Edible Vaccines

Dr. Mahmoud Sakr

1-Prof. Plant Biotechnology, NRC 2-Vice President of the Academy, ASRT 3 President 3-President of Arab Biotechnology Association 4-Editor-in-Cehif, Journal of Genetic Engineering & Biotechnology

Contents 1-Vaccines: 1 Vaccines: an overview 2-DNA vaccine (Gene based vaccines,GBV) i GBV) 3-Biotechnology in Agriculture: gy g C Why plants? 4-Plant 4 Plant transformation 5-Edible vaccines 6-Open discussion

Avaccineisanypreparationintendedto produceimmunitytoadiseasebystimulating theproductionofantibodies.

Themostcommonmethodofadministering vaccinesisby injection, butsomearegivenby p y mouth or nasalspray.

vaccination,"after"vacca,"theLatinwordfor cow,andthesubstance usedtovaccinatewas calleda"vaccine.

History: Hi t
Long before the causes of disease were known and long before the processes of recovery were understood, an interesting thing was observed: If people recovered from a disease, rather than succumbing to it, they appeared to be immune from a second bout with , y pp the same illness. It was these types of observations that led the Chinese to try to prevent smallpox By "variolation, *One person who experienced variolation as a child in the late 1700s was Edward Jenner

Edward Jenner noticed that farmers who treated horses with grease lesions often saw the development of cowpox in their cows, complete with fluidfiled blisters similar to fluid filed those seen in smallpox infection. Unlike lethal smallpox, however, the cowpox blisters eventually disappeared, leaving only a small scar at the site of each blister. Jenner noted that there were many people like the milkmaid people who milked cows and who did not get smallpox even when exposed h t t ll h d repeatedly.

EdwardJenner: Englishphysician

Blistersofcowpox

With this in mind, Jenner undertook a daring experiment in 1796: he i f t d a young b with cowpox i h infected boy ith in hopes of preventing subsequent smallpox infection. After allowing the boy to recover fully from cowpox, Jenner in an experiment that would be considered unethical by today's scientific community intentionally infected the boy with smallpox by injecting pus from a smallpox lesion directly under his skin. As Jenner had predicted, the boy did not contract smallpox smallpox.
Mortality3050%

Totallyeradicatedin1980

How vaccines work in the body?

Vaccines are given to provide immunity against a particular disease disease. They use a harmless form of a pathogen or its toxin to make antibodies without the individual suffering the symptoms of the disease disease. If your body is then invaded by harmful pathogen, it produce antibodies to fight them . On the surface of bacteria and viruses are substance called antigen Antibodies will attack antigens when they invade the body Your body produce antibodies for every bacteria and virus it meets meets, therefore a different vaccine is needed for every different pathogen .

TypesofVaccines: fV i
Therearefourmainwaystodevelopvaccines: Liveattenuatedvaccines:containbacteriaorvirusesthathave beenalteredsotheycan'tcausedisease. y Killedvaccines:containkilledbacteriaorinactivatedviruses. Toxoid vaccines: contain toxins (or poisons) produced by the Toxoidvaccines:containtoxins(orpoisons)producedbythe germthathavebeenmadeharmless. Componentvaccines:containpartsofthewholebacteriaor viruses.

*AdvantagesofAttenuatedVaccine: * d f d
Activatesallphasesofimmunesystem.Cangethumoral IgGandlocalIgA. IgG and local IgA Raisesimmuneresponsetoallprotectiveantigens. Inactivationmayalterantigenicity. Moredurableimmunity;morecrossreactive. Lowcost. Low cost Quickimmunityinmajorityofvaccines. Incaseofpolioandadenovaccines,easyadministration. Easytransportinfield. Canleadtoeliminationofwildtypevirusfromthe community.

ImmunoglobulinG I l b li G

DisadvantagesofLiveAttenuatedVaccine: Mutation;reversiontovirulence(oftenfrequent). Spreadtocontactsofvaccinewhohavenotconsentedtobevaccinated S d f i h h d b i d (couldalsobeanadvantageincommunitieswherevaccinationisnot 100%) Spreadvaccinenotstandardizedmaybebackmutated Poor"take"intropics. Probleminimmunodeficiencydisease(mayspreadtothesepatients

*Advantagesofinactivatedvaccines Givessufficienthumoralimmunityifboostersgiven. Nomutationorreversion. p Canbeusedwithimmunodeficientpatients. Sometimesbetterintropics. *Disadvantages of inactivated vaccines Disadvantagesofinactivatedvaccines Manyvaccinesdonotraiseimmunity. Boostersneeded. d d Nolocalimmunity(important). Highercost. Shortageofmonkeys(polio). Failureininactivationandimmunizationwithvirulentvirus.

Toxoidvaccines Toxoid vaccines

Toxoidvaccinesaremadebytreatingtoxins (orpoisons) Toxoid vaccines are made by treating toxins (or poisons) producedbygermswithheatorchemicals,suchasformalin,to destroytheirabilitytocauseillness. Eventhoughtoxoids donotcausedisease,theystimulatethe bodytoproduceprotectiveimmunityjustlikethegerms natural body to produce protective immunity just like the germs'natural toxins. *Examples : Examples: Diphtheriatoxoid vaccine Tetanustoxoid vaccine

Componentvaccines C t i
somevaccinesaremadebyusingonlypartsofthevirusesorbacteria. Thesevaccinescannotcausedisease,buttheycanstimulatethebody toproduceanimmuneresponsethatprotectsagainstinfectionwiththe wholegerm. h l

Contents 1-Vaccines: an overview 1V i i 2 DNA 2-DNA vaccine (Gene based vaccine, GBV) 3-Biotechnology 3 Biotechnology in Agriculture: C Why p y plants? 4-Plant transformation 5-Edible 5 Edible vaccines 6-Open discussion

DNA vaccination is vaccination by by injecting the organism h with genetically engineered to produce an immunological g response. DNA vaccines have been applied to a number li d b of viral, bacterial and parasite models of diseases as well as several tumor models.

Advantages of DNA vaccines:

1Subunit vaccination with no risk for infection i f ti 2Immune response focused only on antigen of interest 3Ease of development and production 4 Stability of vaccine f 4 S bili f i for storage and d shipping 5Costeffectiveness 5 Cost effectiveness 6Obviates need for peptide synthesis, expression and purification of recombinant proteins and the use of toxic adjuvant 6 Long term 6Longterm persistence of immunogen 7In vivo expression ensures protein more closely resembles normal eukaryotic structure, with accompanying post translational modifications

Contents 1-Vaccines: an overview 2-Gene b d vaccines (GBV) 2G based i 3-Biotechnology in Agriculture: gy g C Why plants? 4-Plant 4 Plant transformation 5-Edible vaccines 6-Open discussion

Biotechnology in agriculture Two Categories T C t i

(1) "Improvements" to existing crops. (2) Development of entirely new uses for plants. The term "improvements", include input traits such as crops with extra resistance to insect attack and improved weed control. i t t i t tt k d i d d t l These "GM" or "GMO" crops, are modified food crops made more commercially viable.

Examples: "Roundup ready" Soya and Canola, Drought resistant Canola or "Frosttolerant" T t l t" Tomatoes. t

WHYPLANTS?
Plants,beinghighereukaryotesarebetter Pl b i hi h k b equippedtoproperlyprocessandfoldproteins thanothersystems. Cropplantscansynthesizeawidevarietyof proteinsthatarefreeofmammaliantoxinsand pathogens. Cropplantsproducelargeamountsofbiomassat lowcostwithoutbeingrestrictedbyphysical facilities. Althoughextractionandpurificationcostsmaybe similartoothersystems,thebasiccostof producingrawplantmaterialwillbesignificantly d i l t t i l ill b i ifi tl lowerthanthatoffermentation. Seedsandfruitsprovidesterilepackaging Seeds and fruits provide sterile packaging containersforthevaluabletherapeuticsand guaranteeacertainstoragelife.

WHYPLANTS? WHY PLANTS?

Bioencpsulation Tough cell wall protects antigen from harsh gastric secretions. Antigens are released, taken up by M cells in intestinal lining and gutassociated lymphoid tissue and passed on to macrophages , generating IgG and IgE responses and local IgA response and memory cells, which would promptly neutralize the attack by the real infectious agent.

AvailableExpressionSystems
Expressions System Yeast Bacteria Plant viruses Transgenic Plants Animal Cell Cultures Transgenic Animals

Cost of maintaining

inexpensive

inexpensive

inexpensive

inexpensive

expensive

expensive

Type of storage

-2.0C

-2.0C

-2.0C

RT*

N2**

N/A

Gene size (protein) restriction

Unknown

Unknown

Limited

Not limited

Limited

Limited

Production cost

Medium

Medium

Low

Low

High

High

Protein yield

High

Medium

Very high

High

Medium to high

High

Therapeutic risk

Unknown

yes

Unknown

Low

yes

yes

* RT room temperature. ** N2 culture must be maintained under nitrogen gas.

Contents 1-Vaccines: 1 Vaccines: an overview 2-Gene based vaccines (GBV) C 3-Biotechnology in Agriculture: Why plants? 4-Plant transformation 5-Edible vaccines d bl 6 Open 6-Open discussion

Allstabletransformationmethodsconsist ofthreesteps:
Delivery of DNA into a single plant cell DeliveryofDNAintoasingleplantcell. IntegrationoftheDNAintotheplantcell genome. Conversion of the transformed cell into a Conversionofthetransformedcellintoa wholeplant.

MahmoudSakr,NRC,Egypt

Differentmethodsofgenetransfer Different methods of gene transfer

MahmoudSakr,NRC,Egypt

Agrobacteriummediated Agrobacteriummediated Transformation

MahmoudSakr,NRC,Egypt

MahmoudSakr,NRC,Egypt

TiPlasmid Ti Plasmid

MahmoudSakr,NRC,Egypt

MahmoudSakr,NRC,Egypt

Whatarethecomponentsof transformationvectorplasmids? f i l id ?
Remarkablyfew y Kanamycinresistance,nptII

Aselectablemarkergene
NOSPROMOTER NPTII NOS3'POLYADENYLATION SEQUENCE

Hygromycinresistance,hpt Bar gene (for resistance to herbicide Bargene(forresistancetoherbicide phosphinothricin) DHFR gene (for resistance to methotrexate) DHFRgene(forresistancetomethotrexate) ESPSgene(forresistancetoRoundupherbicide)
MahmoudSakr,NRC,Egypt

Ascorablemarkergene(optional,butvery useful)
NPTIIactivity NPT II activity Opineproduction betaGlucuronidaseactivity(GUS) Greenfluorescentprotein(GFP) Green fluorescent protein (GFP) Luciferaseactivity

MahmoudSakr,NRC,Egypt

Geneofinterest
Cancomefromanysource Usuallychimeric(madeupfrompartsof differentgenes) different genes) Promoter:CaMV35S,others Terminator:Nos,CaMV35S,others Other elements Otherelements

MahmoudSakr,NRC,Egypt

HowisPlantTransformationwithAgrobacterium Done?
CocultivatetheengineeredA.tumefaciens strain containingthegeneofinterestwithanexplant fromwhichregeneratedplantscanbeobtained. Culturetheexplantonregenerationmediuminthe presenceofaselectiveagent(suchaskanamycinif theTDNAcontainsNPTII)andanantobiotictokill theAgrobacterium orslowitsgrowth(carbinecillin, cefotaxime,timitin).

MahmoudSakr,NRC,Egypt

HowisPlantTransformationwithAgrobacterium Done? Aftersomeperiodoftime,select g ( y ) regeneratedshoots(orembryos)thatare resistant. Screentheregeneratedshootsfor Screen the regenerated shoots for expressionofthescorablemarker. Rootshootsandproduceplants

MahmoudSakr,NRC,Egypt

HowisPlantTransformationwithAgrobacterium Done? Testthetransgenicplantsforthepresence p g andexpressionoftheintroducedgenes. Produceprogenyofthetransgenicplantand determinewhethertheintroducedgeneis determine whether the introduced gene is heritable.

MahmoudSakr,NRC,Egypt

Importantpoints: Important points:

Theprocessisinefficient;onlyasmallfraction g oftargetcellsaretransformed. DNAmustbeintegratedintoachromosome forstabletransformation. for stable transformation Eachtransformedcellisunique.

MahmoudSakr,NRC,Egypt

MahmoudSakr,NRC,Egypt

MahmoudSakr,NRC,Egypt

Inplanta transformationofArabidopsis In planta transformation of Arabidopsis

MahmoudSakr,NRC,Egypt

Vacuuminfiltration Vacuum infiltration

Vacuuminfiltrationwasdonebyplacingflowering plantsupsidedowninabeakerwithasolution containingAgrobacteriumtumefaciensand5% sucrose.Plantswereplacedinsuchawaythatonly inflorescencesweresubmerged. Thisbeakerandplantswereplacedinvacuum chamberandthevacuum(0.05bar)washeldfor severalminutes.

MahmoudSakr,NRC,Egypt

Floraldip Floral dip

Plantswereplacedinasimilarwayasvacuum pp infiltrationbutnovacuumwasapplied. Plantswerekeptinthesolutionforseveral minutesbeforeremoval. minutes before removal

MahmoudSakr,NRC,Egypt

Floralspray Floral spray

FloralspraywasdonebysprayingArabidopsis g flowerswithAgrobacteriumsolutions. Itismuchlessharmfultotheplantsandcan beappliedmanytimes. be applied many times

MahmoudSakr,NRC,Egypt

 Allofthemethodstransformfemalereproductive tissue. ti Reportsshowedthatonlytheapplicationof Agrobacteriumtopollenacceptorwillproduce transformants. ReportsalsoshowedthatGUSexpressioncanonly beobservedinovariesafterflowerswereinfected withAgrobacteriumcarryingACT11gusAIntron.

MahmoudSakr,NRC,Egypt

 Root elongation zone is the most highly Rootelongationzoneisthemosthighly susceptibleareatoAgrobacteriummediated transformation. t f ti Forfloraldip/spraymethods,visibleimmature p p y floralbudsarethemostsusceptibleto transformation. transformation

MahmoudSakr,NRC,Egypt

DirectDNATransfer
Microparticle bombardment Microparticlebombardment PEG/electroporationofprotoplasts Others

MahmoudSakr,NRC,Egypt

BiolisticTransformation Biolistic Transformation

DNAismixedwithmicroscopic(1to10micron )p g g diameter)particlesoftungstenorgoldand theDNAisprecipitatedontotheparticles. The DNA coated particles are placed on the TheDNAcoatedparticlesareplacedonthe endofalargerplasticbullet.

MahmoudSakr,NRC,Egypt

BiolisticTransformation Biolistic Transformation

Thebulletisloadedintoagunbarrelandthe g p y target planttissuethatyouwantto transform ispositionedattheendofthe barrel. barrel Thegunisfired,acceleratingthebullettothe endofthebarrel. f h b l

MahmoudSakr,NRC,Egypt

BiolisticTransformation Biolistic Transformation

Aplatewithasmallholeintheendstopsthe p plasticbullet. Thesmall,DNAcoatedparticlesmaintain momentumandpassthroughtheholeand momentum and pass through the hole and strikethetarget.

MahmoudSakr,NRC,Egypt

MahmoudSakr,NRC,Egypt

MahmoudSakr,NRC,Egypt

BiolisticTransformation Biolistic Transformation

Someoftheparticles willpassthroughthe cellwallandenter individualcells.

MahmoudSakr,NRC,Egypt

BiolisticTransformation Biolistic Transformation

SomeoftheDNAwillbereleasedfromthe p particle,endupinthenucleusandintegrate p g intoachromosome,resultingin transformation. transformation

MahmoudSakr,NRC,Egypt

BiolisticTransformation Biolistic Transformation

Multiplecopiesoftransgenecanleadto g silencing Consumablesareexpensive

MahmoudSakr,NRC,Egypt

ImportantPoints: Important Points:

Unlimitedhostrange. The tissue does not have to be totipotent. Thetissuedoesnothavetobetotipotent.

MahmoudSakr,NRC,Egypt

PEGMediatedTransformation PEG Mediated Transformation

Digestcellswithcellulasetogetprotoplasts PEGinducesreversiblepermeabilizationofthe p plasmamembrane Low viability of protoplasts Lowviabilityofprotoplasts Lowtransformationefficiency(12%) Difficulttoregenerate

MahmoudSakr,NRC,Egypt

MahmoudSakr,NRC,Egypt

 Therequirementofinvitro regenerationfor p protoplastandgeneguntransformationmay p g g y alsoleadtosomaclonalchanges,whichcanbe problematic. problematic

MahmoudSakr,NRC,Egypt

 Liposome
TargetedDNAencapsulatedinasphericallipid bilayertermedaliposome InthepresenceofPEG,endocytosisoccurs. After endocytosis the DNA is free to recombine Afterendocytosis,theDNAisfreetorecombine andintegratewiththehostgenome

MahmoudSakr,NRC,Egypt

 Electroporation
Intensive electrical field leads to pores on plasma Intensiveelectricalfieldleadstoporesonplasma membrane,allowingDNAtoenter Protoplast regeneration is still a problem Protoplastregenerationisstillaproblem Fieldstrength=Volt/cm(volumeofcuvette) p ( ) / Capacitance(F)=Resistance/time

MahmoudSakr,NRC,Egypt

 SiliconCarbideFibers
Usesiliconcarbidefiberstopunchholesthrough culturedplantcells Siliconcarbidefibersandculturedplantcellsare addedtoatubeandvortexedvigorously g y Themechanicalforcegeneratedbythevortex drivesthefibersintothecell drives the fibers into the cell

MahmoudSakr,NRC,Egypt

 Microinjection j
UsesfineglassneedlestoinjecttheforeignDNA directlyintothehostcell directly into the host cell DevelopedtoinjectDNAintoprotoplasts,cultured embryoniccellsuspensionsand embryonic cell suspensions and multicellularstructures Ti Timeconsuming i

MahmoudSakr,NRC,Egypt

 Desiccation
Driedembryoscanbemixedwithanutrient solutioncontainingtheforeignDNA solution containing the foreign DNA TheDNAshouldbetakenupastheembryo rehydratesandseedlingscanbegerminatedinthe rehydrates and seedlings can be germinated in the presenceofaselectionmediumtoassessthe incorporationoftheforeignDNA incorporation of the foreign DNA

MahmoudSakr,NRC,Egypt

Contents 1-Vaccines: 1 Vaccines: an overview 2-Gene based vaccines (GBV) C 3-Biotechnology in Agriculture: Why plants? 4-Plant transformation 5-Edible vaccines d bl 6 Open 6-Open discussion

3 3 EdibleVaccine
Ediblevaccinesareparticularity attractive in providing mucosal attractiveinprovidingmucosal immunity.Mucosalimmunity immunity.Mucosalimmunity isthe termfortheproductionofantibodies inthoseregionsofthebodythatare g y exposedtotheenvironmentsuchas themouth,stomachandintestines. themouth,stomachandintestines. Vaccinationswillbeassimpleand Vaccinations will be as simple and painlessaseatingapieceoffood!

Historicalbackground Thefirstreportoftheproductionof ediblevaccine(asurfaceproteinfrom Streptococcus)intobacco,at0.02%of St t )i t b t 0 02% f total leaf protein level, appeared in 1990 in the form of a patent in the form of a patent application published under the International Patent Cooperation Treaty Subsequently,anumberofattempts Subsequently a number of attempts were made to express various antigensinplants antigens in plants

Arntzen, director of the Arizona Biodesign Institute, is in the vanguardofthemovementtowardediblevaccines. Hehasconcludedthreeearlystageclinicaltrials usingpotatoes bearingvaccinesagainsthepatitisB,E.coliandtheNorwalkvirus. bearing vaccines against hepatitis B E coli and the Norwalk virus Concludedthatvaccine ladenfoodcantriggertheproductionof Concluded that vaccineladen food can trigger the production of antibodies,thebody'svirusfighters,buthehasyettoprovethey dosoataratecomparabletothatofaninjection(dose).

Others have followed Arntzen's lead. In Others have followed Arntzen s lead In 1998 researchers at researchers at California's Loma Linda University reduced the symptoms of juvenile diabetes in mice by 50% after feeding them potatoes carryinginsulin. Iowa State is developing corn that staves off intestinal I St t i d l i th t t ff i t ti l pathogens. MeristemTherapeutics,basedinFrance,isinclinicaltrialswith corngearedtowardcysticfibrosis.

DowChemicalandMonsantohavetakena slightly different approach by looking at waystogrowantibodiesincorn.

SanDiegobasedEpicyte Pharmaceutical g p y willbeginearlystageclinicaltrialsthisyear withaherpesmonoclonalantibodygrown incorn.

Potatoes,tomatoes,andbananas arecurrentlythetopthree candidates for transformation into edible vaccines. Each of thesethreevectorshasadvantagesanddisadvantages. th th t h d t d di d t

Potatoes, whichareeasilytransformedandpropagated,have dominated the clinical trials performed thus far. One problem withpotatoesisthatmanypeopledonotliketoeatthemraw, andcookingthemdenaturestheantigens,effectivelydestroying theirabilitytoinduceanimmuneresponse. their ability to induce an immune response

Tomato is a popular candidate because it is also easily transformed.Threeotherfactorsinthetomato'sfavorarethefact t f d Th th f t i th t t ' f th f t thatitgrowsrelativelyquickly,itcanbeeatenraw,andithasthe highestlevelofvitaminAamongthethreecandidates.VitaminA highest level of vitamin A among the three candidates. Vitamin A regulates transcription of the genes for antibody synthesis; this hadledtospeculationthatitsadministrationalongwithvaccines may boost immune response.

HIVantigenshavebeenexpressedtohighlevels(uptoapproximately40%ofthe plantstotalprotein)inplastidsoftobacco,anonfoodcrop,andtomato,afood cropwithanediblefruit.

Chebolu S,Daniell H.Chloroplastderivedvaccineantigensandbiopharmaceuticals:expression,folding,assemblyandfunctionality.Curr TopMicrobiol Immunol.2009;332:3354.

Banana, which has been advanced as a candidate because of Banana which has been advanced as a candidate because of its popularity with children, who are the main targets of the vaccines being developed. Bananas are also inexpensive to g p p produce, and they are normally eaten raw. In addition, bananas are native to many developing countries. One drawbacktobananasisthattheytaketwelvemonthstobear fruitaftertheyaretransformed.

Contents 1-Vaccines: an overview 2-Gene based vaccines (GBV) 3-Biotechnology in Agriculture: C Why plants? 4 Plant 4-Plant transformation 5-Edible vaccines 6-Open discussion 6O di i