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UTILISING SALMONELLA TO DELIVER
HETROLOGOUS VACCINE ANTIGEN
A THESIS SUBMITTED IN TOTAL FULFILLMENT OF THE REQUIREMENT
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
MANVENDRA SAXENA
M.Sc
School of Applied Sciences Science, Engineering and Technology Portfolio
RMIT University 2007
Declaration
I certify that except where due acknowledgement has been made, the work is
that of the author alone; the work has not been submitted previously, in whole
or in part, to qualify for any other academic award; the content of the thesis is
the result of work which has been carried out since the official
commencement date of the approved research program; and, any editorial
work, paid or unpaid, carried out by a third party is acknowledged.
...........................................................................
Manvendra Saxena
...........................................................................
ii
Acknowledgement
I would like to thank, first and foremost my supervisors, Dr. Peter Smooker
and Professor Peter Coloe. Your guidance, advice, encouragement and moral
support throughout my PhD have been invaluable and memorable. I greatly
appreciate that you really “work hard for me” on the correction of my thesis.
My sincere gratitude to you for making my PhD study becomes possible.
Thanks to my RMIT Biotech lab colleagues Hao, Luke, Fiona, Emily, Natalie,
Viraj, Amber and Tagrid for your helpful discussion, valuable friendship, and
humor. Sincere thanks to Hao, Fiona, Luke, Emily and Natalie for proof
reading of my thesis chapters. Special thanks to Kathryn from ecotoxicology
for helping me with SPSS. Many thanks to Ms.Megan Selman for proof-
reading part of my thesis.
Special gratitude to my beloved wife Ripa for her love and support and being
always beside me during my hard times. Special thanks to my father and
mother who sacrificed through out their life for this special day. Thanks to my
brothers and parent in laws for their unconditional love and support.
Thanks to RMIT International for providing me financial support through RUIS
scholarship.
iii
TABLE OF CONTENTS
List of abbreviations .....................................................................xiii
List of tables .................................................................................xvi
List of figures ...............................................................................xvii
Summary ........................................................................................1
Chapter-1- Literature Review .........................................................5
1.1 Introduction.............................................................................................5
1.2 Types of Vaccines ..................................................................................8
1.3 Killed organisms .................................................................................8
1.4 Purified microbial component vaccines ................................................11
1.5 Synthetic peptide Vaccines ..................................................................11
1.6 Subunit vaccines ..................................................................................11
1.7 DNA vaccines.......................................................................................12
1.8 Live attenuated vaccines......................................................................12
1.9 Salmonella as a candidate ...................................................................15
1.9.1 Pathogenesis of Salmonella ..........................................................16
1.9.2 Immune response against Salmonella ...........................................17
1.9.2.1 Role of Antigen presenting cells in Salmonella immunity ........17
1.9.2.2 T cell responses ......................................................................19
1.9.2.3 Antigen cross presentation......................................................21
1.9.2.4 B cell response........................................................................24
1.9.2.5 Immune responses against a passenger antigen....................25
iv
1.9.2.5.1 Cellular immune responses against heterologous vaccine
antigen.............................................................................................25
1.9.2.5.2 Humoral Immune responses against heterologous vaccine
antigen.............................................................................................26
1.10 Salmonella Typhimurium Mutants ......................................................27
1.10.1 galE Mutants................................................................................27
1.10.2 aro and pur mutants.....................................................................29
1.10.3 Cyclic AMP regulation Mutants ....................................................33
1.10.4 phoP and ompR mutants .............................................................34
1.10.5 Plasmid-cured strains ..................................................................35
1.10.6 Streptomycin-Dependent Mutants ...............................................35
1.10.7 Aspartate Semialdehyde Dehydrogenase Mutants......................36
1.10.8 Temperature-Sensitive Mutants...................................................37
1.10.9 Attenuated Salmonella Strains and their utility.............................37
1.11 Factors that influence the use of Salmonella as a vector to deliver
heterologous antigen..................................................................................40
1.11.1 Stabilisation of antigen expression in attenuated Salmonella strain
................................................................................................................40
1.11.1.1 Use of in vivo inducible promoters.........................................41
1.11.1.2 Chromosomal integration of genes encoding antigenic protein
............................................................................................................42
1.11.2 Effect of preexisting immune response against the bacterial vector
................................................................................................................46
1.12 Conclusion..........................................................................................48
v
Chapter 2 -Materials and Methods ...............................................50
2.1 General Procedures .............................................................................50
2.2 General Chemicals and Equipment......................................................50
2.3 Bacteriological Methods .......................................................................58
2.3.1 Media and Antibiotics.....................................................................58
2.3.1.1 Antibiotic stock solutions .........................................................58
2.3.1.2 Luria Bertoni Agar ...................................................................59
2.3.1.3 Luria Bertoni Broth ..................................................................59
2.3.1.4 Muller Hinton Agar...................................................................59
2.3.1.5 Muller Hinton Broth..................................................................59
2.3.1.6 Horse Blood Agar ....................................................................59
2.3.2 Bacterial Strains and Plasmids ......................................................60
Genotype or description ......................................................................60
Reference/Source ...............................................................................60
2.3.3 Storage of Bacterial Strains and Phage.........................................65
2.3.4 Bacterial Culture Conditions ..........................................................65
2.4 DNA Methods. ......................................................................................65
2.4.1 DNA Extraction Techniques...........................................................65
2.4.1.1 Buffers and Reagents..............................................................65
2.4.1.2 Small Scale Plasmid DNA Extraction Using Qiaprep Mini Spin
Kit ........................................................................................................70
2.4.1.3 Alkaline Lysis Miniprep............................................................71
2.4.1.4 Small-Scale Genomic DNA Extraction ....................................72
2.4.1.5 Large-Scale Genomic DNA Extraction ....................................72
2.4.1.6 Ethanol precipitation................................................................74
vi
2.4.1.7 Purification of DNA from agarose............................................74
2.4.1.8 Purification of PCR amplified DNA ..........................................75
2.4.1.9 Determination of DNA concentration .......................................75
2.4.2 Polymerase chain Reaction. ..........................................................76
2.4.2.1 Primer Design. ........................................................................76
2.4.2.2 General PCR procedure..........................................................76
2.2.2.3 Expand Long Template PCR...................................................77
2.4.2.4 DNA sequence analysis. .........................................................78
2.4.3 Recombinant DNA techniques.......................................................83
2.4.3.1 Cloning of PCR product...........................................................83
2.4.3.2 Restriction Enzyme Digestion .................................................83
2.4.3.3 Phophatase Treatment of vectors ...........................................85
2.4.3.4 Ligation of DNA. ......................................................................86
2.4.3.5 Transformation of DNA............................................................86
2.4.3.5.1 Preparation of Competent Cells........................................86
2.4.3.5.2 Electroporation of Competent Cells ..................................87
2.4.3.5.3 Chemical Transformation..................................................87
2.4.3.6 Chromosomal Integration of Constructs. .................................88
2.4.3.6.1 Lambda Red System ........................................................88
2.4.3.7 Pulsed Field Gel Electrophoresis. ...........................................89
2.4.3.8 Southern Blotting.....................................................................90
2.4.3.8.1 Probe Synthesis................................................................90
2.4.3.8.2 Transfer of DNA to the Nylon Membrane..........................91
2.4.3.8.3 DNA Hybridisation ............................................................92
2.4.3.8.4 Washing and Development of Membrane.........................93
vii
2.5 Protein Methods ...................................................................................94
2.5.1 Buffer and Solutions ......................................................................94
2.5.2 Protein Extraction Method..............................................................96
2.5.2.1 Sonication. ..............................................................................96
2.5.2.2 Preparation of Whole Cell Lysates ..........................................97
2.5.3 Determination of Protein Concentration. ........................................97
2.5.3.1 Lowry Assay............................................................................97
2.5.3.2 Bradford Assay........................................................................98
2.5.4 Recombinant Protein Expression...................................................98
2.5.5 Polyacrylamide Gel Electrophoresis. .............................................99
2.5.5.1 SDS-PAGE..............................................................................99
2.5.6 Visualisation of Proteins...............................................................100
2.5.6.1 Coomassie Staining. .............................................................100
2.5.6.2 Western Blot..........................................................................101
2.5.6.2.1 Transfer of Protein to Nitrocellulose Membrane..............101
2.5.6.2.2 Development of nitrocellulose membrane.......................101
2.6 Animal Experimentation Methods.......................................................102
2.6.1 Buffers and Reagents. .................................................................102
2.6.2 Vaccination with recombinant STM-1 expressing heterologous
antigenic protein. ..................................................................................103
2.6.3 Blood collection............................................................................104
2.6.4 Preparation of Faecal pellets for ELISA. ......................................104
2.6.5 ELISA to detect antibody levels. ..................................................105
2.6.6 ELISPOT assay for cytokine producing cells. ..............................106
2.6.6.1 Collection of Spleen Cells. ....................................................106
viii
2.6.6.2 ELISPOT assay.....................................................................106
Chapter 3 - Characterisation of the STM-1 strain of Salmonella
enterica serovar Typhimurium....................................................109
3.1 Introduction.........................................................................................109
3.2 Results ...............................................................................................112
3.2.1 Amplification of PflB-aroA region .................................................112
3.2.2 Sequence analysis of amplified PCR products ............................115
3.2.3 Amplification of the serC, ycaO, ycaP, fcoA, pflB and sopD genes
..............................................................................................................118
3.2.4 Amplification of the pflB-aroA region with pflB and aroA170 bp
primers..................................................................................................121
3.2.5 Amplification of the gtrA gene ......................................................123
3.2.6 Amplification of pflb-aroA region using gtrA and aorA 170 bp
primers and pflB and gtrA primers ........................................................124
3.2.6 Amplification of pflb-aroA region using gtrA and aorA 170 bp
primers and pflB and gtrA primers ........................................................125
3.2.7 Southern Blot analysis .................................................................127
3.2.8 PFGE analysis of STM-1 genomic DNA ......................................130
3.2.9 PFGE-Southern blot analysis of STM-1 genomic DNA................132
3.2.10 Amplification of the aroA-rpsA region.........................................135
3.3 Discussion ..........................................................................................137
ix
Chapter 4 - Construction of STM-1 as a vector to deliver
heterologous vaccine antigen.....................................................142
4.1 Introduction.........................................................................................142
4.1.1 Stabilisation of antigen expression in attenuated Salmonella strain
..............................................................................................................143
4.1.2 The use of in vivo inducible promoters ........................................144
4.1.3 Integration of constructs in the chromosomal location .................145
4.2 Results ...............................................................................................149
4.2.1 Ligation of the pagC and nirB promoters into the pCR2.1 plasmid
..............................................................................................................149
4.2.2 Amplification of the C. jejuni major outer membrane protein and
chicken ovalbumin genes .....................................................................151
4.2.3 Cloning of the C. jejuni Momp and the chicken ovalbumin genes
with pagC and nirB promoter ................................................................153
4.2.4 DNA Sequence analysis of the assembled constructs.................156
4.2.5 Analysis of protein expression from the constructs by western
blotting ..................................................................................................157
4.2.6 Ligation of constructs from pCR2.1 into pMW2 vector .................159
4.2.7 Lambda Red System for the chromosomal integration of constructs
..............................................................................................................161
4.2.7.1 Ligation of Constructs into the pKD13 plasmid......................163
4.2.7.2 Amplification of the constructs with the antibiotic resistance
marker from the pKD13 vector for chromosomal integration. ............167
4.2.7.3 Integration of the PCR product into the aroA gene of STM-1 169
x
4.2.8 Analysis of in vitro protein expression..........................................171
4.3 Discussion ..........................................................................................175
Chapter 5 - In vivo immune responses induced in mice against
heterologous antigen expressed in STM-1.................................179
5.1 Introduction.........................................................................................179
5.2 Results ...............................................................................................182
5.2.1 Immunisation of mice...................................................................182
5.2.2 Analysis of humoral immune responses against the vectored
ovalbumin antigen.................................................................................184
5.2.3 Analysis of Secretory IgA response from the faecal sample ........188
5.2.4 Analysis of cell mediated immune response against chicken
ovalbumin antigen.................................................................................190
5.2.5 Analysis of humoral responses against C. jejuni Momp antigen in
vaccinated mice ....................................................................................192
5.2.5 Analysis of humoral responses against C. jejuni Momp antigen in
vaccinated mice ....................................................................................193
5.2.6 Analysis of cell mediated responses against C. jejuni Momp antigen
in vaccinated mice ................................................................................196
5.3 Discussion ..........................................................................................200
Chapter 6 - The effect of prior immunological experience against
the bacterial vector on the immune responded against a
heterologous antigen..................................................................204
xi
6.1 Introduction.........................................................................................204
6.2 Results ...............................................................................................207
6.2.1 Initial evaluation of humoral immune responses against ovalbumin.
..............................................................................................................209
6.2.2 Evaluation of humoral immune responses against the ovalbumin
antigen from individual sera..................................................................211
6.2.3 The effect of a longer time period between exposure and
vaccination with vectored antigen .........................................................213
6.2.3 The effect of a longer time period between exposure and
vaccination with vectored antigen .........................................................214
6.2.4 Effect of prior immunological experience with carrier or related
strains; cellular immune responses.......................................................217
6.3 Discussion ..........................................................................................222
Chapter 7 - Conclusion...............................................................225
Chapter-8 - References..............................................................230
xii
List of abbreviations
Amp Ampicillin
ANGIS Australian National Genomic Information Service
APC Antigen presenting cell
bp Base pairs
BSA Bovine serum albumin
°C Degrees centigrade
CFU Colony forming units
CIP Calf intestinal phophatase
CMI Cell-mediated immunity
CO2 Carbon dioxide
CTAB Hexadecyltrimethyl ammonium bromide
CTL Cytotoxic T lymphocyte
Da Dalton
DC Dendritic cell
DIG Digoxigenin
DNA Deoxyribonuclease
DNTPs Deoxynucleoside triphosphates
EDTA Ethylenediamine tetra acetic acid, disodium salt
ELISA Enzyme-linked immunosorbent assay
EtBr Ethidium bromide
FRT Flp recombination target
g Gram
g Relative centrifugal force
GALT Gut-associated lymphoid tissue
h Hour
H2O Water
H2O2 Hydrogen peroxide
HCl Hydrochloric acid
HRP Horse radish peroxidase
Ig Immunoglobulin
IFN-γ Interferon gamma
xiii
IL 4 Interleukin 4
I.P. Intra-peritoneal
IPTG Isopropyl-thiogalactoside
kb Kilobase pairs
kDa One thousand Daltons
Kan Kanamycin
LB Luria Bertani broth
LPS Lipopolysacharide
M Molarity
MCS Multiple cloning site
Mg2+ Magnesium
mg Milligram
min Minutes
mL Millilitre
mM Millimolar
MW Molecular weight
NaCl Sodium chloride
NaOH Sodium hydroxide
NCS New born calf serum
ng Nanograms
NK Natural killer cells
nM Nanomolar
OD Optical density
OMP Outer membrane protein
PAGE Polyacrylamide gel electrophoresis
PBS Phosphate buffered saline
PCR Polymerase chain reaction
pH Negative algorithm of hydrogen ion concentration R Resistance to antibiotic
RES Reticuloendothelial system
RNase Ribonuclease
rpm Revolutions per minute
s Seconds
(S) Sensitivity to antibiotic
xiv
SDS Sodium dodecyl sulphate
SPF Specific pathogen free
SPI Salmonella pathogenicity island
TEMED N,N,N’,N’-tetramethylethylenediamine
Tet Tetracycline
Tm°C Melting temperature
Tris tris (hydroxymethyl) amino methane
U units
UV Ultraviolet
UV Ultraviolet
V Voltage
X-gal 5-bromo-4-chloro-3-indoyl-b-D-galactopyranoside
λ Lambda phage DNA
µg Microgram
µL Microlitre
µm Micrometer
xv
List of tables
Table 1.1: Features of effective vaccines......................................................7
Table 1.2: Classification of common human vaccines ..................................9
Table 2.1: Bacterial strains and bacteriophage used in this study ..............60
Table 2.2: Plamids used in this study..........................................................62
Table 2.3: PCR primers used in this study..................................................80
Table 2.4: Restriction Enzymes ..................................................................85
Table 5.1: Vaccine trial #1 ........................................................................183
Table 5.2: Vaccine trial #2 ........................................................................194
Table 6.1: Protocol for the vaccine trial to test the effect of pre-existing
immunity ...................................................................................................208
xvi
List of figures
Chapter 1
Figure 1.1 Diagramatic representation of T cell response.....................23
Figure 1.2 Metabolism of galactose ......................................................28
Figure 1.3 Aromatic biosynthetic pathway ............................................30
Figure 1.4 Purine biosynthetic pathway ................................................32
Chapter 3
Figure 3.1a Circular map of the S. Typhimurium.................................113
Figure 3.1b Map of aroA region in S. Typhimurium.............................113
Figure 3.1c Amplification of the pflb-aroA region ................................114
Figure 3.2a Sequence analysis of STM-1around the aroA region ......116
Figure 3.2b S. Typhimurium sequence from location 29 at
10 minutes ........................................................................117
Figure 3.3a Amplification of the serC gene .........................................119
Figure 3.3b Amplification of the ycaP and ycaO genes.......................119
Figure 3.3c Amplification of the focA gene..........................................120
Figure 3.3d Amplification of the sopD and pflB genes ........................120
Figure 3.4 Amplification of pflB-aroA region with pflB forward and
170 bp aroA reverse primers..............................................122
Figure 3.5 Amplification of the gtrA gene ............................................124
xvii
Figure 3.6 Amplification of the pflB-aroA region with gtrA forward
and 170 bp aroA reverse primers ......................................126
Figure 3.7 Southern blots with pflB, aroA and gtrA probes .................129
Figure 3.8 PFGE with XbaI .................................................................131
Figure 3.9a PFGE with XbaI, SpeI and NotI .......................................133
Figure 3.9b Southern blot analysis with plfB probe
after PFGE with XbaI, SpeI and NotI................................134
Figure 3.10 Amplification of aroA-rpsA region with aroA
400 bp forward and rpsA reverse primers ........................136
Chapter 4
Figure 4.1 Rationale for construction of targeting constructs
and chromosomal integration of expression cassettes......148
Figure 4.2a Amplification of nirB promoter..........................................150
Figure 4.2b Amplification of pagC promoter........................................150
Figure 4.2c Ligation of pagC and nirB promoters in
pCR2.1 plasmid ...............................................................150
Figure 4.3a Amplification of C.jejuni Momp gene................................152
Figure 4.3b Amplification of chicken ovalbumin gene .........................152
Figure 4.4a Digestion of pagCMomp constructs with
EcoRI and SpeI ................................................................154
Figure 4.4b Digestion of pagCova constructs with
EcoRI and EcoRV ............................................................154
xviii
Figure 4.4c Digestion of nirBMomp constructs with
EcoRI and SpeI ................................................................155
Figure 4.4d Digestion of nirBovaconstructs with
EcoRI and EcoRV ............................................................155
Figure 4.5a Expression of Momp from pCR2.1 constructs..................158
Figure 4.5b Expression of Ovalbumin from pCR2.1 constructs ..........158
Figure 4.6 Restriction of pMW2 plasmid with BamHI enzyme
to select positive clone .......................................................160
Figure 4.7 The pKD46 plasmid from which the Lambda Red
genes are expressed .........................................................162
Figure 4.8 The pKD13 plasmid with R6K origin of replication .............164
Figure 4.9a Ligation of pagCmomp constructs in pKD13 plasmid ......165
Figure 4.9b Ligation of nirBMomp constructs in pKD13 plasmid.........165
Figure 4.9c Ligation of pagCova contructs in pKD13 plasmid.............166
Figure 4.9d Ligation of nirBova constructs in pKD13 plasmid.............166
Figure 4.10 Amplification of constructs from pKD13
plasmid template.............................................................168
Figure 4.11 Amplification of constructs with gtrA forward primer
and aroA reverse primer after chromosomal integration ..170
Figure 4.12a Analysis of C. jejuni Momp antigen
expression by western blotting.......................................173
Figure 4.12b Analysis of ovalbumin antigen expression
by western blotting .........................................................174
xix
Chapter 5
Figure 5.1 Analysis of the IgG response against ovalbumin antigen
from pooled sera collected 3 weeks after the
final vaccination .................................................................186
Figure 5.2 Humoral responses elicited after vaccination
with ovalbumin constructs ..................................................187
Figure 5.3 Analysis of secretory IgA response in mice
from pooled faecal samples ...............................................189
Figure 5.4 Enumeration of IL4-secreting splenocytes
from mice vaccinated with ovalbumin constructs ...............191
Figure 5.5 Enumeration of IFN- γ-secreting splenocytes
from mice vaccinated with ovalbumin constructs ..............192
Figure 5.6 Induction of IgG responses against
C. jejuni Momp antigen in vaccinated mice ........................195
Figure 5.7 Enumeration of IL4-secreting splenocytes
from mice vaccinated with Momp constructs......................198
Figure 5.8 Enumeration of IFN- γ-secreting splenocytes
from mice vaccinated with Momp constructs.....................199
Chapter 6
Figure 6.1 Estimation of IgG responses at week 6 in
all groups ..........................................................................210
Figure 6.2 Evaluation of humoral immune responses against
the carrier antigen (chicken ovalbumin) at week 6 in groups 1-4
..........................................................................................213
xx
Figure 6.3 Evaluation of humoral immune responses against
the carrier antigen (chicken ovalbumin) at week 15
in groups 1-4 ....................................................................215
Figure 6.4 Evaluation of humoral immune responses against
the carrier antigen (chicken ovalbumin) at week 15
in groups 5-8 .....................................................................216
Figure 6.5 Evaluation of IL4-secreting splenocytes from
vaccinated mice in groups 1-8...........................................220
Figure 6.6 Evaluation of IFN- γ-secreting splenocytes from
vaccinated mice in groups 1-8...........................................221
xxi
Summary
Live attenuated Salmonella vectors provide unique alternative in terms of
antigen presentation by acting as a vector for heterologous antigens. The
efficiency of any live bacterial vector rests with its ability to present sufficient
foreign antigen to the human or animal immune system to initiate the
desirable protective immune response.
Salmonella vectors encoding heterologous protective antigens can elicit the
relevant immune responses, be it humoral, mucosal or cell-mediated. STM-1
is a Salmonella mutant developed by RMIT, harbours a mutation in the aroA
gene that renders it attenuated, and is a well characterised vaccine strain
currently in use to protect livestock against Salmonella infection. In previous
work in this laboratory, STM1 was shown to be capable of eliciting immune
responses in mouse to plasmid-borne antigens.
In this study STM-1 was analysed for its ability to vector chicken ovalbumin
model antigen and C. jejuni major outer membrane test antigen using in vivo
inducible promoters such as pagC and nirB from the plasmid location. Also
the expression and immune induction of these constructs was characterised
from the chromosomal location using the same promoter constructs. Induction
of immune responses, both humoral and cell mediated, was analysed to test
the ability of STM1 to present heterologous antigens to the host immune
system. Another issue addressed in this study was effect of pre-existing
immune responses in the animal host against the vector or related strains and
1
the effects on generation of immune responses against the subsequently
vectored antigen.
The aroA gene region was the point of insertion for the designed constructs.
However, this region was uncharacterised in STM-1 mutant, and so an
analysis of this region was first completed. The lesion in STM-1 was created
by an insertion of aroA554::Tn10 in the wild type Salmonella strain.
The sequence analysis and PCR results of STM-1 characterisation reveal that
there is an insertion and a deletion in STM-1, with respect to the parent strain.
The IS10 left hand side (IS10 LH) of the Tn10 element (approximately 1.4 kb)
is inserted adjacent to aroA. Several genes including serC, ycaP, ycoA, fcoA
and part of the pflB have been deleted. Also deleted is the first 90 bp of the
aroA gene. The presence of the composite Tn10 element has also induced a
rearrangement in STM-1 chromosome. This rearrangement is not an inversion
but may be a crossover between two points, not due to specific target
sequence but due to the conserved three dimensional structure of the
bacterial chromosome.
In chapter 4 constructs were designed and created, with genes encoding
chicken ovalbumin (the model antigen) and C. jejuni major outer membrane
protein (Momp, test antigen) put under the control of an in vivo inducible
promoter and incorporated into a plasmid. Similar constructs were integrated
in the aroA gene of STM-1 using the Lambda Red system, based on phage
recombination. Expression was analysed for both ovalbumin and Momp
2
antigens using western blotting. A similar level of expression from the two in
vivo inducible promoters, from both plasmid and chromosomal locations, was
achieved. However, the expression analysis using western blotting is an
indicative measure, with the induction of immune responses after vaccination
is a more realistic measure of in vivo expression.
Analysis of immune responses in mice vaccinated with STM-1 expressing
ovalbumin under the pagC promoter and the nirB promoter, when compared
with mice vaccinated with either STM-1 only or STM-1 harbouring empty
plasmid, yielded significantly increased humoral and T cells responses
(P<0.001). As with the in vitro expression results, no difference was observed
in humoral or T cell responses when the efficiency of the two promoters to
induce responses was compared. Similarly no difference in the induction of
immune responses was observed when comparing constructs that expressed
antigen from the chromosomal location or the plasmid location, indicating no
advantage of multiple gene dosage. Immune responses from STM-1
expressing ovalbumin under control of the nirB promoter were higher,
although not significantly, especially when the expression was from the
chromosomal location. Therefore in the second vaccine trials STM-1
constructs expressing Momp driven from the nirB promoter were used for the
vaccination.
The analysis of anti-Momp humoral and T cell immune responses
demonstrated significantly higher responses in the vaccinated mice when
compared with controls (P<0.001). No difference was observed between the
3
groups receiving STM-1 expressing Momp antigen from the plasmid location
and the chromosomal location.
The impact of pre-existing immune responses was analysed after the ability of
STM-1 to vector heterologous antigen was established. If STM-1 is to be used
in field, any negative impact on immune responses against the vectored
antigens due to pre-existing immunity against the bacterial vector or related
strains would be a significant problem. However, the results from this study
indicate that pre-existing immune responses against the bacterial vector or a
related strain do in fact enhance both humoral and T cell responses against
the heterologous antigen.
In conclusion, the determination of the architecture around the lesion in STM-
1 allowed the development of constructs expressing heterlologous antigen
from the chromosome. Humoral and cellular immune responses to vectored
ovalbumin and C. jejuni Momp antigens were observed following vaccination
with STM-1, when antigens were expressed from either the plasmid or
chromosomal location. Up-regulation of immune responses, both humoral and
cell mediated was observed against the vectored antigens in animals which
were pre-exposed to either the bacterial vector or related strains. These
results indicate that STM-1 has the potential to be used as a vector to deliver
heterologous vaccine antigens from a single copy gene in the field.
4
Chapter-1
Literature Review
1.1 Introduction
Since the origin of human life, the struggle against disease-causing
microorganisms has continued. Several methods have been tried and tested
for preventing or overcoming infections, such as chemical treatments,
antibiotics and vaccination. Some of these methods were used with great
success and some were not so successful. However, the methods which were
successful, such as the use of antibiotics, have limited application as they are
only effective in the case of bacterial diseases. Even the use of antibiotics is
becoming less effective, due to the continuing problem of bacterial drug
resistance. The presence of plasmids carrying antibiotic resistance genes
helps disseminate antibiotic resistance strains, compromising the treatment of
severe infections (Sherratt 2001).
Edward Jenner and Louis Pasteur pioneered the use of vaccines, which was
one of the most notable achievements in the field of medical science. These
vaccines have helped in overcoming many debilitating and deadly diseases.
Since then vaccine development has made some remarkable progress, but
the demand for improved vaccines against pathogenic organisms continues.
Despite the successful use of vaccines, modern medicine was confronted with
new challenges during the last half of the 20th century because of the
emergence of pathogens such as the Human Immunodeficiency virus and the
5
re-emergence of diseases such as tuberculosis, which were believed to be
conquered. While there is a great need to develop new vaccines against
these highly pathogenic organisms, there is also some interest in the
development of vaccines in the field of veterinary science, the food industry
and against the adverse use of pathogens as weapons (Kochi 2003; Atkins et
al. 2006). The requirement to develop new and advanced vaccines has
changed the approach to vaccine development. Several new techniques, such
as vaccination with DNA or subunit vaccines have been developed by using
molecular and immuno-biological techniques.
Vaccines need to satisfy some important criteria (Table 1.1) before being
used in the general population. Safety is a major concern if vaccines are to be
administered in large population therefore even a low level of toxicity is
unacceptable. Secondly, long lasting immune responses which includes both
cellular and humoral immune response must be induced following
administration of these vaccines (Atkins et al. 2006). Finally, a vaccine must
be inexpensive if it is to be administered on a large population, particularly in
a third world country. Similar constraints apply to veterinary vaccines.
6
Table1.1: Features of effective vaccines (Janeway 2001)
Safe
Protective
Protection
Induces neutralizing antibody
Induces appropriate responses
Practical Considerations
Vaccine must not itself cause illness or
death
Vaccine must protect against illness
resulting from exposure to live pathogen
Protection against illness must last for
several years or lifelong
Some pathogens (such as poliovirus)
infect cells that cannot be replaced (e.g.
neurons). Neutralizing antibody is
essential to prevent infection of such cells.
Some pathogens, particularly intracellular,
are more effectively dealt with by cell-
mediated immune responses
Low-cost per dose
Biological stability
Ease of administration
Few side effects
7
1.2 Types of Vaccines
Jenner pioneered the use of vaccines against pathogenic organisms and
since that time, the term vaccine has been used for any preparation of dead
or attenuated pathogens or their products, where these preparations when
introduced into the body stimulates the production of protective immune
responses without causing the disease. Most of the vaccines available at
present are either attenuated or killed (inactivated) organisms (Table 1.2)
(Goldsby 2000).
1.3 Killed organisms
The killed organisms are non-infective and the whole preparation is used as a
vaccine. Killed bacterial vaccines have been accepted since their
development in the 1890s. There was no other alternative to prevent and treat
certain serious infections apart from this method, especially in the case of
typhoid fever which was wide-spread throughout the world (Mims 1998).
However, the major disadvantage of killed vaccines is their efficacy. These
vaccines are particularly good in inducing both cellular and humoral
responses, However booster dose or addition of adjuvant is required for the
generation of long lasting immune responses.
8
Table1.2 Classification of common human vaccines (Goldsby
et al. 2000; Farlane et al. 2003)
Disease or pathogen Type of vaccine
Whole organism
Bacterial Cells
Anthrax Inactivated
Cholera Inactivated
Plague Inactivated
Pertusis Inactivated
Tuberculosis Live attenuated BCG
Typhoid Live attenuated
Viral particles
Hepatitis A Inactivated
Influenza Inactivated
Measles Live attenuated
Mumps Live attenuated
Polio (sabin) Live attenuated
Polio (salk) Inactivated
Rabies Inactivated
Rubella Inactivated
Varicella Zoster (chickenpox) Live attenuated
Yellow fever Live attenuated
Purified macromolecules
9
Toxoid
Diphtheria Inactivated exotoxin
Tetanus Inactivated exotoxin
Capsular Polysaccharides
Hemophilus influenza type B Polysaccharide +protein carrier
Neisseria meningiditis Polysaccharide
Streptococcus pneumoniae 23 distinct capsular polysaccharide
Subunit Vaccines
Hepatitis B vaccine Surface antigen
10
1.4 Purified microbial component vaccines
The use of cell surface macromolecules and cytosolic macromolecules
derived from pathogens for vaccine purposes eliminates some of the risks
associated with killed vaccines. The cellular components of cells such as
capsular polysaccharide, proteins or inactivated exotoxins can be used as
vaccine preparations. A commonly used microbial component vaccine is a
preparation to protect against Neisseria meningitides, a common cause of
bacterial meningitis (van den Dobbelsteen et al. 1995) and for Streptococcus
pneumoniae (Vila et al. 2004). These vaccines are formulated with capsular
polysaccharide component of bacterial cell wall. However, an inability to
stimulate robust T cell responses is one serious limitation of these vaccines.
1.5 Synthetic peptide Vaccines
The generation of synthetic peptide based antigens is an alternative path for
vaccine development. However, these synthetic peptides are often not as
immunogenic as the whole protein, therefore need to be optimised for the
level of immunogenicity. Several methods such as coupling the peptide with
micro-particles or fusions with other molecules can be used to enhance the
immunogenicity of synthetic peptide vaccines.
1.6 Subunit vaccines
Subunit vaccines are based on specific purified or semi purified antigens.
They can be native antigens, as in the case of toxoids, or recombinantly
produced. In this case a gene-coding antigen is cloned into a vector. This type
11
of approach has led to the development of a hepatitis B vaccine (André et al.
1990).
1.7 DNA vaccines
DNA vaccines are one of the latest developments in the field of vaccine
technology. DNA vaccines have been extensively studied in animal models in
the past decade for their effectiveness and mode of action. A study by Tang et
al. (1992) used a gene gun to deliver plasmid DNA into the epidermis of mice
and found that an immune response was generated against the encoded
protein. In addition, Ulmer (1993) showed that vaccination with DNA encoding
the influenza-A nucleoprotein generated a protective immune response in
mice, not only against homologous virus, but also against heterologous
strains. These experiments demonstrated the effectiveness of DNA vaccines
in generating broad immune responses including CTL responses.
1.8 Live attenuated vaccines
The prospective of live, attenuated vaccines has been acknowledged during
the earlier studies. Calmette and Guerin (1921) first used this technology with
Mycobacterium bovis. They passaged the bacteria in culture to reduce its
virulence. Live attenuated strains provide exceptional choice for the delivery
of antigens not only against homologous pathogenic organisms but can also
be used as a vector to deliver passenger antigens. One of the most
successful live attenuated vaccines produced is the Ty21a live S. Typhi
vaccine. Chemical mutagenesis process was used for inducing attenuation,
which eliminated the O antigen from the LPS molecule. This live oral bacterial
12
vaccine generated antigen-specific CD4+ and CD8+ memory T cells (Lundin et
al. 2002). The live attenuated bacteria as a vaccine and vector offers many
advantages compared with killed and subunit vaccines. Live attenuated
strains can be delivered by a simple oral mode of inoculation and may confer
protection following a single dose due to replication (albeit limited) in the host
(Kochi 2003). In addition they are economical to develop (Trach et al. 2002)
can be manufactured commercially on a large scale (Lockman et al. 1999).
The use of live oral vaccines also enhances the repertoire of antigens
presented to the mucosal immune system compared with the antigens
presented in complexes of killed organisms. Killed bacterial vaccines which
are prepared for parental or oral administration are subjected to acetone,
formalin, or heat to kill the organism, all of which have the potential to
denature relevant immunologic determinants. The attenuated organisms also
provide long lasting immunity as the intracellular organism can only be
eliminated by elicitation of strong cellular responses, including CD4+ and
CD8+ T lymphocytes (Kaufmann 1998).
The early live attenuated vaccines, for example Salmonella typhi Ty21a
(Germanier and Fuer 1975) and Mycobacterium- BCG (Guleria 1996) were
developed by randomly generated undefined mutations. However, application
of modern DNA technology have led to the generation live attenuated
vaccines where the organisms are attenuated by the deletion of genes
encoding metabolic functions. These attenuated organisms can effectively
colonise and undergo several round of replication inside the host cell.
13
Another advantage of live attenuated organisms is that they can be
genetically engineered to express a heterologous antigen. These live
attenuated bacterial vectors not only confer long lasting immunological
memory against the homologous antigens but also against the heterologous
antigens presented by the bacterial vectors (Wyszynska et al. 2004).
Despite the fact that a number of organisms have shown potential to be used
as attenuated vaccine, only a few can be effectively used. Major
apprehension in using attenuated organisms as a vector, apart from
immunogenicity, is safety (Kochi et al. 2003). This requires development of
strains that have restricted survival in a host and in the environment, yet still
induce protective immune response. Only a few genera such as Shigella,
Salmonella, Leisteria, Mycobacterium and Vibrio have shown the ability
required to be used as a vector for the delivery of heterologous antigen for
vaccine purpose and proved effective in terms of antigen delivery and immune
presentation. Salmonella, due to an extensive knowledge of the organism’s
biology, have been used most widely as a vector to deliver heterologous
vaccine antigen from both prokaryotic and eukaryotic origin (Mastroeni et al.
2001; Lundin et al. 2002; Baud et al. 2004; Berndt and Methner 2004; Liu et
al. 2006).
14
1.9 Salmonella as a candidate
Salmonella are Gram-negative, facultative anaerobic, motile, non-lactose
fermenting rods, belonging to the family of Enterobacteriaceae. Salmonella
infect humans and animals generally by the oro-fecal route (Curtiss et al.
1993). They have a wide range of animal hosts and can be frequently found in
sewage, sea and river water and contaminated food (Coburn et al. 2007).
Initially the use of S. Typhi Ty21a, as vaccine against the typhoid fever
developed great interest in using Salmonella genus to deliver antigenic
proteins, however, lack of reliable animal models for this species shifted the
attention of researchers towards the use of S. enterica var. Typhimurium to
deliver antigenic determinants (Levine et al. 1997). However, recently several
studies have been conducted using S. Typhi as a vector to deliver
heterologous vaccine antigen in both animals and humans where it has
shown some promise as a live attenuated vector (Capozzo et al. 2004;
Metzger et al. 2004). The progress of serovar Typhimurium as vectors is
based on the concept that the survival of organism in the intestine may induce
mucosal and systemic immune responses (Angelakopoulos and Hohmann
2000).
15
1.9.1 Pathogenesis of Salmonella
Salmonella infects humans and animals generally by oral route. The organism
has the capability to resist the low pH of the stomach allowing it to reach the
distal ileum and the caecum. The bacteria then invades the mucosa, and
replication takes place in the sub-mucosa and the peyer’s-patches (Carter
and Collins 1974). Invasion occurs via M cells with the contribution of
products of the invasion genes encoded within pathogenicity island 1 (SPI1).
After entering the peyer’s patches, bacteria are transported via the lymphatic
and blood streams to the mesenteric lymph nodes, liver and spleen (Darwin
and Miller 1999), or via an alternative pathway which involves systemic
dissemination, if the bacteria are present within CD18+ cells (Vazquez-Torres
et al. 1999). In systemic infection, the bacteria are engulfed by the phagocytes
of the spleen, the liver and the bone marrow. Salmonella is phagocytosed by
APC’s due to the presence of opsonising antibodies and complement
activation at the bacterial surface (Liang-Takasaki et al. 1983; Saxen et al.
1987).
Virulent property of Salmonella is due to its ability to grow within
macrophages, DC’s and polymorphonuclear phagocytes during systemic
infection. (Hoiseth and Stocker 1981; Lissner et al. 1983; Fields et al. 1986;
Dunlap et al., 1991; Mastroeni et al. 1995; Richter-Dahlfors et al. 1997).
16
1.9.2 Immune response against Salmonella
Cellular immune responses are crucial both for protective immunity against
salmonellosis and for the immunogenicity of oral vaccines which are based on
avirulent live Salmonella as antigen carriers. A primary infection leads to the
induction of antibodies against antigens such as lipopolysaccharide (LPS),
flagellin and lipo-protein with the induction of Th1 or Th2 type T-cell memory
and CTL responses (Hormaeche et al. 1991).
The initial stages of infection are characterised by an innate immune response
triggered by the host recognition of several microbial structures (Wick 2004)
which is followed by the induction of antigen specific CD4+, CD8+ and B cells
all of which contribute to protective immunity (John et al. 2002).
1.9.2.1 Role of Antigen presenting cells in Salmonella
immunity
As S. Typhimurium is an intracellular pathogen, T cells are critical
components of the specific immune response. The bacterium specific CD4
and CD8 effector T cells are essential for a complete immune response
against S. Typhimurium (Mittrucker and Kaufmann 2000). Macrophages and
immature dendritic cells (DC) play an important role in the development of
innate and adaptive immune responses to bacterial infections. They act as
antigen presenting cells (APC), capturing and degrading bacteria in a defined
17
proteolytic pathway. The processed bacterial antigens are displayed on the
APC surface by the major histocompatibility complex (MHC) (Watts 1997).
Non-overlapping antigenic peptides which are accessible to CD8+ CTL and
CD4+ helper T cells (TH) are presented by MHC Class I and the Class II
molecules expressed on APC’s (Gotch et al. 1987; Babbitt et al. 2005). The
Class I molecules containing degraded antigenic fragments are transported to
the endoplasmic reticulum before being transferred to the MHC molecule
before being recognised by CTL (Germain 1995; Heemels and Ploegh 1995).
Majority of Class II complex peptides accessible to TH cells are membrane
proteins. These degraded exogenous products after endocytosis fuse with
lysosomes before entering Class II molecules pathway (Cresswell 1994;
Teyton 1994). Yrlid et al. (2001) showed that three major phagocytic cells,
which include neutrophils, macrophages and dendritic cells are all associated
with the induction of S. Typhimurium specific T cell responses. However, DC’s
have a superior capacity to stimulate naïve T cells in comparison to
macrophages.
Immature DCs are efficient in capturing and processing antigen but lack the
capacity to induce naïve T cells. However, after maturation they are an
effective T cell activator (Banchereau et al. 2000). Several studies also
suggest the involvement of DC in M cell-independent transport of non-
invasive Salmonella from the intestine (Rescigno et al. 2001). A potential role
in mediating bacterial transit across the intestinal epithelium is supported by
the observation that CD8α- CD11b- DC are in peyer’s patches and in the
18
follicle associated epithelium and can be in close contact with M cells (Iwasaki
and Kelsall 2001). These data suggest a role of intestinal DC in sampling
intestinal bacteria and facilitating their penetration across the gut. Once
Salmonella penetrates the intestine, other organs which are subsequently
infected include the mesenteric lymph nodes, the spleen and the liver.
The presentation of Salmonella antigens to T cells by infected DC occurs
when the DC are infected with Salmonella that do no induce death in the cells.
However, Salmonella-induced apoptosis of macrophages provides a reservoir
of Salmonella antigens t hat can be presented by bystander DC’s (Yrlid and
Wick 2000). Interestingly, the capacity to act as a bystander APC appears to
be a unique feature of DC, as bystander macrophages ingest Salmonella-
induced apoptotic cells but do not present peptides from Salmonella antigen
(Banchereau et al.1998). Thus, depending upon the type of Salmonella DC
encounter, Salmonella infected DC can process Salmonella for direct
presentation of Salmonella antigen to T cells or can act as bystander APC
that engulf the antigenic material from neighbouring cells that have undergone
Salmonella-induced apoptotic death.
1.9.2.2 T cell responses
Infection with live S. Typhimurium can result in broad activation of CD4+ and
CD8+ T cells and generation of Salmonella specific IFN-γ producing T cells.
Mittrucker and Kaufmann (2000) also showed that the induction IFN-γ
producing CD4+ and CD8+ T cells, particularly the CD4+ subset, and IFN-γ in
immunity to Salmonella. However, the frequency of specific IFN-γ producing
19
CD4+ T cells was significantly greater than that of CD8+ cells. This has been
also indicated by an experiment in transgenic mice model which were nude, T
cell receptor αβ-deficient, and MHC Class II deficient. When infected with
attenuated Salmonella, these mice could not protect themselves from
infection (Hess et al. 1996; Sinha et al. 1997), whereas wild type mice were
not infected by the same Salmonella strain. In contrast transgenic mice
lacking MHC Class I restricted T cells displayed no difference from the wild
type and therefore were not infected with the Salmonella mutant (Hess et al.
1996; Lo et al. 1999). These data suggests that a strong CD4+ response is
critical for immunity against Salmonella.
Another study with MHC Class I deficient and B cell deficient transgenic mice
also showed they are not infected by attenuated Salmonella. However, unlike
wild type mice, these mice did not provide any protection against challenge
with wild type Salmonella (Lo et al. 1999; Mastroeni et al. 2000). These
results indicate that for protection against wild type Salmonella a fully
functioning immune system is required, whereas CD4 cells may be sufficient
to resist attenuated strains.
It has been shown that vaccination of mice with attenuated Salmonella
provides immunity against re-challenge with virulent Salmonella (Hoiseth and
Stocker 1981). However, the protective immunity could not be transferred to
naïve recipients unless both T cells and serum antibodies were transferred
(Mastroeni et al. 1993). Again, this indicates the role of CD4+, CD8+ and B cell
responses for the induction of complete immunity against Salmonella. One
20
reason for the above discrepancy in the analysis of CD8+ response might be
due to cross presentation of antigen during activation of CTL’s.
1.9.2.3 Antigen cross presentation
Phagocytosis of pathogens by DCs Induce cytotoxic T-cell immune response
(Guermonprez et al. 2002). In the classical pathway for antigen presentation
endogenous antigens are presented by MHC Class I molecules to stimulate
CTL responses, and exogenous antigens are presented by MHC Class II
molecules to stimulate a helper T cell response. However, there can be some
variety in this classical pathway in that DC’s can present exogenous antigens
on MHC Class I molecules via a process known as “cross presentation” (Sigal
et al. 1999).
Antigens from pathogenic organisms are exported into the cytosol after
phagocytosis and degraded by the proteasome (Norbury et al. 1997;
Rodriguez et al. 1999). The processed peptides are translocated into the
endoplasmic reticulum (ER) lumen by specific transporters associated with
antigen presentation (TAP) to form MHC Class I peptide complexes which are
transported to the cells surface through the secretory pathway. Guermonprez
et al. (2003) showed that during the formation of phagosomes, or soon after,
they fuse with the ER. After the antigen is exported to the cytosol and
degradated by the proteasome, peptides are translocated by TAP into the
lumen of the same phagosome, and loaded onto phagosomal MHC Class I
21
molecules. Therefore cross-presentation in dendritic cells occurs in a
specialised, self sufficient, ER-phagosome compartment.
The phagosome plays a very important role in antigen cross presentation
(Houde et al. 2003). Though there is a well defined segregation between the
MHC Class I and II pathways, antigens from intracellular pathogens such as
S. Typhimurium, Mycobacteria, and Leshmanaia have been shown to elicit
an MHC Class I dependent CD8+ T cell response because phagocytosis in
the APCs proceeds via the ER at the cell surface, a process referred to as
ER-mediated phagocytosis (Pfeifer et al. 1993; Oliveira and Splitter 1995;
Canaday et al. 1999). In this way an antigen from an intracellular pathogen
could have direct access within the phagosome to the ER machinery needed
for MHC Class I presentation (Houde et al. 2003).
22
Figure 1.1 Diagramatic representation of the induction of T cell response. In the
endogenous pathway, proteins from intracellular pathogens, such as viruses are degraded
by the proteasome and the resulting peptides are shuttled into the endoplasmic reticulum
(ER) by TAP proteins. These peptides are loaded onto MHC Class I molecules and the
complex is delivered to the cell surface, where it stimulates cytotoxic T lymphocytes
(CTLs) that kill the infected cells. In contrast, extracellular pathogens are engulfed by
phagosomes (exogenous pathway). Inside the phagosome, the pathogen-derived peptides
are loaded directly onto MHC Class II molecules, which activate helper T cells that
stimulate the production of antibodies. But some peptides from extracellular antigens can
also be 'presented' on MHC Class I molecules. How this cross-presentation occurs has now
been explained. It seems that by fusing with the ER, the phagosome gains the machinery
necessary to load peptides onto MHC class I molecules. Figure and Legend reproduced (Roy
2003)
23
1.9.2.4 B cell response
The critical role of T cells in the development of protective immunity against
Salmonella challenge has been well characterised (Canaday et al. 1999; Lo et
al. 1999; Bumann 2001; Iwasaki and Kelsall 2001; Yrlid et al. 2001; Yrlid et al.
2001; Wick 2002; Bumann 2003; Wick 2003; Wick 2004). However, the role of
B cells in generating Salmonella specific immune response is not so well
studied. Most of the earlier studies report the generation of serum IgG and
mucosal IgA responses against bacterial LPS and other antigenic
determinants following immunisation of mice with Salmonella (Alderton et al.
1991; Hormaeche et al. 1991).
Sinha et al. (1997) in one of his experiments with T cell deficient mice showed
that the deficient mice infected with attenuated Salmonella did not survive. He
also showed that the humoral response in those mice was limited to IgM and
IgG3 antibody responses, with little or no IgG1, IgG1a and IgG2b. In another
study by Berndt and Methner (2004) it was shown that IgM and IgA responses
are of importance in the caeca of chickens against Salmonella infection.
Recent studies (Mastroeni et al. 2000; Mittrucker et al. 2000; al-Ramadi et al.
2006; Menager et al. 2007) on mice deficient for the generation of B cell
responses, has indicated that the B cell response plays an important role in
the development of complete immunity against Salmonella.
24
1.9.2.5 Immune responses against a passenger antigen
The rationale behind using Salmonella as a vector to deliver heterologous
vaccine antigen is that it can induce both local and systemic immune
responses. The understanding of S. Typhimurium pathogenicity and its
interaction with the host immune system and associated virulence factors led
to the generation of attenuated Salmonella vaccines and their use for the
delivery of heterologous vaccine antigen (Bachtiar et al. 2003; Bowe et al.
2003; Foynes et al. 2003; Baud et al. 2004; Berndt and Methner 2004;
Capozzo et al. 2004).
1.9.2.5.1 Cellular immune responses against heterologous vaccine
antigen
The efficacy of any live bacterial vaccine rests in its ability to present the
passenger antigen to generate the desired protective immune responses
against the foreign antigen. Heterologous antigens expressed from
Salmonella can induce Th1, Th2 and CTL responses (Du and Wang 2005;
Atkins et al. 2006; Chiu et al. 2006). However, several factors such as the
type of strains, type of mutant and the type of antigenic protein expressed
play a critical role in the generation of complete T cell responses (Bowe et al.
2003; Spreng et al. 2006). The activation of a Th1 response is characterised
by the secretion of IFN-γ and IgG2a antibody, whereas the induction of a Th2
response is characterised by IgG1 and IL4 (Kang and Curtiss 2003). Some
studies also demonstrate the activation of CTL responses to the guest
antigens (Methner et al. 2004; Atkins et al. 2006; Chiu et al. 2006).
25
1.9.2.5.2 Humoral Immune responses against heterologous
vaccine antigen
The protective humoral response against a heterologous antigen has been
reported in some studies against bacterial, viral and parasitic antigens carried
by different Salmonella mutants (Karem et al. 1995; Kohler et al. 2000; Lillard
et al. 2001; Chen and Schifferli 2003; Fouts et al. 2003; Foynes et al. 2003;
Morton et al. 2004). Oral immunisation can lead to the induction of IgA
antibody responses against heterologous antigen in the gut, saliva and lung
washes (Berndt and Methner 2004; Capozzo et al. 2004; Jiang et al. 2004;
Wyszynska et al. 2004). Other modes of inoculation such as intranasal
inoculation, rectal or vaginal inoculation also leads to the development of
protective IgA responses against the antigenic protein (van den Dobbelsteen
and van Rees 1995; Garmory et al. 2003; Garmory et al. 2003; Morton et al.
2004). Serum IgG responses against the heterologous antigen have also
been reported following oral vaccination and also when the vaccination was
given via other routes (Harokopakis et al. 1997; Kang et al. 2002; Wyszynska
et al. 2004; Stratford et al. 2005). The induction of subtype IgG1 or IgG2a has
also been observed and the presence of these subtypes is indicative of the
activation of polarised T cell immune responses, as IgG1 an indicative of a
Th2 response and IgG2a is indicative of a Th1 response (Harokopakis et al.
1997; Berndt and Methner 2004; Capozzo et al. 2004; Chiu et al. 2006).
These studies suggest that Salmonella can be used as a vector for the
delivery of heterologous vaccine antigen to target T and B cell responses.
26
1.10 Salmonella Typhimurium Mutants
Bacon, Burrows et al. (1950; 1951) were first to describe attenuated
Salmonella strains. The mutation in these strains was due to the disruption of
genes encoding for purine, p-aminobenzoic acid, and aspartate. These
mutants exhibited reduced virulence in mice due to exogenous requirement of
the metabolites.
1.10.1 galE Mutants
Nitrosoguanidine treatment was used to induce a galE mutation in S.
Typhimurium and S. typhi. These mutants were avirulent but still
immunogenic in mice and humans (Germanier and Fuer 1975). In the
absence of galatose S. typhi was found to be avirulent due to loss of
capability to synthesise complete LPS. However, LPS components were
shown to be synthesised after the addition of galatose to the medium as
shown in Figure 1.2 (Nikaido 1961). The exact nature of avirulent properties
due to disruption of galE genes is still not understood, but almost certainly
involves the loss of the ability to synthesis complete LPS molecules and
galactose sensitivity.
27
Phosphatase galE
Galactose Galactose Gal-1-P UDPGal UDPGlu Glu-1-p GLU-6-P
Permease Kinase+ ATP Transferease Epimerase Synthetase Mutase
Figure1.2 Metabolism of galactose. The
galE mutants are unable to synthesise
glucose from galactose because of the
deficiency of enzyme uridine diphosphate
(UDP)-galactose-4-eipmerase. (Garmory et
al. 2002)
28
1.10.2 aro and pur mutants
Salmonella strains harbouring mutation in the aroA gene were found to be
nonvirulent for invasive infection due to their requirement for the aromatic
metabolites paraaminobenzoate (for synthesising folate) and
dihydroxybenzoate (for synthesising enterochelin) which are unavailable in
the host tissues (Smith et al. 1984). The aroA gene directs the expression of
the enzyme 3-enolpyruvylshikimate-5-phosphate synthetase, this enzyme
plays a major role in the conversion of phosphoenolpyruvate and shikimate 3-
phosphate into 5-enolpyrovoylshikimate-3-phosphate which leads to the
synthesis of chorismate, a common intermediate compound in the synthesis
of aromatic amino acids (Figure 1.3).
S. Typhimurium and S. dublin mutants with an aroA defect have been
successfully used as a vector to deliver heterologous antigen for vaccination
(Guzman 1991; Stocker 2000; Jespersgaard et al. 2001; Mollenkopf 2001).
Strains harbouring mutations in the genes that affect other metabolic
pathways have also been designed.
29
aroA
Shikimic Acid Chorismic acid
Figure 1.3 Aromatic bio
aroA mutants lacks the
chorismic acid from sh
1984).
30
Essential and Available
Essential and not Available2,3 dihydroxybenzoic acid para-aminobenzoic acid
Enterochelin Folate
Ubiquinone
s
c
ik
Not
ynthetic pathw
apability to syn
imic acid (Smit
Tyrosine PhenylalanineTryptophan
Vitamin K
ay
th
h
Parahydroxybenzoic acid o-succinyl benzoic
. The
esise
et al.
Salmonella strains harbouring mutation in the purA gene was developed
(McFarland and Stocker 1987). The purA gene encodes adenylosuccinate
synthetase, the enzyme that catalyzes the conversion of IMP to AMP in the in
the purine synthesis pathway (Figure 1.4). A secondary mutation was created
by deleting the purA gene in existing aroA mutants to make them safer.
However, it was found that strains harbouring a mutation in either in the purA
gene or both in purA and aroA genes were poorly immunogenic (O'Callaghan
et al. 1988; Sigwart et al. 1989).
31
E P
PRPP + glutamine
pur
IMP
A
32
Xanthylate
e
AdenylosuccinatGM
AMP
purFigure 1.4 Purine biosynthetic pathway. The
purA gene encodes adenylosuccinate
synthetase, the enzyme that catalyzes the
first step of the conversion of IMP to AMP
(Garmory et al. 2002).
1.10.3 Cyclic AMP regulation Mutants
A mutation in the genes encoding enzymes for the synthesis of cyclic AMP
and cyclic AMP receptor protein renders Salmonella spp. avirulent. The
complex these protein forms acts as a positive regulator for the transcription
of multiple genes that possess catabolite sensitive promoters (Pastan and
Adhya 1976).
Strains carrying a mutation in cya gene (adenylate cyclase) and the crp gene
(cyclic AMP receptor protein), were found to be less virulent. However, the
stability to these mutants was in question as either cya or crp, reverted back
to wild type with a frequency of 1 in 2 x 107. This is too high a frequency for
vaccine use.
Strains harbouring mutations in both the genes were stable, whereas strains
that had deletion in a single gene had the possibility of reverting to wild type.
Also in comparison to aro or gal mutants the cya and crp mutant strains were
not efficient in causing systemic infection in mice, and due to this there is poor
induction of immune responses (Curtiss and Kelly 1987; Curtiss et al. 1988;
Curtiss et al. 1989).
33
1.10.4 phoP and ompR mutants
PhoP–PhoQ is a two component regulatory system that controls expression of
several virulence properties in S. Typhimurium (Fields et al. 1989; Miller et al.
1989; Miller and Mekalanos 1990). The virulence properties that the PhoP-
PhoQ system controls include the ability to survive inside macrophages
(Fields et al. 1986), resistance of killing by host defence antimicrobial peptides
(Fields et al. 1989; Groisman et al. 1992; Groisman et al. 1992), withstanding
of acidic pH (Foster and Hall 1990), invasion of epithelial cells (Behlau and
Miller 1993), formation of spacious vacuoles and the ability to alter antigen
presentation (Wick et al. 1995).
Angelakopoulos and Hohmann (2000) successfully used a S. Typhimurium
PhoP-PhoQ mutant to deliver H. pylori urease as a vaccine antigen in mice.
The product of ompR gene regulates the expression of component outer
membrane proteins and other proteins which are uncharacterised (Dorman et
al. 1989). The mutation in this gene renders S. Typhimurium strains avirulent.
34
1.10.5 Plasmid-cured strains
Some non-typhoid Salmonella strains such as S. Dublin (Terakado et al.
1983; Chikami et al. 1985; Nakamura et al. 1985), S. Enteritidis (Nakamura et
al. 1985), and S. gallinarum (Barrow et al. 1987; Barrow and Lovell 1988)
have been found to be virulent due to the presence of high molecular weight
plasmids. When these strains are treated to eliminate these plasmids, thay
are rendered avirulent. Before these cured strains can be used as vaccines
however, it will be important to completely characterise plasmid genes and
determine the full growth profiles of the strains.
1.10.6 Streptomycin-Dependent Mutants
A Salmonella typhi mutant developed after repeated culture in the presence of
an antibiotic such as streptomycin has been reported by Reitman et al. (1954)
and Mel et al. (1974). These mutants require the presence of antibiotic for
subsequent growth. The generation of Salmonella mutants by such processes
allows the generation of mutant Salmonella strains without exposing them to
harsh mutagenic treatments, which might result in multiple mutations, and
potentially destroy some key antigenic determinants.
Efficacy of these mutants was tested on human volunteers and were shown to
induce moderate protective immunity and did not result in any illness (Levine,
et al. (1976).
35
1.10.7 Aspartate Semialdehyde Dehydrogenase Mutants
Salmonella strains harbouring a mutation in the asd gene, which encodes for
the enzyme aspartate semialdehyde dehyrogenase, were shown to be
avirulent. The enzyme aspartate semialdehyde dehyrogenase catalyses the
conversion of aspartate-4-phosphate to aspartate semialdehyde which leads
to the synthesis of threonine, methionine, and lysine and diominopimilic acid
(Cohen 1987). These mutants lack the capability to synthesise normal
peptidoglycan, which is an essential component in the synthesis of the
bacterial cell wall. However these mutant are significantly attenuated, and
were unable to induce significant immunogenicity due to a limited survival
time in GALT (Curtiss 1987).
36
1.10.8 Temperature-Sensitive Mutants
S. dansyz was mutated by nitrosoguanidine treatment, and become a
temperature-sensitive variant which was unable to grow at 37°C. These
mutants were able to elicit immune responses after oral or intraperitoneal
inoculation, protecting animals upon challenge with wild type (Fahey and
Cooper 1970).
In a more recent study, temperature sensitive mutants were developed by
exposing the organism to UV rays. The majority of the mutants generated by
this method reverted to wild type with the exception of one strain, which did
not revert to wild type however, generated deficient immune responses in a
mouse animal model (Ohta et al. 1987).
1.10.9 Attenuated Salmonella Strains and their utility
Understanding of biochemical pathways in Salmonella has led to the
development of attenuated Salmonella strains for vaccine purposes either
against the pathogenic strains (Hoiseth and Stocker 1981) or as a carrier for
heterologous antigen (van den Dobbelsteen and van Rees 1995; Titball et al.
1997; Wang HW 2003 Aug; Srinivasan et al. 2004; Wyszynska et al. 2004;
Spreng et al. 2006).
The most widely used metabolically attenuated strains are mutants which lack
the capability of biosynthesis of aromatic amino acids (aroA, aroC and aroD)
(Hoiseth and Stocker 1981; Smith et al. 1984), purine-dependent mutants
37
(purA, purE) (McFarland and Stocker 1987; O'Callaghan et al. 1988; Sigwart
et al. 1989), or mutants deficient in the production of adenylate cyclase (cya)
or cyclic AMP receptor protein (crp)(Curtiss et al. 1988). Other mutants which
are well characterised for the delivery of heterologous antigens include
mutants harbouring mutations in the two component system, PhoP/PhoQ or in
Salmonella pathogenicity island 2 (Angelakopoulos and Hohmann 2000; Baud
et al. 2004). The efficacy of these live vaccines relies on the ability of minimal
survival and maximal immunogenicity.
The disruption of genes encoding for enzymes in important metabolic
pathways necessary for the survival of the organism in vivo leads to the
generation of mutants, which are safe and have minimal survival in the host.
Their effects on the course of infection may vary, thereby altering the quality
of induced immune responses (Dunstan et al. 1998). Medina et al. (1999)
compared an aroA mutant with two Salmonella Pathogenicity Island-2 (SPI-2)
mutants of S. Typhimurium harbouring mutations in the sseC and sseD genes
for the ability of these mutants to induce vectored β-galactosidase specific
immune responses. Both the SPI-2 mutants induced mucosal and systemic
immune response against the passenger antigen that was either similar to or
even stronger than induced by an aroA mutant. In a similar study by Valentine
et al. (Valentine et al. 1998) the S. Typhimurium CL288, CL401, and CL554
mutants exhibited better immune responses when compared to an aroA
mutant. (Dunstan et al. 1998) analysed mutants such as aroA, aroAD, purA,
ompR, htrA, and cya crp to compare their capability to colonise and induce a
vectored antigen specific immune response. All the mutants were able to
38
colonise and persist in murine peyer’s patches and the spleen, albeit at
different levels. The immune response was strongly dependent on the type of
mutant.
In an other study, oral administration of a PhoP mutant exhibited strong innate
immune responses, whereas immunisation with an aroA mutant led to the
development of strong humoral and T cell responses, specifically a Th1 type
response (VanCott et al. 1998). These studies suggest that although the
mutations in genes encoding for metabolic pathway enzymes might lead to
the development of a mutant which is safe to use as live vaccine, their
capacity to carry heterologous vaccine antigen and present it to the host
immune response varies.
39
1.11 Factors that influence the use of
Salmonella as a vector to deliver heterologous
antigen
The use of attenuated Salmonella as a vector to deliver heterologous antigen
is a technique with a significant potential to influence the management of
infectious diseases on a large scale, not only with vaccines against enteric
bacterial pathogens but also with vaccines against a variety of other bacteria,
viruses and parasites. However, some important issues influence the utility of
this approach for vaccine development.
1. stabilisation of antigen expression in the Salmonella carrier, and
2. the effect of pre-existing immune responses against bacterial vector
1.11.1 Stabilisation of antigen expression in attenuated
Salmonella strain
The generation of recombinant Salmonella as a vaccine carrier requires the
introduction of foreign genes on plasmid cloning vectors. However, major
disadvantage of using plasmids to deliver heterologous antigens is, these
plasmids generally harbour antibiotic resistance markers to facilitate selective
pressure in favour of plasmid retention. Secondly, the commonly used cloning
vectors induce metabolic burden on the bacterial replication machinery due to
their size, copy number, type of repicon they harbour and the complexity. Also
due to the expression level of plasmid-encoded genes, which affects the
40
metabolic stability of the bacterial cells (Dunstan et al. 2003). Although the in
vitro stability of these plasmid has been found to be sufficient, in vivo stability
in the absence of antibiotic pressure is doubtful (Cardenas and Clements
1993). Other problems include the possibility of plasmid transfer from the
vaccine strain to human and animal bacterial flora or to environmental
bacteria. For these reasons it is highly unlikely that plasmid borne antigens
will be used outside of the research setting.
1.11.1.1 Use of in vivo inducible promoters
Different strategies for addressing the issue of plasmid instability and avoiding
the need for administration of a stabilising antibiotic have been proposed to
enhance the stable expression of heterologous antigen, and therefore induce
stronger immune responses. One strategy involves the use of in vivo inducible
promoters which allows expression of the heterologous antigen only when the
recombinant Salmonella strains is phagocytised by APC’s, indicating that
promoters activated upon Salmonella invasion could be useful tools to
optimise the expression of heterologous antigens in this bacterial vector
(Hohmann et al. 1995; Chabalgoity et al. 1996). However, the use of in vivo
inducible promoters will not affect the stability of plasmids.
Chatfield et al. (1992) first reported the application of an in vivo inducible-
promoter (nirB) in attenuated Salmonella. Subsequently, the use of in vivo
inducible promoters has been shown to enhance stable expression and
immunogenicity of foreign antigens expressed in Salmonella, while reducing
41
the metabolic load during in vitro propagation (Newton 1995.; Dunstan et al.
1999; Bullifent et al. 2000; Bumann 2001).
In vivo-inducible antigen expression, rather than constitutive expression,
minimally interferes with bacterial viability. This allows the maintenance of
plasmids in recombinant Salmonella culture. Once the Salmonella reach the
appropriate location in the tissue, these in vivo inducible promoters up-
regulates antigen expression, resulting in the efficient induction of an immune
response to the antigen under the control of the promoters (Newton et al.
1995). This concept has been validated in several studies demonstrating an
enhanced stability of such expression plasmids and an increased
immunogenicity against the heterologous antigen when expressed from a
recombinant live Salmonella carrier (Dunstan et al. 1999; Bumann 2001).
1.11.1.2 Chromosomal integration of genes encoding
antigenic protein
Integration of foreign gene into the chromosome of a carrier strains is an
alternative method to achieve stable expression of foreign antigen. (Hone et
al. 1988; Flynn et al. 1990; Strugnell et al. 1990). Hone et al. (1988)
developed one system where heterologous DNA was recombined in the
chromosome of Salmonella by homologous recombination using suicide
plasmid. The gene from E. coli was integrated into the chromosome of mutant
S. Typhimurium using this method. Strugnell, et al. (1990) used a similar
42
strategy to integrate a heterologous gene into the aroC gene of
S. Typhimurium.
The most recently developed method, known as the Lambda Red System
developed by Murphy et al. (1998), takes advantage of phage in the
recombination system. Efficient recombination between short, linear DNA
fragments and the bacterial chromosome can be catalysed by using the
phage recombination system (Court et al. 2002). Homologous recombination
is very efficient in organisms such as yeast. For example, short PCR products
have been extensively used to generate knock-out mutants in yeast
Saccharomyces cerevisiae (Que and Winzeler 2002), where very short region
of identity (from 25-50 bp) are sufficient for efficient recombination. In
contrast, short linear DNA fragments can not recombine efficiently into
bacterial genome.
The phage Lambda Red locus allows homologous recombination of linear
DNA with the aid of enzymes encoded from genes: bet (aka β), exo and gam
(aka γ) (Datsenko and Wanner 2000; Poteete and Fenton 2000; Yu et al.
2000; Poteete et al. 2004). “Exo is 5’-3’ exonuclease that degrades the 5’ end
of linear DNA, bet is a single stranded DNA binding protein, which binds to the
single-stranded 3’ end generated by exo and promotes the annealing of
complementary DNA, and gam binds to the host RecBCD complex and
inhibits its exonuclease activity” (Murphy 2000; Poteete and Fenton 2000; Yu
et al. 2000).
43
This technology was extensively used in E. coli and S. Typhimurium to
generate knockout mutants. The method involves generation of a PCR
product containing a very short region of similarity (35-50 bp). This method
uses the advantage of the phage recombination system to integrate the linear
fragment of DNA into the chromosome. In the year 2000, Datsenko and
Wanner designed temperature sensitive, low copy number plasmids pKD20
and pKD46 (with the ampicilin resistance marker). The Lambda Red genes
bet (aka β), exo and gam (aka γ) were expressed under the control of an
arabinose inducible promoter from these plasmids for efficient recombination
of linear DNA molecules.
Although the Lambda Red recombination system allows recombination of
short linear DNA fragments, the frequency of recombination is low and
therefore a selection is required to identify desired recombinants. To allow the
selection of recombinants, the plasmid templates have been constructed for
the generation of PCR products flanking a selectable antibiotic resistant
marker. Three Pi dependent suicide plasmids (pKD3, pKD4, pKD13) which
lack similarity to any sequence in the E. coli chromosome were constructed to
use as templates for PCR amplification (Datsenko and Wanner 2000).
The recombinase-based system also offers the possibility to precisely
remove the antibiotic resistance marker from the chromosome (Cherepanov
and Wackernagel 1995). The method initially described by (Szybalski 1993;
Posfai et al. 1997) is based on the yeast FLP/FRT recombination system in
which the FLP gene product targets FRT site (FLP recombination target), a 68
44
bp recognition sequence of the yeast FLP gene product and precisely
removes the segment of DNA which exists between the two site. The
antibiotic marker in these Pi dependent plasmids (pKD3, pKD4, pKD13) is
flanked by a FRT site. The plasmid pCP20 is a temperature sensitive replicon,
expressing the FLP recombinase enzyme of S. Cerevisiae, which targets the
FRT sites and precisely removes the antibiotic marker from the chromosome
allowing the generation of recombinants without antibiotic selection markers
(Martinez-Morales et al. 1999; Uzzau et al. 2001). This system has also been
shown to be effective in the development of recombinant Salmonella strains
for vaccine purposes, by allowing the integration of genes at the desired
chromosomal location. (Figure 4.1, Husseiny, et al. 2005).
45
1.11.2 Effect of preexisting immune response against the
bacterial vector
One aspect that must be considered in using mutant Salmonella strains for
the purpose of antigen delivery is the affect of prior immunological memory
against the vector strain or a related strain in the animal host. This is
particularly in case if the same vector is used to consecutively deliver different
antigens. A negative impact of prior immunological memory might limit the use
of intracellular bacterial for the purpose of vaccine delivery, especially in third
world countries where bacterial infections are common and immunity against
the vectors may be high.
Several studies have been conducted to examine the effect of pre-existing
immune response against the bacterial vector. Bao et al. (1991) first reported
that both serum and mucosal antibody responses in mice primed with either
the homologous or the heterologous serotype strain were not significantly
different.
One of the studies by Whittle et al. (1997) reported that mice primed with the
carrier strain demonstrated enhanced serum IgG titers against the foreign
antigen. This immune enhancement was observed up to approximately 6
months after priming. In their findings, they suggest that previous
immunological experience with Salmonella does not limit the immune
response to a foreign antigen carried by the same organism. In fact, prior
46
exposure to Salmonella appears to enhance the response to the foreign
antigen.
Vindurampulle et al. (2003) in his studies suggested that the factors such as
type of Salmonella strain and antigenic determinant that is vectored by the
bacterium, might have an influence on the outcome of the effect of prior
immunological memory. These authors did conclude that prior immune
responses against the bacterial vector may remain a serious limitation in
vectored vaccination. Similar concerns were also raised by Attridge et al.
(1997). However, as mentioned above there are contradictory reports that
indicate a positive effect of prior immunological responses against the
bacterial vector, therefore, it is probable that each vector and antigen
combination will need to be individually evaluated.
47
1.12 Conclusion
The use of attenuated Salmonella strains as vaccine delivery vehicles for
heterologous antigen has been extensively studied in a number of animal
species. The use of live attenuated Salmonella strains as a delivery vector to
express heterologous antigens to the secretory immune system constitutes a
promising approach for the development of new vaccines against a number of
diseases. These mutants are able to establish a limited infection in the host,
and during the natural course of infection, the bacterial vector deliver a series
of in vivo synthesized antigens directly to B and T lymphocytes.
RMIT University developed an attenuated Salmonella vaccine to protect
poultry, calves and adult cattle against Salmonella challenge. This vaccine is
a derivative of S. Typhimurium and is denoted as STM-1 (Salmonella
Typhimurium Mutant-1). The STM-1 mutant harbours an aroA mutation, which
confers complete, non-reverting aromatic biosynthesis (aro) defects (Alderton
et al.1981). The aroA mutation renders STM-1 non-virulent in respect to
invasive infection, as aromatic metabolites paraaminobenzoate (for
synthesising folate) and dihydroxybenzoate (for synthesising enterochelin) are
not available in host tissues (Smith et al. 1984). The STM-1 derivative has
also been used successfully for the delivery of DNA vaccines (Bachtiar et al.
2003).
48
The aim of this project was to evaluate the efficiency of STM-1 as a vector to
deliver heterologous antigen for vaccine purposes. Chicken ovalbumin was
used as a model antigen and Campylobacter jejuni major outer membrane
protein (Momp) as a test antigen to test the efficacy of STM-1 as a vector.
Two areas of concern which might influence the utility of this approach to
vaccine development were also investigated (i) stabilisation of antigen
expression in the Salmonella carrier (in chapters 4 and 5) and (ii) effect of pre-
existing immune responses against the bacterial vector (Chapter 6).
49
Chapter 2
Materials and Methods
2.1 General Procedures
All solutions were prepared in deionised H2O filtered through a Millipore Milli-
Q® water system unless otherwise specified. All media and glassware was
sterilised by autoclaving at 121°C for 20 mins unless otherwise specified. All
solutions from 0.02 µL to 10 mL were dispensed using Finnpipette
micropipettes for all volumes from above 10 mL were measured using
measuring cylinder. Glassware was washed using Pyroneg detergent followed
by a rinse in tap water then a final rinse in deionised H2O.
2.2 General Chemicals and Equipment
10X PCR Buffer Invitrogen, USA
10X Ligase Buffer Promega, USA
Acetic Acid, Glacial BDH Chemicals, Australia
Acrylamide-bis-Acrylamide (40%) BDH Chemicals, Australia
Acetone BDH Chemicals, Australia
Agarose (DNA Grade) Progen Industries, Australia
Albumin, Bovine serum Sigma-Aldritch Pty. Ltd., USA
Ammonium acetate Ajax Chemicals Ltd, Australia
50
Ammonium chloride BDH Chemicals, Australia
Ammonium hydroxide BDH Chemicals, Australia
Ammonium persulfate Bio-Rad Laboratories, USA
Ampicillin CSL, Melbourne
Anaerogen Oxoid Australia Pty. Ltd.
Bacteriological agar Oxoid Australia Pty. Ltd.
Balance
(i) Analytical Balance Sartorius GMBH, Germany
(ii) Balance (0.1-500g) U-Lab, Australia
Bromophenol blue BDH Chemicals, Australia
Balb/C mice ARC, Australia
Capillary tubes BectonDickenson & Co., USA
Carrier ampholytes Bio-Rad laboratories, USA
Cell counter Propper MFG Co., USA
Cell strainer BD Biosciences, USA
Centrifuge
(i) Microcentrifuge Zentrifugen, Germany
(ii) Bench Top Centrifuge Beckman, USA
(iii) High Speed Centrifuge Beckman, USA
Centrifuge Tubes
(i) 1.5 mL Eppendorf Tubes Sarstedt, Germany
(ii) 10 mL Centrifuge Tubes Greiner Labortechnik, Germany
(ii) 15 mL Centrifuge Tubes Greiner Labortechnik, Germany
(iii) 50 mL Centrifuge Tubes Greiner Labortechnik, Germany
4-Chloro-1-napthanol Sigma-Aldritch Pty. Ltd., USA
51
Coomassie brilliant blue R-250 Bio-Rad Laboratories, USA
Coomassie brilliant blue G-250 Bio-Rad Laboratories, USA
Copper sulfate Ajax Chemicals Ltd., Australia
Coverslips Mediglass, Australia
Criterion Bio-Rad Laboratories, USA
Cryovials (1.8 mL) Nalgene Company, USA
L-Cysteine BDH Chemicals, Australia
Deoxynucleoside triphosphates (dNTPs) Roche Diagnostics, Germany
DIG DNA Labelling and Detection Kit Roche Diagnostics, Germany
Dithiothreitol (DTT) Bio-Rad Laboratories, USA
DNA ligase (T4) Roche Diagnostics, Germany
DNA polymerase (Amplitaq) Perkin Elmer, USA
Dye-terminator Sequencing Mix V3.1 Monash University, Australia
Electrophoresis Units
(i) DNA
a) Mini-gel Pharmacia LKB, Sweden
b) Midi-gel Bio-Rad Laboratories, USA
c) Maxi-gel Pharmacia LKB, Sweden
(ii) Protein
a) Mini Protean II gel system Bio-Rad Laboratories, USA
b) Maxi Protean gel system Bio-Rad Laboratories, USA
c) Protean IEF Cell Bio-Rad Laboratories, USA
ELISA 96 well plate Nunc, Denmark
ELISPOT plates Millipore, USA
Ethylenediamine tetra acetic acid (EDTA) BDH Chemicals, Australia
52
Ethanol BDH Chemicals, Australia
Ethidium Bromide Roche Diagnostics, Germany
0.2µm, 0.45µm Filter (Acrodisc) Gelman Sciences USA
Folin-Ciocalteus’ reagent Ajax Chemicals Ltd., Australia
D-Glucose BDH Chemicals, Australia
Glycerol BDH Chemicals, Australia
Glycine BDH Chemicals, Australia
Goat anti-mouse IgG Bio-Rad Laboratories, USA
Goat anti-rabbit IgG Bio-Rad Laboratories, USA
HEPES Buffer Cytosystem Pty Ltd, Australia
Hydrochloric Acid (32%) v/v Eqicell products Pty Ltd,
Australia
Hydrogen peroxide (30%) BDH Chemicals, Australia
IL-4 capture antibody BD Pharmingen, USA
IL-4 biotin conjugated antibody BD Pharmingen, USA
Incubator, for tissue culture Forma Scientific, USA
IFN-γ capture antibody BD Pharmingen, USA
IFN-γ biotin conjugated antibody BD Pharmingen, USA
Imidazole Sigma-Aldritch Pty. Ltd., USA
Iodoacetamide Bio-Rad Laboratories, USA
IPTG Sigma-Aldritch Pty. Ltd., USA
Isoamyl alcohol BDH Chemicals, Australia
Isopropanol BDH Chemicals, Australia
Kanamycin Sigma-Aldritch Pty. Ltd., USA
53
Klenow enzyme Roche Diagnostics, Germany
Lambda DNA Pharmacia LKB, Australia
Lithium chloride BDH Chemicals, Australia
Low melting point agarose Progen Industries, Australia
Lysozyme Roche Diagnostics, Germany
Magnesium chloride Perkin Elmer, USA
Maleic acid BDH Chemicals, Australia
β-mercaptoethanol Bio-Rad Laboratories, USA
Methanol BDH Chemicals, Australia
Mulleur Hinton broth Oxoid Australia Pty. Ltd.
Mulleur Hinton agar Oxoid Australia Pty. Ltd.
Microscopes
(i) Light Microscope Olympus Optical
(ii) Phase Contrast Microscope Nikon Kogaku KK, Japan
Microscope slides LOMB scientific Co. Australia
Microtitre plate (96 well, flat bottom) Nunc, Denmark
NBT/BCIP substrate solution Roche Diagnostics, Germany
Needle (19g, 21g) Terumo Pty. Ltd., Australia
New Calf Serum Cytosystems Pty Ltd, Australia
Nitrocellulose membrane (Hybond-C) Amersham, USA
Nylon Membrane (Hybond-N) Amersham, USA
Parafilm Pechiney Plastic Packaging,
Inc., USA
Penicillin/Streptomycin Thermo Trace, USA
Petri Dish Nunc, Denmark
54
pH meter Radiometer, Denmark
Phenol BDH Chemicals, Australia
Phenol/Chloroform/Isoamyl Alcohol BDH Chemicals, Australia
Phenylmethylsulfonylfluoride Sigma-Aldritch Pty. Ltd., USA
Phosphate Buffered Saline (PBS) Oxoid Australia Pty. Ltd.
Polyethylene glycol 8000 Sigma-Aldritch Pty. Ltd., USA
Potassium acetate BDH Chemicals, Austalia
Potassium chloride BDH Chemicals, Australia
Potassium hydrogen carbonate BDH Chemicals, Australia
Restriction Enzymes
BamHI, EcoRI, KpnI, Promega, USA
SpeI, XhoI, PstI, Promega, USA
MfeI New England Biolab, USA
RNase Roche Diagnostics, Germany
RPMI Thermo Trace, USA
Salmon Sperm DNA Roche Diagnostics, Germany
SB-14 Bio-Rad laboratories, USA
SeeBlue Protein Standards Bio-Rad Laboratories, USA
Sheep blood Oxoid Australia Pty. Ltd.
Silver nitrate Sigma-Aldritch Pty. Ltd., USA
Skim milk Bonlac Foods Ltd., Australia
Sodium chloride BDH Chemicals, Australia
Sodium citrate BDH Chemicals, Australia
Sodium dodecyl sulphate (SDS) BDH Chemicals, Australia
Sodium hydroxide BDH Chemicals, Australia
55
Sodium lauryl sulfate BDH Chemicals, Australia
Sonicator Branson Sonic Power Co.,
USA
Spectinomycin UpJohn, Australia
Sucrose BDH Chemicals, Australia
Sulfuric Acid BDH Chemicals, Australia
Syringe (1 mL, 3 mL, 5 mL, Terumo, Pty. Ltd., Australia
10 mL, 20 mL, 50 mL)
TEMED Bio-Rad laboratories, USA
TMB substrate reagent BD Pharmingen, USA
Tissue Culture Flask (25 cm2, 50 cm2, 75 cm2) Nunc, Denmark
Trans-blot electrophoretic transfer cell Bio-Rad laboratories, USA
Transilluminator (UV) Bio-Rad laboratories, USA
Tributylphosphine Sigma-Aldritch Pty. Ltd., USA
Tris-Base Roche Diagnostics, Germany
Tris-HCl Roche Diagnostics, Germany
Triton-X 100 Sigma-Aldritch Pty. Ltd., USA
Triton-X 114 Sigma-Aldritch Pty. Ltd., USA
Trypan Blue Sigma-Aldritch Pty. Ltd., USA
Trypticase soya broth Oxoid Australia Pty. Ltd.
Tryptone Oxoid Australia Pty. Ltd.
Tween® 20 Sigma-Aldritch Pty. Ltd., USA
Yeast extract Oxoid Australia Pty. Ltd.
Whatman paper Whatman, England
X-Gal Sigma-Aldritch Pty. Ltd., USA
56
XLD Medium Oxoid Australia Pty. Ltd.
Xylene cyanol Bio-Rad Laboratories, USA
57
2.3 Bacteriological Methods
2.3.1 Media and Antibiotics
2.3.1.1 Antibiotic stock solutions
Ampicillin. Ampicillin (500 mg/vial) was dissolved in 5 mL of sterile water,
giving a stock solution of 100 mg/mL. One mL aliquots were stored frozen at
–20°C. The ampicillin was added to media or other solution at the final
concentration of 100 µg/mL.
Kanamycin. Kanamycin was made up as a stock solution of 50 mg/mL in
sterile water and stored in 1 mL aliquots at -20°C. The working concentration
of Kanamycin was 50 µg/mL.
Gentamycin sulfate. Gentamycin sulfate was made up as a stock solution of
10 mg/mL in sterile water and stored in 1 mL aliquots at -20°C. The working
concentration was 50 µg/mL
Tetracycline. Crystalline tetracycline was made up as a stock solution of 50
mg/mL in sterile water and stored at -20°C. The working concentration was
50µg/mL.
58
2.3.1.2 Luria Bertoni Agar
Tryptone (1% w/v), Yeast Extract (0.5% w/v), NaCl (0.5% w/v) and Bacteriological
Agar (1% w/v) were dissolved in deionised H2O and autoclaved at standard
conditions.
2.3.1.3 Luria Bertoni Broth
Tryptone (1% w/v), Yeast Extract (0.5% w/v), and NaCl (0.5% w/v) was dissolved in
deionised H2O and autoclaved at standard conditions.
2.3.1.4 Muller Hinton Agar
Muller Hinton Agar (2.1% w/v) powder was dissolved in deionised H2O and
autoclaved at standard conditions, and then dispensed into petri dishes.
2.3.1.5 Muller Hinton Broth
Muller Hinton Broth powder was dissolved in deionised H2O (2.1% w/v) and
autoclaved at standard conditions.
2.3.1.6 Horse Blood Agar
Trypticase Soya Broth (3% w/v) and Bacteriological Agar (1% w/v) were
dissolved in deionised H2O and autoclaved at standard conditions. After the
agar had cooled, Horse blood (5% w/v) was added to the agar and the
medium poured into petri dishes.
59
2.3.2 Bacterial Strains and Plasmids
The bacterial strains and plasmids used in this study are listed in Table 2.1
and 2.2.
Table 2.1. Bacterial strains and bacteriophage used in this study.
Bacterial Strain/plasmids Genotype or description Reference/Source
Escherichia coli BW25141
BW25113
SY327
∆(araD-
araB)567,∆lacZ4787(::rrnB-
3),lacIp-4000(lacIQ),∆(phoB-
phoR)580,λ-
,galU95,∆uidA3::pir+,
recA1,endA9(del-ins)::FRT,rph-
1, ∆(rhaD-rhaB)568, rrnB-3,
hsdR514
∆(araD-araB)567,
∆lacZ4787(::rrnB-3), lacIp-
4000(lacIQ), rph-1, ∆(rhaD-
rhaB)568, hsdR514
F araD ∆(lac-proAB)
argE(Am) rif nalA recA56
[Datsenko and Wanner, 2000] [Datsenko and Wanner, 2000] [Goldberg, 1986 ]
DH5α supE ∆lacU169 (ф80
lacZ∆M15) hsdR17 recA1
endA1 gyrA96 thi-1 relA1
[Hanahan, 1983 ]
60
TOP10F’ F' [lacIq, Tn10(TetR)], mcrA,
D(mrr-hsdRMS-mcrBC),
f80lacZDM15, DlacX74, deoR,
recA1, araD139 D(ara-
leu)7697, galK, rpsL(StrR),
endA1, nupG.
Invitrogen
Salmonella Enteritidis
SEM2 ∆aroA ,∆aroD [Moutafis, 1996]
Salmonella Typhimurium LT2-9121 leu hsdL trpD2 rpsL120 ilv452
metE551 metA22 hsdA hsdB
Prof P. Reeves,
Department of Microbiology,
University of Sydney
82/6915 Wild type parent strain of STM-
1
RMIT Biotechnology Dept
STM-1 ∆aroA- ∆serc- RMIT Biotechnology Dept
In the table below antibiotics are abbreviated as Amp, Ampicillin; Kan,
Kanamycin; Tet, Tetracycline. Resistance to antibiotic is abbreviated as R and
sensitivity is abbreviated as (S). N/A denotes information not available.
61
Table 2.2. Plasmids used in this study.
Vector Description Reference/Source pBluescript IIKS 2.96 kb phagemid, LacZ, AmpR Stratagene, USA
pCR2.1
3.9 kb PCR cloning plasmid, AmpR,
KanR 3’ T-overhang
Invitrogen, Australia
pCRMomp pCR2.1 containing the C. jejuni major
outer membrane gene
This study
pCRnirB pCR2.1 containing nirB promoter This study
pCRova pCR2.1 containing the chicken
ovalbumin gene
This study
pCRpagC pCR2.1 containing pagC promoter This study
pCRnirmo pCR2.1 containing the C. jejuni major
outer membrane gene under the
control of the nirB promoter
This study
pCRnirova pCR2.1 containing the chicken
ovalbumin gene under the control of
the nirB promoter
This study
pCRpagmo pCR2.1 containing the C. jejuni major
outer membrane gene under the
control of the pagC promoter.
This study
pCRpagova pCR2.1 containing the chicken
ovalbumin gene under the control of
the pagC promoter
This study
pMW2 4.4kb phagemid, LacZ, AmpR, KanR
pMnirmo pMW2 containing the C. jejuni major This study
62
outer membrane gene under the
control of the nirB promoter
pMnirova pMW2 containing the chicken
ovalbumin gene under the control of
the nirB promoter
This study
pMpagmo pMW2 containing the C. jejuni major
outer membrane gene under the
control of the pagC promoter.
This study
pMpagova pMW2 containing the chicken
ovalbumin gene under the control of
the pagC promoter
This study
pKD13 3.4kb plasmid, oriR6Kγ, tL3λ(Ter),
bla(ApR), kan, rgn(Ter)
[Datsenko and
Wanner 2000].
pKnirmo
pKD13 plasmid carrying the C .jejuni
major outer membrane gene under
the control of the nirB promoter
This study
pKnirova
pKD13 containing the chicken
ovalbumin gene under the control of
the nirB promoter
This study
pKpagmo pKD13 plasmid carrying the C. jejuni
major outer membrane gene under
the control of the pagC promoter
This study
pKpacova pKD13 containing the chicken
ovalbumin gene under the control of
the pagC promoter
This study
63
pKD46 6.3kb plasmid, repA101(ts), araBp-
gam-bet-exo, bla(ApR), oriR101
[Detsenko and
Wanner 2000]
64
2.3.3 Storage of Bacterial Strains and Phage
Long term. All bacterial strains were stored at -70°C using the Protect bead
storage system (Technical Service Consultants Ltd., United Kingdom).
Short term. All bacterial strains were stored on LB or MH agar plates at 4°C.
The bacteria were passaged every 4 weeks from single colonies.
2.3.4 Bacterial Culture Conditions
All E. coli and Salmonella strains were grown on solid microbiological media
under aerobic conditions at 37°C for 16 h. In instances where broth cultures
were used, the strains were grown aerobically at 37° C for 16 h on a Ratek
orbital shaker at 120-150x g.
2.4 DNA Methods.
2.4.1 DNA Extraction Techniques.
2.4.1.1 Buffers and Reagents
6x Loading Buffer. Sucrose (40% w/v), Bromophenol Blue (0.25% w/v) and
Xylene Cyanol (0.25% w/v) dissolved in deionised, sterile H2O.
65
Anti-DIG Antibody Solution. Anti-DIG antibody (1:5000 Dilution) in Blocking
solution.
Alkaline Lysis Solution I. 50mMGlucose, 10mM EDTA, 25mM Tris-Cl pH 8.
Alkaline Lysis Solution II. 0.2N NaOH, 1% SDS.
Alkaline Lysis Solution III. 5M Potassium acetate.
Blocking reagent Stock Solution. Blocking Reagent (10 g) was added to
100 mL Maleic Acid Buffer. The Blocking Reagent was dissolved on a heat
plate for 1 h at 65°C with constant stirring. After the Blocking Reagent has
completely dissolved, the solution was autoclaved and stored at 4°C.
Blocking Solution. 1% Blocking reagent (From 10% Stock Solution) diluted
in Maleic Acid Buffer.
CTAB/NaCl. Dissolved 4.1 g of NaCl in 80 mL H2O and added 10 g CTAB
while heating to 65°C.
Detection Buffer. Tris-HCl (100mM, pH 9.5) and 100 mM NaCl dissolved in
dH2O.
Denaturation Solution. Sodium Hydroxide (0.5 M) and Sodium Chloride (1.5
M).
66
dissolved in dH2O.
Depurination Solution. 0.25 M HCl diluted in dH2O.
ESP solution. EDTA (0.5 M), N-lauryl sarcosine (1% w/v) and Proteinase K
(1 mg/mL), dissolved in H2O.
Fluoride/TE solution (PMSF). Phenyl methyl sulfonyl fluoride (18.6 mg/mL)
was added to 1 mL of isopropanol. TE (25 mL) was added aseptically, and
the solution was warmed at 37°C for 15 min.
FSB solution. MnCl2·4H2O (45 mM), CaCl2·2H2O (10 mM), KCl, (100 mM),
Hexamminecobalt Chloride (3 mM), Potassium Acetate (10 mM), Glycerol
(10% v/v) dissolved in H2O, pH to 6.4, filter sterilised, and stored at 4°C.
Isoproply-β-D thiogalactopyranoside (IPTG) (0.1 M) Stock Solution. IPTG
(1.2 g) was dissolved in 50 mL of sterile water, filter sterilised and stored at
4°C.
Maleic Acid Buffer. Maleic Acid (0.1 M) and Sodium Chloride (0.15 M)
dissolved in dH2O and autoclaved.
Maleic Acid Wash Buffer. Tween® 20 (0.3% w/v) in Maleic Acid Buffer.
67
Neutralisation solution. Tris-HCl (0.5 M ,pH 7.5) and Sodium Chloride (3 M)
dissolved in dH2O.
PFGE lysis buffer. Tris-HCl (6 mM), NaCl (1 M) and EDTA (100 mM)
dissolved in dH2O. Store at 4C and sterilise before use. To complete buffer
prior to use, add Sodium Deoxycholate (0.2% w/v), N-lauryl sarcosine (0.5%
w/v) and Lysozyme (1 mg/mL).
PIV buffer. Tris base (10mM) and NaCl (1 M) dissolved in deionised H2O, pH
8.0, and autoclaved.
Standard Hybridisation Buffer. SSC (5X), 0.1% N-lauroylsarcosine, 0.02%
SDS and 1% Blocking Reagent (From 10% stock solution) diluted in dH2O.
20X SSC. Sodium Chloride (3 M) and Sodium Citrate (0.3 M) dissolved in
dH2O, adjusted to pH 7.0 and autoclaved.
2X SSC wash buffer. SSC (2X) and 0.1% SDS diluted in dH2O.
0.5X SSC wash buffer. SSC (0.5X ) and 0.1% SDS Diluted in dH2O.
Substrate Solution. 200 µL NBT/BCIP substrate solution diluted in 10 mL of
detection buffer.
68
TAE Buffer 50X. Tris-base (24.2% w/v), glacial acetic acid (5.17% w/v) and
EDTA (1.86% w/v) were dissolved in deionised water. This was diluted to 1X
before use.
TBE Buffer 5X. Tris base (0.445 M), Boric acid (0.445 M) and EDTA (12.5
mM) dissolved in H2O and adjusted to pH 8.0.
TE buffer. Tris-base (10 mM) and EDTA (1 mM) was dissolved in water. The
pH was adjusted as required with HCl then autoclaved.
X-gal Stock solution. X-gal (100 mg) was dissolved in 5 mL of N,N’-
dimethylformamide and stored at –20°C.
69
2.4.1.2 Small Scale Plasmid DNA Extraction Using Qiaprep
Mini Spin Kit
Small-scale plasmid DNA extraction was achieved by using the Qiaprep Mini
Spin Kit (Qiagen, Germany) as per the manufacturer’s instruction. The
purification procedure was performed according to the manufacturer’s
instructions.
Briefly, 1.5 mL of an overnight culture in LB broth with the appropriate
antibiotic was collected by centrifugation at 5,000 x g for 5 min. The bacterial
pellet was resuspended in 250 µL of buffer P1. Two hundred and fifty
microlitres of buffer P2 was then added and the microcentrifuge tube was
inverted several times and incubated for no more than 5 minutes at RT to lyse
the cells. Protein and chromosomal DNA were precipitated with the addition
of 350 µL of buffer N3 and then the supernatant was collected after
centrifugation for 10 min at 14,000 x g. The supernatant was then applied to
a QIAprep spin column and spin for 30 s in the centrifuge and the flow-
through was discarded. The DNA in the spin column was washed with 750 µL
of wash buffer PE and spin for 30 s in the centrifuge. The flow through was
discarded and the residual wash buffer was removed from the column after
additional 1 min centrifugation. The QIAprep column was placed in a new
microcentrifuge tube and the DNA was eluted with 50 µL of 10 mM Tris, pH
8.0
70
2.4.1.3 Alkaline Lysis Miniprep
The alkaline lysis method was used as an alternative to the Qiaprep method
to isolate plasmids for the purpose of enzyme restriction analysis and is based
on the method of Birnboim and Doly (1979). Five millilitres of LB broth
containing the appropriate antibiotic was inoculated with a single colony of
bacteria and grown overnight on a shaker at 37°C. One and a half millilitres
of the culture was placed in a microcentrifuge tube and centrifuged at 5,000 x
g for 5 min. The pellet was resuspended in 100 µL of ice-cold alkaline lysis
solution I and incubated at room temperature for 5 min followed by lysis with
200 µL of alkaline lysis solution II. Chromosomal DNA and protein were
precipitated with 150 µL of ice-cold alkaline lysis solution III , the tube was
inverted several times and cooled on ice for 5 min. The supernatant was
collected after mixture the centrifugation of mixture for 10 min at 14,000 x g
and the supernatant was then transferred to a new microcentrifuge tube. An
equal volume of phenol/chloroform (24 chloroform: 1 isoamyl alcohol) was
added and the contents mixed vigorously by inversion. The supernatant was
collected after the centrifugation at 14,000 x g for 3 min. The DNA was
precipitated with an equal volume of isopropanol and was collected by
centrifugation for 10 min at 14,000 x g. The DNA pellet was washed with 1 mL
of 70% ethanol and then air-dried by placing the tube upside down on tissue
paper for 15 min. The DNA pellet was finally resuspended in 50 µL of 10 mM
Tris, pH 8.0
71
2.4.1.4 Small-Scale Genomic DNA Extraction
Genomic DNA was prepared using the procedure described in Current
Protocols in Molecular Biology. Briefly, 1.5 mL of an overnight culture of
bacteria was collected after centrifugation at 14,000 x g for 2 min. The pellet
was resuspended in 567 µL of TE buffer, 30 µL of SDS and 3 µL of 20 mg/mL
proteinase K. The tube was mixed thoroughly and incubated at 37°C for 1 h.
One hundred microlitres of 5 M NaCl was added and mixed thoroughly
followed by the addition of 80 µL of CTAB/NaCl. The tube was incubated at
65°C for 10 min. An equal volume of CI was added and after mixing the tube
centrifugation was carried at 14,000 x g for 5 min. The aqueous phase was
removed and mixed with an equal volume of PCI, the centrifugation was
carried at 14,000 x g for 5 min. The aqueous phase was again removed and
the DNA was precipitated by combining with 0.6 volumes of isopropanol. The
DNA pellet was collected by centrifugation in a microcentrifuge at 14,000 x g
for 10 min. The pellet was rinsed with 70% ethanol and dried upside down on
a piece of absorbent paper for 15 min. The pellet was resuspended in 100 µL
of 10 mM Tris, pH 8.0
2.4.1.5 Large-Scale Genomic DNA Extraction
This procedure is described in Current Protocols in Molecular Biology and is
essentially a scale-up of the small-scale genomic extraction described above.
One hundred millilitres of bacterial culture was grown in LB, at 37°C overnight.
The bacteria were collected by centrifugation at 4,000 × g using a Beckman
JA-20 rotor. The cells were resuspended in 9.5 mL TE buffer, to which 0.5
72
mL of 10% SDS and 50 µL of 20 mg/mL proteinase K was added. The
solution was mixed and incubated at 37°C for 1 h. Subsequently, 1.8 mL of 5
M NaCl was added followed by 1.5 mL of CTAB/NaCl solution. The mixture
was incubated at 65°C for 20 min after which an equal volume of CI was
added. The solution was mixed thoroughly and the phases were separated
by centrifugation at 6,000 × g for 10 min at RT. The aqueous phase was
transferred to a fresh tube and 0.6 volumes of isopropanol was added. The
DNA precipitate was transferred into a fresh tube containing 1 mL of 70%
ethanol by using a hook made from a Pasteur pipette that had been bent and
sealed in a Bunsen flame. The DNA was collected by centrifugation at 10,000
× g for 5 min. The supernatant was removed and the pellet was dried. Finally
the pellet was resuspended in 10 mM Tris, pH 8.0.
73
2.4.1.6 Ethanol precipitation
This method was used to concentrate DNA samples by decreasing the total
volume. One-fifth of the sample volume of 10 M ammonium acetate and 2.5
volumes of ice-cold absolute ethanol were added to the DNA preparation.
The sample was stored at -70° C for 30 min. The DNA was collected by
centrifugation in an Eppendorf microcentrifuge at 9000 × g for 15 min. The
pellet was washed twice with 70 % (v/v) ethanol and was then air-dried. The
pellet was resuspended in 10 mM Tris, pH 8.0.
2.4.1.7 Purification of DNA from agarose
Purification of DNA from agarose gel using the Geneclean kit was performed
according to the manufacturer’s instruction. Briefly, the DNA band was
excised from the gel and weighed. Sodium iodide (~5 M, 3 gel volumes) and 5
µL of 3 M sodium acetate (pH 5.2) was added to the gel slice, and the
agarose was melted at 55°C. Glassmilk (5 µL) was added, mixed by vortexing
and allowed to stand for 5 min at room temperature with frequent agitation.
The glassmilk was recovered by centrifugation at 10000 x g for 30 s. The
glassmilk was washed 3 times with 500 µL of New Wash solution, and
recovered by centrifugation at 10000 x g for 30 s. To recover the DNA, the
Glassmilk was dried then resuspended in 10µL of dH2O. After 5min at room
temperature, the Glassmilk was collected by centrifugation at 10000 x g for 2
min and the dH2O collected.
74
2.4.1.8 Purification of PCR amplified DNA
The PCR amplified DNA was purified using Geneclean kit according to
manufacturer’s instruction. The volume of the solution was calculated. Sodium
iodide (~5 M, 3 times the volume) and 5µL of 3M sodium acetate (pH 5.2) was
added to the solution. Glassmilk (5 µL) was added, mixed by vortexing and
allowed to stand for 5 min at room temperature with frequent agitation. The
glassmilk was recovered by centrifugation at 10000 x g for 30 s. The glassmilk
was washed 3 times with 500 µL of New Wash solution, and recovered by
centrifugation at 10000 x g for 30 s. To recover the DNA, the glassmilk was
dried then resuspended in 10 µL of dH2O. After 5 min at RT, the glassmilk
was collected by centrifugation at 10000 x g for 2 min and the dH2O collected.
2.4.1.9 Determination of DNA concentration
DNA concentration and purity was determined by measuring the absorbance
of the DNA sample, diluted 1/200, in matching quartz cuvettes at 260 nm
using a Schimadzu UV-160 spectrophotometer. An OD260 of one is equal to a
DNA concentration of 50 µg/mL. Protein contamination of the DNA was
determined by measuring at a second wavelength of 280nm. An OD260/OD280
ratio was used to measure the quality of the DNA. Ratios of 1.8-2.0 indicated
good quality DNA.
75
2.4.2 Polymerase chain Reaction.
2.4.2.1 Primer Design.
Primers were designed using Sci Ed Central Primer Designer 4 V 4.2. Primers
were designed to have a %GC content between 40-60% with a Tm˚C in the
range of 50-80˚C.
2.4.2.2 General PCR procedure.
The PCR amplifications were carried out in 25 to 50 µL reactions containing:
2-4 U Taq DNA polymerase, 1.5 to 2.0 mM MgCl2, 50 mM KCl, 10 mM Tris-Cl
(pH 9.0), 0.2 mM dNTPs, 50 ng to 100 ng genomic DNA or 0.1 ng to 1 ng
plasmid DNA and 0.2 mM of each primer. The primers used in this
investigation are listed in Table 2.4. The PCR cycling conditions were as
follows:
Number of cycles Temperature Duration
1 (denaturation) 94°C 2-5 min
30-35 (denaturation) 94°C 30 s
30-35 (annealing) 50-65°C 30 s
30-35 (extension) 72°C 30 s/kb
1 (extension) 72°C 10 min
76
2.2.2.3 Expand Long Template PCR.
The Expand Long Template DNA polymerase was either used to amplify DNA
segment >4 kb in size or where proof reading activity was required. The PCR
amplifications were carried out in 25 to 50 µL reactions containing: 2-4 U
Expand Long Template DNA polymerase, Either buffer1 or buffer3, 0.2 mM
dNTPs, 50 ng to 100ng genomic DNA or 0.1ng to 1ng plasmid DNA and 0.2
mM each primer. The primers used in this investigation are listed in Table 2.4.
The PCR programme for the amplification of a DNA segment was as follows:
Number of Cycles Temperature Duration
1 (denaturation) 94°C 2-5 min
10 (denaturation) 94°C 10 s
10 (annealing) 50-65 30 s
10 (extension) 68°C 1min/kb
25 (denaturation) 94 10s
25 (annealing) 50-65 30s
25 (extension) 68oC 1min/kb
1 (extension) 68°C 10-15 min
77
2.4.2.4 DNA sequence analysis.
All DNA sequencing was carried out using the ABI PRISM BigDye Terminator
Cycle Sequencing Ready Reaction Kit according to the manufacturer’s
instructions using the following reagents:
Reagent Volume
Terminator Ready Reaction Mix 2.0 µL
Buffer 3.0µL
Template DNA
Double stranded DNA
or
PCR product
x µL (200-500 ng)
x µL (30-90 ng)
Primer (100 ng/mL) 0.5 µL
Milli-Q H2O x µL
Total volume 25.0 µL
78
The PCR programme used for the sequencing of a DNA segment was as
follows:
Number of cycles Temperature Duration
25 94 10s
25 50 5s
25 60 4min
79
Table 2.3. PCR primers used in this study
Primer
Name
Sequence 5’→ 3’ Amplifies/
Purpose
Reference Tm°C *
AROA3.5K
BFWD
TGCTTCTCACTGGTCG
CACTGTCCG
aroA 3.5 kb
downstrean
This study 72
AROAEND AGGCGTACTCATTCG
CGCCAGTTG
aroA end This study 54.7
AROA170B
P REV
CAGGGCATTGAGCAT
ATGGCG
aroA 170 bp
downstream
rev
This study 66
SERCFWD TGACAGACGCTCGAA
GTATTCGCC
serC gene This study 68
YCAP FWD TTGCAACGAATGGCG
CTGGATAACG
ycaPgene This study 70
YCAP REV TTAGCCCGGCTTGCC
TTCGTCCA
ycaPgene This study 72
YCAO FWD AAGACGCCGCGCTGG
AAGACTCCAT
ycaO gene
fwd
This study 74
YCAO REV TTTGACCAGTATGCCG
CTTTCGC
ycaO gene
rev
This study 68
FCAO FWD ATAGACGCCTGCTTCT
TCAGCC
fcaO gene This study 64
PFLB FWD GCGTGAAGGTACGAG
TAATAACGTCC
pflB gene This study 67
80
PFLB REV CACTCCGTATGAGGG
TGACGAGTCC
pflB gene This study 69
SOPD FWD GCGTCGCATTCAACG
CGCAATCAGG
sopD gene This study 73
SOPD REV AGGCTCCATATCAGT
GGGGC
sopD gene This study 65
GTRA FWD GAGCGCCGTTGTTGG
CTGGATGG
gtrA gene This study 72
GTRA REV TAGCAGCATTCGCGC
AGCCTGCC
gtrA gene This study 74
RPSA REV TCCAGGTGCAGAGTA
TCACGCACCG
rpsA gene This study 72
MOMP
FWD
TATTAGATCTTGACAA
GGAGAATTCTCATG
C. jejuni
major outer
membrane
gene
This study 62
MOMP REV GAGCGCTAGCCCGCC
TTGAAGTTAGCTTG
C. jejuni
major outer
membrane
gene
This study 74
OVA FWD GGAATTCCATGGGCT
CCATCGGTGCAG
Chicken
ovalbumin
This study 73
OVA REV GGAATTCTGGGTCTT
GTTGGAAGGG
Chicken
ovalbumin
This study 67
PAGFWD CTCTGGATCCTAATG pagC gene [Dunstan, 64
81
GGTTTATAGCG promoter 1999]
PAGCREV AATAGATCCAATTGCA
ACTCCTTAAT
pagC gene
promoter
[Dunstan,
1999]
60
NIRBFWD GCCGGATCCTTGAAC
ATAGTGGTCCGC
nirB gene
promoter
[Dunstan,
1999 ]
71
NIRBREV GACTTTGCCAATTGTT
GCCTCGATTC
nirB gene
promoter
[Dunstan,
1999]
67
M13 REV CAGGAAACAGCTATG
AC
Sequencing
primer
44.5
M13 –20 GTAAAACGACGGCCA
G
Sequencing
primer
45.8
* Melting temperature was calculated by S.E central software.
Green sequence indicates an EcoRI site.
Blue sequence indicates a MfeI site.
Purple sequence indicates a BamHI site.
.
82
2.4.3 Recombinant DNA techniques
2.4.3.1 Cloning of PCR product
The PCR amplified genes were inserted into pCR2.1 according to the
manufacturer’s instruction. Briefly, the PCR product was analysed by gel
electrophoresis and the concentration of DNA estimated. The PCR product (1
ng) was mixed with 10x ligation buffer (1 µL), pCR2.1 Vector (2 µL), T4 DNA
Ligase (1 µL), and the reaction was made up to 10 µL with dH2O. The ligation
was incubated overnight at 16°C. After brief centrifugation of the ligation
reaction the tubes were placed on ice. Vials of One Shot® competent cells
were thawed on ice. The cells were mixed with 2 µL of the ligation, and
incubated on ice for 30 min. The cells were then heat shocked for 30 s in a
42°C water bath.
Following transformation, 250 µL of SOC was added to the vial, and the cells
were allowed to recover at 37°C for 1 h. Transformations (10 µL and 100 µL)
were then spread plated onto LB agar containing X-gal and either 50µg/mL
Kanamycin or 100 µg/mL Ampicillin. Plates were incubated overnight at 37°C,
and white colonies were selected for analysis.
2.4.3.2 Restriction Enzyme Digestion
Table 2.4 summarises the restriction endonucleases and their DNA
recognition sequences used in this study. Usage of all enzymes was
according to manufacturer’s instructions
83
DNA was digested using the following formula:
Reagent Volume
DNA 100 ng
10X Buffer 4 µL
RE 1 U
H2O Up to 40 µL
Digestions were performed in a total volume of 40 µL and incubated at 37°C
from 4 h to overnight
84
Table 2.4. Restriction endonucleases and their DNA recognition
sequences used in this study
Restriction
Endonuclease
Recognition Sequence
BamHI G/GATCC
DpnI GA/TC
EcoRI G/AATTC
EcoRV G/ATATC
KpnI GGTAC/C
MfeI C/AATTG
NotI GC/GGCCGC
PstI CTGCA/G
SacI GAGCT/C
XbaI T/CTAGA
XhoI C/TCGAG
2.4.3.3 Phophatase Treatment of vectors
Plasmids were treated with alkaline phosphatase incubating 1 µg of digested
plasmid DNA with 0.05 U of calf-intestinal alkaline phosphatase in the
appropriate buffer for 1 h. The reaction was stopped by adding 0.5 M EDTA
and heat inactivating the enzyme at 65°C for 15 min.
85
2.4.3.4 Ligation of DNA.
DNA was ligated using the following formula:
Reagent Volume
Plasmid 100 ng
Insert DNA Variable
10 Ligase Buffer 1 µL
T4 DNA ligase 1 U
Ligations were made up to 10 µL using dH2O. The total amount of insert DNA
was calculated using the following formula.
ng of vector X size of insert (kb) X 3 = ng of insert
size of vector (kb) 1
2.4.3.5 Transformation of DNA
2.4.3.5.1 Preparation of Competent Cells
Bacterial cells were grown in 10 mL of LB broth on a shaker at 37°C for 16 h.
Two millilitres of this culture was used to inoculate 200 mL of pre-warmed LB
broth in a flask and shaken vigorously at 37°C until approximately mid-log
phase, an absorbance at 600 nm of 0.4-0.5, was reached. The cells were
chilled on ice for 30 min and collected by centrifugation at 4000 × g for 10 min
at 4°C. The supernatant was completely drained off before the cells were
washed in 200 mL of ice-cold H2O and collected by centrifugation at 4000 × g
for 10 min. This step was repeated with 100 mL of ice-cold H2O. The cell
pellet was resuspended in 10 mL of ice-cold 10 % glycerol and centrifuged as
86
before. The cells were resuspended in a final volume of 1 mL of ice-cold 10 %
glycerol and divided into aliquots of 40-50 µL each and stored at -70°C.
2.4.3.5.2 Electroporation of Competent Cells
Frozen electro-competent cells were thawed on ice for 15 min and mixed with
DNA in a volume of up to 2 µL and transferred to an ice-cold electrocuvette
with a 0.2 cm gap. The pulse settings used to deliver DNA into the cells were
2.5kV, 25mF and 200Ω. After the pulse, 1 mL of SOC medium was
immediately added to the cells and they were incubated at 37°C for 1 h. One
hundred microliters of transformed culture was plated out onto LB agar
containing the appropriate antibiotic or X-gal plates. The remaining
transformation mixture was concentrated by centrifugation at 5000 x g for 5
min and resuspending in 100 µL of SOC medium. This was also plated onto
LB containing the appropriate antibiotic.
2.4.3.5.3 Chemical Transformation
A 200 mL LB broth was inoculated with 2 mL of an overnight culture. The 200
mL culture was grown for 2h-3h at 37°C until approximately mid-log phase,
until absorbance at 600 nm was 0.4, was reached. Cells were harvested by
centrifugation at 5000 x g for 20min at 4°C. Cells were washed in 40mL of ice-
cold FSB. After centrifugation at 5000 x g for 20 min at 4°C, cells were
resuspended in 2 mL of FSB. Cells were dispensed in aliquots of 200 µL into
microfuge tubes. The fresh competent cells were used for the ligation. Five to
ten microliter of the ligation reaction or 50 ng of plasmid DNA was added to
200 µL competent cells. The cells were mixed by pipetting, and incubated on
87
ice for 30 min. To perform the tranformation, competent cells were transferred
to a 42°C water bath for 2 mins. The transformations were incubated on ice
for 2 min, and then 1 mL of LB broth was added. The transformants were
allowed to recover at 37°C for 1 h, 100 µL of the culture was spread onto an
LB agar plate containing the appropriate antibiotic and incubated at 37°C
overnight.
2.4.3.6 Chromosomal Integration of Constructs.
2.4.3.6.1 Lambda Red System
STM-1 was transformed with the pKD46 plasmid after the plasmid was
passaged through the LT2 strain. Fresh STM-1/pKD46 competent cells were
prepared by growing STM-1/pKD46 overnight in 100 µg/mL ampicillin at 30oC.
One hundred millilitres of overnight culture was used to inoculate 100 mL MH
broth containing 50 mM L-arabinose and 100 µg/mL ampicillin. The culture
was grown at 30oC until approximately mid-log phase, an absorbance at 600
nm of 0.4-0.5, was reached. The competent cells were prepared according to
the method described in section 2.4.4.1. The pKD13 plasmid in which all the
constructs were ligated was used as template to generate PCR products (see
chapter 4). The PCR product was digested with DpnI restriction enzyme to
remove any residual plasmid template. Fifty to one hundred nanograms of
purified PCR product was used to transform fresh STM-1/pkD46 competent
cells. The pulse settings used to deliver DNA into the cells were 2.5 kV, 25
mF and 200 Ω. After the pulse, 1 mL of SOC medium was immediately added
to the cells the transformants were allowed to recover at 30oC for 2 h. One
88
hundred microlitres of the culture was spread onto an MH agar plate
containing 50 µg/mL kanamycin and incubated at 37°C overnight. The
remaining transformation mixture was concentrated by centrifugation at 5000
x g for 5 min and resuspended in 100 µL of SOC medium. This was also
plated onto MH agar plate containing 50 µg/mL kanamycin.
2.4.3.7 Pulsed Field Gel Electrophoresis.
STM-1 and the parent strain was inoculated in 5 mL LB and grown at 37°C for
3-4 h. The bacterial cells were collected after centrifugation. The cells were
washed in PBS. The cells resuspended in 250 µL of PIV buffer on ice.
The cell suspension was mixed with an equal volume of 2% low melting point
agarose (dissolved in PIV buffer) and poured into sterile, pre-chilled moulds.
The plugs were allowed to set, then placed in 0.5 mL PFGE lysis buffer
overnight at 37°C. The following day, the plugs were allowed to harden at 4°C
for 20 min, placed in 0.5 mL of ESP solution and incubated at 50°C overnight.
The following day, the plugs were hardened at 4°C for 20 min, and then
incubated in 0.5 mL of Fluoride/TE solution at 37C for 2 h. The plugs were
then washed 6 times in TE buffer over 6 h at 37°C. Plugs were stored in TE
Buffer at 4°C for up to 6 months.
A portion of the DNA plug was cut and washed in 1 X RE buffer. The plug
portion was then placed in 80 µL of restriction enzyme solution, consisting of
2.5 µL (25U) of XbaI, 8 µL of 10 X RE buffer, 5 µL of 1 mg/mL bovine serum
89
albumin (BSA) and 64.5 µL of sterile deionised H2O. The DNA was digested
for 6-8 h at 37°C. One percent agarose midigel was set in 1X TBE. Following
restriction enzyme digestion, the DNA plugs were inserted into the wells of the
set, chilled agarose gel and the wells were sealed with the 2% low melting
molten agarose. The agarose gel was then subjected to PFGE at 220 V for 22
h with the pulse time ramped from 2.2-63.8 s. The gel was stained in ethidium
bromide and examined by UV transillumination.
2.4.3.8 Southern Blotting
2.4.3.8.1 Probe Synthesis
The aroA, pflB and gtrA gene products were labelled as probes. The genes
were amplified using S. Typhimurium genomic DNA as template and the PCR
product was purified. The labelling was performed using the DIG DNA
Labelling and Detection kit based on the random priming system. All
procedures were carried out according to the manufacturer’s instructions
using digoxigenin (DIG)-dUTP as the label. The concentration of DNA was
estimated and 3 µg of template was diluted to 15 µL with dH20. The mixture
was heated to 100°C to denature the template and then chilled on ice.
Klenow enzyme (1 µL) and 10x DNA labelling mix (2 µL) were then added to
the reaction and incubated at 37°C overnight. After incubation, the probe was
denatured by boiling for 10 min, and diluted in Southern Blot hybridisation
solution at a concentration of 10-25 ng/mL.
90
The concentration of the probe was determined by a spot test with DIG-
labelled control DNA. The DIG-labelled control DNA was diluted to 1 ng/µL.
From this stock, five 1:10 serial dilutions were made, creating a set of
standards ranging from 1 ng/µL to 0.01 pg/ µL. After estimating the
concentration of experimental probe from the DIG System User’s Guide for
Filter Hybridisation, the experimental probe was diluted to approximately 1
ng/µL. From this stock, five 1:10 serial dilutions were made.
A piece of nylon membrane was spotted with 1 µL of these standards in one
row, then with 1 µL of the experimental probe on a second row. The
membrane was then cross-linked under UV light for 5 min. The membrane
was washed in 2 x SSC wash buffer briefly, and then incubated for 30 min in
blocking solution at room temperature. The membrane was then incubated in
Anti-DIG Antibody Solution for 30 min at room temperature. The membrane
was washed twice for 15 min each with wash buffer at room temperature to
remove any unbound antibody. The membrane was equilibrated in detection
buffer for 2 min, and then developed substrate solution. To stop the colour
development, the membrane was washed with dH2O. The intensity of the
experimental probe spots was then compared to the standard DIG-Labelled
control DNA spots to determine the probe concentration.
2.4.3.8.2 Transfer of DNA to the Nylon Membrane
STM-1 and parent strain genomic DNA was digested with SacI restriction
enzyme. Approximately 3-5µg genomic DNA was digested in the total volume
of 50 µL using 10 U of enzyme in a total volume of 50 µL. The digestion was
91
incubated at 37°C for 20 h. The digested DNA was subjected to
electrophoresis in a 1% agarose gel for 2 h at 80 V. After electrophoresis, the
gel was washed in depurination solution for 10 min then rinsed with water.
The gel was washed twice in denaturing solution for 10 min each, followed by
two 15 min washes in neutralisation solution. The DNA was transferred to the
nylon membrane via capillary transfer. The transfer to the nylon membrane
was achieved by placing the membrane on top of the gel followed by at least
four sheets of 3MM Whatman paper, approximately 30 sheets of paper
towels, a glass plate and a 1 kg weight on top of the glass. The baking dish
was filled with 20 x SSC and the Whatman paper was soaked in 20 x SSC for
capillary transfer. The transfer was allowed to proceed overnight.
Following the transfer, the membrane was air dried then wrapped in plastic
sandwich wrap and placed on top of a UV transilluminator for 5 min. to allow
cross linking of DNA to the membrane.
2.4.3.8.3 DNA Hybridisation
The membrane was incubated at 65°C for 3 h in standard hybridisation
solution. The denatured probe was added to the hybridisation solution at a
concentration of 20 ng/mL and the membrane then incubated overnight at
65°C.
92
2.4.3.8.4 Washing and Development of Membrane
The membrane was washed twice with 2 x SSC wash buffer for 5 min at room
temperature, and then washed twice in 0.5 x SSC wash buffer for 15 min at
65°C. Following the stringency washes, the membrane was equilibrated in
maleic acid wash buffer, and then incubated in blocking solution for 1 h at
room temperature. The blocking solution was replaced with Anti-DIG Antibody
Solution and incubated for 1 h at room temperature. Unbound antibody was
removed by two washes in maleic acid wash buffer for 10 min at room
temperature. Substrate solution was then added and the membrane placed in
the dark until a colour precipitate had formed.
93
2.5 Protein Methods
2.5.1 Buffer and Solutions
Ammonium Persulfate (10%). Ammonium Persulfate (100 mg) was
dissolved in 1 mL of dH2O and filter sterilised through a 0.22 µM syringe filter.
The solution was stored at 4°C wrapped in foil for up to 3 months.
Bradford Coomassie Reagent. Coomassie Brilliant Blue R-250 (0.05% w/v)
was dissolved in ethanol (25 mL, 95%) and Phosphoric Acid (50 mL, 88%),
then diluted with water to 500 mL. Store at 4°C, filter before use.
Cell lysate buffer. Tris-HCl (0.1 M, pH 6.8), SDS (2% w/v) and glycerol (15%
v/v) were dissolved in water.
Coomassie Stain. Coomassie Brilliant Blue R-250 (0.05% w/v) was dissolved
in methanol (250 mL) and Acetic Acid (50 mL). After the Coomassie Brilliant
Blue had dissolved, the solution was diluted to 500 mL with dH2O (Final
concentration 50% Methanol, 10% Acetic Acid).
Coomassie Destaining Reagent. Ethanol (10% v/v) and Acetic Acid (10%
v/v) was diluted in dH2O.
Lowry Folin Reagent. Folin-Ciocalteau reagent was mixed with dH2O at a
1:1 ratio.
94
Lowry Reagent A. Sodium Carbonate (2% w/v), Sodium Hydroxide (0.4%
w/v), sodium tartrate (0.16% w/v) and SDS (1% w/v) dissolved is dH2O.
Lowry Reagent B. Copper sulfate (4% w/v) dissolved in dH2O.
Lowry Reagent C. Lowry Reagent A (50 mL) and Lowry Reagent B (0.5 mL)
were mixed before use.
Outer membrane protein (OMP) Extraction Buffer. Tris base (50 mM) and
EDTA (10 mM) was dissolved in deionised H2O, then 0.05% TritonX-114 was
added.
10x PAGE Running Buffer. Tris Base (3% w/v), Glycine (14.4% w/v) and
SDS (1% w/v) was dissolved in dH2O, pH 8.3. Dilution of 1x was used for
electrophoresis.
PAGE Sample Buffer. Glycerol (25% v/v), Tris Base (60 mM), SDS (2% w/v),
β-mercaptoethanol (14.4mM), Bromophenol blue (01%) were dissolved in
dH2O.
PBS. 0.01 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M
sodium chloride, pH 7.4.
95
SDS-PAGE Sample Buffer. Tris-HCl (125 mM, pH 6.8), β-mercaptoethanol
(5% w/v), Bromophenol blue (0.02% w/v) and glycerol (10% v/v) in dH2O.
TBS. Tris Base (10 mM) and NaCl (500 mM) was dissolved in deionised H2O,
pH 7.4, then autoclaved at standard conditions.
TBS/Tween. Tween 20 (0.05%) was added to TBS.
TE Buffer. Tris Base (10 mM) and EDTA (1 mM) were diluted in dH2O, pH
7.5, and autoclaved at standard conditions.
Western Blot Blocking Solution. Skim milk (5%) was dissolved in TBS.
Western Blot Substrate Solution. The substrate, 4-Chloro-1-Naphthol (30
mg), was dissolved in 10 mL of Methanol. This solution was added to 40 mL
Tris (50 mM), and 30 µL of H2O2 pH 7.0.
2.5.2 Protein Extraction Method
2.5.2.1 Sonication.
Cells were harvested by centrifugation at 5000 x g for 10 min and
resuspended in PBS buffer. The cell suspension was then sonicated in an ice
bath for 6 cycles of 30 s on and 30 s off. The lysate was clarified by
centrifugation at 5000 x g for 10 min to remove cellular debris, and the
96
supernatant collected. The protein concentration was determined by the
Bradford assay and the lysate was stored at –20°C if not used immediately.
2.5.2.2 Preparation of Whole Cell Lysates
An overnight culture of bacteria in 10 mL LB broth was collected by
centrifugation and washed in 2 mL of Tris-HCl (10mM, pH 7.4). The cell pellet
was lysed with cell lysate buffer and boiled for 5 min. The supernatant was
collected by centrifugation and transferred to a new tube and the protein
concentration was determined using the Lowry method.
2.5.3 Determination of Protein Concentration.
2.5.3.1 Lowry Assay.
A Lowry assay was used for protein estimation when the presence of SDS
prevented the use of a Bradford assay. A standard curve was set up from a 1
mg/mL BSA stock at 0, 10, 20, 30, 50, 70 and 100 µg/mL in a total volume of
200 µL in H2O. Test samples were diluted 1:200 in a total volume of 190 µL in
H2O. Lowry Reagent C (0.6 mL) was added to each tube and incubated for 20
min at RT. Lowry Folin Reagent (0.06 mL) was added to each tube and
incubated for 30 min at RT. An aliquot (200 µL) of each sample was added to
a 96 well plates and absorbances read at 600 nm.
97
2.5.3.2 Bradford Assay.
A standard curve was set up from a 1 mg/mL BSA stock at 30, 60, 90, 120,
150, 180 and 210 µg/mL in 0.15 M NaCl. Test samples were diluted 1/10 with
0.15 M NaCl in a total volume of 100 µL. Bradford reagent (1mL) was added
to each standard and test solution and incubated at room temperature for 2
min. Samples (200 µL) were loaded in duplicate into 96 well plates, and
absorbances read at 600nm.
2.5.4 Recombinant Protein Expression
Recombinant protein was expression occurred after inducing the nirB or pagC
promoters. To induce the nirB promoter, 10 mL LB broth was inoculated from
a glycerol stock and cultured for 8-10 h. The culture was resuspended in 50
mL LB broth were incubated without aeration at 37°C for 4–6 h (some cultures
were flushed with nitrogen and sealed prior to static incubation).
To induce the pagC promoter 10 mL LB broth was inoculated from a glycerol
stock and cultured for 8-10 h. The log-phase bacteria were washed once in
M9 medium, then resuspended in M9 medium plus supplements (0.1%
Casamino Acids, 38 mM glycerol, aromatic supplements) and grown at 37oC
for 3-5 h under static condition.
98
2.5.5 Polyacrylamide Gel Electrophoresis.
2.5.5.1 SDS-PAGE.
Polyacrylamide mini-gels were prepared according to the following formula:
12.5% Resolving gel.
Reagents Volume
Water 4.22 mL
1.5 M Tris (pH 8.8) 2.5 mL
10% SDS 0.1 mL
Bis-Acrylamide (40%) 3.135 mL
10% Ammonium persulfate 0.05 mL
TEMED 0.01 mL
Total volume 10 mL
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4% Stacking Gel
Reagent Volume
Water 3.580 mL
0.5 M Tris (pH 6.8) 1 mL
10% SDS 0.04 mL
Bis-Acrylamide (40%) 0.375 mL
10% Ammonium persulfate 0.01 mL
TEMED 0.0005 mL
Total 5 mL
SDS-PAGE loading buffer was added to samples to be analysed in a 4:1
sample to buffer ratio. Prepared samples were then heated to 100°C for 10
min in a heat block. SDS-PAGE gel wells were loaded with 20 µL of sample
using a syringe. SDS-PAGE was performed at 80 V for 30 min then 180 V for
50 min.
2.5.6 Visualisation of Proteins.
2.5.6.1 Coomassie Staining.
The gel was stained with Coomassie Blue Stain for at least 1 h with gentle
agitation, and then destained in destaining solution, with several solution
changes, until protein bands were clearly visible.
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2.5.6.2 Western Blot.
2.5.6.2.1 Transfer of Protein to Nitrocellulose Membrane.
Following PAGE, the gel and a piece of nitrocellulose membrane were soaked
in western blot transfer buffer. Two scotch-brite pads and 3 pieces of
Whatman paper were cut slightly larger than the gel and soaked in Western
Blot Transfer Buffer. To assemble the Western Blot, a pad was placed on the
apparatus, followed by the gel, the membrane, the Whatman paper, and
another pad. The air bubbles were removed by using glass rod. The
apparatus was assembled according to the manufacturer’s instructions, and
the western blot was performed at 90 V for at least 1 h.
2.5.6.2.2 Development of nitrocellulose membrane.
The nitrocellulose membrane was blocked for 1 h in TBS/Skim milk (5% v/v)
at RT. The blocking solution was discarded and the membrane was washed 3
times in TBS/Tween (0.05% v/v). The Primary Antibody was diluted in
TBS/Skim Milk (1% v/v), added to the membrane and incubated for at least 1
h at room temperature or overnight at 4°C. The Primary Antibody Solution
was discarded and the membrane washed twice for 10 min in TBS/Tween
(0.05% v/v). The Detection Antibody was diluted in TBS/Skim Milk (1% v/v),
added to the membrane and incubated for 1 h shaking at RT. The membrane
was washed twice in TBS/Tween (0.05% v/v), once in TBS. The membrane
was developed by the addition of western blot substrate solution and
incubated in the dark until colour developed.
101
2.6 Animal Experimentation Methods.
All experiments involving animals were carried out with the approval of the RMIT AEC.
2.6.1 Buffers and Reagents.
ACK Lysing Buffer. NH4Cl (0.15 M), KHCO3 (10 mM) and Na2EDTA (0.1
mM) dissolved in H2O, pH to 7.3 with 1 N HCl, and filter sterilised through a
0.2 µM filter.
Carbonate Buffer 1. NaHCO3 (8.4 g) was added to 100 mL of H2O. Stored at
4°C.
Carbonate Buffer 2. Na2CO3 (10.6 g) was added to 100 mL of H2O. Stored at
4°C.
ELISA Substrate Solution. Equal volumes of reagent A and reagents B of
TMB substrate was mixed immediately before use.
ELISPOT Blocking Buffer. Newborn Calf Serum (5% v/v) was filter sterilised
into sterile PBS. Made fresh as required.
ELISPOT Coating Buffer. Carbonate buffer 1 (4.53 mL) was added to
Carbonate buffer 2 (1.82 mL) and diluted to 100 mL with H2O. The pH was
adjusted to 9.6 with either Carbonate buffer 1 or 2. Made fresh as required.
102
ELISPOT Detection Solution. BCIP/NBT solution, prepared according to the
manufacturers instructions. BCIP (100 µL) and NBT (100 µL) was added to 10
mL of BCIP/NBT Buffer.
PBS. Phosphate buffer (0.01 M), 0.0027 M potassium chloride and 0.137 M
sodium chloride, pH 7.4.
PBS/T. Phosphate buffer (0.01 M), 0.0027 M potassium chloride and 0.137 M
sodium chloride, pH 7.4, autoclaved at standard conditions. Tween 20®
(0.05% v/v) was then added.
RPMI/NCS. Newborn Calf Serum (5% v/v) was filter sterilised, added to RPMI
and stored at 4°C.
RPMI/NCS/Abs. Newborn Calf Serum (5% v/v) was filter sterilised and added
to RPMI. Penicillin/Streptomycin (0.1% v/v) was then added to the RPMI and
stored at 4°C.
2.6.2 Vaccination with recombinant STM-1 expressing
heterologous antigenic protein.
Cultures of recombinant STM-1 were grown for vaccination using the following
method. Five millilitre LB broth containing the appropriate antibiotic was
inoculated from a –70°C Stock of STM-1 harbouring plasmid constructs and
chromosomal integrants and grown overnight at 37°C. The following day, a
103
100 mL LB broth containing the appropriate antibiotic for plasmid constructs
and without antibiotic for chromosomal integrants, was inoculated with 1 mL of
the overnight culture. This culture was incubated for 3-5 h at 37°C, until an
absorbance at 600 nm was 0.3 was obtained. The STM-1 were recovered by
centrifugation at 5000 x g for 15 min at 4°C. The cell pellet was washed twice
in 10 mL of PBS, before being resuspended at a concentration of 2 x 109
cells/mL. Mice were vaccinated with 5 x 108 CFU of recombinant STM-1 for
oral inoculation and 1 x 106 CFU of recombinant STM-1 for intraperitoneal
inoculation.
2.6.3 Blood collection.
Mice were bled by retro-orbital puncture, and the serum was collected by
centrifugation at 1000 x g for 10 min. Typically 100-200 µL of blood was
collected. The serum was seperated and stored at –20°C.
2.6.4 Preparation of Faecal pellets for ELISA.
Fresh faecal pellets were collected three weeks after the final vaccination and
were prepared by the method adapted from deVos and Dick (1991).
Approximately 5-6 fresh faecal pellets were collected from each group and
stored in a microfuge tube. One millilitre of PBS was added for 0.1 g of
faeces, and the tube was vortexed vigorously for 15 min. The supernatant
was collected after centrifugation at 15000x g for 15 min. One millimolar
phenyl methyl sulfonyl fluorise (PMSF) was added and the samples were
stored at -20°C.
104
2.6.5 ELISA to detect antibody levels.
Purified antigen was diluted to 10 µg/mL in Carbonate Coating Buffer and 100
µL was added to 96 well plates and allowed to incubate overnight at 4°C.
Plates were washed in PBS/T 3 times and then blocked for 1 h at RT in
PBS/Skim milk (5% v/v). Serum was diluted in PBS/T and Skim milk (1% v/v)
at the appropriate concentration and 100 µL was added to each well in
duplicate. Serum was incubated for 2 h at 37°C. Plates were washed 3 times
in PBS/T, and any remaining liquid was removed by tapping the plates on
paper towels. Detection antibody (mouse anti-IgG H+L or mouse anti-IgA)
was then diluted in appropriate concentration in PBS/T and 100 µL was added
to each well. The detection antibody was incubated for 1 h at RT. Plates were
then washed 4 times in PBS/T and twice in PBS. Any remaining liquid was
removed from the wells by patting the plates on absorbent paper towels. One
hundred microliters of substrate was added to each well and allowed to
develop for 30 min. The reaction was then stopped by the addition 50 µL of
0.2 N H2SO4. Absorbance were then measured on and ELISA plate reader at
an absorbance of 450 nm.
105
2.6.6 ELISPOT assay for cytokine producing cells.
2.6.6.1 Collection of Spleen Cells.
Mice were sacrificed and their spleens collected in sterile RPMI/NCS/Abs.
Spleens were placed in petri dishes, crushed with the end of a 3 mL syringe
and resuspended in 5 mL of RPMI/NCS and filtered through a cell strainer.
The cells were recovered by centrifugation at 800 x g for 5 min at 4°C and
resuspended in 5 mL of ACK Lysing Buffer. After 5 min, 5 mL of RPMI/NCS
was added and the cells recovered by centrifugation at 800 x g for 5 min at
4°C. The lymphocytes were washed in RPMI/NCS 3 times, and resuspended
in 1 mL of RPMI/NCS. Recovered lymphocytes were counted by diluting 1 µL
of the cell suspension in 89 µL of PBS. The solution to be counted was
prepared by adding 10 µL of Trypan Blue reagent. After 5 min, 10 µL of the
stained cells was loaded onto a haemocytometer, and the number of viable
cells counted under an inverted microscope.
2.6.6.2 ELISPOT assay.
The ELISPOT plate was prepared by wetting the membrane with 100%
Methanol for 5 min, followed by three washes in sterile PBS. Capture antibody
(100 µL) was diluted in ELISPOT Coating Buffer and added to the wells (0.5
µg/mL IL-4, 1 µg/mL IFN-γ) and incubated overnight at 4°C. The capture
antibody was discarded and the wells washed three times with 200 µL of
106
PBS/T. The wells were blocked with 100 µL of ELISPOT blocking buffer and
incubated at room temperature for 3 h.
The wells were washed three times with 200 µL of PBS/T, then 100 µL of
RPMI/NCS (5 % v/v) was added and the plates were incubated for 10 min at
37°C and 5% CO2. The RPMI was discarded and the cells were seeded in
quadruplets with 1x106 cells in 90 µL/well. The fourth well served as an
internal control. Ten microlitre antigen (chicken ovalbumin or C. jejuni
membrane preparation) was added at concentration 250 µg/mL in the three
wells. No antigen was added in the fourth well, and concavalin A was added
to the positive control wells at a concentration of 1µg/mL. The negative control
was unstimulated spleen cells. The spleen cells were incubated with the
mitogen for 20 h at 37°C and 5% CO2.
The following day, the spleen cells were discarded and the plates washed 6
times in PBS/T, and twice with MiliQ water, and any remaining wash solution
removed by tapping the plates on absorbent paper. The biotin labelled
detection antibody was diluted at a concentration of 0.5 µL/mL PBS/NCS (1%
v/v), and 100 µL was added to the wells and incubated at RT. After 2 h of
incubation, the wells were washed in PBS/T 5-6 times and the remaining
solution removed by tapping the plates on absorbent paper towel. After
washing, 100 µL of alkaline phosphatase labelled streptavidin was added to
the wells, and incubated at RT for 2 h. The streptavidin solution was
discarded and the plates were washed 7 times with PBS/T and 3 times in PBS
and any remaining liquid was removed by tapping the plates on absorbent
107
paper. The ELISPOT was developed by the addition of 50 µL of ELISPOT
substrate solution and incubated at room temperature in the dark until blue
spots developed. The reaction was then stopped by washing the plates 3
times in de-ionised water. Spots were counted under a dissecting microscope.
108
Chapter 3
Characterisation of the STM-1 strain of
Salmonella enterica serovar Typhimurium
3.1 Introduction
Salmonella Typhimurium infects a wide variety of animals, and is one of the
causative agents of diarrhoeal diseases in humans, and it also causes
typhoid-like symptoms in mice (Pang et al. 1995). Consumption of food from
animal origin is the major cause of Salmonella spp. infection in humans.
Orally ingested bacteria adhere to, and penetrate the intestinal mucosa
destroying specialised epithelial M cells of the Peyer’s patches causing
gastroenteritis (Jones and Falkow 1996; Finlay and Falkow 1997). During
infection, Salmonella, being an intracellular pathogen, resides and replicates
inside cells, including macrophages and dendritic cells. This changes the
phenotype of the cells due to the recognition of bacterial products, as well as
the action of bacterial virulence factors (Rosenberger et al. 2000) and can
also induce both cellular and humoral immune responses. The induction of
strong humoral and cellular responses makes Salmonella spp. an ideal
candidate for an attenuated bacterial vaccine or for vectored antigen delivery
(Harokopakis et al. 1997; Wijburg et al. 2002; Jiang et al. 2004). A variety of
S. Typhimurium mutants have been tested for their ability to induce an
immune response in mice. S. Typhimurium harbouring mutations in aroA,
109
aroC, aroD, htrA, ompR and phoPQ genes were able to induce both humoral
and cellular immune responses without causing any disease in mice (Dougan
et al. 1987; Valentine et al. 1998; Bondarenko et al. 2002). Salmonella
species have also been found to be successful as vectors capable of
delivering heterologous vaccine antigen, effectively inducing a broad
spectrum of mucosal and systemic immune responses against the antigenic
proteins expressed in the Salmonella vector (Cardenas and Clements 1992;
Hess 2000; Stocker 2000; Bachtiar et al. 2003; Foynes et al. 2003; Garmory
et al. 2003).
STM-1 is a derivative of S. Typhimurium harbouring an aroA mutation, which
causes complete, non-reverting aromatic biosynthesis (aro) defects. This
renders STM-1 non-virulent in respect to invasive infection, as aromatic
metabolites such as paraminobenzoate (for synthesising folate),
dihydroxybenzoate (for synthesising enterochelin) and aromatic amino acids
tyrosine are not available in host tissues (Smith et al. 1984) and so are not
available to STM-1 to support active replication (Figure 1.2).
The aroA attenuation was created in a wild type strain of S. Typhimurium
using Phage P22 packaged with transposon aroA 554::Tn10 (Alderton et al.
1991). The STM-1 derivative has been used as a vaccine to protect poultry,
calves and adult cattle against Salmonella challenge, and has also been used
for the delivery of DNA vaccines (Bachtiar et al. 2003). However, to date the
genetic basis of attenuation of STM-1 has not been fully characterised.
110
To fully utilise STM-1 as a potential delivery vector for vaccine antigens, and
to enable specific site directed insertion of foreign gene(s) into a defined site
to ensure continued heterologous gene expression in STM-1, it was
necessary to determine the exact nature of the attenuation.
Introduction of Tn10 can promote several types of recombination events
other than transposition. Shen et al., (1987) showed that when the Tn10
element is introduced into Salmonella, IS10 right transposition is the most
common recombination event but Tn10 promoted deletion and
deletion/inversion can also occur. The IS10RH transposition targets sites
randomly throughout the genome and the majority (85%) of deletion/inversion
events due to Tn10 occur at sites less than 35kb away from the original Tn10
insertion (Kleckner, et al. 1979; Reichardt and Botstein, 1979). This means
there is a lower possibility of deletion/inversion events at more distant target
sites. However, no valid reason has been found that show that inversions
should be less than 35kb in length (Shen et al. 1987).
The Polymerase Chain Reaction (PCR), Southern blot analysis, and Pulse
Field Gel Electrophoresis (PFGE) were applied to identify the changes in the
chromosome of the STM-1 with respect to the parent strain S. Typhimurium
82/6915.
111
3.2 Results
3.2.1 Amplification of PflB-aroA region
The architecture of S. Typhimurium LT2 is shown in Figure 3.1a (McClelland
et al. 2001) and 3.1b. It was assumed in this study that the parent strain of
STM-1, S. Typhimurium 82/6915 has similar architecture to LT2. Subsequent
experiments have shown this assumption to be correct. All primers used were
designed according to the LT2 sequence.
To characterise the STM-1 sequence upstream of aroA, the PflB-aroA region
was amplified using the pflB forward primer and aroA reverse primer as
shown in Figure 3.1b. The wild type S. Typhimurium parent strain was used
as a positive control for all PCR’s. In the parent strain the amplified product
was 8.2 kb as expected from the LT2 sequence, but the product amplified in
STM-1 was only 4.4 kb, Figure 3.1c.
112
Location 29 at 10 mins
Figure 3.1a. Circular map of the S. Typhimurium LT2 genome (McClelland et al. 2001). The arrows mark the positions at location 29 at 10 min and location 48 at 19 mins.
Position of pflB -aroA region on circular Map. Location 48 at 19 mins
S . T y p h i m u r i u m L T 2 a r o A r e g i o n ( 9 2 0 6 b p s )
2 0 0 0 4 0 0 0 6 0 0 0 8 0 0 0
S T M 0 9 7 2 p f lB f c o A
y c a O
y c a P s e r C a r o A
PflB forward primer aroA reverse
primer
Figure 3.1b. Map of the aroA region in S. Typhimurium
LT2 at 19 minutes at location 48. The parent S.
Typhimurium 82/6915 is assumed to have similar genomic
architecture as LT2 sequence.
113
1 2 3
8.2 kb11.5kb
5.1kb 4.5kb 2.8kb
4.4 kb
Figure 3.1c. Amplification of the Pflb-aroA region. The product in
wild type is 8.2 kb whereas in STM-1 the product is 4.4 kb, a
difference of approximately 3.8 kb. Lanes: 1, lambda x Pst1
marker; 2, wild type; 3, STM-1.
114
3.2.2 Sequence analysis of amplified PCR products
The amplified PCR product was ligated into pCR2.1 (Invitrogen), to
characterise the unknown region upstream of aroA located at 19 minutes on
the S. Typhimurium circular map Figure 3.1a. The chromosomal walking
method was used to sequence the entire 4.4 kb product. The sequence
analysis indicated that there is an insertion and a deletion in STM-1, with
respect to the parent strain, with the IS10 left hand side (IS10 LH) of the Tn10
element (approximately 1.4 kb) inserted adjacent to aroA. Several genes
including serC, ycaP, ycoA, fcoA and part of the pflB have been deleted. Also
deleted is the first 90 bp of the aroA gene (Figure 3.2a).
The sequence analysis also indicated the possibility of a chromosomal
rearrangement, as the sequence adjacent to the IS10 LH completely aligns
with S. Typhimurium LT2 sequence from location 29, at 10 minutes (Figure
3.2b), whereas aroA lies in the location 48 at 19 minutes on the LT2 map, a
difference of approximately 440 kb.
This sequence at location 29 of LT2 at 10 minutes matches with the gtrA gene
of phage P22.
115
The region marked in blue matches with the sequence from location 29 at 10 mins in the LT2 sequence. Referring to Figure 3.2b, aroA lies at location 48 at 19 mins.
STM-1 sequence (4448 bps)
1000 2000 3000 4000
gtrA section of P22
transposase
aroA
The aroA region sequence of STM-1. On the LT2 map this region lies at location 48 at 19 mins.
Figure 3.2a. Sequence analysis of STM-1 around the aroA region. The sequence
region depicted in the red bar matches with the IS10 LH of the Tn10 element. The
sequence depicted in blue is the S. Typhimurium sequence that matches with the
sequence from location 29 approximately at 10 mins in the circular map of S.
Typhimurium LT2. The aroA gene is at 19 mins, location 48, a difference of 440 kb.
116
ce (4160 bps)
1000 2000 3000 4000
XbaI BamHI
STM0555
STM0554
STM0556 STM0557
matches w ith STM-1 aroA region
yfdH
rfbISequence similar to the Phage P22 gtrA gene
Figure 3.2b. S. Typhimurium sequence from location 29 at 10
minutes. The sequence marked in blue matches with the STM-1
sequence from location 48 at 19 mins. The rfbI gene sequence is
similar to the gtrA gene sequence of Phage P22; therefore it has
been termed as gtrA in STM-1 (Figure 3.2a).
S.Typhimurium LT2 sequenFrom location 29 at 10 mins
117
3.2.3 Amplification of the serC, ycaO, ycaP, fcoA, pflB and
sopD genes Sequence analysis indicated that there was deletion of several metabolically
important genes in the STM-1 sequence upstream of the aroA gene. The serC
gene, which encodes an enzyme involved in serine biosynthesis is of
metabolic importance for the growth of the bacterial cell, the pfl gene
encodes pyruvate formate lyase, which is a central enzyme in bacterial
anaerobic metabolism catalysing the reversible reaction of pyruvate and
coenzyme A into acetyl-CoA and formate (Hoiseth and Stocker 1985), ycaP
encodes for a putative inner membrane protein, ycaO encodes for a putative
cytoplasmic protein, and fcoA encodes for a putative formate transporter. All
of these were proposed to be missing in STM-1.
The ycaO, ycaP, fcoA, pflB and sopD genes were amplified individually to
confirm the sequence analysis results (Figure 3.3a, 3.3b, 3.3c and 3.3d). The
PCR results confirmed the deletion of genes serC, ycaP ycaO and focA from
the aroA region in STM-1, a deletion of approximately 5 kb. However, the pflB
and sopD genes initially proposed to be deleted, as determined by sequence
analysis were found to be present after using the PCR to identify individual
genes (Figure 3.3d).
118
11.5 kb 5.0 kb 4.5 kb 2.8 kb
Figure 3.3
confirms
Lanes: 1, la
F
P
S
g
(
1 2 3
3.5 kb
a Amplification of the serC gene. The PCR
the deletion of the serC gene from STM-1.
mbda x PstI marker; 2, parent strain; 3, STM-1.
1 2 3 4 5 6 7
1.7 kb
11.5 kb 5.0 kb 4.5 kb 2.8 kb 1.7 kb 0.8 kb
700 bp
igure 3.3b Amplification of the ycaP and ycaO genes. The
CR confirms the absence of the ycaP and ycaO genes in
TM-1. Lanes: 1, lambda x PstI marker; 2, parent strain (ycaO
ene); 3, STM-1 (ycaO gene); 4, No DNA control; 5, parent strain
ycaP gene); 6, STM-1 (ycaP gene); 7, No DNA control.
119
1 2 3
6.5 kb
Figure 3.3c. Amplification of the fcoA gene. The
PCR confirms the absence of the fcoA gene.
Lanes: 1, lambda x PstI marker; 2, parent strain; 3,
STM-1.
6
1.2kb
Figure 3.3d Amplifi
presence of the pf
analysis of the STM
DNA control; 4, lamb
1 2 3 4 5
cati
lB a
-1 c
da x
11.5 kb 5.0 kb 4.5 kb
11.5 kb
5.0 kb 4.5 kb 2.8 kb 1.9 kb
2.3 kb
on of the sopD and pflb genes. The PCR result illustrates the
nd sopD genes initially thought to be missing after sequence
hromosome. Lanes: 1, parent strain; 2, STM-1(sopD gene); 3, no
PstI marker; 5, parent strain; 6, STM-1(pflB gene).
120
3.2.4 Amplification of the pflB-aroA region with pflB and
aroA170 bp primers
The PCR result for individual genes indicates the presence of the pflB gene in
STM-1, which was earlier thought to be deleted based on the sequence
analysis data. The pflB-aroA region was amplified using the same pflB
forward primer but a different reverse primer designed at 170 bp downstream
of the aroA gene, for further confirmation.
The result in the parent strain was as expected but there was no amplification
in STM-1 (Figure 3.4). The PCR further confirms that although the pflB gene
is present in the STM-1 chromosome it does not exist in the same region as in
the parent strain, which indicates the likelihood of chromosomal
rearrangement, which may be more complex than a simple inversion.
121
1 2 3 4
11.5 kb 5.0 kb 4.5 kb
6.5 kb
Figure 3.4 Amplification of the pflB-aroA region with pflB
forward and 170 bp aroA rev primers. The PCR shows
that the pflB gene, shown to be present in the STM-1
chromosome, does not exist at the same location as in
the parent strain. Lanes: 1 lambda x PstI marker; 2, parent
strain; 3, STM-1; 4, no DNA control.
122
3.2.5 Amplification of the gtrA gene
The sequence analysis of STM-1 also showed that the sequence which exists
at location 29 in the parent strain, now exists adjacent to the aroA gene in
STM-1 which is at location 48. The part of this sequence was termed as gtrA
as it also matches with the gtrA gene of phage P22 (Figure 3.2b).
The PCR was used to confirm the presence of this sequence in both parent
strain and in STM-1. As mentioned earlier the sequence of the parent strain S.
Typhimurium 82/6915 is assumed to align with the S. Typhimurium LT2
sequence, therefore this PCR was performed to confirm the presence of the
gtrA sequence in both the parent strain and STM-1.
The DNA segment amplified by gtrA forward and reverse primers confirms the
presence of the sequence in both parent strain and STM-1 (Fgiure 3.5). This
means that the presence of phage P22 DNA is a normal occurrence in these
strains of S. Typhimurium and is not simply the result of the delivery of
exogenous DNA in the creation of STM-1.
123
1 2 3 11.5 kb 5.0 kb 4.5 kb 2.8 kb 1.7 kb 0.8 kb 0.4 kb 400 bp
Figure 3.5 Amplification of the gtrA gene. The PCR
confirms that the gtrA sequence exists in both the parent
and STM-1 strains. Lanes: 1, lambda x PstI marker; 2,
parent strain; 3, STM-1.
124
3.2.6 Amplification of pflb-aroA region using gtrA and aorA
170 bp primers and pflB and gtrA primers
After confirmation that the gtrA sequence is a part of the parent strain it was
mandatory to confirm the position of that segment in both the parent strain
and STM-1. The pflB and aroA region was amplified using the gtrA forward
primer and aroA 170bp reverse primers. While there was no amplification in
the parent strain, in STM-1 the size of the amplified product was 2.3 kb as
shown in Figure 3.6, which was expected from the sequence analysis. For
further confirmation, the PCR with pflB forward primer and gtrA reverse
primers was performed. There was no product in either parent strain or STM-
1.
These results confirm the existence of a chromosomal segment from location
29 at 10 mins in the parent strain, in location 48 at 19 mins in STM-1. The
result also confirms the possibility of a chromosomal rearrangement of
genomic DNA of STM-1 other than inversion, as PCRs with pflB forward
primers and aroA reverse primers only amplifies the region in the parent
strain, and not in STM-1 indicating that the pflB gene does not exist in the
same region as aroA. Also the PCR with the gtrA forward primer and aroA
reverse primer gives the expected product in STM-1 only as shown in Figure
3.6.
125
1 2 3 4
11
54
2
1
2.3 kb
Figure
forward
the pre
strain a
parent s
.5 kb
.0 kb
.5 kb
.8 kb
.7 kb
3.6. Amplification of the pflB-aroA region with the gtrA
and 170 bp aroA reverse primers. The PCR result confirms
sence of a segment of DNA from location 29 in the parent
t location 48 in STM-1. Lanes; 1, lambda x PstI marker; 2,
train; 3, STM-1; 4, No DNA control.
126
3.2.7 Southern Blot analysis
The sequence analysis and PCR results indicate that there is a deletion of an
approximately 5 kb DNA segment and insertion of approximately 1.4 kb in
STM-1. The PCR of individual genes also confirms the presence of the pflB
gene, but not in the same region as in the parent strain. The sequence
analysis and the PCR results also indicate that the sequence of DNA which
was originally present at location 29 at 10 minutes in the parent strain, now
exists adjacent to the aroA gene in STM-1 which is at location 48 at 19
minutes in STM-1. However, the possibility of the existence of sequence from
location 29 at location 48 required further evidence; it is also required to
investigate if this change in the STM-1 genome is due to a chromosomal
rearrangement rather than a simple inversion, as the inversion of such a large
segment of DNA has never been reported in Salmonella spp. or E coli.
To further analyse the events leading to the STM-1 chromosomal architecture,
Southern blots, using three different probes, (aroA, pflB and gtrA) were
performed. The genomic DNA of both the parent strain and STM-1 was
digested with SacI after analysing the DNA sequence of LT2 from the
database. The digestion with SacI yields a 15 kb fragment in the aroA region
of the LT2 genome. Southern blot analysis with the pflB probe yields a 15 kb
product in both the parent strain and STM-1 (Figure 3.7; lane, 1 &2). The size
of the fragment when probed with aroA in the parent strain was 15 kb but in
STM-1 the fragment size is only 12 kb (Figure 3.7; lane, 4 & 5). The yield of a
15 kb fragment in the parent strain with both aroA and pflB probes further
confirms that the architecture of the parent strain is similar to LT2 as
127
previously indicated by PCR, and also confirms that the pflB gene and the
aroA gene exists in the same region in the parent strain. However, the
difference in the fragment size amplified in STM-1 between the pflB probe and
aroA probe indicates a possible genomic rearrangement as shown by earlier
sequence analysis and PCR results.
The Southern blot with the gtrA probe yields an approximately 4 kb fragment
in the parent strain whereas the size of the fragment in STM-1 is 12 kb (Figure
3.7; Lane 5 &6). This indicates the presence of the gtrA gene adjacent to the
aroA gene in STM-1, as both the probes yield a 12 kb fragment in STM-1.
These Southern blots indicate the following possibilities (1) a chromosomal
rearrangement other than an inversion in the STM-1 genomic DNA, as the
gtrA probe yields a 12 kb fragment in STM-1, the same fragment size that is
observed with the aroA probe, whereas the gtrA probe in the parent strain
yields only a 4 kb fragment as shown in Figure 3.7. This indicates that the
gtrA fragment now exists in a different chromosomal location, presumed to be
in the aroA region at location 48 at 19 minutes. The plfB gene does not exist
in the aroA region in STM-1, as the pflB probe yields a 15 kb fragment in the
STM-1, whereas the aroA probe yields a 12 kb fragment. These southern
blots confirm a chromosomal rearrangement in the STM-1 genomic DNA.
128
6
WT STM-1 WT STM-1 WT STM-1
15 KB
FIGURE 3.7 Southern blot analysis with the pflB, aroA and gtrA probes. The Southern
blot with the pflB probe yields a 15 kb fragment in both the parent strain and STM-1 (Lane
1 & 2), the aroA probe yields a 15 kb fragment in the parent strain however, only a 12 kb
fragment in STM-1 (Lane 3 & 4). This indicates a possible genomic rearrangement in STM-
1. The gtrA probe yields a 4 kb fragment in the parent strain and a 12 kb fragment in STM-
1 ( Lane 5 & 6), which is similar to the size of fragment yielded with the aroA probe. This
confirms the existence of the gtrA gene adjacent to the aroA gene, which are at different
locations in the parent strain. Lanes: 1, parent strain probed with pflB; 2, STM-1 probed with
pflB; 3, parent strain probed with aroA; 4, STM-1 probed with aroA; 5, parent strain probed with
gtrA; 6, STM-1 probed with gtrA.
129
12
1 2 3 4 5
3.2.8 PFGE analysis of STM-1 genomic DNA
The PCR and Southern blot analysis indicates the possibility of a
chromosomal rearrangement. PFGE was used to further analyse the nature of
the rearrangement in STM-1 genomic DNA. The rationale behind this
experiment was to study the pattern of digestion of the genomic DNA of both
parent strain and STM-1, considering the fact that if such a large
rearrangement (approximately 440 kb) has occurred, as the sequence
analysis, PCR and Southern blot analysis indicate, there should be an
observable difference after pulse field electrophoresis. Chromosomal DNA
was digested with XbaI to study the pattern of digestion by PFGE.
The pattern of genomic DNA digestion is similar in both the parent strain and
STM-1 as shown in Figure 3.8, which was not expected considering the fact
that all the above sequence analysis, PCR and Southern blot results indicates
the possibility of large chromosomal rearrangement. Such a large
rearrangement should have produced an observable change in the pattern of
digestion.
130
1 2 3 4 5
388 kb
339.5 kb 291 kb 242.5 kb 194 kb 145.5 kb 97 kb
48.5 kb
Figure 3.8 PFGE after digestion with XbaI. The pattern
of digestion in both the parent strain and STM-1 is
similar, which contradicts the results of sequence
analysis and PCR and Southern blot. Lanes: 1, parent
strain; 2, STM-1; 3, PFGE lambda marker; 4, parent
strain; 5, STM-1.
131
3.2.9 PFGE-Southern blot analysis of STM-1 genomic DNA
For further confirmation, three different restriction enzymes XbaI, SpeI and
NotI were used for further PFGE analysis and PFGE followed by southern
blot. This experiment was planned keeping in mind two possibilities (1) the
rearrangement in the STM-1 genome is of a nature that cannot be visualised
from the parent strain after XbaI digestion, but may be after digestion with
SpeI and NotI (2) even if the pattern of digestion with other enzymes do not
show any visible changes between the parent strain and the STM-1, PFGE
followed by the Southern blotting might be able to show the difference in the
pattern of digestion, if any exists between the parent strain and the STM-1.
The digestion pattern after PFGE did not show any observable differences
between the parent and STM-1 genomic DNA as shown in Figure 3.9a, but
the PFGE followed by Southern blot with the pflB probe, yields different size
products as shown in Figure 3.9b. This confirms that there is a rearrangement
in the STM-1 chromosome due to the insertion of transposon Tn10. The
Southern blot after digestion with XbaI did not yield any fragment in either
parent strain or STM-1. A possible explanation for this is that the fragment of
DNA carrying sequences complementary to the probe may be very large and
did not properly transfer onto the blotting membrane. The reason however
remains unknown.
132
1 2 3 4 5 6 7
339.5 kb 291 kb 242.5 kb 194 kb 145.5 kb 97 kb 48.5 kb
Figure 3.9a PFGE with XbaI, SpeI and NotI. Digestion with the
three different restriction enzymes does not reveal any
difference in the pattern of digestion between the parent strain
and STM-1. Lanes: 1, PFGE lambda marker; 2, parent strain; 3,
STM-1digested with XbaI; 4, parent strain; 5, STM-1 digested with
SpeI; 6, parent strain; 7, STM-1 digested with NotI.
133
1 2 3 4 5 6
200 kb approx
120 kb app
72 kb approx
Figure 3.9b Southern blot analysis with the pflB probe after
PFGE with XbaI, SpeI and NotI. The observation of different
sized fragments detected confirms a rearrangement in the
STM-1 genome. Lanes: 1, parent strain; 2, STM-1 digested with
XbaI; 3, parent strain; 4, STM-1 digested with SpeI; 5, parent strain;
6, STM-1 digested with NotI.
134
97 kbrox
3.2.10 Amplification of the aroA-rpsA region
The PCR was used to determine the sequence organisation downstream of
the aroA gene. The forward primer was designed from the aroA gene at 400
bp and the reverse primer is designed at the end of the rpsA gene. As shown
in Figure 3.10, there was no difference in the size of the amplified product in
either parent strain or STM-1, suggesting that there is no rearrangement in
the segment downstream of the aroA gene.
135
1 2 3 4
11.5 kb 5.0 kb 4.5 kb 2.8 kb 1.7 kb
2.9 kb
Figure 3.10. Amplification of the aroA-rpsA region
with aroA 400 bp fwd and rpsA rev primers. There
is no rearrangement downstream of aroA in the
STM-1 chromosome. Lanes: 1, lambda x PstI marker;
2, parent strain; 3, STM-1; 4, no DNA control.
136
3.3 Discussion
The transposon Tn10 is a composite transposable element flanked by
inverted repeats known as insertion sequences (IS10). The two ends of IS10
are referred to as inside and outside ends and are not genetically identical.
Shen et al. (1987) concluded that IS10 right hand mediated transposition is
the most common event followed by Tn10 promoted alteration of adjacent
DNA, which is either deletion or deletion/inversion and which occurs at a
frequency of 1% the rate of IS10 RH promoted transposition. Approximately
10% of events are complex or/ uninterpretable. The most frequently occurring
Tn10 mediated recombination event involves intramolecular attack of the two
inside IS10 ends resulting in the deletion of non-repeated Tn10 with either a
deletion or inversion of a region of contiguous DNA (Shen et al. 1987).
As explained earlier, STM-1 was created by infecting S. Typhimurium with
Phage P22 packaged with aroA 554:: Tn10 using the method described by
Hoiseth and Stocker (1981). Initial selection was for tetracycline resistance,
which showed integration of TetR into the aroA gene. The mutant was finally
selected for the loss of Tn10 after biochemical selection, so generating a TetS
and aroA auxotroph. Hoiseth and Stocker (1985) characterised the attenuated
strains of S. Typhimurium mutants developed by a similar method and
showed that in the Tets mutants, the effect of chromosomal rearrangement is
either a deletion or inversion. This deletion can be in either aroA or serC or
both the genes and may continue to the pfl gene upstream of the aroA gene,
giving rise to an Aro-, Aro- SerC- and Aro- SerC- Pfl- phenotype all of which are
137
irreversible. They also showed that Tets mutants carry a residual copy of IS10
adjacent to aroA. Kleckner et al. (1979) and Shen et al. (1987) showed that
the major chromosomal rearrangements can be either deletion, inversion or
deletion/inversion in nature. 10% of events are complex or/ uninterpretable.
One example is a major chromosomal rearrangement as shown to occur in
this study.
The sequence analysis and PCR results of the aroA region of STM-1
demonstrates the deletion of a 5 kb DNA segment in this region. The genes
from serC to fcoA have been completely deleted. This deletion also includes
the first 90 bp of the aroA gene. There is an insertion of approximately 1.4 kb
of DNA that is residual IS10.
The other key area of the analysis is to determine the rearrangement in the
chromosome of STM-1. The sequence analysis reveals the presence of a
DNA segment from location 29 at 10 min in the region of aroA, which in the
wild-type exists in location 48 at 19 mins, a difference of 440 kb. The PCR
and Southern blot analysis also confirms the result of the sequence analysis,
as the amplification of the aroA region with the gtrA forward primer and 170
bp aroA reverse primer yields a product only in STM-1, but there was no
product in the parent strain as shown in Figure 3.6. This confirms the
presence of a sequence from location 29 at 10 mins to the location 48 at 19
minutes. Also, the entire region from pflB to aroA cannot be amplified by
forward primers designed from the pflB gene and reverse primer in the aroA
138
gene in STM-1, but the presence of the pflB gene was confirmed by PCR in
the STM-1 strain.
Southern blot analysis with the pflB probe yields a 15 kb fragment in both the
parent strain and the STM-1, while probing with aroA yields a 15 kb fragment
in the parent strain, identical to the pflB probe, as expected as these genes
are predicted from the LT2 sequence to reside on the same fragment.
Southern blot analysis with the aroA probe in STM-1 yields a 12 kb fragment
which is different than that observed when STM-1 genomic DNA was probed
with the pflB probe (Figure 3.7). The difference in the fragment size in STM-1
probed with pflB or aroA indicates a possible genomic rearrangement. The
aroA and grtA probes yield a 12 kb fragment in STM-1 as shown in Figure 3.7,
whereas in the parent strain the product is 4 kb. This confirms the existence
of the gtrA gene adjacent to the aroA gene in STM-1 at location 48. All these
results point towards the possibility of a chromosomal rearrangement other
then an inversion.
The initial PFGE analysis performed after digesting genomic DNA with XbaI,
did not reveal any change in the pattern of digestion between the parent strain
and STM-1 as shown in Figure 3.8. This contradicts the previous sequence
analysis, PCR and Southern blot analysis results, as a large chromosomal
rearrangement should have created a visible change in the pattern of
digestion of STM-1 genomic DNA in comparison to the parent strain.
139
Further PFGE analysis performed after digesting both the parent strain and
STM-1 with the either XbaI, SpeI, or NotI, was performed under the
assumption that the rearrangement in the STM-1 genome is of a nature that it
does not disturb the pattern of XbaI digestion, but other enzymes which also
digest infrequently (but not as infrequently as XbaI) might reveal different
patterns of digestion between parent strain and the STM-1. However, this also
did not reveal any changes in the pattern of digestion between the parent
strain and STM-1 as shown in Figure 3.10a. However, changes were
observed after PFGE followed by Southern blotting. This yields different size
fragments in STM-1 in comparison to the parent strain when the pflB gene
product was used as a probe, as shown in the Figure 3.10b.
This result supports the previous sequence analysis, PCR and Southern blot
results. Therefore the presence of DNA sequence from location 29 at 10
minutes adjacent to the aroA region in STM1 can be explained best by the
phenomenon of a genomic rearrangement other than an inversion, which was
catalysed by the presence of the Tn10 transposable element.
In S. Typhimurium, inversions from 5 kb up to >60 kb have been reported (Bi
and Liu 1996) but the inversion of a very large fragments has never been
reported, and is therefore considered unlikely. The PFGE result shows no
difference between the parent strain and STM-1 after staining of the bands,
but Southern blot analysis of PFGE patterns confirms some chromosomal
rearrangement. A possible explanation of this chromosomal rearrangement in
STM-1 is a crossover between two distant points on the chromosome, not due
140
to a specific target sequence but due to the conserved three dimensional
structure of the bacterial chromosome. This is the phenomenon observed in
E. coli genomic DNA due to the insertion of the Tn10 transposable element
(Fonstein et al. 1994).
In the natural state the chromosome exists in a three dimensional form and is
in a highly twisted state; this can lead to the crossover between two unspecific
target sequences due to the presence of Tn10 element.
This concludes that the introduction of the composite transposable element
Tn10 in the bacterial chromosome can lead to chromosomal rearrangement,
which can be other then a deletion, inversion or simultaneous
deletion/inversion in nature. This can also lead to the insertion of residual
Tn10 or IS10 but can lead to more complex process where the large
segments of DNA can exchange position, which can be sometimes
unpredictable.
141
Chapter 4
Construction of STM-1 as a vector to
deliver heterologous vaccine antigen
4.1 Introduction
The Salmonellae are Gram-negative, facultative anaerobic, motile, non-
lactose fermenting rods, belonging to the family of Enterobacteriaceae.
Salmonella infects humans and animals generally by the oro-fecal route.
Bacon, Burrows et al. (1950; 1951) were first to describe attenuated
Salmonella strains. The mutation in these strains was due to the disruption of
gene encoding for purine, p-aminobenzoic acid, and aspartate. These
mutants exhibited reduced virulence in mice due to exogenous requirement of
the metabolites. Since then the use of various S. Typhimurium mutants has
been studied as potential vector and were found to be effective as a vector to
deliver heterologous antigens from bacteria, viruses and eukaryotes (Dougan
et al. 1987; Dalla Pozza et al. 1998; Dunstan et al. 1998; Bullifent et al. 2000;
Fouts et al. 2003). These mutants have the ability to establish a limited
infection in the host, and during the natural course of infection, the live
Salmonella vector delivers in vivo synthesized antigens to the host immune
system. The capability of live Salmonella vectors to strongly stimulate both the
humoral and cellular arms of the immune system makes them an ideal
142
candidate as a delivery vector (Guzman 1991; Covone et al. 1998; Bullifent et
al. 2000; Grode et al. 2001; Cochlovius et al. 2002; Capozzo et al. 2004).
4.1.1 Stabilisation of antigen expression in attenuated
Salmonella strain
The work described in this chapter was to devise a strategy to stabilise the
expression of heterologous vaccine antigen. The expression of heterologous
antigens at optimum levels to elicit immune responses is a significant problem
in the construction of recombinant Salmonella strains. Inadequately
immunogenic vaccine due low level of protein expression can be enhanced by
the use of a high copy number plasmid which can subsequently solve the
problem of low expression of antigenic protein (Covone et al. 1998). However,
the use of multicopy plasmids to deliver heterologous antigen has either failed
or had a reduced efficacy because of the unstable expression of antigenic
protein (Tarkka et al. 1989; Cardenas and Clements 1993). One major
drawback of these multicopy plasmids is that commonly used cloning vectors
induce metabolic burden on bacterial replication machinery due to the various
reasons such as type of replicon they harbour, copy number, size and
complexity. Also due to the expression encoded level of genes, affect the
retention of a plasmid by bacteria during in vivo growth where drug selection
is no longer applied (Dunstan et al. 2003). In some circumstances expression
of a high level of heterologous antigen has been found to be toxic (Garmory et
al. 2002).
143
Different strategies for addressing the issue of plasmid instability and avoiding
the necessity for the use of stabilising antibiotic have been proposed. One
such strategy involves the use of promoters that allows expression of
heterologous antigen at particular cellular sites, so called in vivo inducible
promoters (Dunstan et al. 1999).
4.1.2 The use of in vivo inducible promoters
Several in vivo inducible promoters have been tested for their efficiency of
protein expression. Ideally the promoters should allow increased expression
in APCs following uptake of the Salmonella carrier. One such promoter is the
nirB promoter, which was isolated from E. coli, where it directs the expression
of an operon which includes the nitrate reductase gene (Jayaraman et al.
1987). The nirB promoter is regulated by nitrate and by the changes in the
oxygen tension of the environment, becoming active under anaerobic
conditions (Cole 1968). The expression of heterologous antigens in
Salmonella under the control of the nirB promoter has been shown to
stimulate higher immune responses when compared with the expression of
the same antigens from the same strain under the control of a constitutive
promoter (Chatfield et al. 1992; McSorley et al. 1997).
Another anaerobically induced promoter, pagC is a macrophage-inducible
promoter isolated from Salmonella (Hohmann et al. 1995). The pagC
promoter is controlled by the PhoP/PhoQ two component regulatory system
144
which plays a major role in Salmonella virulence. PhoP-PhoQ activates the
transcription of genes following phagocytosis by macrophages. The
expression of these genes is necessary for the survival of Salmonella within
the phagosomal environment (Gunn 1998). The expression of antigenic
protein under the control of the pagC promoter provides stable, high-level
expression of heterologous antigen in Salmonella (Hohmann et al. 1995;
Dunstan et al. 1999; Chen and Schifferli 2001; Chiu et al. 2006). The pagC
promoter when compared with other promoters such as nirB, katG, spv, tac,
sasH, ompC was found to be superior in terms of in vivo expression of
heterologous antigen (Bumann 2001) .
4.1.3 Integration of constructs in the chromosomal location
Another reason for the unstable expression of vaccine antigen in vivo is due
to the instability of the plasmid encoding the antigen. For example, in one of
the early experiments, gene encoded on a plasmid expressing the E. coli
heat-labile enterotoxin subunit B was lost in vivo when the Salmonella carrier
colonised the mouse reticular endothelial system (RES) (Maskell et al. 1987).
Studies have aimed to address the problem of genetic instability in different
ways.
An alternative method to obtain stable antigen expression is to integrate the
foreign genes into the chromosome of the carrier strain (Hone et al. 1988;
Flynn et al. 1990; Strugnell et al. 1990). In 1988, Hone et al. developed one
system whereby heterologous DNA can be recombined in the chromosome of
145
Salmonella. The system used a suicide vector to integrate the genes
encoding K88 fimbriae of E. coli into the chromosome of a galE mutant of S.
Typhimurium by homologous recombination. Strugnell et al. (1990) used a
similar strategy to integrate the heterologous gene in the aroC gene of S.
Typhimurium. This system has since been used to insert DNA encoding
various heterologous antigen into the Salmonella chromosome (Strugnell et
al. 1992; Everest et al. 1995).
The most recently developed method, known as Lambda Red System, was
developed by Murphy et al. (1998). The phage Lambda Red locus provides a
platform that promotes homologous recombination of short linear DNA
fragments. The Lambda Red locus includes three genes: bet (aka β), exo and
gam (aka γ) (Datsenko and Wanner 2000; Poteete and Fenton 2000; Yu et al.
2000; Poteete et al. 2004). Exo is 5’-3’ exonuclease that degrades the 5’ end
of linear DNA, bet is a single stranded binding protein which binds to single-
stranded 3’ end generated by exo and promotes the annealing of
complementary DNA, and gam binds to the host RecBCD complex and
inhibits its exonuclease activity (Murphy, 1998 Poteete, 2000 Yu, 2000).
This technology was extensively used in E. coli and S. Typhimurium to
generate knockout mutants. The method involves generation of a PCR
product containing a very short region of identity (35-50 bp), which was
integrated into the bacterial chromosome taking advantage of the phage
recombination system. Datsenko and Wanner (2000) designed temperature
sensitive, low copy number plasmids pKD20 and pKD46 from which the
146
Lambda Red genes bet, exo and gam were expressed under the control of an
arabinose inducible promoter.
The recombinase-based system also offers the possibility to precisely remove
the antibiotic resistance marker from the chromosome (Cherepanov and
Wackernagel 1995). The method initially described (Szybalski 1993; Posfai et
al. 1997) is based on the yeast FLP/FRT recombination system in which the
FLP gene product targets the FRT site (FLP recombination target), a 68 bp
recognition sequence of the yeast FLP gene product, and precisely removes
the segment of DNA which exists between the two sites. This system has
also been shown to be effective in the development of recombinant
Salmonella strains for vaccine purpose by allowing the integration of genes in
the desired chromosomal location. (Husseiny and Hensel 2005) (Figure 4.1).
The work described in this chapter was to devise the strategy to stabilise the
expression of heterologous vaccine antigen. To achieve the stable expression
of heterologous vaccine antigens, which are chicken ovalbumin as a model
antigen and C. jejuni major outer membrane protein as a test antigen, the
genes were put under the control of both the nirB and pagC in vivo inducible
promoters. Both genes were expressed from a plasmid and also integrated
into the aroA gene of S. Typhimurium STM-1 chromosome. Protein
expression was analysed from both the plasmid and chromosomal location.
147
BamHI pKD13
kan
Figure 4.1. Diagrammatic representation of Lambda Red system. (A) designed constructs consist of an
in vivo-activated promoter (Pro), a gene fragment encoding a model vaccine antigen (dotted areas),
expression cassettes for expression of genes encoding ovalbumin as a model antigen or C. jejuni major
outer membrane protein were inserted into the BamHI site of pKD13 plasmid. The pKD13 plasmid
contains the kanamycin resistance gene (hatched areas) flanked by FRT sites and binding sites for
primers (black symbols). (B) The targeting construct consisting of expression cassettes and the
resistance gene were amplified by primers containing 40 bp of identity to the chromosomal target gene
(grey symbols). (C) S. Typhimurium harbouring pKD46 for expression of the Red recombinase was
transformed with linear DNA of the targeting construct, and recombinant colonies with replacements of
a chromosomal target gene (open symbol) by the targeting construct were selected. (D) The gene
encoding antibiotic resistance after the integration of construct can be removed by FLP-mediated
recombination after transformation of recombinant strains with plasmid pCP20. Figure adapted from
Husseiny and Hensel (2005).
148
4.2 Results
4.2.1 Ligation of the pagC and nirB promoters into the pCR2.1
plasmid
The nirB promoter is anareobically induced, regulated by nitrate and by
changes in the oxygen tension of the environment (Cole 1968). The promoter
was amplified from E. coli genomic DNA as a template by using the nirB
forward primer with a BamHI restriction site and the nirB reverse primer with a
MfeI restriction site (Table 2.4) at 58°C annealing temperature (Figure 4.2a).
The pagC promoter, a macrophage inducible promoter (Dunstan et al. 1999),
was amplified from S. Typhimurium genomic DNA as a template by using the
pagC forward primer with a BamHI restriction site and the pagC reverse
primer with a MfeI restriction site at 60°C annealing temperature ( Figure
4.2b). Fresh PCR product was ligated into the pCR2.1 plasmid supplied by
Invitrogen (Figure 4.2c) and transformed into E. coli TOP10F’. The plasmid
with the pagC promoter was termed pCRpagC and with the nirB promoter as
pCRnirB (Table 2.2).
149
11.5 kb 5.1 kb 2.8 kb 1.9 kb 0.8 kb
11.5 kb 5.1 kb 1.9 kb 1.7 kb 0.34 kb
p
p
1 2 3
11.5 kb 5.1 kb 2.8 kb 1.9 kb 0.8 kb 0.34 kb
pCR2.1 plasmid 3.9 kb
0.7 kb0.3 kb
Figure 4.2c Ligation of the pagC and nirB
promoters into pCR2.1 plasmid revealed by
digestion of recombinant clones with EcoRI.
Lanes: 1, lambda x PstI marker; 2, nirB
promoter in pCR2.1 vector; 3, pagC promoter
in pCR2.1. vector. Inserts are indicated.
150
700 b
300 b
1 2
Figure 4.2b Amplification of the
pagC promoter. Lanes: 1, lambda
x PstI marker; 2, pagC promoter.
The 700 bp product is indicated.
Figure 4.2a Amplification of the
nirB promoter. Lanes: 1, lambda x
PstI marker; 2, nirB promoter. The
300 bp product is indicated.
1 2
4.2.2 Amplification of the C. jejuni major outer membrane
protein and chicken ovalbumin genes
The C. jejuni MOMP gene was amplified from the TAMomp plasmid, the
construct designed by Ooi 2002 (Honours thesis, RMIT University), using the
Momp forward primer with an EcoRI restriction site and the Momp reverse
primer (Table 2.3). The gene was amplified at 55°C annealing temperature
(Figure 4.3a). The chicken ovalbumin gene was amplified from the sOVA
plasmid supplied by Boyle et al.(1997) using the ova forward primer and the
ova reverse primer. The primers used for the amplification of the chicken
ovalbumin gene carry an EcoRI restriction site. The amplification was
achieved at 60°C annealing temperature (Figure 4.3b).
The fresh PCR product was ligated into the pCR2.1 plasmid and transformed
into E. coli TOP10F’. The pCR2.1 plasmid carrying the C. jejuni major outer
membrane protein expressing gene was termed pCRMomp and the pCR2.1
plasmid carrying the chicken ovalbumin gene was termed pCRova (Table
2.2).
151
11.5 kb
1.7 kb
1.16 kb
11.5 kb
1.7 kb 1.16 kb
Figure 4.3a Amplification of
the C. jejuni Momp gene.
Lanes: 1, lambda x PstI marker;
2-3, Momp gene; 4, no DNA
control. The 1.2 kb product is
indicated.
152
Figure 4.3b Amplification of
the Chicken ovalbumin gene.
Lanes: 1, lambda x PstI
marker; 2-6, ovalbumin gene.
The 1.2 kb product is
indicated.
1.2 kb
1.2 kb1 2
1 2
4.2.3 Cloning of the C. jejuni Momp and the chicken
ovalbumin genes with pagC and nirB promoter
The pCRMomp and pCRova plasmids were digested with EcoRI, and the
inserts containing Momp and ovalbumin genes were purified. The pCRpagC
and pCRnirB plasmids were digested with MfeI. After inactivation of the
restriction enzyme the plasmids were alkaline phosphatase treated and
purified to minimise self ligation of the vector. The Momp and ovalbumin
genes were then ligated into the pCRpagC and pCRnirB plasmid at the MfeI
restriction site (as MfeI and EcoRI have compatible ends); to yield
pCRpagMomp, pCRnirMomp, pCRpagova and pCRnirova plasmids.
The orientation of each of the four constructs was analysed by restriction
digestion. The putative ovalbumin constructs were digested with EcoRI and
EcoRV, and the Momp constructs were digested with EcoRI and SpeI. EcoRI
digests just at the beginning and at the end of construct, therefore the
digestion releases the entire insert from the plasmid. SpeI digests the Momp
gene at 13 bp, therefore Momp constructs in the right orientation should give
two products, a 1.2 kb product corresponding to the Momp gene and 700 bp
corresponding to the size of pagC promoter (Figure 4.4a), and 300 bp
corresponding to the nirB promoter (Figure 4.4c). EcoRV digests the
ovalbumin gene at 1000 bp so the positive constructs with ovalbumin should
give a 1.7 kb product with the pagC promoter and a 200 bp minor product
(Figure 4.4b) and 1.5 kb product with nirB promoter and a 200 bp minor
product (Figure 4.4d).
153
1 2 3 4 5 6 7 8 9 11.5 kb 5.1 kb 1.7 kb 1.16 kb 0.8 kb
1.2kb
0.7kb
Figure 4.4a Digestion of PagCMomp
constructs with EcoRI and SpeI for the
detection of correct orientation of the
constructs. Lanes: 1, lambda x PstI marker, 2,
3 & 9positive PagCMomp clones; 4, 5, 6, 7, &8,
clone in opposite orientation. Diagnostic
fragments are indicated.
1 2 3 4 5 6 7 811.5 kb
5.1 kb 1.7 kb 1.16 kb 0.34 kb
1.7 kb
Figure 4.4b Digestion of pagCova
constructs with EcoRI and EcoRV for the
detection of correct orientation of the
constructs. Lanes: 1, lambda x PstI marker;
2, 4 & 5, positive pagCova clones; 3, 6, 7 &
8, clones in opposite orientation. The
diagnostic fragment is indicated.
154
1 2 3 4 5 6 7 8 911.5 kb 1.7 kb 1.16 kb 0.34 kb
1.2 kb
0.3 kb
Figure 4.4c Digestion of nirBMomp
constructs with EcoRI and SpeI restriction
enzyme for the detection of correct
orientation of the constructs. Lanes: 1, 4 &
7, positive nirBMomp Clones; 2, 3, 5, 6 & 8
clone in opposite orientation; 9, lambda x PstI
marker. Diagnostic fragments are indicated.
1 2 3 4 5 6 7 11.5 kb
5.1 kb 1.7 kb
1.16 kb
0.34 kb
1.5 kb
Figure 4.4d Digestion of nirBova
constructs with EcoRI and EcoRV
restriction enzyme for the detection of
correct orientation of the constructs.
Lanes: 1, lambda x PstI marker; 4 & 5,
positive nirBova clones; 2, 3, 6, & 7, clones in
opposite orientation. The diagnostic fragment
is indicated.
155
4.2.4 DNA Sequence analysis of the assembled constructs
The restriction digestion pattern was the primary selection procedure to select
clones in the correct orientation. DNA sequencing was employed to further
confirm the integrity of the constructs. Each constructs was sequenced using
the M13-20 forward primer and M13 reverse primer (Table 2.3) according to
the method described in section 2.4.2.4. The sequence was analysed using
the BLAST tool using Clone Manager, scientific and educational software
version 7.11. The sequence analysis in confirmed the integrity of each
constructs.
156
4.2.5 Analysis of protein expression from the constructs by
western blotting
Western blot was used to determine protein expression from the positive
constructs assembled in plasmids to ensure correct functioning of the
constructs prior to further experimentation. E. coli harbouring the constructs
were inoculated into LB broth and incubated at 37°C. The pagC and nirB
promoters were induced as mentioned in section 2.5.4 for protein expression.
Twenty microgram of protein was loaded for each sample after determining
the protein concentration by Lowry assay. To detect Momp, mouse anti-C.
jejuni antibody was used as the primary antibody, and HRP conjugated sheep
anti-mouse, was used as detecting antibody. The expression of Momp was
visible from both pagCMomp and nirBMomp constructs after the final
development (Figure 4.5a). However, the band from samples with the
pagCMomp construct was very faint, and therefore is not visible in the
scanned photograph (Lane 4, Figure 4.4a)
The detection of ovalbumin was achieved by using mouse anti-ovalbumin
primary antibody and HRP conjugated sheep anti-mouse as the secondary
antibody. The 45 KDa product as expected was visualised after development
in cells harbouring both the pagCova and nirBova constructs (Figure 4.5b). E.
coli harbouring empty plasmid was used as negative control in both the
western blots.
157
98 kd 64 kd 50 kd 36 kd 22 kd
148 k98 kd64 kd50 kd 36 kd
1 2 3 4 5
45 KDa
d
1 2 3 4
Figure 4.5a Expression of Momp from plasmid
constructs. Lanes: 1, seeBlue protein marker; 2, C.
jenuni lysate; 3, E. coli carrying empty vector; 4, E. coli
carrying Momp gene under the control of the pagC
promoter; 5, E. coli carrying Momp gene under the
control of the nirB promoter. The protein size is indicated.
Figure 4.5b Expression of ovalbumin from plasmid
constructs. Lanes: 1, seeBlue protein marker; 2, E. coli
carrying ovalbumin gene under the control of the pagC
promoter; 3, E. coli carrying ovalbumin under the control
of the nirB promoter; 4, E. coli carrying empty vector. The
protein size is indicated.
158
45 KDa
4.2.6 Ligation of constructs from pCR2.1 into pMW2 vector
The pMW2 vector was the choice of vector for the delivery of heterologous
vaccine antigen from the plasmid location. In comparison to pCR2.1 vector,
which is a high copy number vector, pMW2 is medium copy number vector.
The constructs were digested from the holding pCR2.1 plasmid with KpnI and
XhoI, and the inserts and pMW2 vector were purified before ligation. The
ligation mixture was transformed into E. coli DH5α by electro-transformation.
Positive clones were selected and plasmids were digested with BamHI. The
estimated size of the digestion product was approximately 2 kb (Figure 4.6).
STM-1 was transformed with the plasmids carrying inserts after these
plasmids were passaged through S. Typhimurium 9121-LT2 strain.
159
1 2 3 4 5
11.5 kb 5.1 kb 2.8 kb
1.7 kb
2 kb
Figure 4.6 Restriction digestion of pMW2 plasmid with
BamHI to select positive clones. Lanes: 1, pagCmomp; 2,
nirBmomp; 3, pagCova; 4, nirBova; 5, lambda x PstI
marker. The diagnostic fragment is indicated.
160
4.2.7 Lambda Red System for the chromosomal integration of
constructs
The Lambda Red System, supplied by the E. coli genetic stock centre USA,
(courtesy Dr B.L. Wanner) was used for the integration of constructs in the
aroA gene region of the STM-1. The system takes the advantage of Phage
Lambda recombination which allows the integration of linear PCR products
with very short regions of identity.
STM-1 was transformed with pKD46 after the plasmid was passaged through
the LT2-9121 strain. The pKD46 plasmid carries a temperature sensitive
conditional replicon, which allows replication of plasmid at 30°C only (Table
2.2). The lambda red genes which drive the recombination of short linear PCR
fragments are expressed under the control of the arabinose promoter (Figure
4.7). The constructs assembled above were ligated into the pKD13 plasmid
which was used as template to generate PCR products using primers with 35-
40 bp of identity with the aroA gene of STM-1 as described in section 2.4.4.4
(Figure 4.1).
161
pKD466329 bps
1000
2000
3000
4000
5000
6000
EcoRVBssHII
Van91IDrdISapIBstEIIMluI
BamHIBanIIEcl136IISacI
NsiIPpu10I
SexAISalIBclI
XcmIBsrGI
DraIIIOliISgrAI
SmaIXmaI
BstXINcoIStyI
BsaAISnaBI
PciI
SpeI
NotIEco52I
BbeIKasINarISfoI
AhdIBsaIBpmI
PvuIXmnI
BssSIAlw 44I
AarIBspMI
araC
gam
bet
exorepA101
amphicilin
Figure 4.7 The pKD46 plasmid from which the
lambda red genes are expressed under the control
of the arabinose promoter. It has a temperature
sensitive replicon, which allows the replication of
plasmid at 30°C.
162
4.2.7.1 Ligation of Constructs into the pKD13 plasmid
The pKD13 plasmid was designed to be used as template for the generation
of PCR products to be integrated in the aroA gene of STM-1. The inserts were
excised from pMW2 with BamHI and ligated into the pKD13 plasmid at the
BamHI site which lies just outside the FRT site. The pKD13 plasmid replicates
under the control of the R6K origin of replication which requires the pir protein,
and the kanamycin antibiotic marker is flanked by FRT (FLP recombination
target) sites (Table 2.2 & Figure 4.8). Although there are other plasmids such
as pKD3 and pKD4 which could have been used for the generation of PCR
products, the pKD13 plasmid was selected due to the availability of a BamHI
restriction site just outside the FRT site, which allowed the ligation of
constructs in the pKD13 plasmid from pMW2 plasmid without further
processing.
After ligation the pKD13 plasmid was transformed into E. coli BW25141,
which is pir+ (Table 2.1). The plasmids carrying constructs were digested with
BamHI to select positive clones (Figure 4.9a, 4.9b, 4.9c and 49.d).
163
pKD133434 bps
500
1000
15002000
2500
3000
BtrIXbaI
RsrIINaeINgoMIV
BssHIIBanII
Tth111I
DrdI
BglIIXcmI
BstAPIEco47III
XbaIAccIHincIISalIBam
BsmBIBbvCIBpu10IBseRI
NheI
PsiI
SnaBINotI
AhdI
BglI
PvuI
ScaITatI
Alw 44I
SspIBspHI
AarIClaI
FRT
kan
FRT
R6k
ampicillin
BamHI restriction site
HI
Figure 4.8. The pKD13 plasmid with the R6K origin of replication. The
kanamycin antibiotic cassette is flanked by FRT sites. This allows the
precise removal of antibiotic marker once the positive chromosomal
integrants were selected. The BamHI site which exists just outside the
FRT site was used to ligate the constructs excised from the pMW2
vector.
164
1 2 3 4 5 6 7 8 9 10 11
11.5 kb 5.1 kb 2.8 kb 1.7 kb 1.9 kb
1.9kb
Figure 4.9a Ligation of pagCmomp
constructs into the pKD13 plasmid.
Lanes: 1, 2, 3, 4, 5, 7, 8, 9, 10 & 11 positive
clones; 6, lambda x PstI marker. The
diagnostic fragment is indicated.
11.5 kb 5.1 kb
2.8 kb 1.9 kb
Figure 4.9b Ligation of nirBMomp constructs
into the pKD13 plasmid. Lanes: 2, 3, 4, 5, 8, 9,
10 & 11 positive clones; 6, lambda x PstI marker.
The diagnostic fragment is indicated.
1 2 3 4 5 6 7 8 9 10 11
165
1 2 3 4 5 6 7 8 9 10 11
11.5 kb
5.1 kb 2.8 kb
1.9 kb
1.9 kb
Figure 4.9c Ligation of pagCova
constructs into the pKD13 plasmid.
Lanes: 1, 2, 3, 4, 7, 8, 10 & 11 positive
clones; 6, lambda x PstI marker The
diagnostic fragment is indicated.
11.5 kb 5.1 kb 2.8 kb 1.7 kb
Figure 4.9d Ligation of nirBova constructs into the
pKD13 plasmid. Lanes: 3, 4, 5, 7, 9 & 10 positive clones;
6, lambda x PstI marker. The diagnostic fragment is
indicated.
1 2 3 4 5 6 7 8 9
166
4.2.7.2 Amplification of the constructs with the antibiotic
resistance marker from the pKD13 vector for chromosomal
integration.
The pKD13 plasmid was used as a template as described in section 4.2.7.1
for the generation of PCR products with 40 bp of identity to the aroA gene of
STM-1. The pk forward and pk reverse primers were used. These primers are
60-62 bp long with 20-22 bp homologous to the plasmid template and 40 bp
homologous to the aroA gene, where the constructs were to be integrated.
The PCR was performed using expand long template DNA polymerase at
65°C annealing temperature ( section 2.4.2.3). The empty vector was used as
an amplification control and the negative control was without any DNA
template. The estimated size of the PCR product from the positive clones is
approximately 3.2 kb including the kanamycin cassette (Figure 4.10).
167
1 2 3 4 5 6 7
11.5 kb 5.1 kb 2.8 kb 1.7 kb 1.16 kb
3.2 kb
1.2 kb
Figure 4.10. Amplification of constructs from
the pKD13 plasmid template. Lanes: 1, empty
vector; 2, pagcmomp; 3, nirBmomp, 4, pagCova;
5, nirBova; 6, no DNA control; 7, lambda x PstI
marker. Diagnostic fragments are indicated.
168
4.2.7.3 Integration of the PCR product into the aroA gene of
STM-1
The PCR reaction was digested with DpnI to eliminate any plasmid template.
Following the restriction digestion, the PCR product was purified using the
Geneclean kit according to the manufacturer’s instruction as described in
section 2.4.1.8. Fresh competent cells of STM-1 harbouring the pKD46
plasmid were transformed by electroporation (refer to section 2.4.4.2). The
transformants were plated onto MH agar containing 50 µg/mL kanamycin and
incubated at 37°C overnight.
The positive transformants were identified by PCR with the gtrA forward
primer and aroA reverse primer using genomic DNA of colonies as template.
STM-1 genomic DNA was used as positive control, no DNA was used as the
negative control. The estimated size of the products in the positive control
was approximately 5.7 kb (Figure 4.11).
The suicide vector system (Hone et al. 1988) was also tested in this study to
integrate the same constructs into the aroA gene of STM-1 to compare the
efficiency of the two different strategies. However, no recombinant STM-1 was
identified even when the bacterial cells were transformed with up to 500 ng of
DNA (data not shown). In contrast 10-15 positive recombinants were identified
per 100 ng of DNA when the Lambda Red system was used for the
integration of constructs.
169
11.5 kb 4.7 kb 2.6 kb 1.7 kb
1 2 3 4 5 6
5.7 kb
3.5 kb
Figure 4.11. Amplification of constructs with gtrA forward
primer and aroA reverse primer after chromosomal
integration. Lanes; 1, lambda x PstI marker; 2, STM-1 positive
control; 3, pagCmomp; 4, nirBmomp; 5, pagCova; 6, nirBova;
7, no DNA negative control. The diagnostic fragments are
indicated.
170
4.2.8 Analysis of in vitro protein expression
In vitro protein expression was analysed by western blotting from plasmid
constructs in the pMW2 vector and from similar constructs integrated into the
chromosome of STM-1. This experiment was primarily performed to identify
any difference in expression of protein from the plasmid or chromosomal
location before in vivo analysis.
The promoters were induced as described in section 2.5.4. The bacterial cells
were collected by centrifugation. The cells were disrupted by sonication
(section 2.5.2.1) and the protein concentration was determined by Bradford
assay (section 2.5.3.2). Approximately 20-25 µg protein was loaded in each
well except in well 2 of Figure 4.12a where approximately 35 µg of protein
was inadvertently loaded. The in vitro expression analysis of C. jejuni Momp
under the control of the pagC or nirB promoters from both plasmid and
chromosomal location show that the level of expression from the nirB
promoter was slightly higher when expressed from the chromosomal location.
However, expression from the pagC promoter was slightly higher when
compared to the expression from the nirB promoter when antigen was
expressed from plasmid location (Figure 4.12a). It should be noted that the
western blot analysis are indicative, rather than quantitative.
The analysis of ovalbumin antigen expression was also analysed by western
blotting. The in vitro protein expression of the ovalbumin antigen under the
control of the nirB promoter from the chromosomal location was slightly higher
in comparison to expression of same antigen under the control of the pagC
171
promoter. However, a similar level of expression was observed from plasmid
location in both promoters (Figure 4.12b). The levels of protein that were
detected in this experiment were low, and hence bands are not visible in the
reproduced figure.
In summary the level of protein expression from the chromosomal location
and the plasmid location was similar from both promoters. However, the
overall expression may be slightly higher when the antigens were expressed
from plasmid vector.
172
1 2 3 4 5 6
148 kd 98 kd 64 kd
50 kd 36 kd 22 kd
45 KDa
Figure 4.12a Analysis of C. jejuni Momp antigen expression by
western blotting. Lanes: 1, STM-1 without plasmid vector; 2,
nirBmomp chromosome; 3, nirBmomp plasmid; 4, pagCmomp
chromosome; 5, pagCmomp plasmid; 6, seeBlue protein marker.
The protein size is indicated.
173
1 2 3 4 5 6 7
45 KDa
Figure 4.12b Analysis of ovalbumin antigen expression by
blotting. Protein expression was observed from all the four c
although it is difficult to observe in the reproduced figure.
nirBova from chromosomal location; 2, nirBova from plasmid lo
pagCova from chromosomal location; 4, pagCova from plasmid l
STM-1 carrying empty vector; 6, STM-1 without vector; 7, seeBl
marker. The protein size is indicated.
174
148 kd 98 kd
64 kd
50 kd 36 kd 22 kd
western
onstructs,
Lanes: 1,
cation; 3,
ocation; 5,
ue protein
4.3 Discussion
Attenuated Salmonella strains have been widely investigated as a live
bacterial vaccine to deliver heterologous vaccine antigen in a number of
animal models (Bullifent et al. 2000; Fayolle et al. 2001; Chen and Schifferli
2003; Chiu et al. 2006). The use of live attenuated Salmonella strains as
delivery vectors to express heterologous antigens to the secretory immune
system constitutes a promising approach for the development of new
vaccines against a number of diseases, and have been found to be safe and
establish a limited infection in the host (Basso et al. 2000; Chen and Schifferli
2000; Bachtiar et al. 2003). These mutants during the natural course of
infection can deliver antigen to stimulate B and T lymphocytes (Berndt and
Methner 2004; Capozzo et al. 2004; Chiu et al. 2006). In the work discussed
in this chapter, a registered salmonellosis vaccine, STM-1 has been evaluated
for the potential to express heterologous antigen.
For the optimal expression of chicken ovalbumin (model antigen) and C. jejuni
Momp (test antigen) antigens, the antigen coding genes were expressed
under the control of the in vivo inducible promoters nirB and pagC. The
antigens were expressed from both the plasmid and chromosomal locations.
The nirB promoter is an anaerobically induced promoter which was isolated
from E. coli; this promoter is regulated by nitrate and changes in the oxygen
tension (Cole 1968). The pagC promoter was isolated from Salmonella, and is
activated once the organism reaches macrophages, and therefore has the
potential to directly deliver heterologous antigen to the APC (Bullifent et al.
2000).
175
Both the promoters have been successfully used for the in vivo expression of
antigenic protein and provide optimum level of stable expression in
Salmonella vector; however, the expression from pagC promoter is reported
to be significantly higher and induce better immune responses in comparison
to the expression from the nirB promoter (Dunstan et al. 1999; Bumann 2001;
Chen and Schifferli 2001).
The constructs were designed to express chicken ovalbumin and C. jejuni
Momp proteins from both nirB and pagC promoters. These constructs were
expressed from a plasmid vector; however, these constructs were also
integrated into the aroA gene of STM-1 to address the issue of instability of
the plasmid encoding the foreign gene. This strategy to introduce the genes
expressing foreign antigens in the chromosome will also eliminate the in vivo
use of antibiotics, which is necessary if the expression is required from an
unstable plasmid vector. Further, it is likely that in any future vaccine use,
plasmid borne antigens will not be acceptable due to the potential of transfer
of such plasmids to host bacteria.
The Lambda Red system (Datsenko and Wanner 2000) and a suicide vector
(Hone et al. 1988) were used for chromosomal integration of the constructs in
the aroA gene of STM-1. The use of a suicide vector to integrate the foreign
genes in the chromosome was unsuccessful (data not shown), even when up
to 500 ng of DNA was used to transform the STM-1. A similar method using a
suicide vector for introducing a foreign gene in the aroD gene of STM-1 was
176
attempted earlier in our laboratory by Moutafis (2003), which was also
unsuccessful. The reason for the failure of suicide vectors for promoting
homologous recombination in STM-1 is not fully understood.
In contrast the lambda red system was successfully used to introduce foreign
genes into the aroA location of STM-1. Also the efficiency of recombination
was high; approximately 10-15 positive recombinants were identified when
transformed with 100 ng of linear DNA (section 4.2.7.3). This system was
primarily developed to create knockout mutants of E. coli and S. Typhimurium
taking advantage of Phage Lambda recombination system (Datsenko and
Wanner 2000; Murphy et al. 2000; Court et al. 2002; Poteete et al. 2004). The
Lambda Red system allows linear DNA molecules with 35-50 bp identity to
efficiently recombine in the chromosomal location of bacterial cells. The other
advantage of this system is the precise removal of antibiotic resistance genes
(Martinez-Morales et al. 1999). This system has been used not only to
generate knock out bacterial mutants, but also other uses such as the epitope
tagging of a chromosomal gene (Uzzau et al. 2001). Recently this system has
been used for the insertion of genes expressing foreign antigen into the
Salmonella genome (Husseiny and Hensel 2005). In our study this system
was efficiently used for the insertion of chicken ovalbumin and C. jejuni Momp
genes into the aroA region of STM-1 in two steps. Although STM-1 has been
found to be a difficult strain for genetic recombination using suicide vectors,
the use of the lambda red system was successful.
177
The semi-quantitiative in vitro analysis of protein expression indicated that the
efficiency of heterologous antigen expression was approximately the same
from both the promoters when expressed from the plasmid vector. A similar
level of expression was also achieved from the chromosomal location in
comparison to expression from the plasmid vector by using these promoters.
This data suggest that expression can be achieved by using a single copy
gene when expressed under the control of the nirB or pagC promoter.
However, it is necessary to evaluate in vivo immune responses against these
antigens when expressed from the two promoters in an animal model.
178
Chapter 5
In vivo immune responses induced in mice
against heterologous antigen expressed in
STM-1
5.1 Introduction
Salmonella strains have been well characterised for the delivery of
heterologous vaccine antigens in several animal models (Bachtiar et al. 2003;
Baud et al. 2004; Ashby et al. 2005). The potential advantages of live
attenuated vaccines, such as the simple mode of inoculation and generation
of effective immune responses makes them an ideal candidate for the delivery
of heterologous antigens (Kochi 2003). These attributes of Salmonella have
prompted extensive research in the field of vaccine technology to evaluate the
efficacy of Salmonella as a potential vector to deliver a variety of heterologous
antigens (Londono-Arcila et al. 2002; Lasaro et al. 2005; Liu et al. 2006).
Salmonella being intracellular can replicate inside APC’s and induce both
humoral and cell mediated responses not only against homologous antigens
but also against the heterologous antigen for which they act as a carrier
(Bumann 2001; Koesling et al. 2001; Lundin et al. 2002; Garmory et al. 2003).
The cellular immune responses are crucial both for protective immunity
against salmonellosis and for the immunogenicity of oral vaccines which are
179
based on avirulent live Salmonella as antigen carriers. A primary infection can
induce the production of antibodies against antigens such as
lipopolysaccharide (LPS), flagellin and lipo-protein followed by Th1 or Th2
type T-cell memory and also CTL responses (Jones and Falkow 1996). Two
types of phagocytic cells, macrophages and immature dendritic cells (DC)
play an important role in the interface between the innate and adaptive
immune responses to Salmonella infections. They act as antigen presenting
cells (APC), capturing and degrading bacteria in a well-orchestrated series of
proteolytic event. These cells process the bacterial protein into peptide
fragments that are displayed on the APC surface by major histocompatibility
complex (MHC) (Yrlid et al. 2001). The MHC class I and the Class II
molecules, which are constitutively expressed on APC are responsible for the
presentation of non-overlapping antigen-derived peptides to CD8+ CTL and
CD4+ helper T cells (TH) respectively (Kirby et al. 2004; Babbitt et al. 2005).
Mucosal surfaces are the site of entry for most enteric pathogens, therefore
the mucosal immunity is important for the first line of defence, and direct
delivery of antigens to a mucosal surface can induce local immune responses
(DiGiandomenico et al. 2004). The gut associated lymphoid tissue (GALT)
has the largest repertoire of lymphoid tissues, and these lymphoid tissues
contains B and T cells which can be directly targeted by oral vaccination,
however it is not always practical or possible either due to handling involved
or due to the toxicity of the antigen to the mucosal surface (Suckow et al.
2000; Kidane et al. 2001). Therefore local immunity can also be induced by
the common mucosal immune system, where all the mucosal sites are linked
180
together and can be primed by IP inoculation (Mutwiri et al. 2005). Several
studies have shown that Salmonella can also induce strong humoral immune
responses against carrier antigens. In particular oral inoculation with
Salmonella expressing heterologous vaccine antigen can lead to the
generation of strong a IgG and IgA response (Lasaro et al. 2005).
Several factors play important roles in the magnitude and type of immune
response induced against the passenger antigen. For example the type of
mutation that leads to the disruption of genes encoding for important
metabolic enzymes necessary for the survival of an organism in vivo, leads to
the generation of mutants which are safe and have minimal survival in the
host. This may have some effects on the course of the infection, thereby
altering the quality of the induced immune response (Dunstan et al. 1998).
Another factor which can play an important role is the type of antigen for
example whether the antigen translocates into the periplasmic membrane or
resides intracellularly and is degraded and presented to the immune system
(Garmory et al. 2002).
In the previous chapter, work leading to the creation of a set of recombinant
STM-1 vaccine strains was described. The aim of work described in this
chapter is to evaluate the ability of the Salmonella STM-1 mutant to deliver
heterologous antigen in vivo and induce both cellular and humoral immune
responses. Chicken ovalbumin was used as a model antigen and the C. jejuni
major outer membrane protein was used as a test antigen.
181
5.2 Results
5.2.1 Immunisation of mice
Two immunisation experiments were designed. In the first experiment the
uitlity of STM-1 as a vehicle for the induction of immune responses against
chicken ovalbumin antigen expressed from both plasmid and chromosomal
locations was evaluated. Also the efficacy of pagC and nirB promoters was
compared in vivo. In the second experiment in vivo immune responses
against C. jejuni major outer membrane protein (Momp) were evaluated from
both plasmid and chromosome locations under the control of the nirB
promoter.
In both experiments groups of 5 mice were vaccinated three times at an
interval of two weeks with STM-1 expressing heterologous antigen either from
plasmid or chromosome, with empty STM-1 vector and STM-1 vector
harbouring empty plasmid as controls. Vaccination was administered both
orally and by IP injection.
Oral vaccination was performed by inoculation of 5 x 108 CFU and IP
vaccination with 1 x 106 CFU. Serum samples were collected prior to the third
vaccination and three weeks after the third vaccination to analyse the humoral
immune response. Mice were sacrificed three weeks after the third
vaccination and splenocytes were collected for the ELISPOT assay.
182
Table 5.1: Vaccine trial # 1. The Table shows the groups, vaccine
constructs and mode of inoculation for the evaluation of immune
responses raised against the ovalbumin protein
Groups Vaccination Mode of
Inoculation
Group 1 STM-1 only IP
Group 2 “ Oral
Group 3 STM-1 harbouring empty plasmid IP
Group 4 “ Oral
Group 5 STM-1 expressing ovalbumin antigen
under the control of the pagC
promoter from plasmid
IP
Group 6 “ Oral
Group 7 STM-1 expressing ovalbumin antigen
under the control of the pagC
promoter from chromosomal location
IP
Group 8 “ Oral
Group 9 STM-1 expressing ovalbumin antigen
under the control of the nirB promoter
from plasmid
IP
Group 10 “ Oral
Group 11 STM-1 expressing ovalbumin antigen
under the control of the nirB promoter
from chromosomal location
IP
Group12 “ Oral
183
5.2.2 Analysis of humoral immune responses against the
vectored ovalbumin antigen
In this experiment the ability of the Salmonella STM-1 mutant to raise immune
responses against chicken ovalbumin expressed from both plasmid and
chromosome was evaluated. Also the efficacy of the pagC and nirB promoters
is compared in vivo for the optimal expression of heterologous antigen,
inferred by immune responses.
The distribution of groups and the mode of inoculation are shown in Table 5.1.
Group 1 to Group 4 are negative controls, group 1 and 2 mice were
vaccinated with STM-1 only and group 3 and 4 mice were vaccinated with
STM-1 harbouring empty plasmid. The evaluation of humoral and cell
mediated immune responses from other groups was compared with Group 1
to Group 4 for analysis.
Humoral responses to ovalbumin were analysed by ELISA to confirm that
STM-1 can deliver heterologous antigen in vivo activating humoral responses.
The reactivity of IgG antibody from sera was tested against the ovalbumin
antigen from the collected sera. Figure 5.1 shows the results from the pooled
sera and Figure 5.2 shows the results from individual mice sera within each
group. The results depicted in Figure 5.1 indicates that anti-ovalbumin
responses are induced in each test group.
Significant humoral responses were observed in mice vaccinated with STM-1
expressing ovalbumin either from plasmid or from chromosome when
184
compared with negative control (p<0.001). However no significant difference
was observed in humoral responses when the expression from the plasmid
location was compared with expression from the chromosomal location
(Figure 5.2). Also no significant difference in humoral responses was
observed when the antigen was delivered from the pagC and the nirB
promoters.
185
0
0.01
0.02
0.03
0.04
0.05
0.06
STM-1
(IP)
(Oral
)
STM-1/
Empty pl
asmid
(IP)
(Oral
)
STM-1/
pagC
Ova pl
asmid
(IP)
(Oral
)
STM-1/
pagC
Ova ch
romos
ome (
IP)
(Oral
)
STM-1/
nirBOva
plas
mid (IP
)
(Oral
)
STM-1/
nirBOva
chrom
osom
e (IP)
(Oral
)
OD
1:1
00
Figure 5.1. Analysis of the IgG response against ovalbumin antigen from
pooled sera collected 3 weeks after the final vaccination. Reactivity was
measured against 1:100 dilution of sera. Low but measurable responses were
observed in sera from all groups in which ovalbumin was delivered. No
response was observed in sera from the control groups 1-4.
186
0
100
200
300
400
500
600
STM-1(IP
)
ORAL
STM-1/Empty
Plasmid
(IP) Oral
pagC
Ova/Plas
mid (IP
)ORAL
pagC
Ova/ch
romos
ome (
IP)Oral
nirBOva
/plas
mid (IP
)Oral
nirBOva
/chrom
osom
e (IP)
Oral
Titre
Figure 5.2. Humoral responses elicited after vaccination with ovalbumin
constructs. Significant humoral responses were observed in mice vaccinated
with STM-1 expressing ovalbumin either from a plasmid or from chromosomal
location when compared with negative control (P<0.001). Each of the titres in
the last eight groups is significantly higher then that in the corresponding
control groups. However, no significant difference was observed in humoral
responses when the titres induced from plasmid expression was compared with
expression from the chromosome. Also no significant difference in humoral
responses was observed when the antigen was expressed from either the pagC
promoter and the nirB promoter.
187
5.2.3 Analysis of Secretory IgA response from the faecal
sample
The above results indicate positive IgG responses in the mice vaccinated with
STM-1 expressing ovalbumin antigen. Faecal samples were collected from
each group 3 weeks after the final vaccination to analyse the secretory IgA
response. Faecal samples were processed as described in section 2.6.4. The
ELISA was performed on these samples to test the IgA response.
Figure 5.3 shows the IgA response in the test group in comparison to the
control group. Positive responses were observed in sera from mice
vaccinated with STM-1 expressing ovalbumin antigen when expressed under
control of the pagC promoter from plasmid and chromosome. Also mice
vaccinated with STM-1 expressing ovalbumin antigen under the nirB promoter
shows higher responses from chromosomal constructs in comparison to
plasmid constructs. In this experiment a higher sIgA response was observed
when the mice were vaccinated by IP inoculation when compared with oral
inoculation, although as these are pooled samples no significance could be
evaluated.
188
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
STM-1(IP)
Oral
pagCOva/plasmid (IP)
Oral
pagCOva/chromosome (IP
)Oral
nirBOva/Plasm
id (IP)
Oral
nirBOva/ch
romosome (IP)
Oral
OD
450
Figure 5.3. Analysis of secretory IgA response in mice from pooled
faecal samples. The IgA levels in test groups when compared with the
control group was higher in all groups vaccinated with ovalbumin
constructs.
189
5.2.4 Analysis of cell mediated immune response against
chicken ovalbumin antigen
It was established in the above experiments that the delivery of ovalbumin
from both plasmid and chromosomal location in STM-1 can induce humoral
immune responses. To analyse the T cell response secretion of IL4 and IFN-γ
by stimulated splenocytes was measured by ELISPOT assay. Splenocytes
that have been primed by vaccination will respond to the antigen. Results are
expressed as the number of spot forming T cells per million splenocytes
(SFC) with cells from each mouse assayed in triplicate.
Mice vaccinated with STM-1 expressing ovalbumin under control of the pagC
promoter from plasmid (IP inoculation only) and from chromosomal location
resulted in significant responses from activated splenocytes secreting IL4
(P<0.001). Significant responses were also observed from the mice
vaccinated with STM-1 expressing ovalbumin under control of the nirB
promoter from both plasmid and the chromosomal location (P<0.001) as
shown in Figure 5.4.
Significant responses were observed for IFN-γ secretion from stimulated
splenocytes isolated from mice vaccinated with STM-1 expressing ovalbumin
antigen under control of the nirB promoter from the chromosome (P<0.001).
Also, mice vaccinated with STM-1 expressing ovalbumin antigen under
control of the pagC promoter from plasmid and from the chromosomal
location (IP inoculation) shows a significant response as shown in Figure 5.5
(P<0.001).
190
0
10
20
30
40
50
60
70
STM-1
(IP)
Oral
STM-1/
Empty pl
asmid
(IP)
Oral
pagC
Ova/Plas
mid (
pagC
O
SFC
per
mill
ion
sple
nocy
tes *Enumeration of
IL4 *
*
Figure 5.4. Enumeration of IL4- secretin
constructs. Spleen cells from vaccinate
the number of positive IL4-secreting ce
vaccinated with ovalbumin constructs, e
location, oral delivery) yielded significan
(* P < 0.05, ** P < 0.001)
1
**
IP)Oral
va/ch
romos
ome (
IP)O
nirBOva
/P
g splenocytes from
d mice were challen
lls determined. Com
xcept those vaccina
tly higher numbers.
91
*
ral
lasmid
nirBOv
mice
ged w
pared
ted wi
**
(IP)
Oral
a/Chro
mosom
e
*
vaccinated w
ith soluble o
with contro
th STM-1/pag
**
(IP)
Oral
ith ovalbumin
valbumin, and
l groups, mice
COva (plasmid
*
*
0
5
10
15
20
25
30
35
40
45
STM-1(
IP)
Oral
STM-1/
Empty pl
asmid
(IP)
Oral
pagC
Ova/pl
asmid
(IP)
Oral
pagC
Ova/ch
romos
ome (
IP)Oral
nirBOva
/plas
mid (IP
)Oral
nirBOva
/chrom
osom
e (IP)
Oral
SFC
per
mill
ion
sple
nocy
tes
** **Enumeration of IFN-γ
**
**
Figure 5.5. Enumeration of IFN-γ- secreting splenocytes from mice vaccinated
with ovalbumin constructs. Spleen cells from vaccinated mice were challenged
with soluble ovalbumin, and the number of positive IFN-γ -secreting cells
determined. Compared with control groups, mice vaccinated with ovalbumin
constructs, except those vaccinated with STM-1/pagCOva (plasmid location and
chromosomal location, oral delivery) and STM-1/nirBOva (plasmid location, both
IP and oral delivery) yielded significantly higher numbers.
(* P < 0.05, ** P < 0.001)
192
5.2.5 Analysis of humoral responses against C. jejuni Momp
antigen in vaccinated mice
In this experiment the ability of STM-1 was tested to deliver C. jejuni Momp
antigen under control of the nirB promoter from both plasmid and
chromosomal location. It was established from the above experiments that
mice vaccinated with STM-1 expressing ovalbumin antigen under the nirB
promoter from the chromosomal location induces both arms of immune
response when inoculated both orally and intraperitoeneally.
The experimental design is shown in Table 5.2. Group 1 to group 4 are
negative controls group 1 and 2 mice were vaccinated with STM-1 only and
group 3 and 4 mice were vaccinated with STM-1 harbouring empty plasmid.
The evaluation of humoral and cell mediated immune responses from other
groups was compared with group 1 to 4 for the analysis.
Serum samples were collected three weeks after the final vaccination for
ELISA to test the humoral immune responses. Reactivity of IgG antibody was
tested against the Momp antigen. Mice vaccinated with STM-1 expressing
Momp antigen under control of the nirB promoter shows a statistically
significant response (P<0.001) when expressed from both plasmid and
chromosome when compared with the control group. However, no significant
difference in the reactivity of IgG was observed when the delivery of antigen
was compared between plasmid encoded expression and chromosomal
encoded expression (Figure 5.6).
193
Table 5.2: Vaccine trial # 2. The shows the groups, vaccine constructs
and mode of inoculation for the evaluation of immune responses raised
against the Momp antigen
Groups Vaccination Mode of inoculation
Group 1 STM-1 IP
Group 2 “ Oral
Group 3 STM-1 harbouring empty plasmid IP
Group 4 “ Oral
Group 5 STM-1 expressing momo under the
nirB promoter from plasmid
IP
Group 6 “ Oral
Group 7 STM-1 expressing momo under the
nirB promoter from chromosome
IP
Group 8 “ Oral
194
0
100
200
300
400
500
600
STM-1 (IP
)Oral
STM-1/Empty
plas
mid (IP
)Oral
nirBMom
p/plas
mid (IP
)Oral
nirBMom
p/chro
mosom
e (IP)
Oral
Titre
** ******
Figure 5.6. Induction of IgG responses against C. jejuni Momp antigen in
vaccinated mice. Serum from vaccinated mice was used to test the
reactivity of IgG against C. jejuni outer membrane preparation. Compared to
control groups, all groups of mice vaccinated with Momp constructs yielded
a significantly higher response.
(** P < 0.001)
195
5.2.6 Analysis of cell mediated responses against C. jejuni
Momp antigen in vaccinated mice
The above result indicates the ability of STM-1 to deliver heterologous antigen
to effectively induce a modest humoral response. To analyse the T cell
response, secretion of IL4 and IFN-γ by stimulated splenocytes was
measured by ELISPOT assay. Cells were re-stimulated with a C. jejuni outer
membrane preparation. Results are expressed as the number of spot forming
T cells per million splenocytes with cells from each mouse assayed in
triplicate.
The result analysis for the secretion of IL4 indicates significant response in
mice vaccinated with STM-1 expressing Momp antigen under control of the
nirB promoter from both plasmid (P<0.001) and from the chromosomal
location (IP inoculation only) at P<0.005 when compared with the control
groups as shown in Figure 5.7.
Analysis of IFN-γ secreting splenocytes indicates statistically significant
response (P<0.001) in mice vaccinated with STM-1 expressing Momp antigen
under control of the nirB promoter from both plasmid and the chromosomal
location when compared with controls as shown in Figure 5.8. However, one
group of mice vaccinated with nirBMomp constructs from plasmid location
(oral inoculation) did not produce any observable response.
196
These results indicate that STM-1 expressing heterologous vaccine antigen
can induce both IL4 and IFN-γ responses, however spot forming counts were
relatively low from the splenocyltes secreting IL4 in the mice vaccinated with
STM-1 expressing Momp antigen from the nirB promoter, with means ranging
from below four to just over eight SFC per million cells. This would suggest
that cells secreting this cytokine are less prevalent after Momp vaccination
than after ovalbumin vaccination (compare Figure 5.7 and 5.4).
197
0
2
4
6
8
10
12
14
16
STM-1(
IP)Oral
STM-1/
Empty pl
asmid
Oral
nirBMom
p/plas
mid (IP
)Oral
nirBMom
p/chro
mosom
e (IP)
Oral
SFC
per
mill
ion
sple
nocy
tes
Enumeration of IL4 when stimulated with Momp antigen
**
**
*
Figure 5.7. Enumeration of IL4- secreting splenocytes from mice vaccinated with
Momp constructs. Spleen cells from vaccinated mice were challenged with
soluble C. jejuni outer membrane preparation, and the number of positive IL4-
secreting cells determined. Compared with control groups, mice vaccinated with
Momp constructs, except those vaccinated with STM-1/nirBMomp (chromosomal
location, IP delivery), yielded significantly higher numbers.
(* P < 0.05, ** P < 0.001)
198
0
5
10
15
20
25
30
35
STM-1
(IP)
Oral
STM-1/
Empty pl
asmid
(IP)
Oral
nirBMom
p/plas
mid (IP
)Oral
nirBMom
p/chro
mosom
e (IP)
Oral
SFC
per
mill
ion
sple
nocy
tes
Enumeration of IFN-γ when stimulated with Momp antigen
**** **
Figure 5.8. Enumeration of IFNγ- secreting splenocytes from mice vaccinated
with Momp constructs. Spleen cells from vaccinated mice were challenged with
soluble C. jejuni outer membrane preparation, and the number of positive IFNγ-
secreting cells determined. Compared with control groups, mice vaccinated
with Momp constructs, except those vaccinated with STM-1/nirBMomp (plasmid
location, oral delivery), yielded significantly higher numbers.
( ** P < 0.001)
199
5.3 Discussion
The utility of live attenuated Salmonella strains as a vector for the delivery of
heterologous antigen has been shown in different studies (Bachtiar et al.
2003; Chen and Schifferli 2003; Ashby et al. 2005). The capability of the
Salmonella vectors to target both cellular and humoral immune responses and
present heterologous antigen makes them an ideal candidate for the delivery
of passenger antigen and induction of the long lasting immune responses
(Kang et al. 2002; Fouts et al. 2003; Foynes et al. 2003; Du and Wang 2005).
The aim of work described in this chapter is to analyse the optimal expression
of passenger antigen in vivo from Salmonella mutant STM-1 in order to
characterise the optimal conditions which can be further utilised for the
efficient delivery of heterologous antigen by using STM-1 as a vector. STM-1
is an aroA mutant of S. Typhimurium, which is a commercially available
livestock vaccine for poultry to protect against Salmonella infection. Further
exploitation of this mutant as a carrier vaccine is a desirable outcome. The
efficiency of two well characterised in vivo inducible promoters was evaluated
to achieve optimal expression from multi-copy plasmids and the chromosomal
location.
As demonstrated by work described in the previous chapter, these promoters
were evaluated for their expression efficiency and stable protein expression
when the expression was allowed from multi-copy plasmid, also their ability to
induce expression from the single copy gene by integrating the constructs in
200
the aroA region of STM-1. Analysis for the expression of proteins in vitro by
western blotting indicates very low expression from both plasmid and
chromosomal location however, in vitro expression may not be the true
indicator of in vivo expression due to difficulty in replicating the anareobic
conditions required to activate these promoters. As it was impractical to
directly measure expression of vectored proteins in vivo, immune responses
were measured and assumed to reflect expression of the antigen.
Animal experiment demonstrates that STM-1 expressing the ovalbumin
antigen under control of the pagC promoter or nirB promoter can induce anti-
ovalbumin immune responses. In the analysis of humoral immune responses,
significant responses were observed in mice vaccinated with STM-1
expressing ovalbumin either from the plasmid or from the chromosome when
compared with negative control (p<0.001). However no significant difference
in humoral responses was observed when the delivery of antigens was
compared between the plasmid and the chromosomal location (Figure 5.2).
Therefore, there was no obvious advantage in having multiple copies of the
gene. Additionally no significant difference in the immune responses was
observed against the delivered antigen from either the pagC and nirB
promoter, which indicates similar expression efficiency from the two
promoters. The IgA response in the test group when compared with the
control group shows positive responses in vaccinated mice. However, the IgA
response in the mice receiving vaccination by IP injection shows higher
responses then mice which received vaccination orally. The reason for this is
unknown as the mice vaccinated orally were expected to show higher IgA
201
responses. However, the establishment of infection is different between the
two routes (reflected by the different CFU given) hence direct comparision is
not possible.
Similarly cellular immune responses, as measured by enumeration of
cytokine-secreting splenocytes indicates significant responses for the
secretion of IL4 and IFN-γ from the stimulated splenocytes isolated from mice
vaccinated with STM-1 expressing ovalbumin. This is appaent from both pagC
and nirB promoter constructs (* P<0.05, **P<0.001). Exception are mice
vaccinated orally with STM-1 expressing ovalbumin from the plasmid location
under control of the pagC promoter, which did not show an IL4 response, and
mice vaccinated both orally and IP with STM-1 expressing ovalbumin from the
plasmid location under control of the nirB promoter, which did not show any
IFN-γ response. However, both IL4 and IFN-γ count was higher in groups
where the antigen was delivered under control of the nirB promoter from the
chromosomal location, therefore the Momp antigens expressed under control
of the nirB promoter was the choice for the second set of vaccine
experiments.
Humoral and cellular responses were evaluated to test the utility of STM-1 to
express C. jejuni major outer membrane protein as test antigen under the nirB
promoter from both plasmid and chromosomal location. Significant IgG
reactivity against the Momp antigen was observed in mice vaccinated with
STM-1 expressing Momp antigen under control of the nirB promoter when
compared with the control groups. However, no difference in the reactivity
202
was observed when the expression between plasmid and chromosomal
location was evaluated (Figure 5.6), again indicating no advantage of multiple
gene dosage.
Evaluation of cell mediated responses from stimulated splenocytes indicates
positive response for the secretion of both IL4 and IFN-γ in the mice
vaccinated with STM-1 expressing Momp from both plasmid and the
chromosomal location, when compared with the control groups. However spot
forming counts were relatively low for IL4 secreting splenocyltes, indicating
that the Momp antigen may preferentially induce T cells that secreted IFN-γ,
indicative of a Th1 type response in mice.
This study indicates that the STM-1 mutant can be used effectively as a
carrier to deliver the heterologous vaccine antigen. The expression of
heterologous vaccine antigen under the control of in vivo inducible promoters
such as pagC and nirB can induce immune responses from both plasmid and
chromosomal location. However, the nirB promoter was found to be more
efficient in the expression of heterologous antigen when the expression was
analysed from the single copy gene from the chromosome.
203
Chapter 6
The effect of prior immunological
experience against the bacterial vector on
the immune responded against a
heterologous antigen
6.1 Introduction
One of the recent advances in the field of vaccine technology is the use of
attenuated Salmonella strains to deliver heterologous vaccine antigen The
possibility that prior immunological experience of animals with homologous or
related strains might compromise the efficacy of Salmonella vector to deliver
foreign antigen has long been considered (Dougan et al. 1987).
Bao et al. (1991) first reported the consequences of prior exposure of animals
to the vector strain and showed that both serum and mucosal antibody
responses against the foreign antigen up-regulated after the animals were
exposed to the vectored strain. Whittle et al. (1997) reported similar findings
where mice immunised intraperitoneally with an S. dublin aroA mutant
expressing heterologous antigen after being exposed to the same vector
showed a higher immune response to the vectored antigen in comparison to
204
mice without any immunological experience against the vector Bao et al.
(1991).
Most of the studies (Kohler et al. 2000; Jespersgaard et al. 2001) indicate the
up-regulation of immune responses after animals have been exposed to either
homologous or related strains of the vector before the delivery of
heterologous antigen in the vector. One of the recently conducted studies by
Metzger, et al. (2004) on human volunteers using S. typhi as a vector
suggested that there was no change in the immune response against the
heterologous antigen in human volunteers who were exposed to empty vector
in comparison the volunteers who did not have prior immunological
experience with the vector strain. Taken together, these results indicate that
pre-existing immunity may not compromise immunogenecity of vectored
antigen at a subsequent vaccination.
However, there are some conflicting reports. Attridge et al. (1997) reported
that a pre-existing immune responses against the bacterial vector prior to the
delivery of antigenic protein in the vector can down-regulate immune
responses in mice against the antigenic protein. Similar concerns were raised
by Roberts, et al. (1999) and Vindurampulle et al. (2003) who reported that
prior exposure of animals to homologous or related strains reduced the
subsequent immune response in mice against the vectored foreign antigen.
These studies indicate that the prior exposure of animals to bacterial vector
can certainly affect the immune responses against the heterologous antigenic
205
protein in animal models. However, most of the studies indicate that the
immune response was up-regulated after the animals were exposed to either
homologous or related strains of the vector before the delivery of
heterologous antigen in the vector.
These findings lead to the conclusion that the pre-existing immune responses
against the bacterial vector or related strains may positively influence immune
responses against the heterologous antigen, however, there are a number of
factors such as the Salmonella carrier strain used and the type of the foreign
antigen which may influence the outcome (Vindurampulle and Attridge 2003).
These findings have only reported the affect on humoral immune responses,
as there are no studies which report the affects on T cell immune responses.
This is important as one of the advantages of using Salmonella (an
intracellular pathogen) is that strong cellular responses can be induced.
Work described in the previous chapter demonstrated that the Salmonella
aroA mutant can be efficiently utilised for the delivery of heterologous antigen
expressed from both the plasmid and chromosomal location. Both humoral
and cellular immune responses were shown to be induced against the
heterologous antigen. In the work described in this chapter, the influence of
pre-existing immunity on such responses is examined. S. Enteritidis was used
as a model for the pre-existing immunity to a related, but not identical vector.
206
6.2 Results
To analyse the impact of pre-existing immune response, both short term and
long term, 6-8 week old female BALB/c mice were divided into eight groups,
each group consisting of five mice. Each mouse was orally inoculated with
5x108 CFU of bacterial culture. The experiment was designed to test immune
responses to vectored antigen in mice with pre-existing immunity to a
homologous or a heterologous strain. Table 6.1 shows the vaccination
protocol. Basically, the effects of pre-existing immunity were tested after a
short period before vaccination (3 weeks) or a longer period (12 weeks).
The first vaccination was administered at week 0, with Groups 1 and 5 as
controls, therefore not receiving inoculation in week 0. These groups were
then inoculated with STM-1 in week 3 and week 12 respectively. Group 2 and
Group 6 were not exposed to either vector strain or related strains, therefore
these groups were also not inoculated on week 0, but were inoculated on
week 3 and week 12 respectively with STM-1 expressing ovalbumin antigen
from the pKKOva plasmid. Group 3 and Group 7 were exposed to STM-1 at
week 0, to stimulate pre-existing immunity against the homologous strain. In
week 3 or 12 these groups were inoculated with STM-1 expressing ovalbumin
from the pKKOva plasmid. Similarly Group 4 and Group 8 were exposed to
the related strain (S. Enteritidis) in week 0 and in weeks 3 or 12 were
inoculated with STM-1 expressing ovalbumin from the pKKOva plasmid.
207
Table 6.1 Protocol for the vaccine trial to test the effect of pre-existing
immunity.
Group Vaccination
1st dose
Vaccination 2nd
dose week 3
Vaccine 2nd dose
week 12
Gruop1 None STM-1 None
Group 2 None STM-1/pKKOVA None
Group3 STM-1 STM-1/pKKOVA None
Group4 S. Enteritidis STM-1/pKKOVA None
Group5 None None STM-1
Group6 None None STM-1/pKKOVA
Group7 STM-1 None STM-1/pKKOVA
Group8 S. Enteritidis None STM-1/pKKOVA
208
6.2.1 Initial evaluation of humoral immune responses against
ovalbumin.
ELISA analysis was performed to detect the IgG responses against the
vectored ovalbumin antigen as described in section 2.6.5. Serum samples
were collected from all groups at week 6. Although the Group 5-8 mice had
not been exposed to ovalbumin (Table 6.1), the serum was collected to
confirm negative immune responses in these groups. Initially serum samples
from each mouse within a group were pooled for detection of IgG antibodies.
There was no reactivity in sera from mice vaccinated with STM-1 only (Group
1), and mice in the groups which did not receive the second dose of
vaccination at week 3 (Group 5 -8). However a positive response was
detected against ovalbumin in groups vaccinated with STM-1 expressing
ovalbumin antigen from the pKKOva plasmid. (Groups 2-4).
These results from pooled sera indicate that immune responses to ovalbumin
are induced in the vaccinated groups in relation to the control group (Figure
6.1). Responses are low however, and as the sera was pooled, titres and
statistical significance was not calculated. However, there appears to be no
obvious difference between the groups which were pre-exposed to the
homologous and related strains when compared to the unexposed group
(group 2).
209
0
0.01
0.02
0.03
0.04
0.05
0.06
Group-1 (N
one/STM-1)
Group-2 (N
one/STM-1pKKOva)
Group-3 (S
TM-1/STM-1pKKOva
)
Group-4 (S
.E/STM-1p
KKOva)
Group-5 (N
one)
Group-6 (N
one)
Group-7 (S
TM-1)
Group-8 (S
.E)
OD
450
Figure 6.1 Estimation of IgG responses at week 6 in all groups. Sera taken
from mice were pooled within a group and the reactivity of IgG to ovalbumin
was measured. The reactivity show a positive response in mice which were
vaccinated with STM-1 expressing ovalbumin in comparison to groups of
mice which were vaccinated with STM-1 only.
210
6.2.2 Evaluation of humoral immune responses against the
ovalbumin antigen from individual sera
The initial assessment of IgG responses against ovalbumin from the pooled
sera did not show any obvious difference between the pre-exposed mice to
either homologous or related strains of Salmonella when compared with the
unexposed groups.
Further analysis was performed from individual serum samples collected 3
weeks after the second dose of vaccination to Group 1 to Group 4 mice (i.e
week 6). Titre was calculated for sera from each mouse. The IgG reactivity
was compared using the non recombinant STM-1 group (Group 1) as control
using student’s T Test (P<0.001 and P<0.05).
A significant difference was observed at the 99% confidence level between
mice vaccinated with STM-1 expressing ovalbumin from the pKKOva plasmid
(Group 2 to Group 4) when compared with the control group which was
vaccinated with non recombinant STM-1 (Group 1). Also a significant
difference was observed between groups pre-exposed to the vector strain
(Group 3) when compared to the unexposed group (Group 2) (Figure 6.2).
However no significant difference was observed between the group which
was exposed to a related strain (Group 4) when compared with the
unexposed group (Group 2). This analysis indicates that the prior exposure of
animals to the vector strain enhances the subsequent humoral immune
211
response to a vectored antigens carried by the same strain, delivered 3 weeks
later.
212
0
50
100
150
200
250
Group-1(None/STM-1)
Group-2(None/STM-1pKKOva)
Group-3 (STM-1/STM-1pKKOva)
Group-4 (S.E/STM-1pKKOva)
Titre
**
**
**
Figure 6.2. Evaluation of humoral immune responses against the carrier antigen (chicken
ovalbumin) at week 6 in groups 1-4. Mice were first vaccinated (week 0) with nothing
(Group 1 and group 2), STM-1 (Group 3) or S. Enteritidis (Group 4), followed by
vaccination at week 3 with non-recombinant STM-1 (Group 1) and STM-1 expressing
ovalbumin from the pKKOva plasmid (Group 2 to Group 4). Serum samples collected 3
weeks after the second vaccination were tested for IgG responses. Significant
differences in titre were observed between Groups 2 –4 and Group 1 (**P<0.001), also a
significant difference was observed in the groups which were pre-exposed to the vector
strain (Group 3) when compared with the unexposed group (Group 2) (**P<0.001).
213
6.2.3 The effect of a longer time period between exposure and
vaccination with vectored antigen
Serum samples from all mice were collected at week 15, which is 3 weeks
after the second vaccination to Group 5- 8 with STM-1 carrying the pKKOva
plasmid. The titre was calculated and the reactivity against chicken ovalbumin
antigen was compared against mice vaccinated with non-recombinant STM-1
as the control using Student’s T-test (P<0.001 and P<0.05).
Analysis of humoral responses induced against the heterologous antigen after
the animals were first vaccinated with the vector strain or a related strain
indicates significant responses in both exposed and unexposed animals when
compared with mice vaccinated with non-recombinant STM-1 (Figure 6.3).
This is similar to the observation at week 6. However, no significant difference
was observed when the exposed groups were compared with the unexposed
groups.
The analysis of longer term exposure of animals to either vector strain or
related strain indicates up-regulation of humoral immune response not only
when compared to the control group (P<0.001), but also when compared with
the unexposed group and mice exposed to STM-1 prior to vaccination
(P<0.05) as shown in Figure 6.4. The immune response in the unexposed
group was also significantly higher when compared with the control group.
214
0
50
100
150
200
250
Group-1(None/STM -1)
Group-2(None/STM-1pKKOva)
Group-3 (STM -1/STM-1pKKOva)
Group 4 (S.E/STM-1 pKKOva)
Titr
e
**
****
Figure 6.3. Evaluation of humoral immune responses against the carrier antigen
(chicken ovalbumin) at week 15 in groups 1-4. Mice were first vaccinated (week 0)
with nothing (Group 1 and group 2), STM-1 (Group 3) or S. Enteritidis (Group 4),
followed by vaccination at week 3 with non-recombinant STM-1 (Group 1) and STM-
1 expressing ovalbumin from the pKKOva plasmid (Group 2 to Group 4). Serum
samples collected 12 weeks after the second vaccination were tested for IgG
responses. Significant differences in titre were observed between Groups 2 –4 and
Group 1. (**P<0.001)
215
0
50
100
150
200
250
300
350
400
450
Group-5 (None/STM -1) Group-6 (None/STM-1pKKOva)
Group-7 (STM -1/STM-1pKKOva)
group-8 (S.E/ STM-1pKKOva)
Titre
**
**
**
Figure 6.4. Evaluation of humoral immune responses against the carrier antigen
(chicken ovalbumin) at week 15 in groups 5-8. Mice were first vaccinated (week 0)
with nothing (Group 5 and 6), STM-1 (Group 7) or S. Enteritidis (Group 8), followed by
vaccination at week 12 with non-recombinant STM-1 (Group 5) and STM-1 expressing
ovalbumin from the pKKOva plasmid (Group 6 to Group 8). Serum samples collected
3 weeks after the second vaccination were tested for IgG responses. Significant
differences in titre were observed between Groups 6-8 and Group 5 (**P<0.001), also a
significant difference was observed in the groups which were pre-exposed to the
vector strain (Group 7) when compared with the unexposed group (Group 2)
(**P<0.05).
216
6.2.4 Effect of prior immunological experience with carrier or
related strains; cellular immune responses
The effect on carrier protein immunogenecity due to pre-existing immune
responses against the homologous or related strains has been previously
analysed (Roberts et al. 1999; Jespersgaard et al. 2001; Vindurampulle and
Attridge 2003; Metzger et al. 2004). Although there are conflicting reports,
most of the studies so far have only reported the effects on humoral
responses. The purpose of using Salmonella strains as a vector to deliver
heterologous vaccine antigen is to target cellular immune responses so that
long lasting immunity can be induced.
Mice were sacrificed in week 15, which is 12 weeks after the second dose of
vaccination in Group 1-4 and 3 weeks after the second dose of vaccination in
Group 5-8. Splenocytes were prepared for the ELISOPT assay as described
in section 2.6.5.1. The number of cytokine secreting cells in each group was
compared using Student’s T test (P<0.001 and P<0.05). Results were
reported as spot forming cells (SFC).
Results of this experiment are presented as for the humoral responses, with
groups 1-4 compared and groups 5-8 compared. For groups 1-4 (designed to
test the effects of prior exposure followed by vaccination 3 weeks later),
exposure of mice to either the vector or related strain results in significantly
increased IL4 response in mice which were exposed to STM-1 at week 0
(Figure 6.5, compares group 3 vs group 2) but not when exposed to the
217
related S. Enteriditis (group4 vs Group 2). All groups (2-4) yielded
significantly higher numbers than the control, group 1.
The analysis of IFN-γ secreting cells indicates a significant increase in
responses in animals which were exposed to either vector strain (group 3) or
related strain (group 4) before being vaccinated with STM-1 expressing
ovalbumin when compared with mice which were not vaccinated at week 0
(group 2) (P<0.001) as shown in Figure 6.6. All groups (2-4) yielded
significantly higher numbers that the control, group 1.
Groups 5-8 received the 2nd dose vaccination in week 12, which was 9 weeks
after being exposed to the vector strain or related strain. All the mice
vaccinated with ovalbumin constructs showed a statistically significant IL4 and
IFN-γ cell count when compared to mice vaccinated with non-recombinant
STM-1 (compares groups 6-8 with group 5) at P<0.001 as shown in Figure 6.5
and Figure 6.6. Additionally exposure to the vector strain or related strain prior
to vaccination with the vectored antigen in week 12 yielded a statistically
significant increase in cytokine secreting cells for both IL4 and IFN-γ
(P<0.001), when compared with mice vaccinated with STM-1 expressing
ovalbumin from the pKKOva plasmid without being exposed (compare groups
7 and 8 with group 6).
These data indicate that exposure to the vector or related strain can
upregulate subsequent responses to a vectored antigen, with little differences
between those in which the vaccination was given 3 or 12 weeks after the
218
exposure (except in the case of exposure to S. Enteriditis, where there was no
significant increase in the IL4 response where the vaccine was given 3 weeks
after exposure)
The result of IL4 and IFN-γ responses in response to challenge with
ovalbumin correlates with the analysis of humoral immune responses in
groups 1-8. Humoral immune responses against the foreign antigen were
significantly higher in mice which were pre-exposed to the vector strain or
related strain, not only in comparison to mice vaccinated with non-
recombinant STM-1 but also in comparison to mice which were vaccinated
with STM-1 expressing ovalbumin from the pKKOva plasmid without being
exposed.
219
0
10
20
30
40
50
60
70
Grou
p 1 (N
one/S
TM -1)
Grou
p 2 (N
one/S
TM-1pKKOva
)
Grou
p 3 (S
TM-1/STM -1pK
KOva)
Grou
p 4 (S
.E/STM-1
pKKOva
)
Grou
p 5 (N
one/S
TM -1)
Grou
p 6 (N
one/
STM-1pKKOva
)
Grou
p 7 (S
TM-1/STM -1pK
KOva)
Grou
p 8 (S
.E/ S
TM-1pKKOva
)
SFC
per
mill
ion
sple
nocy
tes
Enumeration of IL4- secreting splenocytes **
**
****
****
Figure 6.5. Enumeration of IL4- secreting splenocytes from vaccinated mice in groups
1- 8. Spleen cells from vaccinated mice were challenged with soluble ovalbumin, and
the number of positive IL4-secreting cells determined. Compared with the control
groups (1 and 5), mice vaccinated with ovalbumin constructs yielded significantly
higher numbers (groups 2-4 and 6-8) (**P<0.001). A significant difference was also
observed between groups which were pre-exposed to the vector strain (group 3 and 7)
when compared with unexposed groups (2 and6) (**P<0.001) and the groups which
were pre-exposed to the related strain and vaccinated 9 weeks later (group 8) when
compared with group 6 (**P<0.001).
220
0
10
20
30
40
50
60
Grou
p-1 (
None/S
TM-1)
Group-
2 (Non
e/STM
-1pK
KOva)
Group-
3 (STM-1/
STM-1pKKOva
)
Grou
p-4 (
S.E/S
TM-1p
KKOva)
Grou
p 5 (N
one/S
TM -1
)
Grou
p 6 (N
one/S
TM-1
pKKOva
)
Grou
p 7 (S
TM -1/S
TM-1pKKOva
)
Grou
p 8 (S
.E/S
TM-1
pKKOva
)
SFC
per
mill
ion
sple
nocy
tes
Enumeration of IFN-γ- secreting splenocytes **
**
****
****
Figure 6.6. Enumeration of IFN-γ- secreting splenocytes from vaccinated mice in
groups 1-8. Spleen cells from vaccinated mice were challenged with soluble
ovalbumin, and the number of positive IFN-γ -secreting cells determined. Compared
with the control groups (1 and 5), mice vaccinated with ovalbumin constructs yielded
significantly higher numbers (**P<0.001). A significant difference was also observed
between groups pre-exposed to the vector and related strain respectively (group 3 -4
and group 7-8) when compared with group 2 and 6, the unexposed groups (**P<
0.001).
221
6.3 Discussion
The construction of Salmonella based vaccines where the Salmonella strain
can deliver heterologous vaccine antigen to the host immune response is
based on the notion that long term T-cell immune responses can be induced
efficiently against the passenger antigens (Hess et al. 2000; Kang et al.
2002). However, the practicality of this approach still needs to be tested in the
field trials.
Major issues regarding the use of the proposed technique in the
management of infectious disease emerge when considering the effect of pre-
existing immune responses against the bacterial vector or related strains in
the host. Also, the consequences of repeated use of the same vector to
deliver different foreign antigens needs to be considered.
Several studies have shown conflicting results regarding an impact of vector
priming and its effect on the efficiency to generate protective immune
response against the heterologous antigen (Bao and Clements 1991; Attridge
et al. 1997; Roberts et al. 1999; Metzger et al. 2004). Vindurampulle et al.
(2003) in one of his studies showed that several variable factors, such as the
animal model, vector strain, the type of mutation in vector strain etc plays an
important role in the generation of efficient immune responses against the
heterologous antigens after the prior exposure of animals to either carrier
strain or related strain. In all studies conducted to date only report the effect
on the generation of humoral immune responses.
222
The rationale behind using Salmonella to deliver heterologous vaccine
antigen is that as an intracellular pathogen, Salmonella efficiently induces T
cell responses giving rise to Th1 and Th2 helper cells and CTL’s against
Salmonella and vectored foreign antigen (Wang HW 2003 Aug; Wyszynska et
al. 2004).
This study was conducted to mimic the effect of prior exposure of the animal
host to the vector strain or a related strain, and analyse the effect this
exposure has on the induction of both humoral and cellular immune
responses against a passenger antigens. Two parallel experiments were
performed one with a 3 week gap between exposure and vaccination, and
one with 12 week gap. The results indicate that the prior exposure of animals
to the vector strain or related strain, at 3 and 12 weeks prior to vaccination,
significantly enhances the humoral immune responses not only in comparison
to mice vaccinated with non-recombinant STM-1 but also in comparison with
mice vaccinated with STM-1 expressing ovalbumin without prior exposure.
Similarly, cellular immune responses as measured by enumeration of cytokine
secreting splenocytes, are generally increased.
These results suggest that both humoral and cellular immune responses are
up-regulated if the animal host which were exposed to either the vector strain
or related strain when compared to animals with no immunological memory
against the vector or related strain.
223
This study indicates that Salmonella strains may be effective carriers for the
delivery of heterologous antigen to the host immune response in the field. The
immunological memory against the bacterial vector or related strains in target
hosts will not jeopardise the use of Salmonella as a vector to deliver
heterologous antigens. In fact the immunological memory against the bacterial
vector may enhance the immune responses against passenger antigens.
224
Chapter 7
Conclusion
Live attenuated bacterial strains provide unique alternatives in terms of
antigen delivery and immune presentation, however, they also show promise
as potentially useful vectors for heterologous antigen delivery. The efficiency
of any live bacterial vector rests with its ability to present sufficient foreign
antigen to the human or animal immune system to initiate the desirable
protective immune response. The most obvious choice in the case of live
bacterial vaccines can be those candidates, which can strongly activate both
the humoral and cellular immune response. Intracellular residing organisms,
which can invade macrophages or dendritic cells, have the capability to
markedly elevate B and T cell responses.
Attenuated bacterial strains such as Salmonella, Mycobacterium, Shigella and
Listeria spp have been found to be suitable for this purpose. Initial studies
using Salmonella mutants reported that these mutants can establish limited
infection, induce long lasting immune responses and were also suitable for
the delivery of heterologous antigen to the host immune system (Clements
and El-Morshidy 1984; Dalla Pozza et al. 1998; Ward et al. 1999; Wyszynska
et al. 2004). Since then the use of Salmonella mutants has been widely
studied for use as a potential vector to deliver antigenic proteins from
bacteria, viruses, and eukaryotes (Dougan et al. 1987; Dalla Pozza et al.
1998; Dunstan et al. 1998; Coynault and Norel 1999; Bullifent et al. 2000;
Fayolle et al. 2001; Fouts et al. 2003).
225
The STM-1 mutant, developed by RMIT University, harbours a mutation in the
aroA gene and is a well characterised vaccine strain currently in use to protect
livestock against Salmonella infection. This mutant was tested for its potential
to deliver heterologous vaccine antigen expressed from plasmids and found
suitable as vector for this purpose (Bachtiar et al. 2003)
In order to progress to any commercial application, the use of plasmid-borne
antigen shouls be eliminated. This is for two main reasons; (1) to ensure
stability of antigen expression in the absence of antibiotic selection, and (2) to
eliminate the possibility of environmental contamination of resident bacterial
flora by horizontal transmission of plasmids.
However, one limitation in using STM-1 for this study was uncharacterised
nature of the aroA region of this mutant, where the genes of interest were
planned to be integrated. As described in the results from chapter 3 the lesion
in STM-1 is due to the deletion of several genes, including first 90bp of aroA
gene. However, there is also an insertion and chromosomal rearrangement
which is not an inversion, but may be a crossover between two points not due
to any specific target sequences. The exact mechanism of this rearrangement
is not known, but may be due to the conserved three dimensional structure of
bacterial chromosome (Fonstein, et al. 1994)
Once the architecture of the genome in STM-1 was determined, strategies to
incorporate genes into the chromosome were developed. Constructs were
226
designed with genes encoding chicken ovalbumin (model antigen) and C.
jejuni Momp (test antigen) with both genes placed under the control of two
promoters, either pagC or nirB The pagC promoter is a macrophage inducible
promoter found to be most efficient in terms of in vivo expression of
heterologous antigen (Dunstan et al. 1999). The nirB promoter was isolated
from E. coli (Jayaraman et al. 1987), and this promoter has also been shown
to be efficient in term of in vivo expression and antigen delivery (Chatfield et
al. 1992). Therefore these two promoters were the promoters of choice in this
study.
Plasmid constructs were designed with genes encoding ovalbumin and Momp
proteins were put under the control of one of the promoters. Similar constructs
were integrated into the aroA gene of the STM-1 chromosome. The Lambda
Red system which is based on the phage recombination system was used for
the integration of constructs in the STM-1 chromosome (Murphy 1998;
Datsenko and Wanner 2000). This system proved to be relatively efficient at
generating recombinants in STM-1, whereas a parallel strategy employing
suicide vectors was unsuccessful.
The results details in chapter 4 and 5 indicate that expression of heterologous
vaccine antigen, sufficient to induce both humoral and T cell immune
responses, can be achieved using these two promoters from either plasmid or
chromosomal location. Although the pagC promoter has been reported to be
more efficient in comparison to nirB (Dunstan et al. 1999), results from this
study indicates that both the promoters are equally efficient in inducing
227
immune responses and therefore optimal expression of heterologous
antigens. In fact immune responses were some what higher (although not
significant) from chromosomal constructs, incorporating the nirB promoter.
It was thought that the higher gene copy number afforded by the use of
plasmids may be reflected in the induction of higher immune responses,
however this was not observed. This might be due to the inability of the STM-
1 mutant to retain the plasmid in vivo due to the metabolic burden imposed
and the absence of drug selection. As stated above the mutation in STM-1 is
due to the deletion of several metabolically important genes. In vivo
propagation of plasmid will put the additional metabolic burden on bacterial
replication machinery, so there may in effect be selection for the loss of
plasmid in vivo.
The effect of pre-existing immune responses against the bacterial vector or
related strains is a concern which may jeopardise the use of STM-1 vector or
the use of other Salmonella mutants as vector to deliver heterologous vaccine
antigen in the field. Studies conducted to date indicate conflicting reports.
some studies reported up-regulation of humoral immune responses against
heterologous vaccine antigen in animals which were pre-exposed to the
vector strain or related strains (Bao and Clements 1991; Whittle and Verma
1997), while other studies suggests that immune response against the
vaccine antigen might suffer in animals pre-exposed to such strains (Attridge
et al. 1997; Roberts et al. 1999; Vindurampulle and Attridge 2003). Some,
recent studies concerning the impact of vector priming indicates induction of
228
greater humoral immune responses (Kohler et al. 2000; Jespersgaard et al.
2001; Metzger et al. 2004). All studies to date analyse only humoral immune
responses.
The experiments described in Chapter 6 were designed to mimic the effects of
pre-existing immunity. Animals were left either 3 or 12 weeks after exposure
to either the vector strain (STM-1) or a related species, S. Enteritidis, prior to
administration of a second dose with STM-1 expressing ovabumin. Analysis of
the resulting immune responses against ovalbumin indicates that prior
immunological memory against the bacterial vector or related strains
enhances both humoral and cellular immune responses against the
passenger antigen.
Taken together, these results indicate that heterologous antigen can be
expressed from the chromosomal location in STM-1, and immune responses
to these antigens can be induced. The Lambda Red system allows efficient
recombination of inserts into the bacterial chromosome at the desired location
and also provides the option to precisely remove the gene encoding for the
antibiotic resistance. Both these observations should facilitate further research
into the use of STM-1 as a vaccine vector.
229
Chapter-8
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