DNA vaccine and DNA vaccine and Biopharmaceutical Biopharmaceutical

protein protein ProductionProduction

Theerapol Sirinarumitr DVM, P Theerapol Sirinarumitr DVM, Ph.D.h.D.

Department of Pathology, Fac Department of Pathology, Fac ulty of Veterinary Medicine ulty of Veterinary Medicine

Kasetsart University Kasetsart University

Background

TranscriptionTranscrip

tion

Translation

Translation

Prokaryotes Eukaryotes

HISTORYHISTORY

• Skeletal muscle cell can u Skeletal muscle cell can u ptake naked DNA (plasmi ptake naked DNA (plasmi

d carried genes) and expr d carried genes) and expr essed those genes essed those genes (Scienc(Scienc

-e 2 4 7 :1 4 6 5 1 4 6 -e 2 4 7 :1 4 6 5 1 4 6 8 1990, by Wolff et al.) 8 1990, by Wolff et al.)

AdvantagesAdvantages

• Readily delivery Readily delivery• Molecularly defined reagent Molecularly defined reagent• Neither infectious nor capab Neither infectious nor capab

le of replication le of replication• Elicited both humoral and ce Elicited both humoral and ce

- ll mediated immune respons- ll mediated immune responsee

AdvantagesAdvantages• Manipulate the resulting immune r Manipulate the resulting immune r

- esponse through the co deliverly o - esponse through the co deliverly o f genes encoding immunodulating f genes encoding immunodulating

cytokines or costimulatory molecu cytokines or costimulatory moleculesles

• - site directed mutagenesis for enh- site directed mutagenesis for enh ancing antigenic potency of indivi ancing antigenic potency of indivi

dual epitope or to abolish unwante dual epitope or to abolish unwante - d effects of wild type protein - d effects of wild type protein

Purpose of Purpose of vaccinationvaccination

•ProphylaxisProphylaxis•TherapeuticTherapeutic

– infectious agentinfectious agent– cancercancer

Gene delivery and Gene delivery and ExpressionExpression

• Routes of administration Routes of administration– epidermal, mucosal, intra epidermal, mucosal, intra

muscular and intravenous muscular and intravenous

• Skeletal muscle and skin Skeletal muscle and skin (Langerhans cell) (Langerhans cell)

• Plasmid DNA persisted e Plasmid DNA persisted e pisomally in myocytes pisomally in myocytes

• Intramuscular injection and pa Intramuscular injection and pa- rticle mediated delivery of plas- rticle mediated delivery of plas

mid DNA mid DNA• - Particle mediated delivery req- Particle mediated delivery req

uired up to 5000 times less DN uired up to 5000 times less DN A than intramuscular or intrad A than intramuscular or intrad

ermal inoculation of DNA (nano ermal inoculation of DNA (nano - gram vs 25 100 microgram) - gram vs 25 100 microgram)

• Transfection efficiency does not Transfection efficiency does not necessarily correlate with the ef necessarily correlate with the ef

ficiency of immunization ficiency of immunization• Intradermal injection (low trans Intradermal injection (low trans

fection efficiency) can elicit pot fection efficiency) can elicit pot ent immune response when co ent immune response when co

mpared to muscle (higher trans mpared to muscle (higher trans fection efficiency) fection efficiency)

•Site and method of DNA Site and method of DNA delivery may affect the nature delivery may affect the nature of the immune response of the immune response (skeletal muscle and skin)(skeletal muscle and skin)

LC and keratinocyte LC and keratinocyte

Take up and Take up and process antigenprocess antigen

Proinflammatory cytokines: Proinflammatory cytokines: - - -1IL , TNF a & GM CSF- - -1IL , TNF a & GM CSF

Muscle can notMuscle can not

* Dendritic cell * Dendritic cell LL**10 LL**10

• ID or IMinjection induce Th ID or IMinjection induce Th 1 bias of the immune respo 1 bias of the immune respo

- - nse INF G producing CD - - nse INF G producing CD 4 + IgG2 a isotype 4 + IgG2 a isotype

• Genegun(skinor muscl e) i nduces i mmun Genegun(skinor muscl e) i nduces i mmun e responses with Th2 bias e responses with Th2 bias

Muscle controlMuscle controlMuscle uptakeMuscle uptake

DNA vaccine’s DNA vaccine’s immunologyimmunology

Processing and Processing and PresentationPresentation

(8-12 amino acids)(8-12 amino acids)

12-25 amino acid12-25 amino acid

•Protein-based vaccine Protein-based vaccine elicitation of elicitation of strong CTLstrong CTL

• Th1-type Th1-type responsesresponses– CD8+ CTLCD8+ CTL– CD4+ Th1 cellsCD4+ Th1 cells– IgG2a>IgG1IgG2a>IgG1– IFN-g>IL-4,IL-IFN-g>IL-4,IL-

5,IL-105,IL-10– IL-2, IL-12IL-2, IL-12

• Th2-type Th2-type responsesresponses– DTHDTH– IL-4 and IL-IL-4 and IL-

1010

• Influenza,rabies,HIV,HBV,HCV&HSV Influenza,rabies,HIV,HBV,HCV&HSV• Mycobacterium tuberculo Mycobacterium tuberculo

sis, Mycoplasma pulmoni sis, Mycoplasma pulmoni s, and Borrelia burgdorfer s, and Borrelia burgdorfer

ii• Mal ari a andLei shmani a Mal ari a andLei shmani a

anmlinfc.htm

Genetic Genetic engineering of engineering of

immune responseimmune response

Engineering Engineering Immune ResponseImmune Response

• Costimulatory molecule Costimulatory molecule• Immunostimulatory cyto Immunostimulatory cyto

kinekine• Immunostimulatory DNA Immunostimulatory DNA

sequences (ISS) sequences (ISS)• Biological adjuvant Biological adjuvant

Costimulatory mole Costimulatory moleculescules

•B7.1 (CD28)B7.1 (CD28)•B7.2B7.2•CD40 (CD40L)CD40 (CD40L)

Enhance T-cell responseEnhance T-cell responseapoptosisapoptosis

Immunostimulatory Immunostimulatorycytokinescytokines

• - T cell growth factors- T cell growth factors– - LL-L2 & 7- LL-L2 & 7

• - Th 1 biasing cytokines - Th 1 biasing cytokines– - -12IFN a and IL- -12IFN a and IL

Immunomodulatory molecules Immunomodulatory molecules EffectEffect A. Cytokine proteins A. Cytokine proteins

-1IL-1IL Antibody (Ab) Antibody (Ab)-2IL-2IL AbAb-12IL-12IL Th1 (DTH) Th1 (DTH)LLL -LLLL -L Ab, DTH Ab, DTHL L -L LLL L -L LL AbAb

B. Expression plasmids B. Expression plasmids-IL 1 2-IL 1 2 CTL (i.m. & i.n .) CTL (i.m. & i.n .)

DTHDTHAbAb

-GM CSF-GM CSF AbAbCTLCTL

TCA3TCA3 CTL CTLDTHDTHAbAb

-B7 1-B7 1 CTLCTLDTHDTHAbAb

-72B -72B CTLCTLDTHDTHAbAb

CD40(L)CD40(L) AbAbCTLCTL

Immunostimulatory Immunostimulatory DNA sequences DNA sequences

• Unmethylated CpG dinucleotide Unmethylated CpG dinucleotidemotiffmotiff– In mammal DNA, CpG is suppressed by In mammal DNA, CpG is suppressed by

2 0 fold (bacteria and virus DNA= 1 2 0 fold (bacteria and virus DNA= 1/1 6 )/1 6 )

– - stimulateinnatei mmune system(non speci fi c anti mi - stimulateinnatei mmune system(non speci fi c anti mi crobialsystem) crobialsystem)

– sensi ti ze the speci fi c i mmune syst sensi ti ze the speci fi c i mmune syst embothanti body andcel l medi atedi embothanti body andcel l medi atedi

mmune response mmune response

– 1Th bias of immune respo 1Th bias of immune respo - nse IFN g producing C - nse IFN g producing C

L LLLLL LLL LLLLLLLLL L 4 L LLLLL LLL LLLLLLLLL L 4

cellcell– Cis and Trans effect Cis and Trans effect– - - - - -1 6INF a/b, IFN g, TNF a, IL , IL- - - - -1 6INF a/b, IFN g, TNF a, IL , IL

- -, IL 12, IL 18 - -, IL 12, IL 18

• - 12Type I interferons and IL - 12Type I interferons and IL LLLLLLLL LLLLLLLLLLLL LL L L LLLLLLLL LLLLLLLLLLLL LL L L

cells, CD8 + CTL cells, CD8 + CTL• - ’ () (., 199 7) - ’ () (., 199 7)• ISS can eliminate severe adver ISS can eliminate severe adver

se effects of Freund’s adjuvant se effects of Freund’s adjuvant

• at least one CpG (near terminal m at least one CpG (near terminal m uch less effective) uch less effective)

• - Methylation at C 5 position of the - Methylation at C 5 position of thecytosinecytosine

• Poly (dG, dC) activate macrop Poly (dG, dC) activate macrop hage and NK cell and stimulate an hage and NK cell and stimulate an

-tiviral response (IFN a/b) -tiviral response (IFN a/b)• Phosphorothioate modified ODNs Phosphorothioate modified ODNs

- - (ps ODNs) show 200 fold more po- - (ps ODNs) show 200 fold more po tent than Phosphodiester ODNs ( tent than Phosphodiester ODNs (

-po ODNs)-po ODNs)

IFN-g

- -5 ’ pu pu C - -5 ’ pu pu C- - G py py 3’- - G py py 3’

G A G A T T T T

CC

Optimal B cell activationOptimal B cell activation

IL-18IL-18

Class I MHCClass I MHCCD40CD40ICAM-1ICAM-1CD16/32CD16/32

(IgM)(IgM)IL-12

CD40, CD16/32CD40, CD16/32ICAM-1ICAM-1APCAPC

ChloroquineChloroquineTLR-6TLR-6

• DNAvaccine can elicitboth h DNAvaccine can elicitboth h umoral and cellular immune r umoral and cellular immune r

esponse (biolistic Th 2 esponse (biolistic Th 2bias)bias)

• - - Th 1 biasing cytokines (IFN a - - Th 1 biasing cytokines (IFN a - or IL 1 2 ) shiftbiolistic - or IL 1 2 ) shiftbiolistic

cutaneousi mmune response toTh1 cutaneousi mmune response toTh1

• Side effects Side effects– - - -Provocationofasepticshockl i ke syndrome (I L 1,TNF a - - -Provocationofasepticshockl i ke syndrome (I L 1,TNF a

-LL, ) -LL, )– LLLLLL L LLLLL LLLLLLLL L( LLLLLL L LLLLL LLLLLLLL L(

ather than ISS) ather than ISS)• - - -neutrophils, TNF a, IL 6 , MIP - - -neutrophils, TNF a, IL 6 , MIP

2 significantdamage 2 significantdamage

– Autoimmune disease Autoimmune disease• some evidence of bacterial DNA some evidence of bacterial DNA

-SLE (high IL 6 ) -SLE (high IL 6 )• certaintypesof i nfecti onmay contri bute to t certaintypesof i nfecti onmay contri bute to t

he development of autoimmune (exp he development of autoimmune (exp erimenti n l abani mal ) erimenti n l abani mal )

• potentially dangerous under conditio potentially dangerous under conditio ns of extreme autoimmune sensitivit ns of extreme autoimmune sensitivit

y (inhibit B cell apoptosis by ISS) y (inhibit B cell apoptosis by ISS)

– Reduction in antigen Reduction in antigen expression (induce expression (induce interferon)interferon)•too much ISS may inhibit too much ISS may inhibit immune responseimmune response

• Clinical applications Clinical applications– Allergy (Th 1 response Allergy (Th 1 response

LL L LLLLLL2 LL L LLLLLL2))

– - 2Infectious disease (Th bias i - 2Infectious disease (Th bias i mmune response) mmune response)

Biological adjuvant Biological adjuvantss

• Dendritic cell (DC) Dendritic cell (DC)– establishmentof cell culture (mous establishmentof cell culture (mous

-e spleen &bone marrow GM -e spleen &bone marrow GM -CSFand IL 4 ) -CSFand IL 4 )

– -34CD+(cordblood,bonemarrow,peri pheral bl ood) i nGMC -34CD+(cordblood,bonemarrow,peri pheral bl ood) i nGMCLL LLL LLL-L LL LLL LLL-L

– LLLLL L LLLLLLLL LLLLLLLL LL L L -LLLLLL L LLLLLLLL LLLLLLLL LL L L -L -4SFandI L -4SFandI L

DCDC

– Cancer vaccine Cancer vaccine• Cul turedDCwhenadopti vel y transfe Cul turedDCwhenadopti vel y transfe

rredcaneff ecti vel y present tumor a rredcaneff ecti vel y present tumor anti gennti gen

•- -Genebasedvacci ne VS.pepti de pul sed- -Genebasedvacci ne VS.pepti de pul sed– does not require prior knowledge of patient

- - HLA haplotype or of specific T cell epitope– - -may promote both class I and II restricted T

cell responses

• Vaccination with culture DC ex vi Vaccination with culture DC ex vivovo– viralvector(retrovirus,adenovirus,poxvi rus) viralvector(retrovirus,adenovirus,poxvi rus)– pl asmi dDNA pl asmi dDNA– RNARNA– circumvent the neutralizing antibodies circumvent the neutralizing antibodies

which is the problem ofusing adenoviru which is the problem ofusing adenoviru ses in vivo ses in vivo

Virus vectorsVirus vectors• Retrovirus vector

– express throughout the life of the cell– However,needcellsthat undergone cel l di vi si on(chro

mosomali ntegrati on)– CD34+progeni tor cel l– Transductionefficiency(11.5%cellfrombonemarrow&21.2%fromcordbl ood)

Retrovirus vector MFG, Retrovirus vector MFG, MLV LTR,MLV LTR,

in packaging cellin packaging cell Ecotropic CRE packaging cell line Ecotropic CRE packaging cell line

Ecotropic BOSC23 packaging cell line Ecotropic BOSC23 packaging cell line

Amphotropic CRIP packaging cell line Amphotropic CRIP packaging cell line

Amphotropic BING packaging cell line Amphotropic BING packaging cell line

Amphotropic PA317 packaging cell line Amphotropic PA317 packaging cell line

Amphotropic PG13 packaging cell line Amphotropic PG13 packaging cell line

• - HER 2 (Specht et al., 1997)- HER 2 (Specht et al., 1997)– CTL,prophylactic/therapeuticanti tumor i mmuni ty CTL,prophylactic/therapeuticanti tumor i mmuni ty

• -80Human CD and Murine IL -80Human CD and Murine IL 12 (Nishioka et al., 1997) 12 (Nishioka et al., 1997)

– LLLLLLLLL LLLLLL LL LLLLLLLLLLLLL LLLLLL LL LLLL• - 1Human MUC (Henderson e - 1Human MUC (Henderson e

t al., 1996) t al., 1996)– LLLL LLLLLLLLLLLLLL LLLLLLLLLL

•Human MART-1 (Reeves Human MART-1 (Reeves et al., 1996)et al., 1996)– Gene expression and Gene expression and antigen presentation in antigen presentation in vivovivo

• Adenovi ral vectors Adenovi ral vectors– nondividing cell nondividing cell– transient gene expression transient gene expression– immune response against vir immune response against vir

us itself us itself

• Multiplicity of infection (M.O.I) Multiplicity of infection (M.O.I)– -Adv type (100:1) 1 5% -Adv type (100:1) 1 5%– 10,000 95% 10,000 95%

• Physical method for gene Physical method for gene transfer are relatively ine transfer are relatively ine

fficient (DNA/liposome, el fficient (DNA/liposome, el ectroporation and calciu ectroporation and calciu

m phosphate precipitatio m phosphate precipitation)n)

- - - Adenoviral vector (E1 deleted, replication deficient, Ad 5 vector) - - - Adenoviral vector (E1 deleted, replication deficient, Ad 5 vector) CMV promoter/enhancer CMV promoter/enhancer or or LLLL LLLLLLLLLLLLL/ LLLL LLLLLLLLLLLLL/

40SV poly A termination signal 40SV poly A termination signal

• - - 2 7 1IL and IL (Authur et al.,- - 2 7 1IL and IL (Authur et al.,997)997)– gene expression gene expression

• (. ,1 997) (. ,1 997)– - CTL and anti tumor immunity - CTL and anti tumor immunity

• - 1( . ,1 997)- 1( . ,1 997)– - CTL and anti tumor immunity - CTL and anti tumor immunity

• Polyoma middle T antige Polyoma middle T antige n (Wan et al., 1997) n (Wan et al., 1997)

– - CTL and anti tumor immunity - CTL and anti tumor immunity

• Naked DNA Naked DNA– can transfection more than can transfection more than

one gene (immunostimulat one gene (immunostimulat ory cytokine) ory cytokine)

– without immunological inte without immunological inte rference from viral protein rference from viral protein

– no risk of recombination no risk of recombination

– No insertion of foreign DNA in No insertion of foreign DNA in to the genome to the genome

– can readily be produced in lar can readily be produced in larLL LLLLLLLLLL LLL LLLLLLLL LLLLLLLLLL LLL LLLLLL

– lower transduction efficiency lower transduction efficiency butstill can induce CTL butstill can induce CTL

Plasmid construction Plasmid construction

• 1995Humantyrosinase(melanoma) (Alijagicetal., ) 1995Humantyrosinase(melanoma) (Alijagicetal., )• - - 1 17 1MART /Melan A, pmel /gp- - 1 17 1MART /Melan A, pmel /gp

- - 00, , 1 3 ( ., 1998)tyrosinaseMAGE andMAGE Tutingetal - - 00, , 1 3 ( ., 1998)tyrosinaseMAGE andMAGE Tutingetal• - LLL1 6 7 5 3 ( .- LLL1 6 7 5 3 ( .

1997, ) 1997, )• - 1 1997HSV gB (Manickan et al.,- 1 1997HSV gB (Manickan et al.,

))anmlcancer.htm

AdjuvantAdjuvant

• Adjuvants for plasmid DN Adjuvants for plasmid DN A vaccines A vaccines

– Bupivacaine (myotoxin) Bupivacaine (myotoxin)– Polyvinyl pyrollidone (PVP) Polyvinyl pyrollidone (PVP)– Neutral, anionic and cationic l Neutral, anionic and cationic l

LLLLLLLL

•BupivacaineBupivacaine– destroys myofi ber cel l macroph destroys myofi ber cel l macroph

ages cl earance of debri s prol i f ages cl earance of debri s prol i f erati onof muscl e precursor cel l s erati onof muscl e precursor cel l s

– -enhancementonly occurs with a low effi -enhancementonly occurs with a low effi- ciency expressing plasmid- ciency expressing plasmid

– - better to use high expressing naked DN - better to use high expressing naked DN A(CMVpromotor) rather than use bupiv A(CMVpromotor) rather than use bupiv

acaineacaine

• Polyvinylpyrollidone(PVP) & DMRI E:DOPE cat i oni c l i pi d Polyvinylpyrollidone(PVP) & DMRI E:DOPE cat i oni c l i pi d– Controversy (increase or decrease exp Controversy (increase or decrease expression)ression)

• DMRIE:DOPE (enhance immune r DMRIE:DOPE (enhance immune r esponse, decrease transgene ex esponse, decrease transgene ex

L LL LL LLL LL LLLL LL L) L LL LL LLL LL LLLL LL L) elease plasmid DNA for uptake b elease plasmid DNA for uptake b

L L LLLLL LLL LLLLLLL LLLLLL L LLLL L LLLLL LLL LLLLLLL LLLLLL L LLLLLLL

anmlalrgy.htm

DNA vaccine for human medicineDNA vaccine for human medicine

DNA vaccine for DNA vaccine for Veterinary UseVeterinary Use

BiopharmaceuticBiopharmaceutical farmingal farming

Biopharmaceutical Biopharmaceutical farmingfarming

• Transgeni c ani mal Transgeni c ani mal• Edible vaccine and biologica Edible vaccine and biologica

l products l products– Transgenic plants Transgenic plants– Chimeric plant virus Chimeric plant virus

Transgenic anima Transgenic animall

ObjectivesObjectives• Economically important trait i Economically important trait i

mprovementmprovement– -Porcine growth hormone hMt pGH -Porcine growth hormone hMt pGH– - -Sheepkerati n I GF I - -Sheepkerati n I GF I

•BiomedicineBiomedicine– Pharmaceuti cal Protei ns Pharmaceuti cal Protei ns– L( L( hDAFdecay accelerating factor &hC hDAFdecay accelerating factor &hC

D5 9 transgenic pig D5 9 transgenic pig ) )– Recombi nant anti bodi es Recombi nant anti bodi es

Strategies for Strategies for delivery genedelivery gene

• Microinjection Microinjection– - 01Fertilized embryo [( . %) in cattle ( - 01Fertilized embryo [( . %) in cattle (

5 % ) in mice 5 % ) in mice– LL LLLLL LL LLLLLL LLLLLLLL ( ) LL LLLLL LL LLLLLL LLLLLLLL ( )

LLL LLLLLLLLLLLL LLLLLLLLL• Nuclear transfer Nuclear transfer 1(Schnieke et al., 1(Schnieke et al.,

BBBB9 9 7 ; . , 1 9 9 8 ; BBBB9 9 7 ; . , 1 9 9 8 ; 1999isi et al., ) 1999isi et al., )

– embryonic stem cell (ES, EG, EC) embryonic stem cell (ES, EG, EC)– somatic cell (cattle, sheep, goat, pig) somatic cell (cattle, sheep, goat, pig)

• Sperm mediated DNA transfer Sperm mediated DNA transfer (( 1998 19Gandolfi, ; Squires, 1998 19Gandolfi, ; Squires,

9999

• Intracytoplasmic injection ICSI Intracytoplasmic injection ICSI of transgenic sperm heads of transgenic sperm heads (Perryetal., 19 (Perryetal., 1999)99)

• Retroviral vectors either by inje Retroviral vectors either by inje ction or infection of oocytes or ction or infection of oocytes or

embryos embryos (HaskellandBowen, 1995; Chanetal., 1998) (HaskellandBowen, 1995; Chanetal., 1998)

Nuclear transferNuclear transfer : : Somatic cell (fibroblast)Somatic cell (fibroblast)

Control of gene Control of gene expressionexpression

• Induciblegene(metallothioneinpromoter,heatshock promoter,steroi d Induciblegene(metallothioneinpromoter,heatshock promoter,steroi d responsive elements, tetracycl responsive elements, tetracycl

ine regulatable system) ine regulatable system)• I nternal ri bosome entry si t I nternal ri bosome entry si t

es I RESanddi ci stroni c RNAs es I RESanddi ci stroni c RNAs• Artificialchromosomes(YAC,MAC) Artificialchromosomes(YAC,MAC)

Mammary specific Mammary specific regulatory regulatory sequencesequence• Murine whey acid protein Murine whey acid protein

(WAP)(WAP)• -Bovine alpha lactalbumin -Bovine alpha lactalbumin• - -Bovine alpha s1 casein - -Bovine alpha s1 casein• -Ovi ne beta l actogl obul i n -Ovi ne beta l actogl obul i n• -Caprine beta casein -Caprine beta casein

-(Alpha glucosidase)-(Alpha glucosidase)

Species selectionSpecies selection

Transgenic mice is an ideal system to test expression constructs: mammal milk Transgenic mice is an ideal system to test expression constructs: mammal milk promotor sequences capable of expression in mouse promotor sequences capable of expression in mouse

Cost of transgenic a Cost of transgenic a nimal production nimal production

• 12One transgenic mouse (~ US$ 12One transgenic mouse (~ US$ 0) 0)

• 250One transgenic pig (~ US$ , 250One transgenic pig (~ US$ ,00)00)

• ( ~ 6 0 , ( ~ 6 0 ,000)000)

• ( ~ 5 4 6 , ( ~ 5 4 6 ,000)000)

Wall et al., 1992Wall et al., 1992

AnticoagulantAnticoagulant

inactivating factors V inactivating factors Vaa & VIII & VIIIaa

PACE (paired basic amino acid PACE (paired basic amino acid - cleaving enzyme) = subtilisin like - cleaving enzyme) = subtilisin like

serine protease furin serine protease furin

Bigenic mouse

Production of antibodiesProduction of antibodies

Purification of Purification of recombinant recombinant

proteinprotein•Standard dairy procedure (for Standard dairy procedure (for antibody)antibody)– casein-free, fat-free and lactose-free casein-free, fat-free and lactose-free protein concentrate (30-60% purity)protein concentrate (30-60% purity)

– ion exchange chromatography, ion exchange chromatography, hydrophobic interaction hydrophobic interaction chromatography and affinity chromatography and affinity chromatography (Protein A or chromatography (Protein A or Protein G)Protein G)

Transgenic plant Transgenic plant

Trangenic plant Trangenic plant

•Cholera vaccineCholera vaccine•HBV, FMDV, Norwalk HBV, FMDV, Norwalk

virus, RSV, TGEV, Rabies virus, RSV, TGEV, Rabies virus, PRRSVvirus, PRRSV

FMDVFMDV

Chimeric plant virus Chimeric plant virus

•VaccineVaccine– - 141HIV gp , Rabiesvirus, HCV, Cani ne par vovi r us- 141HIV gp , Rabiesvirus, HCV, Cani ne par vovi r us– S. aureus S. aureus , , P. aeruginosa P. aeruginosa

• Monoclonal antibody producti Monoclonal antibody productionon– - - -171 7332CO A(againstcolorectalcancerassociatedantigenGA )- - -171 7332CO A(againstcolorectalcancerassociatedantigenGA )– LLLLLL LLLLLLLLLLL LLLLL

Chimeric plant virus Chimeric plant virus

• TMV(Tobaccomosai c vi rus) TMV(Tobaccomosai c vi rus)• CPMV(Cowpeamosai c vi rus) CPMV(Cowpeamosai c vi rus)• AlMV(Alfalfa mosaic virus) AlMV(Alfalfa mosaic virus)• PPV(Plum poty virus) PPV(Plum poty virus)

HCVHCV

HIV V3 HIV V3 looploop

Rabies virusRabies virus

Outer membrane F protein Outer membrane F protein of of P. aeruginosa P. aeruginosa