VACCINE PLATFORM FOR INFECTION OR AUTOIMMUNE DISEASES USING
AN ETEC FIMBRIAL SCAFFOLD
by
SangMu Jun
A dissertation submitted in partial fulfillment
of the requirement for the degree
of
Doctor of Philosophy
in
Veterinary Molecular Biology
MONTANA STATE UNIVERSITY
Bozeman, Montana
April 2009
© COPYRIGHT
by
SangMu Jun
2009
All Right Reserved
ii
APPROVAL
of a dissertation submitted by
SangMu Jun
This thesis has been read by each member of the thesis committee and has been
found to be satisfactory regarding content, English usage, format, citations, bibliographic
style, and consistency, and is ready for submission to the Division of Graduate Education.
David W. Pascual, Ph.D.
Approved for the Department of Veterinary Molecular Biology
Mark T. Quinn, Ph.D.
Approved for the Division of Graduate Education
Dr. Carl A. Fox
iii
STATEMENT OF PERMISSION TO USE
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degree at the Montana State University, I agree that the Library shall make it available to
borrow under rules of the library. I further agree that copying of this dissertation is
allowable only for scholarly purpose, consistent with “fair use” as prescribed in the U.S.
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distribute my abstract in any format in whole or in part.”
SangMu Jun
April 2009
iv
TABLE OF CONTENTS
1. SALMONELLA: PATHOGENESIS, HOST IMMUNE RESPONSES,
AND ITS ROLE AS A HETEROLOGOUS ANTIGEN CARRIER............................................1
Introduction ............................................................................................................................................1
Salmonella Pathogenesis.......................................................................................................................3
Enteritis ........................................................................................................................................3
Enteric Fever ...............................................................................................................................8
Host Resistance against Salmonella Infection................................................................................. 12
Innate Immunity....................................................................................................................... 12
Acquired Immunity ................................................................................................................. 18
Attenuated Salmonella and Its Role as a Heterologous Vaccine Carrier...................................... 21
Live Attenuated Salmonella Vaccine ..................................................................................... 21
Live Attenuated Salmonella as a Carrier for HeterologousAntigen................................... 24
2. MATERIALS AND METHODS..................................................................................................... 28
EAE Study........................................................................................................................................... 28
Animals..................................................................................................................................... 28
Cloning of PLP 139-151 into CFA/I Fimbriae and Its Epression on Salmonella
Vaccine Vector .........................................................................................................................28
Oral Vaccination with Salmonella Vaccine and PLP139-151 Challenge ..............................29
Histological and Immunohistochemistry Evaluation .........................................................30
Ab ELISA. ...............................................................................................................................32
Cytokine ELISA .....................................................................................................................32
Statistical Analysis ..................................................................................................................33
ETEC/EHEC Vaccine Study ............................................................................................................33
ETEC/EHEC Dual Vaccine Construction ............................................................................... .33
Restriction-, Sequencing-, and Western Blot Analysis .............................................................34
Immunization............................................................................................................................... 35
Ab ELISA ..................................................................................................................................35
Construction of FanC/protein σ1 ExpressionVector ............................................................35
3. ETEC FIMBRIAL (CFA/I) EXPRESSION ON SALMONELLA: ITS IMPACT
ON HOST IMMUNE RESPONSES, AND PROTECTION AGAINST
INFLAMMATORY AUTOIMMUNE DISEASE, EAE.............................................................. 37
Introduction. ....................................................................................................................................... .37
Rational and Aim of Research ..........................................................................................................40
v
TABLE OF CONTENTS – CONTINUED
Protection from EAE via Bystander Suppression with Immunization of SalmonellaCFA/I.-PLP.......................................................................................................................................... 41
Results....................................................................................................................................... 41
Cloning of PLP139-151 into CFA/I Structural Gene cfa/B and Its
Reduced Expression...................................................................................................... 41
Reduced EAE Clinical Development and Resolution by the Oral Immunization
of Salmonella-CFA/1(H696) .................................................................................................. 44
Histopathological Analysis of CNS: Myelin Status and Nucleated Cell Infiltration
to CNS...................................................................................................................................... 46
Salmonella-CFA/I vaccine Halts CD4+ T Cell and Mac-1+ Cell
Influx into the CNS....................................................................................................... 47
PLP139-151-Specific Immune Deviation from Th1 to Th2 Cell by Vaccination
with Salmonella- CFA/I........................................................................................................... 54
Discussion and Conclusions.............................................................................................................. 57
4. MUCOSAL VACCINE DEVELOPMENT AGAINST ENTEROTOXIGENIC
(ETEC)AND ENTEROHEMORRHAGIC (EHEC) E. COLI.................................................... 63
Intranasal ETEC Vaccine Targeting.................................................................................................. 63
Introduction............................................................................................................................... 63
Results....................................................................................................................................... 65
Plasmid Construction for FanC/protein σ1 Fusion Vaccine and its Expression
in E. coli ......................................................................................................................... 65
FanC/protein σ1 Plasmid Construction for Yeast Expression .................................. 65
LiveVaccine for HEC......................................................................................................................... 69
Introduction ......................................................................................................................................... 69
Results.................................................................................................................................................. 71
Expression of EHEC LPS Peptide Mimetic on Salmonella Vaccine Vector..................... 71
Antibody Responses by Salmonella Vaccine with Chimeric K99/LPS
Peptide Mimetic....................................................................................................................... 72
Conclusions......................................................................................................................................... 75
REFERENCES CITED............................................................................................................................ 76
vi
LIST OF FIGURES
Figure
Page
1. 1.
Salmonella pathogenesis islands (SPIs).......................................................................................4
1. 2.
phoP/phoQ regulon involved regulatory cascade .....................................................................11
1. 3.
Schematic diagram of concerted action of NADPH phagocyte oxidase,
myeloperoxidase, and iNOS ...................................................................................................... 16
3. 1.
Construction of pCFA/PLP and Its Expression........................................................................ 42
3. 2.
Anti-CFA/I Ab kinetics in SJL/J mice........................................................................................ 32
3. 3.
Antigen recall assay for Salmonella-CFA/I-PLP Immunized mice ....................................... 44
3. 4.
Clinical manifestation of EAE ................................................................................................... 48
3. 5.
LFB staining of spinal cords....................................................................................................... 50
3. 6.
H&E staining of spinal cords ..................................................................................................... 51
3. 7.
Immunohistochemistry of spinal cords..................................................................................... 53
3. 8.
Cytokine profile from various tissues........................................................................................ 55
4. 1.
Construction of FanC/proteinσ1 E. coli expression plasmid. ................................................. 67
4. 2.
Construction of FanC/protein σ1 yeast expression plasmid and its expression................... .68
4. 3.
EHEC LPS peptide mimetic sequence obtained from phage display ................................... 71
4. 4.
Cloning of EHEC LPS peptide mimetic sequence into K99 minor structural
gene fanH ..................................................................................................................................... 73
4. 5.
Chimeric K99 fimbrial expression............................................................................................. 74
4. 6.
Immunogenecity of LPS peptide mimetic................................................................................ 74
vii
LIST OF TABLES
Table
Page
1. 1.
SPI-1 encoded effector proteins....................................................................................................5
1. 2.
Summary of typhoid fever vaccines and their characteristics................................................. 22
1. 3.
Animal-based foreign antigen delivery studies with Salmonella ........................................... 26
3. 1.
Quantitative comparison of clinical EAE development.......................................................... 49
3. 2.
Histopathological comparison of EAE ..................................................................................... 52
viii
ABBREVIATIONS
•
Samonella Pathogenesis Islands
•
TTSS: type III secretion system
•
GEFs: guanine ucleotide exchange factor
•
MAPK: MAP kinase
•
GAPs: GTPase-activating factor
•
PMN: polymorpho nuclear cell
•
LPS: lipopolysaccharide
•
CAMPs: cationic anti-microbial peptides
•
TNF-α: tumor necrosis factor alpha
•
SCV: Salmonella-containing vesicle
•
NK: natural killer
•
DC: dendritic cell
•
iNOS: inducible nitric oxide synthase
•
mAb: monoclonal antibody
•
TGN: glyceryl trinitrite
•
CGD: chrnic granulomatous disease
•
Nramp 1: natural resistance-associated macrophage protein 1
•
TRLs: toll-like receptors
•
TAP: transporter-associated with antigen processing
•
ix
ABBREVIATIONS – CONTINUED
•
DTH: delayed-type hypersensitivity
•
CTL: cytotoxic T lymphocyte
•
PABA: ρ-aminobenzoic acid
•
DHB: 2, 3-dihydroxybenzoate
•
MALT: mucosa-associated lymphoid tissue
•
ETEC: enterotoxigenic E. coli
•
CFA/I: colonization factor antigen I
•
EAE: experimental autoimmune encephalomyelitis
•
PLP: proteolipid protein
•
CFU: colony forming unit
•
PT: pertussis toxin
•
NBF: natural buffered formalin
•
H&E: hematoxylin and eosin
•
LFB: luxol fast blue
•
IHC: immunohistochemistry
•
RT: room temperature
•
DPBS: Dulbeccos’ PBS
•
NSB: normal serum block
•
PP: Peyer’s patches
x
ABBREVIATIONS – CONTINUED
•
CLN: cervical lymph node
•
ANOVA: analysis of variance
•
EHEC: enterohemorrhagic E. coli
•
ELISA: enzyme-linked immunosorbent assay
•
GI: gastro intestinal
•
ELISPOT: enzyme-linked immunospot
•
CNS: central nervous system
•
MS: multiple sclerosis
•
MIP-2: macrophage inflammatory protein-2
•
TCA-3: T cell activation gene-3
•
CFA: complete Freunds’ adjuvant
•
APL: altered peptide ligand
•
MBP: myelin basic protein
•
KLH: keyhole limpet hemocyanin
•
BCG: Mycobacterium bovis strain Calmette-Guèrin
•
ST: heat stable toxin
•
LT: heat labile toxin
•
cAMP: cyclic AMP
•
cGMP: cyclic GMP
•
NALT: nasal-associated lymphoid tissue
xi
ABBREVIATIONS – CONTINUED
•
i.n: intranasal
•
VT: verocell cytotoxin
•
HUS: hemolytic uremic syndrome
•
SLTs: “Shiga-like” toxins
•
A/E: attaching and effacing
•
M6PR: mannose 6-phosphare receptor.
xii
ABSTRACT
The expression of enterotoxigenic Escherichia coli (ETEC) fimbriae (colonization factor
antigen I (CFA/I) or K99) on the surface of a Salmonella vaccine vector confers protection against
ETEC challenge. Application of such fimbriae as a treatment for the proinflammatory disease,
experimental autoimmune encephalomyelitis (EAE), or as a molecular scaffold for heterologous
antigen expression by cloning enterohemorrhagic E. coli (EHEC) LPS peptide mimetics into the
K99 fimbriae to produce a dual vaccine for ETEC/EHEC was investigated.
The expression of CFA/I fimbriae by a Salmonella vaccine vector stimulates a biphasic T
helper (Th) cell response and suppresses proinflammatory responses suggesting that CFA/I fimbriae
may be protective against proinflammatory diseases. To test this hypothesis, SJL/J mice were
vaccinated with Salmonella-CFA/I vaccine 1 or 4 wks prior to induction of EAE induced with
encephalitogenic proteolipid protein (PLP) peptide, PLP139-151. Mice receiving Salmonella-CFA/I
vaccine recovered completely from the mild acute clinical disease and showed only mild
inflammatory infiltrates in the spinal cord. This protective effect was accompanied by a loss of
encephalitogenic IFN-γ secreting Th1 cells and replaced with increases in IL-4-, IL-10-, and IL-13producing Th2 cells. These data suggest that Salmonella-CFA/I is an anti-inflammatory vaccine
capable of suppressing proinflammatory cells to protect against EAE via immune deviation.
To obtain an effective nasal vaccine for ETEC, the fanC gene of ETEC K99 major
structural gene was cloned onto the reovirus adhesin, protein σ1, which has been shown as an M
cell targeting molecule. Although FanC/protein σ1 fusion protein was successfully expressed, this
vaccine failed to elicit immune responses against native FanC protein, presumably because of
improper protein folding.
Using K99 fimbriae as a molecular scaffold, a LPS peptide mimetic for EHEC was cloned
into the fanH gene of K99 fimbriae minor structural gene to enable multiple antigenic peptide
expression, resulting in an ETEC/EHEC dual vaccine. Insertion of peptide mimetic into fanH gene
had no adverse effect on the formation of polymerized K99 fimbriae. However, various oral
immunization regimens failed to induce the protective secretory IgA responses against the LPS
mimetic peptide, although serum IgG antibodies were induced.
1
CHAPTER ONE
SALMONELLA: PATHOGENESIS, HOST IMMUNE RESPONSES, AND ITS ROLE AS A
HETEROLOGOUS VACCINE CARRIER
Introduction
The genus Salmonella is a member of the family Enterobacteriaceae. This genus is
composed of bacteria related to each other both phenotypically and genotypically. The bacteria of
the genus are also related to each other by DNA sequence (1). Before the DNA based taxonomy (2),
Salmonella classification was based on clinical manifestations (Salmonella typhi, Salmonella
cholerae-suis, and Salmonella abortus-ovis), serological analysis, the geographical origin of the first
isolated strain of the newly discovered serovars (S. London, S. panama, S. stanleyville), or the host
specificity (S. typhimurium). Using these traditional methods, newly identified Salmonella species
were each considered as a new species (3). Currently, to reduce the increasing numbers of proposed
species, it is accepted that all Salmonella serovars form a single DNA hybridization group, i.e., a
single species composed of seven subspecies. Therefore, a genus Salmonella consists of single
species (Salmonella enterica) with seven subspecies (enterica for subspecies I, salamae II, arizonae
IIIa, diarizonae IIIb, houtenae IV, bongori V, and indica VI) (3). For the convenience of physicians
and epidemiologist, the common serovars names are kept only for the subspecies I strains, which
consist of more than 99.5% of Salmonella strains isolated from humans and other warm-blooded
animals (1, 3). According to this taxonomic rule, Salmonella typhimurium designated as Salmonella
enterica subspe. Enterica serovar Typhimurium, in practice, Salmonella ser. Typhimurium or S.
Typhimurium.
2
The habitat for Salmonella is confined to the digestive tracts of humans and animals, and
some Salmonella species show strict host specificity. S. Typhi,
Paratyphi A, and Sendai are strict human serovars, but Typhimurium resides both in humans and
mice (4-7).
The ingestion of Salmonella from contaminated foods or water initiates infection. The
major clinical syndromes associated with Salmonella infections in human are enteric (typhoid)
fever and gastroenteritis (8). S. Typhi is the causative agent of enteric fever. In contrast to selflimited gastroenteritis by S. Typhimurium, S. Typhi spreads through the reticulo-endothelial system,
including the intestinal Peyer’s patch, mesenteric lymph node, spleen, and bone marrow, by the
migration of the infected macrophage via lymphatics and blood (9). The release of bacteria and
endotoxin at the mesenteric lymph node initiates the septicemic phase of enteric fever and
cardiovascular “colapsus tuphos”, which is the origin of the name of typhoid (10).
Typhoid fever causes severe health problem around the world, especially in
many developing countries, with an approximate 33 million incidences and one-million
deaths a year (11. 12). Consequently, in an effort to prevent typhoid fever, two different
types of vaccines are available: an oral vaccine and a parental vaccine (12. 13).
There are two kinds of parental vaccines. One consists of inactivated S. Typhi virulent strain
and the other is purified S. Typhi virulence factor, Vi polysaccharide (13. 14). Parental vaccination
using inactivated bacteria is effective but is not recommended due to the co-administration of O
antigen (endotoxin) that resulted in unwanted systemic and local reactions (13). Parental
vaccination with Vi polysaccharide antigen showed efficacy of 72% at 17 months and 64% at 24
3
months from the pilot scale field studies in Nepal and South Africa (14). It was licensed in U.S in
1994 (13, 14).
Licensed in 1991 (12), the oral typhoid fever vaccine Ty21a was created by the introduction
of a stable mutation to the pathogenic S. Typhi strain, resulting in a lack of the enzyme UDPgalactose-4-epimerase and Vi capsular polysaccharide (12). The Ty21a vaccine showed 96%
efficacy against confirmed typhoid fever (14). The loss of UDP-galactose-4-epimerase was
considered the major reason for this attenuation, but in later experiments performed with galE+
complementation and galE mutation failed to show the recovery of virulence or attenuation,
respectively, and suggested an unknown cause of Ty21a’s attenuation (15, 16). To develop better
typhoid vaccines and adapt them as heterologous antigen carriers, genetically defined attenuations
of Salmonella was adopted. To implement these attenuations, a better understanding of Salmonella
pathogenesis and host resistance mechanisms would be required to obtain highly attenuated
Salmonella strains while retaining its immunogenicity. The following section presents a brief
summary of Salmonella pathogenesis and host immune response.
Salmonella Pathogenesis
Enteritis
Salmonella pathogenesis can be divided according to two different disease syndrome
features, enteritis and enteric fever (8, 17). The enteritis is caused by the entry of Salmonella into the
intestinal epithelial cell followed by the induction of fluid secretion and inflammation (8). However,
the enteric fever is initiated by the invasion of Salmonella into the macrophage and reproduction in
the macrophage phagosome (17). The Salmonella genes that govern the invasion into host epithelial
4
cell and survival in phagocytic cells are clustered in the Salmonella chromosome, called Salmonella
pathogenesis islands (SPIs) (18-21). The genes of different SPIs have unique functions at different
stages of pathogenesis. For example, SPI-1 encodes genes responsible for the entry of Salmonella
into intestinal epithelium and effector proteins, but SPI-2 encodes genes necessary for the survival
in the macrophage phagosome and systemic infections (21, 22). Figure 1.1 shows the locations of
SPIs in the Salmonella chromosome (21).
The entry of Salmonella into intestinal epithelium can be divided into five distinct steps: 1)
delivery of effector molecules via the type III secretion system (TTSS) into intestinal epithelium,
with both effector molecules and TTSS encoded in SPI-1; 2) stimulation of host signal transduction
pathways; 3) initiation of actin cytoskeleton rearrangement; 4) localized ruffling of intestinal
epithelial cells’ membrane; 5) and shutting off of the signal transduction pathway following the
entry of Salmonella into the intestinal epithelial cell (24-30).
SPI 4 92
SPI 3 83
Chromosome
SPI 1 63
sopE 61
SPI 5 25
phoP/phoQ 27.4
SPI 2 31
sirA 42.4
Figure 1.1: Salmonella pathogenesis islands (SPIs). Schematic representation of S, Typhimurium
chromosome, which is divided into 100 cs. Known SPIs are shown on the outside of the
chromosomal circle. Modified from Marcus, et. al., (21).
5
Table 1.1: SPI-1-encoded effector proteins (24).
Protein
Biochemical activity
AvrA
unknown
SipA
stabilization of F-actin , reduction of the
critical
concentration
for
actin
polymerization
SipB
binding of caspase-1
In vivo function
unknown
actin polymerization and reorganization
translocation of SPI-1 effectors and
induction of apoptosis
actin nucleation and bundling
translocation of SPI-1 effectors and actin
polymerization
unknown
translocation of SIP-1 effectors.
unknown
putative host adaptation factor
unknown
induction of enteritis
inositol phophatase
chloride secretion and actin polymerization
unknown
induction of enteritis
small G protein GEF
actin polymerization
small G protein GEF
actin polymerization
unknown
lethal infection in calves
small G protein GAP and tyrosine recovery
of
actin
cytoskeleton
phosphatase
rearrangement
SipC
SipD
SlrP
SopA
SopB
SopD
SopE
SopE2
SspH1
SptP
The TTSS of SPI-1 transfers Salmonella proteins, which are encoded within the SPI-1 or
other segments of Salmonella chromosome, into the intestinal epithelial cell. The TTSS consists of
several proteins encoded in SPI-1; for example, the base of the TTSS structure is composed of InvG,
PrgH and PrgK, and the main component is PrgI (31). Together with the base proteins and the main
needle protein, the characteristic TTSS needle-like structure is assembled (31). Through the TTSS,
the effector proteins are injected into the host and trigger signal transduction pathways that lead to a
variety of host cellular responses (24). The functions of effector proteins are summarized in Table
1.1 (24).
It has been known that the Rho family of GTP binding proteins (Rho, CDC42, and Rac)
regulates signal transduction in response to extracellular signals and results in actin cytoskeleton
6
rearrangement by cycling between a biologically inactive GDP-bound and an active GTP-bound
conformation (32, 33). Among the SPI-1 TTSS delivered proteins, SopE and SopE2 activate
directly both CDC42 and Rac by acting as a phosphate exchange factor (34, 35). The SopE proteins
exert their activity by binding to target Rho GTPases and inhibiting their GTP hydrolyzing activity,
resulting in permanent activation of downstream signal transduction (34). However, the Rho
GTPase regulation by SopE is different from the other bacterial toxins with noncovalent
modification of its target, and this noncovalent modification of the target closely resembles
eukaryotic guanine nucleotide exchange factors (GEFs) (34). Unlike the SopE, SopE2 is present in
every Typhimurium and has a phosphate exchange factor activity for Rho family GTP-binding
proteins (35). Mutation and complementation of SopE2 demonstrates its role in membrane
rearrangement and host cell invasion (35). Activated CDC42 induces the formation of filopodia
(36). Rac1 promotes the subsequent formation of lamellopodia and membrane ruffling (37). In
addition, activation of CDC42 results in the activation of downstream MAP kinase (MAPK)
mediated by the recruitment of Ras-GRF to the membrane, a GEF for the Ras family of small GTPbinding proteins. However, there is no evidence of direct interaction between CDC42 and Ras-GRF
(38). Activation of MAPK, JNK, p38-MAPK, and IκBα kinase is followed by the proteolysis of
IκBα in proteosome and results in release of NFκB. The translocation of NFκB to the nucleus
stimulates the production and secretion of IL-8, which directs the neutrophil to the site of
Salmonella infection and causes the distinctive inflammatory diarrhea of Typhimurium infection
(39, 40).
Following the induction of cytoskeleton rearrangement and initiation of membrane ruffling
by the activation of CDC42 and Rac, spatial localization and more extended ruffles are needed to
7
internalize Salmonella. Another SPI-1 TTSS-transferred protein, SipA, seems to responsible for the
localization and enhancement of cytoskeleton rearrangement by decreasing the critical
concentration of actin polymerization (27, 28, 41). The decreased critical concentration of actin
polymerization by binding of the SipA to actin results in inhibition of actin filament
depolymerization and further initiates Salmonella induced actin polymerization (28, 41). The null
SipA mutant Salmonella infected cell shows a reduced amount of F actin and suggests that the
function of SipA is to stabilize actin filaments during Salmonella infection (28). SipA mutant strain
of Salmonella is less effective in inducing cytoskeleton rearrangement and the rearrangement is
scattered rather than localized (41). In addition, SipA mutant shows impaired entry in cell a culture
system (41).
As a final step of Salmonella invasion into intestinal epithelial cells, the recovery of host
cytoskeleton structure is required. Another SPI-1 TTSS-delivered effector, SptP, is responsible for
the cytoskeleton recovery of the host by acting as a specific antagonist of CDC42 and Rac-1 (29, 30,
42, and 43). As a member of GTPase-activating proteins (GAPs), SptP preferentially binds to the
active form of CDC42 (GTP-bound) and Rac, and activates their intrinsic GTPase activity (42). The
specific SptP binding activity to its cognate G protein was confirmed by the experiment with a
constitutively active GST fusion form of GST-Rac1 (Rac1 (L61)). The SptP only binds to Rac1
(L61), but does not bind to GST-Rac1 or GST alone (42). In addition to GAP activity, SptP
carboxyl-terminal tyrosine phosphatase activity is involved in reversing MAPK activity (30).
Activation of GTPase activity of CDC42 and Rac1, and with its tyrosine phosphatase activity, SptP
downregulates the cytoskeleton rearrangement induced by SopE, as well as a pro-inflammatory
response caused by the induction of the MAP-kinase pathway (30, 42).
8
The whole concerted process of manipulating the host signal transduction process by the
SPI-1-encoded TTSS and effector proteins explains the Salmonella strategy for invasion and
survival in the host intestinal epithelial cell. The invasion of Salmonella into intestinal epithelial
cells causes enteritis characterized by the severe loss of chloride ion and polymorphonuclear cell
(PMN) accumulation at the intestinal lumen (44-46). The loss of chloride ion is the result of
blocking the inositol phosphate signaling pathway by SopB with its inositol phosphate phosphatase
activity. SopB hydrolyzes phosphatidylinositol 3, 4, 5-triphosphate, an inhibitor of Ca2+-dependent
chloride secretion, resulting in severe loss of chloride ion (44).
The transepithelial PMN accumulation is thought to be induced by the secretion of IL-8 as a
result of the nuclear response triggered by TTSS effector protein (39, 40, 46). However, IL-8
neutralization, transfer, and induction experiments suggest the involvement of other factors besides
IL-8 which are necessary for the PMN transepithelial migration (45).
Enteric Fever
The survival and replication of Salmonella in the host macrophage phagosome causes
systemic infection and results in enteric fever. To survive and replicate in the macrophage
phagosome, Salmonella is endowed with several resistance mechanisms, including the PhoP/PhoQ
two component regulatory system and SPI-2 encoded TTSS and effectors (47-49).
In response to macrophage phagosomal microenvironment, especially low concentration of
Mg++ ion, Salmonella regulates its gene transcription through PhoP/PhoQ two component
regulatory systems in which phosphorylation and dephospholylation are involved (51). The
PhoP/PhoQ system consists of phospho- transmitter (PhoQ) and phospho-receiver (PhoP), also
called sensor kinase and effector protein, respectively (47). In addition to regulating downstream
9
expression, PhoP/PhoQ expression is also regulated in a transcriptionaly auto-regulated fashion
with two different phoP/phoQ promoters: PhoP/PhoQ-independent and PhoP/PhoQ-dependent
(52). For example, the PhoP protein acts as a transcriptional activator of phoP/phoQ when it is
phosphorylated by PhoQ in response to microenvironmental signals. Then, the phosphorylated
PhoP (PhoP-p) activates the PhoP/PhoQ-dependent promoter of phoP/phoQ regulon, resulting in
increased concentration of PhoP-p and further increase of PhoP-p downstream activation (51, 52).
However, the PhoP/PhoQ independent promoter of the phoP/phoQ regulon activates very low basal
level of PhoP expression (52). One of the well-known PhoP/PhoQ system-dependent event is
modification of the lipid A structure of Salmonella lipopolysaccharide (LPS), a host signaling
moiety of LPS, by PagP protein, with the transferring palmitate to 2-hydroxy myristate on lipid A
(53, 54). The addition of palmitate and the resulting charge distribution change on LPS hinders the
interaction between LPS and cationic anti-microbial peptides (CAMPs) and further confers the
Salmonella resistance against membrane damage (54). In addition, the PhoP/PhoQ system also
activates other two component system, PmrA/PmrB (50). The activation of PmrA/PmrB twocomponent system by phoP/phoQ modifies lipid A with the addition of aminoarabinose mediated
by PmrE and PmrF and confers Salmonella resistance to the anti-microbial peptide, polymyxin (55).
The modification of lipid A structure also alters LPS-mediated host responses. For example,
reduced expression of E-selectin and tumor necrosis factor-α (TNF-α) was observed in the host
infected with E. coli which had modified lipid A without myristic acid (56). In Salmonella, PhoP
constitutive phenotype (PhoPc), which constitutively activates downstream genes, modifies the lipid
A structure by the addition of aminoarabinose and 2-hydroxymyristate (57). The PhoPc infected
endothelial cell (HUVEC) and adherent mononuclear cell show reduced induction of E-selectin and
10
TNF-α, respectively (57). This reduced expression of the adhesion molecule and proinflammatory
cytokine by the host implicates a reduced migration of macrophages to the site of inflammation and
killing activity of phagocytes, but increases viability of Salmonella in the host (56, 57) Taken
together, the membrane remodeling by the PhoP/PhoQ two-component system plays an important
role in resisting the host innate immune system by providing Salmonella resistance to CAMPs and
modifying host cell adhesion molecule and effector expression (54, 55, 57-60). Figure 1.2 shows
the illustrated summary of the PhoP/PhoQ regulation cascade (50).
The phagosomal condition induces another TTSS and effector expression encoded by SPI2 (21, 22, 49). The SPI-2 TTSS and effectors are required for Salmonella virulence and proliferation
in macrophages (49). Among the SPI-2 TTSS effectors, the role of SpiC protein in pathogenesis has
been well-characterized (61, 62). SpiC has no homologues in the sequence data base and motifs that
predict function (62). However, animal studies with spiC mutant Salmonella show the requirement
of spiC for virulence, with an increase of LD50 more than 3.6 x 105 times and defective
intramacrophage survival (62). Complementation of the spiC mutant with spiC gene containing
plasmid shows the recovery of intramacrophage survival (62). The functional SpiC protein exerts its
activity by inhibiting phagosome-lysosome fusion and interfering with normal vesicle trafficking
devoid of Salmonella (62). In addition to SpiC protein, other SPI-2 encoded genes, such as ssrA,
ssaJ, sssV, and sseB, are required for virulence (63).The functions of the above genes were
confirmed by the experiments with gp91phox phagocyte NADPH oxidase knockout mice (64).
They antagonize the NADPH phagocyte oxidase, the most potent antimicrobial tool of phagocytic
cells, by interfering with trafficking of oxidase-containing vesicles to the Salmonella containing
11
vesicle (SCV) (49, 63, and 64). Taken together, it is suggested that the SpiC and other SPI-2 effector
proteins block fusion of vesicles harboring NADPH oxidase with SCV.
Environmental Signals
(macrophage phagosome; low
Mg++, Ca++)
Environmental Signals
(pH, Fe++/+++)
OM
IM
PmrB
PhoQ
PhoP
PhoP-P
PmrA
+
+
+
pagP
Lipid Apalmitoylation
CAMP resistance
PmrA-P
pagB
pmrA
pmrB
pmrE
pmrF
Lipid A aminoarabinose
and ethanolamine addition
Core modification
Polymyxin Resistance
Figure 1.2: phoP/phoQ regulon involved regulatory cascade. Diagram of the regulatory cascade
involving PhoP/PhoQ that results in PhoP-activated (pag) or PhoQ-repressed (prg) gene expression.
Upon entry of bacteria into macrophage phagosome, environmental signals are sensed by PhoQ,
which activates PhoQ through a phosphor-relay system. Revised from Ernst, et. al., (50).
12
Host Resistance against Salmonella Infection
Understanding host defense mechanisms against Salmonella infection, including innate and
acquired immunity is important for the design of a Salmonella vaccine strain, as well as to exploit
Salmonella as a carrier for heterologous antigen. The host responses can be divided into two parts;
innate and acquired immune response. The following sections summarize host defense mechanisms
against Salmonella infection.
Innate Immunity
The macrophage is the central controlling cell in infection with Salmonella, as well as with
other intracellular bacteria (65). The early innate immunity by macrophages and natural killer (NK)
cells is important for the successful clearance of Salmonella at initial stages of invasion. Upon
response to Salmonella, macrophages and dendritic cells (DCs) produce IL-12 and IL-18 (6672). In response to IL-12 and IL-18 stimulation, NK cells produce IFN-γ and activate macrophages
(66, 67, 73, and 74). Furthermore, macrophages also produce IFN-γ in response to their own
IL-12 and IL-18, which sustains increased levels of IL-12 in the surrounding microenvironment to
ensure elevated levels of IFN-γ (67). At early stages of Salmonella infection, IFN-γ confers
restriction of intracellular bacterial replication (75). The importance of IFN-γ for bacteriostatic
activity at initial stages of infection was confirmed with experiments of IFN-γ depletion with
specific monoclonal antibody (mAb) treatment, IFN-γ knockout mice (IFN-γ-/-), and administration
of recombinant IFN-γ (76-80). However, this bacteriostatic action of IFN-γ failed to provide
bacterial clearance in the later elimination stage of attenuated Salmonella (∆aroA) infection (79, 80),
13
suggesting the bacteriostatic role of IFN-γ in the initial infection stages rather than bacteriocidal
activity in the later clearance stages. IFN-γ-/- mice with oral Salmonella challenge resulted in
disseminated septicemia 2 weeks later with a 100-fold increase of specific systemic and local
antibody titers (78). In contrast to aroA¯ -Salmonella infection to the IFN-γ-/- mice, phoP¯Salmonella was eliminated from the IFN-γ-/- mice. This suggests the differential role of IFN-γ
against Salmonella infection according to Salmonella virulence gene attenuation. IFN-γ exerts its
antimicrobial activity through the induction iNOS and phagocyte NADPH oxidase (81, 82). iNOS
synthesizes antimicrobial reactive nitric oxide radical (NO·), which acts as an oxidizing eagent and
further forms toxic peroxynitrite (ONOO¯) through its interaction with superoxide (O·2¯). These
iNOS products have different anti-Salmonella activity according to their redox state. For example,
co-culture of Salmonella with NO· donor, diethylenetriamine-nitric oxide, does not exhibit
antibacterial activity, but Salmonella culture with the OONO¯ producer, 3-morpholinosydnonimine
hydrochloride shows oxygen-dependent Salmonella killing (83). It seems that DNA is an important
target for reactive nitrogen derivatives (81). For example, treatment of DNA repair-deficient
Salmonella (His¯) with NO donor, glyceryl trinitrite (TGN), shows a significant increase in His+
Salmonella revertants compared to normal His¯ Salmonella by the transition of DNA base from C to
T at the HisG46 target codon CCC (84). Reactive oxygen species are produced by the NADPH
phagocyte oxidase, such as, superoxide (O2¯), hydrogen peroxide (H2O2) and hypochlorous acid
(HOCl). The phagocyte NADPH oxidase consists of several subunits, including membrane-bound
(gp91PHOX and p22 PHOX), cytosolic (p67PHOX, p47PHOX and p40PHOX) and a low-molecular-weight G
protein (rac2 or rac1) (85). Assembly of the functional NADPH oxidase initiates with the
phosphorylation of a cytosolic component, p47PHOX. The activated p47PHOX is translocated with
14
other cytosolic subunits to membrane-bound components and resulting in a functional NADPH
oxidase assembly (85, 86). Regarding the cells from X-linked (gp91PHOX mutation) chronic
granulomatous disease (CGD) patients, the translocation of activated p47PHOX and other cytosolic
components to the membrane is normal. However, due to the lack of gp91PHOX, stable membrane
association of p47PHOX and p67PHOX can not occur, and therefore functional NADPH phagocyte
oxidase assembly is hindered (86). This indicates that the requirements of gp91PHOX and p22 PHOX
(flavocytochrome b) for the stable association of cytosolic p47PHOX and p67PHOX to the membrane
and further formation of functional NADPH oxidase (86). The Rac protein is also translocated with
cytosolic components of NADPH oxidase, and Rac translocation is associated with NADPH
phagocyte activation (87). The function of another NADPH oxidase cytosolic component, p67PHOX,
is not clear, but appears to be involved in electron transfer from NADPH to oxygen by regulating
electron flow from NADPH to flavin in flavocytchrome b (85, 88). Both the NADPH oxidase and
iNOS are required for Salmonella killing in macrophages, but they seem to have different
Salmonella killing kinetics according to the Salmonella infection stage (89, 90). Experiments with
macrophages from the gp91phox-/-, iNOS-/-, and gp91phox-/-/iNOS-/- mice show the contribution of
NADPH oxidase and iNOS for Salmonella killing at different stage of infection (90). Macrophages
from gp91phox-/- mice show impaired Salmonella killing, as indicated by almost 100% survival
from the initial times of infection and a prolonged Salmonella burden at the later stage (89).
However, iNOS-/- macrophages show the initial percentage decrease in survival rate of Salmonella,
but uncontrolled Salmonella replication at later times of infection (89). Macrophages from
gp91phox-/-/iNOS-/- mice show total failure in controlling Salmonella replication (82). Mice
challenged with Salmonella also show different susceptibility according to their genetic background,
15
gp91phox-/- or iNOS-/-. The bacterial counts in the spleen and liver of gp91phox-/- were greatly
increased, as early as 1 day of infection and resulted in death in 5 days. In contrast, iNOS-/- mice had
a bacterial count increase after 1 week of infection (90). Taken together, the suggestion is that
concerted NADPH oxidase and iNOS activity is required for controlling Salmonella replication at
different stages of infection (81, 89, 90). Figure 1.3 represents the potential interaction between
NADPH oxidase derived reactive oxygens species and iNOS nitrogen intermediates (81).
Along with reactive oxygen species, natural resistance-associated macrophage protein 1
(Nramp1), previously known as the Bcg/Lsh/Ity genes plays an important role in innate host
resistance against Salmonella infection, as well as other intracellular bacteria (91, 92). Nramp1
protein contains a divalent metal binding motif (Fe++ and Mn++) and is supposed to function as a
divalent metal efflux pump (91, 92).
16
Phagocyte
Oxidase
MPO
H2O2
NO•
HOCl
•O2¯
H2O2
O2
Intracellular
O2
NO•
HO2•
H2O2
Extracellular
NO
Synthase
H2O2
? NO2•
NO2•
NO• OONO¯
O2
H2O2
GSNO
HOONO
HOONO
RSNO
++
Fe
Reactive Oxidants
•OH
OONO¯
NO2•
OXIDATION
GSH
NAD+/metal/O2
O2 NO•
RSH/Fe
N2O3
DNIC
N2O4
NITROSATION
Figure 1.3: Schematic diagram of concerted action of NADPH phagocyte oxidase,
myeloperoxidase, and iNOS. Potential interactions between phagocyte-derived reactive oxygen and
nitrogen intermediates. Some possible reactions of products originating from NO synthase,
phagocyte oxidase, and myeloperoxidase are shown in relation to hypothetical microbes situated
within a phagolysosome. “Extracellular” refers to the phagolysomal compartment, and
“intracellular” refers to the microbial cytosol. Adapted from Fang (81).
17
Functional analysis of Nramp1 with murine macrophage cell line RAW264.7, which
contains a homozygous mutation in Nramp1, revealed that when intact Nramp1 was used to
transfect these cells, Salmonella replication was inhibited in contrast to untransfected RAW264.7
cells (93). Further, Nramp1 phagosomal recruitment was confirmed (93, 94). RAW264.7 (Nramp1/-
) cells transfected with plasmid expressing Nramp1-c-myc tag showed the phagosomal localization
of Nramp1 (93). Phagosomal localization of Nramp1 was also assessed in peritoneal macrophages
from 129/sv mice (Nramp1-/-) and wild-type mice, 129/sv. Macrophages from 129/sv wild-type
mice showed the phagosomal co-localization of Nramp1 with fluorescent coated latex bead, in
contrast to the 129/sv (Nramp1-/-) (94). Taken together, this suggests that Nramp1 is recruited to
Salmonella-containing phagosomal membranes, then deprived of phagosomal cations, which are
required as cofactors for the Salmonella catalase and superoxide dismutase, resulting in control of
Salmonella replication (91-94). In addition, Nramp1’s role in regulation of SCV maturation is
known (95). The acquisition of mannose 6-phosphate receptor (M6PR), which cycles between the
trans-Golgi network and the prelysosomal compartment of the endocytic pathway, and
externally supplied labeled dextran by SCV was remarkably enhanced in a Nramp-transfected
Nramp1-/- macrophage cell line and in macrophages from Nramp1+/+ mice, in contrast to the
Nramp1-/- macrophage line and macrophages from Nramp1 null (Nramp1-/-) mice (95). This
indicates that Nramp1 regulates SCV maturation by modulating endocytic vesicular trafficking (95).
Some Toll-like receptors (TRLs), TLR4 and TLR5, host LPS sensing factor and flagellin
receptor, respectively, also influence Salmonella susceptibility (96-98). TLRs were originally
characterized as Drosophila factors involved in dorso-ventral polarization of embryos and in
resistance to fungal infection (99, 100). Mice having point mutations in the TLR4 gene (C3H/HeJ)
18
or homozygous null mutation of TRL4 (C57BL/10ScCr) were resistant to LPS-induced shock and
death (96, 101), implying that the impaired LPS signaling in these mutant strains of mice was due to
altered TLR4 function. However, the TLR4 mutant strain was highly susceptible to Salmonella
infection despite its resistance to LPS-induced shock (102). TLR5 is a Toll-like molecule that
recognizes flagellin from both Gram(-) and Gram(+) bacteria (97, 98). Activation of TLR5
mobilizes NF-kB and stimulates TNF-α (96, 102). Murine TLR5 lies within a locus that is
associated with susceptibility to Salmonella (104). The expression level of TLR5 between
Salmonella-susceptible MOLF/Ei mice and Salmonella-resistant 129/Sv mice is remarkably
different in response to Salmonella infection and suggests the role of TLR5 in response to
Salmonella or Gram (-) bacterial infection (104).
Acquired Immunity
Two types of phagocytic cells (macrophages and immature DCs) are critical in the interface
between innate and adaptive immunity. DCs are especially involved in the initiation of adaptive
immune responses by priming naïve T cells.
Both macrophages and DCs process Salmonella antigen for peptide presentation on MHC-I,
as well as MHC-II (226, 227). For MHC-I presentation of antigen, macrophage takes alternative
antigen presentation routes without involving the classical (cytosolic) MHC-I component, but using
post-Golgi MHC-I molecules, e.g. functional transporter associated with antigen processing (TAP)independent (228, 229). However, DCs use a cytosolic pathway for Salmonella-encoded antigen
presentation on MHC-I (230-232). In addition to direct Salmonella antigen presentation by
macrophages and DCs, bystander Salmonella antigen presentation also occurred by bystander DCs,
with uptake of Salmonella-induced apoptotic or necrotic debris of macrophages or DCs (233).
19
Although, bystander macrophages can internalize apoptotic debris of Salmonella, the peptides are
not presented (233). The DC encounter with Salmonella also induces DC maturation, as evidenced
by increased surface expression of MHC-I, MHC-II, CD40, CD 54, CD80, CD86, and TNF-α (230,
234, 235). DCs show reduced Salmonella phagocytosis and antigen presentation (230). The
coupled high expression of signaling molecules (230) and TNF-α (234, 235) and lowered
subsequent Salmonella antigen presentation (230) suggest the migration of mature DC to secondary
lymphoid organs and the optimal capacity to stimulate naïve T cells for the initiation of immune
responses. DC migration seems to be mediated by the alteration of chemokine and chemokine
receptor production (236). The critical role of DC for T cell priming was suggested by an
experiment in which DC loaded with heat-killed or viable Salmonella can prime both CD4+ and
CD8+ Salmonella-specific T cells on transfer into naïve mice (237).
T cell-mediated specific immune responses play a critical role in controlling Salmonella
infection through the induction of cell-mediated or humoral immune responses. CD28-dependent
activation of CD4 is critical for the clearance of bacteria (238). The role of TCRγδ T cells in
Salmonella infection is controversial (112, 113). However, γδ T cells seem to confer resistance to
Salmonella in itys (Nramp1s) mice (112, 114,115). The major CD4+ T cell subset required for
protection against Salmonella infection is Th1 cells, as evidenced by delayed-type hypersensitivity
(DTH) responses (239) and the dominant production of IL-2 and IFN-γ (105, 106, 238, 240).
Salmonella antigen-specific CD4+ Th1 cells exert their regulatory role through IFN-γ (105, 106,
238). IFN-γ activates macrophages and cytotoxic T lymphocytes (CTLs), and is a B cell switch
factor stimulating the production of murine IgG2a and IgG2b, the potent opsonizing antibodies, for
clearing Salmonella from infected tissues (105-110). The innate immune cytokine network is also
20
an important factor for stimulating and sustaining adaptive Th1 responses (66, 67, 73, 74). IL-12
exerts its regulatory role through IL-12 receptors on Th1 cells, resulting in the upregulation of IFN-γ
production (67). By contrast, administration of heat-killed Salmonella or purified antigen induced
Th2-type responses with predominant production of IL-4 and elevated levels of antigen specific
IgG1 antibodies, resulting in lower DTH responses (241, 242). This suggests that the involvement
of different subsets of DCs for live and dead Salmonella, e.g. CD11c+CD8α+MHC-II+ for Th1 vs.
CD11c+CD8α–MHC-II+ for Th2 (243). CD8+ T cell-mediated CTL is also a very important
acquired immune response. It requires MHC1-mediated Salmonella antigen presentation and
specific CD8+ T cell stimulation (111). CD8+ T cell functions via the secretion of perforin,
granzyme, and IFN-γ and results in lysis of Salmonella-infected cells (107, 111).
B cells also play a role in resistance to Salmonella infection (110, 116, 117), cooperating
with T cells which modulate humoral responses during Salmonella infection. For example, nu/nu
(T cell-deficient) and CD28 –/– (impaired T cell activation and reduced T- and B cell signaling)
mice elicited minimal to no Salmonella-specific IgG subclass antibodies, and only low IgM and
IgG3 levels (244, 245). The protection against wild-type Salmonella challenge after vaccination
with attenuated Salmonella is B cell dependent (116, 117). B cell-deficient mice immunized with
attenuated Salmonella fail to survive with wild-type Salmonella challenge (116, 117). In addition,
CD4+ T cells from the B cell-deficient mice immunized with attenuated Salmonella showed
diminished production of IL-2 and IFN-γ after in vitro stimulation (116). However, in contrast to the
critical role of B cells for the vaccine-induced clearance of Salmonella, resolution of attenuated
Salmonella only depends on CD4+ CD28+ T cells (309). Taken together, with the major role of T
cells in controlling Salmonella infection, B cells also have an important part in controlling
21
Salmonella secondary infection after immunization, but not in initial clearance of attenuated
Salmonella.
Attenuated Salmonella Vaccine and
Its Role as a Heterologous Antigen Carrier
Live Attenuated Salmonella Vaccine
The vaccination strategy based on live attenuated microorganisms has higher efficacy over
subunit and DNA vaccines. During the course of vaccination, live attenuated microorganisms
induce immune responses similar to the natural infection because they have antigenic diversity, as
seen in wild-type microorganisms and take a natural antigen processing and presentation pathway.
In addition, with viability, they could supply antigens for an extended period of time. Therefore, in
many cases, a single immunization would be enough for inducing protective immunity (118).
Salmonella is one of the most extensively studied microorganisms as a live attenuated
vaccine and recently as a heterologous antigen carrier. For use as a vaccine or a foreign antigen
carrier, attenuation of Salmonella is crucial. Attenuation can be divided in two different categories
based upon the method of mutagenesis. Production of Salmonella mutants can be accomplished in
an undefined method such as chemical treatment or UV irradiation (12). In contrast, genetic
recombination can be used to mutate metabolic or virulence genes (119-133). In the discussion
below, a summary of the development of live attenuated Salmonella-based typhoid fever vaccine is
described. Table 1. 2 presents the characteristics of live vaccines developed against typhoid fever.
The first generation of typhoid fever vaccine, S. typhi Ty21a, was obtained by random
mutagenesis with nitrosoguanidine (NTG) treatment (12). Ty21a shows galE¯ phenotype, the loss
22
of galactose-4-epimerase activity (15, 16). It was assumed that the loss of this enzyme activity was
the major cause of Ty21a attenuation. However, in subsequent galE+ complementation and galE
mutation experiments, these failed to recover the virulence or attenuation, respectively (15, 16).
Since this failed to confirm the specific mutation, the impetus for adapting genetic attenuation for
Salmonella was significantly increased.
Table 1.2: Summary of typhoid fever vaccines and their characteristics (134).
Strains
Mutation
Safety
Imumunogenicity
Ty21a
undefined
safe
IgG, IgA, IgA ASC , α4β7
ASC
541Ty, 543Ty
aroA purA Vi
safe
IgG, IgA ASC (<Ty21a)
CVD906 or CVD908
Bacteremia
and IgG, IgA, IgA ASC T
aroC aroD
fever
CVD906-htrA, CVD908- aroC aroD htrA diarrhea and fever
IgG IgA ASC, T
htrA
Ty455
safe
Non immunogenic
aroA
phoP/phoQ
Chi3927
bacteremia
IgG, IgA ASC
cya crp
Ty800
safe
IgG, IgA, IgA ASC
phoP/phoQ
Chi4073
safe
IgG, IgA ASC
cya crp cdt
ACS; antibody secreting cell, T; proliferative responses of peripheral blood lymphocyte
The Salmonella strain with metabolic gene mutation was developed on the basis of an
aroA transposon-induced deletion mutant. This Salmonella (∆aroA) depended on aromatic
compounds, such as ρ-aminobenzoic acid (PABA) or 2, 3-dihydroxybenzoate (DHB), for its
growth, and showed higher attenuation (106-fold increase of LD50) (120-125). In addition,
Salmonella (∆aroA) was highly immunogenic and protective against lethal challenge (120, 121).
The first Salmonella (∆aroA) strains were Ty-2 and CDC10-80 (121, 135). Further, to guarantee the
safety and to protect against reverse mutation, the purine biosynthesis-controlling purA gene was
deleted at the same time. The resulting Salmonella strains were referred to as 541Ty and 543Ty (Vi
23
antigen mutant of 541Ty) (135). The clinical study with these strains showed high attenuation, but
less immunogenicity than Ty2 (135). In addition, the poor immunogenicity was linked to purA
mutation (135). This suggested that the double mutation in two different biosynthetic pathways was
not desirable in inducing high immunogenicity. In an effort to develop a Salmonella strain-with a
nonreverting single biosynthetic pathway mutant, ∆aroA and ∆aroD, containing CVD906 and
CVD908, were generated from the Salmonella strains of ISP1820 and Ty2 (125, 136), respectively.
In clinical trials, both CVD906 and CVD908 were highly immunogenic with a single oral
immunization, but these were also reactogenic, with significantly different severity of bacteremia
and adverse febrile reaction (310, 311). The differences in reactogenecity between CVD906 and
CVD908 seemed to originate from the parental strains. In fact, in CVD908, which has been shown
to produce minor bacteremia without adverse febrile reaction (311), an extra-aro mutation was
found (137). The extra-aro mutation in CVD908 was identified at rpoS, the alternative sigma factor
σS, involved in general stress resistance, survival under stress conditions, and virulence in mice
(137). The rpoS mutation in CVD908 is identical to that of Ty21a (137), a live oral typhoid vaccine
derived from Ty2. Thus, it is suggested that the rpoS mutation had occurred before the Ty21a
development during the long time of laboratory transfer and adaptation since its isolation in early
20th century. With an introduction of htrA gene mutation, which encodes for a heat shock protein
(138), further derivatives of CVD906 and CVD908, called CVD906-htrA and CVD908-htrA,
respectively, were developed (139). In a clinical study, both of these were highly attenuated, but less
immunogenic than their parental strains (139). It seems that the htrA mutation conferred increased
attenuation of CVD906-htrA and CVD908-htrA due to its influence of htrA on Salmonella
24
pathogenesis. In fact, it was shown that the loss of htrA protease activity greatly reduced Salmonella
survival in in vitro and in vivo experiments (138).
The attenuated Salmonella strains were also obtained by induction of the mutation in
virulence genes. In contrast to metabolism-related gene mutations, virulence gene mutation did not
affect bacterial growth in vivo. Ty800 Typhi vaccine strain was produced by the induction of
mutation in the phoP/pohQ-two component regulatory systems that modifies lipid A structure of
LPS in response to phagosomal microenvironments (47, 53, 54, 129, 140). In a clinical trial, Ty800
was safe and highly immunogenic, with the induction of Salmonella-specific IgA-producing B cells
in peripheral blood 7 days after vaccination (140). However, in contrast to the licensed Ty21a, the SIgA antibody in the mucosal sites was rarely induced (140). Using a different approach, the Ty445
strain was developed which has mutations both in metabolic (∆aroA) and virulence (∆phoP/phoQ)
genes. It was highly attenuated, but poorly immunogenic, possibly due to its over-attenuation (141).
In the same vein, the Chi4037 strain contained both metabolic and virulence gene mutations
together; ∆cya, which is responsible for the biosynthesis of adenylate cyclase, ∆crp, which is cAMP
receptor; and ∆cdt, which is responsible for the colonization of Salmonella in deep tissues in the
mucosa-associated lymphoid tissue (MALT) (127, 142). The Chi4037 showed similar responses to
Ty800 and both of them are in the field trials.
Live Attenuated Salmonella as a
Carrier for Heterologous Antigens
Previous research has shown that live attenuated Salmonella vaccines are effective in
protecting against wild-type Salmonella challenge (S. Typhi and S. Typhimurium) in human
25
volunteer trials and animal studies (134). In addition to using live attenuated Salmonella as a
vaccine, it has received attention as a heterologous antigen carrier, along with its innate character of
inducing strong and sustained humoral and cellular responses in both mucosal and systemic
compartments after oral administration. In fact, attenuated S. Typhimurium-based studies for
foreign antigen carrier have been extensively performed as a model system instead of the S. Typhibased one (143). In fact, an extensive number of bacterial (e.g., Yersinia. pestis, Bordetella. pertusis,
Helicobacter. pyroli), viral (e.g., HIV-1, HSV, influenza, HBV), and parasitic antigens (e.g., P.
palcifarum, L. major, S. mansoni) have been expressed in attenuated S. Typhimurium. Table 1.3
summarizes the attenuated Salmonella-based foreign antigen delivery studies.
A single oral dose of S. Typhimurium (∆aroA, ∆aroD) vaccine strain BRD509 expressing B.
pertussis antigen, pertactin, induced strong antigen specific Th1 cell responses and conferred a
significant level of protection (144, 145). S. Typhimuriun with gp63, a major leishmanial antigen,
elicited antigen-specific Th1 cell responses, as well as protective immunity against Leishmania
challenge in a Nramp1-dependent manner (146-148). MHC-1 mediated CD8+ T cell response
(CTL) was also induced subsequent to S. Typhimurium vaccination with bacterial, viral, and
parasite antigens (143). In addition, S, Typhimurium vectors appear to be able to induce
immunological memory. S, Typhimurium expressing the hagB gene of P. gingivalis induced IgA
and IgG responses after 52 weeks of single oral immunization (149). Taken together, with an
induction of mucosal and systemic immune responses including humoral, CTL, and long-term
memory, live attenuated Salmonella-based heterologous antigen delivery has multiple advantages
over nonviable subunit vaccines. In the next chapter, influences of passenger antigen on host
26
immune responses are described with emphasis on Salmonella surface expression of the
enterotoxigenic E.coli (ETEC) fimbrial antigen, colonization factor antigen I (CFA/1). In addition,
its application to protection against inflammatory autoimmune disease, experimental autoimmune
encephalomyelitis (EAE), is suggested.
Table 1.3: Animal based foreign antigen delivery study with Salmonella (143).A. Viral antigens
(143).
A. Viral antigens
Strains and Mutation
Typhimurium; aroA
Typhimurium or Typhi;
cya/crp, phoPc or aroA
Typhimurium or Dublin;
aroA
Typhimurium or Dublin;
aroA
Typhimurium; cya/crp
Passenger Antigen
HA, NP (Influenza)
pre-S core and peptide antigen
(HBV)
gp120, gp41, Gag (HIV-1,
HIV-2)
Nef, Gag (SIV)
Immunogenicity
H, C (Th1)
IgG, IgA (M, S)
Assay
CTLs
ND
IgG, IgA (M, S), C
(Th1)
H, C
ND
IgG, IgA (M, S)
ND
Typhimurium; aroA
Typhimurium; aroA
Typhimurium; htrA
S protein (Gastroenteritis
virus)
VP1 (Poliovirus)
HPV type16
gpD (HSV)
H
H, C
H
ND
Virus N
Virus N, P
B. Bacterial antigens
Strain and Mutation
Passenger Antigen
Immunogenicity
Assay
Typhi, Typhimurium,
ETEC LTB
Enteritidis and Dublin; galE,
aroA, or aroA/purA
IgG and IgA (M,S),
C
toxin N
Typhimurium, Enteritidis or
Typhi; aroA or galE
Typhi; galE
Typhi; galE
Typhimurium; aroA or
cya/crp
Typhimurium or Typhi;
aroA, aroC/aroD
Typhimurium; aroA
Typhimurium; aroA
Typhimurium or Dublin;
aroA or cya/crp
IgG, IgA (M, S), C
P
H
H
IgA, IgG (M,S), C
(DHT)
IgA, IgG (M,S), C
(Th1; DHT)
H, C
H, C
IgA, IgG (M,S), C
PP
ND
ND
CFA/1, CS3, K88 and K99
fimbriae
O antigen (Shigella. spp)
O antigen (V. cholerae)
β-galactosidase (E.coli)
Tetanus toxin C
Pertactin, S1 (B.pertussis)
F1, V antigen (Y. pestis)
SpaA, M protein
(Streptococcus.spp)
CTLs
P
P
PP
P
27
Table 1.3 CONTINUED
Typhimurium; aroA
Typhimurium; cya/crp
Alkaline phosphatase (E.coli)
HA (P.gingivalis)
H
H
ND
ND
Typhimurium or Dublin;
aroA
Typhimurium; aroA
Exoprotein A, OMP1
(P.aerouginosa)
p60, Hly, SOD (L.
monocytogenes)
IgA (M)
P
C
CD8+
CTL P
Typhimurium; phoPc
Typhimurium; aroA
urease (H. pyroli )
MOMP (C. trachomonas)
H, C
H
P
ND
C. Parasite antigens
Strains and Mutation
Typhimurium, Dublin or
Typhi; aroA
Typhi; cya/crp
Typhimurium; aroA
Passenger Antigen
Immunogenicity
Assay
CSP, MSP, RESA
H, C
CD8+
(Plasmodium)
CTL, P
SREHP (E. histolitica)
H
ND
Fatty acid-binding protein (E. H, C (Th1 and Th2) ND
granulosus)
Typhimurium; aroA
GST (S. mansoni)
H
ND
N; neutralization, P; protection, PP; partial protection, ND; non detected, M; mucosal immune
response, S; systemic immune response, C; cellular immune response, H; humoral immune
response.
28
CHAPTER TWO
MATERIALS AND METHODS
EAE Study
Animals
Female SJL/J mice 6-8 wks old were obtained from The Jackson Laboratories (Bar Harbor,
ME). All mice were maintained at the Montana State University Animal Resource Center under
pathogen-free conditions in individually ventilated cages under HEPA-filter barrier conditions and
were fed sterile food and water ad libitum. The mice were free of bacterial and viral pathogens, as
determined by antibody screening and histopathologic analysis of major organs and tissues. All
experiments adhered to the “Guide for the Care and Use of Laboratory Animals,” prepared by the
Committee on Care and Use of laboratory Animal Resources, National Research Council (NIH
Publication No, 86-23, Revised (1985) ) and were approved by MSU’s IACUC.
Cloning of PLP139-151 into CFA/I Fimbriae
and Its Expression on Salmonella Vaccine Vector
To exploit the immunomodulatory nature of Salmonella-CFA/I for protection against EAE,
encephalitogenic PLP139-151 coding sequence was cloned into the 315 base position of CFA/I
fimbrial structural gene, cfaB, by PCR mutagenesis. Briefly, with two different primers sets, two
different cfaB fragments bearing PLP139-151 were amplified (F1: TCT AGA ATG AAA TTT AAA
AAA ACT ATT GGT GCA ATG, R1: GGG TGG CCA AGC CAT TTC CCC AGG GAG TGC
ACA CTG ATA GGC ATT TGA, F2: GCT TGG CCA AGC CCC GGA TAA ATT TTC ATG
GGG AGG ACA AGT A, R2: CTA AGC TTT CAG GAT CCC AAA GTC ATT ACG). After Msc
29
I restriction digestion, these fragments were ligated, then cloned into TA vector and designated as
pTACP. After Dra I and Bam H I restriction digestion of pTACP, cfaB/ PLP139-151 DNA fragment
was subcloned into pUC19 and designated as pUCPLP1. The DNA fragment between BamH I and
Sph I of pJGX15C-asd plasmid was amplified and cloned into pUCPLP1 (BamH I/Sph I) and
designated as pUCPLP2. The EcoR I fragment from pJGX15C-asd (155) then replaced with EcoR
I fragment, and resulting in pCFA/PLP, which encodes chimeric CFA/I with PLP139-151. The E.coli
and Salmonella strains expressing CFA/I-PLP139-151 were designated as JYL001 and JYL002,
respectably. The chimeric CFA/I expression was probed with polyclonal rabbit-anti CFA/I serum.
Oral Vaccination with Salmonella
Vaccines and PLP139-151 Challenge
The ∆aroA ∆asd S. enterica serovar Typhimurium-CFA/I vector vaccine, strain H696,
expressed functional CFA/I fimbriae on the vector’s cell surface (155). This phenotype was
maintained by a plasmid bearing a functional asd gene to complement the lethal chromosomal asd
mutation in the parent Salmonella strain, allowing stabilized
fimbrial expression in the absence of antibiotic selection (162). Groups of five female SJL/J mice
(five/group), pretreated with an oral 50% saturated sodium bicarbonate solution, received a single
oral dose of ~5x109 colony-forming units (CFU) of the Salmonella-CFA/I construct, SalmonellaCFA/I-PLP139-151 or its isogenic control strain H647, which lacked the CFA/I operon (162). Control
mice received PBS only.
The
encephalitogenic
proteolipid
protein
(PLP)
peptide
(PLP139-151)
(HSLGKWLGHPDKF) was synthesized by Global Peptide Services, LLC (Fort Collins, CO), and
30
HPLC-purified to > 90%. One or four weeks after oral immunization with the Salmonella vaccines,
or PBS, mice were given s.c challenge with 200 µl of 100 µg of PLP139-151 emulsified in a modified
Freund’s adjuvant containing 1.5 mg/ml of dead Mycobacterium tuberculosis strain H37RA (Difco
Laboratories, Detroit, MI) per ml of incomplete Freund’s adjuvant. The mice also received i.p 200
ng of B. pertussis toxin (PT; List Biological Laboratories, Campbell, CA) on days 0 and 2, relative
to the day of challenge. Mice were monitored daily for clinical signs, and clinical scores were
assigned as follows (216): 0, normal; 1, a limp tail; 2, hind limb weakness; 3, hind limb paresis; 4,
quadriplegia; 5, death.
Histological and Immunohistochemistry Evaluation
For histological evaluation of tissue pathology, spinal cords were removed, fixed with
neutral buffered formalin (NBF), routinely processed, embedded in paraffin,
and sectioned at 5 micron cross (transverse) sections from the spinal cord lumbar region were
stained with hematoxylin & eosin (H&E) for pathological changes and inflammatory cell
infiltration. Adjacent sections were stained with luxol fast blue (LFB; 217) and examined for loss of
myelin. Pathological manifestations were scored separately for cell infiltration and demyelination.
Each H&E section was scored from 0 to 4 (216): 0, normal; 1, cell infiltrate into meninges; 2, one to
four small focal perivascular infiltration; 3, five or more small focal perivascular infiltrates and/or
one or more large infiltrates invading the parenchyma; 4, extensive cell infiltrates involving 20% or
more of the white matter. In each LFB stained section, demyelination was also scored from 0 to 4
(216): 0, normal; 1, one small focal area of demyelination; 2, two to three small focal areas of
demyelination; 3, one to two large areas of demyelination; 4, extensive demyelination involving
20% or more of the white matte.
31
To identify infiltrating lymphocytes, immunohistochemistry (IHC) was performed on
cryosections of spinal cord from SJL/J mice between days 11 and 12 after PLP139-151 challenge from
each immunization group. Mice were euthanized, and spinal cords were removed via saline
injection into the spinal column. Lumbar regions of cords were embedded in O.C.T.®
cryoembedding media (Sakura Finetek, Torrance, CA) and snap frozen with a dry ice/2-methyl
butane slurry (-90ºC). Lumbar regions of cords were transversely sectioned at -16ºC in a cryostat
and mounted on Plus Charge® (Erie Scientific, Portsmouth, NH). Frozen sections were air dried
overnight at room temperature (RT) and fixed the next day in a 75% acetone/25% absolute ethanol
mixture for 5 min at RT and rinsed immediately in three changes of Dulbeccos’ PBS (DPBS).
Appropriate rinsing was done between all IHC steps using a rinse buffer (DPBS with 0.2% goat
serum and 0.05% Tween 20). Normal spleens were used as positive control for CD4, CD8, and
SK208 staining; for Mac-1+ (CD11b) macrophage staining, a Salmonella infected spleen was used.
Endogenous peroxidase was blocked 10 min with Peroxidase Block (#S2001, DakoCytomation,
Carpinteria, CA) followed by an endogenous biotin block (according to manufacturer’s instruction,
Avidin/Biotin Blocking Kit, Vector Laboratories, Inc., Burlingame, CA) and a normal serum block
(NSB, 10% goat and 2.5% mouse sera in rinse buffer) for 30 min at RT. The primary Abs were
diluted in NSB, and all were incubated for 30 min at RT. Biotinylated rat anti-mouse Abs (BD
Pharmingen, San Diego, CA) specific for CD4 (clone GK1.5, IgG2a) and Mac-1 (CD11b, clone
M1-70, IgG2b) were used at1.0 µg/ml. Isotype-matched biotinylated Abs were used as negative
controls. To stain for neutrophils, an indirect staining procedure was used in which rat anti-mouse
neutrophil (SK208; compliments of Dr. Mark Jutila, Montana State University; [218, 249]) mAb
32
was applied to sections as an undiluted hybridoma supernatant for 30 min at RT, followed by the
secondary biotinylated F(ab')2 fragments of goat anti-rat IgG adsorbed to mouse at 2.0 µg/ml for 30
min. Rat IgG (10µg/ml; Jackson ImmunoResarch Laboratory, West Grove, PA) was used as a
negative control. Biotinylated Abs were detected with 1.0 µg/ml of Strepavidin-HRP
(Biosource/TAGO, Camarillo, CA) in rinse buffer for 20 min at RT. Following a DPBS/0.05%
Tween 20 buffer rinse, AEC+ chromogen (DakoCytomation) was applied and color developed
using microscopic monitoring. Color reaction was halted with DPBS, followed by a water rinse, a
light hematoxylin counterstain, and coverslipping with an aqueous mounting media.
Ab ELISA
CFA/I fimbriae-specific endpoint titers from dilution of immune sera or fecal extract were
measured by an ELISA, as previously described, using purified CFA/I fimbriae Ag (162) as a
coating Ag. Specific reactivity to CFA/I fimbriae was determined using HRP conjugates of goat
anti-mouse IgG-, IgA-, IgG1-, and IgG2a-specific Abs (1µg/ml;
Southern Biotechnology
Associates, Birmingham, AL), and ABTS (Moss, Inc., Pasadena, CA) enzyme substrates were used
to develop color reaction. The absorbance was measured at 415 nm on a Kinetics Reader model
EL312 (Bio-Tek Instruments, Winooski, VT). Endpoint titers were expressed as the reciprocal
dilution of the last sample dilution, giving absorbance ≥ 0.1 OD units above the OD415 of negative
control after one hour incubation.
Cytokine ELISA
Lymphocytes from various tissues (spleens, Peyer’s patches [PP], cervical-lymph nodes
[CLN], and spinal cords) from the different immunization groups (PBS, H647, and H696)
33
following PLP139-151 challenge were cultured at 5 x 106/ml in medium alone or in the presence of
OVA (10 µg/ml), CFA/I fimbriae (10 µg/ml), or PLP139-151 (30 µg/ml) in a total volume of 1.5 ml in
a 24-well tissue culture plate. Lymphocytes were cultured for 60 hrs at 37ºC. The culture
supernatant was collected by centrifugation and saved at -80ºC until assayed. IFN-γ, IL-4, IL-10,
and IL-13 were measured on a duplicate set of samples by capture ELISA, as previously described
(219). For IL-13 ELISA, 2.0 µg/ml of rat anti-mouse IL-13 mAb (clone 38213) were used as the
capture Ab, and 0.2 µg/ml of biotinylated goat anti-mouse IL-13 Ab was used as the detecting Ab
(R & D Systems, Minneapolis, MN). The color reaction was developed using goat anti-biotin HRP
conjugated Ab (Vector Lab, Inc.) and ABTS (Moss, Inc.), as previously described (219). Cytokine
concentrations were extrapolated from standard curves generated by recombinant murine cytokines
IFN-γ, IL-4, IL-10 (R & D Systems), and IL-13 (Peprotec, Inc., Rocky Hills, NJ).
Statistical Analysis
The statistical significance of the differences in clinical scores between groups was assessed
by a one-way ANOVA followed by with a post-hoc Tukey test when more than two groups were
analyzed. The significance of cytokine production and histopathology from the control group was
determined by Student’s t test. A value of p < 0.05 was considered significant.
ETEC/EHEC Vaccine Study
ETEC/EHEC Dual Vaccine Construction
To take advantage of the repetitive nature of K99 fimbriae, oligonucleotide for a 10 amino
acids peptide mimetic to enterohemorrhagic E. coli (EHEC) LPS was cloned into plasmid
34
pMAK99-asd+ (250). Briefly, fanH gene of the K99 operon was amplified and subcloned into
pUC18 vector using fanH Sac I-forward (TAG AGC TCA TCA AAA CCT TTT GAG CGG) and
fanH Hind III-reverse (TAA AGC TTT GAA TTT CTT ATT CCC CTC) primers: pfanH.
Subsquently, MH6083 (dam-) cell was transformed with pfanH. Bcl I restriction digestion product
of pfanH was ligated with LPS peptide mimetic oligonucleotides with Bcl I sequence: pfanH/LPS.
Oligonucleotides were annealed in vitro. fanH gene with LPS mimetic sequence was amplified
(forward: ATG CAT ATC AAA ACC TTT TGA GCG G, reverse: ATG CAT TGA ATT TCT TAT
TCC CCT C) and cloned into TA vector: pTA/LPS. Nsi I treated fragment (fanH/LPS) from
pTA/LPS was cloned into Nsi I (5899 and 6466 of K99 operon) treated pMAK99-asd+: pFH/LPS.
H681 (asd-) E.coli and H683 (asd-) Salmonella strains were transformed with pFH/LPS, resulting
in JY001 and JY002, respectively.
Restriction-, Sequencing- and Western Blot Analysis
Comparative Nsi I restriction analysis was done with pMAK99-asd+, pFH/LPS, and Nsi I
treated self-ligation plasmid of pMAK99-asd+ to examine the size differences of Nsi I treated
fragments compatible to LPS mimetic sequence cloning. Sequencing of pFH/LPS was done with
facilities at University of Montana (Missoula, MT). Western blot analysis was done to examine the
expression of K99 fimbriae with LPS peptide mimetic. Proteins were transferred from the SDSPAGE (15% polyacrylamide) gel to 0.2 µm-pore-size nitrocellulose membrane (Bio-Rad). The
membrane was probed with the rabbit polyclonal K99 fimbrial antiserum and then with a goat antirabbit IgG, conjugated to horse radish peroxidase (Southern Biotechnology Associates).
35
Immunization
Two groups of BALB/c mice (5 mice/group), pretreated with 50% saturated sodium
bicarbonate solution, received single oral dose of 5 X 109 or 5 X 1010 CFU of S. Typhimurium,
JY002. Fimbrial expression was examined by an agglutination test with K99-specific antiserum.
Ab ELISA
Antibody titers in serum and fecal samples were determined by an ELISA adapted from
previously described methods (250). Briefly, K99 fimbriae (1 µg/ml) or LPS peptide mimetic
conjugated to OVA (EC-OVA: 10 µg/ml) in sterile PBS was used to coat Maxisorp Immunoplate II
microtiter plates (Nunc) at 50 µl/well, and the plates were incubated overnight at the RT. Various
dilutions of immune mouse serum or fecal extracts were diluted in ELISA buffer and incubated
overnight at 4ºC. Specific reactivity to K99 or peptide mimetic was determined with horseradish
peroxidase conjugates of the following detecting antibodies (1 µg/ml): goat anti-mouse IgG and IgA
antibodies (SBA). Following 90 min of incubation at 37ºC and a washing step, the specific
reactivity was determined by the addition of an enzyme substrate, ABTS (Moss, Inc.), and
absorbance was measured at 415 nm. End point titers were expressed as the reciprocal log2 of the
last sample dilution giving an absorbance of ≥ 0.1 optical density units above negative controls after
1 hour of incubation.
Construction of FanC/protein σ1 Expression Vector
Three FanC/protein σ1 expression constructs were made, two for E. coli expression, and the
other for yeast expression. Briefly, single or triplicates of the fanC fimbrial gene was cloned in
between maltose binding protein/protein σ1 fusion expression construct, using BamH I/Xba I
36
restriction sites for single FanC/protein σ1 (p1FS) and EcoR I/Sal I for triple FanC/protein σ1
(p3FS), for E. coli (251). Cloning was confirmed by restriction analysis and nucleotide sequencing.
For yeast expression, single fanC/σ1 nucleotides were amplified by PCR with EcoR Iforward (ATG AAT TCA TGA ATA CAG GTA CTA TTA AC) and Kpn I-reverse (TAG GTA ACC
ATA TAA GTG AC) primers, and TA cloned. TA cloning was selected on ampicillin plate. The TA
plasmid with fanC/protein σ1 was treated with EcoR I and Kpn I and cloned into yeast shuttle
vector pPICZ A (pPI1FS) compatible sites. Subsequently, yeast Pichia pastoris was transformed
with pPI1FS, and selected on zeocin plates. FanC/σ1 protein expression was determined with antiK99 poly clonal rabbit antibody (250). FanC/σ1 fusion protein was purified using Ni++-NTA affinity
column chromatography.
37
CHAPTER THREE
ETEC FIMBRIAL (CFA/I) EXPRESSION ON SALMONELLA: ITS IMPACT ON HOST
IMMUNERESPONSES, AND PROTECTION AGAINST INFLAMMATORY
AUTOIMMUNE DISEASE, EAE
Introduction
ETEC remains problematic in developing countries, particularly for young children who
have not acquired immunity and for travelers to endemic areas (reviewed in 220). Enteric
symptoms of E. coli infection are often referred to as “traveler’s diarrhea.” E. coli becomes
enterotoxigenic upon acquisition of a plasmid or plasmids containing the heat-stable enterotoxin
(221) or the cholera-like exotoxin, which is commonly, termed the heat-labile enterotoxin (222,
223). Both toxins are responsible for inducing fluid loss and electrolyte imbalance. Virulence by
ETEC is also partly contributed by acquisition of the plasmid for the pili or CFAs, which enhances
the colonization of E. coli in the GI tract. The CFA pili are a heterogenous group of fimbrial
adhesins and are responsible for adherence to small intestinal epithelium via their fimbriae or long,
hair-like projections extending from the bacterial cell surface, to epithelial mannose-containing
glycoproteins (150). Previous studies show that oral delivery of purified CFAs fails to induce
significant serum IgG or S-IgA Abs (152, 153), and these anti-fimbriae Ab titers (152, 154) fail to
protect human volunteers from pathogenic ETEC challenge (152). Thus, live vaccines may be
necessary to retain immunogenicity of CFAs (155).
Previous work shows that oral immunization with Salmonella vaccine vector expressing the
ETEC fimbriae, CFA/I, elicits high Ab titers, as evidenced by elevated serum IgG1 and mucosal
IgA (162). These elevated Ab titers are supported by a biphasic Th cell response in which Th2 cells
38
are rapidly induced and precede development of Th1 cells (162). Such findings contrast to the
immune responses obtained with conventional Salmonella vaccine vectors, which generally
stimulate Th1 cell-dominating responses toward both Salmonella and passenger Ags (156-161).
These responses are characterized by elevated IFN-γ-regulated IgG2a production and suboptimal
mucosal IgA responses. It has also been observed that the Salmonella-CFA/I vaccine fails to elicit
proinflammatory cytokines upon infection of macrophages (163). While the isogenic Salmonella
vector strain H647 could elicit elevated levels of TNF-α, IL-1, and IL-6, the Salmonella-CFA/I
vaccine remains, as if it were stealth, and fails to elicit these cytokines despite identical infectivity
with the isogenic Salmonella strain.
EAE is an animal model of inflammatory, demyelinating human disease of the central
nervous system (CNS), multiple sclerosis (MS) (164-166). It shares many features with MS, such
as ascending clinical paralysis, T cell immunity to neuroantigen, CNS mononuclear infiltration, and
epitope spreading (167-170). EAE can be induced by either the immunization against specific
myelin antigens, MBP, PLP, myelin oligodendrocyte glycoprotein (MOG), or by passive transfer of
activated myelin-specific CD4+ Th1 lymphocytes (171-173). Encephalitogenic T cells secrete Th1type cytokines, IFN-γ and IL-2, and induce local macrophage and microglial activation, infiltration
of inflammatory cells from peripheral lymphoid tissue, and demyelination (175-181). EAE can be
suppressed by adoptive transfer of myelin-specific Th2 cells, which secrete IL-4, IL-10, and IL-13
and inhibit Th1 cell development (182-185). The protective effect of Th2-type cytokines has also
been demonstrated by direct delivery into the CNS using retrovirally transduced T cells (186, 187)
and experiments with IL-4 knockout mice (188, 189).
39
These studies clearly indicate that induction of Th2 cell-like responses is a viable strategy
for treatment of diseases dominated by Th1-mediated pathophysiology. Several experimental
strategies for induction of Th2 cell-dependent protection in EAE have been reported, including oral
administration of myelin antigens for oral tolerance, immunization using altered peptide ligands
bearing encephalitogenic T cell epitopes, and immunization with Ags that selectively induce
responses (199-203). Low dose tolerance, believed to act by suppressing the induction of
encephalitogenic T cells by Th2 cells, has also been adapted for MBP-specific Th2 cells (204). In
addition, high dose oral tolerance, believed to result in clonal deletion/anergy of encephalitogenic T
cells, has also been tested (205). It has also been observed that MBP-specific Th2 cell responses
suppressed PLP-specific EAE, suggesting that the bystander suppression alters the T cell
microenvironment to become Th2-type dominated (200). Immunization with an altered peptide
ligand for PLP139-151 induced Th2 cells specific for the immunogen and suppressed EAE upon PLP
challenge (202). Other myelin Ag-specific EAE challenges have also been suppressed, supporting
a role for bystander suppression in this model. Protection to EAE could also be achieved upon
immunization with non-self Ags associated with down regulation of CD4 co-receptor at the time of
EAE onset and/or EAE-specific immune deviation from Th1- to Th2-type by the change in the
cytokine microenvironment in which encephalitogenic T cells developed (203, 215). Collectively,
these observations clearly provide precedence for developing strategies for the treatment of
autoimmune diseases using non-self Ags to induce bystander Th2 cell-mediated suppression.
We questioned whether we could adapt bystander or active suppression of EAE by using
our previously described diarrheal vaccine or exploiting it as a molecular scaffold for the
encephalitogenic T cell epitope to induce EAE-protecting Th2 cells, respectively. (162).We
40
hypothesized that oral immunization with Salmonella-CFA/I or Salmonella-CFA/I-PLP could
protect against the development of EAE via different mechanisms, bystander suppression and
active induction of epitope-specific Th2 cells.
Rationale and Aim of Research
As an intracellular parasite, most attenuated Salmonella-based vaccine induced
Th1-type inflammatory responses toward both Salmonella vehicle and passenger antigens.
However, oral immunization with Salmonella vaccine expressing ETEC fimbriae CFA/I
rapidly induced strong Th2-type immune responses in advance of Th1-type development.
These Th2-type responses were evidenced by elevated serum IgG1 and mucosal IgA.
Furthermore, Salmonella-CFA/I failed to induce proinflammatory cytokines on infection
of macrophages despite identical infectivity with the isogenic Salmonella vaccine.
EAE is a pro-inflammatory CNS disease mediated by myelin-specific Th1 cells, induced
by either immunization with myelin antigen or passive transfer of myelin-specific Th1 cells. Studies
have shown the suppression of EAE with provision of Th2-type cytokines by adoptive transfer of
myelin-specific Th2 cells or direct delivery into the CNS. Experimental induction of Th2 cells for
the treatment of EAE was achieved in either an antigen-specific or –nonspecific manner represented
by oral tolerance and bystander suppression. Based on the studies outlined above, we hypothesized
that oral immunization with Salmonella-CFA/I or Salmonella-CFA/I bearing encephalitogenic T
cell epitope (Salmonella-CFA/I-PLP) could protect against the development of EAE via either the
bystander suppression or induction of epitope-specific Th2 cells, respectively.
41
In the following section, we examined the EAE-prophylactic efficacy of Salmonella-CFA/I
and Salmonella-CFA/I-PLP. To carry out this analysis, we compared clinical development,
histology and immunopathology, and cytokine profile among different immunization groups (PBS,
isogenic Salmonella strain [H647], Salmonella-CFA/I [H696], or Salmonella-CFA/I-PLP
[JYL002]).
Protection from EAE via Bystander Suppression with Immunization
of Salmonella-CFA/I and Salmonella-CFA/I-PLP
Results
Cloning of PLP139-151 into CFA/I Structural Gene cfa/B and Its Expression. To obtain
encephalitogenic epitope-specific anti-inflammatory Th2 cell-inducing vaccine, we cloned
encephalitogenic PLP139-151 into CFA/I fimbrial structural gene cfaB for expression by Salmonella.
Mutagenesis of cfaB gene with PLP139-151-specific oligonucleotides does not affect the formation of
CFA/I fimbriae, as confirmed by the CFA/I-specific Western blotting. Figure 3.1 describes the
cloning procedure and fimbrial expression.
42
.
A
α
α
7k
6k
7.213k
7k
6k
7.174k
2k
4k
asd
Tetracycline s
β
4k
β
CFA/I operon
ori
PLP139-151
2k
α: Xho I
pCFA/PLP
pJGX15C-asd
PLP139-151
B
1
F1
β: Sac I
R1
C
R2
480
315
F2
PLP139-151
21 kd
Dra I
EcoR I BamH I
EcoR I
Sph I
H696 JYL002
Figure 3.1: Construction of pCFA/PLP and its expression. Encephalitogenic PLP139-151
oligonucleotides were cloned into cfaB gene by PCR mutagenesis method and subsequently
inserted into 315 DNA position of cfaB gene of pJGXC-asd plasmid, and designated as pCFA/PLP
(A, B). The expression of pCFA/PLP was confirmed by probing with polyclonal rabbit anti-CFA/I
serum (C). The cloning procedure is described in detail in materials and methods fimbriae chapter.
H696 (Salmonella strain expressing pJGX15C-asd. JYL002 (Salmonella strain expressing
pCFA/PLP).
To determine whether mice were effectively immunized by Salmonella-CFA/I-PLP, CFA/Ispecific copro-IgA and serum IgG endpoint titers at week 1 and 2 were measured by ELISA
without PLP139-151 challenge. CFA/I-specific Ab titers (Fig 3.2A, B) were similar to endpoint titers
obtained in BALB/c mice as was the dominant IgG1 subclass responses when compared to IgG2a.
43
The Salmonella-CFA/I-PLP dosed group showed similar clinical EAE course and resolution to the
Salmonella-CFA/I vaccinated group (Fig 3.4A, B). However, in an antigen recall assay with various
tissues from Salmonella-CFA/I-PLP immunized mice, PLP139-151-specific cytokines were not
detected, suggesting the failure of PLP139-151 antigen presentation (Fig 3.3). Thus, further studies
with Salmonella-CFA/I-PLP were halted.
20
16
IgA
IgG
20
Anti-CFA/I Titer (log2)
Anti-CFA/I Titer (log2)
A
16
B
IgGsubtype
IgG1
IgG2a
12
12
Figure 3.2: Immunization with Salmonella-CFA/I (H696) and Salmonella-CFA/I-PLP (JYL002)
8 BALB/c mice. Copro-IgA, serum IgG
shows8 similar responses to Salmonella-CFA/I immunized
titers (A) and IgG subtype (B) were examined either after 2weeks of Salmonella-CFA/I
4
immunization
in 1wk and 4weeks post immunization4challenge group, or 1wk and 2wks after
Salmonella-CFA/I-PLP immunized group without PLP139-151 challenge.
0
0
1wk p.i 4wks p.i 1wk 2wks
1wk p.i 4wks p.i 1wk
2wks
JYL002
H696
JYL002
H696
Figure 3.2: Immunization with Salmonella-CFA/I (H696) and Salmonella-CFA/I-PLP (JYL002)
shows similar responses to Salmonella-CFA/I immunized BALB/c mice. Copro-IgA, serum IgG
titers (A) and IgG subtype (B) were examined either after 2weeks of Salmonella-CFA/I
immunization in 1wk and 4weeks post immunization challenge group, or 1wk and 2wks after
Salmonella-CFA/I-PLP immunized group without PLP139-151 challenge.
44
JYL002 Vaccinated Group
5
ng/ml
3
*
4
*
*
1
0
5
5
M
OVA
CFA/I
PLP139-151
4
2
IL-10
IL-4
IFN-γ
Spleen CLN PP
*
*
*
4
3
3
2
2
1
1
0
*
*
*
0
Spleen CLN PP
Spleen CLN PP
Figure 3.3: Lymphocytes from Salmonella-CFA/I-PLP vaccinated mice failed to respond to PLP139151 peptide stimulation. Lymphocytes were harvested at day 14 from JYL002- vaccinated mice
without PLP139-151 challenge (spleen, CLN, and PP). Data depict the mean of two experiments. * P
< 0.0001 for M vs. CFA/I..
Reduced EAE Clinical Development and Resolution
by the Oral Immunization of Salmonella-CFA/I (H696)
To test whether the bystander suppression hypothesis with oral vaccination of Th2 cellpromoting Salmonella-CFA/I vaccine could alter the course of EAE development, three groups of
SJL/J mice were orally vaccinated with PBS, the isogenic empty Salmonella vaccine vector (H647),
or Salmonella-CFA/I (H696). One or four weeks after oral immunization, each group of mice was
challenged with PLP139-151 and PT. To determine whether mice were effectively immunized by
Salmonella-CFA/I, CFA/I-specific copro-IgA and serum IgG antibody endpoint titers at week 2
were measured by ELISA. CFA/I-specific Ab titers (Fig. 3.2A) were similar to endpoint titers
obtained in BALB/c mice (162). In addition, IgG subtype titer was also determined, and it showed
strong Th2 bias, as indicated by elevated levels of CFA/I-specific IgG1 Ab (Fig. 3.2B). The
45
unvaccinated control group showed the expected disease course with a mean day onset of disease of
8.8 ± 1.2 days (Table 3.1) with disease peaking between days 12 – 20 after PLP139-151 challenge (Fig.
3.4A). Mice orally vaccinated with the Salmonella vector showed similar disease kinetics (Fig.
3.4A) with a mean day onset of 7.83 ± 1.37 days (Table 3.1). However, while the SalmonellaCFA/I vaccinated mice did not show a significant delay in disease onset (Fig. 3.4A and Table 3.1),
the overall clinical outcome was significantly milder in these mice (P < 0.001). These data suggest
that at the peak of the Th2 cell dominance, previously observed for vaccination with strain H696
(162), the disease course was clearly altered, with these mice recovering by 21 days after EAE
induction. Unvaccinated and Salmonella control groups showed disease persistence throughout the
observation period.
To determine whether Salmonella-CFA/I was capable of providing protection during the
IFN-γ dominated phase, a second set of experiments was performed in which oral immunization
occurred 4 weeks prior to EAE induction (Fig. 3.4B). We have previously shown that at this time
point, the Th2 cell response was reduced, while the Th1 cell responses became elevated (162). To
test this, SJL/J mice were orally vaccinated with PBS, H647 or H696 strains and challenged
(immunized) with PLP139-151 on day 28 and evaluated for clinical disease for 33 days (Fig. 3.4B).
As with the 1-wk vaccination study, the unvaccinated control group developed a similar, but more
severe course (Table 3.1). Both the PBS- and H647-vaccinated groups showed similar mean day
onset of clinical disease (Table 3.1 and Fig. 3.4B). In contrast, the Salmonella-CFA/I-vaccinated
groups showed a significantly delayed mean day onset of 10.5 ± 1.5 days when compared to PBSor H647-dosed mice (P < 0.001). Although the Salmonella-CFA/I-vaccinated mice showed higher
clinical scores than mice vaccinated one wk prior to challenge, all the vaccinated mice resolved
46
their EAE by 24 days after challenge. The H647-vaccinated mice, while showing reduced disease
compared to the PBS-dosed group, had elevated clinical scores when compared to the H647-dosed
mice in the one week post-immunization challenge. The PBS-dosed group also showed elevated
clinical scores when compared to PBS-dosed mice in the one week post-immunization challenge.
The elevated clinical scores in the four weeks post-immunization group may reflect the influence of
the age of the mice at the time of challenge on the severity of clinical EAE expression (224, 225).
Thus, the protective effect of Salmonella-CFA/I strain persists throughout the Th2 and Th1 cell
phases of vaccine-specific immune responses.
Histopathological Analysis of CNS: Myelin Status and
Nucleated Cell Infiltration to CNS
Typically, EAE is characterized by CNS inflammatory cell infiltration and loss of myelin. To
determine the extent of demyelination and inflammatory cell infiltration among the one week
vaccination groups, sections of spinal cord were stained with LFB and H&E. A striking difference
in the extent of demyelination was observed among the different treatment groups when compared
to normal SJL/J mouse spinal cord (Fig. 3.5). H696-immunized mice showed no demyelination or
only mild demyelination (Fig. 3.5). Spinal cord sections from PBS- and H647-immunized mice
showed demyelination with the H696-vaccinated mice, revealing the most mild myelin loss
compared with PBS- and H647-vaccinated mice (Table 3.2; P < 0.001). Since demyelination is
caused the inflammatory cells, the degree of inflammatory cell infiltration in the CNS (Fig. 3.6) was
evaluated in spinal cord sections stained with H & E (Fig. 3.6). The H696-vaccinated group showed
little or no inflammation similar to that observed in normal mice. By contrast, PBS- and H647vaccinated mice had clear inflammatory infiltrates in the meninges and CNS parenchyma regions.
47
The degree of cellular infiltration in the spinal cords is summarized in Table 3. 2. The H696vaccinated group scored 0.4, which represented either no infiltration or minimal meningeal
infiltration. PBS- and H647-vaccinated groups had scores in the 2.5 - 2.7 range, indicating
between one to four small perivascular infiltrations and more than four small perivascular/or
large parenchymal invasions. Thus, protection from clinical disease in H696-vaccinated mice was
clearly associated with reduction in inflammatory CNS infiltrates.
Salmonella-CFA/I Vaccine Halts CD4+ T Cell and Mac-1+ Cell Influx into the CNS. To
determine the general composition of the inflammatory infiltrates, immunohistochemistry was
performed to identify cell types typically present in EAE tissue (Fig. 3.7). Infiltrates in spinal cord
sections from PBS-vaccinated mice consisted of CD4+ T cells, Mac-1+ cells, and neutrophiles
(SK208+). By contrast, spinal cords from H647-vaccinated mice showed staining for a small
number of Mac-1+ cells and only a few neutrophils. Finally, CD4+ T cells and SK208+ neutrophiles
were absent from infiltrates in Salmonella-CFA/I-vaccinated mice; only a few Mac-1+ cells were
detected. These data suggested that H696 vaccination suppressed development of myelin-specific,
pathogenic Th1 cells and/or prevented migration of inflammatory cells to the CNS.
48
Clinical Score
5
1-w k Post-Im m unization
A
PB S
H 647
H 696
E pitope
4
3
2
*
1
**
0
0
Clinical Score
5
B
5
10
15
20
25
D ays Post-challenge
3
2
35
4-w k Post-Im m unization
PB S
H 647
H 696
E pitope
4
30
1
*
**
0
0
5
10
15
20
25
30
D ays Post-C hallenge
35
Figure 3.4: Oral vaccination with Salmonella-CFA/I (H696) or Salmonella-CFA/I-PLP (JYL002;
designated as Epitope in legend) was protective against PLP139-151 challenge in SJL/J mice. Mice
were challenged with PLP139-151 one (A) or four wks (B) after oral vaccination with Salmonella
vaccines or PBS and given pertussis toxin on day 0 and 2 of challenge. Salmonella-CFA/I- or
Salmonella-CFA/I-PLP-immunized mice showed significantly reduced EAE in both 1 and 4 wks
post-vaccinated groups and delayed onset in the 4-wk group (Table 3.1). All H696- or JYL002vaccinated mice recovered. The vector control, H647-immunized group also showed reduced
disease when compared to PBS-immunized mice; however, clinical disease persisted, and mice did
not recover. Depicted are the results from two combined experiments each * P < 0.001 for PBS vs.
H647, PBS vs. H696, and PBS vs. JYL002.
49
Table 3.1: Vaccination with Salmonella-CFA/I (H696) one week prior to PLP139-151 challenge
protects SJL/J mice from EAEa
5.
6.
2. Vaccinationn 3. EAE/Totalb 4. Onsetc
Maximum
Cumulative
d
Score
Score e
7. PBS
8. 10/10
9. 8.8 ± 1.2
10. 5
11. 61.22
12. H647
13. 9/9
14. 7.83 ± 1.37
15. 4
16. 33.54*
17. H696
18. 9/9
19. 10.13 ± 3.8
20. 2
21. 6.32*, *
Vaccination with Salmonella-CFA/I (H696) four weeks prior to PLP139-151 challenge protects SJL/J
mice from EAE
24.
25.
26.
22. Vaccination 23. EAE/Totalb
Onsetc
Maximum
Cumulative
Scored
Scoree
27. PBS
28. 10/10
29. 8.0 ± 0.57
30. 5
31. 91.09
32. H647
33. 8/8
34. 8.0 ± 0.8
35. 5
36. 53.07*
,
41. 11.47*, *
37. H696
38. 9/10
39. 10.5 ± 1.5* * 40. 2
a
SJL/J mice were challenged s.c. with 200 µg PLP139-151 in complete Freund’s adjuvant 1 wk postvaccination with PBS, H647, or H696. Mice received i.p. 200 ng PT on days 0 and 2.
b
Number of mice with clinical EAE over total in the group.
c
Mean day of clinical disease onset.
d
Maximum daily clinical score.
e
Cumulative clinical scores were calculated as the sum of all clinical scores from disease onset to
the day of sacrifice (25 - 27 days), divided by the number of mice in each group. *P < 0.001 for
PBS vs. H647, PBS vs. H696, and H647 vs H696.
f
EAE was induced by immunization against PLP139-151 at 4 wks post-vaccination with PBS, H647,
and H696. MDO: *P < 0.001 for PBS vs. H696 and H647 vs. H696.
50
Normal
PBS
H647
H696
Figure 3.5: Spinal cord sections from different immunization regimens were stained with LFB to
detect demyelination. Spinal cord sections from PBS- and H647-immunized mice showed
significant demyelination (indicated by arrows); however, the H696-dosed group showed minimal
to no pathology.
51
Normal
H647
PBS
H696
Figure 3.6: To visualize cell infiltration into the CNS, spinal cord sections from the different
immunization groups were stained with H&E. Inflammation of the meningeal and parenchymal
regions of CNS were found in PBS- and H647-immunized mice (see arrows). Spinal cord sections
from H696-immunized mice showed absence of inflammation and resembled normal spinal cord.
Samples were collected between day 11 and 12 after PLP139-151 challenge.
52
Table 3. 2: Histopathological changes in vaccinated mice with PLP139-151 induced EAEa
Nc
Vaccinationb
Inflammation
Demyelination
PBS
H647
H696
a
6
6
6
2.5 ± 0.7
2.67 ± 0.27
0.33 ± 0.27*
2..67 ± 0.6
2.5 ± 0.3
0.17 ± 0.16*
Spinal cord sections were stained with H&E to evaluate cellular infiltrates, and LFB to assess
demyelination. The sections were scored separately for mononuclear cell infiltrates, and for the
presence of demyelinating lesions. Inflammatory cell infiltrates: 0, normal; 1, meningeal
mononuclear infiltrates; 2, between one and four small perivascular infiltrates/section; 3, more than
four small perivascular infiltrates and/or one or more large infiltrates invading parenchyma; 4,
extensive mononuclear infiltrates involving 20% or more of the white matter/section. Myelin
damage: 0, normal; 1, one small area of demyelination/section; 2, two to three small area of
demyelination; 3, one to two large demyelinating areas; 4, extensive demyelination involving 20%
or more of the white matter/ section.
b
Mice were challenged s.c. with 200 µg PLP139-151 in complete Freund’s adjuvant 1 wk postvaccination with PBS, H647, or H696. On days 0 and 2 post-challenge, they received i.p. 200 ng
PT.
c
Number of mice examined per group. *P < 0.001 for PBS vs. H696
53
CD4+
Mac-1+
SK208
PBS
40x
400x
H647
H696
Figure 3.7: To identify the types of inflammatory cells infiltrating CNS, IHC was performed. Spinal
cords from PBS-dosed mice showed mostly CD4 cells and macrophages, evidenced of minimal
neutrophil infiltration (indicated by Mac-1+ and SK208 mAb staining). Spinal cords from H647vaccinated mice showed mostly Mac-1 and only few SK208 cells. Only a few Mac-1+ cells were
detected in spinal cords from H696-vaccinated mice. Mice were sacrificed between days 11 and 12
after PLP139-151 challenge. Depicted are representative examples of 6 mice /group.
54
PLP139-151 -Specific Immune Deviation from
Th1 to Th2 Cell by Vaccination with Salmonella-CFA/I.
The participation of Th1/Th2-type cytokines in the development of organ-specific
autoimmune disease has been well-documented (246, 247). To determine whether the protective
effects of Salmonella-CFA/I vaccination is Ag-specific, cytokine responses were measured ex vivo
in lymphocytes isolated from spleens, PP, CLN, or spinal cords from vaccinated and unvaccinated
mice, and pulsed in vitro with OVA, CFA/I fimbriae, or PLP139-151 (Fig. 3.8). Unstimulated
lymphocytes were included as controls. As we previously observed (162), cytokine responses were
clearly Th2 cell-dominant in H696-vaccinated mice, exceeding IFN-γ production (Fig. 3.8).
Unstimulated lymphocytes were included as controls. Not having previously measured IL-13, we
observed considerable enhancement in H696-vaccinated mice, but not in the PBS-dosed mice.
Some splenic and CLN IL-13 was induced in mice orally vaccinated with the Salmonella vector,
but not to the levels observed with H696-vaccinated mice. Upon restimulation with PLP139-151
peptides, the IFN-γ levels in H696-vaccinated mice were significantly reduced in all tissues
examined when compared to unvaccinated controls or H647-vaccinated mice.
The major
production of PLP139-151-specific IFN-γ was primarily observed with CLN from PBS- and H647vaccinated mice, suggesting that this was the site of T cell activation. Conversely, the PBS- and
H647-vaccinated mice showed minimal-to-no Th2-type cytokine production in any of the tissues
further examined, suggesting that the Salmonella-CFA/I vaccine imparted a Th2-type bias and, in
an Ag-independent fashion, biased the development of PLP139-151 CD4+ T cells to becoming Th2type. Thus, oral immunization with Salmonella-CFA/I vaccine induced PLP139-151-specific immune
deviation for ultimate protection against EAE.
55
Figure 3.8 (A) and (B)
56
Figure 3.8(C) and (D)
Figure 3.8: Lymphocytes from Salmonella-CFA/I-vaccinated mice showed increased production of
Th2-type cytokines and reduced IFN-γ secretion following in vitro restimulation with PLP139-151
peptide. Lymphocytes were collected from mice vaccinated for one wk and harvested between 11
and 12 days post-EAE induction. Lymphocytes from (A) spleen, (B) CLN, and (C) PP of H696vaccinated mice showed significant increases in the production of Th2-type cytokines (IL-4, IL-10,
and IL-13) in response to PLP139-151 restimulation compared to similar cultures from PBS- and
H647-immunized mice. In contrast, PBS- and H647-vaccinated mice showed greater production of
IFN-γ including CLN and spinal cord (SC) than lymphocytes obtained from the H696-vaccinated
mice. Unstimulated cultures (M) or cultures restimulated with nonspecific Ag (OVA) showed
minimal IFN-γ secretion. Data depict the mean of three experiments. *P < 0.001, ***P < 0.018
depict differences between PLP139-151 restimulated cultures from PBS-dosed mice versus cultures
from H647- or H696-vaccinated mice.
57
Discussions and Conclusions
Previous works show that Salmonella-CFA/I vaccine fails to elicit proinflammatory
cytokines from infected macrophages unlike its isogenic strain, which, in a dose-depend fashion,
induces the expected elevation in TNF-α, IL-1α, IL-1β, and IL-6 (163). It is apparent from this
study that the presence of the CFA/I fimbriae impedes the expected proinflammatory response. This
lack of an inflammatory response is not attributed to increased production of IL-10 or IL-12p40,
since these did not significantly differ from those levels induced by the Salmonella vector strain
(163). Further evidence that the CFA/I fimbriae alters the Salmonella’s behavior is supported by in
vivo studies in which CFA/I fimbria-specific Th2 cells were dominated by IL-4, IL-5, IL-6, and IL10 and accompanied by elevated serum IgG1 and mucosal IgA Ab titers (162). We hypothesized
that this property of the Salmonella-CFA/I vaccine could be exploited to develop an antiinflammatory vaccine.
To test this hypothesis, the EAE-susceptible SJL/J mice received the anti-inflammatory
vaccine, which was engineered to express encephalitogenic PLP139-151, its isogenic construct
(lacking the CFA/I operon), or PBS one or fours wks prior to EAE induction by active
immunization against PLP139-151. We first questioned whether SJL mice would make an appropriate
immune response to the vaccine, as previously shown for BALB/c (162) and B6 mice (218). The
data clearly indicate that SJL/J mice are vaccine responders generating a robust anti-CFA/I Ab
response that is similar in magnitude and kinetics to those exhibited by similarly vaccinated
BALB/c and B6 mice. However, antigen recall experiments with various tissues from the mice
immunized with Salmonella-CFA/I-PLP, which was originally designed to induce PLP139-151-
58
specific EAE suppression, failed to stimulate PLP139-151-cytokine production. The next series of
studies showed that Salmonella-CFA/I-vaccination significantly reduced clinical and
histopathological EAE in PLP139-151-immunized mice. The duration of acute disease was shorter;
peak clinical scores were lower; and, more importantly, all of the vaccinated mice recovered from
the acute phase of disease. By contrast, disease in control mice receiving the Salmonella vector
strain alone or PBS was sustained throughout the 33 day observation period, with minimal recovery
after the acute phase. Although the severity of EAE observed in mice receiving Salmonella-CFA/I
vaccine 4 weeks prior to PLP139-151 immunization was worse than observed in mice with EAE
induced 1 week post-vaccination, all vaccinated mice in both vaccination regimens recovered
completely. It was not surprising that the Salmonella-CFA/I-vaccinated mice in the one week
regimen showed less severe clinical disease, since the Th2 cell response is dominant during the
early stages of Salmonella inflection, and since CFA/I fimbria-specific Th1 cells generally do not
peak until three or four wks post-infection (162). We also noted a difference in the magnitude of
EAE in PBS-dosed mice, which may be related to the differences in age of the challenged mice.
Such differences may be attributed to age, as others have previously suggested that EAE is agedependent (224, 225).
Surprisingly, mice vaccinated with the Salmonella vector strain H647 also showed reduced
EAE in both dosing regimens, which was a consistent finding in 4/4 experiments conducted
independently. In addition, this occurred in the presence of an intact IFN-γ response and in the
absence of a clear Th2 cell deviation. An explanation for this finding may be that pro-inflammatory
responses induced by Salmonella infection alone may be competing with PLP139-151-induced
59
inflammation. In all four experiments, this was consistently observed. Similar observations have
been made for EAE protection (206) and treatment of relapsing-remitting MS in humans (207-209)
following vaccination with BCG. BCG, a Th1-type promoting bacterium, caused the formation of
granulomas in spleen and liver of infected mice, and the investigators demonstrated that MOGspecific Th1 cells were recruited into the liver granulomas rather than to the CNS (206). This
suggests that vaccination with Th1 cell-inducing microbial agents may redirect a Th1 cell response
away from an Ag-targeted site of inflammation. Additional studies are warranted to directly address
this possibility in the Salmonella-CFA/I vaccination model.
In addition to reduction in clinical disease, mice receiving oral Salmonella vaccine also
exhiibited clear evidence of reduced tissue pathology. Salmonella-CFA/I-vaccinated mice showed
minimal demyelination and inflammatory cell infiltration during peak EAE, and the composition of
inflammatory cell infiltration differed significantly among the three experimental groups. CD4+ T
cells and Mac-1+ cells and some neutrophils dominated the spinal cord infiltrates in PBS-vaccinated
mice, while infiltrates in Salmonella vaccinated mice consisted almost exclusively of Mac-1+ cells
(not neutrophils). Thus, a reduction in T cells may account for the observed reduced pathology in
Salmonella-vector vaccinated mice. The lack of T cells in spinal cord infiltrates limited the ability
to study T cell cytokines in the CNS of Salmonella-CFA/I-vaccinated mice. This evidence supports
the notion that oral vaccination with Salmonella-CFA/I vaccine can reduce peripheral or distal
inflammation in a tissue other than the mucosal compartment. This ability to reduce inflammation
in the CNS occurred in an Ag-independent fashion because there was no primary structural
homology between PLP139-151 and CFA/I fimbria.
60
Since no primary structural homology could be identified between PLP139-151 and CFA/I
fimbrial subunit, we hypothesized that the mechanism for the observed efficacy against EAE
involved immune deviation. As previously indicated, the CFA/I fimbriae alter how the host
recognizes the Salmonella vaccine, and it is evident that potent Th2 cells were induced despite the
proinflammatory challenge produced by the EAE regimen. In this context, the development of
inflammatory PLP139-151-specific Th cells was diminished, as indicated by the reduced PLP139-151specific IFN-γ-producing T cells in Salmonlla-CFA/I-vaccinated mice. Instead, PLP139-151-specific
Th2 cells were induced and sustained for at least four weeks. In contrast, the PBS- and Salmonella
vector-immunized mice showed elevated levels of IFN-γ and minimal to no production of Th2-type
cytokines. Thus, it appears that the Salmonella-CFA/I vaccine altered the microenvironment,
possibly in the draining LN of the head and neck, to prevent the development of encephalitogenic
Th1 cells.
We were also surprised to find the quite robust IL-13 responses by Ag-restimulated
lymphocytes since we had not previously evaluated this Th2-type cytokine. IL-13 is a signature
Th2-type cytokine that exhibits functions similar to those observed for IL-4. It was evident in this
study that while IL-13 was induced against the CFA/I fimbriae, the majority was induced by
PLP139-151, especially in the CLN. It has been reported that IL-13 can suppress EAE, using
transfection methods (194) or adoptive transfer of Th2 cells (184), and it has been suggested IL-13
suppresses EAE by inhibiting the production of proinflammatory cytokines, such as IL-1β and
TNF-α, from macrophage/monocyte (196, 197) or nitric oxide by glial cells (198). Although IL-13
and IL-4 share a receptor, the ability of IL-13 to suppress EAE appears distinct from that of IL-4
(195). In our studies, the concomitant stimulation of IL-4 and IL-13 by oral vaccination with a live
61
vaccine suggested a clear advantage for our Salmonella-CFA/I vaccination strategy to prevent
inflammatory autoimmune diseases.
In addition to the stimulation of IL-4 and IL-13, the Salmonella-CFA/I vaccine also
stimulated production of IL-10 (162), which has a well-documented beneficial impact on EAE (184,
190-192). IL-10 delays disease onset and reduces clinical disease when directly administered at the
time of disease induction (190) and in transgenic mice over-expressing IL-10 (183, 191). Moreover,
targeted disruption of IL-10 results in loss of EAE suppression (183, 191). IL-10 acts during the
induction, rather than in the effector stage of disease, since it fails to suppress EAE induced by
adoptive transfer of myelin-specific cells (193).
Administration of IL-10 may actually be
detrimental in the adoptive transfer model (193). In our study, IL-10 was already present at the time
of disease induction and may have contributed to deviation of PLP139-151-specific CD4+ T cells
toward a Th2 cell phenotype, either independently or in conjunction with IL-4 and IL-13.
Data from other infection models suggest that preconditioning the host with a Th2-inducing
organism may bias and alter the development of encephalitogenic T cells. Infection with S.
mansoni initially produces a Th1 cell response, which then shifts to Th2-type response during egg
production, implying that the eggs are responsible for inducing Th2-type responses (211). However,
the mechanism for protection by S. mansoni differs between a live infection or an exposure to eggs
alone. S. mansoni infection results in the down-regulation of IFN-γ and IL-12p40, but leaves IL-10
and IL-4 unchanged, suggesting mechanisms other than Th2 cell activation in EAE protection, and
possibly involving macrophages (212). By contrast, immunization with S. mansoni eggs results in
EAE protection that is accompanied by the down-regulation of IFN-γ and increases production of
IL-4, IL-10 (213, 214), and TGF-β (213). Consequently, adapting the host to a Th2 cell-dominated
62
environment by helminth infection is currently being considered as a strategy for the treatment of
MS (210, 248).
In conclusion, this study showed that oral administration with the anti-inflammatory
vaccine, Salmonella-CFA/I, in an Ag-independent fashion, can prevent the development of the
inflammatory murine disease, EAE. Expression of the ETEC fimbriae was necessary since mice
receiving the “naked” Salmonella vector showed persistent clinical disease throughout the
observation period. Although mice exposed to naked vector showed less disease severity than PBS
treated controls, the Salmonella-CFA/I-vaccinated mice completely resolved EAE as early as 20
days after disease induction. Protection from EAE in Salmonella-CFA/I, but not naked vectorvaccinated mice, was accompanied by elevated production of PLP139-151-induced Th2-type
cytokines and diminished IFN-γ production. Protected animals showed reduced infiltration of
inflammatory cells, especially CD4+ T cells and macrophages, in the CNS. Thus, encephalitogenic
T cells were converted from a pathogenic Th1 to a protective Th2 phenotype that was clearly
capable of preventing CNS immune infiltration and clinical disease. Future studies will address the
ability of this vaccination strategy to induce long-term changes in cytokine environment and
whether it can treat ongoing disease in a treatment paradigm.
63
CHAPTER FOUR
MUCOSAL VACCINE DEVELOPMENT AGAINST ENTEROTOXIGEIC (ETEC) - AND
ENTEROHEMORRHAGIC (EHEC) E.COLI
Intranasal ETEC Vaccine Targeting
Introduction
In the early 1970s, several diarrhea causing E. coli were isolated in humans with various
serotypes, producing an enterotoxin similar to cholera toxin (252-254). Later, two species of cholera
toxin-like enterotoxins were determined and designated as heat stable enterotoxin (ST) and heat
labile enterotoxin (LT). The ST is stable in boiling for 30 minutes, while the LT is destroyed by such
treatment (221-223, and 255). The E. coli strains producing either one or both toxins were classified
as ETEC.
LT is 85kD protein consists of a single 240 residue A subunit and five identical B subunits
(222, 223, and 256). The specific binding of B subunits to GM1 glycolipid ganglioside on the brush
border of small intestine form a pentameric structure with a central pore in which the C-terminal tail
of the A subunit extends to hold the A/B5 holotoxin together (256-258). The A subunit functions as a
permanant adenylate cyclase activator and results in the increase of cyclic AMP (cAMP) levels in
the enterocyte which causes loss of electrolytes, as well as a water, into intestinal lumen (259). The
ST is 18 amino acids, 18 kD small protein (221, 260). The binding of ST to the brush border of the
host initiates the activation of guanylate cyclase with concomitant increase of
intracellular cyclic GMP (cGMP) levels (221, 260). This chain reaction causes the lowering of the
sodium and chloride levels in cytosol, and resulting in fluid loss (221, 261).
64
Along with toxin production, ETEC virulence depends on the presence fimbrial adhesins or
colonization factors, which confer binding and colonization of bacteria. Fimbriae provides tissue
tropism and host specificity. For example: K88 is porcine-specific, K99 is for bovine- and porcinespecific, and CFAs are human-specific (250, 262). The receptors for the fimbriae tend to be very
specific for carbohydrate or glycolipid moieties (150, 263).
ETEC is an important cause of diarrhea in humans as well as in animals. In developing
countries, ETEC contributes to 380,000 infant deaths annually (264) and causes diarrhea for
travelers to endemic areas (220, 264). For animals, it is one of the leading causes of diarrhea scours
for calves and piglets and is a considerable factor in mortality of piglets and newborn calves (250).
Previous studies show that immunization with cholera toxin subunit B or ST-LT conjugate confers
protection against ETEC in human and animal studies (265-267). Fimbrial adhesins were also
investigated as a vaccine target. Oral immunization of calves with purified K99 failed to induce
significant immune responses (151), and volunteers immunized with purified CFAs failed to protect
against ETEC challenge in human trial (152). In addition, oral vaccination with microsphereencapsulated purified CFA/I, which is designed to protect antigen from the gastric environment,
induced strong and sustained antigen-specific serum IgG, but rarely induced protective S-IgA (153).
Thus, novel delivery systems are needed to protect antigens from gastric environment, as well as for
proper targeting to induce protective immunity. Previous studies in our lab show that oral
Salmonella vaccine vector with CFA/I (162) or K99 (250) elicits strong antigen-specific serum IgG
and S-IgA, and confers protection against ETEC challenge (250). Another mucosal targeting means
was also investigated in our lab with reovirus protein σ1. Reovirus is an enteric pathogen that infects
host through the M cell binding (301), and the binding is mediated by reovirus adhesin, protein σ1
65
(301). Previous studies showed that the recombinant protein σ1 binds to its receptor on rodent and
human cell lines (251) and nasal-associated lymphoid tissue (NALT) M cells (302). Intranasal (i.n)
immunization with a protein σ1 polylysine (PL) model DNA vaccine complexes elicited enhanced
S-IgA and cytotoxic T lymphocyte responses when compared to naked DNA immunization (302,
303).
Intranasal (i.n) immunization has several attractive features over conventional vaccination.
It has proven to be effective for inducing both upper and lower respiratory immunity, as well as
distal mucosal immunity with less vaccine requirement and higher S-IgA induction when compared
to conventional immunization (304-307). Thus, we hypothesize that i.n vaccination with K99 major
structural subunit, FanC, as a protein σ1 recombinant conjugate can elicit protective S-IgA
responses against ETEC challenge.
Results
Plasmid Construction for FanC/protein σ1 Fusion Vaccine and Its Expression in E coli .
To obtain the protein σ1/FanC fusion protein, we cloned single- (p1FS) or 3x fanC (p3FS) under
N-terminus of protein σ1 to exploit C-terminus M cell targeting property. Sequencing (data not
shown) and restriction analysis confirms the adequate construction of p1FS or p3FS. Fig 4.1
describes the proposed p1FS and p3FS restriction map and representative p1FS restriction analysis.
However, we experienced difficulties in purification of FanC/protein σ1 fusion protein from the E.
coli expression system due to the formation of inclusion bodies.
66
FanC/protein σ1 Plasmid Construction for Yeast Expression. To overcome the problem of
inclusion body formation, we adapted yeast (Pichia pastoris) expression system to exploit its slow
and regulated protein expression. To obtain the yeast clone that expresses FanC/protein σ1 fusion
vaccine, we cloned fanC/protein σ1 into yeast expression vector, pPICZ A, multi-cloning sites
(EcoR I and Kpn I, Fig 4.2A), and named it as pPI1FS. Positive yeast clones were selected on
zeocin/YPD plates. The successful expression of FanC/protein σ1 fusion vaccine was determined
by SDS-PAGE and Western blotting probed with a monoclonal anti-K99 antibody. Fig 4.2
describes the restriction map of yeast expression vector, pPICZ A, and fanC/protein σ1 yeast
expression construct, pPI1FS, and its expression. However, despite expression in yeast, the
suspected malformed protein conformation disallowed any anti-K99 antibodies to be induced. Thus,
we terminated this project.
67
a b c
A
d
e
f
g
h
p3FS
3X K99 fanC
3X Xa
malE
Protein σ1
p1FS
a b c
a: Sac I
b: Ava I c: Xmn I
a
d e
d: EcoR I
b
f g
e: BamH I
c
h
f: Xba I g: Sal I
h: Hind III
d
B
2.0 kb
1.5 kb
1 kb
0.5 kb
a: construct
b: EcoR I/Xba I
c: EcoR I/Hind III
d: Sal I/Hind III
Figure 4.1: Construction of protein FanC/protein σ1 expression plasmid. (A) represents the
proposed plasmid map for single- or 3x fanC/protein σ1 fusion construct. (B) shows the restriction
digestion of p1FS and confirms appropriate DNA fragments.
68
Apa I
Not I
Sac II
Xho I
Kpn I
Asp718 I
BsmB I
Sfi I
Pml I
EcoR I
Sfu I
A
MCS
c-myc epitope
Stop
6x His
pPICZ A
AOX1 TT
5΄AOX1
PTEF1 PEM7
a b
Zeocin Cyc1 TT pUC ori
cdefg
pPI1FS
5΄AOX1
a: Sfu I
B
fanC
b: EcoR I c: Kpn I
1
Protein σ1
d: Xho I
c-myc 6x His
e: Sac II f: Not I
1
2
Stop
g: Apa I
2
75 kd
75 kd
SDS-PAGE
Western blot
Fig 4.2: Construction of yeast expression vector and its expression. (A) Yeast expression construct
pPI1FS constructed by cloning fanC/protein σ1 gene into pPICZ A multi cloning site (MCS) (EcoR
I/Kpn I). (B) FanC/protein σ1 fusion protein was purified by using Ni++ -NTA column
chromatography and confirmed with SDS-PAGE and Western blotting with K99-specific
monoclonal antibody. Lane 1; blank, lane 2; purified FanC/protein σ1.
69
Live Vaccine for EHEC
Introduction
In late 1977, “Vero” cell active cytotoxin, which was distinct from ST and LT, was
identified from several E. coli strains (268). Later, the Vero cell cytotoxin (VT) producing E. coli
and its association with hemolytic uremic syndrome (HUS) and hemorrhagic colitis were
determined, and it was identified as O157:H7 serotype (269-271). The EHEC VT was found to be
similar to Shiga toxin from HUS-causing Shigella dystentriae type 1, and thus was called “shigalike” toxin (SLT) (272, 273). Two different SLT were idenified: SLT I and SLT II (273, 274).
SLTs are phage encoded and consist of 6 subunits, 1x A and 5x B (274). SLTs act on protein
synthesis. The binding of pentameric B subunits to globotriaosylceramide (Gb3)-lipid complex on
cell surface causes endocytosis of SLT (275-277). Once in cytosol, the A subunit is released, and
targets to ribosome, N-glycosidase activity of A subunit cleaves N-glycosidic bond of the adenine
(position: 2324) of 28S ribosomal RNA (rRNA), and causes subsequent inactivation of 60S
ribosome (278). Previous research showed that the primary target for SLTs is the vascular
endothelium (279-282). In fact, the first signal of EHEC infection is bloody diarrhea due to the
failure of renewing endothelium in gut resulting from protein synthesis inhibition by SLTs, which
eventually lead to a breakdown of lining and hemorrhage (283). In severe cases, SLTs target to
glomerular endothelial cells of the kidney and cause HUS (281, 282).
Another virulence factor of EHEC is intimin, encoded by the eae gene in the chromosome
(284), producing an attaching and effacing (A/E) region in the small intestine brush border (285).
Intimin is also found in Citrobacter rodentium (286), which causes transmissible murine colonic
70
hyperplasia in laboratory mice (287). Mutation of eae causes the failure of colonization and disease
induction by EHEC or C. rodentium (284, 288, and 289).
Cattle have been identified as a major reservoir of EHEC (290-292). Deer (293), sheep
(294), and water (295-297), which is contaminated by EHEC from the fecal waste of cattle, are
minor ones. Ground beef has been responsible for cases of EHEC-related disease outbreaks (298).
The infectious dose in a food-born outbreak of EHEC is very low (2-2000 CFU) due to its acid
tolerance (299). Thus, preventing the colonization of EHEC in cattle intestine has been considered
as the most effective means of preventing human disease. However, past experiments have shown
that cattle preexposed to EHEC were not protected from reinfection, even with prompt and
sustained Ab production to EHEC’s LPS and SLT I (300). This lack of protection is ascribed to poor
mucosal colonization of EHEC in adult cattle as evidenced by the lack of attaching/effacing region
(291, 292), and it is conceivable that the specific mucosal immune responses were not efficiently
stimulated by natural infection. Thus, we hypothesized that by adapting the targeting capabilities of
a live vector system with an EHEC-specific antigenic epitope, we will be able to effectively
stimulate mucosal tissues for optimal induction of S-IgA for protection against EHEC colonization.
To express EHEC antigenic epitope on the Salmonella vaccine, we exploited K99 fimbriae, which
was described previously (250), as a molecular scaffold. The K99 operon is encoded by a virulence
plasmid consists of 8 different genes, referred to as fan ABCDEFGH. These genes have unique
functions in the biogenesis of K99 fimbriae, such as positive regulator (A and B), major fimbrial
protein (C), translocator (D), molecular chaperon (E), and minor fmbrial subunits (F, G, and H).
Previous studies by other groups have shown that mutation in fanG or fanH had no influence on the
71
formation of K99 fimbriae (308). Therefore, we decided to clone the EHEC epitope to a selected
region of fanH.
Results
Expression of EHEC LPS Peptide Mimetic on Salmonella Vaccine Vector. The consensus
peptide sequence that mimics EHEC LPS structure was identified by LigoCyte Pharmaceuticals
(Bozeman, MT) by using a phage display system. Fig 4.3 shows the EHEC peptide mimetic
sequence.
11: GRGQFEGLAA
14: DVTGQFLGLAA
15: NQSGQFVGLAR
22: ADNGQFFELGR
CON: SGQFXGLARV
Fig 4.3: EHEC peptide mimetic sequence. Following three rounds of affinity selection and
amplification, immunoreactive sequence was selected that bind specifically with EHEC LPSspecific monoclonal EC114. Four representative clones are shown with a numbrical designation.
From these, a consensus (con) sequence was identified (LigoCyte Pharmaceuticals., Bozemanm
MT).
To clone and express this mimetic, we first analyzed prokaryotic codon usage by using the
E. coli Codon Usage Analysis 2.0 program (Morris Maduro) and identified optimal sequences for
prokaryotic expression (TCT GGC CAG TTC GAA GGC CTG GCG CGT GTT GAA TCT
TGC). The oligonucleotides were synthesized and annealed in vitro and cloned into the K99
fimbriae minor structural protein gene fanH. Fig 4.4 shows the EHEC LPS peptide mimetic
expression construct (pFH:LPS). The cloning was confirmed with sequencing (data not shown),
restriction analysis, and Western blotting with polyclonal anti-K99 Ab. Fig 4.5 shows the restriction-
72
and Western blot analysis. Restriction digestion with Nsi I reveals the corresponding size increase of
LPS mimetic sequence insertion. Western blot with polyclonal anti-K99 Ab tells us that the
mutagenesis in the fanH gene with LPS mimetic sequence did not influence the formation of K99
fimbriae and suggested that this region can be exploited as a further cloning site for other antigenic
epitopes. The Salmonella vaccine with pFH:LPS is designated as JY002.
Antibody Responses by Salmonella Vaccine with Chimeric K99/LPS Peptide Mimetic. To
of the EHEC peptide mimetic, we immunized mice with 5 x 109 (Fig 4.6 A) or 1 x 1010 (Fig 4.6 B)
CFU of Salmonella vaccine (JY002). Both low and high dose regimens elicited significant serum
IgG titers toward K99 fimbriae, as well as the LPS mimetic, but failed to induce S-IgA against the
LPS mimetic. Even boost with 5 x 109
CFU at week 5 in low dose immunization did not
influence S-IgA induction (Fig 4.6 A: indicated by the arrow). The importance of S-IgA for the
protection of the colonization of EHEC was previously described (300). Thus, without eliciting LPS
mimetic-specific S-IgA, we terminated further studies.
73
β
Ptrc
β
α
α
ori
α
β
pMAK99-asd
pFH:LPS
asd
lacZ
K99 operon
Ptrc promotor
α: BamHI
ori
LPS mimetic
B
C
D
E
F
ori
α
10.54 kb
10.50 kb
A
β
Ptrc
β: Bgl II
G
H
K99 Operon
Figure 4.4: Cloning of EHEC LPS peptide mimetic into K99 minor structural gene fanH. Briefly,
fanH gene was amplified with fanH Sac I-forward (TAG AGC TCA AAA CCT TTT GAG CGG)
and Hind III-reverse (TAA AGC TTT GAA TTT CTT ATT CCC CTC) primers using pMAK99asd as a template. The amplified fanH fragment was cloned into pUC18 vector, and designated it as
pFanH. Further, the pFanH was transferred into MH6083 E.coli strain (dam-) to obtain
unmethylated Bcl I EHEC LPS mimetic cloning site because Bcl I restriction enzyme is
methylation-sensitive. The corresponding peptide mimetic nucleotides were annealed in vitro
(contain Bcl I site and Bcl I-treated) and inserted into Bcl I-treated pFanH: pFanH:LPS. Then, the
LPS mimetic containing fragment of pFanH:LPS was amplified ( fanH Nsi I-forward: TAT ATG
CAT ATC AAA ACC TTT TGA GCG, fanH Nsi I-reverse: TAT ATG CAT TGA ATT TCT TAT
TCC) and cloned into TA vector: pTA/fanH:LPS. The Nsi I-treated LPS mimetic-containing
fragment from pTA/fanH:LPS was replaced with corresponding Nsi I fragment from pMAK99-asd,
and resulted in pFH:LPS.
74
A
B
0.1kb 1
2 3 4 1kb
MW
1
2
3
0.5 kb
20.5 kd
Figure 4.5: Restriction analysis of pFH:LPS and expression of chimeric K99 fimbriae in
Salmonella vaccine, JY002. There are two Nsi I restriction sites in pMAK99-asd+ plasmid, located
in 5899 and 6466 of K99 operon from the starting codon. Restriction digestion of pMAK99-asd+
and pFH:LPS shows size difference corresponding to the cloned LPS peptide mimetic nucleotides
(A). Lane 1; pMAK99-asd+, lane 2, 4; pFH:LPS, lane 3; self-ligated Nsi I treated pMAK99-asd+.
The chimeric K99 expression was confirmed by probing and developing with polyclonal rabbit
anti-K99 serum and goat anti-rabbit IgG HRP-conjugated antibody (B). Lane 1, 2; K99, lane 3;
chimeric K99 with LPS mimetic.
A
B
25
25
20
IgA-EC/OVA
IgA-EC/OVA
IgA-K99
IgG-EC/OVA
IgG-K99
20
IgG-EC/OVA
IgG-K99
15
15
10
10
5
5
0
0
Titer (log2)
IgA-K99
2
4
6
8
Wks Post Immunization
Titer (log2)
2
4
6
Wks Post Immunization
Figure 4.6: Immunogenecity of LPS peptide mimetic. (A): 5 x 109 JY002 CFU dose and arrow
indicates the boost at wk 6 with same amount of CFU. (B): 1 x 1010 CFU vaccination. ELISA plates
were coated with EC-OVA (ovalbumin-LPS mimetic peptide conjugates) and purified K99
fimbriae.
75
Conclusions
In an effort to develop a smart subunit vaccine for ETEC with exploitation of reovirus
adhesin protein σ1, initially, we made recombinant ETEC K99 fanC/ protein σ1 expression plasmid
in E. coli. When this fusion protein expressed in E. coli, it formed an inclusion body, thus making it
difficult to purify for i.n immunizations. To overcome this obstacle, we turned to a yeast expression
system and obtained FanC/protein σ1 fusion protein without forming inclusion bodies. However, i.n.
vaccination with this protein failed to elicit any immune responses supposedly due to the
malformation either in FanC or protein σ1. The FanC/protein σ1 binding assay to nasal epithelial
cells would tell whether protein σ1 retains its mucosal targeting capacity or not when it presents in
chimeric form. Further, above binding assay also could show protein σ1’s influence on antigenecity
of target proteins. We temporalily halted this work at this point.
Using another approach to induce S-IgA to inhibit EHEC colonization, we cloned and
expressed the peptide mimicking EHEC LPS structure on Salmonella vaccine using ETEC K99
fimbriae as a molecular scaffold. Cloning of peptide mimetic into K99 minor structural gene, fanH,
did not influence for the formation of K99 fimbriae. However, immunization with Salmonella
having chimeric K99 fimbriae (K99 with LPS mimetic) failed to elicit S-IgA against the peptide
mimetic, but induced serum IgG. At this point, we stopped further investigation, because without
peptide-specific S-IgA induction, inhibition of EHEC colonization can not be expected.
76
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