response in the intestinal epithelium of animals after oral intake (Figure 2). Genetically modified crops \u2014 still at the experimental, not commercial, level \u2014 used to create \u201cedible vaccines<\/a>\u201d range from potato, tomato, lettuce, papaya, carrot and rice to quinoa, alfalfa, banana and algae. They have focused on hepatitis B, rotavirus, Norwalk virus, malaria, cholera and autoimmune diseases, among others.<\/p>\n This route was the one chosen by Daniel Garza, a young biotechnologist and entrepreneur with a research stay at the Institute of Biotechnology of the Autonomous University of Nuevo Le\u00f3n (UANL) in Mexico, as an approach to developing a vaccine against COVID-19. \u201cThe development of an edible vaccine against SARS-CoV-2 has so far been a little-explored alternative, even though the benefits are evident,\u201d Garza said in an interview with the Cornell Alliance for Science. \u201cUnder this premise, this problem would be addressed with the focus of developing a fusion protein with the characteristics of a vaccine to be expressed in tomato plants.\u201d<\/p>\n Garza, together with a multidisciplinary group of researchers, use bioinformatics and computational genetic engineering in applying a reverse vaccination strategy. More specifically, by using bioinformatics tools, they identify the antigens most likely to be vaccine candidates to induce an immune response through \u201cin silico\u201d analysis of the pathogen genome.<\/p>\n \u201cThe development of vaccines using conventional techniques depends on a large number of biochemical, immunological and microbiological methods that require a large amount of time and that imply higher production costs,\u201d Garza said. \u201cThe reverse vaccination strategy offers the possibility of identifying a greater number of proteins for each pathogen and selecting the best candidate vaccine antigens. This allows the development of vaccines that were previously difficult or impossible to manufacture.\u201d<\/p>\n Researchers from Garza\u2019s laboratory have been working with this approach since 2018 to search for new candidate antigens for an Ebola vaccine \u2014 research\u00a0they published<\/a>\u00a0at the end of 2019 in the UANL magazine\u00a0Planta<\/em>. \u201cThe results observed so far allow us to identify new epitopes in the regions of the sequence of the VP40 protein of the Ebola virus with characteristics of immunogenicity, antigenicity, hydrophilicity and accessibility that make them a vaccine candidate,\u201d Garza said.<\/p>\n Once the candidate sequence has been identified, they continue optimizing the nucleotide sequence in the tomato plant and the genetic transformation by\u00a0Agrobacterium tumefaciens<\/em>. \u201cThe expression in tomato plants with the new identified epitopes allows us to obtain high levels of expression of the recombinant protein\u201d Garza added. In simple terms, prior bioinformatic modeling saves effort and works with antigens that have a high protective response against the pathogen, a useful antigen for the development of a viable and scalable vaccine.<\/p>\n However, due to the contingency and severity of the SARS-CoV-2 outbreak, Garza\u2019s group decided to dedicate its efforts to work on the bioinformatic modeling of a potential vaccine for this pathogen, using the same strategy used against the Ebola virus through the development of an edible tomato as an immunization method.<\/p>\n The only similar work that can be found in the bibliography is the development of a\u00a0tomato with SARS-CoV antigens<\/a>, which was responsible for severe acute respiratory syndrome (SARS) in Southeast Asian countries in 2002-2003 and has 70 percent genomic similarity to the pathogen behind the current pandemic. Although mice immunized orally with this transgenic tomato revealed significantly high levels of specific antibodies against SARS-CoV-1, there was no further progress towards clinical phases.<\/p>\n \u201cWe are at the analysis stage, using the genomic and proteomic sequences of SARS-CoV-2 and using bioinformatic tools that allow us to identify the antigens most likely to be candidates to induce an immune response,\u201d Garza said in regard to the current status of efforts to develop a vaccine against SARS-CoV-2, the virus that causes COVID-19, in tomato plants.<\/p>\n \u201cThe candidate epitopes are selected based on the prediction of their function, such as accessibility and secretion, and then they are cloned, expressed and analyzed to subsequently confirm their cellular location in vitro. The use of animal models will allow us to evaluate their immunogenicity and protective capacity,\u201d Garza added.<\/p>\n As Garza explained, this research is currently in the stage of analysis and identification of potential regions for the development of a vaccine. His research team is now applying its project to a \u201cgeneral call\u201d\u00a0addressed to Mexican researchers \u00a0working on the development of drugs against COVID-19 that was issued by\u00a0the Mexican government, which\u00a0finances the expenses for a collaboration<\/a>\u00a0with the Paul Scherrer Institute of Switzerland. The next phase of the project will be the expression of the candidate antigens in tomato and evaluating their immunogenic and protective capacity in animal models. As the project progresses, links with companies or research centers will be evaluated to bring the candidate vaccine to the clinical phase.<\/p>\n Beyond eliminating the annoying \u201cprick\u201d of a needle, using fruits or edible plants to immunize people against diseases offers several benefits, including reduced production costs since there is no need for treatment or purification prior to oral administration.<\/p>\n The direct consumption of a raw material \u2014 either through the fruit itself or its lyophilized biomass encapsulated in gelatin pills or tablets \u2014 is a\u00a0clear advantage\u00a0<\/a>as it reduces the cost of antigen processing and purification, as well as the degradation of antigens in the gastrointestinal tract due to the protective role of plant cells within the stomach.<\/p>\n Additionally, the expression of the antigen in the seeds allows\u00a0maintenance and stability for longer periods<\/a>. Edible vaccines can also produce complex multimeric proteins that cannot be expressed by microbial systems and are a safe and effective method of vaccination.<\/p>\n The fact that edible vaccine formulations don\u2019t require antigen purification probably would be the main factor contributing to their low-cost, which is necessary to achieve broad vaccination coverage in developing and low- income countries.<\/p>\n Statistics show, for example, that\u00a0only 40 acres<\/a>\u00a0would be required to produce all the annual hepatitis B vaccines for the entire population of China, and only\u00a0about 200 acres<\/a>\u00a0to produce edible vaccines for all children globally. The final objective of this type of technology would be to deliver not only \u201cvaccines\u201d but also real \u201cmedicinal foods\u201d \u00a0\u2014 not in the alternative or marketing sense, but in a literally curative sense \u2014 through plants and fruits that reinforce overall health and protect the immune system against pathogens, cancer or autoimmune diseases. This is especially important in underdeveloped countries, where it is difficult to obtain treatments or procedures that require complex equipment and conventional vaccines are hard to store and transport.<\/p>\n Regulatory and biosafety obstacles, rather than technical and experimental constraints, could delay the arrival of edible vaccines to our tables and hospitals, especially in the countries most in need.<\/p>\n Although many countries on all continents develop, or have developed, GM crops on an experimental level,\u00a0only 26 nations<\/a>\u00a0currently have regulations implemented for their commercial use. The fact that so many countries lack legislation, or use backward and cumbersome regulatory frameworks,\u00a0such as the one employed by the European Union<\/a>, could increase the final cost of bringing the edible vaccine from the laboratory to the market, making it difficult for small and medium-sized companies or public institutions to develop this technology.<\/p>\n In the case of Mexico, where they are already working on the development of an edible vaccine in tomato plants against COVID-19, local scientists are\u00a0dealing with difficult times<\/a>\u00a0under the mandate of a president who has repeatedly declared himself against the use of GM crops and who appointed a scientist\u00a0famous for being a staunch GM opponent<\/a>\u00a0as\u00a0director of CONACYT, the government entity that regulates the national science budget. Nor can we forget the recent\u00a0problem of Mexican cotton growers<\/a>, who aren\u2019t receiving new permits for the cultivation of GMOs by the current government..<\/p>\n \u201cGiven the current contingency situation for the COVID-19 we are experiencing, it will undoubtedly make us rethink the legislation of the GMOs that apply not only in Mexico but in Latin America,\u201d Garza noted. \u201cWhat is currently happening allows us to rethink whether we are really capable as countries of being able to face a pandemic of such magnitude without using the full potential that GMOs offer us for the development of vaccines, especially for developing countries \u2026 The benefits of biotechnology must be shown to society not as an evil, but as an effective solution to many of the problems that we currently have in the region.\u201d<\/p>\n Paradoxically, if the investigation into this promising edible vaccine \u2014 created in the Mexican public sector \u2014 progresses successfully, it is highly likely that its development towards the clinical phase and productive escalation would move north, to the US or Canada, where companies are already working in molecular pharming aimed at COVID-19 and have the world\u2019s most agile GMO regulatory frameworks. This could happen despite high-level research centers and top scientist working in agricultural biotechnology in Mexico, such as CIMMYT, CINVESTAV and INIFAP, as well as local universities with biopharming capabilities.<\/p>\n Facing the background of regulatory agencies that haven\u2019t developed regulations for plant-based pharmaceutical compounds, and the imminent arrival of some vaccine of plant origin for COVID-19, the Mexican researcher\u00a0Sergio Rosales Mendoza<\/a>, who investigates recombinant vaccines in plants and algae in the Autonomous University of San Luis de Potosi (UASLP), concluded an\u00a0editorial<\/a>\u00a0in the journal\u00a0Expert Opinion on Biological Therapy<\/em>\u00a0with key questions:<\/p>\n \u201cWill regulatory agencies display flexibility to validate and approve anti-SARS-CoV-2 virus plant-made biopharmaceuticals (e.g. by adapting\/simplifying the regulatory requirements for this specific technology)? Will regulatory agencies accelerate the evaluation process of plant-made biopharmaceuticals against SARS-CoV-2 virus? Will the existing clinical trials of plant-made vaccines against influenza (with encouraging findings) and the enzyme already approved make the approval pathway smoother? Will the developing and low-income countries ultimately benefit from the plant-based technologies in the fight against COVID-19?\u201d<\/p>\n A root problem for edible vaccines is the popular misconception still ingrained in many people that GM crops \u201care harmful to health or the environment,\u201d despite\u00a0thousands of studies<\/a>\u00a0and\u00a0reviews<\/a>, public statements from more than\u00a0250 scientific\/technical institutions<\/a>\u00a0that corroborate its safety and more than two decades of consuming GM crops with no reported adverse effects. A central challenge is to continue delivering this information, especially to the public and law-makers in developing countries, at pivotal moments, such as when we see that new \u201cplant-based\u201d technologies are making their niche in other fields, such as revolutionary\u00a0lab-meat<\/a>.<\/p>\n Will we tell our grandchildren and great-grandchildren stories from the past of painful injections given to us when we were children? Will they live in a future where a couple of lyophilized carrot and lettuce capsules will immunize them against all the killer pathogens of the 19th, 20th and 21st centuries? Or will a delicious vaccine-tomato salad be enough to protect them against some new virulent strain that doesn\u2019t yet exist?<\/p>\n Perhaps this race against time and the urgency of finding a viable vaccine for COVID-19 will enable GM plants to save millions of lives and clear their reputations, unfairly stained and demonized by fear and disinformation, once and for all.<\/p>\n Rosales-Mendoza, S.; M\u00e1rquez-Escobar, V.A.; Gonz\u00e1lez-Ortega, O.; Nieto-G\u00f3mez, R.; Ar\u00e9valo-Villalobos, J.I. 2020. What Does Plant-Based Vaccine Technology Offer to the Fight against COVID-19?\u00a0Vaccines<\/em>,\u00a08<\/em>, 183<\/a>.<\/p>\n Capell T.: Twyman, R.; Armario-Najera, V.; K.-C. Ma, J.; Schillberg, S.; Christou, P. 2020. Potential Applications of Plant Biotechnology against SARS-CoV-2.\u00a0Trend in Plant Science<\/em><\/a>.<\/p>\n Balke, I., & Zeltins, A. 2020. Recent Advances in the Use of Plant Virus-Like Particles as Vaccines.\u00a0Viruses<\/em>,\u00a012<\/em>(3), 270.<\/a><\/p>\n Kurup, V. M., & Thomas, J. 2020. Edible Vaccines: Promises and Challenges.\u00a0Molecular biotechnology<\/em>,\u00a062<\/em>(2), 79\u201390.<\/a><\/p>\n Concha C, Canas R, Macuer J, Torres MJ, Herrada AA, Jamett F, Ibanez C. 2017. Disease prevention: an opportunity to expand edible plant-based vaccines?\u00a0Vaccines (Basel) 5:14.<\/a><\/p>\n Pogrebnyak, N.; Golovkin, M.; Andrianov, V.; Spitsin, S.; Smirnov, Y.; Egolf, R.; Koprowski, H. 2005. Severe acute respiratory syndrome (SARS) S protein production in plants: Development of recombinant vaccine.\u00a0Proc. Natl. Acad. Sci. USA<\/em>,<\/strong>\u00a0102<\/em>, 9062\u20139067.<\/a><\/p>\nPlants as vaccine biofactories<\/h3>\n
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Efforts in COVID-19 from the public and private sectors<\/h3>\n
What if the vaccine could be \u201ceaten\u201d instead of injected?<\/h3>\n
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Garza\u2019s research<\/h3>\n
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Benefits of an edible vaccine<\/h3>\n
Pending challenges<\/h3>\n
RECOMMENDED REFERENCES<\/strong><\/h4>\n