tug-mm;

71‘ oliglq‘v‘v

gas-um

msnv-u-uyn-vmgn

‘!"i.

«y‘-

2’

m. -u..,

n
u
m.
m

.u c1-.vglr .cm .u. . .

."

nn “.9."- .x

 

 

m“ UBRARY
Michigan State

U 3 -3 awn:*\l
Ul IIVUI any

 

 

 

 

This is to certify that the
dissertation entitled

DESIGN AND EVALUATION OF IMMUNOGENIC
Escherichia coli HEAT- STABLE ENTEROTOXIN (STa)
AND'CHARACTERIZATION OF THE IMMUNE RESPONSE
IN LABORATORY ANIMALS

presented by

Nasr-Eldin Mohamed M. Aref

has been accepted towards fulfillment
of the requirements for the

PhD. degree in Lane Animal Clinical Sciences

 

 

W/uwg/

Major Professor’s Signature

03’.» 07- 0g

Date

 

MSU is an affinnative-action, equal-opportunity employer

 

..-—-—-—-—.—.—.-o--o-o---.—--—-—-.—-—._-—-n—.— .—

.uauo—a-o-o-I-o-n-u-o-I-u-u-I—.-.

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

5/08 K1/Prolecc8Pres/CIRCIDateDue.nndd

 

DESIGN AND EVALUATION OF IMMUNOGENIC
ESCHERICHIA COLI HEAT- STABLE ENTEROTOXIN (STa) AND
CHARACTERIZATION OF THE IMMUNE RESPONSE IN
LABORATORY ANIMALS

BY

Nasr—Eldin Mohamed M. Aref

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Large Animal Clinical Sciences

2008

ABSTRACT

DESIGN AND EVALUATION OF IMMUNOGENIC Escherichia coli HEAT-
STABLE ENTEROTOXIN (STa) AND CHARACTERIZATION OF THE
IMMUNE RESPONSE IN LABORATORY ANIMALS
By
Nasr-Eldin Mohamed M. Aref

Strains of Enterotoxigenic Escherichia coli (ETEC) that produce heat-stable
enterotox'm (STa) are an important cause of diarrheal disease in humans and animals.
They are responsible for a significant proportion of diarrheal cases among infants,
neonatal animals, and travelers going from non-endemic to endemic areas. The
development of effective strategies to reduce the incidence and severity of ETEC-
caused diarrhea has been hampered by the lack of an effective vaccine or
immunotherapeutic agents against this enteric pathogen. The lack of an effective
vaccine against the ETEC is largely due to the diverse antigenic structure of the
ETEC strains and the poor immunogenicity of the STa enterotoxin, which is the
predominant enterotoxin in 75% of ETEC strains and is the immediate mediator of
ETEC-caused diarrhea.

Attempts to induce an appropriate immune response against the STa involve
the design of immunogenic STa-carrier conjugates. However, based on the protocols
reported in previous studies, only low titers of STa-antisera have been obtained. In
this study, we used highly purified E. coli STa to produce STa-carrier conjugates
through covalently cross-linking the STa to a modified bovine serum albumin (BSA)

carrier protein. Four different conjugation protocols were evaluated. The most

 

effective STa-conjugate was selected based on its retention of STa biological activity,
conjugation efficiency, and stability.

The selected STa-BSA conjugate was used to immunize rabbits for antibody
production. This STa-conjugate induced a high immune response against the native
STa. The harvested rabbit sera showed a neutralization capacity of 3 x 104 STa mouse
units/ml serum using a suckling mouse assay, and a specific binding titer of 10'6 using
indirect ELISA. These levels of STa neutralization and binding capacity are higher
than what has been reported in previous studies. This study demonstrated the
feasibility of designing an effective immunogenic ETEC-STa conjugate that can be
used for the development of immunotherapeutic reagents and /or a potential vaccine

against the ETEC-STa.

Copyright by
Nasr—Eldin Mohamed M. Aref

2008

DEDICATION
To my almighty and beloved ALLAH who has been my shield, my strength, and my
very present help in time of need. Thank you my LORD for helping me throughout

this dissertation and for giving me a hope beyond hopes.

ACKNOWLEDGMENTS

I am indebted to my guidance committee members, their contribution made
this work possible. A special thanks to my major advisor, Dr. A. Mahdi Saeed, for his
continuous intellectual advisement, encouragement, and support. He is a great
mentor, colleague and friend.

No words can express my sincere appreciation to my Ph.D. guidance
committee members, Drs. Patricia Ganey, Robert Roth, Ronald Erskine and Daniel
Grooms, for their great mentorship. Their suggestions and constructive feedback
while conducting various parts of my research and writing my dissertation were vital
for the completion of my project.

I gratefully acknowledge Dr. Joseph Leykam and Eric Lund, Research
Technology Support Facility at Michigan State University for their help on mass
spectroscopy and amino acid sequencing.

I would like to thank the staff in the Containment Facility, Michigan State
University for their help. In particular, Cathy Tyler and Randy Shoemaker, for their
assistance in taking care of animals used in this research.

Many thanks to our laboratory staff, Kristin Evon and Nicole Crisp, our
summer interns, Jessica Malay and Amanda Audo, and our undergraduate students,
Laya Kevan, Alyssa DiFilippo, Kyle Korolowicz, Narges Rahman, Krystal
Snodgrass, Tressa Fountain, Cassy Nguyen, Josephine Wee, Ashley Walters, Amy
LaRose, Symone Coleman, I Rin, Diane Sinawi and Hiu-Lam Lau, for their great

assistance throughout this research.

vi

Many friends and colleagues at Michigan State University provided
substantial advice, support and encouragement during this work, in particular, Drs.
Seongbeom Cho, Mokhtar Arshad and Muhammad Younus.

I would also like to thank the administrative staff at the College of Veterinary
Medicine and National Food Safety Toxicology Center, Michigan State University, in
particular, Faith Peterson, Jennifer Sysak, Susan Dies and Margaret Nicolas, for their
support.

My special gratitude and sincere appreciation goes to my wife and children for
their continuous prayers, encouragement, support, patience and tolerance during this
critical stage in my life.

Special thanks are extended to Assiut University, Egyptian High Education
Ministry, Egyptian Cultural Educational Bureau (Washington DC.) and
Microbiological Research Unit-National Institute of Health (MRU-NIH-USA) for

their financial support.

vii

TABLE OF CONTENTS

LIST OF TABLES ............................................................................ xi
LIST OF FIGURES ........................................................................... xii
LIST OF ABBREVIATIONS .............................................................. xv
CHAPTER I: Introduction
Statement of the Issue and Rationale ................................................. 1
General Objectives ....................................................................... 3
Specific Aims ............................................................................. 4
Potential Benefit of the Study .......................................................... 4
References ................................................................................. 6

CHAPTER II: Review of Literature

Magnitude and impact of diarrheal diseases ................................................ 8

General overview of Escherichia coli ....................................................... 9

Virulence Attributes of ETEC ................................................................. 9
Structure, Classification, and Antigenic Types of ETEC ................................. 11
Pilus or F imbrial Antigen (F antigen) ................................................ 11
Somatic (0) antigen ..................................................................... 13
Capsular (K) antigens ..................................................................... 14
Enterotoxins of Enterotoxigenic E. coli ................................................... 15
Heat-Stable Enterotoxins (STs) ............................................................... 15
Differences between STa and STb ........................................................... 16
Enterotoxigenic E. coli STa (STI) ........................................................... 17
The STa family of toxins ................................................................ 18
Structural determinants and biochemical properties of STa ........................ 19
Antigencity and antigenic determinants of E. coli STa ............................... 21
Structural-Function Relationship of E. coli STa ...................................... 22
Receptor of ETEC-STa: Guanylyl Cyclase C (GC-C) ................................. 23
Intestinal fluid and electrolyte homeostasis ............................................. 29
Intestinal absorptive mechanisms ...................................................... 29
Intestinal secretory mechanisms ........................................................ 30
Role of cyclic nucleotide in intestinal homeostasis ........................ ‘ .......... 3 O
Pathogenesis of ETEC Diarrhea ........................................................... 31
Infection with ETEC ..................................................................... 31
Intestinal colonization ................................................................... 32
Elaboration of enterotoxin: Pathophysiology of STa action ....................... 33
Potential systemic effect of ETEC-STa diarrhea ..................................... 37
Current Approaches for Controlling ETEC-STa Diarrhea .......................... 38
References ...................................................................................... 4]

viii

CHAPTER III: PURIFCIATION AND CHARACTERIZATION OF E. coli
HEAT-STABLE ENTEROTOXIN

Introduction .............................................................................. 56
Materials and methods ............................................................... 57
Animals .............................................................................. 57

Reagents ............................................................................ 57
Verifying the ETEC K99+ Strain ............................................... 57

Bacteria] Strain ................................................................. 57

DNA Extraction (Template) by boiling lysis .............................. 58

Primer selection and preparation ............................................ 58

PCR program .................................................................. 58

PCR reaction .................................................................. 59

Agarose gel electrophoresis analysis of PCR product .................... 59

Purification and characterization of E. coli STa ....................................... 59
Seed culture and frozen stock preparation of ETEC ................................... 60
Batch medium (asparagine salt medium) and growth condition ..................... 60
Preparation of cell free filtrate ............................................................ 61
Amberlite XAD-2 Batch Adsorption Chromatography ............................... 61
Acetone Fractionation ..................................................................... 62
Reversed-Phase Batch Adsorption Chromatography .................................. 62
Preparative RP—HPLC ..................................................................... 63
STa assessment for biological activity ................................................... 63
Criteria for homogeneity of the purified STa ........................................... 64
Results and Discussion ....................................................................... 64
Conclusion ...................................................................................... 67
References ...................................................................................... 72

CHAPTER IV: DESIGN AND CHARACTERIZATION OF AN
IMMUNOGENIC E. coli HEAT-STABLE ENTEROTOXIIN (STa)

Introduction ................................................................................... 74
Background and principle of peptide-carrier conjugation ................................ 75
Cross-linking reagents .................................................................. 75
Carrier proteins ........................................................................... 76
Materials and methods ....................................................................... 78
Reagents ..................................................................................... 78
Chemical modification of bovine serum albumin................................... 79
Succinylation of BSA ............................................................... 79
Hyper-succinylation of BSA ...................................................... 80
Coupling of E. coli STa to modified BSA .......................................... 81
Protocol 1: DMF method. ......................................................... 81

Protocol 2: Imidazole method .................................................... 82

Protocol 3: Hyper-succinylation method ........................................ 83

Protocol 4: Conventional method ................................................. 83

Dialysis ..................................................................................... 84

Gel permeation chromatography ........................................................ 84

Size exclusion chromatography ......................................................... 85

ix

Amino acid compositional analysis ................................................... 85
Matrix assisted laser desorption ionization-time of flight mass spectroscopy... 85

Protein Assay ............................................................................. 86
STa-carrier conjugate activity bioassay ............................................... 86
Results ........................................................................................... 86
Discussion ....................................................................................... 88
References ...................................................................................... 98

CAHPT ER V. ANTIBODY PRODUCTION AND CHARACTERIZATION
THE IMMUNE RESPONSE AGAINST E. coli HEAT-STABLE

ENTEROTOXIN
Introduction .................................................................................... 1 03
Materials and methods ...................................................................... 104
Reagents and instruments ............................................................... 104
Animals .................................................................................... 105
Immunization procedures ............................................................... 105
Animal bleeding .......................................................................... 105
STa— Serum Neutralization Assay ...................................................... 106
Kinetics of rabbit immune response to E. coli heat-stable enterotoxin ............ 107
Antibody-capture ELISA for screening sera .................................... 107
Avidity ELISA ...................................................................... 108
Statistical analysis ............................................................................ 109
Results ........................................................................................... 109
Discussion ....................................................................................... 111
References ....................................................................................... 126
Summary and conclusions .................................................................. 129

LIST OF TABLES

Table 1. Difference between E. coli Heat-Stable Enterotoxin A (STa) and Heat-

Stable Enterotoxin B (STb) ................................................................... 16
TableIZ. The STa Family of Toxins .......................................................... 18
Table 3. PCR primers used to detect STa gene .............................................. 58
Table 4. PCR running conditions for detection of STa gene .............................. 58
Table 5. PCR reaction for detection of STa gene ........................................... 59
Table 6. Composition of optimal minimal medium for STa elaboration ................ 60

Table 7. Summary of the purification scheme of E. coli heat-stable enterotoxin per
batch ............................................................................................... 71

Table 8. Summary of conjugation experiments: Evaluation of four different
conjugation protocols ........................................................................... 92

Table 9. Amino acid compositional analysis of E. coli heat-stable enterotoxin
peptide— BSA carrier molecule ................................................................. 93

Table 10. Approximate contribution of STa molecules to one molecule of modified
BSA: calculation of the conjugation ratio .................................................... 93

Table 11. E. coli STa-specific serum antibody end titer: Mean OD i SD value of
group 1, 2 and 3 rabbits after 24 weeks post immunization at various serum

dilutions ........................................................................................................................ 1 24

Table 12. Summary of STa- ELISA binding and neutralization end titers of rabbit
sera immunized with STa-suBSA conjugate after the primary immunization and
during the boosting intervals .................................................................. 124

Table 13. Summary of the development of STa antibody affinity after multiple
boosters with the STa conjugate: Five molar ammonium thiocyanate elution ......... 125

Table 14. Neutralization capacity of sera from animals immunized with several

STa-immuogenes and the end titers of the STa-neutralizing antibodies as reported
in the literature .................................................................................. 125

xi

LIST OF FIGURES

Figure 1: Structural determinants of Heat-Stable Enterotoxin of Enterotoxigenic

E. coli .............................................................................................. 20
Figure 2. Intramolecular cysteine—disulfide linkage for STaP and STaH ................ 24
Figure 3. Domain structure of guanylyl cyclases ........................................... 26
Figure 4. Pathophysiology of E. coli heat-stable enterotoxin diarrhea. ................. 36
Figure 5. Agarose gel electrophoresis of PCR product analysis .......................... 68

Figure 6. Growth kinetic of ETEC on 36L batch ASM under different pH using
Bellco bioreactor .................................................................................. 68

Figure 7. Revere-phase-high performance liquid chromatography elution profile of
60% HPLC-grade methanol- MCI gel -STa-rich fraction on preparative C8 Vaydac
column .............................................................................................. 69

Figure 8. Elution profile of biologically active E. coli heat-stable enterotoxin peaks
on analytic aquapore reverse-phase C 8 Perkin Elmer column ........................... 69

Figure 9. Matrix assisted laser desorption ionization-time of flight mass
spectroscopy profile of E. coli heat-stable enterotoxin .................................... 70

Figure 10. Illustration showing the conjugate of bovine serum albumin carrier
protein- E. coli STa peptide ................................................................... 79

Figure 11. The action of succinic anhydride upon the amino groups of a protein. . . 80

Figure 12. Size exclusion chromatography profile of native and succinylated BSA
molecule ........................................................................................... 94

Figure 13. Matrix- assisted laser desorption ionization-time of flight mass
spectroscopy profile of succinylated BSA molecule ....................................... 94

Figure 14. Matrix- assisted laser desorption ionization-time of flight mass
spectroscopy profile of E. coli heat-stable enterotoxin peptide-succinylated BSA
carrier conjugate. ................................................................................ 95

Figure 15. Matrix- assisted laser desorption ionization-time of flight mass

spectroscopy profile of E. coli heat-stable enterotoxin peptide-hypersuccinylated
BSA carrier conjugate ........................................................................... 96

xii

Figure 16. Calculation of A molecular weight and conjugation ratio of the E. coli

heat-stable enterotoxin peptide- BSA carrier conjugate .................................. 97
Figure 17. Illustration of antibody capture indirect ELISA protocol ..................... 114
Figure 18. E. coli STa-specific serum antibody neutralization bioassay: Group 1

rabbits ............................................................................................. 115
Figure 19. Neutralization capacity of E. coli STa-specific serum antibody ............. 115

Figure 20. E. coli STa-specific serum antibody neutralization bioassay: All
rabbits ............................................................................................. 116

Figure 21. E. coli STa ELISA Optimization: Screening serum dilution for optimal
E. coli STa-STa antibody interaction ........................................................ 117

Figure 22. E. coli STa -specific serum antibody: 104 serum dilution of group 1
rabbits ............................................................................................. 117

Figure 23. E. coli STa-specific serum antibody: 10'4 serum dilution of group 2
rabbits ..................................................... . ........................................ 118

Figure 24. E. coli STa-specific serum antibody: 104 serum dilution of group 3
rabbits .............................................................................................. 118

Figure 25. Mean OD value of group 1 rabbits after 20 weeks post immunization at
various serum dilutions .......................................................................... 119

Figure 26. . Mean OD value of group 2 rabbits after 20 weeks post immunization at
various serum dilutions .. ............................................................................................. 119

Figure 27. . Mean OD value of group 1 rabbits after 24 weeks post immunization at
various serum dilutions ......................................................................... 120

Figure 28. End titer of the E. coli STa-specific serum antibody: 24 weeks post
immunization from 8 rabblts 120

Figure 29. E. coli STa-specific serum antibody end titer: Mean OD value of group
1, 2 and 3 rabbits after 24 weeks post immunization at various serum dilutions ....... 121

Figure 30. Time-course evaluation of the E. coli STa-specific serum antibody
avidity using ammonium thiocyanate dose response ....................................... 122

Figure 31. Five molar thiocyanate elution profile of E. coli STa-STa serum antibody
complex: Mean OD of treated serum from group 1, 2 and 3 of rabbits .................. 123

xiii

Figure 32. Avidity index E. coli STa-specific serum antibody of the group, 1, 2 and
3 of rabbits ....................................................................................... 123

xiv

LIST OF ABBREVIATIONS
ANPR = Atrial natriuretic peptide receptors
BSA = Bovine serum albumin
cAMP = Cyclic adenosine monophosphate
2CIATP = 2 choloradenosine trihosphate
CFA = Colonization fimbriae of human ETEC strains
CFT R = Cystic fibrosis transmembrane conductance regulator
cGMP = Cyclic guanosine monophosphate
CI' = Cholride ion
CS = Coli surface antigen
CT = Cholera toxin
EAEC = Enteroaggressive E. coli
EAST] = Enteroaggregative heat-stable enterotoxin
ECD = Extracellular domain
EDAC =1—ethyl-3[3-dimethylaminopropyl] carbodiimide hydrochloride
ETEC = Enterotoxigenic E. coli
GC-C = Guanylyl cyclase type C
GTP = Guanosine triphosphate
HCO3' = Bicarbonate ion
HPLC = High performance liquid chromatography
ICD = Intracellular domain
K+ =Potassium ion

LT = Heat-labile enterotoxin

XV

LT = Heat-labile toxin

MU = Mouse unit

MWCO = Molecular weight cutoff

Na+ = Sodium ion

NMR = Nuclear magnetic resonance

PBS = Phosphate buffer solution

PDE3 = phosphodiesterase

PKC = Protein Kinase

SMA = Suckling mouse assay

STs = Heat-stable enterotoxins

STb = Heat-stable enterotoxin B

STa = Heat-stable enterotoxin isolated primarily from bovine
STp = Heat-stable enterotoxin isolated primarily from porcine
STh = Heat-stable enterotoxin isolated primarily from human
SuBSA = Succinylated bovine serum albumin

TD = Transmembrane domain

TFA = Triflouroacetic acid

Amino Acid Abbreviations

A ALA Alanine

CYS Cysteine

ASP Aspartic Acid

GLU Glutamic Acid

'11P! U G

PHE Phynylalanine

xvi

 

 

G GLY Glycine

HIS Histidine

I

ILE Isoleucine

“

LYS Lysine
LEU Leucine

MET Methionine

zgt-x

ASN Asparagine

PRO Proline

"U

GLN Glutamine

we

ARG Arginine

SER Serine

U)

T THR Threonine
V VAL Valine
W TRP Tryptophan

Y TYR Tyrosine

xvii

CHAPTER I
INTRODUCTION
Statement of the Issue and Rationale

Enterotoxigenic Escherichia coli (ETEC) is a major enteropathogen that
causes potentially fatal diarrhea in both human and animal neonates (Moon and Bunn
1993, Tacket et al. 1994). It is also responsible for a large proportion of diarrheal
disease among adult travelers (Tacket et a1. 1994). Therefore, strategies to reduce the
incidence and severity of ETEC diarrhea have been considered an important public
health priority (Tacket et a1. 1994). A large proportion of ETEC diarrhea is caused by
heat-stable enterotoxin (STa), a small peptide (2kD), which is an important virulence
determinant in enterotoxin-mediated diseases (Sears & Kaper 1996 and Giannella &
Elizabeth 2003). Upon infection, the STa producing- ETEC adheres to the epithelium
of the small intestine via one or more colonization factor antigens or pili surface
proteins. Once established, ETEC elaborates heat-stable enterotoxin (STa), which acts
on a specific intestinal membrane bound receptor, guanylyl cyclase C, initiating a
cascade of altered metabolic pathways. This may result in secretory diarrhea among
affected adults but can cause fatal dehydration in neonates.

Methods for the treatment and control of ETEC diarrhea are still a matter of
debate among veterinarians, livestock producers and in the animal industry in general.
The use of sub-therapeutic doses of antibiotics may help protect animals from some,
but not all, of these bacterial strains. Moreover, the use of antimicrobials at sub-
therapeutic levels has been linked to the problem of emerging antibiotic resistance

among several bacterial species, including ETEC strains.

“en-

While there are several reagents that are in use against ETEC diarrheal disease
in animals, most of these reagents are based on surface structures of the ETE strains.
However, the development of a broad-spectrum vaccine against ETEC remains
elusive (Walker et al. 2007). Two major technical problems contribute to this
deficiency. The first involves the production of immunogenic preparations of antigens
with the ability to confer broad-spectrum protection against ETEC infections. The
second is the challenge of achieving effective mucosal immunization (Walker et a1.
2007) due to the multiplicity, antigenic diversity, and high prevalence of
unidentifiable forms of specific colonization antigens responsible for mucosal
adherence (Deneke et a1. 1981, Levine et al. 1980, Thomas and Rowe 1982). Against
this background, there is an urgent need to define a new common antigenic
determinant that could provide broad protection against ETEC-STa—induced diarrhea.

Saeed et a1. (1985) demonstrated that calf scour could be experimentally
induced by a highly purified STa preparation, supporting the notion that ETEC STa is
the immediate mediator of diarrhea in claves. Additionally, several studies have
demonstrated a significant correlation between STa-producing ETEC strains and
diarrhea, and that 75% of ETEC strains produce STa either alone or in combination
with heat-labile enterotoxin (LT) (Wolf 1997). Thus, the inclusion of STa in
colonization factor-based ETEC vaccines or the production of neutralizing STa-
antibodies would potentially offer immune protection against ETEC—caused diarrhea.
However, this approach has been a challenge, partly because of the haptenic nature
of STa (molecular weight <2 kDa), which fails to elicit an antibody response (De

Weck 1974 and Pereira et a1. 2001 and Boedeker 2005). Additionally, the correlation

between STa toxicity and antigenicity (Takeda et a1. 1993) hampers the ability to
produce a safe STa/CFAs vaccine. However, it was hypothesized that the poor
immunogencity associated with the STa molecule could be improved by conjugation
of the STa to a suitable macromolecule (carrier protein) (Elanger 1980 and Pauillac et
al. 1998).

Therefore, antibody-based therapy (passive immunization) targeting the STa
antigen could be used to reduce the impact of ETEC-STa induced diarrhea and avoid
the safety issue associated with active immunization with CFA/toxin based—vaccine.
Attempts to conjugate the STa to a carrier protein have been reported (Clements,
l 990; Houghten et al. 1984, 1985; Klipstein et a1. 1982, 1983; Sanchez et al. 1986,
l 988), however no sufficient details were presented on the efficiency and /or the

Characteristics of these conjugates.

In this study, STa was purified to homogeneity and its properties were
ChaJacterized. The purified STa was then covalently cross-linked to modified BSA
using 4 different protocols of peptide-carrier conjugation. We have characterized the
STa-conjugates for biological activity, conjugation efficiency, and stability of the
conjugates. The best STa-conjugate was then used for immunological studies.
\General Objectives

' Purify and characterize E. coli STa.
I Design and characterize an immunogenic E. coli STa.
I Produce and characterize specific antibodies against E. coli STa in laboratory

animal models.

 

 

Specific Aims

1. Production of high performance liquid chromatography - purified E. coli STa
I Verify bacterial strain as an ETEC STa-positive strain by identification of the
STa encoding gene.
I Adoption of bioreactor E. coli culture method for the production of STa and
study the growth kinetics at different pH levels.
I Biological and molecular confirmation of STa specificity and identity.
II. Design and characterization of an immunogenic E. coli STa .
I Evaluate four different protocols of peptide conjugation to carrier protein.
I Molecular characterization of the most effective STa— conjugates.
III- Production of specific antibodies with high neutralizing capacity against
E coli STa
I Study the neutralization capacity of harvested polyclonal antibodies against
E. coli STa.
I Study the kinetics of the E. coli STa antigen-antibody interaction:
- ELISA STa-IgG binding capacity assay.

- Determination affinity and avidity index of STa antibodies.

L’O. tential Benefits of the Study

Enterotoxigenic E. coli (ETEC) that produces STa is a major cause of severe
diarrhea in neonatal animals, children, and adult travelers. The production of specific
aIltibodies with a high neutralization capacity against STa can help to reduce the

incidence and economic losses associated with ETEC STa-induced diarrhea. An

 

effective immunogenic STa may have potential as an ETEC vaccine that may confer

broad protection against ETEC-STa-induced diarrhea.

 

References

Boedeker, E. C. 2005. Vaccines for enterotoxigenic Escherichia coli: current status.
Current Opinion in Gastroenterology. 21 (1):] 5-1 9.

Clements, J. D. 1990. Construction of a nontoxic fusion peptide for immunization
against Escherichia coli strains that produce heat-labile and heat-stable
enterotoxins. Infect Immun 58, 1 159-1 1 66.

De Week, A. L. 1974. Low molecular weight antigens. In: M. Sela (Ed.), The
Antigens, Vol. II. Academic Press, New York, Chapter 3.

Deneke, C.F; Throne, GM. and Gorbach. S.L. 1981 Serotypes of attachment pili of
enterotoxigenic Eschericia coli isolated from humans. Infect immun 32: 1254-
1260.

Elanger, BE. 1980. The preparation of antigenic-hapten-cam'er conjugates: a survey.
Methods Enzymol. 70 (A), 85.

Giannella, R. A. and Elizabeth, A. M. 2003. E. coli heat-stable enterotoxin and
guanylyl cyclase C: New functions and suspected actions. Trans Amr Clin
Climato Ass. 114, 67-85.

Houghten, R. A., Engert, R. F ., Ostresh, J. M., Hoffman, S. R. & Klipstein, F. A.
1985. A completely synthetic toxoid vaccine containing Escherichia coli heat-
stable toxin and antigenic determinants of the heat-labile toxin B subunit.
Infect Immun 48, 735- 740.

Houghten, R. A., Ostresh, J. M. & Klipstein, F. A. 1984. Chemical synthesis of an
octadecapeptide with the biological and immunological properties of human
heat-stable Escherichia coli enterotoxin. Eur J Biochem 145, 157-162.

Klipstein, F. A., Engert, R. F. & Clements, J. D. 1982. Development of a vaccine of
cross-linked heat-stable and heat-labile enterotoxins that protects against
Escherichia coli producing either enterotoxin. Infect Immun 3 7, 55 0-55 7.

Klipstein, F. A., Engert, R. F ., Clements, J. D. & Houghten, R. A.1983. Vaccine for
enterotoxigenic Escherichia coli based on synthetic heat-stable toxin crossed-
linked to the B subunit of heat-labile toxin. J Infect Dis 147, 318-326.

Levine M.M; Rennels, M.B. Daya, V. and Hughes, T.P. 1980. Hemagglutination and
colonization factors in enterotoxigenic and enteropathogenic Eschercia coli
that cause diarrhea. J. Infect. Dis. 141: 733- 73 7.

Moon, H.W. and Bunn, TO. 1993. Vaccinne for preventing enetrotoxigenic
Escherichia coli infections in farm animals. Vacine, 11: 213-220.

 

Pauillac, S; Naar, J; Branaa, P. and Chinain M. 1998. An improved method for the
production of antibodies to lipophlic carboxlic hapten using small amount of
hapten-carrier conjugate. Journal of Immunological Methods 220: 105-1 14.

Pereira, C.M; Guth, B.E.C; Aleida, ME. and Castilho, BA. 2001. Antibody response
against Escherichia coli heat-stable enterotoxin expressed as fusions to
flagellin. Microbiology, 147, 861 -86 7.

Saeed, A. M; Magnuson, N. S; Gay, C. C. and Greenberg, R. N. 1985.
Characterization of heat-stable enterotoxin from a hypertoxigenic Escherichia
coli for cattle. Microbiology and Therapy, 15: 221 -229.

Sanchez, J ., Svennerholm, A-M. & Holmgren, J. 1988. Genetic fusion of a non-toxic
heat-stable enterotoxin-related decapeptide antigen to cholera toxin B-subunit.
FEBS Lett 241, 110-114.

Sanchez, J ., Uhlin, B. E., Grundstrom, T., Holmgren, J. & Hirst, T. R. 1986.
Immunoactive chimeric ST-LT enterotoxins of Escherichia coli generated by
in vitro gene fusion. F EBS Lett 208, 194-198.

Sears, C. L; and Kaper. J. B. 1996. Enteric bacterial toxins: mechanisms of action and
linkage to intestinal secretion. Microbiol. Rev. 60:167-215.

Tacket, C.O; Reid, R.H; Boedeker, E.C; Losonsky, G; Nataro, J .P; Bhagat, H. and
Edelman, R. 1994. Enteral immunization and challenge of volunteers given
enterotoxigenic E. coli CF A/II encapsulated in biodegradable microspheres.
Vaccine 12:1270-1274.

Takeda, T; Nair, G. B; Suzuki, K; Zhe, H. X; Yokoo, Y; Hemelhof, W; Butzler, J. B;
Takeda, Y; and Shimonishi, Y. 1993. Epitope mapping and characterization of
antigenic determinants of heat-stable enterotoxin (STh) of enterotoxigenic
Escherichia coli by using monoclonal antibodies. Infection and Immunity

61 :289-294.

Thomas, L.V. and Rowe, B. 1982. The occurrence of colonisation factors (CFAI,
CF All and E8775) in enterotoxigenic Escherichia coli from various
copuntries in south east Asia. Med Microbiol Immunol 1 71: 85-90.

Walker, R.I; Steele, D; Aguado, T. and Ad Hoc ETEC Technical Expert Committee
2007. Analysis of strategies to successfully vaccinate infants in developing
countries against ETEC disease. Vaccine 25, 2545-25 66.

Wolf, MK. 1997. Occurrence, distribution and association of O and H serogroups,
colonization factor antigens, and toxin of enterotoxigenic Escxheichia coli. Cli
Microbiol Rev 10: 5 69-584.

 

CHAPTER II
REVIEW OF LITERATURES

Magnitude and impact of diarrheal diseases

Diarrhea] diseases are one of the major causes of human death on a global
scale. They are the leading cause of childhood death, resulting in 18% of all deaths in
children under the age of five (Bryce, 2005). In some populous developing areas, they
are responsible for more years of potential life lost than all other causes combined
(WHO, 1990). According to the Global Burden of Disease, in 1990, diarrheal
diseases were a leading cause of disability-adjusted life year (DALY), second only to
lower respiratory infections (est. 99.2 million DALYs lost) (Murray, 1997).

Additionally, the National Animal Health Monitoring System (NAHMS, 1994
&1996) survey and the USDA (1997) identified diarrheal diseases as the most
common infectious cause of neonatal calf mortalities (est.75% of mortalities).
Decades of research have been dedicated to obtaining a better understanding of the
neonatal calf diarrhea complex. However, despite improvements in the identification
of the infectious agents, management practices, and treatment and prevention
strategies, the complex remains the most common and costly (est. $120 million
annually) disease affecting newborn calves in the United States (NAHMS, 1996).

The list of enteropathogens that can potentially cause diarrhea is quite large
and diverse. There are six major pathogens that cause diarrhea in newborn animals:
enterotoxigenic Escherichia coli, Rotavirus, Coronavirus, Cryptosporidium parvum,

Salmonella spp., and Clostridium perfringens type C (Snodgrass et al. 1986).

 

Diarrhea caused by Enterotoxigenic Escherichia coli (ETEC) is problematic
in both animal and human populations. In animal neonates it causes severe profuse
watery diarrhea, potentially fatal dehydration and metabolic acidosis (Argenzio,
1985). Additionally, ETEC is a major cause of traveler’s diarrhea (Gorbach et al.
1975) and a leading enterpathogen responsible for gastroenteritis outbreaks on cruise
ships (Addiss et al. 1989 and Koo et al. 1996).

M] Overview of Escherichia coli

Escherichia coli is a common member of the normal flora of the large
intestine. Most E. coli strains are avirulent and remain benign commensals because
they lack specific genetic elements encoding for virulence factors. However, strains
become virulent upon acquiring bacteriophage plasmid DNA encoding enterotoxins
or invasion factors. The mechanisms by which virulent E. coli strains acquire such
genetic elements seem to be horizontal genetic transfer including phage transudation,
transposition, conjugation of plasmids and simple recombination (Seifert and DiRita,
2006). Virulent E. coli strains cause a variety of disease conditions including plain,
watery diarrhea, inflarrunatory dysentery, hemolytic uremic syndrome, septicemia,
pneumonia and meningitis (Salyers and Witt 1994).

Virulence Attributes of ETEC

Pathogenicity of E. coli is a complex multifactorial mechanism involving a
large number of virulence factors that vary according to the pathotype. They include
attachment functions, host cell surface modifying factors, invasins, and many
different toxins as well as secretion systems which export toxins and other virulence

factors and pilot them to the target host cells. These virulence attributes are often

organized into large genetic blocks on the chromosome, known as pathogenicity
islands, on a large plasmid, or on phage DNA and can be transmitted horizontally
between strains (Seifert and DiRita, 2006). In this regard, E. coli becomes virulent
upon the acquisition of plasmids that encode genes for two virulence determinants,
namely colonization factors (adhesins), and enterotoxins. Both enterotoxins and CPS
work in concert to cause diarrhea in both humans and animals.

Two classification schemes are currently used to classify the different strains
of E. coli, serotyping and virotyping (Lior 1996 and Levine 1987). Serotyping of E.
coli occupies a central place in the history of these pathogens and is mainly based on
antigenic differences in the highly variable bacterial surface molecules such as
lipopolysaccharides (O antigens) and flagella . (H antigen) (Lior 1996). Specific
combinations of O and H antigens define the serotype of an E. coli strain and are
widely used for tracing outbreaks of enteric diseases. More than 170 different “0”-
specific somatic antigens, 56 different types of flagellar “H” antigens and 103
different capsular “K” antigens in E. coli have been characterized (Nataro and Kaper,
1998). Some E. coli, particularly the ETEC strains, produce mannose resistant (MR)
fimbriae that may be used for serological identification. Some of these MR fimbriae
(e.g. K88 and K99) were once identified as K antigens before their chemical
composition was known. They are presently defined as F (fimbrial) antigens, which
also include the mannose sensitive type 1 fimbriae group (Levine et al. 1983).

Virotyping is a phenotypic classification scheme based on virulence
characteristics of E. coli that includes patterns of bacterial attachments on host cells,

the effect of attachments on host cells, production of toxins and invasiveness.

10

Currently there are six virotypes: Enterotoxigenic E. coli (ETEC), Enteropathogenic
E. coli or Attaching and Effacing E. coli (EPEH, AEEC), Enterohemorrhagic E. coli,
Shiga toxin producing E. coli or Virotoxigenic E. coli (EHEC, STEC, VTEC),
Enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC) and diffusely
adherent E. coli (DAEC) (Nataro and Kaper, 1998). In this literature review, we will
largely cover the different characteristics of ETEC strains.

Structure, Classification, and Antigenic Types of ETEC

ETEC organisms are Gram-negative, short rods, not visibly different fiom the
E. coli found in the normal flora of the large intestine (Bettelheim 1994). Virulence-
associated attributes are fimbriae and enterotoxins. Genes for both attributes are
carried on a plasmid and play a crucial role in the pathogenesis of calf scours (Levine
et al. 1983 and Nataro and Kaper 1998). All ETEC strains contain plasmids, but this
is not a distinguishing feature unless gene probe techniques are used to detect specific
virulence-associated genes on these plasmids (N ataro and Kaper, 1998).

Pilus or Fimbrial Antigen (E antigen)

A common trait of ETEC strains is the expression of several surface-
associated proteins, antigenically unrelated to O and H antigens, called F imbriae
(Pili) antigens. Fimbriae, which can be seen in electron micrographs as fuzzy coat, are
filamentous structures that protrude as hair-like structures from the bacterial surface
which are much thinner (3-4 nm) and usually more rigid than flagella (Follett and
Gordon, 1980). They are composed of proteins (pilin) that are tightly packed into an
array that is shaped like a helical cylinder and their assembly occurs on the outer cell

membrane of the bacteria through a specialized pathway of the general secretion

11

system, the alternate chaperone/ usher pathway (Pitek et al. 2004 & Planet et al.
2006)

Commensal E. coli strains usually produce so-called common pili, while
pathogenic ETEC possess specialized host-specific pili (Levine et al. 1983).
Virulence-associated fimbriae are antigenically unrelated to common pili. They are
too small to be seen by light microscopy and act as ligands to bind specific complex
carbohydrate receptors on the epithelial cell surfaces of the small intestine. Since this
interaction results in colonization of the intestine by ETEC, with subsequent
multiplication on the gut surface, these pili are termed adhesins or colonization
factors antigens (CFAs).

A variety of CFs has been described in ETEC strains of animal origin. F4
(K88), F5 (K99), F6 (987P), F41, F42, F165, F17 and F18 are produced by E. coli
that cause acute diarrhea in domestic animals. ETEC strains expressing K-99 are
pathogenic for calves, lambs and pig, whereas K-88 expressing organisms are able to
cause disease only in pigs (Cassels & Wolf 1995). Human ETEC strains also possess
an array of colonization fimbriae (CFAI, CFAII and CFAIV) (de Craaf & Gaastra
1994)

Nearly all ETEC from calves produce F5 (CFA-K99) and STa (Gyles 1996).
In addition to K99, several other fimbriae designated as F41, F92b, Att25, and F210
have been identified on calf ETEC but are much less common (Morris et al. 1983).
F41 has been characterized and it mediates attachment to calf enteroytes in vitro and
to the small intestine of newborn lambs (Morris et al. 1983). There may be a

relatively small group of other fimbriae that also mediate adhesion to calf enterocytes,

12

although K99 is the most common attachment factor on bovine ETEC. This is not
surprising, since a similar situation occurs in ETEC isolated from pigs, where K88
and 987P, and at least two other fimbriae known as K99 and F41, also mediate
adhesion to pig enterocytes (Moon et al. 1977). Age-dependent resistance to diarrhea
caused by F5 positive ETEC develops rapidly in calves (Runnels et al. 1980).

Genes coding for the production of CFAs reside on the ETEC virulence
transmissible plasmids, usually on the same plasmids that carry the genes for one or
both of the two types of E. coli enterotoxin, heat-labile enterotoxin (LT) and heat-
stable enterotoxin (ST). Most F4 (K88) strains produce LT while most F5 (K99) and
F6 (987P) ETEC strains produce ST. Two types of STs are produced, the majority of
ETEC strains produce STa thought a few produce STb. Therefore, most cases of
ETEC diarrhea in newborn calves are caused by E. coli possessing a F5 (K99) and an
ST gene (Gyles, 1996).

Somatic (O) Antigen

Somatic antigens are specific heat-stable and composed of polysaccharide
chains linked to the core lipopolysaccharide (LPS) complex common to all Gram-
negative bacteria. “0” antigen specificity is determined by sugar or amino-sugar
composition and by the sequence of these outer polysaccharide chains (Morris et al.
1983).

In normal smooth strains, the core LPS is buried beneath the 0 antigen while
O-minus mutants, called rough strains, the core LPS is exposed. “O” antigens are not
single antigens, but they are composed of several antigenic components and,

therefore, are called 0 group antigens. Different 0 groups may share some of these

13

antigenic components; hence, there is considerable cross-reactivity among E. coli 0
antigens; also, many 0 groups of E. coli are cross-reactive or identical with specific 0
groups of Shigella, Salmonella, or Klebsiella (Gyles, 1996).

O antigens form the basis of serotyping classification of E. coli. More than
170 different O-specific antigens have been defined but only groups 8, 9, 20, 26, 101
and 141 are common on calf ETEC (Nataro and Kaper, 1998).

The role of “0” antigen in the pathogenesis of diarrhea is not clear, however,
it is assumed that strains that possess these antigens offer unknown advantageous
conditions for the plasmids that carry the genetic elements encoding for enterotoxin
and fimbrial production (Evans et al. 1977).

Capsular (K) antigens

Capsular antigens are acidic polysaccharides that encapsulate the somatic
antigen on some but not all ETEC (Acres, 1985). They appear as a dense mat of
fibrous projections under electron microscope (Hadad and Gyles, 1982a). The process
of E. coli serotyping has encountered difficulty because K antigens prevent the
agglutination of the “O” antigens by the homologous sera. They are further
subdivided into three groups based on their heat stability; (i) The L-type K antigens
are completely destroyed by heat at 100 °C for 1 hour, thus rendering the 0 antigen
agglutinable in O antisera. They retain no antigenicity and lose their ability to
combine with homologous K antisera. (ii) The B-type K antigens are also destroyed
by heat at 100 °C for 1 hour and lose their antigenicity. However, they retain their
ability to bind or combine with their homologous antisera. (iii) A-type K antigens are

not inactivated by heat at 100 °C, but require heat at 121 °C for 2.5 hr before the O

14

antigen becomes agglutinable. There are 103 different K antigens recognized in E.
coli, however only 11 have been identified commonly on calf ETEC strains (Orskov
et al. 1977). The role of K antigens in infection by ETEC is not known, but they
appear to be important in extra-intestinal infections.

Enterotoxins of Epterotoxigenic E. coli

Two enterotoxins have been identified among the extracellular products of
E. coli isolated from human and other mammals with diarrheal diseases- one is a high
molecular- weight type (86.500 kD), namely heat-labile enterotoxin (LT) and the
other is a low-molecular weight type called heat-stable enterotoxin (STs). The LT is
closely related to cholera toxin (CT), sharing approximately 80% protein sequence
identity in the A and B subunits and like CT toxin, stimulates adenylate cyclase and is
inactivated by heating. On the other hand, STs completely differ from CT, they act
more rapidly, and resist inactivation by heating (Levine et al. 1983; Saeed et al 1984;
Sears & Kaper 1996 and Nataro & Kaper, 1998).

Heat— Stable Enterotoxins (STs)

ETEC strains produce a diverse family of closely related potent peptides
toxins that induce secretory diarrhea in humans and animals. STs were initially
defined by their resistance to inactivation by boiling for 30 min (Stine and Nataro,
2006). They are classified into two structurally, functionally and immunologically
unrelated subtypes, STa (STI) and STb (STII). Table 1 shows the basic differences

between STa and STb (Sears and Kaper, 1996 & Nataro and Kaper, 1998).

15

Table 1. Difference between E. coli Heat-Stable Enterotoxin A (STa) and
Heat-Stable Enterotoxin B (STb)

 

Properties

STa = STl

STb = STII

 

Size

<2kDa

5.1kDa

 

Number of amino
acid residues

18-19 amino acids

48 amino acids

 

Mechanism of
synthesis and

Pre-pro form 72 amino-acid
precursor followed by two
consecutive peptidase cleavages

71 amino-acid precursor processed
into 48 amino acid mature toxin
secreted extracellularly without

 

secretion before extracellular diffusion of furth .

18-19 amino acid mature toxin er p rocessmg
Number of cysteine Six cysteine residues F our cysteine residues
residues Three S-S bonds Two S-S bonds

 

Toxic domain

Hydrophobic: l l-l4 amino acid
espgcialy Ala 13

Charged amino acids: especially
lys-22, lys 23, arg 29 and asp-30

 

 

 

 

 

 

 

 

 

 

Solubility Methanol soluble Methanol Insoluble
Inactive in suckling mice but active
. . Active in mice, infant, piglet and In rats and ligated piglet mtestrnal
Actrvrty calves segment.
Have no effect on human small
intestine.
Trypsin effect Resistant Sensitive
Does not act on cyclic nucleotides.
Mechanism of action Act on guanylyl cyclase C Ca, PGE2 and serotonin may be its
mode of action
Loss of villus epithelial cells and
Effect on partial villus atrophy (Nagy and
enterocytes: No effect Fekete 1999)
STaP (procine isolates) and STaH
Prototypes (human isolate) None
Producing strains ETEC and other bacteria Only ETEC

 

16

 

Genes encoding for both classes are found predominantly on plasmids, and
some STa-encoding genes have been found on transposons. So and colleagues (1979,
1980 & 1981) demonstrated that the STa gene is embedded within a functional
transposon, and the presence of STa on widely variable plasmids supports this as the
dominant mechanism for ongoing transfer.

Enterotoxigenic E. coli STa (ST 1)

There are two slightly different toxin prototypes under this designation: STIa
(STp) and Sle (STh). Both variants are structurally, functionally and antigenically
related. They share a core 13 amino acid-sequence that is necessary and sufficient for
enterotoxic activity but differ in their N-terminal sequence. While STp is produced by
porcine, bovine and human ETEC, STh is onlyproduced by human isolates (Nataro
and Kaper, 1998).

A large family of STa-related toxins has been also identified in various
organisms. All share similar size (ranging from 15-30 amino acids) and a highly
conserved 13-amino-acid C- terminal region, which is essential for their toxicity
(Table 2) (Stine and Nataro, 2006). The biochemical, physiOlogical and
immunological properties of the various STa enterotoxins are remarkably similar.
They have four conserved amino acids (N-P-A-C) (Stine and Nataro, 2006) that
mediate binding to the membrane guanylyl cyclase-C (Greenbreg et al. 1997). Most
of them have six cysteine residues located in the same relative positions and linked
intrarnolecularly by three disulfide bonds, which suggests that these enterotoxins have

similar tertiary structure (Shimonishi et al. 1987). The secretory potency and heat

17

stability of the STa enterotoxins are determined by conserved core sequence

(Yoshimura et al. 1985 and Shimonishi et al. 1987).

Table 2. The STa Family of Toxins

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Toxin and No. Sequence Reference
host Amino

acids
STaH ETEC 19 N-S-S-N-Y-C-C-E-L-C-C-N-P-A-C-T-G-C-Y Aimoto et al 1982
STaP ETEC 18 N-T-F-Y-C-C-E-L-C—C-N-P-A-C—A-G-C-Y Takao et al 1983
STa ETEC 18 N-T-F-Y-C-C-E-L—C-C-N-P-A-C-A-G-C-Y Saeed et a1 1984
(bovine)
C itrobacter 18 N-T-F-Y-C-C-E-L-C-C-N-P-A-C-A-G-C-Y Guarino et a1 1989
Freundii
Yersinia 30 S-S-D-Y-D-C-C-D-Y-C-C-N-P-A-C-A-G-C Takao et a1 1985
enterocolitica
V. cholera l7 l-D-C-C-E-l-C-C-N-P-A- C-F-G-C-L-N Yoshimura et a1
Non-01 1986
V.cholera non- 18 L-l-D-C-C-E-l-C-C-N-P-A-C-F—G-C-L-N Arita et al 1991
01 Hataka
V. mimicus l7 l-D-C-C-E-l-C-C-N-P—A-C-F-G-C-L-N Arita et al 1991
E. coli EAST -l 38 ...A-S-S-Y-A-S-C-l-W-C-T---T-A-C-A-S—C-H-G Savarino et a1 1993
Conus l3 E-C-C-N-P—A-C-G-R-H-Y-S-C Gray et a1 1981
geographus I3
Guanylin '15 P—G-T-C-E-l-C-C-AY-A-A-C-T-G-C Greenberg et al
(human) 1997

 

Adopted from Nataro & Stine (2006)

18

 

Structural determinants and biochemical properties of STa (Eigpre l)

STa is a small peptide (molecular weight of less than 2 kDa) that has 18- or
19- amino acids (Sato & Shimonishi, 2004). STa is produced as a 72 amino-acid
precursor that is cleaved by signal peptidase l to a 53-amino- acid-peptide (pro-STa)
(Rasheed et al. 1990). Prior to secretion by the bacteria, three intramolecular disulfide
bonds crucial to toxin activity are formed in the periplasm by DsbA, chromosomally
encoded protein (Yamanaka et al. 1994). A second undefined proteolytic event occurs
extracellularly to process the pro-STa to produce biologically active STa of 18 or 19
amino acids which is released by diffusion across the outer membrane (Rasheed et al.
1990)

The structural characteristics of biologically active STa were extensively
studied (Gariepy et al. 1986; and Osaki et al. 1991) on weakly toxic and nontoxic STa
analogs (Sato et al. 1994). Using X-ray diffraction analysis, Sato and Shimonishi,
(2004) studied the molecular structure of the toxic domain of fully active STa
molecule, which consists of the native sequence from Cys5 to Cysl7 and revealed that
the peptide molecule forms a ring-shaped peptide hexamer. The outer surface of this
peptide is occupied exclusively by the central portion of the side chains of Leus,
Asnl ', Pro”, and Ala”, proposed to be a binding site to the receptor protein, guanylyl
cyclase C, GC-C while the inner surface of the peptide hexamer consists of a single
invariant Gly residue (Osaki et al. 1991; Sato and Shimonishi, 2004).

Overall the STa molecule has a hydrophobic nature with certain structural
elements that are functionally critical. One of which is the occurrence of six cysteine

residues participating in three disulfide bridges (Garpiey et a1. 1987; Okamoto 1987;

19

Figure 1. Structural Determinants of Escherichia coli STa

’ ETEC STa ‘

 

 

Arrrino acid
sequence

 

 

 

 

 

 

 

 

 

 

 

_ Small MW
Structural Determinants ]—— 2kDa

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

INTFYCCELCCNPACAGCY”; 1 Non
immunogenic
Hydrophohrc NMR & X ray
Crystallography
‘ 6-Cysteine residues | L 3-beta turns 1
3-disulfide bonds Bl turn 132 turn B3 turn
Cys6- Asn1 1- CycM-
Cys9 Cysl4 Cysl7
l Cyss-Cysl0 I Cysé-Cysl4 l Cys9-Cys17 11-14 residues
NPAC esp Ala13

 

 

 

 

 

 

 

 

 

 

 

 

Stability and binding activity sites

20

 

Osaki et al. 1991; Sato et al. 1994 and Sato & Shimonishi, 2004) suggesting that this
toxin has tertiary structure (Shimonishi et al. 1987). Listed in descending order of
importance in terms of their respective contribution to enterotoxic activity, the
disulfide linkages between Cys6 and Cys”, CysS and Cys'o, and Cys9 and Cysl7 are all
necessary for stabilizing the STa molecule and expressing full STa toxic activity
(Garpiey et al. 1987). In addition, the second B-turn at residues 11 to 14 (especially
Ala”), is proposed to be crucial to the STa toxicity and its interaction with GC-C
(Osaki et al. 1991 and Sato et al. 1994).

Amino acid sequence of STa purified from several strains of bovine ETEC
was reported by Saeed and colleagues (1983, 1985a & b). STa from bovine ETEC
was found to consist of 18 amino acids and was similar to STa purified from ETEC of
human and porcine origin.

ETEC (STa) is methanol and acetone soluble. It resists proteolytic (pronase
and trypsin) enzymes (Jacks and Wu, 1974), heat and acids but not alkalines. The
toxin loses biological activity on treatment with reducing agents, such as p-mercapto-
ethanol, dithiothreitol or after performic acid oxidation suggesting the presence of

disulfide bridges (Eldeib et al. 1986).

Antigenicity and antigenic determinants of E. coli STa

 

E. coli STa is poorly immunogenic because it has low molecular weight
(Gyles 1971; Evans et al. 1973; and Alderete & Robertson, 1978). The antigenic
determinants of STa have been analyzed (Takeda et al. 1993). Three distinct antigenic
sites of STa have been recognized, one near the N-terminus, another in the core

functional region, and the third in the C-terminal, Asn-Tyr‘l-Leu8 -Asn”-Pro'2-Alal3-

21

Cys'4-Tyr18. Characterization of various STa epitopes revealed that the N—terminus
residues, which are not essential for the biological activity of STa in suckling mice
(Aimoto et al. 1982; Shimonishi et al. 1987 and Yoshimura et al. 1985 & 1987), but
possess an important antigenic determinant (Takeda et a1. 1993).
Structural-Function Relatioyhip of E. coli S'I‘ja

Four characteristic structural features of the STa molecule greatly contribute
to its function: hydrophobicity, single invariant glycine residue, conserved 6-cysteine
residues, and geometrical shape of this molecule (Sato and Shimonishi, 2004).

Hydrophobicity of the STa creates a favorable situation for an interaction with
GC-C on biological membranes. The side chains of the amino acids of the binding
region of ST (Asnl 1, Pro‘2 and Ala”), which are constant for numerous bacteria, form
a prominent, isolated cluster that projects from the surface to the outside of the
molecule and allows binding with GC-C because of their hydrophobic nature. Site
directed mutagenesis studied the contribution of these residues to the toxic activity of
the STa molecule and found that replacement of Ala at position 13 by Gly or Leu
residue led to loss of enterotoxigenic activity of the toxin (Sato and Shimonishi,
2004) suggesting that Ala13 has a key role in the interaction of STa with its receptor
domain. Other amino acid residues were tested for their role in STa-receptor binding
(Takeda et al. 1991 & 1993 and Yoshino et a1. 1994).

The inner surface of the peptide hexamer is occupied by a single invariant
Gly. The backbone conformation in this region is unique for the Gly residue and other
amino acid substitution may disrupt the formation of hexamer structure by steric

hindrance (Sato and Shimonishi, 2004).

22

STa has 6-cystiene residues connected with each other though three
intramolecular disulfide bonds (Figure 2) which are crucial for the spatial structure of
STa and for the expression of toxicity. Yamasaki et al. (1990) studied the contribution
of each disulfide bond to the toxic pr0perty of STa. The authors found that peptides
with only one disulfide bond are not biologically active, but peptides with a disulfide
bond in the second B-turn and one other disulfide bond had distinct activity. A peptide
that lacks a disulfide bond in the second [3 turn is not toxic. Clearly, disulfide bonds
are essential to stabilize the spatial structure of STa are necessary for STa toxicity and
stability.

The finding that native ST forms a self-associated hexamer is consistent with
other structure-activity relationships, which are difficult to rationalize using the
monomeric structure of STa analogous (Sato and Shimonishi, 2004).

Mpg" of ETEC-STa: valyl Cyclase C (GC-C)

The major receptor for STa is a membrane-bound (particulate) guanylyl
cyclase C (GC-C), (Field et al. 1978; Hughes et al. 1978; Schulz et al. 1990 and
Vaandrager, 2002, Giannella and Elizabeth 2003) which belongs to a group of
receptor-linked enzymes that catalyzes the conversion of guanosine 5'-triphosphate
(GTP) to second messenger cyclic guanosine 5'-monophosphate (cGMP) in response
to diverse signals, such as peptide hormones. In addition to GC-C, this family
includes also the atrial natriuretic peptide receptors (GC-A & GC-B) (Lowe et al.
1990 a&b) and the olfactory (GC-D) (Yang et al. 1996) and retinal cyclases, (GC-E,

& GC-F) (Oliveira et al. 1994 and Yang et a1. 1996).

23

Amide backbone Side chains binding site

 

 

 

 

l l l l
Cyss-Cys‘S-Glu-CLeu8 -Cys9 -Cysl0 -Asn"-Pro12 - Alau’ -Cys"—Ala'5-Glyl6-Cysl7
I J l l l l
lSt B turn 2nd B turn 3"I B turn

Crucial Structure of STa

 

STaP: N-T-F-Y-C51-C6-E-L-C9-CLLN-P-A-CI‘ll-A-G-Cl7-Y

 

 

 

 

 

 

STaH: N-S-S-N-Y-p6-C7-E-L-C”-91l-N-P-A-Clls-T-G-Cls-Y

 

 

 

 

 

Figure 2. Intramolecular cysteine-disuflide linkage for STaP and STaH
(Sato et al. 1994)

24

All these GCs are receptor-linked enzymes with one membrane-spanning region and
share a conserved intracellular catalytic domain (ICD) but they differ in their
extracellular ligand-binding domains (ECD) because they are activated by different
signals (Lucas et al. 2000; Schulz et a1. 1990 and Vaandrager, 2002) (Figure 3).

The size and nature of the STa receptors have been an area of intensive
investigation resulting in the identification of at least one definitive STa receptors
located in the apical membrane of the intestinal epithelial cells (Schulz et al. 1990;
Thompson & Giannella, 1990; and Cohen et al. 1993). Binding of ligands (STa,
guanylin and uroguanylin) to the ECD stimulates the intracellular enzymatic activity.
Molecular cloning from various mammalian species revealed that the primary
structure of GC-C (120-kDa protein unglycosylated and a 140 to 160-kDa protein
after N-linked glycosylation) is composed of an extracellular N—terminal receptor
domain (ECD), a single transmembrane domain (TMD) and a cytosolic domain
(ICD). ICD comprises a kinase homology domain (KHD) which is linked by an or-
helical putative dimerization domain to the C-terminal catalytic domain (Schulz et al.
1990; de Sauvage et a1. 1991; Garbers, 1999; Lucas et al. 2000).

GC—C contains a unique ECD comprising approximately 40% of the total
protein, which contains 8-10 N-linked glycosylation sites and functions as the binding
site for GCC-ligands (Schulz et al. 1990 and Lucas et al. 2000). The ligand
recognition sites and the importance of carbohydrate moieties on GC-C were studied
by site directed mutational analysis of the ECD and revealed that the N-linked
carbohydrate moiety at N379 plays an essential role in stabilizing the functional

structure of ECD.

25

 

 

ANPCR ch

Amino terminal

nun—nun—
.—

 
 
  

ECD

TMD

1H

KHD

 

Hinge region

 

Catalytic
Domain

 

 

Carboxyl terminal

 

Figure. 3. Domain structure of guanylyl cyclases (Lucas et al. 2000)

26

 

It also revealed that the ligand specifically binds to the region residue 387-
393. Both the N—linked carbohydrate moiety at N379 and residue 387 to residue 393
regions are located close to the transmembrane portion of GC-C on the external
cellular surface which explain their pivotal role in ligand binding and signaling
processes (Hasegawa et a1. 1999 and Hasegawa & Shimonishi, 2005).

Solubilization and STa receptors cross-linking experiments with intestinal
tissues have been repeatedly identified several species of both small and large STa-
binding proteins, 45 to 80kD and 120-160kD respectively (Cohen et a1 1993; de
Sauvage et al. 1992, Hakki et al. 1993; Ivens et al. 1990; Thompson & Giannella,
1990 and Vaandrager & de Jonge 1994). Study of biogenesis, cellular localization and
functional activation of STa receptors (Runder et al. 1996) demonstrated that only the
full length of STa receptor is translocated to the cell surface and is thus the only
species available to interact directly with the extracellular environment. The smaller
protein forms are likely to represent premature termination products of STa receptor
that are not transported to the cell surface. The generation of antibodies to GC-C
furthermore suggested that low molecular weight STa binding proteins were
proteolytic fragments of GC-C (de Sauvage et al. 1991 and Vaandrager et al. 1993).
The study of STa receptor biogenesis also showed that ligand stimulation of GC-C
does not lead to recruitment of additional receptor to the cell surface. This proves that
STa receptor is rapidly internalized and recycled back to the cell surface after binding
STa (Runder et al. 1996 and Urbanski et al. 1995).

STa-GCC receptor binding kinetics has been studied (Giannella et al. 1983

Frantz et al. 1984; Carpick & Gariepy 1991 & 1993; and Al-Majali et al. 1999 a&b).

27

Giannella (1995) showed that the binding constant of STa is two log orders less than
that of CT for its receptor and accounts for the rapid reversibility of STa-induced
secretion and the relative "permanency" of CT-induced secretion (Fasano, 1999).
Previous studies of affinity chromatography fail to recover the functional STa
receptors from affinity matrices which contain STa covalently cross-linked to a solid
support (Thompson, 1987 and Hugues & Waldman 1992). This suggests that the STa-
receptor interaction is an irreversible process. However, Hugues et al. (1992) showed
that bile salts completely dissociated ST-receptor interaction despite the low recovery
of total ST binding activity, thus supporting the theory of reversibility of STa-
receptor interaction.

Several groups of workers have studied the age-dependence, density and
distribution of STa receptors in different models including human, pig, rat, rabbit and
calf intestines (Cohen et al. 1986; Forte et al. 1989; Jaso-Friedman et al. 1992; Katwa
and White 1992; Krause et al. 1994 and Al-Majali et al. 1999 a, b&c). In all of these
models, a consistent increase in STa receptor density along the brush border
membrane was observed in immature host. This coincides with the period of high
susceptibility to STa-induced diarrhea that occurs in the early life of human and
animals. Study of distribution and characterization of STa-specific receptors on
enterocytes and brush border membrane vesicles (BBMVs) from different intestinal
segments of newborn calves found that higher density and affinity of STa receptors
on enterocytes and BBMVs derived from the ileum than other intestinal segments
(Al-Majali et a1. 1999 a&b). Moreover, ileal villous epithelial cells have

approximately twice as many receptors as crypt cells for the enterotoxin (Giannella et

28

al. 1983 and Frantz et al. 1984). This strongly explains why STa act more rapidly than
LT.

The endogenous agonist for GC-C is 15- amino- acid hormone called
guanylin, which contains four cysteines and is less potent than STa in activating GC-
C and stimulating chloride secretion (Carpick & Gariepy 1993; Currie et al. 1992 and
Forte & Currie 1995). Guanylin presumably plays a role in basal gut homeostasis, and
STa opportunistically utilize GC-C to alter ion transport in the gut. The STa
enterotoxin brings its secretory effect through the GC/CGMP pathway.

Intestinal fluid and electrolyte homeostasis

Most of enteropathogens, including ETEC, target normal processes of
intestinal water and electrolyte transport. Consequently, an understanding of the
physiology of intestinal fluid and ion transport is essential.

Fluid is both secreted, to provide an environment that promotes the digestion
and absorption of ingested nutrients, and absorbed, to avoid excessive fluid loss and
resulting dehydration (Beme & Levy 1996 and Montrose et al. 1999).

Intestinal absorptive mechanisms

Sodium ions play a central role in net nutrient and water intestinal absorption
(Chang & Bookstein 2000). Glucose, amino acids and other nutrients are absorbed,
through an ion selective channel, or by a process coupled transport of Na+ via a
Naif/H)r cation exchanger and Cl'/HC03 anion exchanger. All of these pathways
involve Na/K-ATPase pumps on the basolateral membrane of the enterocyts, (Chang

& Bookstein 2000).

29

Intestinal secretory mechanisms

The primary driving force for secretory fluid fluxes in the intestine is the
movement of Cl' from the blood circulation to the intestinal lumen. Chloride
accumulate in the cytosol above its electrochemical equilibrium in response to the
sodium concentration gradient established by the basolateral Na, K- ATPase and exit
across the apical membrane when Cl‘ channels at that site Open in response to changes
in second messengers. The most important of these Cl' channels is the cystic fibrosis
transmembrane conductance regulator (CFTR) (Barrett and Bertelsen, 2003).

Role of cyclic nucleotides in intestinal homeostasis

The important role of both cAMP and cGMP in the control of intestinal
epithelial ion transport has been documented (Rao et al. 1981 and Donowitz and
Welsh 1986). Cyclic AMP (cAMP) is generated from ATP in response to the
activation of adenylate cyclase while cGMP is generated from GTP through the
action of guanylyl cyclase. Both cyclases are membrane bound enzyme and are
activated in the presence of appropriate agonists.

The cAMP-dependent Cl' secretion is induced through protein kinase A
(PKA) (Berger et al. 1993, Shlatz et al. 1979) while cGMP-induced Cl' secretion has
been documented to occur via a protein kinase GII (PKGII) pathway (Selveraj et al.
2000). Both PKA and PKGII appear to be capable of phosphorylating and thereby
activating of CF TR in the presence of appropriate levels of intracellular signaling.

Absorptive mechanisms may also be affected by changes in cyclic nucleotide,
although in this case it can be inhibitory. Na+/H+ exchangers (NHE3) are also

regulated by phosphorylation events via cAMP-induced PKA action; however this

30

activation has an inhibitory effect on NHE3 with the subsequent inhibition of Na
absorption. Therefore, activation of PKA leads to inhibition of NHE3 and activation
of CFTR stimulating Cl' secretion and inhibiting neutral Na+/Cl' absorption (Yun et
al. 1995).

Pathogenesis of ETEC-STa Diarrlle_a_

ETEC diarrhea occurs in the first few days of life, particularly in the most
colostrum-deprived calves. Three criteria must be met for ETEC to produce illness:
the ETEC must express adhesive factors that allow it to adhere to the small intestine;
the ETEC must possess the plasmid that encodes for the enterotoxin(s), and sufficient
numbers of the ETEC must be ingested. The pathogenesis of ETEC diarrhea involves
three steps: establishment of infection, intestinal colonization, and finally elaboration
of diarrheagenic enterotoxin(s) leading to an acute profuse watery diarrhea and
potentially fatal dehydration, metabolic acidosis and electrolyte imbalances (Argenzio
1985).

Infection with ETEC

Calves contract infection with E. coli from the environment during or shortly
after birth, often by fecal-oral route of transmission (Smith, 1965 and Butler &
Clarke, 1994). Under normal circumstances, nonpathogenic types of E. coli are
among the first bacterial species to gain access the intestinal tract, and they are
prevalent throughout the intestine by the end of the first day of life with marked
increase in number from the proximal to the distal portion of the small intestine
(Smith, 1965 and Acres, 1985). In healthy calves, the majority of these

nonpathogenic E. coli are suspended in intestinal contents and are constantly

31

propelled caudally by peristaltic and flow of ingesta and their number rarely exceed
107/g intestinal content. However, when ETEC are ingested they multiply and
colonize the small intestine in larger numbers. Rapid establishment of E. coli
infection is favored by several characteristics of newborn calves including a relatively
high abomasal pH, sluggish intestinal motility and the absence of competing
microflora (Acres, 1985).
Intestinal colonization

The ability to produce enterotoxin alone is not sufficient for ETEC to cause
diarrhea. The bacteria must also able to colonize the mucosal surface of epithelial
cells of the small intestine. This depends on adhesive factors that are composed of
nonflagellar, flimentous fimbriae that are protein in nature. These fimbriae bind to
specific receptors in the membrane of cells of the small intestine and allow ETEC to
overcome the peristaltic cleansing mechanisms of the intestine. In normal calves, the
majority of E. coli are in the lurnenal content and only 10-20 % attached to the
mucosa. In contrast, in claves with enteric colibacillosis the situation is reversed and
80 % to 90% of ETEC are attached the mucosa (Hadad and Gyles, 1982a&b). The
dynamics of this process have been studied in colostrums-deprived calves where it
appears that colonization begins at the ileal-cecal junction within 3 hours of infection
and progress anteriorly to involve up to 60% of the small intestine by 16 hours post
infection.

The precise molecular mechanism of attachment is not well understood,
however bacterial fimbrial are definitely involved in this process (Smith & Huggins,

1978 and Hadad & Gyles, 1982a&b). Both the surface of ETEC bacterial cells and

32

intestinal epithelial cell possess a negative net charge which tends to repel each other.
To overcome this physicochemical property, adhesive organelles, fimbriae K99,
protrude from the bacteria cell surface and initiate attachment by extending across the
zone of repulsion to reach specific complex receptors on the epithelial cell surfaces of
the small intestine and act as a ligand to bind the bacterial cell to the intestinal surface
(Acres, 1985 and Donnenberg, 2000). Receptors are probably sugar residues located
in the cell membrane of the epithelial cells and those which bind to K99 or F41
fimbriae appears to contain sialic acid (Paris et al. 1980). Following primary
attachment, ETEC multiply and form rnicrocolonies that cover the intestinal villi
(Chan et al. 1982 and Bellamy & Acres, 1979). Colonization of the posterior half of
the intestine is the central event in the ileum and there may be as many as 109-10l0
ETEC/g of intestinal contents (Smith and Huggins, 1978).

The degree of intestinal colonization varies among ETEC strains and is
probably affected by serotype as well as physiological and nutritional factors in the
small intestine (Chan et al. 1982, Isaacson, 1980 and Bellamy & Acres, 1979).
Maximum colonization seems to occur with strains that are fimbriated and
encapsulated (Smith & Huggins, 1978 and Hadad & Gyles, 1982b). In addition, some
strains posses only one type of fimbriae whereas others posses more than one type
which may intensify colonization and increase virulence (Orskov et al. 1977).
Elaboration of eJnterotoxin: Pathophysiology of STa action

When large numbers of ETEC colonize the small intestine, sufficient STa is
produced to cause diarrhea. Presumably, enterotoxin is released by the bacteria and

diffuses toward the brush border where it binds to receptor sites on the membrane of

33

the intestinal epithelial cells. Enterotoxin alters normal movement of ions and water
across the intestinal mucosa by exerting a hormone-like effect on the enterocytes
(Moon, 1974 & 1978). Fluid and electrolytes constantly move across the intestinal
mucosa in two directions: from the intestinal lumen to the blood (absorption), and
from blood to intestinal lumen (secretion). Secretory and absorptive fluxes occur
simultaneously, and the net movement of fluid is the difference between the two
processes (Finco et al. 1973 and Moon 1994).

The villus-crypt unit is the functional apparatus through which these fluxes
occur (F inco et al. 1973). Immature enterocytes in the crypt are the main secretory
cells, whereas more mature villous enterocytes are responsible for digestion and
absorption. In normal calves, absorptive fluxes exceed secretory fluxes creating a
state of net absorption (F inco et al. 1973 and Acres, 1985). STa appears to act by
stimulating secretion as well as reducing absorption, and this dual effect reverses the
normal pattern causing a net secretion of fluid into the small intestine and resulting in
hypersecretory diarrhea (Rao et al. 1980).The total volume of each unidirectional flux
in normal calves 4-6 week of age is between 80-144 liters/day (Bywater, 1973).
Calves infected with ETEC may lose between 1.0-2.7 liters of diarrhea] fluid in 24
hours, which represents only 1-2% of the normal unidirectional flux volume (Fisher
and Martinez, 1975). This illustrates that only a small percentage change of the
normal capacity of the small intestine is required to cause severe diarrhea.

STa induces secretory diarrhea by activating guanylyl cyclase-C and
increasing cGMP (Figure 4). Disruption of the gene encoding GC-C in mice resulted

in resistance to ST-induced diarrhea, demonstrating that GC-C is absolutely required

34

for ST-induced intestinal secretion (Mann et al. 1997 and Schulz et al. 1997). Cystic
fibrosis transmembrane conductance regulator (CFTR), chloride channel, is a key
component mediating the enterotoxigenic effect of STa (Golin—Bisello et a1. 2005). In
the absence of functional CFTR, ST and cGMP analogs fail to induce diarrhea
(Quinton, 1990). Cyclic GMP activates CFTR and promotes chloride efflux, which
presumably drives water transport into the lumen of the intestine. The intermediate
steps involved in the CFTR phosphorylation are controversial, though the roles for
both cGMP-dependent kinases and cAMP-dependent kinases have been reported
(Sears and Kaper, 1996).

GMP-dependent kinases (PKG) appear to be the principal molecular target of
cGMP in the signal sequence leading to CFTR activation. Both PKG-Ia and PKG-H
phosphorylate CFTR, in vitro, with similar kinetics, suggesting the absence of a
specific PKG-mediated function in this process (French et a1. 1995). However, PKG-
II., but not PKG-Ia colocalizes with GC-C in brush borders of enterocytes and
activates CFTR in excised membrane patches of various cell lines transfected with
CFTR (Lohmann et al. 1997) suggesting that PKG-II is a major physiological
mediator of CFTR activation in small intestine (Pfeifer et a1. 1996; Vaandrager et al.
1997 & 2000). The role cAMP-dependent kinases (PKA) are also reported. They
may be activated either directly by cGMP or indirectly by local accumulation of
cAMP in response to inhibition of PDE3 by cGMP and leads to CFTR activation.
Vaandrager et al (2000) reported that STa induce electrogenic chloride secretion in

the colon and jejunum of PKG II-deficient mice.

35

 

  
 
 

Lumen V 1, fl STa

Mucosa
Brush border

Serosa

 

 

 

Figure 4. Pathophysiology of E. coli STa diarrhea. “Enterotoxigenic E. coli, containing plasmids
encoding a member of the homologous peptide family of STs colonize the intestine after the
consumption of contaminated food and/or water. Bacterial colonization leads to production of
ST in the gut lumen, which specifically recognizes and binds to the extracellular domain of
GC-C, expressed in the brush border membranes of intestinal mucosa cells from the
duodenum to the rectum. Interaction of ST to the extracellular domain of GC-C is translated
across the plasma membrane into activation of the cytoplasmic catalytic domain resulting in
the production and accumulation of [cGMP]. This cyclic nucleotide binds to and activates
PKG II, also localized in the intestinal cell brush border membrane. Also, cGMP may activate
PKA, either directly or by inhibiting a cAMP-specific PDE and inducing the accumulation of
cAMP. The CFTR that is colocalized with GC-C and PKG II in brush border membranes is a
substrate for that protein kinase and PKA. CFTR is a chloride channel, and its
phosphorylation by PKA or PKG results in a persistent open state, permitting chloride to flow
down its concentration gradient from the intracellular to the extracellular compartment. Other
ion channels and transporters in the cell maintain the electroneutrality of ST-induced chloride
efflux. Vectoral water flux from the basolateral to the apical surface is driven by these ionic
conductances, resulting in the accumulation of fluid and electrolytes in the intestinal lumen
and secretory diarrhea” (Lucas et al. 2000)

36

In addition to CF TR activation, recent evidence suggests a role for inhibition
of brush border membrane electroneutral sodium absorption, possibly mediated by a
Nail/H+ exchanger, in mechanisms underlying STa-induced fluid and electrolyte
secretion (V aandrager et al. 2000). Lucas and colleagues (2001 & 2005) reported that
STa reduced fluid absorption mediated by a Na+/H+ exchanger but does not act by
causing increased fluid secretion. Hence, the STa-induced diarrhea is the result of an
increase in net fluid secretion due to stimulation of chloride secretion in response to
CFTR activation and electrogenic chloride transport. Whether ST affects the Na+/H+
exchange in the small intestine and hence modifies acidification in the lumen is still
unknown (Laohachai et al. 2003).

Potential systemic effect of ETEC-STa diarrhea .

The systemic effects of ETEC-STa diarrhea, which eventually contribute to
deaths, are precipitated by a single event, the loss of extracellular fluid. (ECF). Four
major potentially fatal can be identified due to the loss of ECF: dehydration,
electrolyte imbalances, metabolic acidosis and negative energy balance (Fisher, 1965,
Barber et a1. 1975; Moon et al. 1978; Argenzio, 1985; Booth and Naylor, 1987;
Groutides and Michell 1990 and Grove-White, 1996).

Metabolic acidosis is an important consequence of ETEC diarrhea and a
nmnber of factors contribute to its development. A major factor is the loss of
bicarbonate ions in feces and additionally, an increase in the production of lactic acid
in poorly perfused tissue and reduction in renal hydrogen ions excretion secondary to

dehydration (Booth and Naylor, 1987).

37

Hyperkalemia is a complex sequence to ETEC diarrhea and occurs despite a
significant loss of potassium ions (K+) in the feces. This paradoxical situation is the
result of acidosis and compromised renal function. A shift of potassium ions from
intracellular to ECF compartment in exchange with the excess extracellular hydrogen
ions occurs to maintain cell electroneutrality (Lewis and Phillips, 1972). This
redistribution of K+ ions in response to acidosis causes a reduction in the resting
membrane potential leading to serious and eventually lethal effects on cardiac muscle
fimction (Fisher 1965, Fisher and McEwan, 1967 and Phillips and Knox, 1969).

Hyponatremia and hypochloremia result from significant fecal loss of sodium
and chloride ions. Hypoglycemia frequently occurs in the last stage severe ETEC
diarrhea near death. Anorexia, decreased absorption of nutrients, minimal glycogen
reserves, inhibited gluconeogenesis, and increased anaerobic glycolysis in poorly
perfused tissues may all contribute to the negative energy balance (Hall et al. 1992).
Current Approaches for Controlling ETEC-STa- induced Diarrhea

Several approaches for the treatment and control of ETEC diarrhea are briefly
described below:

Antibiotic-based therapy: The use of sub-therapeutic doses of antibiotics may help
protect animals from some but not all of the bacterial strains. However, the use of
antimicrobial agents at sub-therapeutic levels has been linked to the problem of
emerging antibiotic resistance among several bacterial species, including ETEC
strains.

Mechanism-based therapy: Therapeutic trails were also explored in an attempt to

approach ETEC-STa diarrhea through its putative receptors (Mechanism-based

38

therapy). Mann et al. 1997 and Schulz et a1. 1997 showed that disruption of the gene
encoding GC-C in mice resulted in resistance to ST-induced diarrhea. However
disruption of genes involved in the regulation of intestinal secretion may have adverse
effect on other host’s cell function. One study showed the GCC knockout- mice were
resistant to STa—induced diarrhea but they suffered of over 33 percent stunted growth
compared to normal mice (Pfeifer et al. 1996).

Another study targeted the path of the signaling cascade mediated by STa
using 2-chloroadenosine triphosphate (2-chloroATP) to interrupt STa stimulation of
chloride current and water secretion in Caco-2 cells (Zhang et al. 1999). However,
models of STa-induced secretion developed in vivo are particularly unsuited to
examine the therapeutic efficacy of prodrug (2C1Ado) that requires time-dependent
transport and metabolic conversion to the active moiety of 2-chloroATP. Moreover,
the pharmacokinetic barriers to maintain this active moiety in enterocytes in vivo limit
examination of the utility of this approach to prevent STa-induced diarrhea.
Immunity-based therapy
Active immunization

Active immunization through vaccination is an effective way to control
infectious agents. However, the current strategy for development of a broad-spectrum
vaccine against ETEC targeting the pili antigen has been challenged by the numerous
intestinal colonization factors expressed by ETEC and continuous antigenic drift of

pili antigens (Boedeker, 2005 and Walker et al. 2007).

39

Passive immunization

Antibody-based therapy, which includes pathogen-specific and non pathogen-
specific modalities, plays an important role in modern therapy against several
diseases, including ETEC. However, due to the lack of common ETEC antigen
candidates, there are no immunotherapeutic reagents that can confer broad protection
against the wide array of ETEC strains. The use of STa toxin as a common antigen to
confer a broad protection against 75% of ETEC is rationalized. However, antibodies
raised against STa toxin have been limited by several factors including the poor
immunogenicity of the STa, and the fact that the design of an effective immunogenic
STa is dependant on a successful conjugation reaction. Antibody-based therapy
against STa is also challenged by the large amount of STa antibodies required to

neutralize the local effect of the STa (Svennerholm and Steele, 2004).

40

References

Acres, S. D. 1985. Enterotoxigenic Escherichia coli infections in newborn calves: A
review. Journal of Dairy Science, 68, 229-256.

Addiss, D. G; Yashuk, J. C; Clapp, D. E. and Blake, P. A.1989. Outbreaks of
diarrheal illness on passenger cruise ships, 1975-1985. Epidemiol Infect. 103:
63- 72.

Argenzio, R. A. 1985. Pathophysiology of neonatal calf diarrhea. Vet Clin of Noth
American. Food Animal Practice. 1: 461-469.

Alderete, J. F and Robertson, D. C.1978. Purification and chemical characterization
of the heat-stable enterotoxin produced by porcine strains of enterotoxigenic
Escherichia coli. Infection and Immunity 19:1021-1030.

Al-Majali, A. M; Robinson, J. P; Asem, E. K; Lamar, C; Freeman, M. J; and Saeed,
A. M. 1999a. Use of flow cytometry to measure the interaction between
Escherichia coli heat-stable enterotoxin and its intestinal receptor in mice. J
Immunol Methods 222(1-2): 65- 72.

Al-Majali, A. M; Robinson, J. P; Asem, E. K; Lamar, C. H; Freeman, M. J; and
Saeed, A. M. 1999b. Characterization of the interaction of Escherichia coli
heat-stable enterotoxixn (STa) with its intestinal putative receptor in various
age groups of mice, using flow cytometry and binding assays. Lab Anim Sci.3:
25 4-259.

Al-Majali, A, M; Robinson, J. P; Asem, E. K; Lamar, C; Freeman, M. J; and Saeed;
A. M. 1999c. Age-dependent variation in the density and affinity of
Escherichia coli heat-stable enterotoxin receptors in mice. Adv Exp Med
Biol.4 73:13 7-45.

Aimoto, S; Takae, T; Shimonishi, Y; Hara, S; Takeda, T; Takeda, Y; and Miwatani.
T. 1982. Amino-acid sequence of heat —stable enterotoxin produced by human
enterotoxigenic Escherichia coli, Eur. J. Biochem. 129: 257-263.

Arita, M; Honda, T; Miwatani, T; Takeda, T; Takao, T; and Shimonishi. Y. 1991.
Purification and characterization of a heat-stable enterotoxin produced by
Vibrio mimicus. FEMS Microbiol. Lett. 63: 105-110.

Barber, D. M. L; Doxey, D. L; and Maclennan, W. 1975. Biochemical studies of the
collapse syndrome in suckled calves. Vet. Rec 29: 424-428.

Barrett, K. M. and Bertelsen, L. S. 2003. Physiological regulation of gastrointestinal

ion transport. In Microbial pathogenesis and the intestinal epithelial cell. Ed.
by G. Hecht, ASM Press Washington D. C.

41

Bellamy, J. EC; and Acres, S. D. 1979. Enterotoxigenic colibacillosis in colostrums-
fed calves: pathologic changes. American Journal of Veterinary Research, 40,
1 391 -1 3 9 7.

Berger, H.A; Travis, SM. and Welsh, M.J. 1993. Regulation of the cystic fibrosis
transmembrane conductance regulator Cl channel by specific protein kinases
and protein phosphatases. J Biol Chem 268: 2037-2047.

Beme, E. W. and Levy, M. N.1996. Principles of physiology. p 43 7-498. Mosby, St
Louis.

Bettelheim, K. A. 1994. Biochemical characteristics of Escherichia coli. P3-30 In
Gyles (ed). Escherichia coli in domestic animals and humans. CAB
International. Wallingfard. UK.

Boedeker, E. C. 2005. Vaccines for enterotoxigenic Escherichia coli: current status.
Current Opinion in Gastraenteralagy. 1:15-19.

Booth, A. J. and Naylor, J. M. 1987. Correction of metabolic acidosis in diarrheal
calves by oral administration of electrolyte solution with or without
bicarbonate. J Am Vet Med Ass. 191: 62-68. _

Bryce, J. 2005. WHO estimates of the causes of death in children; Lancet, 365: p
1 14 7.

Butler, D. G. and Clarke, R. C. 1994. Diarrhea and dysentery in calves. Escherichia
coli in domestic animals and humans, p. 91-116. Editor: CL. Gyles. CAB

International, Walingford, UK.

Bywater, R. J. 1973. Pathophysiological aspects of unidirectional water and sodium
transport in calf intestine. Ann. Rech Vet. 4. 125.

Carpick, B.W. and Gariepy, J. 1991. Structural characterization of functionally

important regionsof Escherichia coli heat-stable enterotoxin STIb.
Biochemistry 3 0: 4803-4809.

Carpick, B.W. and Gariepy, J .1993. Escherichia coli heat-stable enterotoxin is a long-
lived super agonist of guanylin. Infection and Immunity, 61 :4 71 0-4715.

Cassels, F. J. and Wolf, M. K.1995. Colonization factors of diarrheagenic E.coli and
their intestinal receptors. J Ind Microbiol 33 :2894-2898.

Chan, R; Acres, S. D; and Costerton, J. W. 1982. Use of specific antibody to
demonstrate glycocalyx, K99 pili, and spatial relationship of K99+

enterotoxigenic Escherichia coli in the ileum of colotrum-fed calves. Infection
and immunity, 3 7, I 1 70-1 180.

42

Chang E. B. and Bookstein, C. 2000. Mechanisms of intestinal absorption and
secretion: an abbreviated review and update. In: Domschke W, Stoll R, eds.
Intestinal mucosa and its diseases-pathaphysiolagy and clinics (F alk
Symposium 110). The Netherlands: Kluwer Academic publishers, 1999.

Cohen, M. B; Moyer, M. S; Luttrel, M; and Giannella, R. A.1986.The immature rat
small intestine exhibits an increased sensitivity and response to Escherichia
coli heat-stable enterotoxin. Pediatr Res 20:555-5 60.

Cohen, ME; Jensen, N.J; Hawkins, J .A; Mann, E.A; Thompson, MR; Lentze, M.J.
and Giannella, RA. 1993. Receptors for Escherichia coli heat-stable
enterotoxin in human intestine and in a human intestinal cell line (Caco-2).

J. Cell Physio. 1 5 6: 138-144.

Currie, M. G; Fok, K. F; Kato, J; Moore, R. J; Hmara, F. K; Duffin, K. L; and Smith,
C. E. 1992. Guanylin: An endogenous activator of intestinal guanylate
cyclase. Prac Nat Acad Sci USA 89:94 7-951.

de Craaf, F. K; and Gaastra, W. 1994. F imbriae of enterotoxigenic Escherichia coli.
P. 58-83 InP. K. Klemm (ed.), F imbriae: adhesion, genetics, biogenesis and
vaccines. CRC Press, Inc, Boca Ratan, F la. ~

de Sauvage F. J; Camerato, T. R; and Goeddel, D. V. 1991. Primary structure and

functional expression of the human receptor for Escherichia coli heat-stable
enterotoxin J Biol Chem 266: 1 7912-1 7918.

de Sauvage, F. J; Horuk, R; Bennett, G; Quan, C; Burnier, J. P; and Goeddel. D.
V. 1992. Characterization of the recombinant human receptor for Escherichia
coli heat-stable enterotoxin. J. Biol. Chem. 26 7.64 79-6482.

Donnenberg, M. S. 2000. Pathogenic strategies of enteric bacteria. Nature 406: 768-
774.

Donowitz, M. and Welsh, M.J. 1986. Ca“ and cyclic AMP in regulation of intestinal
Na+, KL and Cl' transport. Annu Rev Physiol 48: 135-150.

Eldeib, M. M; Dove, C. R; Parker, C. D; Veum, T. L; Zinn, G. M; and White,
A.A.1986. Reversal of the biological activity of Escherichia coli heat-stable
enterotoxin by disulfide-reducing agents. Infection and Immunity, 51: 24-30.

Evans, D. G; Evans, D. J; and Pierce, N. F. 1973. Differences in response of rabbit

small intestine to heat-labile and heat-stable enterotoxins of Escherichia coli
cultures. Infection and Immunity 7:8 73-880.

43

Evans, D. G; Evans, D. J; DuPont H. L; Orskve, F; and Orskov, I. 1977. Patterns of
loss of enterotoxigenicity by Escherichia coli isolated from adults with
diarrhea: suggestive evidence for interrelationship with serotype. Infection
and Immunity, I 7, 105.

Faris, A; Lindahl, M; and Wadstrom, T. 1980. GM2-like glycoconjugate as possible
erythrocyte receptor for CFA/I and K99 haemagglutinins of enterotoxigenic
Escherichia coli. FEMS Microbiological Letters, 7, 265.

Fasano, A.1999. Cellular microbiology: can we learn cell physiology from
microorganisms? Am J Physio] Cell physio] 276: C 765-C 776.

Field, M; Graf, L. H. J; Laird, W. J; and Smith, P. L. 1978. Heat-stable enterotoxin of
Escherichia Cali: In vitro effects on guanylate cyclase activity, cyclic GMP
concentration and ion transport in small intestine. Prac. Nat] Acad Sci USA
75 :2800-2804.

F inco, D. R; Duncan, J. R; and Schall, W. D. 1973. Chronic enteric disease and
hypoproteinnemia in dogs. Journal of American Veterinary Medical
Association, 163, 262-271.

Fisher, E. D. 1965. Death in neonatal calf diarrhea. British VetJ. 121: 132-138.

Fisher, E. D; and McEwan, A. D. 1967. Death in neonatal calves diarrhea. Part II:
The role of oxygen and potassium. British Vet J 123: 4- 7.

Fisher, E. D; and Martinez, A. A. 1975 Studies of neonatal calf diarrhea British
Veterinary Journal, 131, 190.

F ollett, E. AC; and Gordon, J. 1980. An electron microscope study of Vibrio flagella.
J Gen Microbial 32: 235-239.

Forte, L. R; and Currie, M. G. 1995. Guanylin: A peptide regulator of epithelial
transport. FASEB J 9:643-650.

Forte, L. R; Krause, W. J; and Freeman, R. H. 1989. Escherichia coli heat-stable
enterotoxins receptors: Localization in opossum kidney, intestine and testis.
Am J Physiol 275: F 874-881.

Frantz, J. C; Jaso-Friedman, L; and Robertson, D. C. 1984 Binding of Escherichia

coli heat-stable enterotoxin to rat intestinal cells and brush border membranes.
Infect Immun. F eb;43 (2) .' 622-3 0.

44

French, P. J; Bijman, J; Edixhoven, M; Vaandrager; A. B; Scholte, B. J; Lohman, S.
M; Naim, A. C. and de Jonge, H. R. 1995. Isotype-specific activation of cystic
fibrosis transmembrane conductance regulator-chloride channels by cGMP-
dependent protein kinase II. J. Biol Chem 270: 26626-26631.

Garbers, D. L.1999. The guanylyl cyclase receptors. Methods 19: 477-484.

Gariepy, J; Lane, A; Freeman, F; Wilbur, D; Robien, W; Schoolnik, G. K; and
Jardetzky, O. 1986. Structure of the toxic domain of the Escherichia coli heat-
stable enterotoxin STI. Biochemistry 25: 7854- 7866.

Garpiey, J; Judd A. K; and Schoolnik G. K. 1987. Importance of disulfide bridges in
the structure and activity of Escherichia coli enterotoxin STIb. Prac. Natl.
Acd. Sci. USA 84: 8907-89011.

Giannella, R. A; Luttrell, M; and Thompspon, M .1983 Binding of Escherichia coli
heat-stable enterotoxin to receptors on rat intestinal cells. American Journal of
Physiology. 245: 6492-0498.

Giannella, R. A. 1995. Escherichia coli heat-stable enterotoxins, guanylins and their
receptors: what are they and what do they do? J.Lab. Clin. Med. 125: 1 73-
181. '

Giannella, R. A. and Elizabeth, A. M. 2003. E. coli heat stable enterotoxin and
guanylyl cyclase C: New functions and suspected actions. Trans Amr Clin
Climato Ass. 1 I 4, 6 7-85.

Golin—Bisello, F; Bradbury, N. and Ameen, N. 2005. STa and cGMP stimulate CF TR
translocation to the surface of villus enterocytes in rat jejunum and is
regulated by protein kinase G. Am J Physiol Cell Physiol 289: 708- 716.

Gorbach, S. L; Kaen, B. H; Evans, D. G; and Bessudo, D. 1975. Travelers’ diarrhea
and toxigenic E. coli. N Engl J Med 1975; 292: 933-93 6.

Gray, W. R; Luque, A; Olivera, B. M; Barrett J; and Cruz; L. J .1981. Peptide toxins
from Conus geographyus venom J. Biol. Chem. 156: 4 734-4 7.

Greenberg, R. N; Hill, M; Krytzer, J; Krause, W. J; Eber, S. L; Hamra, F. K. and
Forte, L. R.1997. Comparison effects of uroguanylin, guanylin, and
Escherichia coli heat-stable enterotoxin STa in mouse intestine and kidney:
evidence that uroguanylin is an intestinal natriuretic hormone. J. Investig.

Med. 45: 276-282.

Groutides, C. P; and Michell, A. R. 1990. Change in plasma composition in calves
surviving or dying from diarrhea. Britich Vet J 146:205-21 0.

45

Grove-White D. H. 1996. Pathophysiology and treatment of metabolic acidosis in
diarrheic calf. Proceeding of XIX Buiatrics Congress of Vet Association
(BC VA) Edinburg, Calf Diseases 1 : 102-107.

Guarino, A; Giannella, R. and Thompson, M. R. 1989. Citrobacter freundii produces
an 18-amino acid heat-stable enterotoxin identical to the 18-amino acid
Escherichia coli heat-stable enterotoxin (STIa). Infect. Immun. 5 7: 649-652.

Gyles, C. L. 1971. Heat-labile and heat-stable forms of enterotoxin from Escherichia
coli strains enteropathogenic for pigs. Ann NY Acad Sci. 1 76:314-322.

Gyles, C. L. 1996. Escherichia coli in domestic animals and humans. p 31-72 CAB
International. Wallingfard. UK.

Hadad, J. J. and Gyles, C. L. 1982a. Scanning and transmission electron microscopic
study of the small intestine of colostrums-fed calves infected with selected
strains of Escherichia coli. American Journal of Veterinary Research, 43, 41 -
49.

Hadad, J. J. and Gyles, C. L.1982b. The role of K antigens of enteropathogenic
Escherichia coli in colonization of the small intestine of claves. Canadian
Journal of Comparative Medicine, 46, 21 -26.

Hakki, S; Crane, M; Hugues, M; O'Hanley, P; and Waldman, S. A. 1993.
Solubilization and characterization of functionally coupled Escherichia coli
heat-stable toxin receptors and particulate guanylate cyclase associated with
the cytoskeleton compartment of intestinal membranes. Int J Biochem 25 :

557-566.

Hall, G. A; Jones, P. W; and Morgen, J. H. 1992. Calf Diarrhea. In Bovine Medicine
Diseases and Husbandry of Cattle by Andrews A. H. 1” Ed Oxford Blackwell
Scientific Pp. 154-180.

Hasegawa, M. and Shimonishi, Y. 2005. Recognition and signal transduction
mechanism of Escherichia coli heat-stable enterotoxin and its receptor,
guanylyl cyclase C. J. Peptide Res, 65 .' 261-2 71.

Hasegawa, M; Hidaka, Y; Wada, A; Hirayama, T; and Shimonishi, Y. 1999 The
relevance of N-linked glycosylation to the binding of a ligand to guanylate
cyclase C. Eur J Biochem 263: 338-346.

Hughes, J. M; Murad, F; Chang, B; and Guerrant, R. L.1978. Role of cyclic GMP in

the action of heat-stable enterotoxin of Escherichia coli. Nature (Land)
271: 755- 756.

46

Hugues, M; and Waldman, S. A. 1992. Molecular mechanisms underlying activation
of particulate guanylyl cyclase and GMP accumulation induced by
Escherichia coli heat-stable enterotoxin. Biochem Life Science Adv 10:103-
1 12.

Hugues, M; Crane, M. R; Thomas, B. R; Robertson, D; Gazzano, H’ O'Hanley, P;
and Waldman S. A. 1992. Affinity purification of functional receptors for
Escherichia coli heat-stable enterotoxin from rat intestine. Biochemistry. Jan
14:31:12-16. '

Isaacson, R. E. 1980. Factors affecting expression of Escherichia coli K99 pilus.
Infection and Immunity, 28, 190-194.

Ivens, K; Gazzano, H; O'Hanley, P. and Waldman, S. A. 1990. Heterogeneity of
intestinal receptors for Escherichia coli heat-stable enterotoxin. Infect Immun
58: I81 7-1820.

Jacks, T. M. and Wu, B. J. 1974. Biochemical properties of Escherichia coli low
molecular weight heat-stable enterotoxin. Infection and Immunity 9342-34 7.

Jaso-Friedman, L; Drefus, L. A; Whipp, S. C; and Robertson, D. C. 1992. Effect of
age on activation of porcine guanylate cyclase and binding of Escherichia coli

heat-stable enterotoxin (STa) to porcine intestinal cells and brush border
membranes. Am J Vet Res 53 :2251 -2258.

Katwa, L. C; and White, A. A. 1992. Presence of functional receptors for Escherichia
coli heat-stable enterotoxin in the gastrointestinal tract of chicken. Infect
immune 60:3546—3551.

Koo, D; Maloney, K; and Tauxe, R. 1996. Epidemiology of diarrhea] diseases
outbreaks on cruise ships, 1986 through 1993. JAMA, 275: 545-54 7.

Krause, W. J; Cullingford, G. L; Freeman, R. H; Ber, S. L; Richardson, K. C; F ok, K.
F; Currie, M. G; and Forte LR 1994. Distribution of heat-stable
enterotoxin/guanylin receptors in the intestinal tract of manand other
mammals. J. Anat 184:404-417.

Laohachai, K. N; Bahadi, R; Hardo, M. B; Hardo, P. G; and Kourie, J. I. 2003. The
role of bacterial and non-bacterial toxins in the induction of changes in
membrane transport: implication for diarrhea. Toxican 42: 687- 707.

Levine, M. M. 1987. Escherichia coli that cause diarrhea: Enterotoxigenic,

enteropathogenic, enteroinvasive, enterohemorrhagic and enterodherent.
Journal of Infectious Diseases 155: 33 7-389.

47

Levine, M. M. Kaper, J. B; Black, R. E; and Clements, M. L. 1983. New knowledge
on pathogenesis of bacterial enteric infections as applied to vaccine
development. Microbiological Reviews 4 7:51 0-5 5 0.

Lewis, L. D; and Phillips, R. W. 1972. Water and electrolyte losses in neonatal calves
with acute diarrhea: A complete balance study. Cornell Vet. 62: 596-60 7.

Lior, H. 1996. Classification of Escherichia coli. pp. 31-72. In C.L. Gyles (ed).
Escherichia coli in domestic animals and humans. CAB International.
Wallingford. UK.

Lohmann, S. M; Vaandrager, A. B; Smolenski A, Walter, U. and De Jonge H. R.
1997. Distinct and specific functions of cGMP- dependent kinase. Trends
Biochem Sci 22: 307-312.

Lowe, D. G; Camerato, T. R. and Goeddel, D. V. 1990a. cDNA sequence of the
human atrial natriuretic peptide clearance receptor. Nucleic Acids Res 18:
3412

Lowe, D. G; Klisak , I; Sparkes, R. S; Mohandas, T. and Goeddel, D. V.1990b.
Chromosomal distribution of three members of the human natriuretic peptide
receptor/guanylyl cyclase gene family. Genamics.

Lucas, K. A; Pitari, G. M; Kazerounian, S; Ruiz-Stewart, 1; Park, J; Schulz, S;
Chepenik, K. P; and Waldman, S. A. 2000. Guanylyl Cyclase and Signaling
by Cyclic GMP. Pharmaca] Rev 52: 3 75-413.

Lucas, M. L. 2001. A reconsideration of the evidence for Escherichia coli STa (heat-
stable) enterotoxin-driven fluid secretion: a new view of STa action and a new
paradigm for fluid absorption. Journal of Applied Microbiology 90: 7-26.

Lucas, M. L; Thom, M. M. M; Bradley, J. M; O’Reilly, M and Nelson, Y. B. 2005.
Escherichia coli heat-stable enterotoxin and the upper small intestine: Lack of
evidence in vivo for net fluid secretion. J Membrane Bio]. 206: 29-42.

Mann, E. A; Jump, M.L; Wu, J; Yee, B. and Giannella, R. A. 1997. Mice lacking the
guanylyl cyclase C receptor are resistant to STa-induced intestinal secretion.
Biochem Biophys Res Commun 239:463-466.

Montrose, M. H; Keely, S. J. and Barrett, K. E. 1999. Secretion and absorption. small
intestine and colon, p. 320- 355. In T. Yarnada, DH Alpers, L. Latine, C.
Owyang, and D. W. Powell (ed) Textbook of Gastraenteralogy, 3lrd ed
Lippincatt, Williams and Wilkins, Philadelphia, PA.

Moon, H.W. 1994. Pathophysiology of viral diarrhea. In: Viral infections. of the
gastrointestinal tract, p. 27-52. editor: A.Z. Kapikian, CIP, New York.

48

Moon, H. W. 1974. Pathogenesis of enteric diseases caused by Escherichia coli. In:
Advances in veterinary science and comparative medicine, pp. 179-211.
Editors: C.A. Brandly and CE. Cornelius. Academic Press, New York.

Moon, H.W. 1978. Mechanisms in the pathogenesis of diarrhea: A review. Journal of
American Veterinary Medicine Association, 172, 443-448.

Moon, H. W; Nagy, B; Isaacson, R. E; and Orskov, I. 1977. Occurrence of K99
antigen on Escherichia coli isolated from pigs and colonization of pig ileum
by K99+ enterotoxigenic E. coli from calves and pigs. Infection and Immunity
15: 614-620.

Morris, J. A; Thome, C. J; Wells, G. A; Scott, A. C. and Sojka, W. J. 1983. The
production of F 41 fimbriae by piglet strain of enterotoxigenic Escherichia coli
that lack K88, K99 and 987P fimbriae. Journal of Genreal Microbiology, 129,
2753.

Murray, C. J. L.1997. Global mortality, disability, and the contribution of risk factors:
Global Burden of Disease Study; Lancet,349: 143 6.

Nagy, B. and F ekete, P. Z. 1999. Enterotoxigenic Escherichia coli in farm animals.
Vet Res. 30 (2-3) :25 9-84.

NAHMS 1994. USDA Part III: Beef cow/calf health and health managements. US.
Dept of Agriculture Animal and Plant Inspection Service, NAHMS, Veterinary
Services, Fort Collins, Colorado, 37.

NAHMS 1996. USDA Part 11: Changes in the US. Dairy Industry. 1991-1996. US.
Dept of Agriculture Animal and Plant Inspection Service, NAHMS Veterinary
Services, Fort Collins, Colorado. 17-21.

Nataro, J. P. and Kaper, J. B. 1998. Diarrheogenic Escherichia coli. Clin Microbialo
Rev 11: 142-301.

Okamoto, K; Yukitake, J; Kawarnoto, Y; and Miyama, A. 1987. Substitution of
cysteine residues of Escherichia coli heat-stable enterotoxin by
oligonucleotide-directed mutagenesis. Infect. Immun. 55: 2121-2125.

Oliveira, L; Minion, P; Viegas-Pequignot, E; Rozet, J. M; Dollfus, H. and Pittler, S. J.
1994. Human retinal guanylate cyclase (GUC2D) maps to chromosome
17p13.1. Genomics 22: 478-481.

Orskov, I; Orskov, F; Jann, B; and Jann, K. 1977. Serology, chemistry, and genetics
of O and K antigens of Escherichia coli. Bacteriological Reviews, 41, 667.

49

Osaki, H; Sato, T; Kubota, H; Hata, Y; Katsube, Y; and Shimonishi, Y.1991.
Molecular structure of the toxic domain of heat-stable produced by a
pathogenic strain of Escherichia coli. J.Bial. Chem. 266:5 934-5 94 1 .

Pfeifer, A; Aszodi, A; Seidler, U; Ruth, P; Hofinann, F; and F assler, R. 1996.
Intestinal secretory defects and dwarfism in mice-lacking cGMP-dependent
kinase II. Science (Wash DC) 274:2082-2086.

Phillips, R.W. and Knox, K.L.l969. Diarrheie acidosis in calves. J Comp Lab Med
3:1 -3.

Pitek, R; Zalewska, B; Bury, K; and Kur, J. 2004. The chaperone-usher pathway of
bacterial adhesin biogenesis -from molecular mechanism to strategies of anti-
bacterial prevention and modern vaccine design, International Review
Conference on Biotechnology, Vienna,Austria.

Planet, P. J; Figurski, D. H; and DeSalle 2006. Function, evolution and classification
of macromolecular transport systems. In Evolution of Microbial pathogen,
Edited by Seifert HS. and DiRita V.J. ASM Press, Washington DC.

Quinton P. M.1990. Cystic fibrosis: A disease in electrolyte transport. FASEB J 4:
2709-2 71 7.

Rao, M.C; Guandalini, S; Smith, P. L; and Field, M. 1980. Mode of action of heat-
stable Escherichia coli enterotoxin. Biochemical et Biophysica, 632, 35-46.

Rao, M.C; Field, M; Orellana, S.A; Robertson, DC. and Giannella, RA. 1981.
Comparison of the biological actions of three purified heat-stable
enterotoxins: effects on ion transport and guanylate cyclase activity in rabbit
ileum in vitro. Infect immune 33: 1 6 7.

Rasheed, J. K; Guzman-Verduzco, L-M; and Kupersztoch, Y. M. 1990. Two
precursors of the heat-stable enterotoxin of Escherichia coli: evidence of
extracellular processing. Mol. Microbiol. 4: 265-273.

Runder, X. L., Nicehitta, C. and Almenoff, J. S. 1996. Biogenesis, cellular
localization and functional activation of heat-stable enterotoxin
receptors(Guanylyl cyclase C). Biochemistry 35: 10680-10686.

Runnels, P. L; Moon, H. W. and Schneider, R. A.1980. Development of resistance
with host age to adhesion of K99+ Escherichia coli to isolated intestinal
epithelial cells. Infection and Immunity 28: 298-3 00.

Saeed, A. M; Srianganathan, N; Cosand, W; and Burger, D. 1983. Purification and

characterization of heat-stable enterotoxin from bovine enterotoxigenic
Escherichia coli. Infection and Immunity, 40: 701-701.

50

Saeed, A. M; Magnuson, N. S; Sriranganathan, N; Burger, D; Cosand, W. 1984.
Molecular homogeneity of heat-stable enterotoxins produced by bovine
enterotoxigenic Escherichia coli. Infect Immun 45:242-247.

Saeed, M. A; and Greenberg, R.N. 1985a. Preparative purification of Escherichia coli
Heat-stable Enterotoxin. Anal. Bia.151: 431-43 7.

Saeed, A. M; Magnuson, N. S; Gay, C. C. and Greenberg, R. N. 1985b.
Characterization of heat-stable enterotoxin from a hypertoxigenic Escherichia
coli for cattle. Microbiology and Therapy, 15: 221 -229.

Salyers, A. A; and Witt, D. D.1994. Bacterial pathogenesis: A molecular approach,
ASM Press, Washington DC.

Sato,T; and Shimonishi, Y. 2004 Structural features of Escherichia coli heat-stable
enterotoxin that activates membrane-associated guanylyl cyclase. J. Peptide
Res 63:200-206.

Sato, T., Ozaki, H; Kitagawa, Y; Katsube, Y; and Shimonishi, Y. 1994. Structural
characteristics for biological activity of heat-stable enterotoxin produced by
enterotoxigenic Escherichia coli: X-ray crystallography of weakly toxic and
nontoxic analogs. Biochemisty 33: 8641-8650.

Savarino, S.J; Fasano, A; Watson, J; Martin, B. M; Levine, M. M. Guandalini, S; and
Guerry, P. 1993. Enteroaggregative Escherichia coli heat-stable enterotoxin 1
represents another subfamily of E. coli heat-stable toxin. Proc. Natl. Acad Sci.

USA 90:3 093 -3 09 7.

Schulz, S; Green, C. K; Yuen, P. S. T; and Garbers, D. L. 1990. Guanylyl cyclase is a
heat-stable enterotoxin receptor. Cell 63: 941-948.

Schulz, S; Lopez, M. J; Kuhn, M; and Garbers D. L. 1997. Disruption of the guanylyl
cyclase-C gene leads to a paradoxical phenotype of viable but heat-stable
enterotoxin-resistant mice. J C [in Invest 100: 1590-1595.

Sears, C. L; and Kaper. J. B.1996. Enteric bacterial toxins: mechanisms of action and
linkage to intestinal secretion. Microbial. Rev. 60:167-215.

Seifert, H. S. and DiRita, V.J. 2006. Evolution of Microbial pathogen, ASM Press,
Washington DC.

Selveraj, N. G; Prasad, R; Goldstein, L. and Rao, M. C. 2000. Evidence for the

presence of cGMP-dependent protein kinase-II in human distal colon and in
T84, the colonic cell line. Biochem. Biophys. Acta 1498: 32-43.

51

Shimonishi, Y; Hidaka, Y, Koizumi, M; Hane, M; Arimoto, S; Takeda, T;, Miwatani,
T; and Takeda, Y. 1987. Mode of disulfide bond formation of heat-stable
enterotoxin (STh) produced by human strain of enterotoxigenic Esherichia
coli. FEBS Lett. 215: 165-1 70.

Shlatz L. J; Kimberg, D. V. and Cattieu, K. A. 1979. Phosphorylation of specific rat
intestinal microvillus and basal-lateral membrane proteins by cyclic
mucleotides. Gastaenterology 76: 293.

Smith, H. W. 1965. The development of flora of the alimentary tract in young
animals. Journal of Pathological Bacteriology, 90, 495.

Smith, H.W; and Huggins, M. B. 1978. The influence of plasmid-deterrnined and
other characteristics of enteropathogenic Escherichia coli on their ability to
proliferate in the alimentary tracts of piglets, claves and lambs. Journal of
Medical Biochemistry, 11, 471.

Snodgrass, D. R; Terzolo, H. R; Campbell, D; Sherwood, I; Menzies, J. D; and
Synge, B. A. 1986. Aetiology of diarrhoea in young calves. Vet Record,
1 19:31-34.

So, M; and McCarthy B. J. 1980. Nucleotide sequence of the bacterial transposon
Tn1681 encoding a heat-stable (ST) toxin and its identification in
enterotoxigenic Escherichia coli strains. Prac Nat] Acad Sci USA 77:4 01 1-
4015.

So, M; Heffron, F; and McCarthy B. J. 1979. The Ecali gene encoding a heat-stable
toxin is a bacterial transposon flanked by inverted repeats of IS 1. Nature 277:
453-45 6.

So, M; R. Atchison, Falkow S, Moseley and McCarthy B. J. 1981. A study of the
dissemination of Tn1681: a bacterial transposon encoding a heat-stable
enterotoxin among genetic Escherichia coli isolates. Cod Spring Harbor

Symp. Quant. Biol. 45: (Pt. I ).' 53-58.

Stine, CC; and Nataro, J. P. 2006. The evolution of bacterial toxins. In: Evolution of
Microbial pathogen, Eited by HS. Seifert and VJ. DiRita 2006 ASM Press,
Washington DC.

Svennerholm, A. Steele D. 2004. Progress in enteric vaccine development. Best
Practice & Research Clinical Gastroenteralog, I8: 421 — 445.

Takao, T; T.Hitouji, T; Aimoto, A; Shimonishi, Y; Hara, S; Takeda, T; Takeda, Y;

and Miwatani. T. 1983. Amino acid sequence of a heat-stable enterotoxin
isolated from enterotoxigenic E. coli strain 18D. FEBS Lett. 152: 1-5.

52

Takao, T; Tominaga, T; Shimonishi, Y; Hara, S; Inoue, T; and Miyama. A.1985.
Isolation, primary structure and synthesis of heat-stable enterotoxin produced
by Yersinia enteracalitica. Eur. J. Biach. I52: I99-206.

Takeda, T; Nair, G. B; Suzuki, K; Zhe, H. X; Yokoo, Y; Hemelhof, W; Butzler, J. B;
Takeda, Y; and Shimonishi, Y. 1993. Epitope mapping and characterization of
antigenic determinants of heat-stable enterotoxin (STh) of enterotoxigenic
Escherichia coli by using monoclonal antibodies. Infection and Immunity
61 :289-294.

Takeda, Y; Yamasaki, S; Hirayama, T; and Shimonishi, Y. 1991. Heat-stable
enterotoxins produced by enteric bacteria. In: Molecular pathogenesis of
gastrointestinal Infections (F ederatian European Microbiological Societies
Symposium Series, No 58) (Wadstrom T, Makela PH, Svennerhalm AM and
Wolf-Watz H., eds) pp 125-138. Plenum Press, NY

Thompson, M. R. 1987. Escherichia coli heat-stable enterotoxins and their receptors.
Pathal Immunapatha] Res 6:1. 3-1 I 6

Thompson, M.R., and Giannella, R. A. 1990. Different cross linking agents identify
distinctly different putative Escherichia coli heat-stable enterotoxin rat
intestinal cell receptor proteins. J. Receptor Res. 10: 97-11 7.

Urbanski, R; Carrithers, S. L. and Waldman, S. A. 1995. Intemalization of E. coli ST
mediated by guanylyl cyclase C in T84 human colon carcinoma cells. Biachim
Biophys Acta 1245: 29-3 6.

USDA, NASS&APHIS 1997. Cattle and Calves Death Loss.

Vaandrager, A. B; Schulz, S; De Jonge, H. R; and Garbers, D. L. 1993. Guanylyl
cyclase C is an N-linked glycoprotein receptor that accounts for multiple heat-
stable enterotoxin-binding proteins in the intestine. J Biol Chem 268: 21 74-
21 79.

Vaandrager, A. B. and de Jonge, H. R. 1994. Effect of cyclic GMP on intestinal
transport. Adv Pharmaca] 26:253-283.

Vaandrager, A. B. Bot, A. G; and de Jonge, H. R. 1997. Guanosine 3',5'-cyclic
monophosphate-dependent protein kinase II mediates heat-stable enterotoxin-
provoked chloride secretion in rat intestine. Gastraenteralagy 112: 437-443.

Vaandrager, A. B; Bot, A. G; Ruth, P; Pfeifer, A; Hofrnann, F. and de Jonge, H. R.
2000. Differential role of cyclic GMP-dependent protein kinase II in ion

transport in murine small intestine and colon. Gastraenterolagy 118: 108-
I 1 4.

53

Vaandrager, A. B. 2002. Structure and function of the heat-stable enterotoxin
receptors/guanylyl cyclase C. Molecular and Cellular Biochemistry 230: 73-
83.

Walker, R.I; Steele, D; Aguado, T. and Ad Hoc ETEC Technical Expert Committee.
2007. Analysis of strategies to successfully vaccinate infants in developing countries
against ETEC disease. Vaccine 25, 2545-2566.

World Health Organization 1990. Tropical diseases. ASM News, 1990, 56: 358-359.

Yamanaka, H; Kameyama, M; Baba, T; Fujii, Y; and Okamoto, K. 1994. Maturation
pathway of Escherichia coli heat-stable enterotoxin 1. Requirement of DsbA
for disulfide bond formation. J. Bacteriol. 1 79: 2906-2913.

Yamasaki, S; Sato, T; Hidaka, Y; Ozaki, H; Ito, H; Hirayama, T; Takeda, T;
Sugimura, T; Tai, A; and Shimonishi, Y. 1990. Structural-activity relation
ship of Escherichia coli heat-stable enterotoxin: role of Ala residue at position
14 in toxin-receptor interaction. Bulletin of Chemistry Society of Japan, 63,
2063.

Yang, R.B; F iille H. J; and Garbers, D.L. 1996. Chromosomal localization and
genomic organization of genes encoding guanylyl cyclase receptors expressed
in olfactory sensory neurons and retina. Genomics. 31: 3 6 7-3 72.

Yoshimura, S; Ikemura, Y; Watanabe, H; Aimoto, S; himonishi, Y; Hara, S; Takeda,
T; Miwatani, T; and Takeda, Y. 1985. Essential structure for enterotoxigenic

activity of heat-stable enterotoxin produced by enterotoxigenic Escherichia
coli. FEBS Lett. 181: 138-142.

Yoshimura, S., Hidaka, Y; Aimoto, Shimonishi, S; Takeda T; Miwatani, T; and
Takeda, Y. 1987. Structural-activity relationship of a heat-stable enterotoxin
produced by Yersinia enteracalitica. Bull Chem Soc Jpn 60:2481-2489.

Yoshimura, S., Takao, T; Shimonishi, Y; Hara, S; Arita, M; Takeda, T; Irnaishi, H;
Honda, T; and Miwatani, T. 1986. A heat-stable enterotoxin of Vibrio
cholerae non-OI: Chemical synthesis, and biological and physicochemical
properties. Biopalymers 25 (Suppl. ): S69-S83.

Yoshino K, Huang; X; Miyachi, M; Hong, Y-M; Takao, T; Nakao, H; Takeda, T; and
Shimonishi, Y. 1994. Amino acid sequence of a novel heat-stable enterotoxin
produced by a yst gene-negative strain of Yersinia enterocalitica. Lett Pept Sci

1: 95-105.
Yun, C. H. C; Tse, C. M; Nath, S. K; Levine, S. A; Brant, S. R. and Donowitz, M.

1995. Mammalian Na+/H+ exchanger gene family: structure and function
studies. Am J Physiol 269: G] -GI 1.

54

Zhang, W; Mannan I; Schulz, S; Parkinson . J; Alekseev, A. E; Gomez L. A; Terzic;
A. and Waldman SA. 1999. Interruption of transmembrane signaling as a

novel antisecretory strategy to treat enterotoxigenic diarrhea. FASEB J 13:
913-922.

55

CHAPTER III
PURIFICATION AND CHARACTERIZATION OF HEAT-STABLE
ENTEROTOXIN (STa) OF ENTEROTOIXIGENIC Escherichia coli

Introduction

Enterotoxigenic strains of Escherichia coli (ETEC) have been implicated as
causative agents of diarrhea in human infants (Guerrant et al. 975 and Sack et al.
1975), diarrhea in neonatal domestic animals (Smith and Halls, 1967 and Whipp et al.
1975) and in “travelers” diarrhea (Jiang et al. 2000). ETEC strains are capable of
producing at least two distinct forms of extracellular enterotoxins. One form is called
heat-labile enterotoxin (LT), which is structurally and functionally similar to cholera
toxin (CT) (Gyles 1971 and Evans et al. 1973),. and the other form is heat-stable
enterotoxin (STa), which is a low-molecular-weight (< 2kD) peptide that is non-
immunogenic and is not neutralized by antibodies specific to CT (Gyles 1971 and
Evans et al. 1973 and Naline et al. 1974). Seventy five percent of tested ETEC strains
have been found to produce STa alone or in combination of LT (Wolf, 1997).
Therefore, STa is considered to be an important virulence determinant in enterotoxin-
mediated diarrheal diseases caused by E. coli (Sears & Kaper, 1996 and Giannella &
Elizabeth, 2003).

The purification and characterization of STa have been complicated by the
complexity of various growth media used for toxin production and by laborious
purification procedures such as salt precipitation, ultrafiltration, ion exchange and

molecular sieve chromatography. These procedures are time consuming and have a

56

relatively low recovery yield of homogenous STa (Staples et al. 1980, Saeed et a1.
1983, and Saeed & Greenberg 1985).

In this study, we adopted the growth medium and purification methods
reported by Staples et al. (1980) and modified by Saeed et al. (1983) and Saeed and
Greenberg (1985) under controlled growth conditions using a 36 L Bellco bioreactor.
Material and methods
Animals

Swiss Webster Mice: A group of 20 Swiss-Webster (fifteen females and five
males) was used to establish a colony as a source of suckling mice for STa bioassay.
Exhausted females and males were continuously replaced with younger animals to
ensure production efficiency of infant mice litters by the colony.

Reagents

All reagents were obtained from commercial sources and were of analytical
grade. Mobile phases used for purification of STa include HPLC-grade methanol,
triflouroacetic acid, as well as the other chemical ingredients listed under this section.
Verifying the ETEC K99+ Strain

Basic PCR protocol described by Olsvick and Strockbine (1993) and
Salvadori et al. (2003) was used to detect the STa gene and verify the strain as STa-
producing E. coli.

Bacteria] Strains: An ETEC strain was isolated from a clinically diarrheic neonatal
calf and was provided by A. M. Saeed (Molecular Epidemiology Laboratory,

National Food Safety Toxicology Center (NFST), Michigan State University (MSU),

57

East Lansing MI. A control strain (K-12 E. coli) was kindly obtained from the
Bacterial Evolution Laboratory, NF ST, MSU, East Lansing- MI.

DNA Extraction (Template) by boiling lysis: ETEC and K-12 strains were grown on
Tripticase Soy Agar slants overnight at 37°C. A uniform bacterial colony from both
strains was taken and suspended in 1 ml sterile Milli Q water and boiled for 10 min,
then left in ice for 5 min, followed by eentrifugation at 13,000 rpm for 4 min. The
supernatant was taken and kept at -20 °C until use (Holmes and Quigley,198 l ).
Primer selection and preparation: Two different sizes of STa primer, 244bp and
127bp (Table 3), were obtained from Integrated DNA Technology Inc. (Coralville,
IA). Both primers were prepared according to the manufacturer’s instructions.

Table 3. PCR primers used to detect STa gene

 

 

 

Primer Sequence (5-3) Base Reference
pair
STa 5’- TCC GTG AAA CAA CAT GAC GG-3’ 244 Salvadori et al.
5’- ATA ACA TCC AGC ACA GGC AG-3’ 2003
STI 5’-TTA ATA GCA CCC GGT ACA AGC AGG-3’ 127 Olsvick, and
5’-CTT GAC TCT TCA AAA GAG AAA ATT AC-3’ Strockbine 1993

 

 

 

 

 

 

PCR program: PCR running conditions for detection of the STa gene are presented in
Table 4 using PCT-100 Programmable Thermal Controller (MJ Research Inc).

Table 4. PCR running conditions for detection of STa gene

 

 

 

 

 

 

 

 

Sig) Temperature (°C) Time (min)
Pre-denature 95 5
Denature 95 1
Annealing 60 1
Extension 72 1
Final extension 72 10
Storage 4 24 hours

 

 

 

 

Number of PCR cycles 29 before storage

58

PCR reaction: The PCR reaction was performed as described in table 5 using Fisher
exACTGene Complete PCR kit.

Table 5. PCR reaction for detection of STa gene

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Component Amount/sample # of Amount/two rxns
(11') Samples Volume (pl)

10x buffer A 2 2.5 5
dNTP (1 OmM) Q4 25 1
Sta-thouM) 0.4 2.5 1
Sta-R (20 PM) 0.4 2.5 1
25 mM MgClz 0 0 0
Fisher Taq 0.1 2.5 1
polymerase
Ultrapure water 14.7 2. 5 36.75
Total volume 20 45
Template 2

 

Agarose Gel Electrophoresis Analysis of PCR Products: The analysis of the PCR
products was performed in 2% agarose gel electrophoresis using the Horizontal Gel
Electrophoresis System, Life Technology (Cat # 11068-012). Briefly, two percent of
agarose was prepared (1.5 gm/75 m1 leAE electrophoretic sequence grade) and
ethidium bromide was added at concentration 3 ul/ 50 ml. The reagent was poured
into the electrophoretic chamber and filled with leAE. Five volumes of PCR
product were mixed with 1 volume of gel loading buffer and loaded into the wells
along with a 1.5 kb ladder. The agarose gel was left to run at the appropriate voltage
(100-160 volts) for 30-45 min and then examined under UV light.
Purification and Characterization of E. coli STa

STa was purified according to the protocol reported by Staples et al. (1980)

and modified by Saeed et al. (1983) and Saeed and Greenberg (1985).

59

Seed culture and frozen stock of E T E C preparation

Casamino acid-yeast extract-salts (CAYE) seed culture was used for optimal
growth of the ETEC strain (Giannella, 1976). The ETEC strain was grown on 500 m1
of CAYE, incubated at 39 0C for 24 hours on a rotary shaker at 120 rpm, then mixed
with glycerol at a final concentration of 15%, then aliquoted into 10 ml samples and
frozen at -80 °C (frozen stock).
Batch medium (Asparagine salt medium) and growth conditions (Staples et al.
1980):

Table 6. Composition of optimal minimal medium for STa production (g/L)

 

 

 

 

NaCl 2.52 Na2SO4 0.14
Na acetate 10.00 MgSO4 0.05
K2HPO4.3H20 8.12 Man 1% 0.5 ml
Asparagine 5.00 F eCl3 1% 0.5 ml

 

 

 

 

 

 

We used the Asparagine-salt medium (ASM) reported by Staples et a1. (1980)
that was compared with other grth media by Saeed et a1. (1983) and found to offer
several advantages, including high level of STa production along with minimal
contaminating proteins that facilitated the STa-purification process. Each batch
consisted of 30 liters of culture-innoculated ASM grown in a 36L omni vessel under
different pH conditions (7.4, 8 and 8.6) using a Bellco bioreactor (Bellco Glass Inc.
Vineland NJ). Preeulture was prepared by inoculating 10 ml of frozen stock of ETEC
into 1L of CAYE broth and was incubated at 39 °C for 24 hours on a rotary shaker at
120 rpm. The preculture medium was then transferred into 30 liters of batch medium

and kept at 39°C under continuous agitation at 120 rpm, aeration and oxygenation

60

were at a rate of 5 L/min and 600 ml/min, respectively, through a sintered metal
dispersion ring. Foam, speed of agitation, temperature and P02 were controlled using
respective Bellco control modules. Samples were taken every two hours to determine
the growth kinetics under various pH levels.
Preparation of cell free filtrate

After 24 hours of incubation, the growth medium was immediately filtered by
tangential flow filtration through a 02-11 cassette in Millipore Pellicon System
(Millipore Crop, Bedford, MA). Cell free filtrate was kept on ice throughout the time
of filtration to minimize bacterial grth and enzymatic activity. Samples from the
cell-free filtrate were collected for determination of total protein and STa content
using suckling mouse assay.
Amberlite XAD-2 Batch Adsorption Chromatograph

Cell free filtrate was desalted and the hydrophobic STa was concentrated
using Amberlite XAD-2 batch adsorption chromatography. Amberlite XAD-2 resin
was first washed extensively with purified water to remove any preservative and
powdery contaminants. Then 500g were suspended into 15 L of cell free filtrate in a
20- L carboy and kept overnight at 4 °C under gentle stirring. Resin was poured from
the carboy into a 40 cm long glass column and washed with 5 L of Milli Q water. The
contaminants loosely bound to the resin were eluted with 1L of 1% acetic acid in 20%
methanol/water (v/v). A stepwise elution system was applied to elute the STa starting
with 1L of 1% acetic acid in 80% methanol/water (v/v) followed by 1L of 1% acetic
acid in 99% methanol/water (v/v) and finally 1 L of 50% acetone/water (v/v). The last

three fractions were pooled and concentrated by flash evaporation & freeze-drying.

61

The resin was degassed for 5 minutes after each solvent was added to drain
completely before further addition of solvent. Samples were collected for
determination of the total protein and testing for STa biological activity in suckling
mouse.

Acetone Fractionation

Lyophilized crude STa was dissolved in 20 ml of 25% of acetic acid. Acetone
was added to bring the final volume to 100-150 ml. After standing 1 hour at 4 °C, the
sample was centrifuged at 10,000g for 30 minutes at 4 oC. The supernatant fraction
was evaporated to remove the acetone and was then freeze-dried. Samples were
collected for protein determination and STa biological activity.

Reversed-Phase Batch Adsorption Chromatography (MCI-gel)

An intermediate purification step was applied to the acetone STa-rich fraction
to achieve a further level of STa purity. The lyophilized crude STa was solubilized in
100 ml of 0.1% of 20% HPLC-grade methanol. To this solution, 100g of Reverse-
Phase Methacrylate Adsorbent Polymer Resin (340 °A 30 um Mitsubishi Chemical
Corporation, Cat # CHP2MGY-01L) was slowly added under gentle mixing and the
slurry was kept at 4 °C for 2 hours under gentle shaking. The slurry was poured into a
10-mm-i.d.x 25-cm-long glass column. The column was washed with 300ml of
0.1%TFA/1120 (v/v). Stepwise elution of the proteins was performed with 100 ml of
0.1%TFA of 20, 40, 60, 80 &100% MeOH (v/v). Fractions were collected separately
from each elution step. The methanol and TFA were evaporated and the residues were

tested for protein and STa biological activity.

62

Preparative Reverse-Phase High Performance Liquid Chromatography (RP-
HPLC)

RP-HPLC was performed on Waters Associate Liquid Chromatography
System equipped with multi-solvent delivery pumps, an automated gradient
programmer 6OOS controller, Model 486 tunable absorbance detector using 7 pm, 300
A, 25cm x 10 mm i.d Vydac C8 preparative columns (Sorbent Technologies, Inc,
Atlanta, GA). Samples from RP-methacrylate adsorbent polymer resin were applied
on RP-C8 column and STa was eluted by gradient system with 0.1% TFA in water as
solvent A & 0.1%TFA in 80% methanol as solvent B (0-30% 5min and 30-80% in 80
min). The UV absorbing peaks were detected at 214 nm. Peaks were collected
separately and the methanol was evaporated and then freeze-dried. The freeze-dried
substance was reconstituted into physiological saline and tested for protein content
and STa biological activity.

S T a assessment for biological activity

Detecting and quantifying STa biological activity was done using a reference
standard in vivo model test, suckling mouse assay, according to Dean et al. (1972)
and Giannella, (1976). Newborn Swiss Albino suckling mice (2-3 days old) were
randomly divided into groups (three each). Samples from RP-HPLC were serially
diluted 1/ 100; 1/ 10,000 & 100,000 and 10 ul of 0.2% Evans blue (w/v) was added per
ml. Each suckling mouse was inoculated orally by IOO-ul sample using a 1 ml syringe
and a 20-p-diameter polyethylene tube. Each sample dilution was tested in triplicate.

After 2-hour incubation at room temperature, the mice were euthanized by carbon

63

dioxide in a C02 chamber and the intestine (not including the stomach) from each
newborn mouse was removed and weighed.

The ratios of intestinal weight to remaining body weight of the three mice
were determined. Animals with no dye in the intestine or with dye within the
peritoneal cavity at autopsy were discarded. One unit of ST activity (one mouse unit)
is defined as the minimal amount of toxin that produces an intestinal weight/carcass
ratio of 2 0.083.

Criteria for homogeneity of purified S T a

Homogeneity of the purified STa from preparative runs was validated by
analytic aquapore RP-300A Perkin Elmer C8 column. Additionally, the exact
molecular weight of STa was determined by. matrix-assisted laser desorption
ionization-time of flight mass spectroscopy (MALDI-TOF/MS). The purified STa
was then submitted for amino acid sequencing.

Results and Discussion
Detection of S T a gene

PCR amplification verified that the tested strain carried the gene encoding for
STa after analyzing the product on gel electrophoresis. Two amplicon bands of 127
bp and 244 bp were detected under UV light for the tested strain, which were not
apparent for the control strain (E. coli K-12) (Figure 5).

Culture analysis

Growth kinetics experiments were conducted on 30L batch cultures under

various pH values (7.4, 8 and 8.5). Samples were taken every two hours and the

growth pattern was determined by counting the total cell count (CFU/ml) using a

64

robotic spiral plate and computer linked camera (Q counter). As Figure 6 indicates,
tested ETEC growth was maximal in medium in which the initial pH was adjusted to
7.4. This level of grth was associated with higher level of crude STa as verified by
the suckling mouse assay.
Purification and Characterization of E. coli STa

Table 7 shows the summary of the purification scheme of STa for the ETEC
E. coli in 30 L batch culture.
Amberlite XAD-2 Batch Adsorption Chromatography

This step yields a high specific activity of the crude STa (8.70 x 103 MU/mg
protein) compared with the STa specific activity in the cell free filtrate (1.22x103
MU/mg protein).
Acetone fractionation

Acetone fractionation resulted in further purification of the STa by removing
additional amount of non-STa protein that was precipitated in acetone. Samples were
taken for protein determination and STa biological activity. Specific activity of STa
increased to 88.7 x 103 MU/mg protein. V
Reversed Phase-Batch Adsorption Chromatography (MCI-gel)

Specific activity of the STa at this step of purification increased from 88.7 x
103 to 112 x 103 MU/mg protein. This step allowed for a larger sample load on
preparative RP-HPLC. Up to 15 mg of the crude STa cleaned by this procedure could
be used as a single load in RP-HPLC without overloading the column or losing the
resolution. This has led to a considerable reduction in the number of HPLC runs

needed to purify STa.

65

Preparative Reverse-Phase HPLC Chromatography (RP-HPL C):

Sixty percent methanol MCI-gel STa-rich fractions were loaded on a
preparative C8 column for further purification. Figures 7 and 8 describe the elution
profiles of STa. Elution with an increasing methanol gradient resulted in number of
absorbance peaks at 214 nm (Figure 7), the last of which was found to contain
enterotoxin activity. The enterotoxin peak began to elute at approximately 5 5-60%
methanol after 35 minutes retention time. This peak was collected and after methanol
evaporation, was freeze-dried. It was then reconstituted into physiological saline and
tested for STa biological activity and protein concentration. Further improvement in
the STa specific activity (885 x 104 MU/mg) was achieved in this step. The biological
activity was demonstrated to be 0.113 ng perone mouse unit of STa minimal
effective dose (MED) in 2-3 day-old inoculated Swiss Webster suckling mice.
Criteria for homogeneity of purified STa

I Analytic C8 column: Pooled peaks from several preparative RP-HPLC runs
were tested on an analytic aquapore RP-300A Perkin Elmer C8 column to
demonstrate a single symmetrical peak (Figure 8).

I Mr-value determination using Matrix assisted laser desorption ionization-
time of flighflmass spectroscopy: A lyophilized HPLC-purified sample was
analyzed by MALDI-TOF/MS to determine the molecular weight and the result
is shown in Figure 9. The observed signal at 100% MS intensity with m/c =
1972.1 indicates that the Mr of the purified product is 1972.1 Da, which is
compatible with the Mr (1969-1972) value calculated from amino acid

composition of the STa, confirming the purity and identity of the purified

66

product as the STa molecule. This was in agreement with the findings of Takao
et al. (1983).
I Amino acid sequence: Further confirmation of the homogeneity and identity of
the purified product was performed by determination of amino acid sequence.
A lyophilized HPLC-purified sample was submitted for amino acid sequence
analysis and the results showed the 18 amino acid residues of the STa molecule
were matching the reported sequence.
Conclusion
This protocol includes concentrating the cell free filtrate using Amberlite
XAD-2 batch adsorption chromatography (BAC), acetone fractionation, and
Methacrylate polymer resin BAC and finally through RP-HPLC. Chemical analysis
of the purified preparations matched the reported structure for this type of
enterotoxin. The biological activity was demonstrated to be less than 0.2 ng per one
Mouse Unit ‘of the STa in 2-3 day-old inoculated Swiss Webster suckling mice. In
summary, purification of STa to homogeneity was accomplished and the purity of the
produced STa was documented through amino acid sequencing, and mass

spectroscopy.

67

28 sees B 2:

0 EB 582 3 8m .m EB 83 an 82 .< 33m 55,

eves as: sea :8 .m 3:80 .m ESE 3 SN 5? Baa anon :8 .m EEC .4 BEE 35 E: can:
as: gas :8 .m 3550 .m 55E 35 e? Baa anon :8 .m aoazo .m cause <28 secs 3 82 ._

88855 oozom was: mm Bodegas
down: 35¢. :83 gem co omhm mo 26:2 €380

H2605 mow
mo $223502“. 6w 8989‘. .m oSwE

 

 

Esta—=92: Leta 2.5:

A: :5 s 5:5 l
G.» :5 c 6:5 11.

ex :5 m 8:511

 

8de

2:25..»

mgws. r

mo+mon.w

mom—co.“

gen .N

gmgd

SE36

Ill/{MD

 

 

68

 

 

 

5:28 Lea—m :28;
w 0 895-355“ Baggy. £335“ :0
mason 5288528 03933: :8 .m 26.8
mzeflwofifi mo pica nous—m .w 2:?»

 

 

 

 

 

 

 

5:38 ouch;

mo 3:8ng :0 cozoafi autumn—1w

- Em H02 493508 3590.5: $8
mo oEoE 20:20 Emwuwoumaofio 25c:
vegetated nwfiomgmougom .N. 8sz

 

 

 

are . .
Seared “V m. L;
: new m ..

 

 

 

69

Exoaobfio 2

383.3:

:8 .m we 2on Ecomoboomm mama Ema mo 08:-eoseNEom 83983 59: 62%.? x532 d PEME

 

 

     

08v cord coon Saw

8.2038:
8,8

camp

 

 

«N.

3r

3 a 2“""§”‘"é“

.. ,1“ -«w—a and-..“ 3.

on

8 8

.2
. 8.

£885 .x.

 

 

70

Table 7. Summary of the purification of E. coli STa per batch

 

 

 

 

 

 

 

iRP-HPLC

 

 

 

 

 

 

 

Volume . Total PM“ SP A“ MED Purification

Step Titer 7 Cone MU x

ml MU/ 10 3 ng fold

/mg 10 /mg

Cell Free 3 -2
filtrate 30 x 10 10 3 24,660 1.22 822 1
Amberlite 4
XAD_2 BAC 120 10 1.2 1,378 8.70 114.9 7.13
New?" . 6O 10'5 6 676.8 88.7 11.28 72.67
Fractionation
60%MCI-gel F 30 10‘5 3 267.6 112.10 8.92 91.88

80 10'6 80 90.4 8849.56 0.113 7253.28

 

MU = Mouse Unit = minimal amount of toxin producing intestinal weight to

remaining body weight ratio 3 0.085

Sp Ac = Specific activity = total mouse unit/protein concentration

MED = Minimal effective dose = protein concentration per mg/total mouse unit per

million

BAC = Batch absorption chromatography

Purification fold = specific activity of STa from each step/ specific activity of STa in

the cell free filtrate

Protein assay was done by Lowery method (Lowery 1951) using Perkin Elmer
Spectrophotometer

71

 

References

Dean, A; Chang, Y-C; Williams, R; and Harden, L B. 1972. Test for Escherichia coli
enterotoxin using infant mice: Application in a study of diarrhea in children in
Honolulu. The Journal of Infectious Diseases 125: 40 7-41 I .

Evans, D.G; Evans, DJ; and Perce, NP. 1973. Differences in the response of rabbit
small intestine to heat-labile and heat-stable enterotoxins of E. coli. Infection
and Immunity. 7:8 73-880.

Giannella, R. A. 1976. Suckling mouse model for detection of heat-stable Escherichia
coli enterotoxin: Characteristics of the model. Infection and Immunity I4: 95-
99.

Giannella, R. A. and Elizabeth, A. M. 2003. E. coli heat stable enterotoxin and
guanylyl cyclase C: New functions and suspected actions. Trans Amr Clin
Climato Ass. 1 14, 6 7-85.

Guerrant R.L; Moore, R.A; Kirshenfed, RM. and Sande, M. S. 1975. Role of
toxigenic and invasive bacteria in acute diarrhea of childhood. N. Egl. J. Med
293 :5 6 7-5 73.

Gyles, C. L. 1971. Heat-labile and heat-stable forms of enterotoxin from Escherichia
coli strains enteropathogenic for pigs. Ann NY Acad Sci. 1 76:314-322.

Holmes, D. S. and Quigley, M. 1981. A rapid boiling method for the preparation of
bacterial plasmids. Anal. Biochem. I 14, 193—1 9.

Jiang, Z.D; Mathewson, J .J ; Ericsson, C.D; Svennerholm, A.M; Pulido, C. and
DuPont H.L. 2000. Characterization of enterotoxigenic Escherichia coli

strains in patients with travelers’ diarrhea acquired in Guadalajara, Mexico,
1992-1997. J Infect Dis. 181: 779-82.

Naline. D.R; Bhattachaijee AK. and Richardson, SH. 1974. Cholera-like toxin effect
of culture filtrates of Escherichia coli. J. Infec Dis. 130: 595-601.

Olsvick, O and Strockbine NA. 1993. PCR detection of heat-stable, heat-labile and
Shiga-like toxin genes in Escherichia coli. In Diagnostic Molecular Biology
by Persing, DH; Smith, T.S; T enover, FC. and White, T.J. American Society
for Microbiol Washinghton DC.

Sack, R.B; Hirschhorn, N; Brownlee, I; Cash, R.A; Woodward, (W.E and Sack, DA.

1975. Enterotoxigenic Escherichia coli-associated diarrheal disease in Apache
children. N.Engl.J. Med. 292:1041-1045.

72

Saeed, A. M; Srianganathan, N; Cosand, W; and Burger, D. 1983. Purification and
characterization of heat-stable enterotoxin from bovine enterotoxigenic
Escherichia coli. Infection and Immunity, 40: 701-701.

Saeed, M. A; and Greenberg, RN. 1985. Preparative purification of Escherichia coli
heat-stable enterotoxin. Anal. Bio.151: 431-43 7.

Salvadori, M.R. Valadares, G.F; Leite D.D; Blanco, J. and Yano, T. 2003. Virulence
factors of Escherichia coli isolated from calves with diarrhea in Barzil.
Brazillian J Microbiology, 3 4: 23 0-235.

Sears, C. L; and Kaper. J. B. 1996. Enteric bacterial toxins: mechanisms of action and
linkage to intestinal secretion. Microbiol. Rev. 60:167-215.

Smith, H.W; and Halls, S. 1967. Observation by the ligated intestinal segment and
oral inoculation methods on Escherichia coli infections in pigs, calves, lambs
and rabbits. J.Pathol.bacteriol. 93 :499-529.

Staples, 8.]; Asher, SE; and Giannella, RA. 1980. Purification and characterization
of heat-stable enterotoxin produced by a strain of E. coli pathogenic for man
J. Biol. Chem. 155, 10: 4 71 6—4 721.

Takao, T; Hitouji, T; Aimoto, S; Shimonishi Y; Hara, S; Takeda, Y. and Miwatani, T.
1983. Amino acid sequence of heat-stable enterotoxin isolated from
enterotoxigenic Escherichia coli strain 18 D. FEBS Letters, 152: 1-5.

Whipp, S.C; Moon H.W. and Lyon, NC. 1975. Heat-stable Escherichia coli
enterotoxin production in vivo. Infect. Immun 12:240-244.

Wolf, MK. 1997. Occurrence, distribution and associations of O and H serogroups

colonization factor antigens and toxins of enterotoxigenic Escherichia coli.
Rev Infect Dis. 1: 918-926.

73

CHAPTER IV

DESIGN AND CHARACTERIZATION OF AN IMMUNOGENIC
Escherichia coli HEAT-STABLE ENTEROTOXIN (STa)I

Introduction

Heat-stable enterotoxin (STa) is an important virulence determinant in
enterotoxin-mediated diseases caused by E. coli (Sears & Kaper, 1996; Nataro &
Kaper, 1998 and Giannella & Elizabeth, 2003). The immune system has difficulty in
eliciting antibody against STa because of its small molecular weight (<2 kDa) (De
Week, 1974 and Pereira et al. 2001). However, proteins of molecular size LOGO-3,000
Da can be made immunogenic by conjugation to a suitable macromolecule (carrier
protein) (Elanger, 1980 and Pauillac et al. 1998). A wide range of carrier proteins and
several procedures for conjugation have been reported for the preparation of peptide-
carrier conjugates using various cross-linkers. The selection depends on the chemistry
of conjugation and the location of antigenic determinants (epitopes) within the native
peptide.

Conjugation of STa to several types of carrier protein have been reported,
however, no sufficient details were presented on the efficiency and the characteristics
of these conjugates (Houghten et al. 1984, 1985; Klipstein et al. 1982, 1983; Sanchez
et al. 1986, 1988; Clements, 1990 and Thompson and Giannella, 1990). In this study,
purified STa peptide was covalently cross-linked to the modified bovine serum
albumin (BSA) through the amino terminal of the STa to preserve the biologically

active moiety that is perceived to be associated with carboxyl terminus of the peptide.

 

' Manuscript of this chapter is under preparation

74

This study aims to design and characterize an efficient immunogenic STa
conjugates based on the evaluation of four different conjugation protocols.
Background and principles of peptide -carrier conjugation

Conjugation of proteins involves the linkage of two or more molecules to
form a novel complex which has the combined properties or features of its individual
components (Hermanson, 1996). Ideally, there are two critical criteria that should be
met in any conjugation procedure. The first is that the coupling process should
provide a high yield of well-defined and reproducible composition without
inactivation of the peptide molecule. Secondly, it should yield a stable linkage
between the peptide and carrier (Pauillac et al. 1998 and Fuentes et al. 2005).

The strategies of the coupling process rely on the presence of functional
moieties, which can be used to facilitate the conjugation of the peptide to a carrier
protein. These moieties include carboxylic acids, amines, thiols, anhydrides, N-
maleimides, imino esters and others. Depending on the functional moiety used, the
reactive group may be the e-amino group of a lysine residue, a-amino of an N-
terrninal (NH3,), sufhydryl [SH] group of cysteine, or the carboxylic acid [COO']
groups (Asp, Glu, or alpha-carboxyl). These moieties are capable of forming covalent
bonds between two protein species (Dick & Beurret 1989 and Wong, 1991).

Cross- linking reagents

Cross-linkers contain reactive ends to specific functional groups (primary

amines, sulfhydryls, etc.) on protein molecules (Wong, 1991 and Hermanson, 1996).

Several cross-linkers are available and can be used to covalently link one molecule to

75

another. Most commonly used are carbodiimide derivatives, glutaraldehyde (GA),
and m-maleimidobenzoyl—N-hydroxysuccinimide ester (MBS).

Carbodiimide derivatives, both the water soluble derivative, 1-ethyl-3-(3-
dimethylaminopropyl carbodiimide hydrochloride (EDAC) and the organic soluble
derivative, N,N-dicyclohexylcarbodiimide (DCC) are commonly used. Carbodiimides
are heterobifunctional reagents that are mainly used for coupling —COOH and —NH2
groups (Goodfriend et al. 1964 and Kurzer et al. 1967). They can activate the side
chain carboxylic groups of aspartic and glutamic acid, as well as the carboxyl
terminal group, to make reactive sites for coupling with primary amines (Yamada et
al. 1981).

Glutaraldehyde is a bifunctional coupling reagent that links two compounds
through their amino groups (Habeeb & Hiramoto, 1968; Richard & Knowles, 1968
and Russel and Hopwood, 1976). Although glutaraldehyde provides a highly flexible
spacer between the peptide and carrier protein for favorable presentation to the
immune system, it is a very reactive compound and will react with Cys, Tyr and His
to a limited extent and the result is a poorly defined conjugate (Molin et al. 1978).

The m-Maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) is a hetero-
bifunctional reagent that can be used to link peptides to carrier proteins via cysteines
(Carlson et al. 1978; King et al. 1978 and Yoshitaki et al. 1979). The coupling takes
place with the thiol group (SH) of cysteine residues.

Carrier Proteins
Carrier proteins are macromolecules capable of stimulating the immune cells

to elicit an antibody response (Harlow and Lane, 1988). Peptides lack the T-cell

76

epitopes, which are essential for priming CD4+ T helper cells. The carrier molecule
provides the T-cell antigenic determinants for T-cell signaling, proliferation and
release of mediators which activate specific B cells to stimulate antibody production
in response to the immunogenic peptide-carrier conjugate (De Silva et al. 1999).
Structurally, peptide-carrier conjugates have native antigenic determinants of the
carrier as well as the peptide epiotpes. Introducing this conjugate into a suitable host
generates peptide-specific antibodies (Brinkley, 1992) as well as carrier antibodies.

Peptides have been coupled to several carrier proteins. The most commonly
selected carriers are bovine serum albumin (BSA) and keyhole limpet hemacyanin
(KLH). BSA is one of the most widely used carriers for the design of peptide-carrier
conjugates. It is highly antigenic (M.W. ~67 kD) and can be easily modified to
introduce a new moiety for specific coupling procedures (Habeeb 1967a&b).
However, BSA is used as a blocking agent in many experimental immunoassays
(ELISA), so antibodies raised against peptide-BSA conjugates will limit the
application of the standard ELISA procedure and different blocking agents are
needed.

Although KLH is large (1.3 x 107 Da) and immunogenic, it may precipitate
during cross-linking making it difficult to use in some cases. Moreover, the serum
antibody response to this carrier ofien obscures that of the antigen of interest (De
Silva et al. 1999) resulting in a reduced antigen-specific antibody concentration in the
IgG fraction. Ovalbumin (OVA), rabbit serum albumin (RSA) thyroglobulin (TG) or
synthetic carriers such as multiple antigenic peptides (MAPS) can also be used as

carrier proteins (Wong, 1991 and Hermanson, 1996).

77

It is important to recognize that the immune system reacts to the peptide-
protein carrier as a whole and that there will be a portion of response directed against
the conjugated peptide as well as the carrier protein. Thus, coupling should be done to
keep the antigen of interest in as native a condition as possible (Harlow and Lane,
1988). De Silva et al. (1999) showed that during preparation of peptide-carrier
conjugates, one must strive to preserve the integrity of the antigen structure since
many applications involving the antibodies require discrimination with very minimal
conformational differences.

In this study, BSA was modified in order to incorporate extra functional
carboxyl groups and used as a carrier to cross-link the STa molecules through their
amino terminals (Figure 10). Four different conjugation protocols were evaluated to
design a successful conjugate that retained high biological activity for further use in
the study of its immunogenicity.

Materials and methods
Reagents

All reagents were obtained from commercial sources and were of analytical
grade. Bovine serum albumin (BSA), succinic anhydride (SA), dioxane, N,N-
dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl carbodiimide
hydrochloride (EDAC), N-methy-imidazole, dimethyformamide (DMF), 2—(N-
morpholino) ethanesulfonic acid buffer (MES), triethylamine (ET3N), p-nitrophenol,
sodium azide (NaNg) and phosphate buffer saline (PBS) tablets were obtained from

Sigma Chemical Co. (St. Louis, MO). STa was purified and a detailed methodology

was described in the previous chapter.

78

Carbodiimide

 

C-terminal

   

N—terminal

 

 

 

 

 

 

Figure 10. Illustration showing the conjugate of bovine serum albumin
carrier protein- E. coli STa peptide

Procedure for covalently cross-linking STa with modified BSA
Chemical modification of bovine serum albumin
Bovine serum albumin was chemically modified to introduce new carboxyl
moieties using two different protocols:
I Succinylation
I Hyper-succinylation
Succinylation of Bovine Serum Albumin
Basis of reaction: Succinic anhydride (SA) reacts rapidly with the e-amino
groups of lysines and the a-amino groups of the N -termini of proteins at pH 7-9,
forming an amide bond by replacing the amino group with a carboxyl (Riordan &
Vallee 1964, Gounaris & Perlmann 1976 and Merck & Co., Inc 1996) (Figure 11).
Thus, introducing a succinic anhydride moiety on BSA will afford a protein

derivative with more carboxyl groups and hence increase the possibility to link STa

79

from its amino terminal and preserve its antigenic determinants associated with the

carboxyl terminal of the molecule.

(:1
3 4
+ O _—... protein
2
1 2 3 4
0 NH 1
NH2 \[r CH2CH2 ----- COOH
\j 0 Coverts amine to acid

Figure 11. The action of succinic anhydride upon the amino
groups of a protein

Procedure: The methods followed were those described by Habeeb (1967a,
b). Briefly, one gram of BSA was dissolved in 200 ml of 0.2 M borate buffer, pH 9.3.
A 20 ml solution of dioxane with 5.4 g succinic anhydride was added in small
aliquots over a period 30 minutes, the reaction mixture was stirred magnetically while
maintaining the pH at 9.3 through the addition of 3 M NaOH. Following the last
addition of succinic anhydride, the acylation reaction was allowed to continue for 45
minutes. The solution was then dialyzed at 4°C against several changes of 0.01 M
triethylamine using dialysis tubing with a M.W. cutoff of 12-14 KD. The dialyzed
preparation was first freeze-dried and then further dried in a dessecator over
phosphorous pentoxide (P205). Samples were taken and reconstituted in PBS buffer
(pH 6.8) for size exclusion chromatography and mass spectroscopy.
Hyper-succinylated Bovine Serum Albumin (HS-BSA)

Basis of reaction: An extensive modification of the BSA by introducing a

large number of succinyl moieties [COO'] on the carrier protein could be achieved in

80

the hyper-succinylation reaction. The hyper-succinylation reaction was carried out in
two steps. The first step involves the production of hyperaminated BSA by
conversion of all free carboxyl groups on the BSA (aspartic and glutamic acids) into
amino groups. The second step was the production of hypersuccinylated BSA by
addition of succinic anhydride to the hyperaminated BSA to convert all amino groups
(newly introduced, free, and N—terminal) into carboxyl groups.

Procedure: Native BSA was treated with lmM of ethylenediamine at pH 4.75
in the presence of 10 mM EDAC. This hyperaminated protein molecule was then
treated with 100 moles of succinic anhydride at pH 8.0 for two hours to produce a
hyper-succinylated BSA molecule (Fuentes et al. 2005).

Coupling of E. coli STa to modified BSA

Four different conjugation protocols were used to covalently cross-linking
STa to modified BSA. They were evaluated on the basis of stability of covalent bond,
retained STa biological activity and the conjugation efficiency.

Protocol 1: Using Dimethyformamide (DMF) as a solubilizer for the peptide and
carrier protein

Atassi (1981) described the use of DMF to solubilize several synthetic
peptides before cross-linking them to carrier proteins. This protocol enhances the
coupling of the amino terminal ends of synthetic peptides to the carrier protein. In this
study, the carrier protein (suBSA) was solubilized in DMF and then treated with p-
nitrophenol to activate the carboxyl groups on the carrier.

Procedure: Applying this procedure, we have used the purified native STa peptide
and have solubilized it in DMF prior to addition to the modified suBSA. The mixture

was kept stirring overnight at room temperature. This design was meant to encourage

81

the STa coupling through its amino terminals based on nucleophilic attack at the
reactive ester groups of the modified suBSA forming amide linkages. In a typical
reaction, we used 140 mg of suBSA suspended in 10 ml of anhydrous DMF and
stirred it magnetically in a tight-capped foil-wrapped bottle for 3-4 hours. A solution
of p—nitrophenol (65mg/0.5m1) in DMF was added and magnetic stirring continued
for 15 minutes. A solution of DCC (50 mg/O.5ml) was added to suBSA at a molar
ratio of 120:] and the reaction was allowed to continue stirring at room temperature
for 3 hours. One hundred mg of STa in 1 ml DMF was added to the activated suBSA
at a molar ratio of 24:1. Shortly after, 1 ml of triethylamine was added. The reaction
mixture was stirred overnight, at room temperature, while protected from direct light.
The next day, 30 ml of Milli Q water was added and the mixture was dialyzed
extensively against distilled water at 4°C using a dialysis membrane with a 12-14 kD
M.W. cutoff and then freeze-dried. Samples were taken for measurement of the STa
biological activity of the conjugate using suckling mice assay (SMA), protein
determination, biochemical and molecular characterization.
Protocol 2: Imidazole-based protocol

One coupling procedure (Dean et al. 1990) described the use of imidazole to
stabilize the carrier protein and minimize the formation of polymers due to the
acylation process when the cross linker and the peptides intended for cross-linking are
added.

Procedure: We used 0.5 M N-methyl-imidazole pH (6.0) to dissolve the STa
peptide and the carrier protein suBSA at a molar ratio (100:1). After the addition of

EDAC (molar ratio: 50 mol EDAC/mol STa), the mixture was stirred for 30 minutes

82

at room temperature followed by dialysis (M.W. cutoff 12-14 kD) against distilled
water at 4°C.
Protocol 3: Hyper-succinylated BSA-based protocol

Fuentes et al. (2005) reported that an increase in the numbers of succinyl
groups [COO'] on the carrier protein can enhance the cross linking of the peptides
from their amino terminal. The procedure for hyper-succinylation of BSA was
described above.

Procedure: In this coupling protocol, 3 mg hypersuccinylated-BSA was
dissolved in 2.5 ml of 5 mM sodium phosphate buffer pH 7 and mixed with 2.5 ml of
the STa peptide (0.5 mg/ml) in dioxane. EDAC was then added gradually to reach a
concentration of 100 mM. After that, the conjugated composite was dialyzed using a
tube with a M.W. cutoff 12-14 kD against distilled water at 4°C.

Protocol 4: Conventional peptide-carrier coupling protocol

In this coupling procedure, suBSA, EDAC and MES buffer were used
(Uptima interchim, 2007).

Procedure: SuBSA carrier protein was dissolved in 0.1M MES buffer pH 5 to
a final concentration of 10mg/ml. Two milligrams of STa peptide were added to 2 mg
of suBSA carrier protein. Then, EDAC (10 mg /ml in cold distilled water) was added
at a ratio of 0.5 mg of EDAC per mg of total protein. The reaction was kept stirring
for 2-3 hours at room temperature before dialysis at 4 °C against PBS using a tube of

12-14 kD M.W. cutoff.

83

Dialysis

The products of BSA modification and STa-modified BSA conjugation
reactions were subjected to extensive dialysis to remove the small molecular weight
reactants (<14 kD). Dialysis tubing with nominal M.W. cut-off 12-14 kD was
purchased from Fisher Scientific (Pittsburg, PA). STa-SuBSA conjugate was
subjected to extensive dialysis against Milli Q purified water using a dialysis
membrane of 12-14kD M.W. cutoff. Molecular species of l4kD or higher were
retained inside the dialysis tube and all other reactants below 14 kD including
uncoupled (free) toxin were dialyzed out.

Gel Filtration Chromatography (GFC)

PD-lO columns, Sephadex G-25M packed columns, of a nominal molecular
mass exclusion limit of 5000 for protein were purchased from GE. Healthcare
(Buckingharnshire, UK). These columns are designed to separate proteins based on
their molecular weight. The columns were equilibrated and developed by following
the manufacturer’s instructions. The dialyzed STa conjugate samples were passed
through PD-lO Sephadex G-25 GFC column to purify the STa-suBSA conjugates
from free STa, then assessed for biological activity and protein concentration.

A freeze-dried STa-suBSA conjugate sample was reconstituted into 2.5 ml of
PBS and passed through a PD-lO column to separate the unconjugated STa peptide
from the portion that was successfully cross linked to the carrier. The STa-carrier
conjugate was then eluted with 3.5 ml PBS and the effluent was collected and tested

for biological activity in a suckling mouse assay.

84

Size Exclusion-High Performance Liquid Chromatography

SE-HPLC was performed to compare the molecular size of both native and
modified BSA. Bio-Sil® SEC-125 HPLC 300 x 7.8mm filtration column (Cat # 125-
0060) was purchased from BioRad (Hercules, CA) and hooked to the Waters
Associate Liquid Chromatography System equipped with multi-solvent delivery
pumps, an automated gradient programmer 6008 controller and a tunable absorbance
detector (Model 486). The column was equilibrated with 0.05 M sodium phosphate
(diabasic), 0.05M sodium phosphate (monobasic), 0.15M NaCl and 0.01 M NaN3 at
pH 6.8. Isocratic elution system was applied at flow rate 1 ml/min. The peak
absorbance was monitored at UV 280 nm.

Amino acid compositional analysis

To determine the conjugation ratio of STa to the modified BSA, a freeze-dried
STa-suBSA conjugate sample was subjected to amino acid compositional analysis at
the Research Technology Support Facility (RTSF) at Michigan State University.
Matrix-assisted laser desorption ionization/time of flight mass spectroscopy
(MALDI-TOF/MS)

Two hundreds micrograms of freeze-dried STa-suBSA conjugate sample were
subjected to MALDI-TOF/MS analysis at the MSU-RTSF laboratory to determine
precisely the molecular weight of the conjugate and use this figure to calculate the
number of STa molecules that covalently cross-linked to one BSA molecule

(conjugation ratio).

85

Protein Assay

Protein assays were performed according to the Lowery method (Lowry et al.
1951), using a Perkin Elmer Lambda 3A UV/U S Spectrophotometer.
STa-suBSA conjugates activity bioassay

The biological activity of the STa-suBSA conjugates was determined using
the suckling mouse assay as described by Dean et al. (1972), Giannella, (1976) and
modified by Saeed et al. (1983).
M

Table 8 shows the summary of conjugation experiments and their evaluation.
More details on the DMF conjugation protocol are presented below.
Characteristics of the modified BSA

The results from SE-HPLC showed that modified BSA with succinic
anhydride was eluted faster (RT= 2.99 min) than native BSA (RT= 4.20 min),
suggesting that the molecular weight of suBSA had undergone a significance change
(Figure 12). Change in the molecular weight of the succinylated BSA was also
confirmed by MALDI-TOF/ MS. It was found that suBSA has a M.W. of 72.40 kD in
comparison to native BSA which has a M.W. of ~67.00 kD (Figure 13).
Characteristics of E. coli STa-suBSA conjugates
Dialysis and Gel Filtration Chromatography

A summary of the results is presented in Table 8. The conjugation protocol
based on the DMF method showed a higher rate of conjugation efficiency and higher

level of retained STa biological activity compared to other conjugates (Table 8).

86

Further characterization of the DMF-based conjugation protocol is described
belowAmino acid compositional analysis

A dialyzed conjugate sample based on the DMF protocol was subjected to
amino acid compositional analysis. Table 9 shows the pmole concentration and
retention time of each amino acid residue.
Calculation of S T a Peptide to suBSA Ratio

The conjugation ratio is defined as the number of STa molecules covalently
cross-linked to one molecule of succinylated BSA. The amino acid composition of
the conjugate was empirically determined by measuring the pmoles of each amino
acid detected in a known volume of sample. Well-recovered residues, arginine and
methionine, were used to quantify the concentration of each residue (pmole) in the
conjugate sample. The number of STa molecules in the conjugate sample was
calculated using the Arg & Meth residues not present within the sequence of the STa
peptide. Table 10 gives the approximate number of coupled STa molecules to one
molecule of modified BSA. Based on the data of amino acid compositional analysis,
it was found that approximately 4-5 STa molecules were coupled to each molecule of
suBSA.
S T a Conjugate Analysis by MLDI-TOFMS

Lyophilized conjugate samples based on DMF and HS-BSA protocols were
subjected to MALDI-TOF/ MS to accurately determine the molecular weight (Mr
Value). Both samples showed median Mr Values over 80 kDa (Figure 14 & 15).
Figure 16 shows the molecular weight differences for the modified BSA before and

after STa peptide conjugation (AM.W. = 8342.5 Da). This difference was attributed to

87

the contribution of the STa molecules. Based on this data, the median number of STa
molecules successfully crossed linked to one molecule of suBSA was calculated from
the following equation: A M.W. /STa M.W. = 8342.5/ 1959 = 4 -5. Four to five STa
molecules were successfully crossed linked to one molecule of modified BSA based
on DMF and HS-BSA protocols.
S T a conjugate activity bioassay & Conjugation efficiency

We concluded that the STa biological activity and the conjugation efficiency
of the conjugate were highest in the DMF protocol. A summary of conjugation
efficiency based on the tested protocols expressed largely by the conjugation ratios
and the retained STa biological activities of the conjugates is presented in Table 8.
Discussion

Numerous attempts have been made to render STa immunogenic, including
chemical coupling and genetic fusions to appropriate carrier proteins (Houghten et al.
1984, 1985, Sanchez et al. 1986, 1988, Clements, 1990). However, results of these
studies showed limited success since the uncontrolled cross-linking process led to the
loss of the biological activity of STa as a part of the conjugation process (Pereira et
al. 2001). Additionally, these studies showed no sufficient details on the efficiency
and the characteristics of the produced STa conjugates.

The objective of this study was to design and characterize a well-defined,
stable and active STa conjugate for further study of its immunogenicity in laboratory
animal models. We have evaluated several conjugation protocols to achieve a stable

biologically active STa conjugate through carefully planned cross-linking of the STa

88

peptide to a modified carrier protein using BSA, carbodiimide derivatives and
different solvents.

Given the perceived molecular structure of the STa peptide and the desire to
crosslink it through its amino terminus, we have selected carbodiimide coupling
reagents. Other coupling reagents, glutaraldehyde and m-Maleimidobenzoyl-N-
hydroxysuccinimide ester (MBS), may affect the 3-demientional structure of the STa
peptide. Glutaraldehyde binds non—specific amino groups and this leads to
polymerization of peptide and/ or carrier protein, which results in a poorly defined
conjugate (Molin et al. 1978). Cysteine residues on the STa peptide play a crucial role
in the biological activity and the stability of STa peptide. Thus, using MBS as a
hetero-bifimctional reagent targeting thiol group on cysteine residue (Carlson et al.
1978; King et al. 1978 and Yoshitaki et al. 1979) may disrupt the disulfide bonds and
affect the biological moieties on the STa peptide. BSA is widely used as a carrier
protein in conjugation reactions because it is highly antigenic and can be easily
modified to introduce a new moiety for specific coupling procedures (Habeeb
1967a&b). Therefore, the use of carbodiimide and BSA, in our conjugation reaction
was justified based on a thorough understanding of the molecular structure of the STa
peptide.

In this study, BSA was modified by introducing succinic moieties, and its
modification was confirmed using size exclusion chromatography and MALDI-
TOF/MS. The data showed a 5000 Da difference in the molecular size between the
modified and native BSA molecules, indicating an 8% increases in the M.W. of the

modified BSA. This suggests that an extensive modification of the free amino groups

89

was achieved with the addition of succinic anhydride (Habeeb, 1967b). The
subsequent step in the design of STa-BSA conjugate was the cross-linking of STa to
the modified BSA. This reaction was initiated by incubation of the modified BSA
with p—nitrophenol and DCC for three hours to provide reactive ester groups that
could easily attach the STa from its amino terminal, forming amide linkages. The use
of DMF was shown to enhance the solubility of reactants including peptides and
carrier proteins ((Lateef, 2007). We believe that the use of DMF as a solvent reagent
may have facilitated the solubility of the hydrophobic STa molecules, solving a
problem encountered with the other solvents and coupling media.

The STa-conjugate was tested for its protein content and biological activity.
Based on the protein estimation, there was a conjugation efficiency of 52-64%, which
is higher than previously reported (Frantz & Robertson 19981; F rantz et al. 1987; and
Thompson and Giannella, 1990). Moreover, this conjugate showed a higher
biological activity than any activity reported in the previous STa-conjugates (Table
8). Based on these results, it is clear that most of the biological activity of the STa
introduced into this reaction was retained in the conjugate even after extensive
dialysis, GFC and SEC.

Covalent attachment of STa molecules to modified BSA was documented by
amino acid composition analysis and MALDI-TOF/ MS. A median value for the
conjugation ratio of 4-5:1 STazsuBSA has been determined based on amino acid
analysis and MALDI-TOF/MS (Figure 16). Based on the results of the biological
activity of this conjugate, we believe strongly that STa molecules may have been

more efficiently oriented on the BSA carrier molecule via linkage through their amino

9O

terminals. Such orientation, achieved through the DMF protocol, has preserved the
biologically active moiety of the STa and may offer an explanation for the relatively
low yield of STa conjugate produced by other protocols in this study. The ineffective
preservation and presentation of the STa biologically active moiety on previously
studied STa conjugates may also explain the sub-optimal immune response against
STa in laboratory animals (Alderete and Robertson 1978; Lockwood and Robertson
1984; Houghten et al. 1984, 1985; Sanchez et al. 1986, 1988; Clements, 1990;
Lowenadler et al. 1991 and Pereira et al. 2001).

In summary, we have designed a well-defined STa-conjugate based on a
thorough understanding of the molecular structure of the STa peptide. Afier careful
evaluation of several peptide-carrier conjugation protocols, we have defined the
conditions for a conjugate that expressed a high STa biological activity in suckling
mice. Its stability and biochemical attributes were characterized using GFC, amino

acid analysis and MALDI-TOF/mass spectroscopy.

91

3388...-.. 182....
<mm Bum—Emoozflogm “mm {mm mops—>583 H<wm=m

683 22... 668.389.885.285.3818: .682. 38.32... 18 .oanéeaeoefi “835
88.5er 625.688 222853.886-ciao-.188 8.55.8.8 338.636 .z.z noon.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

8cm 35.x... 82 ..o 5 Son. 88 .o .o 6.35. :3. .o a .382 853.3.
A .23.. .8. 2o...
.82 as. $82 :8. .5552. 35.28...
:ouamigu
EH80.— 033
$22 $99. 2 son 2 seam? 3.5.8....
:83”:an
I 1 TV Tmnv 6:9- :Oflfl =.=OU
Ewen. .5858 .Emetm... .Emeoo. N38 522..
mesa .25. .25... m.2 x o. :2 ohm .58.
<83 82
<83 8. mm + .8 we 2 <8-8 we N. + ohm 8.: m .85 Q + <83 3:582 “.53
wE N + «Hm wE N
we 3 + new we 2
m S a 3 .81
Avoammoévomzazv . . 52.52
. 0 CNN .5
822:. . F. 8.25m 82022
G<8v Gee
OEFEEOPEU o .E: O .50 0 D “WMDwm—v o .E: o .50 o = “WMAMWV OBS—$609.50 hog—E: macho
6323 as? .6. ...c A. 3 . c 3 u. ...U .. 3 . . B 6323 6.390
<85 <83 <8-8 <83 3.50
.5562 85...: 3.3:. .352...
2.5.3250 288.5385 8288 828285

 

£3895 flotawsmneo 39.22.. .58 no Etna—gm.— ufiuofitonxo notawinoo no baa—Haw .a oEaH

92

Table 9. Amino acid compositional analysis of E. coli STa-su BSA carrier conjugate

 

 

 

 

 

 

 

 

 

 

Serial # Name RT 3:3:nt Serial # Name RT 3;:th
l Gln 1.78 5.983 10 Tyr 37.09 499.248
2 Glu 20.63 549.781 11 Cys 37.53 509.796
3 Ser 25.61 147.557 12 Val 37.85 297.137
4 Gly 27.42 329.482 13 Meth 39.214 38.214
5 His 29.29 174.214 14 [1e 43.65 118.619
6 Thr 30.47 31 1.339 15 Leu 44.80 576.721
7 Arg 31.57 219.833 16 Lys 46.87 # 333.965
8 Ala 31.886 507.974 17 Phe 38.22 386.2

9 Pro 33.18 377.778 18

 

 

 

 

 

 

 

 

 

 

Table 10. Approximate contribution of STa molecules to one molecule of modified
BSA: calculation of the conjugation ratio

 

 

 

 

 

Total # of residue # of residue # of residue
Residue in STa-suBSA lone BSA lone STa Conjugation Ratio
conjugate sample molecule molecule
Threonine 3113;39800 = 34 residues 1 38.9-34 = 4-5
Cysteine 5097:3200 = 35 residues 6 63.6-35 = 28.6/6 =4-5
Leucine 576.721/8.00 = 72 65 residues 1 72-65 = 7
Alanine 50796739115300 = 48 residues 2 63.5-48= 15.5/2 = 7-8

 

 

 

 

 

 

 

*Brown, 1975; Patterson and Geller, 1977; McGillivray et al. 1979; Reed et al. 1975 and Hirayama et
a1. 1990.

93

2:029:

 

 

 

 

 

 

 

 

 

 

 

 

23208 <mm 38.23.88 20 050E 388588 82: <mm 88.23288 28 025m: 20 0589
Ema mo 08380228252 gouacomow .88. @888 x532 .2 839m 239288822... 2828—88 8% .2 239m
0920385.
88.2.. .808 88.2 88.. 808. .288
i... .. -- i . . . t . . , . . . - - _ i - mo
. We.
2 . . .2
2.2.22 .22. 222. 21222. 22222 222 2_ 2...
22.22 22.2. 222.2 22
22.22 2.2 a
.. 2 2 o 228%
*2 , 2-9.
2 2e
2. 2
I 2 won
2. _ .
2.2.. we 2
.222. 2- 8 cm... lb. 3% 1.5.
_. H <mm 0382 «<QO
22 2.82
m 382 5.885 .x.

 

 

 

 

94

 

Sawing 8:80 <mm BEEEoosméEEom 2588285 03833: :8 M mo 05th
230888on 322: Ema mo oEfiéEENEB 28:39.8 8mg 33.2mm“ 23me .3 233m

 

 

QOchmms.

oooom oooow oooon oooom

2.222.. 222.22 2.22.. ”22%;.222

222...... 6330

2. 2..
- 2.2 - 0:
2.223322. .22

2..
2. 2.2

0823.2 2. . , Fe“;
$0.2... mM.-WWW... T .2222? 32o“

wbvnom

.22...

2.222.222... 1 .
222.222.222.222;

m. Nah;-

oooom oooov

wow

 

W 82
365:: 2x.

 

 

95

 

flawacoo .558 <mm 35255835255405.5593 25.88525 2.3% «so: .58 N 5° .5an

 

 

 

30.58.8qu 32: Ema mo oEuéBENEE 58:982. 58.: 5.898.. .5532 m5 ohswfl
mmficoammz
cocoa roooow 00005 0800 oooom. .o.oo.ov oooom: oooom
I. J I, . .Il A.AA . .va AI... 1‘? VIA . If .I . ....:.VA ...A .1. VII. . A III r. .A > . IL IIII>II4§1IIAII :P. .\ .A .k 1 O
5 o.
5...... 3.5.... 5........... 1.. .15.........§..?£J.§.&3f+~1¥2¥5K1$.5§..5 ON
8.3. 5...MW .55.. w .88 m. mNmN ,8
. 55...... 3.. , r
m 35.5.. m 9. 55.5. N SEN...
..
_. .. o. 0.82.
.. 5. mom
5.. 5
... ._ .,
. moo %
.. .
.5. . .
._. .,. . 0.5.
5 .. ..
. .8
5. $35 I...
5
005
b.3055 .x.

.11.
mémoow

 

 

ozfiméo: :8 .m 0:... mo 0:8 :oflwmsEoo was 5303 33032: < mo nous—HSHU .3 oSmE

Emma—.38 8E8 <mm=méEEoQ Exogouoio

 

 

00000

 

f;

_ ‘

00ng _
F_.oo 3

999.9392

00005 00000 oooom

»

       

383.

5.133?va 333 fgé ,
_ mmfio m. om

~

:

.

o. Aim
,w 03
oa‘wggxfi 5t. ,

wdSbw QVOVNN

     

mNmN—uv

oooov

m om
T

r

floor
€235 .x.

 

 

 

97

References

Alderete J .F. and Robertson, DC. 1978. Purification and chemical characterization of

the heat-stable enterotoxin produced by porcine strains of enterotoxigenic
Escherichia coli. Infection and Immunity 19: 1021-1030.

Atassi, M. Z; Kazim, L. and Sakata, S. 1981. High yield coupling of peptide carriers.
Biochimica et Biophysica Acta, 670: 3 00-3 0.

Brinkley, M. 1992. A brief survey of methods for preparing protein conjugates with
dyes, haptens and cross-linking reagents. Bioconjugates Chem 3 :2-13.

Brown, J .R. 1975. Structure of Bovine serum albumin. Fed. Proc. 34; 591 -5 91 .

Carlson, J; Drevin, H. and Axen, R. 1978 Protein thiolationand reversible
proteinprotein conjugation N-succinimidyl 3-(2pyridyldithio)propionate a new
hetero-bifiinctional reagent. Biochem J. 1 73, 723.

Clements, J. D. 1990. Construction of a nontoxic filsion peptide for immunization
against Escherichia coli strains that produce heat-labile and heat-stable
enterotoxins. Infect Immun 5 8, 1 159-1 1 66.

De Silva, B.S; Egodage, K. L. and Wilson, G. S. 1999. Purified protein derivatives
(PPD) as an immunogen carrier elicits high antigen specificity to haptens.
Bioconjugate Chem. 10: 496- 501.

De Week, A. L. 1974. Low molecular weight antigens. In: M. Sela (Ed.), The
Antigens, Vol. H. Academic Press, New York, Chapter 3.

Dean, A; Chang, Y-C; Williams, R; and Harden, L B.1972. Test for Escherichia coli
enterotoxin using infant mice: Application in a study of diarrhea in children in
Honolulu. The Journal of Infectious Diseases 125: 407-411.

Dean, C; Claassen, E; Gerritsie, K; Zegers, N. D. and Boersma, W.J.A. 1990. Novel
carbodiimide coupling methods for systhetic peptides chanced anti-peptide

antibody responses. Journal of Immunological Methods, 129: 1 19-125.

Dick, W. E. Jr. and Beurret, M. 1989. Conjugate vaccines. Contrib. Microbiol.
Immunol. IOpgs 48-114. Cruse, J. M. and Lewis, R. E., Jr. eds., Basel.

Elanger B.F. 1980. The preparation of antigenic-hapten-carrier conjugates: a survey.
Methods Enzymol. 70 (A), 85.

98

F rantz, J. C and Robertson, DC. 1981. Immunological properties of Escherichia coli
heat-stable enterotoxins: development of a radioimmunoassay specific for
heat- stable enterotoxins with suckling mouse activity. Infec. Immun. 33:193-
198.

F rantz, J .C; Bhatnagar, R.K; Brown, Albert, L. and Garrett, L.K. 1987 Investigation
of synthetic Escherichia coli heat-stable enterotoxin as an immunogen for
swine and cattle. Infect and Immu 55, 1077-1084.

Fuentes, M; Palomo, J .M; Mateo, C; Venteo, A; Sanz, A; F emandez-Lafuente, R. and
Guisan, J ..M. 2005. Optimization of the modification of carrier proteins with
aminated haptens. Journal of Immunological Methods 307: 144-149.

Giannella, R. A. 1976. Suckling mouse model for detection of heat-stable Escherichia
coli enterotoxin: Characteristics of the model. Infection and Immunity 14: 95-
99.

Giannella, R. A. and Elizabeth, A. M. 2003. E. coli heat stable enterotoxin and
guanylyl cyclase C: New functions and suspected actions. Trans Amr Clin
Climato Ass. 1 I 4, 6 7-85.

Goodfriend, T; Levine, L. and Fasman, G. 1964. Antibodies to bradykinin and
angiotensin: A use of carbodiimides in immunology. Science 144, 1344.

Gounaris, AD. and Perlmann, GE. 1976. Succcinylation of pepsinogen. J.Biol.
Chem. 242:2739—2745.

Habeeb, A. F. 1967a. Quantitation of conformational changes or chemical
modification of proteins: Use of succinylated proteins as a model. Arch.
Biochem. Biophys. 121, 652.

Habeeb, A.F. and Hiramoto, R. 1968. Reaction of proteins with glutaraldehyde. Arch
Biochem Biophys 126316.

Habeeb, A.F.1967b. Antigenicity of chemically modified bovine serum albumin. The
Journal of Immunology 99: 1264-1276.

Harlow, E. and Lane, D. 1988. Antibodies a laboratory manual. Cold Harbor
Laboratory New York.

Hermanson, G. T. 1996. Bioconjugation Techniques. Academic Press; 1 st edition.
Hirayama, K; Akashi, S; Furuya, M; and Fukuhara, K. I. 1990. Rapid confirmation

and revision of the primary structure of bovine serum albumin by ESIMS and
FRIT-FAB LC/MS. Biochem. Biophys. Res. Commun.173; 639-646.

99

 

Houghten, R. A., Ostresh, J. M. & Klipstein, F. A. 1984. Chemical synthesis of an
octadecapeptide with the biological and immunological properties of human
heat-stable Escherichia coli enterotoxin. Eur J Biochem I 45, 157-162.

Houghten, R. A., Engert, R. F ., Ostresh, J. M., Hoffman, S. R. & Klipstein, F. A.
1985. A completely synthetic toxoid vaccine containing Escherichia coli heat-
stable toxin and antigenic determinants of the heat-labile toxin B subunit.
Infect Immun 48, 735- 740.

King, T. P; Li, Y. and Kouchoumian, L. 1978. Preparation of protein conjugates via
intermolecular disulfide bond formation. Biochemiistry 1 7, 1499.

Klipstein, F. A., Engert, R. F. & Clements, J. D. 1982. Development of a vaccine of
cross-linked heat-stable and heat-labile enterotoxins that protects against
Escherichia coli producing either enterotoxin. Infect Immun 3 7, 550-55 7.

Klipstein, F. A., Engert, R. F ., Clements, J. D. & Houghten, R. A.1983. Vaccine for
enterotoxigenic Escherichia coli based on synthetic heat-stable toxin crossed-
linked to the B subunit of heat-labile toxin. J Infect Dis 14 7, 318-326.

Kurzer, F. and Dourghi-Zadeh, K. 1967. Advances in chemistry of carbodiimides.
Chem Rev. 67.107.

Lateef, S.S; Gupta, S; Jayathilaka, L.P; Krishnanchettiar, S; Huang J—S. and Lee, B-
S. 2007. An Improved Protocol for Coupling Synthetic Peptides to Carrier
Proteins for Antibody Production Using DMF to Solubilize Peptides. Journal
of Biomolecular Techniques. 18:] 73-1 76.

Lockwood, DE. and Robertson, D.C.1984. Development of a competitive enzyme-
linked immunosorobent assay (ELISA) for Escherichia coli heat-stable
enterotoxin (STa). Journal of Immunological Methods, 75: 295-3 0 7.

Lowenadler, B. Lake M. Elmblad A. Holmgren E. Holmgren J. Karlstrom A and
Svennerholm, A-M. 1991. A recombinant Escherichia coli heat-stable

enterotoxiin (STa) fusion protein eliciting anti-STa neutralizing antibodies.
FEMS Microbiology Letters, 82: 271-2 78.

Lowery, O.H; Rosebrough, N.J; Farr, LA. and Randall, R.J 1951. Protein
measurement with the folin phenol reagent. Iournal Biol. Chem. 193: 265-
275.

McGillivray, R.T.A; Chung, D.W. and Davie, B.W. 1979. Biosynthesis of bovine

plasma proteins in a cell-free system. Amino-terminal sequence of
preproalbumin. Eur. J. Biochem. 98: 4 77-485.

100

Merck & Co., Inc 1996. Compound 9039, P.1515. In Merck Index, 12th ed. Merck &
Co., In., Rahway, NJ.

Molin, S-O; Nygren, H. and Dolonius, L. 1978. A new method for the study of
glutaraldehyde-induced cosslinking properties in proteins with special
reference to the reaction with amino groups. The Journal of Histochemistry
and cytochemistry, 26: 412-414.

Nataro, J. P. and Kaper, J. B. 1998. Diarrheogenic Escherichia coli. Clin Microbiolo
Rev 11: 142-301.

Patterson, J .E. and Geller, D.M. 1977. Bovine microsomal albumin: Amino terminal
sequence of bovine proalbumin. Biochem. Biophys. Res. Commun.74;1220-
1226.

Pauillac, S; Naar, J; Branaa, P. and Chinain M. 1998. An improed method for the
production of antibodies to lipophlic carboxlic hapten using small amount of
hapten-carrier conjugate. Journal of Immunological Methods 22 0: 105-1 14.

Pereira, C.M., Guth, B.E.C., Aleida, ME. and Castilho, BA. 2001. Antibody
response against Escherichia coli heat-stable enterotoxin expressed as fusions
to flagellin. Microbiology, 147, 861 -86 7.

Reed, R. G; F eldhoff, R. C; Clute, O. L. and Peters, T., Jr. 1975. Fragments of Bovine
Serum Albumin Produced by Limited Proteolysis. Conformation and Ligand
Binding. Biochemistry 14; 45 78-4583.

Richard, RM. and Knowles, J .R.l968. Glutaraldehyde as a protein crosslinking
reagent. Mol Biol 3 7:23].

Riordan, J .F . and Vallee, BL. 1964. Succinylcarboxypeptidase. Biochemistry
11:1 768-1 774.

Russel, AD. and Hopwood, D. 1976. The biological uses and importance of
glutaraldehyde. Progress in Medical Chemistry. Edited by GP Ellis and GB
West North Holland Publ C 0 13:2 74.

Saeed, A. M; Srianganathan, N; Cosand, W; and Burger, D. 1983. Purification and
characterization of heat-stable enterotoxin from bovine enterotoxigenic
Escherichia coli. Infection and Immunity, 40: 701-701.

Sanchez, J ., Svennerholm, A-M. & Holmgren, J. 1988. Genetic fusion of a non-toxic

heat-stable enterotoxin-related decapeptide antigen to cholera toxin B-subunit.
FEBS Lett 241, 110-114.

101

Sanchez, J ., Uhlin, B. E., Grundstrom, T., Holmgren, J. & Hirst, T. R. 1986.
Immunoactive chimeric ST-LT enterotoxins of Escherichia coli generated by
in vitro gene fusion. F EBS Lett 208, 194-198.

Sears, C. L. and Kaper. J. B. 1996. Enteric bacterial toxins: mechanisms of action and
linkage to intestinal secretion. Microbiol. Rev. 60:16 7-21 5.

Thompson, M.R., and Giannella, R. A. 1990. Different cross linking agents identify
distinctly different putative Escherichia coli heat-stable enterotoxin rat
intestinal cell receptor proteins. J. Receptor Res. 10: 97-11 7.

Uptima interchim. 2007. EDC conjugation protocol
http://www.interchim.com/interchim/bio/mduits_uptima/tech_sheet/FT-
UP52005(EDCLDdf ‘

 

Wong, S. S. 1991. Chemically introduced reactive groups in Textbook Chemistry of
Protein Conjugation and Cross-Linking, CRC Press, Inc. pp14-2 7.

Yamada, H; Imoto, T; Fujiita, K; Okazaki, K. and Motomura, M. 1981. Selective
modification of aspartic acid-101 in lysozyme by carbodiimide reaction.
Biochemistry 20, 48363.

Yoshitaki, S; Yamada, Y; Ishikawa, E. and Masseyeff, R. 1979. Conjugation of
glucose oxidase from aspergillus niger and rabbit antibodies using n-
hydroxysuinimide ester of n-(4-carboxyxyxlohexylmethy)-maleimide. Eur J
Biochem 101, 395.

102

CHAPTER V
PRODUCTION OF SPECIFIC ANTIBODIES AND CHARACTERIZATION
OF THE IMMUNE RESPONSE AGAINST Escherichia coli HEAT-STABLE
ENTEROTOXIN‘ '
Introduction

Strains of Enterotoxigenic Escherichia coli (ETEC) that produce STa are
among the most important causes of diarrheal disease in children, travelers and
neonatal animals (Gyles, 1996, Kosek et al. 2003, Svennerholm and Steele, 2004).
The changing epidemiology of ETEC strains including the emergence of new
pathotypes (toxin/CFs combinations) presents serious challenges for vaccine
development. The antigenic diversity of ETEC strains and the nonimmunogenic
property of STa account for a high prevalence of ETEC-STa-diarrheal cases in
endemic areas. ETEC strains are incriminated as the cause of 280-400 million
diarrheal episodes in children below 5 years and additional 100 million episodes in
children aged 4-14 years (Svennerholm and Steele, 2004) each year.

Currently, there are no effective vaccines or immunotherapeutic reagents that
confer a broad protection against ETEC-induced diarrhea. Intestinal colonization by
ETEC is a prerequisite for the initiation of diarrhea. However, the colonization factors
that facilitate the ETEC adhesion may undergo an antigenic evolution causing failure
of the currently used CFA-based vaccines. Therefore targeting STa, as the immediate
mediator of ETEC-induced diarrhea, may be the logical approach for the development
of a new immunotherapeutic reagent or an effective vaccine against ETEC diarrhea

(Boedeker, 2005 and Walker et al. 2007). Numerous attempts were made to render

STa immunogenic, including chemical coupling and genetic fusions to appropriate

 

‘ Manuscript of this chapter is under preparation

103

carrier proteins (Houghten et al. 1984, 1985, Sanchez et al. 1986, 1988 and Clements,
1990) but the described chemical coupling reduced the STa-associated toxicity as a
result of the STa conjugation to a carrier protein. The use of reported STa-conjugates
did not result in the production of quality STa-neutralizing antibody from the
immunized animals (Cardenas & Clements, 1993 and Pereira et al. 2001). In this
study, we have developed a biochemically-defmed STa-suBSA conjugate and used it
for immunization of New Zealand rabbits. The immune response during the
immunization process was monitored by the specific binding and neutralization
capacities of the produced antisera against purified native ETEC-STa.
Materials and methods
Reagents and instruments

STa- suBSA conjugates were designed and characterized as described in the
previous chapter. All buffers ingredients, Freund’s complete and incomplete adjuvant,
alkaline phosphatase labeled goat-anti-rabbit IgG antibodies, p-nitrophenyl
phosphate, fish gelatin, Tween-20 and ammonium thiocyanate (NH48CN) were
obtained from Sigma Chemical Co. (St. Louis, MO). Costar 3590- 96-well] microtiter
plates were obtained from Fisher Scientific (F airlawn, NJ). Molecular Devices
ThemoMax Microplate reader equipped with SOFT max Pro 2.6.1 was used to read
the ELISA plate. A bleeding set (coagulant vacutainer tubes, adaptors and 20 gauge
vacutainer needles) was obtained from B-D. (Franklin Lakes, NJ) and used for rabbit

bleeding.

104

 

I! I Am v.‘_.-

 

Animals

Ten female New-Zealand albino rabbits (2-4 kg) were obtained from Charles
River Laboratories (Wilmington, MA) and were housed in approved-size-single cages
at the Containment Facility of Michigan State University. Temperature was kept at
20°C :1: 4°C, with 55% humidity. Rabbits were checked on a daily basis for their
health status by qualified staff and veterinarians.

Immunization procedure

Standard operating procedures for handling and immunization of rabbits in
compliance with the guidelines and recommendations for the Institutional Animal
Care & Use Committee (IACUC) of Michigan State University were used.

A water-in-oil emulsion of STa-conjugate in F reund’s adjuvant was prepared
as follows: 20 mg of freeze-dried STa-suBSA conjugate was reconstituted in 10ml
0.01M PBS-pH 7.0 and added to 10 ml of Freund’s complete adjuvant (primary
immunization). The mixture was homogenized with a polytron at 15,000 rpm for up
to 10 minutes or until a stable water-in-oil emulsion was obtained. The ten rabbits
were inoculated at multiple intradermal sites as described by Vaitukaitis (1981) with
0.5 ml of the described emulsion. Each rabbit was similarly inoculated with a booster
dose at three-week intervals with 0.5 ml of STa conjugates mixed with Freund’s
incomplete adjuvant. Rabbits received boosters until STa-neutralizing antibodies
titers were detected.

Animal bleeding
Pre-immunization blood samples were collected after one weak of adaptation

from the central ear artery according to Gordon (1981) using serum separator ED-

105

 

 

vacutainer tubes to obtain a reference baseline for serum titers. Blood samples were
then collected three weeks after the primary immunization, approximately 4-5 days
after each booster immunization. After collection, blood was allowed to clot for 60
min. at 37°C. The clot was then separated from the sides of the collection vessel and
allowed to contract at 4°C overnight. The separated sera were collected by
centrifugation at 2000 rpm for 30 min, aliquoted and kept at -20°C. The sera were
tested for neutralization and binding capacity against STa using suckling mouse assay
(SMA) and indirect ELISA binding assay respectively.
STa- Serum Neutralization Assay

The STa-serum neutralization capacity was determined using the SMA as
described by Frantz et al. (1987). Briefly, three 50 ul aliquots of serum sample with
were incubated with 25, 50 and 75 ul of STa (20 Mouse Units per ul) at 37°C for 2
hours. The contents of each tube were brought to a final volume of 0.5 ml with PBS,
before bioassay. Three mice were used for each sample. In addition, two controls
were included; one has the 25 ul STa mixed with similar volumes of PBS instead of
the tested sera and the 2"d control had 25 ul STa mixed with similar volume of base
line serum of the corresponding rabbit. All samples were treated similarly.

Neutralization end titer of tested serum is defined as the highest serum
dilution that neutralized one mouse unit of STa. Neutralization capacity is defined as
the total mouse units of STa that were neutralized per one ml serum. Neutralization
specific activity is defined as the total mouse units of STa that were neutralized per

one mg serum protein.

106

Kinetics of the Rabbit Immune Response to ETEC STa
Antibody-capture ELISA for screening sera

An indirect antibody-capture ELISA in which STa antigen was bound to a
solid phase and reaction with antibody-containing samples was allowed (Figure 20) in
order to monitor the presence of STa antibodies in rabbit sera as described under
ELISA protocol (Lefltovits, 1997).

The ELISA plates were coated with 2.5 ug STa a 100 pl 0.05M carbonate
buffer, pH 9.6 (plate coating buffer) and incubated overnight at 4°C. Plates were
washed four times with 0.01M PBS-0.05% Tween-20 and blotted dry. One hundred
ul of 0.5% cold fish gelatin- 0.01M PBS-0.1%Tween-20 (blocking buffer) was added
to each well to block nonspecific binding sites on the plastic surface and incubated at
37°C for 30 min. Plates were then washed with 0.01M PBS-0.1%Tween-20 (washing
buffer) and blotted. Serum samples collected from all rabbits over the period of
immunization were screened at ten-fold dilution with PBS-0.1% Tween-20. One
hundred pl of each serum dilution (103, 104, 105, & 10°) was added to each well, in
triplicate, and incubated at 37°C for 45 minutes. After four washings and blotting, 100
pl of lOOO-fold diluted alkaline phosphatase-conjugated goat anti-rabbit IgG
antibodies (1 til/ml) was added to each well, incubated at 37°C for 45 min. Plates
were washed as previously described and 100 pl of freshly prepared substrate
solution (one tablet set of p-nitrophenyl phosphate (pNPP/PBS) / 5m] in 0.1M
diethanolamine buffer, pH 9.8) was added to each well. The reaction was allowed to
develop for 30 minutes at 37°C. The developed color was read at 405 nm with an

ELISA plate reader (Molecular Devices “ThemoMax” Microplate Reader with SOFT

107

 

max Pro 2.6.1). Reciprocal value of the maximal dilution of serum that had a mean
plus 2 Standard Deviations (§+ 2 SD) O.D value or higher than the OD of the

baseline serum sample, was reported as antibody end titer for each tested serum
sample.
Avidity ELISA

Avidity (Functional Affinity) is the measure of the overall strength of
interaction of an antigen with many antigenic determinants with multivalent
antibodies (Goldblatt, 2001). In this study, the STa-antibody avidity was measured by
comparing the amount of antibodies that could bind the STa antigen in the presence
or absence of increasing concentrations of a chaotropic agent. In this test, a mild
chaotropic agents, ammonium thiocyanate, was added in increasing molar
concentrations to the antibody-antigen mixture. Antibodies of low avidity are more
likely to dissociate from antigen-antibody complexes than those of higher avidity as
described by Ferreira & Katzin (1995).

The procedure was performed in similar steps to the ELISA protocol
described above. However, after the final incubation of the tested sera, four washings
and blotting, 100 pl of three different molar concentrations of ammonium thiocyanate
in PBS (5M, 2.5M, and 1.25M) were added to each well, in duplicates, incubated at
37°C for 15 minutes. Plates were processed then as described under the ELISA
protocol. Avidity index was calculated as a percentage of the OD of the sample with
different concentrations of ammonium thiocyanate and the OD of the sample without
treatment. Serum samples obtained from rabbits before the initial immunization were

used as baseline controls.

108

Statistical analysis
Generated ELISA OD data were adjusted and subjected to statistical analysis for the
calculation of the mean and standard deviation. Plotting of the ELISA OD data were
performed using Microsoft Excel (2003).
M“;
Characterization of serum antibody response in STa-conjugate-immunized
rabbits

Sera from immunized rabbits were tested for STa-serum neutralization
capacity as described under the methods section. Tested sera from three rabbits
showed a positive neutralization capacity in suckling mouse assay (gut weight to
body weight ratio = 0.06 d: 0.001) (Figure 18) and up to 3 x104 mouse units of STa
could be completely neutralized by one ml ”of serum (Figure 19). Neutralizing
antibodies were first detected among these rabbits after 12 weeks post immunization
(4th immunization). These 3 rabbits were grouped retrospectively as “group one”
based on the onset of the detectable neutralizing ST-antibody titers. At week l7-post
immunization, another three rabbits had a detectable neutralization titer. These rabbits
were grouped as “group 2”. At week twenty-four, two more rabbits had mild
neutralization capacity against E. coli STa. These rabbits were grouped as “group 3”.
Figure 20 shows E. coli STa-specific serum antibody neutralization bioassay from all
rabbits.
Binding activity

Indirect ELISA was used to determine the binding capacity of anti-STa IgG.

Figures 21-29 shows the results of binding data for sera from seven bleedings of all

109

 

rabbits that were giving antibodies against STa at various serum dilutions (103-10°).
The 10‘4 dilution was recognized as the optimal dilution for screening the tested sera
for STa-binding capacity Figure 21 (equivalence point of antigen antibody
interaction).

Three weeks after the primary immunization, detectable neutralizing IgG-
antibodies against STa were not observed. However, based on ELISA assay, slight
binding titers were detected. Higher binding antibody titers were only detected 12
weeks post immunization (Figure 22). Other three rabbits showed late immune
response at week 17 post immunization (Figure 23), however higher antibody binding
titers (10°) and STa-neutralizing antibodies (3 x 104 MU/ml serum) were then
detected. By 24 weeks post immunization, other two rabbits mounted mild
neutralization and binding titer against E. coli STa (Figure 24). Figures 25-29 shows
the end titer of E. coli STa-specific serum antibody. Mean OD i SD values of STa-
specific sermn antibody end titer of group 1, 2 and 3 rabbits after 24-week post
immunization at various serum dilutions are shown in Table 11. Summary of STa-
ELISA binding and neutralization end titers of rabbit sera immunized with STa-
suBSA conjugate after the primary immunization and during the boosting intervals
are presented in Table 12.

STa- binding avidity of the rabbit immune sera

The dissociation effect of ammonium thiocyanate on the STa-binding to its
specific antibodies was demonstrated. The dissociation effect of the chaotropic agent
on the STa-antibody binding using sera from a rabbit with the highest neutralizing

antibody titer, is shown in Figure 30. Sera from other rabbits demonstrated similar

110

 

avidity patterns. The figure shows the time course evaluation of STa-IgG avidity
using ammonium thiocyanate dose response. The increasing molar concentration of
ammonium thiocyanate (1.25 M-5 M) needed to dissociate STa-IgG complex is
depicted suggesting that the strength of the STa-IgG avidity developed gradually after
multiple boosters with the STa-conjugate. A 5 M concentration of the chaotropic
agent was determined to be the appropriate cutoff point in order to demonstrate the
strength of the avidity of STa-specific serum antibodies. Mean values of the ODs of
serum ELISA for the 3 groups of rabbits assayed using 5 M of the ammonium
thiocyanate are depicted in Figure 31 and summarized in Table 13. It is noted that
there is some variation in the patterns of dissociation of the STa- antibodies from sera
of the 3 groups of rabbits, which are largely corresponding to the STa-neutralization
and binding titers established for these sera. .

The avidity index which is calculated by dividing the OD of the sera in the
binding ELISA with 5 M of chaotrpic agent by the OD of the ELISA binding result
for the same sera without treatment with the chaotrpic agent (Figure 32 & Table 13).
By week 24-post immunization, sera from all 3 groups of rabbits demonstrated
variable avidity indices. It was noted that sera from the first group of rabbits had the
highest avidity index, which is associated with the high STa-neutralization and
binding titers demonstrated for these sera.

Discussion

Construction of immunogenic ETEC STa has been reported by several

investigators who used different chemical coupling protocols to link the STa to carrier

proteins as well as the genetic expression of the STa with flagellin as a fusion protein

111

(Houghten et al. 1984, 1985; Sanchez et a1. 1986, 1988; Clements, 1990; Klipstein et
a1. 1982, 1983 and Pereira et al. 2001). However, limited success in producing high
titers STa antisera was reported in those studies. This could be attributed to the
uncontrolled cross-linking process of the STa to the carrier proteins which led to the
reduction or loss of the STa biological activity as a part of the conjugate (Pereira et al.
2001). An important objective of this study was to design and characterize an
effective immunogenic STa conjugate using the major different peptide-carrier
conjugation protocols. Based on the evaluation of four different conjugation
procedures, a well-defined STa conjugate with high STa biological activity was
produced.

Immunization of ten rabbits with this STa-conjugate led to the production of
STa-specific neutralizing antibodies by eight animals. The STa-neutralization and
specific binding titers of these sera were higher than previously reported by other
investigators (Alderete and Robertson 1978; Frantz and Robertson 1981; Lockwood
and Robertson 1984; Lowenadler et a1. 1991 and Pereira et a1 2001). However some
variations in the onset and quality of the immune responses were noticed. Two rabbits
demonstrated weaker STa neutralizing titer 20 weeks post immunization. It is not
fully understood why some rabbits differed in their immune response to the STa
immunogen. Such individual variations may worth additional investigation in future
studies. Comparing data on the STa binding and STa neutralization titers of the sera
produced in this study with data from previous reports is presented in Table 14.

Measurement of the avidity of the STa-antibodies using a chaotropic agent,

ammonium thiocyanate at several increasing molar concentrations suggested that the

112

avidity of the STa neutralizing antibodies improved throughout the series of boosters
administered to the rabbits. Comparison of the avidity of the serum antibodies
demonstrated that the strength in the STa antibody avidity developed in time
corresponding to the development of the STa-neutralizing and ELISA binding titers
of the tested sera. This is consistent with the common knowledge about maturation of
antibodies after immunization and continuous boosting protocols (Goldblatt 2001).

In summary, we have described the design of a highly defined STa-conjugate
and its use in the induction of high STa neutralization and ELISA binding serum titers
in immunized rabbits. The carefully designed STa-conjugate and the produced high
STa-neutralizing serum antibodies can be evaluated for the design of effective
vaccine and / or immunotherapeutic reagents against the STa-producing E. coli strains

that are associated with a significant proportion of diarrheal disease worldwide.

113

55m 0336 ages/V .2 2%;

 

2.3 oi as :8 a? House
BmEEEEXaEOEofL moo_>oQ
5823.0 02.2 <95 3.. £5

’ Jfl 4 = i 3558 3 How Do mm a 08383 05
I; 40 4 088:0:0 :95 some 8 0000a 83 9.2

 

 

Ed mow pm ~28 0002050 20 08m , H. . .. - M.0 C 83% 0836 28.5002: 00_
.EE .

2 0.. 2 a 8:385 23 :03 be
some 8 000?». we? 3&va 20028 8 3:20 05 8-523. Ax..n..0.0
88033 023on £030 .8 E 00_ -mmm E 00.0 53> 3:5 Bow :3?

b0 32% 33 Cuban

wE 00 -503 o . - .

ow 0883 98 3-525. $8.0 .x Law—WW 0mm WWW. mewmw “flow

-mmm 320.0 0:3 88: 58 fig? .
.9

58 we 50 00 mm as 02335 b0 8 3320

£25 :08 8 825 mm? 03 ESE can 8-523. $3.0-me

-55 “wow cowmwsfiooomfimammonm $20.0 H33 880 500 :33

 

2E8?“ 085:0 28-003031 00“
P. v

“a images £3 28 Gowns 9:88
823 0.0 mm .5003 828080
28.0 E a: 84.9221 2:
55 0880 mm? Baa «aim 2:.

be
8 cocoa 05 8-593 $8.0
.20 5:00 £5 380 :50 :83

 

 

 

 

114

 

STa-IgG neutralization

 

0.140
0.120
0.100
0.080
0.060
0.040
0.020
0.000

GWIRBW

 

wk1 wk3 wk6 wk12 wk17 wk20 wk24
Week Post immnization

 

 

 

 

Figure 18. E. coli STa-specific serum antibody neutralization bioassay: Group 1
(rabbits. 1, 2, and 3). Straight horizontal line at 0.083 Gut weight: remaining body
weight ratio, signifies the cut off value for STa- positive SAM.

 

 

Neutrallztion Capacity of IgG
100000
10000 ‘

i

w 1000
‘8
a

g 100
O
z

10

0 5 10 15 20 25 30
weeks post Immunization

 

 

 

Figure 19. Neutralization capacity of E. coli STa-specific serum antibody. Points represent
the mean of neutralization titers from sera of 3 rabbits

115

 

STa-IgG neutralization bioassay

0.16

0.14
-+- Rabbil#1

_._ Rabbit# 2
_.— Rabbit#3

   
 
 
 
 
  
  
 

0.12

3 °-1 —-— Rabbit#4
g o 08 _._ Rabbi1# 5
E V j + Rabbit#6

+ Rabbit#7
' 7 —-— Rabbit# 8
—I- RabbiflfiQ
—I-— Rabbit#10

 

 

 
   

wk1 wk 12 wk 17 wk 20 wk 24

weeks postinmunization

 

 

 

Figure 20. E. coli STa-specific serum antibody neutralization bioassay: All rabbits

Note: Three rabbits showed STa neutralizing antibodies by 12 weeks, another 3 rabbits
showed STa neutralizing antibodies by 17 weeks and finally 2 rabbits started to show

STa neutralizing antibodies by 20 weeks post immunization
GW= Gut weight, RBW= Remaining body weight

116

 

 

Ablnecxess

OD405nm

 

10‘3 10‘4 10‘5 10‘6

Reciprocal serum dilution

 

 

Figure 21. E. coli STa-binding ELISA optimization: screening serum dilution for
optimal E .coli STa-STa antibody interaction

 

ST a-igG Ending Assay
10M serum dilution

  
  

 

    

&seine wk3 wk6 wk 12 wki5 wk 17 wk20 wk24
weeks Fl

 

 

 

Figure 22. E. coli STa-specific serum antibody: 10‘4 serum dilution of group 1
rabbits

117

 

—I— Rabbiw 4

+ Rabbi1# 5
+ Rabbii# 6

OD 405 nm

 

Baseline wk 3 wk 6 wk 12 wk 17 wk 20 wk 24
Weeks Pl

 

 

 

Figure 23. E. coli STa-specific serum antibody: ‘10'4 serum dilution of group 2 rabbits

 

2.500

+ Rabbiflf- 7
+ Rabbiflf 8

2 .000

     

 

1 .500

1.000

OD 405 nm

0.500

    

0.000

Baseline wk 3 wk 6 wk 12 wk 17 wk 20 wk 24

weeks Pi

 

 

 

Figure 24. E. coli STa-specific serum antibody: 10'4 serum dilution of group 3 rabbits

118

 

 

   

 

 

 

 

 

 

 

 

 

 

 

i
E
; 2.500 —
1 +61 PI Rabbit
2.000 - +G1 Baseline
2 1.500 —
In
0
v
o _
O 1.000
0.500 —
0.000 a . . . . . J .
10"3 10M 10"5 10"6
Reciprocal of serum Dilution

Figure 25. Mean OD value of group 1 rabbits after 20 weeks post immunization at
various serum dilutions

 

 

 

 

 

 

 

 

 

2.5 .
+G2 Rabbit
2 +G2 Baseline
E 1.5 —
In
O
<-
0 -
O 1
0.5 -
0 , . I 14"".
10"3 10M 10"5 10"6
Reciprocal of serum dilution

 

 

 

Figure 26. Mean OD value of group 2 rabbits after 20 weeks post immunization at
various serum dilutions

119

 

 

+63 Pl Rabbits
2.000 - +63 Baseline
g 1.500 -
8 1.000 -
0.500 —
0.000 K t, =

2.500 -

 

 

10M 10"5
Reciprocal of serum Dilution

10‘6

 

 

Figure 27. Mean OD value of group 3_ rabbits afier 24 weeks post
immunization at various serum dilutions

 

 

OD 405 nm

2.500

2.000

.5
0'
o
0

§

0.500

0.000
10"3

 

10"4 10"5
Reciprocal of Serum Dilution

 

-I— Rabbit# 1
—¢-— Rabbil# 2
-l- Rabbit# 3
—l- Rabbifll 4
-I— Rabbit# 5
+ Rabbi’di 6
--—- Rabbit# 7
-—l— Rabbit# 8

 

 

10‘6

 

Figure 28. End titer of E. coli STa- specific serum antibody: 24 weeks post
immunization from 8 rabbits

120

 

 

 

2.500 —

 

 

 

 

   

 

 

2 000 + Group 1
' q A + Group 2
+Group 3
+ Baseline
E 1.500 ~
C
In
0
V
o
o 1.000 .
0.500 -
0.000 . . T . . T a
10"3 10M 10"5 10"6

Reciprocal of Serum Dilution

 

Figure 29. E. coli STa-specific serum antibody end titer: Mean OD value of group
1, 2 and 3 rabbits after 24 weeks post immunization at various serum dilutions

121

 

 

 

OD 405 nm

2.5

    

+ umtreated

0 3 6 12 17 24
weeks postimmunization

 

Figure 30. Time-course evaluation of the avidity of E. coli STa-specific serum
antibody using ammonium thiocyanate dose response

122

 

 

 

 

 

OD 405 nm

 

0 3 6 17 24

weeks postimmunization

Figure 31. Five molar thiocyanate elution profile of E. coli STa- STa serum
antibody complex: Mean OD of treated serum from group 1, 2 and 3 of rabbits

 

Avidity index of three groups

 

0 3 6 17 24

weeks post im m unlzation

 

 

Figure 32. Avidity index of E. coli STa-specific serum antibody from
group 1, 2 and 3 of rabbits using avidity ELISA procedure

123

Table 11: E. coli STa-specific serum antibody end titer: Mean OD 1: SD value of
group 1, 2 and 3 rabbits after 24— week post immunization at various serum dilutions

 

 

 

 

 

 

 

 

 

Grow/serum 10'3 10“ 10'5 10“
dilution
G1 1.933 :t 1.923 :t 1.237 i 0.379 :1:
0.013 0.014 0.568 0.352
G2 1.964 t 1.969 :1: 1.550 :t 0.374 :t
0.035 0.031 0.010 0.173
G3 1.936 1 1.656 :1: 0.354 d: 0.040 :t
0.006 0.035 0.015 0.010
Baseline 0.864 :1: 0.232 d: 0.033 :t 0.039 :1:
0.318 0.155 0.048 0.060

 

 

 

Table 12. Summary of STa- ELISA binding and neutralization end titers of rabbit sera
immunized with STa-suBSA conjugate after the primary immunization and during the
boosting intervals. Data were generated by STa binding ELISA and STa

neutralization using suckling mouse assay.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Anti-STa Response Neutralization
Blee din Protein Specific
g ELISA Neutralized Assay Activity
Titer STa MU/ml mg/ml
serum (Titerl

Baseline 0 0 64.5 -
Week 3 PI 0 0 66.8 -
Week 6 PI 0 0 65.7 -
Week 12 PI 10,000 2000 83.5 23.95
Week 15 PI 100,000 15,000 69.6 215.52
Week 20 PI 1,000,000 20,000 65.00 307.69
Week 24 PI 1,000,000 20,000 65.00 307.69
Week 28 PI 1,000,000 30,000 65.00 461.54

 

124

 

Table 13. Summary of the development of STa antibody avidity after multiple
boosters with the STa conjugate using 5 M ammonium thiocyanate ELISA
dissociation assay.

 

 

 

 

 

 

 

 

 

 

 

Baseline Week 3 Week 6 1273‘ Week 24
Avidity Index (AI) % afier measurement OD at 405 nm

Gm 1 0.017 0.043 0.001 0.578 0.927
P (4.70%) (8.91%) (0.03%) (25.94%) (48.21%)

Gm 2 0.043 0 0.022 0.027 0.762
P (3%) (0%) (2.24%) (1.53%) (38.71%)

Gm“ 3 0.04 0 0.095 0.151 0.352
P (0%) (0%) (1 1.36%) (12.05%) (21.30%)

 

 

 

 

 

Table 14. Neutralization capacity of sera from animals immunized with several STa-
immuogenes and the end titers of the STa-neutralizing antibodies as reported in the
literature.

 

 

 

 

 

 

 

 

 

 

 

 

 

. Neutralization
Max1mum Amount of .
References neutralization titer ‘ SMU cap acity
Total MU/ml serum
Liiwenadler et al. .
1991 1.55 30 1665
Lockwood and ,
Robertson 1984 1'100 8 800
Frantz and , .
Robertson 1981 1.5000 to l.10,000 1 5000 to 10,000
Alderete and _ -
Robertson 1978 1'2’300 1 2’500
1:50 for
immunogene using 1 50
. native STa fusion
Pereira et al. protein
2001 1:4000 using
mutated STa fusion 1 4,000
protein
This study 1:30.000 1 30,000

 

125

 

References

Alderete J .F. and Robertson, DC. 1978. Purification and chemical characterization of
the heat-stable enterotoxin produced by porcine strains of enterotoxigenic
Escherichia coli. Infection and Immunity 19: 1021-1030.

Boedeker, E. C. 2005. Vaccines for enterotoxigenic Escherichia coli: current status.
Current Opinion in Gastroenterology. 21 (1):] 5-1 9.

Cardenas, L and Clements, J .D. 1993. Development of mucosal protection against the
heat-stable enterotoxin (ST) of Escherichia coli by oral immunization with a
genetic fusion delivered by a bacterial vector. Infect Immun. 61: 4629—463 6.

Clements, J. D. 1990. Construction of a nontoxic fusion peptide for immunization
against Escherichia coli strains that produce heat-labile and heat-stable
enterotoxins. Infect Immun 5 8, 1 159-1 1 66.

Ferreira, M.U. and Katzin, A.M. 1995. The assessment of antibody distribution by
hocyanate elution: a simple dose-response approach. Journal of
Immunological Methods 1 8 7:29 7-3 05.

Frantz, J. C and Robertson, DC. 1981. Immunological properties of Escherichia coli
heat-stable enterotoxins: development of a radioimmunoassay specific for
heat- stable enterotoxins with suckling mouse activity. Infection and

Immunity. 33: 193-198.

Frantz, J .C; Bhatnagar, P.K; Brown, A.L; Garrett, L.K.and Hughes, J .L. 1987.
Investigation of synthetic Escherichia coli heat-stable enterotoxin as an
immunogen for swine and cattle. Infection and Immunity. 55: 1077—1084.

Goldblatt, D. 2001. Affinity of antigen-antibody interactions. Encyclopedia of Life
Sceinences. John Wiley &Sons, Ltd. [-5

Gordon, L.K. 1981. A reliable method for repetitively bleeding rabbits from the
central artery of the ear. Journal of immunological methods, 44: 241 -24.

Gyles, C. L. (1996). In Escherichia coli in domestic animals and humans. pp. 31-72
CAB International. Wallingford. UK.

Houghten, R. A., Engert, R. F., Ostresh, J. M., Hoffman, S. R. & Klipstein, F. A.
1985. A completely synthetic toxoid vaccine containing Escherichia coli heat-
stable toxin and antigenic determinants of the heat-labile toxin B subunit.
Infect Immun 48, 735- 740.

126

Houghten, R. A., Ostresh, J. M. & Klipstein, F. A. 1984. Chemical synthesis of an
octadecapeptide with the biological and immunological properties of human
heat-stable Escherichia coli enterotoxin. Eur J Biochem 145, 157-162.

Klipstein, F. A., Engert, R. F. & Clements, J. D. 1982. Development of a vaccine of
cross-linked heat-stable and heat-labile enterotoxins that protects against
Escherichia coli producing either enterotoxin. Infect Immun 3 7, 55 0-55 7.

Klipstein, F. A., Engert, R. F ., Clements, J. D. & Houghten, R. A.1983. Vaccine for
enterotoxigenic Escherichia coli based on synthetic heat-stable toxin crossed-
linked to the B subunit of heat-labile toxin. J Infect Dis 147, 318-326.

Kosek, M., Bern, C., Guerrant, KL 2003. The magnitude of global burden of
diarrhoea] disease from studies published 1992-2000. Bulletin of the World
Health Organization, 81: p19 7-204.

Lefl<ovits, I. 1997. Immunology methods manual. Vol 4. Academic Press. Harcout
Brace & Company, Publishers. San Diego, London, Boston, New York,
Sydney, Tokyo, Toronto.

Lockwood, DE. and Robertson, D.C.1984. Development of a competitive enzyme-
linked immunosorobent assay (ELISA) for Escherichia coli heat-stable
enterotoxin (STa). Journal of Immunological Methods, 75: 295-307.

Lowenadler, B. Lake M. Elmblad A. Holmgren E. Holmgren J. Karlstrom A and
Svennerholm, A-M. 1991. A recombinant Escherichia coli heat-stable
enterotoxiin (STa) fusion protein eliciting anti-STa neutralizing antibodies.
FEMS Microbiology Letters, 82: 271 -2 78.

Pereira, C.M., Guth, B.E.C., Aleida, M.E. and Castilho, BA. 2001. Antibody
response against Escherichia coli heat-stable enterotoxin expressed as fusions
to flagellin. Microbiology, 147, 861-86.

Sanchez, J., Svennerholm, A-M. & Holmgren, J. 1988. Genetic fusion of a non-toxic
heat-stable enterotoxin-related decapeptide antigen to cholera toxin B-subunit.

FEBS Lett 241, 110-114.
Sanchez, J ., Uhlin, B. E., Grundstrom, T., Holmgren, J. & Hirst, T. R. 1986.
Immunoactive chimeric ST-LT enterotoxins of Escherichia coli generated by

in vitro gene fusion. FEBS Lett 208, 194-198

Svennerholm, A. Steele D. 2004. Progress in enteric vaccine development. Best
Practice & Research Clinical Gastroenterolog, 18: 421 — 445.

Vaitukaitis, J .L.1981. Production of antisera with small doses of Immunogen:
Multiple intraderrnal injections. Methods in Enzymology 73: 46-52.

127

Walker, R.I; Steele, D; Aguado, T. and Ad Hoc ETEC Technical Expert Committee
2007. Analysis of strategies to successfully vaccinate infants in developing
countries against ETEC disease. Vaccine 25, 2545-2566.

128

 

SUMMARY AND CONCLUSIONS

Enterotoxigenic strains of E. coli (ETEC) that produce heat-stable enterotoxin
(STa) are a major cause of diarrheal disease in humans and animals. Economically,
ETEC is among the important causes of diarrheal diseases in animals, including
neonatal calves, pigs and lambs. Additionally, ETEC strains have been recognized to
cause a significant proportion of “travelers” diarrhea. Currently, there are no effective
vaccines or immunotherapeutic reagents that confer broad protection against STa-
induced diarrheal disease. This is largely due to the antigenic diversity of surface
antigens and the nonimmunogenic property of STa, which is the immediate mediator
of ETEC diarrhea. To reduce the impact of STa-caused diarrhea, it was logical
therefore to investigate the feasibility of designing an immunogenic STa conjugate to
produce immunotherapeutic reagents and /i or potential vaccine to combat the
diarrheal disease caused by these enteropathogens.

This study was based on the hypothesis that the design of an effective STa
immunogen is feasible, if based on the understanding of its molecular structure and
the preservation of its bioactive configuration. This study has supported the stated
hypothesis through the achievement of the objectives listed below.

1. Production of purified STa from Enterotoxigenic Escherichia coli

STa was purified to homogeneity and its purity was documented using amino
acid sequence analysis, and MALDI-TOF mass spectroscopy. The minimal effective
dose of the purified STa was less than 0.2 ng per one Mouse Unit using 2-3 day-old

inoculated Swiss Webster suckling mice.

129

2. Design and evaluation of immunogenic Enterotoxigenic Escherichia coli STa

Conjugation of a STa -carrier has been reported. However, no sufficient
details were presented on the efficiency and the characteristics of the produced STa
conjugates. An important objective of this study was to design and characterize an
effective immunogenic STa conjugate using the major different peptide-carrier
conjugation protocols. Based on the evaluation of four different conjugation
procedures, a well defined STa conjugate with high STa biological activity was
produced. We confirmed the stability of the molecular structure of this conjugate
using amino acid compositional analysis and MALDI-TOF mass spectroscopy.

3. Immunization of rabbits and the production of high titers of STa neutralizing
and specific ELISA binding serum antibodies.

Numerous attempts have been made to render STa immunogenic, including
chemical coupling to carrier proteins and genetically produced fusions proteins.
However, only limited success was reported for the immunogenic potency of the STa-
conjugates and the quality of the STa-specific antibody titers produced in immunized
laboratory animals. We have used the produced STa-conjugate to immunize a group
of New Zealand albino rabbits. The immunized rabbits produced higher STa-
neutralizing serum titers than reported in previous studies within 15 weeks post
immunization, indicating the effective immunogenicity of the designed STa-
conjugate. Additionally, the kinetics of immune response of the rabbits to this
conjugate, the STa-neutralization capacity and the quality of the antibodies of the
produced sera, were characterized. Results of this study validated the value of the

designed STa-suBSA conjugate as a potent immunogen.

130

 

Strength of the study

Previously reported STa conjugation protocols did not consider the
preservation of the STa biological activity in the conjugation process which,
when used for immunization of laboratory animals, may have led to
suboptimal quality of the produced STa antibodies. This study was based on
thorough understanding of the molecular structure of the STa that was
considered in the planning of the conjugation process of the STa to modified
BSA. This approach ensured the effective crosslinking of the STa through its
amino terminal end and thus had preserved its biologically active moiety.

Additionally, the conjugation design may have enhanced the presentation of
STa on the BSA carrier that was modified to have longer reactive groups for
crosslinking with the STa peptide. This was demonstrated by the high
retention of the STa biological activity in the STa conjugate using the suckling
mouse assay, prior to the immunization process. Finally, the produced STa
conjugate was proved to be highly immunogenic as it elicited higher STa
neutralizing antibody titers in immunized rabbits than previously reported by

other investigators.

Limitations

The yield of STa from one batch of ETEC culture is not sufficient to enable
extensive experimentation and evaluation of several methods for optimized
protocols to produce the desired STa conjugates. Therefore, numerous batches

of cultures were required.

131

We have evaluated the crosslinking protocols that were reported for several
synthetic peptides to carrier proteins. Those peptides were not structurally
similar to the STa peptide. Therefore, we had to use large amounts of the
precious STa to optimize the conjugation methods. Several initial attempts
were not successful.

The use of the produced STa conjugate was only evaluated in rabbits. Its use
in immunizing other animal species could have further revealed its potential

as a possible vaccine against ETEC-induced diarrhea.

132

3 1293 03063 049

 

III
I"
u
u
"I
"I
V
III"
.1“
N
u
H
'l”
'11“