Plant derived vaccine

ABSTRACT

Biotechnologists in recent years have come up with a new concept. This new concept is about edible vaccine. Edible vaccines are composed of antigenic proteins and do not contain pathogenic genes (because obviously they use attenuated strains). Thus, they have no way of establishinginfectionand safety is assured. Edible vaccines hold great promise as a cost-effective, easy-to-administer, easy-to-store, fail-safe and socio culturally readily acceptable vaccine delivery system, especially for the poor developing countries. It involves introduction of selected desired genes into plants and then inducing these altered plants to manufacture the encoded proteins. Resistance to genetically modified foods may affect the future of edible vaccines. This Review article include many aspects related to plant derived vaccine like Molecular farming, Process of development of plant derived vaccines. Mechanism of action .recent research regarding development of edible vaccines against cholera, malaria, Hepatitis B, rabies etc. The review article gives a overall picture of Plant derived vaccine.

Key words: Plant, Vaccines, Protein, Antigen, Diseases, Tissue culture

Introduction

The plant-based vaccine production method works by isolating a specific antigen protein, one that triggers a human immune response from the targeted virus. A gene from the protein is transferred to bacteria, which are then used to “infect” plant cells. The plants then start producing the exact protein that will be used for vaccinations. The flexibility of the plant expressed vaccine system, combined with its low cost and ability to massively scale, may provide vaccine protection not only to citizens of the United States, but to many parts of the world that cannot currently afford vaccines.Subunit vaccines that consist of one or more antigenic epitopes or proteins are often preferred to traditional vaccines made of killed or attenuated organisms. Mammalian, yeast and insect cell cultures are used to produce subunit vaccines because of their ability to process recombinant proteins in a manner similar to that of the native organism. However, expensive media and the purification steps needed for recovering recombinant proteins expressed in these organisms increase the cost of producing these subunit vaccines. In addition, most subunit vaccines produced in these systems are heat sensitive and require parenteral delivery. This restricts use of subunit vaccines in the poorly funded health systems of developing countries.

A promising alternative is to transform plants with a gene(s) encoding an immunogenic protein capable of preventing infection by a pathogenic agent. The production of vaccines in transgenic plants overcomes the risk of contamination with mammalian pathogens and can enable oral delivery. These characteristics simplify vaccine delivery and decrease the cost of an immunization program.

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History of plant derived vaccine

By the late 1990s an international campaign to immunize all the world’s children against six devastating diseases was reportedly reaching 80 percent of infants (up from about 5 percent in the mid-1970s) and was reducing the annual death toll from those infections by roughly three million. Vaccines trigger and prepare our body’s defense mechanisms so that the system is able to fight and eliminate the pathogens when encountered due to natural infection. Recent progress in vaccine technology has improved public health to a remarkable extent. Each year, millions of children in underdeveloped countries have no access to immunization. The traditional vaccines are expensive and require special conditions for storage, distribution and & dispensing. Moreover, the supplies of traditional vaccines are limited and they are in short supply. The first report of the production of edible vaccine (a surface protein from Streptococcus) in tobacco, at 0.02% of total leaf protein level, appeared in 1990 in the form of a patent application published under the International Patent Cooperation Treaty. Fifteen years later, the first technical proteins produced in transgenic plants are on the market, and proof of concept has been established for the production of many therapeutic proteins, including antibodies, blood products, cytokines, growth factors, hormones, recombinant enzymes and human and veterinary vaccines.Subsequently, a number of attempts were made to express various antigens in plants10-12.Arntzen and John Clements, of Tulane University Medical School, launched an immunological battle against gut-invading bugs in 1991 using "bio-pharmed" tobacco to target a form of Escherichia coli (E-coli), a diarrhea-causing bacterium that kills approximately three million infants each year. In the past five years experiments conducted by Arntzen (who moved to the Boyce Thompson Institute for Plant Research at Cornell University in 1995) and his collaborators have demonstrated that tomato or potato plants can synthesize antigens from the Norwalk virus, Enterotoxigenic, E. coli, V.cholerae and the Hepatitis B .

Molecular farming

Plant biologists had developed a plan of introducing selected genes (the blueprints for proteins) into plants and inducing the manipulated, or transgenic, plants to manufacture the encoded proteins. For making edible vaccines against the different pathogens, it is necessary to find out pathogen associated antigenic epitopes or surface antigens. The antigenic epitopes are proteins or peptides that are encoded by genomic sequences. The basic methodology includes identification, selection and isolation of desirable genes from the pathogen that encodes the surface antigen proteins. The isolated gene can be then cloned in a suitable vector for gene transfer. The selected vector should possess all the unique characteristics of an ideal vector. The molecular markers present in vectors can be used for screening transformed host cells from untransformed. After integration of desirable gene in host genome, the cells can be checked for cloned gene expressions using eliza that ultimately uses antigen specific monoclonal antibodies. The transformed cells with positive cloned gene expressions allow them for propagation using plant tissue culture (Subbirhussain, 2011).

As molecular farming has come of age, there have been technological developments on many levels, including transformation methods, control of gene expression, protein targeting and accumulation, the use of different crops as production platforms (Twyman et al, 2003,2005), and modifications to alter the structural and functional properties of the product. One of the most important driving factors has been yield improvement, as product yield has a significant impact on economic feasibility.Strategies to improve the recombinant protein yield in plants include the development of novel promoters, the improvement of protein stability and accumulation through the use of signals that target the protein to intracellular compartments, and the improvement of downstream processing technologies. 13

Considering the cost of protein purification as comparable savings in the upstream components make the production cost of a commercially important protein in plants substantially less than other systems. Genetically modified plants can be grown in large area. 14,15.The cost of goods sold(COGS) for bulk production of recombinant protein in plants has been estimated to be 1/10th to 1/50th of bacteria lfermentation16.Therefore it is economically sound to use transgenic plants for antigen production. A variety of gene expression and protein localization systems now available for plants allows stable accumulation of the recombinant proteins in target plant tissue (Fig.1.).A plant ideal for oral vaccine production should have the following features:

(i) A men ability to transformation,
(ii) Expression in edible tissue that can be consumed un cooked since vaccine antigens are heat sensitive
(iii) Targeted tissue to be rich in protein because vaccine protein will only be a small percentage of the total protein.
(iv) Targeted tissue should not produce toxic molecules and would allow correct folding of the antigen protein and desired post translational modifications. Expression of antigen in edible tissue offers a convenient and inexpensive source to deliver a vaccine.

Expression of commercially important proteins in leaf tissue is not a good strategy17,18,19 on account of the following reasons:

(i)Over all protein content in leaf tissue is low.
(ii) Leaves have high protease activity.
(iii)The presence of pigments and phenolic makes purification of recombinant protein from leaves

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Figure 1.Curtesy :Tiwari et al.,2009. Steps in the production of plant-derived vaccine

Manufacturing practices becomes more difficult due to the need for handling large volume and biomass. The expression levels of plant-derived bio-pharmaceuticals need to be increased before commercial production would accomplished20,21 on economically competitive basis. The expression levels of their combinant proteins in the transgenic plants are also influenced by environmental factors. High expression levels could be best achieved in cell suspension, hairy root cultures (in vitro) and seeds(in vivo).Seed tissue represents potentially a very promising target for producing pharmaceutically important proteins for extraction at commercial level. There combinant seeds also offer the possibility of direct use as an edible vaccine. Single chain antibodies expressed in seeds of rice and wheat showed high biological activities and remained stable for several years 22.Thus,the proteins expressed in seeds are highly stable. Long term storage and easy transportability of seeds is possible due to very low moisture content of mature seeds23 Other tissues like hairy root and cell suspension cultures could be useful target issues to express recombinant proteins24-26,though the establishment and running costs of such in vitro systems are higher.

One of the most important aspects in molecular bio-farming is the selection of promoter to achieve high level expression of the antigen coding gene. The choice of promoters affects trans gene transcription, resulting in change snot only in concentration, but also in the stage, tissue and cell specificity of its expression. Cauliflower mosaic virus 35S(CaMV35S)promoter has widely been used because of its strong and constitutive expression. High level protein expression is essential to develop economically competitive plant-based process for cultivation of the transgenic variety within confines fields with controlled environmental and contained biosafety conditions highly expressing and yet tightly controlled promoters are desirable for bio-farming proteins from plants27.Expression of a protein with CaMV35S promoter does not permit regulated gene expression. Though CaMV35S promoter expresses genes at a relatively high level in leaves and roots, low level of total protein .Many therapeutic proteins can be expressed in stable or transient state in whole plants, plant tissues or cell suspension cultures. There is growing acceptance of transgenic crops in both developed and developing countries. The future of edible vaccines depends on acceptability for genetically modified foods. Successful implementation of edible vaccines depend on how well we overcome various technical complications, regulatory issues and non-scientific challenges. .The World Health Organization (WHO) has called for new strategies to deliver vaccines. WHO estimates that 10 million children die in developing countries each year from infectious diseases that could be prevented with vaccines.28

Approach for Production of Edible Vaccines

Creating edible vaccines involves introduction of selected desired genes into plants or animal and then inducing these altered plants or animals to manufacture the encoded proteins 29,30. This process is known as "transformation," and the altered plants or animals are called "transgenic plants" or “transgenic animals”. Edible vaccines are similar to conventional subunit vaccines as they are composed of antigenic proteins andare devoid of pathogenic genes, Hence, they cannot cause infection and can be safely used in patients with weak immune system. Immunization is done by feeding animals or humans with food derived from edible parts of transgenic plants or animals in which an orally active antigen of the target pathogen is expressed and accumulated. Immunoglobulinmolecules have been successfully synthesized in tobacco plants using the same technology

Thedesiredgenewhichcodesforantigenisisolatedfromthemicrobesandcouldbehandledintwodifferentways:

1.Appropiateplantvirusisgeneticallyengineeredtoexpressthedesiredpeptidesproteins. Therecombinantvirusistheninoculatedintotheplant.Theresultantplantediblevaccinesareutilizedforimmunologicalpurpose
2 .In another approach, the geneofinterest is integrated withplant vector by transformation Avarietyoftechniqueshavebeenusedtointroducetransgeneintoplantcell.

Thesecouldbe groupedintofollowingcategories:

A: Agro bacteriummediatedgene transfer:

TheappropriategeneconstructisinsertedintotheTregionofadisarmedTiplasmidofAgrobacterium.TherecombinantDNAisplacedintoAgrobacterium;aplantpathogenwhichiscoculturedwiththeplantcellsortissuestobetransformed.31Thedrawbackofthismethodisthatitgiveslowyieldandtheprocessisslow.Thismethodworksespeciallyfordicotelydenousplantslikepotato,tomatoandtobacco.Studieshavealsoproved that thegenes are expressed by this method in experimental animalsandplants.18‐20

B: Biolistic method:

The gene containing DNA coated metal (e.g.gold, tungsten)particlesare firedat theplantcellsusinggenegun.21ThoseplantcellsthattakeuptheDNAarethenallowedtogrowinnewplants,andareclonedtoproducelargenumberof genetically identical crop.This method is quite attractivebecauseDNAcanbedeliveredintocellsofplantwhichmakesgene transfer independent of regeneration ability of thespecies. But the chief limitation is the need for costly device particlegun.

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