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6/2006 6/2006
JUSSI JO
ENSU
U Production of F4 Fim
brial Adhesin in Plants: A
Model for O
ral Porcine Vaccine against Enterotoxigenic Escherichia coli
Production of F4 Fimbrial Adhesin in Plants:A Model for Oral Porcine Vaccine against
Enterotoxigenic Escherichia coli
Dissertationes bioscientiarum molecularium Universitatis Helsingiensis in Viikki
JUSSI JOENSUU
Department of Applied BiologyFaculty of Agriculture and Forestry
andDepartment of Biological and Environmental Sciences
Faculty of Biosciencesand
Viikki Graduate School in BiosciencesUniversity of Helsinki
Recent Publications in this Series:
10/2005 Katja PihlainenLiquid Chromatography and Atmospheric Pressure Ionisation Mass Spectrometry in Analysing Drug Seizures11/2005 Pietri PuustinenPosttranslational Modifications of Potato Virus A Movement Related Proteins CP and VPg12/2005 Irmgard SuominenPaenibacillus and Bacillus Related to Paper and Spruce Tree13/2005 Heidi HyytiäinenRegulatory Networks Controlling Virulence in the Plant Pathogen Erwinia Carotovora Ssp. Carotovora14/2005 Sanna JanhunenDifferent Responses of the Nigrostriatal and Mesolimbic Dopaminergic Pathways to Nicotinic Receptor Agonists15/2005 Denis KainovPackaging Motors of Cystoviruses16/2005 Ivan PavlovHeparin-Binding Growth-Associated Molecule (HB-GAM) in Activity-Dependent Neuronal Plasticity inHippocampus17/2005 Laura SeppäRegulation of Heat Shock Response in Yeast and Mammalian Cells18/2005 Veli-Pekka JaakolaFunctional and Structural Studies on Heptahelical Membrane Proteins19/2005 Anssi RantakariCharacterisation of the Type Three Secretion System in Erwinia carotovora20/2005 Sari AiraksinenRole of Excipients in Moisture Sorption and Physical Stability of Solid Pharmaceutical Formulations21/2005 Tiina HildenAffinity and Avidity of the LFA-1 Integrin is Regulated by Phosphorylation22/2005 Ari Pekka MähönenCytokinins Regulate Vascular Morphogenesis in the Arabidopsis thaliana Root23/2005 Matias PalvaInteractions Among Neuronal Oscillations in the Developing and Adult Brain24/2005 Juha T. HuiskonenStructure and Assembly of Membrane-Containing dsDNA Bacteriophages25/2005 Michael StefanidakisCell-Surface Association between Progelatinases and ß2 Integrins: Role of the Complexes in LeukocyteMigration26/2005 Heli KansanahoImplementation of the Principles of Patient Counselling into Practice in Finnish Community Pharmacies1/2006 Julia PerttiläExpression, Enzymatic Activities and Subcellular Localization of Hepatitis E Virus and Semliki Forest VirusReplicase Proteins2/2006 Tero WennbergComputer-Assisted Separation and Primary Screening of Bioactive Compounds3/2006 Katri MäkeläinenLost in Translation: Translation Mechanisms in Production of Cocksfoot Mottle Virus Proteins4/2006 Kari KreanderA Study on Bacteria-Targeted Screening and in vitro Safety Assessment of Natural Products5/2006 Gudrun WahlströmFrom Actin Monomers to Bundles: The Role of Twinfilin and a-Actinin in Drosophila melanogasterDevelopment
Helsinki 2006 ISSN 1795-7079 ISBN 952-10-3097-6
Production of F4 Fimbrial Adhesin in Plants: a Model for Oral Porcine Vaccine against
Enterotoxigenic Escherichia coli
Jussi Joensuu
Department of Applied BiologyFaculty of Agriculture and Forestry
andDepartment of Biological and Environmental Sciences
Division of GeneticsFaculty of Biosciences
andViikki Graduate School in Biosciences
University of HelsinkiFinland
Academic dissertation
To be presented, with the permission of the Faculty of Biosciences of the University of Helsinki, for public criticism, in the auditorium 1041 at Viikki Biocenter (Viikinkaari 5, Helsinki), on May 26th 2006, at 12 o’clock noon.
Supervised by Docent Viola Niklander-Teeri Department of Applied Biology University of Helsinki Finland
Reviewed by FaT Anneli Ritala VTT Biotechnology Espoo Finland
and
Professor Airi Palva Department of Basic Veterinary Sciences University of Helsinki Finland
Opponent Professor Paul Christou Department of Plant Production and Forest Science University of Lleida Spain
Cover: Detection of FaeG protein in isolated chloroplasts of transgenic (left) and nontransgenic plants (right).
ISBN: 952-10-3097-6 (paperback)ISBN: 952-10-3098-4 (PDF, online)ISSN: 1795-7079 (paperback)ISSN: 1795-8229 (PDF, online)
Edita Prima OyHelsinki 2006
Anima sana in corpore sano.
CONTENTS
ABBREVIATIONS .................................................................................................. 6LIST OF ORIGINAL PUBLICATIONS ............................................................... 8ABSTRACT .............................................................................................................. 9TIIVISTELMÄ ...................................................................................................... 101 INTRODUCTION ............................................................................................112 LITERATURE REVIEW ................................................................................ 12
2.1 Plant-produced vaccine antigens for animal use .................................. 122.1.1 Preface .............................................................................................. 122.1.2 Vaccine antigen targets ..................................................................... 252.1.3 Plant species utilized for vaccine antigen expression ....................... 252.1.4 Properties of host plant tissues for antigen production ..................... 262.1.5 Plant vaccine production platforms .................................................. 26
2.1.5.1 Transgenic plants ..................................................................... 272.1.5.2 Transient expression with viral vectors ................................... 282.1.5.3 Transient expression with agroinfi ltration ............................... 28
2.1.6 Optimizing antigen yield in transgenic plants .................................. 292.1.6.1 Transcription and mRNA stability ........................................... 292.1.6.2 Translation ............................................................................... 302.1.6.3 Subcellular targeting: optimal yield and glycosylation .......... 312.1.6.4 Fusion proteins ......................................................................... 322.1.6.5 Transplastomy .......................................................................... 32
2.1.7 Plant-produced candidate vaccines for animal health ...................... 332.1.7.1 Applications for avian diseases ................................................ 332.1.7.2 Applications for bovine diseases .............................................. 342.1.7.3 Applications for canine diseases .............................................. 352.1.7.4 Applications for porcine diseases ............................................. 362.1.7.5 Applications for other animal diseases .................................... 36
2.2 Escherichia coli postweaning diarrhea in pigs ...................................... 382.2.1 Preface .............................................................................................. 382.2.2 Virulence markers of ETEC in PWD ............................................... 392.2.3 Virulence factors of ETEC in PWD ................................................. 39
2.2.3.1 Colonization factors ................................................................. 392.2.3.2 Toxins ....................................................................................... 40
2.2.4 Prevention of porcine PWD .............................................................. 412.2.4.1 Hygiene and diet ...................................................................... 422.2.4.2 Breeding of resistant pigs ........................................................ 422.2.4.3 Antimicrobial medication ........................................................ 432.2.4.4 Passive immunotherapy ........................................................... 432.2.4.5 Vaccination .............................................................................. 43
2.2.5 ETEC F4 fi mbria ............................................................................. 442.2.5.1 Structure and assembly ............................................................ 442.2.5.2 F4 variants ................................................................................ 462.2.5.3 F4 receptor ............................................................................... 472.2.5.4 Immunogenicity of F4 fi mbriae ............................................... 48
3 AIMS OF THE STUDY ................................................................................... 524 MATERIAL AND METHODS ....................................................................... 535 RESULTS AND DISCUSSION ....................................................................... 54
5.1 Candidate plant species for this study ................................................... 545.1.1 Tobacco ............................................................................................. 545.1.2 Alfalfa ............................................................................................... 545.1.3 Barley ............................................................................................... 54
5.2 Analysis of transgenic plants producing FaeG protein ........................ 555.2.1 Tobacco: optimization of FaeG production by subcellular targeting .. 55
5.2.1.1 Transgene copy number ........................................................... 555.2.1.2 mRNA accumulation ................................................................ 555.2.1.3 FaeG accumulation .................................................................. 575.2.1.4 Subcellular localization of FaeG .............................................. 585.2.1.5 N-glycosylation of FaeG protein ............................................. 58
5.2.2 Alfalfa: accumulation of FaeG in an edible crop plant ..................... 605.2.3 Barley: FaeG production in seed endosperm .................................... 615.2.4 FaeG production yield compared to other vaccine antigens
expressed in plants ........................................................................... 635.3 Stability of F4 fi mbriae and FaeG in gastrointestinal conditions ....... 64
5.3.1 pH in the porcine stomach ................................................................ 645.3.2 pH in the porcine intestine ................................................................ 645.3.3 F4 stability in low pH ....................................................................... 645.3.4 F4 stability in simulated gastric fl uid .............................................. 655.3.5 F4 stability in simulated intestinal fl uid ........................................... 655.3.6 Stability of plant-produced FaeG in simulated gastric fl uid ............. 655.3.7 Stability of plant-produced FaeG in simulated intestinal fl uid ......... 66
5.4 Receptor binding of F4 fi mbriae and pFaeG ........................................ 675.5 Immunogenicity of plant-produced FaeG ............................................ 69
5.5.1 Immunogenicity of N-glycosylated erFaeG ..................................... 695.5.2 Immunogenicity of pFaeG ............................................................... 69
5.5.2.1 Systemic immune response ...................................................... 705.5.2.2 Mucosal immune response ....................................................... 71
6 CONCLUSIONS AND FUTURE PROSPECTS ........................................... 74ACKNOWLEDGMENTS ..................................................................................... 76REFERENCES ....................................................................................................... 77
6
ABBREVIATIONS
AlMV Alfalfa mosaic virusAPC antigen presenting cellapFaeG apoplast-targeted FaeG expressed in plant
BHV Bovine herpesvirus
cAMP cyclic adenosine monophosphateCaMV Caulifl over mosaic viruscGMP cyclic guanosine monophosphateCPMV Cowpea mosaic virusCPV Canine parvovirusCT cholera toxin of Vibrio choleraeCT-A cholera toxin A subunit of Vibrio choleraeCT-B cholera toxin B subunit of Vibrio cholerae
DNA deoxyribonucleic aciddppi days post primary immunization
E. coli Escherichia coliEAST1 heat-stabile enterotoxin of Escherichia coliEHEC enterohemorrhagic Escherichia coliEPEC enteropathogenic Escherichia coliER endoplasmic reticulumerFaeG ER-targeted FaeG expressed in plantETEC enterotoxigenic Escherichia coli
FaeG major subunit and adhesin of Escherichia coli F4 fi mbriaF4 F4 fi mbria of Escherichia coliF4+ ETEC enterotoxigenic Escherichia coli possesing F4 fi mbriaF4R pig intestinal receptor for F4 fi mbriaFMDV Foot-and-mouth disease virus
GFP green fl uorescence protein of Aequorea victoriaGUS beta glucuronidase of Escherichia coli
HBsAg Hepatitis B virus surface antigen
IBDV Infectious bursal disease virusIBV Infectious bronchitis virus
LT heat-labile enterotoxin of Escherichia coliLT-A heat-labile enterotoxin subunit A of Escherichia coliLT-B heat-labile enterotoxin subunit B of Escherichia coli
7
MHC major histocompatibility complexmRNA messenger RNA
PAGE polyacrylamide gel electrophoresisPEDV Porcine epidemic diarrhea viruspFaeG plastid-targeted (chloroplast) FaeG expressed in plantPPV Plum pox potyvirusPVX Potato virus XPWD postweaning diarrhea
rFaeG recombinant FaeG expressed in cytoplasm of E. coliRHDV Rabbit hemorraghic disease virusRNA ribonucleic acid
SDS sodium dodecyl sulfateSGF simulated gastric fl uidSIF simulated intestinal fl uidSTa heat-stabile enterotoxin of Escherichia coliSTb heat-stabile enterotoxin of Escherichia coli
TEV Tobacco etch virusTGEV Transmissible gastroenteritis virusTMV Tobacco mosaic virusTSP total soluble protein
vsp vegetative storage protein of soybean
Amino acids are described by three letter code.
8
LIST OF ORIGINAL PUBLICATIONS
This thesis is based on the following original articles referred in the text by their Roman numerals. Some unpublished data is also included.
I
Snoeck V, Cox E, Verdonck F, Joensuu JJ and Goddeeris BM (2004) Infl uence of pH and gastric digestion on antigenicity of F4 fi mbriae for oral immunization. Veterinary Microbiology 98:45-53.
II
Joensuu JJ, Kotiaho M, Riipi T, Snoeck V, Palva ET, Teeri TH, Lång H, Cox E, Goddeeris BM and Niklander-Teeri V (2004) Fimbrial subunit protein FaeG expressed in transgenic Tobacco inhibits the binding of F4ac enterotoxigenic Escherichia coli to porcine enterocytes. Transgenic Research 13: 295-298.
III
Joensuu JJ, Kotiaho M, Teeri TH, Valmu L, Nuutila AM, Oksman-Caldentey K-M and Niklander-Teeri V (2006) Glycosylated F4 (K88) fi mbrial adhesin FaeG expressed in barley endosperm induces ETEC-neutralizing antibodies in mice. Transgenic Research, in press.
IV
Joensuu JJ, Verdonck F, Ehrström A, Peltola M, Siljander-Rasi H, Nuutila AM, Oksman-Caldentey K-M, Teeri TH, Cox E, Goddeeris BM and Niklander-Teeri V (2006) F4 (K88) fi mbrial adhesin FaeG expressed in alfalfa reduces F4+ enterotoxigenic Escherichia coli excretion in weaned piglets. Vaccine 24:2387-2394.
Publications reprinted with a kind permission from Elsevier Science (I and IV) and Springer Science and Business Media (II and III).
9
ABSTRACT
F4 fi mbriae of enterotoxigenic Escherichia coli (ETEC) are highly stable multimeric structures with a capacity to evoke mucosal immune responses. With these characters F4 offer a unique model system to study oral vaccination against ETEC-induced porcine postweaning diarrhea. Postweaning diarrhea is a major problem in piggeries worldwide and results in signifi cant economic losses. No vaccine is currently available to protect weaned piglets against ETEC infections. Transgenic plants provide an economically feasible platform for large-scale production of vaccine antigens for animal health.
In this study, the capacity of transgenic plants to produce FaeG protein, the major structural subunit and adhesin of F4 fi mbria, was evaluated. Using the model plant tobacco, the optimal subcellular location for FaeG accumulation was examined. Targeting of FaeG into chloroplasts offered a superior accumulation level of 1% of total soluble proteins (TSP) over the other investigated subcellular locations, namely, the endoplasmic reticulum and the apoplast. Moreover, we determined whether the FaeG protein, when isolated from its fi mbrial background and produced in a plant cell, would retain the key properties of an oral vaccine, i.e. stability in gastrointestinal conditions, binding to porcine intestinal F4 receptors (F4R), and inhibition of the F4-possessing (F4+) ETEC attachment to F4R. The chloroplast-derived FaeG protein did show resistance against low pH and proteolysis in the simulated gastrointestinal conditions and was able to bind to the F4R, subsequently inhibiting the F4+ ETEC binding in a dose-dependent manner.
To investigate the oral immunogenicity of FaeG protein, the edible crop plant alfalfa was transformed with the chloroplast-targeting construct and equally to tobacco plants, a high-yield FaeG accumulation of 1% of TSP was obtained. A similar yield was also obtained in the seeds of barley, a valuable crop plant, when the FaeG-encoding gene was expressed under an endosperm-specifi c promoter and subcellularly targeted into the endoplasmic reticulum. Furthermore, desiccated alfalfa plants and barley grains were shown to have a capacity to store FaeG protein in a stable form for years. When the transgenic alfalfa plants were administred orally to weaned piglets, slight F4-specifi c systemic and mucosal immune responses were induced. Co-administration of the transgenic alfalfa and the mucosal adjuvant cholera toxin enhanced the F4-specifi c immune response; the duration and number of F4+ E. coli excretion following F4+ ETEC challenge were signifi cantly reduced as compared with pigs that had received nontransgenic plant material. In conclusion, the results suggest that transgenic plants producing the FaeG subunit protein could be used for production and delivery of oral vaccines against porcine F4+ ETEC infections. The fi ndings here thus present new approaches to develop the vaccination strategy against porcine postweaning diarrhea.
10
TIIVISTELMÄ
Kasveissa tuotettu syötävä mallirokote porsaiden vierotusripuliin
Enterotoksigeeniset Escherichia coli -kannat (ETEC) ovat yleisiä porsas- ja sikaripulin aiheuttajia ympäri maailmaa. Ripuli aiheuttaa paitsi kärsimystä porsaille, myös tuotannon heikkenemistä kasvun hidastuessa ja ääritapauksissa porsaskuolemia. ETEC-kantojen tuottamat myrkyt muuttavat suolen nestetasapainoa ja aiheuttavat ripulin. Avainasemassa taudin puhkeamisessa on ETEC-bakteereiden kyky tarttua suoliston pintaan hiusmaisten proteiinirakenteiden, fi mbrioiden avulla. F4-fi mbria on yleisin porsasripulia aiheuttavista ETEC-kannoista tavattava fi mbriatyyppi. Se koostuu sadoista yhteenketjuttuneista FaeG-alayksikköproteiineista. Imetysporsaiden ripuli-infektioita pystytään ehkäisemään rokottamalla emakoita, jolloin ternimaidossa välittyvät vasta-aineet suojaavat porsaiden suolistoa passiivisesti. Vierotusporsaiden tavanomainen rokottaminen F4-koliripulia vastaan on tehotonta, koska se ei käynnistä suolistoa suojaavaa paikallista vasta-ainetuotantoa. Sen sijaan aiempi infektio tai suun kautta annettu F4-fi mbria- tai FaeG-alayksikköproteiini aktivoivat suoliston vasta-ainetuotannonja ehkäisevät F4-koli-infektioita.
Tässä työssä tuotettiin E. colin F4-fi mbrian FaeG-alayksikköproteiinia siirtogeenisissä kasveissa. Ensin määritettiin FaeG-tuottotason kannalta optimaalinen paikka kasvisolussa tupakkakasvin avulla (siirtogeenitekniikan mallikasvi). Kun rokoteproteiini ohjattiin viherhiukkasiin, siirtogeeniset tupakkakasvit tuottivat sitä 1% kaikesta liukoisesta proteiinistaan. Kasvissa tuotettu rokoteproteiini osoittautui kestäväksi maha- ja suolinestekokeissa ja se pystyi tarttumaan suolinukan F4-reseptoreihin ja ehkäisi samalla F4-kolibakteereiden kiinnittymistä suoliepiteeliin. Porsaiden syöttökokeita varten FaeG-rokoteproteiinia tuotettiin siirtogeenisessä sinimailasessa. Sinimailaset tuottivat rokoteproteiinia viherhiukkasissa 1% liukoisesta proteiinistaan. Ohran jyvissä saavutettiin yhtä korkea tuottotaso, kun FaeG kohdennettiin tärkkelysendospermiin solukkospesifi sen geenisäätelyalueen avulla. Kasvissa tuotettu rokoteproteiini todettiin kestäväksi ja se säilyi muuttumattomana kuivatussa sinimailasessa ja ohran jyvissä ainakin kahden vuoden ajan. Porsaskokeessa vierotetuille porsaille annettiin rokoteproteiinia sisältävää sinimailasta. Kasvirokote sai porsaissa aikaan heikon F4-spesifi sen seerumin vasta-ainereaktion, jota pystyttiin vahvistamaan tehosteaineella. Kun koeporsaat altistettiin tautia-aiheuttavalla F4 ETEC-kannalla, tehostettu rokoteproteiini pystyi myös vähentämään ulosteessa erittyvien F4-kolibakteereiden määrää merkittävästi. Yhteenvetona todetaan, että FaeG-rokoteproteiinia pystyttiin tuottamaan tehokkaasti rehukasveissa siirtogeenitekniikan avulla. Kasvissa tuotettu syötävä mallirokote vähensi vierotusporsaiden F4-koli-infektioiden vakavuutta. Nämä tulokset avaavat uusia mahdollisuuksia porsaiden vierotusripulin ehkäisemiseksi tulevaisuudessa.
11
1 INTRODUCTION
Postweaning diarrhea (PWD) caused by enterotoxigenic Escherichia coli possessing F4 fi mbriae (F4+ ETEC) is a common problem on pig farms worldwide, leading to severe economic losses due to mortality and reduced growth rates. F4 fi mbriae are long proteinaceous appendages on the surface of F4+ ETEC that are mainly composed of identical repeating protein subunits called FaeG. The major subunit FaeG is also an adhesin and allows ETEC to adhere to F4-specifi c receptors on porcine small intestinal enterocytes, resulting in colonization, toxin production, and subsequent diarrhea.
To prevent the outbreak of ETEC infections in suckling piglets, sows can be vaccinated parenterally with attenuated/killed ETEC bacteria or purifi ed F4 fi mbriae to enable protective IgA antibodies to be transmitted via colostrum. Because this passive protection no longer exists for weaned piglets, they become susceptible to ETEC infections. Parenteral vaccination of weaned piglets is not effi cient since it stimulates a systemic rather than an intestinal F4-specifi c immune response. By contrast, oral administration of piglets with purifi ed F4 fi mbriae or adhesin FaeG induces a F4-specifi c mucosal immune response.
Here, it was evaluated whether immunogenic F4 adhesin FaeG could be produced in transgenic plants, and whether crop plants carrying this antigen could be used as a delivery vehicle for an oral vaccine against PWD. The literature review covers the current status of plant-produced vaccines and concentrates on the applications developed for animal health. Porcine PWD caused by F4+ ETEC is also reviewed.
Introduction
12
Literature review
2 LITERATURE REVIEW
2.1 Plant-produced vaccine antigens for animal use
2.1.1 PrefaceFor more than 2000 years, plants have provided the material for the majority of our medicinal products. Plants have unique metabolic diversity and produce thousands of proteins and metabolites as protection against their own pests and diseases. Many of these proteins include potentially important therapeutic treatments for human and animal diseases. The era of modern biotechnology has afforded us the opportunity to develop and produce protein pharmaceuticals in plants. Plants offer a means of inexpensive biopharmaceutical production without sacrifi cing product quality or safety. Following the footsteps of the fi rst plant-produced technical proteins (β-glucuronidase, avidin, trypsin (Prodigene, www.prodigene.com) and interferons α-2a and α-2ab (Large Scale Biology, www.lsbc.com), the fi rst pharmaceuticals will soon be available for commercial production. Medically relevant proteins expressed in plants can roughly be divided into three functional categories: (i) vaccines, (ii) recombinant antibodies, and (iii) other medical proteins, including blood products, growth factors, cytokines, enzymes, and structural proteins. The two latter cathegories will not be discussed in any detail here.
After the introduction of the concept of transgenic plants as edible vaccines in 1992 by Arntzen and colleagues, the fi eld of plant-based vaccines has increasingly attracted the attention of scientists. To date, over 150 articles describing original results on the expression of vaccine
antigens in plants have been published (see Table 1). Six phase I clinical human studies with plant-produced oral vaccine candidates have been established (Tacket et al., 1998, Kapusta et al., 1999, Tacket et al., 2000, Yusibov et al., 2002, Tacket et al., 2004, Thanavala et al., 2005). After initial rush, it soon became clear that the goal of locally produced edible plant vaccines (e.g. in bananas) to relieve the health issues in developing countries has many obstacles to conquer. Firstly, the transgenic plants must be shown to be a competitive production platform offering advantages over the current manufacturing practices. The benefi ts of the easy upscaling and the low cost of the starting material are clear, but before commercialization the plant platform has several technical issues, such as the mammalian-like glycosylation, to overcome. Secondly, the intellectual property rights and the regulatory framework concerning the transgenic plants as a production platform and the derived medical products require clarifi cation.
Although the majority of putative vaccine candidates expressed in transgenic plants are against human pathogens, the stringent quality standards for human medical products and the expensive and time-consuming phase II and III clinical trials for human vaccine candidates will likely result in the fi rst plant-produced vaccines reaching the market being for veterinary use, since the regulatory burden of such products is
13
Literature review
smaller (Moulin, 2005). Indeed, the company Dow AgroSciences (www.dowagro.com) has forecasted the release of a plant-produced poultry vaccine sometime in 2006. To be successful, the dosage cost of veterinary vaccines must be minimal. Moreover, economical production and delivery are critical in the development of such products (especially against diseases that are not lethal but diminish animal welfare and growth rate). Animal enteric diseases are a particularly attractive target for edible plant-produced vaccines. Optimal protective mucosal immune response will be achieved when the vaccine is administered by the same route as the
infection enters the body (Dougan, 1994). Vaccine antigens for animal health can be produced cost-effectively in feed plants, and the material can be delivered without extensive purifi cation, which is unlikely to be the case with products intended for human use. Published studies of vaccine candidates expressed in plants are summarized in Table 1. Applications for human health are included in the Table 1, but are not thoroughly discussed in the text. The following review will concentrate on the potential of plant-produced vaccines for animal health, and the aspects of vaccine production in plants will be discussed in this manner.
14
Literature review
Path
ogen
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20
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Imm
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enic
in m
ice
and
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s afte
r pa
rent
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adm
inis
tratio
n. R
educ
ed
sym
ptom
s in
cow
s cha
lleng
ed w
ith th
e vi
rus
Pere
z Fi
lgue
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t al.,
200
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aren
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assi
ve
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rote
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pup
s
Pere
z Fi
lgue
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t al.,
200
4
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4 as
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on w
ith G
US
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aves
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Imm
unog
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and
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in m
ice
afte
r ora
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tera
l adm
inis
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n.
Pass
ive
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ogen
ic p
rote
ctio
n of
pup
s
Wig
doro
vitz
et a
l., 2
004
Can
ine
parv
ovir
us/
dogs
VP2
epi
tope
PPV
vec
tors
in
toba
cco
Dis
play
on
vira
l par
ticle
s as
par
t of C
PN
RIm
mun
ogen
ic in
mic
e an
d ra
bbits
afte
r pa
rent
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adm
istra
tion.
Neu
traliz
ing
antib
odie
s in
mic
e
Fern
ande
z-Fe
rnan
dez
et
al.,
1998
CPM
V v
ecto
rs in
co
wpe
aD
ispl
ay o
n vi
ral p
artic
les
as p
art o
f CP
NR
Prot
ectiv
e ag
ains
t let
hal c
halle
nge
in
dogs
afte
r par
ente
ral a
dmin
istra
tion
Lang
evel
d et
al.,
200
1
CPM
V v
ecto
rs in
co
wpe
aD
ispl
ay o
n vi
ral p
artic
les
as p
art o
f CP
NR
Neu
traliz
ing
antib
odie
s in
mic
e af
ter
pare
nter
al o
r nas
al a
dmin
istra
tion
Nic
hola
s et a
l., 2
002
VP2
epi
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as
fusi
on w
ith G
US
Ara
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bsis
le
aves
p35S
-3´n
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SPIm
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ogen
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tera
l or
oral
adm
inis
tratio
nG
il et
al.,
200
1
VP2
2L2
1 ep
itope
as
fusi
on w
ith
chol
era
toxi
n B
su
buni
t, or
with
gr
een fl u
ores
cent
pr
otei
n
Toba
cco
leav
es
(chl
orop
last
)pr
rn-5
´psb
A-3
´psb
A22
.6 a
nd
33.1
% T
SPN
eutra
lizin
g an
tibod
ies i
n m
ice
and
rabb
its a
fter p
aren
tera
l adm
inis
tratio
n.
Imm
unog
enic
but
not
neu
traliz
ing
in
mic
e af
ter o
ral a
dmin
istra
tion
Mol
ina
et a
l., 2
004,
M
olin
a et
al.,
200
5
App
licat
ions
aga
inst
ani
mal
vir
us p
atho
gens
Tabl
e 1.
Sub
unit
vacc
ine
cand
idat
es e
xpre
ssed
by
trans
geni
c pl
ants
or p
lant
viru
ses.
15
Literature review
Foot
-and
-mou
th
dise
ase
viru
s/fa
rmed
ani
mal
s
VP1
epi
tope
CPM
V v
ecto
rs in
co
wpe
aD
ispl
ay o
n vi
ral p
artic
les
as p
art o
f CP
NR
ND
Ush
a et
al.,
199
3
Ara
bido
bsis
le
aves
p35S
-3´n
osN
RIm
mun
ogen
ic in
mic
e af
ter p
aren
tera
l ad
min
istra
tion.
Mic
e pr
otec
ted
agai
nst
vira
l cha
lleng
e
Car
rillo
et a
l., 1
998
TMV
vec
tors
in
toba
cco
Pept
ide
expr
essi
on u
nder
C
P su
bgen
omic
pro
mot
er15
0µg/
g FW
Imm
unog
enic
in m
ice
afte
r par
ente
ral
adm
inis
tratio
n. M
ice
prot
ecte
d ag
ains
t vi
ral c
halle
nge
Wig
doro
vitz
et a
l., 19
99
Alfa
lfa le
aves
p35S
-3´n
osN
RIm
mun
ogen
ic in
mic
e fo
llow
ing
pare
nter
al o
r ora
l adm
inis
tratio
n. M
ice
prot
ecte
d ag
ains
t vira
l cha
lleng
e
Wig
doro
vitz
et a
l., 19
99
p35S
-3´n
os0.
01%
TSP
Imm
unog
enic
in m
ice
follo
win
g pa
rent
eral
adm
inis
tratio
n. M
ice
prot
ecte
d ag
ains
t vira
l cha
lleng
e
Dus
San
tos a
nd
Wig
doro
vitz
, 200
5
Pota
to le
aves
p35S
-3´n
os3
2x p
35S-
3´no
s0.
01%
TSP
3Im
mun
ogen
ic a
nd p
rote
ctiv
e in
mic
e af
ter p
aren
tera
l adm
inis
tratio
n C
arril
lo e
t al.,
200
1
VP1
epi
tope
as
fusi
on w
ith G
US
Alfa
lfa le
aves
p35S
-3´n
os0.
1% T
SPIm
mun
ogen
ic in
mic
e fo
llow
ing
pare
nter
al a
dmin
istra
tion.
Mic
e pr
otec
ted
agai
nst v
iral c
halle
nge
Dus
San
tos e
t al.,
200
2
Infe
ctio
us b
ronc
hitis
vi
rus/
poul
tryS1
gly
copr
otei
nPo
tato
tube
rp3
5S-3
´nos
0.22
% T
SP,
2.53
µg/g
FW
Imm
unog
enic
in c
hick
ens a
fter
pare
nter
al o
r ora
l adm
inis
tratio
n.
Neu
traliz
ing
antib
odie
s pro
tect
ed
chic
kens
aga
inst
vira
l cha
lleng
e
Zhou
et a
l., 2
003,
Zh
ou e
t al.,
200
4
Infe
ctio
us b
ursa
l di
seas
e vi
rus/
poul
tryV
P2 p
rote
inA
rabi
dobs
is
leav
esN
R4.
8% T
SPIm
mun
ogen
ic a
nd p
rote
ctiv
e in
chi
cken
s af
ter o
ral a
dmin
istra
tion
Wu
et a
l., 2
004
16
Literature review
Path
ogen
/hos
tA
ntig
enPr
od. s
yste
mE
xpre
ssio
n sy
stem
1Y
ield
2Im
mun
e re
spon
seR
efer
ence
s
Min
k en
teri
tis v
irus
/m
ink,
dog
s, ca
tsV
P2 e
pito
peC
PMV
vec
tors
in
cow
pea
Dis
play
on
vira
l par
ticle
s as
par
t of C
P12
00µg
/g F
WIm
mun
ogen
ic a
nd p
rote
ctiv
e in
min
ks
follo
win
g pa
rent
eral
adm
inis
tratio
nD
alsg
aard
et a
l., 1
997
Mur
ine
hepa
titis
vi
rus/
mic
eG
lyco
prot
ein
S 5B
19 e
pito
peTM
V v
ecto
rs in
to
bacc
oD
ispl
ay o
n vi
ral p
artic
les
as p
art o
f CP
NR
Imm
unog
enic
and
pro
tect
ive
in m
ice
follo
win
g pa
rent
eral
or n
asal
ad
min
istra
tion
Koo
et a
l., 1
999
New
cast
le d
isea
se
viru
s/po
ultry
, wild
bi
rds
F an
d H
N su
rfac
e gl
ycop
rote
ins
Pota
to le
aves
p35S
-5´Ω
-3´n
os0.
06%
TSP
Imm
unog
enic
in m
ice
follo
win
g pa
rent
eral
or o
ral a
dmin
istra
tion
Ber
inst
ein
et a
l., 2
005
F an
d H
N e
pito
pes
CM
V v
ecto
rs in
to
bacc
oD
ispl
ay o
n vi
ral p
artic
les
as p
art o
f CP
430µ
g/g
FWN
DZh
ao a
nd H
amm
ond,
200
5
Pest
e-de
s-pe
tits-
rum
inan
t vir
us/
dom
estic
- and
wild
an
imal
s
Hem
aglu
tinin
-ne
uram
idas
ePi
geon
pea
leav
esp3
5S-3
´nos
NR
ND
Pras
ad e
t al.,
200
4
Porc
ine
epid
emic
di
arrh
ea v
irus
/pig
sSp
ike
prot
ein
Toba
cco
leav
esN
R20
µg/g
FW
Syst
emic
and
muc
osal
ant
ibod
ies i
n m
ice
afte
r ora
l adm
inis
tratio
nB
ae e
t al.,
200
3
Toba
cco
leav
esp3
5S-5
´Ω-3
´nos
0.1%
TSP
ND
Kan
g et
al.,
200
5a,
Kan
g et
al.,
200
5b
Pota
to tu
bers
p35S
-5´Ω
-SEK
DEL
-3´n
os0.
1% T
SPN
DK
im e
t al.,
200
5
Rabb
it he
mor
rhag
ic
dise
ase
viru
s/ra
bbits
VP6
0 pr
otei
nPo
tato
leav
esp3
5S-3
´nos
0.16
% T
SPIm
mun
ogen
ic in
rabb
its a
fter p
aren
tera
l ad
min
istra
tion.
Neu
traliz
ing
antib
odie
s pr
otec
ted
rabb
its a
gain
st le
thal
ch
alle
nge
Cas
tano
n et
al.,
199
9
PPV
vec
tors
in
toba
cco
Prot
ein
expr
essi
on a
s par
t of
vira
l pol
ypro
tein
(p
rote
in re
leas
ed b
y vi
ral
prot
ease
)
NR
Imm
unog
enic
in m
ice
and
rabb
its a
fter
pare
nter
al a
dmin
istra
tion.
Neu
traliz
ing
antib
odie
s pro
tect
ed ra
bbits
aga
inst
le
thal
cha
lleng
e
Fern
ande
z-Fe
rnan
dez
et
al.,
2001
Rabi
es v
irus
/dom
estic
- and
wild
ani
mal
s (s
ee v
acci
nes f
or h
uman
hea
lth b
elow
)
Rind
erpe
st v
irus
/ca
ttle
Hem
aggl
utin
in
prot
ein
Pige
on p
ea le
aves
p35S
-3´n
os0.
49%
TSP
ND
Saty
avat
hi e
t al.,
200
3
Pean
ut le
aves
p35S
-3´n
os0.
5% T
SPN
eutra
lizin
g an
tibod
ies i
n m
ice
afte
r or
al a
nd p
aren
tera
l adm
inis
tratio
nK
hand
elw
al e
t al.,
200
4
App
licat
ions
aga
inst
ani
mal
vir
us p
atho
gens
17
Literature review
Tran
smis
sibl
e ga
stro
ente
ritis
vi
rus/
pigs
Spik
e (S
) gl
ycop
rote
inA
rabi
dobs
is
leav
esp3
5S-3
´nos
0.06
% T
SPN
eutra
lizin
g an
tibod
ies i
n m
ice
afte
r pa
rent
eral
adm
inis
tratio
nG
omez
et a
l., 1
998
Pota
to tu
bers
35S-
3´no
s 0.
07%
TSP
Imm
unog
enic
in m
ice
afte
r par
ente
ral o
r or
al a
dmin
istra
tion
Gom
ez e
t al.,
200
0
Toba
cco
leav
espS
UPE
R-S
P(Pr
1a)-
3´no
spS
UPE
R-3
´nos
30.
2% T
SP3
Neu
traliz
ing
antib
odie
s in
pigs
afte
r pa
rent
eral
adm
inis
tratio
nTu
boly
et a
l., 2
000
Mai
ze se
eds
NR
2% T
SPIm
mun
ogen
ic a
nd p
rote
ctiv
e in
pig
lets
af
ter o
ral a
dmin
istra
tion
Stre
atfi e
ld e
t al.,
200
1,
Lam
phea
r et a
l., 2
002,
St
reatfi e
ld a
nd H
owar
d,
2003
b
App
licat
ions
aga
inst
ani
mal
pat
hoge
ns o
ther
than
vir
us
Path
ogen
/hos
tA
ntig
enPr
od. s
yste
mE
xpre
ssio
n sy
stem
1Y
ield
2Im
mun
e re
spon
seR
efer
ence
s
Man
nhei
mia
ha
emol
ytic
a/ca
ttle
A1
leuc
otox
in 5
0 as
a
fusi
on w
ith G
FPW
hite
clo
ver
leav
esp3
5S-S
P-3´
nos
(orig
in o
f SP
NR
)1%
TSP
Neu
traliz
ing
antib
odie
s in
rabb
its
follo
win
g pa
rent
eral
adm
inis
tratio
nLe
e et
al.,
200
1
Alfa
lfa le
aves
p35S
-SP-
HD
EL-3
´nos
prbc
S-SP
-HD
EL-3
´nos
(orig
in o
f SP
NR
)
NR
ND
Ziau
ddin
et a
l., 2
004
Esch
eric
hia
coli,
en
tero
toxi
geni
c/pi
gs
F4 fi
mbr
ial a
dhes
in
FaeG
Toba
cco
leav
esp3
5S-3
´nos
0.15
% T
SPIm
mun
ogen
ic in
mic
e af
ter p
aren
tera
l ad
min
istra
tion
Hua
ng e
t al.,
200
3
p35S
-TP-
3´no
s1%
TSP
Rec
epto
r bin
ding
in v
itro
II
Alfa
lfa le
aves
p35S
-TP(
rbcS
)-3´
nos
1% T
SPIm
mun
ogen
ic a
nd p
artia
lly p
rote
ctiv
e in
pi
gs a
fter o
ral a
dmin
istra
tion
IV
Bar
ley
seed
spT
I-SP
(TI)
-SEK
DEL
-3´
nos
1% T
SPN
eutra
lizin
g an
tibod
ies i
n m
ice
afte
r pa
rent
eral
adm
inis
tratio
n II
I
Esch
eric
hia
coli,
en
tero
toxi
geni
c/pi
gs,
cattl
e, c
hick
en
F5 fi
mbr
ial a
dhes
in
FanC
Soyb
ean
leav
esp3
5S-5
´TEV
-3´3
5S0.
5% T
SPIm
mun
ogen
ic in
mic
e af
ter
pare
nter
al a
dmin
istra
tion
Pille
r et a
l., 2
005
18
Literature review
Path
ogen
Ant
igen
Prod
. sys
tem
Exp
ress
ion
syst
em1
Yie
ld2
Imm
une
resp
onse
Ref
eren
ces
Hep
atiti
s B v
irus
Su
rfac
e an
tigen
(H
BsA
g)To
bacc
o le
aves
p35S
-3´n
os2x
p35
S-5´
TEV-
3´no
s30.
0066
% T
SP3
Imm
unog
enic
in m
ice
afte
r par
ente
ral
adm
inis
tratio
nM
ason
et a
l., 1
992,
Th
anav
ala
et a
l., 1
995
Toba
cco
and
soyb
ean
cell
susp
ensi
on
cultu
res
2x p
35S-
5´TE
V-3´
nos
6.5
and
45µg
/g
FWN
DSm
ith e
t al.,
200
2
Toba
cco
and
soyb
ean
cell
susp
ensi
on
cultu
res
2x p
35S-
5´TE
V-3´
nos
6.5
and
45µg
/g
FWN
DSm
ith e
t al.,
200
2
Pota
to tu
bers
p35S
-5´T
EV-3
´nos
p35S
-5´T
EV-S
EKD
EL-3
´nos
p35S
-5´T
EV-S
P(vs
pαS)
-3´n
osp3
5S-5
´TEV
-SP(
vspα
L)-3
´nos
p35S
-5´T
EV-T
P(rb
cS)-
3´no
sp3
5S-5
´TEV
-3´p
in23,
4
p35S
-5´T
EV-3
´vsp
p35S
-5´Ω
-3´n
ospp
atat
in-3
´nos
4
16µg
/g F
W3
Imm
unog
enic
in m
ice
afte
r ora
l ad
min
istra
tion
Ric
hter
et a
l., 2
000,
K
ong
et a
l., 2
001
Pota
to tu
bers
2x p
35S-
3´oc
s3
ppat
atin
-3´o
cs0.
035%
TSP
3N
DSh
ulga
et a
l., 2
004
Pota
to tu
bers
NR
8.5µ
g/g
FWIm
mun
ogen
ic in
hum
ans a
fter o
ral
adm
inis
tratio
nTh
anav
ala
et a
l., 2
005
Che
rry
tom
atill
o le
aves
and
frui
tp3
5-3´
nos
0.3µ
g/g
FW,
0.01
µg/g
FW
Imm
unog
enic
in p
aren
tera
lly p
rimed
m
ice
afte
r ora
l adm
inis
tratio
nG
ao e
t al.,
200
3
Ban
ana
leav
espE
FE-3
´nos
3
pEFE
-SEK
DEL
-3´n
ospU
BQ
3-3´
nos
pUB
Q3-
SEK
DEL
-3´n
os
0.03
8µg/
g FW
3N
DK
umar
et a
l., 2
005
Tom
ato
frui
tsN
R0.
3µg/
g D
WSp
ecifi
c se
rum
and
muc
osal
ant
ibod
ies
dete
cted
in m
ice
afte
r ora
l ad
min
istra
tion
Shch
elku
nov
et a
l.,
2005
App
licat
ions
aga
inst
hum
an v
irus
pat
hoge
ns
19
Literature review
HB
sAg
expr
esse
d as
fu
sion
with
soyb
ean
vege
tativ
e pr
otei
n vs
pαS
Toba
cco
leav
esp3
5S-3
´vsp
p35S
-SP(
vspα
S)-3
´vsp
3
p35S
-SP(
vspα
S)-S
EKD
EL-3
´vsp
p35S
-3´v
spp3
5S- S
P(vs
pαL)
-3´v
spp3
5S- S
P vs
pαL)
-SEK
DEL
-3´v
sp
0.02
3% T
SP3
Imm
unog
enic
in m
ice
afte
r par
ente
ral
adm
inis
tratio
n. F
usio
n pr
otei
n ge
nera
ted
high
er le
vels
of s
erum
IgG
than
the
nativ
e H
BsA
g
Sojik
ul e
t al.,
200
3
S an
d Pr
eS su
rfac
e an
tigen
s si
mul
tane
ousl
y
Pota
to tu
bers
2x p
35S-
5´A
lMV-
3´no
s0.
08 a
nd
0.01
2% T
SPIm
mun
ogen
ic in
ora
lly im
mun
ized
mic
e af
ter p
aren
tera
l boo
stin
gJo
ung
et a
l., 2
004
Hep
atiti
s C v
irus
H
VR
1 ep
itope
of E
2 en
velo
pe p
rote
in a
s fu
sion
with
CT-
B
TMV
vec
tors
in
toba
cco
Pept
ide
expr
essi
on u
nder
CP
subg
enom
ic p
rom
oter
0.04
% T
SPFu
sion
pro
tein
imm
unog
enic
in m
ice
afte
r nas
al a
dmin
istra
tion
Nem
chin
ov e
t al.,
20
00
HV
R1
epito
pe o
f E2
enve
lope
pro
tein
CM
V v
ecto
rs in
to
bacc
o le
aves
Dis
play
on
vira
l par
ticle
s as p
art
of C
P10
0µg/
g FW
Cro
ss re
activ
e w
ith a
wid
e ra
nge
of
hum
an a
nti H
VR
-1 a
ntib
odie
sN
atill
a et
al.,
200
4
Dis
play
on
vira
l par
ticle
s as p
art
of C
PN
RIm
mun
ogen
ic in
rabb
its a
fter p
aren
tera
l ad
min
istra
tion.
Ant
igen
-spe
cifi c
CD
8 T-
cell
resp
onse
in H
CV
infe
cted
hum
ans
Piaz
zolla
et a
l., 2
005
Hep
atiti
s E v
irus
O
RF2
pro
tein
Tom
ato
leav
es
and
frui
tp3
5S-5
´Ω-3
´nos
0.48
µg/g
and
0.
61µg
/g F
WN
DM
a et
al.,
200
3
Pota
to tu
bers
p35S
-5´T
EV-3
´vsp
30µg
/g F
WN
ot im
mun
ogen
ic in
mic
e fo
llow
ing
oral
ad
min
istra
tion
Mal
oney
et a
l., 2
005
Hum
an
cyto
meg
alov
irus
Gly
copr
otei
n B
Toba
cco
seed
spG
T3-S
P(G
T3)-
3´no
s0.
0146
% a
nd
1% T
SPN
DTa
ckab
erry
et al
., 19
99,
Tac
kabe
rry et
al.,
2003
Hum
an
imm
unod
efi c
ienc
y vi
rus (
type
1)
p120
pro
tein
AlM
V v
ecto
rs
in to
bacc
oD
ispl
ay o
n vi
ral p
artic
les a
s par
t of
CP
NR
Neu
traliz
ing
antib
odie
s in
mic
e af
ter
pare
nter
al a
dmin
istra
tion
Yusi
bov
et a
l., 1
997
P120
pro
tein
TBSV
vec
tors
in
toba
cco
Dis
play
on
vira
l par
ticle
s as p
art
of C
P90
0µg/
g FW
Imm
unog
enic
in m
ice
afte
r par
ente
ral
adm
inis
tratio
nJo
elso
n et
al.,
199
7
p24
prot
ein
TBSV
vec
tors
in
toba
cco
Dis
play
on
vira
l par
ticle
s as p
art
of C
P0.
4% T
SPN
DZh
ang
et a
l., 2
000,
Zh
ang
et a
l., 2
002
ELD
KW
A e
pito
pe
of g
p41
prot
ein
PVX
vec
tors
in
toba
cco
Dis
play
on
vira
l par
ticle
s as p
art
of C
PN
RIm
mun
ogen
ic in
mic
e af
ter p
aren
tera
l or
nasa
l adm
inis
tratio
n. N
eutra
lizin
g an
tibod
ies i
nduc
ed in
mic
e
Mar
usic
et a
l., 2
001
TBI e
pito
peTo
mat
o fr
uits
NR
0.3µ
g/g
DW
Spec
ifi c
seru
m a
nd m
ucos
al a
ntib
odie
s de
tect
ed in
mic
e af
ter o
ral
adm
inis
tratio
n
Shch
elku
nov
et a
l.,
2005
20
Literature review
App
licat
ions
aga
inst
hum
an v
irus
pat
hoge
ns
Path
ogen
Ant
igen
Prod
. sys
tem
Exp
ress
ion
syst
em1
Yie
ld2
Imm
une
resp
onse
Ref
eren
ces
Hum
an p
apill
oma
viru
s (ty
pe 1
1)L1
pro
tein
Pota
to tu
bers
2x 3
5S-5
´TEV
-3´v
sp0.
023µ
g/g
FWIm
mun
ogen
ic in
ora
lly im
mun
ized
mic
e af
ter p
aren
tera
l boo
stin
g W
arze
cha
et a
l., 2
003
Hum
an p
apill
oma
viru
s (ty
pe 1
6)E7
pro
tein
PVX
vec
tors
in
toba
cco
Prot
ein
expr
essi
on u
nder
C
P su
bgen
omic
pro
mot
er4µ
g/g
FWIm
mun
ogen
ic a
nd p
rote
ctiv
e in
mic
e af
ter p
aren
tera
l adm
insi
tratio
nFr
anco
ni e
t al.,
200
2
L1 p
rote
inTo
bacc
o le
aves
, po
tato
tube
rsp3
5S-5
´Ω-3
´ocs
0.5%
TSP
and
0.
2% T
SPIm
mun
ogen
ic in
mic
e w
hen
adm
inis
tere
d or
ally
follo
win
g a
pare
nter
al b
oost
Bie
mel
t et a
l., 2
003
Toba
cco
leav
esp3
5S-3
´ocs
0.00
4µg/
g FW
Imm
unog
enic
in ra
bbits
afte
r par
ente
ral
adm
inis
tratio
nVa
rsan
i et a
l., 2
003
Hum
an rh
inov
irus
(ty
pe 1
4)V
P 1
epito
peC
PMV
vec
tors
in
cow
pea
Dis
play
on
vira
l par
ticle
s as
par
t of C
PN
RIm
mun
ogen
ic in
rabb
its a
fter p
aren
tera
l ad
min
istra
tion
Porta
et a
l., 1
994
Mea
sles
vir
usH
emag
glut
inin
Toba
cco
leav
esp3
5S-5
´TEV
-3´3
5S p
35S-
5´TE
V-K
DEL
-3´3
5S2
p35S
-5´T
EV-S
S(PR
1a)-
KD
EL-3
´35S
NR
Neu
traliz
ing
antib
odie
s in
mic
e af
ter
pare
nter
al o
r ora
l del
iver
y H
uang
et a
l., 2
001,
W
ebst
er e
t al.,
200
2,
Web
ster
et a
l., 2
005
Car
rot l
eave
s and
ta
proo
ts2x
p35
S-5´
TEV-
3´35
SN
RN
eutra
lizin
g an
tibod
ies i
n m
ice
afte
r pa
rent
eral
adm
inis
tratio
nB
ouch
e et
al.,
200
3,
Mar
quet
-Blo
uin
et a
l.,
2003
Hem
aggl
utin
in a
nd
teta
nus t
oxoi
d po
lyep
itope
s
Car
rot l
eave
s2x
p35
S-5´
TEV-
3´35
SN
RN
eutra
lizin
g an
tibod
ies i
n m
ice
afte
r pa
rent
eral
adm
inis
tratio
nB
ouch
e et
al.,
200
5
Nor
wal
k vi
rus
Cap
sid
prot
ein
Toba
cco
leav
es,
pota
to tu
bers
p35S
-3´n
os2x
p35
S-5´
TEV-
3´no
spp
atat
in-5
´TEV
-3´n
os3
0.23
% T
SP3
and
20µg
/g
FW3
Seru
m a
nd m
ucos
al a
ntib
odie
s in
mic
e an
d hu
man
s afte
r ora
l adm
inis
tratio
nM
ason
et a
l., 1
996,
Ta
cket
et a
l., 2
000
Rabi
es v
irus
G
lyco
prot
ein
Tom
ato
leav
es
and
frui
tp3
5S-3
´nos
0.00
1% T
SPN
DM
cGar
vey
et a
l., 1
995
Toba
cco
leav
esp3
5S-S
P(Pr
1a)-
SEK
DEL
-3´
nos
0.38
% T
SPPr
otec
tive
antib
odie
s in
mic
e af
ter
pare
nter
al a
dmin
istra
tion
Ash
raf e
t al.,
200
5
Gly
copr
otei
n an
d nu
cleo
prot
ein
epito
pes
AlM
V v
ecto
rs in
to
bacc
oD
ispl
ay o
n vi
ral p
artic
les
as p
art o
f CP
NR
Neu
traliz
ing
antib
odie
s in
mic
e af
ter
pare
nter
al a
dmin
istra
tion
Yusi
bov
et a
l., 1
997
AlM
V v
ecto
rs in
to
bacc
o an
d sp
inac
h
Dis
play
on
vira
l par
ticle
s as
par
t of C
PN
RPr
otec
tive
antib
odie
s in
mic
e af
ter
pare
nter
al a
nd o
ral a
dmin
istra
tion
Mod
elsk
a et
al.,
199
8
21
Literature review
Gly
copr
otei
n an
d nu
cleo
prot
ein
epito
pes
AlM
V v
ecto
rs in
to
bacc
o an
d sp
inac
h
Dis
play
on
vira
l par
ticle
s as
par
t of C
P30
µg/g
FW
Pr
otec
tive
antib
odie
s in
mic
e af
ter
pare
nter
al a
dmin
istra
tion.
Imm
unog
enic
in
hum
ans a
fter o
ral a
dmin
istra
tion
Yusi
bov
et a
l., 2
002
Resp
irat
ory
sync
ytia
l vir
usR
SV-f
usio
n pr
otei
nTo
mat
o fr
uits
p35S
-3´n
ospE
8-3´
nos3
35.5
µg/g
FW
3Im
mun
ogen
ic in
mic
e af
ter o
ral
adm
inis
tratio
nSa
ndhu
et a
l., 2
000
Epito
pes o
f G a
nd F
pr
otei
ns
AlM
V v
ecto
rs in
to
bacc
oD
ispl
ay o
n vi
ral p
artic
les
as p
art o
f CP
500µ
g/g
FW
Prot
ectiv
e an
tibod
ies i
n m
ice
afte
r pa
rent
eral
adm
inst
ratio
n B
elan
ger e
t al.,
200
0
Rota
viru
sV
P6 p
rote
inPV
X v
ecto
rs in
to
bacc
oPr
otei
n fu
sion
with
CP
and
prot
ein
expr
essi
on
unde
r CP
subg
enom
ic
prom
oter
3
50µg
/g F
W3
ND
O
’Brie
n et
al.,
200
0
Tom
ato
cell
susp
ensi
on
cultu
re
pCsV
MV-
3´no
s4.
3 µg
/g F
W2
ND
Chu
ng e
t al.,
200
0,
Kim
et a
l., 2
001
Pota
to tu
bers
pmas
2-SE
KD
EL-3
´g7
0.01
% T
SPSe
rum
and
muc
osal
ant
ibod
ies i
n m
ice
afte
r ora
l del
iver
yYu
and
Lan
grid
ge, 2
003
Alfa
lfa le
aves
2x p
35S-
5´TE
V-SE
KD
EL-3
´nos
0.28
% T
SPSe
rum
and
muc
osal
ant
ibod
ies i
n m
ice
afte
r ora
l del
iver
y. R
educ
ed sy
mpt
oms
in ro
tavi
rus c
halle
nged
pup
s
Don
g et
al.,
200
5
NSP
4 ep
itope
as
fusi
on w
ith C
T-B
Pota
to tu
bers
pmas
1&2-
SEK
DEL
-3´
ocs/
3´g7
0.1%
TSP
GM
1 ga
nglio
side
bin
ding
in v
itro
Ara
kaw
a et
al.,
200
1
NSP
4 ep
itope
as
fusi
on w
ith C
T-B
co
-exp
ress
ed w
ith
CFA
I CT-
A2
fusi
on
Pota
to tu
bers
pmas
1&2-
SEK
DEL
/K
DEL
-3´o
cs/3
´g7
3.3µ
g/g
FWIm
mun
ogen
ic in
mic
e ag
ains
t ETE
C,
rota
viru
s, an
d V.
cho
lera
e af
ter o
ral
deliv
ery
Red
uced
sym
ptom
s in
rota
viru
s ch
alle
nged
pup
s
Yu a
nd L
angr
idge
, 200
1
VP7
pro
tein
Pota
to tu
bers
p35S
-SEK
DEL
-3´n
osN
RN
eutra
lizin
g an
tibod
ies i
n m
ice
follo
win
g or
al a
dmin
istra
tion
Wu
et a
l., 2
003
SARS
vir
usS1
frag
men
t of
spik
e pr
otei
nTo
bacc
o le
aves
, to
mat
o fr
uits
pSU
PER
-5´T
oEV-
HD
ELN
RIm
mun
ogen
ic in
mic
e af
ter o
ral a
nd
pare
nter
al a
dmin
istra
tion
Pogr
ebny
ak e
t al.,
200
4
Sim
ian/
hum
an
imm
uno
defi c
ienc
y vi
rus
89.6
pTat
as f
usio
n w
ith C
T-B
Pota
to tu
bers
pmas
1&2-
SEK
DEL
-3´
g4/3
´g7
0.00
7% T
SPG
M1
gang
liosi
de b
indi
ng in
vitr
oK
im e
t al.,
200
4b
Gag
cap
sid
prot
ein
Pota
to tu
bers
pmas
2-SE
KD
EL-3
´g7
0.01
4% T
SPN
DK
im e
t al.,
200
4a
22
Literature review
Path
ogen
Ant
igen
Prod
. sys
tem
Exp
ress
ion
syst
em1
Yie
ld2
Imm
une
resp
onse
Ref
eren
ces
Baci
llus a
ntra
cis
Prot
ectiv
e an
tigen
(P
A)
Tom
ato
leav
es,
Toba
cco
leav
es
(chl
orop
last
)
NR
and
prrn
-5´a
adA
-3´p
sbA
3N
R a
nd 8
%
TSP3
Neu
traliz
ing
antib
odie
s in
mic
e af
ter p
aren
tera
l adm
inis
tratio
n of
to
mat
o le
af T
SP
Azi
z et
al.,
200
5
PA-4
Ds e
pito
peA
lMV
vec
tors
in
toba
cco
Dis
play
on
vira
l par
ticle
s as p
art
of C
P30
0µg/
g FW
Imm
unog
enic
in m
ice
afte
r pa
rent
eral
adm
inis
tratio
nB
rodz
ik e
t al.,
200
5
Toba
cco
leav
esp3
5S-3
´ocs
NR
Cyt
otox
icity
on
mac
roph
ages
in
vitro
Azi
z et
al.,
200
2
Clo
stri
dium
teta
niTe
tanu
s tox
in
frag
men
t C (T
etC
)To
bacc
o le
aves
(chl
orop
last
)pr
rn-5
´T7g
ene1
0-3´
rbcL
prrn
-5´a
tpB
-3´r
bcL3
25%
TSP
3Im
mun
ogen
ic in
mic
e af
ter n
asal
an
d or
al a
dmin
istra
tion.
Pr
otec
tive
in m
ice
afte
r nas
al
adm
inis
tratio
n
Treg
onin
g et
al.,
200
3,
Treg
onin
g et
al.,
200
4,
Treg
onin
g et
al.,
200
5
TetC
as f
usio
n w
ith
TetC
spec
ifi c
antib
ody
Toba
cco
leav
esp3
5S-S
P(m
Ig)-
3´no
s0.
8% T
SPIm
mun
ogen
ic a
nd p
rote
ctiv
e in
m
ice
afte
r par
ente
ral
adm
inis
tratio
n
Cha
rgel
egue
et a
l., 2
005
Esch
eric
hia
coli,
en
tero
toxi
geni
cC
FAI e
xpre
ssed
as
fusi
on w
ith C
T-A
2 su
buni
t and
co-
expr
esse
d w
ith C
T-B
ro
tavi
rus N
SP4
fusi
on
Pota
to tu
bers
pmas
1&2-
SEK
DEL
/KD
EL-
3´oc
s/3´
g73.
3µg/
g FW
Imm
unog
enic
in m
ice
agai
nst
ETEC
, rot
aviru
s, an
d V.
cho
lera
e af
ter o
ral d
eliv
ery
Red
uced
sy
mpt
oms i
n ro
tavi
rus
chal
leng
ed p
ups
Yu a
nd L
angr
idge
, 200
1
CFA
I as f
usio
n w
ith
CT
A c
o-ex
pres
sed
with
NSP
-CT
B fu
sion
Pota
to tu
bers
pmas
1&2-
SEK
DEL
/KD
EL-
3´oc
s/3´
g70.
002%
TSP
Imm
unog
enic
in m
ice
afte
r ora
l im
mun
izat
ion.
Ant
iser
a re
duce
d ET
EC a
dhes
ion
to e
nter
ocyt
es in
vi
tro
Lee
et a
l., 2
004
Hea
t lab
ile to
xin,
B
subu
nit
Pota
to tu
bers
p35S
-5´T
EV-3
´vsp
p35S
-5´T
EV-S
EKD
EL-3
´vsp
30.
01%
TSP
3N
eutra
lizin
g se
rum
and
muc
osal
an
tibod
ies i
n m
ice
afte
r ora
l ad
min
istra
tion
Haq
et a
l., 1
995
p35S
-5´T
EV-3
´vsp
17.2
µg/
g FW
Imm
unog
enic
and
par
tially
pr
otec
tive
in m
ice
afte
r ora
l ad
min
istra
tion
Mas
on e
t al.,
199
8
p35S
-5´T
EV-3
´vsp
15.7
µg/g
FW
Spec
ifi c
seru
m a
nd m
ucos
al
antib
odie
s in
hum
ans a
fter o
ral
adm
inis
tratio
n
Tack
et e
t al.,
199
8
ppat
atin
-3´n
os13
µg/g
FW
Imm
unog
enic
in p
rimed
mic
e af
ter o
ral a
dmin
istra
tion
Laut
ersl
ager
et a
l., 2
001
Mai
ze se
eds
NR
NR
Prot
ectiv
e an
tibod
ies i
n m
ice
afte
r ora
l adm
inis
tratio
nSt
reatfi e
ld e
t al.,
200
1
App
licat
ions
aga
inst
hum
an p
atho
gens
oth
er th
an v
irus
23
Literature review
NR
10%
TSP
Seru
m a
nd m
ucos
al a
ntib
odie
s in
mic
e af
ter o
ral a
dmin
istra
tion
Lam
phea
r et a
l., 2
002,
St
reatfi e
ld an
d How
ard,
200
3b,
Stre
atfi e
ld e
t al.,
200
3
pγ-z
ein-
5´TE
V-3´
vsp
pγ-z
ein-
5´TE
V-SE
KD
EL-3
´vsp
3
p35S
-5´T
EV-3
´vsp
p35S
-5´T
EV-S
EKD
EL-3
´vsp
3.7%
TSP
3Se
rum
and
muc
osal
ant
ibod
ies i
n m
ice
afte
r ora
l adm
inis
tratio
nC
hikw
amba
et a
l., 2
002a
, C
hikw
amba
et a
l., 2
002b
NR
NR
Muc
osal
and
seru
m a
ntib
odie
s in
hum
ans a
fter o
ral a
dmin
istra
tion
Tack
et e
t al.,
200
4
Toba
cco
leav
esp3
5S-3
´nos
pUB
I-3´
nos3
3.3%
TSP
3G
M1
gang
liosi
de b
indi
ng in
vitr
oK
ang
et a
l., 2
005c
Non
-toxi
c m
utan
t of
LT-B
Toba
cco
leav
es
(chl
orop
last
)pr
rn-3
´psb
A3.
7% T
SPG
M1
gang
liosi
de b
indi
ng in
vitr
oK
ang
et a
l., 2
004b
LT-B
exp
ress
ed a
s fu
sion
with
im
mun
ocon
trace
ptiv
e an
tigen
Tom
ato
frui
t and
le
aves
p35S
-5´T
EV-3
´vsp
65µg
/g D
W,
10µg
/g F
WN
DW
alm
sley
et a
l., 2
003
Esch
eric
hia
coli,
en
tero
hem
orrh
agei
cIn
timin
of O
157:
H7
Toba
cco
leav
esp3
5S-5
´TEV
-3´v
sp4
p35S
-5´T
EV-S
P(vs
pαS)
-3´v
sp3
3µg/
g FW
3Im
mun
ogen
ic4 a
nd p
artia
lly
prot
ectiv
e in
par
ente
rally
prim
ed
mic
e af
ter o
ral a
dmin
istra
tion
Judg
e et
al.,
200
4
Esch
eric
hia
coli,
en
tero
path
ogen
icB
undl
e fo
rmin
g pi
lus
antig
en A
Toba
cco
leav
esp3
5S-3
´vsp
7.7%
TSP
Spec
ifi c
seru
m a
nd m
ucos
al
antib
odie
s in
mic
e af
ter o
ral
adm
inis
tratio
n
Vie
ira d
a Si
lva
et a
l., 2
002
Hel
icob
acte
r pyl
ori
Ure
B a
ntig
enTo
bacc
o le
aves
p35S
-3´n
osN
RN
DG
u et
al.,
200
5
Plas
mod
ium
fa
lcip
arum
Circ
umsp
oroz
oite
pr
otei
n ep
itope
TMV
vec
tors
in
toba
cco
1200
µg/g
FW
ND
Turp
en e
t al.,
199
5
PfM
SP1
prot
ein
Toba
cco
leav
esp3
5S-3
´nos
0.00
35%
TS
PN
DG
hosh
et a
l., 2
002
Pseu
dom
onas
ae
rugi
nosa
Mem
bran
e pr
otei
n F
epito
pes
CPM
V v
ecto
rs
in c
owpe
aD
ispl
ay o
n vi
ral p
artic
les a
s par
t of
CP
1200
µg/g
FW
Imm
unog
enic
and
pro
tect
ive
in
mic
e fo
llow
ing
pare
nter
al
adm
inis
tratio
n
Bre
nnan
et a
l., 1
999a
, B
renn
an e
t al.,
199
9b,
Gill
elan
d et
al.,
200
0
TMV
vec
tors
in
toba
cco
Dis
play
on
vira
l par
ticle
s as p
art
of C
PN
RIm
mun
ogen
ic a
nd p
rote
ctiv
e in
m
ice
follo
win
g pa
rent
eral
ad
min
istra
tion
Gill
elan
d et
al.,
200
0,
Stac
zek
et a
l., 2
000
24
Literature review
Path
ogen
Ant
igen
Prod
. sys
tem
Exp
ress
ion
syst
em1
Yie
ld2
Imm
une
resp
onse
Ref
eren
ces
Shig
ella
fl ex
neri
IpaC
pro
tein
Ara
bido
bsis
le
aves
p35S
-3´n
os0.
2% T
SPN
DM
acR
ae e
t al.,
200
4
Stap
hylo
cocc
us
aure
usD
2 ep
itope
of
fi bro
nect
in-b
indi
ng
prot
ein
CPM
V v
ecto
rs in
co
wpe
a an
d PV
X
vect
ors i
n to
bacc
o
Dis
play
on
vira
l par
ticle
s as
par
t of C
P12
00µg
/g F
W
and
200µ
g/g
FW
Neu
traliz
ing
antib
odie
s in
mic
e an
d ra
ts
follo
win
g pa
rent
eral
del
iver
yB
renn
an e
t al.,
199
9c
Vibr
io c
hole
rae
Cho
lera
toxi
n su
buni
t BPo
tato
tube
rspm
as2-
SEK
DEL
-3´g
70.
3%TS
PIm
mun
ogen
ic in
mic
e af
ter o
ral
adm
inis
tratio
nA
raka
wa
et a
l., 1
997
Toba
cco
leav
es(c
hlor
opla
st)
prrn
-3´p
sbA
4.1%
TSP
GM
1 ga
nglio
side
bin
ding
in v
itro
Dan
iell
et a
l., 2
001
Toba
cco
leav
esp3
5S-Ω
-KD
EL-3
´nos
3
p35S
-SEK
DEL
-3´n
os4
1.5%
3 and
0.
02%
TSP
Imm
unun
ogen
ic4 i
n m
ice
afte
r pa
rent
eral
adm
inis
tratio
nJa
ni e
t al.,
200
4,
Kan
g et
al.,
200
4a
Tom
ato
leav
es
and
frui
tp3
5S-S
EKD
EL-3
´nos
0.02
and
0.
04%
TSP
GM
1 ga
nglio
side
bin
ding
in v
itro
Jani
et a
l., 2
002
1 on
ly h
eter
olog
ous s
igna
l pep
tides
are
repo
rted;
2 m
axim
um a
ccum
ulat
ion
leve
l as r
epor
ted
in th
e lit
erat
ure;
3 rep
orte
d yi
eld
obta
ined
with
this
exp
ress
ion
syst
em;
4 rep
orte
d im
mun
e re
spon
se o
btai
ned
with
ant
igen
der
ived
from
this
exp
ress
ion
syst
em; 2
x p3
5S, 3
5S p
rom
oter
with
dou
ble
enha
ncer
regi
on; 3
´, po
lyad
enyl
atio
n si
gnal
s and
site
; 5´,
5´
untra
nsla
ted
regi
on; a
ad, a
min
ogly
cosi
de 3′-a
deny
lyltr
ansf
eras
e; A
lMV,
Alfa
lfa m
osai
c vi
rus;
atp
B, A
TP sy
ntha
se su
buni
t B; C
MV,
Cuc
umbe
r mos
aic
viru
s; C
P, c
oat p
rote
in; C
PMV
C
owpe
a m
osai
c vi
rus;
CsV
MV,
Cas
sava
vei
n m
osai
c vi
rus;
DW
, dry
wei
ght;
g4, A
grob
acte
rium
gen
e 4;
g7,
Agr
obac
teriu
m g
ene
7; G
T3, r
ice
glut
elin
3; γ
-zei
n, m
aize
stor
age
prot
ein;
E8
, tom
ato
ethy
lene
synt
hesi
s reg
ulat
ory
prot
ein;
EFE
, ban
ana
ethy
lene
form
ing
enzy
me;
FW
, fre
sh w
eigh
t; la
cZ, E
. col
i β-g
alac
tosi
dase
; m Ig
, mou
se im
mun
oglo
bulin
; mas
, Ag
roba
cter
ium
man
nopi
ne sy
ntha
se; N
D, n
ot d
etec
ted;
nos
, Agr
obac
teri
um n
opal
ine
synt
hase
; NR
, not
repo
rted;
Ω, u
ntra
nsla
ted
5´ le
ader
from
TM
V; o
cs, A
grob
acte
rium
oct
opin
e sy
ntha
se;
p, p
rom
oter
; pat
atin
, pot
ato
stor
age
prot
ein;
pin
2, p
otat
o pr
otei
nase
inhi
bito
r 2; P
PV, P
lum
pox
pot
yvir
us; P
r1a,
toba
cco
path
ogen
esis
rela
ted
prot
ein
1a; p
sbA
, pho
tosy
stem
II
subu
nit B
; PV
X, P
otat
o vi
rus X
; rbc
L, ru
bisc
o la
rge
subu
nit;
rbcS
, rub
isco
smal
l sub
unit;
rrn,
16S
rRN
A; (
SE)K
/HD
EL, e
ndop
lasm
ic re
ticul
um re
tain
sign
al; S
P, si
gnal
pep
tide
for
secr
eted
pro
tein
s; S
UPE
R, s
ynth
etic
pro
mot
er b
ased
on
ocs a
nd m
as p
rom
oter
s; T
7gen
e10,
bac
terio
phag
e T7
gen
e 10
; TI,
barle
y try
psin
inhi
bito
r; trc
, E. c
oli t
rypt
opha
n op
eron
; TEV
, un
trans
late
d 5´
lead
er fr
om T
obac
co e
tch
viru
s; T
BSV
, Tom
ato
bush
y st
unt v
irus
; TM
V, T
obac
co m
osai
c vi
rus;
ToE
V, u
ntra
nsla
ted
5´ le
ader
from
Tom
ato
etch
vir
us; T
P, c
hlor
opla
st
trans
it pe
ptid
e; T
SP, t
otal
solu
ble
prot
ein;
35S
, Cau
lifl o
wer
mos
aic
viru
s 35S
; UB
Q3,
ara
bido
bsis
ubi
quiti
n 3;
vsp
, soy
bean
veg
etat
ive
stor
age
prot
ein;
vspαL
, ful
l vsp
sign
al p
eptid
e (v
acuo
le ta
rget
ing)
; vspαS
, tru
ncat
ed v
sp si
gnal
pep
tide
(ER
targ
etin
g)
App
licat
ions
aga
inst
hum
an p
atho
gens
oth
er th
an v
irus
25
Literature review
2.1.2 Vaccine antigen targets
Currently, the majority of licensed bacterial and viral vaccines are either live-attenuated or killed, while plant-produced vaccines are subunit vaccines. To conquer a disease with a subunit vaccine, identifi cation of a suitable antigen with the ability to elicit a protective immune response is a critical early step. Known protective antigens can be expressed in transgenic plants, which would increase the possibilities for vaccination (e.g. injections vs. oral applications) due to the low costs of production and delivery. On the other hand, when new antigens must be discovered, plant-produced transient expression systems are competitive to the other available production platforms in terms of time and scalability.
2.1.3 Plant species utilized for vaccine antigen expressionThe plant species and tissue are selected for expression hosts according to the planned delivery system. In the case of oral delivery, edible food or feed crops are preferable, whereas nonfood plants can be used in applications where the antigen is purifi ed prior to use. The most common plant species selected to serve as an expression host for vaccine antigens is tobacco (see Table 1), for which well-established transformation and regeneration techniques are available. Tobacco is also easy to propagate and grow on a laboratory scale, rapidly producing a large biomass, and it has no special growth condition requirements. A drawback of the tobacco plant is the production of toxic secondary metabolites, particularly nicotine alkaloids, which limits its use for oral delivery. Edible leafy crops selected for vaccine antigen
production include alfalfa (Wigdorovitz et al., 1999), lettuce (Kapusta et al., 1999), spinach (Modelska et al., 1998), and white clover (Lee et al., 2001). The legumes alfalfa and white clover are high biomass feed plants that are cultivated widely. They are capable of Rhizobium-related nitrogen fi xation and possess a high protein content in terms of reduced fertilization costs. Lettuce and spinach are regularly consumed by humans and thus, are suitable delivery vehicles for human oral vaccines. Also the easily transformable genetic model plant arabidobsis (Gomez et al., 1998) has been utilized for vaccine antigen production, but the tiny size of this nonfood plant restricts its use to small-scale proof of principle studies.
Fruit and vegetable crops have been utilized in antigen production for oral vaccines since they can be consumed raw or processed to various palatable forms without cooking. Reported examples include bananas (Kumar et al., 2005), tomatoes (McGarvey et al., 1995), cherry tomatillos (Gao et al., 2003), potatoes (Haq et al., 1995), and carrots (Bouche et al., 2003). The tomato is a major vegetable plant grown worldwide that posseses a high fruit biomass yield per hectare. Another widely cultivated high-yield Solanaceae-family crop plant is the potato. Well-established and effi cient techniques for potato transformation are available, and the potato has served as a host for several vaccine antigens (see Table 1). Although potato tubers can be consumed as raw, they are not very palatable for human or animal use without cooking. In contrast to potato tubers, carrot taproots are regularly consumed raw as part of human and animal diets and the carrot has been used as a host plant for some vaccine candidates. Sweet banana fruits are an
26
Literature review
attractive choice for human oral vaccination, also being a pleasant delivery host for young children. The cherry tomatillo is a fruit plant regularly consumed in Mexican and Central American diets.
The leguminous plants pigeon pea (Satyavathi et al., 2003), soybean (Piller et al., 2005), and peanut (Khandelwal et al., 2005) have been reported to express vaccine antigens. Although these plants can be considered seed-based production and delivery systems, thus far vaccine antigens have only been produced in the leaf tissue. To date, maize is the only crop plant that has been used as a seed-based vaccine antigen production system (Streatfi eld et al., 2001).
2.1.4 Properties of host plant tissues for antigen productionVaccine antigens have been expressed in fresh tissues such as mature leaves (Mason et al., 1992), fruits (McGarvey et al., 1995), tubers (Haq et al., 1995), and taproots (Bouche et al., 2003), and in dry tissues, including mature seeds (Streatfi eld et al., 2001). Harvested fresh plant tissues usually need processing to preserve the antigen in a stable form, while mature seeds are desiccated and allow long-term storage at ambient temperatures. However, fruits, tubers, and taproots can be stored for a limited time, especially in a chilled environment. Vaccine antigens have also been expressed in undifferentiated plant cell cultures such as, cell suspensions (Smith et al., 2002) and callus cultures (Kapusta et al., 1999), although these systems are not competitive in the bulk-scale production needed for oral vaccine applications.
A high protein content of the target plant tissue selected for antigen expression is desirable. This demand is
fulfi lled by leguminous leafy crops, such as alfalfa and white clover, and in applications were the antigen is targeted to seeds. However, only a minor proportion of the fruit and tuber tissues is protein, limiting the amount of antigen which can be expressed and delivered in these tissues. To increase long-term storage and to concentrate the antigen, these tissues can be processed with such techniques as freeze-drying, followed by grinding or powdering. Antigen expression levels of individual potato tubers (Tacket et al., 2000) and tomato fruits (Sandhu et al., 2000) have been reported to vary. Batch processing homogenizes the raw material and allows products with a uniform antigen dosage to be manufactured. Similarly, the harvesting and batch processing of leafy crops homogenize the starting plant material. If the vaccine antigen is produced in the grains, the batch-to-batch variation can be monitored directly from the harvested seeds, and the material can be delivered even in unprocessed form to animals. However, carefully selected processing techniques can be used to increase the palatability and antigen concentration of the material. The processing must avoid exessive physical forces, such as temperature and pressure, to preserve the antigen in an effective fold. Streadfi eld and colleagues (2003) have shown how transgenic maize seeds expressing Transmissible gastroenteritis virus (TGEV) antigen can be processed effectively with existing milling techniques.
2.1.5 Plant vaccine production platformsIn the context of using plants as antigen production and delivery systems, there are three main options for engineering a
27
Literature review
plant to produce immunologically active peptides. The fi rst is to integrate the DNA encoding the gene of interest into the nuclear or plant organelle genome to generate a stably transformed transgenic plant that expresses the antigen either constitutively or in specifi c tissues. The second is to integrate the genetic material encoding the immunologically active protein or peptide into the genome of a plant virus used to infect plants. This transient expression system is initiated by virus inoculation of plants. The protein or peptide is then expressed either on the surface of the virus particle as a fusion epitope with the viral coat protein or as an autonomous protein produced as a by-product of the virus infection. The third option is a transient gene expression system known as agroinfi ltration. The main characteristics and the advantages and disadvantages of these production platforms are discussed below.
2.1.5.1 Transgenic plantsThe major advantage of stably transformed plants over transient protein production systems is that the protein production trait is heritable and can be stable over multiple generations, making upscaling simple, and allowing the establishment of seed stock to ensure long-term availability of starting material. The production of antigenic proteins in edible tissues of transgenic plants presents the very attractive possibility that these tissues can be consumed directly, providing an “edible vaccine” which obviates the need to purify the vaccine protein. As purifi cation of pharmaceutical proteins can account for more than 50% of the fi nal cost, it is often the limiting step in commercialization of such products (Streatfi eld and Howard, 2003a). Cost-effectiveness acts utmost
importance in the vaccination of domestic animals. Oral delivery of vaccine antigens within coarsely processed feed plants is an attractive way to increase animal health.
Drawbacks of using transgenic plants as a production platform include laborious and tedious regeneration processes to obtain the transformants. The generation of suffi cient transgenic plant material for protein analysis takes months, and despite advancements in the fi eld some plant species have remained diffi cult to transform (especially some monocotyledoneous plants). Another problem with transgenic plants has been the low production yield of the antigenic protein (see Table 1). However, recent progress in plastid transformation has enabled very high-level expression of foreign proteins in transgenic plants. These transplastomic plants have been reported to accumulate immunologically active peptides in amounts of up to 33.1% of total soluble protein (Molina et al., 2004). Plastid transformation currently works effi ciently only in solanaceous plants, such as tobacco (Svab and Maliga, 1993), tomato (Ruf et al., 2001), and potato (Sidorov et al., 1999), but it has been introduced into other crop species, including carrot (Kumar et al., 2004a), cotton (Kumar et al., 2004b), soybean (Dufourmantel et al., 2004) and oilseed rape (Hou et al., 2003). For cost-effective production of vaccine antigens, transgenic plants need to be grown on a fi eld scale. Applications intented to open-fi eld cultivation might evoke potential risks that must be assessed case-by-case. Two major risks are: (i) the risk of transgene escape and (ii) the risk of unintended exposure to the vaccine subunit protein (Commandeur et al., 2003). These risks can be controlled by various physical and biological means that are not discussed further here.
28
Literature review
2.1.5.2 Transient expression with viral vectors
Plant viral expression systems have generally been more effi cient than transgenic plant-based production systems in terms of the amounts of foreign protein that can be expressed in plant tissue. When virus-based production systems are used for antigen production, the chimeric virus particles or free foreign protein must usually be purifi ed from infected leaf tissues. If the immunogenic peptide is expressed in the form of an epitope fused to the plant viral coat protein, purifi cation of plant viruses from infected tissues is often a straightforward process. In addition, virions are often remarkably stable structures. However, strict limitations are present in the size of the insert that can be introduced into the virus genome and successfully assembled into virions (Yusibov et al., 1997). This is usually not the case with peptides or proteins expressed as an autonomous by-product of the virus infection or in transgenic plants. In contrast to transgenic plant material (particularly seeds, tubers, fruits), storing leaf tissue of virus-infected plants is not very practical. However, purifi ed plant virus material can be stored for long periods of time under appropriate conditions. Advantages of plant virus platform expression include savings in time and labor. Various genetic constructs can be tested and scaled up in a matter of weeks. This is an important feature for a vaccine production system against emerging new pathogenic variants. However, the bulk-scale production of antigens is inconvenient since the recombinant plant viruses need to be generated in vitro and inoculated into individual plants, and genetic stability of the virus must be monitored through multiple passages.
While it is possible to inoculate fi eld-grown plants with the viral vectors to transiently express the vaccine antigen, the risk of spread of the modifi ed plant viruses would be very high, and the use of this system must therefore be limited to contained environments such as greenhouses. To prevent the the unintended spread of viral vectors, the inoculation must be carried out in contained environment such as greenhouses. Examples in which epitopes of vaccine antigens have been successfully displayed as a part of chimeric plant viruses include Cowpea mosaic virus (CPMV) (Dalsgaard et al., 1997), Tobacco mosaic virus (TMV) (Koo et al., 1999), Alfalfa mosaic virus (AlMV) (Yusibov et al., 2005), Plum pox potyvirus (PPV) (Fernandez-Fernandez et al., 1998), and Potato virus X (PVX) (Marusic et al., 2001). Successful co-expression of antigens has been employed with TMV (Wigdorovitz et al., 1999), PVX (Franconi et al., 2002), and PPV (Fernandez-Fernandez et al., 2001).
2.1.5.3 Transient expression with agroinfi ltrationIn agroinfi ltration, the Agrobacterium culture is forced into intact or harvested plant leaf tissues by pressure, the transgene being expressed in plant cells for some hours to a couple of days and subsequently harvested for the foreign protein (Kapila et al., 1997). This technique is very convenient for preliminary laboratory-scale testing for transformation vector capacity, and for production of small amounts of purifi ed recombinant proteins, and is now being scaled up to milligram amounts, thus becoming an effi cient production platform (Twyman et al., 2005). Recently, inhibition of gene silencing in
29
Literature review
agroinfi ltrated leaves was shown to increase recombinant protein yields up to 50-fold (Voinnet et al., 2003). Large-scale agroinfi ltration is currently being utilized by the Canadian biotechnology company Medicaco (www.medicaco.com) that is processing alfalfa material (D’Aoust et al., 2005). No large-scale production of candidate vaccine antigens with this technique has thus far been reported, but agroinfi ltration has been utilized for preliminary testing purposes (Lee et al., 2001). Recently, the German company Icon Genetics (www.icongenetics.com) has established an agroinfi ltration-based application which combines the transfection effi ciency of Agrobacterium and the high expression yield of viral vectors (Marillonnet et al., 2005).
2.1.6 Optimizing antigen yield in transgenic plantsOral immunization of herds of farm animals with subunit vaccines requires bulk-scale production of the antigens. Equally as important as for adequate-sized oral doses is high expression level for cost-effective purifi cation of vaccine antigens. The overall biomass yield of the crop species and the intrinsic protein content of the plant tissue defi ne the capacity of the chosen production system. However, multiple factors determine the fi nal antigen content of the production platform. To achieve high yields, the expression construct design must include all stages of gene expression, from transcription to protein stability. Approaches that have been utilized to give high levels of expression of foreign proteins in transgenic plants are discussed below.
2.1.6.1 Transcription and mRNA stability
The promoter selected to control the transcription of the transgene is perhaps the most important component of the expression construct. Several promoters have been identifi ed to provide high levels of gene expression in plants. The most commonly used promoter to control antigen expression in dicotyledonous plant species is the strong and constitutive Caulifl ower mosaic virus 35S (CaMV 35S), promoter; its enhancer region is sometimes used as a duplicate to further amplify gene expression (Smith et al., 2002, Warzecha et al., 2003, Dong et al., 2005). Another strong candidate promoter is the synthetic Super promoter (Ni et al., 1995), which has also been successfully used to drive vaccine antigen expression in plants (Tuboly et al., 2000, Pogrebnyak et al., 2005). For expression of vaccine antigens in the potato, the auxin-inducible mannopine synthase (mas) P1, P2 dual promoter system has been widely used by Langridge and colleagues (Arakawa et al., 1997, Arakawa et al., 2001, Yu and Langridge, 2001), while other groups have preferred the tuber-specifi c patatin promoter (Mason et al., 1996, Lauterslager et al., 2001). In maize, the only monocotyledonous plant in which the expression of vaccine antigens has thus far been reported, the CaMV 35S promoter can be used to accumulate LT-B in seeds (Chikwamba et al., 2002b). However, more effi cient antigen production has been achieved with maize seed-specifi c globulin-1 and γ-zein promoters (Chikwamba et al., 2002b, Streatfi eld and Howard, 2003a). In general, the most widely used promoter to control transgene expression in monocotyledonous plants has been the constitutive maize ubiquitin-1 promoter (Christensen and Quail, 1996).
30
Literature review
After transcription, the mature transcript has to be protected from premature degradation and transported effi ciently to the cytoplasmic translation machinery. The related posttranscriptional eukaryotic processing events (capping, splicing, polyadenylation) can have a major effect on the levels of protein produced in the plant cell (Gutierrez et al., 1999). Introduction of introns into the expression construct has been demonstrated to increase the stability of mRNA (Topfer et al., 1993). Particularly in monocot plants, the introduction of introns has been demonstrated to elevate the expression levels of marker genes (Callis et al., 1987, Maas et al., 1991). Polyadenylation signals also strongly infl uence the stability of mRNA and the level of gene expression in plant cells (Ingelbrecht et al., 1989, Hunt, 1994). Widely used polyadenylation signals in vaccine applications include those from the CaMV 35S transcript (Huang et al., 2001, Piller et al., 2005), the soybean vegetative storage protein (vsp) (Richter et al., 2000, Viera da Silva et al., 2002), and the Agrobacterium nopaline synthase (nos) and octopine synthase (ocs) genes (Mason et al., 1992, Aziz et al., 2002). Richter et al. (2000) did a side-by-side comparison of three different polyadenylation signals in potato and found that plants with vsp and potato proteinase inhibitor 2 (pin2) polyadenylation signals accumulated Hepatitis B virus surface antigen (HBsAg) in quantities several-fold higher than plants equipped with nos-terminator indicating that posttranscriptional effects contribute strongly to the enhanced expression of HBsAg. Generation of the synthetic antigen-encoding genes enables the elimination of putative internal methylation and polyadenylation sites, mRNA secondary structure hairpins, and
premature transcriptional termination sites to ensure effi cient transcription and stability of the generated transcript (Dong et al., 2005).
2.1.6.2 TranslationInitiation of mRNA translation is typically the rate-limiting step in polypeptide synthesis in plants (Kawaguchi and Bailey-Serres, 2002). The initiation can be optimized by confi rming that the translational start-site matches the Kozak consensus for plants (Joshi et al., 1997). After initiation, the rate of translation may become limited by a lack of suitable tRNAs. Indeed, genes of foreign origin may have an aberrant codon composition for plant translation machinery, and increased levels of protein expression have been reported in plants after modifi cation of gene-coding sequences of bacterial origin (Perlak et al., 1991, Adang et al., 1993, Horvath et al., 2000). Similarly, optimization of gene sequences for plant codon usage has enhanced the expression of various vaccine antigens such as E. coli heat-labile enterotoxin subunit B (LT-B) (Mason et al., 1998), cholera toxin subunit B (CT-B) (Kang et al., 2004a) and TGEV spike glycoprotein (Tuboly et al., 2000, Streatfi eld et al., 2001). For example, LT-B accumulation was enhanced 5- to 40-fold in potato tubers when a codon-optimized gene was used instead of an unmodifi ed LT-B gene (Mason et al., 1998).
RNA leader sequences of plant viral origin have been identifi ed to boost antigen expression in plants (Dowson Day et al., 1993). Most widely used 5´ untranslated regions used in antigen applications include 5´ leader sequences from Tobacco etch virus (TEV) (Thanavala et al., 1995, Mason et al.,
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1996, Richter et al., 2000, Dong et al., 2005) and TMV Ω element (Richter et al., 2000, Matsumura et al., 2002, Biemelt et al., 2003).
2.1.6.3 Subcellular targeting: optimal yield and glycosylation The subcellular environment in which the recombinant protein accumulates infl uences its folding, assembly, and posttranslational modifi cation. Factors such as surrounding pH and presence of appropriate chaperones or degrading proteinases may greatly affect antigen stability and subsequent accumulation level. Recombinant proteins can be directed to the secretory pathway by a N-terminal signal peptide, which can be either of plant or foreign origin. Such proteins are co-translationally synthesized in the endoplasmic reticulum (ER) and transported by default through the Golgi network to the apoplast, or in the presence of a suitable signal directed to the vacuole (Matsuoka and Nakamura, 1991). In the apoplast, depending on its size and structure, the protein can be retained in the cell wall matrix or secreted from the cell. The ER is an oxidizing environment with an abundance of molecular chaperones and a few proteases. Comparative analysis with recombinant antibodies has shown that they accumulate more effi ciently when targeted to the secretory pathway than to the cell cytoplasm (Schillberg et al., 1999). This has also been shown with vaccine antigens; Richter et al. (2000) targeted the synthesis of HBsAg to the apoplast or vacuoles and found 2- to 7-fold accumulation levels compared with cytoplasm-targeted potato plants. Streadfi eld et al. (2003) studied the subcellular targeting of LT-B in maize seeds, and reported that targeting to the
apoplast and vacuoles increased the expression level 3080-fold and 20 000-fold, respectively, with expression in the cytoplasm. Moreover, the ER-targeted proteins can be retained in the ER by using a C-terminal (SE)H/KDEL peptide (Munro and Pelham, 1987). This has led into even greater accumulation of recombinant antibodies than the targeting into secretory pathway (Conrad and Fiedler, 1998). Similarly, a fourfold increase in LT-B accumulation was shown in tobacco and potato plants when the secretion pathway-targeted protein was retained in the ER (Haq et al., 1995). Maize seeds, by contrast accumulated more than tenfold less LT-B when retained in the ER than targeted to the secretion pathway (Streatfi eld et al., 2003).
Recombinant proteins can also be targeted to intracellular plant organelles, namely, mitochondria and plastids. This is achieved by adding N-terminal transit peptides, which are recognized by the organelle transport machinery delivering the proteins to organelles (Glaser and Soll, 2004). Richter et al. (2000) were unable to detect viral protein HBsAg when it was targeted to chloroplasts. In maize seeds, bacterial LT-B plastid targeting led to a sevenfold increase compared with cytosolic protein, but did not reach the levels obtained with apoplast, ER, or vacuolar targeting (Streatfi eld et al., 2003). Candidate vaccine antigens are typically proteins or peptides of viral or bacterial origin. Proteins of bacterial origin lack glycosylation natively, but many viral surface proteins are heavily glycosylated by the host cell. A suitable glycosylation of vaccine antigens can be achieved by appropriate subcellular targeting. When glycosylation is desired, the antigen should be directed to the ER. The level of glycosylation can be affected by retaining
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the antigen peptide in the ER instead of being secreted through the Golgi apparatus, where further carbohydrate groups will be added (Faye et al., 2005). Correct glycosylation can be a prerequisite (Yelverton et al., 1983) or alter (Judge et al., 2004) the immunogenicity of vaccine antigens. Glycosylation can be avoided by targeting the accumulation of the antigen to the cytoplasm or to intracellular plant organelles. Alternatively, the addition of carbohydrates can be prevented by mutating the putative glycosylation sites on the antigen peptide. Mammals and plants have a similar structure of core high-mannose glycans, but some differences in glycosylation do exist. Plant glycans use α-1,3 fucose linkage rather than the α-1,6 fucose linkage found in mammals, have additional β-1,2 xylose linkages, and lack the sialic acid moieties typical of mammalian glycosylation (Faye et al., 2005). Completely mammalized plant glycosylation has not been reported to date, but has been of a considerable interest in research (Wenderoth and von Schaewen, 2000, Bakker et al., 2001, Shah et al., 2003, Strasser et al., 2004).
2.1.6.4 Fusion proteinsFusing peptides of whole proteins of poor stability to other known stable proteins can improve the stability of the selected antigen in the plant tissues and in the subsequent vaccine delivery. This approach is commonly used for peptides produced with the plant virus expression system, and has also been used to optimize the antigen expression in stably transformed plants. Such vaccine antigen fusion partners include green fl uorescence protein (GFP) (Molina et al., 2004, Ziauddin et al., 2004), CT-B (Arakawa et al., 2001, Yu and Langridge, 2001, Kim
et al., 2004b, Lee et al., 2004, Molina et al., 2004, Molina et al., 2005), CT-A (Yu and Langridge, 2001), and β-glucuronidase (GUS) (Gil et al., 2001, Dus Santos et al., 2002). Antigen fusion with marker genes also allows antigen production to be screened conveniently. The Canadian company Sembiosys Genetics Inc. (www.sembiosys.ca) has established a production system in which recombinant proteins are expressed in oilseed crops as a fusion with oleosin. Fusion proteins accumulate in oil bodies and can be extracted using a simple extraction procedure and the recombinant protein can be released from its fusion partner by proteolytic cleavage. Another application based on protein fusions has been introduced by the Spanish company ERA Plantech (www.eraplantech.com). It has introduced a production system in which recombinant proteins are fused with peptides derived from storage proteins and accumulate host-independently in ER-derived protein bodies that can be separated by their high density.
2.1.6.5 TransplastomyChloroplast transformation can provide better gene expression capacity than nuclear genetic engineering. This is mainly because of the high transgene copy number; a single cell can possess up to 10 000 copies of the plastid genome (Daniell et al., 2004). Transgenes are introduced into the chloroplast genome by site-specifi c integration, and despite the high accumulation level of transcripts, transgene silencing has not been reported in transplastomic plants (Lee et al., 2003, Dhingra et al., 2004). Similarly, lack of posttranscriptional gene silencing was evident with the accumulation of bacterial Cry toxins in over 46% of total soluble protein (TSP) in transplastomic tobacco
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Literature review
lines (De Cosa et al., 2001). In addition, chloroplasts offer a contained environment within a plant cell where potentially plant-toxic compounds have been successfully expressed (Lee et al., 2003, Leelavathi et al., 2003). This approach has also been applied for vaccine antigen expression in tobacco chloroplasts. Successful examples include CT-B (Daniell et al., 2001), the TetC fragment of the tetanus toxoid (Tregoning et al., 2003) and an anthrax protective antigen (Aziz et al., 2005) in tobacco chloroplasts. Chloroplasts cannot complete protein posttranscriptional modifi cation, but mammal glycosylation machinery is being introduced into the chloroplast genome (H. Daniell, personal communication).
2.1.7 Plant-produced candidate vaccines for animal healthStudies of vaccine candidates expressed in plants for animal health (until the end of the year 2005) are summarized in Table 1. The majority of these applications are against viral pathogens; applications against other pathogens are scarce. The likely reason for this is not a lack of potential bacterial pathogens but a lack of cost-effective means to treat animal virus infections, whereas bacterial infections can usually be controlled by antibiotics. Most of these plant-produced vaccines have only preliminary been tested in laboratory animals (mainly mice), but the most advantageous examples show protection in the target species after oral delivery (Lamphear et al., 2002, Wu et al., 2004, Zhou et al., 2004). The main results of plant-based vaccine applications for animal health are summarized in the text below.
2.1.7.1 Applications for avian diseases
A Chinese research group has successfully developed an edible potato-based vaccine against Infectious bronchitis virus (IBV) (Zhou et al., 2003, Zhou et al., 2004). Sliced tubers were administered to chickens in three doses over two weeks, and a week after the last administration the chickens were challenged with IBV. Orally immunized chickens developed a virus-specifi c antibody response and were protected against IBV. Wu et al. (2004) succeeded in vaccinating chickens against Infectious bursal disease virus (IBDV). Chickens orally immunized with arabidobsis crude extracts were protected in a manner similar to their counterparts, who had received a commercial injectable vaccine. The effi cacy of this vaccine was verifi ed in three replicate experiments (ten chickens per group) with identical results.
Two recent publications report achievements in the development of a plant-produced vaccine against avian Newcastle disease virus. Cucumber mosaic virus was utilized as a platform to display epitopes from virus surface glycoproteins. Chimeric virus particles were assembled in tobacco leaves, but their immunogenicity was not determined (Zhao and Hammond, 2005). Berinstein et al. (2005) had another approach, using transgenic potato plants to express the same surface antigens in full length. Mice fed with the potato leaves or injected with leaf extracts showed virus-specifi c antibody responses. For an application against diarrhea caused by F5-positive ETEC, see section 2.1.7.4.
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2.1.7.2 Applications for bovine diseases
A TMV-based vaccine against Bovine herpesvirus (BHV) is the only example of a bovine disease where an immune response in cattle has been reported (Perez Filgueira et al., 2003). Immunogenic glycoprotein D was produced as a by-product in TMV-inoculated tobacco plants, and the crude plant extract emulsifi ed in oil and subsequently injected into cows was able to raise specifi c humoral and cellular immune responses. Most importantly, these animals were protected against BHV to a similar level as cows vaccinated with the commercial vaccine.
Foot -and -mouth disease virus (FMDV) infects many meat- and milk-producing domestic animals, including cows. In an Argentinean laboratory, a vaccine against FMDV has been extensively developed. This vaccine is based on the viral structural VP1 protein, and expression has been reported in arabidobsis (Carrillo et al., 1998), potato tubers (Carrillo et al., 2001), and alfalfa leaves (Wigdorovitz et al., 1999, Dus Santos et al., 2002, Dus Santos and Wigdorovitz, 2005). In their latest report, the presence of FMDV particles in transgenic alfalfa was speculated, but the data were not shown (Dus Santos and Wigdorovitz, 2005). Protective immunogenicity of orally or parenterally delivered plant material has been reported in mice (Carrillo et al., 1998, Wigdorovitz et al., 1999, Carrillo et al., 2001, Dus Santos et al., 2002, Dus Santos and Wigdorovitz, 2005), although the production level of vaccine antigen has been low (0.01% of TSP in alfalfa) (Dus Santos and Wigdorovitz, 2005). The same authors have also extensively developed a plant-produced vaccine against Bovine
rotavirus infections. They have expressed epitopes of VP4 protein with a TMV-based transient system (Perez Filgueira et al., 2004) and in transgenic alfalfa plants (Wigdorovitz et al., 2004). Immunogenicity was determined in a mouse model. Most importantly, alfalfa-fed mice developed a virus-specifi c antibody response, with pups subsequently being protected against viral challenge by passive lactogenic immunity (Wigdorovitz et al., 2004). Furthermore, the expression of Bovine rotavirus VP6 protein has been reported in transplastomic tobacco plants (Birch-Machin et al., 2004) and potato tubers (Matsumura et al., 2002). Birch-Machin et al. (2004) reported a high-level expression (3% of TSP) in young tobacco plants, but lower levels in older plants, suggesting low stability of the VP6 protein in chloroplasts. An Indian research group has been developing a plant-produced vaccine against cattle Rinderpest virus. Hemaglutinin of the virus was produced in the leaves of the edible plant species pigeon pea (Satyavathi et al., 2003) and peanut (Khandelwal et al., 2004). In the latter case, the immunogenicity of plant-produced hemaglutinin was assessed in a mouse model. Both oral and systemic immunizations raised in vitro virus-neutralizing antibodies.
A Canadian group has investigated a plant-produced vaccine against bovine pneumonic pasteurellosis caused by Mannheimia haemolytica. Transgenic white clover (Lee et al., 2001) and alfalfa (Ziauddin et al., 2004) plants expressing a fragment of leucotoxin fused with GFP were generated, and the immunogenicity of this fusion protein was established in rabbits after intramuscular injection. The generated antibodies also neutralized a related leucotoxin in vitro.
The colonization factor of O157:H7
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Literature review
bovine diarrhea-causing enterohemorraghic E. coli (EHEC) was expressed in tobacco plants (Judge et al., 2004). His-tagged intimin was purifi ed from plant extracts and injected intraperitoneally in mice. Alternatively, mice were fed the transgenic plant material. In the following EHEC challenge, reduced E. coli shedding was observed in the parenterally immunized group as well as in mice that had received an oral boost after being parenterally primed. For an application against diarrhea caused by F5-positive ETEC, see section 2.1.7.4.
2.1.7.3 Applications for canine diseasesDevelopment of a plant-produced vaccine against Canine parvovirus (CPV) has been the objective of several studies. Epitopes of the VP2 major structural protein have been displayed on plant viral particles (Fernandez-Fernandez et al., 1998, Langeveld et al., 2001, Nicholas et al., 2002) and expressed in transgenic arabidobsis (Gil et al., 2001) and tobacco plants (Molina et al., 2004, Molina et al., 2005).
Chimeric CPMV particles expressing the CPV VP2 epitope were used to immunize mice intranasally or subcutaneously, and the subsequent immune response was characterized (Nicholas et al., 2002). Intranasal delivery activated antigen-specifi c IgA secretion into mucosal fl uids. Moreover, serum antibodies from both subcutaneously and intranasally vaccinated mice showed neutralizing activity against CPV in vitro. Langeweld et al. (2001) achieved similar results with a purifi ed CPMV-based vaccine in a natural CPV host species. After two subcutaneous injections, the dogs were challenged with a lethal dose of CPV; the group than had received the
plant-produced vaccine was completely protected.
CPV-specifi c serum antibody response was seen in mice following parenteral injections or after oral administration of crude protein extracts from transgenic arabidobsis plants expressing the VP2 epitope fused to the GUS marker gene (Gil et al., 2001). Molina et al. (2004) generated transplastomic tobacco plants that accumulated the VP2 epitope fused to CT-B or GFP in 33.1% or 22.6% of TSP, respectively. Plant extracts with CT-B fused vaccine antigen were able to raise in vitro virus-neutralizing antibodies after parenteral delivery. The oral delivery of pulverized plant material also raised virus-specifi c antibodies, but they were not neutralizing (Molina et al., 2005).
Rabies virus has a wide host range, including domestic and wild animals as well as humans. The canine species are an important carrier and source of rabies infections, and therefore plant-produced vaccine applications against rabies are discussed in this section. Antigenic rabies surface glycoprotein has been expressed in transgenic tobacco (Ashraf et al., 2005) and tomato (McGarvey et al., 1995) plants and the related epitopes have been displayed on AlMV particles (Yusibov et al., 1997, Modelska et al., 1998). The immunogenicity of these plant-produced antigens was assessed in a mouse model. The chimeric AlMV particles as well as the surface glycoprotein expressed in tobacco raised a protective immune response after a parenteral (Modelska et al., 1998, Ashraf et al., 2005) or oral (Modelska et al., 1998) immunization. The oral immunogenicity of chimeric virus particles has also been studied in humans (Yusibov et al., 2002).
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Literature review
2.1.7.4 Applications for porcine diseases
Development of a plant-produced vaccine against porcine TGEV has been carried out by several research groups. Neutralizing virus spike protein antigens have been expressed in the leaf tissue of transgenic tobacco (Tuboly et al., 2000) and arabidobsis plants (Gomez et al., 1998), in potato tubers (Gomez et al., 2000), and in maize seeds (Streatfi eld et al., 2001). Plant-produced spike protein antigens were immunogenic in mice following parenteral (Gomez et al., 1998, Gomez et al., 2000) or oral (Gomez et al., 2000) administration. Tuboly et al. (2000) immunized weaned piglets intraperitoneally with crude tobacco extracts and detected virus-specifi c neutralizing antibodies after three injections. The American biotechnology company Prodigene (www.prodigene.com) has extensively studied the use of maize seeds as an edible delivery vehicle against TGEV. They have convincingly presented the effi cacy of an edible maize vaccine in multiple experiments with piglets (Streatfi eld et al., 2001, Lamphear et al., 2002). In addition, they found that the antigen was stable during storage in various conditions and were able to concentrate the antigen with milling techniques (Lamphear et al., 2002). This is perhaps the most advanced plant-produced vaccine reported up to date.
Development against a plant-produced vaccine against Porcine epidemic diarrhea virus (PEDV) has been established by a Korean research group. PEDV spike protein has been expressed in tobacco (Bae et al., 2003, Kang et al., 2005b) and potato (Kim et al., 2005). The tobacco-derived protein was reported to raise virus-specifi c antibodies in mice after an oral delivery (Bae et al., 2003).
ETEC expressing F5 fi mbriae causes diarrhea in various farm animals, including pigs, chickens, and cows, whereas F4+ ETEC is pathogenic only to pigs. Major subunit proteins of these fi mbriae have been expressed in the leaves of tobacco (F4) (Huang et al., 2003) and soybean (F5) (Piller et al., 2005). The immunogenicity of these plant-produced subunit proteins was confi rmed by vaccinating mice parenterally with crude leaf extracts. In addition to these candidate vaccines based on colonization factors, the expression of ETEC heat-labile toxin subunit B in plants has been widely studied. LT-B forms homopentamers to mediate the binding of the toxin to enterocytes. The autoassembly of these pentamers has been observed when the LT-B encoding gene was expressed in transgenic plants. Successful examples include tobacco (Kang et al., 2005c), maize seeds (Streatfi eld et al., 2001, Chikwamba et al., 2002b) and potato tubers (Haq et al., 1995, Mason et al., 1998, Lauterslager et al., 2001). Immunogenicity studies of plant-produced LT-B have been carried out with mice (Haq et al., 1995, Mason et al., 1998, Lauterslager et al., 2001, Streatfi eld et al., 2001, Streatfi eld et al., 2002) and humans (Tacket et al., 1998, Tacket et al., 2004). With the mouse model, the induced immune response has been reported to protect against subsequent challenge with the LT-holotoxin (Mason et al., 1998, Streatfi eld et al., 2001, Chikwamba et al., 2002a).
2.1.7.5 Applications for other animal diseasesHemaglutinin neuramidase of Peste-des-petits-ruminant virus was expressed in pigeon pea plants, but the immunogenicity of the plant-produced antigen was not
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studied (Prasad et al., 2004). A Plant-produced vaccine against Rabbit hemorraghic disease virus (RHDV) has been developed by expressing the VP60 major structural protein in potato leaves (Castanon et al., 1999) or by displaying the related epitopes on PPV particles (Fernandez-Fernandez et al., 2001). In both applications, the parenteral vaccination with plant-produced antigens protected the rabbits against lethal RHDV challenge. The VP2 coat protein-derived epitope of Mink enteritis virus was displayed on the CPMV particles (Dalsgaard et al., 1997). Minks parenterally vaccinated with the chimeric virus particles were protected against the related virus challenge. Mice were
similarly protected after being parenterally or nasally immunized with TMV-derived surface glycoprotein of Murine hepatitis virus (Koo et al., 1999).
In some instances, vaccination of animals also serves to protect human health. Rabies and O157:H7 EHEC are examples of target diseases for which plant-produced vaccines have been developed and that would offer protection to both animals and humans. For detailed description of these applications, refer to sections 2.1.7.3 and 2.1.7.4. Other examples of animal pathogens potentially pathogenic to humans include Salmonella and avian infl uenza; however, these have not yet been the focus of plant-based vaccination strategies.
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2.2 Escherichia coli postweaning diarrhea in pigs
2.2.1 PrefaceEscherichia coli is a Gram-negative, facultatively anaerobic, fl agellated, and rod-shaped bacterium with a diameter of about 1 µm. It is a common resident in the large intestine of mammals and birds but also a pathogen causing intestinal as well as extra-intestinal infections such as urinary tract infections, cystitis, pyelonephritis, meningitis, peritonitis, mastitis, septicaemia, and Gram-negative pneumonia. Intestinally pathogenic E. coli strains are classifi ed on the basis of virulence properties into enterotoxigenic (ETEC), enteropathogenic (EPEC), enteroinvasive (EIEC), verotoxigenic (VTEC), enterohemorrhagic (EHEC), enteroaggregative (EAggEC), and diffusively adherent (DAEC) E. coli (Lee and Mekalanos, 1999). The main characteristics of these strains are summarized in Table 2.
E. coli is a major cause of porcine postweaning diarrhea in Finland (Laine et al., 2004) and worldwide (Fairbrother et al., 2005). This diarrhea, occurring typically during the fi rst two weeks after weaning, is also referred to as postweaning enteric colibacillosis. It hampers animal welfare and leads to signifi cant economic losses due to reduced growth and mortality. Multiple factors, such as weaning stress, disappearance of maternal antibodies in the gut, and dietary changes, affect the severity and incidence of this disease. E. coli PWD is mainly caused by ETEC, a pathotype described by the virulence factors of two types: (i) adhesins mediating the bacterial adherence to the intestine and (ii) enterotoxins causing symptoms of diarrhea. Another type of E. coli related to porcine PWD is EPEC, but this type has only a minor role (accounting for 1.4-6% of E. coli PWD) (Frydendahl, 2002, Fairbrother et al., 2005). Some of the PWD-causing ETEC strains possess an additional gene encoding a verotoxin that induces edema
Table 2. Characterization of enteric Escherichia coli pathogens. B, bovine; C, cat; G, goat; H, human; O, ovine; P, porcine; S, sheep. Adapted from Lee and Mekalanos, 1999; Van den Broeck et al., 2000; Kaper, 2004.
Pathogenic E. coli Hosts Pathogenic mechanism DiseaseEnterotoxigenic (ETEC)
B, G, H, O, P, S
Adherence by various colonization factors and secretion of enterotoxins
Colonization of intestine and noninfl ammatory diarrhea
Enteroinvasive(EIEC)
H Entry into M-cell cytosol and spread into adjacent enterocytes
Necrosis on intestinal cells and infl ammatory diarrhea
Enteropathogenic (EPEC)
C, H, P Intimate adherence to epithelium and effacement of enterocytes
Alteration of enterocyte brush border and noninfl ammatory diarrhea
Enterohemorraghic(EHEC)
B, G, H Intimate attachment to, and effacement of enterocytes, production of Shiga-like toxins
Damage on vascular function and cause hemorrhagic complications on epithelium, infl ammatory, bloody diarrhea
Enteroaggregative (EAggEC)
H Adherence to epithelium and effacement of enterocytes, release of enterotoxins
Colonization of intestine and noninfl ammatory diarrhea
Diffusively adherent (DAEC)
H Dispersed adherence to enterocytes via afi mbrial or fi mbrial adhesins
Colonization of intestine and noninfl ammatory diarrhea
Verotoxigenic (VTEC)
B, C, P Adherence to epithelium and release of Shiga-like verotoxins
Colonization of intestine and oedema disease
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Literature review
disease. EPEC and edema disease are not discussed further here. The following review provides an overview of the ETEC pathotype related to porcine PWD and summarizes deployment strategies to prevent infection. F4 fi mbria, one of the main virulence factors found in porcine ETEC, is described in detail and discussed as an oral vaccine candidate against PWD.
2.2.2 Virulence markers of ETEC in PWDClassically, identifi cation of pathogenic E. coli strains is based on serotyping, i.e. a series of bacterial agglutination tests with specifi c antisera. Complete serotyping comprises determination of several different serogroups, including O (outer membrane lipopolysacharide), K (capsule), H (fl agellar), and F (fi mbrial) antigens (Nataro and Kaper, 1998). Current diagnostic serotyping of PWD-associated E. coli typically covers the O, H, and F serogroups, the latter also being a virulence factor (Fairbrother et al., 2005). Although serotyping was developed prior to the identifi cation of ETEC virulence factors, it is still a valuable diagnostic tool and epidemiological marker, showing a high correlation with certain virulence traits (Bertschinger and Fairbrother, 1999). However, the virulence factor-specifi c genotyping is quickly becoming a preferred method to identify PWD-associated E. coli (Frydendahl, 2002). The predominant serogroup detected worldwide in PWD-causing ETEC in pigs is O149. O149-associated H antigens include H10, H19, and H43, and the most common related fi mbrial antigen is F4ac (Fairbrother et al., 2005).
In addition to virulence factors, ETEC often carries a hlyA gene encoding
α-hemolysin (Fairbrother et al., 2005). The presence of α-hemolysin is easy to confirm, indicated as clear zones of hemolysis surrounding colonies grown on blood agar. Frydendahl (2002) found a strong positive correlation between hemolysin production and virulence factors of ETEC strains in Denmark; however, this was not the case in China (Chen et al., 2004). Hemolytic activity can also be detected on other pathogenic E. coli strains (Fairbrother et al., 2005), and although it can serve as a useful marker, its use as the sole tool for ETEC diagnosis is not recommended (Bertschinger and Fairbrother, 1999).
2.2.3 Virulence factors of ETEC in PWD
2.2.3.1 Colonization factors
The nomenclature of E. coli surface adhesins is confusing and in many cases parallel nomination is used to describe colonization factors. In 1983, Ørskov and Ørskov introduced a simplifi ed nomenclature for E. coli fi mbrial adhesins. In this designation, “F” (for fi mbrial) is followed by consecutive numbers in the order of their discovery. This nomenclature has been widely established to designate fi mbrial antigens found in E. coli strains infecting animals, and it is also used here (on the fi rst mention, synonyms are indicated in parentheses). In contrast, F nomenclature has not been generally adapted for describing colonization factors mediating the attachment of E. coli strains pathogenic to humans. The most commonly used nomenclature in this fi eld is available in a review of Nataro and Kaper (1998).
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Literature review
The ETEC causing porcine PWD most often carries the F4 (K88) or F18 (F107, 2143P, 8813, Av24) fi mbriae. These two fi mbria types have been the most prevalent in Denmark (Frydendahl, 2002), Germany (Wittig et al., 1995), Hungary (Nagy et al., 1996), Poland (Osek, 1999), South Dakota, USA (Francis, 2002), and Quebec, Canada (Fairbrother et al., 2005). Other fi mbria types occasionally associated with porcine PWD include F5 (K99), F6 (987P), and F41, but these fi mbriae are more often found in E. coli isolates causing neonatal diarrhea (Harel et al., 1991, Ojeniyi et al., 1994). Recently, an afi mbrial adhesin, AIDA, has been detected in E. coli isolates associated with porcine PWD (Niewerth et al., 2001, Ha et al., 2003, Ngeleka et al., 2003). Another surface protein, Paa, has also been proposed as a potential adhesin of ETEC in porcine PWD (Batisson et al., 2003). In general, only one adhesin type is present in a single ETEC isolate, but ETEC strains can sometimes carry two or three different adhesins simultaneously (Bertschinger and Fairbrother, 1999).
2.2.3.2 ToxinsHeat-labile enterotoxins LTI and LTIITwo distinct antigenic variants of E. coli heat-labile enterotoxin, LTI and LTII, have been identifi ed. LTII is not associated with the disease (Holmes et al., 1986), and thus, is not discussed here. In this review, LT in the text refers to LTI. LT is composed of B subunits forming a homopentameric structure that attaches the toxin to the host cell and an enzymatically active subunit A1 that is attached to the ring of B subunits by an A2 fragment (Sixma et al., 1991). LT toxin binds to monosialoganglioside GM1, as well as to some other
glycostructures found on the surface of mammalian cells, by the pentameric ring of B subunits (Teneberg et al., 1994). It is endocytosed by invagination of the plasma membrane, leading to the release of A1 subunit into the cytoplasm of the host cell (Lencer et al., 1995). The A1 subunit possesses adenosine diphosphate-ribosyl transferase activity and stimulates the Gs protein, which upregulates the catalytic activity of adenylate cyclase. As a result, an excessive level of cyclic adenosine monophosphate (cAMP) is converted from adenosine triphosphate by the adenylate cyclase. Elevated cAMP activates protein kinase A, which stimulates the chloride channels and transporters (Sears and Kaper, 1996), leading to increased secretion of chloride ions and subsequent massive water loss into the intestinal lumen.
LT has a high structural similarity to the CT toxin of Vibrio cholerae. Both, besides being active mucosal antigens, are potent immunomodulators, and their use as mucosal adjuvants has been comprehensively investigated (Elson and Dertzbaugh, 1999). Indeed, LT and CT have been found to induce strong mucosal and systemic immune responses, and to provide a long-term memory for mucosally co-administered proteins (Vajdy and Lycke, 1993, Rappuoli et al., 1999). However, these toxins must be administered simultaneously with the antigen and by the same route in order to act as mucosal adjuvants (Lycke and Holmgren, 1986). Moreover, the purifi ed or recombinant B subunits of CT and LT can enhance immune responses not only to directly conjugated antigens delivered by the oral route but also to antigens mixed with the LT or CT B subunits (Williams et al., 1999). However, they can also suppress immune responses and induce tolerance to orally or nasally
41
Literature review
delivered antigens (Sun et al., 1994, Williams et al., 1999). Interestingly, LT and CT do not induce antibody responses to the food antigens expected to be present in the intestine at the time of administration (Nedrud and Sigmund, 1990).
Heat-stabile enterotoxins: STa, EAST1, and STbEnterotoxins STa and STb found in PWD-causing ETEC strains are low molecular weight peptides (STa ca. 2 kDa, STb 5 kDa) stabilized by intramolecular disulfi de bonds (3 in STa and 4 in STb) (Lee et al., 1983, Picken et al., 1983, Thompson and Giannella, 1985, Gariepy et al., 1986, Fujii et al., 1991, Ozaki et al., 1991). As implied by their designation, they are resistant to boiling (15 min). STa is also very stable against low pH and proteolytic enzymes, STa and STb both are inactivated by chemicals that destroy disulfi de bonds (Dreyfus et al., 1983). Heat-stabile enterotoxin EAST1 was originally detected in enteroaggregative E. coli and shares a high similarity to STa, but possesses only two intramolecular disulfi de bonds (Menard and Dubreuil, 2002). STa toxins bind the membrane-spanning protein gyanylyl cyclase S on enterocytes and activate cytoplasmic guanylate cyclase leading to elevated levels of cyclic guanosine monophosphate (cGMP) inside the enterocytes (Waldman et al., 1986, Crane et al., 1992, de Sauvage et al., 1992, Jaso-Friedmann et al., 1992). Similarly to the elevation of cellular cAMP caused by LT, the accumulation of cGMP leads to secretion of chloride ions, followed by passage of fl uid into the gut lumen. The mechanisms by which STb causes accumulation of fl uid in the intestine are poorly known. They are not related to raised levels of cyclic monophosphates, but appear to
involve elevated prostaglandin E2 levels in the intestine (Menard and Dubreuil, 2002).
STb is the most frequently detected enterotoxin in PWD-related E. coli strains (Moon et al., 1986, Frydendahl, 2002), and is usually found in combination with other toxins (Post et al., 2000, Francis, 2002). Indeed, the gene encoding STb is frequently present on a plasmid that also encodes LT (Francis, 2002). STa is often associated with the ETEC that causes neonatal infections (Flores-Abuxapqui et al., 1997), and occasionally it is found in the ETEC implicated with porcine PWD, but rarely as the sole enterotoxin (Frydendahl, 2002). EAST1 has also been shown to be widespread among porcine ETEC (Yamamoto and Nakazawa, 1997, Choi et al., 2001, Frydendahl, 2002, Noamani et al., 2003, Osek, 2003), commonly being detected in F4+ ETEC with LT toxin (Yamamoto and Nakazawa, 1997), as well as F18+ ETEC (Frydendahl, 2002).
2.2.4 Prevention of porcine PWDDehydration caused by PWD can be effi ciently treated with electrolyte replacement solutions (Bywater and Woode, 1980), but postweaning syndromes with complicated shock are often fatal (Bertschinger and Fairbrother, 1999). Mortality of and morbidity of PWD can be reduced by postponing the weaning since older piglets are not as vulnerable to infection and have a better prognosis. Because multiple factors implicate the outbreak of PWD, no single preventative strategy has been completely effective thus far (Fairbrother et al., 2005). Current measures deployed to defeat PWD are summarized below.
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Literature review
2.2.4.1 Hygiene and diet
Improved hygiene standards can limit the spread of PWD, but even high-grade isolation with specifi c pathogen-free animals is not always suffi cient to prevent E. coli infections (Bertschinger and Fairbrother, 1999). Diet is one of the most important factors affecting the outbreak of PWD. Antibiotics were previously routinely added to creep and weaners’ feed to control bacterial infections. Since this unavoidably leads to generation of resistant pathogenic strains, in 1999 the EU countries banned the use of growth promoters and prophylactic antibiotics in feed.
A weaners’ feed supplemented with animal-source proteins such as dried plasma reduces the duration of period of lowered feed intake immediately after weaning and promotes tolerance to incidence of PWD (Van Dijk et al., 2002). Later, restricted feeding and increased fi ber content in feed stabilizes gastrointestinal transit and can diminish PWD infections (Bertschinger et al., 1978). The addition of organic acidifi ers to feed lowers stomach pH and decreases the viability of ETEC and the incidence of PWD (Giesting and Easter, 1985). Protease supplements such as bromelain may similarly decrease the incidence (Mynott et al., 1996). Moreover, supplementation of feed with zinc oxide (antibacterial effect) to levels of 2400-3500 ppm reduces diarrhea and mortality among weaned piglets (Holm and Poulsen, 1996). However, regular use of high amounts of potentially toxic zinc is unacceptable for environmental reasons. Göransson et al. (1993) reported that antisecretory factors in blood plasma can diminish the effects of PWD. They also reported that these antisecretory factors can be increased by supplementing the
feed with glucose and some amino acids (Göransson et al., 1993).
A soybean-containing diet has been proposed to cause hypersensitivity in porcine gut and increase the risk of PWD outbreak (Dreau et al., 1994). A diet supplemented with Rhizopus- and Bacillus-fermented soybean meal, by contrast, has been found to reduce the incidence and symptoms of PWD (Kiers et al., 2003). Other bacterial probiotics that have been reported to reduce PWD include the viable spores of Bacillus licheniformis and Bacillus toyoi (Kyriakis et al., 1999). Recently, the Finnish feed company Suomen Rehu introduced a feed for weaned piglets with yeast-derived probiotics proclaimed to diminish the incidence of PWD (www.suomenrehu.com). Although the onset of PWD can be decreased by dietary means, this may only delay the ETEC infections since outbreaks of PWD have recently been detected three or even six to eight weeks after weaning (Fairbrother et al., 2005).
2.2.4.2 Breeding of resistant pigsAttachment of ETEC to intestinal enterocytes is a prerequisite for the colonization and outbreak of PWD. This attachment is mediated by particulate receptors present in the intestine. Pigs lacking these receptors exist naturally and are resistant to related infections (Van den Broeck et al., 1999a, Meijerink et al., 2000). Expression of these receptors is genetically determined, with presence being dominant (Gibbons et al., 1977). Breeding of F18-resistant pigs has shown some success in Switzerland (Stranzinger, 2004), but the genetic and molecular origins of F4 receptor(s) (F4R) are poorly characterized, and reliable methods for selection of breeding stock are not available (see section 2.2.5.3).
43
Literature review
Even should breeding of F4-resistant pigs become dependable, it should be kept in mind that receptor-negative sows do not transfer F4-specifi c lactogenic antibodies to their piglets; thus, outbreaks of neonatal F4+ ETEC infections will likely increase. In breeding resistant animals, selection pressure is created, and additional types of adhesive fi mbria or new variants of the prevalent types may therefore emerge.
2.2.4.3 Antimicrobial medication Recently, an increase in the incidence of outbreaks of severe F4+ ETEC-associated diarrhea has been observed worldwide (Fairbrother et al., 2005). Meanwhile, the prophylactic use of antibiotics has supported the development of resistant bacterial strains. It is hardly therefore surprising that E. coli isolates associated with PWD often show resistance to three or more antibiotics, making the medical therapy challenging (Lanz et al., 2003, Maynard et al., 2003). While antimicrobial drugs remain a valuable tool to treat infected animals, their routine use should be limited and more precisely targeted means of controlling the incidence of PWD are needed.
2.2.4.4 Passive immunotherapyWhile induction of a protective immune response by vaccination takes several days to develop and must be deployed prior to the infection, passive immunotherapy acts immediately and can also be used to treat infected individuals. The neutralizing antibodies transmitted by the sow’s milk are the basis for preventing ETEC infections in neonatal pigs (Rutter and Jones, 1973, Moon and Bunn, 1993). In the same manner, weaned piglets can be protected by supplementing their diet
with ETEC neutralizing antibodies in sow’s milk (Deprez et al., 1986). Other deployed approaches include the feeding of spray-dried plasma derived from parenterally vaccinated donor pigs (Owusu-Asiedu et al., 2002) and the supplementation of diet with chicken egg yolk derived from hens immunized with F4 fi mbriae (Marquardt et al., 1999, Owusu-Asiedu et al., 2002, Owusu-Asiedu et al., 2003). However, to be effi cient, passive immunotherapy must be continuous and large oral dosages are needed, making these applications economically nonfeasible. Edible transgenic plants can offer a cost-effective means of producing and delivering antibodies for prophylactic use. Applications to treat porcine PWD are already underway. In the United States, Diversa Corporation (www.diversa.com) has produced F4-specifi c antibodies in transgenic corn, but the current product is speculated to be insuffi ently cost-effective to reach the market (P. Heifetz, personal communication). A similar product is being developed by the German company Novoplant (www.novoplant.de), which is producing anti-F4-scFv antibodies in transgenic pea plants (G. Hensel, personal communication).
2.2.4.5 Vaccination Neonatal ETEC infections have effectively been controlled for more than two decades by parenteral vaccination of sows, and effi cient killed whole-cell or purifi ed fi mbrial vaccines are well established (Rutter et al., 1976, Morgan et al., 1978, Nagy et al., 1985, Riising et al., 2005). Vaccination of newborn or weaned piglets would be a desirable means of controlling PWD. However, injectable vaccines, such as those administered to sows, tend to
44
Literature review
stimulate systemic rather than protective mucosal immune responses (Van den Broeck et al., 1999a), and no effective vaccine against porcine PWD is currently available on the market. Parenteral vaccines have even been shown to suppress the mucosal immune responses (Bianchi et al., 1996). The oral delivery of live nontoxigenic ETEC or related virulence factors can, however, evoke mucosal immune responses (Bianchi et al., 1996, Van den Broeck et al., 1999a, Bozic et al., 2003).
F4 fi mbria is a well-characterized and prevalent colonization factor, and one of the most promising candidates for an oral ETEC PWD vaccine since it is able to induce a protective mucosal F4-specifi c immune response (Van den Broeck et al., 1999a, Van den Broeck et al., 2000). The last part of this review concentrates on F4 as a model immunogen for an oral vaccine against PWD and summarizes the relevant information to date. Other potential vaccine candidates for PWD are not discussed.
2.2.5 ETEC F4 fi mbria E. coli colonization factors mediating binding to host cells are classifi ed into two categories by their morphology, namely, pili and fi mbriae. Fimbriae are thin, fl exible structures with a diameter of about 2-4 nm and do not possess an axial hole, whereas pili are hollow, more rigid structures with a diameter of 7-8 nm (de Graaf and Mooi, 1986). Both are composed of repeating major subunits and additional minor units encoded by multiple genes located in a single operon. F4 antigens are classifi ed as fi mbriae and are thin and fl exible threads with a length of 0.1-1 µm and a diameter of 2.1 nm (de Graaf and Mooi, 1986).
2.2.5.1 Structure and assembly
F4 fi mbria is composed of hundreds of identical subunits connected to each other, and on SDS-PAGE, purifi ed F4 fi mbriae appear as a single protein band representing the major fi mbrial component (Klemm, 1981). Analysis of the F4 gene cluster has revealed that the faeG gene encodes this major subunit, designated accordingly as FaeG, with a calculated molecular mass of 27.5 kDa (Klemm, 1981). In addition to the major subunit FaeG, F4 fi mbria is composed of minor subunits encoded by the F4 operon (Figure 1). These minor subunits exist in small amounts and include FaeC, FaeF, FaeH, and probably FaeI and FaeJ (Oudega et al., 1989, van Doorn et al., 1982). FaeE, a molecular chaperone present as a homodimer in the periplasm forms heterotrimeric complexes with the F4 fi mbrial subunits (Mol et al., 1994, Mol et al., 1995, Mol and Oudega, 1996, Van Molle et al., 2005), which are targeted to outer membrane usher FaeD (van Doorn et al., 1982). FaeA and FaeB do not participate in the assembly of F4 fi mbria, but have a regulatory function for the F4 operon (Huisman et al., 1994, Huisman and de Graaf, 1995). The F4 operon is located in the large plasmids with a molecular mass of 50-177 MDa (Shipley et al., 1978, de Graaf, 1990, Wasteson and Olsvik, 1991, Mainil et al., 1998). The genes encoding the LT and ST toxins are often on the same plasmids (de Graaf, 1990, Mainil et al., 1998). In addition, these plasmids frequently carry genes coding for raffi nose-degrading enzymes (Shipley et al., 1978, Mooi et al., 1979). These enzymes may offer a selective advantage to ETEC since raffi noses present in the intestine can compete with the fi mbrial receptor sites.
45
Literature review
The assembly of F4 fi mbriae occurs in three stages (Verdonck et al., 2004a). Firstly, the fi mbrial subunits are translocated across the cell membrane into the periplasm. Secondly, the subunits interact with their periplasmic chaperone FaeE. Thirdly, these chaperone-subunit complexes are transported to outer membrane usher FaeD, which transports the subunits across the outer membrane, assembles the fi mbria, and anchors it to the outer membrane (Figure 1).
The N-terminal part of the F4 fi mbrial proteins contains a signal peptide that mediates translocation of these proteins across the cytoplasmic membrane by secretion machinery (Pugsley, 1993, Pugsley et al., 1997). In the periplasm, the signal peptide is cleaved off and the
chaperone FaeE forms homodimers, subsequently, binding fi mbrial subunits FaeG, FaeH, or FaeI and protecting them from degradation, aggregation, and premature fi ber formation by capping their assembly surfaces (Mol et al., 1995). The minor subunit FaeF does not form a complex with the chaperone FaeE, but is found as a free protein in the periplasm (van Doorn et al., 1982). Since FaeC shows a high similarity to FaeF, it also does not interact with FaeE (Mol et al., 2001).
Assembly of F4 fi mbria is initiated when the fi mbrial subunits are targeted to the outer membrane usher FaeD (van Doorn et al., 1982). The minor subunit FaeC can directly interact with FaeD, and this is assumed to initiate the assembly of fi mbria (Mol et al., 2001). Indeed, this
Figure 1. Genetic organization of the F4 operon and a model of synthesis of F4 fi mbria. The fi mbrial proteins are transported to the periplasmic space, and F4 assembly is initiated at the outer membrane usher FaeD by subunits FaeC and FaeF. Chaperone FaeE binds subunits FaeG, FaeH, and FaeI and delivers them to the usher FaeD to elongate the fi mbria. See text for details. Adapted from Van den Broeck et al., 2000, Verdonck et al., 2004a.
faeA faeB faeC faeE faeF faeG faeH faeI faeJfaeD7.4 12.5 16.9 82.1 24.8 15.2 27.5 25.5 24.8 25.1 kDa
minorunit
minorunits
usher/anchor
chaperone
F G H IC
EE
EE EE
D
GGG
G
GGGHF
GGGGFC
EE
outer membrane
inner membrane
F4 operon
periplasm
cytoplasm
receptorbindingsite
major unit
?
regulators ?
46
Literature review
subunit is present at the tip of mature fi mbria (Oudega et al., 1989). The next subunit to interact with the FaeD-FaeC complex is FaeF, which enables binding of major FaeG subunits to the complex, allowing elongation of fi mbria to commence (Mol et al., 2001). After addition of a limited amount of FaeG subunits, incorporation of FaeH, FaeF and probably also FaeI is needed for further elongation of fi mbria with FaeG subunits (Bakker et al., 1992b). Whether FaeJ is produced or incorporated into F4 fi mbria is not known. Finally, usher FaeD anchors the fi mbria to the outer membrane. Affi nity of various subunits to the usher FaeD is supposed to play an important role in the organization of F4 biogenesis (Verdonck et al., 2004a). However, the relative concentration of different subunits in the periplasm is also likely to have a role. Moreover, since the F4 operon from faeB to faeJ is thought to be transcribed as a single polycistronic RNA, posttranscriptional regulation probably determines the level of expression of different F4 subunits (Verdonck et al., 2004a).
The conserved amino acids and sequence motifs of F4 fi mbrial subunits (Verdonck et al., 2004a, Verdonck et al., 2004b) and the periplasmic chaperone FaeE (Bakker et al., 1991) suggest a donor strand complementation and donor strand exchange mechanisms in F4 assembly (Verdonck et al., 2004b). In the donor strand complementation, being well characterized with type 1 and P pili (Hung and Hultgren, 1998, Thanassi et al., 1998), the periplasmic chaperone interacts with the C-terminal part of the subunit and the donated chaperone β-sheet protects the assembly surfaces of the subunit by completing its immunoglobulin(Ig)-like fold (Choudhury et al., 1999, Sauer et al., 1999). In the
following donor strand exchange, the donated chaperone β-sheet is replaced by an N-terminal β-sheet of another fi mbrial subunit. This fi nishes the Ig-fold of the subunit and connects the subunits to each other (Justice et al., 2003). The subunit-subunit conformation is energetically more favorable than the chaperone-subunit interaction enabling the independent function of fi mbrial chaperones without cellular energy (Jacob-Dubuisson et al., 1994).
2.2.5.2 F4 variantsIn 1964, Ørskov et al. distinguished two serological variants of F4 fi mbriae, namely F4ab and F4ac, and a third serological variant, F4ad, was discovered later (Guinee and Jansen, 1979). With sequence analysis of F4 variants (FaeG of F4ab and F4ad are 264 amino acids long, whereas F4ac variant is 262 amino acids long), F4 fi mbriae were found to contain conserved regions designated “a” and variable regions forming “b”, “c”, and “d” determinants (Klemm, 1981, Gaastra et al., 1983, Josephsen et al., 1984, Verdonck et al., 2004b). Three conserved regions at 1-37, 105-132, and 236-264 were identifi ed between the three antigenic variants, whereas 163-173 and 208-218 were designated as variable regions. Mapping of the “a”, “b”, “c”, and “d” epitopes along the FaeG sequence has been elucidated by sequence analysis (Gaastra et al., 1983, Bakker et al., 1992a), by oligopeptide-directed site-specifi c blocking of hemagglutinating activity (Jacobs et al., 1987), and by monoclonal antibodies (van Zijderveld et al., 1990, Sun et al., 2000). The “c” determining epitope apparently lies partly in the variable region 163-173 (Bijlsma et al., 1982, Bakker et al., 1992a, Verdonck et al., 2004b), but since the 3D
47
Literature review
structure of FaeG or the F4R binding site has not yet been characterized, elucidating and positioning the “a”, “b”, and “d” -determining regions is diffi cult. The common “a” epitopes are likely located in the conserved regions and involved in functional mechanisms such as subunit-subunit interaction. The receptor binding site is thought to be related to both common and serotype-specifi c antigenic determinants of FaeG (Bakker et al., 1992a). F4ac is by far the predominant variant associated with F4+ ETEC worldwide (Westerman et al., 1988, Gonzalez et al., 1995, Choi and Chae, 1999).
2.2.5.3 F4 receptorCorresponding to the three antigenic F4 variants, six different porcine phenotypes, designated by letters A to F, can be distinguished based on variability in adhesiveness to F4ab-, F4ac-, and F4ad-positive E. coli, indicating the presence of multiple F4 receptors (Bijlsma et al., 1982, Baker et al., 1997, Erickson et al., 1997). Several putative receptors for F4 adhesins have been identifi ed on porcine epithelial cells. Characterization of the enteric glycostructures mediating F4
binding is rather complicated, and the term F4 receptor(s) is used to describe the F4+ ETEC binding site(s) present in the brush border of small intestinal epithelial cells. Examples of putative F4R include intestinal mucin-type sialoglycoproteins, which bind F4ab and F4ac (Erickson et al., 1992, Erickson et al., 1994); porcine enterocyte transferrin (Grange and Mouricout, 1996), which binds F4ab; and a neutral glycosphingolipid, which binds F4ad (Grange et al., 1999). The following model of F4 receptors is presented according to the observed porcine phenotypes (Van den Broeck et al., 2000).
• receptor bcd binds all F4 variants and is present in phenotype A
• receptor bc binds variants F4ab and F4ac and is found in phenotypes A and B
• receptor d binds F4ad and is present in phenotypes C and D
• receptor b binds F4ab and is found in phenotype F
This receptor binding model is summarized in Table 3. β-galactose has been shown to interfere with F4 fi mbria-receptor interaction (Sellwood, 1980),
Table 3. Characterization of different putative F4-adherent porcine phenotypes. Adapted from Van den Broeck et al., 2000.
Identifi cation of receptorPhenotype Adhesiveness Receptor Characterization Mol. mass
(kDa)References
A ab, ac, ad bcd Glycoproteins 45-70 Willemsen and de Graaf, 1992bc Sialoglycoproteins 210, 240 Erickson et al., 1992, Erickson
et al., 1994, Billey et al., 1998B ab, ac bc Sialoglycoproteins 210, 240 Billey et al., 1998C ab, ad d Glycosphingolipid ? Grange et al., 1999D ad d Glycosphingolipid ? Grange et al., 1999E - - - -F ab b Enterocyte transferrin 74 Grange and Mouricout, 1996
48
Literature review
and the proposed sugar moieties mediating F4 binding include various glycan structures containing galactose, N-acetylglucosamine (GlcNAc), and/or N-acetylgalactosamine (GalNAc), or some combination of these monosaccharides (Anderson et al., 1980, Willemsen and de Graaf, 1992, Blomberg et al., 1993, Payne et al., 1993, Seignole et al., 1994). Furthermore, all F4 adhesin variants have been proposed to share similar core carbohydrate specifi cities, the minimal core carbohydrate structure recognized by the variants being acetylhexosamine (either GalNAcβ(1-4) or GlcNAcβ(1-3)) β-linked to a galactose residue (Grange et al., 2002).
The prevalence of F4 receptor seems to differ between countries; in Australia and Belgium, about 12% and 4% of pigs possessed possessed resistant phenotype E, respectively (Snodgrass et al., 1981, Cox and Houvennaghel, 1988). In the midwestern United States, 20% (Baker et al., 1997), and in the Netherlands up to 50% (Bijlsma et al., 1985) of pigs were F4R-negative.
2.2.5.4 Immunogenicity of F4 fi mbriaeSystemic immunogenicityAlthough porcine neonatal infections caused by F4+ ETEC can effectively be prevented with passive lactogenic immunity by vaccinating the sows parenterally (Rutter and Jones, 1973, Rutter et al., 1976, Logan and Meneely, 1981, Nagy et al., 1985, Moon and Bunn, 1993, Osek et al., 1995, Barman and Sarma, 1999, Riising et al., 2005), such vaccination cannot prevent related PWD since it stimulates in weaned piglets a systemic rather than an intestinal F4-specifi c immune response (Moon and
Bunn, 1993, Bianchi et al., 1996, Van der Stede et al., 2003). However, the systemic IgA immune response following intramuscular immunization with F4 is similar to that seen after oral challenge with pathogenic F4+ ETEC (Van den Broeck et al., 1999a). In the presence of an appropriate adjuvant, namely 1α,25-dihydroxyvitamin D3, this systemic response can be modulated towards a mucosal immune response, and can also reduce F4+ ETEC excretion in a subsequent challenge to some extent (Van der Stede et al., 2003).
Mucosal barrier and mucosal immune responseThe intestinal epithelium has a dual function. It allows penetration of nutrients and macromolecules important to growth and development, while providing a barrier to potentially harmful micro-organisms and macromolecules (Mestecky et al., 2005). The diet of all animals consists of a complex mixture of proteins and other macromolecules that are potentially antigenic. Since these food antigens usually do not elicit immune responses, the mucosal immune system apparently can distinguish between harmful pathogenic antigens and harmless food antigens (Holmgren et al., 2003). Pathogenic antigens tend to elicit immunosurveilance, leading to active immune defence, whereas soluble food antigens usually activate an immunosuppressive state known as oral tolerance. However, certain soluble antigens, including CT, LT, and some lectins that bind enterocytes effi ciently, can elicit immune responses upon oral administration (Strober et al., 1998). The exact mechanisms behind the induction of the opposite mucosal functions are not completely understood, but tolerance is
49
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the default response to soluble antigens (Holmgren et al., 2003). The mucosal immune response can be elicited either by transporting the antigen through specialized antigen sampling cells, designated M-cells, which are present mainly in the follicular associated epithelium overlaying the organized lymphoid structures known as Peyer’s patches, or by the receptor-mediated transport through epithelial enterocytes (Strober et al., 1998, Neutra and Kozlowski, 2006). The transported antigens are captured by underlying antigen-presenting cells (APCs), which are activated by the co-stimulatory signals and can elicit the immune response at immunocompetent sites (Neutra and Kozlowski, 2006). In addition, dendrite cells can capture microorganisms directly from the luminal content of the intestine (Rescigno et al., 2001, Rimoldi and Rescigno, 2005). The basic mechanism for the mucosal immune response is presented in Figure 2.
Oral immunogenicityA successful vaccine against PWD must stimulate the mucosal immune response in the gut, leading to the production of secretory immunoglobulins, which can effi ciently agglutinate pathogens and neutralize their antigens at the mucosal surfaces, and must also provide protection against subsequent infections. In F4R-defi cient animals, F4 fi mbriae seem to act as normal food antigens (Van den Broeck et al., 2002), whereas in pigs possessing F4R, oral immunization with F4 induces F4-specifi c systemic and mucosal immune responses (Van den Broeck et al., 1999a). Most importantly, this mucosal immune response is able to protect pigs against subsequent F4+ ETEC challenge (Van den Broeck et al.,
1999a, Verdonck et al., 2004c). Oral immunogenicity of F4 apparently relies on F4R binding and subsequent uptake by porcine small intestinal enterocytes. Indeed, F4 fi mbriae are endocytosed and translocated from the gut lumen by the epithelial and follicular enterocytes expressing F4R as well as M-cells, but not by enterocytes derived from an animal lacking F4R (Snoeck, 2004). However, equal intestinal immune responses can be evoked in F4R- and F4R+ animals via local injection of F4 into the lamina propria (Snoeck et al., 2006). This demonstrates the importance of F4R and consequent transepithelial transport for the induction of F4-specifi c mucosal immune responses (Snoeck, 2004). Apparently, this transport is mainly carried out by enterocytes, the major cell type of the intestinal epithelium. While it is unlikely that porcine enterocytes act as APCs to the underlying lymphocytes since they do not express MHC class II molecules (Stokes et al., 1996), they may activate professional APCs in lamina propria by secreting chemokines and cytokines (Eckmann et al., 1997, Stadnyk, 2002). Jejunal Peyer’s patches appear to be the major induction sites for the mucosal immune response to F4 fi mbriae (Snoeck et al., 2006). In addition, the activated APCs migrate to mesenteric lymph nodes, where they activate lymphocytes to evoke the F4-specifi c systemic immune responses (Van den Broeck et al., 1999a).
Towards a F4 subunit vaccineF4 fi mbriae are mainly composed of the subunit FaeG (Van den Broeck et al., 2000), and F4-specifi c antibody response is directed against this major subunit. As FaeG also constitutes an adhesin and F4-induced antibodies can neutralize the
50
Literature review
Figure 2. Induction of a mucosal immune response at the intestine. (A) Structure of Peyer’s patches. (B) Antigens are transported across the mucosal barrier by M-cells, enterocytes, or dendritic cells, and antigen-presenting cells induce activation of an antigen-specifi c mucosal and systemic immune responses. The main protection at mucosal surfaces is provided by the secretory immunoglobulins transported to the lumen. Adapted from Neutra and Kozlowski, 2006.
h
(B)follicle-associated epithelium
corona
germinalcenter
dome
lamina propria
gut lumenvillus
(A)
T-cellzone
B-cellzone
(B)
MUCOSALEFFECTOR SITE
MUCOSALINDUCTION SITE
enterocyteM cell
dendritic cell antigenpresenting cells
YY
YY
YY
YY
Y
Y
YY
NEUTRALIZATION
by sIgA and sIgM
mesentriclymph nodes
ANTIGEN UPTAKE
YY
YY
Y
YY YY
Y
macrophage
B cell
T cell
antigen samplingand endocytosis
by M cells
receptor-mediatedendocytosis
by enterocytescapture
by dendrites
activation ofB- and T-cells
homing to mucosalsites (mainly intestine)
systemiccirculation(e.g. spleen)
LYMPH BLOOD
transport
vesicle YY
PEYER’S PATCH LAMINA PROPRIA
51
Literature review
fi mbria-receptor interaction (Yokoyama et al., 1992, Van den Broeck et al., 1999b, Sun et al., 2000), FaeG is an ideal candidate for subunit vaccine. Indeed, orally delivered recombinant FaeG was recently reported to induce an F4-specifi c mucosal antibody response in F4R+ piglets, reducing also the excretion of fecal F4+ ETEC following the challenge (Verdonck et al., 2004c, Verdonck et al., 2005b). In addition to FaeG, the capacity of FaeG-encoding DNA to prime a systemic F4-specifi c immune response in pigs has been exploited, but data on mucosal response were not presented (Verfaillie et al., 2004). F4 fi mbriae have been shown to have the potential to function as a mucosal carrier molecule for coupled heterologous protein when delivered orally to F4R+ pigs (Verdonck et al., 2005a). Preliminary data also suggest that the major subunit FaeG alone could serve as a mucosal carrier for genetically fused peptides (Verdonck, 2004). This is an important fi nding since currently only a few mucosal carriers have
been identifi ed. This could open new avenues for mucosal vaccination, also allowing simultaneous vaccination against multiple enteropathogens. However, this carrier function can be exploited only in animals possessing the F4R.
To conclude, FaeG is a promising subunit vaccine candidate with a unique ability to bind F4R and induce a mucosal immune response upon oral delivery. Stimulation of mucosal and/or serum immune responses by oral immunization requires large amounts of antigens (milligrams per immunization) and effi cient as well as cost-effective manufacturing practices. This is particularly true with applications for animal health, such as PWD, where the economic benefi t is one of the major driving forces for the vaccination. Transgenic plants that express antigens in their edible tissues offer an attractive means for bulk-scale production of such antigens, in addition to providing an elegant delivery system.
Aims of the study
52
3 AIMS OF THE STUDY
The initial aim of the present study was to determine the capacity of transgenic plants to produce FaeG protein in their tissues. Furthermore, the potential of plant-derived FaeG as a mucosal vaccine candidate against porcine F4+ ETEC diarrhea was evaluated.
In this purpose, the following specifi c questions were addressed:
• Does subcellular targeting have an infl uence on the accumulation level or form of FaeG protein, when the faeG gene is expressed in transgenic tobacco plants under the CaMV 35S promoter?
• Can crop plants alfalfa and barley accumulate and store FaeG protein in the levels required for oral vaccine applications?
• What is the infl uence of pH and proteolytic conditions present in piglet gastrointestinal tract on the stability of F4 fi mbria and plant-produced FaeG?
• Can plants fold FaeG protein in a conformation with a F4 receptor binding capacity?
• Does N-glycosylation, have an effect on FaeG stability in conditions simulating piglet gastric fl uid, and does it abolish the immunogenicity of FaeG protein?
• Does plant-produced FaeG protein induce a F4-specifi c immune response when administered orally to weaned piglets, and does this immune response protect piglets against following ETEC challenge?
53
Material and methods
4 MATERIAL AND METHODS
This section summarizes briefl y the materials and methods used. For more detail, please refer to the original publications (I-IV).
Table 4. Gene constructs used in this study.
Plasmid Gene expression construct Destination Plant RefecencepEKH6 pCaMV 35S-SP-faeG-3´nos Apoplast (apFaeG) Tobacco Figure 3pEKH7 pCaMV 35S-SP-faeG-SEKDEL-3´nos ER (erFaeG) Tobacco Figure 3pJJJ60 pCaMV 35S-TP-faeG-3´nos Plastid (pFaeG) Tobacco,
AlfalfaFigure 3; II, Figure 1A; IV
pJJJ33 pTI-SP-faeG-SEKDEL-3´nos ER (erFaeG) Barley III, Figure 1
faeG, FaeG encoding gene from ETEC strain 5/95; 3´nos, nopaline synthase gene polyadenylation signal from Agrobacterium; SEKDEL, ER-retaining amino acid signal; SP, ER-targeting signal peptide from barley trypsin inhibitor; pCaMV35S, Caulifl ower mosaic virus 35S promoter; TP, chloroplast-targeting transit peptide from pea rubisco small subunit
Table 5. Methods used in this study.
Method UsedAgrobacterium-mediated transformation of tobacco and alfalfa II, IIIBarley transformation by particle bombardment IIIChloroplast isolation* IICloning of faeG transformation constructs II, IIIComparison of endosperm-specifi c promoters by transient gene expression IIIConfocal immunofl uorescence microscopy IIDNA-hybridization II, III, IVEndosperm-specifi c promoter isolation by PCR from barley IIIExpression and purifi cation of FaeG-6xHis in E. coli IIF4 and FaeG binding to F4R in vitro IIF4 purifi cation III, IVF4 ELISA III, IVFaeG glycostaining* IIIFaeG immunoblotting II, III, IVfaeG isolation by PCR form E. coli IIFaeG MALDI-TOF analysis* IIIFaeG purifi cation from plants by column chromatography II, IIIImmunohistostaining IIIn vitro SGF and SIF analyses for F4 and FaeG I, II, IIIMice immunization IIIPiglet immunization IVProduction of FaeG antisera in rabbits* II, III, IVRNA-hybridization* II, IV
* not conducted by the author
54
Results and discussion
5 RESULTS AND DISCUSSION
5.1 Candidate plant species for this study
Selection of a proper candidate plant species for expression of an edible vaccine antigen was important in the early phase of this project. Three plant species, namely tobacco (Nicotiana tabacum L.), alfalfa (Medicaco sativa L. ssp), and barley (Hordeum vulgare L.), were selected as hosts for FaeG expression. The properties that make these species desirable for this application are discussed briefl y below.
5.1.1 TobaccoTobacco is a plant for which well-established transformation and regeneration techniques are available (Horsch et al., 1985). It accumulates high biomass effi ciently and can be grown in laboratory and greenhouse conditions easily and safely. Tobacco cannot survive in the Finnish climate and has no wild relatives in Finland. Tobacco was utilized as a model plant with different genetic constructs being tested to evaluate and optimize the expression of FaeG protein in plant tissue.
5.1.2 AlfalfaTransformation techniques are also available for alfalfa (Saunders and Bingham, 1972, Shetty and McKersie, 1993), which is a legume feed plant with a high protein content. In the Finnish climate, alfalfa does not overwinter well and is sparsely cultivated for feed in the southern part of the country. Regular, large-scale seed production of alfalfa is not established in Finland. Due to its poor
overwintering capacity, alfalfa does not widely populate the Finnish fauna. A few plants can occasionally be found by the roadside, in meadows, and on the western coast, but permanent spread of the species is restricted to the cultivated fields (Hämet-Ahti et al., 1998). Alfalfa isa bee-pollinating species. However, cultivated alfalfa has only one wild relative species in Finland, namely Medicaco lupulina L., which do not cross-pollinate with cultivated alfalfa (Hämet-Ahti et al., 1998) Thus, the Finnish environment and climate offer natural containment for controlled fi eld-scale cultivation of alfalfa. This, together with the high biomass production capacity, makes alfalfa an interesting production and delivery vehicle for plant-produced oral vaccine.
5.1.3 BarleyExpression of candidate vaccine proteins in cereal grains, as compared with leafy crops, has several attractive features. Seeds have evolved to accumulate high levels of storage proteins in a stable environment. Mature seeds are desiccated, allowing long-term storage at ambient temperatures. Barley transformation has been studied at our laboratory (Ritala, 1995), and barley is the main cereal crop cultivated in Scandinavia and is a major constituent of pig feed. Barley is mainly a self-pollinating plant and has no signifi cant wild relatives in Finland. With suitable cultivation practices, transgenic barley can be safely grown in Finnish open-fi eld conditions (Ritala et al., 2002), and thus, could serve as an ideal candidate production and delivery system for an edible porcine vaccine.
55
Results and discussion
5.2 Analysis of transgenic plants producing FaeG protein
Specifi c yield of recombinant protein per unit of plant biomass is infl uenced by the optimization of transgene expression, which is obtained by expression construct design. Perhaps the most important component of the expression construct is the promoter used to control the transcription of the transgene. In dicotyledonous plant species, a powerful and constitutive Caulifl ower mosaic virus 35S promoter (CaMV 35S) is widely used for transgene expression (see Table 1) and was also selected here to drive faeG expression in transgenic tobacco and alfalfa plants. In barley, the aim was to accumulate the FaeG protein in seeds and an appropriate tissue-specifi c promoter was selected.
Subcellular targeting of recombinant protein in a plant cell can have a major role in protein accumulation as well as in folding and posttranslational modifi cations. The optimal compartment depends on the various properties of each protein and has to be empirically evaluated.
5.2.1 Tobacco: optimization of FaeG production by subcellular targeting To determine the optimal environment for FaeG accumulation, three different gene constructs were designed to target FaeG protein to three different cellular destinations: the apoplast (apFaeG; with a signal peptide) via the secretion pathway, the endoplasmic reticulum (erFaeG; with a signal peptide + ER retain signal), and the plastid (chloroplast) (pFaeG; with a transit peptide), respectively. The regenerated transgenic tobacco plants (ten plants with each construct) were characterized at the DNA, RNA, and protein levels (Figure 3).
5.2.1.1 Transgene copy number
DNA hybridization studies confi rmed the presence of the faeG gene in regenerated tobacco plants. The copy number of the transgene varied between one and seven with the apoplast-targeting construct, and between one and fi ve with the ER-targeting construct. Only a single copy of the transgene was detected in the majority of plants (one transformant with two copies) transformed with the chloroplast-targeting construct (Figure 3A). This variation in transgene copy number can be explained by the different protocols utilized for plant transformation. Agrotransformations of tobacco plants with apoplast- and ER-targeting constructs were performed with a binary vector system in which the gene cassette is not fused to the tumor-inducing (Ti) plasmid but is delivered in a separate multi-copy plasmid (De Framond et al., 1983, Hoekema et al., 1983). The transformation with the chloroplast-targeting construct utilized the co-integration protocol in which the designed gene cassette is integrated into the Ti plasmid by homologous recombination (Van Haute et al., 1983). These results suggest that in co-integration-based agrotransformation the foreign gene is inserted to plant genome at the same low copy number as that of Ti plasmid in Agrobacterium and favors the generation of single-copy transformants (Gelvin, 2003). Generally, a single transformation event is a desired character, enabling straightforward breeding of homozygous plant material and minimizing the risk of gene silencing in subsequent generations.
5.2.1.2 mRNA accumulationLike the copy number of the transgene, the accumulation of faeG-specifi c mRNA
Results and discussion
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56
57
Results and discussion
in transgenic plants obtained with the apoplast- and ER-targeting constructs also varied more than the mRNA level in plants transformed with the chloroplast-targeting construct (Figure 3B). This might refl ect RNA silencing in high-copy transformants (Vaucheret et al., 1998), although the low mRNA level did not directly correlate with the high transgene copy number in the plants transformed with apoplast- and ER-targeting constructs. This is in agreement with previous observations with transgenic tobacco plants (Leech et al., 1998). Whether the multicopies of the transgene were in a single locus or spread in the genome was not determined since only the T0 generation of tobacco plants was studied. The expression level of a transgene can be affected by the random insertion site in the genome (the position effect) (Peach and Velten, 1991). T-DNA can integrate near or far from transcriptional activating elements or enhancers, resulting in the activation (or lack thereof) of T-DNA-carried transgenes (Beilmann et al., 1992, Springer et al., 1995, Sundaresan et al., 1995, Campisi et al., 1999). The random insertion of the transgene is also a likely explanation for the variation in faeG-specifi c mRNA levels observed here.
5.2.1.3 FaeG accumulationAccumulation of FaeG protein in transgenic plants was studied with FaeG-specifi c immunoblotting (Figure 3C). Immunoblotting of TSP of plants transformed with apoplast- and ER-targeting constructs revealed multiple FaeG-specifi c bands of different mobility. By comparing the intensity of these bands by densitometry with FaeG samples of known concentration, the best lines obtained with apoplast- (clone 6.5) and
ER-targeting (clone 7.9) gene constructs were calculated to accumulate 0.025% of apFaeG and 0.015% of erFaeG in their TSP, respectively (Kotiaho, 2003). Correspondingly, the plants transformed with the chloroplast-targeting construct accumulated pFaeG protein up to 1% of TSP (clone 60.21). This 40- to 67-fold increase in FaeG level indicates that the chloroplast is a superior environment to the apoplast or ER for accumulation of FaeG.
When the FaeG yield was compared with the amount of faeG-specifi c mRNA within each construct, a correlation was observed; plants with reduced levels of mRNA also accumulated less FaeG protein (Figures 3B and 3C). However, no difference was observed in the maximum level of mRNA between the three constructs when the high-yield plants (6.9, 7.9, and 60.21) from each construct were compared side-by-side on the same northern blot (Kotiaho, 2003). This suggests that FaeG accumulation in the apoplast or ER is not limited by transcription or mRNA stability but rather by translation, translocation, or FaeG folding and stability at the destination site.
Recently, Huang et al. (2003) cloned an FaeG-encoding gene of F4ad fi mbria under the CaMV 35S promoter without an encoding region of ER-targeting signal peptide. This gene construct was expressed in tobacco plants, which accumulated FaeG protein up to 0.15% of TSP. The cellular localization of FaeG was not studied, but presumably proteins expressed without any signal peptide would accumulate in the cell cytosol.
Our results together with those of Huang et al. (2003) suggest that the cytosol might be a more favorable place for the accumulation of FaeG than the apoplast or ER since 6- to 10-fold more
58
Results and discussion
FaeG was detected from plants transformed without a signal peptide (0.15% TSP) (Huang et al., 2003) than from plants transformed with the apoplast- (0.025% TSP) or ER-targeting (0.015% TSP) constructs (our study). This contradicts fi ndings of more effi cient accumulation of recombinant proteins in plants when the protein was targeted to the ER than to the cell cytosol (Wandelt et al., 1992, Schillberg et al., 1999, Richter et al., 2000, Yang et al., 2002, Judge et al., 2004). Moreover, an ER-retaining C-terminal hexapeptide, SEKDEL, has been shown to promote the accumulation of recombinant proteins in plants (Wandelt et al., 1992, Haq et al., 1995, Schouten et al., 1996, Huang et al., 2001). However, in our study less erFaeG (0.015% TSP) than apFaeG (0.025% TSP) accumulated. Since FaeG does not contain cysteine residues (Klemm, 1981), the assembly of disulfi de bonds cannot affect its folding or stability in different subcellular compartments.
To conclude, the chloroplast stroma seems to be a more favorable environment for FaeG accumulation than the cytosol, ER, or apoplast, where the FaeG probably fails to fold correctly and is subsequently digested by cellular proteases. Alternatively, within the chloroplast there might be some chaperone-like proteins similar to FaeE helping FaeG to fold in a native-like conformation that serves to stabilize FaeG and prevent its degradation.
5.2.1.4 Subcellular localization of FaeGTo confi rm the subcellular localization of apFaeG, erFaeG, and pFaeG proteins in tobacco plants, leaf sections were dyed with a FaeG-specifi c immunofl uorescence staining and subsequently examined by
confocal laser scanning microscopy. Results of this study are summarized in Figure 4.
The plants in which FaeG was targeted to the secretion pathway (line 6.5) showed a weak FaeG-specifi c signal in the apoplast. The plants transformed with the chloroplast-targeting construct (line 60.21) showed a clear FaeG-specifi c signal in the chloroplasts. No FaeG-specifi c signal was detected in plants transformed with the ER-targeting construct (line 7.9). This might be due to the detection limit of the immunostaining protocol used, since the accumulation level of erFaeG protein in these plants was lower than in the plants accumulating apFaeG or pFaeG (Figure 3C). This might also refl ect the poor penetration of antibodies into the plant tissue with the immunostaining protocol since the plants transformed with the apoplast- or chloroplast-targeting construct showed the FaeG-specifi c signal only in cells next to the cutting edge. The results of pFaeG immunostaining were further confi rmed with chloroplasts isolated from the plants transformed with the chloroplast-targeting construct (II). Chloroplasts derived from the transgenic plants showed a clear FaeG-specifi c signal, while chloroplasts from nontransgenic plants remained unstained (II, Figure 1B, cover of this thesis).
5.2.1.5 N-glycosylation of FaeG proteinEukaryotic proteins are cotranslationally N-glycosylated in the ER on the amino acid triplet Asn-X-Ser/Thr, X being any amino acid other than proline or serine. Multiple N-glycosylation sites present in a protein give rise to various glycoforms (Lerouge et al., 1998). The FaeG amino acid sequence has three potential N-
59
Results and discussion
Figure 4. Cellular localization of FaeG protein in the transgenic plants transformed with apoplast- (line 6.5), ER- (line 7.9), and chloroplast-targeting constructs (line 60.21). Leaf sections of transgenic plants were dyed with FaeG-specifi c immunofl uorescence staining and subsequently studied by confocal laser scanning microscopy. Immunostained leaf sections were excited at wavelength of 488 nm, and the emission was simultaneously monitored at two wavelengths. A) The emission at wavelength 680 nm (red light) corresponds to the autofl uorescence of chloroplasts. B) The emission at 530 nm (green light) is specifi c to the fl uorescein isothiocyanate-labeled antibody used in FaeG immunostaining. A+B) Overlay of the two monitored wavelengths.
apoplast-targeted FaeG ER-targeted FaeG chloroplast-targeted FaeG 6.5 7.9 60.21 nontransgenic plant
A)
B)
A+B)
60
Results and discussion
glycosylation sites at Asn7, Asn128, and Asn200 (Kotiaho, 2003), but is not glycosylated in its native form in bacteria (III, Table 2). N-glycosylation was expected to occur in the apFaeG and erFaeG in plants because FaeG was preceded with a signal peptide. Indeed, multiple FaeG-specifi c bands with a lower mobility than their bacterial counterpart (27.5 kDa) were observed from tobacco plants transformed with apoplast- and ER-targeting constructs (Figure 3C). In contrast, when FaeG was targeted to the chloroplast, one major band with a size consistent with bacterial FaeG was detected and shown to be nonglycosylated by MALDI-TOF analysis (J. Joensuu, unpublished results). A minor FaeG-specifi c band with higher mobility was also observed (Figure 3C). The estimated size of this band corresponded to the calculated size of FaeG when the chloroplast transit peptide is not cleaved off (Kotiaho, 2003). In addition to glycosylation, incorrect cleavage of the signal peptide may also partially account for the heterogeneity of FaeG bands detected from plants transformed with apoplast- and ER-targeting constructs. However, due to poor expression level, the presence of glycans in the apFaeG or erFaeG could not be confi rmed and the nature of these multiple FaeG bands was not studied further.
As revealed here, the level of FaeG accumulation refl ected the effi ciency of FaeG antiserum to recognize proteins on western blots. In theory, glycans attached to the apFaeG and erFaeG might interfere with the binding of anti-FaeG antibodies to the protein, leading to an underestimate of the amount of FaeG present in these plants. However, this is unlikely since polyclonal antiserum detects multiple epitopes on FaeG. Furthermore, N-
glycosylation did not interfere with the immunodetection of erFaeG extracted from barley endosperms (III). The erFaeG purifi ed from barley grains was detected by the FaeG antiserum in denaturating western analyses (III, Figure 3) as well as in nondenaturating ELISA analyses (J. Joensuu, unpublished results) in quantities equal to the nonglycosylated forms of FaeG. These data were consistent with band intensities visualized on Coomassie-stained SDS PAGE gels (J. Joensuu, unpublished results).
5.2.2 Alfalfa: accumulation of FaeG in an edible crop plantThe targeting of FaeG protein to the chloroplast of tobacco plants resulted in higher accumulation levels than the other subcellular locations studied. To investigate the oral immunogenicity of FaeG protein, the edible crop plant alfalfa was transformed with the same chloroplast-targeting gene construct (Figure 3; II, Figure 1A).
DNA hybridization analysis of regenerated alfalfa plants revealed that one to three copies of the faeG gene were integrated into the genome of transgenic plants (IV, Figure 1A). This is in agreement with the corresponding tobacco results (Figure 3A), in which one to two copies of the transgene were observed. The transgene copy number did not have an effect on the accumulation level of pFaeG protein in the alfalfa plants since a uniform accumulation level of 1% of TSP was detected within all fi ve transgenic lines studied. The observed accumulation level is also in accord with the corresponding transgenic tobacco results (see section 5.2.1.3). Furthermore, the molecular size of the pFaeG band detected by immunoblotting in alfalfa was identical to that in tobacco plants
61
Results and discussion
transformed with the same construct. Accumulation of FaeG-specifi c mRNA and the subcellular localization of pFaeG protein was assumed to be identical to the corresponding tobacco plants (II, Figure 1B), and was not further confi rmed. Easy processing and high stability during storage are important characters for a plant-based vaccine candidate, enabling the use of the plant material as a delivery vehicle without expensive purifi cation of the antigen. To investigate whether the harvested transgenic alfalfa material could preserve the pFaeG protein in a stable form, the plants were desiccated at room temperature or in an oven at 37°C. pFaeG did not decompose during the desiccation process. A similar amount of recombinant protein was detected by immunoblotting from the dried material as from the fresh tissue (IV, Figure 2). Furthermore, the amount of pFaeG was unaffected by a two-year storage period at ambient room temperature.
5.2.3 Barley: FaeG production in seed endospermTo determine whether FaeG could be produced in seeds of a cereal plant, barley was transformed with apoplast- and ER-targeting constructs in which the faeG gene was placed under the control of an endosperm-specifi c barley trypsin inhibitor (TI) promoter (III, Figure 1).
Only a single transgenic line with two copies of the transgene was obtained with the apoplast-targeting gene construct (J. Joensuu, unpublished results), whereas three transgenic barley lines were regenerated with the ER-targeting construct (III). In these plants, the transgene copy number varied from three to multiple copies. FaeG protein was detected only from grains of barley plants
transformed with the ER-targeting construct. The erFaeG accumulation was endosperm-specifi c since no FaeG was detected in the seed coat, embryo, or leaf tissues (III, Figure 4A). Accumulation of FaeG-specifi c mRNA in the developing endosperm was not analyzed.
FaeG immunodetection of grain TSP revealed fi ve bands in the mass range of 25-33 kDa (III, Figure 3), none of these, however, representing the exact size of the bacterial FaeG (27.5 kDa). SDS-PAGE gel glycostaining of purifi ed erFaeG samples suggested the presence of glycans on the protein (III, Figure 5). To confi rm these results, tryptic peptides of multiple FaeG bands were studied with MALDI-TOF mass spectrometric analysis. Findings indicated that heterogeneous N-glycosylation and N-terminal cleavage are possible reasons for the presence of multiple FaeG bands in barley grain TSP (III, Table 2). When intensity of these FaeG bands was compared with FaeG samples of known concentration by densitometry, erFaeG was calculated to account for up to 1% of TSP in barley grains. Correspondingly, when FaeG was targeted to the ER of tobacco plants, only 0.015% of erFaeG accumulated in leaf TSP. The reason for this 60-fold increase in FaeG accumulation in barley grains was not studied further, but three partially overlapping interpretations for this phenomenon can be suggested. (i) In tobacco plants, faeG expression was controlled by the constitutive CaMV 35S promoter, whereas an endosperm-specifi c TI promoter was used in barley. Seed-specifi c promoters have been reported to offer signifi cant advantages over the CaMV 35S promoter in recombinant protein accumulation in seeds (Chikwamba et al., 2002b, De Jaeger et al., 2002). However, our results with transient gene expression suggest that the
62
Results and discussion
high accumulation of erFaeG protein in barley endosperm was not due to the TI promoter since the studies with 13- to 15-days after pollination-old barley endosperms revealed that the CaMV 35S promoter drives the uidA-marker gene expression more effi ciently than the TI promoter (J. Joensuu, unpublished results). (ii) The higher accumulation of erFaeG might be the result of supplementation of barley transformation constructs with 5´untranslated elements, which have been reported to enhance transgene expression in monocot plants, namely the epsilon element from the Cocksfoot mottle virus (Mäkeläinen, 2006) and the 5´ untranslated exon fused with the fi rst intron of the maize ubiquitin (Christensen and Quail, 1996, Vain et al., 1996) (III, Figure 1). No 5´untranslated elements were used with faeG expression in tobacco, although these elements have been reported to enhance transgene expression level also in dicotyledonous plants (Gallie et al., 1989, Gallie, 1996, Mason et al., 1996, Matsumura et al., 2002). Biemelt et al. (2003) did report that the insertion of the TMV Ω element was essential for accumulation of Human papilloma virus major capsid protein L1 in transgenic tobacco and potato plants. No L1 mRNA was detected prior to addition of the TMV Ω element. Nonetheless, poor transcription level and mRNA instability are unlikely reasons for low accumulation of FaeG protein in tobacco plants transformed with the ER-targeting construct since mRNA levels were equal to those detected in plants transformed with apoplast- and chloroplast-targeting constructs (Figure 3B). Accumulation of FaeG-specifi c mRNA was not studied in barley endosperms, and thus mRNA stability and level cannot be compared with tobacco data. (iii) The maturing barley endosperm tissue may offer
conditions more favorable for folding and accumulation of FaeG protein than those present in the ER of vegetative tissues of tobacco plants. Indeed, the seeds are plant organs that have evolved to accumulate storage proteins in a stable environment and lack many of the proteases present in vegetative tissues (Müntz, 1998). Similar erFaeG levels were detected in developing and mature dessicated barley grains, indicating the high stability of accumulated erFaeG protein (III, Figure 4B). In addition, one-year storage of barley grains did not decrease the amount of erFaeG. Subcellular localization of endospermic erFaeG protein was not confi rmed in our study, and it remains unclear how the erFaeG protein is localized in developing and mature barley endosperm.
The most benign subcellular environment for FaeG accumulation in barley endosperm could not be evaluated since transgenic plants producing FaeG protein were obtained only with the ER-targeting gene construct. Subcellular targeting has been thoroughly studied in maize seeds with the LT-B protein, the highest accumulation levels being detected with apoplast and vacuolar targeting (Streatfi eld et al., 2003).
Endospermic accumulation of recombinant protein should not affect the vegetative growth or fertility of transgenic plants. However, in our study, the transgenic barley plants (T0, T1 and T2 generations) showed retarded growth and poor seed production. Perhaps the random insertion of the transgene into the barley genome disturbed the metabolism of the plants, thereby affecting the growth and development. Consistent with our fi nding fi eld-tested transgenic barley plants have been demonstrated to have reduced grain weight and somewhat reduced growth rates (Horvath et al.,
63
Results and discussion
2000). In contrast to transgenic barley plants, we observed no visible changes in the phenotype of transgenic FaeG-producing tobacco or alfalfa plants.
5.2.4 FaeG production yield compared to other vaccine antigens expressed in plantsReliable and unambiguous methods to quantify the yield of plant-produced antigens are not currently available. Different research groups have reported their yields of recombinant proteins in various ways, including percentage of TSP, yield per gram fresh or dry weight, and units of enzyme activity per gram fresh or dry weight. In this study, accumulation of FaeG protein was characterized as percentage of TSP since solubility was assumed to be a prerequisite for oral immunogenicity of the FaeG (see sections 5.3.6 and 5.4). It is diffi cult to directly compare the FaeG accumulation with amounts of other plant-produced antigens reported in variable units. Therefore, below the accumulation level of FaeG protein is compared only with plant-produced vaccine antigens whose accumulation was determined as a percentage TSP. Furthermore, it must be kept in mind that the chemical composition of the protein extraction buffer crucially infl uences the amount and quality of proteins released from the plant tissue. Such compounds as Triton X-100 (Mason et al., 1998), SDS (Brodzik et al., 2005), or Tween (Dong et al., 2005) are often added to protein extraction buffers. Here, soluble FaeG protein was extracted with a buffer containing no detergents to preserve FaeG in a folded conformation. Roughly half of the total FaeG amount present in the plant material (tobacco and alfalfa leaves, barley seeds) was estimated to be
released by the utilized buffer (J. Joensuu, unpublished results).
Here, FaeG accumulation level of 0.015-1% of TSP was detected when the FaeG-encoding gene was introduced into the nuclear genome of tobacco plants. This lies within the range of 0.0035-7.7% of TSP reported for other vaccine antigens expressed in such transgenic tobacco plants. In general, the expression level of vaccine antigens has been lower than 1% of TSP (see Table 1). However, transplastomic tobacco plants have usually accumulated recombinant protein accounting for more than 1% of TSP, and an accumulation level of up to 25% of TSP has been reported with the TetC fragment of tetanus toxin (Tregoning et al., 2003).
To date, only a few vaccine antigens have been expressed in alfalfa plants (see Table 1). Two recent publications have reported antigen accumulation levels in transgenic alfalfa plants (Wigdorovitz et al., 2004, Dong et al., 2005). In these studies, rotavirus antigens accumulated to levels of 0.04% and 0.28% of TSP. By comparison, the FaeG accumulation level of 1% of TSP obtained in our study (IV) is the highest expression level reported in transgenic alfalfa plants thus far.
Expression of vaccine antigens in transgenic barley plants has not previously been described. Other recombinant proteins have been expressed in barley grains at levels of 0.025-5.4% of TSP (Jensen et al., 1998, Nuutila et al., 1999, Horvath et al., 2000, Patel et al., 2000, Schunmann et al., 2002). The FaeG accumulation of 1% of TSP achieved in our study (IV) is the second highest accumulation level of recombinant proteins reported in barley grains to date. In addition to reports with maize seeds (Streatfi eld et al., 2001, Chikwamba et al., 2002a, Lamphear et al., 2002), our
64
Results and discussion
results with the crop plant barley as a seed-based production system for vaccine antigen are unique.
5.3 Stability of F4 fi mbriae and FaeG in gastrointestinal conditionsF4 fi mbriae are highly immunogenic when delivered orally to weaned piglets and can raise a protective F4-specifi c mucosal immune response (Van den Broeck et al., 1999a). The resistance of orally delivered proteins to low pH and to gastric and intestinal fl uid digestion is a prerequisite for their antigenicity (McGhee et al., 1999). It was therefore crucial to determine the conditions present in the piglet gastrointestinal tract around the time of weaning and to study the stability of F4 fi mbriae and plant-produced FaeG protein under these conditions in order to design a model oral subunit vaccine against PWD.
5.3.1 pH in the porcine stomachSince pH is one of the main aspects affecting protein stability and the activity of proteolytic enzymes in the gastrointestinal tract, the pH in different compartments of the porcine gastrointestinal tract was analyzed (I). In suckling piglets, the pH was measured in different parts of the stomach (I, Figure 1) and ranged from 3.0 ± 0.3 (fundus gland region; mean ± S.D.) to 4.1 ± 0.1 (torus pyloricus). On the day of weaning and at one and two weeks postweaning, the pH was measured only in the fundus gland region, where the lowest pH values in suckling piglets were observed, being 3.4 ± 0.8, 3.5 ± 1.3, and 2.9 ± 0.9, respectively (I, Figure 2).
5.3.2 pH in the porcine intestine
In the jejunum, which is the major inductive site for F4-specifi c immune response in the small intestine (Snoeck et al., 2006), the average pH was 6.3-6.5 in suckling piglets; 6.1-6.8 on the day of weaning and 5.8-6.8 and 5.5-6.7 at one and two weeks postweaning, respectively (I, Figure 2). In general, the variations in pH observed at any point along the mid and caudal small and large intestine were much smaller than in the stomach, indicating that the digesta, which enter this part of the intestine are already effectively buffered by pancreatic secretions and bile (Braude et al., 1976).
5.3.3 F4 stability in low pHF4 fi mbriae from the GIS26 strain appear in multimers when analyzed in SDS-PAGE without previous boiling of the samples. Heat denaturation of the F4 fi mbriae leads to degradation of the multimeric character of the fi mbriae to FaeG monomers (Verdonck et al., 2004d). To study the effect of low pH values on the degradation of the F4 multimeric structure, the fi mbriae were incubated at pH 1.5, 2, 3, and 7 for time periods of 1, 2, 2.5, and 3.5 h and detected on SDS-PAGE followed by immunoblotting (I, Figure 3). A One- or two-hour incubation of the F4 fi mbriae at low pH did not lead to considerable degradation: after 1 h at pH 1.5, only a very small proportion of the multimeric F4 fi mbriae was converted into monomers, after 2 h, monomeric forms were detected at pH 1.5 and 2, and after 2.5 h also, at pH 3. However, after 2.5 h at pH 1.5 and 3.5 h at pH 1.5 and 2, only di- and monomers of the major subunit were found, whereas after 3.5 h at pH 3 most of the F4 fi mbriae were still in multimers (I, Figure 3).
65
Results and discussion
5.3.4 F4 stability in simulated gastric fl uid
Since the acidity present at the piglet gastrointestinal tract alone had no major effect on the stability of F4 fi mbriae, an in vitro assay simulating piglet gastric fl uid (SGF) was developed here and standardized by measurement of its proteolytic activity on hemoglobin (Ryle, 1984). When F4 fi mbriae from strain GIS26 were incubated in SGF at pH 1.5 and 2, degradation of F4 multimers was observed after 15-min incubation, with complete disappearance of F4 fi mbriae at 3 h. At pH 3 and 4, the multimeric structure of F4 was not greatly affected in SGF, and only a slight decrease in total amount of F4 was observed after 3-h digestion (I, Figure 4). Reports indicate that 50% of ingested liquids and solid pellets pass through the porcine stomach in 1.5-2 h (Clemens et al., 1975, Gregory et al., 1990, Davis et al., 2001). During the suckling period the stomach can be almost completely emptied within 2 h (Kidder and Manners, 1968). Our results suggest that the structure of F4 fi mbriae is highly stable, being unaffected by the pH values and gastric conditions typically present in the porcine stomach, allowing F4 fimbriae to pass into the small intestine, where the F4R and mucosal inductive sites are located. However, Snoeck et al. (2004) reported that the stress of weaning prolongs the gastrointestinal transit, with shortly after weaning being the period when a most of the F4 fi mbriae might be digested in the stomach.
5.3.5 F4 stability in simulated intestinal fl uidTo study the stability of F4 fi mbriae in the intestine, an in vitro assay simulating piglet intestinal conditions (SIF) was
developed here and standardized by measurement of its proteolytic activity on casein (Twining, 1984). No degradation of F4 fi mbriae was found in these conditions after a 1-h incubation (J. Joensuu, unpublished results). Snoeck (2004) studied the uptake of F4 with intestinal loops in vivo and observed that F4 is endocytosed by the F4R-possessing epithelial cells already after a 15-min incubation. The observed stability of F4 in the simulated intestinal conditions and the fast endocytosis suggest that the low-pH proteolytic conditions present in the stomach are a major limiting factor in the induction of the F4-specifi c mucosal immune response.
5.3.6 Stability of plant-produced FaeG in simulated gastric fl uidTo study the stability of plant-produced FaeG proteins in piglet gastric conditions, crude plant extracts were subjected to SFG treatment at pH 3.5. This value was selected to represent the average gastric pH in piglets around weaning (I, Figure 2). pFaeG protein in the crude extract of tobacco plants was clearly detectable on the immunoblots after a 2-h treatment in SGF, while most of the other plant proteins were degraded within a few minutes, indicating that the amount of protease was not the limiting factor in this assay (II, Figure 2A). However, pFaeG protein was partially digested and its proteolytic resistance in SGF was inferior to that of F4 fi mbriae under similar conditions (unaffected by 2-h treatment in SGF at pH 3-4, I). N-glycosylation has been reported to protect human gastric lipase (Wicker-Planquart et al., 1999) and mouse IgG1 (Wilson et al., 2002) from pepsin digestion. To determine whether N-glycosylation could further improve the stability of FaeG
66
Results and discussion
protein against pepsin digestion, the TSP from transgenic barley grains expressing N-glycosylated erFaeG was exposed to simulated porcine gastric fl uid (III, Figure 6). Results of this study suggest that N-glycosylation of erFaeG protein does not confer further protection on the FaeG against pepsin digestion; similar results were seen with a chloroplast-derived nonglycosylated pFaeG protein (II, Figure 2A). At lower pH values, pFaeG was more effi ciently digested than at pH 3.5 (J. Joensuu, unpublished results), suggesting that the entry of plant-produced FaeG into the small intestine may be more limited than F4 fi mbriae in the extremely low pH conditions of the stomach. This might be due to incomplete folding of the plant-produced FaeG protein or better accessibility of proteases to plant-produced FaeG than to multimeric F4 fi mbriae.
To fold correctly in F4+ ETEC, the molecular chaperone FaeE forms a periplasmic complex with FaeG and prevents the subunits from degradation (Mol et al., 1995, Mol and Oudega, 1996). The chaperone FaeE is essential for the stability and folding of subunits; faeE deletion mutants do not contain FaeG subunits (Mooi et al., 1982). The recombinant overexpression of FaeG protein in the cytoplasm of E. coli (rFaeG) leads to the formation of cytoplasmic inclusion bodies. To solubilize these protein aggregates, highly denaturing solvents like urea or guanidine hydrochloride have to be used (Verdonck et al., 2004c, J . Joensuu, unpublished results). Removal of these denaturing agents by dialysis leads to aggregation of FaeG protein. Furthermore, neither the use of non-ionic detergents (Tween, Triton-X-100), sonication, or altered pH was successful in refolding the rFaeG protein (Verdonck et al., 2004c). Similarly, high-level expression of the type I fi mbrial
adhesin FimH and the P-pili adhesin PapG in the cytoplasm resulted in formation of inclusion bodies and aggregation when the denaturant urea was removed by dialysis (Kariyawasam et al., 2002). X-ray diffractions of type I and P-pili subunits revealed an immunoglobulin-like domain, but lacking the seventh β-stand, exposing the hydrophobic core of the subunit to the surface. However, the groove along the surface of these subunits is fi lled by a β-strand of the chaperone in the periplasm or by a β-strand of a subunit in the fi mbriae (Choudhury et al., 1999, Sauer et al., 1999). The conformation of the F4 fi mbrial chaperone FaeE (Van Molle et al., 2005), and the conserved amino acid regions in the F4 fi mbrial subunits suggest a similar interaction in F4 fi mbria (Verdonck et al., 2004a). Therefore, a hydrophobic core of rFaeG could have caused the aggregation during refolding (Verdonck, 2004). However, the inclusion body-derived rFaeG refolded into a soluble and native-like receptor binding conformation in the presence of SDS (Verdonck et al., 2004c). The authors suggested that the hydrophobic core of rFaeG was masked by SDS, preventing the aggregation. The stability of this SDS-refolded rFaeG was reported to be low, even when stored at 4°C (Verdonck, 2004).
In tobacco and alfalfa chloroplasts (II, IV) and in ER of barley grains (III), the FaeG protein folded into a soluble and stabile form without the help of chaperone protein FaeE. Perhaps this folding is aided by a plant chaperone with similarity to FaeE.
5.3.7 Stability of plant-produced FaeG in simulated intestinal fl uidA 2-h digestion in SIF had no major impact on the pFaeG protein, whereas most
67
Results and discussion
plant proteins showed clear fragmentation under these conditions (II, Figure 4B). Similarly, the reFaeG was unaffected by SIF (J. Joensuu, unpublished results). These results suggest that gastric digestion is a limiting step in stability of plant-produced FaeG along the gastrointestinal tract.
To conclude, plant-produced FaeG protein showed resistance under harsh gastrointestinal conditions. This is the fi rst prerequisite for a plant-produced oral subunit vaccine. However, at extremely low pH stomach conditions, the entry of plant-produced FaeG protein into the mucosal inductive sites of the intestine is likely to be more limited than that of F4 fi mbriae. The plant cell matrix has been proposed to provide protection to plant-produced vaccine antigens upon oral administration (Modelska et al., 1998, Molina et al., 2005). However, the protective role of the plant matrix has not been confi rmed by detailed studies. In our study, the plant matrix did not have any signifi cant protective effect on plant-produced FaeG protein under SGF conditions (J. Joensuu, unpublished results).
5.4 Receptor binding of F4 fi mbriae and pFaeGIn contrast to harmless food antigens, which activate immunosuppressive mechanisms resulting in oral tolerance, effective oral antigens need to be actively transported through the mucosal barrier. This can be mediated by binding to specifi c receptors present on epithelial cells (Elson and Dertzbaugh, 1999). Indeed, to evoke a mucosal immune response, orally delivered F4 fi mbriae or FaeG protein need to bind to the F4 receptor present on the porcine small intestinal enterocytes (Van den Broeck et al., 1999a, Verdonck et al., 2004c). By contrast, in piglets lacking F4R, orally
delivered F4 fi mbriae act as normal food antigens, inducing oral tolerance (Van den Broeck et al., 1999a, Van den Broeck et al., 2002).
Because the oral vaccination of suckling piglets with F4 is not very effi cient (Snoeck et al., 2003) and the induction of an F4-specifi c mucosal immune response in weaned piglets takes at least a week (Van den Broeck et al., 1999a, Verdonck et al., 2002), shortly after weaning, there is a gap in which an oral F4 vaccine cannot provide active protection against F4+ ETEC infections. This is also the period when piglets are most vulnerable to ETEC infections (Fairbrother et al., 2005). F4 fi mbriae have been reported to be able to bind to F4R in vitro, subsequently inhibiting the binding of F4+ ETEC to the villous brush border (Van den Broeck et al., 1999b). In addition to evoking an active mucosal immune response, an ideal oral ETEC vaccine would passively protect the weaned piglets during the fi rst days postweaning. Edible transgenic plants could function as an effi cient delivery system for an oral vaccine, competing for F4R binding sites with ETEC until neutralizing IgA antibodies would be present in the intestine. It was therefore important to assess the F4R binding capacity of pFaeG protein and compare it with that of F4 fi mbriae (II, Figure 2C; Figure 5). The F4R binding was studied in vitro with piglet villi samples in a protocol where the ability of a protein sample to inhibit subsequent F4+ ETEC binding was determined (Van den Broeck et al., 1999a).
The native-like folding of FaeG appears to be obligatory for F4R binding and immunogenicity of the FaeG protein since a urea-refolded aggregating rFaeG protein did not bind F4R (Verdonck et al., 2004c, J. Joensuu, unpublished results) and also failed to induce a
68
Results and discussion
mucosal immune response (Verdonck et al., 2004c). The soluble pFaeG protein (the faeG gene derived from ETEC strain 5/95) produced in tobacco chloroplasts was able to inhibit ETEC binding in a dose-dependent manner (II, Figure 2C). However, this inhibition was not as effi cient as with the corresponding F4 fi mbriae (Figure 5). At a concentration of 200 µg of FaeG /ml, pFaeG inhibited ETEC adhesion 70.5 ± 3.0% (mean ± SEM), while 82.0 ± 5.2% inhibition was seen with 5/95 F4 fi mbriae. Previous reports indicate that 200 µg/ml of GIS26
F4 fi mbriae inhibited adhesion of homologous ETEC almost completely, i.e. by 96.3 ± 2.3% and 97.7 ± 3.2% (Van den Broeck et al., 1999b, Verdonck et al., 2004c). Furthermore, Verdonck et al. (2004) reported 94.7 ± 4.9% inhibition of ETEC adhesion with 800 µg of FaeG/ml with an SDS-refolded rFaeG derived from strain GIS26. However the complete inhibition of ETEC adhesion with pFaeG was not achieved, even when concentrations as high as 1400 µg of FaeG/ml were used (J. Joensuu, unpublished results). The reason for incomplete inhibition of ETEC adhesion even at high pFaeG concentrations remains obscure. FaeG amino acid sequence alignment of GIS26 and 5/95 revealed seven differences between the strains (Table 6). None of these differences are, however, located on amino acids 134, 136, 147, 150, 155, 163-174, or 216, which are proposed to be related to F4ac receptor binding (Jacobs et al., 1987, Bakker et al., 1992a). Because the structure of the FaeG protein and the exact receptor binding site are still unknown, it is impossible to elucidate which of the deviating amino acids are responsible for the observed differences in F4R binding.
To conclude, these data confi rm that, in addition to whole F4 fi mbriae (Erickson et al., 1992, Van den Broeck et al., 1999b), purifi ed F4 fi mbrial FaeG (Van den Broeck et al., 1999b), and SDS-refolded rFaeG (Verdonck et al., 2004c), plant-produced pFaeG is also able to bind to F4R present at the surface of small intestinal villi and inhibit subsequent binding of F4+ ETEC bacteria. This suggests that it might be possible to passively protect piglets with pFaeG protein immediately after weaning and simultaneously evoke an F4-specifi c mucosal immune response in weaned piglets.
Table 6. Deviating amino acids in the FaeG protein between two F4ac+ ETEC strains.
Amino acid position
ETECGIS 26
ETEC5/95
97 Asn Lys105 Lys Asn118 Glu Gly177 Glu Ala188 Asn Asp201 Ile Val221 Thr Asn
30
40
50
60
70
80
90
100
10 100 1000FaeG concentration (µg/ml)
% in
hibi
tion
of E
TE
C a
dhes
ion
F4
pFaeG
Figure 5. Inhibition of the F4+ ETEC (strain 5/95) adhesion to villous brush borders (mean ± SEM, n=4) by incubating the villi with increasing concentrations of plant-produced FaeG protein or F4 fi mbriae derived from ETEC 5/95.
69
Results and discussion
5.5 Immunogenicity of plant-produced FaeG
5.5.1 Immunogenicity of N-glycosylated erFaeG
Evaluating the immunogenic capacity of barley endosperm-derived erFaeG protein by oral immunization in a pig model would have been interesting. However, due to legislation, the transgenic barley plants had to be cultivated under contained greenhouse conditions, and in the framework of this project we did not have suffi cient greenhouse capacity or labor to multiply the seed material into the amounts needed to orally immunize piglets. Therefore, the immunogenicity of erFaeG was studied in mice (III). However, the murine gut does not posses F4 receptors, and oral immunization of mice with F4 or erFaeG did not induce F4-specifi c antibody responses (J. Joensuu, unpublished results). Hence, the immunogenicity of the N-glycosylated erFaeG protein was confi rmed in a mouse model by subcutaneous injection of TSP extracted from barley grains (III). The N-glycosylation did not abolish the immunogenic capacity of FaeG protein, as evidenced in the F4-specifi c antibodies detected from the sera of erFaeG-immunized mice by ELISA and immunoblotting (III, Figure 7).
However, erFaeG was not as immunogenic as F4 fi mbriae, inducing a weaker and delayed serum antibody response. As multimeric antigens tend to induce more vigorous immune responses than their monomeric counterparts (Miller et al., 1998), the murine immune system likely recognizes multimeric F4 fi mbriae more effi ciently than the monomeric erFaeG, making it diffi cult to evaluate whether N-glycosylation further reduced the immunogenicity of erFaeG.
Huang and co-workers (2003) also reported delayed and weaker immune response with the recombinant FaeG than with the related F4ad fi mbriae. The immunization schedule and the dose of the antigen used in our study are consistent with those of Huang et al. (2003), where mean F4-specifi c antibody titers of up to 4 (log 10 scale at 56 days post primary immunization (dppi)) were observed in the serum of mice immunized with the nonglycosylated FaeG protein expressed in the cytoplasm of transgenic tobacco plants. In our study, when inverted to a log 10 scale, a mean 2.4 anti-F4-titer was recorded on day 56 dppi with erFaeG (III, Figure 7). Although some differences were present in the immunization methods and between the mouse and F4+ ETEC strains used, this result might suggest a negative effect of glycosylation on the immunogenicity of the FaeG. Furthermore, the F4+ ETEC-neutralizing capacity of erFaeG-immunized mice sera was confi rmed in vitro. The sera were shown to inhibit the adhesion of F4ac+ ETEC bacteria to F4R in piglet brush borders in a dose-dependent manner (III, Figure 8).
5.5.2 Immunogenicity of pFaeG The fi nal goal for the model plant-produced ETEC vaccine was to determine whether alfalfa-derived pFaeG could evoke a mucosal immune response in a pig model and protect the piglets against subsequent ETEC challenge (IV).
To study the immunogenicity of pFaeG protein, transgenic alfalfa was administered intragastrically to weaned piglets with and without a known mucosal adjuvant cholera toxin (CT). As a positive control, the piglets were immunized with purifi ed 5/95 F4 fi mbriae delivered together with nontransgenic
70
Results and discussion
alfalfa. Nontransgenic alfalfa served as a negative control. Because the amount of available transgenic plant material was limited, it was administered intragastrically. The intragastric delivery method enabled equal administration of the antigen as well as simultaneous neutralization of gastric acid with a buffer solution. Although pFaeG showed considerable stability against gastric digestion in vitro at pH 3.5 (II, Figure 2A), it was partially digested and was not as stable as the F4 fi mbriae under similar conditions (I, Figure 4). Therefore, gastric neutralization was used in this proof-of-principle experiment. Verdonck et al. (2004) recently demonstrated that F4 fi mbriae tend to stimulate a higher immune response than monomeric FaeG, and hence, a ten times higher amount of pFaeG (20 mg) than F4 fi mbriae (2 mg) was used to immunize the piglets.
5.5.2.1 Systemic immune responseThe systemic immune response in piglets was elucidated by analyzing the F4-specifi c antibodies of isotypes IgM, IgA, and IgG in the blood samples collected weekly. Following the fi rst intragastric immunization of newly weaned piglets (0, 1, and 2 dppi), only a very low F4-specifi c IgM titer was observed in the positive control group immunized with purifi ed F4 fi mbriae (C+F4 group; mean titer 17) at 7 dppi (VI, Figure 3). The booster immunization induced a weak secondary F4-specifi c systemic immune response in the C+F4 group, with the F4-specifi c IgM antibody titer decreasing and the IgA and IgG titers increasing one week following the boost. Intragastric immunization with pFaeG also induced an F4-specifi c immune response; low F4-specifi c IgG titers were detected in the
pFaeG-immunized pigs at 21 dppi. Induction was improved when pFaeG was co-administered with CT as a mucosal adjuvant (pFaeG+CT group). These data indicate that intragastric immunization of newly weaned piglets with pFaeG does activate a weak systemic F4-specifi c immune response. Verdonck et al. (2004) reported clearer, even though a low systemic F4-specifi c immune with SDS-refolded monomeric rFaeG. However, when CT was used as an adjuvant with rFaeG, a signifi cant systemic response was induced (Verdonck et al., 2005b).
Perhaps the oral delivery method used by Verdonck and colleagues stimulates the systemic immune response better than the intragastic delivery used in our study. Snoeck et al. (2006) reported that the F4-specifi c serum antibody response after oral vaccination with F4 fi mbriae in solution might not only be the result of an immune response in the small intestine, but also of an induced response in the mount or pharynx region. This is supported by the observation that 5/95 F4 fi mbriae also tend to induce a higher serum antibody response with oral administration (Figure 6) than with intragastric administration (IV). By contrast, in a mouse model the systemic and mucosal immune responses have been proposed to be stimulated more effi ciently by intragastric than oral delivery of antigens (Lauterslager and Hilgers, 2002).
5.5.2.2 Mucosal immune responseAfter immunization, the piglets were exposed to a pathogenic F4+ ETEC 5/95 strain, and the induction of protective mucosal immune response was monitored by following the severity of infection via
71
Results and discussion
3
4
5
6
7
8
9
10
0 7 14 21 28 35
F4-s
peci
fic s
erum
ant
ibod
y tit
er (l
og2) F4-
F4+
0
1
2
3
4
5
6
7
8
1 2 3 4 5 6
F4+
E.c
oli
per
g fe
ces
(log
10) -F4
+F4
0
1
2
3
4
5
6
7
8
1 2 3 4 5 6
F4+
E.c
olip
er g
fece
s (lo
g 10
) - F4 +F4
3
4
5
6
7
8
9
10
0 7 14 21 28 35
F4-s
peci
fic s
erum
antib
ody
titer
(log2
)
F4-F4+
ETEC GIS26 ETEC 5/95
A)
B)
dppi dppi
dpc dpc
secretion of the F4+ ETEC in feces (IV, Figure 4). It has been previously shown with F4- and rFaeG-immunized piglets that the reduction in the number of F4+ ETEC secreted following challenge correlates with the local indicators of F4-specifi c mucosal immune response, i.e. the appearance of F4-specifi c antibody-secreting cells at mucosal effector sites and the presence of F4-specifi c IgM and IgA in the intestinal content (Van den Broeck et al., 1999a, Verdonck et al., 2004c).
Oral immunization with pFaeG reduced the excretion of F4+ E. coli slightly following challenge but did not shorten excretion time (IV, Figure 4). Co-administration of pFaeG and CT did, however, signifi cantly reduce the number of excreted F4+ E. coli as well as the
excretion time, compared with the negative control group. Indeed, F4+ E. coli excretion in the pFaeG+CT group was identical to that in the positive control group that had received F4 fi mbriae. These results indicate that intragastric immunization of piglets with pFaeG induces a mucosal F4-specifi c immune response, which is improved by co-administration of pFaeG and CT. This is in agreement with the fi ndings of Verdonck et al. (2005), who reported a higher induction of F4-specifi c mucosal antibody response with rFaeG when co-administered with CT. CT supplementation also resulted in the induction of CT-specifi c antibodies at the moment of ETEC challenge (IV, Verdonck et al., 2005b). Although CT-specifi c antibodies may affect LT-induced diarrhea, they will not
Figure 6. A) Mean F4-specifi c serum antibody titers (± SEM) at 0, 7, 14, 21, 25, 28, and 35 days post primary immunization (dppi) of F4R+ piglets orally immunized with F4 fi mbriae derived from ETEC strains GIS26 and 5/95. The black arrows represent immunization and the white arrows the F4+ ETEC challenge. B) Mean F4+ Escherichia coli excretion per gram feces (± SEM) after oral challenge with the homologous ETEC strain. +F4, immunized animals (n=3); –F4, nonimmunized animals (n=2); dpc, days post challenge.
72
Results and discussion
reduce F4+ ETEC colonization (Nataro and Kaper, 1998, Verdonck et al., 2005b).
The F4-specifi c serum antibody titers following F4+ ETEC challenge are consistent with the results of F4+ E. coli excretion (IV, Figures 3 and 4). Infection of the negative control group (C) induced a primary F4-specifi c antibody response characterized by high F4-specifi c IgM titers during the fi rst week following challenge and the subsequent appearance of F4-specifi c IgA and IgG antibodies. A similar F4-specifi c IgM response was observed in the pFaeG group. This is not surprising since the F4+ E. coli excretion was only slightly lower in this group than in the negative control group. However, the faster appearance and higher amounts of F4-specifi c IgA antibodies in the pFaeG group suggest a priming of the immune system following pFaeG immunization. On the other hand, the presence of protective F4-specifi c mucosal antibodies at the moment of challenge will reduce bacterial proliferation and result in reduced stimulation of the immune system. Indeed, the lowest F4-specifi c serum antibody titers following challenge were observed in the pFaeG+CT group. These results confi rm the ability of plant-produced FaeG to induce a protective F4-specifi c immune response.
However, the effi cacy of pFaeG (derived from ETEC 5/95) was lower than the SDS-refolded recombinant bacterial rFaeG (derived from ETEC GIS26) (Verdonck et al., 2004c, Verdonck et al., 2005b). This might refl ect differences in the primary structure and folding of these proteins (Table 6, see also sections 5.3.6 and 5.4). Moreover, Verdonck and co-workers (2004c, 2005b) blocked the secretion of gastric acid medically by rabeprazolum while in our study pFaeG was delivered in a neutralizing buffer
solution (IV). Perhaps the medical approach is more effi cient and offers a longer rise in gastric pH, partly explaining the higher effi cacy of rFaeG than pFaeG. Similarly, a higher effi cacy has been observed following oral immunization with purifi ed GIS26 F4 fi mbriae (Verdonck et al., 2004c) than with purifi ed 5/95 fi mbriae (Figure 6; IV, Figure 4). However, in these studies, the animals were challenged only with homologous ETEC strains. To confi rm these results, challenge experiments with heterologous ETEC strains should be performed.
The lower immunogenicity of 5/95 F4 might be related to a difference in FaeG polymerization and subunit folding between these strains. In contrast to other F4ac strains, the nondenatured F4 fi mbriae of ETEC strain 5/95 did not appear as multimers on SDS-PAGE (Verdonck, 2004, Verdonck et al., 2004b). In addition, an electron micrograph of purifi ed 5/95 F4 samples revealed that most of the protein was present in aggregates rather than fi mbrial structures (Verdonck, 2004). Since 5/95 ETEC strain was able to bind F4R (Figure 5) and was pathogenic (Figure 6; IV, Figure 4), it must express F4 fi mbriae on its surface. Indeed, F4 multimers from 5/95 strain were detected when fi mbriae were analyzed on native PAGE gels (J. Joensuu, unpublished results). According these results, the 5/95 strain may express F4 fi mbriae in which the interaction of FaeG subunits is weaker, leading to dissociation of most of the fi mbriae during the purifi cation process or exposure to SDS. However, this aggregation did not render 5/95 F4 more susceptible to gastric digestion since the stability of 5/95 F4 fi mbriae in simulated porcine gastric fl uid was comparable with that of GIS26 F4 fi mbriae (J. Joensuu, unpublished results). Perhaps the more defi ned polymeric
73
Results and discussion
appearance of GIS26 F4 enables a higher avidity of binding to F4R as compared with 5/95 F4 fi mbriae. Miller et al. (1998) reported the importance of the multimeric nature of Yersinia pestis F1 antigen, in inducing better protection following immunization than its
monomeric form. However, this does not explain the differences in the effi cacy of related recombinant FaeG proteins (pFaeG and rFaeG); the F4R binding might be related to the primary structure of FaeG as well.
74
Conclusions and future prospects
6 CONCLUSIONS AND FUTURE PROSPECTS
We investigated a novel approach in which transgenic crop plants are utilized as a production and delivery system for an oral veterinary vaccine. F4 fi mbriae of enterotoxigenic Escherichia coli are highly stable multimeric structures with a capacity to evoke mucosal immune responses, offering a unique model system to study oral vaccination against ETEC-induced porcine postweaning diarrhea which is a major problem in pig houses worldwide. Transgenic plants can offer an economically feasible platform for large-scale production of vaccine antigens for animal health.
FaeG, the major subunit protein of E. coli F4 fi mbria and a promising candidate for an oral vaccine against porcine PWD, can be produced in transgenic tobacco, alfalfa, and barley plants. When FaeG protein was subcellularly targeted to the tobacco or alfalfa chloroplast, a high-yield production of 1% TSP was achieved. A similar yield was obtained in the seeds of barley, a valuable crop plant. Moreover, desiccated alfalfa plants and barley grains have the capacity to store FaeG protein in a stable form for years. It was estimated that 100 g of barley seeds or dried alfalfa plants possess 10 and 67 mg of soluble FaeG protein, respectively. The obtained production yield is suffi cient for oral vaccine applications in which a few milligrams of antigens typically need to be administered to evoke detectable immune responses. This proposes that transgenic plant material could therefore serve as a production platform as well as a storage and delivery system for an oral porcine ETEC vaccine against PWD.
The unique ability of F4 fi mbriae to raise a mucosal immune response is mediated by the F4R present in the
porcine intestine, while in F4R-defective animals F4 acts like most orally delivered soluble nonreplicating antigens and tends to induce oral tolerance. To reach the inductive sites in the small intestine, the F4 must pass through the stomach and survive the harsh gastric conditions. We demonstrated that the conditions present in the porcine gastrointestinal tract around the moment of weaning are not a major limiting factor for F4 integrity. The plant-produced FaeG protein also showed resistance in gastrointestinal conditions. However, the extremely low pH proteolytic stomach conditions might limit the passage of FaeG to the intestinal F4R. Glycosylation can be a prerequisite for proteolytic stability of some proteins. The F4 fi mbrial FaeG is not glycosylated in the native form, but contains three putative N-glycosylation sites, which were glycosylated by the plant cell glycosylation machinery when FaeG was targeted to the ER and produced in barley seed endosperm. However, the N-glycosylation was unable to improve the stability of plant-produced FaeG protein against gastric digestion. Furthermore, it was evaluated in a mouse model that the N-glycosylation did not abolish the immunogenicity of plant-produced FaeG protein.
Most importantly, the plant-produced FaeG was able to evoke F4-specifi c systemic and mucosal immune responses following an oral delivery of transgenic alfalfa to weaned piglets. When co-administered with the mucosal adjuvant CT, the effi cacy of this plant-based subunit vaccine was identical to the corresponding purifi ed F4 fi mbriae (derived from ETEC 5/95). However, neither of them could not protect the piglets completely against F4+ ETEC
75
Conclusions and future prospects
infection, as has been reported before for the oral administration of purifi ed F4 fi mbriae derived from ETEC GIS26.
Infections with F4+ ETEC are a major cause of diarrhea and mortality in newly weaned piglets, resulting in signifi cant economic losses. No vaccine is currently available to protect weaned piglets against ETEC infections. The fi ndings here thus present new approaches to develop a vaccination strategy against PWD. However, the stability and immunogenicity of plant-produced FaeG should be improved to neutralize F4+ ETEC colonization more effi ciently. Our observations indicate that the FaeG used in this study (derived from ETEC stain 5/95) might not have the ideal features for a subunit vaccine since the corresponding F4 fi mbriae were dissociated into FaeG monomers by the purifi cation process and were not as immunogenic as their GIS26 counterparts. Perhaps the transformation of plants with the FaeG-encoding gene from strain GIS26 would lead to formation of FaeG multimers. The polymeric appearance of FaeG subunits in purifi ed F4 fi mbriae may enable a higher avidity of binding to the F4R and better stability against gastric digestion than the monomeric FaeG. Further research is needed to determine whether these FaeG polymers could be produced by plants. On the other hand, the aberrant properties of 5/95 F4 fi mbriae might provide a means of characterizing regions in the amino acid sequence of FaeG that are essential to subunit-subunit interaction or receptor binding. This might open possibilities to design a more stable and immunogenic recombinant F4 fi mbriae or derived subunit complexes. To this end, elucidating the crystallographic structure of FaeG and characterizing the assembly of F4 fi mbria would be useful.
The outbreaks of PWD caused by ETEC typically occur during the fi rst two weeks of postweaning, when the maternal protection has dissapeared and the piglets encounter physical, nutritional, and social stresses leading to slow gastrointestinal transit, which facilitates ETEC colonization. The oral vaccination of suckling piglets has previously been demonstrated to be rather ineffi cient compared with that of weaned piglets due to the immature mucosal immune system of suckling piglets and the blocking effect of maternal antibodies present in the milk. When the piglets are orally administered with F4 around the moment of weaning, it takes about a week to develop a protective F4-specifi c immune response in the small intestine, and during this time the piglets are not protected by passive lactogenic immunity, or by active immunity. F4R binding is a prerequisite for the uptake of F4 and for the induction of an F4-specifi c mucosal immune response. Here we showed that plant chloroplasts were able to fold the FaeG protein to a bioactive conformation, enabling the binding to F4R present on the villous enterocytes and subsequently inhibiting the adhesion of F4+ ETEC to F4R. This ‘probiotic effect’ of plant material expressing FaeG protein warrants further study to determine the optimal dosage and delivery schedule. In an ideal situation, creep or weaners’ feed supplemented with FaeG could protect piglets passively during the fi rst days of postweaning and induce an F4-specifi c protective mucosal immune response. An effi cient plant-based production and delivery system of vaccine antigens would make this application economically feasible.
76
ACKNOWLEDGMENTS
My deepest gratitude is due to my supervisor Docent Viola Niklander-Teeri as well as to Professor Teemu Teeri for providing me with the opportunity to conduct this project and for generously offering their time and expertise over these six years. The reviewers Dr. Anneli Ritala and Professor Airi Palva are acknowledged for critical evaluation and for constructive comments on the manuscript. Carol Ann Pelli is thanked for editing the language of the manuscript. Piia Ouri is acknowledged for editing the layout of the manuscript.
I warmly thank the current and former members of the Plant Vaccine Group: Andrea, Chris, Eerika, Johanna, Kaisa, Lilia, Mikko, Mirkka, Minna, Tero, and Veronica. Without your hard work at the lab, this thesis would not exist. I especially want to thank Lilia and Mirkka for thorough work with the plants. Collaborators Dr. Hannu Lång and Dr. Leena Valmu are thanked for providing their help and know-how on this research. Professor Jari Valkonen is thanked for guidance into the world of scientifi c thinking and writing, and for both scientifi c and non-scientifi c conversations during the creative process.
The people at Gerbera Lab and the staff at Department of Applied Biology are acknowledged for providing a pleasant work atmosphere. Marja H., I especially want to thank you for all your advice on cloning.
Dr. Kirsi-Marja Oksman-Caldentey, Dr. Anna Maria Nuutila, and the staff at VTT Biotechnology are thanked for introducing me to the secrets of barley and alfalfa transformation.
Dr. Hilkka Siljander-Rasi and the staff at MTT research station, I thank for generous collaboration, instrumental to the success of my work with the piglets. Tapio, your contribution to the experiments was irreplaceable.
I am grateful to Professors Eric Cox and Bruno Goddeeris for offering me the opportunity to visit their lab at Gent University. My visits to the Laboratory of Veterinary Immunology were a pleasant experience and made this thesis possible. I’m thankful to the entire lab team for hands-on guidance into the world of animals. Dr. Veerle Snoeck, thanks for fi nding time to help despite your busy schedule. Dr. Frank Verdonck is warmly acknowledged for fruitful discussions, ideas, and friendship. It has been a priviledge to collaborate with you.
This research was supported by the Academy of Finland, Finnish Cultural Foundation, Raisio Feed Ltd, Raisio Plc Research Foundation, TEKES, Tervakoski Research Foundation for Student Grants, University of Helsinki, and Viikki Graduate School.
My heartfelt thanks go to my loving wife Anita; your tender care and patience have not ceased even in my worst moments. I am also indebted to my parents Eero and Marjatta, sister Essi, and parents-in-law Jalo and Lilja for ongoing support and encouragement.
77
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