Plant vaccine

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Information about Plant vaccine

Published on February 18, 2009

Author: pangjai


Slide 1: Transgenic tomatoes Expressing human beta - amyloid for use as a vaccine against Alzheimer’s disease From Youm, J.W. et al. Biotechnol. Lett. (2008) 30: 1839–1845 By Wuthipong Pangjai 1 Slide 2: Forgetfulness Loss of language Inability to find simple solutions Changes in personality Difficulty performing routine daily tasks Alzheimer’s disease (AD) 2 Slide 3: Intracellular neurofibrillary tangles Extracellular amyloid plaque. Aggregation of misfolded proteins ? 3 Slide 4: beta-amyloid (A?) - Toxic protein - Neuronal death EFFECT of beta-amylod neurodegenerative 4 Slide 5: Alzheimer’s Normal Brain Cross-Sections 5 Slide 6: - Inhibits the degeneration of the nervous system - Suppression of A? generation by vaccine Prevention - treatment 6 Recombinant vaccine : Recombinant vaccine (antigen) in E. coli or yeast purification Use as a vaccine Capture Chromatography 7 Slide 8: It was difficult to produce A? - Toxicity of the protein 8 Slide 9: Plants are alternative system for production of recombinant proteins. Antibodies Antigens ? 9 Slide 10: Eatable beta-amyloid vaccine idea 10 Objectives : Objectives Produce transgenic A? tomato Examine immunogenicity respond to A? extracted in mice 11 Works Process : Works Process Construction of expression vectors 12 in vivo test Plant selection & confirmation Transformation & regeneration - PCR - Northern blot - Western blot - ELISA Slide 13: Construction of expression vectors pMBP3A? ? pMBP4A? ? pMBP5A? ? CaMV 35S A? 13 Works Process : Works Process Construction of expression vectors Transformation & regeneration Plant selection & confirmation - PCR - Northern blot - Western blot - ELISA in vivo test 14 Slide 15: Plant material fully expanded cotyledons (in tissue culture system) Tomato cultivar “Housechallenge” ? 15 Slide 16: Transformation of tomato Agrobacterium-mediated transformation Kanamycin + Carbenicillin Lines 3A? Lines 4A? Lines 5A? NT line pMBP3A? ? pMBP4A? ? pMBP5A? ? 16 Slide 17: In vitro shoots regenerating from cotyledon-disc callus MS medium + Kanamycin Established in the greenhouse Transgenic tomato regeneration 4 weeks after co-cultivation > 20 individual no phenotypic abnormalities 17 ? Works Process : Works Process Construction of expression vectors Transformation & regeneration Plants selection & confirmation - PCR ? A? gene - Northern blot ? A? transcript - Western blot ? A? protein - ELISA ? A? protein quantitative in vivo test 18 Slide 19: PCR analysis for the presence of A? gene DNA : leaf extracts Primers specifically amplify a 3A?, 4A?, 5A? 1kp Marker 100 bp Marker ? ? 450 bp 600 bp 750 bp 19 Slide 20: Detection of A? mRNA (Northern blot) 1.0% formaldehyde agarose gel hybridized by an A? specific Dig-labeled probe ? ? Quite different levels. 20 Slide 21: Anti-A? antibody Western blot analysis A chemiluminiscence kit 3A?-6 3A?-9 21 PC Slide 22: Quantitative analysis of expressed 3A? using direct ELISA N Plate were coated with anti-A? antibody NT 3A? 6 3A? 9 22 Works Process : Works Process Construction of expression vectors Transformation & regeneration Plant selection & Confirmation in vivo test - Vaccine preparation - BALB/c mice preparation - Vaccination 23 Vaccine preparation : Vaccine preparation Cholera Toxin B subunit (CTB) ? Adjuvant The protein was extracted from 5 g tomato plants - pMBP3A?-6 line - NT line 24 Immunization of BALB/c mice : Immunization of BALB/c mice - sodium bicarbonate 15 month old BALB/C mice - gastric gavage Neutralize stomach acidity 25 ? ? Slide 26: 1 2 3 4 5 6 7 8 9 10 Week Tomato extract + Adjuvant booster - A? peptide emulsified in alum. Sera ELISA Immunization times 26 Slide 27: anti-A? antibody production (ELISA) NT pMBP3A?-6 line 1st Bleeding time 2nd 3rd ? ? ? Significant increase in antibody against A? 27 Slide 28: Discussions 28 For further use of plant edible vaccine - Stronger promoters Plant-derived leader sequences or signal peptides Plant optimized synthetic genes - Targeting the protein for retention in the appropriate tissue Tomato have low protein content Slide 29: High level of A? transcript was found in all transgenic lines 3A? Conclusions 29 A? successfully introduced to tomato via Agrobacterium-mediated transformation A? were found in line 3A?, 4A? and 5A? Slide 30: Highest A? protein expression was found in line 3A?-6 Conclusions 30 Protein extract from transgenic tomato can trigger antibody against A? Slide 31: 31 Assoc. Prof. Amara Thongpan Acknowledgement Slide 32: 32 Thank you! for your attention Slide 33: Anemia depression effect The chicken which eats the food with which the vaccine was mixed Food with which the vaccine was mixed 33 Slide 34: 34 Stronger promoters Plant-derived leader sequences Signal peptides Optimized synthetic genes Targeting the protein to appropriate tissue Slide 35: Discussions 35 Advantage Oral delivery - Use as raw food or dry powder - No need for “cold chain” - Cost efficiency - Optimised expression system - Ease of genetic manipulation F. Sala et al., 2003 Slide 36: 36 F. Sala et al. / Vaccine 21 (2003) 803–808 Slide 37: F. Sala et al. / Vaccine 21 (2003) 803–808 37 Slide 38: Plants such as tobacco and alfalfa often produce proteins in their green leaf tissues, but these tend to contain high concentrations of phenolic and other potentially toxic compounds. Seeds are likely to have fewer phenolic compounds and a less complex mixture of proteins and lipids than green leaves, which might be an advantage during purification. 38 Slide 39: 39 M cells form pockets that contain clusters of leucocytes Slide 40: 40 Slide 41: 3-5 Multimer of A? Cut with BamHI Agarose gel stained with ethidium bromide pMBP3A? pMBP4A? pMBP5A? ? ? ? 41 Slide 42: The cause of Alzheimer’s is unknown.  Dominant mutation in three different genes have been found to account for a small number of cases of familial early-onset AD.  For the most common form of late onset, ApoE is the only clearly established susceptibility gene.  All four genes can have mutations or variants that confer increased risk for AD, but this only accounts for thirty percent of the genetic picture. 42 Slide 43: Beta Amyloid 1-40 peptide Sequence DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV 3-letter-code Asp - Ala - Glu - Phe - Arg - His - Asp - Ser - Gly - Tyr - Glu - Val - His - His - Gln - Lys - Leu - Val - Phe - Phe - Ala - Glu - Asp - Val - Gly - Ser - Asn - Lys - Gly - Ala - Ile - Ile - Gly - Leu - Met - Val - Gly - Gly - Val - Val Molecular weight 4329.86 Counter ion TFA Description Beta Amyloid peptides are derived from amyloid precursor protein (APP) thought to play a role in the development of the senile plaques associated with Alzheimer's Disease. 43 Slide 44: 44 The adaptive immune system is composed of highly specialized, systemic cells and processes that eliminate or prevent pathogenic challenges. Adaptive immunity is triggered in vertebrates when a pathogen evades the innate immune system and generates a threshold level of antigen.[1] The major functions of the adaptive immune system include: The recognition of specific “non-self” antigens in the presence of “self”, during the process of antigen presentation. The generation of responses that are tailored to maximally eliminate specific pathogens or pathogen infected cells. The development of immunological memory, in which each pathogen is “remembered” by a signature antigen. These memory cells can be called upon to quickly eliminate a pathogen should subsequent infections occur. Slide 45: 45 Slide 46: 46 Slide 47: 47 Slide 48: 48 Slide 49: 49 Slide 50: F. Sala et al. / Vaccine 21 (2003) 803–808 50 Slide 51: F. Sala et al. / Vaccine 21 (2003) 803–808 51 Slide 52: 52 Slide 53: The humble tomato could be a suitable carrier for an oral vaccine against Alzheimer’s disease, according to HyunSoon Kim from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) in Korea and colleagues from Digital Biotech Inc. and the Department of Biological Science at Wonkwang University. Although their research is still in the early stages, it is a promising first step towards finding an edible vaccine against the neurodegenerative disease. Alzheimer’s disease is the most common cause of dementia and it progresses over a long period of time. It is thought to be caused by the accumulation of human beta-amyloid, a toxic insoluble fibrous protein in the brain, which leads to the death of neurons. Reducing the accumulation of beta-amyloid may inhibit the degeneration of the nervous system and therefore prevent or delay the onset of Alzheimer’s disease. One approach is to stimulate the immune system to reduce beta-amyloid in the brain. Kim and colleagues’ aim was to develop a plant-derived vaccine against Alzheimer’s disease, since beta-amyloid is toxic to animal cells. Tomatoes are an attractive candidate as a vaccine carrier because they can be eaten without heat treatment, which reduces the risk of destroying the immune stimulation potential of the foreign protein. The researchers inserted the beta-amyloid gene into the tomato genome and measured the immune responses to the tomato-derived toxic protein in a group of 15-month-old mice. They immunized the mice orally with the transgenic tomato plants once a week for three weeks, and also gave the mice a booster seven weeks after the first tomato feed. Blood analyses showed a strong immune response after the booster, with the production of antibodies to the human foreign protein. The authors conclude: “Although we did not reveal a reduction of existing plaques in the brain of mice challenged with tomato-derived beta-amyloid…this study represents a unique approach in which transgenic plants expressing beta-amyloid protein are used to produce a vaccine.” The team is currently looking at strategies to increase the potency of the tomato-based vaccine, because fresh tomatoes contain only 0.7% protein and levels of foreign protein are even lower. 53 Slide 54: What needs to be done now? Is the vaccine toxic and are there side-effects?There were no signs of toxicity over the time period studied in the mice. Nevertheless, scientists will have to check if there are long-term side-effects in the mice. Then studies will be performed on humans after an extensive approval process. Concerns such as toxic side effects need to be addressed. Can the immune response that fights the beta amyloid protein be sustained?Getting the immune system to battle something that's not really foreign to our bodies is difficult. This is, for example, why the immune system fails to fight some cancerous tumor cells. If all goes well, the U.S. Food and Drug Administration will be asked to approve this treatment as a human vaccine. What is the role of plaques and tangles?In Alzheimer's, plaques are not the only abnormality in the brain. Neurofibrillary tangles also play a role in the disease process. Scientists do not know how plaques and tangles each contribute to the disease. If tangles turn out to cause most of the damage, being able to reduce plaques may not help treat the disease in humans. Will the vaccine work in humans?This research was performed using laboratory mice. It is unknown whether the same approach will work for humans. Although mice develop amyloid plaques just like AD patients, the mice do not show symptoms like AD so one cannot say that AD in mice has been cured. One can only say that the amount of plaques was reduced. But this work promises to steer Alzheimer's research in a new direction. 54 DNA Vaccine : DNA Vaccine 55 Slide 56: 56 Slide 57: 57 Slide 58: 58 Slide 59: 59 Slide 60: 60 Slide 61: 61 Slide 62: 62

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