PhD Fusarium
217
SELECTION, CHARACTERIZATION, AND APPLICATION OF A MYCOTOXIN-SPECIFIC SINGLE DOMAIN ANTIBODY FRAGMENT. A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by PATRICK JOHN DOYLE In partial fulfillment of the requirements for the degree of Doctor of Philosophy December, 2009 © Patrick Doyle, 2009
description
Fusarium
Transcript of PhD Fusarium
i
SELECTION, CHARACTERIZATION, AND APPLICATION OF A
MYCOTOXIN-SPECIFIC SINGLE DOMAIN ANTIBODY FRAGMENT.
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by
PATRICK JOHN DOYLE
In partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
December, 2009
© Patrick Doyle, 2009
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ABSTRACT
SELECTION, CHARACTERIZATION, AND APPLICATION OF A
MYCOTOXIN-SPECIFIC SINGLE DOMAIN ANTIBODY FRAGMENT.
Patrick John Doyle Advisor:
University of Guelph, 2009 Professor J.C. Hall
15-Acetyl-deoxynivalenol (15-AcDON) is a low molecular weight (M.W. = 338
Da) sesquiterpenoid trichothecene mycotoxin associated with Fusarium Head Blight
(FHB), a global disease of small grain cereals. Estimated economic losses from the most
recent Canadian and US FHB epidemic during the mid 1990s range from $1.3 to $3.0
billion USD, depending on valuations assigned to reduced crop yield and mycotoxin
contamination of food and feed. The accumulation of DON and related trichothecene
mycotoxins within harvested grain is subject to stringent regulation as both toxins pose
dietary health risks to humans and animals. A property of 15-AcDON, and structurally-
similar trichothecene mycotoxins that are intimately involved in FHB pathogenesis, is the
propensity to inhibit peptidyl transferase activity and thus inhibit protein synthesis within
infected host-plant cells. This effect, coupled with secondary phytotoxic effects, act as
mechanisms of virulence for pathogenic Fusarium species.
A single-domain variable heavy chain (VHH) recombinant antibody (rAb) fragment
specific to 15-AcDON was isolated from a hyper-immunized phagemid library. The
dominant clone (NAT-267) was expressed and purified as a VHH monomer with a
competitive binding affinity of 1.24 µM. The gene encoding the NAT-267 camelid
single-domain antibody fragment (VHH) was expressed within the methylotropic yeast
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Pichia pastoris. Mycotoxin-mediated cytotoxicity was assessed by continuous
measurement of cellular growth over time. At equivalent doses, 15-AcDON was
significantly more toxic to wild-type P. pastoris than was DON, which, in turn, was more
toxic than 3-AcDON. Intracellular expression of this toxin-specific VHH „intrabody‟
within P. pastoris conveyed significant (p = 0.01) resistance to 15-AcDON cytotoxicity at
doses ranging from 20 to 100 µg·mL-1
, making this the first report of VHH-based
sequestration of a haptenic mycotoxin. Furthermore, a biochemical transformation of
DON to 15-AcDON was documented, which explained significant attenuation of the
efficacy of DON at 100 and 200 µg·mL-1
. This “proof of concept” model suggests that in
planta VHH expression may lead to enhanced tolerance to mycotoxins and thereby serve
as a novel tool to help limit Fusarium infection of commercial agricultural crops such as
wheat and barley that are engineered to express this mycotoxin-specific VHH.
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PREFACE
This dissertation contains three manuscripts prepared for publication within a peer-
reviewed text book and two scientific journals. Each manuscript is listed below with the
respective publication status.
CHAPTER TWO
Doyle, P.J., Arbabi-Ghahroudi, M., Sheedy, C., Yau, K., Hall, J.C. 2008. Antibody
Engineering in Agricultural Biotechnology. Chapter 3. In: Immunoassays in
Agricultural Biotechnology. G. Shan (ed.). John Wiley & Sons, New York, NY. (In
Press).
CHAPTER FOUR
Doyle, P.J., Arbabi-Ghahroudi, M., Gaudette, N., Furzer, G., Savard, M.E., Gleddie, S.,
McLean, M.D., MacKenzie, C.R., Hall, J.C. 2008. Cloning, Expression, and
Characterization of a Single Domain Antibody Fragment with Affinity for 15-Acetyl-
Deoxynivalenol. Molec. Imm. 45: 3703 - 3713.
CHAPTER FIVE
Doyle, P.J., Saeed, H., Hermans, A., Gleddie, S.C., Hussack, G., Arbabi-Ghahroudi, M.,
Seguin, C., Savard, M.E., MacKenzie, C.R., Hall, J.C. 2009. Intracellular Expression
of a Single-Domain Antibody Reduces Cytotoxicity of 15-Acetyl-Deoxynivalenol in
Yeast. J. Biol. Chem. 284: 35029-35039. Manuscript M109.045047.
http://www.jbc.org/content/early/2009/09/25/jbc.M109.045047
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ACKNOWLEDGEMENTS
I would like to thank my graduate committee for their trust, wisdom, and patience in
helping to guide me through this degree. Chris, Marc, Roger, and Steve… it has been a
privilege to learn from you and work in your labs. Thank-you for your friendship and
help along the journey! Also, in alphabetical order… special regards and sincere thanks
to: Anne Hermans, Barb Blackwell, Charles Seguin, Christian Luebbert, Claudia Sheedy,
Garth Gourlay, Ginette Dubuc, Gord Furzer, Greg Hussack, Hanaa Saeed, Hong Tong-
Sevinc, Jamshid Tanha, Joy Roberts, Karen Gourlay, Keith Solomon, Larry Erickson,
Linda Veldhuis, Mehdi Arbabi-Ghahroudi, Mike McLean, Nathalie Gaudette, Nina
Weisser, “Osama-Bin Llama”, Shannon Ryan, Tomoko Hirama, and Yonghong Guan.
Sincere gratitude and thanks to my friends at Syngenta Crop Protection Canada Inc.
for the tremendous support and encouragement. Special thanks to Marian Stypa. You
believed in me and were always there for me (and my family) right from the start! To
Norm Dreger, thanks for adopting me into the “Business Development” family and for
the huge… patience, support, and of course for the valuable insights that you have taught
me about people. I would also like to thank Ms. Laurie Vescio and Mr. Jay Bradshaw for
the great support and words of kind encouragement over these six + years.
Finally, to my family. Thank-you so much… Nadine, you held our family together
all those times that I was away. Even today, I never worry about the most important stuff
because I always know that you have it covered. Mom and Dad… thanks for all the
advice and insights provided during all those late-night phone calls to Calgary. Finally, to
my kids… Liam and Danielle, I have watched you grow alongside this PhD project. I am
very sad that doing this took a lot of my time away from you. Danielle and Liam, you
always were, and will always be in my heart. All those times that I was working
on”school stuff”, you were always the light and my joy that kept me going forward. I
love you both with all my heart...!
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PICTURE
Freelton, Ontario. April 17, 2005.
Left to Right: “Osama-Bin” Llama; Danielle (hiding), Liam, and Nadine
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NOTE ABOUT WHY
In the preface of: Fusarium Head Blight of Wheat and Barley, the editors (Kurt J.
Leonard and William R. Bushnell) make the following dedication.
“This book is dedicated to those scientists who labor to solve the Fusarium head
blight problem, despite knowing full well that easier paths may be taken in the pursuit
of peer recognition and scientific acclaim.”
I do not believe that this dedication was a challenge (or „snub‟) to the challenges,
relevance, or prestige of the vast number of other disciplines and technical pursuits within
modern science. To me, Drs. Leonard and Bushnell were simply trying to acknowledge
what “bad actors” FHB and its toxin problem(s): i.e., DON... + acetylated “brother‟s”,
15-AcDON and 3AcDON can be.
For the record, my interest in FHB goes back to the epidemic in 1993/1994/1995
(esp. 1994!) when I ran a small research farm in southern Manitoba. FHB may be a
cyclical disease that comes along only once in a long while, but when it gets going, it is a
huge problem that affects many people, especially farmers. Like Leonard and Bushnell, I
take my hat off to, and admire, those scientists who contribute to this field.
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LIST OF ABBREVIATIONS
15-AcDON 15-Acetyl-Deoxynivalenol
15-DON-BSA Bovine Serum Albumin Conjugated to C-15 of DON
15-DON-HRP Horse Radish Peroxidase Conjugated to C-15 of DON
15-DON-OVA Ovalbumin Conjugated to C-15 of DON
3-AcDON 3-Acetyl-Deoxynivalenol
AAFC Agriculture and AgriFood Canada
Ab Antibody
Ag Antigen
AOX Alcohol Oxidase Promoter
AP Alkaline Phosphatase
ARM Antibody-Ribosome-mRNA Complex
BCIP 5-Bromo-4-Chloro-3-Indolyl Phosphate
BSA Bovine Serum Albumin
CD-ELISA Competitive Direct ELISA
cDNA Complementary DNA
CDR Complementarity Determining Region
CH Constant Domain of the Heavy Chain
cIgG Conventional Immunoglobulin Gamma (γ) Isotype Abs
CL Constant Domain of the Light Chain
Da Dalton
DAI Days After Inoculation
DAPI 4′,6-diamidino-2-phenylindole dihydro-chloride
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DAS Diacetoxyscirpenol
DMAP Dimethylaminopyridine
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic Acid
DON Deoxynivalenol
dsFv Disulfide-Stabilized Fv
ECORC Eastern Cereal and Oilseed Research Center
EDTA Ethylenediaminetetraacetic acid
ELISA Enzyme-Linked Immunosorbent Assay
Fab Fragment, Antigen Binding Region of an Antibody
FDK Fusarium Damaged Kernels
Ff Filamentous Bacteriophage
FHB Fusarium Head Blight
FITC Fluorescein Isothiocyanate
FP Fluorescence Polarization
FPA Fluorescence Polarization Assay
FR Framework Region
Fv Variable Fragment of an Antibody
GC/MS Gas Chromatogram / Mass Spectrometry
HCAb Heavy Chain Antibody
HEPES N-(2-hydroxyethyl)-piperazine-N-2-ethanesulfonic acid
HPI Hours Post Infection
HPLC High Pressure Liquid Chromatography
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HRP Horseradish Peroxidase
IC50 Half Maximal Inhibitory Concentration
IgA Immunoglobulin Alpha (α) Isotype Antibody
IgM Immunoglobulin Mu (μ) Isotype Antibody
IMAC Immobilized Metal Affinity Chromatography
IPTG isopropyl β-D-1-thiogalactopyranoside
JECFA Joint Expert Committee on Food Additives
KD Equilibrium Dissociation Constant
kDa kilodalton
LD50 Median Lethal Dose
LSD Least Significant Difference
mAb Monoclonal Antibody
MCS Multiple Cloning Site
MGYH Minimum Glycerol Medium with Histidine
MMH Minimal Methanol Medium
mRNA Messenger RNA
MW Molecular Weight
NBT Nitro Blue Tetrazolium Chloride
NEO Neosolaniol
NMR Nuclear Magnetic Resonance
NRCC National Research Council of Canada
NSERC National Sciences and Engineering Research Council
OAc Acetyl Ester (-OCH3)
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OD620 Optical Density at 620nm
OIsoval Isovalerate Ester (-OCOCH2CH(CH3)2)
OMAFRA Ontario Ministry of Agriculture, Food and Rural Affairs
OVA Ovalbumin
PAGE Polyacrylamide Gel Electrophoresis
PBL Peripheral Blood Lymphocytes
PBL Peripheral Blood Lymphocytes
PBS Phosphate Buffered Saline
PBST Phosphate Buffered Saline Tween
PCR Polymerase Chain Reaction
PEG Polyethylene Glycol
pIII Minor Coat Protein of Filamentous Phage
PMSF Phenylmethylsulphonyl Fluoride
PMTDI Provisional Maximum Tolerable Daily Intake
PVDF Polyvinylidene Fluoride
pVIII Major Coat Protein of Filamentous Phage
rAb Recombinant Antibody
RNA Ribonucleic Acid
RPL3 Ribosomal Protein L3
SAR Systemic Acquired Resistance
scFv Single Chain Fv
sdAb Single Domain Ab
SDS Sodium Dodecyl Sulfate
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SPR Surface Plasmon Resonance
ssDNA Single-Stranded DNA
StEP Staggered Extension Process
TBST Tween Buffered Saline
TEA Triethylamine
TFAA Trifluoroacetic anhydride
TLC Thin Layer Chromatography
UGT UDP Glycosyltransferase
USD United States Dollars
VH Variable Domain of the Heavy Chain
VHH Variable Region on Single-Domain Heavy Chain Ab
VL Variable Domain of the Light Chain
YPD Yeast Potato Dextrose media
YPDS Yeast Extract Peptone Dextrose Sorbitol Media
ΔAUC Differential Area Under Curve
x
TABLE OF CONTENTS
ABSTRACT ........................................................................................................................ i
PREFACE .......................................................................................................................... ii
ACKNOWLEDGEMENTS ............................................................................................. ii
PICTURE .......................................................................................................................... iii
NOTE ABOUT WHY ...................................................................................................... iv
LIST OF ABBREVIATIONS ...........................................................................................v
TABLE OF CONTENTS ..................................................................................................x
LIST OF TABLES ........................................................................................................ xvii
LIST OF FIGURES ..................................................................................................... xviii
1 INTRODUCTION, OBJECTIVES, AND HYPOTHESIS ......................................1
2 LITERATURE REVIEW 1: FUSARIUM HEAD BLIGHT AND
TRICHOTHECENE MYCOTOXINS: NOVEL TARGETS FOR
RECOMBINANT ANTIBODY TECHNOLOGY ....................................................6
2.1 Introduction ...........................................................................................................6
2.2 Fusarium Head Blight ...........................................................................................8
2.2.1 Disease Cycle ...........................................................................................8
2.2.1.1 Inoculum ...................................................................................10
2.2.1.2 Environment ..............................................................................11
2.2.1.3 Host Crop ..................................................................................11
2.2.2 Pathways of FHB Pathogenesis ..............................................................13
2.2.2.1 Fungal Colonization ..................................................................13
2.2.2.2 Systemic Infection .....................................................................15
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2.2.3 FHB Control Strategies ..........................................................................16
2.2.3.1 Agronomic and Cultural Practices ............................................16
2.2.3.2 Fungicides .................................................................................17
2.2.3.3 Biological Control Agents ........................................................17
2.2.3.4 Crop Breeding ...........................................................................18
2.2.4 Classifications of FHB Resistance .........................................................19
2.3 Trichothecene Mycotoxins ..................................................................................20
2.3.1 Mode of Action ......................................................................................21
2.3.2 Mechanisms of Fungal Protection ..........................................................22
2.3.3 Effects on Plants .....................................................................................23
2.4 DON, 3-AcDON, and 15-AcDON ......................................................................24
2.4.1 Contamination of Food and Feed ...........................................................25
2.4.2 Mechanism of Virulence for FHB ..........................................................26
2.5 Transgenic Approaches to Limit Trichothecene Toxicity ...................................28
2.5.1 Inhibition of Trichothecene Biosynthesis ...............................................29
2.5.2 Alteration of Target Protein ....................................................................30
2.5.3 Metabolic Trichothecene Detoxification ................................................31
2.5.4 Reduction of Intracellular Concentrations ..............................................33
2.5.5 Plantibodies and Mimotopes ..................................................................33
3 LITERATURE REVIEW 2: ANTIBODY ENGINEERING IN
AGRICULTURAL BIOTECHNOLOGY ...............................................................36
3.1 Introduction .........................................................................................................36
3.2 Recombinant Antibody Fragments .....................................................................37
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3.2.1 Fab Fragment ..........................................................................................38
3.2.2 scFv Fragment ........................................................................................39
3.2.3 VHH Fragment ........................................................................................42
3.3 Development and Types of Antibody Libraries ..................................................44
3.3.1 Immunized Libraries...............................................................................44
3.3.2 Naïve Libraries .......................................................................................45
3.3.3 Synthetic / Semi-Synthetic Libraries ......................................................46
3.4 Display Systems ..................................................................................................47
3.4.1 Phage Display .........................................................................................47
3.4.1.1 Filamentous Bacteriophage .......................................................48
3.4.1.2 Phage vs. Phagemid Vectors .....................................................49
3.4.1.3 Panning .....................................................................................52
3.4.2 Bacteria Display .....................................................................................54
3.4.3 Yeast Display ..........................................................................................54
3.4.4 Ribosome Display ..................................................................................55
3.5 Optimization of Recombinant Antibodies ..........................................................57
3.5.1 Optimized Panning Procedures ..............................................................57
3.5.2 In vitro Affinity Maturation ....................................................................58
3.5.2.1 Random Mutagenesis ................................................................59
3.5.2.1.1 Error-Prone PCR ...................................................59
3.5.2.1.2 Bacterial Mutator Strains .......................................59
3.5.2.2 Focused Mutagenesis ................................................................60
3.5.2.2.1 Site-Directed Mutagenesis .....................................60
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3.5.2.2.2 Mutational Hot Spots.............................................61
3.5.2.2.3 Parsimonious Mutagenesis ....................................62
3.5.3 Optimized Panning Procedures ..............................................................63
3.5.3.1 Chain Shuffling .........................................................................63
3.5.3.2 DNA Shuffling by Random Fragmentation and
Reassembly .......................................................................................64
3.5.3.3 Staggered Extension Process (StEP) .........................................65
3.6 Expression and Purification ................................................................................65
3.6.1 Prokaryotic Systems ...............................................................................66
3.6.1.1 E. coli Cytoplasmic Expression ................................................66
3.6.1.2 E. coli Periplasmic Expression .................................................67
3.6.2 Eukaryotic Systems ................................................................................68
3.6.2.1 Yeast Expression .......................................................................68
3.6.2.2 Insect Cell Expression ...............................................................71
3.6.2.3 In planta Expression .................................................................73
3.7 Application to Immunoassays and Agricultural Biotechnology .........................77
3.7.1 Immunoassays ........................................................................................77
3.7.2 Research Applications to Agricultural Biotechnology ...........................80
4 RESEARCH CHAPTER 1: CLONING, EXPRESSION, AND
CHARACTERIZATION OF A SINGLE DOMAIN ANTIBODY FRAGMENT
WITH AFFNIITY FOR 15-ACETYL-DEOXYNIVALENOL..............................82
4.1 Abstract ...............................................................................................................82
4.2 Introduction .........................................................................................................83
4.3 Materials and Methods ........................................................................................86
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4.3.1 Reagents and Solutions ..........................................................................86
4.3.2 Immunogen and Llama Immunization....................................................87
4.3.3 Fractionation of Polyclonal Serum .........................................................88
4.3.4 Confirmation of Titer .............................................................................89
4.3.5 Library Construction ...............................................................................90
4.3.6 VHH Library Panning .............................................................................92
4.3.7 Phage ELISA ..........................................................................................93
4.3.8 Colony PCR and Sequencing .................................................................94
4.3.9 Monomer Expression and Purification ...................................................94
4.3.10 Pentamerization of VHH Monomer .......................................................95
4.3.11 Pentamer Expression and Purification ..................................................96
4.3.12 Determination of Binding Affinity ........................................................96
4.3.12.1 Fluorescence Polarization Assay..............................................96
4.3.12.2 Surface Plasmon Resonance ....................................................98
4.3.12.3 Competitive Inhibition ELISA .................................................99
4.4 Results ...............................................................................................................100
4.4.1 Polyclonal Response ..............................................................................100
4.4.1.1 Direct ELISA ..........................................................................100
4.4.1.2 Fluorescence Polarization .......................................................101
4.4.2 HCAb and cIgG Response .....................................................................102
4.4.2.1 Protein A and G Chromatography...........................................102
4.4.2.2 ELISA on Polyclonal Serum Fractions ...................................102
4.4.3 Library Construction ..............................................................................103
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4.4.4 Panning and Phage ELISA ....................................................................103
4.4.5 VHH Sequence Results ..........................................................................104
4.4.6 Expression of Soluble VHH Protein ......................................................105
4.4.7 VHH Affinity Measurements .................................................................105
4.4.7.1 Fluorescence Polarization .......................................................105
4.4.7.2 Surface Plasmon Resonance ...................................................106
4.4.7.3 CI ELISA ................................................................................110
4.5 Discussion .........................................................................................................110
4.6 Conclusions .......................................................................................................118
4.7 Acknowledgements ...........................................................................................119
5 RESEARCH CHAPTER 2: INTRACELLULAR EXPRESSION OF A
SINGLE-DOMAIN ANTIBODY REDUCES CYTOTOXICITY OF 15-
ACETYL-DEOXYNIVALENOL IN YEAST. ......................................................120
5.1 Abstract .............................................................................................................120
5.2 Introduction .......................................................................................................121
5.3 Experimental Procedures ..................................................................................124
5.3.1 VHH Genes ............................................................................................124
5.3.2 Transformation ......................................................................................125
5.3.3 Induction of VHH Expression ................................................................126
5.3.4 Preparation of Soluble VHH Extracts and Western Blot Analysis ........126
5.3.5 Cytotoxicity Assays ...............................................................................127
5.3.6 Data Analysis .........................................................................................128
5.3.7 Quantitative Western Blot Analysis ......................................................129
5.3.8 VHH Immunolocalization ......................................................................131
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5.3.9 Mycotoxin Biotransformation Assays ...................................................132
5.4 Results ...............................................................................................................134
5.4.1 Sensitivity to Ribotoxin Treatments ......................................................134
5.4.2 Transformation and VHH Intrabody Expression ....................................135
5.4.3 Quantitative Western Blot Assay ..........................................................135
5.4.4 Cytotoxicity Assays ...............................................................................137
5.4.4.1 15-AcDON ..............................................................................137
5.4.4.2 DON, 3-AcDON, Cycloheximide ...........................................139
5.4.4.3 B-24 VHH Control ...................................................................141
5.4.5 VHH Immunolocalization ......................................................................141
5.4.6 Mycotoxin Biotransformation Assays ...................................................141
5.5 Discussion .........................................................................................................145
5.6 Acknowledgements ...........................................................................................153
5.7 Supplemental.....................................................................................................154
6 CONCLUSIONS ......................................................................................................159
7 REFERENCES .........................................................................................................162
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LIST OF TABLES
Table 4.1 Nucleotide sequences of primers for PCR amplification of HCAb VHH
genes……………………………………………………………………...91
Table 4.2 HCAb and cIgG fractions of llama serum. Volumes of pre-immune, 42-
day and 70-day samples = 12.1, 12.5 and 15.5 mL, respectively............. 102
Table 5.1 Sensitivity of P. pastoris (KM71H) pPICZB transformants to various
ribotoxin treatments tested. Standard deviations (σ) for each mean value
shown in brackets..................................................................................... 134
Table 5.2 Mean VHH intrabody concentration (t = 0 h) within P. pastoris
transformants used in cytotoxicity assays.................................................136
Table 5.S1 Raw data and calculations of VHH intrabody expression in Pichia pastoris
transformants derived from quantitative Western blot analysis........155/156
Table 5.S2 HPLC retention times of trichothecene metabolites evaluated after 24 h of
growth of P. pastoris transformants cultured in YPD media supplemented
with 100 µg·mL-1
of highly-purified DON...............................................158
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LIST OF FIGURES
Figure 1.1 Structure, composition, and molecular weight of trichothecene mycotoxins
used within this study................................................................................... 2
Figure 2.1 Photos of wheat showing progressive signs of FDK (Fusarium damaged
kernels) in wheat.......................................................................................... 6
Figure 2.2 Disease Cycle of FHB infection of small grain cereals................................9
Figure 2.3 The grass floret. A: Partly dissected at anthesis. The ovary and two
stigmas comprise the pistil. B: Transverse diagram showing overlap of
lemma and palea.........................................................................................12
Figure 2.4 Scanning electron micrographs of F. culmorum colonization of wheat (cv.
Agent).........................................................................................................13
Figure 2.5 Penetration of lemma of wheat (cv. Agent) by F. culmorum.....................14
Figure 2.6 Schematic diagram of the first two florets of a wheat spikelet, showing the
relationship among the ovary (O), lemma (L), glume (G), palea (P),
rachilla (RL), Rachis Node (RN), and Rachis (R) in terms of initial
Fusarium infection sites (red arrowheads) and systemic infection pathways
(blue arrows) of F. culmorum.................................................................... 15
Figure 2.7 Diagram of the general structure, numbering system of the tetracyclic
trichothecene nucleus, and specific side-chain groups of selected naturally-
occurring trichothecnes of the genus Fusarium..........................................20
Figure 2.8 Structure of DON, 15-AcDON, and 3-AcDON......................................... 25
Figure 2.9 Immunogold localization of Fusarium toxins in infected host tissue (cv.
Agent) by F. culmorum.............................................................................. 27
Figure 3.1 Schematic representation (left) and ribbon diagram (right) of recombinant
antibody fragment (Fab)............................................................................. 39
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Figure 3.2 Schematic representation (left) and ribbon diagram (right) of recombinant
single chain variable fragment (scFv) antibody.........................................40
Figure 3.3 Schematic representation (left) and ribbon diagram (right) of recombinant
HCAb variable domain (VHH) of antibody “AMD9”................................ 42
Figure 3.4 Schematic representation of a filamentous bacteriophage and its protein
coats...........................................................................................................50
Figure 3.5 Phage (left) and phagemid (right) vectors to construct rAb phage display
libraries through the minor coat protein pIII..............................................51
Figure 3.6 Phage display panning cycle.......................................................................53
Figure 3.7 Ribosome display panning cycle................................................................ 56
Figure 4.1 ELISA of polyclonal llama serum response to 250 μg 15-DON-BSA
conjugate...................................................................................................101
Figure 4.2 Amino acid sequence of NAT-267 VHH from fourth round of panning..104
Figure 4.3 FP inhibition of mNAT-267 and pNAT-267 VHH binding and 56-day
polyclonal llama serum binding to DON-fluorescein tracer with DON
toxin (A); 3-AcDON (B); and 15-AcDON (C) from 5 μg∙mL-1
to 9.8
ng∙mL-1
..................................................................................................... 107
Figure 4.4 Overlay sensorgrams of mNAT-267 VHH steady state (Req) binding to 15-
DON-HRP conjugate................................................................................108
Figure 4.5 (A) Steady state (Req) affinity binding (RU) mNAT-267 VHH to 15-DON-
HRP conjugate for ten monomer concentrations, as shown in Figure 4.4.
(B) Scatchard plot of steady state „Req‟ binding (RU) relative to Req / C
(mNAT-267 VHH concentration) (RU/μM)............................................. 109
Figure 4.6 Structure, composition, and molecular weight of trichothecene mycotoxins
used in this study...................................................................................... 114
xx
Figure 5.1 Structure, composition, and molecular weight of trichothecene mycotoxins
used in this study...................................................................................... 122
Figure 5.2 Diagram of pPICZB expression vector (3745 bp) with VHH gene (NAT-
267 = 387 bp, B-24 = 390 bp)................................................................. 125
Figure 5.3 Quantitative Western blot analysis of VHH intrabody expression within P.
pastoris cytotoxicity assays...................................................................... 135
Figure 5.4 Cytotoxicity assay of P. pastoris pPICZB and NAT-267 VHH transformant
growth measured by optical density at 620 nm (OD620) to 15-AcDON at
doses of 20 to 200 µg·mL-1
.......................................................................138
Figure 5.5 Cytotoxicity assay of P. pastoris pPICZB and NAT-267 VHH transformant
growth measured by optical density at 620 nm (OD620) to DON and
cycloheximide at doses of 50 to 200 µg·mL-1
.......................................... 140
Figure 5.6 Cytotoxicity assays showing the effect of various concentrations (50 to
200 µg·mL-1
) of 15-AcDON (A) and cycloheximide (B) on the growth of
P. pastoris pPICZB and non-specific B-24 VHH transformants as
measured by optical density at 620 nm (OD620)....................................... 142
Figure 5.7 Representative confocal microscopy photographs of P. pastoris (KM71H)
cells immuno-probed with anti-HA epitope mAb-FITC conjugate and
DAPI stain. Transformants expressing pPICZB empty vector (control) (A)
and NAT-267 VHH intrabody (B).............................................................143
Figure 5.8 Representative 500 mHz 1H NMR spectrum of A = 15-AcDON Standard.
B = HPLC fraction of P. pastoris cell lysate sample (24 hrs).................. 144
Figure 5.S1 Homology chart of DNA and amino acid sequence of codon-optimized
NAT-267 and control B-24 VHH fragments.............................................154
1
1 INTRODUCTION, OBJECTIVES, AND HYPOTHESIS
Fusarium head blight (FHB) of cereals is caused by morphologically similar species
(F. graminearum, F. culmorum, etc.) common throughout global agricultural regions.
With few exceptions, Fusarium epidemics are characterized by cyclical and highly
aggressive infection of cereal crops with economic impacts on food and feed industries
that are immediate and far reaching (Demcey Johnson et al., 2003; McMullen, 2003;
Miller, 2008).
Trichothecene mycotoxins are a toxin class commonly found within agricultural
commodities infected by Fusarium. Trichothecene toxins represent a large group of >180
sesquiterpenoids that have molecular weights typically ranging from 200-500 Da and are
characterized by a tricyclic ring structure containing a double bond at C-9,10 and an
epoxide group at C-13 (reviewed in Desjardins, 2006; Foroud and Eudes, 2009; Mirocha
et al., 2003). Regardless of size and structural composition, trichothecenes are potent
inhibitors of eukaryotic protein synthesis with specific activity on ribosomal protein L3
(RPL3) within the 60S subunit resulting in inhibition of peptidyl transferase activity
(Carter and Cannon, 1977; Rocha et al., 2005). Although the capacity to inhibit protein
synthesis is regarded as central to trichothecene cytotoxicity (Ueno, 1984; Rotter et al.,
1996), adverse effects on eukaryotic cells may actually be attributed to dysregulation of
cellular signaling and alterations in downstream gene expression (Pestka, 2008).
Deoxynivalenol (3,7,15-trihydroxy, 12,13-epoxy-trichothec-9-en-8-one), or DON, is
one of many members of the trichothecene group of mycotoxins produced by Fusarium
species. DON is a low molecular weight hapten (MW = 296 Da) containing one primary
and two secondary hydroxyl groups conferring limited solubility in water and high
2
solubility in various polar solvents such as methanol and acetonitrile (reviewed in
Desjardins, 2006; Mirocha et al., 2003). Of the trichothecene mycotoxins that
accumulate within cereal grain, DON and its closely related-acetylated analogues (i.e.,
15-Acetyl-Deoxynivalenol (15-AcDON) and 3-Acetyl-Deoxynivalenol (3-AcDON))
(Figure 1.1) are considered among the most common and widely distributed food and
feed contaminants (Abramson et al., 2001; D‟Mello et al., 1999; Greenhalgh et al.,
1986; Snijders, 1990a).
Trichothecene MW
a
(Da)
Oxygenation and Esterification at Position
C-3 C-15
3-Acetyl-DON 338 O-CO-CH3 OH
15-Acetyl-DON 338 OH O-CO-CH3
Deoxynivalenol 296 OH OH a MW = Molecular Weight
Figure 1.1 Structure, composition, and molecular weight of DON, 15-AcDON, and 3-
AcDON (Desjardins, 2006).
There is a demonstrated correlation between in planta DON accumulation and
Fusarium virulence in susceptible cultivars of wheat (Mesterházy, 2002) and maize
(Harris et al., 1999a). Based on these findings, mechanisms which convey innate and
3
acquired host plant resistance to DON and other trichothecene toxins have received
considerable attention. To date, in planta trichothecene resistance has been achieved
through mechanisms that alter targeted proteins within host cell ribosomes (e.g., Harris
and Gleddie, 2001; Mitterbauer et al., 2004), promote metabolic transformation to less
toxic forms: e.g., DON-glucosyl conjugate (Poppenberger et al., 2003) or to 3-AcDON
(Alexander, 2008), and/or reduce intracellular concentrations to effectively limit
mycotoxin exposure to sensitive cellular targets. Collectively, such research can be
applied to assess and impart novel mechanisms of trichothecene resistance within
eukaryotic test systems such as yeast or Arabidopsis (Mitterbauer and Adam, 2002) for
eventual application in higher order plants such as cereals and maize.
Current applications of antibody (Ab)-based technology involving FHB and
trichothecene toxins such as DON, 3-AcDON, and 15-AcDON are generally limited to
immunoassays based on polyclonal and monoclonal antibodies (mAb) to detect
mycotoxins within grain, food, feed, and animal serum (e.g., Maragos and McCormick,
2000; Nicol et al., 1993; Sinha et al., 1995). Although these tests provide robust and
precise results, mAb development requires highly specialized equipment and labor-
intensive procedures to select, culture, and maintain optimal hybridoma cell lines.
Because of these limitations, recombinant Ab and phage display technologies are often
used to generate target-specific recombinant antibody (rAb) fragments from Ab cell lines.
In this thesis, these two technologies were used to develop and quantify the efficacy of a
single-domain heavy chain antibody fragment (i.e., VHH) with affinity for 15-AcDON.
4
VHH fragments are derived from the camelidae heavy chain IgG subfamily and are
among the smallest functional rAb fragments known. VHH fragments exhibit the same
exquisite specificity as larger immunoglobulins, and confer added biochemical
advantages of high solubility, stability, and robust expression in various recombinant
systems (Holliger and Hudson, 2005; Muyldermans, 2001). VHH fragments have been
generated against a wide variety of epitopes and antigen (Ag) types including low
molecular weight ligands (haptens) (Nguyen et al., 2000; Sheedy et al., 2006; van der
Linden et al., 2000) and toxins (e.g., Doyle et al., 2008).
The advantages associated with ease of expression, stability, and solubility of VHH
fragments can be attributed to key differences when compared to standard rAb fragments
(e.g., scFv, Fv, Fab, etc.). VHH fragments are small (~ 14 – 15 kDa), typically have
hydrophilic amino acid substitutions within the second framework region (FR-2), and by
their single-domain nature, do not require the linker associated with the standard scFv
format which can lead to problems associated with aggregation, proteolytic degradation,
and variable expression within recombinant hosts (Arbabi-Ghahroudi et al., 2005).
Improved functionality over various temperature and solvent regimes (Goldman et al.,
2006; van der Linden et al., 1999) coupled with high levels of recombinant expression
combine to make VHH fragments useful reagents within next-generation immunoassays
(Goldman et al., 2006, Ladenson et al., 2006). When expressed as intrabody fragments
within recombinant hosts, VHH fragments can be used as Ag-specific reagents to
elucidate potential in vivo mechanisms of immunomodulation with applications ranging
5
from pharmacology (Holliger and Hudson, 2005; Saerens et al., 2008) to plant science
(Jobling et al., 2003).
The research summarized within this Ph.D. project was based on a two related
objectives. Initial experiments were established to identify, characterize, and assess
binding affinity of a trichothecene-specific recombinant VHH fragment that was isolated
from a hyper-immunized phage-display library. The second group of experiments were
focused on an in vivo assessment of a VHH intrabody immunomodulation of the cytotoxic
effects of the target mycotoxin within a model eukaryotic system based on Pichia
pastoris. Collectively, this research was established as a „proof of concept‟ model to
demonstrate that intrabody expression, i.e. expression of recombinant VHH fragments,
could impart a novel means of mycotoxin-specific resistance in susceptible eukaryotic
organisms.
Hypothesis
Expression of a mycotoxin-specific VHH antibody fragment within a model
eukaryotic test system will limit mycotoxin-specific cytotoxicity and inherently
reduce disease symptoms associated with FHB pathogenesis.
6
2 LITERATURE REVIEW 1: FUSARIUM HEAD BLIGHT AND
TRICHOTHECENE MYCOTOXINS: NOVEL TARGETS FOR
RECOMBINANT ANTIBODY TECHNOLOGY
2.1 Introduction
Fusarium head blight (FHB) of small grain cereals is a fungal disease caused by a
range of Fusarium species common throughout the world‟s agricultural regions.
Epidemics are characterized by highly cyclical and aggressive plant pathogenesis with
few known mechanisms of control. Despite causing significant yield loss, the principal
economic impact of a FHB epidemic usually results in harvested grain with severely
reduced quality since Fusarium-damaged kernels (FDK) (Figure 2.1) are often shrunken
and shriveled with little economic value. Reduced seed quality and value is exacerbated
by the fact that infected grain is often contaminated with toxic fungal metabolites, or
mycotoxins, which affect food and feed quality (Bai and Shaner, 1994; Dickson, 1942;
Foroud and Eudes, 2009; McMullen, 2003; Miller, 2008; Parry et al., 1995; etc.).
Figure 2.1 Photos of wheat showing progressive signs of FDK (Fusarium damaged
kernels) in wheat (Harvest samples from Syngenta Field Trials, 2004).
The two major classes of mycotoxins common to Fusarium infected small grain
cereals are trichothecenes and zearalenone. Trichothecenes are tricyclic sesquiterpenes
that contain a 12,13-epoxide ring and a double bond between carbons 9 and 10.
7
Zearalenones are an unrelated group of Fusarium mycotoxins derived by different
cyclizations and modifications of nonaketide precursors. Like trichothecenes,
zearalenones are low molecular weight, heat-stable mycotoxins commonly found within
Fusarium-infected cereals and maize (Desjardins, 2006).
Unlike zearalenones, which are not generally considered to be phytotoxic
compounds, trichothecenes can produce a wide array of phytotoxic effects within plant
tissue. Some of the first evidence to link trichothecenes as a mechanism of virulence for
Fusarium infection of plant tissue was provided by gene disruption experiments. Various
researchers showed that loss-of-function mutations in the Tri5 gene (key enzyme within
the trichothecene biosynthesis pathway) within virulent Fusarium spp. resulted in reduced
fungal pathogenesis and colonization of plant tissue (Desjardins et al., 1996; Eudes et al.,
2001; Proctor et al., 1995). Subsequent research, as outlined within this chapter,
demonstrated that various other techniques to limit trichothecene (particularly
deoxynivalenol) phytotoxicity could limit Fusarium infection of small grain cereals and
maize. In addition, research has shown that the production of DON within plant tissue is
commonly associated with the necrotrophic stage of Fusarium pathogenesis during which
host-plant tissue dies as the fungus advances and proliferates within infected cereal tissue
(Mesterházy, 2002; Muehlbauer and Bushnell, 2003; Proctor et al., 2002).
The following chapter provides an overview of the general aspects related to the
occurrence of, and control strategies associated with, FHB infection and trichothecene
contamination of agricultural crops. Given the vast array and enormous scope of
publications covering this subject matter, this introductory chapter is intended to be a
8
concise and general overview to support, and align with subsequent research chapters
within this thesis.
2.2 Fusarium Head Blight
FHB has the most dramatic and widespread effect on cereal production during
major epidemics; however once established within an area, even at baseline infestation
levels, FHB can be a major limitation to cereal production (Diaz de Ackermann and
Kohli, 1997a; Mesterházy, 1997; Sutton, 1982; Wang, 1997). Arguably, the best
documented, and perhaps the most costly FHB epidemic in North America occurred in
the upper Great Plains of the USA and within the provinces of Manitoba, Ontario, and
Quebec, Canada between 1993 and 1998 (Gilbert et al., 1994; McMullen, 2003;
McMullen et al., 1997; Windels, 2000; etc.). Estimated economic losses associated with
lost crop yield and reduced harvest quality over the course of the epidemic range from
$1.3 to nearly $3.0 billion USD (Demcey Johnson et al., 2003; Windels, 2000).
2.2.1 Disease Cycle
The critical factors or conditions which must exist for FHB to occur are described
by the classic plant pathologist „Disease Triangle‟ denoted by: 1.) inoculum, 2.)
susceptible host, and 3.) the right mix of environmental conditions (Shaner, 2003). More
specifically, the establishment of FHB can be attributed to the release of inoculum from
partially-degraded crop residues throughout periods of warm, windy, humid weather
during anthesis (i.e. flowering) of susceptible host crops (Figure 2.2).
FHB is a monocyclic disease that is initiated by inoculum carried within air currents
or splashing water (Sutton, 1982). The quantity of primary inoculum therefore directly
9
influences the intensity and extent of disease development during infection (Bai and
Shaner, 1994; Khonga and Sutton, 1986, 1988). Although secondary infections are
theoretically possible, the monocyclic nature of FHB can be attributed to the relatively
short period of crop susceptibility and limited window of favorable environmental
conditions which favor FHB establishment (Fernando et al., 1997; Shaner, 2003).
Figure 2.2 Disease Cycle of FHB infection of small grain cereals (from Bergstrom,
1993).
Fusarium seedling blight, or Fusarium crown and foot rot, is a related seed-borne
disease of cereals (Figure 2.2). Affected seedlings emerge stunted and yellow with the
crown, roots and/or lower stem having a brown to red-brown appearance (Sutton, 1992).
Although FHB is not regarded as a systemic disease based on an infection that develops
from Fusarium infected seed, seedling blight may serve as a source of secondary
inoculum within the canopy of the growing crop (Parry et al., 1995).
Conidia
(asexual)
Ascospores
(sexual)
Moist, Warm Conditions at
Anthesis
Infected Seed
Fusarium
Seedling Blight
Reduced
tillage
Crop debris (wheat, maize, barley)
bearing perithecia and conidia
Fusarium
Head
Blight
10
2.2.1.1 Inoculum
While several species of Fusarium cause FHB around the world, including F.
graminearum Schw. (Teleomorph – Gibberella zeae Schw. Petch), F. culmorum (Wm. G.
Smith) Sacc., and F. avenaceum (Corda ex Fr.) Sacc. (Teleomorph - G. avenacea
(Cook)), etc., F. graminearum is the most common within North America, Europe and
Asia (McMullen et al., 1997; Parry et al., 1995; Sutton, 1982; Tekauz et al., 2000;
Wilcoxson et al., 1988).
Infected crop residue (i.e., debris of previously-harvested wheat, barley, maize) is
the principle source of primary F. graminearum inoculum for FHB infestations (Figure
2.2). Therefore, inoculum levels are related to the amount of crop residue and the degree
to which crop debris is infected on the surface of soil (Khonga and Sutton, 1988: Sutton,
1982). Challenges associated with inoculum build-up are exacerbated by reduced tillage
practices, which result in larger amounts of, and longer degradation profiles for, crop
residues within the top layer of soil (Shaner, 2003). Primary types of inoculum of
virulent Fusarium species include ascospores (F. graminearum), as well as conidia,
hyphal fragments, and chlamydospores (F. culmorum, F. avenaceum, etc.). Environment
has a direct impact on inoculum production and release since warm, moist, and windy
weather disseminate Fusarium inocula by rain-splash and air currents to enable direct
contact of sexual and asexual innoculum with susceptible tissue of host plants (Menzies
and Gilbert, 2003) (Figure 2.2).
11
2.2.1.2 Environment
The role of environment in FHB epidemiology is a complex subject that has been
extensively reviewed (Andersen, 1948; Sutton, 1982; Parry et al., 1995; Shaner, 2003).
The inherent complexity of the disease is associated primarily with temperature and
duration of continuous moisture, which are both tied to rainfall and prevailing humidity
levels and are inherently variable factors that affect both inoculum production and
dispersal as well as the duration of host crop susceptibility to Fusarium infection.
Therefore, the establishment of the FHB disease cycle (Figure 2.2) is dependent upon
prolonged precipitation and/or periods of high humidity (e.g., 92 - 94% R.H.) in
conjunction with warm temperatures during anthesis of host crops (Sutton, 1982).
Prevailing moisture has a greater role than temperature in terms of inoculum production,
distribution, and colonization and pathogen development within infected heads because
favorable temperatures for FHB establishment are generally common when cereal crops
are flowering (Shaner, 2003; Sutton, 1982). However, it is also well established that
longer periods of prevailing moisture are required for FHB infection when temperatures
are lower or higher than 25°C (Andersen, 1948).
2.2.1.3 Host Crop
The initial point of Fusarium infection associated with FHB of cereals is fungal
colonization of floret and caryopsis tissue. Cereal florets are composed of an inner palea
(Figure 2.3A) and an outer lemma (Figure 2.3B), which overlap and partially enclose the
edge of the palea. Together, the lemma and palea enclose the pistil, stamens, lodicules,
and an ovary to form the caryopsis (Figure 2.3) (Bushnell et al., 2003).
12
Floret and caryopsis host tissues are most susceptible to Fusarium infection within a
3 - 5 day period corresponding to flowering or anthesis (i.e., BBCH = 61 - 69). However,
depending on prevailing environmental conditions and innoculum levels, colonization
can occur from mid inflorescence (i.e., BBCH = 55) to the soft dough growth stage (i.e.,
BBCH = 85) (Paulitz et al., 1997). After fungal colonization, disease severity progresses
rapidly to a necrotrophic systemic infection which results in widespread Fusarium
infection throughout the cereal spike (Shaner, 2003).
Figure 2.3 The grass floret/caryopsis. A: Partly dissected at anthesis. The ovary and
two stigmas comprise the pistil. B: Transverse diagram showing overlap
of lemma and palea (adapted from Bushnell et al., 2003).
lemma
lodicule
palea
anther
ovary
lodicule
Figure 2.3B
Figure 2.3A
ovary rachilla
stigma
filament
anther
ventral
crease
13
2.2.2 Pathways of FHB Pathogenesis
2.2.2.1 Fungal Colonization
Upon initial germination of ascospores (i.e., first ~ six hours), Fusarium hyphae
rapidly elongate over the abaxial (exterior) surfaces of glumes and florets to form a
complex mycelial network across the surface of colonized host tissue (Figure 2.4A).
Figure 2.4 Scanning electron micrographs of F. culmorum colonization of wheat (cv.
Agent). A, Hyphal network between pollen grains (PG) on brush hairs at
the top of the ovary (48 hours post infection (HPI)). B, Hyphae (H) within
digested host cuticular layer on the inner surface of palea next to plant hair
structure (48 HPI). C, Hyphae (H) growing upward over the edge of the
glume from germinating macroconidia (M). (Kang and Buchenauer, 2000).
From initial stages of infection, hyphae initiate penetration of host tissue through
enzymatic degradation of abaxial surfaces of the caryopsis (Baláza and Bagi, 1997;
Urbanek, 1989; etc.) (Figure 2.4B). Seventy-six HPI, hyphae can progress across the
outer surfaces of the lemma and glume to reach the developing caryopsis (Figure 2.4C)
A
5 µm
H H
M
C
5 µm
B
20 µm
H
14
(Kang and Buchenauer, 2000). Throughout the later stages of this biotrophic infection
process, mycelial growth progresses to the point where fungal reproductive structures
(conidiophores) are established on the surface of infected tissue (Pritsch et al., 2000).
Fusarium hyphae follow various routes to reach interior surfaces of infected plant
tissue (Bushnell et al., 2003). These routes of entry include direct wall penetration of the
epidermal cell wall by a hyphal peg (Figure 2.5A), entry through stomates (Figure 2.5B),
or colonization of openings between lemma and palea during the brief period of
dehiscence when cereal florets are open (Lewandowski and Bushnell, 2001). Following
primary penetration, Fusarium hyphae exhibit a subcuticular growth pattern with flat,
branched hyphae growing between the cuticle and epidermal cell walls of host tissue
(Pritsch et al., 2000).
Figure 2.5 Penetration of lemma of wheat (variety = Agent) by F. culmorum. A,
Transmission electron micrograph (TEM) showing penetration peg
(arrowhead) from an infection hyphae (IH) invading epidermal cell wall
(EW) at 36 HPI. B, Scanning electron micrograph (SEM) showing growth
of hyphae (H) through a stomatal (S) opening on the inner surface of the
lemma 48 HPI (Kang and Buchenauer, 2000).
A B
S
H
1 µm 5 µm
15
2.2.2.2 Systemic Infection
Once fungal colonization is established, Fusarium hyphae infect adjacent florets on
the same spikelet through a systemic and necrotic infection of the rachis and rachis node
(Figure 2.6). Systemic infection is marked by increased fungal colonization and a rapid
necrotic deterioration of host-cell tissue because invasive hyphae progress from floret to
floret (Ribichich et al., 2000; Schroeder and Christensen, 1963). Thereafter, infected
florets cease to develop as host cell tissue and organelles become completely fragmented
as hyphae spread vertically and horizontally within the rachis and rachis node (Figure
2.6).
Figure 2.6 Schematic diagram of the first two florets of a wheat spikelet, showing the
relationship among the ovary (O), lemma (L), glume (G), palea (P),
rachilla (RL), rachis node (RN), and rachis (R) in terms of initial
Fusarium infection sites (red arrowheads) and systemic infection
pathways (blue arrows) of F. culmorum. Dotted lines and dotted areas
indicate vascular tissues within the RN (Kang and Buchenauer, 2000).
O
RL
O P
G L
RN
R
16
Continued necrotic infection leads to a degeneration of plant cytoplasts, cell walls,
chloroplasts and other plastids, which limit the ability of the phloem and xylem to deliver
nutrients to developing tissue. FHB infection is characterized by premature death of
infected spikelets, resulting in bleached and shriveled kernels often referred to as
tombstones or Fusarium damaged kernels (Figure 2.1).
2.2.3 FHB Control Strategies
2.2.3.1 Agronomic and Cultural Practices
FHB epidemics are initiated by inoculum (mostly ascospores for F. graminearum)
produced within crop residues on the soil surface. Any measure to reduce crop residue
accumulation and longevity on the soil surface such as plowing or burning excess residue
has the potential to reduce primary inoculum levels (Dill-Macky and Jones, 2000;
Fernandez et al., 1993). Furthermore, agronomic techniques to stagger seeding dates and
maintain long rotation periods between susceptible crops (e.g. cereals or maize) within a
given area are also recommended practices to reduce inoculum levels and limit FHB
development (Dill-Macky and Jones, 2000; McMullen et al., 1997).
Unfortunately, it is difficult to predict the effectiveness of such practices to limit
FHB (Meier et al., 2000) as disease establishment within a susceptible crop is highly
dependent on climate and irregular in terms of disease incidence and severity. In many
cases, environmental conditions associated with the year of production tend to be the
single largest factor associated with variation in disease impact (Schaafsma et al., 2001).
17
2.2.3.2 Fungicides
Fungicide applications are commonly used to limit yield losses associated with FHB
infection and mycotoxin contamination of small grain cereals (reviewed by Mesterházy,
2003b). Among the currently-available fungicides registered for FHB control and
mycotoxin suppression, products based on triazole chemistry (e.g., tebuconazole,
metconazole, or prothioconazole) are considered to be the most efficacious (Jennings,
2002; Mesterházy, 2003b; etc.). However, commercial experience and extensive trial
data from various sources have shown that fungicide efficacy is variable at best (e.g.,
D‟Mello et al., 1998; Homdork et al., 2000; Jennings et al., 2000; Simpson et al., 2001).
Chemical disease control is dependent on timing and method of application, host crop
FHB susceptibility, as well as prevailing environmental conditions, and Fusarium
inoculum levels (Magan et al., 2002; Mesterházy, 2003b). Given the complexity
associated with all of the variables that affect FHB pathogenesis, fungicide treatments are,
therefore, recommended and considered most effective when used as part of an integrated
crop management strategy (Milus and Parsons, 1994; Teich, 1989) that is based on an
accurate disease forecasting system and sound agronomy (McMullen et al., 1997;
Schaafsma et al., 2001).
2.2.3.3 Biological Control Agents
A common approach to biological control strategies is to apply non-pathogenic
organisms in advance of Fusarium infection of cereals as part of a competitive exclusion
strategy to disrupt the FHB disease cycle upon spikelet colonization and infection
(reviewed by da Luz et al., 2003). Numerous research groups have tested a wide range of
18
biological control agents against diseases attributed to Fusarium spp. (e.g., Huang et al.,
1993; Kempf and Wolf, 1989; Shi and Wang, 1991; Stockwell et al., 2000; etc.);
however, very few researchers have reported successful large-scale applications of this
strategy. Additional work and screening is required to identify biological control strains
and application technologies to limit FHB colonization and mycotoxin accumulation
under various field conditions (Chen et al., 2000; da Luz et al., 2003).
2.2.3.4 Crop Breeding
The standard approach to develop cultivars with enhanced FHB resistance has been
to cross cultivars with resistance or enhanced tolerance to Fusarium infection with a
susceptible, but agronomically superior and locally adapted, cultivars or crop varieties to
generate progeny with a favorable phenotype. Unfortunately, progress to date in
developing cereal cultivars with enhanced resistance to FHB-related yield loss and
mycotoxin contamination has been limited (reviewed in: Bai et al., 2003; Miedaner,
2002; Mesterházy, 2003a; Steffenson, 2003). Lack of progress using classical plant
breeding efforts has been attributed to: i) limitations associated with practical techniques
to evaluate FHB resistance within large breeding programs, ii) difficulties in establishing
consistent levels of Fusarium inoculum within FHB nurseries on a cost-effective basis,
iii) limited availability of cereal germplasm with high levels of innate resistance, and iv)
inconsistent financial support for public research (Dill-Macky, 2003; Sutton, 1982).
Sources of resistance to FHB in wheat are based on spring wheat genotypes with
innate resistance. Varieties such as: „Sumai 3‟, „Nobeoka Bozu‟, „Shanghai 7-31B‟,
„Nyubai‟, „Ning 7840‟, and „Frontana‟ are commonly use in breeding programs around
19
the world to develop regionally-adapted cultivars with enhanced FHB and mycotoxin
resistance (Buerstmayr et al., 1996; Diaz de Ackermann and Kohli, 1997b; Gilchrist et
al., 1997; Snijders, 1990b; etc.). Relatively few barley cultivars have been identified with
a high level of innate resistance to FHB and mycotoxin accumulation (Steffenson, 2003).
Unfortunately, most accessions from Fusarium-resistant varieties exhibit unfavorable
agronomic traits which render them largely unsuitable for widespread adoption within
FHB affected regions (Chen et al., 2000). Furthermore, most new accessions produced by
backcrossing resistant varieties with regionally-adapted germplasm tend to have limited
and often unstable FHB resistance (Mesterházy, 2003a).
2.2.4 Classifications of FHB Resistance
Classic descriptions of host-plant resistance to initial infection and to spread of
Fusarium within the spike were initially categorized as „Type 1‟ and „Type 2‟, resistance,
respectively (Schroeder and Christensen, 1963). Type 1 resistance is regarded as an
important component of head blight resistance of barley, since disease resistance given by
percentage of florets infected is inherently more important than spread from floret to
floret. Type 2 resistance is believed to be more important for wheat as FHB tends to
spread more readily from spikelet to spikelet (Bushnell et al., 2003).
Since the development of this classification system, three other types of resistance
have been postulated; „Type 3‟ resistance, which is based on an ability to limit
accumulation of trichothecene toxins in kernels (Miller et al., 1985), „Type 4‟ resistance
based on kernel infection (Mesterházy, 1995), and „Type 5‟ resistance based on increased
tolerance to yield loss in the presence of FHB (Mesterházy, 1995). Unfortunately, beyond
20
the descriptions of Types 1 and 2, there is little agreement on the order and general
designation of various mechanisms of FHB resistance in cereals.
2.3 Trichothecene Mycotoxins
Trichothecenes are low molecular weight sesquiterpenoid mycotoxins characterized
by a tricyclic ring structure with a double bond at C-9,10 and an epoxide group at C-12,13
(Mirocha et al., 2003) (Figure 2.7).
Trichothecene MW Oxygenation and Esterification at Position
b
R1 R2 R3 R4 R5
T-2 Toxin 466 -OH -OAC -OAC -H -OIsoval
HT-2 Toxin 424 -OH -OH -OAC -H -OIsoval
4,15-Diacetoxyscripenol 366 -OH -OAC -OAC -H -H
Nivalenol 312 -OH -OH -OH -OH =O
DON 296 -OH -H -OH -OH =O
15-AcDON 338 -OH -H -OCH3 -OH =O
3-AcDON 338 -OCH3 -H -OH -OH =O a MW = Molecular Weight (Daltons).
b OAc (Acetyl Ester = - O-CO-CH3), OIsoval (Isovalerate Ester = -OCOCH2CH(CH3)2).
Figure 2.7 Diagram of the general structure, numbering system of the tetracyclic
trichothecene nucleus, and specific side-chain groups of naturally occurring
trichothecenes of the genus Fusarium (Desjardins, 2006).
Type A and B structural classes can be grouped based on type of substituent group
at C-8 (Chelkowski, 1989; Desjardins, 2006; Foroud and Eudes, 2009; Mirocha et al.,
1
6
16
R5
10
11
6
7 8
9
R3
15
13
5
14
12
2 O
1 H
O
3
4
R2
R1
H
H
R4
21
2003; Perkowski et al., 1995). Type A trichothecenes (e.g., T-2 toxin, HT-2 toxin,
diacetoxyscirpenol) have hydrogen, hydroxyl, or ester groups at C-8 position. Type B
trichothecenes (e.g., nivalenol, DON, 15-AcDON) have a keto substituent at C-8 (Figure
2.7).
The name denoted to trichothecene mycotoxins is derived from the fungus
Trichothecium, which produces the natural product called trichothecin (Freeman and
Morrison, 1948). In pure crystalline form, trichothecenes tend to be colourless,
odourless, relatively soluble in polar solvents, and very stable within a wide range of
matrices (Scott, 1991; Trenholm et al., 1991; 1992).
2.3.1 Mode of Action
The biochemical mode of action and cytotoxic effects of trichothecene mycotoxins
within eukaryotic cells has been extensively reviewed (e.g., Canady et al., 2001;
Desjardins, 2006; Prelusky et al., 1994; Rotter et al., 1996; Ueno, 1983). In general,
trichothecene cytotoxicity is attributed to the disruption of peptidyl-transferase activity
given by mycotoxin binding to ribosomal protein L3 (RPL3) within the 60S subunit of
eukaryotic ribosomes (Feinberg and McLaughlin, 1989; McLaughlin et al., 1977).
Although binding to RPL3 has been specifically attributed to the presence of the C-
12,13 epoxide group common to all trichothecenes (Figure 2.7) (Carter and Cannon,
1977; Rocha et al., 2005; Rotter et al., 1996), the number and position of substituent
groups on the tetracyclic nucleus (Figure 2.7) has a profound influence on the relative
toxicity of various trichothecene mycotoxins. Given the inherent diversity of the various
side chain substituents that comprise both Type A and B trichothecenes, a wide range of
22
physico-chemical properties and cytotoxic effects of trichothecenes has been exhibited on
various types of eukaryotic cells (Desjardins, 2006; Desjardins and Hohn, 1997; Kuiper-
Goodman, 1994; Mirocha, 1983; Nishiuchi et al., 2006; Ohtsubo, 1983; Otokawa, 1983;
Prelusky et al., 1994; Terao, 1983; Ueno, 1983; etc.).
The presence of different side chains on the trichothecene nucleus is, therefore, a
key determinant of relative cytotoxicity values and specific cytotoxic effects for
trichothecene mycotoxins. Depending on the test system, different substituents can lead
to diverse effects including inhibition of protein synthesis during translation,
transcription, or elongation as well as various secondary effects such as inhibition of
DNA and RNA synthesis, necrosis and inhibition of cell division (Feinberg and
McLaughlin, 1989), inhibition of mitochondria enzymes, immunotoxicity (Creppy, 2002;
Sudakin, 2003).
2.3.2 Mechanisms of Fungal Protection
Although trichothecenes have no function related to the growth of the producing
organism, they are involved in the biological adaptation and ecology of Fusarium species
to various environments (Desjardins, 2006). Part of this adaptation mechanism involves
various specialized genetic adaptations by the producing organism during the biosynthesis
of trichothecenes as a means of „self-preservation‟ (Cundliffe, 1989).
Genes that encode for detoxification mechanisms and/or molecular efflux pumps to
limit cytotoxicity to the parent organism are believed to be the principal means by which
virulent Fusarium species protect themselves from trichothecene-specific cytotoxicity.
For example, the F. sporotrichioides gene Tri12, which encodes an efflux pump, is
23
responsible for pumping trichothecenes out of, and away from, the fungal cell (Alexander
et al., 1999). Genes to help limit the metabolic cytotoxicity of trichothecenes have also
been demonstrated as a defense response within Fusarium species. For example, the gene
Tri101 encodes an acetyl-transferase enzyme responsible for the addition of an acetyl-
ester side-chain group (R-OHCH3) to trichothecene mycotoxins such as DON as a
mechanism to reduce molecular cytotoxicity to the parent organism (Kimura et al.,
1998b).
2.3.3 Effects on Plants
The toxicity of trichothecenes to plant cells was first reported in 1961 (Brian et al.,
1961). Since this initial work, trichothecene toxins are well established as phytotoxic
compounds that produce wilting, chlorosis, necrosis, and various other non-specific
effects within plant tissue (Cutler and Jarvis, 1985, Desjardins, 2006; Nishiuchi, et al.,
2006; Packa, 1991; Wakulinski, 1989, etc.). Inhibition of ribosomal protein synthesis is a
common mechanism of phytotoxicity; however, some trichothecenes affect electron
transport chains, cellular membranes, leaf chlorophyll synthesis and other functions
within plant tissue. However, many aspects related to the specific effects of
trichothecenes within plant systems, particularly host cell defense mechanisms, remain
unknown (Desjardins, 2006).
Genetic disruption techniques to produce „knock-out‟ strains of virulent Fusarium
species have been invaluable tools to clarify the relationship between Fusarium
pathogenicity and trichothecene production within host plant cell tissue. Desjardins et al.
(1989) demonstrated that inoculation of parsnip root discs with a T-2 producing wild type
24
strain of F. sporotrichioides caused extensive root rot, while the Tri4 mutant, which
produced little or no trichothecene, caused significantly less root tissue damage. Similar
studies conducted with a mutant strain of F. graminearum, where the trichodiene
synthase (Tri5) gene was altered to impair trichothecene biosynthesis, were initiated to
examine the effect of trichothecene production on seedling emergence and height of
Fusarium-inoculated wheat, maize, oat and rye (Proctor et al., 1995). Relative to
trichothecene producing (Tri5+) wild-type strains, non-trichothecene producing (Tri5-)
mutant strains had reduced incidence and severity of disease (i.e., bleaching of heads,
yield reduction, etc.) and reduced fungal spread from single spikelet inoculations
(Desjardins et al., 1996; Mirocha et al., 1997; Proctor et al., 1995). Similar results were
noted by Harris et al. (1999a) who found that mutant strains of F. graminearum, which
did not produce trichothecene, were less virulent on inoculated maize. A comprehensive
summary of these as well as subsequent Tri5 disruption studies provided by Desjardins
(2006) clearly demonstrates that trichothecenes act to enhance the ability of Fusarium to
cause disease on susceptible host plants.
2.4 DON, 3-AcDON, and 15-AcDON
Most phytopathogenic F. graminearum species are believed to produce 15-AcDON
or 3-AcDON as labile, acetylated precursors to DON (Figure 2.8). Production of either
acetylated compound is geography-dependent since 3-AcDON chemotypes tend to
dominate in Asia and Europe, while Fusarium species which produce 15-AcDON have
been generally more prevalent in North America (Goswami and Kistler, 2004; Moss and
Thrane, 2004). Therefore, the accumulation of DON within plant tissue is believed to be
25
a metabolic process conferred by fungal and plant carboxyl-esterases, which continuously
deacetylate either 15-AcDON or 3-AcDON within plant tissue (Evans et al., 1997;
Mitterbauer and Adam, 2002; Scott et al., 1984).
Figure 2.8 Structure of DON, 15-AcDON, and 3-AcDON (Desjardins, 2006).
2.4.1 Contamination of Food and Feed
After FHB infection of cereals, DON, 15-AcDON and 3-AcDON accumulate within
grain as thermally stable and persistent contaminants through storage, handling and
processing of food and feed (Canady et al., 2001; Desjardins, 2006; D'Mello et al., 1999,
Visconti and De Girolamo, 2002). Therefore, DON and either 3-AcDON or 15-AcDON
are considered food- and feed-borne contaminants that are hazardous to human and
animal health (reviewed in Canady et al., 2001; D'Mello et al., 1999; Desjardins, 2006).
The effects of chronic exposure to DON within small-grain cereals has been widely
tested within domestic livestock. Among animals tested, swine are the most sensitive to
DON exposure (Trenholm et al., 1994). In fact, swine producers termed DON
“vomitoxin” when they associated observed emesis (vomiting) following the
26
consumption of Fusarium-infected maize well before the biochemical effects were clearly
evident (Mirocha, 1983). In subsequent research, more sophisticated chronic feeding
studies with swine have shown decreased skin temperature, depressed feed intake,
reduced thyroid size, altered stomach conditions, increased albumin levels, and decreased
-globulin levels after consumption of dry feed with as little as 1 - 3 mg·kg-1
DON
(Jacobsen et al., 1993; Rotter et al., 1994).
Although type B mycotoxins are among the least acutely toxic trichothecenes (e.g.,
DON, LD50 = 46 mg·kg-1
mice, oral), chronic exposure is considered a significant risk for
both humans and domestic animals (Canady et al., 2001). Most countries involved in the
export and import of grain have imposed strict guidelines to regulate the trade of DON-
contaminated cereals. In March, 2004, the Codex Committee on Food Additives and
Contaminants reaffirmed the 2001 Joint FAO/WHO Expert Committee on Food
Additives (JECFA) provisional maximum tolerable daily intake (PMTDI) for DON of 1
µg·kg-1
·day-1
for cereals for use within human food or food processing
(http://ec.europa.eu/food/fs/ifsi/eupositions/ccfac/archives/ccfac_04366_item14g_en.pdf).
This regulatory limit for DON, and closely-related mycotoxins, will undoubtedly continue
to evolve as more accurate risk-assessment models are developed based on more precise
toxicological end-points (Miller, 2008).
2.4.2 Mechanism of Virulence for FHB
DON is regarded as a principal component of the necrotrophic phase of Fusarium
pathogenesis of plant tissue (Atanasoff et al., 1994; Desjardins et al., 1996; Desjardins et
al., 2000; Desjardins, 2006; Harris et al., 1999a; McCormick, 2003; Mesterházy, 2002;
27
Miller, 1994; Miller et al., 1985; Mirocha et al., 1997; Mudge, et al., 2006; Proctor et al.,
1995; 1997; Snijders and Perkowski, 1990; Wang and Miller, 1987; 1988; etc.). Aside
from inhibition of protein synthesis, as previously discussed, DON can induce a range of
phytotoxic symptoms including light-dependent bleaching of detached plant tissues based
on loss of chlorophyll, carotenoids (Bushnell et al., 2002; Seeland and Bushnell, 2001),
and electrolytes within exposed plant-cell tissues (Cossette and Miller, 1995).
Electron microscopy and immunogold labeling techniques provide anecdotal
evidence of DON, 15-AcDON and 3-AcDON toxicity in Fusarium pathogenicity (Kang
and Buchenauer, 1999). Toxin diffusion into and within susceptible plant tissue occurs
well in advance of Fusarium hyphal invasion (Figure 2.9). Without exception, toxin
accumulation was well correlated with symptoms of Fusarium-related plant cell damage.
Figure 2.9 Immunogold localization of DON, 3-AcDON, and 15-AcDON within wheat
spikelet (cv. Agent) infected with F. culmorum. Toxins detected within: A,
Plant host cell wall (HCW) and ahead (see arrow) of hyphal fungal cell (FC)
1.5 days after inoculation (DAI). B, HCW of plant cells within the rachis 6
DAI. C, Parenchyma cells (PC) within a young kernel located three
spikelets above inoculated spikelets 10 DAI. Secondary thickening (ST)
noted within PC (Bars = 0.5 µm (Kang and Buchenauer, 1999).
5 µm 5 µm 5 µm
A B C
28
Analytical methods to measure DON concentrations have also provided valuable
input in terms of quantifying DON within developing cereal tissue. For example, Sinha
and Savard (1997) used a competitive direct ELISA technique to measure DON
concentrations within developing wheat heads and found that the rachis had the highest
DON concentrations (93 ppm), followed by floral chaff (50 ppm), kernels (25 ppm), and
peduncles (16 ppm). In a follow-up study, DON was detected below the site of injection
into single florets at concentrations of hundreds of ppm, i.e. it was found within the
kernels and rachis (Savard et al., 2000). The results of these studies are complemented by
fungal colonization experiments based upon quantitative PCR assays of plant tissue
before and after Fusarium pathogenesis. Nicholson et al. (1998) used quantitative PCR
based on oligonucleotides from known F. graminearum DNA sequences to demonstrate
greater fungal colonization of plant tissue by trichothecene-producing than non-producing
mutant strains.
2.5 Transgenic Approaches to Limit Trichothecene Toxicity
The study and subsequent application of innate and transgenic mechanisms to instill
enhanced “Type III” resistance to DON and structurally-similar trichothecenes within
higher order plants is a complex and difficult task. Many variables (both known and
unknown) affect host plant responses to Fusarium pathogenesis (Desjardins, 2006;
Foroud and Eudes, 2009; Muehlbauer and Bushnell, 2003). For this reason, novel
trichothecene-specific mechanisms are often initially evaluated within yeast and/or
Arabidopsis as model eukaryotic test systems prior to evaluation within higher order
plants. Such model eukaryotic test systems offer increased efficiency in terms of overall
29
convenience and decreased cost with minimal complexity, variability, and time to initial
evaluation (Mitterbauer and Adam, 2002; Urban et al., 2002).
Four general mechanisms have been proposed as transgenic approaches to limit
trichothecene, particularly DON, cytotoxicity within plants and eukaryotic test systems;
1) inhibition of trichothecene biosynthesis, 2) alteration of the target ribosomal site, 3)
metabolic trichothecene detoxification, and 4) inhibition of trichothecene biosynthesis
(McCormick, 2003; Mitterbauer and Adam, 2002). A fifth mechanism attributed to
plantibodies and mimotopes as been added to this literature review based on novel
applications of recombinant antibody engineering technology to limit mycotoxin-specific
cytotoxicity.
2.5.1 Inhibition of Trichothecene Biosynthesis
Biosynthesis of trichothecenes within in vitro test systems can be blocked by the
addition of flavonoids, furanocoumarins, and various other naturally-occurring soluble
phenolic compounds derived from plants (Bakan et al,. 2003; Desjardins et al., 1988).
For example, xanthotoxin and various other furanocoumarins, which are produced by a
variety of plants, have been shown to be effective inhibitors of trichothecene biosynthesis
at the preliminary step of trichodiene oxygenation. Unfortunately, the efficacy of such
compounds is often limited as many fungi are capable of expressing enzymes that
detoxify and limit the effectiveness of trichothecene biosynthesis inhibiting compounds
(Spencer et al., 1990).
30
2.5.2 Alteration of Target Protein
Given that trichothecene mycotoxins inhibit protein synthesis by binding to RPL3, a
possible mechanism to attenuate trichothecene cytotoxicity would be to develop
ribosomal genes whose products do not bind trichothecene toxins. Some of the earliest
work in this area was reported by Hobden and Cundliffe (1980) who reported ribosomal
resistance to trichothecene produced by Myrothecium verrucaria. In fact, this work
coupled with related research, initially confirmed that the RPL3 protein was the primary
target of trichothecenes (Fernandez-Lobato et al., 1990; Fried and Warner, 1981).
Harris et al. (1999b) introduced an altered RPL3 protein from rice (Oryza saliva)
into tobacco (Nicotiana tabacum) cells to instill enhanced mycotoxin resistance.
Expression of a rice cDNA encoded with a modified RPL3 based on a change from
tryptophan to cysteine at residue 258 provided enhanced trichothecene resistance within
tobacco (Harris and Gleddie, 2000; 2001, US Patent 606046). A similar approach was
reported by Adam et al. (2000) who engineered a mutant tomato Rpl3 gene to provide
resistance to DON in yeast. Unfortunately, results based on modified plant Rpl3 cDNA
expression within transgenic tobacco plants were less promising as constitutive
mycotoxin resistance was not achieved. Limited efficacy was attributed to a post-
transcriptional effect which led to a preferential utilization of endogenous RPL3 protein
as well as limitations associated with stoichiometry of RPL3 protein expression relative
to rapidly elevated mycotoxin levels within infected tissue (Mitterbauer et al., 2004).
31
2.5.3 Metabolic Trichothecene Detoxification
In planta detoxification of DON, and structurally similar trichothecenes, based on
expression of trichothecene detoxification and resistance genes is a rapidly-emerging and
important field of study with many practical commercial applications. Comprehensive
reviews of enhanced metabolic resistance to Fusarium mycotoxins within plants is
described in additional detail by Berthiller et al. (2007) and Boutigny et al. (2008).
In planta expression of metabolic detoxification genes such as Tri101 is a common
approach to instill enhanced trichothecene tolerance. Tri101 is a gene located outside the
principal trichothecene biosynthetic gene cluster that encodes trichothecene C-3
acetyltransferase, an enzyme which adds an acetylester group to the C-3 hydroxyl group
on trichothecenes (outlined in Section 2.3.2). Constitutive expression of Tri101 within
plants and other model eukaryotic organisms enables a metabolic detoxification of DON
to 3-AcDON, a compound with reduced eukaryotic cytotoxicity (Kimura et al., 1998a;
1998b). Tri101 has been expressed in a range of plants with promising results to show
decreased trichothecene cytotoxicity and prevention of FHB yield loss (reviewed in:
Alexander, 2008; Kimura et al., 2006). Unfortunately, expression of Tri101 alone may
not provide complete resistance as both Fusarium and plants commonly produce
deacetylation enzymes that can convert 3-AcDON back to DON. In fact, Mitterbauer and
Adam (2002) stated that the efficacy of Tri101 expression within plants could be limited
by a „futile cycling‟ whereby plant carboxylesterases, as well as fungal enzymes such as
Tri8 (McCormick and Alexander, 2002), could play a significant part in the metabolic
32
conversion of acetylated compounds back to more toxic C-3 hydroxyl precursors (e.g., 3-
AcDON to DON).
The identification of trichothecene detoxification genes is not solely limited to those
isolated from Fusarium species. Enzymes capable of trichothecene metabolism have also
been isolated from other organisms. For example, potato tissue can rapidly metabolize,
and limit the cytotoxicity of the trichothecene diacetoxyscirpenol based on an initial
removal of the acetyl groups followed by cleavage of the trichothecene skeleton
(Desjardins et al., 1992). In addition, constitutive over-expression of a UDP
glycosyltransferases (UGTs) isolated from Arabidopsis thaliana can also decrease plant
sensitivity to DON and 15-AcDON. Expression of UGTs enables the conjugation of
sugars to trichothecenes thereby forming less toxic DON-3O-glucoside and 15-AcDON-
3O-glucoside conjugates (Poppenberger et al., 2003). This work is supported by the
earlier work of Miller and Arnison (1986), which showed that FHB-resistant wheat
cultivars (e.g., „Frontana‟) metabolized DON to a less toxic compound based on a
biochemical conversion to at least three metabolites, one of which was a DON-glycoside
conjugate.
Another means of enhanced, and perhaps more complete, metabolic resistance may
involve the expression of bacterial genes responsible for the cleavage of the trichothecene
epoxide group at C-12,13 (Figure 2.7). Although such de-epoxidation activity has been
successfully demonstrated in bacteria (Fuchs et al., 2002), application of this technology
within plants to consistently yield favorable results remains an area of future research.
33
2.5.4 Reduction of Intracellular Concentrations
The identification and application of candidate genes that encode for molecular
efflux pumps may also be used to limit trichothecene cytotoxicity within plant cells. For
example, Adam and Lemmens (1996) demonstrated enhanced trichothecene resistance
based on in planta expression of the ABC (ATP Binding Cassette) transporter protein
encoded by a Pleiotropic Drug Resistance gene (i.e. Pdr5) isolated from S. cerevisae
(Balzi et al., 1994). Pdr5 is one of nine members of the (NBF-TMS6)2 subclass of ABC
encoding genes present within the yeast genome. ABC transporter proteins are located in
the plasma membrane of host cells and remove toxic substances using the energy of ATP
hydrolysis (Decottignies and Goffeau, 1997; McCormick et al., 1999).
Expression of Pdr5 genes is a promising strategy to complement other approaches.
Muhitch et al. (2000) successfully demonstrated that co-expression of the Fusarium
acetylation enzyme (TRI101), and Saccharomyces transporter protein (PDR5) resulted in
increased resistance to the trichothecene 4,15-diacetoxysciprenol during germination and
plantlet development of transgenic tobacco.
2.5.5 Plantibodies and Mimotopes
The expression of functional full-size antibodies and antibody fragments within
plants has received extensive research interest within the last 15 years. Plant-produced
antibodies, or “plantibodies” have been evaluated and applied mainly for production of
biopharmaceuticals and immunodiagnostic agents as well as biocontrol agents to improve
resistance against plant pathogens and cytotoxic herbicides (reviewed in Section 3.6.2.3).
34
Various antibodies with specificity to a wide range of mycotoxins are commonly
used within standard immunoassay formats (e.g., Enzyme-Linked Immunosorbent
Assays) to assess mycotoxin levels within contaminated grain (reviewed in Mirocha et al.,
2003). However, despite reports of favorable efficacy, very few have been used as
plantibodies to instill enhanced in planta resistance against Fusarium pathogenesis
(Peschen et al, 2004). Of those tested, most are Fusarium-specific plantibodies expressed
as fusion proteins to antifungal peptides (reviewed in Hu et al., 2008). In these cases, the
Fusarium-specific antibody targets the pathogen while the fusion protein destroys it.
Surprisingly, only a very limited number of recombinant plantibodies have been
applied to limit, or immuno-modulate the cytotoxic effects of low molecular weight
mycotoxins. Yuan et al. (2000) reported the first in planta expression of an anti-
mycotoxin gene. The functional, zearalenone-specific scFv fragment produced within the
cytoplasm of transgenic Arabidopsis-leaf tissue was shown to have a similar binding
affinity to a bacterially-produced scFv and its parent monoclonal Ab. Likewise, Yuan et
al. (1999) were the first to report expression of two functional anti-idiotype peptides, or
mimotope peptides for a low molecular weight mycotoxin (DON). Both mimotopes were
antagonistic to DON-induced protein synthesis inhibition and cellular damage within a
model eukaryotic test system.
These preliminary studies provide insights into the vast potential that antibody
engineering can provide to design and express new and novel peptides that reduce (or
perhaps eliminate) mycotoxin-specific cytotoxicity. The next literature review chapter
provides a comprehensive overview of antibody engineering as applied to agricultural
35
biotechnology. Subsequent research chapters describe experiments at the heart of this
Ph.D. project which were designed to develop, isolate and assess the efficacy of a unique
single-domain antibody fragment with specificity for the mycotoxin 15-AcDON.
36
3 LITERATURE REVIEW 2: ANTIBODY ENGINEERING IN
AGRICULTURAL BIOTECHNOLOGY
3.1 Introduction
Antibodies (Abs) are gamma globulin proteins produced by the immune system of
vertebrates to recognize and bind to a virtually limitless repertoire of antigens (Ags). Abs
are inherently unique, as they possess high affinity and binding specificity for a target Ag.
These properties have made Abs invaluable reagents within various biotechnological and
biomedical applications ranging from standard diagnostic immunoassays (e.g., ELISA) to
integrated immunoaffinity-based systems designed to isolate, enrich, and purify specific
target reagents (e.g., proteins, immuno-conjugates, etc.).
Initial efforts to produce in vitro Ag-specific Abs led to the introduction of
hybridoma technology in 1975 in which Ag-stimulated B-cells were fused with their
myloma counterparts to generate stable hybrid or “hybridoma” cell lines to enable large
scale production of Ag-specific monoclonal Abs (mAb) (Köhler and Milstein, 1975).
Subsequent advances in the fields of genetics, molecular biology and immunology have
greatly enhanced applications of novel technologies required for the development,
isolation, and application of recombinant Ab (rAb) fragments for an expanded range of
applications. For example, routine use of phage display technology to screen
combinatorial rAb libraries (reviewed in Winter et al., 1994; Hoogenboom, 2005)
coupled with new software tools to support an expanding structure-function database
(e.g., Harding et al., 2004) has opened unlimited opportunities to develop tailor-made rAb
fragments. Moreover, on-going improvements associated with rAb optimization and
subsequent expression in recombinant hosts offers the possibility of large-scale and low-
37
cost production platforms as an alternative to more expensive, laborious and often
cumbersome methods associated with the mAb development using mammalian cell lines
Antibody engineering has emerged as a new discipline focused on the identification,
cloning, optimization and expression of Ag-specific rAb genes within various prokaryotic
and eukaryotic host systems (Filpula, 2007; Holliger and Hudson, 2005; Jain et al., 2007;
Lo, 2004; Maynard and Georgiou, 2000). This review presents an overview of
established and emerging technologies available for the de novo generation of Ag-
specific, rAb fragments as an alternative to traditional immunoassays based on
monoclonal antibodies or polyclonal serum. Applications and future research needs
specific to agricultural biotechnology and genetically-modified plants are presented as
concluding remarks.
Key Words
Antibody; antigen; immunoassay; immunoaffinity; ELISA; recombinant antibody (rAb);
antibody engineering; rAb fragment; phage display; combinatorial libraries; rAb
optimization; rAb expression; Ab engineering.
3.2 Recombinant Antibody Fragments
The production of Ag-specific rAb fragments derived from full-size conventional
Abs was first reported in the late 1980‟s following the discovery of the genetic
mechanism associated with Ab gene rearrangements. Concurrent advances in subcloning
and expression systems coupled with the introduction of polymerase chain reaction (PCR)
technology further enabled the recombinant expression of rAb fragments within bacterial
hosts (initial reviews in Plückthun and Skerra, 1989; Winter and Milstein, 1991). In
38
general terms, the production of rAb fragments through these new processes involved the
isolation of mRNA for specific Ab genes derived from either hybridoma, spleen cells, or
lymph node leukocytes followed by reverse transcription PCR (RT-PCR) to form
complementary DNA (cDNA) and eventual PCR amplification by using gene-specific
primer sets to generate a complete Ab gene sequence (Maynard and Georgiou, 2000).
Given the inherent reliance on PCR amplification, the most significant initial
advancements were related to the application of optimized oligonucleotides or so-called
primers to anneal and amplify target rAb genes. Within ten years of adoption of PCR,
numerous primer sets were published based on N-terminal sequences of isolated rAb
genes (Benhar and Pastan, 1994), Ab leader sequences (Larrick et al., 1989), and known
variable domain framework sequences.
A wide variety of rAb fragments have been produced, characterized, and applied in
various biological systems (reviewed in Hudson, 1998; 1999; Little et al., 2000; Holliger
and Hudson, 2005; Yau et al., 2003a). The following section provides a general
description of the three most common and well documented rAb fragments with potential
applications in agricultural biotechnology.
3.2.1 Fab Fragment
The term „Fab‟, or fragment for antigen binding (Better et al., 1988), refers to a
relatively large (MW ≈ 50 – 55 kDa) heterodimer molecule comprised of two polypeptide
chains, one containing the constant and variable light chain domains (CL + VL) of a parent
Ab paired with its corresponding variable and first constant regions (CH1 + VH) (Jeffrey
et al., 1993) (Figure 3.1).
39
Figure 3.1 Schematic representation (left) and ribbon diagram (right) of recombinant
antibody fragment (Fab). Constant and variable light chain domains (CL +
VL) and corresponding first constant and variable heavy chain domains
(CH1 + VH), and target Ag (i.e., Digoxin) are labeled accordingly (adapted
from Joosten et al., 2003 and Jeffrey et al., 1993).
Fabs have the advantage of enhanced durability within in situ applications, as they
are inherently more stable and less prone to dissociation and proteolytic degradation
relative to other conventional rAb fragment formats (e.g., scFv). Enhanced stability is
attributed to the presence of a covalent disulfide bond which serves to retain tertiary
folding and to facilitate more extensive interface and non-covalent interactions between
the two paired immunoglobulin chains or four Ab domains (Maynard and Georgiou,
2000). Despite their relatively large size relative to other rAb fragment types, Fab
proteins are amendable to standard Ab engineering techniques to improve attributes
associated with biological function, recombinant expression, affinity for target Ag, etc.
(reviewed by: Filpula, 2007; Holliger and Hudson, 2005; Maynard and Georgiou, 2000).
3.2.2 scFv Fragment
The term single-chain variable fragment (scFv) refers to a rAb fragment in which
the variable heavy and light domains (VH and VL, respectively) of a parent
immunoglobulin (reviewed in Chapter 1) are joined by a hydrophilic and flexible peptide
40
linker (Figure 3.2) (Bird et al., 1989; Huston et al., 1988). The order of the domains can
be designed for expression as either VH-linker-VL or VL-linker-VH (Huston et al., 1991;
1995). In most cases, the length of the peptide linker is 15 amino acids based on a
(Gly4Ser)3 configuration (reviewed in Huston et al., 1995).
Figure 3.2 Schematic representation (left) and ribbon diagram (right) of recombinant
single chain variable fragment (scFv) antibody. Variable light and heavy
chain domains (VL and VH, respectively) and synthetic peptide linker are
labeled accordingly (adapted from Joosten et al., 2003 and Filpula, 2007).
Specific length and composition of the peptide linker can have a dramatic impact on
post-translational folding and valency of scFv fragments (Robinson and Sauer, 1998;
Turner et al., 1997). For example, Atwell et al., (1999) demonstrated that three amino
acid linkers often form scFv dimers whereas linkers with two residues or less induce the
formation of trimers. In general, increasing the length of linker will lead to monomeric
variants. However, monomeric and multimeric scFv variants have been reported with
linkers of up to 30 residues (Desplancq et al., 1994; Nieba et al., 1997).
41
Once valency is accounted for, one cannot assume that all scFv fragments possess
comparable Ag-binding affinity to larger Ab formats (e.g., mAb and Fab) because they all
have the same VH / VL domain pairing. Numerous publications, based on various Ags,
have demonstrated that relative scFv affinity is best described on case by case basis. For
example, Choi et al. (2004) noted that a scFv fragment raised against deoxynivalenol
(haptenic Ag, MW ≈ 296 Da) had a binding affinity that was approximately two orders of
magnitude less than the parent mAb. However, based on a similar comparison for the
same Ag, Wang et al. (2007) noted no differences between mAb and scFv binding
affinity.
Similar variability has been noted for scFv stability and efficiency of expression. In
general terms, post-translational scFv folding and function is greatly influenced by
specific expression systems and growth conditions (i.e., choice of expression vector and
recombinant host, etc.) as well as method of purification. In addition, factors such as
proteolytic degradation of the linker sequence (Whitlow et al., 1993) and formation of
insoluble inclusion bodies (Choi et al., 2004) have great influence on scFv stability and
expression.
Various techniques have been used to overcome such limitations. For example,
Reiter et al. (1994) reported on the formation of disulfide-stabilized Fvs (i.e., dsFvs)
through the introduction of cysteine residues between the VL and VH domains which
resulted in the formation of an inter-chain disulfide bond which thereby conferred greater
rAb stability. As well, molecular chaperones have been used as part of scFv expression
42
strategy to assist in post translational scFv folding and to increase efficiency of
expression (reviewed in Arbabi-Ghahroudi et al., 2005).
3.2.3 VHH Fragment
The term “VHH” refers to the variable domain of camelidae family heavy chain Abs
(HCAbs). VHH fragments are among the smallest intact Ag-binding fragments known (
average 14 to 15 kDa, 118 - 136 amino acid residues) (Figure 3.3).
Figure 3.3 Schematic representation (left) and ribbon diagram (right) of recombinant
HCAb variable domain (VHH) of antibody “AMD9”. CDR 1, 2, and 3 in
ribbon diagram are labeled accordingly (adapted from Joosten et al., 2003
and Desmyter et al., 2002).
The hallmark of VHH domain resides in key hydrophobic to hydrophilic amino acid
substitutions at former VL interface which imparts increased solubility and stability (e.g.,
resistance to aggregation). Despite the absence of VL and VH combinatorial diversity,
single domain VHH fragments have demonstrated high affinity binding to a wide range of
Ag types (Ghahroudi et al. 1997; Muyldermans, 2001). HCAbs are believed to
compensate for the loss of VL domain through extended CDR3 loop regions and a higher
rate of somatic hypermutations to form a VHH paratope that can form a large antigen-
43
binding repertoire and additionally can penetrate into and conform to clefts and cavities
of Ag epitopes (reviewed in Muyldermans, 2001). An example of this unique ability is
VHH binding to active sites of enzymes that are not typically recognized by conventional
rAb fragments (e.g., Desmyter et al., 1996; 2002). Numerous reviews are available which
document Ag binding characteristics and potential applications of VHH fragments (e.g.,
Holliger and Hudson, 2005; Joosten et al., 2003; Muyldermans, 2001).
Single domain VHH fragments offer a number of advantages relative to larger and
more complex rAb fragments such as scFv or Fab. DNA encoding the variable domain of
HCAb can be readily isolated from peripheral leukocytes and subcloned into various
phage display vectors for highly efficient VHH library generation since no synthetic linker
is required for the pairing of VH-VL domains (Ghahroudi et al., 1997). Another beneficial
characteristic of VHH is its hydrophilic amino acid substitutions within FRs
(Muyldermans, 2001). Consequently, expression in hosts such as E. coli tends to produce
high yields of soluble, stable VHH protein that is inherently free of problems associated
with protein aggregation and proteolytic degradation common to scFv expression
(Arbabi-Ghahroudi et al., 2005). Finally, recent experiments have demonstrated that
VHH fragments possess enhanced functionality and stability over a range of adverse
temperature and solvent regimes where current immunoassays do not typically function
(e.g., Goldman et al., 2006; Ladenson et al., 2006).
The understanding of amino acid sequences of VHH fragments provides interesting
and useful techniques to improve expression of non-dromedary Abs. For example, it is
possible to replace hydrophobic residues on the VH side of a conventional rAb fragment
44
with „VHH-like‟ residues to minimize limitations associated with insolubility and non-
specific binding. This process is known as „camelization‟ and can be used with VH
fragments derived from various animal sources (Davies and Riechmann, 1994; Davies
and Riechmann, 1996a; Tanha et al., 2001).
3.3 Development and Types of Antibody Libraries
The concept of Ab libraries was established following the elucidation of the genetic
mechanisms associated with the creation of immunoglobulin gene diversity (Tonegawa,
1983) and the advancement of technologies associated with PCR for amplification of the
variable domains of immunoglobulin genes (Orlandi et al., 1989; Songsivilai et al., 1990).
In principle, rAb libraries may be classified as immunized, naïve, semi-synthetic, or
synthetic according to the genetic material used for library construction.
3.3.1 Immunized Libraries
Immunized rAb libraries are constructed from DNA isolated from peripheral
leukocytes of a host that has been repeatedly exposed to a single target immunogen or
group of immunogens. Library construction occurs through a stepwise process whereby
the B cell genes encoding Ab variable domain regions are amplified by PCR and
subcloned into vectors to enable expression of different rAb fragment formats including
VHH (Ghahroudi et al., 1997; Goldman et al., 2006; Ladenson et al., 2006), scFv
(Clackson et al., 1991; McCafferty et al., 1990) and Fab (Huse et al., 1989; Orum et al.,
1993; Persson et al., 1991).
The inherent advantage of an immunized library format resides in the fact that
peripheral leukocytes, and therefore high concentration of corresponding mRNA used for
45
library construction, are the product of the process of affinity maturation (Hoogenboom et
al., 1998). Although the development of a rAb library based on an immunogen-specific
response improves the probability of isolating Ag-specific rAbs, overall library size and
diversity is often limited relative to other non-biased library formats (Burton et al., 1991;
Clackson et al., 1991).
Depending on the nature of the Ag and overall test system, the development of
hyper-immunized libraries can present a number of technical challenges and potential
drawbacks associated with isolating rAbs against self-Ags or against Ags that are
extremely toxic or chemically unstable. Immunological tolerance is another factor which
can reduce the diversity of natural in vivo repertoire. In addition, one must account for
the various logistical, financial and ethical considerations associated with the
immunization protocol to ensure a robust and predictable response to the Ag of interest
(Willats, 2002; Hoogenboom, 1997).
3.3.2 Naïve Libraries
Naïve libraries are prepared according to the same methodology used for
construction of immune libraries except that the animal has not been hyperimmunized
with the Ag of interest. The major source of immunoglobulin genes are peripheral blood
lymphocytes (PBLs), bone marrow, tonsils, and spleen cells. IgM is the preferred
immunoglobulin isotype as it is more diverse and has not been subjected to bias or
previous Ag selection (Dörsam et al., 1997; Griffiths et al., 1993; Little et al., 1993;
Marks et al., 1991; Schier et al., 1995).
46
Library size plays a vital role in successfully isolating Ag-specific rAb fragments.
This concept has been proven in several reports in which naïve libraries of medium size
have only resulted in the isolation of Abs with micromolar affinities (Marks, et al., 1991;
Griffiths et al., 1994). Accordingly, a library size of approximately 1010
is likely to yield
Abs with affinities in the low nanomolar range (Vaughan et al., 1996). These results
demonstrate that size and heterogeneity of such libraries have critical roles in isolating
high affinity Abs without prior immunization. An advantage of naïve libraries, in
particular larger-sized ones, is that they are often not antigenically pre-disposed to self-
antigens. As such, large, diverse naïve libraries are a fit as a universal source of Ab
binders (Vaughan et al., 1996).
3.3.3 Synthetic / Semi-Synthetic Libraries
Methodologies associated with synthetic libraries combine the advantages of naïve
libraries (e.g., structural diversity, etc.) with on-going advancements associated with
improved knowledge of rAb structure and function. Accordingly, these technologies
enable in vitro production of high affinity rAbs with application against a range of target
Ags that are not affected by inherent controls which may limit expression or bias an
immunogenic response to a target Ag (Fellouse et al., 2007; Knappik et al., 2000; Winter
and Milstein, 1991).
One of the first semi-synthetic libraries was generated by using a repertoire of VH
genes, combined with a synthetic CDR3 construct, and displayed on phage surface as
either scFv (Hoogenboom et al., 1992) or Fab (Barbas et al., 1992) fragments. The
complexity and diversity was further expanded using a repertoire of VL genes in
47
conjunction with initial library formats (de Kruif et al., 1995; Nissim et al., 1994).
Second generation libraries evolved from the development of synthetic Ab scaffolds that
express well in E. coli to help overcome limitations associated with post-translational rAb
folding (Knappik and Plückthun, 1995). It is also possible to construct fully synthetic
rAb libraries using available DNA sequences of human immunoglobulins (Andris et al.,
1995; Cook and Tomlinson, 1995; Tomlinson et al., 1992; Winter, 1998) followed by
total gene synthesis for expression in E. coli based on optimized rAb scaffolds (Griffiths
et al., 1994; Knappik et al., 2000; Krebs et al., 2001).
3.4 Display Systems
High throughput screening of rAb libraries is made possible through techniques that
establish a direct physico-chemical link between a gene (i.e., genotype) and the resultant
rAb fragment (i.e., phenotype). This linkage allows isolation of an Ab fragment with
desired Ag binding properties from a large, pooled rAb repertoire, while the genetic
material is available for further isolation and engineering. rAb fragments are most
commonly displayed on phage, bacteria, yeast, and ribosomes (Li, 2000; Yau et al.,
2003a).
3.4.1 Phage Display
Phage display was first demonstrated by Smith (1985) who reported expression of
foreign peptides as a fusion with pIII viral coat protein on the surface of non-lytic
filamentous phage. Within five years of Smith‟s demonstration of a direct link between
phenotype, i.e., as protein expressed on the phage surface, to genotype, i.e., as foreign
DNA within phage genome, McCafferty et al. (1990) used phage display techniques to
48
isolate Ag-specific scFv fragments from a diverse filamentous phage-scFv display
repertoire. Since the time of these reports, phage display technology has matured into a
widely adopted technique for selection of rAb fragments from very large phage-display
libraries.
Phage display techniques are now commonly used to produce Ab fragments with
optimized binding affinity, stability, activity (reviewed by Forrer et al., 1999) and to
isolate Abs which were previously considered to be very difficult to obtain through
conventional methods (Griffiths et al., 1993; Hoogenboom and Chames, 2000).
Numerous reviews highlight the diverse applications of phage-display technologies (e.g.,
Hoogenboom et al., 1998; Paschke, 2006; Smith and Petrenko, 1997; Willats, 2002; etc.).
3.4.1.1 Filamentous Bacteriophage
Filamentous bacteriophage (Ff) are viruses (e.g., F1, M13, fd, etc.) that can infect
and replicate within gram-negative bacteria (e.g., E. coli) without killing the host cell
(Russel et al., 2004). These viruses are ideal for applications within rAb engineering
since the Ff genome is inherently small and tolerates insertions into non-essential regions
without loss of bacterial infectivity. During phage assembly, fusion proteins are
expressed on a particular viral coat protein while genetic information encoding the
displayed protein is packaged within the single-stranded DNA (ssDNA) of nascent phage
particles (Smith and Pertrenko, 1997). Direct genotype to phenotype coupling ensures
that phage produced within the same infected bacterial cell are comprised of identical
clones (Paschke, 2006).
49
The defining characteristic of Ff structure is a circular 6.4 kbp ssDNA genome
encased by a long, flexible tube comprised of ≈ 2,700 copies major coat protein (pVIII),
with a minor coat proteins (pIII, pVI, pVII, and pIX) expressed at phage tips (Figure 3.4)
(Yau et al., 2003a). Of these coat proteins, pIII is the most extensively used and well
characterized phage-display format since it is more amendable than the other coat
proteins to large protein insertions without loss of phage infectivity (reviewed in
Hoogenboom et al., 1998; Hoogenboom and Chames, 2000; Russel et al., 2004; Smith
and Pertrenko, 1997; etc.).
3.4.1.2 Phage vs. Phagemid Vectors
Phage display rAb libraries are constructed by cloning a diverse PCR amplified Ab
repertoire into either a phage or phagemid vector (Hoogenboom et al., 1992; Winter et al.,
1994). Regardless of library type (i.e., phage or phagemid), the salient goal is to
construct a heterogeneous and robust mixture of phage clones which collectively display
rAb–phage fusions capable of target or “Ag-specific” recognition and replication to
produce identical progeny phage in large scale (Smith and Petrenko, 1997).
50
Figure 3.4 Schematic representation of a filamentous bacteriophage and its protein
coats (adapted from Willats, 2002; Yau et al., 2003a).
Both phage and phagemid vectors are designed so that the target rAb gene is
inserted between a signal sequence and the coat protein gene (e.g., pIII). Likewise, both
types of vectors carry antibiotic resistance selectable markers, as well as a phage-derived
origin of replication to enables packaging of foreign DNA into viral ssDNA and
production of nascent phage progeny which display rAb fused to surface phage coat
protein (Figure 3.5) (Russel et al., 2004; Smith and Pertrenko, 1997).
Phage vectors are derived directly from the Ff genome whereby the recombinant
protein is cloned as a fusion to phage coat protein (e.g., pIII) for production of multivalent
rAb phage protein progeny. Conversely, a phagemid vector is a plasmid-based sequence
harbouring gene III and intergenic region of Fd phage fusion gene under control of a weak
to moderate bacterial promoter such as LacZ. Phagemid vectors also have a second
51
origin of replication to allow propagation as a plasmid within host E. coli (Figure 3.5).
Many phagemid vectors also have an amber stop codon inserted between end of desired
protein and pIII gene to enable soluble rAb expression within non-suppressor strains of E.
coli (e.g., HB2151) (Russel et al., 2004; Smith and Pertrenko, 1997).
Figure 3.5 Phage (left) and phagemid (right) vectors to construct rAb phage display
libraries through the minor coat protein pIII. Ff ori = filamentous phage
origin of replication. Plasmid ori = plasmid origin of replication. TAG =
Amber stop codon. Sig. = filamentous phage leader sequence (adapted
from Russel et al., 2004).
Phage production using phagemid vectors is only possible when additional phage
proteins are provided by helper phages through a process known as “phage-rescue”
(reviewed within Paschke, 2006, etc.) (Figure 3.5). Use of helper phage is, therefore,
52
very critical as improves the relative proportion of univalent hybrid phage progeny
carrying rAb-phagemid genome to wild-type or “bald” phage based only on phage
genome (Russel et al., 2004). Helper phage can also impact resultant phage valency
within pIII phagemid systems to promote production of monovalent phages carrying only
one rAb fragment pIII fusion protein per phage particle (Paschke, 2006; Russel et al.,
2004).
Finally, regardless of vector type, during library construction, it is essential to
maintain stringent control of fusion protein expression using controllable bacterial
promoter such as LacZ (for example by using Glucose as repressor) through selectable
markers and consistent techniques during all propagation steps as continuous expression
of rAb-phage coat fusion proteins increases the metabolic burden to host E. coli cells.
Such techniques are important to ensure consistent selection pressure to limit any
ecological advantage for non-functional clones which limit library efficiency and function
(Paschke, 2006).
3.4.1.3 Panning
The selection of Ag-specific Abs from a phage display library occurs through a
procedure known as „bio-panning‟ or simply „panning‟. Although this process can
involve many variations and modifications, it relies mainly on the stepwise isolation and
amplification of phage displaying Ag-binding Ab fragments (reviewed in Maynard and
Georgiou, 2000; Paschke, 2006; Smith and Pertrenko, 1997; etc.).
After exposure of the full phage-display rAb repertoire to the target Ag (e.g., coated
onto a solid surface or present in solution or even on the cell surface), washing steps are
53
used to remove phage carrying non-specific Abs from those displaying Ag-specific Abs.
The latter are desorbed through low or high pH elution prior to amplification in phage
host (typically E. coli) (Figure 3.6). After each round of panning, amplified phage are
subject to increased selection pressure and washing to continue phage enrichment and
amplification based on Ag-binding properties of phage-displayed rAbs (Paschke, 2006).
Figure 3.6 Phage display panning cycle: 1.) rAb-displaying phages are added to
immunotubes coated with target Ag; 2.) Washing is performed to remove
non-binding phages; 3.) Positive-binding phage are eluted; 4.) Eluted
phage are used to infect E. coli; 5.) Positive clones are plated as E. coli
plaques or colonies; 6.) Helper phage (if phagemid system) is added for
phage rescue to amplify positive clones; 7.) Positive phage are purified for
reintroduction into next round of panning (de Bruin et al., 1999).
High affinity phage display Abs are commonly selected within three to four rounds
of panning (de Bruin et al., 1999). After panning, rAb fragments are typically expressed
in E. coli and purified for subsequent characterization, and application.
54
3.4.2 Bacteria Display
Peptide display on the surface of bacteria was first described well over a decade ago
(Georgiou, et al., 1993). Since the initial reports of recombinant peptide expression in
prokaryotes, fully functional Ab fragments have been displayed on surface of E. coli
(Francisco et al., 1993; Fuchs et al., 1991). As noted for phage display, bacterial display
libraries can also be used to select rAbs with improved affinity for target Ag (Daugherty
et al., 1998; 1999).
A limitation associated with the expression of foreign peptides on the surface of
gram-negative bacteria (e.g., E. coli) is the physiological barrier associated with an
extensive network of macromolecules within the bilayer cell envelope. However, this
limitation can be circumvented: peptidoglycan chaperones have been used to facilitate
transport and anchoring of recombinant Ab fragments through both the inner and outer
membranes without causing an adverse effect on cell growth and viability (Fuchs et al.,
1991). Another way to address this problem is to select an appropriate leader sequence
and a surface-localized carrier protein (Yau et al., 2003a).
It is also possible to express rAbs on the surface of gram-positive bacteria. For
example, scFv expression has been reported on the surface of Staphylococcus xylosus and
S. carnosus (Gunneriusson et al., 1996) Ab display on gram-positive bacteria has been
shown to provide efficient rAb secretion and post-translational folding.
3.4.3 Yeast Display
As in bacterial surface display, the phenotype and genotype linkage for yeast surface
display models is comprised by the protein on the cell surface and the corresponding
55
DNA within the cell. Given this direct linkage, an additional subcloning step is not
required after selection of an Ag-binding cell (Yau et al., 2003a).
Yeast display of rAbs offers many of the advantages of bacterial display. Yeast has
a thick, rigid cell wall which enables stable maintenance of surface-displayed proteins
(Georgiou et al., 1997). In contrast to typical prokaryotic expression systems (e.g., E.
coli), yeast possess protein folding and secretory mechanisms of higher order eukaryotic
(e.g., mammalian) systems. As such, yeast display alleviates some biases associated with
prokayotic protein expression and folding (Boder and Wittrup, 1997).
Despite these advantages, experience has shown that yeast-display Ab libraries tend
to have limited transformation efficiency, leading to smaller and less diverse repertoires
relative to phage or bacterial display (Yau et al., 2003a). Nevertheless, high affinity rAb
fragments have been isolated from yeast display libraries. For example, Boder and
Wittrup (1997) successfully used yeast-surface display to screen a diverse peptide library
to select binders to a soluble fluorescein tracer. Likewise, Kieke et al., (1997) reported
successful isolation of an anti-T cell receptor scFv using a yeast-surface display library.
3.4.4 Ribosome Display
Ribosome display represents an alternative to expression of Ab fragments on the
surface of phage, yeast or bacterial cells. As in other display systems, ribosome display
libraries are designed based on a direct link between peptide / Ab phenotype and
genotype. Unlike other library formats, ribosome display is entirely in vitro or cell-free
(reviewed in Hanes and Plückthun, 1997; Schaffitzel et al., 1999). Ribosome display is
unique as the phenotype to genotype construct is designed to include a spacer sequence
56
with no stop codon such that, when the nascent Ab fragment is translated from mRNA,
both the mRNA and the Ab remain bound to the ribosome molecule to form an „ARM‟
(Antibody-Ribosome-mRNA) complex (Figure 3.7) (He and Taussig, 1997).
Figure 3.7 Ribosome display panning cycle. 1.) Double-stranded DNA (dsDNA)
transcription to form messenger RNA (mRNA). 2.) In vitro translation of
mRNA to form ARM (Antibody-Ribosome-mRNA) complexes. 3.)
Exposure of ARM complexes to immobilized target Ag for affinity
selection or Ag-specific panning. 4.) Elution and isolation of mRNA from
Ag-specific ARM complex(es). 5.) Reverse transcription PCR
amplification of target mRNA to form dsDNA for continued panning or
transformation into host organism (e.g., E. coli) (adopted from Yau et al.,
2003a).
A key advantage of cell-free systems is that very large libraries can be handled since
transformation efficiency is not a limiting factor as it is for other systems. In addition, in
vitro systems are not impacted by ongoing random mutation of foreign DNA imparted by
57
the host organism which may limit library diversity. As an example, the typical size limit
associated with transformation efficiency for bacterial display libraries ranges from 1010
–
1011
clones (Yau et al., 2003a). With ribosome display, however, libraries with up to 1015
members can be created and screened (Roberts, 1999).
Ribosome display also enables the incorporation of continuous diversification
during each panning cycle using PCR-based mutagenesis. As a result, ribosome display
is a good format to select Ab fragments with improved KD (dissociation constants) and
robust Ag-binding affinities (Yau et al., 2003a). For example, Hanes et al. (1998)
reported the isolation of scFvs from a ribosome-display library with affinities to target Ag
in the picomolar range.
3.5 Optimization of Recombinant Antibodies
Ab fragments such as scFv, Fab, and VHH often display lower affinity than their
parental, full-size counterparts (Adams and Schier, 1999; Huston et al., 1996). A number
of strategies can be used to overcome this limitation, thus enabling isolation of high-
affinity Ag-specific rAb fragments. For example, panning procedures vs. target Ag can
be optimized and in vitro mutagenesis (where the natural affinity maturation process is
mimicked) can be performed (Adams and Schier, 1999).
3.5.1 Optimized Panning Procedures
Although optimization of the selection or panning process (Figure 3.6) does not
inherently improve rAb characteristics, it allows for more efficient recovery or selection
of high affinity Ag-specific rAb fragments from a diverse library. Panning strategies used
to increase the probability of isolating high-affinity rAbs have generally consisted of
58
gradually decreasing the Ag concentration during successive rounds of panning (Strachan
et al., 2002). In the case of panning against low molecular weight haptenic Ags, panning
strategies often use soluble or free hapten to elute the rAb from target Ag. Likewise,
structurally-similar hapten analogues can be preincubated with the phage-display rAbs
prior to exposure to the target hapten conjugate. Finally, subtractive panning, where the
rAbs are incubated with the carrier protein prior to incubation with the hapten, can also be
used to improve the probability of selecting Ag-specific, high affinity rAbs (reviewed in
Sheedy et al., 2007).
As more knowledge is gained on Ab structure through X-ray crystallography,
specific mutations can be targeted to modify and improve rAb specificity and affinity for
target Ag (Korpimäki et al., 2003; Lamminmäki et al., 1999). Ab engineering provides
excellent tools to tailor the properties of rAb fragments with respect to affinity, specificity
and performance for different applications (Valjakka et al., 2002).
3.5.2 In vitro Affinity Maturation
Because of the relatively low affinity of rAbs isolated from naïve libraries, the
process of somatic hypermutation must be mimicked through antigen-driven affinity
maturation (Irving et al., 1996). In vitro affinity maturation by site-directed or random
mutagenesis is not limited by the restrictions inherent to the natural somatic
hypermutation mechanisms (Parhami-Seren et al., 2002). Affinity maturation in vitro
can, therefore, generate extended and functional variability in rAb molecules (Borrebaeck
and Ohlin, 2002), and lead to selection of rAb fragments with increased affinity and
specificity for target Ag (Roberts et al., 1987; Wu et al., 1998; 1999).
59
Strategies to affinity mature Abs and fragments thereof in vitro can be grouped into
the following categories: random mutagenesis, site-directed mutagenesis, and shuffling.
Random mutagenesis consists, as its name implies, of the introduction of mutations
randomly throughout the gene. Random mutagenesis can be subgrouped into error-prone
PCR and bacterial mutator strains. Site-directed mutagenesis includes mutational hot
spots and parsimonious mutagenesis, as well as any mutational strategy which assigns the
mutations to given positions or residues within an Ab‟s wild-type gene. Shuffling
encompasses chain shuffling, DNA shuffling and staggered extension process. The most
frequently used strategies are those of site-directed mutagenesis, where mutations are
directed to specific CDRs or framework regions (Irving et al., 1996), and error-prone
PCR, where mutations are randomly introduced across the gene.
3.5.2.1 Random Mutagenesis
3.5.2.1.1 Error-Prone PCR
Error-prone PCR uses low-fidelity polymerization PCR conditions to introduce
point mutations randomly over a gene sequence (Stemmer, 1994). The introduction of
random mutations in vitro is an efficient method for increasing both specificity and
affinity, as long as a strong selection pressure is applied during the panning stage
(Miyazaki et al., 1999).
3.5.2.1.2 Bacterial Mutator Strains
Affinity maturation of rAbs and increase in their expression levels has been
achieved by using E. coli mutator cells (Coia et al., 2001a; 2001b; Irving et al., 1996;
2002). This approach consists of the selection of rAbs from a library followed by gene
60
diversification or mutagenesis through amplification in a bacterial mutator strain of E.
coli such as MutD5, a conditional mutant which produces single base substitutions
(transversions or transitions) at high frequency compared to normal E. coli cells. Such
mutator strains can produce a large number of mutant rAbs which can then be selected by
phage-display or other methods (Irving et al., 1996). For example, the affinity of an scFv
for the glycoprotein glycophorin was increased by passaging the rAb construct through
such a bacterial mutator strain. A single point mutation resulted in a 10-fold increase in
the scFv binding affinity, and another point mutation located in a framework region near
the CDR3 of the VL resulted in a 1,000-fold increase in affinity (Irving et al., 1996). The
effects of these point mutations illustrate the power of random mutagenesis in generating
high-affinity mutants that could not be obtained by site-directed mutagenesis. However,
four to ten rounds of mutation or passage through mutator cells are required to obtain
high affinity mutants (Coia et al., 2001a).
3.5.2.2 Focused Mutagenesis
3.5.2.2.1 Site-Directed Mutagenesis
Improvements in affinity can be achieved in vitro by site-directed mutagenesis,
where specific or selected residues are mutated at predetermined locations within target
rAb genes. Following mutagenesis, a library is constructed and screened for high-affinity
mutants. For example, Davies and Riechmann (1996b) have investigated the effect of
randomizing CDRs 1 and 2 residues in VH domains specific for both haptens and protein
antigens. This research was successful in terms of generating randomized repertoires
which were displayed on phage and affinity selected to improve Ag binding.
61
3.5.2.2.2 Mutational Hot Spots
Chowdhury and Pastan (1999) have developed a strategy that enables the isolation
of rAb fragments with increased affinity from small phage-display libraries. Their
approach is based on the fact that the DNA encoding the variable domains of Abs
contains “mutational hot spots”, or nucleotide sequences naturally prone to
hypermutations during the in vivo affinity maturation process. Several types of hot spots
have been suggested, such as direct and inverted repeats, palindromes, secondary
structures and certain consensus sequences (Chowdhury and Pastan, 1999). Several
consensus hot spot sequences have been studied in great details; one of these sequences is
the tetranucleotide A/G-G-C/T-A/T (Chowdhury and Pastan, 1999). The serine codons
AGY, where Y can be either C or T, are other hot spot consensus sequences found in Ab
genes that can be used as targets for mutagenesis.
Hot spot mutagenesis techniques have been used to affinity mature rAb fragments
as a precursorary step to develop Ag-specific high affinity rAbs (e.g., Chowdhury and
Pastan, 1999; Li et al., 2001). For example, hot spot mutagenesis was used to generate a
scFv fragments with high affinity to mesothelin, a cancer protein Ag. The affinity of the
mesothelin-specific scFv PE38 was found to be 11 nM (Chowdhury and Pastan, 1999).
Improved affinity was desired to increase its cytotoxicity so that it could serve as a potent
immunotoxin. DNA sequences present in the rAb variable domains (hot spots) were
selected and random mutations were introduced in the light chain CDR3 (Chowdhury and
Pastan, 1999). Thirty-two hot spots were identified in the scFv, 14 being located in the
variable heavy domains, and 18 in the variable light domains. A library with the affinity-
62
matured clones was constructed and panning of the library yielded several mutants with a
15- to 50-fold increase in affinity. Mutagenesis of the same original library but outside of
the hotspots resulted in a mutated library from which the highest-affinity clone selected
only had a four-fold increased affinity compared to the wild-type Ab (Chowdhury and
Pastan, 1999).
3.5.2.2.3 Parsimonious Mutagenesis
Parsimonious mutagenesis was developed by Balint and Larrick (1993) as a
technique whereby all three CDRs of a variable region gene are simultaneously and
thoroughly searched for improved variants in libraries of manageable size (Balint and
Larrick, 1993; Schier et al., 1996a; 1996b; 1996c). In parsimonious mutagenesis,
synthetic codons are used to mutate about 50 % of all targeted amino acids while keeping
the other 50% of targeted residues intact (wild type) (Chames et al., 1998). rAb libraries
can be constructed with low-redundancy “doping” codons and biased nucleotide mixtures
designed to maximize the abundance of combining sites with predetermined proportions
of pre-selected sets of alternative amino acids (Balint and Larrick, 1993).
This technique has several advantages over standard mutagenesis procedures, one of
which is the total number of substitutions per Ab gene is reduced thereby resulting in a
larger number of well-folded and potentially active binders in the library (Chames et al.,
1998). Random mutagenesis may target residues essential to Ag-binding, leading to a
library of mutants that will be mostly non functional, whereas parsimonious mutagenesis
will preserve these essential residues, resulting in a library of mainly functional clones
(Chames et al., 1998).
63
3.5.3 Optimized Panning Procedures
Shuffling of rAb genes can be accomplished in several ways: chain shuffling, DNA
shuffling and staggered-extension process (StEP), and through variations of these
techniques. Chain shuffling consists in shuffling heavy and light chain variable regions
of Abs. In DNA shuffling, the Ab DNA is digested with DNase I, and randomly
reassembled and amplified by PCR. StEP is also a PCR-based method, where template
switching, caused by shortened extension time, shuffles various portions of several
parental Ab genes.
3.5.3.1 Chain Shuffling
Chain shuffling can generate high-affinity rAbs (e.g., scFv) from immunized
animals quite rapidly (Kang et al., 1991). Since shuffling approaches mimic somatic
hypermutation, they are believed to be more efficient than random or site-directed
mutagenesis in producing functional rAbs (Ness et al., 1999; 2002). The heavy and light
chains of rAbs isolated from an immune library can be recombined, thereby generating a
vast number of functional rAbs from an initially limited genetic repertoire (Kang et al.,
1991).
For chain shuffling to be an effective mutagenesis strategy, the pre-selection of Ag-
binding rAbs from an immune library is required prior to invoking the shuffling process.
In other words, chain shuffling is only feasible to improve rAb affinity within immunized
libraries (Park et al., 2000). Accordingly, chain shuffling is not applicable to naïve rAb
libraries since heavy and light chains available within the library have not been exposed
to target Ag. Moreover, experiments have shown that individual chains might
64
discriminate among partners, rendering the shuffling process difficult between naïve and
somatically mutated chains (Kang et al., 1991).
3.5.3.2 DNA Shuffling by Random Fragmentation and Reassembly
DNA shuffling (Stemmer, 1994; Stemmer et al., 1995) is based on repeated cycles
of point mutagenesis, recombination and selection, which should allow in vitro molecular
evolution of complex sequences such as proteins. It somewhat mimics the natural
mechanisms of molecular evolution (Minshull and Stemmer, 1999), but with a faster rate
(Patten et al., 1997; Stemmer, 1994).
The method involves the digestion with DNase I of a large Ab gene to create a pool
of random DNA fragments. These fragments can then be reassembled into full-length
genes by repeated cycles of annealing in the presence of DNA polymerase (Stemmer,
1994). The fragments prime each other based on homology, and recombination occurs
when fragments from one copy of a gene prime another copy, causing a template switch.
Because of the template switching events, a certain level of homology is required among
the parental genes to be suitable for this method (Stemmer, 1994).
DNA shuffling offers several advantages over more traditional mutagenesis
strategies. Compared to methods such as error-prone PCR and site-directed mutagenesis,
DNA shuffling can be used with longer DNA sequences, and also allows for the obtention
and selection of clones with mutations outside of the binding or active site of the protein,
whereas site-directed mutagenesis is limited to a given region of the protein due to the
limitation in library size that can be efficiently transformed to construct the mutant library
(Stemmer, 1994).
65
DNA shuffling was first performed with 1 kb-long interleukin 1β genes. The genes
were broken into 10- to 50-bp fragments and reassembled to their original size and
function (Stemmer, 1994). Sequencing of clones following the shuffling process revealed
that reassembly introduced point mutations at a rate of 0.7%, a rate similar to that of
error-prone PCR. A similar method was used to shuffle β-lactamase genes, where three
cycles of shuffling followed by selection yielded a mutant with 32,000-fold increase in
enzymatic activity. Error-prone mutagenesis of the same genes resulted only in a 16-fold
increase (Stemmer, 1994).
3.5.3.3 Staggered Extension Process (StEP)
DNA shuffling by staggered extension process was developed in 1998 by Zhao et
al., (1998). StEP consists in the priming of template sequences followed by several
cycles of denaturation and shortened annealing/polymerase-catalyzed extension. During
each cycle, the DNA fragments can anneal to different templates based on sequence
complementarity, and extend further to create recombinant genes. Because of the
template switching, the growing polynucleotide chains can contain information from one
or various parental clones (Zhao et al., 1998). The whole process can be performed in a
single PCR reaction, and results in a pool of mutants, the majority of which are
functional.
3.6 Expression and Purification
There is a growing demand for rAbs for treatment of human diseases, in vitro
diagnostic tests, and affinity purification methods, placing pressure on current production
capacity which is based largely on bacterial and tissue culture techniques. Alternative
66
systems, such as yeast (Horwitz et al., 1988), baculovirus (zu Putlitz et al., 1990) and
plants (Hiatt and Ma, 1992), are very attractive, both for cost effectiveness and individual
advantages such as scalability and safety for instance, that are absent in bacterial or
mammalian systems. Several plant-produced Abs (plantibodies) are undergoing clinical
trials and the first commercial approval could be only a few years away. The
performance of the first generation of products has been very encouraging so far (Stoger
et al., 2005). Ongoing studies are addressing further biochemical constraints, and aim to
further improve yields, homogeneity and authenticity (e.g. its binding characteristics
compared to parent Abs). There is no universal expression system that can guarantee
high yields of recombinant product, as every Ab will pose unique challenges. The choice
of an expression system will depend on many factors: the type of rAb being expressed,
sequence of the individual rAb and the investigator‟s preferences.
Given that the focus of this book is application of immunoassays within agricultural
biotechnology, studies and experimental findings which describe mammalian expression
and post-translational modification, etc. support rAbs as therapeutic treatments, will not
be addressed.
3.6.1 Prokaryotic Systems
3.6.1.1 E. coli Cytoplasmic Expression
The expression of rAb fragments in the reducing environment of the cytoplasm of
E. coli often leads to the formation of insoluble inclusion bodies, which contain unfolded
protein (Verma et al., 1998). This necessitates the development of refolding protocols to
recover functional Abs. In addition, accumulation of foreign protein in bacterial cells
67
may lead to their poor growth. Inducible promoters can, therefore, reduce the risk of cell
toxicity. lac, trp, and their hybrid tac promoters, which are regulated by the lac repressor
and induced by IPTG, are such examples. Another popular promoter is the λPL promoter
responsible for the transcription of the λ DNA molecule, which is regulated by a
temperature sensitive repressor. The T7 RNA promoter can also be used to obtain tightly
controlled, high level expression. Together with the T7 RNA polymerase (encoded by T7
gene1), it is one of the most powerful systems currently employed for recombinant
protein productions. The efficiency of T7 RNA polymerase in processing gene transcripts
is superior to that of native E. coli translation machinery which has made it a very
attractive for high level production of recombinant proteins (see review by Terpe, 2006).
Even under optimized expression conditions with a suitable promoter system,
cytoplasmic expression of functional rAbs and fusion proteins can be very challenging.
For example Peipp et al. (2004) reported that functional rAbs and fusion proteins was
only obtained in a minority of the cases for which it has been attempted.
3.6.1.2 E. coli Periplasmic Expression
Expression of Ab fragments into the periplasmic space of E. coli is the most
promising route to produce functional rAb fragments. This methodology was first
developed for Fv (Skerra and Plückthun, 1988) and Fab (Better et al., 1988; Plückthun
and Skerra, 1989) fragments. The periplasmic route is similar to protein synthesis
pathway in eukaryotes, in which the nascent proteins go through the endoplasmic
reticulum (ER) and into the Golgi apparatus. The transport of secretory proteins to the
periplasm of E. coli is comparable to that of ER and can lead to proper folding. Signal
68
sequences such as pelB (pectate lyase), derived from the pelB gene of Erwinia carotovora
are added in frame with the genes encoding the H and L chains of Ab fragments, resulting
in simultaneous secretion of both chains into the periplasmic space (Plückthun and
Skerra, 1989).
The advantage of periplasmic secretion over cytoplasmic expression is that it leads
to the production of an assembled, fully functional product without having to refold the
protein in vitro. The oxidative environment of the periplasm allows for disulfide bond
formation and the low protease environment permits stable folding of the recombinant
proteins (Better et al., 1988). Both intra- and inter-chain protein folding and heterodimer
associations occur in the periplasm, which is necessary for proper folding of a functional
Ab (Plückthun and Skerra, 1989). The bacterial environment, however, is not efficient in
producing full length Ab molecules.
3.6.2 Eukaryotic Systems
Production of Ab fragments in eukaryotes has also been widely studied, from
unicellular yeast cultures (Freyre et al., 2000; Gach et al., 2007) to higher plants (Hiatt et
al., 1989). In this section, different eukaryotic expression systems used for recombinant
antibody production will be described and their individual pros and cons will be
discussed.
3.6.2.1 Yeast Expression
The main advantages of yeast over bacterial systems are related to the fact that yeast
is both a microorganism and a eukaryote. Frenken et al. produced both scFv (1998) and
VHH (2000) in E. coli and Saccharomyces cerevisiae and found that VHH production
69
levels in the latter organism was up to 100 mg·L-1
, i.e., 1000-fold higher than E. coli.
Furthermore, E. coli-produced VHH exhibited less specific activity (higher fraction of
incorrectly folded soluble species present in the periplasmic preparation) than yeast. In
another study (Huang and Shusta, 2006), two scFvs were expressed at 0.25 mg·L-1
in E.
coli, while yields in Pichia pastoris were between 60 - 250 mg·L-1
.
Yeast provides folding pathways for heterologous proteins and, when yeast signal
sequences are used, correctly folded proteins can be secreted into culture medium.
Moreover, yeasts rapidly grow on simple growth media, and, therefore, represent an
attractive option for industrial-scale production of rAbs. Proteins which may accumulate
as insoluble inclusion bodies in E. coli are often soluble when expressed in yeast (Freyre
et al., 2000). In addition, the degradation of heterologous proteins, often a problem in E.
coli, is usually reduced in yeast. Another unique feature of yeast is that it has the option
of stable transformation without the use of antibiotic resistance, like ampicillin or
kanamycin resistance commonly used with E. coli. E. coli uses episomal vectors which
propagate extra-chromosomally, whereas in yeast, the vector carrying the gene of interests
is integrated into the host genome by homologous recombination (Joosten et al., 2003).
Secretion of functional Fab and whole immunoglobulin (IgG) Abs in yeast was first
demonstrated by Horwitz et al. (1988) in S. cerevisiae, although expression levels were
rather low (approx. 200 ng Fab·mL-1
). Many efforts have been devoted to improving the
expression level of Ab in yeast cells. Yeast secretion yields have been reported to be as
high as 1.2 g·L-1
for scFv fragment in fed batch fermentation cultures (Freyre et al.,
2000). Elements that control the expression level of foreign protein in yeast have been
70
well studied. The yeast invertase signal sequence, the PGK promoter and the
polyadenlylation signal were used in the study of Fab and IgG expression (Horwitz et al.,
1988). The proteins were correctly folded and whole Ab and Fab were purified from the
culture supernatants. The antibodies behaved indistinguishably from their lymphoid cell-
derived counterparts in both direct and competition binding assay. The chimeric whole
Ab from yeast exhibited the same ADCC activity as the chimeric Ab from Sp2/0 cells.
Codon optimization and lower repetitive AT and GC content also improve the expression
levels of some rAbs by 5- to 10- fold in standard shake flask cultures (Sinclair and Choy,
2002; Woo et al., 2002), but this is not the case for all rAbs (Outchkourov et al., 2002).
Non-conventional species of yeasts and filamentous fungi have been tested for
expression of Abs and rAb fragments. Abdel-Salam et al. (2001) reported the expression
of Fab in Hansenula polymorpha. Nevertheless, Ab monomers (kappa and gamma
chains) did not assemble into a heterodimer and were poorly secreted. Yarrowia
lipolytica and Kluyveromyces lactis have also been reported for scFv production
(reviewed by Joosten et al., 2003). Filamentous fungus Trichoderma reesei was also
reported to have secreted 1 mg·mL-1
of Fab into the growth medium under shake-flask
conditions and the yield could be increased to 150 mg·L-1
in bioreactor cultures by fusing
the heavy Fv chain to T. reesei cellubiohydrolase I (CBHI) enzyme (Nyyssönen et al.,
1993; Nyyssönen and Keränen, 1995).
A strong preference has been given to P. pastoris for its aerobic growth; a key
physiological trait that greatly facilitates culturing at high cell densities compared with
the fermentative baker‟s yeast S. cerevisiae (Cregg et al., 1993). An scFv-green
71
fluorescent protein was expressed by methylotrophic yeast P. pastoris under the
methanol-inducible alcohol oxidase 1 (AOX1) promoter and secreted into the growth
medium. The soluble fusion protein was properly folded without additional renaturation
or solubilization (Petrausch et al., 2007). Rahbarizadeh et al. (2006) found that high
inoculum densities limit growth potential but gave rise to a higher level of VHH
production in P. pastoris. Medium composition and pre-induction osmotic stress were
found to have the greatest influence on yield, but can be improved when casamino acid or
EDTA are included in growth medium. However, glycerol supplementation during
induction resulted in increased growth rates and biomass accumulation, but expression of
scFv was repressed (Hellwig et al., 2001). Gasser et al. (2006) first engineered the
protein disulfide isomerase (PDI) and the unfolded protein response (UPR) transcription
factor HAC1 to be constitutively over-expressed in P. pastoris (Mattanovich et al., 2004).
While the over-expression of HAC1 led to a moderate increase of Fab secretion, i.e., 1.3-
fold, PDI enabled an increase of the Fab secretion level by 1.9 fold. Hence, the formation
of interchain disulfide bonds can be seen as a major rate limiting factor to Fab assembly
and subsequent secretion.
3.6.2.2 Insect Cell Expression
Insect cell expression systems have been shown to be a viable alternative to
standard microbial and mammalian systems designed to express rAb fragments and full-
size mAbs, particularly for Abs designed for use as therapeutic proteins (Demangel et al.,
2005; Guttieri et al., 2000; Verma et al., 1998). The baculovirus-mediated gene
expression in insect cells not only produces large amounts of the foreign protein while
72
allowing it to retain its functional activity, it has a highly restricted host range and is less
likely to have contamination during the downstream process and cause harmful effects in
end users. Between 1 to 500 mg of recombinant protein per liter of infected cells have
been reported (Filpula, 2007; Guttieri et al., 2000). However, the success of a foreign
gene expression in insect cells depends on a number of factors. High quality growth
media and careful culturing is required and, for optimal results, the insect cells should be
highly viable and in the log phase of growth. Unfortunately, expression of the foreign
protein is controlled by a very late viral promoter and peaks while the infection
culminates in death of the host cell, thereby allowing for only transient expression of the
rAb. Previous studies also suggested that, during the late phase of baculovirus infection,
the host‟s secretory pathway can become impaired (Reavy et al., 2000).
An alternative to baculovirus-mediated expression is based on stable transformation
of the gene, under the control of an appropriate promoter, into insect cells. For example,
the Drosophila metallothionein promoter has been used to control expression and found
to be tightly regulated, directing high levels of transcription when induced by heavy
metals, such as cadmium or copper (Guttieri et al., 2000). Moreover, the polyhedrin
promoter is considerably stronger than most other eukaryotic promoters, and therefore
enables the gene to be transcribed at a high level, causing recombinant proteins to be
secreted in the insect cell culture in large amounts (Tan and Lam, 1999). To allow for the
continuous expression of Ab genes, Ab DNA can be inserted into insect cell
chromosomes under the control of the baculovirus immediate to early gene promoter
(IEI), which is recognized by the insect cell RNA polymerase (Guttieri et al., 2000). The
73
concentration of IgG recovered from the transformed insect cell culture medium was
considerably lower (approx. 0.06 µg of IgG·mL-1
) than the level generated by infection
with the baculovirus recombinant (approx. 9 µg of IgG·mL-1
) and with the predicted yield
from hybridoma cells (1 – 10 µg of IgG·mL-1
). Nevertheless, the baculovirus system is
still considered a viable alternative to microbial expression systems for whole Ab
molecule expression and when post-translational modification, e.g. glycosylation, is
required.
3.6.2.3 In planta Expression
“Plantibodies”, a contemporary term to describe full size Ab or rAb fragments
expressed in plant tissues, was first reported almost twenty years ago by Hiatt et al.,
(1989). Since then many researchers have been trying to express and scale up the
production of Ab or fragments in plants (reviewed within Fischer et al., 1999; Peeters et
al., 2001; Stoger et al., 2002). Using plants as bioreactors presents many advantages
related with manufacturing facilities, production costs and biosafety, among others.
However, despite the production costs reduction and the biocomparability of plantibodies
with their conventional Ab counterparts, contamination risk exists with mycotoxins,
alkaloids, allergenic and immunogenic proteins, especially if the expression host is a
tobacco plant. However, the favorable properties of plants are likely to make them a
useful alternative for small, medium and large scale production throughout the
development of new Ab-based pharmaceuticals or diagnostic reagents. For example,
Khoudi et al. (1999) reported the production of a functional, purified anti-human IgG
mAb through expression of its encoding genes in perennial transgenic alfalfa.
74
The deposition and storage of a scFv rAb fragment in pea seeds was reported using
a seed specific USP promoter (Saalbach et al., 2001), in cereals (Stoger et al., 2000),
tobacco plants (Ko and Koprowski, 2005) or suspension cells (Yano et al., 2004). In
particular, seeds offer special advantages, such as ease of handling and long-term storage
stability. Nevertheless, most of the plantibody studies have focused on expression in
leaves. Arguably, there may not be a high enough demand for diagnostic Ab that requires
the large-scale Ab production possible within plant expression, nonetheless, the idea of
expressing functional rAb molecules in plant has already provided a new way of
understanding plant/virus interaction (Sudarshana et al., 2007). The studying and/or
altering the function of an antigen in vivo, also termed immuno-modulation, has also
gained new insights into plant physiology (Artsaenko et al., 1995; 1998; Conrad and
Fiedler, 1998; Tavlodoraki et al., 1993), plant pathogen resistance (Peschen et al, 2004)
or potentially confers herbicide resistance to plants (Almquist et al., 2004; Weiss et al.,
2006).
Currently, Agrobacterium and particle bombardment are the most commonly used
technologies for plant transformation. A single plant binary vector carrying genes
encoding heavy and light chains under two different promoters has been used to express
both genes and to assemble functional full-size Ab (Düring et al., 1990). The major
drawback of the binary vector is low transformation efficiency due to the large size of the
Ab DNA, in addition to the enormous binary vector required (Komori et al., 2007).
Instead of molecularly stacking the heavy and light Ab genes in the same vector, crossing
two transgenic lines separately expressing the heavy and light chain provides an
75
alternative that is less demanding in terms of molecular biology (Schillberg et al., 2003).
The particle bombardment approach can insert genes into the genome of the plant nucleus
or the plastid genome, depending on the vector construct. Chloroplasts have been used
for stable expression of mAb in the chloroplast genome. Advantages of targeting the
chloroplast genome include no position effect, no gene silencing, high
expression/accumulation, and minimized environmental concerns (Daniell et al., 2004).
Chloroplasts can process proteins with disulfide bridges, which is required for proper
folding of proteins. Although stably transformed (transgenic) plants are able to express
correctly folded and functional Ab of both the IgG and IgA classes (Ma et al., 1995) or its
fragments (Owen et al., 1992), yields are generally very low (usually in the range of 1-40
µg·g-1
of fresh biomass). In addition, the time necessary to generate the first grams of
research Ab material is very long, requiring > 2 years (Giritch et al., 2006).
To shorten the time required to obtain the functional antibodies from plants,
transient expression using agro-infiltration or plant viral vector system have been used
(Giritch et al., 2006; Ko and Koprowski, 2005). Using the subgenomic promoter of TMV
coat protein and tobacco mild green mosaic virus variant U5 coat protein, two separate
virus vectors were used to co-infect Nicotiana benthamiana leaf cells. Full length heavy
chain and light chain proteins were produced and assembled into glycosylated functional
full size Ab (Verch et al., 1998). Regeneration time is much shorter than that for stable
transformation, and different host plants can be infected by the same viral vector,
allowing time-efficient screening for recombinant gene expression. More importantly, it
76
saves the labor-intensive step of crossing different transgenic plants producing different
Ab subunits.
Many different Ab formats have been expressed successfully in plants. These
include full-size antibodies (Giritch et al., 2006; Ma et al., 1995), camelid heavy-chain
antibodies (Ismaili et al., 2007), Fab fragments (Weiss et al., 2006; Yano et al., 2004),
and scFvs (Owen et al., 1992; Saalbach et al., 2001; Stoger et al., 2000). Regardless of
which form of Ab fragments are being produced by the plant systems, ER is an important
site of the major bio-functions of synthesis, assembly, and glycosylation of these protein
molecules. Antibodies are often targeted to subcellular compartments or the apoplastic
space since most proteins are more stable in the subcellular compartment than in the
cytosol. The greatest accumulation of full size antibodies has been obtained by targeting
to the apoplastic space and the greatest accumulation of scFv antibodies was obtained
when the antibodies were retained in the ER (Daniell et al., 2004).
The average yield of the recombinant protein using plant secretion sequence (SS) is
usually 0.1 - 2% of total soluble protein (Yano et al., 2004). So far, three different leader
sequences have been tested: human derived leader sequence (LS), dicotyledonous
calreticulum derived SS, and monocotyledonous hordothionin derived SS. The latter did
not consistently result in mAb expression, while plants transformed with the dicot SS
construct grew more vigorously and expressed the antibodies more consistently than
transgenic cells made with human LS. The promoter is another critical element
determining the expression level, for examples, Cauliflower Mosaic virus 35S
(CaMV35S) promoter for dicotyledonous plants (McLean et al., 2007; Olea-Popelka et
77
al., 2005) and the maize ubiquitin-1 promoter preferable in monocotyledonous plants.
Petruccelli et al. (2006) successfully produced a full length anti-rabies mAb molecule in
transgenic tobacco using CaMV35S to drive the heavy chain, while the potato proteinase
inhibitor II (pin2) promoter controlling light chain gene produced functional mAb. An
effective plant production system for recombinant Ab fragments also requires the
appropriate control of post-translational processing of recombinant products. The ER
retention signal sequence (KDEL) has little effect on the accumulation of Ab in the
transgenic leaves, but leads to higher Ab yields in seeds. The proteins purified from
leaves contain complex N-glycans, including Lewis a epitopes, as typically found in
extracellular glycoproteins and consistent with an efficient ER retention and the cis-Golgi
retrieval of the Ab. The glycosylated proteins purified from the seed were partially
secreted and sorted to protein storage vacuoles (PSVs) in seeds and not found in the ER
(Ramirez et al., 2003). More importantly, full size antibody (McLean et al., 2007) or
scFv fragments (Almquist et al., 2004, 2006; Makvandi-Nejad et al., 2005) produced in
Tobacco have been proved to be functional. This further supports the fact that
“plantibodies” could be an alternate source of diagnostic as well as therapeutic antibodies.
3.7 Application to Immunoassays and Agricultural Biotechnology
3.7.1 Immunoassays
Given the widespread adoption of genetically modified (GM) crops within global
agriculture and strict regulations governing their co-existence with non-GM crops
(Demont and Devos, 2008), mandatory product labeling (e.g., CODEX alimentarius, EU,
2003; etc.) and international trade, there is an increased need to standardize and
78
continuously upgrade the analytical methods used to monitor and verify the presence of
GM traits within food and feed (Dong et al., 2008).
Immunoassays for the detection of protein expression within GM crops are currently
limited to enzyme linked immunosorbent assays (ELISA) based on either trait-specific
polyclonal serum or mAbs, as well as rapid lateral-flow / immuno-chromatographic
diagnostic strip kits (typically based on polyclonal serum) (e.g., Lin et al., 2001; Ma et al.,
2005; Tripathi, 2005; Van den Bulcke et al., 2007). Both immunoassay formats are
widely used in the US, Canada, and other countries which produce and export GM crops
as a primary method for determining minimum thresholds of GM products within
commercial food and feed shipments (Stave, 2002).
A wide array of immunoassay products are currently available to assess GM
expression within agricultural crops. These testing formats are developed within private,
or in-house programs or are available as commercial products from companies such as:
Agdia Inc. (Elkart, IN, USA), EnviroLogix Inc. (Portland, ME, USA), and Strategic
Diagnostics Inc. (Newark, DE, USA). Although these platforms are very useful
qualitative tools to detect the presence of GM traits within commercial crops, the results
generated by current immunoassay products are often limited by detection limit and
overall precision, or consistency, of results. For example, Ma et al. (2005) reported that
both ELISA and DNA-based (PCR) tests were capable of distinguishing samples with
GM concentrations between 0.1 to 0.5%, but the precision at this range was very low as
results were highly inconsistent. Another consideration is the disparity between the time,
costs, and resources required to develop GM-specific immunoassay reagents based on
79
traditional mAb and polyclonal platforms relative to the rapid progression of GM crops
toward newer events based upon multiple / stacked traits.
Antibody engineering has evolved rapidly over the past 20 years to the point where
it is possible to isolate and optimize high-affinity and Ag-specific rAb fragments as a
cost-effective alternative to established mAb technologies (biomedical applications
reviewed in Holliger and Husdon, 2005). In this regard, rAb fragments (i.e., Fab, scFv,
VHH) could represent a means to complement, and potentially displace, polyclonal serum
and mAbs used within immunoassay formats. In fact, one could argue that the
application of rAb technology to detect GM proteins within plant matrices would be a
natural extension of published research which demonstrated effective use of rAb
technology within immunoassay formats to detect ligands ranging from: mycotoxins
(Wang et al., 2007; 2008) to caffeine (Ladenson, 2006) to large complex protein toxins
such as cholera toxin, ricin and staphylococcal enterotoxin B (Goldman et al., 2006). The
application of rAb technology within current ELISA and lateral flow stick GM
immunoassay formats would obviously depend on any benefits, or advantages, imparted
by this new technology. The following is an initial (and by no means exhaustive) list of
“key traits” that rAb fragments could exhibit relative to current mAb and polyclonal
technology: enhanced GM trait specificity based on recognition of a single target protein;
improved sensitivity to target protein; decreased development costs and timelines; ability
to combine rAb reagents to enable simultaneous and customized detection of stacked GM
traits; improved durability and speed of deployment impart convenient and real-time
immunoassay results.
80
Apart from the advantages associated with improved affinity, cost-effective
expression and Ag-specificity (as discussed within this chapter), rAb technology also
imparts unique opportunities to optimize the physical characteristics of rAb fragments to
suit the intended use pattern. For example, it is possible to express single-domain VHH
fragments in a multimeric pentamer format as a means of increasing antibody avidity
(Zhang et al., 2004) with potential use in lateral flow devices and ELISA kits. Depending
on the intended application and immunoassay design, it is also possible to couple rAb
fragments to larger indicator molecules (e.g., fluorophores, etc.) to impart visual GM
detection in real time. Finally, given that VHH fragments have been shown to have high
affinity for large, complex enzymes (e.g., Desmyter et al., 1996; 2002), such fragments
may be particularly suited for detection of GM traits which express enzymes which confer
tolerance to the herbicide glyphosate (e.g., GAT, EPSPS, etc.).
3.7.2 Research Applications to Agricultural Biotechnology
Apart from standard immunoassay detection procedures, it is also possible to apply
rAb technology to study structure-function relationships between GM traits and the target
organism. For example, Fernández et al. (2008) reported the use of scFv phage-display
libraries and various bio-panning techniques to characterize epitopes that mediate binding
of Cry1Ab and Cry11Aa toxins with target protein receptors within Manduca sextra and
Aedes aegypti, respectively. Such results could provide insights into the mechanism of
insect specificity and mode of action of Cry toxins to enable strategies designed to
improve target insect toxicity and specificity. Ongoing research in this area could be
useful to study, and potentially improve upon, the precise mechanism and toxicity by
81
which target transgenic events work within the host crop and efficacy versus the target
organism. Finally, rAbs are also well suited to study and modify metabolic pathways
within plants as a means of developing new transgenic traits and GM products (reviewed
in Nölke et al., 2006).
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4 RESEARCH CHAPTER 1: CLONING, EXPRESSION, AND
CHARACTERIZATION OF A SINGLE DOMAIN ANTIBODY
FRAGMENT WITH AFFNIITY FOR 15-ACETYL-DEOXYNIVALENOL.
4.1 Abstract
A single-domain variable heavy chain (VHH) antibody fragment specific to the
mycotoxin 15-acetyldeoxynivalenol (15-AcDON) was obtained after immunization of a
llama (Llama glama) with the protein conjugate 15-DON-BSA plus TiterMax™ Classic
adjuvant. After confirmation of a polyclonal response to DON toxin in both conventional
(cIgG) and heavy chain antibody (HCAb) fractions, a VHH library was constructed from
amplified cDNA by nested PCR. VHH fragments with binding affinity for the mycotoxin
were selected by panning of the phagemid library against microtiter plates coated with 15-
DON-OVA. The dominant clone (NAT-267) was expressed in E. coli and was purified
as a VHH monomer (mNAT-267) at a final concentration of 1.3 mg∙mL-1
. Isolated NAT-
267 VHH DNA was fused to the homopentamerization domain of the B subunit of
verotoxin to generate the pentabody format of single-domain antibody (sAb). The VHH
pentamer (pNAT-267) was expressed in E. coli and was purified at a final concentration
of 1.0 mg∙mL-1
. Surface Plasmon Resonance (SPR) analysis of soluble mNAT-267
binding kinetics to immobilized 15-DON-HRP (Horse Radish Peroxidase) indicated a
dissociation constant (KD) of 5 μM. Competitive Direct Enzyme-Linked Immunosorbent
Assay (CD-ELISA) and Fluorescence Polarization Assay (FPA) inhibition experiments
with monomer and pentamer confirmed binding to 15-AcDON. Competitive inhibition
FPA tests with mNAT-267 and pNAT-267 IC50 values for 15-AcDON hapten were 1.24
μM and 0.50 μM, respectively. These values were similar to the IC50 value of 1.42 μM
for 15-AcDON given by polyclonal llama serum sampled 56 days after immunization.
83
Competition formats for structurally related trichothecenes resulted in no cross reactivity
to: DON; 3-acetyldeoxynivalenol (3-ADON); neosolaniol (NEO); diacetoxyscirpenol
(DAS); and T-2 toxin. Our study confirmed that recombinant VHH fragments capable of
binding low molecular weight haptens can be produced through the creation and panning
of hyper-immunized single domain (sdAb) libraries.
Key Words
VHH, Hapten, Mycotoxin, 15-acetyl-deoxynivalenol, Phagemid Library, Surface
Plasmon Resonance, CD-ELISA, Fluorescence Polarization Assay.
4.2 Introduction
Deoxynivalenol (3,7,15-trihydroxy, 12,13-epoxy-trichothec-9-en-8-one), or DON, is
one of many members of the trichothecene group of mycotoxins produced by Fusarium
species. DON is a low molecular weight hapten (M.W. 296 Da) containing one primary
and two secondary hydroxyl groups conferring limited solubility in water and high
solubility in various polar solvents such as methanol and acetonitrile (Desjardins, 2006).
Of the trichothecene mycotoxins that accumulate within cereal grain, DON toxin and its
acetylated metabolites (i.e., 15-acetyl-deoxynivalenol (15-AcDON) and 3-acetyl-
deoxynivalenol (3-AcDON)) are considered among the most commonly and widely
distributed food and feed contaminants (Abramson et al., 2001; Greenhalgh et al., 1986).
Immunoassay techniques using polyclonal and monoclonal antibodies (mAb) have
been developed to detect DON and other mycotoxins within grain, food, feed, and animal
serum (e.g., Maragos and McCormick, 2000; Sinha et al., 1995). These systems typically
use anti-DON mAbs from hybridoma cell lines producing murine immunoglobulins.
84
Although these tests provide robust and precise results, mAb development requires highly
specialized equipment and labor-intensive procedures to select, culture, and maintain
optimal hybridoma cell lines (Sinha et al., 1995).
Heavy chain antibodies found within sera of the camelidae (i.e., camels,
dromedaries and llamas) (Hamers-Casterman et al., 1993) are unique in that they are
devoid of the variable light chain (VL) and first constant region (CH1) which are common
to larger conventional immunoglobulin (cIgG) structures (Nguyen et al., 1999). The
paratope of HCAbs is comprised of a single heavy chain variable domain (often called
„VHH‟) which is directly linked to the second constant domain (CH2) by a unique hinge
region (Hamers-Casterman et al., 1993).
Extended hinge regions coupled with deletion of the CH1 domain, and amino acid
substitutions within the variable domain are believed to impart increased hydrophilicity
and solubility of HCAbs within serum (Desmyter et al., 2001; Nguyen et al., 2000; Vu et
al., 1997). These properties coupled with expanded complementarity determining region
(CDR) loops within the VHH paratope may expand the antigen (Ag) - binding repertoire
within a camelid immune system by enabling HCAbs to bind otherwise inaccessible
epitopes (Muyldermans, 2001; Nguyen et al., 2000).
Despite their lack of variable light and heavy chain pairing, single domain
antibodies (sdAb) recognize and bind a wide variety of epitopes and Ag types (Nguyen et
al., 2000; Sheedy et al., 2006; van der Linden et al., 2000). Recombinant sdAb fragments
derived from phage libraries are regarded as among the smallest intact Ag-binding
fragments currently known (Muyldermans, 2001) and demonstrate robust and stable
85
expression in bacteria (e.g., Desmyter et al., 2001; Ghahroudi et al., 1997; Sheedy et al.,
2006; etc.) and yeast (e.g., Dolk et al., 2005; Frenken et al., 2000; etc.) relative to other
rAb formats (e.g., Fab, Fv, scFv, mAb, etc.).
The advantages associated with ease of expression, stability, and solubility of VHH
fragments can be attributed to key differences relative to standard rAb fragments (e.g.,
scFv, Fv, Fab, etc.). VHH fragments are small (≈ 14 – 15 kDa), typically have hydro-
philic amino acid substitutions within the second framework region (FR-2) and by their
single-domain nature, do not require the linker associated with the standard scFv format
which can lead to problems associated with aggregation, proteolytic degradation and
variable expression within recombinant hosts (Arbabi-Ghahroudi et al., 2005). Improved
functionality over various temperature and solvent regimes (Goldman et al., 2006; van
der Linden et al., 1999) coupled with high levels of recombinant expression combine to
make VHH fragments useful reagents within next-generation immunoassays (Goldman et
al., 2006, Ladenson et al., 2006) and for expression within recombinant hosts to evaluate
potential in vivo mechanisms of immunomodulation (Muyldermans, 2001).
As with other rAb fragments, VHH fragments isolated from hyper-immunized or
naïve libraries are highly specific based on the recognition of unique epitopes on target
Ags (Ghahroudi et al., 1997; Goldman et al., 2006; Ladenson et al., 2006; Sheedy et al.,
2006; van der Linden et al., 2000). A wide-range of affinities has been published for Ags
ranging from large immunogenic proteins to enzymes to low-molecular weight haptens
(reviewed in Muyldermans, 2001). A growing number of VHH fragments have been
isolated which demonstrate a range of binding affinities against an array of different
86
hapten types (Alvarez-Rueda et al., 2007; Goldman et al., 2006; Ladenson et al., 2006;
Sheedy et al., 2006; Spinelli et al., 2000; 2001; Yau et al., 2003b). These results serve to
validate potential uses of this new technology in terms of small molecule binding.
The objective of this research was to isolate and characterize mycotoxin-specific
recombinant VHH fragments from a hyper-immunized phage-display library. The
identification of mycotoxin-specific fragments will provide additional information to
further characterize the nature and binding kinetics of VHH antibody fragments to
haptens. Such fragments will also aid in the development of low cost, durable reagents
required for effective and robust toxin detection systems (Goldman et al., 2006; Ladenson
et al., 2006). Finally, with favorable binding affinity and expression, mycotoxin-specific
VHH fragments can also be used in eukaryotic systems in vivo (e.g., yeast and plant cells)
to evaluate their potential for immunomodulation of the cytotoxic effects of DON and
related trichothecene mycotoxins.
4.3 Materials and Methods
4.3.1 Reagents and Solutions
Restriction enzymes were purchased from New England Biolabs (Mississauga, ON).
Ampicillin, kanamycin, bacto-tryptone, bacto-yeast extract, isopropyl β-D-1-
thiogalactopyranoside (IPTG), phenylmethylsulphonyl fluoride (PMSF), isopropanol, 6-
aminofluorescein (fluoresceinamine isomer II), bovine serum albumin (BSA), ovalbumin
(OVA), TiterMax™ Classic, N-(2-hydroxyethyl)-piperazine-N-2-ethanesulfonic acid
(HEPES), Tris HCl, sodium dodecyl sulfate (SDS), triethylamine, polyethylene glycol
(PEG), and polyoxyethylene sorbitan mono-laurate (Tween 20) were purchased from
87
Sigma Chemical Co. (St. Louis, MO). Skim milk was purchased from Difco (Oakville,
ON). PCR primers were synthesized by Gibco BRL (Burlington, ON). Mouse anti-6-His
monoclonal IgG and goat anti-mouse alkaline phosphatase conjugate were purchased
from Qiagen (Mississauga, ON). PVDF membrane and NBT (nitro blue tetrazolium
chloride) / BCIP (5-Bromo-4-chloro-3-indolyl phosphate, toluidine salt) were purchased
from Roche Diagnostics (Laval, PQ). 96 well Maxisorp™ microtiter-plates were
purchased from Nalge Nunc Inc. (Naperville, IL). Rabbit anti-6-HIS-HRP conjugate was
purchased from QED Bioscience Inc (San Diego, CA). AP and HRP development
substrates were purchased from Kirkegaard & Perry Laboratories (Gaithersburg, MD).
4.3.2 Immunogen and Llama Immunization
All DON-based reagents were obtained from the Eastern Oilseed and Cereal
Research Center (ECORC) of Agriculture and AgriFood Canada (AAFC) Ottawa,
Ontario, Canada. DON was isolated from large-scale fermentations of Fusarium
graminearum liquid culture followed by purification as described by (Greenhalgh et al.,
1986). 15-AcDON was prepared by selective hydrolysis of 3,15-diacetyl-DON, which
was obtained by acetylation of DON or 3-AcDON (Savard, 1991). Neosolaniol and T-2
Toxin were isolated according to standard methods (Greenhalgh et al., 1986).
Diacetoxyscirpenol (DAS) toxin was purchased from Sigma-Aldrich (Oakville, Ontario).
Synthesis of DON-protein conjugates 15-DON-BSA, 15-DON-OVA, 3-DON-HRP and
15-DON-HRP was based on standard methods (Bauminger and Wilcheck, 1980) with
modifications as previously described (Sinha et al., 1995). DON-protein conjugates were
88
purified by size exclusion chromatography on Sephadex G-15 (2.5 x 21-cm column) with
distilled deionized water at a flow rate of 4 mL∙min-1
.
A two-year-old male llama was immunized by sub-cutaneous (Sub-Q) lower-back
injection of 15-DON-BSA (250 μg∙mL-1
) conjugate in 1.0 mL PBS + 1.0 mL of TiterMax
Classic adjuvant. The llama was boosted with a second injection three weeks after the
first immunization followed by three subsequent injections every two weeks for a total of
five immunizations over a nine-week period.
Pre-immune serum was collected as a negative control, and 15.0 mL of blood was
collected one week after each immunization to measure titer at 7, 28, 42, 56 and 70 days
after the first immunization. Serum was prepared by storing blood samples overnight at
4ºC followed by centrifugation at 2,700 x g for 10 minutes at 4ºC. Samples of fresh
blood (20 mL) were collected one week after the third and fifth immunizations from
which leukocytes where isolated, i.e., 42 and 70 days after immunization, respectively.
The isolation of RNA from peripheral leukocytes was performed using a QIAamp RNA
Blood Mini Kit (Cat.No. 52304 – Qiagen Inc., Mississauga, Canada).
4.3.3 Fractionation of Polyclonal Serum
Llama serum from a pre-immune sample and from samples taken one week after the
third and fifth immunizations (42 and 70 day samples, respectively) were fractionated by
protein G and protein A chromatography (Hi Trap, Pharmacia, Upsala, Sweden) and
separated by acidic elution as previously described (Hamers-Casterman et al., 1993).
Serum fractions A1 (HCAb), A2 (HCAb), G1 (HCAb), and G2 (cIgG) from each
collection date were dialyzed against PBS and subsequently stored at 4°C with 0.01%
89
NaN3 as a preservative. The IgG content of the isolated serum fractions was measured
using the BCA-protein assay (Pierce, Rockford, IL, USA).
4.3.4 Confirmation of Titer
Polyclonal serum was screened against 15-DON-OVA or OVA (control) by indirect
ELISA as based on a modified standard protocol (Sinha et al., 1995). Briefly, a standard
96-well microtiter plate was coated with 100 μL 15-DON-OVA (5 μg∙mL-1
in 0.15 M
carbonate-bicarbonate buffer, pH 9.6). Blank wells were coated with 100 µL OVA (20
μg∙mL-1
in PBS). After incubation overnight, plates were washed with PBST, and
blocked with 1.0% OVA (2 hours, 37°C) followed by washing with PBST. Non-
immunized (control) wells for 15-DON-OVA and OVA (blank) were coated with pre-
immune polyclonal serum. Treatment wells were coated with 42- and 70-day serum
samples. Immune and non-immune polyclonal sera were diluted serially from 1:3 to
1:59,049 in PBS for both 15-DON-OVA and OVA (blank) coated wells. After
incubation at room temperature (1.5 hours) and washing with PBST, 100 μL of goat anti-
llama IgG (1:1,000 dilution PBS) was added. Plates were incubated at 37°C for 1 hr and
washed five times with PBST. Swine anti-goat HRP conjugate (100 µL; 1:3,000 dilution
in PBS) was added followed by incubation and washing as described. Titer was
determined with standard colourimetric buffer followed by 1 M H3PO4 and measurement
of optical density at 450 nm on a standard ELISA plate reader (Dynatech MR5000). Best
titers were defined as the greatest sample dilution giving twice the background
absorbance of OVA control.
90
Titer of cIgG and HCAb fractions were tested against 15-DON-OVA or OVA
(control) by indirect ELISA as described above with one modification. As an additional
blocking step, equal volumes and concentrations of processed cIgG and HCAb fractions
were „pre-incubated‟ with 2% OVA in PBS. Antibody fractions were added to 15-DON-
OVA and control wells and diluted in PBS to establish a dilution series from 1:2 to
1:1,024 and processed according to the previously described method (Sinha et al., 1995).
4.3.5 Library Construction
Total RNA was isolated from the leukocyte fraction of Peripheral Blood
Lymphoctes (PBL) of whole blood samples collected one week after the third and fifth
immunizations (42 and 70 days, resp.) using a QIAamp RNA Blood Mini Kit (Cat. No.
52304 – QIAGEN Inc., Mississauga, Canada). EDTA (dipotassium salt) at 1-2 mg ml-1
blood was used as an anticoagulant. The VHH library was constructed according to
Ghahroudi et al., (1997) with minor modifications. Briefly, first strand complementary
DNA (cDNA) was derived from 33 µL of total RNA (approx. 3 µg∙20 µL-1
) isolated from
42 and 70 day PBLs using oligo dT primer and a „First-Strand cDNA Synthesis Kit‟
(Amersham Biosciences, Buckinghamshire, UK). Resultant synthesis of VH and VHH
dsDNA occurred through nested PCR using proofreading Taq polymerase with sense and
anti-sense hinge-specific primer mixtures “MJ1.2.3 Back”, and “CH2 + CH2b3”,
respectively (Table 4.1). After amplification, doublet PCR bands between 620 – 670 bp
corresponding to VHH – CH2 hinge HCAb dsDNA were agarose gel-purified. Gene-
specific primers: MJ7 (Sense) and MJ8 (Antisense) (Table 4.1) were used to amplify the
91
VHH region of HCAb and add two SfiI restriction sites flanking the 5‟ and 3‟ termini of
VHH coding sequences. PCR products were then pooled and agarose gel-purified.
Table 4.1 Nucleotide sequences of primers for PCR amplification of HCAb VHH
genes.
Type Name Nucleotide Sequencea
PCR Isolation and Amplification
Forward
MJ1.2.3
MJ1 Back 5’-GCCCAGCCGGCCATGGCCSMKGTGCAGCTGGTGGAKTCTGGGGGA-3’
MJ2 Back 5’-GCCCAGCCGGCCATGGCCCAGGTAAAGCTGGAGGAGTCTGGGGGA-3’
MJ3 Back 5’-GCCCAGCCGGCCATGGCCCAGGCTCAGGTACAGCTGGTGGAGTCT-3’
Reverse CH2 5’-CGCCATCAAGGTACCAGTTGA-3’
CH2b3 5’-GGGGTACCTGTCATCCACGGACCAGCTGA-3’
Addition of SfiI
Forward MJ7 5’-CATGTGTAGACTCGCGGCCCAGCCGGCCATGGCC-3’
Reverse MJ8 5’-CATGTGTAGATTCCTGGCCGGCCTGGCCTGAGGAGACGGTGACCTGG-3’
Colony PCR
Forward PN2 5’-CCCTCATAGTTAAGCGTAACGATCT-3’
Reverse MJ13RP 5’-CAGGAAACAGCTATGAC-3’
a Key to symbols: M=A+C, K=G+T, S=G+C.
VHH dsDNA was ligated into pMED1 phagemid vector (Ghahroudi et al., 1997)
using the LigaFast Rapid DNA ligation system (Promega) according to standard methods.
Briefly, amplified VHH fragments were digested with SfiI restriction enzyme along with
pMED1 phagemid DNA overnight at 50°C. To reduce the probability of self-ligation of
the phagemid vector and to improve the functional size of the VHH library, pMED1 DNA
was further digested with PstI and XhoI restriction enzymes (2 hours, 37°C). Once the
ratio of insert to vector was optimized, VHH leukocyte DNA was ligated into the pMED1
phagemid vector at the SfiI restriction site prior to ligation to create the library.
92
After transformation into TG1 E. coli (Stratagene), VHH library size and diversity
were estimated by plating on LB + ampicillin (100 mg∙mL-1
) agar plates followed by
colony PCR using universal reverse primer “MJ13RP” and forward primer “PN2” (Table
4.1). 30 positive clones which produced a PCR product of approximately 600 bp (400 bp
VHH plus 200 bp oligo DNA) were nucleotide sequenced. Positive clones were also
digested with the random 4-mer restriction endonuclease enzyme BstN1 followed by
agarose gel electrophoresis. Clones were regarded as unique if the pattern of digestion
produced a DNA „fingerprint‟ that was different from other randomly selected clones.
4.3.6 VHH Library Panning
The VHH library was subjected to four rounds of panning on 96-well Maxisorp
plates. Target Ag was coated as: 20.0 µg, 17.5 µg, 15 µg, and 10.0 µg 15-DON-OVA per
well for rounds 1, 2, 3, and 4, respectively. Two replicates of target Ag were used in each
round of panning. Blank wells (one replicate) were maintained during panning by coating
wells with PBS. Antigen and control wells for all panning rounds were coated with 100
μL∙well-1
and incubated overnight at 4°C. Blocking buffer (2% w/v OVA in PBS) was
used for panning rounds 1 and 2. To limit non-specific binding for rounds 3 and 4,
blocking buffer was switched to casein (1% w/v in PBS). As well, phage from each
round of panning were also pre-incubated with the respective blocking solution (100 µL)
for 1 hour at 37°C before 100 μL of phage were added to 15-DON-OVA and PBS coated
wells. After incubation of phage for 2 hours at 37°C, antigen and control wells were
washed with PBST. Stringency of washing was increased by washing 5, 6, 7, and 8 times
with 200 µL PBST for rounds 1, 2, 3, and 4 of panning, respectively.
93
Bound phage were eluted by adding 100 µL of freshly prepared triethylamine
solution (35 µL TEA + 2.50 mL sterile H2O). Eluant was neutralized after incubation for
10 minutes by adding 200 µL of 1.0 M Tris HCl. Eluted phages after each round of
panning were used to infect 2 mL E. coli TG1 cells for subsequent amplification as
described in (Ghahroudi et al., 1997). Briefly, infected cells were incubated at 37°C for
30 min without shaking. A 2 μL aliquot was taken for phage titration, the remaining
culture volume was transferred to 6 mL 2xYT medium + ampicillin (100 μg∙mL-1
)
followed by incubation and shaking (37°C, 30-60 min). The culture was subsequently
superinfected with 900 μL of helper phage M13 K07 (1 x 10 11
cfu∙mL-1
) (New England
Biolabs) followed by incubation with no shaking (37°C, 15 min.) and incubation with
shaking (37°C, 30-60 min). Cells were transferred to 92 ml 2xYT medium containing
ampicillin and kanamycin (final concentrations of 100 mg∙mL-1
and 50 μg∙mL-1
,
respectively). After amplification of eluted phages overnight (37°C with shaking), phage
titers were estimated by plating infected cells on 2xYT + ampicillin 100 mg∙mL-1
agar
plates. Input and eluted phage were quantified by serial dilution with PBS and plating on
LB-AMP (data not shown). The number of colonies in each plate were counted and
compared with the colony numbers from subsequent rounds of panning.
4.3.7 Phage ELISA
Individual colonies obtained after four rounds of panning were tested against 15-
DON-OVA conjugate in a phage ELISA according standard procedures (Ghahroudi et al.,
1997). Briefly, clones were grown in 2xYT medium + ampicillin (100 mg∙mL-1
) + 0.1 %
glucose medium to OD600 = 0.3 – 0.5, and infected with M13K07 helper phage (37°C no
94
shaking, 30 min) followed by kanamycin (50 μg∙mL-1
) and amplification overnight (37°C
with shaking). Cultures were centrifuged (5,000 rpm, 20 min., 4°C) to pellet the cells,
followed by the addition of 100 μL of supernatant (recombinant phage particles) to pre-
coated microtiter plate wells. Treatments were two replicates of 15-DON-OVA (0.5
μg∙well-1
in PBS or 0.15 M carbonate-bicarbonate buffer). Blank wells were OVA (2.0
μg∙well-1
PBS) and BSA (2.0 μg∙well-1
PBS). Negative control was 100 μg∙well-1
of
M13K07 in PBS. After 2-hour incubation at 37°C, microtiter plate wells were washed
five times with PBST followed by addition of anti-M13 HRP conjugate (1:5000). VHH-
phages bound to DON were detected by the addition of 100 μL of HRP substrate (KDL)
followed by colourimetric development and assessment as previously described.
4.3.8 Colony PCR and Sequencing
Universal primers M13R and PN2 were used to amplify the VHH fragments of the
positive clones determined by phage ELISA. Sequencing was carried out using the ABI
PRISM™ Big Dye® Terminator Cycle Sequencing Reaction Kit (Perkin-Elmer,
Wellesley, MA) on a 373 DNA sequencer apparatus (Perkin-Elmer). Sequencing data
were aligned and numbered according to the Kabat numbering system (Kabat et al.,
1991). The unique nucleotide sequence for the dominant clone (repeated in 38 of 100
clones) that bound to 15-DON-OVA and not to OVA control was termed “NAT-267”.
4.3.9 Monomer Expression and Purification
Restriction enzyme sites BbsI and BamHI were added to the 5‟ and 3‟ ends of the
NAT-267 VHH DNA fragment using a PCR involving gene-specific sense primer VHH-
BbsI (5‟-TATGAAGACACCAGGCCCAGGTGCAGCTGGTGGAGTCT-3‟) and anti-
95
sense primer VHH-BamHI (5‟-CGCGGGATCCTGAGGAGACGGTGACCTGGGT-3‟).
The amplified DNA was then digested with BbsI and BamHI restriction enzymes and
ligated into digested pSJF2 vector using standard techniques (Tanha et al., 2003).
Competent E. coli TG1 cells were transformed and clones expressing DON-specific
recombinant VHH were grown in 1-liter cultures of 2xYT medium + ampicillin (100
mg∙mL-1
) with 0.1% glucose to an OD600 of 0.8. Cultures were induced with 0.5 mM
IPTG and grown overnight on a rotary shaker at 28°C. After confirmation of expression
by SDS-PAGE and Western blotting, recombinant mNAT-267 VHH protein was extracted
from the bacterial periplasm by standard methods (Anand et al., 1991) and purified by
immobilized metal affinity chromatography (IMAC) and quantified as described
elsewhere (MacKenzie et al., 1994). The state of aggregation of the purified protein was
checked by size exclusion chromatography on Superdex 200 (Amersham Biosciences).
4.3.10 Pentamerization of VHH Monomer
Restriction enzyme sites BbsI and ApaI were added to the 5‟ and 3‟ ends of the
NAT-267 VHH DNA fragment using a PCR involving the gene-specific sense primer
VHH-BbsI and antisense primer VHH-ApaI (5‟-ATTATTATGGGCCCTGAGGAGACG
GTGACCTGGGTC-3‟). Amplified NAT-267 DNA was digested with BbsI and ApaI
restriction enzymes and ligated into digested pVT2 using techniques as described in
(Zhang et al., 2004). A VHH pentamer of the dominant clone was formed by fusion of
monomer DNA to the verotoxin B subunit encoded within the pVT2 expression vector.
pNAT-267 DNA was then used to transform E. coli TG1 according to standard
techniques.
96
4.3.11 Pentamer Expression and Purification
The same conditions which were used to express the mNAT-267 VHH protein, were
applied for expression of pNAT-267 VHH. In accordance with standard protocols (Zhang
et al., 2004), expressed proteins were extracted by whole-cell lysis. Briefly, cells were
pelleted by centrifugation (20 min, 6,000 xg, 4°C) and resuspended in 25 mL of ice-cold
lysis buffer (10 mM Hepes, 500 mM NaCl; 20 mM Imidazole; pH 7.4) followed by
centrifugation (1 hour, 6,000xg, 4°C). Supernatants were filtered (0.22 µm Millipore
filter) and protein extracts were centrifuged again (1 hour, 6,000 xg, 4°C). After
confirmation of protein expression by SDS-PAGE and Western Blotting, pentabodies
were purified by IMAC as described above. The state of aggregation was checked by size
exclusion chromatography on Superdex 200 (Amersham Biosciences).
4.3.12 Determination of Binding Affinity
4.3.12.1 Fluorescence Polarization Assay
DON-fluorescein tracer was prepared according to standard methods (Maragos et
al., 2002). Briefly, DON (0.625 mg in 100 μL acetone) was added to 1,1-carbonyl-
diimidazole (2 mg) and allowed to mix (2 hours, 24C). 6-aminofluorescein (10 mg) was
dissolved in 1 mL sodium carbonate (1 mL, 0.1 M). 6-aminofluorescein (100 µL) was
added to DON-1,1-carbonyl-diimidazole, final volume 200 μL. After reaction for 48
hours at 2 - 8C, DON– fluorescein solution (20 μL; approx. 20 mM) was spotted onto
LK6F silica gel plates (1,000 m Preparative layer thickness 20 x 20 cm; Whatman)
along with samples of DON (20 mM), DON-1,1-carbonyl-diimidazole (20 mM), and 6-
aminofluorescein (20 mM), as reference standards. Thin layer chromatography (TLC)
97
plates were developed in chloroform:methanol:acetic acid (90:10:1 v/v/v). Each product
visualized at 366 nm was scraped from the plate into a vial containing 0.2 mL methanol
(2 - 8C) incubated over-night and stored at -20C until required.
Fluorescence polarization was measured with a Perkin Elmer Envision 2100
multilabel reader. To determine the operating dilution of fluorescent tracer within the
assay, DON-fluorescein was serially diluted 1:2 in 1 mg∙mL-1
BSA from 1:100 to
1:102,400 and analyzed by fluorescence polarization (100 μL∙well-1
) with an excitation
wavelength of 488 nm and an emission wavelength of 530 nm. The operating dilution of
DON-fluorescein conjugate was the dilution yielding a total fluorescence within the linear
range of the above titration, nearest the lower plateau to represent the lowest accurately-
measurable concentration of DON-fluorescein conjugate, thus requiring the least amount
of VHH to saturate the system. To determine the dilution of mNAT-267 and pNAT-267
VHH fragments, DON- fluorescein conjugate was diluted in 1 mg∙mL-1
BSA and mixed
with serial dilutions of VHH in PBS. These mixtures were allowed to react (10 min.,
22C) in the dark and followed by fluorescence polarization (100 μL∙well-1
) analysis with
excitation and emission wavelengths of 488 nm and 530 nm, respectively.
A standard curve of competition between DON-fluorescein (1:8,000 dilution in
PBS) and free DON in solution at concentrations ranging from 5 μg∙mL-1
to 9.8 ng∙mL-1
was prepared using a competition assay. VHH antibody fragments were diluted 1:50 (v/v)
in PBS containing DON serially diluted 1:2 (v/v) from an initial concentration of 10
μL∙mL-1
(six replicates for each concentration). Mixtures were allowed to react for 1
hour at 22C after which they were applied (80 μL∙well-1
) to a black 96-well microtitre
98
plate. DON-fluorescein was diluted 1:640 (v/v) in 1 mg∙mL-1
BSA and added (20
μL∙well-1
) to the serum-DON solution in each well. Mixtures reacted for 10 minutes at
22C in the dark and were analyzed by FPA. Cross reactivity with other trichothecene
mycotoxins (i.e., 3-AcDON, 15-AcDON, DAS, T2, and NEO) was determined by
substitution of DON with each mycotoxin within the serial dilutions as previously noted.
4.3.12.2 Surface Plasmon Resonance
The interaction of mNAT-267 VHH and immobilized 15-DON-HRP conjugate was
determined by SPR using the BIACORE
3000 biosensor system (Biacore, Inc.,
Piscataway, NJ). Data were analyzed with BIAevaluation 4.1 software. Approximately
500 resonance units (RUs) of 15-DON-HRP conjugate and 190 RUs HRP (control) were
immobilized on a CM5 research grade sensorchip (BIACORE), respectively.
Immobilizations were carried out at protein concentrations of 50 μg∙mL-1
for conjugate
and 200 μg∙mL-1
for HRP in 10 mM acetate (pH 4.0) using an amine coupling kit
supplied by the manufacturer. Before analysis, the mNAT-267 VHH was passed through
a Superdex 75 (GE Healthcare) column. mNAT-267 VHH was injected over HRP
reference and 15-DON-HRP surfaces at concentrations of: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10
μM for 6 sec. All analyses were carried out at 25ºC in 10 mM HEPES, pH 7.4,
containing 150 mM NaCl, 3 mM EDTA and 0.005% surfactant P20 at a flow rate of 100
μL∙min-1
with 6-sec pulse injection of mNAT-267 VHH followed by 6-sec. injection of
10 mM sodium borate, pH 8.5, in 1 M NaCl (BIACORE) for regeneration. Steady state
binding (Req) at each concentration of VHH monomer was determined for KD analysis.
99
To evaluate competitive inhibition of mNAT-267 to free DON, 15-DON-HRP
conjugate and 190 RUs of HRP (control) were immobilized on a CM5 research grade
sensorchip (BIACORE) as noted above. All analyses were conducted at 25°C in 10 mM
HEPES, pH 7.4, containing 150 mM NaCl, 3 mM EDTA and 0.005% surfactant P20 at a
100 μL∙min-1
flow rate with 6 sec pulse injection of VHH monomer in absence or presence
of free DON (50 and 500 μM).
pNAT-267 VHH and control pentabody „C3C-BSA8‟ were captured on a Ni2+
charged sensorchip NTA (BIACORE) at densities of 10,000 RUs and 6,000 RUs,
respectively. DON toxin was injected over both surfaces at a flow rate of 20 μg∙min-1
at
concentrations ranging from 0.1 - 2 mM. Pentamers were regenerated with 1-min
injection of 350 mM EDTA in 10 mM HEPES pH 8.5 containing 150 mM NaCl and
0.005% P20 surfactant. All analyses were conducted at 25ºC in 10 mM HEPES, pH 7.4
containing 150 mM NaCl and 0.005% surfactant P20.
4.3.12.3 Competitive Inhibition ELISA
Microtiter plates were coated with either 15- DON-OVA conjugate (0.5 μg∙well-1
,
0.15 M bicarbonate buffer) or OVA (2.0 μg∙well-1
, PBS) and blocked with 1.0% casein
(PBS) (2 hours, 37°C). Eleven serial dilutions of DON ranging from 0.005 to 10.00
μg∙mL-1
(17 nM to 34 µM, respectively) were prepared in PBS. Purified soluble mNAT-
267 or pNAT-267 VHH (100µL) were added in equal volumes to each dilution series for a
final DON concentration ranging from 0.0025 to 5 μg∙mL-1
(or 8.4 nM to 17 µM,
respectively). After vortexing and incubation (2 hours, 37°C), DON–VHH solutions were
added to the ELISA-plate wells and incubated (1 hour, 37°C) followed by five washes
100
with PBST. Rabbit anti-6 His-HRP conjugate (100 µL; 1:5,000 in PBS) was added to
each well followed by incubation (1 hour, 25°C). HRP substrate (KPL, Gaithersburg,
MD) was added followed by the addition of 1 M H3PO4. Absorbance at (450) nm was
measured with an ELISA plate reader. Cross reactivity with other trichothecene
mycotoxins (i.e., 3-AcDON, 15-AcDON, DAS, T2, and NEO) was tested by substitution
of DON-standards with each mycotoxin using the serial dilutions previously noted.
4.4 Results
4.4.1 Polyclonal Response
4.4.1.1 Direct ELISA
ELISAs using polyclonal llama sera sampled either 42 or 70 days after initial
immunization with 15-DON-BSA + TiterMax Adjuvant confirmed a robust humoral
response over a dilution series ranging from 1:1 to 1:2,097,152 (Figure 4.1). Polyclonal
titer, expressed as 50% of maximum absorbance for pre-immune, 42, and 70 day sera
were estimated to be 1:250; 1:70,800; and 1: 56,200, respectively. Overall response was
regarded as mycotoxin-specific as the response of polyclonal sera to control antigen (2%
OVA) was markedly less than to wells coated with 15-DON-OVA (Figure 4.1).
101
0.0
0.5
1.0
1.5
2.0
0.00000010.0000010.000010.00010.0010.010.11
Log of Dilution
Absorbance
(450 nm)
Pre: 15-DON-OVA
42d IgG: 15-DON-OVA
72d IgG: OVA Control
72d IgG: 15-DON-OVA
Figure 4.1 ELISA of polyclonal llama serum response to 250 μg 15-DON-BSA
conjugate. 42-and 70-day serum samples each taken one week after third
and fifth immunizations, respectively. X-axis shows dilutions from 1 to
1:2,097,152 in PBS. Antigen wells were coated with 15-DON-OVA (0.5
μg•well-1
). Control wells were coated with OVA (2.0 μg·well-1
).
4.4.1.2 Fluorescence Polarization
TLC of DON-fluorescein conjugate yielded three distinct bands (Rf = 0.29, 0.24 and
0.18). Llama anti-DON serum had affinity for each conjugate using FPA (data not
shown). DON-fluorescein conjugate corresponding to Rf = 0.29 was used for the
development of an optimized FP assay (FPA) since it was the most plentiful product and
had the most consistent results.
A competitive polyclonal FPA was established with 56-day polyclonal serum based
on a plot of polarization/maximum polarization observed (i.e. P/Po) against DON
concentration. The linear working range of the FPA was 10 – 5,000 ng∙mL-1
with limits
of detection and quantitation of 19 ng∙mL-1
and 50 ng∙mL-1
, respectively (data not
shown). The IC50 from the competitive FPA to DON was 229 ng∙mL-1
, a value which is
102
significantly higher than previously published values based on similar polyclonal assays
to assess hapten binding (e.g., Furzer et al., 2006; Maragos et al., 2002).
4.4.2 HCAb and cIgG Response
4.4.2.1 Protein A and G Chromatography
Fractionation of pre-immune, 42- and 70-day serum samples confirmed an increase
in HCAb and cIgG content after immunization with 15-DON-BSA. A greater proportion
of HCAb relative to cIgG was noted within fractionated pre-immune serum, while
immunization with 15-DON-BSA increased the relative proportion of cIgG in the 42- and
70-day hyper-immunized sera (Table 4.2). Absolute and relative quantities of HCAb and
cIgG fractions were in agreement with published values (Muyldermans, 2001; van der
Linden et al., 2000).
Table 4.2 HCAb and cIgG fractions of llama serum. Volumes of pre-immune, 42-
day and 70-day samples = 12.1, 12.5 and 15.5 mL, respectively.
IgG G1 (HCAb) G2 (cIgG) A1 (HCAb) A2 (HCAb)
Pre 42d 70d Pre 42d 70d Pre 42d 70d Pre 42d 70d
Vol. (mL) 2.3 2.5 2.8 6.0 6.4 8.5 1.8 1.9 2.1 2.0 1.8 1.7
Ratio* (% v/v) 19.0 19.8 18.3 49.6 50.8 56.4 14.9 15.1 14.0 16.5 14.3 11.3
Conc. (mg·mL-1
) 0.11 0.17 0.13 0.05 0.71 0.21 0.10 0.10 0.14 0.16 0.12 0.16
Amount (mg) ** 0.25 0.43 0.36 0.30 4.54 1.79 0.18 0.19 0.29 0.32 0.22 0.27
* % v/v ratio calculated as volume of fraction ÷ total volume.
** Amount = Conc. (mg·mL-1
) x Vol. (mL).
4.4.2.2 ELISA on Polyclonal Serum Fractions
Relative to pre-immune fractions, both cIgG and HCAb fractions from 42- and 70-
day sera had significantly greater binding to 15-DON-OVA than to OVA. The cIgG
103
fractions of 42- and 70-day serum samples had the greatest overall binding to 15-DON-
OVA. However, HCAb fractions also showed significant binding to 15-DON-OVA (data
not shown). Based on the positive HCAb ELISA results, a recombinant VHH phage-
display library was constructed using HCAb cDNA from both the 42- and 70-day sera.
4.4.3 Library Construction
The initial step in VHH library construction was PCR amplification of a mixture of
42- and 70-day variable heavy chain leukocyte DNA. Sense primer mixture “MJ1.2.3
Back” and hinge-specific anti-sense primer mixture “CH2b3 + CH2” (Table 4.1) annealed
to conserved regions of cIgG and HCAb DNA to produce PCR fragments of 620 – 690 bp
and 900 bp, respectively. VH/VHH–specific primers MJ7 and MJ8 (Table 4.1) amplified
620 – 690 bp HCAb DNA and to add SfiI restriction sites to produce 400 – 450 bp PCR
products which were gel purified and ligated into pMED1 phagemid. Library diversity
was determined by sequencing of 30 recombinant clones and by concurrent digestion of
PCR-amplified VHH fragments from individual colonies with 4-mer BstN1 restriction
endonuclease. Library size was determined to be approx. 109 primary recombinants.
4.4.4 Panning and Phage ELISA
The recombinant phage-displayed VHH library was panned against 15-DON-OVA
and OVA (control) through four rounds of panning. Favorable ratios of input and output
phage specific to the target mycotoxin were maintained through a continuous reduction in
15-DON-OVA conjugate coating for each round of panning and a switch in blocking
agents from OVA to casein between panning rounds two and three.
104
Phage ELISA conducted on ten randomly selected clones from the fourth round of
panning revealed three clones (phage # 2, 6, and 7) that bound to 15-DON-OVA, but not
to control proteins (i.e., OVA, BSA). Further phage ELISA screening of 100 randomly
selected output clones from the fourth round of panning produced 38 clones that bound to
15-DON-OVA, but not to control proteins, which suggested that ~ 40% of the phage
clones were hapten-specific (data not shown).
4.4.5 VHH Sequence Results
Nucleotide sequencing of positive phage ELISA clones revealed a single dominant
nucleotide sequence („NAT-267‟) encoding a VHH which bound to 15-DON-OVA, but
not protein controls. Relative to previously published VHH sequences, the NAT-267
sequence was unique (Figure 4.2).
FR-1 (1-25)
CDR-1 (26-35)
FR-2 (36-49)
CDR-2 (50-65)
1 10 20
.........│.........│.....
30
....│.....
40
....│.........
50 60
│..a.......│.....
QVQLEESGGGLVQAGGSLRLSCAAS GRTSSNTFVG WFRQAPGKEREFVA AIRRSDDRTYYAASVRG
FR-3 (66-94)
CDR-3 (95-102)
FR-4 (103-113)
70 80 90
....│.........│..abc.......│....
100
.....│abcdef..
110
.......│...
RFTISGDSAKNVVALQMSSLRPEDTAVYYCAA TRTWLVTGQSDYPY WGQGTQVTVSS
Figure 4.2 Amino acid sequence of NAT-267 VHH from fourth round of panning.
Recombinant VHH CDR (underlined) and FR delimitations as well as
amino acid insertions (52a, 82a,b,c, 100a,b,c,d,e,f) defined according to
Kabat numbering scheme (Kabat et al., 1991).
Although the sequence of the Framework Region (FR) was similar to that of the
hapten binder „RR6-R2‟ (Spinelli et al., 2001), residues within the three complementarity
determining regions (CDR) were novel. As commonly observed for VHH hapten binders,
105
NAT-267 did not contain cysteine residues within CDR 1 or 3, which is commonly
believed to serve as the basis of an interloop disulfide bridge within recombinant VHH
protein structure (Figure 4.2). Except for clones that bound only to control proteins, all
VHH sequences isolated had relatively long predicted CDR-3 regions (12 – 18 amino
acids) (data not shown).
4.4.6 Expression of Soluble VHH Protein
Recombinant protein fractions of approximately 16 kDa in size, containing the
mNAT-267 VHH were pooled. From the absorption measurement at 280 nm, a yield of 3
to 6 μg of purified protein per liter of E. coli culture was calculated. The purified protein
was concentrated, by ultrafiltration, to 1.3 mg∙mL-1
in PBS; no aggregation was observed.
Purification of pNAT-267 VHH of approximately 130 kDa in size was successful
using standard IMAC procedures. Aggregated pentabodies were removed by size
exclusion chromatography on Superdex 200 column. Overall yield of pNAT-267 was
determined to be 3 to 9 μg∙L-1
of E. coli culture. Ultrafiltration was used to obtain a final
pentamer concentration of 1.0 mg∙mL-1
in PBS; no aggregation was observed.
4.4.7 VHH Affinity Measurements
4.4.7.1 Fluorescence Polarization
Initial FPA analysis based on step-wise titrations of both mNAT-267 and pNAT-
267 confirmed identical binding of both recombinant proteins to DON-fluorescein tracer
diluted 1:8,000 in skim milk (1 mg∙mL-1
) (data not shown). Plots, based on P/PO versus
concentration of free hapten, were used to determine IC50 values, or concentration
required for 50% inhibition of binding. Competitive FP inhibition assays determined an
106
IC50 value of 0.82 μM for free DON versus 56-day polyclonal llama serum (Figure 4.3A).
Neither the monomer nor pentamer bound to soluble DON. Likewise, FP inhibition with
3-AcDON determined an IC50 value of 0.62 μM for 56-day polyclonal llama serum, but
no binding for NAT-267 monomer or pentamer (Figure 4.3B). FP inhibition with 15-
AcDON determined an IC50 value of 1.42 μM for 56-day polyclonal serum, and IC50
values of 1.24 and 0.50 μM for NAT-267 monomer and pentamer VHH, respectively
(Figure 4.3C). FP inhibition assays with diacetoxyscirpenol (DAS), neosolaniol (NEO),
and T-2 trichothecene toxins indicated no specific binding to polyclonal serum, mNAT-
267 or pNAT-267 VHH (data not shown).
4.4.7.2 Surface Plasmon Resonance
Binding kinetics of mNAT-267 VHH binding to 15-DON-HRP were determined by
SPR (BiaCore™) analysis. Monomer was injected over HRP control and 15-DON-HRP
bound to a SPR sensor chips at concentrations of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 μM to
generate an overlay sensogram of mNAT-267 binding to 15-DON-HRP conjugate (Figure
4.4). Trace amounts of aggregate mNAT-267 VHH contamination were likely responsible
for second phase binding to 15-DON-HRP conjugate, which could be seen on sensorgram
traces as trends with slightly positive slope. To minimize this effect, data were collected
at the highest flow rate (100 μL∙min-1
), and binding kinetics were measured as soon as
first phase binding reached steady state, or „Req‟ (Figure 4.4).
107
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.00.00
0.25
0.50
0.75
1.00
1.25Serum
Monomer
Pentamer
Log[DON ng/mL]
P/P
o
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.00.00
0.25
0.50
0.75
1.00
1.25Serum
Monomer
Pentamer
Log[DON ng/mL]
P/P
o
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.00.00
0.25
0.50
0.75
1.00
1.25Serum
Monomer
Pentamer
Log[DON ng/mL]
P/P
o
Figure 4.3 FP inhibition of mNAT-267 and pNAT-267 VHH binding and 56-day
polyclonal llama serum binding to DON-fluorescein tracer with DON
toxin (A); 3-AcDON (B); and 15-AcDON (C) from 5 μg∙mL-1
to 9.8
ng∙mL-1
. IC50 of llama serum binding to: DON; 3-AcDON; and 15-
AcDON = 0.82 μM; 0.62 μM; and 1.42 µM, respectively. IC50 for mNAT-
267 and pNAT-267 binding to 15-AcDON = 1.24 μM and 0.50 μM,
respectfully. For each assay, error bars represent ±1 SD, number of
replicates = 6. DON- fluorescein tracer within each assay diluted 1:8,000
in PBS.
A
B
C
108
0 5 10 15 20
0
50
100
150
200
Time (s)
Re
sp
on
se
(R
U)
Figure 4.4 Overlay sensorgrams of mNAT-267 VHH steady state (Req) binding to 15-
DON-HRP conjugate. mNAT-267 VHH injected over HRP reference and
15-DON-HRP surfaces at the concentrations of 1 (lowest trace), 2, 3, 4, 5,
6, 7, 8, 9 and 10 (highest trace) μM for 6 seconds.
An assessment of steady state binding (Req) relative to concentration of mNAT-267
VHH confirmed first-order binding kinetics (Figure 4.5A). A Scatchard plot of Req
binding at each mNAT-267 VHH concentration relative to actual binding demonstrated
binding to immobilized 15-DON-HRP with a dissociation constant (KD) of 5 µM (Figure
4.5B). Independent association and dissociation rates were too fast to calculate.
Free DON (50 and 500 μM) was mixed with mNAT-267 VHH (5 μM) to test
competitive inhibition of monomer binding to bound 15-DON-HRP. No competitive
inhibition was observed indicating that mNAT-267 had no affinity for soluble DON.
This observation was further confirmed by SPR analysis of the interaction between
pNAT-267 VHH and soluble DON. After capture of pNAT-267 to the SPR sensor chip,
no binding interaction was noted between the pentamer and concentrations of soluble
DON ranging from 0.1 to 2 mM (data not shown).
Req
109
0 2 4 6 8 10 120
50
100
150
200
Conc (M)
Ste
ad
y s
tate
bin
din
g (
RU
)
0 25 50 75 100 125 150 1750
10
20
30
40
50
Steady state binding (RU)
Ste
ad
y s
tate
bin
din
g/C
(R
U/
M)
Figure 4.5 (A) Steady state (Req) affinity binding (RU) mNAT-267 VHH to 15-DON-
HRP conjugate for ten monomer concentrations, as shown in Figure 4.4.
(B) Scatchard plot of steady state „Req‟ binding (RU) relative to Req / C
(mNAT-267 VHH concentration) (RU·μM-1
). Dissociation constant (KD)
= 5 μM.
SPR analysis was not used to assess interaction of mNAT-267 or pNAT-267 to 3-
AcDON and 15-AcDON. Unfortunately, it was not possible to prepare functional
solutions since the relative water solubilities of these acetylated toxins were much lower
than DON. Furthermore, it was impractical to dissolve the acetylated toxins in organic
solvent (e.g., acetone) followed by dilution in PBS since the solvent adversely affects
SPR measurement relative to control assessments.
110
4.4.7.3 CI ELISA
Checkerboard immunoassay titrations confirmed optimum binding of NAT-267
VHH (relative to OVA controls) based on dilution of monomer and pentamer at 0.38
μg∙mL-1
within ELISA wells coated with 15-DON-OVA at 0.5 μg∙well-1
. Although CD
ELISA consistently showed that 15-AcDON inhibited mNAT-267 and pNAT-267
binding to 15-DON-OVA, it was not possible to precisely define the respective IC50
values below 10 µM (data not shown). Binding of monomer and pentamer to 15-DON-
OVA coated plates was not affected by competition with serial dilutions of DON or 3-
AcDON at concentrations ranging from 8.4 nM (0.0025 μg∙mL-1
) to 17 μM (5.0 μg∙mL-1
).
4.5 Discussion
DON toxin and its acetylated metabolites are low molecular weight (296 to 338 Da)
highly stable trichothecene mycotoxin contaminants of maize and small grain cereals
(Desjardins, 2006). Trichothecene mycotoxins, a group of over 148 related compounds,
are known to cause severe toxicosis in humans and farm animals. For example, swine
vomit and display a wide variety of other symptoms of toxicosis including
immunosuppression, after consumption of contaminated feed (Canady et al., 2001). Most
countries have legislation and strict guidelines regarding acceptable levels of mycotoxins
in contaminated cereals (Rotter et al., 1996).
Immunoassay techniques that can detect and quantify DON and structurally-similar
mycotoxins use polyclonal serum and monoclonal antibodies. These assays provide
highly accurate mycotoxin measurement. However, the development of toxin-specific
immunoglobulins is difficult since it typically involves the repeated immunization of
111
animals and the use of highly specialized, laborious techniques to produce specific
polyclonal sera or mAbs with high specificities and affinities.
The development of toxin-specific recombinant antibody (rAb) fragments such as
single-chain variable fragment (scFv) and VHH provides an alternative means of
producing low-cost and abundant quantities of high affinity binders within common
recombinant expression hosts. Although scFv fragments often display binding affinities
equivalent to the parent mAb (e.g., Wang et al., 2007), in practical terms, difficulties in
obtaining purified protein, and challenges associated with protein degradation and the
formation of insoluble aggregates and inclusion bodies within bacterial cultures (Choi et
al., 2004), has limited large-scale application of this technology (Arbabi-Ghahroudi et al.,
2005). We pursued the development of DON-specific single domain VHH fragments as
an alternative to the conventional IgG mAb and scFv rAb formats, as VHH are smaller
and generally have favorable expression from microbial cells.
This study describes the successful isolation and cloning of a single-domain VHH
gene from a hyperimmunized llama library, followed by soluble expression as a
monomeric and pentameric VHH protein, and characterization of binding kinetics to
various DON toxins and their respective protein conjugates. To our knowledge, this is
the first example of the development of a single-domain VHH binder to a mycotoxin.
Before VHH library construction and panning, we confirmed a robust and toxin-
specific response to immunization with 15-DON-BSA within peripheral leukocytes
(Figure 4.1). Serial dilution of whole and cIgG and HCAb fractionated serum tested by
ELISA confirmed a polyclonal response specific for 15-DON-OVA. An initial
112
competitive FP inhibition assay confirmed that whole llama serum had affinity for free
DON toxin (IC50 = 229 ng·mL-1
). Based on these results, we concluded that the methods
and reagents used to generate a polyclonal response were optimized. Although the
fractionated llama serum demonstrated a greater increase in cIgG content relative to
HCAb fractions (Table 4.2), we concluded that the HCAb response was pronounced
enough to warrant construction of a hyper-immunized VHH library.
Peripheral leukocytes were used as sources of mRNA for the cloning of VHH coding
sequences to construct the hyperimmunized library (reviewed in Muyldermans, 2001).
Variable regions of HCAb and IgG immunoglobulins were amplified by nested PCR
based on hinge-specific primers. VHH-specific PCR products were excised from an
agarose gel and re-amplified with hinge-specific primers. The latter primers added the
required SfiI restriction endonuclease enzyme site onto amplified VHH DNA to enable
digestion and ligation into the pMED1 phagemid system. Results based on colony PCR,
DNA fingerprinting, and successive plate dilutions of transformed TG1 E. coli showed
the VHH library to be diverse and of sufficient size to proceed with biopanning.
Four rounds of panning against decreasing concentrations of 15-DON-OVA coupled
with increased washing stringency yielded a single dominant VHH clone (termed NAT
267) at a frequency of 38%. Subsequent phage ELISA confirmed that NAT-267 bound to
15-DON-OVA with little or no binding to OVA (control). The panning process was
deemed successful despite the fact that it was not based on a process of affinity selection
through elution with free DON hapten which may have improved the selection of high
affinity toxin-specific VHH binders (Sheedy et al., 2007).
113
The NAT-267 VHH gene was well expressed in monomeric and pentameric forms
within E. coli. The relatively high concentrations of expressed VHH are comparable to
the values published for scFv and Fab (Maynard and Georgiou, 2000). The high yield of
monomer and pentamer forms of NAT-267 was attributed to the soluble and inherently
robust nature of recombinant VHH proteins (Ghahroudi et al., 1997; Goldman et al., 2006;
Ladenson et al., 2006). Direct ELISA assays confirmed that soluble VHH monomer and
pentamer bound to 15-DON-OVA, but not to OVA (control). Given identical microtiter
plate dilution regimes, the ELISA signal for the pentamer was consistently higher than for
equivalent monomer concentrations. This result suggested that the dominant VHH clone
was selected based on avidity of a multivalent phage during panning to hapten-protein
conjugate rather than affinity to free hapten, which is a limitation when panning with
multivalent Ab-displaying phage.
A series of assays to determine VHH monomer and pentamer binding specificity
confirmed that purified NAT-267 monomer and pentamer fragments recognized 15-
AcDON (Figure 4.3C) but not DON (Figure 4.3A), 3-AcDON (Figure 4.3B), or the other
trichothecene toxins tested (diacetoxyscirpenol, neosolaniol, and T-2 toxin).
The free hydroxyl group at position 15 of DON and 3-AcDON may have interfered
with NAT-267 VHH binding. Conversely, the presence of an acetyl ester group (≈ 42 Da)
at position 15 of the trichothecene structure (Figure 4.6) may have altered the molecular
structure and conformation to impart VHH binding to 15-AcDON. Given that the DON
toxin was conjugated to carrier proteins by a hemi-succinate linker at position 15 for both
the antigen (15-DON-BSA) and the coating conjugate (15-DON-OVA) used for panning,
114
it was postulated that the VHH paratope recognized an epitope at or in close proximity to
position 15 of the DON molecule.
Trichothecene MW
a
(Da)
Oxygenation and Esterification at Positionb
C-3 (R1) C-4 (R2) C-7 (R3) C-8 (R4) C-15 (R5)
3-Acetyl-DON 338 OAc H OH =O CH2-OH
15-Acetyl-DON 338 OH H OH =O CH2-OAc
Deoxynivalenol 296 OH H OH =O CH2-OH
Diacetoxyscirpenol 366 OH OAc H H CH2-OAc
Neosolaniol 382 OH OAc H OH CH2-OAc
T-2 Toxin 466 OH OAc H OIsoval CH2-OAc a MW = Molecular Weight
b OAc = acetyl ester. OIsoval = isovalerate ester.
Figure 4.6 Structure, composition, and molecular weight of trichothecene mycotoxins
used in this study (Desjardins, 2006).
Highly-specific VHH binding to 15-AcDON was therefore regarded as a function of
both the linker used to fix DON to carrier proteins and the panning methods, while the
physico-chemical properties of these ligands, such as water solubility and molecular size,
may have had little impact on VHH specificity. It would be interesting to test this
hypothesis by panning the VHH library against a DON-protein conjugate with different
linker chemistry and/or a different conjugation position (e.g., 3-DON-OVA).
115
Based on competitive FP assays, IC50 values determined by competition with 15-
AcDON were 1.24 and 0.50 μM for the VHH monomer and pentamer, respectively.
These values are comparable to IC50 of 1.42 µM for 15-AcDON using 56-day polyclonal
llama serum in a competitive FP assay (Figure 4.3C). Unfortunately, it was not possible
to validate FP results by CD ELISA since 15-AcDON inhibition values for both the
monomer and pentamer could not be precisely determined. The inherent difficulty in
establishing consistent IC50 values by CD ELISA was attributed to the relatively fast
dissociation rate of both the NAT-267 monomer and pentamer coupled with the
continuous removal of VHH from the non-equilibrium binding system by successive
washing steps after initial binding. Furthermore, the limited solubility of 15-AcDON
prevented SPR analysis of its binding to chip-immobilized NAT-267 VHH. However,
SPR analysis of NAT-267 monomer binding to chip-immobilized 15-DON-HRP yielded
a dissociation constant (KD) of 5 µM, which is similar to the IC50 determined in the
competitive FP assay using NAT-267 (1.24 μM).
Unlike reports associated with poor scFv expression and isolation (Choi et al.,
2004), our monomeric and pentameric VHH fragments were found to be well expressed,
stable and not prone to aggregation or formation of insoluble inclusion bodies.
Furthermore, unlike scFv for DON toxins, NAT-267 VHH was highly specific to the 15-
AcDON moiety with no cross-reactivity to 3-AcDON or DON. Such specificity could aid
in toxin-specific detection as a compliment to existing mAb assays. For example, the
mAb (IgG) developed by (Maragos and McCormick, 2000) was reported to be very
specific for DON and 3-AcDON with little or no affinity for 15-AcDON. When used in
116
conjunction this mAb-based ELISA, NAT-267 VHH would allow quantification of all
three toxins (i.e., DON, 3-AcDON, and 15-AcDON). Furthermore, the mAb-based assay
developed by Sinha et al. (1995), which detects both 15-AcDON and DON (i.e., 15-
AcDON + DON), could be used in conjunction with NAT-267 VHH to determine the
exact concentration of 15-AcDON as well as DON.
We compared NAT-267 VHH in terms of affinity and specificity with the other
„hapten-specific‟ VHH fragments that have been isolated from both naïve (Sheedy et al.,
2006; Yau et al., 2003b) and hyper-immunized llama libraries (Alvarez-Rueda et al.,
2007; Frenken et al., 2000; Ladenson et al., 2006; Spinelli et al., 2000). For example,
the indoleacetic acid (IAA)-binding VHH CSF2A, isolated from a naïve llama library, had
a KD of 20 µM for free IAA (Sheedy et al., 2006). Unlike NAT-267, CSF2A cross-
reacted to structurally-similar analogues. Furthermore, a picloram-specific VHH, 3-ID2,
isolated from a naïve ribosome-display llama library had an IC50 of 800 µM for the
hapten picloram (Yau et al., 2003b). In both cases, these naïve-library-based VHHs had
much lower affinities than did NAT-267.
Unlike the hapten-specific VHHs isolated from naïve libraries (Sheedy et al., 2006;
Yau et al., 2003b), the VHH sequences isolated from hyper-immunized llama libraries
(e.g., Alvarez-Rueda et al., 2007; Ladenson et al., 2006; Spinelli et al., 2000) were in
agreement with our work in that each VHH isolated was found to be hapten-specific with
minimum cross-reactivity to structurally-similar analogues and their IC50 values as
determined by competition with free hapten were in the µM range. For example, a
caffeine-specific VHH, VSA2, had an IC50 of ca. 154 µM with minimum cross-reactivity
117
to structurally-similar haptens (Ladenson et al., 2006). Similarly, seven methotrexate
(MTX)-specific VHHs isolated from a hyper-immunized llama library (Alvarez-Rueda et
al., 2007) had nanomolar affinities (KD = 29 - 515 nM) for MTX-BSA; however, we
estimate the affinity for the free hapten, which was not determined by the authors, to
range from 10 and 70 µM based on IC50 values (Alvarez-Rueda et al., 2007). Finally, the
KD for an azo dye (Reactive-Red-6; M.W. = 728 Da)-specific VHH, known as RR6-R2,
was determined to be 22 nM. Although the affinity of this VHH is much higher than
NAT-267, the unusually high affinity of RR6-RR2 was attributed to a co-ordination bond
between the copper of the dye and a histidine moiety in the VHH, rather than to a normal
VHH-hapten interaction (Spinelli et al., 2000). Therefore, based upon the results of these
authors (Alvarez-Rueda et al., 2007; Ladenson et al., 2006; Sheedy et al., 2006; Spinelli
et al., 2000; Yau et al., 2003b), NAT-267 has the lowest hapten-based IC50 of any hapten-
specific VHH yet discussed in the literature.
The four scaffold FR residues within the NAT-267 VHH sequence had over 80%
similarity with the VHH gene sequences of CSF2A (Sheedy et al., 2006), VSA2
(Ladenson et al., 2006), and RR6-R2 (Spinelli et al., 2000). Based on nucleotide acid
identity, NAT-267 VHH belongs to camelid VHH sub-family I (Harmsen et al., 2000).
Furthermore, each of the three CDRs of „NAT-267‟ has unique amino acid residues.
Contrary to published VHH sequences which have been shown to bind to large protein
antigens, the CDR3 of NAT-267 is not constrained by an inter-loop disulfide bond to
CDR1 since no cysteine residues were present within either region. Therefore, other than
for the disulfide bridge formed between FR Cys-22 and Cys-92 (Figure 4.2), it is unlikely
118
that other disulfide bonds are present within NAT-267. Based on these observations, we
concluded that the CDR1 to CDR3 inter-loop disulfide bonds present within many
published sequences of VHHs are important for facilitating VHH binding to large
antigenic proteins but not to low molecular weight haptens (i.e. < 600 Da). Therefore, we
hypothesize that the highly-specific binding of NAT-267 VHH to the haptens 15-AcDON
and DON-fluorescein as well as to the protein conjugates 15-DON-OVA and 15-DON-
HRP is likely conveyed by novel sequences within the non-crosslinked CDR residues.
Furthermore, since the VHH paratope is not regarded to be a flat surface (Spinelli et al.,
1996), we further hypothesized that high affinity interaction of NAT-267 VHH with the
two haptens and coating conjugates occurs within flexible grooves or cavities between the
three CDR regions (Spinelli et al., 1996; 2000; 2001).
4.6 Conclusions
This research further confirms that hapten-specific recombinant VHH fragments can
be selected from a hyper-immunized llama VHH library. Both monomer and pentamer
forms of the NAT-267 dominant clone selected by four rounds of panning was specific to
the mycotoxin 15-AcDON as determined by competitive binding assay, and there was no
cross-reactivity to closely related moieties of DON toxin. Although the dominant clone
was both novel and highly specific to an acetylated moiety of the target mycotoxin, the
nucleotide sequences for all four framework regions were similar to previously-isolated
hapten-specific VHH sequences. Further research to assess different panning strategies
and various affinity maturation strategies as a means of improving cross reactivity and
overall binding affinity are now being pursued. Furthermore, since 15-AcDON has been
119
shown to have a similar eukaryotic cytotoxicity as DON (Binder et al., 1997;
Poppenberger et al., 2003), we are focusing on expression of NAT-267 VHH in
eukaryotic test systems such as yeast and Arabidopsis thaliana to determine whether the
negative effects of the toxin may be reduced or eliminated.
4.7 Acknowledgements
The authors of this paper would like to thank Tomoko Hirama (NRCC, Ottawa,
Canada) for conducting the SPR analysis and Hong Tong-Sevinc (NRCC, Ottawa,
Canada) for expression and purification of the VHH clones. This research was supported
by the Natural Sciences and Engineering Research Council (NSERC) of Canada, the
Canada Research Chairs Program, the National Research Council (NRC) of Canada, and
the Ontario Ministry of Agriculture and Food (OMAF).
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5 RESEARCH CHAPTER 2: INTRACELLULAR EXPRESSION OF A
SINGLE-DOMAIN ANTIBODY REDUCES CYTOTOXICITY OF 15-
ACETYL-DEOXYNIVALENOL IN YEAST.
5.1 Abstract
15-Acetyl-Deoxynivalenol (15-AcDON) is a low molecular weight sesquiterpenoid
trichothecene mycotoxin associated with Fusarium Ear Rot of maize and Fusarium Head
Blight of small grain cereals. The accumulation of mycotoxins such as deoxynivalenol
(DON) and 15-AcDON within harvested grain is subject to stringent regulation as both
toxins pose dietary health risks to humans and animals. These toxins inhibit peptidyl
transferase activity which in turn limits eukaryotic protein synthesis. To assess the ability
of intracellular antibodies (intrabodies) to modulate mycotoxin-specific cytotoxocity, a
gene encoding a camelid single-domain antibody fragment (VHH) with specificity and
affinity for 15-AcDON was expressed in the methylotropic yeast Pichia pastoris.
Cytotoxicity and VHH immunomodulation were assessed by continuous measurement of
cellular growth. At equivalent doses, 15-AcDON was significantly more toxic to wild-
type P. pastoris than was DON. In turn, DON was orders of magnitude more toxic than
3-AcDON. Intracellular expression of a mycotoxin-specific VHH within P. pastoris
conveyed significant (p = 0.01) resistance to 15-AcDON cytotoxicity at doses ranging
from 20 to 100 µg∙mL-1
. We also documented a biochemical transformation of DON to
15-AcDON to account for the attenuation of DON cytotoxicity at 100 and 200 µg∙mL-1
.
The proof of concept established within this eukaryotic system suggests that in planta
VHH expression may lead to enhanced tolerance to mycotoxins and thereby limit
Fusarium infection of commercial agricultural crops.
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5.2 Introduction
Fusarium head blight (FHB) of cereals and Fusarium Ear Rot of maize are caused
by morphologically similar species (F. graminearum, F. culmorum, etc.) common
throughout global agricultural regions. With few exceptions, Fusarium epidemics are
characterized by cyclical and highly aggressive infection of commercial crops with
economic impacts on food and feed industries that are immediate and far reaching. For
example, losses associated with the most recent Fusarium outbreak in North America in
the 1990s were estimated to range from $1.3 to 3.0 billion USD (Demcey Johnson et al.,
2003).
A toxin class commonly found within agricultural commodities infected by
Fusarium are trichothecene mycotoxins. Trichothecenes represent a highly diverse group
of over 180 sesquiterpenoid low molecular weight (typically 200-500 Da) mycotoxins
characterized by a tricyclic ring structure containing a double bond at C-9,10 and an
epoxide group at C-13 (Desjardins, 2006). Regardless of size and structural composition,
trichothecenes are potent inhibitors of eukaryotic protein synthesis with specific activity
on ribosomal protein L3 within the 60S subunit resulting in inhibition of peptidyl
transferase activity (Carter and Cannon, 1977; Rocha et al., 2005). Although the
capacity to inhibit protein synthesis is regarded as central to trichothecene cytotoxicity
(Rotter et al., 1996; Ueno, 1984), adverse effects on eukaryotic cells may actually be
attributed to dysregulation of cellular signaling and alterations in downstream gene
expression (Pestka, 2008). As a result, trichothecenes such as deoxynivalenol (DON),
15-acetyl-deoxynivalenol (15-AcDON), and 3-acetyl-deoxynivalenol (3-AcDON) (Figure
122
5.1) are considered to be inherently hazardous food and feed borne contaminants
(Desjardins, 2006; D‟Mello et al., 1999).
Trichothecene MW
a
(Da)
Oxygenation and Esterification at Position
C-3 C-15
3-Acetyl-DON 338 O-CO-CH3 OH
15-Acetyl-DON 338 OH O-CO-CH3
Deoxynivalenol 296 OH OH a MW = Molecular Weight
Figure 5.1 Structure, composition, and molecular weight of trichothecene mycotoxins
used within this study (Desjardins, 2006).
Numerous studies have demonstrated a correlation between in planta DON
accumulation and Fusarium virulence in susceptible cultivars of wheat (Mesterházy,
2002) and maize (Harris et al., 1999a). Based on these findings, mechanisms which
convey innate and acquired host plant resistance to DON and other trichothecene toxins
have received considerable attention. To date, in planta trichothecene resistance has been
achieved through mechanisms which alter targeted proteins within host cell ribosomes
(e.g., Harris and Gleddie, 2001; Mitterbauer et al., 2004), promote metabolic
123
transformation to less toxic forms: e.g., DON-glucosyl conjugate (Poppenberger et al.,
2003) or to 3-AcDON (Alexander, 2008), and/or reduce intracellular concentrations to
effectively limit mycotoxin exposure to sensitive cellular targets. Collectively, such
research can be applied to impart novel mechanisms of trichothecene resistance in higher
order plants.
Yeast is well suited as a eukaryotic model organism to identify and validate
mechanisms involved in host plant resistance to mycotoxins (Mitterbauer and Adam,
2002; Mitterbauer et al., 2004; Poppenberger et al., 2003). Test systems based on yeast
offer cost-effective convenience and flexibility as one can validate a wide range of novel
detoxification mechanisms within a short period of time at a minimal cost using common
/ non-specialized lab equipment. Assessment of mycotoxin resistance mechanisms is
likewise straightforward as reproducible treatment-specific effects can be precisely
determined based on simple measurements of cellular growth and function over time.
Single-domain heavy chain antibody fragments (i.e., VHH) from the camelidae
heavy chain IgG subfamily, are among the smallest functional recombinant antibody
(rAb) fragments at 14-15 kDa. VHH fragments exhibit the same exquisite specificity as
larger immunoglobulins, with added biochemical advantages of high solubility, stability,
and robust expression in various recombinant systems (Holliger and Hudson, 2005;
Muyldermans, 2001). VHH fragments have been generated against low molecular weight
ligands (haptens) and toxins (e.g., Doyle et al., 2008). Based on favorable physico-
chemical properties and efficacy against a wide range of antigens, single domain rAb
fragments have been developed and tested as immuno-therapeutic reagents with
124
applications ranging from pharmacology (Holliger and Hudson, 2005; Saerens et al.,
2008) to plant science (Jobling et al., 2003).
This paper demonstrates that intrabody expression of a VHH fragment isolated from
a hyper-immunized phagemid library with affinity for 15-AcDON (Doyle et al., 2008) can
impart real-time immunomodulation of mycotoxin-specific cytotoxicity within a model
eukaryotic system. Pichia pastoris was selected as the host organism based on an
expected high level expression of functional VHH intrabody fragments and anticipated
sensitivity to 15-AcDON. This system was established as a „proof of concept‟ to
demonstrate that intrabody expression of recombinant VHH fragments could impart a
novel means of mycotoxin-specific resistance.
5.3 Experimental Procedures
5.3.1 VHH Genes
NAT-267 VHH DNA sequence (GenBank: EU676170.1) (Doyle et al., 2008) was
used to design a P. pastoris codon optimized version of the gene to which was added 3‟
HA and 6His epitope tags, as well as EcoRI and XbaI cloning sites at the respective 5‟
and 3‟ ends (GeneArtTM
, Toronto, ON, Canada). A non-specific VHH gene (B-24)
isolated from a hyper-immunized phagemid llama library with confirmed non-specificity
for 15-AcDON, or any other trichothecene mycotoxin, was used as a VHH intrabody
control. Both VHH fragments were of the same immunoglobulin family and had similar
molecular weights and isoelectric points. Like NAT-267, B-24 VHH DNA was PCR
amplified to include 3‟ HA- and 6His- epitope tags and restriction cloning sites EcoRI
and XbaI with the following gene-specific primers: VHH-B24For (5‟-GGAATTCCATG
125
CAGGTAAAGCTGGAGGAG-5‟) and VHH-B24Rev (5‟-GGGTCTAGACCCTATGCT
CGGCCGGAACCGTAG-3‟). A homology chart of nucleotide and peptide sequences of
NAT-267 and B-24 VHHs is provided to show respective framework (FR) and
complementarity determining regions (CDR), endonuclease sites and epitope tags (Fig.
S1 in Supplementary Information). VHH DNA inserts (~ 0.46 kb) were ligated into
pPICZB vector (Invitrogen Inc., Carlsbad, CA, USA). Constructs were sequenced by
StemCore Labs (Ottawa, ON, Canada) using pPICZB-specific primers: 5‟ AOX1For (5 ‟-
GACTGGTTCCAATTGACAAGC-3‟) and 3‟ AOX1Rev (5‟-GCAAATGGCATTCTGA
CATCC-3‟).
5.3.2 Transformation
Expression constructs (Figure 5.2) were linearized with SacI and electroporated into
P. pastoris strain KM71H (Invitrogen Inc., Carlsbad, CA, USA). Transformants
Figure 5.2 Diagram of pPICZB expression vector (3745 bp) with VHH gene (NAT-
267 = 387 bp, B-24 = 390 bp). Legend in the box shows schematic
representation for each component of transgenic DNA for expression in P.
pastoris.
were plated onto yeast extract, peptone, dextrose, sorbitol (YPDS) agar containing 100
µg∙mL-1
zeocin and incubated for 3 days at 30°C until colonies formed. Ten colonies
5’ 3’ EcoRI XbaI
5’AOX1
Promoter
VHH DNA HA-Tag
6His-Tag 3’AOX1
Terminator
Zeocin Resistance
Gene
126
from each transformant were re-streaked on yeast extract, peptone and dextrose (YPD)
agar plates with 100 µg∙mL-1
zeocin to ensure pure clonal isolates.
5.3.3 Induction of VHH Expression
Single colonies of VHH and pPICZB control transformants were used to inoculate 5
mL YPD media. Cultures were grown overnight (30°C, 300 rpm). One mL of each
culture was used to inoculate 100 mL of minimal glycerol medium with histidine
(MGYH) and cultured in 1-L baffled flasks (30°C, 250-300 rpm) for 1 day. Cells were
harvested with centrifugation (3,000g x 5 min.) at room temp. To induce VHH
expression, Pichia cells were resuspended in 20 mL of minimal methanol medium with
histidine (MMH) and transferred to 125-mL baffled flasks and incubated at 30°C with
300 rpm shaking. Methanol (100%) was added to final conc. of 0.5% (% v/v) every 24
hours. Western blot analysis of ten VHH (NAT-267 and B-24) transformants from 0, 24,
48, 72, 96, and 120 hours post-induction was used to select clones and induction time
points corresponding to the highest overall protein expression.
5.3.4 Preparation of Soluble VHH Extracts and Western Blot Analysis
Cell pellets were thawed on ice and resuspended in 100 µL lysis buffer (50 mM
Tris, pH 7.5, 1mM EDTA, 0.5% Triton-X 100, 1 mM DTT, 1X Roche‟s Complete
Protease Inhibitor Cocktail). Equal volumes of acid-washed 0.5-mm glass beads were
added to each resuspended pellet, followed by successive 30-second cycles of vortexing
and incubation on ice for a total of ten cycles. Cell lysate samples were clarified by
centrifugation (14,000 rpm x 10 min.) at 4°C. One-hundred microliters of 2X-SDS
loading buffer (BioRad Laboratories, Mississauga, ON, Canada) was added to the
127
supernatant followed by boiling (95°C x 5 min.) and electrophoresis on 12% SDS-PAGE
gels. Samples were transferred to PVDF membrane (BioRad, Mississauga, ON Canada)
and blocked overnight in 1% blocking reagent (Roche, Laval, QC, Canada) in TBS at
4°C. VHH intrabody fragments were detected by probing for 1 h with rabbit anti-HA IgG
primary antibody (Sigma, St. Louis, MO) diluted 1:1000 in 0.5% blocking buffer
followed by 2 washes with TBS + 0.1% Tween (TBST) and 2 washes with TBS + 0.5%
blocking reagent. Secondary antibody (mouse anti-rabbit IgG horseradish peroxidase
conjugate; Jackson, Immuno-Researcher Labs, West Grove, PA, USA), diluted 1:50,000
in 0.5% blocking buffer, was used to probe the membrane for 1 h, followed by 4
consecutive 15 min washes with TBST. VHH proteins were visualized with ECL Plus
Western Blotting Detection System (GE Healthcare, Baie d‟Urfé, QC, Canada).
Transformants with the highest VHH intrabody expression levels were used in subsequent
in vivo cytotoxicity assays.
5.3.5 Cytotoxicity Assays
Cells were induced as previously described. Forty-eight hours post induction, P.
pastoris cells were diluted to an OD600 of 0.25 in YPD and incubated for 4 hours (30°C,
300 rpm). After this “recovery period”, cells were diluted to an OD600 of 0.1 in YPD, and
immediately transferred (250 µL·well-1
) to an F96 microwell assay plate (Nalge Nunc
Inc., Naperville, IL, USA). Pre-calibrated concentrations of the four ribotoxin treatments
(15-AcDON, DON, 3-AcDON, or cycloheximide) prepared in DMSO and a DMSO only
control were added to P. pastoris cells contained in the wells of assay plates. All
treatments, respective controls, and cell-free wells containing only YPD media (i.e. blank
128
wells) were established in triplicate on each plate. Plates were sealed with Progene
pressure-sensitive optical sealing film (Ultident, St.-Laurent, QC, Canada) prior to
initiation of cytotoxicity assays. Cellular growth (30°C, 300 rpm) measured in real time
based on measurement of OD620 values at 25 min intervals, following 5 minutes of
shaking (600 rpm) in a Polarstar Optima Microplate Reader (BMG Labtech, GMbH,
Offenberg, Germany). Assay results were considered valid if similar and reproducible
effects were observed in three separate experiments, conducted in triplicate under the
same test parameters.
5.3.6 Data Analysis
Results were presented as P. pastoris growth curves over a 24-h time period. Mean
(n = 3) OD620 values for VHH and pPICZB (empty vector) transformants used in ribotoxin
and control treatments, minus OD620 values of YPD media (blank) wells, were plotted
against time (i.e. 25-min intervals). Standard error values and paired t-tests (p = 0.05 and
0.01) were used to assess statistically significant differences at the end of each 25-min
time interval.
Time to doubling of initial P. pastoris OD620 cellular growth values were calculated
from cytotoxicity assay data to assess relative toxicity of each ribotoxin treatment. A
comparative ranking of various ribotoxin treatments on P. pastoris growth was calculated
based on time required to double OD620 values of t = 0.0h pPICZB empty vector
transformants.
Differential area under curve (ΔAUC) values were calculated to quantify relative
differences in P. pastoris growth over the full time course of each cytotoxicity assay.
129
Relative differences in cellular growth were established by subtracting mean OD620
values of pPICZB (empty vector) wells from corresponding values for VHH-
transformants. ΔAUC values for each toxophore treatment were calculated based on
addition of differences in cellular growth between VHH and control transformants at each
25 min time interval across the full time course of each assay.
5.3.7 Quantitative Western Blot Analysis
20-mL cultures were centrifuged (1500g, 5 min, 4°C) 48 hours after methanol
induction. Culture pellets were lysed and prepared for SDS-PAGE as described above.
VHH lysate samples (500 µL) were diluted 1:5; 1:25; and 1:50 in non-reducing SDS
sample buffer (BioRad, Mississauga, ON, Canada). pPICZB vector only (control)
samples were not diluted. A reference VHH rAb fragment of a precisely-defined
concentration, and of similar size to NAT-267 and B-24 VHH with C-terminal HA and 6-
His epitope tags, was used to establish a standard dilution series at final protein
concentrations of: 300.0; 150; 75; 37.5; and 18.75 ng·lane-1
. To decrease the bias of
Western blot band intensities, VHH standard dilution series was prepared in P. pastoris
cell lysate from pPICZB empty-vector sample.
Samples were loaded onto 12.5% SDS-PAGE gels and electrophoresed followed by
transfer to a PVDF membrane (Millipore, Billerica, MA, USA) followed by a 1-h
blocking with 3% non-fat skimmed milk in PBST. Mouse anti-6His IgG primary
antibody (GE Healthcare, Piscataway, NJ, USA) diluted 1:2,500 in 3% milk was added to
membranes for 1 h followed by 1 h of blocking and 3 consecutive washes with PBST.
Alkaline phosphatase labeled goat-anti-mouse IgG conjugate (Cedarlane Laboratories,
130
Burlington, ON, Canada), diluted 1:3000 in blocking buffer, was used to probe the
membrane for 1 h followed by 3 consecutive washes with PBST. VHH proteins were
visualized using an alkaline phosphatase conjugate substrate kit (Bio-Rad Laboratories,
Hercules, CA, USA).
Quantitative Western blots were dried and photographed with an AlphaImager 3400
(Alpha Innotech Corporation, San Leandro, CA). Analysis was performed using the
AlphaEaseFc software package (Version 7.0.1, Alpha Innotech Corp.). Densometric
(Denso) values for a standard five data-point VHH dilution series (18.75 - 300 ng VHH ·
lane-1
) were used to produce regression equations. Denso values measured for 1:5; 1:25;
and 1:50 dilutions of NAT-267 or B-24 VHH samples were expressed as: „VHH mass per
µL of P. pastoris cell pellet (ng·µL-1
), based on: VHH quantity (ng) (from standard
regression equation) divided by cell pellet lysis volume loaded into each well (µL).
Cellular densities for each cytotoxicity assay were determined by multiplication of OD600
of 1.0 mL culture by 5 x 107 cells per mL culture (constant). VHH concentration (fg·cell
-
1) was calculated as VHH mass per µL cell pellet (ng·µL
-1) (above) divided by cell density
(number of cells·µL-1
culture). VHH intrabody concentration expressed as attoMole
VHH per cell (aMol·cell-1
) was calculated based on: VHH mass per cell (fg·cell-1
) divided
by molecular weight of NAT-267 or B-24 VHH proteins (16637 and 16976 Da,
respectively). Finally, intracellular VHH molarity (µMoles VHH ·L-1
) was calculated as
VHH concentration (aMol·cell-1
) divided by cell volume. P. pastoris cell volume was
estimated to be 29 femtoliters per cell (fL·cell-1
) based on average of independent
measurements of generic yeast-cell volume: 42, 37, 70, and 83 fL (Jorgensen et al., 2002;
131
Tyson et al., 1979; Sherman, 2000; and Tamaki et al., 2005; respectively) multiplied by
0.5 to account for an estimate that the cytosol represents 50% of total yeast cell volume
(Biswas et al., 2003).
Mean and standard error values of intracellular VHH expression values were
calculated based on five independent quantitative Western blot assays. Additional details
are summarized in supplemental information (Table 5.S1).
5.3.8 VHH Immunolocalization
Cultures of P. pastoris cells expressing either NAT-267 VHH or pPICZB (empty
vector) were induced with methanol (as described). Cells (20 OD600 units) were pelleted
and washed three times with PBS and resuspended in 500 µL PBS. Fifty-µL aliquots of
P. pastoris cells were incubated in 500 µL fixative solution (1:1 acetone:methanol) for 20
minutes at -20°C. Cells were pelleted (1000g x 1 min), washed three times with PBS and
resuspended in 500 µL PBS supplemented with Lyticase (5 U·µL-1
). After incubation
(30°C, 15 min.), cells were pelleted (as described) and incubated in 500 µL
permeabilization solution (3% BSA (bovine serum albumin), 0.5% Triton X-100 in PBS)
for 30 minutes at room temp. After washing with incubation solution (1% BSA, 0.5%
Tween 20), cells were probed with a 1:100 dilution of monoclonal anti-HA-FITC
antibody (Sigma-Aldrich, Oakville, ON, Canada) for 1-2 h at room temp. Cells were
mounted in PBS supplemented with DAPI (4′,6-diamidino-2-phenylindole dihydro-
chloride) stain (Sigma-Aldrich, Oakville, ON, Canada) at a final concentration of 1.3
µg·ml-1
. Cells were observed after 5 minutes using a Carl Zeiss LSM 510 Meta confocal
microscope equipped with Diode 405 nm and Argon/2 line 488 nm lasers for excitation.
132
Emission images were taken 420-480 nm and 505-565 nm for DAPI and FITC,
respectively.
5.3.9 Mycotoxin Biotransformation Assays
After 48 hours of induction (see above), cultures were diluted to an OD600 of 0.25 in
YPD and incubated for 4 h (30°C, 300 rpm). Cultures were diluted to OD600 0.1 in YPD,
and 2.5-mL aliquots of each culture were transferred to 50-mL tubes. Highly purified
DON was added to respective samples to a final concentration of 100 µg·mL-1
followed
by incubation (30°C, 300 rpm). Culture samples (0.5 mL) were taken at intervals of 30
min., 16 and 24 h after mycotoxin addition and used to measure OD600 and culture pH.
Samples were pelleted (3500g, 5 minutes). Supernatant was removed and transferred to
2.0-mL tubes. Samples were frozen in liquid nitrogen and stored at -80°C. Lysate
samples were prepared from cell pellets (as described above). All samples were split to
enable concurrent anti-HA Western blot analysis (as described above) and mycotoxin bio-
transformation assays.
Supernatant, cell lysate, and pellet of selected samples were washed with dd H2O
and passed through 0.8 µm filter (Millipore). Filtrates were passed through Chromosep
C18 columns (C18 Sep-Park Cartridge, Waters Corp., Millford, MA), pre-washed with 10
mL of 100 % (w/v) HPLC grade methanol and 10 mL of dd H2O. After washing with dd
water (10 mL), mycotoxin fractions were eluted with 10 ml of 100 % (v/v) HPLC grade
methanol and dried under a stream of N2. Selected culture samples (as described in Table
5.S2) were diluted in 0.5 mL HPLC grade methanol (100%) and 50.0-µL subsamples
were used for HPLC analysis.
133
Trichothecenes were separated on a Shimadzu HPLC system equipped with a 150 x
4.6 mm C18 column (5 µm packing size) and a Hichrome UV flow cell. Standards and
samples were eluted at a flow rate of 1.0 mL∙min
-1 with a 20 min linear gradient of 95%
solvent A (degassed dd H2O): 5% solvent B (acetonitrile) to 40% solvent A: 60% solvent
B. The column was washed from 20-25 min by ramping the gradient to 100% solvent B,
holding at 100% B for 5 min and then returning to the starting conditions over 2 min.
Assessment of DON bio-transformation was based on assessment of retention times of
sample peaks relative to trichothecene standard peaks.
GC/MS analysis, based on a standard Canadian Food Inspection Agency approved
method (Schwadorf and Müller, 1991), was used to further characterize and validate
HPLC samples for structural composition of trichothecene analytes. Biotransformation
samples from HPLC analysis were derivatized with 1.5 mg·mL-1
dimethylaminopyridine
(DMAP) in toluene:acetonitrile (95:5) solution and trifluoroacetic anhydride (TFAA).
This solution was heated at 60°C for 30 minutes, and neutralized twice with 5% KH2PO4.
Derivatized samples were analyzed on a Saturn 2000 GC/Ion-trap mass spectrometer
equipped for chemical ionization using acetonitrile.
1H NMR spectra of pooled P. pastoris lysate samples and subsequent HPLC
fractions of those samples were obtained on a Bruker AM 500 NMR spectrometer in
CDCl3. Chemical shifts are referenced to residual CHCl3 at 7.24 ppm for 1H spectra and
reported (δ) relative to TMS.
134
5.4 Results
5.4.1 Sensitivity to Ribotoxin Treatments
The structure, composition, and molecular weight of the mycotoxins used in these
experiments are shown in Figure 5.1. The sensitivity of wild-type P. pastoris to
trichothecene and cycloheximide (control) ribotoxin treatments, was governed by dose
and chemical structure tested. Treatments based on 15-AcDON resulted in the most
immediate and largest overall reduction of cellular growth (Table 5.1).
Table 5.1 Sensitivity of P. pastoris (KM71H) pPICZB transformants to various
ribotoxin treatments tested. Standard deviations (σ) for each mean value
shown in brackets.
Dose
(µg∙mL-1
)
Time to doubling of initial OD620 cellular growth (minutes)
15-AcDON DON 3-AcDON Cycloheximide L.S.D. ††
0† 205 (2.2) 221 (14.4) 219 (21.7) 221 (14.4) 14.5
20 322 (4.4) n.d. n.d. n.d. -
30 384 (4.4) n.d. n.d. n.d. -
40 439 (6.6) n.d. n.d. n.d. -
50 494 (8.8) 308 (7.2) 190 (124) 283 (7.2) 71.7
100 642 (138) 417 (47.3) 194 (119) 310 (20.1) 99.3
200 996 (312) 590 (58.1) 217 (13.0) 341 (7.2) 166.6
L.S.D. 117.2 40.2 91.3 14.0
n.d. not determined
† DMSO, ribotoxin-free media.
††
Least Significant Difference (p=0.05)
P. pastoris was comparatively less sensitive to equivalent doses of DON and
cycloheximide. It was not possible to establish a dose-specific sensitivity to 3-AcDON
135
relative to toxin-free DMSO media as cellular growth was not inhibited at the highest
concentration (200 µg·mL-1
) (Table 5.1).
5.4.2 Transformation and VHH Intrabody Expression
P. pastoris KM71H cells were successfully transformed with linearized NAT-267
(treatment) or B-24 (control) VHH DNA ligated into expression vector pPICZB (Figure
5.2). Lysate fractions of methanol-induced transformants were assessed by anti-HA
epitope Western blot analysis and robust intracellular VHH expression was confirmed by
the presence of a 17 kDa band at 48 and 72 hours post methanol-induction (data not
shown).
5.4.3 Quantitative Western Blot Assay
Mean levels of soluble intracellular VHH expression (t = 0h) transformants tested
within cytotoxicity assays were determined by quantitative western blot analysis (Figure
5.3). The initial concentration of NAT-267 VHH in P. pastoris cells within cytotoxicity
assays of 15-AcDON (Figure 5.4) was 4.01 ± 0.31 aMol VHH·cell-1
(Table 5.2).
Figure 5.3 Quantitative Western blot analysis of VHH intrabody expression within
cytotoxicity assays. Blot 1 is VHH standard dilution series from 300.0 to
18.75 ng·well-1
. Blot 2 is NAT-267 VHH expression for cytotoxicity assay
#1 (Figure 5.4). Blot 3 is NAT-267 VHH expression for cytotoxicity assay
#2 (Figure 5.5). Blot 4 is B-24 (control) VHH expression for cytotoxicity
assay #3 (Figure 5.6). BioRad Molecular weight marker (MW) indicated
on the left. Protein dilutions are shown along bottom of blots 2 to 4.
VHH Standard (ng)
300 150
75
37.5
18.8
MW
15 kDa
20 kDa
NAT-267
Assay #1
1/ 5 1/ 25 1/50
NAT-267
Assay #2
1/ 5 1/ 25 1/50
B-24
Assay #3
1/ 5 1/ 25 1/50
136
Accounting for an estimated mean P. pastoris cytosol volume of 29 fL·cell-1
(as
previously described), initial expression of NAT-267 VHH was equivalent to an
intracellular concentration of 138 ± 10.8 µMoles VHH·L-1
. Likewise, mean NAT-267
intrabody concentration within cells used in cytotoxicity assays of DON, 3-AcDON, and
cycloheximide (Figure 5.5) was 3.18 ± 0.42 aMol·cell-1
or 110 ± 14.4 µMol·L-1
(Table
5.2). The average initial concentration of B-24 control VHH intrabody in cytotoxicity
assays of 15-AcDON and cycloheximide (Figure 5.6) was 1.93 ± 0.14 aMol·cell-1
or 66.6
± 4.3 µMol·L-1
(Table 5.2).
Table 5.2 Mean VHH intrabody concentration (t = 0 h) within P. pastoris transformants
used in cytotoxicity assays (Figures: 5.4, 5.5, 5.6). Values derived from data
generated from quantitative Western blot assays (Figure 5.3) as summarized
in Table 5.S1. VHH concentration values expressed in femtogram and
attoMol VHH per cell, as well as µMol·L-1
VHH equivalent within P.
pastoris cytosol. Standard Error for all means (n = 9) is shown in
parenthesis.
VHH
Gene
Cytotoxicity
Assay
VHH Intrabody Concentration
fg·cell-1
aMol·cell-1
µmol·L-1
NAT-267 15-AcDON
(Figure 5.4) 66.8 (± 5.2) 4.01 (± 0.31) 138 (± 10.8)
NAT-267
DON,
3-AcDON
Cycloheximide
(Figure 5.5)
53.0 (± 7.0) 3.18 (± 0.42) 110 (± 14.4)
B-24 15-AcDON
Cycloheximide
(Figure 5.6)
32.8 (± 2.3) 1.93 (± 0.14) 66.6 (± 4.3)
137
5.4.4 Cytotoxicity Assays
Intrabody VHH expression did not influence the cellular growth of transformed P.
pastoris cultures in mycotoxin-free DMSO media, as OD620 values and overall growth
rate of NAT-267 and B-24 VHH transformants were similar to pPICZB empty-vector
control cells (Figures: 5.4, 5.5, 5.6).
5.4.4.1 15-AcDON
Significant differences (p = 0.01) in cellular growth were observed between NAT-
267 VHH and pPICZB control transformants grown in media spiked with 20-100 µg·mL-1
15-AcDON (Figure 5.4). The impact of NAT-267 VHH intrabody expression on cellular
growth was dependent on the concentration of 15-AcDON used. At the lowest dose (20
µg·mL-1
), growth of NAT-267 VHH expressing cells was similar to cell lines grown in
DMSO (control) media.
Time to significantly improved (p=0.01) growth of NAT-267 VHH intrabody
transformants was dose-dependent. Significant (p=0.01) differences in OD620 values
were observed after 7.5 hours of growth for cells cultured in medium supplemented with
15-AcDON at both 20 and 50 µg·mL-1
(Figure 5.4). Assays spiked with 30, 40, and 100
µg·mL-1
15-AcDON had significant differences in OD620 values after 4.6, 2.9, and 11.3
hours of growth, respectively. No differences in OD620 values were observed between
NAT-267 VHH and control transformants at 200 µg·mL-1
15-AcDON (Figure 5.4).
Differential area under curve (ΔAUC) analysis was used to quantify relative
differences in cellular growth between NAT-267 and B-24 (control) VHH transformants.
138
Figure 5.4 Cytotoxicity assays showing the effect of various concentrations (20 to 200 µg·mL
-
1) of 15-AcDON on the growth of P. pastoris pPICZB and NAT-267 VHH
transformants as measured by optical density at 620 nm (OD620). Differential Area
Under Curve (ΔAUC) values for NAT-267 VHH minus pPICZB empty vector are
shown on each graph. At all concentration of 15-AcDON (except 200 µg·mL-1
)
where blue versus red symbols are used, there was a significant difference (p=0.01)
at each time between NAT-267 and pPICZB transformants.
NAT-267, DMSO
pPICZB, DMSO pPICZB, 15-AcDON
NAT-267, 15-AcDON
pPICZB, 15-AcDON (p=0.01)
NAT-267, 15-AcDON (p=0.01)
0.000
0.200
0.400
0.600
0.800
1.000
1.200
0 2 3 5 7 8 10 12 13 15 17 18 20 22 23
0.000
0.200
0.400
0.600
0.800
1.000
1.200
0 2 3 5 7 8 10 12 13 15 17 18 20 22 23
20 µg·mL-1
30 µg·mL-1
OD620 OD620
0.000
0.200
0.400
0.600
0.800
1.000
1.200
0 2 3 5 7 8 10 12 13 15 17 18 20 22 23
40 µg·mL-1
0.000
0.200
0.400
0.600
0.800
1.000
1.200
0 2 3 5 7 8 10 12 13 15 17 18 20 22 23
0.000
0.200
0.400
0.600
0.800
1.000
1.200
0 2 3 5 7 8 10 12 13 15 17 18 20 22 23
100 µg·mL-1
200 µg·mL-1
Time (hours) Time (hours)
ΔAUC = 4.48
ΔAUC = 5.25
ΔAUC = 4.50
ΔAUC = 1.20
ΔAUC = 0.00
0.000
0.200
0.400
0.600
0.800
1.000
1.200
0 2 3 5 7 8 10 12 13 15 17 18 20 22 23
50 µg·mL-1
ΔAUC = 4.45
139
The largest ΔAUC value (5.25) corresponded to NAT-267 VHH expressing P. pastoris
grown with 15-AcDON at 30 µg·mL-1
. Cytotoxicity assays based on 15-AcDON at 20,
40, and 50 µg·mL-1
had similar ΔAUC values (4.48; 4.50; and 4.45, respectively) (Figure
5.4). Accordingly, assays at 100 µg·mL-1
produced a substantially lower ΔAUC value
(1.20) while NAT-267 VHH intrabody expression conferred no immunomodulation when
spiked with 200 µg·mL-1
15-AcDON (Figure 5.4).
5.4.4.2 DON, 3-AcDON, Cycloheximide
NAT-267 VHH intrabody expression resulted in a dose-dependent response to DON.
Although no beneficial impact on cellular growth was observed in cultures supplemented
with 50 µg·mL-1
DON, NAT-267 VHH resulted in significantly-improved cellular growth
at 100 and 200 µg·mL-1
DON relative to pPICZB control transformants. Comparative
differences in growth were greater for yeast spiked with 100 relative to 200 µg·mL-1
DON (ΔAUC values = 2.11 and 1.68, respectively). In addition, time to significant
differences in OD620 values was several hours later for the higher dose (Figure 5.5A).
Differences in cellular growth between NAT-267 VHH and pPICZB control P.
pastoris cells could not be established for cytotoxicity assays spiked with 3-AcDON
because this trichothecene had little or no adverse effect on P. pastoris growth (Table
5.1). Assays based on cycloheximide (control ribotoxin) exhibited a dose-dependent
effect on cellular growth; however, no differences were observed between P. pastoris
NAT-267 VHH and pPICZB transformants spiked with doses of 50, 100, and 200 µg·mL-1
(Figure 5.5B).
140
Figure 5.5 Cytotoxicity assays showing the effect of various concentrations (50 to 200
µg·mL-1
) of DON (A) and cycloheximide (B) on the growth of P. pastoris
pPICZB and NAT-267 VHH transformants as measured by optical density at
620 nm (OD620). Differential Area Under Curve (ΔAUC) values for NAT-
267 VHH minus pPICZB empty vector are shown for each concentration of
DON. ΔAUC values for cycloheximide treatments were nil. At all
concentrations of DON where green versus red symbols are used, there was
a significant difference (p=0.01) at each time between the NAT-267 and
pPICZB transformants; this was not the case for all cycloheximide
treatments.
A
0.000
0.200
0.400
0.600
0.800
1.000
1.200
0 2 3 5 7 8 10 12 13 15 17 18 20 22 23
Time (hours)
OD620
ΔAUC = 1.20
ΔAUC = 2.11
ΔAUC = 1.68
Cycloheximide
Time (hours)
OD620
pPICZB, DMSO
NAT-267, DMSO
.
pPICZB, Cycloheximide
+ NAT-267, Cycloheximide
pPICZB, DMSO
NAT-267, DMSO
NAT-267, DON
pPICZB, DON
pPICZB, DON (p=0.01)
NAT-267, DON (p=0.01)
200 µg·mL-1
100 µg·mL-1
50 µg·mL-1
200 µg·mL-1
100 µg·mL-1
50 µg·mL-1
0.000
0.200
0.400
0.600
0.800
1.000
1.200
0 2 3 5 7 8 10 12 13 15 17 18 20 22 23
B
Deoxynivalenol
141
5.4.4.3 B-24 VHH Control
Expression of B-24 control VHH intrabody had no effect on cellular growth rate
relative to pPICZB control transformants in DMSO or in media supplemented with either
15-AcDON or Cycloheximide (Figure 5.6). These results suggest that trichothecene-
specific immunomodulation imparted by NAT-267 VHH intrabody expression was
trichothecene-specific, as Pichia cells expressing a non-specific VHH intrabody were
equally as sensitive as pPICZB control transformants.
5.4.5 VHH Immunolocalization
NAT-267 VHH intrabody expression and distribution within P. pastoris
transformants was further validated with fluorescent confocal microscopy (Figure 5.7).
Detection of the HA-tag on NAT-267 VHH intrabody fragments within transformants
grown in YPD media to OD600 = 20 further confirmed robust intrabody expression within
our test system.
5.4.6 Mycotoxin Biotransformation Assays
Given that NAT-267 VHH was confirmed previously to have binding affinity
limited to 15-AcDON (Doyle et al., 2008), we were surprised to observe a dose-
dependent amelioration to DON cytotoxicity within NAT-267 VHH transformants (Figure
5.5A). To validate this observation and re-confirm in vivo antigen specificity of NAT-
267 VHH intrabody expression, we assessed the potential biotransformation of DON
within our P. pastoris test system by spiking with DON. HPLC analysis of 16-h and 24-h
cultures grown in DON-supplemented YPD media consistently yielded another peak in
addition to the DON peak at ~ 5.85 min (Table 5.S2). This peak appeared at ~
142
Figure 5.6 Cytotoxicity assays showing the effect of various concentrations (50 to 200
µg·mL-1
) of 15-AcDON (A) and cycloheximide (B) on the growth of P.
pastoris pPICZB and non-specific B-24 VHH transformants as measured by
optical density at 620 nm (OD620). Differential Area Under Curve (ΔAUC)
values for B-24 VHH minus pPICZB empty vector for both15-AcDON and
cycloheximide are nil. At all concentrations of DON and cycloheximide
there was no significant difference (p=0.01) at each time between the B-24
and pPICZB transformants.
OD620
100 µg·mL-1
200 µg·mL-1
pPICZB, DMSO
B-24, DMSO
. pPICZB, Cycloheximide
+ B-24, Cycloheximide
0.000
0.200
0.400
0.600
0.800
1.000
1 3 5 6 8 10 11 13 15 16 18 20 21 23
Cycloheximide
Time (hours)
200 µg·mL-1
50 µg·mL-1
100 µg·mL-1
OD620
0.000
0.200
0.400
0.600
0.800
1.000
1 3 5 6 8 10 11 13 15 16 18 20 21 23
15-AcDON
B-24, 15-AcDON
pPICZB, 15-AcDON
pPICZB, DMSO
B-24, DMSO
Time (hours)
A
B
143
Figure 5.7 Representative confocal microscopy photographs of P. pastoris (KM71H)
cells isolated, washed and immuno-probed with anti-HA epitope mAb-FITC
conjugate and DAPI stain after growing in YPD media to OD600 = 20. A =
Control, pPICZB empty vector (control). B = P. pastoris transformants
expressing NAT-267 VHH intrabody shown by fluorescent green cells.
9.5 min and matched the retention time of 15-AcDON (Table 5.S2). HPLC
chromatograms of cell lysate and supernatant samples of pPICZB and NAT-267 VHH
transformants provided clear evidence of a metabolic conversion of DON to 15-AcDON.
No trichothecenes were found within cell pellet fractions (Table 5.S2). HPLC analysis
revealed no evidence of DON-glucosyl metabolites or any structurally-similar compounds
within samples tested. GC/MS analysis confirmed the presence of 15-AcDON within
pooled cell lysate samples (data not shown). The GC retention time and mass spectrum
of the peak confirmed the presence of 15-AcDON and absence of 3-AcDON within
samples tested (data not shown). Finally, the 1H NMR spectra of the total filtrate extract
144
was dominated by DON and other impurities from the medium. However, the presence
of 15-AcDON was confirmed in the NMR spectrum of the HPLC fraction isolated at ~9.5
min from the pooled lysate samples (Figure 5.8).
Figure 5.8 Representative 500 mHz 1H NMR spectrum of A = 15-AcDON
Standard. B = HPLC fraction of P. pastoris cell lysate sample (24 hrs).
Unique chemical shifts of 15-AcDON are labeled due to H-11 (4.87
ppm), H-7 (4.81 ppm), and H-15 (4.21 ppm) protons which confirm the
presence of 15-AcDON within cell lysate.
145
5.5 Discussion
Various strategies have been used to develop plants with enhanced resistance to
trichothecene cytotoxicity and associated Fusarium pathogenesis. These approaches tend
to be based on mechanisms which alter host cellular targets (Harris and Gleddie, 2001;
Mitterbauer et al., 2004), reduce mycotoxin cytotoxicity (Alexander, 2008; Poppenberger
et al., 2003), or enhance innate host resistance through crop breeding techniques
(Snijders, 2004). Our goal was to evaluate the ability of a mycotoxin-specific VHH
intrabody to immuno-modulate, or attenuate, the cytotoxic effects of 15-AcDON using a
yeast model.
Aside from application as a bioassay-indicator organism (Abolmaali et al., 2008;
Binder, 1999), several species of yeast have been used to evaluate the in situ function of
various trichothecene-specific genes (Alexander et al., 1999; Poppenberger et al., 2003)
or assess efficacy of mycotoxin-specific transgenes prior to in planta application
(Mitterbauer and Adam, 2002; Mitterbauer et al., 2004; Poppenberger et al., 2003). The
methylotropic yeast P. pastoris (strain KM71H) was selected as our eukaryotic model
based on expected sensitivity to target ribotoxins and known capacity to express high
levels of functional heterologous proteins (Cereghino and Cregg, 2000). We
demonstrated that it was possible to transform, assess, and validate P. pastoris cells
within a matter of weeks, thus creating an ideal platform for screening mycotoxin-specific
constructs before in planta evaluation.
Trichothecene cytotoxicity is determined by the C-12,13 epoxide group common to
this class of mycotoxins (Carter and Cannon, 1977; Rocha et al., 2005; Rotter et al.,
146
1996). However, the number and position of hydroxyl and acetyl-ester groups on the
trichothecene structure can also influence the mechanism of protein synthesis inhibition
and relative toxicities within eukaryotic cells (Betina, 1989; Desjardins et al., 2007;
Ehrlich and Daigle, 1987). In this regard, we found that the position of an acetyl-ester
group on either carbon 3 or 15 of DON (Figure 5.1) had a profound effect on their
cytotoxicity to P. pastoris (Table 5.1). Our findings align with results established in other
yeast species where 15-AcDON was significantly more toxic than DON (Mitterbauer et
al., 2004; Poppenberger et al., 2003), which was more toxic than 3-AcDON (Binder et al.,
1997). These results also support findings of structure-activity studies established in
planta that demonstrated 15-AcDON is more toxic than DON (Desjardins et al., 2007;
Poppenberger et al., 2003) and 3-AcDON (Alexander, 2008; Desjardins, 2006). Our
observation of no measurable cytotoxicity of 3-AcDON is in agreement with previous
studies which demonstrated that addition of an acetyl group at C-3 (Figure 5.1) serves to
eliminate cytotoxicity as part of a metabolic defence mechanism during trichothecene
synthesis (Kimura et al., 1998a; Kimura et al., 2006).
Our results also quantify relative differences in trichothecene cytotoxicity over time,
not at a specific assay endpoint. Our P. pastoris model system enables real-time
evaluation of cellular effects during the initial phases of ribotoxin exposure. We propose
that this biological system is very sensitive (i.e. compared to whole plant systems) and
shows clear effects on cellular growth within the first hours of exposure thereby
indicating the potency of trichothecene-mediated cytotoxicity.
147
A sequential two-step process was required before commencement of each
cytotoxicity assay. First, transformants were induced with methanol for 48h. After
induction of VHH intrabody expression, P. pastoris cells were transferred to YPD media
for a 4-h „recovery period‟ to re-stimulate cellular growth. Mycotoxin or cycloheximide
(control) treatments were then added (at t = 0h) to initiate each cytotoxicity assay. Given
the well-established negative impact on eukaryotic protein synthesis, it was an obvious
necessity to induce VHH expression prior to addition of ribotoxin treatments. The 4-h
recovery in YPD media was required because induction conditions did not support a
optimal cellular division and growth over time. We also noted that, by virtue of its
design, our test system ensured a fixed ratio of VHH antibody to ribotoxin hapten
throughout the course of each cytotoxicity assay. We recognized the potential
disadvantage of culturing induced cells in YPD as leading to a continuous dilution of
VHH concentration within the cytosol of rapidly dividing cells. However, we sought to
maintain a fixed ratio of Ab:Ag within each microtiter well to ensure unbiased
measurements of overall cellular growth.
Soluble NAT-267 and B-24 control VHH intrabody fragments were well expressed
within P. pastoris. Average protein expression levels (Table 5.2) were found to be much
higher than previously reported in P. pastoris (Gurkan et al., 2004; Omidfar et al., 2007;
Rahbarizadeh et al., 2006). Furthermore, images generated by confocal immuno-
microscopy confirmed that NAT-267 VHH fragments were well distributed within the
cytosol of P. pastoris transformants even after growth to a very high OD600 (Figure 5.7).
148
Our principal hypothesis was that the expression of mycotoxin-specific VHH
intrabody fragments would effectively sequester (i.e., neutralize) bio-available 15-
AcDON concentrations within the cell and thereby limit cellular toxicity, thereby
resulting in enhanced growth of P. pastoris. VHH-mediated mycotoxin binding was
assessed by time-to-significant immunomodulation and ΔAUC values between VHH
transformants and pPICZB (control) cell lines for each ribotoxin treatment tested (Figures
5.4 – 5.6). The initial doses of 15-AcDON tested are generally equivalent to those found
during Fusarium infection of agricultural crops. For example, intracellular levels of
DON have been reported at concentrations ranging from 13.3 to 88.7 µg·g-1
within F.
graminearum inoculated cereal tissue (Del Ponte et al., 2007; Mudge et al., 2006).
Attenuation of 15-AcDON activity clearly demonstrates that NAT-267 VHH intrabody
expression conveys significant (p = 0.01) resistance to mycotoxin-specific cytotoxicity
(Figure 5.4) by regulating the availability of free and VHH-bound 15-AcDON.
Consequently, results were dose-dependent as time to immumodulation was optimal for
30 and 40 µg·mL-1
15-AcDON, when compared to the lower (20 µg·mL-1
) and higher
concentrations (i.e., 50 and 100 µg·mL-1
) of the toxophore. Sequestration of 15-AcDON
was substantially reduced and delayed at concentrations of 100 µg·mL-1
while at 200
µg·mL-1
there was no benefit associated with VHH expression (Figure 5.4).
NAT-267 VHH intrabody immuno-modulation was trichothecene-specific as no
attenuation of 15-AcDON toxicity was observed in assays supplemented with control
ribotoxin (cycloheximide) (Figure 5.5). It was not possible to assess the effect of NAT-
267 VHH on the cytotoxicity of 3-AcDON because all doses tested were not toxic to P.
149
pastoris cells (Table 5.1). Further confirmation of NAT-267 VHH specificity was shown
with cytotoxicity assays using P. pastoris transformants expressing a control VHH
intrabody, i.e., B-24. Furthermore, there was no immunomodulation of either 15-
AcDON- or cycloheximide-specific cytotoxicity when pPICZB control cells were used
(Figure 5.6).
The attenuation of DON cytotoxicity at 100 and 200 µg·mL-1
(Figure 5.5) was an
unexpected result since we previously-reported that NAT-267 VHH has no affinity for
DON (Doyle et al., 2008). In vivo sequestration of DON was explained by confirmation
of bio-transformation of DON to 15-AcDON in P. pastoris cell lysate and culture
supernatant samples of NAT-267 and pPICZB transformants (Table 5.S2; Figure 5.8).
Data mining of the genome of NRRL Y-11430 P. pastoris (the parent strain of KM71H)
by Integrated Genomics Inc. (Chicago, IL, USA) revealed no similarities or related
homologs to a previously characterized fungal gene responsible for acetylation of DON to
15-AcDON (McCormick et al., 1996). We therefore attributed this biochemical
conversion to a previously-uncharacterized acetyl-transferase gene within the P. pastoris
genome.
To predict the impact of NAT-267 VHH intrabody binding to reduce effective 15-
AcDON toxin concentrations within the cytosol, we adopted a previously described
(Almquist et al., 2004) mass-balance model based on the following equation.
[Bound Toxin] =
[VHH] [Total Toxin] x
KD [VHH] + ( )
150
We assumed linear, dose-independent binding of VHH to 15-AcDON within an
aqueous environment where 1 g = 1 mL. The dissociation constant (KD) of NAT-267
VHH was taken as1.24 µM (Doyle et al., 2008) with 1:1 stoichiometry of Ab:Ag binding.
NAT-267 ([VHH]) concentration within the P. pastoris cytosol was estimated to be 138
µmol·L-1
(Tables 5.2, 5.S1). We assumed optimal disulfide bond formation and post-
translational VHH folding within the endoplasmic reticulum and cytosol (Ellgaard, 2004)
with no NAT-267 VHH intrabody leakage from the P. pastoris cell membrane. The
concentration of 15-AcDON within the cytosol was taken as equivalent to culture media,
and we also assumed that there were no trichothecene targets outside of the cells.
Based on total 15-AcDON concentration of 50 µg·mL-1
(or 148 µM) (Figure 5.4), this
model predicts (138 µM x 148 µM) ÷ (138 µM + 1.24 µM) = 147 µM of 15-AcDON (or
> 99.9%) toxin was bound and ~ 1.3µM (or < 0.1%) of 15-AcDON was free. This simple
model indicates that 15-AcDON cytotoxicity at 50 µg·mL-1
should be completely
eliminated by expression of NAT-267 VHH; however, although the effects of 15-AcDON
were significantly ameliorated by VHH expression at this concentration, they were not
completely eliminated (Figure 5.4).
A more complete assessment of NAT-267 VHH immunomodulation should also
account for in situ competition effects and binding affinity between 15-AcDON and its
major cellular target, i.e., ribosome binding. Binding affinity is also an important
consideration because NAT-267 VHH has a relatively low affinity (KD = 1.24 µM) for 15-
AcDON that is mediated by weak, non-covalent interactions (e.g., van der Waals forces,
hydrogen bonding, etc.) (Maynard and Georgiou, 2000). Due to the rapid dissociation rate
151
constant for NAT-267, we hypothesize that 15-AcDON is subject to continuous turnover
in terms of mycotoxin binding to the VHH. In other words, the efficiency of NAT-267
VHH in terms of limiting the cytotoxicity of 15-AcDON is limited by the short “residence
time” of hapten binding to the VHH (Copeland et al., 2006). Further evidence for this
hypothesis resides in the fact that even when the VHH intrabody is expressed in excess
(138 µmol·L-1
, Table 5.2) compared to 15-AcDON, i.e. at 89 μM (30 µg mL-1
), NAT-267
cannot fully immunomodulate the cytotoxic effects of 15-AcDON when compared to
controls (Figure 5.4). Thus, at higher doses ≥30 µg·mL-1
of 15-AcDON, we postulate
that the VHH simply cannot compete with a stronger and quite possibly longer residence
time of 15-AcDON on the 60S ribosomal protein subunit L3 (Rpl3) of P. pastoris.
We also assert that, at very high doses, 15-AcDON may cause other potentially
irreversible effects such as membrane disruption, inhibited RNA and DNA synthesis, and
various other apoptotic effects (reviewed in Rocha et al., 2005; Rotter et al., 1996; Ueno,
1984) which may also severely limit cellular growth. Thus, to accurately determine the
efficacy of NAT-267 VHH intrabody in P. pastoris cells one must account for the
dynamic nature of mycotoxin-mediated cytotoxicity and in vivo binding kinetics of 15-
AcDON for other cellular binding targets. Our assertion that attenuation of
trichothecene-specific cytotoxicity is not a simple process is in agreement with previous
work which demonstrated only partial remediation of DON cytotoxicity based on in
planta expression of an altered Rpl3 target protein (Harris and Gleddie, 2001;
Mitterbauer et al., 2004) or the expression of Tri101 (Alexander, 2008, Kimura et al.,
1998a).
152
Most phytopathogenic Fusarium species are believed to produce 15-AcDON or 3-
AcDON as acetylated precursors to DON (Mitterbauer and Adam, 2002). Production of
each acetylated compound has been described as geography-dependent; 3-AcDON
chemotypes dominate in Asia and Europe, while Fusarium species which produce 15-
AcDON are more prevalent in North America (Goswami and Kistler, 2004; Moss and
Thrane, 2004). Therefore, the accumulation of DON within plant tissue is believed to be
a metabolic process conferred by fungal and plant carboxyl-esterases which continuously
deacetylate either 15-AcDON or 3-AcDON within plant tissue (Mitterbauer and Adam,
2002). Confirmation of NAT-267 efficacy observed in this work is very significant since
15-AcDON was the most cytotoxic compound tested. If 15-AcDON is as toxic to plants
as it is to yeast then expressing NAT-267 VHH within the cytosol before trichothecene
accumulation may help limit in vivo pathogenesis and metabolism to DON during
Fusarium infection of plants such as corn and wheat.
Future experiments will focus on the development of an anti-15-AcDON VHH with
an improved dissociation constant (KD) to ensure a stronger association between the VHH
and target ligand for improved in vivo efficacy (Copeland et al., 2006). It would also be
of great interest to develop and test novel VHH fragments with affinity for various other
trichothecenes (e.g., neosolaniol, diacetoxyscripenol, T-2 toxin, etc.) and various other
mycotoxin classes (e.g., fumonisins, aflatoxins, etc.) using this test system. A logical
subsequent application would be constitutive in planta expression of optimized
mycotoxin-specific VHH fragments, possibly with catalytic activity, to bind and
deactivate/degrade mycotoxins during critical initial periods of plant pathogenesis.
153
5.6 Acknowledgements
We would like to express our sincere appreciation to Dr. Barbara Blackwell at
Agriculture and Agri-Food Canada Ottawa for assistance, insights, and expertise in
HPLC, GC/MS, and NMR assays to characterize mycotoxin biotransformation samples.
We are also indebted to colleagues at the Canadian Food Inspection Agency for GC/MS
analysis of biotransformation samples. This research was supported by grants to JCH
from the Natural Sciences and Engineering Research Council of Canada, the Canada
Research Chairs Program, and the Ontario Ministry of Agriculture, Food, and Rural
Affairs.
154
5.7 Supplemental
EcoRI
NAT-
267
AAGCTTGAATTCGGTACCATGCAAGTCCAATTGGAAGAATCCGGTGGTGGTTTAGTTCAA 60
M__Q__V__Q__L__E__E__S__G__G__G__L__V__Q_
B-24 A__Q__V__K__L__E__E__F__G__G__R__L__V__Q_
AAGCTTGAATTCGGTACCGCCCAGGTAAAGCTGGAGGAGTTTGGGGGAAGATTGGTGCAG 60
EcoRI
NAT-
267
GCCGGTGGTTCCTTGAGATTGTCTTGTGCTGCTTCTGGTAGAACTTCCTCCAACACCTTC 120
_A__G__G__S__L__R__L__S__C__A__A__S__G__R__T__S__S__N__T__F_
B-24 _A__G__G__S__L__R__L__S__C__A__A__S__V__R__T__F__S__N__R__P_
GCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGTACGCACCTTCAGTAACAGACCC 120
NAT-
267
GTCGGTTGGTTTAGACAAGCCCCAGGTAAAGAAAGAGAATTTGTCGCCGCCATTAGAAGA 180
_V__G__W__F__R__Q__A__P__G__K__E__R__E__F__V__A__A__I__R__R_
B-24 _M__G__W__F__R__Q__T__P__G__N__E__R__E__F__V__T__A__I__S__S_
ATGGGCTGGTTCCGCCAAACTCCAGGGAATGAGCGTGAGTTTGTAACAGCTATTAGTTCG 180
NAT-
267
TCCGACGACAGAACTTACTACGCTGCCTCTGTCAGAGGAAGATTCACCATTTCCGGTGAT 240
_S__D__D__R__T__Y__Y__A__A__S__V__R__G__R__F__T__I__S__G__D_
B-24 _S__G__A__S__T__R__Y__A__D__S__V__K__G__R__F__T__I__S__R__D_
AGTGGTGCGAGCACACGCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGAC 240
NAT-
267
TCCGCTAAGAACGTCGTTGCTTTGCAAATGTCCTCCTTAAGACCAGAAGATACCGCCGTT 300
_S__A__K__N__V__V__A__L__Q__M__S__S__L__R__P__E__D__T__A__V_
B-24 _N__A__R__N__T__V__Y__L__Q__M__N__S__L__K__P__E__D__T__A__V_
AACGCCAGGAACACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAAGACACGGCCGTT 300
NAT-
267
TACTACTGTGCTGCTACCAGAACTTGGTTGGTTACCGGTCAATCCGATTACCCATACXXX 360
_Y__Y__C__A__A__T__R__T__W__L__V__T__G__Q__S__D__Y__P__Y__X_
B-24 _Y__Y__C__A__A__S__Y__Y__S__G__T__P__T__T__S__S__A__Y__D__Y_
TATTACTGTGCAGCCTCATACTATAGTGGTACTCCCACCACCAGCAGCGCGTATGACTAC 360
NAT-
267
TGGGGTCAAGGTACTCAAGTTACCGTTTCCTCTGGTCAAGCCGGTCAACATCATCACCAT 420
_W__G__Q__G__T__Q__V__T__V__S__S__G__Q__A__G__Q__H__H__H__H_
B-24 _W__G__Q__G__T__Q__V__T__V__S__S__G__Q__A__G__Q__H__H__H__H_ 420
TGGGGCCAGGGGACCCAGGTCACCGTCTCCTCAGGCCAGGCCGGCCAGCACCATCACCAT
NAT-
267
CACCACGTGTCCTCTGGTAGATACCCATACGATGTCCCAGATTACGGTTCTGGTAGAGCC
_H__H__V__S__S__G__R__Y__P__Y__D__V__P__D__Y__G__S__G__R__A_
B-24 _H__H__V__S__S__G__R__Y__P__Y__D__V__P__D__Y__G__S__G__R__A_ 480
CACCATGTCTCCAGCGGCCGCTACCCGTACGACGTTCCGGACTACGGTTCCGGCCGAGCA
XbaI
NAT-
267
TAAGTCGACTCTAGAGCTC 486
_*_
B-24 _*_
TAGGTCGACTCTAGAGCTC 486
XbaI
Figure 5.S1 Homology chart of NAT-267 and B-24 VHH fragments. Framework regions (FR)
are outlined in black while complementarity determining regions (CDR) 1, 2, and 3
are outlined in blue. Cysteine residues at C23 and C97 responsible for disulfide
bond VHH folding are shown in red. XXX denotes extra codon within CDR3 of
NAT-267 gene. Restriction endonuclease sites, epitope tags (6HIS = green, HA =
pink) and stop codons (*) are marked accordingly. NAT-267: Theoretical pI for
NAT-267 and B-24 VHH = 9.00 and 9.17, respectively (ExPASy).
155
Table 5.S1: Raw data and calculations of VHH intrabody expression in Pichia pastoris
transformants derived from quantitative Western blot analysis (Figure 6.3) and as
summarized in Table 6.2.
Sample
Dilution
Total VHH
Lysis* (ng)
VHH, 1 mL cell †
(µg·mL-1
)
VHH, 1 mL pellet††
(µg·mL-1
)
Calculated Intracellular VHH Expression
(fg·cell-1
) ††
(aMol·cell-1
) ‡‡
µMol·L-1☺
Cytotoxicity Assay-1: NAT-267 vs. 15-AcDON (Figure 6.4)
Blot 1
1 / 5 435.8 217.9 77.35 71.96 4.325 149.1
1 / 25 71.2 178.0 63.19 58.78 3.533 121.8
1 / 50 n.d. n.d. n.d. n.d. n.d. n.d.
Blot 2
1 / 5 322.2 161.1 57.19 53.20 3.198 110.3
1 / 25 122.5 281.1 99.80 92.84 5.580 192.4
1 / 50 48.40 242.0 85.91 79.92 4.803 165.6
Blot 3
1 / 5 192.3 96.15 34.13 31.75 1.908 65.8
1 / 25 60.90 152.3 54.05 50.28 3.022 104.2
1 / 50 36.60 183.0 64.97 60.43 3.632 125.3
Blot 4
1 / 5 305.30 152.65 54.19 50.41 3.030 104.5
1 / 25 114.11 285.27 101.27 94.20 5.662 195.2
1 / 50 53.84 269.19 95.56 88.90 5.343 184.2
Blot 5
1 / 5 272.60 136.30 48.39 45.01 2.705 93.3
1 / 25 88.57 221.43 78.61 73.12 4.395 151.6
1 / 50 50.67 253.33 89.93 83.66 5.028 173.4
Mean (n=14) 66.75 4.01 138
Standard Error (n=14) 5.23 0.31 10.8
Cytotoxicity Assay-2: NAT-267 vs. DON, 3-AcDON, Cycloheximide (Figure 6.5)
Blot 1
1 / 5 127.9 63.94 22.70 19.48 1.171 40.4
1 / 25 25.11 62.78 22.29 19.13 1.150 39.6
1 / 50 n.d. n.d. n.d. n.d. n.d. n.d.
Blot 2
1 / 5 238.9 119.5 42.42 36.41 2.188 75.5
1 / 25 107.5 268.8 95.41 81.89 4.922 169.7
1 / 50 69.19 346.0 122.8 105.42 6.336 218.5
Blot 3
1 / 5 148.6 74.3 26.38 22.64 1.361 46.9
1 / 25 85.36 213.4 75.75 65.03 3.908 134.8
1 / 50 31.07 155.4 55.15 47.34 2.845 98.1
Blot 4
1 / 5 287.32 143.66 51.00 43.78 2.631 90.7
1 / 25 79.42 198.54 70.48 60.50 3.636 125.4
1 / 50 54.85 274.26 97.36 83.57 5.023 173.2
Blot 5
1 / 5 233.25 116.62 41.40 35.54 2.136 73.7
1 / 25 88.57 221.43 78.61 67.47 4.055 139.8
1 / 50 35.04 175.20 62.20 53.39 3.209 110.7
Mean (n=14) 52.97 3.18 109.8
Standard Deviation (n=14) 6.96 0.42 14.4
156
Table 5.S1, Continued: Raw data and calculations of VHH intrabody expression in Pichia
pastoris transformants derived from quantitative Western blot analysis (Figure
6.3) and as summarized in Table 6.2.
Sample
Dilution
Total VHH
Lysis* (ng)
VHH, 1 mL cell †
(µg·mL-1
)
VHH, 1 mL pellet††
(µg·mL-1
)
Calculated Intracellular VHH Expression
(fg·cell-1
) ‡
(aMol·cell-1
) ‡‡
µMol·L-1☺
Cytotoxicity Assay-3: B-24 vs. 15-AcDON and Cycloheximide (Figure 6.6)
Blot 1
1 / 5 137.9 68.95 24.48 26.46 1.559 53.8
1 / 25 n.d. n.d. n.d. n.d. n.d. n.d.
1 / 50 n.d. n.d. n.d. n.d. n.d. n.d.
Blot 2
1 / 5 179.8 89.9 31.91 34.50 2.032 70.1
1 / 25 35.24 88.1 31.28 33.81 1.992 68.7
1 / 50 24.29 121.5 43.11 46.61 2.746 94.7
Blot 3
1 / 5 137.5 68.75 24.41 26.39 1.554 53.6
1 / 25 33.21 83.03 29.47 31.86 1.877 64.7
1 / 50 15.54 77.70 27.58 29.82 1.757 60.6
Blot 4
1 / 5 187.29 93.65 33.24 35.94 2.117 73.0
1 / 25 51.31 128.27 45.53 49.23 2.900 100.0
1 / 50 n.d. n.d. n.d. n.d. n.d. n.d.
Blot 5
1 / 5 129.08 64.54 22.91 24.77 1.459 50.3
1 / 25 28.10 70.24 24.94 26.96 1.588 54.8
1 / 50 13.92 69.59 24.70 26.71 1.573 54.3
Mean (n=12) 32.76 1.93 66.6
Standard Deviation (n=12) 2.30 0.14 4.3
Calculations, Table 5.S1:
* Total VHH Lysis (ng) of diluted VHH samples from each assay derived from regression equation analysis
based on a five point dilution series (300, 150, 75.0, 37.5, 18.75 ng·lane-1
) of a reference VHH fragment
(as described in EXPERIMENTAL PROCEDURES).
x = Calculated mass of VHH in cell lysate (ng).
y = Measured densometric value of unknown sample (densometric units).
Blot 1: x = [(y - 3.292) ÷ 0.3387], R2 = 0.9555
Blot 2: x = [(y + 1.8174) ÷ 0.0913], R2 = 0.9815
Blot 3: x = [(y + 3.700) ÷ 0.5600], R2 = 0.9902
Blot 4: x = [(y + 17.561) ÷ 0.3949], R2 =0.9856
Blot 5: x = [(y + 2.9104) ÷ 0.3456], R2 =0.9975
† VHH from 1 mL Cell Lysis (µg · mL
-1) = Est. VHH Quantity (ng) . x 1,000 (µL·mL
-1)
Total Vol. Cell Pellet Loaded (µL) 1,000 (ng·µg-1
)
†† VHH from 1 mL Cell Pellet (µg · mL
-1) = VHH from 1 mL Cell Lysis (µg) .
Lysis Volume per 1 mL Cell Culture (µL)
157
Calculations, Table 5.S1 (Continued):
‡ VHH per cell (fg·cell
-1) = VHH from 1 mL Cell Pellet (µg·mL
-1) . x 1.0 x 10
9 fg
P. pastoris cell density (# cells· 1mL culture-1
) 1.0 µg
‡‡
VHH per cell (aMol·cell-1
) = Intracellular VHH per cell (fg·cell-1
) x 1.0 x 103 aMol
VHH MW (fg·fMol-1
) 1.0 fMol
☺ VHH Concentration within P. pastoris cytosol = VHH per cell (a Mol · cell
-1) ÷ 29 femto L · cell
-1
Constants, Table 5.S1:
- Total Volume of Cell Pellet Loaded for 1 / 5; 1/25 and 1/50 dilutions = 2.0; 0.4 and 0.2 µL, resp.
- Lysis Volume per 1.0 mL Cell Culture = 355 µL
- Mean OD600 of 1.0 mL P. pastoris culture for Cytotoxicity Assay-1 (Figure 5.4) = 21.5
- Mean OD600 of 1.0 mL P. pastoris culture for Cytotoxicity Assay-2 (Figure 5.5) = 23.3
- Mean OD600 of 1.0 mL P. pastoris culture for Cytotoxicity Assay-3 (Figure 5.6) = 18.5
- For Pichia pastoris, 1 OD600 = 5 x 107 cells·mL
-1
- For Pichia pastoris, Volume of one cell = 29 femtoliters (2.9 x 10-14
Liters)
Therefore,
- Mean P. pastoris cell density for Cytotoxicity Assay-1 = (5 x 107 cells · mL
-1) X 21.5 = 1.075 x 10
9
- Mean P. pastoris cell density for Cytotoxicity Assay-2 = (5 x 107 cells · mL
-1) X 23.3 = 1.165 x 10
9
- Mean P. pastoris cell density for Cytotoxicity Assay-3 = (5 x 107 cells · mL
-1) X 18.5 = 9.250 x 10
8
Molecular Weights (www.expasy.ch/)
- NAT-267 (treatment) VHH = 16637.3 Da (fg · fMol-1
)
- B-24 (control) VHH = 16975.6 Da (fg · fMol-1
)
158
Table 5.S2: HPLC retention times of trichothecene metabolites evaluated after 24 h of growth
of P. pastoris transformants cultured in YPD media supplemented with 100
µg·mL-1
of highly-purified DON. Transformation of DON (5.750 minutes) to 15-
AcDON (9.625 minutes) within supernatant (S/N), cell lysate (LYS), and cell
pellet debris (DEBS) shown in 30-minute, 16-h and duplicate 24-h samples of
NAT-267 VHH and pPICZB empty vector transformants.
Sample Description Peaks (Retention time, min)
DON 15-AcDON
Supernatant (S/N) Fraction
NAT-267 VHH, S/N, 30min. 5.850 H 9.542 NP
NAT-267 VHH, S/N, 16h 5.825 H 9.517 H
NAT-267 VHH, S/N, 24h, Rep 1 5.833 VH 9.533 VH
NAT-267 VHH, S/N, 24h, Rep 2 5.808 H 9.550 H
pPICZB (empty vector), S/N, 30min. 5.975 H 9.542 L
pPICZB (empty vector), S/N, 16h 5.817 VH 9.525 VH
pPICZB (empty vector), S/N, 24h, Rep 1 5.825 VH 9.542 H
pPICZB (empty vector), S/N, 24h, Rep 2 5.825 VH 9.533 H
Cell Lysate (LYS) Fraction
NAT-267 VHH, LYS, 24h, Rep 1 5.858 M 9.575 M
NAT-267 VHH, LYS, 24h, Rep 2 5.875 H 9.567 M
pPICZB (empty vector), LYS, 24h, Rep 1 5.817 M 9.542 M
pPICZB (empty vector), LYS, 24h, Rep 2 5.817 M 9.525 L
Cell Debris (DEBS) Fraction
NAT-267 VHH, DEBS, 24h 5.550 NP 9.567 NP
pPICZB (empty vector), DEBS, 24h 5.855 NP 9.525 NP
- No peak (NP) observed
- Low (L) = very small or no peak observed.
- Medium (M) = medium or measurable peak observed.
- High (H) = large peak observed.
- Very High (VH) = very large peak observed.
159
6 CONCLUSIONS
The research and results summarized within this thesis reaffirms the vast potential
of recombinant antibody engineering as a discipline with wide ranging applications. Our
specific goal was to develop a rAb fragment with binding affinity to DON, a key
mycotoxin produced during FHB pathogenesis. This was a proof of concept to apply a
novel technology platform commonly used in pharmaceutical research toward an untested
discipline, plant pathology. Our hypothesis was based on the notion that expression of an
engineered rAb fragment within a model eukaryotic test system could bind a target
mycotoxin and inherently limit in situ cytotoxicity to provide enhanced tolerance to FHB
pathogenesis. Although the results of this dissertation are well summarized within
Chapters # 4 and # 5, the following provides an overall summary of key conclusions and
outlines future research interests and potential technological applications.
Immunization of a llama with 15-DON-BSA conjugate resulted in a robust
polyclonal response to the hapten complex (i.e., DON, 15-AcDON, 3-AcDON). A hyper-
immunized, phage-display VHH library was successfully created from peripheral blood
lymphocytes based on standard procedures (Ghahroudi et al., 1997). A single dominant
VHH clone known as „NAT-267‟ was selected after four rounds of library panning against
15-DON-OVA conjugate. Both NAT-267 VHH monomer and pentamer demonstrated
absolute binding specificity to 15-AcDON. NAT-267 VHH nucleotide sequence results
(GenBank: EU676170.1 http://lifesciencedb.jp/ddbj/ff_view.cgi?accession=EU676170)
confirmed that Ag specificity was generated by somatic hypermutations within the CDR
regions. The NAT-267 VHH fragment was highly specific to 15-AcDON as it did not
160
recognize structurally similar trichothecenes, particularly DON and 3-AcDON. We
attributed the exquisite specificity of NAT-267 VHH to the fact that both the immunogen
and panning mycotoxin protein conjugates (BSA and OVA, respectively) were linked to
DON at carbon 15 using a common hemi-succinate linker.
The methylotropic yeast Pichia pastoris was selected as a model eukaryotic test
system based on expected sensitivity to 15-AcDON and well-established capacity to
produce well-characterized quantities of functional protein. The P. pastoris system was a
convenient and cost-effective system to generate reproducible data to assess the ability of
intracellular antibodies (intrabodies) to modulate mycotoxin-specific cytotoxocity as a
precursor to transformation of higher order plants. Mycotoxin-mediated cytotoxicity was
assessed by continuous measurement of cellular growth in real time which allowed for
precise measurements of cytotoxicity during initial exposure to each ribotoxin treatment.
At equivalent doses, 15-AcDON was significantly more toxic to wild-type P. pastoris
than was DON which, in turn, was more toxic than 3-AcDON. The order of
trichothecene toxicity established within our work aligned with results previously
published in other species of yeast and model plant systems. Our observation of no
measureable cytotoxicity of 3-AcDON was in agreement with previous studies which
demonstrated that the addition of an acetyl ester group at C-3 significantly limits DON
cytotoxicity. Intracellular expression of toxin-specific VHH within P. pastoris conveyed
significant (p = 0.01) resistance to 15-AcDON cytotoxicity at doses ranging from 20 to
100 µg·mL-1
. A key limitation of NAT-267 VHH efficacy was that rAb binding to 15-
AcDON was of low affinity. The specific limitation was that the Ab-Ag complex most
161
likely had a short „residence time‟ which limited VHH efficacy, especially at higher doses
of 15-AcDON. Furthermore, we documented a biochemical transformation of DON to
15-AcDON which explained significant attenuation to the phytotoxic effects of DON at
100 and 200 µg·mL-1
. This “proof of concept” model established in this work suggests
that in planta VHH expression may lead to enhanced tolerance to mycotoxins and thereby
serve as a novel tool to help reduce the impact of Fusarium infection of commercial
agricultural crops. Furthermore, based on these results, we accept our hypothesis in
affirming that the expression of a recombinant VHH antibody fragment within a model
eukaryotic test system can limit mycotoxin-specific cytotoxicity. However, we reiterate
that applications of mycotoxin-specific rAb fragments would be most effectively applied
as a tool to complement (and hopefully enable) other means of enhanced FHB resistance
in higher order plants.
We recommend that future experiments and next steps be focused on the
development of VHH rAbs with improved dissociation constants (KD) to ensure a longer
residence time supported by stronger association between VHH and the target ligand for
improved efficacy (Copeland et al., 2006). It would also be of interest to develop and test
novel VHH fragments for various other trichothecenes (e.g., neosolaniol,
diacetoxyscripenol, T-2 toxin, etc.) and perhaps other classes of mycotoxin (e.g.,
fumonisins, aflatoxins, etc.). A logical subsequent application would be constitutive in
planta expression of optimized mycotoxin-specific VHH fragments, possibly with
catalytic activity, to bind and deactivate/degrade mycotoxins during critical initial periods
of Fusarium pathogenesis.
162
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