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Plant Based Expression of Dengue Subunit VaccinesBY: Hareepriyaw M | Category: Biotechnology-products | Submitted: 2013-12-05 07:28:55
Article Summary: "A well-defined uniform antigen represents the subunit vaccine. The subunit vaccine does not involve any risk of addition of any genetic material to the person inoculated with it. .."
A well-defined uniform antigen represents the subunit vaccine. The subunit vaccine does not involve any risk of addition of any genetic material to the person inoculated with it. When the Rhesus monkey was inoculated with subunit vaccine consisting of dengue E protein as one of the primary trials, the results were very much encouraging. The disadvantage of dengue subunit vaccine is low level expression of dengue proteins in insect and mammalian cells.
Plants are observed as an alternative method of expressing bio-pharmaceutical products which include the bacterial, viral and fungal antigens. In the present review study, the researchers have focused on discussing about the method involved in the expression of bio-pharmaceutical products like dengue subunit vaccine in plants.
Dengue is the most important viral borne disease occurring in humans. Dengue initially causes mild fever. In some cases, it might cause flu-like fever associated with headache, muscle and joint pain, rashes and vomiting. Most severe forms of this disease are called as Dengue hemorrhagic fever, which is characterized by extensive plasma leakage during defervescence. The conditions of hypotension and tachycardia coupled with plasma leakage are described as Dengue shock syndrome.
The distribution of dengue with fatal results allows dengue as the main candidate for development of vaccine as an aid to eliminate the disease. Dengue is caused by viruses (DENVs) that are transmitted to the humans by the bite of female mosquito Aedes aegypti and Aedes albopictus. The dengue viruses consist of antigens named as DENV-1 to DENV-4. Infection with DENV serotype will trigger immune protection all through the life against reinfection with homotypic virus.
The cause of secondary infections is associated with disease severity explained by a critical component named as antibody dependent enhancement (ADE) of infection. Dengue vaccine development is hindered by the process of ADE. A vaccine should have the capacity to generate complete protection against all the four serotypes.
Vaccine development for Dengue - Subunit vaccines
Dengue viruses belong to the family of viruses called Flaviviridae with the genus Flavivirus. They are closely associated with Yellow fever virus (YFV). Hence, the vaccine produced against YFV is a successful vaccine against mosquito borne virus. The original vaccine for YFV is a live attenuated vaccine which can be represented by the Dengue subunit vaccine. The major disadvantage of producing Dengue "subunit" vaccine is requirement of large quantities of purified antigen and the use of adjuvants in the vaccine which increase the cost of it.
The most vital advantage of Dengue "subunit" vaccine is that it possesses the capacity to balance the level and immunogenicity of four components of the vaccine. So, this vaccine can offer complete protection against all the serotypes.
Plant expression system
It is very important to find the systems that can produce large quantities of vaccine as well as support post translational modification of expressed proteins. Scaling to large amounts of vaccine production can be done with bacterial systems as well. Post translational modification like glycosylation cannot be done with bacterial systems. Yeast systems are also beneficial for large scale production of the vaccines but lack in hyper glycosylation. Mammalian cell cultures are found to be helpful in post translational modification and are expensive to be maintained. All the above systems can be replaced effectively by plant expression systems. Transgenic tobacco plants were used for expressing hepatitis B surface antigen in 1992. Many pharmaceutically important products like vaccines and antibodies were expressed in plant hosts.
Tobacco plants were used for successful application of technologies for its manipulation and for the higher biomass yield. Crops like potatoes, rice, maize, soya beans and tomatoes were used for vaccine production. The crops will have reduced downstream purification costs and will be less affected by plant immunogenic substances. Oral vaccines are found to be effectively produced in vegetable and fruit crops.
Tissue target: Seeds of the plant are considered as effective tissues for high protein expression and low costs of downstream purification compared to the leaves.
Expression strategies: Transient expression systems were found to have shorter time to express the proteins compared with the chloroplast or nuclear expression systems. Expression of transgene involves a viral vector or a binary vector of the plant. The Binary vector based expression is carried out by Agrobacterium mediated gene transfer which is injected into the plant intracellular spaces. The DNA and RNA viral backbones of cow pea mosaic virus, tobacco mosaic virus, alfalfa mosaic virus, plum pox virus, and potato virus X accompanied by many cloning and expression methodologies were used to produce pharmaceutically vital products.
Production of Dengue subunit vaccines in plants
Till now, there are three research studies published to produce plant based dengue subunit vaccine. The initial study by Saejung et al. dealt with TMV based vector made to infect Nicotiana benthamiana to express D2EIII (DENV-2 E domain III coding for amino acids 298 to 400). The protein was expressed with TMV coat protein sub genomic promoter. The subunit created was successful in working as antigen in mice which provided evidence for the synthesis of dengue subunit vaccine.
The second study constituted an expression of truncated EB4 consisting of DENV-2 E protein binding region with amino acids from 379 to 423 in Cucumis melo. This subunit was fused with cucumber green mottle mosaic virus coat protein (CGMMV CP). The recombinant CGMMV CP containing EB4 epitope or dengue virus epitope was expressed in plants in large quantities.
The third study showed the efficiency of transfection of binary vectors by Agrobacterium showing higher expression of antigens or higher yield of plant RNA virus. The results showed that 0.4 to 0.6mg of corresponding antigens per gram fresh weight was analyzed 7 to 10 days after inoculation.
Plant as bioreactors in producing antigen is a practical approach to the development of Yersinia and Influenza vaccines and the dengue subunit vaccine prepared from plants are competitively immunogenic.
Yun-Kiam Yap and Duncan R. Smith. Strategies for the plant based expression of dengue subunit vaccines. Biotechnol. Appl. Biochem. (2010) 57, 47-53. DOI: 10.1042/BA20100248.
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