JP2002536339A - Compositions and methods for treating and preventing pathogenic bacterial infections based on the essential role of DNA methylation in bacterial toxicity - Google Patents

Abstract

  (57) [Summary] The present invention relates to vaccine compositions comprising pathogenic bacteria such as Salmonella having non-reverting mutations that alter the activity of DNA adenine methylase (Dam), and methods of using these compositions to elicit an immune response About. The present invention also provides methods for preparing vaccines, as well as screening methods for identifying agents that may have antibacterial activity. Typically, the pathogenic bacteria include S. aureus. typhimurium, S.P. enteritidis, S.M. typhi, S.M. abortusovi, S.A. abortus-equi, S.A. dublin, S .; gallinarum, and S. gallinarum. pullorum.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This patent application is related to US Provisional Patent Application No. U.S. patent application Ser. No. 09 / 241,951, which has been modified from US Pat.
On February 2) and US Provisional Patent Application No. 09/3 changed from No.
No. 05,603 (filed May 5, 1999).

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH This invention was made with grant number AI36373 (M) awarded by the National Institutes of Health.
. Mahan) and AI23348 (to D. Low) with government support. The government may have certain rights in the invention.

FIELD OF THE INVENTION [0003] The present invention relates to vaccines useful for preventing or modifying microbial pathogenesis. In particular, the invention relates generally to immunogenic compositions comprising a pathogenic bacterium (eg, Salmonella) containing a mutation affecting DNA adenine methylase (Dam). The invention also relates to methods of eliciting an immune response using these compositions, and screening methods.

BACKGROUND OF THE INVENTION Food-borne diseases represent a serious threat to our health, the safety of our national food supply, and the agricultural industry. More than 80 million Americans suffer from food poisoning each year, with costs estimated at between $ 5 billion and $ 23 billion annually in medical procedures and wages spent (Sny)
dman, D.A. R. , Food poisoning. : Infectious
Diseases (second edition), Gorbash, S .; L. Ed., 768-78
1 (1998)). Human defenses against food-borne diseases have failed. This is because new pathogens have emerged that can cause more forms of disease that debilitate humans and / or can no longer be controlled by available antibiotics; for example, Escherichia coli (E. coli) O1
57: H7, Salmonella enteritidis (S. enter
Itidis), and S. a. Typhimurium DT104 (Alterkruse, SF, et al., Emerging food bone).
e disease, 3: July-September (1997)). Salmonellosis is one of the major food-caused diseases in the United States, valued at between 1 and 4 million cases per year (Shere, KD et al., Salmon.
ella infections: infectious Diseases,
Second Edition, Gorbach, S .; L. Ed., 699-712 (1998)). The disease is caused by exposure to the consumption of salmonella contaminated products (animal products (eg, eggs), milk, poultry, or food (including fruits and vegetables) exposed to animal feces). Due to large-scale manufacturing and distribution practices, the occurrence of salmonellosis affected a large population (Tauxe, R.
. V. Et al., Emerging food bone diseases: an
evolving public health challenge. Em
erging infectious diseases, 3: October to December (1997)).

[0005] Salmonella is a major example of pathogenic microorganisms of which various species are responsible for a spectrum of clinical diseases including acute gastroenteritis and intestinal fever. Salmonella infection is obtained by ingestion. Salmonella infection is obtained by ingestion. After the microorganisms cross the stomach, they invade and replicate intestinal mucosal cells. Hornik et al. Eng. J. Med. , 283: 686 (1970).
checking ... Some species (eg, S. typhi) spread through this mucosal barrier and, via Peyer's patches, into the lamina propria and localized lymph nodes. Salmonella typhi, which only infects humans, is the cause of typhoid fever and remains an important public health problem for residents in non-developed countries.

[0006] Urinary tract infections (UTIs) are among the most common bacterial infections. It is estimated that about 20% of women will experience at least one UTI during their lifetime. Women are major targets of UTI, but men and children can also be affected by the disease. About 70% of all UTIs are uropathogeni
c) Caused by Escherichia coli. This disease
It may be restricted to the lower urinary tract (cystitis) or may be involved in the renal pelvis (pyelonephritis). E. coli isolated from a woman suffering from pyelonephritis More than 90% of E. coli contains pyelonephritis-associated hair
pili) (pap) gene cluster (O'Hanley, PM et al., N. Engl. J. Med. 313: 414-447 (1985)). E. FIG. c
Most patients with pyelonephritis caused by oli mount a strong immune response against Pap hair. Pap hair contains, at their tips, adhesins that allow these bacteria to colonize the urinary tract. Most Pap hair-adhesin complexes bind to P-type blood group receptors. This receptor is located in the gut,
It is expressed on epithelial cells on the wall of the bladder and ureter. Despite our understanding of the role of adhesion in the pathogenesis of UTI, vaccines are not available for UTI. This is also true for many other important microbial pathogens that cause significant morbidity and mortality.

[0007] Microbial pathogens or disease-producing microorganisms can infect a host by one of several mechanisms. They can penetrate through wounds in the skin, they can be introduced by vector transfer, or they can interact with mucosal surfaces. If the pathogen is fully expressed in its ability to disrupt normal bodily function, the disease will follow following infection of the host.

[0008] Each disease-producing microorganism carries a collection of toxic factors that enhances their pathogenicity and allows them to invade host or human tissues and destroy normal bodily functions. . Infectious disease has been a major killer over the past millennia, and while vaccines and antibacterial agents play a significant role in the dramatic reduction in the incidence of infectious disease, infectious disease is still a global phenomenon. It is the number one cause of death.

[0009] Environmental conditions within the host are responsible for regulating the expression of most known virulence factors (Mekalanos, JJ, Bacteriol. 174: 1 (1992).
)). In the past, scientists have attempted to mimic the environmental conditions in the host in vitro in an attempt to identify the gene that encodes a toxic factor and is responsible for the production of that factor. As a result, the identification of many virulence factors depends on and is limited by the ability of researchers to mimic host environmental factors in the laboratory. However, Ma
han, M .; J. With the advent of the in vivo expression technology (IVET) discovered by U.S. Pat. No. 5,434,065 and disclosed in US Pat. It is now possible to decide. As a result, the molecular mechanisms of certain pathogenic microorganisms have been elucidated, which allow them to evade the host (human body) immune system and initiate physiological changes inherent in the disease process, thereby elucidating the pathogenicity It allows to develop better therapeutic and diagnostic approaches to microorganisms.

[0010] Along with water sanitation, the prevention of infectious diseases by vaccination is the most efficient, economical and practical way of disease prevention. Other treatment modalities (even antibiotics) did not have such a major effect on mortality reduction and population growth. The impact of vaccination on the health of the world's people is exaggerated. Vaccination, at least in parts of the world, controlled nine major diseases: smallpox,
Diphtheria, tetanus, yellow fever, whooping cough, polio, measles, mumps and rubella. In the case of smallpox, the disease was all eradicated from the world. The effectiveness of a vaccine depends on its ability to elicit a protective immune response, described generally below.

[0011] The means by which vertebrates, especially birds and mammals, overcome microbial pathogenesis are complex. Pathogens that invade the host cause many highly diversified and protective systems. Microbial pathogens or their toxins successfully penetrate defenses outside the body,
And when it reaches the bloodstream, the lymphoid tissues of the spleen, liver and bone marrow remove and destroy foreign substances as blood circulation through these organs. Lymphoid tissue is mainly composed of a network of overlapping reticular cells and reticular fibers. Adhesion to the interstitial spaces of tissues can be caused by a large number of leukocytes, more particularly lymphocytes, and other cells at various stages of differentiation (eg, plasma cells, lymphoblasts, monocyte-macrophages,
Eosinophils and mast cells). Two major lymphocytes (T cells and B cells)
Have distinct and complementary roles in mediating antigen-specific immune responses.

[0012] The immune response is a very complex and valuable homeostasis mechanism with the ability to recognize foreign pathogens. The initial response to a 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 the first line of defense against foreign pathogens until an "adaptive immune response" can be generated. Both T cells and B cells are involved in the adaptive immune response. Various mechanisms are involved in generating an adaptive immune response. A discussion of all possible mechanisms that generate an adaptive immune response
Some mechanisms that are beyond the scope of this section but are well characterized are B cell recognition of antigens and subsequent T cells by binding to activated and antigen presenting cells that secrete antigen-specific antibodies. Including activation.

[0013] Microorganisms can have cell membranes that are recognized as foreign by the immune system. In addition, microorganisms can also produce toxins or proteins that are also regarded as foreign by the host's immune system. The first mechanism described above involves the binding of antigens (eg, bacterial cell walls or bacterial toxins) to surface immunoglobulin receptors on B cells. Receptor binding transmits a signal inside the B cell. This is what is commonly referred to in the art as "first signal". In some cases, only one signal is needed to activate B cells. Those antigens that can activate B cells without relying 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 that bind to B cells. T
When cellular help is required for activation of B cells to a particular antigen, the antigen is referred to as a T-dependent antigen (or thymus-dependent antigen). In addition to binding to surface receptors on B cells, this antigen is also internalized by B cells, then digested into smaller fragments within B cells, and the antigenic peptide-M
It may be presented on the surface of B cells in the context of HC class II molecules. These peptide-MHC class II molecules are recognized by T helper cells that bind to B cells to provide a "second signal" required for some antigens. Once the B cells are activated, they begin to secrete antibodies to the antigen, which ultimately leads to inactivation of the antigen. Another mode of B cells to be activated is follicular dendritic cells (follicular dendritic cells) in the germinal centers of the lymph nodes and spleen.
by contact with a lactic cell (FDC). Follicular dendritic cells capture antigen-antibody (Ag-Ab) complexes circulating through the lymph nodes and spleen, and FDC presents them to B cells and activates them.

Another well-characterized mechanism of an adaptive immune response to an antigen is the activation of T cells by binding to antigen presenting cells (eg, macrophages and dendritic cells). Macrophages and dendritic cells are powerful antigen presenting cells. Macrophages have various receptors that recognize microbial components such as the macrophage mannose receptor and scavenger receptor. These receptors bind microorganisms and macrophages engulf them and degrade microorganisms in endosomes and lysosomes. Some microorganisms are destroyed directly in this manner. Other microorganisms are digested into small peptides, which are then presented to T cells on the surface of macrophages in the context of MHC class II-peptide complexes. T cells that bind to these complexes are activated. Dendritic cells are also potent antigen-presenting cells, and require peptide-MHC class I molecules and peptide-MHC class I to activate T cells.
Present the I molecule.

When a B cell binds to an antigen that has never been encountered, it undergoes a developmental pathway called “isotype switching”. During developmental changes, plasma cells
It switches from producing general IgM type antibodies to producing highly specific IgG type antibodies. In this population of cells, some undergo repetitive divisions in a process called "clonal expansion." These cells mature and become antibody factories that release immunoglobulins into the blood. If they are sufficiently mature, they can generally release plasma cells (about 2,000 identical antibodies per second until they die) within two or three days after reaching maturity Cells) were identified. Other cells in this group of clones do not produce antibodies, but function as memory cells that recognize and bind to a particular antigen when encountering it.

[0016] As a result of the initial challenge with the antigen, there are much more cells identical to the original B cells or the parent cells, each of which, as the original B cells, in some fashion, Can respond. Consequently, when the antigen appears a second time, it immediately encounters one of the correct B cells, and since these B cells are programmed for a particular IgG antibody, this The immune response starts immediately, accelerates rapidly, is more specific, and produces more antibodies.
This event is considered a second or past response. FIG. 1 shows a comparison of antibody titers present as a result of primary and secondary responses. Immunity can last for years. Because memory cells survive months or years, and also foreign substances are occasionally reintroduced in small doses that are sufficient to continually elicit low levels of immune response . In this manner, memory cells are replenished periodically.

After a first exposure to the antigen, the response often produces antibodies slowly and the amount of antibodies produced is small (ie, primary immunization). Upon a second challenge with the same antigen, the response (ie, secondary immunization) is more rapid and very strong, whereby pathogenic microorganisms are targets that should be induced by the vaccine. Achieves an immune status equivalent to an enhanced secondary response after re-infection with E. coli.

In general, active vaccines fall into two general classes: subunit vaccines and whole organism vaccines. Subunit vaccines are prepared from components of whole organisms and are usually present in whole organism vaccines to avoid the use of living organisms that can cause disease, as discussed in more detail below. Developed to avoid toxic components. H. in humans. in
The use of the purified capsule polysaccharide material of this organism as a vaccine against meningitis caused by dluenza type b is an example of a vaccine based on antigenic components. Parks et al. Inf. Dis. 136 (supplement): 551 (1997); Anderson et al. Inf. Dis. 136 (
Addendum): 563 (1977); and Makela et al. Inf. Dis. 1
36 (Addendum): 543 (1977).

Classically, subunit vaccines have been prepared by chemical inactivation of partially purified toxins and are therefore called toxoids. Formaldehyde or glutaraldehyde has been the chemical of choice for detoxifying bacterial toxins. Both diphtheria and tetanus toxin have been successfully inactivated with formaldehyde, resulting in a safe and effective toxoid vaccine that has been used for over 40 years to control diphtheria and tetanus. Papp
enheimer, A .; M. Diphtheria. : Bacterial
Vaccines (R. Germanier, eds.), Academic Pre
ss, Orlando, FL. 1-36 (1984); Bizzini, B .;
, Tetanus. Ibid., Pp. 37-68. However, chemical toxoids are not the desired properties. In fact, this type of vaccine can be more difficult to develop. Because the protective antigen must first be identified, then a procedure must be developed to efficiently isolate the antigen. In addition, in some cases, subunit vaccines may contain exogenous substances (eg, membrane or endotoxin) that are more immunogenic due to masking of protective antigens or removal of substances that are immunodominant. ) Does not elicit an immune response of the same strength as a whole organism vaccine elicits due to the lack of a).

On the other hand, whole organism vaccines utilize whole organisms for vaccination. The organism can be killed or live (usually attenuated) depending on the requirements to elicit protective immunity. Pertussis vaccines are, for example, killed and whole cell vaccines are prepared by treatment of Bordetella pertussis cells with formamide. Bacteria B. pertussis is a highly contagious respiratory disease in humans that colonizes the epithelial lining of the respiratory tract and has the highest morbidity and mortality in infants and young children (pertussis).
) Or whooping cough. Colony formation was further enhanced by B.I. Pertussis produces local tissue damage and systemic effects caused mostly by toxins produced by pertussis. Manclarck et al., P.
ertussis, ibid., pages 64-106. These toxins include endotoxins or lipopolysaccharides, peptidoglycan fragments called tracheal cytotoxins, thermolabile cutaneous necrotic toxins, adenylate cyclase toxins, and protein exotoxins pertussis toxins. Vaccination is the most effective way to control pertussis, and killed whole-cell vaccines administered with diphtheria and tetanus toxoid (DPT vaccine) are effective in controlling disease in many countries It was a target. Fine et al., Reflections on
the Efficiency of Pertussis Vaccines, R
ev. Infect. Dis. , 9: 866-883 (1987). Unfortunately, many children experience adverse reactions upon injection due to the large amounts of endogenous products contained in the pertussis vaccine. Endotoxin, an essential component of the outer membrane of Gram-negative organisms (as well as all other Gram-negative organisms), has a wide range of side effects, including mild to severe, including fever, shock, leukocytosis, and miscarriage. Can be induced. The side effects associated with pertussis vaccines are usually mild, but they can be very severe. However, the toxin component present in influenza virus vaccines can induce a strong pyrogen response and is responsible for the generation of Guillain-Barre syndrome. As influenza vaccines are prepared by propagation of the virus in chick embryos, embryo or egg components appear to contribute to this toxicity.

[0021] The use of the killed vaccine is also important for Bordetella br
U.S. Pat. No. 4,016,253 describes Switcher et al., which adapted such a method for the preparation of a vaccine against Onchiseptica infection. Brown et al., Br. Med. J. 1: 263 (1959), the use of whole killed cells is described in Haemophilus influ.
Disclosed is the preparation of a vaccine against chronic bronchitis caused by enzae. However, the use of killed cells is usually achieved by a concomitant loss of immunogenic capacity. This is because the process of killing can usually destroy or alter many surface determinants required for the induction of specific antibodies in the host. The killed vaccine is available from many pathogenic bacteria (Salmonella).
spp. And V. cholerae) or no effect. Parental killed whole cell vaccine in current use for Salmonella typhi is only moderately effective and causes unacceptably high frequency of significant systemic and local adverse reactions .

In the case of intracellular pathogens such as Salmonella, a vaccine based on live but attenuated microorganisms (live vaccine) can induce a very effective type of immune response, Generally agreed. Live attenuated vaccines are live organisms that are benign but typically are capable of replicating in host tissues, and likely express many natural target immunogens that are processed and presented to the immune system, preferably similar to a natural infection Consists of 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 by S. spp. Has been implemented. Because they establish infection by direct interaction with the gut-associated lymphoid tissue (GALT), resulting in a strong humoral immune response. They can also invade host cells and thus elicit strong cell-mediated responses. Eisenstein
(1999) Intracellular Bacterial Vaccin
e Vectors (edited by Paterson, Wiley-Liss, Inc.)
51-109; Hone et al. (1999) Intracellular Bac.
terminal Vaccine Vectors (Edited by Paterson, Wil
eye-Liss, Inc. ) Pp. 171-221; Sirard et al. (1999) I.
mmun. Rev .. 171: 5-26. Ideally, these attenuated microorganisms
While maintaining the integrity of the cell surface components necessary for specific antibody induction, they still cannot cause disease. For example, they do not produce toxic factors, grow very slowly, or do not grow at all in the host. Furthermore, these attenuated strains should have substantially no potential to revert virulent wild-type strains. Traditionally, live vaccines have been used to isolate antigenically related viruses from another species, to select for passage and adaptive attenuation in non-target species or tissue culture, or to use temperature-sensitive variants. Was obtained by either of the following methods: The first approach was to use an Edward Je using bovine poxvirus to vaccinate humans against smallpox.
Used by Nner.

Selecting attenuation by lineage passage in non-target species is a 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 lineage expansion in green monkey kidney cells. Measles vaccine
Prepared by passage of the virus in chicken embryo fibroblasts. Vaccines against polio, hepatitis A, Japanese encephalitis B, dengue, and cytomegalovirus have all been prepared according to similar procedures.

Although animal models, particularly monkeys, are useful in developing live vaccines by lineage passage and selection, significant uncertainty as to whether the vaccine is truly non-pathogenic remains until inoculated in humans . For example, the yellow fever Daker strain produced from infected suckling mouse brain induced encephalitis in 1% of the vaccine. Another significant problem is the possible vaccine contamination by exogenous viruses during passage in cell cultures or animals, especially monkeys. This choice to develop a live vaccine is highly questionable, given more recent findings on the potential dangers of the virus that can be transmitted from animals to humans.

In contrast to the somewhat dangerous approach of selecting live vaccines described above, modern development approaches introduce specific mutations in the pathogen's genome that affect the ability of the pathogen to induce disease. I do. Defined genetic manipulation is the current approach taken in an attempt to develop live vaccines against various diseases caused by pathogenic microorganisms.

In an effort to develop a live vaccine that is safer and elicits a higher immune response, researchers have focused on efforts to develop a live vaccine with specific genetic mutations. Curtiss in US Patent No. 5,294,441,
S. typhi can be attenuated by constructing a deletion (Δ) of either or both the cya (adenylate cyclase) gene and the crp (cyclic 3 ′, 5′-AMP [cAMP] reporter protein) gene. Is disclosed.
The cAMP and cAMP receptor proteins (the products of the pleiotropic genes cya and crp, respectively) work together to form a regulatory complex, which affects the transcription of many genes and operons. As a result, by mutating any of these genes, an attenuated microorganism is obtained. In addition, microorganisms having a single mutation in either the cya or crp genes cannot compensate for the deficiency by clearing these gene products from the host to be vaccinated. The crp gene product is not available in mammalian tissues, while the metabolites produced by the cya gene product (
cAMP) is present in mammalian cells. The concentration of typhi present in the invading cells is lower than the concentration required for cya to mutate and express the wild-type phenotype. Curtiss et al., Infect. Immun. , 55: 3035-3.
043 (1987).

Since cAMP is present in host tissues at some level, Curtiss et al. Stabilized the Δcya microorganism by introducing mutations into the crp gene. Ticket et al., Infect. Immun. , 60 (2): 563-5
41 (1992) conducted a study in healthy adult inpatient volunteers,
Attenuated S. aureus having deletions in the cya and crp genes. typhi has been shown to have a tendency to produce heat and bacteremia (bacteria in the blood).

A similar approach in an attempt to develop a live vaccine is described in Dr. B. A
. D. Made by Stocker. The gene mutated by Stocker produces a product that is also non-viable in host tissue. Stock
In U.S. Patent No. 5,210,035, er is described in US Pat. The construction of a microbial-derived vaccine strain is described. Specifically, Stocker states that Salmonella has an auxotrophy for p-aminobenzoic acid (PABA) and 2,3-dihydroxybenzoate (a substance that is not available to bacteria in mammalian tissues) (i.e., By interrupting the pathway for the biosynthesis of aromatic (aro) metabolites that make teach that typhi can be attenuated. These a
The ro mutation makes the synthesis of chorismate (a precursor of the aromatic compounds PABA and 2,3-dihydroxybenzoate) impossible, and Salmonella states that
There are no other routes that can overcome this deficiency. As a result of this auxotrophy,
Aro-deleted bacteria cannot grow in hosts; however, they survive and grow in cells long enough to stimulate a protective immune response. The above-mentioned Takee
In a technical paper written by T. et al. The attenuated strain of typhi is also S
It was constructed for use as a vaccine by introducing deletions into the aroC and aroD genes according to the Tocker. However, these attenuated strains administered to healthy inpatient volunteers have a tendency to produce heat and bacteremia (Hone et al. (1987), Hormaeche et al. (1996) Vacci.
ne 14: 251-259; Hassan and Curtiss (1997).
Avian Dis. 41: 783-791; and Miller et al. (199
0) Res. Microbiol. 141: 817-821).

[0029] Comparative studies between these vaccines have not been rigorously tested, and thus the efficacy of these current strains against each other remains unclear. Moreover, the toxicity of current live bacterial vaccine candidates (e.g., symptoms such as diarrhea), and the reality that many individuals in the human population are immunocompromised, provide better protection and last longer, And it clearly justifies the search for additional vaccines with lower toxicity.

Another important problem with vaccine development is the fact that many pathogenic species consist of multiple serotypes that can cause disease in animal hosts vaccinated against similar pathogenic strains. . Previous attempts at developing long-term, mutually protective Salmonella vaccines have often been questioned. For example, raw attenuated aro
A Salmonella strain is a heterologous serotype (eg, a group B strain (typhim).
urium) and group D strains (enteritidis and dublin)) have been shown to elicit a mutual protective response,
After the vaccine has been removed from the immunized animal, it is virtually eliminated. H
ormaeche et al. (1996).

Like many cellular macromolecules, DNA is exposed to “modification” after synthesis by adding small chemical moieties to the intact polymer. In various organisms,
This is because, as shown in FIG. 2, involves the N6-position of C5 position or adenosine cytosine in either the enzymatic addition of methyl (-CH ₃₎ group to DNA. Enzymes responsible for the addition of methyl groups to DNA include DNA methyltransferase or DN
Known as A methylase. DNA methylases can be divided into two classes: (1) those that methylate cytosines (DNA cytosine methylases);
And (2) those that methylate adenine (DNA adenine methylase).

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 base pair repair, and transfer ( Marinus, Ec
Oli and Salmonella: Cellular and Moli
cultural biology, 2nd edition, 782-791 (1996)). Further, E.I. In E. coli, Dam regulates the expression of operons such as pyelonephritis-associated hair (pap), which is an important toxicity determinant of upper urinary tract infection (Rogerts
J. et al. Urol. 133: 1068-1075 (1985); van de.
r Woude et al., Trends Microbiol. 4: 5-9 (199
6)). The latter regulatory mechanism involves the formation of an inherited DNA methylation pattern, which controls gene expression by altering the binding of regulatory proteins.

[0033] A significant need remains for vaccines prepared from live, pathogenic microorganisms that are safe and, when administered to a host, induce an effective type of immune response in the host. ing. It is also highly desirable to develop a single vaccine strain that can stimulate an immune response against different strains (ie, heteroserotypes or species). There is also a further need for safe and effective antibacterial drugs that can be used to treat patients suffering from diseases caused by pathogenic microorganisms.

[0034] All references and patent applications cited in the present application are hereby incorporated by reference in their entirety.

SUMMARY OF THE INVENTION The present invention relates to the discovery that DNA adenine methylase (Dam) is essential for the pathogenesis of Salmonella, and that Dam ^- Salmonella is effective as a live attenuated vaccine against typhoid fever in mice and Salmonel
based on the finding that it elicits an immune response against a second species of la. Since DNA adenine methylase is highly conserved in many pathogenic bacteria that cause significant morbidity and mortality, Dam derivatives of these pathogens can be effective as live attenuated vaccines. Furthermore, since methylation of DNA adenine residues is essential for bacterial toxicity, it alters the expression of DNA adenine methylase,
Or drugs that inhibit its activity probably have broad antibacterial activity,
Genes encoding adenine methylase and its products are promising targets for antimicrobial drug development.

Accordingly, it is an object of the present invention to provide a live vaccine for vaccinating a host against a spectrum of pathogenic or similar pathogenic microorganisms.

A further object of the invention is to serve as a carrier for antigens, preferably immunogens of other pathogens, in particular microorganisms, including viruses, prokaryotes and eukaryotes.
To provide a live vaccine.

[0038] Yet another object of the present invention is to provide an antibacterial drug that specifically inhibits DNA adenine methylase and genes responsible for producing DNA adenine methylase. Further, the composition of the present invention comprises (i) a DNA adenine methylase enzyme activity,
ii) DNA adenine methylase expression, (iii) DNA adenine methylase activator, (iv) an activating compound for DNA adenine methylase receptor, and / or (v) a toxic factor regulated by DNA adenine methylase As well as natural and synthetic molecules.

Accordingly, one aspect of the present invention provides an immunogenic composition comprising a live attenuated pathogenic bacterium in a pharmaceutically acceptable excipient, wherein the pathogenic bacterium comprises a DNA adenine. It contains mutations that alter methylase (Dam) activity, resulting in attenuated pathogenic bacteria.

In another aspect, the present invention provides an immunogenic composition comprising a killed pathogenic bacterium in a pharmaceutically acceptable excipient, wherein the pathogenic bacterium comprises a mutant DNA adenine. Includes mutations that alter methylase (Dam) activity.

In another aspect, the invention provides an attenuated strain of a pathogenic bacterium, wherein the bacterium comprises:
It contains mutations that alter Dam activity, resulting in attenuated bacteria.

[0042] In another aspect, the invention provides a method of raising an immune response in an individual, the method comprising: Administration that involves administering to the individual in an appropriate amount.

In another aspect, the present invention provides a method of preventing infection by an pathogenic bacterium in an individual, the method comprising the steps of: Administering to an individual in an amount sufficient to reduce symptoms associated with infection by a pathogenic bacterium.

[0044] In another aspect, the invention provides a method of treating an infection by a pathogenic bacterium in an individual, the method comprising: administering any of the immunogenic compositions described herein to a pathogenic bacterium. Administering to the individual in an amount sufficient to reduce the symptoms associated with infection by the individual in the individual.

In another aspect, the invention provides a method of treating an individual infected with a pathogenic bacterium, comprising administering to the individual a composition containing an agent that alters Dam activity. I do.

In another aspect, the present invention provides a method of eliciting an immune response to a second species of Salmonella in an individual, the method comprising an attenuated first species Salm.
comprising administering to an individual an immunogenic composition containing onella, wherein the first comprises a mutation that alters Dam activity, thereby resulting in Salmonella.
Is attenuated.

[0047] In another aspect, the present invention also provides a screening method. The present invention includes a method of identifying an agent that may have antimicrobial activity, the method using an in vitro transcription system, the level of transcription from the dam gene when the agent is added to the in vitro transcription system. Detecting a drug that changes the level of transcription, wherein the drug is identified by its ability to change the level of transcription from the dam gene as compared to the level of transcription when the drug is not added.

In another aspect, the invention provides a method of identifying an agent that may have antimicrobial activity, the method comprising using an in vitro transcription system, wherein the agent is added to the in vitro transcription system. Detecting a drug that alters the level of translation from the RNA transcript encoding Dam, wherein the drug encodes Dam relative to the level of translation when the drug is not added. Identified by its ability to alter the level of translation from an RNA transcript.

In another aspect, the invention provides a method of identifying an agent that may have antibacterial activity, the method comprising determining whether the agent binds Dam.
Here, the agent is identified by its ability to bind Dam.

In another aspect, the present invention provides a method for identifying an agent that may have antibacterial activity, comprising: (a) an unmethylated oligo that includes a Dam binding site. The nucleotides were replaced with Dam, S-adenosylmethionine (S-adeno).
and incubating with an agent, wherein the unmethylated oligonucleotide further comprises a signal.
(B) digesting all unmethylated target sites, thereby releasing the unmethylated oligonucleotide; and (c) the signal resulting from digestion of the unmethylated target sites. Detecting the inhibition of DNA adenine methylase, wherein the agent comprises the steps (a), (b),
And c) a step identified by its ability to cause an increase in signal as compared to performing in the absence of the agent.

In another aspect, the present invention provides a method for identifying an agent that may have antibacterial activity, comprising the steps of: (a) identifying the agent to be tested with Dam
Contacting with a suitable functional host cell; and (b) analyzing at least one characteristic associated with a change in Dam function, wherein the agent elicits at least one of the characteristics. A process identified by its capabilities.

[0052] The invention also provides a method of preparing the vaccines and strains described herein. In one aspect, the present invention provides a method of preparing an immunogenic composition described herein, comprising the steps of: administering a pharmaceutical excipient to a DNA adenine methylase (
Dam) combining with a pathogenic bacterium containing a mutation that alters activity,
As a result, the pathogenic bacteria are attenuated.

In another aspect, the invention provides a method for preparing an attenuated bacterium capable of eliciting an immune response by a host susceptible to a disease caused by a corresponding or similar pathogenic microorganism. The method comprises constructing at least one mutation in the pathogenic bacterium, wherein the first mutation results in altered Dam function.

Another object of the present invention is a method wherein the vaccine can be produced by altering the expression of an overall regulator of a toxic gene, and more specifically by altering the expression of DNA adenine methylase. It is to provide.

Another object of the present invention is to provide a method wherein a vaccine can be produced by altering the expression of a gene regulated by DNA adenine methylase.

In another aspect, the present invention provides a method for preparing a live vaccine from a virulent pathogenic bacterium such as Salmonella, wherein the microorganism comprises a microorganism against which it is desired to be vaccinated. Or the expression of DNA adenine methylase in a virulent strain of a pathogenic bacterium
Alternatively, the method includes a step of changing the expression of a gene regulated by DNA adenine methylase.

A still further object of the invention is to administer to a vertebrate a compound that alters the expression of DNA adenine methylase or inhibits its activity.
It is an object of the present invention to provide a method for treating a host such as a vertebrate infected with a pathogen.

Further objects, advantages and novel features of the invention will be set forth in part in the detailed description which follows, and in part will become apparent to those skilled in the art upon inspection of the following specification, Or it may be known by the practice of the present invention. The objects and advantages of the invention will be realized and attained by means of the instruments, combinations, compositions, and methods particularly pointed out in the appended claims.

(Detailed Description) The present inventors have determined that the dam gene and its product enzyme DNA adenine methylase (
Dam) was found to be necessary for bacterial toxicity. Despite previous research efforts on Dam function, there is no report on the important role of Dam in bacterial virulence, the inventive significance of this role, and the ability of Dam ^- mutant vaccines to elicit a protective immune response. All previous reports of dam mutations from other laboratories used Salmonella strain LT2, which is at least 1000-fold less toxic than wild-type when delivered intraperitoneally. In conjunction with the knowledge of this finding, the present invention relates to the invention of (a) either genes that alter functions such as expression of DNA adenine methylase, and / or (ii) genes that are regulated by DNA adenine methylase. A vaccine having a non-reverting mutation; (b
) (I) a gene encoding DNA adenine methylase and / or DNA adenine methylase, (ii) an activator of DNA adenine methylase activator and / or a repressor of DNA adenine methylase, and (iii) regulated by Dam. A class of inhibitors that are natural and / or synthetic molecules with binding specificity for the virulence factors to be treated;
Methods for preparing vaccines and inhibitors based on the knowledge that adenine methylase is important for bacterial etiology; (d) eliciting an immune response using the immunogenic compositions described herein Treating a vertebrate with (e) the vaccine of the present invention before being infected with the pathogenic microorganism, or (ii) using the inhibitor of the present invention after being infected with the pathogenic microorganism. (F) methods of preventing infection using the immunogenic compositions described herein; and (g) screening methods to identify compounds that may be useful therapeutic agents. About.

As described in the examples, the S. cerevisiae required to kill 50% of animals is required.
The oral lethal dose (LD ₅₀ ) of the Typhimurium Dam ⁻ mutant (generated by insertion into the dam gene (Mud-Cm)) was 10,10 compared to the wild type.
The intraperitoneal (ip) LD ₅₀ increased by more than 000-fold,
It increased more than 2,000-fold (Example 1; Table 1). Furthermore, the highly attenuated Dam ⁻ mutant was found to confer a protective immune response in an art accepted model of typhoid fever in mice (Example 2; Table 2). S. typhi
All 17 mice immunized with the murium Dam ⁻ insert survived a 10 ⁺⁴ wild-type challenge above the LD ₅₀ , while all 12 non-immunized mice died after the challenge. Dam ⁺ Salmonella and Dam ^- Salm
Survival studies comparing onella showed that the Dam ^- bacteria were completely superior in colonizing mucosal sites (Pierre's patches), but showed significant defects in colonizing deeper tissue sites (performed Example 2; FIG. 6). Without wishing to be bound by theory, we believe that one possible explanation for the reason that Dam ⁻ elicits a protective immune response is that mutant bacteria have long enough intestinal tracts to elicit an immune response. Note that it grows in the mucosa but cannot penetrate and / or colonize deeper tissues.

[0061] Even more surprising is that especially vaccines containing one species of Salmonella cannot elicit an immune response against a second species of Salmonella, or at least a significant lasting immune response against a second strain. In view of the widely supported view in the art, our data shows such cross protection, especially when these species are attenuated due to mutations in a single gene.
Dam ^- S. The mice immunized with S. typhimurium (serotype B) were immunized 11 weeks after immunization. enteritidis and S. et al. dublin (serotype D)
In it protected against heterologous challenge (100~1000 LD ₅₀₎ (Example 3
Table 3). This protection persisted for more than 6 weeks after the vaccine strain was removed from the immunized animal (ie, even after more than 6 weeks, the Dam ^- organism remained in the Peyer's patches, mesenteric lymph nodes, liver and spleen). Could not be detected). Salmo
In contrast to Nella cross protection, 5 weeks after immunization Yersinia pseudo
No protection was observed against douberculosis. Similarly, Da
m ^- S. immunization with S. enteritidis is performed by S. enteritidis. typhimurium and S. typhimurium. Cross protection against dublin was provided. Raw attenuated Salmonel
la strain is a group B (typhimurium) strain and a group D (enteritidis)
And dublin) strains (attributed to a shared common LPS antigenic determinant), which have been shown to elicit a cross-protective response with a very short lifespan and a 10-fold response to immunity. After 12 weeks, it is virtually eliminated. Hormaeche et al. (
1996) Vaccine 251-259.

Ectopic expression of Dam derivatives as described in Examples 1 and 3 (ie,
Expression of normally repressed proteins) has widespread applications for vaccine development. Ectopic expression in Dam derivatives of many pathogens can result in protection against parental toxic organisms and / or a cross-protective response. Salmonella Dam
Derivatives may have utility as platforms for expressing passenger bacterial and viral antigens that elicit a strong protective immune response against cognate pathogens. Dam, ^- so immunized mice can eliminate lethal bacterial load full toxicity of Salmonella organisms, Dam, ^- vaccines may have therapeutic utility for effectively treating pre-existing infection. Dam ^- derivatives ectopically express complex proteins, opening up the possibility that vaccines can be constructed in strains that are less harmful to humans, which poses a risk of infection for immunocompromised individuals. While developing the high level of protection benefits induced by live vaccines.

The fact that DNA adenine methylase is essential, for example, for the bacterial etiology of Salmonella is also very important and has many implications. First
In some cases, the dam gene is highly conserved in pathogenic bacteria, i.e., the gene sequence of dam in one microorganism shares sequence identity with the dam gene of another microorganism not only in the same species but across the genus of bacteria. And secondly, the dam gene regulates many genes involved in toxicity. DNA adenine methylase is used by many pathogenic bacteria (eg, Vibrio c) to cause significant morbidity and mortality.
holerae (Bandyopadhyay and Das, Gene, 1
40: 67-71 (1994)), Salmonella typhi (199).
9-3, Sanger Center), pathogenic E. coli. Coli (Blattne
r et al., Science, 277: 1453-1474 (1977), Yersi.
nia pestis (1999-3, Sanger Center), Hae
mofilus influenzae (Fleischmann et al., Sci.
ence, 269: 496-512 (1995), and Treponema.
pallidum (Fraser et al., Science, 281: 375-388).
(1998))), the Dam derivatives of these pathogens may be effective as live attenuated vaccines. In addition, since Dam is essential for bacterial toxicity, Dam inhibitors are believed to have a wide range of antibacterial effects, and therefore, any gene that alters Dam or Dam expression will be required for the development of antibacterial drugs. Is a promising target.

The implications are as follows: (1) (i) a DNA adenine methylase and / or dam gene, (ii) an active compound for Dam activator and / or Dam repressor, and (iii) It is now possible to rationally develop a class of inhibitors, natural and / or synthetic molecules with binding specificity for toxic factors regulated by Dam; and (2) (i) DNA It is now possible to produce vaccines with non-reverting mutations in either genes that alter the expression of adenine methylase and / or (ii) toxic genes regulated by DNA adenine methylase. Dam is a global regulator of gene expression, and because many of these regulated genes are conserved in various species and genera, inhibitors and vaccines based on DNA adenine methylase may confer cross protection. Very possible. Thus, as discussed above, one strain, species, serotype and / or
Or an inhibitor or vaccine against a group provides protection against different strains of the pathogen.

[0065] The compositions described herein can be used for administration to an individual. They can be administered, for example, for experimental purposes or to obtain a source of an antimicrobial antibody such as a Salmonella antibody. They may also be used to elicit an immune response in the individual and to protect the individual from infection, or Salmon.
can be administered to treat individuals infected with toxic bacteria such as ella.

General Techniques The practice of the present invention will employ, unless otherwise indicated: molecular biology (including recombinant techniques), microbiology, cell biology, within the skill of the art. Traditional techniques of biochemistry and immunology. Such techniques are explained fully, for example, in the following documents:
Molecular Cloning: A Laboratory Manua
1, Second Edition (Sambrook et al., 1989); Oligonucleotides.
e Synthesis (MJ Gait, eds., 1984); Animal C
cell Culture (edited by RI Freshney, 1987); Meth
ods in Enzymology (Academic Press, Inc.)
. ); Handbook of Experimental Immunolo
gy (DM Wei & CC Blackwell ed.); Gene Tr
ansfer Vector et al. for Mammarian Cells (J
. M. Miller & M. P. Calo et al., 1987); Current
Protocols in Molecular Biology (FM.
Ausubel et al., Eds., 1987); PCR: The Polymerase C
hain Reaction (ed. by Mullis et al., 1994); Current
Protocols in Immunology (JE Colligan)
Ed., 1991); Short Protocols in Molecula.
r Biology (Wiley & Sons, 1999).

Definitions “DNA adenine methylase” (Dam) is defined as a group of enzymes that can methylate adenine residues in DNA. The Dam gene and the Dam product encoded by the dam gene are known in the art and this definition is defined in E.C. co
li (gi 118682) and Salmonella (gi 250015)
7) Includes the Dam enzyme, which shares the same important amino acids as the DNA adenine methylase from DNA and preferentially methylates the DNA sequence "GATC" and methylates the N-6 position of adenine. In particular, the highly conserved D that encodes the region of Dam
The NA sequences are shown in SEQ ID NOs: 1-4 as described herein. According to art-recognized notation, “dam” or “dam gene” is DN
A indicates the gene encoding A adenine methylase, and "Dam" indicates the DNA
Adenine methylase (ie, the polypeptide) is shown. For the purposes of the present invention, a gene is defined to include coding and / or regulatory regions.

Dam “activity” or “function” means any biological activity associated with dam expression or non-expression. Dam activity is described herein. For example, dam
Non-expression leads to repression (or, alternatively, re-suppression) of certain genes regulated by Dam; thus, repression (or de-repression) of any of these genes is Dam activity. As another example, methylation of adenine in DNA (eg, methylation of GATC) is active on dam expression and the resulting Dam product; thus, adenine methylation is Dam active. Thus, Dam "activity" or "function" encompasses any one or more of the biological 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 Dam activity, including an increase or decrease in Dam activity. Bacteria containing mutations that alter Dam activity are commonly referred to as "Dam derivatives."

“Expression” includes transcription and / or translation, as well as any factor or event that affects expression (eg, an upstream event such as a second gene that affects expression).

A “vaccine” is a pharmaceutical composition for human or animal use, in particular,
Administered with the intent of conferring on the recipient some degree of specific immunological reactivity to a particular target or group of targets (ie, eliciting and / or enhancing an immune response to a particular target or group of targets). Immunogenic compositions. This immunoreactivity or response can be caused by antibodies or cells that are immunologically reactive to the target, especially B cells, plasma cells, T helper cells, and cytotoxic T lymphocytes,
And their precursors), or any combination thereof. For the purposes of the present invention, the target is primarily a toxic bacterium such as Salmonella. If an attenuated bacterium is used as a carrier, this target may be another antigen described herein. Immunological reactivity may be desired for experimental purposes, treatment of certain conditions, removal of certain substances, and / or prevention.

“Pathogenic” bacteria are bacteria that can cause disease. "Toxicity" is an index of the degree of virulence that can be expressed numerically as the ratio of the number of cases of overt infection to the total number of infections. It is understood that the pathogenic bacteria used in the vaccines described herein are other than non-toxic strains commonly used in the laboratory, and are known and / or can cause disease.

A “attenuated” bacterium used in the compositions described herein is a bacterium that exhibits reduced toxicity. As is well understood in the art and described above,
Toxicity is the extent to which bacteria can cause disease in a given population. For the purposes of the present invention, attenuated bacteria are generally designated as directed by appropriate governmental agencies,
Has toxicity that is reduced to an appropriate and acceptable safety level. The degree of attenuation that is acceptable depends, inter alia, on the recipient (ie, human or non-human) and the various regulations and standards provided by regulatory agencies, such as the United States Food and Drug Administration (FDA). Most preferably, especially for human use, the attenuated bacteria are non-toxic, meaning that administration of these organisms will not cause disease symptoms. As is well understood in the art, attenuated bacteria are alive at least at the time of administration.

“Antigen” means a substance that is specifically recognized and bound by an antibody or T cell antigen receptor. As is well understood in the art, antigens
It may include peptides, proteins, glycoproteins, polysaccharides, gangliosides and lipids, and parts and / or combinations thereof. The antigen may be a naturally-occurring antigen or it may be synthetic.

An “adjuvant” is a chemical or biological agent provided in combination with an attenuated bacterium described herein to enhance the immunogenicity of the attenuated bacterium. is there. As known in the art, "adjuvants"
Are substances that non-specifically enhance or enhance the immune response of a recipient (host) to an antigen when added to the antigen.

“Stimulate” an immune response, which may be a B cell response and / or a T cell response
, "Trigger", or "trigger" means an increase in response, which may result from eliciting and / or enhancing a response.

“Heterologous” means derived from and / or different from the compared entities. For example, a "heterologous" antigen for a bacterial strain is an antigen that is not normally and naturally associated with the strain.

An “effective amount” is an amount sufficient to effect beneficial or desired results, including clinical results, and as such, an “effective amount” will depend on the context in which it is applied. Effective dose is
It can be administered in one or more doses. For purposes of the present invention, an effective amount of a Dam derivative bacterium (or a composition comprising a Dam derivative bacterium) is an amount that induces an immune response.
For treatment, an effective amount is an amount sufficient to slow, ameliorate, stabilize, reverse, or slow the progression of a bacterial disease, or otherwise reduce the pathological consequences of the disease. For prophylaxis, an effective amount is an amount sufficient to reduce (or completely eliminate) one or more symptoms upon exposure and infection.

“Treatment” is a method for obtaining beneficial or desired clinical results. Beneficial or desired clinical results include alleviation of symptoms, reduction in the extent of the disease, a stabilized (ie, not exacerbated) condition of the disease, prevention of the spread of the disease, slowing or slowing the progression of the disease, a disease condition But is not limited thereto.

“Prevention” of a disease or infection is one of the infections of an individual who receives a composition described herein, as compared to other same conditions, except for receiving the composition. It means a decrease in the above symptoms (including but not limited to elimination). As understood in the art, "prevention" of an infection may include mild symptoms, but does not necessarily mean elimination of symptoms associated with the infection.

An “individual” used interchangeably with a “host” is a vertebrate, preferably a mammal, more preferably a human. As mammals, livestock animals (eg, cattle)
, Sports animals, and pets. "individual"
Also include poultry, such as chickens. A "host" may be uninfected or infected by a bacterium.

“Agent” means a biological compound or chemical compound, such as a simple or complex organic or inorganic molecule, polypeptide, polynucleotide, carbohydrate or lipoprotein. A vast number of compounds can be synthesized (eg, oligomers such as oligopeptides and oligonucleotides, and synthetic organic compounds based on various core structures), which are also included in the term “drug”. In addition, various natural sources can provide compounds for screening, such as plant or animal extracts and the like. The compounds can be tested alone or in combination with one another.

“Antimicrobial activity” or “toxic control” means that an agent can negatively affect the ability of a bacterium to cause disease. For the purposes of the present invention, agents that can control toxicity are agents that alter Dam activity and can be selected by the screening methods described herein, and furthermore, in further studies, reduce bacterial toxicity. It can prove to control and even exert therapeutic activity.

The term “comprising” and its cognates are understood to include “including
ing).

“A,” “an,” and “the” include plural references unless otherwise indicated. For example, "a" Dam means any one or more DNA adenine methylase.

Compositions of the Invention The compositions described are useful for raising an immune response and / or for treating or preventing a disease associated with a bacterial infection, especially a Salmonella infection. Vaccines prepared from live, pathogenic bacteria can be produced by the corresponding pathogenic bacteria, by similar pathogenic bacteria of the same strain, species, serotype and / or group, or by different strains, species, sera. Due to different types and / or groups of bacteria,
Provided for immunization or treatment of a host susceptible to the resulting disease. The live vaccines generated herein can also serve as carriers for antigens (eg, immunogens of other pathogens), thereby generating multiple immunogenic reactions.

Thus, in one embodiment, the present invention provides a live attenuated pathogenic bacterium (eg, S
almonella) and a pharmaceutically acceptable excipient. The pathogenic bacterium contains (has) a mutation that alters DNA adenine methylase (Dam) activity, thereby attenuating the pathogenic bacterium. In some embodiments, the mutation is a DNA adenine methylase (Da
m), in which the mutation alters DNA adenine methylase activity.

[0088] Dam activity can be increased or decreased, and Dam activity can be altered at any level, including transcription and / or translation. For example, with respect to translation, activity can be altered in a number of ways, including the amount of protein produced and / or the nature (ie, structure) of the protein produced. For example, the mutation may result in an increase or decrease in the amount of Dam produced by the cell (due to its effect on translational and / or post-transcriptional events); or, the mutation may have altered activity This can result in altered Dam. Mutations and variants that alter Dam activity are produced using techniques well known in the art. As an example, the Dam product can be reduced by using a promoter known to initiate transcription at lower levels. Assays for determining the level of transcription from a given transcriptional regulatory element (eg, a promoter) are well-known in the art. Native dam promoters can be replaced with the promoter of the lower translation activity; or, dam - ^(here, the native dam gene has been removed)
Can be used as a basis for integrating a re-engineered dam gene containing a less active promoter and can be integrated into the genome. Alternatively, a different dam gene (eg, T4 dam) can be used. An example of a dam overproducer, E. coli at 100 times wild type level. pTP producing E. coli Dam
A 166 plasmid can be used. Mutations can occur in the Dam gene itself (including transcriptional and / or translational regulatory elements), as well as in Dam production and / or
Or it may be in the gene (s) that affects Dam activity.

[0089] Any pathogenic (preferably virulent) bacterial strain can be used in the immunogenic compositions described herein. In some embodiments, the E.C. co
Pathogenic bacteria other than li are used. In other embodiments, the pathogenic Esch
erichia (preferably E. coli) is used. The dam gene may or may not be essential (ie, the deletion of dam may or may not be lethal), since overexpression of dam can lead to a useful vaccine.

The present invention relates to any known group, species or strain, more preferably group A, B or D
Particularly suitable for a wide variety of Salmonella, including most species that are particular pathogens of particular vertebrate hosts. S from which a live vaccine can be produced
almonella include, for example, S. almonda. typhimurium;
S. enteritidis, S.M. typhi; abortus-ovi;
S. Abortus-equi; dublin; gallinarum
S .; and others known or may be found to cause infection in mammals.

Other organisms in which the present invention may be used include Yersinia species (especially Y. p.
estis), Vibrio species (especially V. cholerae), Shigell
a species (especially S. flexneri and S. sonei); Haemophi
rus species (especially H. influenzae, more particularly type B), Bordet
ella (particularly B. pertussis); Neisseria (particularly N. me)
Ningitidis and N. et al. gonorrohoeae); Pasteur
ella (especially P. multicida), pathogenic E. coli. coli and Tre
ponema (eg, T. pallidum); and other organisms known or found to cause infection in mammals.

[0092] In another embodiment, the invention provides a vaccine used to vaccinate a host. The vaccine comprises a pharmaceutically acceptable excipient and an attenuated form of the pathogenic bacterium. Here, the attenuation can be due to at least one mutation. Here, the first mutation alters either (i) the expression or activity of one or more DNA adenine methylases, or (ii) the expression of one or more genes regulated by DNA adenine methylase. The first modification is preferably non-reverting, and in some embodiments, the gene product is constructed in a gene that activates one or more of the DNA adenine methylases. The first modification may be constructed in a gene whose gene product inactivates or attenuates one or more activities of the DNA adenine methylase. In another embodiment, the first modification is such that the gene product is constructed in a gene that suppresses expression of the DNA adenine methylase, and the gene product is capable of suppressing Dam. The vaccine may further comprise a second mutation independent of the first mutation, wherein the second mutation comprises:
This produces attenuated microorganisms. This second mutation is preferably non-reverted.

In another embodiment, the invention provides a vaccine for eliciting an immunological response in a host to be vaccinated. The vaccine includes a bacterial cell having a mutation introduced into the gene that abolishes the ability of the bacterial cell to regulate the expression of DNA adenine methylase (Dam) expressed by the dam gene.

[0094] Ectopic expression of multiple proteins in the Dam-vaccine indicates that the killed Da
This suggests that m-organisms may elicit a significantly stronger protective immune response than killed Dam + organisms. Accordingly, in some embodiments, the invention provides an immunogenic composition comprising a killed pathogenic bacterium (including a mutation that alters Dam activity) and a pharmaceutically acceptable excipient. I do. Preferably, the mutation is d
It can result in a decrease or increase in Dam activity, as in the am gene, and as described herein. In some embodiments, the dam mutation results in bacterial death. In another embodiment, the mutation is attenuated and the bacterium comprises
Killing is achieved by using methods well known in the art (eg, sodium azide treatment and / or exposure to UV). If the mutation is lethal, the bacteria can be further treated for killing (eg, using sodium azide and / or UV). Examples of bacteria suitable for these vaccines include Salmonella,
Vibrio (including V. cholerae) and Yersinia (Y.p.
pseudotuberculosis), but is not limited thereto.

Preferably, the composition comprises a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are relatively inert substances that facilitate administration of a pharmaceutically effective substance. For example, an excipient may provide form or consistency for the vaccine composition, or act as a diluent. Suitable excipients include stabilizers,
Examples include, but are not limited to, wetting and emulsifying agents, salts for altering osmotic pressure, encapsulating agents, buffers, and skin penetration enhancers. Examples of pharmaceutically acceptable excipients include Remington's Pharmaceutical S
sciences (edited by Alfonso R. Gennaro, 19th edition, 1995)
)It is described in.

The present invention also provides an immunogenic composition comprising any combination of the mutant strains described herein (whether weakened or killed) for a given genus (eg, Salmonella). Including things. Two different vaccine strains (eg, Da
m- and Dam overproducers) are two of the potentially protective antigens.
Use in these combinations can elicit a superior immune response, as two different repertoires can be generated.

A pathogenic bacterium according to the present invention is attenuated, preferably detoxified, as a result of a non-revertive mutation made in at least one gene. This alters the function of DNA adenine methylase. In essence, live vaccines provided according to preferred embodiments of the invention originate from pathogenic bacteria. Non-revertive mutations are introduced into the gene of the pathogen, thus altering the expression of DNA adenine methylase. "
"Non-reverting" mutations generally refer to less than about 1 in 10 ⁸ , preferably less than about 1 in 10 ¹⁰ , or preferably less than about 1 in 10 ¹⁵ , even more preferably about 10 in 10 ^20. Return to less than 1. Preferably, this mutation is non-leaky; however, regulation of the gene by Dam appears to be very sensitive to Dam concentration. Thus, overexpression of Dam and underexpression of Dam result in attenuation of the pathogen. This mutation is preferably made in the dam gene itself, but in another embodiment of the present invention, discussed in more detail below, the vaccine according to the present invention has a product that activates the dam gene. It is contemplated that it can be produced by mutating the related gene (s) either "upstream" or "downstream" of dam. Or, alternatively, the mutation is constructed in at least one virulence gene that is regulated by DNA adenine methylase. Mutations are non-reverting. Because restoration of normal gene function can only occur by the random, accidental occurrence of more than one event, each of which is very rare. For example, D
Am methylase activity can be down-regulated and / or an antisense oligonucleotide that blocks expression of the dam gene by introducing a deletion into the promoter or coding region, inserting a transposon or intervening DNA into the promoter or coding region Or the use of ribozymes that prevent dam gene expression. Alternatively, the mutation may be an insertion and / or deletion that is sufficient to cause non-reversion.

In the case of a deletion mutation, restoration of genetic information is performed by restoring lost genetic information.
In tandem, requires many simultaneous random nucleotide insertions. In the case of insertion plus inversion, restoration of gene function requires simultaneous deletion of the inserted sequence and correct reinversion of the adjacent inverted sequence. Each of these events has a very low frequency of expression and is undetectably low. Thus, each of the two types of "non-reverting" mutations has substantially no reversion to prototrophy.

[0099] Other methods of constructing insertions in the Dam gene are well known and apparent to those skilled in the art.

While a single non-revertant provides a high degree of safety against reversion to toxicity, there are still events (albeit rare) that have a finite likelihood of occurrence.
If the microorganism is present in a host that can transfer heritability to a non-toxic organism by conjugation, an opportunity for reversion exists. Alternatively, there may be a small alternative pathway for the production of DNA adenine methylase, which may be operable by rare mutations or under stress. Thus, in some embodiments, the attenuated bacterium described herein further comprises a second mutant. Live vaccines with two separate and unrelated mutations can survive and live reasonably long in the host, provide a strong immune response upon administration to the host, and It can also act as a carrier for pathogen antigens (eg, antigens of other pathogens) and provide immune protection from such pathogens.

Salmonel Can Serve as Second Mutation for Live Attenuated Vaccine Candidate
Examples of la typhimurium attenuated mutants include galE (galactose-induced toxicity), pur and aro (aromatic compounds not available in vivo), cr
p and cya (overall changes in gene expression through catabolite regulation), and phoP (overall changes in toxic gene expression) (Hone et al. (1)
987), Hormaeche et al. (1996); Hassan and Curti.
ss (1997); and Miller et al. (1990)). Comparative studies between these vaccines have not been rigorously tested, so the efficacy of these current strains against each other remains elusive. In addition, the toxicity of current live bacterial vaccine candidates (eg, symptoms such as diarrhea), and the fact that many individuals within the human population are clearly immunocompromised, provide good protection and Is permanent,
And it is a good reason to search for additional vaccines with low toxicity.

[0102] In addition to the above mutations, it may be desirable that the bacterium for use as a live vaccine has one or more genetic "marker characteristics". This "marker feature" makes this bacterium easily distinguishable from other bacteria of the same species, either wild-type or other live vaccine strains. Thus, desirably, a strain of pathogen is selected that has a marker to identify the Dam-mutant to be generated from other members of the strain. Alternatively, such markers can be introduced into vaccine strains. As discussed previously, various markers may be used. The marker used must not affect the immunogenic characteristics of the bacterium and must not interfere with the processing of the bacterium to produce a live vaccine. This marker only alters the phenotype to allow recognition of the bacteria of interest. For example, the Dam mutant is a base analog 2-
Sensitive to aminopurines (Miller “Experiments in
Molecular Genetics "CSHL 1972). Since the dam gene is genetically linked to cysG, one of skill in the art can use a pool of transposon insertions to transduce cysG- recipients to cysG +. These prototrophy are screened for 2-aminopurine sensitivity. To ensure that the insertion is present in the dam gene, the insertion is cloned and the flanking regions are sequenced. This marker may also be some other nutritional requirement. Such markers are useful in differentiating vaccine strains from wild-type strains.

The subject bacteria are then treated to provide one or more non-revertants. The first mutation is preferably (but not necessarily) performed by mutating the dam gene to produce Da
Alters m function (eg, expression). If a second mutation is desired, the gene whose loss is known to cause attenuation is further mutated. This mutation is a deletion,
It can be insertion, or inversion, or a combination thereof. Various technologies are available
Can be used to introduce deletions, insertions, or inversions to obtain bacteria with the desired "non-leaky" non-reverting mutation that results in altered expression of The presence of two completely independent mutants, each of which is significantly less likely to revert, almost completely guarantees that the vaccine strain cannot become virulent.

A number of well-known techniques that can be used to abolish or mutate a gene, such as PCR techniques, translocation elements, mutagenic elements, transducing phage, and DNA-mediated transformation, and / or conjugation , Use) exists. Recombinant DN
Other methods also known to those skilled in the art, such as the A technique, can be used to successfully introduce one or more mutant genes into a single host strain to be used as a vaccine.

After engineering a bacterium to introduce one or more non-reverting mutations into some member of the population, the bacterium may be subjected to such conditions under conditions that facilitate isolation of the desired mutant. Grow either under conditions where the mutation has a selective advantage over the parental bacterium, or under conditions that allow easy recognition of the desired bacterium from unmodified bacterium or other type of mutant. The isolated autotrophic mutant is then cloned and screened for virulence, inability to revert, and the ability of the mutant to protect the host from virulent pathogenic strains.

The vaccine can be used in a wide variety of livestock animals and humans. Among livestock animals that are or can be treated today (if susceptible to bacterial diseases) with vaccines include chickens, cows, pigs, horses, goats and sheep (to name more important livestock animals). It is.

According to the invention, this vaccine is produced by introducing a non-reverting mutation in at least one gene. Here, each mutation is a mutation of a sufficient number of bases in tandem to ensure that the likelihood of reversion is substantially zero. Preferably, the mutation results in the non-expression of the respective mutated gene in the sense of its total inability to determine the production of the active protein. However, as described herein, Dam overproducers can also be made. Furthermore, the gene selected is involved in the expression of DNA adenine methylase, and preferably the gene is dam.

The resulting strain is a live, non-toxic vaccine with the desired immunogenicity. That is, the mutation does not affect the production of the antigen that elicits the host's natural immune response. Typically, when a wild-type pathogen reaches a particular tissue in a host, a specific virulence factor or set of virulence factors is expressed as a result of the specific environment to which the pathogen is exposed. Dam-mutants are thought to express all of the many virulence factors simultaneously and constitutively, rather than in a particular tissue. Because the physiological effects of many virulence factors are tissue-specific, virulence factors constitutively expressed in bad tissues do not initiate intrinsic physiological changes in the disease process. However, these virulence factors
Elicit an immune response from the host. Thus, the immune system encounters these factors in the environment. Here, the factor cannot initiate the necessary physiological changes in the host to produce the disease, and the host can raise the immune response.

In another embodiment of the invention, the vaccine is produced by introducing non-reverting mutations in at least two genes. Here, the mutations are large enough to ensure that each mutation has substantially zero chance of reversion and the certainty of non-expression of each mutant gene. The first gene selected is involved, either directly or indirectly, in the expression of DNA adenine methylase. The second gene selected can also result in attenuation, regardless of the attenuating effect of the first gene mutation; however, this second mutation cannot affect the protective effect of the first mutation . Mutations in the first and second genes can be achieved as previously discussed.

Thus, the present invention provides a vaccine for eliciting (eliciting) an immune response in a host to be vaccinated, comprising: A bacterium having one mutation; and a second mutation in the bacterium that attenuates the microorganism independently of the first mutation.

In another embodiment, the invention provides a live vaccine that can be used as a vector or carrier for an antigen. The antigen can be any antigen, including antigens of bacterial genus or species other than those used in non-toxic pathogenic vaccines. The antigen can be added as a mixture attached or bound to the bacteria, or one or more structural genes encoding the desired antigen can be introduced into a non-toxic pathogenic vaccine as an expression cassette. Accordingly, any of the mutant bacteria described for use in the vaccines described herein may further include an expression cassette having one or more structural genes encoding the desired antigen. The expression cassette contains the structural gene (s) of interest under the regulatory control of the transcription initiation and translation initiation and termination regions (naturally adjacent to or heterologous to the structural gene of interest). Where bacterial or bacteriophage structural genes are included, natural or wild-type regulatory regions are usually (although not always) sufficient. It may be necessary to link regulatory regions recognized by non-toxic pathogens to structural genes of antigens isolated from eukaryotes (sometimes prokaryotes). As antigens, C fragment of tetanus toxin, B subunit of cholera toxin,
Examples include, but are not limited to, hepatitis B surface antigen, Vibrio cholerae LPS, HIV antigen and / or Shigella sonei LPS.

The expression cassette may be a recombinant construct or may be in the form of a part of a naturally occurring plasmid. When the expression cassette is a recombinant construct, the expression cassette may be linked to a replication system for episomal maintenance or may be non-recombinant under conditions for recombination and integration into non-toxic pathogenic chromosomal DNA. It may be introduced into toxic pathogenic bacteria. The structural gene of the antigen of interest can be a bacterial protein (eg, a toxin subunit), a viral protein (eg, a capsid), or an enzymatic pathway (eg, a pathway involved in the synthesis of a carbohydrate antigen such as lipopolysaccharide (LPS)). ). For example, among the antigens expressed in other live attenuated Salmonella vaccines, include fragment C of tetanus toxin, B subunit of cholera toxin, hepatitis B surface antigen, and Vibrio cholerae LP
There is S. In addition, the HIV antigens GP120 and GAG are attenuated Mycob
activium bovis BCG and expressed in Shig.
gella soneii LPS is attenuated Vibrio cholerae
Is expressed in The construct or vector can be introduced into a host strain via a number of well-known methods (eg, transduction, ligation, transformation, electroporation, transfection, etc.).

In another embodiment, a live vaccine prepared according to the invention is prepared to have an irreversible mutation in a gene that is regulated by DNA adenine methylase, preferably by DNA adenine methylase (Dam). These irreversible mutations can be adjusted as previously described.

[0114] In another embodiment, a vaccine is provided. Here, the bacteria are preferably produced by overproducing DNA adenine methylase (Dam),
have mutations that result in overproduction of am. Methods of production that overproduce bacterial genes are described herein and are known in the art, and additional dam
Gene; a mutation in the promoter that controls the transcription of dam; including the addition of a plasmid carrying a mutation in the dam gene that results in reduced responsiveness to feedback inhibition, which may or may not be integrated. , But not limited to them.

The immunogenic compositions described herein can be used with adjuvants that increase the immune response against limiting bacteria such as Salmonella. Adjuvants are particularly suitable for killed vaccines, but need not be limited to this use. Suitable adjuvants are known in the art and include:
Aluminum hydroxide, alum, QS-21 (U.S. Pat. No. 5,057,540)
DHEA (US Pat. Nos. 5,407,684 and 5,077,28).
No. 4) and derivatives and precursors thereof (eg, DHEA-S), β-2 microglobulin (WO 91/16924), muramyl dipeptide, muramyl tripeptide (US Pat. No. 5,171,568) and monophosphoryl lipid A (US Pat. No. 4,436,728; WO 92/16231) and its derivatives (eg, DETOX ^™ ), and BCG (US Pat. No. 4,726,947). Other suitable adjuvants include, but are not limited to:
Aluminum salt, squalene mixture (SAF-1), muramyl peptide, saponin derivative, mycobacterial wall preparation, mycolic acid derivative, nonionic block copolymer surfactant, Quil A, cholera toxin B subunit, polyphosphazene and derivatives And immunostimulatory complexes (ISCOM) (eg, Taka
hashis (1990) Nature 344: 873-875). For veterinary applications and for the production of antibodies in animals, mitogenic components of Freund's adjuvant can be used. The choice of adjuvant will depend in part on the stability of the vaccine in the presence of the adjuvant, the route of administration, and the acceptability of modulating the adjuvant, especially if intended for human use. For example, alum is approved by the US Food and Drug Administration (FDA) for use as an adjuvant in humans.

[0116] In some embodiments, the immunogenic composition can also include a carrier molecule (with or without adjuvant). Carriers are known in the art. Plt
okin, Vaccines, 3rd edition, Philadelphia, WB Su
anders Co. (1990). Bacterial carriers (ie, bacterial-derived carriers) include cholera toxin B subunit (CTB); diphtheria toxin mutant (CRM197); diphtheria toxin; group B streptococcal αC protein; meningococcal outer membrane protein (OMPC) ; Diphtheria; Haemophilu indistinguishable
s influenzae (eg, P6) outer membrane protein; recombinant class 3
Protein (group B meningococcal rPorBP); heat-killed Burcella ab
ortus; heat-killed bacteria Listeria monocytogeneis; and Pseudomonas aeruginosa recombinant exoprotein A.
Another carrier is keyhole limpet hemocyanin (KLH).

The vaccines of the present invention are systemically administered to an individual in unit dosage form, sterile parenteral solutions or suspensions, sterile parenteral solutions or oral solutions or suspensions, oil-in-water or water-in-oil emulsions, and the like. Suitable for Formulations or parenteral, parenteral drug delivery are known in the art and are subject to Remington's Pharmaceutical
technical Sciences, 19th Edition, Mack Publishing
(1995). The vaccine may be administered parenterally, for example, by subcutaneous, intramuscular, intraperitoneal or intradermal injection. Administration can also be oral, intranasal,
It can be in the lung (ie, by aerosol) and intravenously. Additional formulations suitable for other forms of administration include suppositories and, in some cases, oral formulations. The route of administration depends on the condition of the individual and the desired clinical effect. For administration to livestock animals (eg, chickens), the preferred administration is an oral formulation. Formulations for live vaccines can widely and desirably alter those that provide an increased immunogenic response.

The vaccines and antimicrobial agents of the present invention can be used on a wide variety of vertebrates. The vaccines and antimicrobial agents of the present invention find particular use in mammals (eg, humans and domestic animals). Domestic animals include cattle, sheep, pigs, horses,
Goats, poultry, rabbits (Leporidate) (for example, rabbits
)), Or other animals that may be susceptible to, or be vectors for, diseases affecting domestic vertebrates. Individuals suitable for administration are those who are at risk for or have exposure to bacteria (eg, Salmonella (S. spp.)) Or are at risk or expected to be at risk. Individuals and individuals who have been exposed and / or infected. The mode of application of the vaccine or antimicrobial can vary widely, and any of the conventional methods for administration are applicable. These include oral application in solid physiologically acceptable bases or in physiologically acceptable dispersions, parenteral application by injection, and the like. The dosage of the vaccine or antimicrobial will depend, inter alia, on the route of administration and will vary according to the species to be protected. One or more administrations can be provided as booster dosages, usually at conventional intervals (eg, 2-3 weeks). Since DNA adenine methylase is not present in vertebrates, inhibitors of DNA adenine methylase appear to show zero or low toxicity when administered to vertebrates. In addition, since DNA adenine methylases are enzymes, they are present at low concentrations in cells; thus, requiring the administration of lower concentrations of inhibitors and the potential for inhibiting all DNA adenine methylases. increase.

Kits and Strains The present invention also provides attenuated strains as described herein. A preferred strain is a Salmonella strain that contains one or more mutations that alter Dam activity. Similar strains are described herein. Accordingly, in one embodiment, the invention provides an attenuated strain of a pathogenic bacterium, wherein the strain has an altered Dam activity,
As a result, it contains mutations that attenuate bacteria. The mutation can be any of the mutations described herein. Preferably, this strain is a Salmonella strain.

The invention also includes a kit comprising any one or more of the strains and / or vaccine formulations described herein in stable packaging. The kit optionally provides instructions (eg, for administration). In some embodiments, the instructions are for administration to a non-human (eg, a chicken, or other livestock animal). In another embodiment, the instructions are for human administration.

Methods of the Invention The present invention also provides methods of using the immunogenic compositions described herein (Dam
Screening methods to identify potentially useful agents that alter activity, and methods of preparing the immunogenic compositions described herein).

For any method involving administration of any of the compositions described herein, any one or more of the compositions may be administered (ie, the compositions may be administered alone or with each other). It is understood that they can be administered in combination. Further, the compositions can be used alone or in combination with other modalities (ie, clinical intervention) for prophylactic and / or therapeutic purposes.

Use of Immunogenic Compositions for Raising an Immune Response, for Prevention of a Disease, and for Treating a Disease In some embodiments, the present invention provides that an immune response is generated in an individual. Provided are methods of using the immunogenic compositions described herein to elicit. In general,
These methods include the step of administering to an individual an amount of any one or more of the immunogenic compositions described herein in an amount sufficient to elicit an immune response. The immune response in the composition can be an immune response against a particular species and / or strain of bacteria, or in other embodiments, an immune response against a second species and / or strain.

The immune response can be a B cell response and / or a T cell response. Preferably, the response is antigen-specific (ie, the response detects a response to the bacterium used in the immunogenic composition (ie, a response to an antigen associated with the bacterium used). Preferably, the immune response is sustained in the absence of the vaccine composition. Thus, in some embodiments, the immune response persists after administration of the immunogenic compositions described herein for about any of the following (when given in multiple dosage forms): , Preferably after the most recent administration): 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 6 months, 1 year.

To determine the effect of administration of an immunogenic composition described herein, an individual is treated with either an antibody (humoral) or a cellular immune response against bacteria, or a combination thereof. It can be monitored by techniques standard in the art. Alternatively, such monitoring may not be necessary if it has already been demonstrated to elicit such a response.

For the purpose of raising an immune response, the immunogenic compositions described herein can be administered in unmodified forms. It may sometimes be preferred to modify the bacteria to improve immunogenicity. As used herein, and as known in the art, "immunogenic" refers to the ability to elicit a specific antibody (B cell) or cellular (T cell) immune response, Or both abilities. Methods to improve immunogenicity include, inter alia, crosslinking with reagents such as glutaraldehyde or bifunctional couplers or coupling to multivalent platform molecules. Immunogenicity can also be improved by coupling to a protein carrier, especially a carrier comprising a T-cell epitope and / or a B-cell epitope.

Suitable individuals for receiving the compositions have been described above, and also apply to these methods. Generally, such individuals will have symptoms associated with the infection and / or
Or is susceptible to exposure to a disease state, is exposed to these symptoms and / or disease states, and / or indicates these symptoms and / or disease states. The individual may or may not have been exposed to Salmonella at the time of administration, and thus, at the time of administration,
ella may or may not be infected. Preferably, the individual has not been exposed to Salmonella.

[0128] In some embodiments, the present invention provides a method for administering Salmonell to an individual.
A method for raising an immune response against a second species, strain, serotype, and / or group of a is provided. The method comprises administering to any individual an immunogenic composition described herein in an amount sufficient to elicit an immune response against a second species, strain, serotype, and / or group of Salmonella. And administering to the subject. This individual is Sa
A second species, strain, serotype, and / or group of lmonella may or may not have been previously exposed. In some embodiments, the second Salmonella in which an immune response is elicited is from a second group, such as Group A, Group B, or Group D (compared to the first serotype administered). It is. In other embodiments, the second Salmonella in which an immune response is elicited is from a second serotype (compared to the first serotype administered).

The first and second species can be any species of Salmonella, some of which are described above. In some embodiments, the first species is S. aureus.
typhimurium, and the second species is S. typhimurium. enteritidi
s. In some embodiments, the first species is S. aureus. typhimuri
um, and the second species is S. dublin. In another embodiment, the first species is S. aureus. enteritidis, and the second species is S. aureus.
typhimurium. In still other embodiments, the first species is S
. enteritidis, and the second species is S. aureus. dublin. Similarly, the first group is a Salmonell, such as Group A, Group B, or Group D.
a can be any of the known groups. The second group can be any of the known groups, such as Group A, Group B, or Group D, provided that the second group is selected from the group consisting of the first. Is different). In other embodiments, the first serotype is different from the second serotype. Salmonella serotypes are known in the art.

[0130] The immune response may include one or more additional antigens (ie, one or more additional Sal).
It is understood that the strain may be raised against S. monella strains, groups, serotypes and / or species). Therefore, the present invention provides that the immune response is the third, fourth, fifth, etc. Sa
and methods raised against strains, groups, serotypes, and / or species of Imonella.

[0131] The present invention also provides a method for producing, in an individual, a second species, strain, serotype, and / or
Alternatively, a method of raising an immune response against the group is provided. The method comprises administering to an individual an immunogenic composition comprising an attenuated bacterium in an amount of a Dam derivative sufficient to elicit an immune response against a second species, strain, serotype, and / or group of pathogenic bacteria. And administering to the subject.

The present invention also relates to a method for producing a bacterium (preferably, for example, Salmonel) in an individual.
la) Methods for treating infection are provided. In some embodiments, the present invention provides a method for inhibiting disease symptoms associated with infection with a virulent bacterium (Salmonella). The method should be performed in an amount sufficient to control disease symptoms associated with the infection.
And administering any one or more of the compositions described herein. Preferably, the infection is due to Salmonella. In another embodiment, the infection is due to Escherichia (preferably, E. coli). In other embodiments, the methods comprise any one or more of the methods described herein in an amount that reduces the amount of a pathogenic bacterium (eg, Salmonella) in an individual (as compared to non-administration). Administering the composition of

The vaccine will be administered in a manner compatible with the dosage formulation, and for example, in a therapeutically effective amount. The amount to be administered depends on the individual to be treated, the ability of the individual's immune system to synthesize antibodies, the route of administration, and the degree of protection desired. Precise amounts of active ingredient that need to be administered may depend on the judgment of the practitioner managing the treatment, and may be peculiar to the individual.

In one embodiment, the invention provides a method of treating an individual infected with a pathogenic bacterium, comprising administering to the individual a composition comprising an agent that alters Dam activity. In another embodiment, the present invention provides a method of treating a host infected with a pathogenic microorganism (bacteria), comprising: (a) administering the compound to the host. Here, the compound alters the expression or activity of one or more DNA adenine methylases. The compound may (a) bind to one or more DNA adenine methylases and thereby alter the activity of DNA adenine methylase; (b
) Can bind to one or more genes that express the DNA adenine methylase, thereby altering the expression of the DNA adenine methylase. The expression of this DNA adenine methylase is overactive. Alternatively, the expression of this DNA adenine methylase is suppressed. In some embodiments, the compound is an antisense oligonucleotide having a sequence that is complementary to one or more DNA adenine methylase gene sequences.

The present invention also provides a pathogenic microorganism (bacteria) comprising administering a compound to a host.
Methods for treating a host infected with Here, the compound binds to one or more virulence factors regulated by DNA adenine methylase.

[0136] In some embodiments, the invention provides methods for preventing a bacterial infection, such as a Salmonella infection. In these embodiments, the immune response elicited by the immunogenic composition is such that the recipient of the immunogenic composition has one or more reduced symptoms of the infection as compared to an individual that does not receive the composition. Defensive in the sense of presenting. In other embodiments, protection is provided by reducing the amount of a bacterium, such as Salmonella, in an individual receiving the composition, as compared to an individual not receiving the composition.

[0137] In some embodiments, the invention provides a method of inhibiting a condition associated with a bacterial infection in an individual (or a method of treating a bacterial infection). The method comprises the step of administering to the individual a composition comprising an agent that alters Dam activity. The bacterium can be any of the bacterium described herein (particularly Salmonella).

In another embodiment, antimicrobial agents according to the present invention are prepared. This is a DN
Inhibits A adenine methylase, preferably DNA adenine methylase (Dam). The following discussion focuses specifically on the dam gene and its product Dam, but it is understood that this specificity is for simplicity and clarity purposes only. The methods and compositions discussed below include (i) any gene that expresses DNA adenine methylase, (ii) any gene or gene product that modulates a DNA adenine methylase gene, (iii) DNA adenine methylase. It is intended to be applicable to any gene and / or (iv) DNA methylase. Thus, while specific genes and gene products, dam and Dam, are discussed below, other DNA adenine methylase genes and DNA adenine methylases are intended to be equivalents of dam and Dam, respectively. Thus, the following discussion is interchangeable.

Inhibition of Dam can be achieved by the use of antisense oligonucleotides to inhibit dam gene translation, direct inhibitors of Dam enzymatic activity, inhibitory compounds for Dam activator and / or activating compounds for Dam repressor. It can be performed by a number of approaches, including reducing Dam levels upon detachment, as well as targeting dam-regulated virulence factors. This antisense approach
It was previously used to inhibit cytosine methyltransferase (MeTase) from mammalian cells (MacLeod, AR and Szyf, M. et al.
, J. et al. Biol. Chem. , 7: 8037-8043 (1995)). Transfection of antisense nucleic acids into adrenocortical cells resulted in DNA demethylation and reduced tumorigenic potential for MeTase activity.

In another embodiment, the antimicrobial activates Dam. Such compounds may be effective for such activation, for example, by stimulating the dam promoter, inactivating the repressor, and / or increasing the half-life of Dam.

Screening Assays The present invention may also have antibacterial activity based on its ability to alter Dam activity (
Thus, methods of identifying agents (which can modulate toxicity) are included. These methods are
It can be implemented in various embodiments. We have observed that loss or even an increase in 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, but does not affect host cells. This is especially true since humans do not have homologs to the dam gene. Further, the present inventors have determined that dam is Vibrio ch.
olerae and Yersinia pseudotuberculosis
(Example 7). This means that Dam
Are excellent drug targets in these pathogenic organisms. Thus, agents identified by the methods of the present invention may be used to control bacterial infections, particularly Escherichia
Infection, Salmonella infection, Vibrio infection, and / or Yers
It may be useful for treating inia infection.

[0142] The methods described herein are in vitro screening assays and cell-based screening assays. In an in vitro embodiment, the factor is tested for its ability to modulate the function of this Dam. In a cell-based embodiment, viable cells with Dam function are used to test the factor. For the purposes of the present invention, factors may be identified based on any alteration in the function of Dam, although characteristics preferably relate to the total loss of function of Dam.

In all of these methods, alteration of the function of Dam, whether positive or negative, can occur at any level that affects the function of Dam. The factor is Da
By reducing or interfering with the transcription of m, the function of Dam can be altered. Examples of such factors are those that bind to upstream regulatory regions, including polynucleotide sequences or polypeptides. Factors can alter the function of Dam by increasing the transcription of Dam RNA. An agent may alter the function of Dam by reducing or preventing the translation of Dam RNA. Examples of such agents are agents that bind RNA, such as antisense polynucleotides,
Alternatively, it is a factor that selectively degrades RNA. Antisense attempts to inhibit Dam have been described above. Factors can alter the function of Dam by increasing the translation of Dam RNA. The factor binds to Dam by binding to Da.
m can cause a defect in the function. Examples of such factors are polypeptides or chelators. Factors can impair the function of Dam by affecting the gene expression of genes regulated by Dam. Examples of such factors are those that alter the expression of a Dam regulatory gene at any of the levels discussed above.

The screening method has been described as applicable to any pathogen having the Dam gene.

In Vitro Screening Methods In the in vitro screening assays of the invention, factors are screened in an in vitro system, which can be any of the following: (1) The factor is da
assay to determine whether transcription is inhibited or increased; (2)
Assays for factors that interfere with the translation of Dam RNA or a polynucleotide encoding Dam, or alternatively, factors that specifically increase translation of Dam; (3) Assays for factors that bind Dam.

For assays that determine whether an agent inhibits or increases the transcription of dam, an in vitro transcription system or an in vitro transcription / translation system may be used. These systems are commercially available and generally include a coding sequence as a positive control, preferably an internal control. The polynucleotide encoding Dam is
Introduced and allow transcription to occur. Transcript comparison between the in vitro expression system without any factor (negative control) and the in vitro expression system with the factor indicates whether the factor affects transcription of dam. Control and D
Comparison of transcripts with am indicates that if the factor acts at this level,
Indicates whether it selectively affects transcription (as opposed to affecting transcription in a general, non-selective, or specific manner).

[0147] With respect to assays that determine whether an agent inhibits or increases the translation of a polynucleotide encoding dam RNA or Dam, in vitro transcription / translation assays, such as those described above, except for comparing translation products , Can be used. Comparison of translation products between the in vitro expression system without any factor (negative control) and the in vitro expression system with the factor indicates whether the factor affects the translation of dam. Comparison of translation products between control and dam indicates that this factor
When acting at this level, it indicates whether it selectively affects the translation of dam (as opposed to affecting translation in a general, non-selective, or specific manner).

With respect to assays for factors that bind Dam, Dam is first expressed in prokaryotic or eukaryotic expression systems, either as a native protein or as a native protein.
am is recombinantly expressed as a fusion protein conjugated to a well-characterized epitope or protein. The recombinant Dam is then, for example,
Purified by immunoprecipitation using anti-Dam or anti-epitope antibodies or by binding the conjugate to an immobilized ligand. The mixture of appropriately labeled compounds is then screened using an affinity column made of Dam or a Dam fusion protein. Suitable labels include, but are not limited to, fluorescent dyes, radioisotopes, enzymes and chemiluminescent compounds. The unbound compound and the bound compound are subject to various conditions (eg,
(Eg, high salt, surfactant). Non-specific binding to the affinity column can be minimized by simply pretreating the compound mixture with an affinity column containing the conjugate or epitope. Similar methods can be used to screen for factors that compete for binding to Dam. In addition to affinity chromatography, there are other techniques, such as measuring changes in the melting point or fluorescence anisotropy of a protein that change upon binding to another molecule. For example, a sensor chip that covalently binds to native Dam or Dam fusion protein (Pharmacia Biosensor, Stat)
(supplied by T. et al. (1995) Cell 80: 661-670) can be performed to determine the Dam binding activity of the different factors.

[0149] With respect to Dam binding, it is understood that appropriate fragments of Dam may also be used. For example, if a particular region of Dam is known to be important for binding to DNA, a fragment that includes this region or consists of just this region may be used.

In another embodiment, the in vitro screening assay detects an agent that competes with another substance (roughly a polynucleotide) that binds Dam. For example,
Dam is a specific DNA motif (ie, the GATC which is the Dam target site).
It is known to bind to An assay can be performed, whereby the agent is tested for its ability to compete with binding to this motif. Competitive binding assays are known in the art and need not be described in detail herein. Briefly, such an assay involves measuring the amount of Dam complex formed in the presence of an increased amount of a putative competitor. For these assays, one of the reactants is labeled, for example, with ³² P. One such assay also encompassed by the present invention is described in more detail below.

The isolation of inhibitors or activators of Dam can be accomplished, for example, using a rapid, high-throughput assay for Dam using a chemical library (Neust).
adt et al., Bioorg. Med. Chem. Lett. 8: 2395-239
8 (1998)) or a peptide library (Lam, KS, Antic).
canceler Drug. Res. , 12: 145-167 (1997)). Such inhibitor libraries have previously been shown to be effective in blocking the activity of some enzymes (Carroll, CD, Bioorg. Med. Chem.
Lett. , 8: 3203-3206 (1998)). This Dam assay
It consists of a double-stranded oligonucleotide containing a tethered group (eg, biotin) at one end and a Dam target site (GATC sequence) with a signal at the other end. The signal can be a radioactive compound (eg, phosphorus-32), a fluorescent molecule (eg, fluorescein), or an antigen. The unmethylated oligonucleotide containing the Dam target site is anchored to a solid surface (eg, a 96-well microtiter plate containing avidin). Pre-incubating the Dam enzyme (predetermined to have just enough activity to methylate all GATC sites of the target oligonucleotide) with the inhibitor library,
It is then added to each well in the presence of S-adenosylmethionine (SAM). After the incubation, the sample wells are washed with buffer and the restriction enzyme MboI
To digest all unmethylated GATC sites in the oligonucleotide,
Thus, the signal end of the molecule is released. The plate wells are then counted (
(Radioactive signal), scanned for fluorescence (fluorescent signal), or incubated with a secondary antibody conjugated to an enzyme such as horseradish peroxidase, followed by incubation with the non-radioactive substrate of the enzyme. Dam inhibition is detected as a decrease in signal in the sample wells due to release of unmethylated GATC sites. This assay can be used to rapidly screen chemical and peptide libraries for inhibitory activity. The feasibility of such studies is due to the inhibitor of MeTase activity, sinefung.
Indicated by isolation of in. Sinefungin is an analog of S-adenosyl-L-methionine (SAM) and acts as a competitive inhibitor of DNA methylation. However, sinefungin does not use S as a methyl donor.
This drug is not useful as a chemotherapeutic agent against bacteria because AM blocks all DNA methylases, including mammalian cytosine methylases that are required.

In order to isolate the activator of Dam (including a low percentage of target sites (eg, GATC sites), sufficient activity to methylate a low percentage of target oligonucleotides (eg, 20%) Dam) (predetermined as such) is pre-incubated with one or more factors (including the activator library) and then added to each well in the presence of SAM. Dam
Activation is detected as an increase in signal in the sample wells due to methylation of the target site (eg, GATC) and thus interference with the MboI restriction reaction.

Thus, in some embodiments, the present invention provides a method comprising: (a) anchoring an unmethylated oligonucleotide comprising a DNA adenine methylase target site to a solid surface, wherein the unmethylated oligonucleotide comprises: (B) having a tethered group on the first end and a signal on the second end); (b) incubating a DNA adenine methylase with sufficient activity to generate the unmethylated oligonucleotide using a factor; inhibitor library. Methylating the target site, preferably all of the target site, above; (c) converting the incubated DNA adenine methylase to the tethered unmethylated oligonucleotide in the presence of S-adenosylmethionine. (D) digesting all unmethylated target sites, thereby Releasing the tethered unmethylated oligonucleotide; and (e) increasing the signal as a result of the digestion of the unmethylated target site as D
Provided is a method for identifying an agent that modifies or modulates (ie, an agent that modulates Dam function, preferably inhibits Dam function), comprising a step of detecting inhibition of NA adenine methylase. Preferably, the target sequence is a GATC sequence. The tether can be any suitable moiety known in the art (eg, biotin). This signal can be due to fluorescence, radioactivity, or antigen. In some embodiments, the solid surface is a microtiter plate comprising avidin. The unmethylated target site can be digested with a restriction enzyme (eg, MboI). If an inhibitor library is used as a source of the factor to be tested, the library may contain biomolecules (eg, peptides) or may contain organic or inorganic compounds.

It is also understood that the in vitro screening methods of the invention encompass structural drug design, or theoretical drug design, in which the amino acid sequence of Dam, the three-dimensional atomic structure, or other properties include: It provides a basis for designing factors that are suspected of binding to Dam. In general, the design and / or selection of factors in this context depends on several parameters, such as the understood function of the Dam target (where binding to DNA is one such function. A), the tertiary structure of the Dam target (if known or inferred), and other aspects of theoretical drug design). Combinatorial chemistry techniques can also be used to generate many permutations of a candidate agent. For the purposes of the present invention, factors designed and / or obtained by theoretical drug design can also be tested in the cell-based assays described below.

Cell-Based Screening Methods In a cell-based screening assay, viable cells, preferably bacteria containing a functional dam gene, or viable cells, preferably bacteria containing a polynucleotide construct containing a Dam coding sequence, are expressed as factors. Exposure to In contrast, conventional in vitro drug screening assays (as described above) typically measured the effect of a test agent on an isolated component (eg, an enzyme or other functional protein).

[0156] The cell-based screening assays described herein have several advantages over conventional drug screening assays: 1) Factors enter cells to achieve the desired therapeutic effect. If so, a cell-based assay can provide an indication as to whether the factor can enter cells; 2) a cell-based screening assay can be performed at the stage when the factor is added to the assay system.
Factors that are not effective at altering the function of, but can be identified by factors of the cells once they enter the cell so that they become effective factors; 3
) Most importantly, cell-based assay systems allow the identification of factors that affect any component of the pathway that ultimately produces characteristics associated with altered function of Dam.

In one embodiment, an agent is identified in an appropriate host cell by its ability to elicit features associated with altering the function of Dam. Suitable host cells in this connection are any host cells. In this host cell, the function of Dam can be observed. Preferably, the host cell is a bacterial cell. Suitable host cells include Salmonella, Escherichia, Vi containing the Dam gene.
bryo, Yersinia and any other bacterial genera and species. One example of an assay is described in Using the fimbrial operon system in E. coli, In E. coli, the level of reporter expression is determined. Any bacterial operon system that is responsive to methylation is suitable for bacterial-based assays using any of the many reporter systems known in the art.
The level of transcription and / or translation from such a system in the presence of the factor indicates whether the factor affected Dam activity.

In one embodiment, the present invention provides a method for identifying a factor that can control toxicity, comprising the steps of: (a) replacing at least one factor to be tested with Dam's Contacting a suitable host cell with a function; and (b) at least one feature associated with alteration of the function of Dam in the host cell, which can increase, decrease, or delete the function of Dam. A) wherein the factors have been identified by their ability to elicit at least one such characteristic. For these methods, the host cell can be any cell in which the function of Dam is demonstrated.

For genes that are derepressed in the loss of function of Dam, the loss of function of Dam can be measured using a reporter system, in which the reporter gene sequence is operably linked to the Dam of interest. Linked to a suppressor gene. Such suppressor genes are described herein, including by way of example. As used herein, the term “reporter gene” refers to a gene that encodes a gene product that can be identified (ie, a reporter protein). Reporter genes include, but are not limited to, alkaline phosphatase, chloramphenicol acetyltransferase, β-galactosidase, luciferase, and green fluorescent protein. Identification methods for the reporter gene product include, but are not limited to, enzyme assays and fluorescent assays. Reporter genes and assays for detecting these products are well known in the art and are described, for example, in Current Protocols in Molec.
edited by Green Biology, Ausubel et al., Greene Publis
wing and Wiley-Interscience: New York
(1987) and regular updates, as well as Short Protocols
in Molecular Biology (Wiley and Sons, 1
999). Reporter genes, reporter gene assays and reagent kits are also available from commercial sources (Strategene, Invitrogen).
Etc.).

In another embodiment, these methods include the following steps: (a) Da
introducing a polynucleotide encoding m (or a functional fragment thereof) into a suitable host cell that would otherwise lack the function of Dam, comprising:
am function is restored in the host cell; (b) contacting the cell of step (a) with at least one factor to be tested; (c) associated with a loss of function of Dam. Analyzing at least one feature, wherein the factor is identified by its ability to elicit at least one of the features.

The host cells used for these methods first lack the function of Dam (
That is, the function of Dam is deleted before introduction of the polynucleotide encoding Dam). It may be preferred that the step of losing the function of Dam be complete. Dam
The steps of creating a host cell that lacks the functions of: mutagenesis, recombinant or replacement of a functional portion of the gene, frameshift mutation, conventional or classical genetic techniques for isolating the mutant, or regulatory domains Can be achieved in a variety of ways, including, but not limited to, genetic manipulation such as modification of With respect to cells in which the loss of function of Dam (or a homolog thereof) is lethal, a plasmid containing a wild-type copy of Dam is present in the cell during the disruption or mutagenesis process. If the cell cannot survive without the plasmid containing the wild-type gene, the loss of function of Dam is assumed to be lethal. Example 7 describes an assay for determining whether the Dam gene is essential.

The introduction of a Dam-encoding polynucleotide or a functional fragment thereof will depend on the particular host cell used, and will be determined by many methods known in the art, including spheroplasting, electroporation, electroporation, and the like. CaCl ₂ precipitation, lithium acetate treatment, and lipofectamine treatment).

A polynucleotide introduced into a suitable host cell is a polynucleotide construct that includes a polynucleotide encoding Dam or a functional fragment thereof. These constructs contain elements (ie, functional sequences) that allow for the expression (ie, transcription, translation, and post-translational modification, if any) of the Dam amino acid sequence in the host cell upon introduction of the construct. To The composition of these elements (eg, a suitable selectable marker) will depend on the host cell used.

Restoration of Dam (or a homolog thereof) function in a host cell can be determined by analyzing the host cell for detectable parameters associated with Dam function (ie, wild-type). These parameters will depend on the particular host cell used. For Salmonella, the function of Dam is related to any of the following: (a) repression of the Dam regulatory gene; (b) toxicity; (c) paf
Regulation of pili expression; (d) loss of sensitivity of certain amic acids. Genes known to be repressed in the presence of Dam in Salmonella are described above. If there are methods well known in the art for making reporter constructs (see above), any of these genes can be modified to accommodate the reporter system. Examples of suitable reporter systems are discussed above.

In some embodiments, the polynucleotide encoding Dam is operably linked to an inducible promoter. The use of an inducible promoter provides a means for determining whether an element acts via the Dam pathway. The factor is
If the inducible promoter produces features indicative of a loss of function of Dam that appears in the activated cell, the observation that the factor does not elicit a similar result in cells where the inducible promoter is not activated indicates that At least one of the functions of
To affect one process or aspect. Conversely, when a feature indicative of a loss of Dam function is also observed in cells where the inducible promoter is not activated,
It may be assumed that this factor does not necessarily act simply through the functional pathway of Dam.

The cell-based screening assays of the invention include, for example, reporter proteins (ie, easily assayable proteins such as β-galactosidase, chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP) or luciferase )) Expression can be designed by constructing a cell line that depends on the function of Dam. For example, a gene under Dam control is:
It may have a reporter sequence inserted into the coding region as described in Example 1. The cells are exposed to the test agent, and after sufficient time to effect β-galactosidase expression and to allow depletion of previously expressed β-galactosidase, the cells are replaced with a standard Assayed for production of β-galactosidase under typical assay conditions.

Assay methods generally require comparison to a control sample to which no drug has been added. Furthermore, it may be desirable to use cells that partially or completely lack Dam function as controls. For example, if the drug is Da
When acting along the m pathway, one skilled in the art would expect to see the same phenotype as the dam cells treated with the drug. If the agent did not act along the Dam pathway, one skilled in the art would expect to see other characteristics that occur in dam cells when treated with the agent.

[0168] The above screening methods represent a primary screen that represents detecting any agent that may exhibit antibacterial activity. One skilled in the art will recognize that a secondary test is probably needed to further evaluate the drug. For example, secondary screening is
If the primary screen was performed in a host cell rather than a bacterium, this may include testing the agent in the bacterium of interest. A further screen is to perform an infectivity assay using cells treated with the drug. The infectivity assay using mice is described in Example 1 and other animal models (eg, rats)
Are known in the art. In addition, cytotoxicity assays are performed as a further confirmation that drugs that tested positive in the primary screen are suitable for use in vivo. Any assay for cytotoxicity is suitable for this purpose, including, for example, the MTT assay (Promega
).

Preparation of Vaccines and Attenuated Bacteria The present invention also provides a method of preparing or making a vaccine described herein and a mutant strain (ie, a Dam derivative) described herein. Methods for making are provided. Vaccine preparations are discussed fully above, and these methods are therefore encompassed by the present invention. Any of the mutations described herein (including mutations that increase, decrease, or eliminate Dam activity, including Dam expression)
It can be used in the preparation method of the present invention and is not generally repeated in this section.

In one embodiment, the present invention provides a method for preparing an immunogenic composition comprising an attenuated bacterium with altered Dam function, the method described herein. Combining any of the mutants and / or mutants (ie, Dam derivatives) with a pharmaceutically acceptable excipient. A preferred embodiment comprises a Salmonella strain as described herein. Salmonella strains having a mutation that eliminates Dam activity (eg, deletion mutants described herein) are particularly preferred.

In one embodiment, the invention relates to an attenuated pathogenic bacterium (preferably Salmonella) wherein an individual susceptible to a disease caused by the corresponding bacterium or a similar pathogenic bacterium can elicit an immunological response. Which comprises constructing at least one mutation in the pathogenic bacterium described above, wherein the first mutation alters Dam function (preferably, expression of Dam) Changes). Preferably, the first mutation is introduced into a first gene that expresses Dam. In some embodiments, the first mutation described above is introduced into a first gene, the expression of which suppresses or overactivates the expression of a gene that expresses a DNA adenine methylase enzyme. In some embodiments, the first mutation is introduced into a first gene, the expression of which is regulated by DNA adenine methylase. In other embodiments, the second mutation is made in a gene independent of the first mutation, and the second mutation causes attenuation of the bacterium.

In another embodiment, the present invention provides a method for preparing an attenuated bacterium capable of eliciting an immunological response in a host subject susceptible to a disease caused by a corresponding toxic bacterium, the method comprising: The method comprises (a) constructing at least one mutation in the dam gene of a virulent strain of a pathogenic bacterium. In some embodiments, a second mutation is introduced into a second gene that results in attenuation of the bacterium, independent of the first mutation.

In another embodiment, there is provided a method for preparing an attenuated bacterium in which a host susceptible to a disease caused by a corresponding or similar pathogenic bacterium is capable of eliciting an immunological response. The method comprises (a) constructing a first non-restoring mutation in the pathogenic bacterium, wherein the first non-restoring mutation comprises one or more DNs.
Altering the expression of A-adenine methylase or its activity, and (b)
Constructing a second non-restoring mutation in said pathogenic bacterium, wherein:
The second non-restoring mutation is independent of the first non-restoring mutation and comprises attenuating. In some embodiments, the first non-restoring mutation is constructed in a gene whose gene gene product activates one or more DNA adenine methylases described above. In some embodiments, the gene product activates DNA adenine methylase. In some embodiments, the first non-restoring mutation is constructed in a gene whose gene product suppresses the expression of DNA adenine methylase as described above. In some embodiments, the gene product inhibits DNA adenine methylase. In other embodiments, the first non-restoring mutation is that the gene product inactivates the activity of the one or more adenine methylases by directly binding to the one or more DNA adenine methylases, Or construct in the gene to be reduced. In some embodiments, one of the DNA adenine methylases is a DNA adenine methylase. In some embodiments, the pathogenic bacterium is Salmon
, and preferably Salmonella is a strain of S. ella. typhim
urium, S.M. enteritidis, S.M. typhi, S.M. borutus
-Ovi, S .; abortusequi, S.A. dublin, S .; gallin
arum, S .; pullorum. In another embodiment, the pathogenic bacterium is any one of the following: Yersinia, Vibrio, Shi.
gella, Haemophilus, Bordetella, Neisser
ia, Pasteurella, pathogenic Escherchia, Trepone
ma. The host can be a vertebrate (eg, a mammal), preferably a human or a domestic animal. In some embodiments, the vertebrate is a chicken.

[0174] In some embodiments, the method of preparation involves the addition of an antigen. For example, the antigen may simply be added to the bacteria in the vaccine, or an expression cassette containing one or more structural genes encoding the desired antigen may be inserted into the attenuated bacteria.

Antigens include, but are not limited to, fragment C of tetanus toxin, B subunit of cholera toxin, hepatitis B surface antigen, Vibrio
cholerae LPS, HIV antigens and / or Shigellas
oneii LPS.

In another embodiment, the present invention provides a method of preparing an attenuated microorganism wherein a host susceptible to a disease caused by a corresponding or similar pathogenic microorganism can elicit an immunological response. The method comprises the steps of (a) constructing a first non-restoring mutation in the pathogenic microorganism, wherein the first non-restoring mutation comprises DN
Altering the expression or activity of one or more genes regulated by A methylase, and (b) constructing a second non-restoring mutation in the pathogenic microorganism, wherein the second non-restoring mutation is The two non-restoring mutations are independent of the first non-restoring mutation and comprise attenuating.

[0177] The above disclosure generally describes the present invention. A more complete understanding may be obtained by reference to the following specific examples, which are provided herein by way of example only and are not intended to be limiting.

EXAMPLES The following non-limiting examples provide vaccines prepared from live pathogenic bacteria and target sites for antimicrobial drugs in accordance with the teachings of the present invention, and are illustrative,
Provided in a non-limiting manner. All technical and scientific terms have meanings as understood by a person of ordinary skill in the art. Recombinant DNA technology is now considered to be well known and widespread and conventional. In broadest and general terms, these techniques involve transferring the genetic material of one organism to a second organism,
As a result, the transferred genetic material becomes part of the genetic material of the transferred organism.
This typically comprises a first step of obtaining a piece of DNA from a first organism from either plasmid or chromosomal DNA. A piece of DNA can be of any size and is often obtained by the use of restriction endonuclease enzymes that recognize and cleave DNA at specific base pair sites. After isolating a piece of specific DNA, the DNA is inserted or cloned into a plasmid, phage or cosmid vector to form a recombinant molecule, which is subsequently
Host cells can be transferred by various methods such as transformation, transduction, transfection, and conjugation.

Transformation involves the uptake of naked DNA from the external environment, which can be induced artificially by the presence of various chemical agents (eg, calcium ions) or by electroporation. Transduction involves the packaging of the recombinant DNA into a phage (eg, a transducing phage or cosmid vector). Once the recombinant DNA has been introduced into the microbial host, it can continue to exist as a separate piece, or can be inserted or integrated into the chromosome of the host cell and replicated with the chromosome during cell division. Conjugation involves classical microbial conjugation techniques.

Example 1 Dam Salmonella Derivatives Are Not Toxicity Strain Construction All Salmonella typhimurium strains used were Ame
product Tissue Culture Collection (ATCC
) Strain 14028 (smooth virulent strain of S. typhimurium which is called wild type). Previously, all dam mutations reported from other laboratories used Salmonella strain LT2. This Salmonella strain LT
2 is i. p. At least 1000 times less virulent than wild-type when delivered. See data in Table 1.

[0181] All restriction enzymes and pBR322 are commercially available (eg, Strata).
gene, 11099 North Torrey Pines Rd. , La
Jolla, California 92037) or can be purchased. Electroporation is performed using the BioRad Gene Pulser app
paratus Model No. 1652098. S. ty
Phimurium cells were prepared according to the manufacturer's instructions. An aliquot of the competent cells was 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 with a field strength of 2.5 KV / cm according to the manufacturer's instructions.

(1. Construction of non-polar dam mutation) For the construction of non-polar dam mutation, typhimurium genomic DNA
Was used as a template for PCR using Pfu polymerase (Stratagene). 350 bp, including the first 100 codons of dam
Of the following oligonucleotide pair: 5'-GATTCTCT
AGAGTAGTCTGCGGAGCTTTC-3 ′ (SEQ ID NO: 1) (including an XbaI site at the 5 ′ end) and 5′-GATTCTCGAGGGTGTTGAA
It is amplified by PCR using CTCCTCCGCG-3 ′ (SEQ ID NO: 2) (containing an XhoI site at the 5 ′ end). 92 ° C. in a buffer containing 2.0 mM Mg ²⁺
For 45 seconds, 1 minute at 42 ° C and 1 minute 30 seconds at 72 ° C for 30 cycles.
CR was performed. This procedure was performed using DNA Thermal Cycler # 801-0150 (P
erkin-Elmer Cetus). The PCR product was then double digested with XbaI and XhoI. In the second PCR amplification, d
A 300 bp DNA fragment containing the last 79 codons of am was ligated to the following oligonucleotide primers: 5'-GATTCTCGAGTTTAGCCTG
ACGCAACAAG-3 ′ (SEQ ID NO: 3) (including XhoI site at 5 ′ end)
And 5'-GATGTGCATGCCTCTTCACCCAGGGCAG-3 '
It was synthesized using (SEQ ID NO: 4) (including a SphI site at the 5 ′ end). Then
This PCR product was double digested with XhoI and SphI. Suicide vector pC
VD442 (Donnennberg, MS et al., Infect. Immun.
, 59: 4310-4317 (1991)) was double digested with XbaI and SphI, the band was purified and ligated into a single reaction with two tailored PCR products. An in-frame deletion of 100 internal amino acids of Dam was made, leaving a unique XhoI site at the deletion junction. Then, E. coli DH5α
λ pir was transformed by selecting for ampicillin resistance. The DNA from the appropriate ampicillin resistant construct (confirmed by restriction digestion) was then used to construct S. aureus. ty
phymurium 14028. Then the incorporated pCV
D442 containing constructs were separated on LB 5% sucrose / no salt plates. Isolates were confirmed for ampicillin sensitivity by printing and Dam by streaking on LB plates containing 2-aminopurine (0.6 mg / ml) (Dam mutant pairs are 2-AP sensitive). In addition, PCR was used to confirm deletion by size compared to the wild-type sequence. Finally, the deleted region was replaced with pGP
Cloning at 704 and obtaining sequences near or at the deletion junction (including the XhoI site) confirmed that the deletion was indeed in-frame.

The mutation caused by the dam102 insertion (dam102 :: Mud-Cm discussed above) was driven by P22-mediated transduction of virulent Salmonella strain 14028 to give construct strain 2.

2. Mouse Toxicity Assays typhimurium strain, as described above, the female B
ALB / c mice were tested by intraperitoneal or oral inoculation and the results are shown in Table 1 below.

[0185] Female BALB / c mice were isolated from Charles River Breeding.
Laboratories, Inc. , (Wilmington, Mass.)
And initially challenged at the age of 6-8 weeks. S. typhi
The murium strain was grown overnight at 37 ° C. in luria broth (LB) until stationary phase. Bacteria were washed once with PBS and then diluted with PBS to approximately the appropriate dilution (samples were plated for colony forming units (CFU) in LB to give the correct bacterial count). Mice were challenged with 200 μl of the appropriate bacterial dilution, either intraperitoneally or orally. For inoculation via the mouth, the bacteria are washed and concentrated by centrifugation, then the bacteria are reduced to 0.2 M
Resuspend in Na ₂ HPO ₄ (pH 8.0) to neutralize stomach acid and
Animals were administered as a bolus of 1 under ether anesthesia. For all LD ₅₀ determination, 5 mice were each inoculated per dilution. Control mice received PBS only.

All bacterial strains used in this study were S. typhimurium 14028
It was a derivative of (strain 1). Mutants were isogenic with the wild type and were obtained or constructed as described. (Dam102 :: Mud-Cm and m
The utS121 :: Tn10 allele is in LT2 (strain 7), and as shown in Table 2, the highly attenuated (actually non-pathogenic) strains were, respectively, Dr. Jo
hn Roth (University of Utah) and Dr. Tom
Cebula (The Food and Drug Administra)
these alleles (and further alleles below)
Was transduced into virulent strain 14028 to construct strains 2 and 5, respectively. da
mΔ232 (strain 3) was constructed with an internal oligonucleotide acting as a PCR primer designed to construct an in-frame 300 bp deletion of the defined dam sequence. dcm1 :: Km (Julio, SM et al., Mole
c. Gen. Genet. 258: 178-181 (1998)); the Km resistance determinant is associated with an internal deletion of more than 600 bp of the dcm sequence. lrp31 :: Km is a null insertion in the lrp gene (strain 6).
The Dam overlapping strain (strain 4) is a recombinant plasmid (strain 4) in a wild type background.
pTP 166). E. coli dam (Marinus, et al., Ge
ne, 28: 123-125 (1984).

For in vivo competition studies, bacteria were treated as discussed above, and mutant cells were then mixed with wild-type cells at a 1: 1 ratio (approximate input bacteria were 500
Mutant + 500 wild type). The actual ratio is determined by first plating the input bacteria with LB and then 100% for resistance to the appropriate antibiotic.
Colonies were scored. Bacteria were injected intraperitoneally into at least 5 BALB / C mice (at 1: 1 mutant: wild-type ratio as described) (Conner,
C. P. Proc. Natl. Acad. Sci. USA, 14: 4641.
-4645 (1998)), then, after 4-5 days, if the mice appeared moribund, they were sacrificed and their spleens were isolated, homogenized, diluted and plated. Again, the mutant: wild-type ratio was determined by scoring 100 colonies for the mutant phenotype. The competition index is the ratio of mutant: wild-type bacteria recovered, and essentially reflects how to match the mutant compared to the wild-type strain. Thus, those strains exhibiting a competitive index lower than 0.0001 reflect the fact that the mutant was not recovered from the spleen. In conclusion, the mice died as a result of the wild type.

An advantage of the LD ₅₀ assay is that it quantifies a major toxicity defect. The disadvantage of this is that sensitivity is lacking, and thus a small but significant toxic contribution is often overlooked. The competition index is the mutant: wild-type bacterial ratio recovered from infected tissues after co-inoculation. The competition index is very sensitive and allows the detection of a slight toxicity contribution. However, due to its sensitivity, quantifying the difference in toxicity between the two mutants that give the major deficiency is problematic. Accordingly, the use in cooperation with LD ₅₀ and competition index assay is an effective means to quantify both the large virulence defects and slight toxicity defect. The competition index is a further indicator of how to match the mutant compared to the wild type, but does not necessarily have to directly correlate with complete toxicity.

Table 1 shows the results. LD ₅₀ is the volume required to kill 50% of infected animals. The (LD ₅₀ ) assay for each of these strains was determined using the wild type (strain 1; (ND)
, Not determined)). Gastrointubation
Oral LD50 for all derivatives via (ion) was determined by infecting at least 12 BALB / c mice; intraperitoneal (ip) L.
The D _50, was determined by infecting at least six mice.

[0190]

[Table 1] Since a dam insertion could reduce the expression of downstream genes (polar effect), an in-polar non-polar dam deletion was constructed and shown to have the same reduced toxicity as the dam insertion. Thus, the attenuation was specifically due to the lack of Dam. Furthermore, an equal number of Dam ⁺ Salmonella and Dam ^- Sal
Inoculation of mice with monella intraperitoneally showed that the Dam ⁻ mutant was completely eliminated during growth in mice (competition index assay). Similar results were observed using strain 4 (Table 1), which overproduces Dam from the recombinant plasmid. This suggested that the correct level of Dam methylase was required for complete toxicity. These results indicate for the first time that Dam methylase is essential for bacterial virulence.

Dam is methyl-directed mismatch repair (methyl-directed).
Salmonella toxicity can be affected through an increase in mutation rate caused by the abolition of the missmatch repair (MDMR). Mut
S plays an essential role in MDMR, so mutS Salmonel
It was determined whether la attenuated the toxicity. The data in Table 1 (above) show mutS Salmon in both the oral LD ₅₀ and the competitive indicator toxicity assays.
ella indicates that it was identical to wild type, indicating that Dam does not affect virulence through the MDMR pathway. MutS ^- strains acquire new virulence determinants more easily because MutS ^- strains show higher levels of DNA change between species than Mus ⁺ strains (Marinus, E. coli and).
Salmonella: Cellular and Molecular Bi
ology, 2nd edition, 782-791 (1996)). The fact that the Mus ^- strain is sufficiently virulent is that mutSE. coli and Salmonell
The a variant may explain the high frequency of being found in clinical isolates (LeClerc et al., Science, 274: 1208-1211 (1996)).

Dam and Lrp directly regulate the expression of Pap pili, which are uropathogenic E. coli. essential for E. coli toxicity (O'Hanley
J. et al. Clin. Invest. , 75: 347-360 (1985); and Roberts et al. Urol. , 133: 1068-1075 (1985).
)). Lrp ^- Salmonella was analyzed to determine whether Dam affected Salmonella toxicity through an Lrp-mediated pathway (Table 1).
). Salmonella lacking Lrp was sufficiently virulent based on LD ₅₀ and competition index assay. These data are from Salmonell
a Lrp is not a virulence factor in mice.

The results discussed above indicate that adenine methylation is critical for Salmonella virulence. DNA methylation of cytosine residues appears to be important for the regulation of biological processes in both plants and animals.
Salmonella contains DNA cytosine methylase (Dcm), but the role of cytosine methylation in this organism is unclear. dcm ^- mutant (d
cml :: Km), was virulent in LD ₅₀ and competition index assay (data not shown). These results indicate that adenine but not cytosine residue methylation is required for Salmonella virulence.

Methylation of DNA adenine is described in E. coli. have been shown to directly regulate toxic gene expression in E. coli (Braatin et al., Cell, 76: 577-).
588 (1994)). Therefore, Dam is in vivo inducible (in vivo).
It was determined whether to regulate the Salmonella gene, preferentially expressed in mice, termed induced (iv) gene. Conner
, C.I. P. Proc. Natl. Acad. Sci. USA, 14: 464
1-464 (1998); Heithoff, D .; M. Proc. Nat
l. Acad. Sci. USA. , 94: 934-939 (1997); Mah.
an, M .; J. Et al., Science, 259: 666-668 (1993); M.
ahan, M .; J. 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. When grown in enriched media, Dam significantly increased the expression of more than 20 iv genes (2
~ 18-fold) suppression, eight of these are presented in FIG. Four of the eight fusions are known genes, all of which contribute to toxicity or
Or has been shown to be associated with toxicity: spvB is Salmonella
Present on toxic plasmids and function to facilitate growth at systemic sites of infection (Gulig et al., Mol. Microbiol., 7: 825-830).
(1993); pmrB is involved in resistance to an antimicrobial peptide named defensin (Roland et al., J. Bacteriol., 75:41).
54-4164 (1993); mgtA and entF are involved in the transport of magnesium and iron, respectively (Earhart, Escherichia c.
oli and Salmonella Cellular and Mole
cultural Biology, 2nd ed., 1075-1090 (1996); and Vescovi, G .; Et al., Cell, 84: 165-174 (1996)).
Additional viv genes of unknown function were also Dam regulated. These results indicate that Dam is a global regulator of Salmonella gene expression.

The Salmonella pathogen is known to be controlled by PhoP, a DNA-binding protein that acts as both an inducer and receptor for certain virulence genes (Groisman and Heffron Two-compo).
tent signal transduction, 319-332 (199)
5)). To determine whether the Dam regulatory pathway and the PhoP regulatory pathway share a common gene, the effect of Dam was determined by seven PhoP-activating ivs.
i gene (including spvB, pmrB and mgtA). FIG.
am represses the expression of these three genes by a factor of 2 to 19, and these repressions
This shows that oP protein was not dependent on the protein. Dam, the remaining four PhoP ^-
It did not significantly affect the expression of the activated gene (data not shown). These results indicate that Dam and PhoP constitute an overall regulatory network that controls Salmonella toxicity.

The binding of the regulatory protein to the DNA is determined by a specific Dam target site (GATC sequence (
van der Wood et al. Bacteriol. , 180: 5913
-5920 (1998)) to form a methylation pattern of DNA. Therefore, further studies of the interaction between Dam and PhoP indicate that the binding of PhoP (or a PhoP-regulated protein) to specific DNA sites will block the methylation of these sites by Dam, thereby reducing DNA methylation. Performed to determine whether a change in the chemical pattern would occur. Ph
The analysis for oP ⁺ Salmonella and PhoP ^- Salmonella was
It showed distinct differences in the methylation pattern of NA. Digestion of genomic DNA from PhoP ⁻ bacteria with MboI, which only cuts at unmethylated GATC sites, resulted in the appearance of DNA fragments not present in DNA from PhoP ⁺ bacteria (FIG. 5). , See arrows). These results indicate that Pho
FIG. 7 shows that the P protein (or PhoP regulated gene product) blocks Dam methylation at specific GATC-containing sites in the Salmonella genome. Alternatively, PhoP ⁺ and PhoP ⁻ strains can have different levels of Dam activity, which in turn can affect the methylation pattern of DNA. However, this regulation does not occur at the transcriptional level. Because Dam does not alter PhoP expression,
This is because hoP also does not alter Dam expression (DM Heithoff and MJ Mahan, unpublished material). Further analysis shows that these PhoP-
Whether the defense site is within the regulatory region of the virulence gene, and whether DNA methylation
It is determined whether altering the NA-PhoP interaction directly affects PhoP regulation.

Example 2A: Protective Efficiency of Dam ^- Salmonella Attenuated Strains Strains that showed attenuation as a result of intraperitoneal or oral challenge of BALB / c mice were further purified by 10 ⁵ I. P. Or 10 ⁹ oral doses were tested for protective immunity to subsequent challenge with the wild-type strain. BALB / c mice were ^challenged with 10 ^{+ 9} Dam ^- S. Typhimurium doses were used to orally immunize via gastric intubation. After 5 weeks, immunized mice were ^challenged with 10 ⁺⁹ wild-type S.D. Oral challenge with typhimurium. After 5 weeks, surviving mice were replaced with wild-type 14028 as described in Table 2 (below).
Challenged with strains. Survival for 4 weeks post-challenge was considered adequate protection. These data indicate a potential use of the present invention in vaccine strain development.

Since the Dam ^- mutant was highly attenuated, it was determined whether Dam ^- Salmonella could act as a live attenuated vaccine. Table 2 shows that S.D. typhi
All (17/17) mice immunized with the S. murium Dam ^- insert showed that they survived a 10 ⁺⁴ wild-type challenge with an LD ₅₀ or more, while all non-immunized mice (12/12 ) Died after the challenge.

[0199]

[Table 2] .

Virtually, there was no apparent effect of typhoid fever observed after immunization with Dam ^- Salmonella, and there was no apparent effect after wild-type challenge. In addition, since all (8/8) mice immunized with Salmonella containing the non-polar dam deletion (strain) survived the challenge, these data indicate that protection was specific depending on the absence of Dam methylase. To show that Dam as a vaccine ^- toxicity attenuation and efficacy of mutant (Table 1 and 2) are, Salmonella growth and / or ectopic expression of virulence determinants seem detrimental to survival during infection ( It can be attributed to Tables 3 and 4). Thus, ectopic expression is
It provides an explanation why the Dam mutant is totally attenuated and still provides sufficient protection as a live attenuated vaccine.

(Study on colony formation) Dam ⁺ Salmonella and Dam ^- Salmon in mouse tissues
ella survival was compared. As shown in FIG. 6, the Dam ⁻ bacteria performed well in colonizing mucosal sites (Pier plaques) (profici
ent) showed severe defects in colonization of deeper tissue sites. Five days after infection, we found that in the mesenteric lymph nodes, a three orders of magnitude reduction in the number of Dam ^- Salmonella (relative to the number of Dam ⁺ bacteria), and in the liver and spleen, Dam ^- Salmonella. An 8 order magnitude reduction in the number of was observed. These data indicate that Dam ^- Salmonella survives in Peyer's patches of the mouse small intestine for at least 5 days, providing an opportunity for eliciting a host immune response. But Dam ^- Salmonell
a could not cause disease; they could either invade systemic tissue or could invade but not survive.

[0203] (Example 2B: protective efficacy of a killed Dam derivative) (alive Dam ^- or Dam overexpression bacteria, the determination of whether it is necessary to elicit a sufficient protective response) Dam ^- ectopic expression of multiple proteins (see it above and below) in the vaccine were killed Dam, ^- organism, a killed Dam, ⁺ obtain elicited significantly stronger protective immunity than the organism, therefore, Suggests that it can be used as a mucosal vaccine. S. cerevisiae grown in vitro 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 from the killed organism. The efficiency of whole cell killed vaccine preparations can be determined by using a mucosal adjuvant (eg, cholera toxin, E. coli unstable toxin, or vitamin D).
3 (1,25 (OH) ₂ D ₃ )) with or without test. Therefore, 1
A vaccine preparation containing 0 ¹⁰ killed Dam ^- Salmonella, alone or in combination with a mucosal adjuvant, is used to orally immunize BALB / c mice (as described in the Examples). As in the dose regimen, mice are immunized by gastric intubation once a week for three weeks. Killed wild-type S. t
yphimurium acts as a negative control. Immunized mice were vaccinated two weeks after the last immunization. Typhimurium is challenged orally to determine whether an effective immune response is generated. If an effective immune response is generated, mice immunized with the killed vaccine preparation will also have other pathogenic Salmonella serotypes (eg, entereri
tidis, choleraesuis, dublin), and the induced immunity is cross-protected against relevant strains.
active) and similarly for the case of oral administration of a live Dam ^- Salmonella vaccine. If mice immunized with a dead vaccine preparation are protected two weeks (or three weeks) after the last immunization, whether the elicited immunity is persistent or not is determined by the last immunization. After 7 weeks, it is determined by challenged immunized mice.

[0203] Overexpression of Dam wild type (Dam ⁺⁾ or Dam ^- so can result in potential ectopic expression of a new repertoire of protective antigens not expressed in any of the vaccine strains, killed vaccines The experiments are performed using the overexpressing strain of Dam alone or in combination with the killed Dam ^- organism. Since two different vaccine strains can potentially generate two different repertoires of protective antigens, their use in combination can elicit a predominant immune response.

[0204] (Example ^3: Dam, - cross protection induced by Salmonella) ^(Dam, - immunization with Salmonella induces cross-protective response against heterologous serotypes) shown in FIG. 7 and discussed below Thus, the Dam ⁻ mutant ectopically expresses multiple genes (and possibly proteins) that are normally only expressed during infection. Such ectopic expression of multiple antigens can generate a cross-protective immune response against heterologous serotypes. BALB / c mice were administered orally (via gastric intubation) to give 1 × 10 ⁹ Dam ^- S. Typhimurium (serotype B) and 11 weeks later, virulent S. typhimurium. enteritidis and S. et al. du
We were challenged with Blin (serotype D) (100~1000LD _50). The data in Table 3 show that the mice were protected against a heterologous challenge 11 weeks after immunization. Importantly, cross-protective immunity was not due to persistence of the vaccine strain in mouse tissues. Because mice are heterogeneous for more than 6 weeks after the vaccine strain has been removed from the immunized animal (ie, no Dam-organism can be detected in Peyer's patches, mesenteric lymph nodes, liver and spleen). Because he was protected against the challenge. The induced cross-protection is specific to the Salmonella strain as there is no protection induced against the systemic pathogen Yersinia pseudotuberculosis 5 weeks after immunization.

[0205]

[Table 3] .

Similarly, Dam ^- S. enteritidis (dam102 :: Mud-C
Immunization using the experimental protocol described above) followed by 10 ⁹ S.M. t
yphimurium and 10 ⁹ of S. Cross protection against challenge with dublin is provided, and may be provided for longer periods of time.

(Dam ⁻ derivatives express ectopic multiple proteins in vitro) Ectopic expression of multiple proteins in a Dam ⁻ strain is induced against heterologous serotypes that share a common epitope Can contribute to cross defense. To this end, we have determined that the Dam ⁻ strain has a number of Sa that are normally suppressed in vitro.
1 shows that the lmonella gene is ectopically expressed.

Two-dimensional protein gel electrophoresis was performed using logarithmic S. cerevisiae grown in Luria broth. Typhimurium whole cell protein extract against O'Farr
ell ((1975) J. Biol. Chem. 250: 4007-4021).
Was carried out by the method described above. Ampholine (BioRad Labo) at pH 5-7
ratifications, Hercules, CA).
The operation was performed at 00 V for 17 hours. The two dimensions were run at 175 V for 5.5 hours.
Consisted of a 5% polyacrylamide slab gel. Proteins were visualized by silver staining (Merril et al. (1984) Methods Enzymol. 1).
04: 441-47. ). The result is shown in FIG. This result indicates that the Dam ⁻ strain, the Dam ⁺ (wild type) strain and the Dam overproduction (O
P) two-dimensional gel electrophoretic analysis of strain (2-D protein analysis), Dam, ^- expressed in conditions of, Dam, ⁺ (either wild-type) or Dam, OP (expressing approximately 100 times higher than normal) 2 shows that some proteins were not detected under these conditions. These data indicate that Dam ^- Salmonella ectopically expresses multiple proteins in vitro (and possibly in vivo), where dysregulation of protein expression causes processing and presentation to the immune system. Suggest that it may provide multiple novel protein targets to be done.

The 2-D protein analysis showed that the Salmonella Dam overexpressing strain (plasmid pTP1 overexpressing E. coli Dam at about 100 times wild type level)
S.66. typhimurium ATCC 14028) was obtained under laboratory growth conditions using Dam ⁺ (wild type) Salmonella or Dam ^- Salm.
FIG. 9 shows that many gene products that are not expressed by onella are expressed. We did not detect proteins produced by Dam ⁺ and not produced by Dam ^- or Dam overexpressing strains. Taken together, our observations that the Dam overexpressing strain attenuates and elicits protective immunity, these results indicate that Dam overexpression has a different repertoire of antigens than the antigen produced in the Dam ⁻ strain. Suggests that expression can occur. Therefore, Dam ^- in combination with the strain vaccine consisting Dam overexpressing strain, due to potential ectopic expression of protective antigens of the two different repertoires may be very cross-protective.

(Immunity elicited by Dam ⁻ strains is greater than immunity elicited after wild-type infection) One of the most effective virulence properties of pathogens is the ability to evade the host immune response.
Such a "stealth" strategy is achieved by tightly regulating many of its functions to evade host immune recognition. Thus, many antigens produced by virulent organisms, such as bacterial defense mechanisms,
It does not appear to be produced in sufficient quantities and / or not in sufficient quantities to elicit a host immune response. However, Dam ⁻ bacteria can ectopically express multiple antigens that are processed and presented to the immune system,
Animals immunized with the Dam ^- vaccine are better than animals surviving the natural infection.
It can elicit a stronger immune response.

The immunity elicited by the Dam ^- vaccine was compared to the immunity elicited after natural infection with the wild-type strain. BALB / c mice were transformed with the virulent strain S. aureus. type
himurium (10 ⁺⁵ organisms) (ie, half of the mice survived wild-type immunization) or 10 ⁺⁵ Dam ⁻ organisms were immunized orally with an LD ₅₀ . Five weeks after immunization, immunized mice were challenged with a lethal dose of virulent strain. Table 5 shows that the immunity elicited by the Dam ^- vaccine was not elicited in mice that survived immunization with the wild-type strain (1 out of 10 mice had 1
^Shows that at least 100-fold more (3 out of 10 mice survived the 10 ^{+ 9} challenge) than 0 ^{+ 7} challenge survived.

[0212]

[Table 4] .

[0213] Further, Dam^-Immunizations with organisms are subject to a wide range of challenge doses (10⁺⁷-10^{+ 9} ) Showed relatively similar levels. This is 10⁺⁵Dam^-Bacterial
Immunization dose is required to ensure a protective immune response in all immunized animals
It suggests that there are fewer than the minimum threshold of organisms. Induced by Dam-strain
The enhanced immunity that appears may be due in part to the ectopic expression of the Dam-suppressing antigen.
Yes, this antigen is produced in sufficient quantity and / or duration during wild-type infection
I can't.

Immunized animals inhibit the growth of toxic bacteria in systemic tissues. Dam ^- Salmonella is good enough to colonize Peyer's patches in the small intestine of mice,
Very poor colony formation at deep tissue sites (liver and spleen) was found (Example 1). S. The Typhimurium Dam ^- mutant is also less cytotoxic to M cells, has poor epithelial infiltration, and exhibits defects in protein secretion. Pucciarelli et al. (1999) Proc. Natl.
Acad. Sci. ScL USA 96: 11578-11583. Taken together, these data provide a possible explanation as to why the Dam ^- variant cannot produce disease but elicit a full protective immune response. Compare the fate of wild-type Salmonella in immunized versus non-immunized mice, as mice immunized with Dam ^- Salmonella showed virtually no significant symptoms of the disease after challenge with toxic organisms did. Data in Figure 8, Dam, ^- immunized mice, after the wild-type challenge 10 ⁹ organisms, both in mucosal tissues and systemic tissues, at least 5 days, the toxicity bacterial high-load ⁽¹⁰⁴⁾ Indicates that it is retained. However, immunized mice have the ability to not only inhibit the growth of these toxic organisms,
Immunized mice are capable of clearing toxic organisms from both mucosal and systemic tissues (2 of 4 mice have Peyer's patches, mesenteric lymph nodes 28 days after challenge) , Liver, and spleen to remove all toxic organisms).
This ability to remove 10 ⁴ toxic organisms from the liver and spleen is important given the fact that the intraperitoneal LD ₅₀ is less than 10 organisms. Therefore, immunization with Dam ^- Salmonella inhibits the growth of wild-type organisms in all tissues tested. The ability to eliminate the lethal burden of toxic bacteria systemically suggests that Dam ^- vaccines may have therapeutic applications in the treatment of pre-existing microbial infections.

Example 4: Chicken vaccination against S. enteritidis S. enteritis in poultry A thorough understanding of the kinetics of enteritidis infection is important for S. cerevisiae from laying hens to human consumers. Enteritidis is essential in formulating an effective strategy to prevent infections resulting from eggs. Salmonella
Disease occurs by colonizing and invading the intestinal epithelium. In some cases, extensive dissemination and systemic disease follows Salmonella invading the bloodstream through the intestinal mucosa. S. enteritidis is an invasive serotype in chickens, but did not show any level of pathogenicity to chickens that was distinctly different from other Paratyphi salmonella serotypes. Popiel and Turnbull (1985) Infect. Immun. 47 (3): 78
6-792. Chickens were infected with S. cerevisiae from contaminated feed. enteritidis can easily be infected, colonize the intestine, and internal tissues such as the liver (iss
ue) with both intrusions. Hinton et al. (1989) Vet. Rec
. 124: 223.

Some S. cerevisiae of adult hens Experimental infection with enteritidis strains
Intestinal colonization (persisted for several months), but not other S. aureus. enter
In studies with the S. itidis strain, the duration of excretion was quite short. (Gast and Beard, 1990), Gast and Beard (19
90) Avian Dis. 34: 991-993; Shivaprasad et al. (1990) Avian Dis. 34: 548-557. In one study, birds infected intravenously were longer in S. aureus than birds infected orally. enter
excretes itidis. Shivaprased et al. (1990).

E. coli in eggs and egg products The effectiveness of various methods of destroying enteritidis has become an increasingly important topic for public health authorities and the egg industry. Such information is greatly needed to provide consumers and commercial or institutional users of eggs with instructions on the safe preparation of egg-containing foods. Shivapr
sad et al. (1990) report that S. cerevisiae in eggs by various recipes. enteriti
The time / temperature requirements for destroying dis are described by S.D. typhimuriu
It was observed that m did not differ significantly from similar requirements previously determined. Baker et al. (1983) Pult. Sci. 72: 1211-1216
. Humphrey et al. enteritidis, S.M. ty
phimurium, and S. Senftenberg strains have found that when planted in egg yolk, some of the yolk can survive from a cooked form that remains liquid. Humphrey et al. (1989) epidemiol. Infec
t. 103: 35-45. Furthermore, if the eggs were stored at room temperature for 2 days after planting, Enteritidis populations increased in yolk to levels high enough that none of the standard cooking methods completely eliminated Salmonella.
On the other hand, S.P. Storage of enteritidis cultures has been found to increase sensitivity to heat. Humphrey (1990) J. Am. Ap
pl. Bacteriol. 69: 493-497. In another study, S. cerevisiae in homogenized whole eggs was used. enteritidis phage type 4 comprises phage type 8 or phage type 13a and S. aureus. Typhimurium is more heat resistant,
S. extremely heat resistant It was determined that it was less thermotolerant than the Senftenberg strain 775W. All Salmonella strains tested were more thermotolerant in yolk than in whole eggs or albumin. Humphrey et al. (1990
) Epidemiol. Infect. 104: 237-241.

The vaccines of the present invention, in particular Strain 3, are useful for S. cerevisiae in eggs and egg products. ente
ritidis may be effective. Dam ^- S. typhimur
The ium vaccine is prepared as previously described. The vaccine is introduced into chickens by oral administration (ie, mixed with chicken feed and / or water). Once the vaccine is administered, a toxic factor that is typically suppressed by Dam is expressed, and the chicken induces an immune response. Some of these Dam regulatory genes are enteritidis is a homologue of a gene shared by Dam ^- S. typhimurium, as shown by the data of Example 3, S. typhimurium. cross prot against enteritidis
action).

Example 5 Administration of a Dam-Inducible Salmonella Vaccine to Cattle Salmonella is the most commonly isolated infectious intestinal bacterial pathogen of dairy cows and human, beef It is the most common zoonotic disease associated with the consumption of household goods. In recent years, the incidence and severity of human cases of salmonellosis has, in part, increased the antimicrobial resistance of S. aureus in bovine populations. typhimuriumD
It has been rising due to the emergence of T104. Prevalence studies are conducted on one of the dairy farms in the United States.
Six to 73% are infected with Salmonella, and up to 50% of selected cows are contaminated with Salmonella at the time of slaughter. Salmonella control on the farm is important to reduce production losses and diseases arising from human food.

On a large commercial farm, it is very common for cattle to be exposed to multiple Salmonella serotypes and for calves to be infected shortly after birth. Under these circumstances, it is highly desirable to have a Salmonella vaccine that can stimulate immunity against a heterologous Salmonella serotype.

[0221] (A. need for Dam about Salmonella infections in cattle, and Dam as a live bovine vaccine ^- the effectiveness of the derivative) 1-3-day-old Holstein Yuko cows, used for all experiments. To evaluate calf passive immunity, measurements of total plasma protein were used. Only calves with a total plasma protein of 5.5 or more are used. The source dairy farm Salmonella infection status is determined prior to purchasing calves by culturing fecal and environmental samples for Salmonella. The calm Salmonella negative status is confirmed after purchase by daily Salmonella culture of feces.

The calves were subjected to animal biosafety levels (Animal Biosafety).
y) Rearing and rearing at two facilities. Give the calf 2 quarts of 20:20 milk (
2 times a day) and add fresh calf cereal and fresh water,
Access 24 hours a day. At the time of feeding each day, all calves are given appetite and attitude scores. The appetite score was graded on a scale of 1 to 4 (1 = consumed 2 quarts of milk, 2 = consumed less than 2 quarts and more than 1 quart of milk, 3 =
<1 quart of milk was consumed, and 4 = no milk was consumed). Attitude scores also range from 1 to 4 (1 = standing, 2 = standing with encouragement, 3
= Stand when assisted, 4 = do not stand). Following all challenge experiments, calves were examined three times a day and vital parameters recorded twice a day. Any calves that do not stand are considered terminal and are euthanized. Following the Salmonella challenge, calves are not given any antibacterial or anti-inflammatory treatment to avoid confusion of experimental results.

Determination of the safety of a live Dam ^- Salmonella vaccine in Holstein bulls. Dam ^- S. In 1-3 day old calves. Typhimurium safety is determined as follows. Eighteen 1-3 day old calves are divided into 3 groups of 6 each. Six animals in the first group were used with 10 ⁹ Dam ^- Salmonella, the second group was used with 10 ¹⁰ Dam ^- Salmonella, and the third group was used with 10 ¹¹ -Da.
Challenge orally with m ^- Salmonella. Three weeks after the challenge, each calf in the study is evaluated twice daily to measure pulse and respiratory rate, rectal temperature, appetite, and attitude. For Salmonella culture, a fecal sample is collected daily from each calf. Three weeks after the challenge, the calves were euthanized and the organs (liver, bile, spleen, mesenteric lymph nodes, ileal mucosa, small intestinal contents, cecal mucosa, and cecal contents) were cultured for Salmonella.

Determining whether a Salmonella Dam-based vaccine can colonize mucosal and / or systemic tissues. The kinetics of bovine tissue colonization was determined by oral administration of Dam ⁺ and Dam ^- S. typhimurium. "Bacterial load" in small intestinal contents, ileal mucosa, Peyer's patches, caecal mucosa, caecal contents, mesenteric lymph nodes, liver, and spleen is measured in calves as a function of time post-infection. 24 Holstein cows Yuko, of 10 ⁹ Dam ^- S. t
Challenge orally with yphimurium. Six animals are randomly assigned to 4 groups and euthanized at 24 hours and 5, 14, and 28 days after challenge. Tissue is collected from each calf at necropsy for quantitative Salmonella culture. Dam ⁺ S. of 10 ⁹ . Twenty-four Holstein bulls orally challenged with typhimurium were treated identically and serve as positive controls for these experiments.

For Dam ^- Salmonella to be an ideal bovine vaccine, Dam ^- Salmonella should colonize Peyer's patches, replicate and persist in M cells, and Peyer's patch lymph The antigen should be presented to endogenous immune cells, including follicles (eg, macrophages, B cells, and T cells). Importantly, Dam ^- Salmonella should not colonize deep tissues such as liver and spleen, and should eventually be removed from Peyer's patches. If these criteria are met, perhaps the Salmonella Dam ⁻ mutant will serve as a basis for a safe and effective bovine vaccine.

Dam ^- S. Against allogeneic wild-type challenge. Protective effect of typhimurium vaccination. Twenty calves, one to three days old, are randomly divided into two groups of ten. In the first group, one to three days old, Dam ^- S. typhim
Urine was vaccinated orally. The remaining 10 unvaccinated calves serve as controls. All calves were 5 weeks old,
Toxicity of 10 ¹¹ Challenge orally with typhimurium. For three weeks post-challenge, each calf is evaluated three times a day and pulse and respiration rates, rectal temperature, appetite scores, and attitude scores are recorded twice a day. For Salmonella culture, a fecal sample is collected daily from each calf. All calves that die after the challenge are necropsied and the organs (liver, bile, spleen, mesenteric lymph nodes, ileal mucosa, small intestinal contents, cecal mucosa, and cecal contents) are cultured for Salmonella did. Calves surviving the toxic Salmonella challenge were euthanized, necropsied 3 weeks after the challenge, and organs (liver, bile, spleen, mesenteric lymph nodes, ileal mucosa, small intestinal contents, cecal mucosa, And cecal contents)
Was cultured for Salmonella.

Minimum dosage regimen required for efficacy in calves and reduced vaccine persistence in bovine tissues. Three important characteristics of any vaccine regimen are i) the dose of the vaccine, ii) the age of the animal, and iii) the persistence of the vaccine in the immunized animal. The minimum dose required to induce complete protection (10,000 times the LD _50), and Peyer's patches, mesenteric lymph nodes, a reduction in persistence in tissues of mice, such as the liver and spleen Measure.

B. Dam Derivatives Induce Cross-Protection Against Related (Heterologous Salmonella Serotype) Pathogen Strains (Protective Effect of Dam ^- S. Typhimurium Vaccination Against Heterologous Wild-Type Challenge) Three Similar Virulent Salmonella Challenge experiments are performed using three different challenge organisms. Each experiment was performed at 1 to 3 days of age with Dam ^- S. ty
Oral immunization of calves with phimurium and challenge with toxic Salmonella at 5 weeks of age. In the first experiment, S.D. montevid
eo (serogroup C1) was used as the challenge organism and in a second experiment S. dublin (serogroup D), in the last experiment anatu
m (serogroup E1). A different calf is used for each experiment. For each of these three experiments, 20 1-3 day old calves are randomly divided into two groups of 10. This first group was treated with Dam ^- S. typhi
Vaccination orally using murium. The remaining 10 non-vaccinated calves serve as controls. All calves are challenged orally at 5 weeks of age with 10 ¹¹ virulent Salmonella.

Three weeks after the challenge, each calf was evaluated three times daily and pulse, respiratory rate,
Rectal temperature, appetite score, and attitude score are recorded twice daily. Fecal samples are collected from each calf daily for Salmonella culture. All calves that died after the challenge are necropsied and the organs (liver, bile, spleen, mesenteric lymph nodes, ileal mucosa, small intestinal contents, cecal mucosa and cecal contents) are cultured for Salmonella . Calves surviving the toxic Salmonella challenge were euthanized three weeks after challenge, necropsied and the organs (liver, bile, spleen, mesenteric lymph nodes, ileal mucosa, small intestinal contents, cecal mucosa and cecum) Is cultured for Salmonella. A comparison of the cross-protective immunity induced by the Dam-overproducing strain, alone and in combination with the Dam ^- mutant, is also made.

C. Killed Dam ⁻ Derivatives of Salmonella S. cerevisiae grown in vitro. Typhimurium Dam ^- Bacteria are killed by exposure to sodium azide (0.02%) and / or UV light, after which the antimicrobial is washed or dialyzed from the killed organism. The effect of the killed whole cell vaccine is tested by oral and parenteral administration. For parenteral vaccine group ¹⁰⁶ of Dam were killed ^- Salmonella, mixed with aluminum hydroxide and Quill (quill) A adjuvant and administered to calves via intramuscular injection. For the oral vaccination group, 10 ¹⁰ Dam ^- Salmonella killed are administered orally with vitamin D3 as a mucosal adjuvant. As a dosing regimen, newborn calves are immunized weekly for three weeks. Wild-type S. aureus killed with the same adjuvant and by the same route. typhimurium
Newborn calves served as negative controls. Immunized calves were immunized 2 weeks after the last immunization using the same protocol described above. Typhimurium is used to determine whether an effective immune response is generated. In this case, calves immunized with the killed vaccine preparations were also transferred to other pathogen Salmonella serotypes (eg, montevideo, S. dub).
lin, and S.L. anatum), and this induced immunity
Determine if cross-protective against relevant strains. This experiment is repeated using the Dam overproducing strain alone or in combination with the killed Dam ^- organism.

Since dam overproduction can result in ectopic expression of a new repertoire of potential protective antigens not expressed in either the wild-type (Dam ⁺ ) vaccine strain or the Dam ⁻ vaccine strain, vaccine killing experiments were performed using The overproducing strain is repeated using alone and in combination with the killed Dam ^- organism.

Example 6: Construction of a dam ^- mutant in Vibrio cholerae (A. Construction of a V. cholerae dam mutant) The cholerae dam mutant is not currently available. Known V.I.
Using the cholerae dam sequence, primers are designed to PCR amplify the dam gene. Using this as a probe, cholerae lambda clone bank and hybridized to wild-type cholera
Collect the e dam clone. Both ends of the DNA of the hybridizing clone were sequenced and It is determined whether or not a cholerae dam region is included. Subcloning and further sequencing these subclones from both ends of the vector is described in V.S. The smallest DNA restriction fragment that contains the entire cholerae dam sequence is identified. Irreversible dam deletion mutants associated with antibiotic resistance markers are constructed according to recently established methods (Julio, SM
. Et al., Molec. Gen. Genet. , 258: 178-181 (1998).
)).

V. The role of dam mutants in cholerae virulence has been described by mouse cholera (
Chichijuu mouse model) is tested in two different toxic assayed for LD ₅₀ and competitive indices. These are described in Example 1.

B. Determining the Protective Ability of Dam Mutants for the Purpose of Constructing a Live Attenuated Human Vaccine against V. cholerae As discussed in detail above, Salmonella Dam ⁻ mutants are associated with typhoid fever Serves as a live attenuated vaccine in a mouse model for The purpose of this experiment was to determine whether these desired effects were specific for Salmonella DNA adenine methylation, or whether the Dam ^- mutant also Awareness of providing protection against cholerae, and thus may provide a basis for the new generation of live attenuated vaccines.

[0235] Live attenuated human vaccines are designed to limit the risk of reversion to wild-type and to ensure that these strains do not serve as a reservoir for the spread of antibiotic resistance to emerging pathogens. Must do. Therefore, the next step in this analysis is to construct a suitable non-reverting antibiotic-sensitive derivative. Non-polar deletions in dam (no effect on downstream genes in the operon) were determined by standard PCR.
Construction is performed by removing the internal sequences of these genes by a base-based approach, ligation to a suicide vector, and recovery of the resulting in-frame deletion strain. Deletions of each gene were introduced individually using standard positive selection suicide vector strategies (Donnennberg, MS et al., Infect. Immun., 59: 4).
310-4317 (1991)) to obtain the desired non-reverting, attenuated, antibiotic-sensitive vaccine strain. The efficacy of this vaccine is retested as described above. Strains in which Dam has been constructed to be modified (ie, not completely deleted and / or neutralized) are tested, as well as Dam overproducing strains.

Example 7: Vibrio.cholerae and Yersinia ps
Essentiality of the dam Gene in Eutuberculosis Dam duplication was constructed by incorporating a recombinant plasmid containing the Dam mutant into the wild-type Dam locus. The resulting duplication included two copies of dam (mutant copy and wild type copy). Usually, recombinant plasmids segregate at a given frequency, and the recombinants (isolates) may be approximately equivalent to containing either mutant or wild-type genes. If the gene is essential,
All isolates of this duplication (recombined by this plasmid) are wild-type; recombinants with mutant genes die. If a recombinant plasmid containing this gene is present, the duplication may segregate to either mutant or wild type. Vi
brio. cholerae and Yersinia pseudotuber
For culosis, duplication of this dam gene, including both wild-type and mutant, cannot be segregated into mutants unless a recombinant plasmid providing the wild-type dam gene is present.

The foregoing description is considered only as illustrative of the principles of the present invention. It is not desired to limit the invention to the precise construction and process set forth above, as many further modifications and changes will readily occur to those skilled in the art. Accordingly, all suitable modifications and equivalents may be resorted to as falling within the scope of the invention as defined by the appended claims.

[Brief description of the drawings]

The accompanying drawings, which are incorporated herein and form a part of the specification, aid in illustrating the invention and, together with the description, serve to explain the principles of the invention. The accompanying drawings are not drawn to scale.

FIG. 1 is a graph of the levels of antibodies present according to primary and secondary immune responses.

FIG. 2 is a schematic diagram of the sites of methylation that occur in cytosine and adenine.

FIG. 3 is a graph illustrating that Dam regulates in vivo inducible genes. Dam ⁺ and Dam were grown in LB ^- S. in strains typhimurium
β-galactosidase expression from iv fusion. The vertical axis represents the β- galactosidase activity (.mu. moles of o- nitrophenol formed per minute A ₆₀₀ units per 1ml of the cell suspension × 10 ³ (ONP)).

FIG. 4 is a graph illustrating that Dam expresses a PhoP activated gene. S. cerevisiae grown in minimal medium β-galactosidase expression from typhimurium iv fusion. The vertical axis represents the β- galactosidase activity (.mu. moles of o- nitrophenol formed per minute A ₆₀₀ units per 1ml of the cell suspension × 10 ³ (ONP)). The dam genotype is shown below the horizontal axis, and the phoP genotype is shown as solid squares (PhoP ⁺ ) and gray squares (phoP ⁻ ).

FIG. 5 shows that PhoP affects the formation of Salmonella DNA methylation patterns. DNA methylation patterns formed in PhoP ⁺ and PhoP ⁻ strains growing in minimal medium. The arrow points to PhoP ^- Salmone
Illustrates a DNA fragment present in lla but not in PhoP ⁺ Salmonella.

FIG. 6 is a graph showing the amount and tissue distribution of Salmonella in mice immunized or unimmunized (open squares) with the Dam ⁻ mutant (closed squares) on days 1 and 5. . PP, Peyer's patches; MLN, mesenteric lymph nodes; CFU, colony forming units.

FIG. 7 shows Salmonell in mice immunized or not (open squares) with the Dam ⁻ mutant (closed squares) on days 1, 5, 14, and 28.
It is a graph which shows the amount and tissue distribution of a. PP, Peyer's patches; MLN, mesenteric lymph nodes; CFU, colony forming units.

FIG. 8 (A)-(C) shows the Dam ⁻ strain (dam nonpolar deletion, MT2188; (A
)); Dam ⁺ strains (wild-type, ATCC 14028 (B)); and S. cerevisiae showing proteins produced in Dam ⁺⁺⁺ strains (overproducing strain, MT2128). typhi
2 is a half-tone copy of a 2D gel electrophoresis of a whole cell protein extract of M. murium.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61K 48/00 A61K 48/00 A61P 31/04 A61P 31/04 37/00 37/00 G01N 33/02 G01N 33/02 33/15 33/15 Z 33/50 33/50 Z 33/53 33/53 M 33/566 33/566 (31) Priority claim number 09 / 495,614 (32) Priority date 2000 February 1 (2000.2.1) (33) Priority Claimed States United States (US) (81) Designated States EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR , IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, S Z, TZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CR, CU, CZ, DE, DK, DM, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP , KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Heisoff, Douglas M. United States California 93117, Goleta, Apartment 320, Camino del Sur 785 (72) Inventor Rho, David A. United States California 93117, Goleta, La Goleta Road 6089 (72) Inventor Shinsheimer, Robert El. United States California 93110, Santa Barbara, Via Beyent 4606 F-term (reference) 2G045 AA28 AA29 CB21 DA20 FB03 FB05 FB06 FB07 JA20 4C084 AA13 NA14 ZB072 ZB352 4C085 AA03 BA07 BA20 BA21 BA23 BA24 CC01 CC07 DD23 FF02 FF06

Claims (85)

[Claims] 1. An immunogenic composition comprising a live attenuated pathogenic bacterium in a pharmaceutically acceptable excipient, wherein the pathogenic bacterium is such that the pathogenic bacterium is attenuated. D
An immunogenic composition comprising a mutation that alters NA adenine methylase (Dam) activity. 2. The immunogenic composition of claim 1, wherein said mutation reduces Dam activity. 3. The immunogenic composition of claim 2, wherein said mutation eliminates Dam activity. 4. The immunogenic composition according to claim 3, wherein said mutation is a deletion of a dam gene. 5. The immunogenic composition of claim 1, wherein said mutation causes an increase in expression of Dam. 6. The immunogenic composition according to claim 1, wherein said pathogenic bacterium is Salmonella. 7. The method of claim 7, wherein the pathogenic bacterium is S. aureus. typhimurium, S.P. en
terididis, S .; typhi, S.M. abortusovi, S.A. abo
rtus-equi, S.R. dublin, S .; gallinarum, and S
. The immunogenic composition according to claim 6, wherein the immunogenic composition is selected from the group consisting of pulorum. 8. The immunogenic composition of claim 6, wherein said mutation eliminates Dam activity. 9. The immunogenic composition according to claim 8, wherein the mutation is a deletion of a dam gene. 10. The immunogenic composition of claim 6, wherein said mutation causes an increase in expression of Dam. 11. The method of claim 11, wherein said pathogenic bacterium is S. aureus. The immunogenic composition according to claim 7, which is typhimurium. 12. The immunogenic composition according to claim 11, wherein said mutation is a deletion of a dam gene. 13. The method of claim 13, wherein said pathogenic bacterium is S. aureus. The immunogenic composition according to claim 7, which is dublin. 14. The immunogenic composition according to claim 13, wherein said mutation is a deletion of a dam gene. 15. The method according to claim 15, wherein the pathogenic bacterium is S. aureus. The immunogenic composition according to claim 7, which is enteritidis. 16. The immunogenic composition according to claim 15, wherein said mutation is a deletion of a dam gene. 17. The immunogenic composition of claim 1, wherein said pathogenic bacterium is Escherichia. 18. The method according to claim 18, wherein the pathogenic bacterium is E. coli. 18. The immunogenic composition of claim 17, which is E. coli. 19. The immunogenic composition according to claim 1, wherein said pathogenic bacterium is Vibrio. 20. The method according to claim 19, wherein the bacterium is V. 20. The immunogenic composition of claim 19, which is cholerae. 21. The immunogenic composition of claim 1, wherein said bacterium is Yersinia. 22. The method according to claim 19, wherein the bacterium is Y. 22. The immunogenic composition of claim 21, which is a pseudotubercolosis. 23. The bacterium is of Shigella, Haemophilus,
Bordetella, Neisseria, Pasteurella and T
2. The immunogenic composition of claim 1, wherein the composition is selected from the group consisting of reponema. 24. The immunogenic composition according to claim 1, further comprising an adjuvant. 25. The immunogenic composition of claim 1, further comprising a heterologous antigen. 26. The immunogenic composition of claim 1, wherein said pathogenic bacterium further comprises an expression cassette comprising a polynucleotide sequence encoding a heterologous antigen. 27. The immunogenic composition of claim 1, wherein said mutation is non-revertive. 28. The immunogenic composition of claim 1, wherein said bacterium comprises a second mutation that causes attenuation of said bacterium. 29. A kit comprising the immunogenic composition of claim 1. 30. An immunogenic composition comprising a killed pathogenic bacterium in a pharmaceutically acceptable excipient, wherein the pathogenic bacterium comprises a DNA adenine methylase (Dam).
A.) An immunogenic composition comprising a mutation that alters activity. 31. The immunogenic composition of claim 30, wherein said mutation is non-lethal and attenuates said pathogenic bacteria. 32. The immunogenic composition of claim 30, wherein said mutation is lethal. 33. The pathogenic bacterium is Salmonella.
The immunogenic composition according to 0. 34. The immunogenic composition of claim 33, wherein said mutation deletes a dam gene. 35. The immunogenic composition of claim 33, wherein said mutation causes Dam overexpression. 36. An attenuated strain of a pathogenic bacterium, wherein the bacterium comprises a mutation that alters Dam activity such that the bacterium is attenuated. 37. The attenuated strain of claim 36, wherein said mutation reduces Dam activity. 38. The attenuated strain of claim 37, wherein said mutation eliminates Dam activity. 39. The attenuated strain of claim 38, wherein said mutation is a deletion of the dam gene. 40. The attenuated strain of claim 36, wherein said mutation causes an increase in expression of Dam. 41. The attenuated strain of claim 36, wherein said bacterium is Salmonella. 42. The attenuated strain of claim 39, wherein said bacterium is Salmonella. 43. The attenuated strain of claim 40, wherein said bacterium is Salmonella. 44. A method of eliciting an immune response in an individual comprising administering to the individual an immunogenic composition of claim 1 in an amount sufficient to elicit an immune response. Method. 45. A method of eliciting an immune response in an individual, comprising administering to the individual an immunogenic composition of claim 12 in an amount sufficient to elicit an immune response. Method. 46. A method of eliciting an immune response in an individual, comprising administering to the individual an immunogenic composition of claim 14 in an amount sufficient to elicit an immune response. Method. 47. A method of eliciting an immune response in an individual, comprising the step of administering to the individual an immunogenic composition of claim 16 in an amount sufficient to elicit an immune response. Method. 48. The method of claim 45, wherein said immune response persists for more than about 4 weeks after administration. 49. The method of claim 46, wherein said immune response persists for more than about 4 weeks after administration. 50. The method of claim 47, wherein said immune response persists for more than about 4 weeks after administration. 51. The method of claim 44, wherein said individual is a human. 52. The method of claim 44, wherein said individual is a livestock animal. 53. The method of claim 52, wherein said animal is a chicken. 54. The method of claim 52, wherein said animal is a cow. 55. A method of preventing an infection by a pathogenic bacterium in an individual, wherein the method is in an amount sufficient to reduce the symptoms associated with infection by the pathogenic bacterium upon infection by the pathogenic bacterium. A method comprising the step of administering the immunogenic composition according to 1 to the individual. 56. A method of treating a pathogenic bacterial infection in an individual, the immunogenicity of claim 1 in an amount sufficient to reduce symptoms associated with infection by the pathogenic bacterial in the individual. Administering a composition to the individual.
Method. 57. A method of preventing Salmonella infection in an individual, the method comprising the steps of:
7. A method comprising administering to the individual an immunogenic composition of claim 6 in an amount sufficient to reduce the symptoms associated with an Ila infection. 58. A method of treating a Salmonella infection in an individual, wherein the immunogenic composition of claim 6 is administered to the individual in an amount sufficient to reduce the symptoms associated with the Salmonella infection in the individual. A method comprising the step of administering. 59. A method of treating an individual infected with a pathogenic bacterium, comprising:
administering to the individual a composition comprising an agent that modifies m activity. 60. A method of eliciting an immune response to a second species of Salmonella in an individual, the method comprising administering to the individual an immunogenic composition comprising an attenuated first species of Salmonella. The first species is Salm
comprising a mutation that alters Dam activity such that the first species of onella is attenuated. 61. The first Salomonella sp. typhi
61. The method of claim 60, which is a murium. 62. The first Salmonella sp. enteri
61. The method of claim 60, which is tis. 63. A method for identifying a drug that may have antibacterial activity, comprising using an agent that alters the level of transcription from the dam gene when the drug is added to the in vitro transcription system. And detecting, wherein the agent is identified by its ability to alter the level of transcription from the dam gene as compared to the level of transcription without the agent added. 64. The method of claim 63, wherein said dam gene is from Salmonella. 65. A method for identifying an agent that may have antibacterial activity, comprising the step of altering the level of translation from an RNA transcript encoding Dam when the agent is added to an in vitro transcription system. Detecting using a translation system, wherein the 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 without the agent added. The method comprising the steps of: 66. The Dam according to claim 6, wherein the Dam is derived from Salmonella.
5. The method according to 5. 67. A method for identifying an agent that may have antibacterial activity, comprising determining whether the agent binds Dam, wherein the agent is identified by its ability to bind Dam. The method comprising the steps of: 68. The Dam according to claim 6, wherein the Dam is from Salmonella.
7. The method according to 7. 69. A method for identifying a drug that may have antibacterial activity, comprising the steps of: (a) combining a non-methylated oligonucleotide containing a Dam binding site with Dam, S-adenosylmethionine and a drug. Incubating, wherein the unmethylated oligonucleotide further comprises a signal; (b) digesting all unmethylated target sites, thereby releasing the unmethylated oligonucleotide; c) detecting inhibition of DNA adenine methylase as an increase in the signal due to digestion of the unmethylated target site, wherein the agent is in the absence of the agent. b) and (c)
Identified by its ability to cause an increase in signal as compared to performing the method. 70. The method of claim 69, wherein said oligonucleotide is anchored to a solid surface. 71. The method of claim 70, wherein said solid surface is a microtiter plate comprising avidin and said oligonucleotide comprises biotin. 72. The method of claim 69, wherein said Dam binding site is a GATC sequence. 73. The Dam according to claim 6, wherein the Dam is from Salmonella.
10. The method according to 9. 74. The method of claim 69, wherein said agent is selected from an inhibitor library consisting of a group selected from polypeptides, organic compounds and inorganic compounds. 75. A method for identifying an agent that may have antibacterial activity, comprising the steps of: (a) contacting the agent to be tested with a suitable host cell having Dam function; (B) analyzing at least one characteristic associated with an alteration in Dam function, wherein the agent is identified by its ability to elicit at least one said characteristic;
A method comprising: 76. The method of claim 75, wherein said host cell is a bacterium. 77. The method of claim 76, wherein said bacterium is Salmonella. 78. A method of preparing an immunogenic composition according to claim 1, wherein the pharmaceutical excipient and the DNA adenine methylase (Dam) activity are altered such that the pathogenic bacteria are attenuated. Combining with the pathogenic bacterium containing the mutation. 79. A method for preparing an attenuated bacterium capable of eliciting an immunological response by a host susceptible to a disease caused by a corresponding or similar pathogenic microorganism, wherein said attenuated bacterium comprises at least A method comprising the step of constructing one mutation, wherein the first mutation results in an alteration of Dam function. 80. The method of claim 79, wherein the first mutation is introduced into a dam gene. 81. The method of claim 80, wherein a second mutation is made in a gene independent of said first mutation, said second mutation causing attenuation of said pathogenic bacterium.
The method described in. 82. The method of claim 80, wherein said first mutation alters expression of Dam. 83. The method of claim 82, wherein said first mutation eliminates expression of Dam. 84. The method of claim 80, further comprising inserting an expression cassette comprising one or more structural genes encoding a desired antigen into said attenuated bacterium. 85. The method of claim 85, wherein the desired antigen is tetanus toxin fragment C, cholera toxin B subunit, hepatitis B surface antigen,
Vibrio cholerae LPS, HIV antigen and Shigella
method selected from the group consisting of Soneii LPS.