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Oral immunization with a recombinant Salmonella typhimurium vaccine expressing a hemagglutinin of Porphyromonas gingivalis

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Oral immunization with a recombinant Salmonella typhimurium vaccine expressing a hemagglutinin of Porphyromonas gingivalis
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ORAL IMMUNIZATION WITH A RECOMBINANT
SALMONELLA TYPHIMURIUM VACCINE
EXPRESSING A HEMAGGLUTININ OF
PORPHYROMONAS GINGIVALIS











By

JAMES JOHN KOHLER













A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA 1998




ORAL IMMUNIZATION WITH A RECOMBINANT
SALMONELLA TYPHIMURIUM VACCINE
EXPRESSING A HEMAGGLUTININ OF
PORPHYROMONAS GINGIVALIS
By
JAMES JOHN KOHLER
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE
UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1998


To my parents,
John and Mary Lou Kohler,
and the Department of Oral Biology at the University of Florida.
Thank you very much.


ACKNOWLEDGMENTS
I would like to express my thanks and appreciation for the never-ending support
and encouragement that my mentor, Dr. Thomas A. Brown, provided for me during my
doctoral training. His attention to detail helped instill in me a reproducible scientific
method. A special thank you to our Senior Bioscientist and Lab Supervisor, Latha
Pathangey, who patiently trained me and has offered continued support and friendship for
the past five years. Id like to thank Dr. Arnold S. Bleiweis who introduced me to the
graduate PhD program in the Department of Oral Biology, provided guidance, and served
as an advocate for the overall best interest of the students. I would also like to thank Dr.
William P. McArthur for his expertise in the area of immunology, his friendship, and
helpful suggestions. I would further like to thank the rest of my committee, Dr. Paul A.
Gulig, Dr. Jeffrey D. Hillman, and Dr. Ann Progulske-Fox for their added expertise and
guidance in my scientific training.
I would extend a note of gratitude to the entire faculty of the Department of Oral
Biology who have provided a nurturing environment for all the students in the department.
I thank them all for their wonderful dedication, especially Dr. Michael Humphreys-Beyer
and Dr. Jeannine L. Brady.
Lastly, I want to recognize the support, both academic and personal, that I have
received by all the staff of the Department of Oral Biology (Sherri Sullivan, Elaine Green,
Valerie Brown, Cindy Link, Paula Crowley, Elaine Beem, Amy Shawley, and Janice
Braddy) and the graduate office of the College of Medicine (especially BJ Streetman). In
addition, I would like to recognize the support of our student workers who helped me in
the lab (Adnan Hasona, Louis Bravo, Rod Dietz, Shiela Gillespie, Chris Powell).
iii


TABLE OF CONTENTS
ACKNOWLEDGMENTS m
LIST OF FIGURES vii
ABSTRACT x
CHAPTERS
1 INTRODUCTION AND BACKGROUND
The Pathogen Porphyromoncis gingivalis 1
Protective Immunity to P. gingivalis 2
Mucosal Immunity 3
Mucosal Vaccines 6
Recombinant Salmonella typhimruium Vaccines 8
2 MATERIALS AND METHODS
Bacterial Strains, Plasmids, Media, and Culture Conditions 14
Construction of Plasmids 14
DNA Characterization 16
Electroporation into S. typhimurium 16
Sodium-Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis and
Western Blotting 17
Colony Immunoblotting To Determine Stable Expression of HagB 18
Analysis of in vitro Growth Rate of Recombinant S. typhimurium 18
Mouse Immunization and Sample Collection 19
Colonization of Peyers Patches and Spleen Assay 20
Preparation of Formalin-Killed S. typhimurium Cells (x4072) 20
Preparation of Sonicated S. typhimurium x4072/pYA292 Cells 21
Elispot Assay for Determination of Specific Antibody Secreting Cells 22
Verification and Enumeration of Viable, Isolated Lymphocytes 25
Immunoassay Methods 25
ELISA Assays 25
ELISPOT Assays 27
Amylase Assay 28
Statistical Analysis 29
3 DATA AND RESULTS
Reconstruction and Characterization of the recombinant S. typhimurium
Strain /4072/pDMDl Expressing the hagB gene of P. gingivalis 30
Transformation into S. typhimurium 30
Restriction Endonuclease Analysis of DNA Plasmid 30
De novo HagB Protein Analysis 32
IV


LPS Silver Staining Analysis 32
In vitro Growth Rate 36
Colonization Studies of S. typhimurium Strains in BALB/c Mouse
Model Following A Single Oral Immunization 36
Assay for Stable in vivo Expression of HagB 40
Oral Immunization Study in BALB/c Mice with Boosting at Week 14 43
Experimental Design 43
Detection of Specific Anti-HagB Serum Antibodies After Primary
Oral Immunization and Boosting with S. typhimurium
x4072/pDMDl 45
Detection of Specific Anti-HagB Response in Mucosal Secretions 45
Detection of Specific Anti-Salmonella %4072 antibodies in Serum IgG
and Fecal Extract IgA Levels 50
IgG Subclass Distribution Following Primary Oral Immunization and
Boosting 50
Effect of Boosting at Three Different Time Points with Recombinant S.
typhimurium strain x4072/pDMDl in BALB/c Mice 55
Experimental Design 55
Detection of Specific Anti-HagB Serum Antibodies After Primary Oral
Immunization and Boosting at Different Time Points with S.
typhimurium x4072/pDMDl 58
Detection of Specific Anti-HagB Responses in Mucosal Secretions 61
Effect of Pre-existing Immunity to the Salmonella strain on Subsequent Oral
Immunization in BALB/c Mice 64
Experimental Design 64
Detection of Specific Anti-Salmonella Responses Following Oral
Immunization with Strain x4072/pYA292 at week 0 66
Detection of Specific Anti-HagB Serum and Mucosal Antibodies
After Oral Immunization with Recombinant S. typhimurium
x4072/pDMDl at Week 7 or Week 14 in Groups With or
Without Pre-existing Immunity to the Salmonella Carrier Strain 67
Assessment of the Colonization of Peyers Patches Following Oral
Immunization With the Recombinant S. typhimurium
x4072/pDMDl in Mice With or Without Pre-existing Immunity
to the Salmonella Carrier Strain 67
Studies Investigating Long Term Memory and Potential for
Recall Response Following Boosting at Week 52 (One Year) 72
Experimental Design 72
Determination of Specific Anti-HagB Serum Responses Following
Boosting at Week 52 in Three Separate Groups of BALB/c Mice 75
Determination of Specific Anti-HagB Mucosal Responses Following
Boosting at Week 52 78
ELISPOT Analysis of Specific Antibody-Secreting Cells from Spleen,
Salivary Glands, and Lamina Propria of BALB/c Mice Following
Oral Immunization or Boosting 78
Experimental Design 78
Enumeration of Specific Spot Forming Cells (SFC) 81
4 CONCLUSIONS AND DISCUSSION
Reconstruction and Characterization of the Recombinant S. typhimurium
Strain x4072/pDMDl Expressing the hagB Gene of P. gingivalis 84
Oral Immunization Study in BALB/c Mice with Boosting at Week 14 86
v


Effect of Boosting at Three Different Time Points with Recombinant S.
typhimurium y4072/pDMDl in B ALB/c Mice 89
Effect of Pre-existing Immunity to the Salmonella Strain on Subsequent
Oral Immunization in BALB/c Mice .91
Long Term Memory and Potential for Recall Response Following Boosting after
One Year 92
ELISPOT Analysis of Specific Antibody-Secreting Cells from Spleen,
Salivary Glands, and Lamina Propria of BALB/c Mice Following
Oral Immunization or Boosting 94
LIST OF REFERENCES 96
BIOGRAPHICAL SKETCH 114
vi


LIST OF FIGURES
Figure Page.
1. Plasmid Map of pDMDl 15
2. Restriction digests of plasmid DNA run on agarose gel 31
3. Coomassie blue staining of cell lysates 33
4. Western blot analysis for HagB expression 34
5. LPS analysis by Tricine SDS-PAGE and silver staining 35
6. In vitro growth curve for recombinant S. typhimurium %4072/pDMDl 37
7. Recovery of viable S. typhimurium y4072/pYA292 or x4072/pDMDl cells on
LB vs. MacConkey agars 39
8. Colonization of Peyers patches at 5 days after single oral immunization
with S. typhimurium strains x4072/pYA292 or x4072/pDMDl 41
9. Colony immunoblot of colonies recovered from Peyers patches 5 days
after a single immunization in BALB/c mice 42
10. Immunization & sampling schedule involving oral immunzation and boosting
of BALB/c mice with S. typhimurium y4072/pDMDl 44
11. Serum IgG anti-HagB levels following immunization at week 0 and boosting
at week 14 (arrow) with S. typhimurium x4072/pDMDl 46
12. Serum IgA anti-HagB levels following immunization at week 0 and boosting
at week 14 (arrow) with S. typhimurium x4072/pDMDl 47
13. Salivary IgA anti-HagB levels following immunization at week 0 and boosting
at week 14 (arrow) with S. typhimurium x4072/pDMDl 48
14. Vaginal IgA anti-HagB levels following immunization at week 0 and boosting
at week 14 (arrow) with S. typhimurium y4072/pDMDl 49
15. Fecal IgA anti-HagB levels following immunization at week 0 and boosting
at week 14 (arrow) with S. typhimurium %4072/pDMDl 51


16. Serum IgG anti-Salmonella levels following immunization at week 0 and
boosting at week 14 (arrow) with S. typhimurium x4072/pDMDl 52
17. Fecal IgA anti-Salmonella levels following immunization at week 0 and
boosting at week 14 (arrow) with S. typhimurium %4072/pDMDl 53
18. Serum IgG anti-HagB subclass levels 54
19. Serum IgG anti-5, typhimurium x4072 subclass levels 56
20. Immunization & sampling schedule involving oral immunization and
boosting at three different times with 5. typhimurium
x4072/pDMDl 57
21. Serum IgG anti-HagB levels following immunization at week 0 and
boosting at week 7 (group I), 14 (group II), or 21 (group III) with
5. typhimurium x4072/pDMDl 59
22. Serum IgA anti-HagB levels following immunization at week 0 and
boosting at week 7 (group I), 14 (group II), or 21 (group III) with
5. typhimurium x4072/pDMDl 60
23. Salivary IgA anti-HagB levels following immunization at week 0 and
boosting at week 7 (group I), 14 (group II), or 21 (group III) with
5. typhimurium x4072/pDMDl 62
24. Vaginal IgA anti-HagB levels following immunization at week 0 and
boosting at week 7 (group 1), 14 (group II), or 21 (group III) with
5. typhimurium x4072/pDMDl 63
25. Immunization & sampling schedule investigating effect of pre-existing
immunity to Salmonella carrier strain 65
26. Serum IgG anti-HagB responses from groups of BALB/c mice to
determine effect of pre-existing immunity 68
27. Serum IgA anti-HagB responses from groups of BALB/c mice to
determine effect of pre-existing immunity 69
28. Salivary IgA anti-HagB responses from groups of BALB/c mice to
determine effect of pre-existing immunity 70
29. Vaginal IgA anti-HagB responses from groups of BALB/c mice to
determine effect of pre-existing immunity 71
30. Colonization of Peyers patches to determine the effect of pre-existing
immunity to the Salmonella carrier 73
31. Immunization and sampling schedule investigating long-term memory
and potential for recall responses following boosting at week 52 74
32. Serum IgG anti-HagB levels for three groups following various primary
immunizations and boosting at week 52 76


33. Serum IgA anti-HagB levels for three groups following various primary
immunizations and boosting at week 52 77
34. Salivary IgA anti-HagB levels for three groups following various primary
immunizations and boosting at week 52 79
35. Vaginal IgA anti-HagB levels for three groups following various primary
immunizations and boosting at week 52 80
IX


Abstract of Dissertation Presented to the Graduate School of the University of Florida in
Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
ORAL IMMUNIZATION WITH A RECOMBINANT SALMONELLA TYPHIMURIUM
VACCINE EXPRESSING A HEMAGGLUTININ OF PORPHYROMONAS
GINGIVA LIS
By
James John Kohler
August 1998
Chairman: Thomas A. Brown, Ph.D.
Major Department: Oral Biology
A recombinant, attenuated Salmonella vaccine is one approach which induces both
mucosal and systemic immunity. The object of these studies was to characterize the
immune responses following oral immunization with a recombinant S. typhimurium strain,
x4072/pDMDl, expressing the HagB hemagglutinin of Porphyromonas gingivalis and
investigate the potential effects of boosting. A balanced-lethal construct was employed
using an attenuated Salmonella strain x4072 {Aasd AcyaAcrp) with the hagB gene inserted
into a complementary plasmid pYA292 (encoding the asd gene).
Induced immune responses following oral immunization with the recombinant 5.
typhimurium strain were determined through a set of studies in BALB/c mice. By ELISA,
the kinetics of the primary responses to the Salmonella carrier and HagB were shown to be
parallel. The serum IgG subclass anti-Salmonella and anti-HagB distributions both
x


represented a Thl-type response, suggesting an additional potential induction of cell-
mediated immunity.
The potential for recall responses was also investigated. While a strong recall
response followed boosting at week 14, boosting at an earlier time when the primary
responses were still strong yielded a reduced response. The inhibition of Salmonella
colonization of the Peyers patches due to pre-existing immunity in the gut does not appear
to be a possible explanation. Furthermore, pre-existing immunity to the Salmonella carrier
on subsequent boosting at week 7 or week 14 yielded mixed results. Some reduction in
specific anti-HagB responses was shown in serum IgG at week 14 and salivary IgA at
week 7; the serum IgA and vaginal IgA response levels were not affected. However,
increased responses were shown for all response parameters following boosting at a later
timepoint (week 52) which suggests that any inhibitory effect from pre-existing immunity
to the carrier is transient.
Other studies investigated long-term responses with boosting at one year.
Consistently, strong recall responses, particularly in IgA levels, were shown. These
studies demonstrated not only immunological responsiveness in mice at ~57 weeks of age,
but also support the notion of long-term mucosal memory.
Specific antibody-secreting cells homing to the systemic and mucosal effector sites
were assessed by ELISPOT assay. Specific anti-Salmonella spot-forming cells (SFCs)
were predominantly IgA. In addition, a significant increase in SFCs was shown following
boosting.
xi


CHAPTER 1
INTRODUCTION AND BACKGROUND
The Pathogen Porphxromonas gingivalis
Porphyromonas gingivalis (formerly Bacteroides gingivalis) is characterized as a
gram-negative, non-spore-forming, black-pigmented, anaerobic, rod-shaped bacterium.
More notably, P. gingivalis has been implicated as an etiologic agent in adult periodontitis
(198). Periodontal diseases result in a loss of attachment of the periodontal ligament to the
tooth surface and result in bone loss. Periodontal diseases are bacterial infections with
recent evidence implicating specific organisms such as P. gingivalis, Prevotella intermedia,
Fusobacterium nucleatum, Campylobacter rectus (formerly Wolinella recta), Eikenella
corrodens, and possibly spirochetes (74).
Some recent reports suggest periodontal diseases may present an added clinically
significant risk factor for cardiovascular disease (7, 194). In addition, a case-control study
of 124 pregnant mothers indicated that periodontal diseases may represent a significant risk
factor for preterm low birth weight (160). Together these reports suggest a considerable
impact that P. gingivalis may have on not only oral but overall health, warranting further
investigation of this pathogen.
P. gingivalis possesses numerous adhesin molecules in addition to polysaccharide
capsules, cytotoxins, and extracellular hydrolytic enzymes and proteases which aid in its
colonization, destruction of the periodontal tissues, and evasion of host immune
mechanisms (116, 131). Like other microorganisms that colonize the oral cavity, its
adhesins allow for an initial attachment of the organism to specific receptors in an
environment continuously bathed by saliva that otherwise would be efficiently removed
from the oral cavity. Defined by in vitro assays, P. gingivalis has been shown to adhere to
1


2
saliva-coated and serum-coated hydroxyapatite (196), bind to the protein fibrinogen (117)
and epithelial cells (197), coaggregate with several oral gram-positive and gram-negative
bacteria (196), and agglutinate erythrocytes from a variety of animals (14, 164, 206).
Hemagglutination, demonstrated in vitro as the agglutination of erythrocytes, has been
implicated as a potentially important virulence associated factor in other pathogenic bacteria.
Several studies have reported the characterization of hemagglutinin proteins isolated from
P. gingivalis (53, 119, 157, 165, 174) while others have applied molecular genetic studies
(118, 172, 173). To date, at least five hemagglutinins (hagA, hagB, hagC, hagD, hagE)
have been cloned and characterized (118, 119, 174). One or more of these hemagglutinins
may mediate attachment to host cells.
Protective Immunity to P. gingivalis
Many vaccines have been or are being developed against pathogenic organisms
known to cause disease. The goal of these various vaccines is to reduce or eliminate the
colonization of the known pathogen, interfere with its pathogenic mechanisms and
ultimately prevent disease. Likewise, many investigators believe a vaccine targeted against
one or more virulence factors of P. gingivalis, an implicated pathogenic organism, could
reduce or eliminate colonization of deep gingival tissues and prevent further development of
periodontal disease. In fact, the critical importance of both innate immunity and T-cell-
dependent humoral responses to the prevention of subsequent P. gingivalis infection was
verified in a recent study utilizing various mouse strains with known alterations in immune
system components; normal mice demonstrated a delayed onset and decrease in lesion size
of 15-30% compared to the primary infection, while immunodeficient strains appeared
unable to develop immune protection to a secondary challenge (102).
At the current level of vaccine development, immunization studies with P.
gingivalis cannot be performed in humans. However, several useful animal models do
exist. In the gnotobiotic rat model, for example, the occurrence of periodontal disease and


3
recovery of P. gingivalis have been reported (62, 89, 106). Other models include the
squirrel monkey (Saimir sciureus) (133) and the cynomolgus monkey (Macaca
fascicularis) (169), the mouse subcutaneous abscess model (11), and the mouse
subcutaneous chamber model (73). Various antigen preparations from P. gingivalis have
been used in immunization studies including whole bacterial cells (27, 28, 108, 133),
partially purified fimbriae and purified 43 kD fimbrillin (61, 63, 161), a purified outer
membrane preparation (11), and a hemagglutinin (51, 163).
Continuing development of a periodontitis vaccine has focused on a response that
would prevent colonization and thus target the infection at an early stage (107). While
passive immunization by an oral rinse could effectively prevent exogenous P. gingivalis
from establishing a niche in the dental plaque, it would not significantly interfere with
proliferation of subgingival P. gingivalis. A more effective approach would stimulate
active synthesis of antibodies in serum and saliva and thus protect the mucosal surfaces and
subgingival regions of the oral cavity. Furthermore, a vaccine against P. gingivalis may
need to confer protection against several of its virulence factors and therefore warrant a
combination vaccine.
Mucosal Immunity
While most successful vaccines to date (i.e., rubella, measles, pertussis,
meningococcus, and Haemophilus influenza type b, among others) have been systemic
vaccines that induce serum IgG antibodies and are capable of neutralizing microorganisms
or toxins, the response is effective only after the specific organism has breached a mucosal
barrier and become systemically invasive (115). In truth, the mucosal tissues serve as
primary portals of entry for most infectious diseases, such as HIV-1 (136). Consequently,
the focus of current vaccine development is to stimulate long-lasting antibody responses at
mucosal surfaces and thereby provide protective immunity against microorganisms that
either infect or enter the host through mucosal membranes.


4
The mucosal tissues that line the gastrointestinal, urogenital, and respiratory tracts
constitute a surface area of -400 nr; an enormous surface area when compared to the total
surface area of skin which is only -2m2 (136). In addition, an estimated 1012 organisms
per gram of feces make up the normal flora of the intestine, exceeding the total number of
cells in the body (58). These bacteria not only help prevent colonization by external
pathogens but also prime the estimated 10' lymphocytes/ meter generating cross-reactive
antibodies to bacterial antigens (58). The major immune effector molecule present in
mucosal secretions is the secretory IgA (slgA) molecule. It has been estimated that a
normal individual makes >5g of IgA daily, representing 65 90% of all antibody isotypes
produced, thereby making it clearly the predominate Ig isotype in humans (136, 144). In
addition, immunohistochemical studies of mucosal tissues and glands have established that
the slgA is locally synthesized by plasma cells found in abundance in mucosal tissues and
secretory glands (143). In fact, it has been argued that the intestine is a major lymphoid
organ on the basis that 25% of its cellular make-up consists of lymphoid cells (58).
Many studies have focused on further defining the parameters and components of
the mucosal immune system (see review (58, 114, 137, 153)). Like the systemic immune
system, the mucosal immune system is complex and can be divided into two general sites:
the inductive and effector sites (105, 138). Specific inductive sites include areas where
antigens are first encountered, processed, and presented with subsequent triggering of
specific B and T-cells. Examples of these specialized inductive sites include the Peyers
patches of the gut-associated lymphoreticular tissues (GALT), the palatine and pharyngeal
tonsils of the nasal-associated lymphoreticular tissues (NALT), and large intestinal follicles
of the urogenital mucosal tissues. The inductive sites are equipped with specialized cells,
capable of antigen uptake, which are termed follicle-associated epithelium (FAE) or M cells
(12). As reviewed by McGhee and Kiyono (136), microorganisms or antigens are sampled
and passaged intact in endocytotic vesicles by these M cells and delivered to underlying
lymphoid cells for further antigen processing and presentation. One organism in particular,


5
Salmonella, exploits this feature and specifically uses these M cells as a portal of entry
(166).
Separate from the initial inductive sites, effector sites are areas of mucosal surfaces
and glands where these activated immune cells ultimately accumulate and function due to
specific interactions with receptor molecules on cells located at these sites (referred to as
homing) (58, 97). The effector sites include the lamina propria of the gastrointestinal
tract, the upper respiratory tract (URT) and the genitourinary tract, as well as the exocrine
glands. Unique to these sites is the selective active transport of polymeric IgA into external
secretions which has been extensively studied and reviewed (15, 16, 144, 156, 212).
Briefly, a specific receptor, pig, expressed on the surface of epithelial cells interacts
exclusively with polymeric, J-chain-containing IgA released from the sub-epithelial plasma
cells (16). This complex is then internalized by the epithelial cells, transported in a vesicle
to the apical surface of the epithelial cell, where it is then fused with the apical membrane
(114, 137). This active transport results in the release of a cleaved portion of the pig
receptor, termed the secretory component (SC), as a SC-IgA complex constituting a
secretory IgA (slgA) molecule in an external secretion (156).
It has also been demonstrated in several experimental models that antigenic
stimulation at a local mucosal surface or secretory gland resulted in the production of
specific antibodies, not only at this localized site of induction but also in the circulation and
in remote external secretions (16, 58, 114, 144, 170). This phenomenon has been referred
to as the common mucosal immune system (134). The ability to induce secretion of
specific IgA at multiple mucosal sites following immunization with an antigen at a single
mucosal inductive site has an incredible potential for global protection against a pathogenic
organism at the mucosal surfaces.
Secretory IgA has several functional advantages (19, 114, 177). First of all,
polymeric IgA has been shown to be more effective than monomeric IgA in neutralizing
viruses (209). In addition, slgA has an added resistance to proteolysis due to its


6
association with the SC (103). Inhibition of microbial adherence and prevention of
absorption of antigens from mucosal surfaces has also been associated with slgA (103).
Another functional feature associated with slgA is immune exclusion. Through this
mechanism, IgA limits further absorption of undigested antigenic material and the
potentially harmful formation of circulating immune complexes. These complexes have
been associated predominantly with IgG antibodies as seen in IgA-deficient individuals
(34). An important factor in the preservation of the barrier function of mucosal surfaces is
the inability of slgA to activate complement, either through the classical or alternative
pathways, thereby preventing the induction of local inflammation and enhanced
permeability of the mucosal membranes due to tissue damage.
Mucosal Vaccines
Several approaches have been taken toward the development of mucosal vaccines.
One approach extensively used involves the use of soluble and particulate antigens in
animal models. With the exception of cholera toxin (92), HA of influenza virus (8), and a
few others (145) the general observation has been that higher and repeated administrations
of oral doses are required for induction of immunity, unlike with systemic immunization.
It is believed that the response to oral delivery of antigens is low because most of them are
degraded by enzymes secreted in the gut or only partially absorbed due to their high
molecular weight. One additional measure that has been used to preserve the immunogenic
material from the acid pH of the intestinal environment and proteolysis has been to co
administer large amounts of sodium bicarbonate with the oral antigen.
As mentioned, cholera toxin (CT) which is produced by Vibrio cholerae has proven
to be one exceptionally effective oral antigen (127). CT has a high affinity for binding to a
specific receptor, GM1 ganglioside, found on various nucleated cells including intestinal
epithelium. CT is composed of two subunits, A and B. CTB, composed of five identical
subunits, is directly involved in the binding of the toxin to cell surfaces, while CTA is the


7
enzymatic subunit involved in the toxigenic ADP-ribosylation of the adenylate cyclase
regulatory protein Gs (138). This results in increased cellular cAMP levels and the influx of
cellular secretions (138), yielding the characteristic diarrhea and fluid loss. The CTB
subunit has also been found to be a potent adjuvant for use with poor oral immunogens that
alone had resulted in oral tolerance (59). Several other investigators have confirmed the
adjuvant activity of CT with a variety of other antigens (42, 88, 112, 171, 178, 186). In
addition, a recent study determined CTB to directly induce an up-regulation of TGF-J11
activity and thereby promote the IgA isotype switching and IgA mucosal immunity (104).
Adding to the variety of experimental systems using liposomes to target drugs and
antigens by the systemic route, liposomes have recently been considered for oral delivery
of antigens (148). In early reports, these small, submicron liposomes containing purified
carbohydrate from Streptococcus mutans, for example, demonstrated a better induction of
an IgA response in orally immunized rats than the carbohydrate itself (147). Similarly,
various antigens have been incorporated into biodegradable microspheres (55, 57). These
microspheres containing antigen are composed of co-polymers of glycolic and lactic acids
which degrade into original catabolizable components by hydrolysis. Eldridge et al. have
demonstrated increased antibodies to a Staphylococcal enterotoxin following oral
immunization with the antigen within a microsphere compared with soluble antigen alone
(56). More recently, two novel adjuvants, an oil-in-water microemulsion and a polymeric
microparticle, have been developed and reported (158).
A more effective induction of both local and generalized secretory and systemic
immune responses has been reported with the use of live attenuated viruses (183) and
bacteria expressing a variety of heterologous antigens (136, 137, 141-143). Currently
there are two live viral vaccine delivery systems approved for human use: adeno and
polioviruses. The trivalent oral (Sabin) poliovirus vaccine (OPV) together with the earlier
inactivated (Salk) polio vaccine (IPV) has resulted in the virtual elimination of
endogenously transmitted paralytic disease caused by wild poliovirus in the United States


8
and in many other parts of the world (143). Other attenuated viral vaccines being
investigated include respiratory syncytial virus (RSV) (29, 98), rotavirus (86, 159, 218),
adenovirus (168, 201, 217), rabies virus (185), human immunodeficiency virus (HIV)
(20, 123), influenza virus (78, 207), and vaccinia virus (17, 47, 72, 96, 122, 139, 208,
223, 226). These attenuated vaccines are designed to either induce protective immunity to
the wild type strain or serve as recombinant carriers for expression of heterologous genes
from target pathogens. Aside from the progress of viral vaccine development, one
limitation of this approach is that the introduced recombinant virus may never be cleared
from the immunized host and could revert to a wild-type or a completely new strain.
A long-standing approach has been the use of bacteria and their products in mucosal
immunizations. In fact, Gay first reported on local immunization with bacteria in 1924
(71). Presently, there are over 17 important mucosal and systemic pathogens that have
been used for mucosal immunization (143). Salmonella species have been, by far, the
most studied recombinant vaccine for the heterologous expression of a variety of
pathogenic virulence factors (13, 33, 35, 40, 43, 51, 52, 64, 68, 79, 83, 84, 90, 93, 109,
121, 155, 175, 176, 204, 221, 224). Other important recombinant vaccine carriers include
Vibrio cholerae (92, 110, 181, 205), Shigella species (5, 30, 85, 111, 121), and
Streptococcus species (46, 140). In all these cases, the live attenuated bacterial strain
offers a safer approach than an attenuated virus as the bacterium do not persist, but rather
are cleared from the host shortly after immunization.
Recombinant Salmonella typhimurium Vaccines
As mentioned, Salmonella typhimurium has received much attention and
investigation as a recombinant vaccine carrier. Several characteristics of live attenuated
Salmonella constitutes advantages for its use as a mucosal vaccine (22, 94, 141). First,
Salmonella naturally targets the specialized M cells of the Peyers patches in the gut, a


9
major inductive site of the GALT. In fact, Salmonella has even been reported to induce M-
cell formation in germ-free mice (189). Likewise, Salmonella has been reported to interact
with eucaryotic membranes to trigger internalization into non-phagocytic cells through
induction of selective aggregation and internalization of host cell surface proteins (70). The
multiplication of the live Salmonella leads to a strong humoral immune response.
Salmonella are also facultative intracellular parasites, capable of survival in macrophages,
and thereby can also induce cell-mediated immune responses (49). In addition, live
Salmonella expressing a heterologous gene product provides an economical, stable in situ
synthesis of an antigen. Thus, a recombinant Salmonella overcomes two obstacles
associated with preparation and oral delivery of a purified antigen: excessive time and cost
to purify the antigen, and enzymatic degradation of the antigen while passing through the
harsh gastric environment. Furthermore, a live Salmonella strain could potentially express
multiple gene products simultaneously in one vaccine. In some cases, expression of
multiple gene products and subsequent induction of immunity to several putative virulence
factors may be necessary to result in effective protection against a pathogenic organism.
Many pathogens have been shown to possess multiple virulence factors and in some cases
duplication of functions such as adhesins, for example, through several related genes. S.
typhimurium, for example, has been shown to express multiple fimbrial adhesins.
Elimination of one fimbrial adhesin in a mutant strain has been shown to result in no
significant reduction in virulence (214). Likewise, P. gingivalis has been shown to
express multiple hemagglutinins which may function collectively for adherance and may
warrant a vaccine approach with expression of multiple gene products.
To optimize the efficacy of a live recombinant vaccine, a variety of mutations have
been developed to attenuate the Salmonella species. Initially random undefined mutations
were employed. However, for development as a human vaccine, a defined set of mutations
was desired. The use of temperature sensitive (TS) mutants was attempted.
Unfortunately, it was found that TS mutants could grow in mice in areas of lower body


10
temperature and cause subcutaneous lesions (95). Several other mutations have been
developed for attenuation (38, 141, 191). For example, a strAd mutation results in a
dependency on streptomycin. On the other hand, a galE mutant which specifically affects
the UDP4-galactose epimerase results in a sensitivity to galactose in vivo (77, 94).
Likewise, pur mutations (i.z..purA and purE) have been shown to render the strain
attenuated through deficiencies in purine metabolism enzymes (135). Other Salmonella
strains which have been attenuated through mutations in the prechorismate pathway (25,
91) are referred to as aro mutants, and were first described from studies in S. typhi (3).
These mutant strains do not produce chorismate, an essential intermediate in de novo
synthesis of aromatic compounds including aromatic amino acids, and are therefore
rendered auxotrophic for compounds not available in animal tissues. Other means of
attenuation include deletions in phoP and phoF which have a global effect on the
expression of virulence genes (149, 150). Similarly, a global effect of attenuation has been
reported with deletions in the regulator genes crp and cya, which have global effects on
cyclic AMP levels and subsequent inhibition on expression of genes involved with
transport and breakdown of catabolites (36, 37). In addition, deletional or insertional
mutations have been engineered in the genes pagC (involved in capacity to survive in
macrophage)(151), htrA (encoding a heat shock protease)(26), and cdt (associated with
colonization of deep tissues)(225). Finally, combinations of these mutations have been
engineered to insure stable attenuation with a low probability of a revertant wild-type
Salmonella in the host. One comparative study of several of these mutant Salmonella
strains in mice suggested that the level of total Ig specific for the carried antigen is
determined by the carrier mutation and the background of the S. typhimurium strain (50).
More recently, it wras reported that the level of protective immunity in mice was dependent
on the degree of colonization by the immunizing strain when several combination-mutation
Salmonella vaccine strains were compared (225). Another approach for attenuation
includes curing of the identified virulence plasmid on the basis that genetic analysis of


11
virulence genes encoded on the virulence plasmids of S. typhimurium have a direct effect
on increasing its growth rate (80, 81).
With regard to developing the actual expression of heterologous genes by these
attenuated live Salmonella, several important aspects of molecular biology have been
considered and investigated. A direct approach for introduction of heterologous gene(s)
into Salmonella has been the use of a genetically engineered plasmid encoding the gene(s)
of interest into the attenuated strain. In so doing, considerations have been made including
the stability of the expression system (namely the plasmid), the choice of promoters
together with the gene copy number which will determine the level of expression, the
toxicity of the gene product to Salmonella, and finally the folding and native
immunogenicity of the gene product (39, 192). Antibiotic resistance markers on
expression plasmids, a common approach for in vitro studies in bacterial strains, combined
with the use of the equivalent antibiotic in the media provides a selective pressure to insure
stable maintenance of the plasmid within the recombinant bacterium. However, for vaccine
strains, continued use of antibiotic pressure during immunization is not a favorable
approach. Consequently, Curtiss et al. designed a balanced-lethal construct deletional
mutation in the asd gene of aSalmonella strain (154). The Aasd mutation is lethal to the
gram-negative bacterium in the absence of media containing diaminopimelic acid (DAP) as
it is rendered unable to synthesize {3-aspartate semi-aldehyde dehydrogenase, a critical step
in cell wall synthesis. Without DAP, the cell is unable to create the crosslink bridges of the
cell wall structure, resulting in a leaky cell state. Moreover, the mutant is unable to survive
in vivo. Furthermore, a plasmid was engineered encoding the asd gene of Salmonella
which, when introduced into the Aasd mutant Salmonella strain, provides survival of the
strain in vivo (67). Another approach to overcome the problem of plasmid instability has
been reported by Cardnas in which specialized vectors direct the integration of the
encoded gene into the chromosome of the mutant Salmonella strain (23).


12
Most promoters used in the recombinant Salmonella vaccine experiments, including
lac, tac, and trc, had been previously used for molecular cloning and expression studies in
E. coli. Unlike in E. coli, however, the repressors for these promoters are absent in
Salmonella and therefore are constitutively active (192). While it is speculated that maximal
expression of the carrier gene is desirable for optimal immunogenicity, there is also concern
that over-expression could be detrimental to the Salmonella carrier. A desirable aim,
therefore, would be to identify a promoter which is tightly repressed under simple
cultivation conditions but would be preferentially turned on in a specific environment
within the host. One set of investigators reported the use of an in vivo regulated promoter,
nirB, which is maximally active under anaerobic conditions (24). Subsequently, a different
group suggested the superiority of the htrA promoter over the nirB promoter in an oral
vaccination approach (182). Similarly, another group reported a novel approach which
would control expression of an antigen whereby the antigen was not expressed in vitro but
was expressed in vivo (60). Briefly, this system utilized two plasmids: one plasmid
encoding the trc promoter and the heterologous gene, the other a repressor plasmid
expressing the LacI in trans which would eventually be lost by segregation due to
incompatibility with the first plasmid. Yet another approach reported the use of a strong
promoter located on a DNA fragment which would invert at random. As a result, the
antigen would only be expressed in one particular orientation of the promoter, and,
consequently, a bacterial population harboring the plasmid would consist of a sub
population not producing the antigen and therefore would not be affected in growth or
persistence in the host (211). The mixed population approach with sub-populations of live
recombinant Salmonella strains has also been reported recently by Yan and Meyer (222).
Several investigations have focused on the expression of heterologous proteins on
the surface of recombinant Salmonella cells for optimal immunogenicity. Particularly for
induction of immunity to viral or intracellular pathogenic virulence factors, surface
expression of the heterologous gene is thought to promote appropriate host responses.


13
Using polymerase chain reaction (PCR), one group constructed a single-copy expression
cassette encoding a chimeric outer membrane protein derived from OmpA fused to a
truncated derivative of an HIV gpl20 antigen (220). They reported surface expression of
the OmpA/gpl20 complex in the recombinant Salmonella strain. Similarly, a recent study
reported a ten-fold- higher antibody response to a p67 sporozoite antigen (fused to the C-
terminal secretion signal of Escherichia coli hemolysin) expressed and secreted by S.
dublin than to a non secreted p67 expressed by the same Salmonella strain (75). On the
other hand, other investigators have reported earlier that expression of foreign antigen on
the bacterial surface is not necessarily essential for induction of appropriate immunity due
to the intracellular nature of the Salmonella strain (18). It has been suggested that the most
important aspect of expression of a foreign antigen for development of an immune response
may be the initial amount of antigen that primes the GALT and not persistence of the vector
in tissues (22, 33). In other words, very high synthesis of the antigen during the initial
phase of invasion could be the most critical element.
In summary, many studies to date have led to further development of live
recombinant Salmonella as a candidate vaccine vector. In addition, many critical virulence
factors required for invasion and colonization of the Salmonella in host tissues have been
identified. At the same time, other investigators have been developing various recombinant
Salmonella strains expressing a variety of antigens. Together, these studies have led to
further interest and development of live Salmonella as a vaccine carrier. Nevertheless,
many aspects of Salmonella colonization, expression of heterologous genes, and resultant
immunogenicity remain a mystery awaiting continued investigations in the future. The
focus of these studies was to further investigate the parameters of the host immune
response in BALB/c mice resulting from oral immunization with a recombinant S.
typhimurium expressing a cloned hemagglutinin gene, hagB, from P. gingivalis.


CHAPTER 2
MATERIALS AND METHODS
Bacterial Strains. Plasmids. Media, and Culture Conditions
E. coli x6097 (F'A[lac-pro\ rpsL AasdA4 A[zhf-2::TnlO]thi (J)80 dlacZ AM15), a K-12
derivative, S. typhimurium x4072 (pStSRIOO" gyrA 1816 Acya-1 Acrp-1 AasdAl A[zhf-
4::Tn70]), an SR-11 derivative, and plasmid pYA292 were provided by Roy Curtiss III
(Washington University St. Louis, Mo.)(67). Plasmid pYA292 contains the asd gene from
S. typhimurium, which complements the deletion in x6097 and x4072, influencing the
stable maintenance of the plasmid in the absence of diaminopimelic acid. Plasmid pAX-
HagB containing the hagB gene of P. gingivalis was designed and provided by Ann
Progulske-Fox and Guylaine Lpine (119). Strains were routinely grown at 37C in Luria-
Bertani (LB), SOB-Mg (2% Bacto tryptone, 0.5% Bacto yeast extract, lOmM NaCl,
2.5mM KC1, lOmM MgCl2, lOmM MgS4) or SOC (SOB-Mg + 20mM Glucose) medium
as appropriate (188). Media were supplemented, as necessary, with DL-a,e-
diaminopimelic acid (DAP) (50 |ig/ml). Cultures were maintained at -80C as (20%)
glycerol stocks.
Construction of Plasmids
Plasmid pDMDl containing the hagB gene was constructed by inserting the 1.8 kb
hagB gene from pAX-HagB into pYA292 (Fig. 1) as previously described (52). The
orientation of hagB in pDMDl placed it under control of its own promoter.
14


15
Fig. 1: Plasmid Map of pDMDl.


16
The recombinant plasmid was transformed into competent cells of the E. coli strain
X097 by frozen storage buffer (FSB) -based chemical transformation (87). Positive
clones were characterized and stored as glycerol stocks as described previously (53).
DNA Characterization
DNA was isolated and characterized by standard techniques (188). From the HagB
positive transformant E. coli strains x6097/pDMDl, one clone was selected and grown on
LB medium to isolate plasmid DNA using Wizard Miniprep DNA purification system
(Promega, Madison, WI). To verify the presence of a correctly sized hagB insert, the
plasmid DNA was subjected to restriction endonuclease digestion with £coRI and Hindlll
in a buffered solution for 1.5 hours in a 37C waterbath. The digested DNA mixture was
then electrophoresed through a 1% Agarose gel (SeaKem GTG agarose, FMC
BioProducts, Rockland, ME). Plasmid pYA292 linearized with Smal digest, and DNA
insert pAX-HagB were added in adjacent wells as positive controls.
Electroporation into S. typhimurium.
S. typhimurium strains were transformed by electroporation as described
previously (10). Following plasmid pDMDl purification from the recombinant E. coli
strain y6097/pDMDl, the plasmid was electroporated into the attenuated S. typhimurium
x4072. Competent S. typhimurium cells were prepared and stored as a glycerol stocks for
electroporation (10). Electroporation was carried out with 2pl of DNA (lOOng) and 40|il
of competent cells in a 0.2 cm electrode gap Potter-type cuvette (Bio-Rad Corp.,
Richmond, Ca.) under the following conditions: 1.25 KV, 400 Q resistance, and 25 pf for
9.1 ms. One milliliter of SOC broth with DAP was added and the cells were incubated at
37C for 1 hour with moderate shaking. The resultant cells were plated with direct, 10 ',
10'2, and 103 concentrations on SOB-Mg plates with DAP (positive control for viable cells)
or without DAP. Positive transformants were identified as growth on selective media


17
without DAP. Five clones were selected and isolated on separate LB plates for preparation
of glycerol stocks and further characterization.
We determined that an extra phenol-chloroform extraction was needed in the Wizard
Miniprep plasmid purification from S. typhimurium in order to eliminate degradative
nucleases. Restriction enzyme analysis, once again was performed with a simultaneous
double digestion with EcoRl and Hindlll to verify properly sized insert and vector bands
on a 1% agarose gel. HagB expression was detected by SDS-PAGE and Western blot
analysis. Quantitation of HagB expression was determined using an enzyme-linked
immunosorbent assay (ELISA) as described below.
Lastly, positive clones were tested to check for smooth LPS phenotype using the
Tricine SDS-PAGE Silver Staining procedure (120).
Sodium-Dodecvl-Sulfate Polyacrylamide Gel Electrophoresis and Western Blotting
HagB expression of the constructs was determined by sodium-dodecyl-sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of cell lysates.
Cell lysates were prepared as previously described (52) and appropriate dilutions of
samples were loaded into two duplicate gels of a 4% stacking/10% running SDS
polyacrylamide gel in a discontinuous buffer system (113). The samples were
electrophoresed at 200V for -40 minutes (Bio-Rad mini PROTEAN II). One gel was
stained with Coomassie Brilliant Blue R250 (Bio-Rad) and later dried. The other SDS gel
was electrotransferred to nitrocellulose and analyzed for HagB protein using a rabbit IgG
anti-HagB absorbed with S. typhimurium y4072/pYA292, followed by goat anti-rabbit
immunoglobulin alkaline phosphatase conjugate (Tago, Burlingame, CA). A lysate of
pAX-HagB served as a positive control for each gel.


18
Colony Immunoblotting To Demonstrate Stable Expression of HagB
Colony immunoblots were performed with a modification of the procedure
described by Sambrook (188). Colonies were transferred to a nitrocellulose membrane and
exposed to chloroform fumes for 15-20 minutes to lyse the cells. The membrane was
washed briefly with NET Buffer, pH 7.4 (188), containing 50mM Tris, 150mM NaCl,
5mM EDTA, 0.25% gelatin and 0.05% Triton X-100 and then blocked for an hour with
NET Buffer containing 1% Bovine Serum Albumin (w/o Triton X-100). Colonies
expressing the hagB gene product were detected with the IgG fraction of rabbit anti-HagB
absorbed with S. typhimurium y4072 followed by alkaline phosphatase-labeled goat anti
rabbit immunoglobulin (Tago, Burlingame, CA). The plate was developed with substrate:
nitro blue tetrazolium (NBT, 5% in 70% dimethyl formamide (DMF); Sigma Chemical
Co.), 5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside (BCIP, 5% in 100% DMF;
FisherBiotech) and MgCl2 (5mM) in Tris-saline (0.1 M, pH 9.6).
Analysis of in vitro Growth Rate of Recombinant 5. typhimurium
The in vitro growth rate of the recombinant S. typhimurium y4072/pDMDl was
studied over a 14 hour incubation under controlled conditions. An overnight static culture
of the recombinant strain was prepared from a glycerol stock by inoculation into sterile LB
broth and incubation at 37C. The culture was diluted 1:50 in fresh LB broth and incubated
at 37C in a temperature controlled shaker at 2200 rpm. Aliquot samples were taken from
the growing culture at time 0 and subsequently at 30 minute intervals up to the 10th hour
and a final time at 14 hours. Optical density (OD) readings were taken for each aliquot with
a spectrophotometer of a wavelength of 600nm. Each sample was subsequently plated in
serial dilutions on LB plates for enumeration of viable cells for each timepoint. The growth
rate study was repeated threefold to verify reproducibility of the recoverable cells with
respective OD^ readings and timepoints. From the set of data collected, a growth curve


19
was established with predictable concentrations of cells based on OD^ and approximate
incubation time.
Mouse Immunization and Sample Collection
Female BALB/c VAF/Plus mice, 6-8 weeks of age, (Charles River, Wilmington,
Mass.) were housed in the Infectious Disease Isolation Unit of the University of Florida
Animal Resource Center and given food and water ad libitum. Generally, groups
consisting of six mice were immunized with S. typhimurium strain y4072/pDMDl. The
strain was grown in LB broth overnight at 37C as a static culture, diluted 1/20 in fresh
LB broth, grown 4 hours at 37C to an optical density at 600nm of 0.8, after which the
culture was centrifuged, and resuspended in sterile NaHCC>3(0.1 M) to a density of 1010
cells per ml. The mice food supply was removed and the bedding changed 4 hours prior to
immunization. Mice were immunized by gastric intubation with 109 cells (0.1ml of 1010
cells/ml) with a sterile ball-end needle and Becton-Dickinson tuberculin syringe (Aldrich
Chemical Company, Inc., Milwaukee, Wis.) in three doses on days 1, 3, and 5 of week 0.
Boosting was carried out in the same three dose manner. In colonization studies, however,
mice were immunized with a single dose of the recombinant S. typhimurium followed by
recovery of the bacteria from mouse tissues 5 days later. Serum, saliva, feces, and vaginal
washes were among the samples collected for evaluation of specific antibody directed
against the hemagglutinin and the Salmonella carrier. Mice were anesthetized with sodium
pentobarbital (70mg/kg; Butler, Colombus, OH) intraperitoneally and bled from the retro-
orbital sinus, collecting 200|il with a heparinized capillary tube. Stimulated saliva was
collected using a pipettor after subcutaneous injection of 0.11 ml of sterile pilocarpine
nitrate (1 mg/ml; Sigma, St. Louis, MO). Both saliva and blood samples were centrifuged
at 12,000 x g for 15 minutes to remove particulate matter and to separate plasma from the
red blood cells, and samples were stored at -80C. Fecal extracts were processed by
suspending fecal pellets (10 volumes w/v) in phosphate buffered saline (PBS)(10mM


20
sodium phosphate, and 0.85% NaCl, pH 7.4) containing 0.01% sodium azide,
centrifuged at 12,000 x g for 15 minutes to remove the particulate matter and the samples
were stored at -80C (45). Vaginal washes were collected by flushing the vagina three
times with 80pl of PBS (210). An equal volume of PBS with DTT (0.01M ) was added to
the washes and incubated on ice for 20 minutes to reduce viscosity before centrifuging the
samples at 12,000 x g for 15 minutes. Again, samples were stored at -80C.
Colonization of Peyers Patches and Spleen Assay
For the purpose of investigating the colonization potential of the recombinant strain
a subset of the group of mice was orally immunized with a single dose of recombinant
y4072 containing one of several plasmids. Five days later, the subset of mice was
euthanized with sodium pentobarbital followed by cervical dislocation. The spleen and a
set of five Peyers patches were surgically removed under sterile conditions. Samples from
each mouse were processed separately. Spleen tissues were dispersed in PBS with sterile
glass tissue homogenizers and stored on ice. Peyers patches were dispersed in PBS with
a hand-held homogenizer and sterile disposable pestles and stored on ice. Dilutions of each
sample were plated on LB agar with nalidixic acid (20 pg/ml). Colony forming units
(CFUs) were enumerated and recorded for each sample.
Preparation of Formalin-Killed S. tvphimuriuni Cells (v4072)
Preparation of whole cell proteins for coating on ELISA plates to determine specific
antibody responses to the Salmonella carrier was carried out by the formalin-killed
procedure (48, 146). Briefly, the S. typhimurium strain y4072/pYA292 was grown
overnight at 37C as a static culture in SOB-Mg broth (pH 7.5). The culture was diluted
(1:50) in fresh media and incubated with shaking overnight at 37C. The resultant culture
was centrifuged at 5000 x g for 20 minutes. The cell pellet was washed (2X) in PBS
(0.01M sodium phosphate and 0.85% NaCl, pH 7.4). Then, the cells were resuspended


21
into 2 volumes of 0.05M sodium phosphate and 0.85% NaCl, pH 7.4, containing 0.056%
formalin (1.5 ml/L of 37% stock formaldehyde, Sigma Chemical Co.) and incubated at
room temperature with stirring for 90 minutes. After centrifuging the cells and washing
(2X) with PBS, the cells were resuspended into 1/2 the starting volume and the cell
concentration was determined by the optical density at 600nm. The cell suspension was
stored at 4C with sodium azide added (0.01%). For coating ELISA plates, an aliquot of
the cells was diluted in PBS to a concentration of 101 cells/ml, centrifuged, and
resuspended in the same volume of bicarbonate/carbonate buffer (0.2M, pH 9.6) as
described in Immunoassay methods (see below).
Preparation of Sonicated 5. tvphimurium y4072/pYA292 Cells
Soluble proteins from S. typhimurium yv4072/pYA292 cells were prepared through
a sonication procedure (31). From a glycerol stock (stored at -80C), an aliquot of LB
medium was inoculated and grown as a static culture overnight at 37C. Diluted in fresh
LB medium (1:50), the culture was grown with shaking overnight at 37C. The culture
was then centrifuged at 5000 rpm at 4C for 20 minutes, and the pellet was then washed
(3X) with 0.01M PBS. The washed pellet was resuspended in PBS, concentrated ten-fold
from the original overnight volume. The cell suspension was then sonicated on ice with
glass beads using a large sonicator (Sonic Dismembrator 300, ARTEK Systems Corp.,
Farmingdale, NY) at power setting 60 for 8 bursts at 15 second intervals with 5 minutes
cooling in between. The resultant sonicated cells were then centrifuged at 9000 rpm for 15
minutes at 4C. The supernatant was filtered through a low protein binding sterile acrodisc
filter (0.2 fim, Gelman Sciences, Ann Arbor, MI).
The concentration of the protein was determined with a BCA protein assay (Pierce,
Rockford, IL). Briefly, the standards and samples were diluted from 2000 pg/ml to 25
flg/ml and from direct to 1: 8192 by two fold serial dilutions, respectively. Samples were
then placed in designated wells of an ELISA plate in duplicate. Working reagent from the


22
kit was added to each well, mixed gently, and incubated at 37C for 30 minutes. The plate
was then cooled to room temperature and absorbance measured with MPM titertek (filter
with wavelength = 570 nm). MPM program estimated values, the curve fit of the standards
and the average value of the protein sample. The protein was further diluted to a
concentration of 1.06 mg/ml and aliquots stored at -80C.
Elispot Assay for Determination of Specific Antibody Secreting Cells
For enumeration of the specific antibody secreting cells from specific effector sites
in the mouse, three separate procedures were used for isolation of viable lymphocytes from
spleen, lamina propria, and salivary glands. To ensure viability of the cells during the
various tissue digestions and separation of cells, the cells were continuously supported in
incomplete RPMI 1640 medium (adjusted to pH 7.2 with Bicarbonate/ carbonate buffer and
ImM HEPES) or complete RPMI 1640 media with 10% fetal bovine serum (GIBCO BRL,
Grand Island, NY). Tissues were removed from four euthanized mice (as described
above) for each trial with sterile surgical technique. Pooled tissue types were processed
according to a modification of the following protocols (31,41, 44, 125) and a
communication with Dr. Michael Russell (University of Alabama, Birmingham).
Recovery of viable lymphocytes from the spleens (31) of immunized mice began
with suspension of the removed tissues in cold complete RPMI 1640 media in a sterile
polystyrene petri dish. Using a sterile mesh screen, the spleens were ground across the
screen surface until only a clear membrane remained. The released cells were evenly
dispersed and an equal volume of complete RPMI 1640 media was added. The complete
suspension was centrifuged for 10 minutes at 4 C at 200 x g. The supernatant was
aspirated, and the red pellet was resuspended in cold complete RPMI 1640 medium.
Centrifugation and aspiration of the supernatant was repeated. The red pellet was then
resuspended in ACK lysing buffer (0.15 M NH4C1, 0.1 mM Na,EDTA, and 1.0 mM
KHCO, in distilled water adjusted to pH 1.2-1 A and filter sterilized through 0.2 pm filter


23
and incubated five minutes at room temperature with occasional shaking to lyse the red
blood cells. Afterwards, an equal volume of complete RPMI 1640 medium was added to
block the reaction, and the cell suspension was centrifuged. The supernatant was
discarded, and the now white pellet was washed twice with complete RPMI 1640 medium
and subsequently centrifuged. The remaining viable cells were then resuspended in
incomplete media (pre-warmed to 37C) and purified through a Hystopaque gradient
(density 1.083, Sigma) by centrifugation for 15min at room temperature at 800 x g. The
resultant pellet was resuspended in complete RPMI 1640 media and stored on ice for
subsequent trypan blue staining and enumeration of viable cells.
Recovery of cells from salivary glands from immunized mice, using a modification
of a reported procedure (41) and a communication with Dr. Michael Russell (University of
Alabama, Birmingham), involved surgical removal of the sublingual and submandibular
gland pairs from each mouse. The pooled sets of glands from four mice were collectively
weighed and then minced with a sterile scalpel. The tissue fragments were transferred into
incomplete RPMI 1640 medium (pre-warmed to 37C) and a small magnetic stirbar was
added. The tissue was then digested with hyaluronidase (14 U/mg, Sigma Chemical Co.
St. Louis, Mo), collagenase type IV (2.5 U/mg, Sigma Chemical Co.), deoxyribonuclease
I (1016 U/ 4 sets of glands, Sigma Chemical Co.), and dispase (0.05 mg/ ml of medium,
GIBCO BRL, Grand Island, NY) in a 37C waterbath with stirring for 15 minutes. Then
the supernatant was aspirated after allowing tissue to settle for 5 minutes without stirring.
The digests were repeated an additional two times with fresh enzymes and pre-warmed
incomplete RPMI 1640 media for 30 minute incubations. After these last two digests the
supernatant was collected, blocked with two volumes of complete RPMI 1640 media and
stored on ice. The pooled supernatants were then centrifuged for 5 minutes at 4C at 300 x
g. The supernatant was aspirated, and the pellet was resuspended into incomplete RPMI
1640 media (pre-warmed to 37C) and purified through a 40% Percoll gradient [ 4 parts of
100% stock (9 parts Percoll (Sigma Chemical Co.) with 1 part 10X Dulbeccos solution


24
(Sigma Chemical Co.) in 6 parts incomplete RPMI 1640 with heparin (6000 U/ml)] by
centrifugation (2X) at room temperature for 15 minutes at 2300 x g. The purified pellet
was resuspended to a final concentrated volume in complete RPMI 1640 media and stored
on ice for further determination of viable cell concentration with trypan blue staining.
Recovery of lymphocytes from lamina propria from immunized mice required a
modification of another procedure (44, 125). The small intestines from four mice were
surgically removed. The intestines were soaked in incomplete RPMI 1640 media. Using a
syringe with an intubation needle attached, the intestines were each washed out with
incomplete RPMI 1640 medium, removing the fecal contents. On a surgical board, the
connective tissues and all visible Peyers patches were removed from the intestinal
surfaces. The cleaned intestines were then cut open longitudinally with a sterile disposable
scalpel and then chopped into small 2mm pieces. Collectively the pieces were washed in
incomplete RPMI 1640 medium and drained off (6 times). The intestinal pieces were
incubated with incomplete RPMI 1640 (pre-warmed to 37C and containing 5mM EDTA)
in a 37C waterbath with stirring for 15 minutes to release the epithelial cells. After
allowing the tissue to settle, the spent media was aspirated off and the incubation was
repeated three times with fresh pre-warmed media. The remaining tissue was then blocked
with cold complete RPMI 1640 medium for 15 minutes in a 37C waterbath with stirring.
After aspirating off the spent blocking medium, pre-warmed complete RPMI 1640 and
collagenase VIII (40 U/ml, Sigma Chemical Co.) was added for stationary digestion for 30
minutes in a 37C waterbath. The spent medium was collected and stored on ice. Three
additional digestions in complete RPMI 1640 and collagenase VIII were carried out, but
with stirring. The pooled supernatants were then filtered through glass wool in a glass
Pasteur pipette after priming with complete RPMI 1640 medium to prevent attachment of
the lymphocytes to the glass surface. The filtered supernatants were centrifuged for 10
minutes at 200 x g, and the pellets were resuspended into one volume of incomplete RPMI
1640. The cells were purified again through a 40% Percoll gradient. The final pellet was


25
resuspended into complete RPMI 1640 medium and stored on ice for determination of cell
concentration following trypan blue staining.
Verification and Enumeration of Viable. Isolated Lymphocytes
Putative isolated lymphocytes were first verified by cyto-centrifugation of an aliquot
of the final suspension of cells followed by Wright staining and analysis under a high
powered microscope. Briefly, each aliquot (-20-100 pi) was placed by micropipette in one
of 10 cells of a cyto-centrifuge (Shandon Elliott, Co.) consisting of a microscope slide
(pre-marked with a wax pencil for identification), a poly-resin re-usable cell with a sample
chamber, and a blotter paper between the cell and the microscope slide. Once the samples
were loaded and locked into place they were centrifuged for 5 minutes at setting 10 (-600
RPM). Afterwards, the microscope slides were recovered and the concentrated cell spot
was stained with Wright stain directly for 1 minute, followed by dilution with an equal
volume of dH20 to the point of creating a silver metallic film and incubated an additional 3
minutes. Then the slides were carefully rinsed with dH20 until a reddish-pink film on the
thinner portion of the cellular mass developed. The cells on the slides were then readily
identifiable under a microscope.
Using a hemacytometer, the viable cell concentration (# cells/ ml) from each of the
three tissues were determined from aliquot samples combined with equal volume of trypan
blue stain (0.4%, Gibco BRL) and viewing under a microscope. Cells were then delivered
to ELISPOT wells in direct and 10'1 dilution.
Immunoassay Methods
ELISA Assays
Samples were assayed for antibody to HagB using an enzyme-linked
immunosorbent assay (ELISA). Each sample well was coated with 0.1 ml of PBS


26
containing purified HagB protein (1.25-p.g/ml) isolated by nickel affinity chromatography
of the 6x-histadine tagged protein using the QIAexpress system (Qiagen Inc., Chatsworth,
CA). The plates were blocked with PBS containing bovine serum albumin (BSA, 0.25%,
Gibco BRL). Samples were diluted in the blocking solution and incubated overnight at
4C. After washing, a horseradish peroxidase-conjugated goat anti-mouse anti-isotype
antibody (Fisher Biotech, Pittsburgh, PA.) was diluted in PBS (containing: 0.5% gelatin,
0.05% Tween 20, and 1% Bovine Serum Albumin), added to the wells, and incubated for
3 hours at 4C. Following washing, 0.1 M phosphate buffer, pH 5.0, containing 0.012%
H2O2 and 0.04% o-phenylenediamine dihydrochloride was added and the absorbance at
490nm was recorded on one of two microplate readers: Titertek Multiscan MC model 340
(Flow Laboratories, Helsinki, Finland), or MPM Titertek model 550 (BioRad, Hercules,
CA). Antibody levels were quantitated by comparison with standard amounts of MOPC
315 IgA, or polyclonal mouse IgG as previously described (52). Briefly, standard wells
were coated with the appropriate anti-light chain antisera in order to bind dilutions of the
immunoglobulin standards. Standard and sample wells were developed with the same anti
isotype conjugate. Data were analyzed by comparison with log-logit transformed standard
curves using Microplate manager III (BioRad, Hercules, CA), and results were expressed
as nanograms of immunoglobulin per milliliter of sample.
Specific purified goat anti- mouse IgG subclasses IgGl, IgG2a, IgG2b, and IgG3
and the corresponding mouse IgG subclass standards (Southern Biotechnology Association
Inc., Birmingham, AL) were used to assess specific mouse IgG subclass response to
HagB and 5. typhimurium %4072.
Specific anti-S. typhimurium y4072 IgG subclass activity was measured against a
formalin killed preparation of the whole cells (48, 146). The cells were resuspended to
ODoo of 0.8 (yielding IX109 cells/ml in 0.2M bicarbonate/ carbonate buffer, pH 9.6).


27
Cells were prepared as described previously and lOOpl/well was used for coating ELISA
plates.
ELISPOT Assays
Select wells of a 96-well ELISPOT plate (nitrocellulose bottom, Millipore
Corporation, Bedford, MA) were coated with either purified HagB protein (1.25 pg/ ml),
sonicated S. typhimurium extract (1.25 pg/ml), or unlabeled goat anti-mouse IgA
antibodies (2.5 pg/ml, Southern Biotechnology Associates, Inc.) in PBS and incubated
overnight. Wells were blocked with complete RPMI 1640 for at least 1 hour prior to
adding the purified lymphocytes from the three tissues. Each sample set of cells was
placed in duplicate in wells coated with HagB, S. typhimurium, and goat anti-mouse IgA
antibodies (positive control) for enumeration of specific antibody secreting cells to the
cloned gene and the Salmonella vector. The plate was then incubated at 37C humidified
with CO, (5%) for three hours. After washing the plate three times with PBS containing
Tween 20 (0.05%, FisherBiotech), biotin-labeled goat anti-mouse IgG, or IgA antibodies
(1: 500) in PBS containing BSA (0.25%) were added to designated wells and incubated
overnight at 4C. After washing again three times with PBS/ Tween 20, ExtrAvidin
alkaline phosphatase (1:2000, Sigma Chemical Co.) in AP buffer was added to all wells
and incubated at room temperature for 30 minutes. After one washing with PBS/ Tween
20 and two washings with Tris-saline buffer (0.1 M, pH 9.6), the plate was developed
with substrate: nitro blue tetrazolium (NBT, 5% in 70% dimethyl formamide (DMF);
Sigma Chemical Co.), 5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside (BCIP, 5% in
100% DMF; FisherBiotech) and MgCl, (5mM) in Tris-saline (0.1 M, pH 9.6). The
reaction was stopped with PBS/ Tween 20 containing EDTA (20mM) after -30 minutes or
when violet spots were visible. The plate was then separated to allow for proper drying.
Spots were enumerated in each well under a stereoscopic microscope at 20X
magnification. Countable spots for each sample in duplicate were averaged and then


28
divided by the concentration of viable cells (previously determined by hemacytometer
counting) added to the well. Final concentrations were adjusted to be expressed as colony
forming units (CFU) /106 cells. Specific IgG and IgA anti- HagB and S. typhimurium
CFU were determined from the spleen. Specific IgA anti-HagB and S. typhimurium CFU
were determined from the lamina propria and salivary glands when viable cells were
recovered.
Amylase Assay
Antibody levels in salivary secretions, namely specific secretory IgA, were
standardized by amylase enzyme activity to account for variable dilutions. Briefly, the
enzyme activity for each salivary sample was determined by a modification of an amylase
assay reported by Bernfeld (9). Samples were diluted 1:10 and 1:20 in dH,0. A starch
solution [0.4g Potato starch (Sigma Chemical Co.), 30 ml of incubation medium (24.2 g
Tris, 160 ml of IN HC1, 29 g NaCl, 110 mg CaCl, in 1 liter volume) and 70 ml dH20]
was dissolved with heating in a microwave oven until clear (approximately 2X 1 minute
intervals). An iodine solution [6 ml of IN HC1, 3 ml of iodine stock reagent (3 g iodine in
1L of 3% potassium-iodide) and 191 ml of dH20] was also prepared. The solutions were
pre-warmed in a 37C waterbath for 15 minutes. Aliquots of the diluted samples (25 pi)
were individually added to small tubes containing 2.5 ml of the starch solution, vortexed,
and incubated for 5 minutes at room temperature. Then each incubation solution (100 pi)
was transferred to a large glass tube containing 5 ml of iodine solution and vortexed. The
color change was instantaneous and relatively stable. Each end product was then added to
separate ELISA wells in duplicate and optical density at 570 nm was taken with the MPM
Titertek reader. The individual color values were compared to the blank and standard
controls and the concentration determined by the following calculation:
(Wank sample) mg/ml x 450(constant) x dilution / (# of minutes (5) x 1000)= mg/ min/ ml


29
These values were then used to normalize the salivary specific slgA levels detected for each
sample.
Statistical Analysis
Immune response data from different groups of mice with variable times of boost
immunizations were compared with the Wilcoxon rank-sum test for levels of anti-
Salmonella responses. Colonization data was also compared with the Wilcoxon rank-sum
test. Logarithms of the anti-HagB antibody data were compared with repeated measures
analysis of variance (ANOVA) for each parameter. Multiple comparisons between means
was done with Fishers Least Significant Difference (LSD) method.


CHAPTER 3
DATA AND RESULTS
Reconstruction and Characterization of the Recombinant S. typhimurium strain
v4072/pDMDl Expressing the hagB Gene of P. gingivalis
Transformation into S. typhimurium:
The recombinant strain S. typhimurium x4072/pDMDl expressing the hagB gene
under its own native promoter was originally constructed by Dr. David Dusek in the
laboratory of Dr. Thomas A. Brown (53). Due to loss of immunogenicity, the recombinant
S. typhimurium strain was reconstructed at the beginning of these studies. The plasmid
pDMDl was then electroporated into a fresh batch of competent cells of S. typhimurium
attenuated strain *4072 as described previously (Chapter 2: Materials & Methods). Five
clones were isolated on separate LB plates for use in making glycerol stocks, minipreps for
plasmid verification, cell lysates for verification of HagB protein expression, and LPS
silver staining analysis for identification of rough or smooth recombinant Salmonella
strains.
Restriction Endonuclease Analysis of DNA Plasmid
Purification of the plasmid from the Salmonella strain with the use of the Wizard
miniprep proved to be more complicated than with the well-characterized E. coli strains.
After several trials, it was discovered that an additional, final phenol/ chloroform wash
was necessary in order to eliminate active endonucleases and recover stable, purified
plasmid from the Salmonella strain. A double endonuclease digestion of the plasmid
pDMDl miniprep confirmed 2 bands on a 1% agarose gel (Fig. 2, lane b). One band of
-3.4 kb aligned with the positive control marker of a linear pYA292 plasmid of -3.5 kb
30


31
abed
21.2 kb
5.1 kb^
4.3 kb
3.5 kb
2.0
cb-v.
1.9
cb
1.6
kb
1.41
kb
.9
kb
.8
kb'
.6
cb
3.5 kb
2.9 kb
1.8 kb
Fig. 2: Restriction digests of plasmid DNA run on
agarose gel. Included are the EcoRl Hindlll
digested X standard (lane a), the FcoRI- Hindlll
digested plasmid pDMDl (lane b), the Smal
digested plasmid pYA292 (lane c), and the Hindlll-
Xba 1 digested plasmid pAX-HagB (lane d).


32
(Fig. 2, lane c), and the hagB gene insert of ~ 1.8 kb aligned with the lower band of the
positive control plasmid digest of pAX-HagB (Fig. 2 lane d). The slight differences in the
bands from the positive controls are explained by the small plasmid fragments from the
multiple cloning region that were included in the hagB insert or cut from the plasmid band
due to the restriction endonucleases used (refer to Fig. 1 of Chapter 2, Materials &
Methods).
de novo HagB Protein Analysis
Proteins from cell lysates (prepared as described in Chapter 2: Materials and
Methods) were separated by electrophoresis on SDS gels and stained with Coomassie
Brilliant Blue. A prominent band was detected for all seven clones corresponding with the
positive control HagB protein at 49 kd (Fig. 3). In addition, the identity of the 49 kd band
was confirmed to be the HagB protein by Western Blot analysis (Fig. 4). The cell lysates
were also analyzed by ELISA to quantitate the HagB expression. The expression level of
HagB in the recombinant S. typhimurium %4072/pDMDl was 70 fig/ ml of cell culture
grown to an OD600 = 0.8.
LPS Silver Staining Analysis
LPS silver staining analysis was performed as described previously. All five
clones demonstrated multiple bands (Fig. 5) indicative of a smooth LPS on the cell surface
and a characterisitic smooth appearance on agar plates. Smooth vaccine strains have been
reported to be more immunogenic (100); it is postulated that smooth strains may survive
longer in the host due to a reduced ability of immune components (eg. complement) to
readily attach to their surfaces.


1 2 3 4 5 6 7 8
97.4 kd
66.2 kd^
I &
31 kd w
9
U)
U)
Fig. 3: Coomassie blue staining of cell lysates. Included are SDS-PAGE low
molecular weight standards (lane 1), 7 clones of the recombinant S.
typhimurium %4072/pDMDl (lanes 2-8), and a positive control, purified HagB
protein (lane 9).


1 4 5 6 7 10
97.4 kcL
66.2 kd
42.6 kd
31 kd
21.5 kd
14.4 kd
- 49kd
u>
4^
Fig. 4: Western blot analysis for HagB expression. Included are
biotinylated SDS-PAGE low molecular weight standards (lane 1),
4 recombinant clones of S. typhimurium %4072/pDMDl (lanes 4-7),
and positive control, purified HagB (lane 10). Biotinylated-rabbit
IgG anti-HagB absorbed with S. typhimurium %4072/pYA292 was
used for detection of HagB protein.


35
1 2 3 4 5 SR
Fig. 5: LPS analysis by Tricine SDS-PAGE and silver staining.
Included in the gel are five clones of the reconstructed S.
typhimurium %4072/pDMDl (lanes 1-5), a smooth, positive
control (S) S. typhimurium y4072, and a rough, negative control
(R) E. coli strain JM105.


36
In vitro Growth Rate
The in vitro growth rate of the recombinant S. typhimurium '/4072/pDMDl was
analyzed to establish a growth curve (Fig. 6) for the recombinant strain grown at set
conditions described earlier (Chapter 2: Materials & Methods). Repeated three times, the
growth rates were consistent with a doubling time of ~60 minutes. Earlier studies had
established that a dosage of 109 cells to be sufficient to induce an immune response in the
BALB/c mouse model. Therefore, from the growth curve established from these growth
rate studies, a target concentration of 108 cells/ ml could be consistently achieved for
vaccinations by following the growing cultures to an OD^ of ~0.8-0.85. After further
concentration of the culture 100:1, the net yield was 1010 cells/ ml of which 0.1 ml was
orally delivered, or 109 cells. The growth curve also revealed that cells used for the oral
vaccine were in log phase, which has been recommended for optimum immunogenicity
(personal communication with Dr. Roy Curtiss, III).
Colonization Studies of S. typhimurium Strains in the BALB/c Mouse Model Following A
Single Oral Immunization
In order to determine whether pre-existing intestinal immunity to Salmonella could
interfere with colonization of a recombinant Salmonella strain, several preliminary studies
were performed to examine the colonization potential of the recombinant strain of S.
typhimurium x4072/pDMDl compared to the strain %4072/pYA292. Mice were
immunized with a single oral dose of one of the two strains using the standard method of
preparation and delivery described earlier. The mice were then euthanized with sodium
pentobarbital and the spleens and Peyers patches (average of 5/ mouse) were removed
using sterile technique. Initially the sample supernatants were plated in dilutions on
MacConkey agar plates (containing bile salts) as the selective media to distinguish S.
typhimurium from enteric E. coli contaminants. From this standard procedure, we had
very little recovery of Salmonella (data not shown). We hypothesized that transformants


37
O 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 2.6
OD600
Fig. 6: In vitro growth curve for recombinant S. typhimurium
%4072/pDMDl. Representative graph is based on aliquot sampling
at time intervals during a growth rate study. Optical density (OD)
readings at 600 nm and dilution plating on LB agar plates yielding
colony-forming units (CFU) were determined for each aliquot.
Growth rate was consistent in three separate studies.


38
expressing HagB were more sensitive to the selective agents in MacConkey agar. We then
investigated the use of less selective media for growth of recoverable cells from the animal
tissues. We carried out a comparative study of plating recovered cells on MacConkey agar
versus LB agar with nalidixic acid (the y4072 S. typhimurium strains are resistant to
nalidixic acid).
In this study we measured colony-forming units of in vitro grown cultures for
comparison. Three comparisons were made: viability on LB vs. MacConkey agar, growth
of S. typhimurium strains x4072/pYA292 vs. x4072/pDMDl, and re-suspending the cells
in sodium bicarbonate vs. PBS (Fig 7). It was determined that MacConkey agar had a
limiting effect only on the growth of the recombinant x4072/pDMDl strain. Thus, the
overall state of the recombinant strain appears to be weakened by the expression of the
heterologous protein and growth on a selective medium such as MacConkey agar further
reduces the viability. Therefore, to increase the potential for detecting cells recovered in
colonization studies, we selected for S. typhimurium strains on LB agar plates containing
nalidixic acid. Finally, it was determined that re-suspension in sodium bicarbonate vs.
PBS had no significant effect on the growth of either strain. Therefore, oral immunization
with the recombinant strain re-suspended in 0.1M sodium bicarbonate directly to buffer the
acidic gastric environment would most likely not alter the viability of the vaccine if used
within a relatively short time period.
In addition, preliminary studies were conducted to determine the optimal time to
attempt recover}' of S. typhimurium cells from BALB/c mice following a single oral
immunization. Recovery of viable cells at 3, 5, and 7 days following oral immunization
was tested. From these studies (data not shown) recovery of cells from Peyers patches at
5 days following the oral immunization was found to be the best time point. For all time
points, recovery of viable cells from the spleen were very low or not detectable. It should
be noted that the strain x4072 lacks the virulence plasmid and therefore this finding of a
lack of deep tissue invasion (ie. spleen colonization) is not surprising. So, for comparison


39
o
O
X
ID
fe
U
pYA292 pDMDl
Bicarbonate Buffer
pYA292 pDMDl
PBS
Fig. 7: Recovery of viable S. typhimurium %4072/pYA292 or
%4072/pDMDl cells on LB vs. MacConkey agars. Following in vitro
growth to an OD600 ~ 0.8, both strains were resuspended in sodium
bicarbonate or in phosphate buffered saline (PBS). Average yields from
duplicate plating are expressed as colony forming units (CFU).


40
of colonization potential of the S. typhimurium strains, only recovery of CFUs from
Peyers patches are reported.
Once the parameters were optimized for the colonization of S. typhimurium strains,
a comparative study between the recombinant x4072/pDMDl and %4072/pYA292 parent
strains was conducted. There was a noticeable reduction in the level of CFUs recovered
from Peyers patches for the recombinant strain expressing the HagB gene (Fig 8). The
parent strain y4072/pYA292 had a mean (n=4) of 1 x 105 CFU while the recombinant
strain x4072/pDMDl had a mean (n=4) of only 1 x 102 CFU. One explanation for this
observation could be the fact that expression of a foreign gene might be inhibiting the
ability of the recombinant strain to express other genes that are initiated by in vivo signaling
and have critical, functional roles to survival of the strain in vivo.
Assay for Stable in vivo Expression of HagB
Parallel to the colonization studies of the recombinant S. typhimurium strain
x4072/pDMDl, the stability of in vivo expression of HagB was also investigated. The
recoverable viable colonies on LB agar with nalidixic acid were assayed further by colony
immunoblotting (see Chapter 2: Materials and Methods). All colonies were picked from
primary Peyers patch isolation plates and patched onto an LB/ nalidixic acid plate for
analysis. All colonies were shown to be positive (100 out of 100) for HagB expression
along with the positive control, S. typhimurium x4072/pDMDl from glycerol stock (Fig
9). In addition, a negative control, S. typhimurium x4072/pYA292 was also included to
ensure that there was no non-specific antibody binding. While this assay was actually
performed on recovered colonies grown in vitro and does not necessarily represent in vivo
expression, the results indicate that even after passage through the mouse (in vivo) model,
retention of the heterologous gene was stable in the recombinant strain.


6
4)
V3
3
O
£
fa
u
w)
o
fa
5-
4-
3-


*

2-
1
i
pYA292



i
pDMDl
Fig. 8: Colonization of Peyer's patches at 5 days after single oral immunization with S.
typhimurium strains %4072/pYA292 or %4072/pDMDl. Presented are individual colony forming
units (CFU) from individual mice (diamond) on LB agar with nalidixic acid and the group mean
(horizontal bar).


42
(-)-
(+)
m &
#V,I
J J
*' *
;
i
VW 5'/'
Fig. 9: Colony immunoblot of colonies recovered from Peyers patches
5 days after a single immunization in BALB/c mice. Colonies were
screened for stable production of HagB protein with anti-HagB
antibodies. Included were a negative control strain (-) of S.
typhimuriwn with pYA292 plasmid only, and a positive control (+) with
S. typhimuriwn %4072/ pDMDl from stock .


43
Oral Immunization Study in BALB/c Mice with Boosting at Week 14
Experimental Design
Previously, it had been reported that a recombinant S. typhimurium strain
x4072/pDMDl expressing the HagB protein was immunogenic in mice, inducing systemic
and mucosal immune responses (52). For most vaccines developed to date, boosting one
or two additional times has been found to be necessary for enhancement of immune
responses and the development of a potential for long-term memory responses. Due to the
diverse compositions of mucosal vaccines developed, conflicting reports would argue
whether the potential to induce a long-term memory response exists. In this experiment,
we evaluated the ability to induce systemic and mucosal recall immune responses after oral
immunization and boosting with the cloned hagB gene and examined the IgG sub-class
distribution of anti-HagB and anti-Salmonella antibodies induced at different stages of the
immune response.
The immunization and sampling schedule is provided (Fig 10). Briefly a group of
female BALB/c mice (4-6 weeks old) was sampled for serum, saliva, and fecal extracts at
week 0, pre-immunization. Mice were orally immunized with three doses of 109 cells
(prepared as described in Chapter 2: Materials & Methods) on alternate days at week 0.
Subsequent samples (including vaginal washes beginning at week 7) were taken at two-
week intervals through week 13. In addition to confirming immunogenicity of the re
constructed recombinant strain, the effects of boosting the group of mice with the same
recombinant oral vaccine were investigated. The mice were boosted with the same
Salmonella strain in a similar three-dose manner at week 14 and subsequent samples were
taken at two-week intervals. Samples were analyzed for specific anti-HagB or anti-
Salmonella antibodies by ELISA (described in Chapter 2: Materials & Methods) and group
averages with standard error of the mean for each time point were calculated and reported in
summary graphs.


%4072/pDMDl
X4072/pDMDl
Dose
Week
ttt
rr
0 1
Sampling
A
1 i i r
2 3 4 5
i r
6 7
i
m
r-r-r-rr-r-r-r-r-r-r-r-r-r-r-r-r-r-r-r
8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
A
A
A
Fig. 10: Immunization & sampling schedule involving oral immunization and boosting of
BALB/c mice with S. typhimurium %4072/pDMDl. Immunizations (downward arrows)
were given in three doses on alternate days of week 0( primary) and week 14(boosting).
Sampling (upward arrows) of serum, saliva, fecal extracts and vaginal washes (starting at
week 7) were collected at alternate weeks.


45
Detection of Specific Anti- HagB Serum Antibodies After Primary Oral Immunization and
Boosting with S. typhimurium v4072/pDMDl
Mice immunized with S. typhimurium x4072/pDMDl developed a strong serum-
IgG response to HagB (Fig.l 1). The levels reached a peak at week 5 after primary
immunization. The recall response peaked at week 19, five weeks after the boosting at
week 14. The mice also developed a primary serum IgA response that peaked at week 7
after primary immunization (Fig. 12). A recall response was also seen at week 19
corresponding to the secondary IgG peak. The serum IgA recall response represented a
greater increase over primary than was seen with IgG although the absolute levels were not
as high. Both the IgG and IgA responses persisted at high levels throughout the remaining
eight weeks of the study. In previous studies, we showed that no response to HagB is
detected in mice immunized with S. typhimurium containing the cloning vector alone (52).
Detection of Specific Anti-HagB Response In Mucosal Secretions
To assess the mucosal immune response induced to HagB we examined antibody
levels in saliva, vaginal washes, and in extracts of fecal pellets. Results were expressed as
nanograms of specific IgA antibody per microgram of total IgA in fecal samples and
vaginal washes, or per unit of amylase activity in saliva.
A primary peak of salivary IgA anti-HagB was seen at week 9, which was later
than that seen for IgG and IgA in serum (Fig. 13). A recall response was seen with a peak
at week 17 that was greater than the primary peak at week 9.
Analysis of specific anti-HagB IgA levels in vaginal washes showed detectable
levels following primary immunization. Although it was not possible to determine the
peak response since data were not available for weeks 0-5, levels appear to decline from
week 7 thru 13 indicating the peak has occurred at week 7 or earlier (Fig. 14).
Subsequent studies have verified that pre-immunization levels of anti-HagB antibodies in
vaginal washes are limited to background. A peak in the recall response was seen at week
17 which was earlier than that seen in serum.


46
6
in
c
^ 5-
X!
0 3 5 7 9 11 13 15 17 19 21 23 27
Week
Fig. 11: Serum IgG anti-HagB levels following immunization at week 0
and boosting at week 14 (arrow) with S. typhimurium %4072/pDMDl.
IgG levels expressed as group average (n = 6) determined by ELISA of
individual samples. Error bars represent the standard error of the mean.


47
6
rr
O
X 5
£
W)
s
0 3 5 7 9 11 13 15 17 19 21 23 27
Week
Fig. 12: Serum IgA anti-HagB levels following immunization at week 0
and boosting at week 14 (arrow) with S. typhimurium %4072/pDMDl.
IgA levels expressed as group average (n =6) determined by ELISA of
individual samples. Error bars represent the standard error of the mean.


48
150
M
i T T TT"T TT TTTXX*
0 3 5 7 9 11 13 15 17 19 21 23 27
Week
Fig. 13: Salivary IgA anti-HagB levels following immunization at week 0
and boosting at week 14 (arrow) with S. typhimurium %4072/pDMDl. IgA
levels expressed as group average (n =6) determined by ELISA of individual
samples. Error bars represent the standard error of the mean.


49
Week
Fig. 14: Vaginal IgA anti-HagB levels following immunization at week 0
and boosting at week 14 (arrow) with S. typhimurium %4072/pDMDl.
IgA levels expressed as group average (n =6) determined by ELISA of
individual samples. Error bars represent the standard error of the mean.


50
Fecal pellets were extracted to estimate the level of specific IgA in intestinal
secretions. A primary peak was seen at week 7 and a recall peak at week 15, following'
boosting (Fig. 15). The peak after boosting occurred earlier than that for serum IgG or
IgA. There was no pronounced increase in the total IgA in either vaginal washes or fecal
samples as was seen in saliva.
Detection of Specific Anti-Salmonella v4072 Antibodies In Serum IgG and Fecal Extract
IgA Levels
High levels of IgG anti-Salmonella %4072 were found that followed the time course
of the specific anti-HagB response (Fig. 16). A primary peak was seen at week 7 followed
by a recall response peak at week 19 following boosting. In this case, the recall response
was not greater than the primary response. High levels of specific IgA anti-Salmonella
/4072 were also detected in feces (Fig. 17). A primary peak was seen at week 7 was
followed by a recall response and peak at week 15(1 week after boosting) which far
surpassed the primary response.
IgG Subclass Distribution Following Primary Oral Immunization and Boosting
It has been reported that the predominance of certain subclasses of IgG reflects
either a Thl or Th2 type helper cell response (202). Thl responses are associated with a
predominance of IgG2a and IgG3 while Th2 responses are associated with IgG 1. We
examined the serum IgG subclass response at three time points, representing the primary
peak, just prior to boost, and the recall peak (weeks 5, 13, and 19 respectively) for both
anti-HagB and anti-5, typhimurium antibodies.
The anti-HagB response was dominated by IgG2a, which accounted for 70-85% of
the response at each time point (Fig. 18). This pattern is consistent with a Thl type
response.
The IgG subclass distribution of antibodies to 5. typhimurium *4072 was also
assessed from the serum samples. Serum IgG antibodies specific for 5. typhimurium


Fecal IgA anti-HagB/ total IgA (ng/jug)
51
1.5
1
0.5
0
Fig. 15: Fecal IgA anti-HagB levels following immunization at week 0
and boosting at week 14 (arrow) with S. typhimurium %4072/pDMDl.
IgA levels expressed as group average (n =6) determined by ELISA of
individual samples. Error bars represent the standard error of the mean.
0 3 5 7 9 11 13 15 17 19 21 23 27
Week


Serum IgG anti-Salmonella %4072 (ng/ml) x 10
52
'sO
Week
Fig. 16: Serum IgG an-Salmonella levels following
immunization at week 0 and boosting at week 14 (arrow) with S.
typhimurium %4072/pDMDl. IgG levels expressed as group
average (n = 6) determined by ELISA of individual samples. Error
bars represent the standard error of the mean.


53
fe
Week
Fig. 17: Fecal IgA anti-Salmonella levels following immunization at
week 0 and boosting at week 14 (arrow) with S. typhimurium
%4072/pDMDl. IgA levels expressed as group average (n = 6)
determined by ELISA of individual samples. Error bars represent the
standard error of the mean.


54
100
5 13 19
Week
Fig. 18: Serum IgG anti-HagB subclass levels. Time points correspond to:
primary (week 5), pre-boost (week 13), and secondary peak (week 19) in
mice immunized with S. typhimurium %4072/pDMDl. IgG subclass levels
expressed as percentage (%) of total IgG levels determined by ELISA of
individual samples. Error bars represent standard error of the mean.


55
X4072 underwent a shift in subclass predominance (Fig. 19) from IgG3 (70% decreasing
to 36%) to IgG2a (17% increasing to 40%) while IgGl and IgG2b marginally increased (2
to 5% and 10 to 20%, respectively). Again, the absence of IgGl and the predominance of
IgG2a and IgG3 is consistent with a Thl type response. It is possible that antibodies to O-
polysaccharide may have contributed to the IgG3 levels since antibodies to polysaccharides
are known to occur in that subclass in mice (195). This supports the findings of others that
a Salmonella carrier induces a Thl associated response (18, 32, 187, 193).
Effect of Boosting at Three Different Time Points with Recombinant S. typhimurium strain
Y4072/pDMDl in BALB/c Mice
Experimental Design
Earlier we reported the induction of specific anti-HagB primary and recall responses
in serum and mucosal secretions following oral immunization and boosting at week 14 with
the recombinant S. typhimurium x4072/pDMDl. At the same time, we identified
responses to the carrier strain, S. typhimurium (Figs. 16 & 17). In the area of vaccine
development, the practicality of utilizing the same recombinant Salmonella strain for
boosting or for different vaccines has been a concern because of the response to the carrier
strain that could theoretically inhibit colonization in subsequent immunizations. Again, we
were able to induce recall responses following immunization at week 14 with the same
construct. However, at week 14, the peak of the primary response to the carrier
diminished. Therefore, we investigated whether boosting at an earlier time when the
response to the carrier was at peak would affect the potential for recall responses.
Specifically, we selected three time points for boosting which would represent an
early boosting at week 7 (coinciding with strong primary responses), a later boosting at
week 21, and again at week 14 as a control group. The immunization and sampling
schedule is shown in Fig. 20. Briefly three groups of female BALB/c mice (6-8 weeks
old) were sampled for serum, saliva, and vaginal washes at week 0, pre-immunization.


56
Week
Fig. 19: Serum IgG anti-5, typhimurium %4072 subclass levels. Time
points correspond to : primary (week 5), pre-boost (week 13), and
secondary peak (week 19) in mice immunized with 5. typhimurium
%4072/pDMDl. IgG subclass levels expressed as percentage (%) of
total IgG levels determined by ELISA of individual samples. Error
bars represent standard error of the mean.


Dose
Group A
Week
Sampling
T
t
6 7 8
10
12
i t t t
Dose
Group B
Week
Sampling
Dose
Group C
Week
Sampling
T
I
t
w
rrTTTTTT
13 14 15 17 19
t t t t
20 21 22 24 26
t t t t
Fig. 20: Immunization & sampling schedule involving oral immunization and boosting at three different times
with recombinant S. typhimurium %4072/pDMDl. Immunizations (downward arrows) were given in three doses
on alternate days of week 0 (primary for all 3 groups) and boosting at week 7 (group A), week 14 (group B) or
week 21 (group C). Sampling (upward arrows) of serum, saliva and vaginal washes were collected at specific
intervals as indicated for each group.


58
Due to the low level of specific anti-HagB levels detected from the fecal extracts in the
previous experiment, fecal extracts were not collected in this and subsequent experiments
for detection of specific anti-HagB IgA. Mice were orally immunized with three doses of
109 cells (prepared as described in Chapter 2: Materials & Methods) at week 0.
Subsequent samples were taken at selected time points indicated. All groups were boosted
with the same Salmonella strain in a similar three-dose manner at either week 7, 14, or 21
and subsequent samples were taken in two-week intervals. Samples were analyzed for
specific anti-HagB antibodies by ELISA (described in Chapter 2: Materials & Methods)
and group means for each time point were calculated and reported in summary graphs.
Detection of Specific Anti- HagB Serum Antibodies After Primary Oral Immunization and
Boosting at Different Time points with 5. typhimurium v4072/pDMDl
Using the repeated measures analysis of variance, comparisons were made between
week 6 and each of the 3 measurements after the boost for each parameter and group.
Based on ELISA analysis of the serum samples, we found variation in the amplitude of the
recall response means for each group that was a resultant effect of the time of boosting
(Fig. 21). Specific anti-HagB IgG for Group I went from a mean of 2.5 xlO5 ng/ml at
week 6 to a maximum level from the three post-boost sample times of 4.9 xlO5 ng/ml ( a 2-
fold increase from baseline at week 6) at week 10 (3 weeks post-boost). In comparison,
the specific IgG response in Group II went from 1.5 xlO5 ng/ml at week 6 to 4.1 xlO6
ng/ml (all- fold increase from baseline at week 6) at week 17 (3 weeks post -boost).
Similarly, the specific IgG response in Group III was also increased several fold with a
shift from 1.1 xlO4 ng/ml at week 6 to 2.3 xlO6 ng/ml ( a 68-fold increase from baseline at
week 6) at week 22 (1 week post-boost).
Specific IgA anti-HagB responses in serum samples were also compared (Fig. 22).
Once again, group I averages were lower in magnitude following boost compared to the
other groups. More specifically, the average for Group I specific serum IgA was 304


59
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ea
cc
c
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Cl
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3
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V3
6
5
4
3
2
1
0
0 6 8 10 12
6
5
4
3
2
1
0
6
5
4
3
2 -
1
0
0 6 22 24 26
Week#
- Gp II (Boost@ Wk 14)
I
-J
1
0 6 13 15 17 19
Gp III (Boost @Wk 21)
Fig. 21: Serum IgG anti-HagB levels following immunization at week 0 and
boosting at week 7 (group I), 14 (group II), or 21 (group III) with S. typhimurium
%4072/pDMDl. IgG levels expressed as group average (n=6) determined by
ELISA of individual samples. Error bars represent the standard error of the mean.
Statistically significant increases (p< 0.05) in IgG levels from the primary response
at week 6 are indicated for each group (*).


60
Tf
O
E
"el
e
JD
a
X
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a
<
3
.
in
8
6
4
2
0
8
6
4
2
0
8
6
4
2
0
0 6 20 22 24 26
Week#
Fig. 22: Serum IgA anti-HagB levels following immunization at week 0 and boosting
(arrow) at week 7 (group I), 14 (group II), or 21 (group III) with S. typhimurium
%4072/pDMDl. IgA levels expressed as group average (n=6) determined by ELISA of
individual samples. Error bars represent the standard error of the mean. Statistically
significant increases (p< 0.05) in IgA levels from the primary response at week 6 are
indicated for each group (*).


61
ng/ml at week 6 and only increased to a maximum of 3002 ng/ml (a 10- fold increase from
baseline) at week 12 (5 weeks post -boost). Similarly, Group III IgA response averages
were also only moderate from 226 ng/ml at week 6 to 7116 ng/ml ( a 31- fold increase from
baseline at week 6) at week 26 (5 weeks post-boost). In contrast, the specific IgA
response averages for Group II were greatly increased and demonstrated a strong recall
response with a baseline level of 354 ng/ml at week 6 increasing up to 5.3 xlO4 ng/ml ( a
152- fold increase from baseline) at week 17 (3 weeks post-boost).
Detection of Specific Anti-HagB Responses In Mucosal Secretions
Analysis of the salivary secretions also showed a difference in specific IgA anti-
HagB responses between the groups (Fig. 23). While all three groups demonstrated a
recall response following their respective boosts, Group II once again had a higher overall
magnitude of response. The salivary specific IgA response for Group I went from 14
ng/ml at week 6 to a maximum average level of 211 (a 15-fold increase from baseline) at
week 12 (5 weeks post-boost). The average specific IgA response for Group II was 32
ng/ml at week 6 and increased to 887 ng/ml (a 28-fold increase from baseline) at week 15
(1 week post-boost). The average specific IgA response for Group III went from 3 ng/ml
at week 6 to 356 ng/ml (a 118-fold increase from baseline) at week 26 (5 weeks post
boost). Interestingly, Group II had the highest magnitude of specific IgA response but
Group III had the greatest fold increase from its respective baseline, which incidentally was
the lowest week 6 baseline response of all three groups. Overall, no statistically significant
differences were determined between the groups.
Analysis of the vaginal wash samples for the three groups showed average specific
IgA levels that were relatively consistent, with recall responses for all three groups (Fig.
24). However, none were statistically elevated over week 6. Specifically, the average
vaginal specific IgA response for Group I was 3 ng/ml at week 6 and increased to 13 ng/ml
(a 4-fold increase from baseline) at week 12 (5 weeks post-boost). The average vaginal


62
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1000
750
500
250
0
GpIII (Boost @ Wk21)
i 1 r-
~i 1 1 1 1 _i
i
III
20 22 24 26
Week#
Fig. 23: Salivary IgA anti-HagB levels following immunization at week 0 and
boosting (arrow) at week 7 (group I), 14 (group II), or 21 (group III) with S.
typhimurium x4072/pDMDl. IgA levels expressed as group average (n=6)
determined by ELISA of individual samples. Error bars represent the standard error
of the mean. Statistically significant increases (p< 0.05) in IgA levels from the
primary response at week 6 are indicated for each group (*).


63
w¡
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en

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Week#
Fig. 24: Vaginal IgA anti-HagB levels following immunization at week 0 and
boosting (arrow) at week 7 (group I), 14 (group II), or 21 (group III) withS.
typhimurium %4072/pDMDl. IgA levels expressed as group average (n=6)
determined by ELISA of individual samples. Error bars represent the standard error
of the mean.


64
specific IgA response for Group II went from 2 ng/ml at week 6 up to 24 ng/ml ( a 13-fold
increase from baseline) at week 19 (5 weeks post-boost). Lastly, the average vaginal
specific IgA response for Group III was 2 ng/ml at week 6 and increased to 9 ng/ml (a 4-
fold increase from baseline) at week 26 (5 weeks post-boost).
Effect of Pre-existing Immunity to the Salmonella strain on Subsequent Oral Immunization
in BALB/c Mice
Experimental Design
The previous experiment indicated that mice are refractory to boosting at week 7
during the peak of the the response to the Salmonella carrier. This may be due either to
immune interference or lack of sufficient memory at the earlier time. The goal of this study
was to directly address the effect of pre-existing immunity on both the immune response to
and colonization of x4072/pDMDl.
Two groups of mice (n=10) were orally immunized at week 0 with the carrier strain
containing the vector, 5. typhimurium y4072/pYA292, to induce pre-existing immunity to
the Salmonella carrier. Two additional groups were given saline as parallel control groups.
The immunization and sampling schedule is shown in Fig. 25. Briefly the groups of
female BALB/c mice (6-8 weeks old) were sampled for serum, saliva, and vaginal washes
at week 0, pre-immunization. Subsequent samples were taken at selected time points
indicated (s) for each group and assayed for specific immunity to S. typhimurium. Then,
one group previously immunized with the carrier and one control (saline) group was orally
immunized with the three-dose regimen of S. typhimurium y4072 at week 7 or week 14.
Subsequent samples were analyzed for specific anti-HagB antibodies by ELISA (described
in Chapter 2: Materials & Methods) and group means for each time point were calculated
and reported in summary graphs.


Dose
Week 0
Sampling!
X4072/pYA292
R i fT1
"I r
t x
X4072/pDMDl
4^
t
8
t
10
t
12

X4072/pYA292
Dose
Week
P
0
Group II
T
t x
X4072/pDMDl
1U
Sampling
s
saline
Dose
Week 0
Samplingf
saline
Dose
Week 0
Samplingf
T
Group IV
t x
X4072/pDMDl
~r
13
t
s
i i
14 15
6
t
4^
8
t
tr
10
t
12
t
Group V
t x
X4072/pDMDl
T
i r
2/pDM
1 1 T
13 14 15
t
t
T
Y
i r
17
t
1t9
19
t
Fig. 25: Immunization & sampling schedule investigating effect of pre-existing immunity to Salmonella carrier
strain. Immunizations (downward arrows) were given in three doses on alternate days. Sampling (upward
arrows) of serum, saliva, and vaginal washes were collected at specific intervals as indicated for each group;
where indicated with an S-upward arrow, the samples were assayed for anti-Salmonella antibody levels. In
addition, a subset of mice (X) were killed at five days following a single immunization (single downward arrow)
for recovery colonization studies.


66
Detection of Specific Anti-Salmonella responses Following Oral Immunization with Strain
v4072/pYA292 at week 0.
Non-immune samples from all four groups of BALB/c mice were collected at week
0 prior to immunization with attenuated S. typhimurium y4072/pYA292 (Group I and II) or
saline (Group IV and V). As anticipated, no background immunity to Salmonella
components was detected by ELISA for serum IgG or IgA, as well as in fecal IgA, while
background levels of specific IgA were detected in all vaginal wash samples. Following
primary immunization with %4072/pYA292 or saline during week 0, groups I and IV were
again sampled and assayed for specific anti-Salmonella IgG or IgA levels at week 6, just
prior to their immunization with the recombinant S. typhimurium x4072/pDMDl. As
expected, the group orally immunized with S. typhimurium y4072/pYA292 demonstrated a
strong primary anti-Salmonella response in serum IgG, serum IgA, fecal IgA, and vaginal
IgA levels (data not shown). In contrast, Group IV (saline controls) remained at zero or
background anti-Salmonella levels in all samples (data not shown). Similarly, Group II
and Group V were sampled and assayed for anti-Salmonella Ig levels at week 13, just
prior to their respective immunizations with the recombinant S. typhimurium
x4072/pDMDl. Once again, Group V (saline controls) had no specific anti-Salmonella Ig
levels, while Group II had apparent anti -Salmonella IgG and IgA levels, although overall
levels were lower for Group II at week 13 than for Group I at week 6 (data not shown).
This difference in the anti-Salmonella Ig levels correlates with our previous studies in
which an anti-Salmonella response within a group was very strong at week 6 but had
plateaued at a lower level by week 13. Statistically, Group I and IV were significantly
different (p< 0.004) for all response levels. In addition, Group I and Group II (both
initially immunized with the Salmonella carrier strain) were significantly different (p<
.0001) for the serum IgA levels.


67
Detection of Specific Anti- HagB Serum & Mucosal Antibodies After Oral Immunization
with Recombinant S. typhimuriiun v4072/pDMDl at Week 7 or Week 14 in Groups With
or Without Pre-existing Immunity to the Salmonella Carrier Strain.
Following the oral immunization of Group I and IV at week 7 with 3 doses of S.
typhimurium x4072/pDMDl, the two groups were sampled at 1,3, and 5 weeks post
immunization and assayed by ELISA for specific anti-HagB Ig levels in serum, vaginal
washes, and saliva. Values were determined for all three time points post-immunization,
however only the peak responses at 5 weeks post-immunization were compared using the
Wilcoxon rank sum test.
The anti-HagB IgG response in serum was significantly reduced (p< 0.05) in mice
pre-immunized with the 5. typhimurium carrier and then immunized at week 14 (Fig. 26).
While there was some reduction in mice immunized at week 7, the difference was not
statistically significant. Pre-immunization with the Salmonella carrier had no effect on
serum IgA anti-HagB levels at either week 7 or 14 (Fig. 27).
With regard to mucosal responses, pre-immunization with the carrier strain
significantly reduced the salivary IgA anti-HagB response in animals immunized at week 7
(Fig. 28). Levels were reduced in animals immunized at week 14 but the difference was
not statistically significant. No statistically significant differences were seen in vaginal
wash IgA anti-HagB levels at either week 7 or 14 (Fig. 29).
Assessment of the Colonization of Pevers Patches Following Oral Immunization With the
Recombinant S. typhimurium v4072/pDMDl in Mice With or Without Pre-existing
Immunity to the Salmonella Carrier Strain.
A subset of each group of mice was assessed for the level of colonization of the
spleen and Peyers patches. Following a single oral dose of S. typhimurium
x4072/pDMDl at week 7 or week 14, mice were sacrificed and the spleen and an average
of 5 Peyers patches were removed for homogenization. Following plating on selective
agar plates, the CFUs were enumerated for individual mice within each group. No CFUs


50000
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X
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Pre-immunized at Week 0 with:
Time of Immunization with x4072/pDMDl
Fig. 26: Serum IgG anti-HagB responses from groups of BALB/c mice to determine effect of pre-existing immunity.
Presented are responses at 5 weeks following oral immunization with S. typhimurium x4072/pDMDl at week 7 or 14. IgG
levels expressed as group averages (n=6) determined by ELISA of individual samples. Error bars represent the standard error
of the mean. Statistically significant decreases (p< 0.05) in IgG levels in groups previously immunized at week 0 with S.
typhimurium %4072/pYA292 versus the corresponding control group (saline) are indicated (*).


2000
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1500 -
1000
500
Pre-immunized at Week 0 with:
S. typhimurium carrier strain
Saline
Wk 7
Wk 14
o\
VO
Time of Immunization with x4072/pDMDl
Fig. 27: Serum IgA anti-HagB responses from groups of BALB/c mice to determine effect of pre-existing immunity. Presented
are responses at 5 weeks following oral immunization with S. typhimurium %4072/pDMDl at week 7 or 14. IgA levels
expressed as group averages (n=6) determined by ELISA of individual samples. Error bars represent the standard error of the
mean. Statistically significant decreases (p< 0.05) in IgA levels in groups previously immunized at week 0 with S.
typhimurium x4072/pYA292 versus the corresponding control group (saline) are indicated (*).


1000
Pre-immunized at Week 0 with:
S. typhimurium carrier strain
Saline
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Time of Immunization with %4072/pDMDl
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Fig. 28: Salivary IgA anti-HagB responses from groups of BALB/c mice to determine effect of pre-existing immunity. Presented
are responses at 5 weeks following oral immunization with S. typhimurium %4072/pDMDl at week 7 or 14. IgA levels expressed
as group averages (n=6) determined by ELISA of individual samples. Error bars represent the standard error of the mean.
Statistically significant decreases (p< 0.05) in IgA levels in groups previously immunized at week 0 with S. typhimurium
%4072/pYA292 versus the corresponding control group (saline) are indicated (*).


O
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Fig. 29: Vaginal IgA anti-HagB responses from groups of BALB/c mice to determine effect of pre-existing immunity.
Presented are responses at 5 weeks following oral immunization with S. typhimurium %4072/pDMDl at week 7 or 14.
IgA levels expressed as group averages (n=6) determined by ELISA of individual samples. Error bars represent the
standard error of the mean. Statistically significant decreases (p< 0.05) in IgA levels in groups previously immunized
at week 0 with S. typhimurium %4072/pYA292 versus the corresponding control group (saline) are indicated (*).


72
were recovered from the spleens of any group. From the Peyers patches, within each
group there was some variability between the individual mice (Fig. 30). However, there
was no statistically significant difference between the average CFU/ group; all four groups
had an average of ~3 xlO4 1 xlO5 CFU/mouse. These results demonstrate no inhibition
of colonization due to pre-existing immunity to the carrier strain.
Studies Investigating Long Term Memory and Potential for Recall Response Following
Boosting at Week 52 (One Year)
Experimental Design
In the previous studies we have shown that oral immunization with 5. typhimurium
expressing a foreign antigen can lead to a recall response shortly after a course of primary
immunization (i.e. week 14). The longevity of memory, particularly mucosal memory,
induced by oral Salmonella carriers has not been adequately demonstrated. In this study
we wished to examine whether recombinant oral Salmonella y4072/pDMDl could induce a
lifelong capacity to mount a recall response following challenge. The immunization and
sampling schedule is presented (Fig. 31).
Three groups of mice were examined. Group I was immunized at week 0 and
boosted at week 52. Week 52 was chosen to represent long-term memory since it equals
approximately one-half the lifespan of a BALB/c mouse. Group II was immunized at week
14 and boosted at week 52. Group III was immunized at week 0 and boosted at week 14
as well as week 52. (Note that Group II was immunized in the previous study
investigating pre-existing immunity and kept for week 52 boosting in this study to conserve
mice. Likewise, Group III was immunized as part of the earlier experiment determining the
effect of time of boost on recall responses.)


73
6
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3
2
Gp I Gp IV Gp II Gp V
(X4072/p Y A292) (saline) (X4072/pYA292) (saline)
Group (week 0 immunization)
Fig. 30: Colonization of Peyer's patches to determine the effect of pre
existing immunity to the Salmonella carrier. Represented are colonies
recovered at 5 days following single oral immunization with S. typhimurium
%4072/pDMDl at week 7 (groups I and IV) or 14 (groups II and V) in groups
of mice with prior immunization at week 0 with S. typhimurium
%4072/pYA292 (groups I and II) or saline (groups IV and V). Presented are
individual colony forming units (CFU) on LB agar with nalidixic acid from
individual mice (diamond) and the group mean (horizontal bar).


H4072/pDMD1
Dose HI
Group I
H4072/pDMD 1
III
r-r-r-
Week 0
i
5
W
52 55
1 1 1 1
57 59
Sampling T
t
t t
t t
saline
H4072/pDMDl
K4072/pDMD 1
Dose HI
Group II
III
III
Week 0
i
iillll
13 14 15
i i
17
19
\\
52
55
57
59
Sampling T
t t
t
t
t
t
t
t
H4072/pDMD 1
Dose HI
Group III
H4072/pDMD 1
III
H4072/pDMD1
III
Week 0
rmttr-r
6
13 14 15
i 1
1
17
r
19
1 1 1 1
52
1
55
57
59
SamplingT
t
t t
t
t
t
t
t
t
Fig. 31: Immunization and sampling schedule investigating long-term memory and potential for recall
responses following boosting at week 52. BALB/c mice were orally immunization with recombinant S.
typhimurium %4072/pDMDl and subsequently boosted with the same recombinant S. typhimurium strain .
Immunizations (downward arrows) were given in three doses on alternate days. Sampling (upward arrows)
of serum, saliva, and vaginal washes were collected at specific intervals as indicated for each group.


75
Determination of Specific Anti-HagB Serum Responses Following Boosting at Week 52 in
Three Separate Groups of BALB/c Mice
Serum samples collected at the specified time points for each of the three groups
were analyzed for specific anti-HagB IgG and IgA responses by ELISA (Figs. 32 & 33).
The levels of responses are presented as group means of specific Ig levels. All three
groups showed a recall response in serum IgG and IgA as well as mucosal IgA. Group I,
immunized at week 0, had very little residual response at week 51 (one week pre-boost),
but had a subsequent strong recall response following boosting at week 52 with a serum
IgG anti-HagB level of -180,000 ng/ml (Fig. 32). Although this level of response was
only marginally higher than the primary response at week 5 of -135,000 ng/ml, it was a
significant recall response for one year after primary exposure. In comparison, Group II
which had been immunized at week 14, had a residual IgG response at week 51 of -35,000
ng/ml. Following boosting at week 52, the serum IgG anti-HagB response increased to a
steady level of -75,000 ng/ml. Despite an overall lower level of response within Group II,
a strong recall response following boosting at week 52 was detected. Group III,
previously immunized at week 0 and week 14, again had a low residual IgG response at
week 51. Following boosting at week 52, a recall IgG anti-HagB response was observed
immediately but was not to the amplitude of the response following the first boosting at
week 14. Still, the IgG levels after week 52 were significant at -750,000, higher than for
the other two groups.
More significant were the serum IgA recall responses for all three groups following
oral boosting at week 52 (Fig. 33). A strong IgA recall response, -6,400 ng/ml, for
Group I was detected after only 3 weeks following the week 52 boost; this recall response
far surpassed the primary response of -600 ng/ml. Similarly, Group II had an immediate
IgA recall response, at 3 weeks post- boost, of -9,000 ng/ml which was a ten-fold increase
from the primary peak response of -900 ng/ml. Group III had an IgA recall response level
at -43,000 ng/ml following the boost at week 52 which was comparable to the earlier recall


76
Fig. 32: Serum IgG anti-HagB levels for three groups following various primary
immunizations and boosting at week 52. All immunization timepoints are
indicated (arrow). IgG levels expressed as group average (n =6) as determined
by ELISA of individual samples. Error bars represent the standard error of the
mean.


77
Fig. 33: Serum IgA anti-HagB levels for three groups following various
primary immunizations and boosting at week 52. All immunization timepoints
are indicated (arrow). IgA levels expressed as group average (n =6) as
determined by ELISA of individual samples. Error bars represent the standard
error of the mean.


78
response following the first boosting at week 14. Despite no significant increase in the
recall level of IgA following the second boosting, the absolute level was approximately.
nine times greater than the magnitude of the IgA responses in the other two groups.
Determination of Specific Anti-HagB Mucosal Responses Following Boosting at Week 52
Mucosal responses in the three groups following boosting at week 52 were
determined by ELISA and presented as means of specific anti-HagB slgA levels (Fig. 34 &
35). Overall, the mucosal responses demonstrated a similar pattern of characteristic recall
responses with earlier (within 3 weeks of boosting) and elevated absolute levels of slgA.
Salivary IgA anti-HagB levels (Fig. 34) were greatly elevated in Group I and III following
boosting at week 52 to -120 ng/U and -2800 ng/U, respectively. The recall response
levels represented a 10 to 20-fold increase over the primary responses. Similarly, Group II
demonstrated a marginal increase to -1000 ng/U following the week 52 boost.
Vaginal IgA anti-HagB levels (Fig. 35) were also reflective of a recall response.
Group II and Group III had particular increases in responses following the boost at week
52 with respective levels of -70 ng/pg IgA and -100 ng/jig IgA. Group I had a post-boost
level that was comparable in absolute concentration to the primary response but appeared
within 3 weeks instead of 5 weeks following oral immunization. Surprisingly, the residual
primary IgA response at week 51 was still at considerable levels in all three groups.
ELISPOT Analysis of Specific Antibody- Secreting Cells from Spleen. Salivary Glands.
and Lamina Propria of BALB/c Mice Following Oral Immunization or Boosting
Experimental Design
Collectively the previous set of experiments has determined the immune responses
following oral immunization with respect to concentration of specific immunoglobulin in
circulation or secretions. In this set of experiments we set out to define the specific
immune response following oral immunization or boosting with the recombinant S.


79
Fig. 34: Salivary IgA anti-HagB levels for three groups following various
primary immunizations and boosting at week 52. All immunization timepoints
are indicated (arrow). IgA levels expressed as group average (n =6) as
determined by ELISA of individual samples. Error bars represent the standard
error of the mean.


80
0 6 14
52
Week #
Fig. 35: Vaginal IgA anti-HagB levels for three groups following various
primary immunizations and boosting at week 52. All immunization timepoints
are indicated (arrow). IgA levels expressed as group average (n =6) as
determined by ELISA of individual samples. Error bars represent the standard
error of the mean.


81
typhimurium y4072/pDMDl at the cellular level. Specific antibody-secreting cells from
spleen, salivary glands, and lamina propria of B ALB/c mice were assayed by ELISPOT.
In preliminary studies we examined spot-forming cells (SFC) at weeks 1, 2, 3, 4, and 5
following oral immunization (data not shown). From these studies it was apparent that the
level of anti-HagB specific spots was low, even at the peak of the response, preventing us
from doing a kinetic study. Thus we focused efforts on comparing groups at peak times
only. It was determined that 5 weeks post-primary immunization yielded sufficient spot
forming cells from all three tissues. In addition, it was determined that a minimum group
of four was required to obtain enough viable cells from the pooled samples for plating.
Collectively, four groups (n=4) of BALB/c mice at five weeks following primary
oral immunization and two groups (n=4) at 3 weeks post- boosting (week 14) were
assayed. Viable lymphocytes isolated from the spleen were assayed for specific IgG and
IgA antibody-secreting cells directed towards S. typhimurium y4072 and the HagB protein.
Similarly, isolated cells from the lamina propria and salivary glands were assayed for
specific IgA antibody-secreting cells directed towards S. typhimurium *4072 and the HagB
protein. IgG SFC were not assayed from lamina propria or salivary glands since they are
not found in high numbers in these mucosal tissues. The specific antibody-secreting cells
were enumerated and reported as SFC/ 106 mononuclear cells (MNC). The collective
results of these studies are summarized in Table 1.
Enumeration of Specific Spot Forming Cells (SFC)
The average concentration of specific anti-5, typhimurium IgG and IgA was 6.5
and 81 SFCs/ 106 MNC, respectively. Surprisingly, immune cells stimulated against the
Salmonella carrier strain were predominantly IgA versus IgG with a ratio greater than 12:1.
In contrast, the average concentration of specific anti-HagB IgG and IgA was nearly equal.
Most interesting is the contrast in the concentration of specific antibody-secreting cells from
the group boosted at week 14. In this group, the number of stimulated cells from the


Table 1. Cells secreting antibodies to S. typhimurium and HagB in
various organs of BALB/c mice orally immunized with recombinant
S. typhimurium x4072/pDMDl
SFC/106 MNC secreting:
Cell source
Anti-S. typhimurium
Anti-HaeB
IgG
IgA
IgG
IgA
Spleen (primary)
6.5 1.8
81.2 45
8.5 2.9
6 3.5
Spleen (boost)
5.3 0.9
41.8 3.7
21.4 6.8
46 2.1
Salivary gland (primary)
ND
4.5 2.6
ND
0.5 0.5
Salivary gland (boost)
ND
42.9 5.6
ND
67.4 8.9
Lamina propria (primary)
ND
93.5 34.5
ND
21.5 11.5
Lamina propria (boost)
ND
164.5 68.2
ND
81.5 9.6
ND= not determined


83
spleen directed toward the components of S. typhimurium was similar to the previous
levels in the primary immunized animals. This lack of increase reflects what we observed
in serum in terms of anti-5, typhimurium IgG levels after boosting (Fig. 16). In contrast,
boosting the group of mice resulted in a 4-fold increase in specific IgG and 7-fold increase
in specific IgA SFCs directed to the HagB protein.
Spot-forming cells from the salivary glands following primary immunization were
difficult to determine due to low abundance and low cell yield from salivary glands.
However, following boosting at week 14, the concentration of specific IgA anti-Salmonella
and anti-HagB increased to 42.9 and 67.4 SFC/ 106MNC, respectively.
The isolation of viable cells from the lamina propria was more difficult and only
two groups yielded viable cells for the ELISPOT assay. High numbers of IgA SFC were
detected in lamina propria against both 5. typhimurium (93.5) and HagB (21.5). Once
again, boosting at week 14 resulted in an increase; the specific IgA anti -Salmonella level
increased to 164.5 SFC/ 106MNC and the specific IgA anti-HagB level increased to 81.5
SFC/ 106MNC.


CHAPTER 4
CONCLUSIONS AND DISCUSSION
Reconstruction and Characterization of the Recombinant S. typhimurium Strain
v4072/pDMDl Expressing the hasB Gene of P. gineivalis
The use of a live, attenuated, recombinant Salmonella typhimurium for delivery of
an expressed, heterologous gene is one approach to oral immunization. Salmonella's
specificity for targeting the Peyers patches of the gut provides the appropriate presentation
and stimulation to induce mucosal immunity. At the same time, it is capable of inducing
systemic immunity. As reviewed earlier, several attenuated forms of the S. typhimurium
have been developed and investigated as recombinant vaccine strains. In these studies we
used the strain y4072 which has an asd deletion which can be complemented by the asd
gene encoded on the plasmid pYA292. This balanced-lethal construct, developed by Dr.
Roy Curtiss, III (Washington University), provides a convenient mechanism for ensuring
plasmid stability without the need for continuous antibiotic selective pressure. The
requirement for co-administration of an antibiotic is not desirable in a practical vaccine.
Our vaccine strain expresses the hagB gene, which encodes a hemagglutinin of P.
gingivalis cloned into plasmid pYA292 to yield pDMDl (53).
Although several recombinant constructs of S. typhimurium have been reported,
expression of the heterologous gene and its effect (e.g., toxicity) on the host strain can vary
with each foreign gene. Consequently, it is necessary to characterize the level of
expression and resultant growth rate for each construct. Following the reconstruction of S.
typhimurium y4072/pDMDl, we investigated the level and stability of expression of the
HagB hemagglutinin as well as the growth rate and colonization potential of the
recombinant construct. Through restriction analysis of plasmid minipreps we confirmed
84


85
the stable maintenance of the hagB gene insert in the plasmid pYA292. From cell lysates of
the recombinant strain x4072/pDMDl, we identified strong expression of HagB by both
Coomassie Blue staining and Western blotting. While high expression of a cloned gene is
generally desired for immunogenicity, over-expression could have a deleterious effect on
the carrier strain. The expressed protein itself may be toxic to the bacterial host or may
consume important resources required for expression of its own critical genes (182, 211).
Expression of late genes, or stress-response genes activated in vivo, are particularly
vulnerable to suppression resulting from over-expression of a cloned gene. For example,
the spv genes encoded by Salmonella typhimurium on a virulence plasmid increase the
growth rate of the bacteria within host cells (81), particularly in macrophages (82), and
most-likely are activated in vivo. While our attenuated strain does not contain the virulence
plasmid, other genes may be turned on by in vivo signaling and be susceptible to over
expression of our cloned hagB gene. In fact, one study identified as many as 34 from a
pool of 57 detectable proteins synthesized by S. typhimurium that appeared to be unique to
the intracellular environment (21).
LPS silver staining confirmed a smooth LPS, again, which is favorable for vaccine
strains (76). Rough strains of Salmonella, lacking the repeating oligosaccharide units,
have been shown to have highly reduced pathogenicity in mice (179, 180) and conventional
piglet studies (179). It is postulated that smooth strains may survive longer in the host due
to a reduced ability of immune components to readily attach to their surfaces. Again, even
though the parent S. typhimurium y4072 strain had smooth LPS, the expression of HagB
could have affected this status.
The effects of the heterologous expression of HagB on the attenuated Salmonella
were investigated further. In vitro growth rate was determined for the recombinant strain
X4072/ pDMDl. A reproducible rate with a doubling time of ~60 minutes during
logarithmic growth was determined from three successive studies. This growth rate was
slower compared to the control strain x4072/pYA292, which had an average doubling time


86
of ~40 minutes. A decrease in the growth rate of a recombinant strain is common. A
critical factor is whether this decreased growth rate would inhibit normal colonization and
immunogenicity in vivo, which can only be determined by a preliminary study in an animal
model. We previously demonstrated the ability of S. typhimurium x4072/pDMDl to
induce immunity to HagB (52).
In this study, we have demonstrated that the HagB expression in the recombinant
Salmonella strain is stable even after passage through the BALB/c mice. Colonies
recovered from the Peyers patches five days following oral immunization with the
recombinant S. typhimurium x4072/pDMDl were consistently positive (100%) for HagB
expression by colony immunoblotting. While developing the colonization studies, we
discovered that the recombinant strain recovered from the host tissues was more sensitive
to selective medium components than the control strain, x4072/pYA292. In fact, the use of
MacConkey agar, a standard selective medium used in colonization studies of wild-type
strains, was found to be too harsh for strains expressing HagB (Fig. 7). It is possible that
expression of HagB may affect the outer membrane, making the strain more susceptible to
bile salts and crystal violet, which are used as selective agents in this medium. This
phenomenon offers a caution for other studies involving recombinant constructs and
determination of colonization in vivo.
Oral Immunization Study in BALB/c Mice with Boosting at Week 14
One of the concerns with using live avirulent carriers such as Salmonella has been
that induction of immunity to the carrier may preclude its subsequent use for other vaccines
or for boosting. In one report, it appeared that prior exposure to the Salmonella carrier
enhanced the secondary response to the foreign antigen (4). In our system, we saw recall
responses to HagB in serum in both IgG and IgA antibodies as well as in saliva, vaginal
washes and in fecal extracts, despite the concurrent high anti-Salmonella responses also


87
detected. In particular, high anti -Salmonella IgA levels in the gut might be expected to
interfere with the colonization and persistence of the vaccine strain. However, this
apparently did not occur with the dosage and immunization regimen used in this study.
In saliva, the response after boosting appeared to be minimal when the data were
expressed as nanograms of specific IgA anti-hagB per microgram of total IgA (data not
shown). This was done to compensate for the variable dilution one encounters in mucosal
secretions. However, in saliva, we detected an increase in total IgA after boosting which
tended to damp down the response when expressed in this manner. When the results were
normalized to specific antibody per unit amylase (whose level should not be altered by
immunization) increases could be detected after boosting.
A possible explanation for the increase in total IgA might involve the response to
the Salmonella carrier. The number of anti-Salmonella spot-forming cells (SFC) in salivary
glands found in the ELISPOT studies (Table 1) appears greater than the number of anti-
HagB SFC, at least during the primary response. After boosting, however, anti-HagB
SFC predominate.
Analysis of the serum IgG subclass distribution was performed to infer the T-helper
subset associated with the systemic response. Examination of the IgG subclass distribution
of anti-/iagB antibodies revealed a predominance of IgG2a antibodies. In mice, Thl cells
produce IL-2, IFNy, and TNF-fl and have been demonstrated to induce B cells to secrete
IgG2a (202). Th2 cells on the other hand, preferentially secrete IL-4, IL-5, IL-6, and IL-
10 and are most effective in providing help for B cell responses in the form of IgGl and
IgE (202). Th 1 responses have been also associated with cell-mediated immune and
inflammatory responses (152,203).
We found a similar predominance of IgG2a anti-HagB antibodies in serum samples
collected from BALB/c mice nasally immunized with repeated doses of P. gingivalis W50
strain in an experiment (data not shown) conducted by Dr. Jeannine Brady and Dr. Emil
Kazarov (University of Florida, Gainesville, FL). Both immunizations are directed toward


88
mucosal inductive sites, which may influence the T-helper type response and consequently
the subclass distribution. Immunization with HagB by a strictly systemic route (e.g.,
subcutaneously, in adjuvant) would help answer questions concerning the role of the nature
of the protein, the delivery system, or route of immunization on the subclass response.
In addition, the serum specific anti-HagB responses in the nasally immunized mice
were extremely low compared to the strong responses detected from the orally immunized
mice. One possible explanation is that the native expression of HagB in the W50 strain
may, in fact, be considerably lower than the expression in the recombinant Salmonella
strain. A significant difference in antigen concentration at the effector sites could result in a
decreased induction of a response. In addition, the immunization routes (oral versus nasal)
offer an obvious possible explanation for the differences in the responses. In our mouse
studies, however, the different immunization routes most likely do not explain the
difference in the anti-HagB response levels since the total serum anti-/5, gingivalis
responses were very high following intranasal immunizations (data not shown).
Furthermore, a recent report investigating the kinetics and distribution of immune
responses following nasal versus oral immunizations in humans found both routes of
vaccination to effectively induce strong serum responses (184).
Nevertheless, the results of our study agree with those of others (162, 213), in
which it was found that an antigen expressed in S. typhimurium appears to induce a Thl
type immunoglobulin pattern in the systemic compartment. In contrast, it was shown that
expression of S. mutans Ag I/ll as a fusion protein with cholera toxin B subunit induces a
mixed Thl/Th2 type immunoglobulin pattern (88). In our studies, a definitive answer will
require examination of antigen-specific T-helper cells in both systemic and mucosal sites.
In addition, the implication of a Thl-type association offers the potential for the
induction of a cell mediated immune response. This could be explored through a delayed
type hypersensitivity (DTH) assay (e.g., footpad or ear pinna swelling). The role of T
cells and cell mediated immunity in periodontal disease is still being explored (130), but


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