Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS FOR TREATING AND PREVENTING
PATHOGENIC BACTERIAL INFECTION BASED ON THE ESSENTIAL ROLE
OF DNA METHYLATION IN BACTERIAL VIRULENCE
CROSS-REFERENCE TO OTHER APPLICATIONS
This patent application claims the priority benefit of U.S. patent application
Serial
Nos.09/241,951, filed February 2, 1999, converted to U.S. Provisional Ser. No.
,
and 09/305,603, filed May 5, 1999, converted to U.S. Provisional Ser. No. ,
which are incorporated by reference in their entirety.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY
SPONSORED RESEARCH
This invention was made with Government support under Grant Nos. AI36373 (to
M. Mahan) and AI23348 (to D. Low), awarded by the National Institutes of
Health. The
Government may have certain rights in this invention.
FIELD OF THE INVENTION
The present invention relates to vaccines useful for the prevention or
modification
of microbial pathogenesis. In particular, this invention relates to
immunogenic
compositions generally comprising pathogenic bacteria (e.g., Salmonella) which
contain a
mutation affecting DNA adenine methylase (Dam). The invention also relates to
methods
of eliciting an immune response using these compositions, as well as screening
methods.
BACKGROUND OF THE INVENTION
Food-borne disease presents a serious threat to our health, the safety of the
nation's
food supply, and to the agricultural industry. Each year over 80 million
Americans suffer
from food poisoning, at a cost estimated between $5 and $23 billion annually
in medical
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treatment and lost wages (Snydman, D. R., Food poisoning. In: Infectious
Diseases,
second edition, Gorbach, S. L., et al., eds., 768-781 (1998)). Our defenses
against food-
borne disease are failing as new pathogens have emerged that can cause more
debilitating
forms of disease and/or can no longer be controlled by available antibiotics;
examples
include Escherichia coli (E. coli) 0157:H7, Salmonella enteritidis (S.
enteritidis), and S.
typhimurium DT104 (Alterkruse, S. F., et al., Emerging food borne diseases, 3:
July-Sept.
(1997)). Salmonellosis is one of the major food-borne diseases in the United
States,
estimated at between 1 and 4 million cases/year (Shere, K. D., et al.,
Salmonella infections.
In: Infectious Diseases, second edition, Gorbach, S. L., et al., eds., 699-712
(1998)). This
disease is caused by exposure to products contaminated with Salmonella, e.g.,
animal
products such as eggs, milk, poultry or the ingestion of food products that
have been
exposed to animal feces, including fruits and vegetables. Due to large scale
manufacturing
and distribution practices, salmonellosis outbreaks have affected large
populations (Tauxe,
R. V., et al., Emergingfood borne diseases: an evolvingpublic health
challenge.
Emerging infectious diseases, 3:Oct-Dec (1997)).
Salmonella is a prime example of a pathogenic microorganism whose various
species are the cause of a spectrum of clinical diseases that include acute
gastroenteritis and
enteric fevers. Salmonella infections are acquired by oral ingestion. The
microorganisms
after traversing the stomach, invade and replicate in the intestinal mucosal
cells. See,
Hornik, et al., N. Eng. J. Med., 283:686 (1970). Some species, such as S.
typhi, can pass
through this mucosal barrier and spread via the Peyer's patches to the lamina
propria and
regional lymph nodes. Salmonella typhi, which only infects man, is the cause
of typhoid
fever and continues to be an important public health problem for residents in
the less
developed world.
Urinary tract infections (UTI) are among the most common bacterial infections.
It
is estimated that about 20% of women will experience at least one UTI during
their
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lifetime. Although women are the major target of UTI, men and children can
also contract
this disease. About 70% of all UTI are caused by uropathogenic Escherichia
coli. The
disease may be limited to the lower urinary tract (cystitis) or can involve
the renal pelvis
(pyelonephritis). Over 90% of E. coli isolated from women with pyelonephritis
contain the
pyelonephritis-associated pili (pap) gene cluster (O'Hanley, P. M., et al., N.
Engl. J. Med.,
313:414-447 (1985)). Most patients with pyelonephritis caused by E. coli mount
a strong
immune response to Pap pili. The Pap pili contain adhesins at their tips that
enable these
bacteria to colonize the urinary tract, id. Most Pap pili-adhesin complexes
bind to the P
blood group receptor, which is expressed on epithelial cells lining the gut,
the bladder, and
ureters. Despite our understanding of the role of adhesion in the pathogenesis
of UTI, no
vaccine is available against UTI. This is also true for many other important
microbial
pathogens that cause significant morbidity and mortality.
Microbial pathogens, or disease-producing microorganisms, can infect a host by
one
of several mechanisms. They may enter through a break in the skin, they may be
introduced by vector transmission, or they may interact with a mucosal
surface. Disease
ensues following infection of the host, when the potential of the pathogen to
disrupt normal
bodily functions is fully expressed.
Each disease-producing microorganism possesses a collection of virulence
factors
that enhance their pathogenicity and allow them to invade host or human
tissues and disrupt
normal bodily functions. Infectious diseases have been major killers over the
last several
thousand years, and while vaccines and antimicrobial agents have played an
important role
in the dramatic decrease in the incidence of infectious diseases, infectious
diseases are still
the number one cause of death world-wide.
Environmental conditions within the host are responsible for regulating the
expression of most known virulence factors (Mekalanos, J. J., J. Bacteriol.,
174:1 (1992)).
In the past, scientists would attempt to mimic, in vitro, the environmental
conditions within
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the host in an attempt to identify those genes that encode and are responsible
for producing
virulence factors. As a result, the identification of many virulence factors
was dependent
on, and limited by, the ability of researchers to mimic host environmental
factors in the
laboratory. However, with the advent of in vivo expression technology CIVET)
discovered
by Malian, M. J., et al., and disclosed in U. S. Patent No. 5,434,065 it is
now possible to
determine which genes are expressed within a host and within which tissues of
the host the
genes are expressed. Consequently, the molecular mechanisms of the specific
pathogenic
microorganisms that allow them to circumvent the host's (e.g., human body)
immune
system and initiate the physiological changes inherent in the disease process
can be
elucidated, thus allowing for the development of better therapeutic and
diagnostic
approaches against pathogenic microbes.
Along with water sanitation, prevention of infectious diseases by vaccination
is the
most efficient, cost-effective, and practical method of disease prevention. No
other
modality, not even antibiotics, has had such a major effect on mortality
reduction and
population growth. The impact of vaccination on the health of the world's
people is hard to
exaggerate. Vaccination, at least in parts of the world, has controlled the
following nine
major diseases: smallpox, diphtheria, tetanus, yellow fever, pertussis,
poliomyelitis,
measles, mumps and rubella. In the case of smallpox, the disease has been
totally
eradicated from the world. The effectiveness of a vaccine depends upon its
ability to elicit
a protective immune response, which will be generally described below.
The means by which vertebrates, particularly birds and mammals, overcome
microbial pathogenesis is complex. Pathogens that invade a host provoke a
number of
highly versatile and protective systems. If the microbial pathogen or its
toxins successfully
penetrate the body's outer defenses and reach the bloodstream, then the
lymphoid tissue of
the spleen, liver, and bone marrow will remove and destroy the foreign
material as the
blood circulates through these organs. Lymphoid tissue is composed primarily
of a
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meshwork of interlocking reticular cells and fibers. Clinging to the
interstices of the tissues
are large numbers of leukocytes, more specifically, lymphocyte cells, and
other cells in
various stages of differentiation, such as plasma cells, lymphoblasts,
monocyte-
macrophages, eosinophils and mast cells. The two main lymphocytes, T cells and
B cells,
have different and complementary roles in the mediation of the antigen-
specific immune
response.
The immune response is an exceedingly complex and valuable homeostatic
mechanism that has the ability to recognize foreign pathogens. The initial
response to
foreign pathogen is called "innate immunity" and is characterized by the rapid
migration of
natural killer cells, macrophages, neutrophils, and other leukocytes to the
site of the foreign
pathogen. These cells can either phagocytose, digest, lyse, or secrete
cytokines that lyse
the pathogen in a short period of time. The innate immune response is not
antigen-specific
and is generally regarded as a first line of defense against foreign pathogens
until the
"adaptive immune response" can be generated. Both T cells and B cells
participate in the
adaptive immune response. A variety of mechanisms are involved in generating
the
adaptive immune response. A discussion of all the possible mechanisms of
generating the
adaptive immune response is beyond the scope of this section, however, some
mechanisms
which have been well-characterized include B cell recognition of antigen and
subsequent
activation to secrete antigen-specific antibodies and T cell activation by
binding to antigen
presenting cells.
Microbial organisms can have cell membranes that are recognized as foreign by
the
immune system. In addition, microbial organisms may also produce toxins or
proteins that
are also considered foreign by the host's immune system. The first mechanism
mentioned
above involves the binding of antigen, such as bacterial cell wall or
bacterial toxin, to the
surface immunoglobulin receptors on B cells. The receptor binding transmits a
signal to
the interior of the B cell. This is what is commonly referred in the art as
"first signal". In
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some cases, only one signal is needed to activate the B cells. These antigens
that can
activate B cell without having to rely on T cell help are commonly referred to
as T-
independent antigens (or thymus-independent antigens). In other cases, a
"second signal"
is required and this is usually provided by T helper cells binding to the B
cell. When T cell
help is required for the activation of the B cell to a particular antigen, the
antigen is then
referred to as T-dependent antigen (or thymus-dependent antigen). In addition
to binding
to the surface receptors on the B cells, the antigen can also be internalized
by the B cell and
then digested into smaller fragment within the B cell and presented on the
surface of B cells
in the context of antigenic peptide-MHC class II molecules. These peptide-MHC
class II
molecules are recognized by T helper cells that bind to the B cell to provide
the "second
signal" needed for some antigens. Once the B cell has been activated, the B
cells begin to
secrete antibodies to the antigen that will eventually lead to the
inactivation of the antigen.
Another way for B cells to be activated is by contact with follicular
dendritic cells (FDCs)
within germinal centers of lymph nodes and spleen. The follicular dendritic
cells trap
antigen-antibody (Ag-Ab) complexes that circulate through the lymph node and
spleen and
the FDCs present these to B cells to activate them.
Another well-characterized mechanism of adaptive immune response to antigens
is
the activation of T cells by binding to antigen presenting cells such as
macrophages and
dendritic cells. Macrophages and dendritic cells are potent antigen presenting
cells.
Macrophages have a variety of receptors that recognize microbial constituents
such as
macrophage mannose receptor and the scavenger receptor. These receptors bind
microorganisms and the macrophage engulfs them and degrades the microorganisms
in the
endosomes and lysosomes. Some microorganisms are destroyed directly this way.
Other
microorganisms are digested into small peptides that are then presented to T
cells on the
surface of the macrophages in the context of MHC class II-peptide complexes. T
cells that
bind to these complexes become activated. Dendritic cells are also potent
antigen
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presenting cells and present peptide-MHC class I molecules and peptide-MHC
class II
molecules to activate T cells.
When a B cell binds to an antigen which has never been encountered, the cell
undergoes a developmental pathway called "isotype switching". During the
developmental
changes, the plasma cells switch from producing general IgM type antibodies to
producing
highly specific IgG type antibodies. Within this population of cells, some
undergo repeated
divisions in a process called "clonal expansion". These cells mature to become
antibody
factories that release immunoglobulins into the blood. When they are fully
mature, they
become identified as plasma cells, cells that are capable of releasing about
2,000 identical
antibody molecules per second until they die, generally within 2 or 3 days
after reaching
maturity. Other cells within this group of clones never produce antibodies but
function as
memory cells that will recognize and bind that particular antigen upon
encountering the
antigen.
As a consequence of the initial challenge by an antigen there are now many
more
cells identical to the original B cell or parent cell, each of which is able
to respond in the
same way to the antigen as the original B cell. Consequently, if the antigen
appears a
second time, it will encounter one of the correct B cells sooner, and since
these B cells are
programmed for the specific IgG antibody, the immune response will begin
sooner,
accelerate faster, be more specific and produce greater numbers of antibodies.
This event is
considered a secondary or anamnestic response. Figure 1 shows a comparison of
the
antibody titer present as a result of the primary and secondary responses.
Immunity can
persist for years because memory cells survive for months or years and also
because the
foreign material is sometimes reintroduced in minute doses that are sufficient
to constantly
trigger low-level immune responses. In this way the memory cells are
periodically
replenished.
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Following the first exposure to an antigen the response is often slow to yield
antibody and the amount of antibody produced is small, i.e., the primary
response. On
secondary challenge with the same antigen, the response, i.e., the secondary
response, is
more rapid and of greater magnitude thereby achieving an immune state equal to
the
accelerated secondary response following reinfection with a pathogenic
microorganism,
which is the goal that is sought to be induced by vaccines.
In general, active vaccines can be divided into two general classes: subunit
vaccines
and whole organism vaccines. Subunit vaccines are prepared from components of
the
whole organism and are usually developed in order to avoid the use of live
organisms that
may cause disease or to avoid the toxic components present in whole organism
vaccines, as
discussed in further detail below. The use of purified capsular polysaccharide
material of
H. influenza type b as a vaccine against the meningitis caused by this
organism in humans
is an example of a vaccine based upon an antigenic component. See Parks, et
al., J. Inf.
Dis., 136 (Suppl.):551 (1977), Anderson, et al., J. Inf. Dis., 136
(Suppl.):563 (1977); and
Makela, et al., J. Inf. Dis., 136 (Suppl.):543 (1977).
Classically, subunit vaccines have been prepared by chemical inactivation of
partially purified toxins, and hence have been called toxoids. Formaldehyde or
glutaraldehyde have been the chemicals of choice to detoxify bacterial toxins.
Both
diphtheria and tetanus toxins have been successfully inactivated with
formaldehyde
resulting in a safe and effective toxoid vaccine which has been used for over
40 years to
control diphtheria and tetanus. See, Pappenheimer, A. M., Diphtheria. In:
Bacterial
Vaccines (R. Germanier, ed.), Academic Press, Orlando, FL, pp. 1-36 (1984);
Bizzini, B.,
Tetanus. Id. at 37-68. Chemical toxoids, however, are not without undesirable
properties.
In fact, this type of vaccine can be more difficult to develop since
protective antigens must
first be identified and then procedures must be developed to efficiently
isolate the antigens.
Furthermore, in some cases, subunit vaccines do not elicit as strong an immune
response as
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do whole organism vaccines due to the lack of extraneous materials such as
membranes or
endotoxins that may be more immunogenic due to the removal of materials that
would
otherwise mask the protective antigens or that are immunodominant.
Whole organism vaccines, on the other hand, make use of the entire organism
for
vaccination. The organism may be killed or alive (usually attenuated)
depending upon the
requirements to elicit protective immunity. The pertussis vaccine, for
example, is a killed
whole cell vaccine prepared by treatment of Bordetella pertussis cells with
formaldehyde.
The bacterium B. pertussis colonizes the epithelial lining of the respiratory
tract resulting in
a highly contagious respiratory disease in humans, pertussis or whooping
cough, with
morbidity and mortality rates highest for infants and young children. The
colonization
further results in local tissue damage and systemic effects caused in large
part by toxins
produced by B. pertussis. See, Manclarck, et al., Pertussis., Id. at 64-106.
These toxins
include endotoxin or lipopolysaccharide, a peptidoglycan fragment called
tracheal
cytotoxin, a heat-labile dermonecrotizing protein toxin, an adenylated cyclase
toxin, and
the protein exotoxin pertussis toxin. Vaccination is the most effective method
for
controlling pertussis, and killed whole-cell vaccines administered with
diphtheria and
tetanus toxoids (DPT vaccine) have been effective in controlling disease in
many countries.
See, Fine, et al., Reflections on the Efficacy of Pertussis Vaccines, Rev.
Infect. Dis., 9:866-
883 (1987). Unfortunately, due to the large amounts of endogenous products,
discussed
above, contained in the pertussis vaccine, many children experience adverse
reactions upon
injection. Endotoxin, which is an integral component of the outer membrane of
the gram-
negative organism (as well as all other gram-negative organisms), can induce a
wide range
of mild to severe side effects including fever, shock, leukocytosis, and
abortion. While the
side effects associated with pertussis vaccine usually are mild, they may be
quite severe.
The toxic components present in influenza virus vaccines, however, can induce
a strong
pyrogenic response and have been responsible for the production of Guillain-
Barre
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syndrome. Since influenza vaccines are prepared by growth of the virus in
chick embryos,
it is likely that components of the embryo or egg contributes to this
toxicity.
The use of killed vaccines has also been described by Switzer et al., U.S.
Patent No.
4,016,253, who applied such a method in preparing a vaccine against Bordetella
bronchiseptica infection in swine. In a technical paper by Brown, et al., Br.
Med. J., 1:263
(1959), the use of killed whole cells is disclosed for preparing a vaccine
against chronic
bronchitis caused by Haemophilus influenzae. The use of killed cells, however,
is usually
accompanied by an attendant loss of immunogenic potential, since the process
of killing
usually destroys or alters many of the surface antigenic determinants
necessary for
induction of specific antibodies in the host. Killed vaccines are ineffective
or marginally
effected for a number of pathogenic bacteria including Salmonella spp. and Ij
cholerae.
The parenteral killed whole cell vaccine now in use for Salmonella typhi is
only moderately
effective, and causes marked systemic and local adverse reactions at an
unacceptably high
frequency.
In the case of intracellular pathogens, such as Salmonella, it is generally
agreed that
vaccines based on live but attenuated microorganisms (live vaccines) induce a
highly
effective type of immune response. Live attenuated vaccines are comprised of
living
organisms that are benign but typically can replicate in a host tissues and
presumably
express many natural target immunogens that are processed and presented to the
immune
system similar to a natural infection. This interaction elicits a protective
response as if the
immunized individual had been previously exposed to the disease. Most of the
work
defining attenuating mutations for the construction of live bacterial vaccines
has been
performed in S. spp. since they establish an infection by direct interaction
with the gut
associated lymphoid tissue (GALT), resulting in a strong humoral immune
response. They
also invade host cells and thus are capable of eliciting a strong cell
mediated response.
Eisenstein (1999) Intracellular Bacterial l~accine Vectors (Paterson, ed.,
Wiley-Liss, Inc.)
to
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pp. 51-109; Hone et al. (1999) Intracellular Bacterial Vaccine Vectors
(Paterson, ed.,
Wiley-Liss, Inc.) pp. 171-221; Sirard et al. (1999) Immure. Rev. 171:5-26.
Ideally, these
attenuated microorganisms maintain the full integrity of cell-surface
constituents necessary
for specific antibody induction yet are unable to cause disease, because, for
example, they
fail to produce virulence factors, grow too slowly, or do not grow at all in
the host.
Additionally, these attenuated strains should have substantially no
probability of reverting
to a virulent wild-type strain. Traditionally, live vaccines have been
obtained by either
isolating an antigenically related virus from another species, by selecting
attenuation
through passage and adaptation in a nontargeted species or in tissue cultures,
or by
selection of temperature-sensitive variants. The first approach was that used
by Edward
Jenner who used a bovine poxvirus to vaccinate humans against smallpox.
Selecting attenuation through serial passages in a nontargeted species is the
second
approach that has been widely successful in obtaining live vaccines. For
example,
Parkman, et al., N. Engl. J. Med., 275:569-574 (1966), developed an attenuated
rubella
vaccine after serial multiplication in green monkey kidney cells. A measles
vaccine has
been prepared by passaging the virus in chick embryo fibroblasts. Vaccines
against, polio,
hepatitis A, Japanese B encephalitis, dengue, and cytomegalovirus have all
been prepared
following similar procedures.
While animal models, and especially monkeys, are useful in developing live
vaccines by serial passages and selection, a large uncertainty as to whether a
vaccine is
truly nonpathogenic remains until humans have been inoculated. For example,
the Daker
strain of yellow fever produced from infected suckling mouse brains induced
encephalitis
in 1 % of vaccines. Another crucial problem is the possible contamination of
the vaccine by
exogenous viruses during passages in cell culture or in animals, especially in
monkeys. In
light of the more recent knowledge of the potential danger of viruses that can
be
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transmitted from animals to humans, this choice of developing live vaccines is
highly
questionable.
In contrast to the somewhat haphazard approaches of selecting for live
vaccines,
discussed above, modern developmental approaches introduce specific mutations
into the
genome of the pathogen which affects the ability of that pathogen to induce
disease.
Defined genetic manipulation is the current approach being taken in an attempt
to develop
live vaccines for various diseases caused by pathogenic microorganisms.
In an effort to develop live vaccines which are safer and elicit a higher
immunological response, researchers have focused their efforts to developing
live vaccines
having specific genetic mutations. Curtiss, in U.S. Patent No. 5,294,441,
discloses that S.
typhi can be attenuated by constructing deletions (0) in either or both the
cya (adenylate
cyclase) and crp (cyclic 3', 5 ~-AMP [CAMP] receptor protein) genes. CAMP and
the
cAMP receptor protein, the products of pleiotropic genes cya and crp,
respectively,
function in combination with one another to form a regulatory complex that
affects
transcription of a large number of genes and operons. Consequently, mutating
either of
these genes results in an attenuated microorganism. Furthermore,
microorganisms having
single mutations in either the cya or crp genes can not supplement their
deficiency by
scavenging these gene products from a host to be vaccinated. The crp gene
product is not
available in mammalian tissues, and while the metabolite produced by the cya
gene
product, cAMP, is present in mammalian cells, the concentrations present in
the cells which
S typhi invades are below the concentrations necessary to allow cya mutants to
exhibit a
wild-type phenotype. See, Curtiss, et al., Infect. Immun., 55:3035-3043
(1987).
Since cAMP is present in host tissues at some level, Curtiss et al. stabilized
the
AC~Jcya microorganisms by introducing a mutation into the crp gene. Tacket, et
al., Infect.
Immun., 60(2):563-541 (1992), conducted a study with healthy adult in-patient
volunteers
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which revealed that attenuated S. typhi having deletions in the cya and crp
genes have the
propensity to produce fever and bacteremia (bacteria in the blood).
A similar approach in the attempt to develop live vaccines has been taken by
Dr. B.A.D. Stocker. The genes mutated by Stocker produce products which are
also not
available in host tissues. Stocker, in U.S. Patent No. 5,210,035, describes
the construction
of vaccine strains from pathogenic microorganisms made non-virulent by the
introduction
of complete and non-reverting mutational blocks in the biosynthesis pathways,
causing a
requirement for metabolites not available in host tissues. Specifically,
Stocker teaches that
S. typhi may be attenuated by interrupting the pathway for biosynthesis of
aromatic (aro)
metabolites which renders Salmonella auxotrophic (i.e., nutritionally
dependent) forp-
aminobenzoic acid (PABA) and 2, 3-dihydroxybenzoate, substances not available
to
bacteria in mammalian tissue. These aro-mutants are unable to synthesize
chorismic acid
(a precursor of the aromatic compounds PABA and 2, 3-dihydroxybenzoate), and
no other
pathways in Salmonella exist that can overcome this deficiency. As a
consequence of this
auxotrophy, the aro-deleted bacteria are not capable of proliferation within
the host;
however they reside and grow intracellularly long enough to stimulate
protective immune
responses. In the technical paper authored by Tacket, et al., discussed above,
attenuated
strains of S. typhi were also constructed for use as vaccines by introducing
deletions in the
aroC and aroD genes, according to Stocker. However, these attenuated strains
administered to healthy in-patient volunteers have the propensity to produce
fever and
bacteremia. (Hone et al. (1987), Hormaeche et al. (1996) Vaccine 14:251-259;
Hassan and
Curtiss (1997) Avian Dis. 41:783-791; and Miller et al. (1990) Res.
Microbfiol. 141:817-
821 ).
Comparative studies between these vaccines have not been rigorously tested and
thus the efficacy of these current strains with respect to each other remains
unclear.
Moreover, toxicity (e.g., symptoms such as diarrhea) of current live bacterial
vaccine
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candidates and the reality that many individuals within the human population
are
immunocompromised clearly warrants the search for additional vaccines that
offer better
protection, are longer lasting, and have less toxicity.
Another significant problem with vaccine development is the fact that many
pathogenic species are comprised of multiple serotypes that can cause disease
in animal
hosts vaccinated against a similar pathogenic strain. Previous attempts at
developing a
long-term cross-protective Salmonella vaccine have often been problematic. For
example,
live attenuated aroA Salmonella strains have been shown to elicit a cross-
protective
response against heterologous serotypes (e.g., group B (typhimurium) and Group
D
(entev~itidis and dublin)) strains, but the cross-protective capacity is
virtually eliminated
after the vaccine is cleared from the immunized animals. Hormaeche et al.
(1996).
Like many cellular macromolecules, DNA is subject to postsynthetic
"modification" by addition of small chemical moieties to the intact polymer.
In a variety of
organisms this involves enzymatic addition of methyl (-CH3) groups to DNA,
either at
position CS of cytosine or at position N6 of adenosine, shown in Figure 2. The
enzymes
responsible for the addition of methyl groups to DNA are known as DNA
methyltransferases or DNA methylases. DNA methylases can be divided into two
classes:
(1) those that methylate cytosine (DNA cytosine methylases); and (2) those
that methylate
adenine (DNA adenine methylases).
Methylation at adenine residues by DNA adenine methylase (Dam) controls the
timing and targeting of important biological processes such as DNA
replication, methyl-
directed mismatch repair, and transposition (Marinus, E. coli and Salmonella:
Cellular and
Molecular biology, 2nd ed., 782-791 (1996)). In addition, in E. coli, Dam
regulates the
expression of operons such as pyelonephritis-associated pili (pap) which are
an important
virulence determinant in upper urinary tract infections (Roberts, et al., J.
Urol., 133:1068-
1075 (1985)); van der Woude, et al., Trends Microbfiol., 4:5-9 (1996). The
latter regulatory
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mechanism involves formation of heritable DNA methylation patterns, which
control gene
expression by modulating the binding of regulatory proteins.
There remains a serious need for vaccines that are prepared from live,
pathogenic
microorganisms which are safe and when administered to a host and will induce
an
effective type of immune response in the host. It is also very desirable to
develop a single
vaccine strain that is capable of stimulating an immune response against a
different strain
(i.e., heterologous serotypes or species). There is also a further need for
safe and effective
antimicrobial drugs that may be used to treat patients afflicted by disease
caused by
pathogenic microorganisms.
All references and patent applications cited within this application are
herein
incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
This invention based on the discoveries that DNA adenine methylase (Dam) is
essential for Salmonella pathogenesis and that Dam Salmonella are effective as
live
attenuated vaccines against murine typhoid fever and elicit an immune response
against a
second species of Salmonella. Since DNA adenine methylases are highly
conserved in
many pathogenic bacteria that cause significant morbidity and mortality, Dam
derivatives
of these pathogens may be effective as live attenuated vaccines. Moreover,
since
methylation of DNA adenine residues is essential for bacterial virulence,
drugs that alter
the expression of or inhibit the activity of DNA adenine methylases are likely
to have broad
antimicrobial action and thus genes that encode DNA adenine methylases and
their
products are promising targets for antimicrobial drug development.
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Accordingly, it is an object of this invention to provide live vaccines for
vaccinating
a host against a pathogenic microorganism or a spectrum of similar pathogenic
microorganisms.
It is a further object of this invention to provide live vaccines which serve
as
carriers for antigens, preferably immunogens of other pathogens, particularly
microorganisms, including viruses, prokaryotes, and eukaryotes.
It is yet another object of this invention to provide antimicrobial drugs that
specifically inhibit DNA adenine methylases and the genes responsible for the
production
of DNA adenine methylases. Furthermore, the compositions of the present
invention
comprise natural and synthetic molecules having inhibitory effects on (i) DNA
adenine
methylase enzymatic activities, (ii) expression of DNA adenine methylases,
(iii) DNA
adenine methylase activators, (iv) activating compounds for DNA adenine
methylase
repressors, and/or (v) virulence factors that are regulated by DNA adenine
methylases.
Accordingly, in one aspect the invention provides immunogenic compositions
comprising live attenuated pathogenic bacteria in a pharmaceutically
acceptable excipient,
said pathogenic bacteria containing a mutation which alters DNA adenine
methylase (Dam)
activity such that the pathogenic bacteria are attenuated.
In another aspect, the invention provides immunogenic compositions comprising
killed pathogenic bacteria in a pharmaceutically acceptable excipient, said
pathogenic
bacteria containing a mutation which alters DNA adenine methylase (Dam)
activity.
In another aspect, the invention provides attenuated strains of pathogenic
bacteria,
said bacteria containing a mutation which alters Dam activity such that the
bacteria are
attenuated.
In another aspect, the invention provides methods of eliciting an immune
response
in an individual comprising administering which entail administering any of
the
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compositions described herein to the individual in an amount sufficient to
elicit an immune
response.
In another aspect, the invention provides methods of preventing infection by
pathogenic bacteria in an individual, comprising administering any of the
immunogenic
compositions described herein to the individual in an amount sufficient to
reduce a
symptom associated with infection by the pathogenic bacteria upon infection by
the
pathogenic bacteria.
In another aspect, the invention provides methods of treating a pathogenic
bacterial
infection in an individual, comprising administering any of the immunogenic
compositions
described herein to the individual in an amount sufficient to reduce a symptom
associated
with infection by the pathogenic bacteria in the individual.
In another aspect, the invention provides methods of treating an individual
infected
with a pathogenic bacteria, comprising administering to the individual a
composition
comprising an agent which alters Dam activity.
In another aspect, the invention provides methods of eliciting an immune
response
against a second species of Salmonella in an individual, comprising
administering to the
individual an immunogenic composition comprising an attenuated first species
of
Salmonella, said first species containing a mutation which alters Dam activity
such that the
Salmonella is attenuated.
In another aspect, the invention also provides screening methods. The
invention
includes methods of identifying an agent which may have anti-bacterial
activity comprising
using an in vitro transcription system to detect an agent which alters the
level of
transcription from a dam gene when the agent is added to the in vitro
transcription system,
wherein an agent is identified by its ability to alter the level of
transcription from the dam
gene as compared to the level of transcription when no agent is added.
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In another aspect, the invention provides methods of identifying an agent
which
may have anti-bacterial activity comprising using an in vitro translation
system to detect an
agent which alters the level of translation from an RNA transcript encoding
Dam when the
agent is added to the in vitro transcription system, wherein an agent is
identified by its
ability to alter the level of translation from the RNA transcript encoding Dam
as compared
to the level of translation when no agent is added.
In another aspect, the invention provides methods of identifying an agent
which
may have anti-bacterial activity comprising determining whether the agent
binds to Dam,
wherein an agent is identified by its ability to bind to Dam.
In another aspect, the invention provides methods of identifying an agent
which
may have anti-bacterial activity comprising the steps of: (a) incubating non-
methylated
oligonucleotides comprising a Dam binding site with Dam, S-
adenosylamethionine, and an
agent, wherein said nonmethylated oligonucleotide further comprises a signal;
(b)
digesting all nonmethylated target sites, thereby releasing said nonmethylated
oligonucleotides; and (c) detecting inhibition of DNA adenine methylase as an
increase in
said signal due to digestion of said nonmethylated target sites, wherein an
agent is
identified by its ability to cause an increase in signal compared to
conducting steps (a), (b),
and (c) in absence of agent.
In another aspect, the invention provides methods of identifying an agent
which
may have anti-bacterial activity comprising the steps of: (a) contacting an
agent to be tested
with a suitable host call that has Dam function; and (b) analyzing at least
one characteristic
which is associated with alteration of Dam function, wherein an agent is
identified by its
ability to elicit at least one said characteristic.
The invention also provides methods of preparing the vaccines and strains
described
herein. In one aspect, the invention provides methods of preparing the
immunogenic
compositions described herein, comprising combining a pharmaceutically
excipient with
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pathogenic bacteria containing a mutation which alters DNA adenine methylase
(Dam)
activity such that the pathogenic bacteria are attenuated.
In another aspect, the invention provides methods for preparing attenuated
bacteria
capable of eliciting an immunological response by a host susceptible to
disease caused by
the corresponding or similar pathogenic microorganism comprising constructing
at least
one mutation in said pathogenic bacteria wherein a first mutation results in
altered Dam
function.
Another object of this invention is to provide a method whereby a vaccine may
be
produced by altering the expression of a global regulator of virulence genes
and, more
specifically, by altering the expression of DNA adenine methylases.
Another object of this invention is to provide a method whereby a vaccine may
be
produced by altering the expression of genes regulated by DNA adenine
methylases.
In another aspect, the invention provides methods for preparing a live vaccine
from
a virulent pathogenic bacteria, such as Salmonella, comprising altering the
expression of
DNA adenine methylases and/or the expression of genes that are regulated by
DNA
adenine methylases in a virulent strain of a pathogenic bacteria that is, or
is similar to, the
microorganism desired to be vaccinated against.
It is yet a further object of this invention to provide a method of treating a
host, such
as a vertebrate infected with a pathogen by administering to the vertebrate a
compound or
compounds that alter the expression of or inhibit the activity of DNA adenine
methylases.
Additional objects, advantages and novel features of this invention shall be
set forth
in part in the description that follows, and in part will become apparent to
those skilled in
the art upon examination of the following specification or may be learned by
the practice of
the invention. The objects and advantages of the invention may be realized and
attained by
means of the instrumentalities, combinations, compositions, and methods
particularly
pointed out in the appended claims.
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BRIEF DESCRIPTION OF THE DRAWINGS
The accompanying drawings, which are incorporated herein and form a part of
the
specification, assist in illustrating the present invention and, together with
the description,
serve to explain the principles of the invention. The accompanying drawings
are not drawn
to scale.
Figure 1 is a graphic representation of the levels of antibody present
following the
primary and secondary immune responses.
Figure 2 is a schematic representation of the sites of methylation that occur
on
cytosine and adenine.
Figure 3 is a graphic representation illustrating that Dam regulates in vivo
induced
genes. (3-galactosidase expression from S. typhimurium ivi fusions in Dam+ and
Dam
strains grown in LB. The vertical axis shows [3-galactosidase activities (~-
moles of o-
nitrophenol (ONP) formed per minute per A6oo unit per milliliter of cell
suspension x 103)
Figure 4 is a graphic representation illustrating that Dam represses PhoP
activated
genes. [3-galactosidase expression from S. typhimurium ivi fusions grown in
minimal
medium. The vertical axis shows (3-galactosidase activities (p-moles of o-
nitrophenol
(ONP) formed per minute per A6oo unit per milliliter of cell suspension x
103). The dam
genotype is shown below the horizontal axis, and the phoP genotype is shown as
black
(PhoP+) and gray (PhoP-) boxes.
Figure 5 shows that PhoP affects the formation of Salmonella DNA methylation
patterns. DNA methylation patterns formed in PhoP+ and PhoP- strains grown in
minimal
medium. The arrows depict DNA fragments that are present in PhoP- Salmonella
but are
absent in PhoP+ Salmonella.
Figure 6 are graphs depicting the amount and tissue distribution of Salmonella
in
mice immunized with Dam mutants (solid boxes) or not immunized (open boxes) on
day 1
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and day 5. PP, Peyer's patches; MLN, mesenteric lymph nodes; CFU, colony
forming
units.
Figure 7 are graphs depicting amount and tissue distribution of Salmonella in
mice
immunized with Dam mutants (solid boxes) or not immunized (open boxes) on day
1, 5,
14 and 28. PP, Peyer's patches; MLN, mesenteric lymph nodes; CFU, colony
forming
units.
Figures 8 (A) - (C) are half tone reproductions of 2D gel electrophoresis of
whole-
cell protein abstracts of S. typhimurium showing proteins produced in Dam
strain (dam
non-polar deletion, MT2188; (A)); Dam+ strain (wild type, ATCC 14028 (B)); and
Dam+++
strain (overproducer, MT2128).
DETAILED DESCRIPTION
We have discovered that the dam gene and its product enzyme DNA adenine
methylase (Dam) are required for bacterial virulence. Despite previous
research efforts
directed to Dam functions, the critical role of Dam in bacterial virulence,
the inventive
implications of this role, as well as the ability of a Dam mutant vaccine to
elicit a
protective immune response, have not been reported. Previously, all reported
dam
mutations from other laboratories used Salmonella strain LT2 which is at least
1000-fold
less virulent that than the wild type when delivered intraperitoneally.
Equipped with the
knowledge of this discovery, the present invention is directed towards (a)
vaccines having
non-reverting genetic mutations in either (i) genes that would alter a
function, such as
expression, of DNA adenine methylases and/or (ii) genes that are regulated by
DNA
adenine methylases; (b) a class of inhibitors that are natural and/or
synthetic molecules
having binding specificity for (i) DNA adenine methylases and/or genes that
encode DNA
adenine methylases, (ii) activators of DNA adenine methylases and/or
activating
compounds for repressors of DNA adenine methylases, and (iii) virulence
factors that are
regulated by Dam; (c) methods for preparing vaccines and inhibitors based on
the
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knowledge that DNA adenine methylase is essential for bacterial pathogenesis;
(d) methods
of eliciting an immune response using the immunogenic compositions described
herein;
(e) methods for treating vertebrates with (i) the vaccines of the present
invention prior to
their becoming infected or (ii) the inhibitors of the present invention after
their becoming
infected with a pathogenic microorganism; (f) methods of preventing infection
using the
immunogenic compositions described herein; and (g) screening methods to
identify
compounds which may be useful therapeutic agents.
As described in the Examples, the oral lethal dose of a Dam mutant (created by
an
insertion in the dam gene (Mud-Cm)) in S. typhimurium required to kill 50% of
the animals
(LDso) was increased over 10,000-fold and the intraperitoneal (i.p.) LDso was
increased
over 1,000 fold compared to wild type (Example 1; Table 1). Further, the
highly attenuated
Dam mutants were found to confer a protective immune response in an art-
accepted model
of murine typhoid fever (Example 2; Table 2). All 17 mice immunized with a S.
typhimurium Dam insertion strain survived a wild-type challenge of 10+4 above
the LDso,
whereas all 12 nonimmunized mice died following challenge. Survival studies
comparing
Dam+ to Dam Salmonella showed that Dam bacteria were fully proficient in
colonization
of a mucosal site (Peyer''s patches) but showed severe defects in colonization
of deeper
tissue sites (Example 2; Figure 6). Without wishing to be bound by theory, the
inventors
note that one possible explanation of why Dam elicits protective immune
response is
because the mutant bacteria grow in intestinal mucosa long enough to elicit an
immune
response but are unable to invade and/or colonize deeper tissue.
Even more striking, especially in view of the widely held tenet in the art
that a
vaccine containing one species of Salmonella could not elicit an immune
response against a
second species of Salmonella, or at least a significant, lasting immune
response against a
second strain, especially if the species is attenuated due to mutation in a
single gene, our
data show such cross-protection. Mice immunized with Dam S. typhimurium
(serogroup
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B) were protected against a heterologous challenge (100 to 1000 LDSO) with S.
enteritidis
and S. dublin (serogroup D) eleven weeks post immunization (Example 3; Table
3). This
protection persisted more than six weeks after the vaccine strain was cleared
from the
immunized animals (i.e., more than six weeks after the Dam organisms could not
be
detected in Peyer's patches, mesenteric lymph nodes, liver and spleen). In
contrast to the
Salmonella cross-protection, no protection was observed against Yersinia
pseudotuberculosis five weeks post immunization. Similarly, immunization with
Dam S.
enteritidis conferred cross-protection against S. typhimurium and S. dublin.
Although live
attenuated Salmonella strains have been shown to elicit cross-protection
between group B
(typhimurium) and group D (enteritidis and dublin) strains (attributed to a
shared common
LPS antigenic determinant), the cross-protective response is very short-lived,
and is
virtually eliminated ten to twelve weeks post immunization. Hormaeche et al.
(1996)
l~accine 251-259.
The ectopic expression in Dam derivatives (i.e., expression of proteins that
are
normally repressed) as described in Examples 1 and 3 has broad applications to
vaccine
development. Ectopic expression in Dam derivatives of many pathogens may yield
protective and/or cross-protective responses to the parent virulent organisms.
Salmonella
Dam derivatives may have utility as a platform to express passenger bacterial
and viral
antigens that elicit strong protective immune responses against the cognate
pathogen.
Since Dam immunized mice can clear a lethal bacterial load of fully-virulent
Salmonella
organisms, Dam vaccines may have therapeutic utility to effectively treat a
pre-existing
infection. Since Dam derivatives ectopically express multiple proteins, it
opens the
possibility that vaccines could be constructed in strains that are less
harmful to humans,
which would exploit the benefits of the high levels of protection elicited by
live vaccines
while reducing the risk of infection to immunocompromised individuals.
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The fact that DNA adenine methylase is essential for bacterial pathogenesis,
in, for
example, Salmonella is also of extreme importance, the implications of which
are many.
First, the dam gene is highly conserved in pathogenic bacteria, that is, the
gene sequence of
dam in one microorganism shares sequence identity with the dam gene in another
microorganism not only within the same species but also across bacterial
genera; and
second, the dam gene regulates many genes involved in virulence. Since DNA
adenine
methylases are highly conserved in many pathogenic bacteria that cause
significant
morbidity and mortality, such as Vibrio cholerae (Bandyopadhyay and Das, Gene,
140:67-
71 (1994), Salmonella typhi (1999-3, Sanger Centre), pathogenic E. coli
(Blattner, et al.,
Science, 277:1453-1474 (1997), Yersinia pesos (1999-3, Sanger Centre ),
Haemophilus
influenzae (Fleischmann, et al., Science, 269:496-512 (1995), and Treponema
pallidum
(Fraser, et al., Science, 281:375-388 (1998)), Dam derivatives of these
pathogens may be
effective as live attenuated vaccines. Moreover, since Dam is essential for
bacterial
virulence, Dam inhibitors are likely to have broad antimicrobial action and
thus Dam or
any gene that alters the expression of Dam is a promising target for
antimicrobial drug
development.
The implications of this are as follows: (1) it is now possible to rationally
develop a
class of inhibitors that are natural and/or synthetic molecules having binding
specificity for
(i) DNA adenine methylases and/or the dam gene, (ii) Dam activators and/or
activating
compounds for Dam repressors, and (iii) virulence factors that are regulated
by Dam; and
(2) it is now possible to produce vaccines having non-reverting genetic
mutations in either
(i) genes that would alter the expression of DNA adenine methylases and/or
(ii) virulence
genes that are regulated by DNA adenine methylases. Because Dam is a global
regulator of
gene expression and many of these regulated genes are conserved in various
species and
genera, it is highly probable that inhibitors and vaccines based on DNA
adenine methylase
will give cross-protection. Thus, as discussed above, an inhibitor or a
vaccine against one
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strain, species, serotype and/or group of pathogen would provide protection
against a
different strain of pathogen.
Compositions described herein may be used for administration to individuals.
They
may be administered, for example, for experimental purposes, or to obtain a
source of anti
s bacteria antibody, such as Salmonella antibody. They may also be
administered to elicit an
immune response in an individual as well as to protect an individual from
infection or to
treat an individual infected with a virulent bacteria, such as Salmonella.
General techniques
The practice of the present invention will employ, unless otherwise indicated,
conventional techniques of molecular biology (including recombinant
techniques),
microbiology, cell biology, biochemistry and immunology, which are within the
skill of the
art. Such techniques are explained fully in the literature, such as, Molecular
Cloning: A
Laboratory Manual, second edition (Sambrook et al., 1989); Oligonucleotide
Synthesis
(M.J. Gait, ed., 1984); Animal Cell Culture (R.I. Freshney, ed., 1987);
Methods in
Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M.
Wei &
C.C. Blackwell, eds.); Gene Transfer hectors for Mammalian Cells (J.M. Miller
& M.P.
Calos, eds., 1987); CuYrent Protocols in Molecular Biology (F.M. Ausubel et
al., eds.,
1987); PCR: The Polymerase Chain Reaction (Mullis et al., eds., 1994); Current
Protocols
in Immunology (J.E. Coligan et al., eds., 1991 ); Short Protocols in Molecular
Biology
(Whey & Sons, 1999).
Definitions
"DNA adenine methylases" (Dam) are defined as a group of enzymes which are
able to methylate adenine residues in DNA. Dam genes and Dam products encoded
by
dam genes are known in the art, and the definition includes Dam enzymes that
share
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significant amino acid similarity to the DNA adenine methylase from E. coli
(gi 118682)
and Salmonella (gi 2500157) and that preferentially methylate the sequence
"GATC" on
DNA, methylating the N-6 position of adenine. Particular highly conserved DNA
sequences encoding a region of Dam are depicted in SEQ ID NOS:I-4, as
described herein.
In accordance with art-accepted designations, "dam" or "dam gene" indicates a
gene
encoding a DNA adenine methylase, and "Dam" indicates a DNA adenine methylase
(i.e,
the polypeptide). For purposes of the present invention a gene is defined as
encompassing
the coding regions and/or the regulatory regions.
Dam "activity" or "function" means any bio-activity associated with dam
expression or non-expression. Dam activities are described herein. For
example, non-
expression of dam leads to repression (or, alternatively, de-repression) of
certain genes
regulated by Dam; thus, repression (or de-repression)of any of these genes is
a Dam
activity. As another example, methylation of adenine in DNA (for example,
methylation of
GATC) is an activity associated with dam expression and the resultant Dam
product; thus,
adenine methylation is a Dam activity. Dam "activity" or "function" thus
encompasses any
one or more bio-activities associated with dam expression.
An "alteration" of Dam activity is any change in any Dam activity, as compared
to
wild-type Dam function. An "alteration" may or may not be a complete loss of a
Dam
activity, and includes an increase or decrease of a Dam activity. Bacteria
which contain a
mutation that alters Dam activity are generally referred to as "Dam
derivatives."
"Expression" includes transcription and/or translation, as well as any factor
or event
which affects expression (such as an upstream event, such as a second gene
which affects
expression).
A "vaccine" is a pharmaceutical composition for human or animal use,
particularly
an immunogenic composition which is administered with the intention of
conferring the
recipient with a degree of specific immunological reactivity against a
particular target, or
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group of targets (i.e., elicit and/or enhance an immune response against a
particular target
or group of targets). The immunological reactivity, or response, may be
antibodies or cells
(particularly B cells, plasma cells, T helper cells, and cytotoxic T
lymphocytes, and their
precursors) that are immunologically reactive against the target, or any
combination
thereof. For purposes of this invention, the target is primarily a virulent
bacteria, such as
Salmonella. In instances where an attenuated bacteria is used as a carrier,
the target may be
another antigen as described herein. The immunological reactivity may be
desired for
experimental purposes, for treatment of a particular condition, for the
elimination of a
particular substance, and/or for prophylaxis.
"Pathogenic" bacteria are bacteria that are capable of causing disease.
"Virulence"
is a indicator of the degree of pathogenicity which may be numerically
expressed as the
ratio of the number of cases of overt infection to total number infected. It
is understood
that the pathogenic bacteria used in the vaccines described herein are other
than innocuous
strains commonly used in laboratories, and are known to and/or are capable of
causing
disease.
"Attenuated" bacteria used in the compositions described herein are bacteria
which
exhibit reduced virulence. As is well understood in the art, and described
above, virulence
is the degree to which bacteria are able to cause disease in a given
population. For
purposes of the invention, attenuated bacteria have virulence reduced to a
suitable and
acceptable safety level, as is generally dictated by appropriate government
agencies. The
degree of attenuation which is acceptable depends on, inter alia, the
recipient (i.e., human
or non-human) as well as various regulations and standards which are provided
by
regulatory agencies such as the U.S. Food and Drug Administration (FDA). Most
preferably, especially for human use, attenuated bacteria are avirulent,
meaning that
administration of these organisms cause no disease symptoms. As is well
understood in the
art, attenuated bacteria are alive, at least at the time of administration.
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"Antigen" means a substance that is recognized and bound specifically by an
antibody or by a T cell antigen receptor. As is well understood in the art,
antigens can
include peptides, proteins, glycoproteins, polysaccharides, gangliosides and
lipids, as well
as portions and/or combinations thereof. Antigens can be those found in nature
or can be
synthetic.
An "adjuvant" is a chemical or biological agent given in combination with an
attenuated bacteria as described herein to enhance its immunogenicity. As is
known in the
art, an "adjuvant" is a substance which, when added to an antigen,
nonspecifically enhances
or potentiates an immune response to the antigen in the recipient (host).
"Stimulating", "eliciting", or "provoking" an immune response (which can be a
B
and/or T cell response) means an increase in the response, which can arise
from eliciting
and/or enhancement of a response.
"Heterologous" means derived from and/or different from an entity to which it
is
being compared. For example, a "heterologous" antigen with respect to a
bacterial strain is
an antigen which is not normally or naturally associated with that strain.
An "effective amount" is an amount sufficient to effect a beneficial or
desired result
including a clinical result, and as such, an "effective amount" depends on the
context in
which it is being applied. An effective amount can be administered in one or
more doses.
For purposes of this invention, an effective amount of Dam derivative bacteria
(or a
composition containing Dam derivative bacteria) is an amount that induces an
immune
response. In terms of treatment, an effective amount is amount that is
sufficient to palliate,
ameliorate, stabilize, reverse or slow the progression of a bacterial disease,
or otherwise
reduce the pathological consequences of the disease. In terms of prevention,
an effective
amount is an amount sufficient to reduce (or even eliminate) one or more
symptoms upon
exposure and infection.
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"Treatment" is an approach for obtaining beneficial or desired clinical
results.
Beneficial or desired clinical results include, but are not limited to,
alleviation of
symptoms, diminishment of extent of disease, stabilized (i.e., not worsening)
state of
disease, preventing spread of disease, delay or slowing of disease
progression, amelioration
or palliation of the disease state.
"Preventing" disease or infection means a reduction (including, but not
limited to,
elimination) of one or more symptoms of infection in an individual receiving a
composition
described herein as compared to otherwise same conditions except for receiving
the
composition(s). As understood in the art, "prevention" of infection can
include milder
symptoms and does not necessarily mean elimination of symptoms associated with
infection.
An "individual", used interchangeably with "host", is a vertebrate, preferably
a
mammal, more preferably a human. Mammals include, but are not limited to, farm
animals
(such as cattle), sport animals, and pets. An "individual" also includes fowl,
such as
chickens. A "host" may or may not have been infected with a bacteria.
An "agent" means a biological or chemical compound such as a simple or complex
organic or inorganic molecule, a polypeptide, a polynucleotide, carbohydrate
or lipoprotein.
As vast array of compounds can be synthesized, for example oligomers, such as
oligopeptides and oligonucleotides, and synthetic organic compounds based on
various
core structures, and these are also included in the term "agent". In addition,
various natural
sources can provide compounds for screening, such as plant or animal extracts,
and the
like. Compounds can be tested singly or in combination with one another.
"Anti-bacterial activity" or "controlling virulence" means that an agent may
negatively affect the ability of bacteria to cause disease. For purposes of
the invention, an
agent which may control virulence is one which alters Dam activity, and may be
selected
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by the screening methods described herein, and further may, upon further
study, prove to
control bacterial virulence and may even exert therapeutic activity.
"Comprising" and its cognates mean "including".
"A", "an" and "the" include plural references, unless otherwise indicated. For
example, "a" Dam means any one or more DNA adenine methylases.
Compositions of the invention
The compositions described are useful for eliciting an immune response, and/or
treating or preventing disease associated with bacterial infection, especially
Salmonella
infection. Vaccines prepared from live, pathogenic bacteria are provided for
the
immunization or for the treatment of a host which is susceptible to disease
caused by the
corresponding pathogenic bacteria, by a similar pathogenic bacteria of the
same strain,
species, serotype, and/or group, or by a different bacteria of a different
strain, species,
serotype, and/or group. The live vaccines produced herein may also serve as
carriers for
antigens, such as immunogens of other pathogens thereby producing a multiple
immunogenic response.
Accordingly, in one embodiment, the invention provides an immunogenic
composition comprising live attenuated pathogenic bacteria, such as
Salmonella, and a
pharmaceutically acceptable excipient, said pathogenic bacteria containing
(having) a
mutation which alters DNA adenine methylase (Dam) activity such that the
pathogenic
bacteria are attenuated. In some embodiments, the mutation is in a gene
encoding a DNA
adenine methylase (Dam), wherein the mutation alters DNA adenine methylase
activity.
Dam activity may be increased or decreased, and Dam activity may be altered on
any level, including transcription and/or translation. With respect to
translation, for
example, activity can be altered in any number of ways, including the amount
of protein
produced and/or that nature (i.e., structure) of the protein produced. For
example, a
CA 02359469 2001-07-27
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mutation could result in increasing or reducing the amount of Dam produced by
the cell
(due to affecting transcriptional and/or post-transcriptional events);
alternatively, a
mutation could give rise to an altered Dam with altered activity. Generating
mutations and
mutants which alter Dam activity use techniques well known in the art. As an
example,
Dam production could be lowered by using a promoter which is known to initiate
transcription at a lower level. Assays to determine level of transcription
from a given
transcriptional regulatory element such as a promoter are well known in the
art. The native
dam promoter could be replaced with a promoter of lower transcriptional
activity;
alternatively, a dam- (in which native dam gene has been removed) could be
used as a basis
for integrating a re-engineered dam gene containing a lower activity promoter
to integrate
into the genome. Alternatively, a different dam gene could be used such as a
T4 dam. An
example of a dam over-producer, a pTP166 plasmid that produces E. coli Dam at
100-fold
wild-type level could be used. Mutations can be within the Dam gene itself
(including
transcriptional and/or translational regulatory elements) as well as a gene or
genes which
affect Dam production and/or activity.
Any pathogenic, preferably virulent, strain of bacteria may be used in the
immunogenic compositions described herein. In some embodiments, pathogenic
bacteria
other than E. coli are used. In other embodiments, pathogenic Esche~ichia is
used,
preferably E. coli. Because overexpression of dam can lead to a useful
vaccine, dam gene
may or may not be essential, i.e., deletion of dam may or may not be lethal.
The subject invention is particularly applicable to a wide variety of
Salmonella,
including any of the known groups, species or strains, more preferably groups
A, B, or D,
which includes most species which are specific pathogens of particular
vertebrate hosts.
Illustrative of the Salmonella-causing disease for which live vaccines can be
produced are
S. typhimurium; S. enteritidis, S. typhi; S. abortus-ovi; S. abortus-equi; S.
dublin; S.
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gallinarum; S. pullorum; as well as others which are known or may be
discovered to cause
infections in mammals.
Other organisms for which the subject invention may also be employed include
Yersinia spp., particularly Y. pesos, Vibrio spp., particularly V. cholerae,
Shigella spp.,
. particularly S. flexneri and S. sonnei; Haemophilus spp., particularly H.
influenzae, more
particularly type b; Bordetella, particularly B. pertussis; Neisseria,
particularly N
meningitides and N. gonorrohoeae; Pasteurella, particularly P. multocida,
pathogenic E.
coli, and Treponema such as T. pallidum; as well as others which are known or
may be
discovered to cause infections in mammals.
In another embodiment, the invention provides vaccines used to vaccinate a
host
comprising a pharmaceutically acceptable excipient and an attenuated form of a
pathogenic
bacteria, wherein attenuation is attributable to at least one mutation,
wherein a first
mutation alters either (i) the expression of or the activity of one or more
DNA adenine
methylases or (ii) the expression of one or more genes regulated by a DNA
adenine
methylase. The first mutation is preferably non-reverting, and in some
embodiments is
constructed in a gene whose product activates one or more of said DNA adenine
methylases. The first mutation may be constructed in a gene whose product
inactivates or
decreases the activity of one or more of said DNA adenine methylases. In other
embodiments, the first mutation is constructed in a gene whose product
represses the
expression of said DNA adenine methylases, and the gene product may repress
Dam. The
vaccine may further comprise a second mutation independent of said first
mutation with the
second mutation resulting in an attenuated microorganism. The second mutation
is
preferably non-reverting.
In another embodiment, the invention provides vaccines for provoking an
immunological response in a host to be vaccinated comprising a bacterial cell
having a
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mutation, introduced into a gene that disables the ability of said bacterial
cell to regulate the
expression of a DNA adenine methylase (Dam), which is expressed by the dam
gene.
The ectopic expression of multiple proteins in Dam vaccines suggests the
possibility that killed Dam organisms may elicit significantly stronger
protective immune
responses than killed Dam+ organisms. Accordingly, in some embodiments, the
invention
provides immunogenic compositions comprising killed pathogenic bacteria which
contain a
mutation which alters Dam activity and a pharmaceutically acceptable
excipient.
Preferably, the mutation is in the dam gene, and, as described herein, may
result in
reduction or increase in Dam activity. In some embodiments, the dam mutation
causes
death of the bacteria. In other embodiments, the mutation is attenuating, and
the bacteria
are killed by using methods well known in the art, such as sodium azide
treatment and/or
exposure to UV. In the instance where the mutation is lethal, the bacteria may
further be
treated for killing (e.g., using sodium azide and/or UV). Examples of bacteria
suitable for
these vaccines include, but are not limited to, Salmonella, Vibrio (including
V. cholerae)
and Yersinia (including Y. pseudotuberculosis).
Preferably, the compositions comprise a pharmaceutically acceptable excipient.
A
pharmaceutically acceptable excipient is a relatively inert substance that
facilitates
administration of a pharmacologically effective substance. For example, an
excipient can
give form or consistency to the vaccine composition, or act as a diluent.
Suitable
excipients include but are not limited to stabilizing agents, wetting and
emulsifying agents,
salts for varying osmolarity, encapsulating agents, buffers, and skin
penetration enhancers.
Examples of pharmaceutically acceptable excipients are described in
Remington's
Pharmaceutical Sciences (Alfonso R. Gennaro, ed., 19th edition, 1995).
The invention also comprises immunogenic compositions containing any
combination of the mutant strains described herein (whether attenuated or
killed), for a
given genus, such as Salmonella. Since the two different vaccine strains (such
as a Dam
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and a Dam overproducer) may produce two different repertoires of potentially
protective
antigens, use of them in combination may elicit a superior immune response.
Pathogenic bacteria, according to this invention, are made attenuated,
preferably
avirulent, as a result of a non-reverting mutation that is created in at least
one gene, which
thereby alters the a function of a DNA adenine methylase(s). Essentially, the
live vaccines
provided for, according to the preferred embodiment of the present invention,
originate
with a pathogenic bacteria. A non-reverting mutation is introduced into a gene
of the
pathogen, thus altering the expression of DNA adenine methylases. "Non-
reverting"
mutations generally revert in less than about 1 in 10g, preferably less than
about 1 in 101°,
or preferably less than about 1 in 1015, even more preferably less than 1 in
102° cell
divisions. Preferably, the mutation is non-leaky; however, regulation of genes
by Dam
appears to be exquisitely sensitive to Dam concentration. Therefore, over-
expression of
Dam as well as under expression of Dam results in the attenuation of the
pathogen. The
mutation is preferably made in the dam gene itself, however it is contemplated
in other
embodiments of the present invention, discussed in further detail below, that
the vaccines
according to the present invention may be produced by mutating a related gene
or genes
either "upstream" or "downstream" of dam whose products) activates) or
represses) the
dam gene or, in the alternative, a mutation is constructed in at least one
virulence gene that
is regulated by DNA adenine methylase. The mutation is non-reverting because
restoration
of normal gene function can occur only by random coincidental occurrence of
more than
one event, each such event being very infrequent. For example, Dam methylase
activity
can be down-regulated and/or shut off by introduction of deletions in the
promoter or
coding region, insertion of transposons or intervening DNA sequences into the
promoter or
coding regions, use of an antisense oligonucleotide that blocks expression of
the dam gene,
or use of a ribozyme that prevents dam gene expression. Alternatively, the
mutations) can
be an insertion and/or a deletion to an extent sufficient to cause non-
reversion.
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In the case of a deletion mutation, restoration of genetic information would
require
many coincidental random nucleotide insertions, in tandem, to restore the lost
genetic
information. In the case of an insertion plus inversion, restoration of gene
function would
require coincidence of precise deletion of the inserted sequence and precise
re-inversion of
the adj acent inverted sequence, each of these events having an exceedingly
minute,
undetectably low, frequency of occurrence. Thus, each of the two sorts of "non-
reverting"
mutations has a substantially zero probability of reverting to prototrophy.
Other methods of constructing an insertion in the Dam gene would be well known
and obvious to one skilled in the art.
While a single non-reverting mutation provides a high degree of security
against
possible reversion to virulence, there still remain events which, while
unlikely, have a finite
probability of occurrence. Opportunities for reversion exist where
microorganisms exist in
the host which may transfer by conjugation the genetic capability to the non-
virulent
organism. Alternatively, there may be a cryptic alternative pathway for the
production of
DNA adenine methylases which by rare mutation or under stress could become
operative.
Accordingly, in some embodiments, the attenuated bacteria described herein
further
comprise a second mutation. Live vaccines with two separate and unrelated
mutations
should be viable and reasonably long lived in the host, provide a strong
immune response
upon administration to the host, and they may also serve as a carrier for
antigens, such as
antigens of other pathogens, of other pathogens to provide immune protection
from such
pathogens.
Examples of Salmonella typhimurium attenuating mutations that may serve as
secondary mutations for live attenuated vaccine candidates are galE (galactose
induced
toxicity), pur and aro (aromatic compounds not available in vivo), crp and cya
(global
changes in gene expression via catabolite control), and phoP (global changes
in virulence
gene expression) (Hone, et al. (1987), Hormaeche, et al. (1996); Hassan and
Curtiss
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(1997); and Miller, et al. (1990)). Comparative studies between these vaccines
have not
been rigorously tested and thus the efficacy of these current strains with
respect to each
other remains unclear. Moreover, toxicity (e.g., symptoms such as diarrhea) of
current live
bacterial vaccine candidates and the reality that many individuals within the
human
population are immunocompromised clearly warrants the search for additional
vaccines
that offer better protection, are longer lasting, and have less toxicity.
In addition to the mutations discussed above, it is desirable that the
bacteria for use
as a live vaccine have one or more genetic "marker characters" making it
easily
distinguishable from other bacteria of the same species, either wild strains
or other live
vaccine strains. Accordingly, one chooses a strain of the pathogen which
desirably has a
marker for distinguishing the Dam mutant to be produced from other members of
the
strain. Alternatively, such a marker can be introduced into the vaccine
strain. Various
markers can be employed, as discussed previously. The markers) used should not
affect
the immunogenic character of the bacteria, nor should it interfere with the
processing of the
bacteria to produce the live vaccine. The marker will only alter the
phenotype, to allow for
recognition of the subject bacteria. For example, Dam mutants are sensitive to
the base
analog 2-amino purine (Miller, "Experiments in Molecular Genetics" CSHL 1972).
Since
the dam gene is genetically linked to cyst, one can use a pool of transposon
insertions to
transduce a cyst- recipient to cyst+. These prototrophs are screened for 2-
amino purine
sensitivity. To ensure that the insertion is in the dam gene, the insertion is
cloned and the
flanking region is sequenced. The marker may be some other nutritional
requirements also.
Such markers are useful in distinguishing the vaccine strain from wild type
strains.
The subject bacteria are then processed to provide one or more non-reverting
mutations. The first mutation will alter a Dam function, such as expression,
preferably, but
not necessarily, by mutating the dam gene. If a second mutation is desired, a
gene, the loss
of which is known to result in attenuation, is further mutated. The mutations
may be
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deletions, insertions, or inversions, or combinations thereof. Various
techniques can be
employed for introducing deletions or insertion inversions, so as to achieve a
bacteria
having the desired "non-leaky" non-reverting mutation resulting in an altered
expression of
dam. The presence of two completely independent mutations, each of which has
an
extremely low probability of reversion, provides almost absolute assurance
that the vaccine
strain cannot become virulent.
There are a number of well known techniques which can be employed for
disabling
or mutating genes, such as the employment of PCR techniques, translocatable
elements,
mutagenic agents, transducing phages, and DNA-mediated transformation, and/or
conjugation. Other methods also known to one with ordinary skill in the art
such as
recombinant DNA technology may also be employed to successively introduce one
or more
mutated genes into a single host strain to be used as the vaccine.
After manipulating the bacteria so as to introduce one or more non-reverting
mutations into some members of the population, the bacteria are grown under
conditions
facilitating isolation of the desired mutants, either under conditions under
which such
mutants have a selective advantage over parental bacteria or under conditions
allowing
their easy recognition from unaltered bacteria or mutants of other types. The
isolated
autotrophic mutants are then cloned, screened for virulence, their inability
to revert, and
their ability to protect the host from a virulent pathogenic strain.
The vaccines can be used with a wide variety of domestic animals, as well as
humans. Included among domestic animals which are treated by vaccines today or
could
be treated, if susceptible to bacterial diseases, are chickens, cows, pigs,
horses, goats, and
sheep, to name the more important domestic animals.
In accordance with the subject invention, the vaccines are produced by
introducing
a non-reverting mutation in at least one gene, where each mutation is of a
sufficient number
of bases in tandem to insure a substantially zero probability of reversion.
Preferably, the
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mutations) give rise to non-expression of each mutated gene, in the sense of
its total
inability to determine production of an active protein, although, as described
herein, Dam
overproducers may also be made. In addition, the gene chosen will be involved
in the
expression of a DNA adenine methylase and preferably the gene will be dam.
The resulting strain will be an avirulent live vaccine having the desired
immunogenicity, in that the mutation does not affect the production of the
antigens which
trigger the natural immune response of the host. Typically, when a wild type
pathogen
reaches a specific tissue within the host a specific virulence factor or set
of virulence
factors are expressed as a result of the specific environment to which the
pathogen is
exposed. It is believed that Dam mutants constitutively express many virulence
factors all
at the same time and not within specific tissues. Since the physiological
effect of many
virulence factors is tissue specific, the virulence factors that are
constitutively expressed in
the wrong tissues do not initiate the physiological changes inherent in the
disease process.
These virulence factors do, however, elicit an immune response from the host.
The
immune system thus encounters these factors in an environment where the
factors are not
able to initiate the necessary physiological changes in the host to cause
disease and the host
is able to mount an immune response.
In another embodiment of the present invention, the vaccines are produced by
introducing non-reverting mutations in at least two genes, where each mutation
is large
enough to insure a substantially zero probability of reversion and assurance
of the non-
expression of each mutated gene. The first gene chosen will be either directly
or indirectly
involved in the expression of a DNA adenine methylase. The second gene or
genes chosen
will also result in attenuation regardless of the attenuating effect of the
first gene mutation;
however, the second mutation can not affect the protective effects of the
first mutation.
The mutations in the first and second gene may be accomplished as discussed
previously.
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Accordingly, the invention provides a vaccine for provoking (eliciting) an
immunological response in a host to be vaccinated comprising: a bacteria
having a first
mutation in a first gene that alters the expression of a DNA adenine
methylase; and a
second mutation in said bacteria which renders said microorganism attenuated
independently of said first mutation.
In another embodiment, the invention provides live vaccines which may be used
as
vectors or carriers for an antigen. The antigen may be any antigen, including
an antigen of
a bacteria genus or species other than the bacteria used in the non-virulent
pathogenic
vaccine. The antigen may be added as an admixture, attached or associated with
the
bacteria, or one or more structural genes coding for the desired antigens) may
be
introduced into the non-virulent pathogenic vaccine as an expression cassette.
Accordingly, any of the mutant bacteria described for use in the vaccines
described herein
may further comprise an expression cassette having one or more structural
genes coding for
a desired antigen. The expression cassette comprises the structural gene or
genes of
interest under the regulatory control of the transcriptional and translational
initiation and
termination regions which naturally border the structural gene of interest or
which are
heterologous with respect to the structural gene. Where bacterial or
bacteriophage
structural genes are involved, the natural or wild-type regulatory regions
will usually, but
not always, suffice. It may be necessary to join regulatory regions recognized
by the non-
virulent pathogen to structural genes for antigens isolated from eukaryotes
and occasionally
prokaryotes. Antigens include, but are not limited to, Fragment C of tetanus
toxin, the B
subunit of cholera toxin, the hepatitis B surface antigen, Vibrio cholerae
LPS, HIV antigens
and/or Shigella soneii LPS.
The expression cassette may be a recombinant construct or may be, or form part
of,
a naturally occurring plasmid. If the expression cassette is a recombinant
construct, it may
be joined to a replication system for episomal maintenance or it may be
introduced into the
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non-virulent pathogenic bacteria under conditions for recombination and
integration into
the non-virulent pathogen's chromosomal DNA. Structural genes for antigens of
interest
may encode bacterial proteins such as toxin subunits, viral proteins such as
capsids, or
enzyme pathways such as those involved in synthesis of carbohydrate antigens
such as
lipopolysaccharide (LPS). For example, among the antigens expressed in other
live
attenuated Salmonella vaccines are Fragment C of tetanus toxin, the B subunit
of cholera
toxin, the hepatitis B surface antigen, and Vibrio cholerae LPS. Additionally,
the HIV
antigens GP 120 and GAG have been expressed in attenuated Mycobacterium bovis
BCG
and Shigella soneii LPS has been expressed in attenuated Vibrio cholerae. The
construct or
vector may be introduced into the host strain through a number of well known
methods
such as, transduction, conjugation, transformation, electroporation,
transfection, etc.
In another embodiment, live vaccines prepared in accordance with the present
invention are prepared having non-reverting mutations in genes that are
regulated by an
DNA adenine methylase(s), preferably by DNA adenine methylase (Dam). These non-
reverting mutations may be prepared as described previously.
In another embodiment, a vaccine is provided for, wherein the bacteria have a
mutation which results in the overproduction of Dam, preferably by
overproducing DNA
adenine methylase (Dam). Methods of producing overproducing bacterial genes
are
described herein and are known in the art and include, but are not limited to,
addition of a
plasmid (which may or may not integrate) which carries an additional dam gene;
alteration
of a promoter which controls transcription of dam; alteration in the dam gene
which results
in lowered responsiveness to feedback inhibition.
The immunogenic compositions described herein may be used with an adjuvant
which enhances the immune response against the pathogenic bacteria such as
Salmonella.
Adjuvants are especially suitable for killed vaccines, but need not be limited
to this use.
Suitable adjuvants are known in the art and include aluminum hydroxide, alum,
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(U.S. Pat. No. 5,057,540), DHEA (U.S. Pat. Nos. 5,407,684 and 5,077,284) and
its
derivatives and precursors, e.g., DHEA-S, beta-2 microglobulin (WO 91/16924),
muramyl
dipeptides, muramyl tripeptides (U.S. Pat. No. 5,171,568) and monophosphoryl
lipid A
(U.S. Pat. No. 4,436,728; WO 92/16231) and its derivatives, e.g., DETOXTM, and
BCG
(U.S. Pat. No. 4,726,947). Other suitable adjuvants include, but are not
limited to,
aluminum salts, squalene mixtures (SAF-1), muramyl peptide, saponin
derivatives,
mycobacterium wall preparations, mycolic acid derivatives, nonionic block
copolymer
surfactants, Quil A, cholera toxin B subunit, polyphosphazene and derivatives,
and
immunostimulating complexes (ISCOMs) such as those described by Takahashi et
al.
(1990) Nature 344:873-875. For veterinary use and for production of antibodies
in
animals, mitogenic components of Freund's adjuvant can be used. The choice of
an
adjuvant will depend in part on the stability of the vaccine in the presence
of the adjuvant,
the route of administration, and the regulatory acceptability of the adjuvant,
particularly
when intended for human use. For instance, alum is approved by the United
States Food
and Drug Administration (FDA) for use as an adjuvant in humans.
In some embodiments, the immunogenic composition may also comprise a carrier
molecule (with or without an adjuvant). Carriers are known in the art.
Pltokin, Vaccines
3'd Ed. Philadelphia, WB Suanders Co. (1999). Bacterial carriers (i.e.,
carriers derived
from bacteria) include, but are not limited to, cholera toxin B subunit (CTB);
diphtheria
toxin mutant (CRM197); diphtheria toxoid; group B streptococcus alpha C
protein;
meningococcal outer membrane protein (OMPC); tetanus toxoid; outer membrane
protein
of nontypeable Haemophilus influenzae (such as P6); recombinant class 3 porin
(rPorBP of
group B meningococci; heat-killed Burcella abortus; heat-killed Listeria
monocytogeneis;
and Pseudomonas aeruginosa recombinant exoprotein A. Another carrier is
keyhole limpet
hemocyanin (KLH).
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The vaccines of the present invention are suitable for systemic administration
to
individuals in unit dosage forms, sterile parenteral solutions or suspensions,
sterile non-
parenteral solutions or oral solutions or suspensions, oil in water or water
in oil emulsions
and the like. Formulations or parenteral and nonparental drug delivery are
known in the art
and are set forth in Remington's Pharmaceutical Sciences; 19th Edition, Mack
Publishing
(1995). The vaccines may be administered parenterally, by injection for
example, either
subcutaneously, intramuscularly, intraperitoneally or intradermally.
Administration can
also be oral, intranasal, intrapulmonary (i.e., by aerosol), and intravenous.
Additional
formulations which are suitable for other modes of administration include
suppositories
and, in some cases, oral formulations. The route of administration will depend
upon the
condition of the individual and the desired clinical effect. For
administration to farm
animals, such as chickens, preferred administration is oral formulations. The
formulations
for the live vaccines may be varied widely, desirably the formulation
providing an
enhanced immunogenic response.
The subject vaccines and antimicrobial drugs may be used in a wide variety of
vertebrates. The subject vaccines and antimicrobial drugs will find particular
use with
mammals, such as man, and domestic animals. Domestic animals include bovine,
ovine,
porcine, equine, caprine, domestic fowl, Leporidate e.g., rabbits, or other
animals which
may be held in captivity or may be a vector for a disease affecting a domestic
vertebrate.
Suitable individuals for administration include those who are, or suspected of
being, at risk
or exposure to bacteria, such as Salmonella (S. spp.), as well as those who
have been
exposed and/or infected. The manner of application of the vaccine or
antimicrobial drug
may be varied widely, any of the conventional methods for administering being
applicable.
These include oral application, on a solid physiologically acceptable base or
in a
physiologically acceptable dispersion, parenterally, by injection, or the
like. The dosage of
the vaccine or antimicrobial drug will depend inter alia on route of
administration and will
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vary according to the species to be protected. One or more additional
administrations may
be provided as booster doses, usually at convenient intervals, such as two to
three weeks.
Since DNA adenine methylases are not present in vertebrates, it is likely that
inhibitors of
DNA adenine methylases when administered to a vertebrate will display zero or
low
toxicity. Furthermore, since DNA adenine methylases are enzymes, they will be
present in
low concentrations within the cell; thus, requiring the administration of
lower levels of
inhibitors and increasing the likelihood that all the DNA adenine methylases
will be
inhibited.
Kits and strains
The invention also provides attenuated strains as described herein. Preferred
strains
are Salmonella strains which contain one or more mutations which alter Dam
activity.
Similar strains are described herein. Accordingly, in one embodiment, the
invention
provides attenuated strains of pathogenic bacteria, said bacteria containing a
mutation
which alters Dam activity such that the bacteria are attenuated. The mutation
can be any of
those described herein. Preferably, the strain is a Salmonella strain.
The present invention also encompasses kits containing any one or more of the
strains and/or vaccine formulations described herein in suitable packaging.
The kit may
optionally provide instructions, such as for administration. In some
embodiments, the
instructions are for administration to a non-human, such as chicken or other
farm animal.
In other embodiments, the instruction are for administration to a human.
Methods of the invention
The invention also provides methods using the immunogenic compositions
described herein, screening methods to identify potentially useful agents
which alter Dam
activity, as well as methods of preparing the immunogenic compositions
described herein.
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With respect to any methods involving administration of any of the
compositions
described herein, it is understood that any one or more of the compositions
can be
administered, i.e., the compositions can be administered alone or in
combination with each
other. Further, the compositions can be used alone or in conjunction with
other modalities
(i.e., clinical intervention), for the purpose of prevention and/or treatment.
Use of immunogenic compositions for eliciting an immune response, prevention
of
and heating disease
In some embodiments, the invention provides methods using the immunogenic
compositions described herein to elicit an immune response in an individual.
Generally,
these methods comprise administering any one or more of the immunogenic
compositions
described herein to an individual in an amount sufficient to elicit an immune
response.
The immune response may be against the particular species and/or strain of
bacteria in the
composition, or, in other embodiments, may be against a second species and/or
strain.
The immune response may be a B cell and/or T cell response. Preferably, the
response is antigen-specific, i.e., the response is against the bacteria used
in the
immunogenic composition (i.e., a response against an antigen associated with
the bacteria
used is detected). Preferably, the immune response persists in the absence of
the vaccine
components. Accordingly, in some embodiments, the immune response persists for
about
any of the following after administration of an immunogenic composition
described herein
(if given as multiple administrations, preferably after the most recent
administration): four
weeks, six weeks, eight weeks, three months, four months, six months, one
year.
In order to determine the effect of administration of an immunogenic
composition
described herein, the individual may be monitored for either an antibody
(humoral) or
cellular immune response against the bacteria, or a combination thereof, using
standard
techniques in the art. Alternatively, if an immunogenic composition is already
proven to
elicit such a response, such monitoring may not be necessary.
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For the purpose of raising an immune response, the immunogenic compositions
described herein may be administered in an unmodified form. It may sometimes
be
preferable to modify the bacteria to improve immunogenicity. As used herein,
and as well
known in the art, "immunogenicity" refers to a capability to elicit a specific
antibody (B
cell) or cellular (T cell) immune response, or both. Methods of improving
immunogenicity
include, inter alia, crosslinking with agents such as glutaraldehyde or
bifunctional couplers,
or attachment to a polyvalent platform molecule. Immunogenicity may also be
improved
by coupling to a protein carrier, particularly one that comprises T and/or B
cell epitopes.
Suitable individuals for receiving the compositions have been described above
and
likewise apply to these methods. Generally, such individuals are susceptible
to exposure
to, have been exposed to, and/or display a symptom and/or disease state
associated with
infection. The individual may or may not have been exposed to Salmonella at
the time of
administration, and accordingly may or may not have been infected by
Salmonella at the
time of administration. Preferably, the individual has not been exposed to
Salmonella.
In some embodiments, the invention provides methods of eliciting an immune
response to a second species, strain, serotype, and/or group of Salmonella, in
an individual,
comprising administering to the individual any of the immunogenic compositions
described
herein in an amount sufficient to elicit an immune response to the second
species, strain,
serotype, and/or group of Salmonella. The individual may or may not have been
previously
exposed to the second species, strain, serotype, and/or group of Salmonella.
In some
embodiments, the second Salmonella against which an immune response is
elicited is from
a second group, such as Group A, B, or D (as compared to the first serotype
administered).
In other embodiments, the second Salmonella against which an immune response
is elicited
is from a second serotype (as compared to the first serotype administered).
A first and second species may be any species of Salmonella, some of which
have
been described above. In some embodiments, the first species is S. typhimurium
and the
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second species is S. enteritidis. In some embodiments, the first species is S.
typhimurium
and the second species is S. dublin. In other embodiments, the first species
is S. enteritidis
and the second species is S. typhimurium. In yet other embodiments, the first
species is S.
enteritidis and the second species is S. dublin. Similarly, the first group
may be any of the
known groups of Salmonella, such as Group A, B, or D. The second group may be
any
known, such as Group A, B, or D (provided that the second group is different
from the first
group). In other embodiments, the first serotype is different than the second
serotype.
Serotypes of Salmonella are known in the art.
It is understood that an immune response may be elicited against one or more
additional antigens (i.e., one or more additional Salmonella strains, groups,
serotypes,
and/or species). Thus, the invention encompasses methods by which an immune
response
is elicited against a third, fourth, fifth, etc. Salmonella strain, group,
serotype, and/or
species.
The invention also encompasses methods of eliciting an immune response to a
second species, strain, serotype and/or group of a pathogenic bacteria in an
individual
comprising administering to the individual an immunogenic composition
comprising an
attenuated bacteria which is a Dam derivative amount sufficient to elicit an
immune
response to a second species, strain, serotype and/or group of the pathogenic
bacteria.
The invention also provides methods of treating a bacterial, preferentially,
such as
Salmonella, infection in an individual. In some embodiments, the invention
provides
methods of suppressing a disease symptom associated with infection of a
virulent bacteria,
such as Salmonella. The methods comprise administering any one or more of the
compositions described herein in an amount sufficient to suppress a disease
symptom
associated with infection. Preferentially, the infection is due to Salmonella
In other
embodiments, the infection is due to Escherichia, preferably E. coli. In other
embodiments,
these methods comprise administering any one or more of the compositions
described
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herein in an amount to reduce the amount of pathogenic bacteria, such as
Salmonella, in an
individual (as compared to non-administration).
The vaccines are administered in a manner compatible with the dosage
formulation,
and in such amount as will be therapeutically effective. The quantity to be
administered
depends on the individual to be treated, the capacity of the individual's
immune system to
synthesize antibodies, the route of administration, and the degree of
protection desired.
Precise amounts of active ingredient required to be administered may depend on
the
judgment of the practitioner in charge of treatment and may be peculiar to the
individual.
In one embodiment, the invention provides methods of treating an individual
infected with a pathogenic bacteria, comprising administering to the
individual a
composition comprising an agent which alters Dam activity. In other
embodiments, the
invention provides methods of treating a host infected with a pathogenic
microorganism
(bacteria) comprising (a) administering a compound to the host, wherein said
compound
alters the expression of or activity of one or more DNA adenine methylases.
The
compounds) may (a) bind to one or more DNA adenine methylases thereby altering
the
activity of said DNA adenine methylases; (b) bind to one or more genes that
express a
DNA adenine methylase, thereby altering the expression of said DNA adenine
methylase(s). The expression of said DNA adenine methylase(s) is/are
overactive.
Alternatively the expression of said DNA adenine methylase(s) is/are
repressed. In some
embodiments, the compound is an antisense oligonucleotide having a sequence
complementary to one or more DNA adenine methylase gene sequences.
The invention also provides methods of treating a host infected with a
pathogenic
microorganism (bacteria) comprising administering a compound to the host,
wherein said
compound binds one or more virulence factors that are regulated by DNA adenine
methylases.
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In some embodiments, the invention provides methods of preventing bacterial
infection, such as Salmonella infection. In these embodiments, an immune
response
elicited by the immunogenic compositions) is protective in the sense that a
recipient of the
immunogenic composition displays one or more lessened symptoms of infection
when
compared to an individual not receiving the composition. In other embodiments,
a
protection is conferred by reducing amount of bacteria, such as Salmonella, in
the
individual receiving the composition as compared to not receiving the
composition.
In some embodiments, the invention provides methods of suppressing a symptom
associated with bacterial infection in an individual (or, alternatively,
methods of treating a
bacteria infection) comprising administering to the individual a composition
comprising an
agent which alters Dam activity. A bacteria may be any of those described
herein,
particularly Salmonella.
In another embodiment, an antimicrobial drug in accordance with the present
invention is prepared which inhibits a DNA adenine methylase(s), preferably
DNA adenine
methylase (Dam). While the following discussion focuses specifically on the
dam gene and
its product, Dam, it is to be understood that this specificity is only for the
purpose of
simplicity and clarity. It is contemplated that the methods and compositions
discussed
below are applicable towards (i) any gene that expresses a DNA adenine
methylase, (ii) any
gene or gene product that regulates a DNA adenine methylase gene, (iii) any
gene that is
regulated by a DNA adenine methylase, and/or (iv) DNA methylases.
Consequently, while
a specific gene and gene product, that is dam and Dam, are discussed below, it
is
contemplated that other DNA adenine methylase genes and DNA adenine methylases
are
equivalents of dam and Dam, respectively, and are thus interchangeable with
respect to the
discussion which follow.
Inhibition of Dam could be carried out by a number of approaches including use
of
antisense oligonucleotides to inhibit dam gene translation, direct inhibitors
of Dam
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enzymatic activity, reduction of Dam levels by isolation of inhibitory
compounds for Dam
activators and/or activating compounds for Dam repressors, and targeting of
virulence
factors that are regulated by Dam. The antisense approach has been used
previously to
inhibit the cytosine methyltransferase (MeTase) from mammalian cells (MacLeod,
A. R.
and Szyf, M., J. Biol. Chem., 7:8037-8043 (1995)). Transfection of an
antisense nucleic
acid into adrenocortical cells resulted in DNA demethylation and reduced
tumorigenicity
associated with MeTase activity.
In another embodiment, the anti-microbial drug activates Dam. Such a compound
could effect such activation by, for example, stimulating the dam promoter,
inactivating
repressors, and/or extend half life of Dam.
Screening assays
The present invention also encompasses methods of identifying agents that may
have anti-bacterial activity (and thus may control virulence) based on their
ability to alter
Dam activity. These methods may be practiced in a variety of embodiments. We
have
observed that loss or even increase of Dam function results in significantly
lower
infectivity of Salmonella in an art-accepted mouse model. This suggests that
modulation of
Dam function may result in control of the pathogenesis of various bacteria,
including, but
not limited to, Salmonella, while not affecting host cells. This is especially
true since
humans do not have a homolog to dam genes. Further, we have found that dam is
an
essential gene in Vibrio cholerae and Yersinia pseudotuberculosis (Example 7),
which
indicates that Dam is an excellent drug target in these pathogenic organisms.
Thus, an
agent identified by the methods of the present invention may be useful in the
treatment of
bacterial infection, especially Escherichia, Salmonella, Vibrio, and/or
Yersinia infection.
The methods described herein are in vitro and cell-based screening assays. In
the in
vitro embodiments, an agent is tested for its ability to modulate function of
Dam. In the
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cell-based embodiments, living cells having Dam function are used for testing
agents. For
purposes of this invention, an agent may be identified on the basis of any
alteration of Dam
function, although characteristics associated with total loss of Dam function
may be
preferable.
In all of these methods, alteration of Dam function may occur at any level
that
affects Dam function, whether positively or negatively. An agent may alter Dam
function
by reducing or preventing transcription of Dam. An example of such an agent is
one that
binds to the upstream controlling region, including a polynucleotide sequence
or
polypeptide. An agent may alter Dam function by increasing transcription of
Dam RNA.
An agent may alter Dam function by reducing or preventing translation of Dam
RNA. An
example of such an agent is one that binds to the RNA, such as an anti-sense
polynucleotide, or an agent which selectively degrades the RNA. Anti-sense
approaches to
inhibiting Dam have been described above. An agent may alter Dam function by
increasing translation of Dam RNA. An agent may compromise Dam function by
binding
to Dam. An example of such an agent is a polypeptide or a chelator. An agent
may
compromise Dam function by affecting gene expression of a gene that is
regulated by Dam.
An example of such an agent is one that alters expression of a Dam-regulated
gene on any
of the levels discussed above.
The screening methods described as applicable to any pathogenic bacteria
having a
Dam gene.
In vitro screening methods
In in vitro screening assays of this invention, an agent is screened in an in
vitro
system, which may be any of the following: (1) an assay that determines
whether an agent
is inhibiting or increasing transcription of dam; (2) an assay for an agent
which interferes
with translation of Dam RNA or a polynucleotide encoding Dam, or
alternatively, an agent
which specifically increases translation of dam; (3) an assay for an agent
that binds to Dam.
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For an assay that determines whether an agent inhibits or increases
transcription of
dam, an in vitro transcription or transcription/translation system may be
used. These
systems are available commercially, and generally contain a coding sequence as
a positive,
preferably internal, control. A polynucleotide encoding Dam is introduced and
transcription is allowed to occur. Comparison of transcription products
between an in vitro
expression system that does not contain any agent (negative control) with an
in vitro
expression system that does contain agent indicates whether an agent is
affecting dam
transcription. Comparison of transcription products between control and Dam
indicates
whether the agent, if acting on this level, is selectively affecting
transcription of dam (as
opposed to affecting transcription in a general, non-selective or specific
fashion).
For an assay that determines whether an agent inhibits or increases
translation of
dam RNA or a polynucleotide encoding Dam, an in vitro
transcription/translation assay as
described above may be used, except the translation products are compared.
Comparison
of translation products between an in vitro expression system that does not
contain any
agent (negative control) with an in vitro expression system that does contain
agent indicates
whether an agent is affecting dam translation. Comparison of translation
products between
control and dam indicates whether the agent, if acting on this level, is
selectively affecting
translation of dam (as opposed to affecting translation in a general, non-
selective or specific
fashion).
For an assay for an agent that binds to Dam, Dam is first recombinantly
expressed
in a prokaryotic or eukaryotic expression system as a native or as a fusion
protein in which
Dam is conjugated with a well-characterized epitope or protein. Recombinant
Dam is then
purified by, for instance, immunoprecipitation using anti-Dam antibodies or
anti-epitope
antibodies or by binding to immobilized ligand of the conjugate. An affinity
column made
of Dam or Dam fusion protein is then used to screen a mixture of compounds
which have
been appropriately labeled. Suitable labels include, but are not limited to,
fluorchromes,
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radioisotopes, enzymes and chemiluminescent compounds. The unbound and bound
compounds can be separated by washes using various conditions (e.g. high salt,
detergent )
that are routinely employed by those skilled in the art. Non-specific binding
to the affinity
column can be minimized by pre-clearing the compound mixture using an affinity
column
containing merely the conjugate or the epitope. A similar method can be used
for
screening for agents that competes for binding to Dam. In addition to affinity
chromatography, there are other techniques such as measuring the change of
melting
temperature or the fluorescence anisotropy of a protein which will change upon
binding
another molecule. For example, a BIAcore assay using a sensor chip (supplied
by
Pharmacia Biosensor, Stitt et al. (1995) Cell 80: 661-670) that is covalently
coupled to
native Dam or Dam-fusion proteins, may be performed to determine the Dam
binding
activity of different agents.
With respect to binding Dam, it is understood that suitable fragments of Dam
could
also be used. For example, if it is known that a particular region of Dam is
important for
binding to DNA, then this fragment containing or even consisting of this
region could be
used.
In another embodiment, an in vitro screening assay detects agents that compete
with
another substance (most likely a polynucleotide) that binds Dam. For instance,
it is known
that Dam binds a certain DNA motif, namely GATC, which is a Dam target site.
An assay
could be conducted such that an agent is tested for its ability to compete
with binding to
this motif(s). Competitive binding assays are known in the art and need not be
described in
detail herein. Briefly, such an assay entails measuring the amount of Dam
complex formed
in the presence of increasing amounts of the putative competitor. For these
assays, one of
the reactants is labeled using, for example, 32P. One such assay, also
encompassed by this
invention, is described in more detail below.
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Isolation of inhibitors or activators of Dam could be carried out, for
example, by
screening chemical (Neustadt, et al., Bioorg. Med. Chem. Lett., 8:2395-2398
(1998)) or
peptide libraries (Lam, K.S., Anticancer Drug. Res., 12:145-167 (1997)) using
a rapid, high
throughput assay for Dam. Such inhibitor libraries have already been shown to
be effective
in blocking the activity of several enzymes (Carroll, C.D., Bioorg. Med. Chem.
Lett.,
8:3203-3206 (1998)). This Dam assay consists of a double stranded
oligonucleotide
containing Dam target sites (GATC sequences) with a tethering group on one end
(e.g.
biotin) and a signal at the other end. This signal could be a radioactive
compound such as
phosphorous-32, an fluorescent molecule such as fluorescein, or an antigen.
The
nonmethylated oligonucleotide containing Dam target sites is tethered to a
solid surface
such as a 96-well microtiter plates containing avidin. Dam enzyme
(predetermined to
contain just sufficient activity to methylate all of the GATC sites of the
target
oligonucleotide) is preincubated with inhibitor libraries and then added to
each well in the
presence of S-adenosylmethionine (SAM). Following an incubation period, sample
wells
are rinsed in buffer and restriction enzyme MboI is added to digest all
nonmethylated
GATC sites within the oligonucleotide, thus releasing the signal end of the
molecule. Plate
wells are then counted (radioactive signal), scanned for fluorescence
(fluorescent signal), or
incubated with secondary antibody conjugated to an enzyme such as horse radish
peroxidase, followed by a non-radioactive substrate of the enzyme. Inhibition
of Dam
would be detected as a reduction in signal within a sample well due to release
of
nonmethylated GATC sites. This assay could be used to rapidly screen chemical
and
peptide libraries for inhibitory activity. The feasibility of such studies has
been shown by
the isolation of sinefungin, an inhibitor of MeTase activity. Sinefungin is an
analog of S-
adenosyl-L-methionine (SAM), and acts as a competitive inhibitor of DNA
methylation.
However, because sinefungin would block all DNA methylases including the
mammalian
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cytosine methylase that require SAM as methyl donor, this drug would not be
useful as a
chemotherapeutic agent against bacteria.
To isolate activators of Dam, Dam (predetermined to contain sufficient
activity to
methylate a low percentage of target sites, such as GATC sites, of the target
oligonucleotide, for example, 20%) is preincubated with one or more agents
(including
activator libraries) and then added to each well in the presence of SAM.
Activation of Dam
would be detected as an increase in signal within the sample well due to
methylation of the
target sites (such as GATC) and thus prevention of MboI restriction reaction.
Accordingly, in some embodiments, the invention provides methods of
identifying
an agent which alters or modulates (i.e., an agent which alters Dam function,
preferably
inhibits Dam function), comprising the steps of (a) tethering a nonmethylated
oligonucleotide containing a DNA adenine methylase target site to a solid
surface wherein
said nonmethylated oligonucleotide has a tethering group on a first end and a
signal on a
second end; (b) incubating a DNA adenine methylase having sufficient activity
to
methylate said target sites, preferably all of said target sites, on said
nonmethylated
oligonucleotide with an agent; inhibitor libraries; (c) adding said incubated
DNA adenine
methylase to said tethered nonmethylated oligonucleotide in the presence of S-
adenosylmethionine; (d) digesting all nonmethylated target sites, thereby
releasing said
tethered nonmethylated oligonucleotides; and (e) detecting inhibition of DNA
adenine
methylase as an increase in said signal due to digestion of said nonmethylated
target sites.
Preferably, the target site is a GATC sequence. The tethering group may be any
suitable
moiety known in the art, such as biotin. The signal may be due to
fluorescence,
radioactivity, or an antigen. In some embodiments, the solid surface is a
microtiter plate
containing avidin. A restriction enzyme, such as Mbol, may be used to digest
said
nonmethylated target sites. If an inhibitor library is used as a source of
agents to be tested,
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the library may comprise biomolecules, such as peptides, or may comprise
organic
compounds or inorganic compounds.
It is also understood that the in vitro screening methods of this invention
include
structural, or rational, drug design, in which the amino acid sequence, three-
dimensional
atomic structure or other property (or properties) of Dam provides a basis for
designing an
agent which is expected to bind to Dam. Generally, the design and/or choice of
agents in
this context is governed by several parameters, such as the perceived function
of the Dam
target (here, binding DNA is one such function), its three-dimensional
structure (if known
or surmised), and other aspects of rational drug design. Techniques of
combinatorial
chemistry can also be used to generate numerous permutations of candidate
agents. For
purposes of this invention, an agent designed and/or obtained by rational drug
designed
may also be tested in the cell-based assays described below.
Cell-based screening methods
In cell-based screening assays, a living cell, preferably a bacterium
containing a
functioning dam gene, or a living cell, preferably a bacterium containing a
polynucleotide
construct comprising a Dam encoding sequence, are exposed to an agent. In
contrast,
conventional in vitro drug screening assays (as described above) have
typically measured
the effect of a test agent on an isolated component, such as an enzyme or
other functional
protein.
The cell-based screening assays described herein have several advantages over
conventional drug screening assays: 1) if an agent must enter a cell to
achieve a desired
therapeutic effect, a cell-based assay can give an indication as to whether
the agent can
enter a cell; 2) a cell-based screening assay can identify agents that, in the
state in which
they are added to the assay system are ineffective to alter Dam function, but
that are
modified by cellular components once inside a cell in such a way that they
become
effective agents; 3) most importantly, a cell-based assay system allows
identification of
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agents affecting any component of a pathway that ultimately results in
characteristics that
are associated with alteration of Dam function.
In one embodiment, an agent is identified by its ability to elicit a
characteristic
associated with an alteration of Dam function in a suitable host cell. A
suitable host cell in
this context is any host cell in which a Dam function may be observed.
Preferably, the host
cell is a bacterial cell. Suitable host cells include, but are not limited to,
Salmonella,
Escherichia, Vibrio, Yersinia, and any other bacteria genus and species that
contains a Dam
gene. One example of an assay uses the pili operon system in E. coli, in which
level of
expression of a reporter is determined. Any bacterial operon system which is
responsive to
methylation would be suitable for bacterial-based assays, using any of a
number of reporter
systems known in the art. Levels of transcription and/or translation from such
systems in
the presence of agents) would indicate whether an agent was affecting Dam
activity.
In one embodiment, the invention provides methods for identifying an agent
that
may control virulence comprising the following steps: (a) contacting at least
one agent to
be tested with a suitable host cell that has Dam function; and (b) analyzing
at least one
characteristic which is associated with alteration of Dam function (which can
be increase,
decrease, or loss of Dam function) in said host cell, wherein an agent is
identified by its
ability to elicit at least one such characteristic. For these methods, the
host cell may be any
cell in which Dam function has been demonstrated.
For genes that are de-repressed upon loss of Dam function, loss of Dam
function
may be measured using a reporter system, in which a reporter gene sequence is
operatively
linked to the Dam-repressed gene of interest. Such repressed genes are
described herein,
including the examples. As used herein, the term "reporter gene" means a gene
that
encodes a gene product that can be identified (i. e., a reporter protein).
Reporter genes
include, but are not limited to, alkaline phosphatase, chloramphenicol acetyl
transferase, (3-
galactosidase, luciferase and green fluorescence protein. Identification
methods for the
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products of reporter genes include, but are not limited to, enzymatic assays
and
fluorometric assays. Reporter genes and assays to detect their products are
well known in
the art and are described, for example in Current Protocols in Molecular
Biology, eds.
Ausubel et al., Greene Publishing and Wiley-Interscience: New York (1987) and
periodic
updates, as well as Short Protocols in Molecular Biology (Whey and Sons,
1999). Reporter
genes, reporter gene assays and reagent kits are also readily available from
commercial
sources (Strategene, Invitrogen and etc.)
In another embodiment, these methods comprise the following steps:
(a) introducing a polynucleotide encoding Dam (or a functional fragment
thereof) into a
suitable host cell that otherwise lacks Dam function, wherein Dam function is
restored in
said host cell; (b) contacting said cell of step (a) with at least one agent
to be tested;
(c) analyzing at least one characteristic which is associated with loss of Dam
function,
wherein an agent is identified by its ability to elicit at least one said
characteristic.
The host cell used for these methods initially lacks Dam function (i.e., lacks
Dam
function before introduction of polynucleotide encoding Dam). Lacking Dam
function may
be partial to total. Devising host cells that lack Dam function may be
achieved in a variety
of ways, including, but not limited to, genetic manipulation such as deletion
mutagenesis,
recombinant substitution of a functional portion of the gene, frameshift
mutations,
conventional or classical genetic techniques pertaining to mutant isolation,
or alterations of
the regulatory domains. For cells in which loss of Dam (or its homology
function is lethal,
a plasmid containing a wild type copy of the Dam is in the cell during the
disruption, or
mutagenesis, process. If the cells cannot survive without the plasmid
containing the wild-
type gene, then it is assumed that the loss of Dam function is lethal. Example
7 describes
an assay for determining whether a Dam gene is essential.
Introduction of polynucleotides encoding Dam or a functional fragment thereof
depend on the particular host cell used and may be by any of the many methods
known in
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the art, such as spheroplasting, electroporation, CaCl2 precipitation, lithium
acetate
treatment, and lipofectamine treatment.
Polynucleotides introduced into a suitable host cells) are polynucleotide
constructs
comprising a polynucleotide encoding Dam or a functional fragment thereof.
These
constructs contain elements (i.e., functional sequences) which, upon
introduction of the
construct, allow expression (i.e., transcription, translation, and post-
translational
modifications, if any) of Dam amino acid sequence in the host cell. The
composition of
these elements; such as appropriate selectable markers, will depend upon the
host cell being
used.
Restoring Dam (or its homology function in the host cells) may be determined
by
analyzing the host cells) for detectable parameters associated with Dam
function (i.e., wild
type). These parameters depend upon the particular host cell used. For
Salmonella, Dam
function is associated with any of the following: (a) repression of Dam-
regulated genes;
(b) virulence; (c) regulation of paf pili expression; (d) lack of sensitivity
of certain amino
acids. Genes known to be repressed in the presence of Dam in Salmonella have
been
described above. Given methods well known in the art for making reporter
constructs (see
above), any of these genes could be altered to accommodate a reporter system.
Examples
of suitable reporter systems have been discussed above.
In some embodiments, a polynucleotide encoding Dam is operatively linked to an
inducible promoter. Use of an inducible promoter provides a means to determine
whether
the agent is acting via a Dam pathway. If an agent causes a characteristic
indicative of loss
of Dam function to appear in a cell in which the inducible promoter is
activated, an
observation that the agent fails to elicit the same result in a cell in which
the inducible
promoter is not activated indicates that the agent is affecting at least one
step or aspect of
Dam function. Conversely, if the characteristic indicating loss of Dam
function is also
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observed in a cell in which the inducible promoter is not activated, then it
can be assumed
that the agent is not necessarily acting solely via the Dam functional
pathway.
Cell-based screening assays of the present invention can be designed, e.g., by
constructing cell lines in which the expression of a reporter protein, i. e.,
an easily assayable
protein, such as (3-galactosidase, chloramphenicol acetyltransferase (CAT),
green
fluorescent protein (GFP) or luciferase, is dependent on Dam function. For
example, a
gene under Dam control may have reporter sequences inserted within the coding
region as
described in Example 1. The cell is exposed to a test agent, and, after a time
sufficient to
effect (3-galactosidase expression and sufficient to allow for depletion of
previously
expressed (3-galactosidase, the cells are assayed for the production of (3-
galactosidase under
standard assaying conditions.
Assay methods generally require comparison to a control sample to which no
agent
is added. Additionally, it may be desirable to use a cell partially or
completely lacking
Dam function as a control. For instance, if an agent were acting along a Dam
pathway, one
might expect to see the same phenotype as dam- cells treated with agents. If
an agent were
not acting along a Dam pathway, one may expect to see other characteristics
that occur in
the dam- cells when treated with the agent.
The screening methods described above represent primary screens, designed to
detect any agent that may exhibit anti-bacterial activity. The skilled artisan
will recognize
that secondary tests will likely be necessary in order to evaluate an agent
further. For
example, a secondary screen may comprise testing the agents) in bacteria of
interest if the
initial screen has been performed in a host cell other than those bacteria A
further screen
is to perform an infectivity assay using the cells that have been treated with
the agent(s).
An infectivity assay using mice is described in Example 1, and other animal
models (such
as rat) are known in the art. In addition, a cytotoxicity assay would be
performed as a
further corroboration that an agent which tested positive in a primary screen
would be
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suitable for use in living organisms. Any assay for cytotoxicity would be
suitable for this
purpose, including, for example the MTT assay (Promega).
Preparation of vaccines and attenuated bacteria
The invention also provides methods of preparing, or making, the vaccines
described herein as well as methods of making the mutant strains (i.e., Dam
derivatives)
described herein. Preparation of vaccines has been discussed above and as
such, these
methods are included in the invention. It is understood that any of the
mutations described
herein (including those which increase, decrease, or eliminate Dam activity,
including Dam
expression) may be used in the methods of preparation of the invention, and
are generally
not repeated in this section.
In one embodiment, the invention provides methods for preparing an immunogenic
composition comprising attenuated bacteria with altered Dam function,
comprising
combining any of the mutants and/or mutant strains described herein (i.e., Dam
derivatives)
with a pharmaceutically acceptable excipient. Preferred embodiments include
Salmonella
strains such as those described herein. Particularly preferred are Salmonella
strains which
have mutations which have eliminated Dam activity, such as those deletion
mutants
described herein.
In one embodiment, the invention provides methods for preparing an attenuated
pathogenic bacteria, preferably Salmonella, capable of eliciting an
immunological response
by a individual susceptible to disease caused by the corresponding or similar
pathogenic
bacteria comprising constructing at least one mutation in said pathogenic
bacteria wherein a
first mutation results in alteration of Dam function, preferably the altered
expression of a
Dam. Preferably, the first mutation is introduced into a first gene that
expresses Dam. In
some embodiments, said first mutation is introduced into a first gene, the
expression of
which represses or over activates expression of a gene that expresses a DNA
adenine
methylase enzyme. In some embodiments, said first mutation is introduced into
a first gene
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the expression of which is regulated by a DNA adenine methylase. In other
embodiments,
a second mutation is created in a gene that is independent of said first
mutation, said second
mutation causing attenuation of the bacteria.
In another embodiment, the invention provides methods for preparing an
attenuated
bacteria capable of eliciting an immunological response by a host susceptible
to disease
caused by the corresponding virulent bacteria comprising (a) constructing at
least one
mutation in the dam gene of a virulent strain of the pathogenic bacteria. In
some
embodiments, a second mutation is introduced into a second gene which results
in
attenuation of said bacteria independently of said first mutation.
In another embodiment, the invention provides methods for preparing an
attenuated
bacteria capable of eliciting an immunological response by a host susceptible
to disease
caused by the corresponding or similar pathogenic bacteria comprising (a)
constructing a
first non-reverting mutation in said pathogenic bacteria wherein said first
non-reverting
mutation alters the expression of or the activity of one or more DNA adenine
methylases,
and (b) constructing a second non-reverting mutation in said pathogenic
bacteria wherein
said second non-reverting mutation is independent of said first non-reverting
mutation and
is attenuating. In some embodiments, the first non-reverting mutation is
constructed in a
gene whose product activates one or more of said DNA adenine methylases. In
some
embodiments, the gene product activates DNA adenine methylase. In some
embodiments,
the first non-reverting mutation is constructed in a gene whose product
represses the
expression of said DNA adenine methylases. In some embodiments, said gene
product
represses DNA adenine methylase. In other embodiments, the first non-reverting
mutation
is constructed in a gene whose product inactivates or decreases the activity
of one or more
of said DNA adenine methylases by binding directly to one or more of said DNA
adenine
methylases. In some embodiments, one of said DNA adenine methylases is DNA
adenine
methylase. In some embodiments, the pathogenic bacteria is a strain of
Salmonella,
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preferably Salmonella is S. typhimurium, S. enteritidis, S typhi, S bonus-ovi,
S. abortus-
equi, S. dublin, S gallinarum, S. pullorum. In other embodiments, the
pathogenic bacteria
are any one of the following: Yersinia, Vibrio, Shigella, Haemophilus,
Bordetella,
Neisse~ia, Pasteurella, pathogenic Escherchia, Treponema. The host may be a
vertebrate,
such as a mammal, preferably human or a domestic animal. In some embodiments,
the
vertebrate is a chicken.
In some embodiments, the preparation methods comprise addition of an antigen.
For example, the antigen can be added simply to the bacteria in the vaccine,
or,
alternatively, expression cassette comprising one or more structural genes
coding for a
desired antigen may be inserted into the attenuated bacteria.
Antigens include, but are not limited to, Fragment C of tetanus toxin, the B
subunit
of cholera toxin, the hepatitis B surface antigen, Vibrio cholerae LPS, HIV
antigens and/or
Shigella soneii LPS.
In another embodiment, the invention provides methods for preparing an
attenuated
microorganism capable of eliciting an immunological response by a host
susceptible to
disease caused by the corresponding or similar pathogenic microorganism
comprising the
steps of (a) constructing a first non-reverting mutation in said pathogenic
microorganism
wherein said first non-reverting mutation alters the expression of or activity
of one or more
genes that are regulated by DNA methylases; and (b) constructing a second non-
reverting
mutation in said pathogenic microorganism wherein said second non-reverting
mutation is
independent of said first non-reverting and is attenuating.
The above disclosure generally describes the present invention. A more
complete
understanding can be obtained by reference to the following specific examples
which are
provided herein for purposes of illustration only and are not intended to be
limiting.
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EXAMPLES
The following non-limited examples provide vaccines prepared from live,
pathogenic bacteria and the target sites for antimicrobial drugs according to
the teachings
of the present invention, and are offered only by way of illustration and not
by way of
limitation. All scientific technical terms have the meanings as understood by
one with
ordinary skill in the art. Recombinant DNA techniques are now sufficiently
well known
and widespread so as to be considered routine. In very broad and general
terms, these
techniques entail transferring the genetic material of one organism into a
second
organism so that the transferred genetic material becomes a part of the
genetic material of
the organism to which it is transferred. This typically consists of first
obtaining a piece
of DNA from the first organism either from a plasmid or the chromosomal DNA.
The
piece of DNA may be of any size and is often obtained through the use of
restriction
endonuclease enzymes which recognize and cut DNA at specific base pair sites.
Following the isolation of a particular piece of DNA, the DNA may be inserted
or cloned
into plasmid, phage or cosmid vectors to form recombinant molecules that may
be
subsequently transferred into a host cell by various means such as
transformation,
transduction, transfection, and conjugation.
Transformation involves the uptake of naked DNA from the external
environment, which can be artificially induced by the presence of various
chemical
agents such as calcium ions, or by electroporation. Transduction involves the
packing of
the recombinant DNA within a phage, such as transducing phage or cosmid
vectors.
Once the recombinant DNA is introduced into the microbial host, it may
continue to exist
as a separate piece or it may insert or integrate into the host cell's
chromosome and be
reproduced with the chromosome during cell division. Conjugation involves
classical
microbial mating techniques.
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Example 1: Dam Salmonella derivatives are avirulent
Strain Construction
All Salmonella typhimurium strains used were isogenic with American Tissue
Culture Collection (ATCC) strain 14028, a smooth virulent strain of S.
typhimurium
referred to as "wild type". Previously, all reported dam mutations from other
laboratories
used Salmonella strain LT2 which is at least 1000-fold less virulent than the
wild type
when delivered i.p. See the data in Table 1.
All restriction enzymes and pBR322 were, and can be, purchased from
commercial sources, such as Stratagene, 11099 North Torrey Pines Rd., La
Jolla,
California 92037. Electroporation was carried out with a BioRad Gene Pulser
apparatus
Model No. 1652098. S. typhimurium cells were prepared as per the
manufacturer's
instructions. Aliquots of competent cells were mixed with an aliquot of the
desired
plasmid and placed on ice for 1 minute. The mixture was transferred into a
cuvette-
electrode (0.2 cm) and pulsed once at a field strength of 2.5 KV/cm as per the
manufacturer's instructions.
1. Construction of Nonpolar dam mutant
For construction of a nonpolar dam mutant, S. typhimu~ium genomic DNA was
used as template for the PCR using Pfu polymerase (Stratagene). A 350-by DNA
fragment containing the first 100 codons of dam was amplified by PCR using the
following oligonucleotide pair: 5'-GATTTCTAGAGTAGTCTGCGGAGCTTTC- 3'
(SEQ ID NO. 1) (containing anXbaI site at the 5' end) and 5'-
GATTCTCGAGGGTGTTGAACTCCTCGCG- 3' (SEQ ID NO. 2) (containing an XhoI
site at the 5' end). PCR was carried out in a buffer containing 2.0 mM Mg 2+
for 30
cycles of 45 seconds at 92°C, 1 minute at 42°C and 1 minute 30
seconds at 72°C. This
procedure was carried out in a DNA Thermal Cycler #N801-0150 (Perkin-Elmer
Cetus).
The PCR product was then double-digested with XbaI and XhoI. In a second PCR
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amplification, a 300-by DNA fragment containing the last 79 codons of dam was
synthesized using the following oligonucleotide pair: 5'-
GATTCTCGAGTTTAGCCTGACGCAACAAG- 3' (SEQ ID NO. 3) (containing an
XhoI site at the 5' end) and 5'-GATTGCATGCTCCTTCACCCAGGCGAG-3' (SEQ ID
NO. 4) (containing an SphI site at the 5' end). This PCR product was then
double
digested with XhoI and SphI. The suicide vector pCVD442 (Donnenberg, M. S., et
al.,
Infect. Immun. , 59:4310-4317 ( 1991 )), was double digested with XbaI and
SphI, band
purified, and ligated in a single reaction with the two custom-cut PCR
products. An in-
frame deletion of 100 internal amino acids of Dam was created, leaving a
unique XhoI
site at the deletion join point. E. coli DHSalpha lambda pir was then
transformed
selecting ampicillin resistance. DNA from the appropriate ampicillin resistant
construct
(confirmed by restriction digest) was then used to transform S. typhimurium
14028. The
integrated pCVD442-containing construct was then segregated on LB 5%
sucrose/no salt
plates. Segregants were confirmed ampicillin sensitive by printing and Dam by
streaking on LB plates containing 2-aminopurine (0.6mg/ml) (Dam mutants are 2-
AP
sensitive). Additionally, PCR was used to confirm the deletion by size in
comparison to
wild-type sequences. Lastly, the deleted region was cloned into pGP704 and
sequence
near and at the deletion join point (including the XhoI site) was obtained to
confirm that
the deletion in fact was in-frame.
The mutation caused by the dam102 insertion (dam102::Mud-Cm discussed
above) was moved by P22-mediated transduction into virulent Salmonella strain,
14028
to construct strain 2.
2. Mouse virulent assays
Virulent properties of all the various S typhimurium strains constructed, as
described above, were tested by intraperitoneal or oral inoculations of female
BALB/c
mice and the results are presented in Table 1 below.
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Female BALB/c mice were purchased from Charles River Breeding Laboratories,
Inc., (Wilmington, Mass.) and were 6 to 8 weeks of age at initial challenge.
S.
typhimurium strains were grown overnight at 37°C to stationary phase in
Luria Broth
(LB). Bacteria were washed once with PBS, then diluted in PBS to the
approximate
appropriate dilution (samples were plated for colony forming units (CFUs) on
LB to give
an accurate bacterial count). Mice were challenged with 200 ~,l of the
appropriate
bacterial dilutions either intraperitoneally or perorally. For peroral
inoculations bacteria
were washed and concentrated by centrifugation, the bacteria were then
resuspended in
0.2M Na2HP04 at pH 8.0, to neutralize stomach acid, and administered as a 0.2
ml bolus
to animals under ether anesthesia. For all LDSO determinations, 5 mice each
were
inoculated per dilution. Control mice received PBS only.
All bacterial strains used in this study were derivatives of S. typhimurium
14028
(strain 1). Mutant strains were isogenic to wild type and were obtained or
constructed as
described (dam102::Mud Cm and mutS121::Tn10 alleles are in LT2 (strain 7), a
highly
attenuated (virtually non-pathogenic) strain as shown in Table 2, were
obtained from
Dr. John Roth (University of Utah) and Dr. Tom Cebula (The Food and Drug
Administration), respectively; these alleles (and additional alleles below)
were transduced
into virulent strain, 14028, constructing strains 2 and 5, respectively.
damd232 (strain 3)
was constructed using internal oligonucleotides that serve as PCR primers
designed to
construct an in-frame 300 by deletion of defined dam sequence. dcml::Km was
constructed according to (Julio, S. M., et al., Molec. Gen. Genet., 258: 178-
181 (1998));
the Km resistance determinant is associated with an internal deletion of > 600
by of dcm
sequence. The 1rp31::Km is a null insertion in the lrp gene (strain 6). The
Dam
overproducing strain (strain 4) contains E. coli dam on a recombinant plasmid
(pTP 166)
in a wild-type background (Marinus, et al., Gene, 28:123-125 (1984).
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For in vivo competition studies, bacteria were treated as discussed above,
then
mutant cells were mixed with wild-type cells at a 1:1 ratio (approximate input
bacteria
was 500 mutant + 500 wild type). Actual ratios were determined by first
plating input
bacteria on LB, then scoring one hundred colonies for resistance to
appropriate
antibiotic(s). Bacteria were injected intraperitoneally into at least five
BALB/C mice
(with a one-to-one ratio of mutant to wild type as described (Conner, C. P.,
et al., P~oc.
Natl. Acad. Sci. USA, 14:4641-4645 (1998)), then after 4-5 days, when mice
appeared
moribund, they were sacrificed and their spleens isolated, homogenized,
diluted and
plated. Again, the ratio of mutant to wild-type was determined by scoring one
hundred
colonies for the mutant phenotype. The competitive index is the ratio of
mutant to wild-
type bacteria recovered and essentially reflects how fit the mutant strain is
compared to
the wild-type strain. Thus, those strains that display a competitive index of
less than
0.0001 reflect the fact that no mutant strains were recovered from the
spleens.
Consequently, the mice died as a result of the wild-type strains.
The advantage of the LDSO assay is that it quantitates large virulence
defects. The
disadvantage is that it lacks sensitivity and thus subtle but important
virulence
contributions are often missed. The competitive index is the ratio of mutant
to wildtype
bacteria recovered from infected tissues after co-inoculation. The competitive
index is
very sensitive allowing subtle virulence contributions to be detected.
However, because
of its sensitivity, quantitation of the differences in virulence between two
mutants that
confer large defects is problematic. Thus the use of the LDSO and competitive
index
assays in concert are an effective means to quantitate both large and subtle
virulence
defects. The competitive index is an additional indicator of how fit the
mutant strains are
compared to wild type, but does not necessarily directly correlate with full
virulence.
The results are shown in Table 1. LD;o is the dose required to kill 50% of
infected animals (LDSO) assay for each of these strains was compared to that
of wild type
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(strain 1; (ND, Not determined)). The peroral LDSO via gastrointubation for
all
derivatives was determined by infecting at least twelve BALB/c mice; the
intraperitoneal
(i.p.) LDSO was determined by infecting at least six mice.
TABLE 1
Competitive
Strain Genotype Oral LDSO LP. LDSO Index (LP.)
1 "wild type" >10+5 <10 ---
2 dam 102: : Mud > 10+9 > 10+4 < 10 4
Cm
damd232 >10+9 >1O+4 <10-4
(non-polar deletion)
wild type, (pTP166)10+s >10+4 <10_4
(Dam overproducer)
mutS121: : TnlO 10+5 ND 0.9
6 1rp31: : Km 10+5 ND 10.0
7 LT2 ND 2 x 10+4 ND
Since the dam insertion could decrease the expression of downstream genes
(polar
effects), an in-frame, nonpolar dam deletion was constructed, and was shown to
have the
same reduced virulence as the dam insertion. Thus, the attenuation was
specifically due
to the lack of Dam. Furthermore, intraperitoneal inoculation of mice with
equal numbers
of Dam+ and Dam Salmonella showed that Dam mutants were completely eliminated
during growth in the mouse (competitive index assay). Similar results were
obtained
with strain 4 (Table 1 ) that overproduces Dam from a recombinant plasmid,
suggesting
that precise levels of the Dam methylase are required for full virulence.
These results
show for the first time that the Dam methylase is essential for bacterial
pathogenesis.
Dam could affect Salmonella virulence via an increase in mutation rate caused
by
abrogation of methyl-directed mismatch repair (MDMR). Since MutS plays an
essential
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role in MDMR, it was determined whether mutS Salmonella were attenuated for
virulence. The data in Table 1, above, show that in both the oral LDSO and the
competitive index virulence assays, mutS Salmonella were identical to wild
type,
indicating that Dam does not affect pathogenesis via the MDMR pathway. Since
MutS-
strains show higher levels of DNA exchange between species than MutS+ strains,
they
more readily acquire new virulence determinants (Marinus, E. coli and
Salmonella:
Cellular and Molecular Biology, 2nd ed., 782-791 (1996)). The fact that MutS-
strains
are fully virulent could explain the high frequency at which mutS E. coli and
Salmonella
mutants are found amongst clinical isolates (LeClerc, et al., Science,
274:1208-1211
(1996)).
Dam and Lrp directly regulate the expression of Pap pili, which are essential
for
virulence of uropathogenic E. coli (O'Hanley et al., J. Clin. Invest., 75:347-
360 (1985);
and Roberts, et al., J. Urol., 133:1068-1075 (1985)). To determine if Dam
affects
Salmonella virulence through an Lrp-mediated pathway, Lrp- Salmonella were
analyzed
(Table 1 ). Salmonella lacking Lrp were fully virulent based on the LDSO and
competitive
index assays. These data show that Salmonella Lrp is not a virulence factor in
mice.
The results discussed above show that adenine methylation is critical for
Salmonella pathogenesis. DNA methylation of cytosine residues appears to be
important
for the regulation of biological processes in both plants and animals.
Although
Salmonella contain a DNA cytosine methylase (Dcm), the role of cytosine
methylation in
this organism is unclear. The dcm- mutant (dcml::Km) was virulent in the LDSO
and
competitive index assays, data not shown. These results demonstrate that
methylation of
adenine but not cytosine residues is required for Salmonella pathogenesis.
DNA adenine methylation has been shown to directly control virulence gene
expression in E. coli (Braaten, et al., Cell, 76:577-588 (1994)). Therefore,
it was
determined whether Dam regulates Salmonella genes that are preferentially
expressed in
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the mouse, designated as in vivo induced (ivi) genes. See, Corner, C. P., et
al., Proc.
Natl. Acad. Sci. USA, 14:4641-4645 (1998); Heithoff, D. M., et al., Proc.
Natl. Acad. Sci.
USA., 94:934-939 (1997); Mahan, M. J., et al., Science, 259:666-668 (1993);
Mahan, M.
J., et al., Proc. Natl. Acad Sci. USA, 92:669-673 (1995); and U.S. Patent No.
5,434,065,
all of which are incorporated herein by reference. Dam significantly repressed
the
expression of over 20 ivi genes (2 to 18 fold) when grown in rich medium,
eight of which
are displayed in Figure 3. Four of the eight fusions are in known genes, all
of which have
been shown to be involved, or implicated, in virulence: spvB resides on the
Salmonella
virulence plasmid and functions to facilitate growth at systemic sites of
infection (Gulig,
et al., Mol. Microbiol., 7:825-830 (1993); pmrB is involved in resistance to
antibacterial
peptides termed defensins (Roland, et al., J. Bacteriol., 75:4154-4164 (1993);
mgtA and
entF are involved in the transport of magnesium and iron, respectively
(Earhart,
Escherichia coli and Salmonella Cellular and Molecular Biology, 2nd edition,
1075-1090
(1996); and Vescovi, G., et al., Cell, 84:165-174 (1996)). Additional ivi
genes of
unknown function were also Dam-regulated. These results indicate that Dam is a
global
regulator of Salmonella gene expression.
Salmonella pathogenesis is known to be controlled by PhoP, a DNA binding
protein that acts as both an inducer and repressor of specific virulence genes
(reviewed in
Groisman and Heffron Two-component signal transduction, 319-332 (1995)). To
determine whether the Dam and PhoP regulatory pathways share common genes, the
effect of Dam was tested on seven PhoP-activated ivi genes, including spvB,
pmrB, and
mgtA. Figure 4 shows that Dam repressed the expression of these three genes by
2 to 19
fold, and this repression was not dependent on the PhoP protein. Dam did not
significantly affect the expression of the remaining four PhoP- activated
genes (data not
shown). These results indicate that Dam and PhoP constitute an overlapping
global
regulatory network controlling Salmonella virulence.
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Binding of regulatory proteins to DNA can form DNA methylation patterns by
blocking methylation of specific Dam target sites (GATC sequences (van der
Woude, et
al., J. Bacte~iol., 180:5913-5920 (1998)). Therefore, further investigation of
the
interactions between Dam and PhoP were carried out by determining if binding
of PhoP
(or a PhoP-regulated protein) to specific DNA sites blocks methylation of
these sites by
Dam, resulting in an alteration in the DNA methylation pattern. Analysis of
PhoP+ and
PhoP- Salmonella showed distinct differences in DNA methylation patterns.
Digestion of
genomic DNA from PhoP- bacteria with MboI (which cleaves only at nonmethylated
GATC sites) resulted in the appearance of DNA fragments that were not present
in DNA
from PhoP+ bacteria (Figure 5, see arrows). These results indicate that the
PhoP protein
(or a PhoP-regulated gene product) blocks Dam methylation at specific GATC-
containing
sites in the Salmonella genome. Alternatively, PhoP+ and PhoP- strains may
have
different levels of Dam activity which, in turn, may affect DNA methylation
patterns.
However, this regulation does not occur at the transcriptional level since Dam
does not
alter PhoP expression, nor does PhoP alter Dam expression (D. M. Heithoff and
M. J.
Mahan, unpublished material). Further analysis will determine whether these
PhoP-
protected sites are within regulatory regions of virulence genes, and if DNA
methylation
directly affects the PhoP regulon by altering DNA-PhoP interactions.
Example 2A: Protective efficacy of Dam Salmonella attenuated strains
Strains which demonstrated attenuation as a result of intraperitoneal or oral
challenge of BALB/c mice were further tested for protective immunity against
subsequent challenge by the wild-type strain at 105 LP. or 109 orally. BALB/c
mice were
perorally immunized via gastrointubation with a dose of 10+9 Dam S.
typhimurium. Five
weeks later, the immunized mice were challenged perorally with 10+9 wild-type
S.
typhimurium as described. After five weeks, surviving mice were challenged
with the
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wild-type 14028 strain as noted in Table 2 below. Survival for four weeks post
challenge
was deemed full protection. These data demonstrate the potential use of the
present
invention in developing vaccine strains.
Since Dam mutants were highly attenuated, it was determined whether Dam
Salmonella could serve as a live attenuated vaccine. Table 2 shows that all
(17/17) mice
immunized with a S. typhimurium Dam insertion strain survived a wild-type
challenge of
10+4 above the LDSO, whereas all nonimmunized mice (12/12) died following
challenge.
TABLE 2
Immunization with Challenge with 10+9
Dam S. typhimurium wild-type S. typhimurium
None 12/12 dead
dam102:: Mud-Cm 17/17 alive
damd232 (nonpolar deletion) 8/8 alive
Virtually no visible effects of typhoid fever were observed subsequent to
immunization with Dam Salmonella, nor were there visible effects after the
wild type
challenge. Moreover, because all (8/8) mice immunized with Salmonella
containing the
nonpolar dam deletion (strain ) survived challenge, these data indicate that
protection was
specifically due to the absence of Dam methylase. The virulence attenuation
and
effectiveness of Dam mutants as a vaccine (Tables 1 and 2) could be due to the
ectopic
expression of virulence determinants (Figures 3 and 4) which would likely be
deleterious
to the growth and/or survival of Salmonella during infection. Thus, ectopic
expression
provides an explanation as to why the Dam mutant is totally attenuated yet
still provides
full protection as a live attenuated vaccine.
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Colonization studies
The survival of Dam+ and Dam Salmonella in mouse tissues was compared. As
shown in Figure 6, Dam bacteria were fully proficient in colonization of a
mucosal site
(Peyer's patches) but showed severe defects in colonization of deeper tissue
sites. Five
days after infection, we observed a reduction of three orders of magnitude in
numbers of
Dam Salmonella in the mesenteric lymph nodes (relative to numbers of Dam+
bacteria)
and a reduction of eight orders of magnitude in numbers of Dam Salmonella in
the liver
and spleen. These data show that Dam Salmonella survive in Peyer's patches of
the
mouse small intestine for at least 5 days, providing an opportunity for
elicitation of a host
immune response. Dam Salmonella, however, were unable to cause disease; they
either
were unable to invade systemic tissues or were able to invade but could not
survive.
Example 2B: Protective efficacy of killed Dam derivatives
Determination of whether living Dam or Dam overproducing bacteria are
required to elicit a fully protective response. The ectopic expression of
multiple proteins
in Dam vaccines (see above and below) suggests the possibility that killed Dam
organisms may elicit significantly stronger protective immune responses than
killed
Dam+ organisms and thus be used as mucosal vaccine. In vitro grown S.
typhimurium
Dam bacteria are killed by exposure to sodium azide (0.02%) and/or UV light,
after
which the antimicrobial is either washed or dialyzed away from the killed
organisms.
The efficacy of the whole cell killed vaccine preparation is tested with and
without the
use of mucosal adjuvants such as cholera toxin, E. coli labile toxin, or
vitamin D3
(1,25(OH)2D3). Accordingly, vaccine preparations containing 101° killed
Dam
Salmonella, alone and in combination with mucosal adjuvants, are used to
orally
immunize BALB/c mice (as described in the Examples). As a dosing regimen, mice
are
immunized by gastrointubation once a week for three weeks. Killed wild-type S.
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typhimurium serves as a negative control. The immunized mice are orally
challenged
with virulent S. typhimurium 2 weeks after the last immunization to determine
if an
effective immune response is generated. If so, mice immunized with the killed
vaccine
preparation are also challenged with other pathogenic Salmonella serotypes
(e.g.,
enteritidis, choleraesuis, dublin) to determine if the immunity elicited is
cross-protective
against related strains as is the case for oral administration of Dam
Salmonella live
vaccines. If mice immunized with the dead vaccine preparation are protected
two weeks
after the final immunization (of three), whether the immunity elicited is long-
lasting is
determined by challenging immunized mice 7 weeks after the last immunization.
Since Dam overproduction may result in the ectopic expression of a new
repertoire of potential protective antigens that are not expressed in either
the wild-type
(Dam+) or Dam vaccine strains, the killed vaccine experiments are performed
with Dam
overproducing strains, alone and in combination with killed Dam organisms.
Since the
two different vaccine strains may produce two different repertoires of
potentially
protective antigens, use of them in combination may elicit a superior immune
response.
Example 3: Cross-protection elicited by a Dam- Salmonella
Immunization with Dam- Salmonella elicits a cross protective response to
heterologous se~otypes. As shown in Figure 7 and discussed below, Dam mutants
ectopically express multiple genes (and presumably proteins) that are normally
only
expressed during infection. Such ectopic expression of multiple antigens may
result in
cross-protective immune responses against heterologous serotypes. BALB/c mice
were
immunized with 1 X 109 Dam S. typhimurium (serogroup B) administered orally
(via
gastrointubation) and were challenged eleven weeks later with (100 to 1000
LDso)
virulent S. enteritidis and S. dublin (serogroup D). The data in Table 3 show
the mice
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were protected against a heterologous challenge eleven weeks post
immunization.
Importantly, the cross-protective immunity was not attributed to the
persistence of the
vaccine strain in marine tissues, since mice were protected against
heterologous
challenge greater than six weeks after the vaccine strain was cleared from
immunized
animals (i.e., after Dam organisms could not be detected in Peyer's patches,
mesenteric
lymph nodes, liver and spleen). The cross-protection elicited is specific to
Salmonella
strains as no protection was elicited against the systemic pathogen Yersinia
pseudotuberculosis five weeks post-immunization.
TABLE 3
Immunization with Dam' S. typhimurium confers cross-protective immunity
Oral immunizationOral challengeOral challengeOral challengeOral challenge
with 109 Dam S. with 1 Og with 109 wild-with 1 Og with 109
typhimurium Wild-type type S. dublinwild-type Wild-type S.
S. S.
enteritidis dublin typhimurium
None 17/17 dead 25/25 dead 11/11 dead 10/10 dead
damd232 4/18 alive 4/19 alive 10/19 alive11/11 alive
Similarly, immunization with Dam- S. enteritidis (dam102::Mud-Cm, following
an experimental protocol described above) confers cross-protection against
challenge
with 109 S. typhimurium and 109 S. dublin after five weeks and may confer
cross-
protection for even longer periods.
Dam- derivatives ectopically express multiple proteins in vitro. Ectopic
expression of multiple proteins in Dam- strains may contribute to the cross-
protection
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elicited against heterologous serotypes that share common epitopes. To this
end, we have
shown that Dam- strains ectopically express of a number of Salmonella genes
that are
normally repressed in vitro.
Two-dimensional protein gel electrophoresis was performed by the method of
O'Farrell
((1975) J. Biol. Chem. 250: 4007-4021) on whole-cell protein extracts of log-
phase S.
typhimurium grown in Luria broth. Isoelectric focusing using pH 5-7 ampholines
(BioRad Laboratories, Hercules, CA) was carried out at 800 V for 17 h. The
second
dimension consisted of 12.5% polyacrylamide slab gels which were run for 5.5 h
at 175
V. Proteins were visualized by silver staining (Merril et al. (1984) Methods
Enzymol.
104:441-447.). The results are shown in Figure 7. The results show that two-
dimensional gel electrophoresis analysis (2-D protein analysis) of Dam,- Dam+
(wild
type) and Dam overproducer (OP) strains grown in vitro resulted in the
detection of
several proteins that were expressed under the Dam condition that were not
detected
under either the Dam+ (wild type) or Dam OP (expressing about 100-fold higher
Dam
than normal) conditions. These data indicate that Dam Salmonella ectopically
express
multiple proteins in vitro (and presumably in vivo), suggesting that
dysregulation of
protein expression could provide multiple novel protein targets to be
processes and
presented to the immune system
2-D protein analysis indicates that Dam overproducing strains of Salmonella
(S.
typhimurium ATCC 14028 with plasmid pTP166 that overproduces E. coli Dam at
about
100-fold the wildtype level) express a number of gene products that are not
expressed by
Dam+ (wild type) or Dam Salmonella under laboratory growth conditions. We have
not
detected proteins that are produced by Dam+ that are not produced by Dam or
Dam
overproducing strains. Taken together with our observation that Dam
overproducing
strains are attenuated and elicit protective immunity, these results suggest
that Dam
overproduction may result in the expression of a different repertoire of
antigens than
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what is produced in Dam strains. Thus, vaccines consisting of Dam
overproducing
strains in combination with the Dam strains may be highly cross-protective due
to the
ectopic expression of two different repertoires of potentially protective
antigens.
Immunity elicited by Dam- strains is greater than immunity elicited after a
wild-
type infection. One of the most effective virulence properties of a pathogen
is the ability
to evade host immune responses. Such a "stealth" strategy is achieved by
tightly
regulating many of its functions to avoid host immune recognition. Thus, as a
bacterial
protective mechanism, it is likely that many antigens produced by virulent
organisms are
not produced in sufficient quantities and/or for a sufficient amount of time
to elicit a host
immune response. However, Dam bacteria may ectopically express multiple
antigens
that are processed and presented to the immune system, and thus, animals
immunized
with Dam vaccines may elicit stronger immune responses than animals that
survive a
natural infection.
The immunity elicited by the Dam vaccine was compared to the immunity
elicited after a natural infection with the wild-type strain. BALB/c mice were
orally
immunized at the LDSO of the virulent strain S. typhimurium (10+5 organisms)
(i.e., one
half the mice survived the wild-type immunization) or 10+5 Dam organisms. Five
weeks
post-immunization, the immunized mice were challenged with lethal doses of the
virulent
strain. Table 5 shows that the immunity elicited by the Dam vaccine was at
least 100-
fold greater (3 of 10 mice survived a 10+9 challenge) than the immunity
elicited in mice
that survived an immunization with the wild-type strain (1 of 10 survived a
10+'
challenge).
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Table 5. Mice immunized with Dam- vaccines elicit greater protection than mice
that
survive a wild-type infection.
Oral immunization Oral challenge with Oral challenge with Oral challenge with
10+5 S. typhimurium 10~ 10g 109
wild-type S. wild-type S. S. typhimurium
typhimurium typhimurium
None 10/10 dead 10/10 dead 10/10 dead
Dam+ (at LDS p) 1 / 10 alive 10/ 10 dead 10/ 10 dead
damd232 5/10 alive 4/10 alive 3/10 alive
S Additionally, immunization with Dam organisms showed relatively similar
levels
of protection over a wide range of challenge doses (10+' to 10+9). This
suggests that an
immunizing dose of 10+5 Dam bacteria is below the minimum threshold of
organisms
required to ensure a productive immune response in all immunized animals. It
is possible
that the enhanced immunity elicited by Dam strains may be attributed, in part,
to the
ectopic expression of Dam repressed-antigens, which may not be produced in
sufficient
quantities and/or duration during a wild-type infection.
Immunized animals hinder growth of virulent bacteria in systemic tissues. Dam-
Salmonella were found to be fully proficient in colonization of Peyer's
patches of the
mouse small intestine but were severely deficient in colonization of deeper
tissue sites
(liver and spleen) (Example 1 ). Dam- mutants of S. typhimurium are also less
cytotoxic
to M cells, are deficient in epithelial invasion, and display defects in
protein secretion.
Pucciarelli et al. (1999) Proc. Natl. Acad. Sci. USA 96:11578-11583. Taken
together,
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these data provide a possible explanation as to why Dam mutants are unable to
cause
disease but are able to elicit a full-protective immune response. Since mice
immunized
with Dam Salmonella showed virtually no overt symptoms of disease after
challenge
with virulent organisms, the fate of wild-type Salmonella was compared within
immunized vs. non-immunized mice. The data in Fig. 8 show that Dam immunized
mice
carry high loads (104) of virulent bacteria for at least five days in both
mucosal and
systemic tissues after wild-type challenge of 109 organisms. However, the
immunized
mice have the ability not only to inhibit the growth of these virulent
organisms, they are
capable of clearing them from both mucosal and systemic tissues (2 out of 4
mice have
cleared all virulent organisms from the Peyer's patches, mesenteric lymph
nodes, liver
and spleen 28 days post challenge). This ability to clear 104 virulent
organisms from the
liver and spleen is significant in light of the fact that the i.p. LDso is
less than 10
organisms. Thus, immunization with Dam Salmonella hinders the proliferation of
wild-
type organisms in all tissues tested. The ability to clear a lethal load of
virulent
bacterium from systemic suggests the possibility that Dam vaccines may have
therapeutic application to the treatment of a pre-existing microbial
infections.
Example 4: Vaccination of chicken against S. enteritidis
A thorough understanding of the dynamics of S. enteritidis infection in
poultry is
essential to the formulation of an effective strategy to interrupt the
eggborne transmission
of S. enteritidis from laying hens to human consumers. Salmonellae cause
disease by
colonizing and invading the intestinal epithelium. In some cases, Salmonella
penetration
through the intestinal mucosa to the bloodstream is followed by widespread
dissemination and systemic disease. S. enteritidis is an invasive serotype in
chicks but
has not exhibited a level of pathogenicity for chicks that is markedly
different from that
of other paratyphoid Salmonella serotypes. Popiel and Turnbull (1985) Infect.
Immun.
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47(3):786-792. Chicks can be readily infected, involving both intestinal
colonization and
invasion to reach internal issues such as the liver, with S. enteritidis from
contaminated
feed. Hinton et al. (1989) Vet. Rec. 124:223.
Experimental infections of adult hens with some S enteritidis strains have led
to
intestinal colonization that persisted for several months, although in studies
with other S.
ente~itidis strains the duration of fecal shedding has been considerably
shorter. (Gast and
Beard, 1990), Gast and Beard (1990) Avian Dis. 34:991-993; Shivaprasad et al.
(1990)
Avian Dis. 34:548-557. In one study, intravenously infected birds shed S.
enteritidis for a
longer period than did orally infected birds. Shivaprasad et al. (1990).
The effectiveness of various methods of destroying S enteritidis in eggs and
egg
products has become a topic of increasing importance to public health
authorities and the
egg industry. Such information is vitally needed in order to provide
instructions to
consumers and commercial or institutional users of eggs regarding safe
preparation of
egg-containing foods. Shivaprasad et al. (1990) observed that the
time/temperature
requirements for destroying S enteritidis in eggs by various cooking methods
did not
differ significantly from similar requirements previously determined for S.
typhimurium.
Baker et al. (1983) Poult. Sci. 72:1211-1216. Humphrey et al. found that
strains of phage
type 4 S. enteritidis, S. typhimu~ium, and S. senftenberg, when inoculated
into egg yolk,
could survive forms of cooking in which some of the yolk remained liquid.
Humphrey et
al. (1989) epidemiol. Infect. 103:35-45. Moreover, when eggs were stored at
room
temperature for 2 days after inoculation, the S. enteritidis population grew
to such a high
level in the yolk that no standard cooking method completely eliminated the
Salmonella.
Storage of S enteritidis cultures at refrigerator temperatures, on the other
hand, has been
found to increase their sensitivity to heat. Humphrey (1990) J. Appl.
Bacteriol. 69:493-
497. In another study, S enteritidis phage type 4 in homogenized whole egg was
determined to be more heat resistant than phage types 8 or 13a and S.
typhimurium, but
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less than the highly heat-resistant S. senftenberg strain 775W. All Salmonella
strains
tested were more heat resistant in yolk than in whole egg or albumin. Humphrey
et al.
(1990) Epidemiol. Infect. 104:237-241.
The vaccines of the present invention, specifically Strain 3, may be effective
at
eliminating S. enteritidis in eggs and egg products. A dam- S. typhimurium
vaccine is
prepared as described previously. The vaccine is introduced into the chicken
by way of
oral administration, that is, mixed with the chickens feed and/or water. Once
the vaccine
has been administered the virulence factors typically repressed by Dam will be
expressed
and the chicken will elicit an immune response. Since some of these Dam-
regulated
genes are homologs to those shared by S. enteritidis, the Dam S. typhimurium
may elicit
cross-protection against S. enteritidis, as the data in Example 3 indicate.
Example 5: Administration of Dam derivative Salmonella vaccines to cattle
Salmonella is the most commonly isolated infectious enteric bacterial pathogen
of
dairy cattle and the most common zoonotic disease associated with human
consumption
of beef and dairy products. In recent years there has been a rise in the
incidence and
severity of human cases of salmonellosis, in part due to the emergence of the
antimicrobial resistant S. typhimurium DT104 in cattle populations. Prevalence
studies
indicate 16 to 73% of U.S. dairy farms are infected with Salmonella and up to
50% of
cull dairy cows are contaminated with Salmonella at slaughter. On-farm
Salmonella
control is important to reduce production losses and human food borne disease.
On large commercial dairy farms it is very common for cattle to be exposed to
multiple Salmonella serotypes and for calves to become infected shortly after
birth.
Under these conditions it would be very desirable to have a Salmonella vaccine
capable
of stimulating immunity to heterologous Salmonella serotypes.
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A. Requirement of Dam for Salmonella infection of cattle, and effectiveness of
Dam derivatives as live bovine vaccines
Holstein bull calves 1-3 days of age are used for all of the experiments.
Measurement of total plasma protein is used to assess passive immunity of
calves. Only
calves with a total plasma protein greater than or equal to 5.5 are used. The
Salmonella
infection status of the source dairies is determined prior to purchasing the
calves by
culturing fecal and environmental samples for salmonellae. The Salmonella
negative
status of calves will be confirmed after purchase by daily fecal Salmonella
cultures.
The calves are housed and raised in Animal Biosafety 2 level facilities.
Calves are
fed 2 quarts of 20:20 milk replaced twice a day and have access to fresh calf
grain and
fresh water 24 hours a day. Each day at feeding time all calves are given an
appetite and
attitude score. The appetite score is on a scale of 1 to 4 (1 = consumed 2
quarts of milk, 2
= consumed < 2 but > 1 quart of milk, 3 = consumed < 1 quart of milk, and 4 =
consumed
no milk). The attitude score is also on a scale of 1 - 4 (1 = standing, 2 =
stands with
encouragement, 3 = stands with assistance, 4 = unable to stand). Following all
of the
challenge experiments calves are checked 3 times a day and vital parameters
recorded
twice a day. Any calf that is unable to stand is considered terminal and is
euthanized. No
antimicrobial or anti-inflammatory treatments are administered to calves
following
Salmonella challenge to avoid confounding of the experimental results.
Determination of the safety of live Dam Salmonella vaccines in Holstein bull
calves. The safety of Dam S. typhimurium in 1 - 3 day old calves is determined
as
follows. Eighteen 1 - 3 day old calves are divided into 3 groups of 6. The
first group of
6 calves is challenged orally with 109 Dam Salmonella, the second group with
10'° and
the third with 10' ~. For the 3 weeks following challenge each calf in the
study is
evaluated twice a day to measure pulse and respiratory rate, rectal
temperature, appetite,
and attitude. Fecal samples are collected from each calf daily for Salmonella
culture. At
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3 weeks post challenge the calves are euthanized and organs (liver, bile,
spleen,
mesenteric lymph nodes, ileum mucosa, small intestinal contents, cecum mucosa
and
cecal contents) cultured for salmonellae.
Determination of whether Salmonella Dam based vaccines can colonize mucosal
and/or systemic tissues. The kinetics of colonization of bovine tissues is
determined for
both Dam+ and Dam S. typhimurium after oral administration. The "bacterial
load" in
the small intestinal contents, ileum mucosa, Peyer's patches, cecum mucosa,
cecal
contents, mesenteric lymph nodes, liver, and spleen, is determined in calves,
as a function
of time post infection. Twenty four holstein bull calves are challenged orally
with 109
Dam S. typhimurium. Six calves are randomly assigned to 4 groups to be
euthanized at
24 hours and 5, 14, and 28 days post challenge. Tissues are collected from
each calf at
necropsy for quantitative Salmonella culture. Twenty four holstein bull calves
challenged orally with 109 Dam+ S. typhimurium are processed identically and
serve as a
positive control for these experiments.
For Dam Salmonella to be ideal bovine vaccines, they should colonize the
Peyer's patches, replicate and persist within the M cells, and present
antigens to the
underlying immune cells (e.g., macrophages, B cells and T cells) that comprise
the
Peyer's patch lymphoid follicle. As importantly, they should not colonize
deeper tissue
such as the liver and spleen, and should eventually be cleared from the
Peyer's patches.
If these criteria are met, it is more likely that Salmonella Dam mutants would
serve as
the basis for a safe, effective bovine vaccine.
Protective efficacy of Dam S. typhimurium vaccination against homologous wild
type challenge. Twenty calves 1 - 3 days of age are randomly divided into 2
groups of
10 calves. The first group is vaccinated per os with Dam S. typhimurium at 1 -
3 days of
age. The remaining 10 unvaccinated calves \serve as controls. All calves are
challenged
per os with 10' ~ virulent S. typhimurium at 5 weeks of age. For the 3 weeks
following
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challenge each calf is evaluated three times a day and pulse, respiratory
rate, rectal
temperature, appetite score, and attitude score recorded twice a day. Fecal
samples are
collected from each calf daily for Salmonella culture. All calves that die
following
challenge are necropsied and organs (liver, bile, spleen, mesenteric lymph
nodes, ileum
mucosa, small intestinal contents, cecum mucosa and cecal contents) cultured
for
salmonellae. Calves surviving virulent Salmonella challenge are euthanized 3
weeks post
challenge, necropsied, and organs cultured for salmonellae (liver, bile,
spleen, mesenteric
lymph nodes, ileum mucosa, small intestinal contents, cecum mucosa and cecal
contents).
Minimum dose regimen required for efficacy in calves and reduced vaccine
persistence in bovine tissues. Three important features of any vaccine regimen
are i) the
dose of the vaccine, ii) the age of the animal, iii) and the persistence of
the vaccine in the
immunized animal. Minimum dose required to elicit full protection (at 10,000
times the
LDSO) and reduced persistence in murine tissues such as the Peyer's patches,
mesenteric
lymph nodes, liver, and spleen is determined.
B. Dam derivatives elicit cross-protection against related (heterologous
Salmonella serotypes) pathogenic strains
Protective efficacy of Dam S. typhimurium vaccination against heterologous
wild
type challenge. Three similar virulent Salmonella challenge experiments are
performed
using 3 different challenge organisms. Each experiment involves oral
immunization of
calves with Dam S. typhimurium at 1- 3 days of age and challenge with virulent
Salmonella at 5 weeks of age. In the first experiment S. montevideo (serogroup
C1) is
used as the challenge organism, S. dublin (serogroup D) in the second, S.
anatum
(Serogroup E1) in the last. Different calves are used for each experiment. For
each of
these 3 experiments twenty calves 1 - 3 days of age are randomly divided into
2 groups
of 10 calves. The first group is vaccinated per os with Dam S. typhimurium at
1 - 3 days
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of age. The remaining 10 unvaccinated calves serve as controls. All calves are
challenged per os with 10' ~ virulent Salmonella at 5 weeks of age.
For the 3 weeks following challenge each calf is evaluated three times a day
and
pulse, respiratory rate, rectal temperature, appetite score, and attitude
score recorded
twice a day. Fecal samples are collected from each calf daily for Salmonella
culture. All
calves that die following challenge are necropsied and organs (liver, bile,
spleen,
mesenteric lymph nodes, ileum mucosa, small intestinal contents, cecum mucosa
and
cecal contents) cultured for salmonellae. Calves surviving virulent Salmonella
challenge
are euthanized 3 weeks post challenge, necropsied, and organs (liver, bile,
spleen,
mesenteric lymph nodes, ileum mucosa, small intestinal contents, cecum mucosa
and
cecal contents) cultured for salmonellae. Comparison of cross-protective
immunity
elicited in Dam overproducing strains, alone and in combination with Dam
mutants, is
also performed.
C. Killed Dam derivatives of Salmonella
In vitro grown S. typhimurium Dam bacteria are killed by exposure to sodium
azide (0.02%) and/or UV light, after which the antimicrobial is either washed
or dialyzed
away from the killed organisms. The efficacy of the whole cell killed vaccine
is tested
administered per os (oral) and parenterally. For the parenteral vaccine group
106 killed
Dam Salmonella is mixed with aluminum hydroxide and quill A adjuvants and
administered to calves via intramuscular injection. For the per os vaccination
group 10'°
killed Dam Salmonella is administered per os with Vitamin D3 as a mucosal
adjuvant.
As a dosing regimen, neonatal calves are immunized once a week for three
weeks. Killed
wild-type S. typhimurium administered by the same route and with the same
adjuvants
serve as a negative control. The immunized calves are challenged with virulent
S
typhimurium 2 weeks after the last immunization using the same protocol as
described
above to determine if an effective immune response is generated. If so, calves
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immunized with the killed vaccine preparation are also be challenged with
other
pathogenic Salmonella serotypes (e.g. montevideo, S. dublin, and S. anatum) to
determine
if the immunity elicited is cross-protective against related strains. The
experiment is
repeated using Dam overproducing strains, alone or in combination with killed
Dam
organisms.
Since Dam overproduction may result in the ectopic expression of a new
repertoire of potential protective antigens that are not expressed in either
the wild-type
(Dam+) or Dam vaccine strains the killed vaccine experiments are repeated with
Dam
overproducing strains, alone and in combination with killed Dam organisms.
Example 6: Construction of dam mutants in Vibrio cholerae
A. Construction of V. cholerae dam mutations
Y cholerae dam mutations are not currently available. Known V. cholerae dam
sequence is used to design primers to PCR amplify the dam gene, which is used
as a
probe to hybridize against an Y cholerae lambda clone bank to recover the wild-
type V.
cholerae dam clone. The DNA ends of hybridizing clones are sequenced to
determine
whether they contain the V. cholerae dam region. Subcloning and further
sequencing off
the vector ends of these subclones identifies the smallest DNA restriction
fragment
containing the entire V. cholerae dam sequence. Non-revertible dam deletion
mutations
associated with an antibiotic resistance marker are constructed according to
methods
recently developed (Julio, S. M., et al., Molec. Gen. Genet., 258:178-181
(1998).
The roles) of dam mutants in Y cholerae pathogenesis are tested in two
different
virulence assays for murine cholera (suckling mouse models), the LDSO and the
competitive index, which have been described in Example 1.
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B. Determination of the protective capacity of dam mutants toward the goal of
constructing human live attenuated vaccines against V. cholerae
As discussed in detail above, Salmonella Dam mutants serve as live attenuated
vaccines in a mouse model for typhoid fever. The goal of this experiment is to
discern
whether these desired effects are specific to Salmonella DNA adenine
methylation or
whether Dam mutants also afford protection against V cholerae, and thus may
provide a
foundation for a new generation of live attenuated vaccines.
Human live attenuated vaccines must be designed to limit the risk of reversion
to
wild type and to ensure that these strains will not serve as a reservoir for
the spread of
antibiotic resistance to emerging pathogens. Thus, the next step in this
analysis will be to
construct an appropriate non-reverting, antibiotic sensitive derivative. Non-
polar
deletions (no effect on downstream genes in the operon) in dam are constructed
by
removing internal sequences of these genes by standard PCR-based approaches,
ligation
into a suicide vector, and recovery of the resultant in-frame deletion
strains. Deletions of
each gene are introduced individually using standard positive-selection
suicide vector
strategies (Donnenberg, M. S., et al., Infect. Immun., 59:4310-4317 (1991)),
resulting in
the desired non-reverting, attenuated, antibiotic sensitive vaccine strain.
The efficacy of
this vaccine is retested as described above. Strains constructed such that Dam
is
modified (i.e., not completely deleted and/or disabled) are tested, as are Dam
overproducing strains.
Example 7: Essentiality of dam gene in Vibrio cholerae and Yersinia
pseudotuberculosis
A duplication of dam was constructed by integrating a recombinant plasmid
containing a Dam mutation into the wild type Dam locus. The resulting
duplication
contained two copies of dam: a mutant copy and a wild type copy. Normally, the
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recombinant plasmid segregates at a given frequency, and there is a roughly
equal chance
that the recombinants (segregants) contain either the mutant or the wildtype
gene. If a
gene is essential, all segregants of the duplication (which recombines out of
the plasmid)
is wild type; the recombinants having the mutant gene die. If a recombinant
plasmid
containing the gene is present, the duplication can segregate either to the
mutant or wild
type. For Vibrio cholerae and Yersinia pseudotuberculosis, duplication of the
dam gene
to contain both a wild type and a mutant cannot segregate to the mutant unless
a
recombinant plasmid providing a wild type dam gene is present.
The foregoing description is considered as illustrative only of the principles
of the
invention. Furthermore, since numerous modifications and changes will readily
occur to
those skilled in the art, it is not desired to limit the invention to the
exact construction and
processes shown as described above. Accordingly, all suitable modifications
and
equivalents may be restored to falling within the scope of the invention as
defined by the
I S claims which follow.
88
