WO2015118541A1 - Système génétique pour générer des bactéries inactivées ou atténuées de manière conditionnelle - Google Patents

Système génétique pour générer des bactéries inactivées ou atténuées de manière conditionnelle Download PDF

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WO2015118541A1
WO2015118541A1 PCT/IL2015/050137 IL2015050137W WO2015118541A1 WO 2015118541 A1 WO2015118541 A1 WO 2015118541A1 IL 2015050137 W IL2015050137 W IL 2015050137W WO 2015118541 A1 WO2015118541 A1 WO 2015118541A1
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promoter
bacterium
spp
gene
dna molecule
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Chen Katz
Jacob Pitcovski
Elina Aizenshtein
Caroline NOACH
Avner FINGER
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Gavish-Galilee Bio Applications Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0258—Escherichia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0275—Salmonella
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/36—Adaptation or attenuation of cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5252—Virus inactivated (killed)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5254—Virus avirulent or attenuated
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
    • A61K2039/552—Veterinary vaccine
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates in general to vaccines comprising conditionally inactivated or attenuated bacteria, genetic systems utilized for producing the bacteria and uses thereof.
  • Vaccines are designed to stimulate induction of active immunity and are made using several different processes. These vaccines may contain a live pathogen that has been attenuated; inactivated or killed; inactivated toxins (for bacterial diseases where toxins generated by the bacteria, and not the bacteria themselves, cause illness); or merely segments of the pathogen (this includes both subunit and conjugate vaccines).
  • Live attenuated vaccines have been used extensively and very successfully against a number of animal and human diseases, over an extensive span of years, whereas killed vaccines have often proven to be poorly efficacious.
  • Live attenuated vaccines contain weakened or less virulent forms of the bacterial or viral strains that causes the disease. The concept behind such vaccines is that the pathogens are efficacious enough in stimulating immunity, but too weak to cause diseases. These vaccines activate immune responses that closely mimic a natural infection. Attenuated vaccines activate both innate and acquired immune systems. While stimulating antibody and cellular responses, live attenuated vaccines induce both local and systemic immune responses, activate cellular immunity and long term-memory B and T-cells.
  • live attenuated vaccines in comparison with killed or subunit vaccines: (a) they mimic natural infection, therefore eliciting immune responses that are highly specific, effective, and long-lasting; (b) they can prevent infection by the pathogen, not just disease symptoms; (c) in comparison with highly purified subunit vaccines, they are relatively inexpensive to produce and administer, and do not require sophisticated downstream processing or formulation with adjuvants; (d) several live attenuated vaccines can be administered orally, which has a higher acceptance and better safety profile than injection with syringe and needle, and mimics natural infection better; (e) they stimulate local immune response (e.g. IgA) that confers protection at the site of pathogen entrance; (f) Induce T cytotoxic response more efficiently; and (g) they may provide competitive exclusion.
  • IgA local immune response
  • IgA Induce T cytotoxic response more efficiently
  • Attenuated vaccines are more difficult to generate for bacteria than for viruses. Bacteria have thousands of genes and thus are much harder to control. Indeed, tens of attenuated vaccines for viruses are registered for human use, whereas for bacteria there are only three worldwide. Attenuated bacterial vaccines can be made in several ways. Usually these kinds of vaccines are obtained from homologous closely related strains which are naturally attenuated, or from virulent agents that are attenuated in a stable way following different techniques. The most frequently used attenuation techniques consist of: 1. Passaging the bacteria in specific media until they loses their virulence; 2. passaging the wild-type bacteria through phagocytic cells; 3. using spontaneous chromosomal antibiotic -resistant mutants; and 4. using mutations in genes coding for the biosynthesis of aromatic amino acids or for essential enzymes.
  • the process of attenuation of bacteria may cause modification of epitopes which may be important for invoking an immune response against the bacteria, may in some cases cause the attenuated bacteria to be less immunogenic and less vaccinogenic (i.e. less able to induce a protective or neutralizing immune response), and require extra-chromosomal expression of the immunogenic epitopes.
  • live bacterial vaccines must meet a delicate balance: the strain must reach an appropriate level of attenuation to be safe and be sufficiently vaccinogenic to ensure protective efficacy.
  • Genes that kill bacteria are highly prevalent in nature. These genes encode for a toxic element - either RNA or protein - that is expressed under specific conditions leading to growth arrest or complete cell lysis. Some toxins that inactivate bacteria are derived from extracellular origin, such as bacteriophages, fungi and other bacteria, while others are encoded by intracellular genetic elements such as plasmids, transposons and the bacterial chromosome itself.
  • Bacteriocins are protein-based toxins expressed and secreted by one strain of bacteria, and kill related bacteria that are found in their immediate vicinity. The killing mechanisms are diverse, ranging from membrane depolarization to degradation of macromolecules such as DNA, mRNA and tRNA.
  • an additional immunity protein is expressed, forming high affinity complex with the toxin, leading to its inactivation within the bacteria. These complexes are secreted from the host cells, and disassemble when entering the target cells, resulting in the release of the toxin and subsequent cell death (Cascales et ah, 2007).
  • toxins that are of extracellular origin
  • genetic systems for example, systems known as addiction systems, that are usually designed to kill the bacterial cell that carries them in case a specific gene, which is part of the system, is lost or damaged.
  • the systems are composed of a toxic gene, encoding a toxic protein or in some cases toxic RNA, and an additional antitoxin gene, encoding an RNA or a protein, that inhibits the activity of the toxin.
  • the stability of the toxin is much higher than the stability of the antitoxin, and therefore, when the genetic system is lost, expression of both genes is inhibited, the antitoxin gene product is quickly degraded, releasing the toxin, and resulting in cell death (Yamaguchi et al, 2011).
  • All natural genetic systems - those described above and others - can potentially be manipulated in order to induce bacterial cell death under desired conditions.
  • the gene encoding the toxic component of the genetic system can be fused to an inducible promoter leading to its expression under specific conditions that will lead to cell death. While some toxins are designed to kill the bacteria in which they are expressed, others, such as bacteriocins, that are naturally secreted, can be manipulated in a way that inhibits their secretion leading to their activity inside the host cell.
  • the present invention provides a DNA molecule comprising a nucleic acid sequence including at least one pair of a first and a second differentially controlled transcription units, wherein the first transcription unit includes a first promoter operably linked to a first gene encoding a cytotoxic gene product, the second transcription unit includes a second promoter that is different from the first promoter and is operably linked to a second gene encoding a gene product neutralizing said cytotoxic gene product, at least one of the first and said second promoter is a regulated promoter responsive to a predetermined condition, and the DNA molecule is designed such that under the predetermined condition the amount of the neutralizing gene product is not sufficient to neutralize the cytotoxic effect of the cytotoxic gene product.
  • the present invention provides a synthetic vector comprising the DNA molecule described herein.
  • the present invention is directed to a method for producing a conditionally inactivated or attenuated bacterium, said method comprising introducing to a vaccinogenic and pathogenic live or attenuated bacterium the DNA molecule or the synthetic vector described hereinabove, whereby activation or de-repression of said regulated promoter by said predetermined condition causes bacterial cell death, thereby producing a conditionally inactivated or attenuated bacterium that is inactivated or attenuated in the presence of the predetermined condition.
  • the present invention provides a conditionally inactivated or attenuated bacterium comprising the DNA molecule or the vector carrying the DNA molecule of the invention.
  • conditionally inactivated or attenuated bacterium of the present invention is for use in immunization of an animal, such as poultry or a mammal such as human.
  • the present invention provides a vaccine comprising the conditionally inactivated or attenuated bacterium of the invention.
  • the present invention provides a method of vaccination comprising administering to a subject in need thereof a conditionally inactivated or attenuated vaccinogenic bacterium comprising the DNA molecule described hereinabove, whereby the cytotoxic gene product kills or attenuates said vaccinogenic bacterium after a specific immune response against said conditionally inactivated or attenuated vaccinogenic bacterium has been elicited, thereby vaccinating said subject.
  • the present invention provides a method for prevention of transfer of a pathogenic bacterium from a first animal to a second animal, comprising vaccinating the first animal against the pathogenic bacterium.
  • the vaccination comprises administering to the first animal a conditionally inactivated or attenuated vaccinogenic bacterium comprising the DNA molecule or the synthetic vector described herein, wherein said conditionally inactivated or attenuated vaccinogenic bacterium is of the same or similar species as said pathogenic bacterium, and the cytotoxic gene product kills or attenuates said vaccinogenic bacterium after having induced a specific immune response against said conditionally inactivated or attenuated vaccinogenic bacterium, thereby vaccinating said animal and preventing transfer of said bacterium from said first to said second animal.
  • Fig. 1 shows growth rates of S. enteritidis strains carrying suicidal genetic systems under permissive conditions. Wild type Salmonella enteritidis B3 (filled circles ⁇ ), and S. Enteritidis B3 strains carrying the genetic systems pBAD/Hok (open triangles ⁇ ), pBAD/CeaB (filled triangles A), FNR/CeaB (open squares ⁇ ), PhoP/Hok (crosses X ), FUR-Hok (no symbol), and combined FNR/CeaB -PhoP/Hok (cross filled squares El ) were grown at permissive conditions as described in "Materials and Methods" and optical density was determined every 30 min. The data represent the mean O.D values from two independent experiments each performed in triplicate.
  • FIGs. 2A-2B show survival of E. coli and Salmonella strains carrying suicidal genetic systems under restrictive conditions.
  • E. coli (A) and Salmonella (B) strains carrying the genetic systems (left to right each panel starting from second bar): pBAD/Hok, pBAD/CeaB, PhoP/Hok, FNR/CeaB, the combined system FNR/CeaB -PhoP/Hok (two bars), and FUR/Hok, were grown under restrictive conditions as described in "Materials and Methods" for 24 h.
  • Strains carrying the empty vector pBR322 served as negative control (first bar on the left).
  • the data represent mean log percentage of survival in three independent experiments, each performed in triplicate. Error bars denote the standard deviation of the mean.
  • Figs 3A-3B show long-term persistence of Salmonella under restrictive conditions.
  • Salmonella B3 strains carrying genetic systems of the invention (filled squares) or an empty vector (filled circles) were incubated under restrictive conditions as described in "Materials and Methods" for 10 days. Samples were collected at indicted days, and live bacteria were analyzed by plating and CFU counting.
  • A FNR/CeaB genetic system
  • B PhoP/Hok genetic system. The data represent mean CFU values from two independent experiments, each performed in triplicate.
  • FIG. 4 shows the effect of the genetic systems of the invention on salmonella survival inside macrophages. Wild type Salmonella Enteritidis B3 (filled circles ⁇ ) and S. Enteritidis B3 strains carrying the genetic systems pBAD/Hok (open triangles ⁇ ), pBAD/CeaB (filled triangles A), FNR/CeaB (open squares ⁇ ), PhoP/Hok (crosses X) and combined FNR/CeaB -PhoP/Hok (cross filled squares El ) were tested for intracellular survival inside macrophages cells.
  • CFU colony forming units
  • the present invention is related to a genetic system which can be introduced into live bacteria in order to produce a vaccine bacterial strain that can be safely administered to an animal for the purpose of vaccination against the same bacteria.
  • the genetic system is designed in such a way that death or reduced virulence of the bacteria carrying the genetic system is triggered under predetermined conditions which occur in the host, so that the bacterial pathogen is pacified after inducing a specific immune response but before inflicting disease in the host or exiting the host.
  • the genetic system includes at least one gene encoding a cytotoxic gene product that causes cell death or reduced virulence, and at least a promoter which is responsive to the predetermined conditions, meaning that it is either induced or repressed by the predetermined condition thereby causing bacterial cell death or reduced pathogenicity.
  • promoter is used herein in the normal sense of the art, e.g. including an RNA polymerase binding site.
  • a promoter may be designed as a regulated or a constitutive promoter depending on whether it induces/represses transcription of the gene it is controlling in response to a certain signal or whether it facilitates a constant level of transcription, respectively.
  • promoter is intended as possibly also including recognitions sites or elements for binding regulators, as explained below with reference to regulators and regulated promoters.
  • the gene encoding the cytotoxic gene product may be operably linked to a promoter which is induced by the predetermined condition, such that the expression of the cytotoxic gene is induced under the predetermined conditions, thereby causing bacterial cell death or reduced pathogenicity; or the cytotoxic gene may be operably linked to a promoter that is repressed until the predetermined condition occurs. In the latter case, under the predetermined condition (i.e. lack of the repressor), the expression of the cytotoxic gene is no longer repressed and it is expressed, thereby causing bacterial cell death or reduced pathogenicity.
  • the predetermined condition i.e. lack of the repressor
  • a genetic system that is composed of at least two genes, encoding a toxin and an antitoxin (a neutralizing gene product), that are differentially controlled such that the stoichiometry of the toxin-antitoxin is changed in favor of the toxin under desirable predetermined conditions.
  • an antitoxin a neutralizing gene product
  • a highly potent toxin can be used, and its leakage under conditions which are not the predetermined conditions is controlled by the presence of the antitoxin or equivalent neutralizing entity. Under the predetermined conditions, the relative toxin/antitoxin levels will increase, leading to highly efficient cell death.
  • the genetic system may further include a second gene encoding a second gene product that neutralizes the cytotoxic activity of the cytotoxic gene product.
  • the first gene is operably linked to a first promoter
  • the second gene is operably linked to a second promoter, and at least one of the first or the second promoter is responsive to the predetermined condition.
  • cytotoxic gene product refers to a gene product having bactericidal or bacteriostatic activity.
  • a cytotoxic gene product is also referred to herein as a toxin.
  • the cytotoxic gene product remains in the cytoplasm of the bacteria that produces it until the bacteria is killed.
  • the gene encoding the cytotoxic gene product is lacking elements which are needed for secretion of the cytotoxic gene product outside of the bacteria and therefore the cytotoxic gene product is not secreted from the bacterial cell.
  • the cytotoxic gene product causes bacterial cell death or reduced pathogenicity only of the bacteria carrying the genetic system.
  • the first gene and the second gene are encoded by separate transcription units.
  • transcription unit means a nucleotide sequence that codes for at least one RNA molecule, along with the sequences necessary for their transcription (regulation), normally a promoter, an RNA-coding sequence, and a terminator. It is understood, as explained above, that the term promoter encompasses also promoter regions including recognition sequences for regulators.
  • the present invention therefore provides a DNA molecule comprising a nucleic acid sequence including at least one pair of a first and a second differentially controlled transcription units, wherein the first transcription unit includes a first promoter operably linked to a first gene encoding a cytotoxic gene product, the second transcription unit includes a second promoter that is different from the first promoter and is operably linked to a second gene encoding a gene product neutralizing said cytotoxic gene product, at least one of the first and the second promoter is a regulated promoter responsive to a predetermined condition, and the DNA molecule is designed such that under the predetermined condition the amount of the neutralizing gene product is not sufficient to neutralize the cytotoxic effect of the cytotoxic gene product.
  • the relative expression levels of the cytotoxic gene and the neutralizing gene change such that the expression level of the cytotoxic gene is increased relative to the expression level of the neutralizing gene, and as a result the amount of the neutralizing gene product is no longer sufficient to neutralize the cytotoxic effect of the cytotoxic gene product, whereby the cytotoxic gene product kills or attenuates said vaccinogenic bacterium, thereby eliminating the bacteria during or after vaccination of said subject.
  • the term "differentially controlled" as used herein with reference to a pair of transcription units means that the regulation of the two transcription units is not identical and that at least one regulator exists that affects one transcription unit in a different way than it affects the other transcription unit.
  • a regulator exists that can cause induction of a promoter of one transcription unit of the pair and repression of a promoter of the other transcription unit of the pair, or alternatively cause induction of a promoter of one transcription unit of the pair and not affect the promoter of the other transcription unit of the pair, or alternatively cause repression of a promoter of one transcription unit of the pair and not affect the promoter of the other transcription unit of the pair.
  • a regulator exists that can, for example, cause increased expression from one of the transcription units and no increase in expression, or reduced expression from the other transcription unit.
  • the expression from both transcription units can be coordinated by virtue of their promoters binding to a common regulator (for example, a dual regulator, as defined hereinbelow) or they could be completely unrelated, for example, by one promoter being regulated by one regulator and the other by another, or the other promoter driving constitutive expression.
  • the term "regulator” as used herein refers to a compound, for example a protein or a nucleic acid molecule that is capable of binding to a promoter region and either up-regulating or down-regulating the transcription of the gene regulated by the promoter region.
  • the predetermined condition causes the regulator to be induced or repressed at the transcription level, or alternatively to be activated or inactivated for example by a conformational change or a modification, such as phosphorylation or glycosylation, or otherwise change in concentration levels as a consequence of the predetermined condition, thereby causing a change in the expression level of the gene which is regulated by the aforesaid promoter region.
  • regulated promoter as used herein relates to a promoter which includes at least one element that when bound by a regulator as explained above, causes transcription to be induced or repressed. Therefore, according to the present invention, the regulated promoter is induced or repressed under the predetermined conditions, e.g., the promoter is induced or repressed by the presence of a certain level of a regulator molecule, which level is affected by the predetermined conditions, as explained in more detail below.
  • both of the first and the second promoter are regulated promoters, meaning that both the first and the second promoter are responsive to a predetermined condition.
  • the two promoters may be combined in various ways.
  • the first promoter may be induced by the predetermined condition and the second promoter repressed by the predetermined condition.
  • the first promoter may be repressed until occurrence of the predetermined condition and the second promoter activated in the absence of the predetermined condition.
  • one of the promoters is constitutive and the other is regulated. In any case, the result is that as long as the predetermined condition has not occurred, the second gene is expressed in excess relative to the first gene and therefore the neutralizing gene product is in excess of the cytotoxic gene product and cell death or reduction in viability does not occur.
  • one of the first and the second regulated promoter is activated by the predetermined condition and the other of the first and the second promoter is repressed by the predetermined condition.
  • the first of the first and the second regulated promoter is activated by the predetermined condition thereby causing elevated expression of the cytotoxic gene product and the second of the first and the second promoter is repressed by the predetermined condition thereby causing reduced expression of the second gene product.
  • Dual regulators are molecules which can up-regulate expression from one promoter and down-regulate expression from another promoter. Such dual regulators can be used in the invention in order to increase expression of the cytotoxic gene product and reduce expression of the neutralizing gene product, under the same conditions, or alternatively to reduce expression of the cytotoxic gene product and increase expression of the neutralizing gene product, under the same conditions.
  • Examples for dual regulators are the E. coli regulators FNR (activated under anaerobic conditions), PhoP (activated in response to low levels of divalent cations), and FUR (repressed by the presence of Fe ++ ions).
  • the first and the second promoter are regulated by a single dual regulator.
  • the first promoter is induced by the dual regulator and the second promoter is repressed by the dual regulator.
  • the dual regulator is the Fumarate and Nitrate reduction regulator known in E. coli as FNR, the low divalent ion regulator known in E. coli as PhoP (Groisman, 2001), or the iron-dependent dual-regulator repressor ferric uptake regulator (FUR).
  • the dual regulator is an orthologous dual regulator of FNR, PhoP, or FUR having an amino acid sequence identity of 50% or more with the respective E. coli dual regulator.
  • one of the first and the second promoter is a constitutive promoter and the other of the first and the second promoter is a regulated promoter, meaning that one promoter is constantly active and the other promoter is responsive to a predetermined condition.
  • the first of the first and the second promoter is a constitutive promoter and the second of the first and the second promoter is a regulated promoter.
  • promoters can be selected according to the desired conditions.
  • the E. coli PhoP is a regulator which is activated in response to low extracellular levels of certain divalent cations, such as Magnesium, Calcium and Manganese (Groisman, 2001) and PhoP can either activate or repress PhoP-regulated promoters;
  • E. coli fumarate and nitrate reductase regulator (FNR) is a regulator inactivated by oxygen (active under anaerobic conditions) and FNR can either activate or repress FNR-regulated promoters, FUR is responsive to Fe ++ ions and was shown to repress FUR-regulated promoters in E.
  • FUR acts as a dual regulator and can regulate a different set of genes as an apo-enzyme (without Fe++) or as a holo-enzyme (when bound to Fe++) (Butcher et al.
  • pBAD is induced by the presence of arabinose-binding protein (AraC) (Lee, 1980);
  • pLAC is repressed by Lacl and can be induced by lactose or isopropyl thiogalactopyranoside (IPTG) (Oehler et al., 1990);
  • TrpP is repressed by TrpR (in the presence of tryptophan), Bass and Yansura, 2000); and TetP is repressed by TetR and is induced by the antibiotics tetracycline (Gossen and Bujard, 1992).
  • constitutive promoters can also be used in this system, for example, a constitutive promoter that is based on the prokaryotic housekeeping promoter sequence having a -35 sequence based on the TTGACA consensus and a -10 sequence based on the TATAAT consensus that are separated by 17 nucleotides.
  • One or more of the consensus nucleotides can be changed in order to affect the transcription efficacy of the promoter. Small adjustments to the promoter sequences are performed in order to enhance or reduce the expression levels of both toxin and antitoxin encoding genes, in order to adjust the kinetics of the bacterial death.
  • Increasing the strength of the toxin promoter or reducing the strength of the antitoxin promoter is expected to increase the kinetics of bacterial death in response to the predetermined restrictive conditions, and vice versa.
  • the adjustments are made by introducing one or more alterations in the nucleotides sequences of the genetic system in a way that either increases or decreases the similarity of the promoters to the known consensus sequence of the prokaryotic promoter.
  • promoters CP2, CP12, CP20, CP22 and CP25 that are based on the consensus prokaryotic sequence ⁇ TTGACA 17bp TATAAT...5-
  • 6bp...A ⁇ were shown to have a range of transcription strengths from very weak to very strong.
  • bacterial ribosome binding site known as the Shine-Dalgarno sequence, having a consensus sequence of - AGGAGG - is located a few nucleotides upstream of the translation start codon ATG, and can be modified for increased or decreased translation by increasing or decreasing the similarities to the consensus sequence. This adjustment is relevant only for toxin and/or antitoxins that are translated proteins.
  • the promoter is based on the promoters or dual regulator promoters listed above and modified as explained in the preceding paragraphs.
  • At least one of the first and the second promoters contains a recognition sequence for a transcription regulator selected from the E. coli regulators PhoP, FNR, FUR, AraC, Lacl, TrpR or TetR, or from orthologs thereof.
  • the promoters containing a recognition sequence can be, for example, pBAD, which contains a recognition sequence for arabinose-binding protein (AraC); pLac, which contains a recognition sequence for Lacl; TrpP, which contains a recognition sequence for TrpR; or TetP, which contains a recognition sequence for TetR.
  • pBAD which contains a recognition sequence for arabinose-binding protein
  • pLac which contains a recognition sequence for Lacl
  • TrpP which contains a recognition sequence for TrpR
  • TetP which contains a recognition sequence for TetR.
  • ortholog thereof relates to a gene product that is an ortholog of a gene product mentioned (e.g. the E. coli dual regulator FNR, FUR,or PhoP), which has essentially the same activity and 50% or higher amino acid sequence identity with the gene product mentioned.
  • a gene product mentioned e.g. the E. coli dual regulator FNR, FUR,or PhoP
  • the cytotoxic gene product and the neutralizing gene product can each be an RNA molecule or a protein.
  • neutralizing gene product means a gene product which neutralizes or counteracts the effect of the cytotoxic gene product.
  • the neutralizing effect can be obtained by various means, for example, by interfering with transcription, degrading transcripts, interfering with translation and degrading or inhibiting the activity of proteins.
  • a neutralizing gene product can be, for example, an immunity protein or as an antitoxin.
  • RNA antitoxin antitoxin system Several bacterial systems are known to encode for a toxin antitoxin system including a cytotoxic gene and a neutralizing gene. Toxin- antitoxin systems are typically classified according to how the antitoxin neutralizes the toxin.
  • Toxin- antitoxin systems are typically classified according to how the antitoxin neutralizes the toxin.
  • mRNA messenger RNA
  • the protein toxin in a type II system is inhibited post-translationally by the binding of a protein antitoxin.
  • a single example of a type III toxin- antitoxin system has been described whereby a protein toxin is bound directly by an RNA molecule.
  • the first gene and the second gene encode a toxin- antitoxin system.
  • the toxin- antitoxin gene system is of type I, and the neutralizing comprises inhibiting translation of the first gene product by the second gene product.
  • An example of a type I toxin antitoxin system is the hok/sok system.
  • the hok/sok toxin antitoxin system is naturally found on E. coli plasmid Rl and prevents the loss of the plasmid during replication.
  • Hok (a non-limiting example of a cytotoxic gene product of the present invention) is a 52 amino acids toxic protein that pierces the cell membrane from within, leading to membrane depolarization and subsequent inhibition of ATP production.
  • Sok (a non-limiting example of a neutralizing gene product of the present invention) is an RNA molecule that is naturally transcribed from the complementary strand of the hok gene, and binds the hok mRNA molecule preventing its translation.
  • the sok RNA serves as an antitoxin in this genetic system.
  • both native hok and sok have constitutive promoters, they are both simultaneously expressed thereby the activity of hok is naturally suppressed.
  • the hok toxin mRNA is stable and remains following cell division, while the sok antitoxin degrades quickly and needs to be transcribed from the plasmid in order for the daughter cell to survive. Consequently, in nature, the toxin kills bacteria which have lost the Rl plasmid while bacteria carrying the Rl plasmid are protected by the antitoxin.
  • the hok/sok system can be genetically separated into two distinct transcription units which can be individually controlled.
  • the stoichiometry of the toxin/antitoxin can be kept in favor of the antitoxin under one set of conditions (resulting in normal bacterial growth) and changed in favor of the toxin under a different set of conditions (leading to bacterial death) by fusing each of the hok and sok genes to a different desirable promoter.
  • the toxin and antitoxin from such systems can be fused to desired promoters and used in the present invention to cause cell death under predetermined conditions.
  • the toxin-antitoxin gene system is the hok/sok gene system.
  • Various promoters as described above can be used with the hok/sok gene system, including a combination of a regulated promoter and a constitutive promoter, two regulated promoter, or a dual regulator system.
  • Another non-limiting example for a system that can be used with the present invention is the endonuclease E colicins ( ⁇ - ⁇ - ⁇ / ⁇ -Me enzymes by another name), which belongs to the bacteriocin family of toxic proteins that are secreted by different naturally occurring E. coli strains and kill other related E. coli strains in the proximal environment.
  • the colicin E proteins have a C-terminal DNAse domain with highly active cytotoxic activity, and an N-terminal domain that directs the protein to secretion and translocates it into other cells with compatible receptors.
  • a small immunity protein is produced by the host, and this antitoxin binds to the DNAse domain of the colicin with ultra-high affinity, forming a DNAse-Immunity protein complex, which neutralizes the DNAse toxin.
  • the expression of the colicin is coupled to the expression of the immunity protein, in order to prevent damage to the host cell. Co-expression of both genes is a fundamental character of these natural genetic systems.
  • the DNAse-Immunity protein complex breaks down during the translocation, releasing the actie DNAase into the target cell resulting in its death.
  • ColE2 (alternative name: ceaB), ColE7 (ceaE7), ColE8 (ceaE8) and ColE9 (CeaE9)
  • Colicin-E2 immunity protein also known as ImmE2 or DCB
  • Colicin-E7 immunity protein ImmE7 or DCE7
  • Colicin-E8 immunity protein ImmE8 or DCE8
  • Colicin-E9 immunity protein ImmE9 or DCE9
  • the 15kDa C-terminal domain of the proteins can be designed to be separately expressed, yielding a highly active intracellular toxin.
  • the DNAse domain is sufficient in order to form the complex with the immunity protein, it is possible to use this system as a kind of artificial toxin/antitoxin genetic system that will kill the host under desired conditions.
  • the present inventors have fused a gene encoding a colicin DNAse domain (an additional non-limiting example of a cytotoxic gene product of the present invention) and a gene encoding an appropriate immunity protein (an additional non-limiting example of a neutralizing gene product of the present invention) to desirable promoters, and as a result, the stoichiometry of the toxin/immunity protein can be kept in favor of the immunity protein under one set of conditions (resulting in normal bacterial growth) and changed in favor of the toxin under a different set of predetermined conditions, leading to bacterial death.
  • the first gene encodes for a bacteriocin or a cytotoxic portion thereof, and the second gene encodes for an immunity protein inactivating said bacteriocin.
  • the bacteriocin is Colicin E2 (ColE2).
  • Various promoters as described above can be used with the bacteriocin/immunity protein gene system, including a combination of a regulated promoter and a constitutive promoter, two regulated promoter, or a dual regulator system.
  • the genetic switching between non-toxic state and toxic state is done by changing the stoichiometry in favor of the toxin. This can be achieved by several mechanisms, for example: i. keeping the cytotoxic gene under constitutive expression and keeping the neutralizing gene under a promoter that is repressed at the predetermined condition;
  • predetermined condition relates to an environmental condition upon which bacterial death or reduced virulence are caused by the genetic system of the invention.
  • predetermined conditions can be, e.g. levels of certain minerals or a nutrients, oxygen levels, etc.
  • predetermined conditions are also referred to hereinbelow as “restrictive conditions”.
  • restrictive conditions are conditions that are not restrictive, i.e. under permissive conditions the bacteria are viable and bacterial death or reduced virulence is not caused by the genetic system of the invention.
  • the predetermined conditions are chosen such that the bacteria are killed or inhibited by the genetic system before causing disease or exiting the host body, and therefore they are safe for administering to a host for the purpose of immunization, without the need for attenuation or otherwise manipulating the bacterial pathogen to reduce or eliminate virulence.
  • a genetic system that under anaerobic conditions induces a cytotoxic gene product and represses its neutralizing gene product will become lethal to the bacteria after a certain number of cell divisions, because the neutralizing gene product is labile and is quickly degraded but no new neutralizing gene product is produced leaving the cytotoxic gene product free to act under anaerobic conditions.
  • the genetic system can be selected from the hok/sok system or the bacteriocin system described above.
  • Another way to keep pathogenic bacteria in check is, for example, to keep them to a certain compartment in which they are not pathogenic but do elicit an immune response. As soon as the bacteria leave the chosen compartment, they are killed by the cytotoxic gene of the genetic system or at least the pathogenicity of the bacteria is reduced. Alternatively, the system may be designed such that the bacteria are killed upon entering a specific compartment.
  • a genetic system that is based on the dual regulator PhoP may be used.
  • the cytotoxic gene is fused to a promoter that is induced by PhoP and the neutralizing gene is fused to a promoter that is repressed by PhoP. Therefore, whenever bacteria are entering the intracellular space, the levels of divalent cations in the bacteria's immediate surrounding are reduced, PhoP is activated causing elevated expression of the cytotoxic gene product and reduced expression of the neutralizing gene product, and the bacteria die.
  • the genetic system can be selected from the hok/sok system or the bacteriocin system described above.
  • a genetic system that is dependent on the FNR dual regulator may be used; however, the cytotoxic gene and the neutralizing gene are fused to the promoters in an inverse way compared to the system described above, such that the cytotoxic gene is fused to a promoter that is repressed by FNR and the neutralizing gene is fused to a promoter that is induced by FNR. Therefore, in the presence of this genetic system, the bacteria can grow well under anaerobic conditions but die when the oxygen concentrations increase.
  • an additional antitoxin encoding gene is fused to an inducer dependent promoter (such as pBAD or pLAC as described above) allowing the growth of the bacteria aerobically only in the presence of the inducer.
  • an inducer dependent promoter such as pBAD or pLAC as described above
  • the above two complementary genetic systems can be combined, on one hand preventing the bacteria from penetrating the epithelial tissue by PhoP regulation, and on the other hand preventing the bacteria from leaving the intestine by inducing death at aerobic conditions, by FNR regulation.
  • Another method for controlling bacteria following administration to a host may be to make it dependent on a compound that is not naturally present in the host. By choosing a promoter system which is responsive to the level of the compound, bacterial death can be guaranteed before the bacteria reach a level in the host that can effectively cause disease.
  • the cytotoxic gene can be fused to a constitutive promoter, for example sigma 70, and the neutralizing gene may be fused to the arabinose-induced pBAD promoter. Since the arabinose is absent from the animal, its concentration in the near vicinity of the bacteria is rapidly reduced after introduction of the bacterial into the animal, the pBAD promoter is turned off leading to reduced levels of neutralizing gene product and leaving the cytotoxic gene product free to act. Thus, the bacteria are kept alive or pathogenic only in the presence of arabinose.
  • the predetermined conditions are certain oxygen levels.
  • high oxygen levels, or aerobic conditions are used as a trigger if bacterial death is desired when it leaves the digestive system.
  • bacterial death may be desired under low oxygen (anaerobic including hypoxic or dysoxic) conditions for other situations, exemplified above.
  • the predetermined condition is selected from high oxygen levels (aerobic conditions, under which bacterial anaerobic respiration is repressed), low oxygen levels (anaerobic conditions, under which bacterial anaerobic respiration is induced), limitations of minerals such as iron, phosphorus, magnesium, manganese and calcium to levels which cause induction of mechanisms that acquire or maintain such minerals in bacteria, acidic pH, high levels of organic compounds (including, but not limited to, amino acids, vitamins, nucleotides, organic acids, fatty acids and secondary metabolites), or low levels of organic compounds.
  • the predetermined condition is a condition existing in compartments where bacterial death is desired.
  • high oxygen levels may correspond to atmospheric oxygen levels if bacterial death upon excretion is desired.
  • minerals such as magnesium and calcium
  • the predetermined condition may correspond to their concentration in the compartment in which the bacterial death is desired, for example, in cells lining the intestine.
  • the PhoP regulator is induced at low magnesium and calcium concentrations, such as concentrations below 10 and 2 milimolar, respectively (Ve 'scovi et al 1996).
  • the cytotoxic gene is fused to a constitutive (housekeeping) promoter.
  • a constitutive (housekeeping) promoter A variety of promoters with different strengths can be readily designed by a person having ordinary skills in the art of molecular biology by slightly changing the consensus sequence (e.g. by changing one or more of the nucleotides in the -35 box or the -10 box, resulting in a sequence with higher or lower similarity to the consensus sequence, for stronger or weaker transcription, respectively).
  • the neutralizing gene in this system is fused to an inducible promoter such as the Arabinose-dependent pBAD promoter or the Lactose-dependent pLAC promoter.
  • the system is designed in a way that in the presence of the inducer, the expression of the neutralizing gene is higher than the expression levels of the cytotoxic gene, resulting in functional live bacteria. When the levels of the inducer are reduced, the expression to the neutralizing gene is repressed, resulting in bacterial death.
  • the predetermined conditions are a low concentration of certain compounds, more specifically organic compounds such as arabinose or lactose, which can be present at a high concentration (for example, above 0.001% for arabinose) prior to immunization and are diluted thereafter because they do not naturally occur in the immunized animal.
  • bacterial death or reduction in virulence can be triggered by high levels of certain compounds. Accordingly, in certain embodiments,
  • the first gene is hok and the second gene is sok, or
  • the first gene is Colicin E2 CeaB and the second gene is Colicin E2 CeiB;
  • first and second promoter are selected from:
  • a promoter activated by and a promoter repressed by the FUR dual regulator or iv. a constitutive promoter, and an inducible or repressible promoter containing a recognition sequence for a transcription regulator selected from PhoP, FNR, FUR, AraC, Lacl, TrpR, or TetR, or from orthologs thereof.
  • SEQ ID NO: 1 - the hok gene fused to an inducible promoter and the sok gene fused to a repressible promoter of the FNR dual regulator promoters for causing bacterial death under anaerobic conditions (FNR/Hok).
  • the hok gene in all constructs has been mutated so that the sok gene encoded within it will not be expressed, and a second sok sequence is independently present in the system.
  • SEQ ID NO: 2 the hok gene fused to an inducible promoter and the sok gene fused to a repressible promoter of the PhoP dual regulator for causing bacterial death under low divalent cation concentrations (PhoP/Hok).
  • SEQ ID NO: 3 the hok gene fused to a constitutive promoter (CP20) and the sok gene fused to the pBAD promoter for causing bacterial death at low arabinose concentrations (pBAD/Hok).
  • SEQ ID NO: 4 the Colicin E2 CeaB gene fused to an inducible promoter of the FNR dual regulator and the ColE2 CeiB immunity protein gene fused to a repressible promoter for causing bacterial death under anaerobic conditions (FNR/CeaB).
  • SEQ ID NO: 5 the Colicin E2 CeaB gene fused to an inducible promoter of the PhoP dual regulator and the ColE2 CeiB immunity protein gene fused to a repressible promoter for causing bacterial death at low divalent cation concentration (PhoP/CeaB).
  • SEQ ID NO: 6 the Colicin E2 CeaB gene fused to a constitutive promoter (CP20) and the ColE2 CeiB immunity protein gene fused to the pBAD promoter for causing bacterial death at low arabinose concentrations (pBAD/CeaB).
  • SEQ ID NO: 7 the hok gene fused to an inducible promoter and the sok gene fused to a repressible promoter of the PhoP dual regulator in combination with the Colicin E2 CeaB gene fused to an inducible promoter and the ColE2 CeiB immunity protein gene fused to a repressible promoter of the FNR dual regulator (PhoP/Hok- FNR/CeaB ) .
  • SEQ ID NO: 8 the hok gene fused to an inducible promoter and the sok gene fused to a repressible promoter of the Fur dual regulator for causing bacterial death under low iron conditions (FUR/Hok).
  • SEQ ID NO: 9 the Colicin E2 CeaB gene fused to an inducible promoter and the ColE2 CeiB immunity protein gene fused to a repressible promoter of the Fur dual regulator for causing bacterial death under low iron conditions (FUR/CeaB).
  • the nucleic acid sequence is selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO:9.
  • the genetic systems of the invention including PhoP/Hok, FNR/Hok, pBAD/Hok, FNR/CeaB and pBAD/CeaB show decreased survival in E. coli and S. enteritidis under the respective restrictive conditions, while growth was not affected under permissive conditions (Example 1).
  • the DNA molecule comprises two pairs of a first and a second differentially controlled transcription units, wherein the promoters of the first pair are responsive to a dual regulator and the promoters of the second pair are responsive to a different dual regulator.
  • at least one of the promoters is responsive to a regulator that is not a dual regulator, and at least one of the promoters is a constitutive promoter.
  • the genes controlled by the promoters can be selected from systems such as the hok/sok system or the bacteriocin system, described above.
  • the DNA molecule comprises a nucleic acid sequence including two pairs of a first and a second differentially controlled transcription units, wherein one promoter of the first pair is activated by an E. coli FNR dual regulator or an ortholog thereof and the other promoter of the first pair is repressed by the E. coli FNR dual regulator or an ortholog thereof; and one promoter of the second pair is activated by an E. coli PhoP dual regulator or an ortholog thereof and the other promoter of the second pair is repressed by the E. coli PhoP promoter or an ortholog thereof.
  • the present invention provides a synthetic vector comprising the DNA molecule described hereinabove.
  • the system can be cloned into any vector suitable for replication and expression in the appropriate bacteria using methods well-known in the art of molecular biology, e.g. as taught in Molecular Cloning: A Laboratory Manual (Green and Sambrook, 2012, Cold Spring Harbor Laboratory Press).
  • such as a vector comprises an origin of replication and transcription elements compatible with the replication and transcription machineries of a variety of bacterial species.
  • origin of replication belonging to the pMB l, pUC (mutated pMB l), pl5, pSClOl, ColEl, pKN402, PI, F, and R6K families.
  • the vector is able to replicate, and the first and second promoters are able to function properly, in more than one strain of a bacterial species.
  • the genetic system of the present invention is intended to be introduced into live bacteria in order to produce a vaccine strain that can be safely administered to an animal for the purpose of vaccination against the live bacteria.
  • the present invention is directed to a method for producing a conditionally inactivated or attenuated bacterium, said method comprising introducing to a live pathogenic bacterium (in case the method is for producing either a conditionally inactivated or attenuated bacterium) or an attenuated bacterium (in the case the method is for producing a conditionally inactivated bacterium) a DNA molecule comprising a nucleic acid sequence including a promoter operably linked to a gene encoding a cytotoxic gene product, wherein said promoter is a regulated promoter responsive to a predetermined condition, whereby activation or de-repression of said regulated promoter by said predetermined condition causes bacterial cell death or reduced virulence, thereby producing a conditionally inactivated or attenuated bacterium that is inactivated or attenuated in the presence of the predetermined condition.
  • the present invention is further directed to the conditionally inactivated or attenuated bacterium
  • the nucleic acid sequence introduced to the live pathogenic bacterium includes the DNA molecule of the invention as described above.
  • the DNA molecule introduced into the bacterium is extra- chromosomal, i.e. it exists in the bacteria outside of the nucleoid region as a circular or linear plasmid and it contains an origin of replication which allows the plasmid to replicate independently of the bacterial chromosome.
  • the cytotoxic gene is not present or expressed in the native wild-type bacterial species of the bacterium.
  • pathogenic is used herein to refer to bacteria which are capable of causing disease in a host. It is noted that if the bacteria is attenuated, then the term pathogenic means that the bacteria was capable of causing disease in a host prior to attenuation, even if the bacteria is no longer capable of causing disease following attenuation, in contrast to a benign bacteria that in its native state does not have the potential to cause disease. In certain embodiments, pathogenic bacteria are capable of causing disease only in specific hosts.
  • conditionally inactivated bacteria and “conditionally attenuated bacteria” as used herein, refer to bacteria that under conditions that are different from the “predetermined condition” are fully viable in comparison with normal wild type bacteria of the same species, and which are killed or their virulence is reduced when encountering the "predetermined condition", respectively.
  • the present invention provides a conditionally inactivated or attenuated bacterium comprising the DNA molecule or vector carrying the genetic system of the invention.
  • the bacterium is vaccinogenic, derived from a live pathogenic or attenuated bacterium and differs from the live pathogenic or attenuated bacterium essentially by the presence of said DNA molecule or vector.
  • Various promoters as described above can be used with the genetic system, including a combination of a regulated promoter and a constitutive promoter, two regulated promoter, or a dual regulator system.
  • the genetic system can be selected from the hok/sok system or the colicin system described above.
  • vaccinogenic is used herein to refer to bacteria that are capable of inducing a protective and/or neutralizing immune response, which is sufficient to neutralize a later infection by bacteria of the same species and protect the host from these later- infecting bacteria.
  • a protective and/or neutralizing immune response it is meant that immune response causes a reduction in severity of any of the effects of a bacterial infection with virulent pathogenic bacteria, a reduction in the number of infecting bacteria, or an increase in specific anti-pathogenic antibodies in comparison with a control animal which was not immunized with the vaccine of the present invention.
  • the system is safe for use even if based on live pathogenic bacteria.
  • the genetic system takes advantage of existing systems such that under the predetermined conditions, the bacterium is dependent on expression of the regulator for its survival, for example, the FNR regulator, which activates genes which are required for survival under anaerobic conditions. Under anaerobic conditions, if the regulator is not expressed then the bacterium cannot survive. However, when it is expressed, the bacteria is killed because of the genetic system design.
  • Another advantage of the system is competitive exclusion, namely that when the modified bacteria of the invention have already occupied the compartment, wild-type pathogenic bacteria of the same species that might attempt to infect the same compartment will fail to do so due to the competitive advantage of the already established conditionally non-viable modified bacteria.
  • the present invention provides a conditionally inactivated or attenuated vaccinogenic bacterium comprising a DNA molecule comprising a nucleic acid sequence including at least one transcription unit comprising a first promoter operably linked to a first gene encoding a cytotoxic gene product, wherein said first promoter is a regulated promoter responsive to a predetermined condition, whereby activation or de-repression of said regulated promoter by said predetermined condition causes bacterial cell death or reduced virulence.
  • the DNA molecule is extra-chromosomal.
  • the bacterial cell death or reduced virulence is caused when the bacterium is present in a specific compartment of the animal in which the predetermined condition exists.
  • the genetic system is designed to restrict the bacteria to the digestive system of the host and thus, the specific compartment in this case may be selected from the digestive system itself, a cell lining the digestive system, a space in between the cells lining the digestive system or outside the host.
  • Toxins can cause cell death by various mechanisms, including piercing the cell membrane, degrading peptidoglycan, degrading DNA, degrading RNA, and modifying essential metabolites.
  • Many systems for self-killing of bacteria kill the cell by causing lysis, a process during which the content of the bacterial cell is spilled, potentially exposing the host to bacterial toxins, or intracellular antigens that can cause harmful immunologic response. This is required, for example, in systems which use the bacteria as carriers for a plasmid encoding an immunogenic protein, and in order to induce immune response, the bacteria are lysed, thereby releasing the immunogen and exposing it to the immune system.
  • the bacterium is not a carrier but is itself the immunogen without the need for a plasmid encoding an immunogenic protein, and therefore lysis is not needed in order to expose immunogenic epitopes. Therefore, in the systems presented in the present invention, bacterial killing does not necessarily happen by lysing the cell. For example, in the hok/sok system, bacterial death is caused by membrane depolarization, and in the colicin system, bacterial death is caused by depolarization of the cytoplasmic membrane, DNase activity, RNase activity, or inhibition of murein synthesis.
  • the cytotoxic gene product does not cause lysis of the bacterial cell.
  • the genetic system does not include sequences encoding for antigens that increase the antigenicity of the carrier bacterium, modify the specificity of an immune response elicited against the carrier bacterium or that are meant for inducing an immune response against the carrier bacterium or against any other pathogen.
  • the cytotoxic gene can cause reduced virulence instead of causing cell death.
  • the cytotoxic gene can transiently inhibit bacterial growth by inhibiting DNA replication or cell division, or any other cellular processes required for bacterial growth, in the host environment, while allowing the bacteria to resume growth outside the host. This could be achieved for example by transient expression of dominant negative alleles of essential genes, or expression of toxic bacteriophages genes such as the lambda ell gene.
  • the cytotoxic gene can also inhibit processes important for bacterial virulence, such as motility and secretion of virulence factors.
  • conditionally inactivated or attenuated bacterium of the invention can be selected from Bacillus Spp., Bordetella Spp., Borrelia Spp., Brucella Spp., Campylobacter Spp., Chlamydiae Spp.
  • Citrobacter Spp. Clostridium Spp., Corynebacterium Spp., Enterobacter Spp., Escherichia coli, Francisella Spp., Haemophilus Paragallinarum, Helicobacter Spp., Klebsiella Spp., Legionella Spp., Listeria Spp., Mycobacterium Spp., and Mycoplasma Spp., Neisseria Spp., Pasteurella Spp., Proteus Spp., Salmonella Spp., Serratia Spp., Shigella Spp., Staphylococcus Spp., Streptococcus Spp., Treponema Spp., Vibrio Spp., and Yersinia pestis.
  • the inactivated or attenuated bacterium is Salmonella Spp.
  • the preparation of a vaccine against a new strain of pathogenic bacteria requires only one step, i.e. the insertion of the genetic system of the invention into the bacteria. Because this genetic system may be carried on extra- chromosomal plasmids and is thus compatible with close species, it enables easy development of vaccines to new strains or related species.
  • the vector as mentioned above, is able to replicate, and the first and second promoter are able to function properly, in more than one strain of a bacterial species.
  • such bacteria can be antigenically identical to the un- manipulated wild-type, infective pathogenic strain with the same infection path and they are easy to propagate and simple to use, compared with presently available systems for attenuating pathogenic bacteria, which require manipulating the bacteria by mutations or selection.
  • At least one of the components of the genetic system is encoded by the bacterial chromosome.
  • the inactivated or attenuated bacterium of the present invention is for use in immunization of an animal, such as poultry or a mammal, e.g. a human.
  • immunosens are used interchangeably herein and refer to the administration of antigenic material (a vaccine) to stimulate a subject's immune system to develop adaptive immunity to a pathogen.
  • vaccine bacteria carrying the genetics systems of the invention are cleared from the chicken body within 10 days. Furthermore, according to Example 9, vaccination with S. enteritidis bacteria carrying the genetic systems of the invention (pBAD/Hok, pBAD/CeaB, Pho/Hok, Pho/Hok-FNR/CeaB), caused enhanced clearance of pathogenic bacteria two weeks following a challenge
  • the present invention provides a vaccine comprising the conditionally inactivated or attenuated bacterium of the invention.
  • the vaccine of the present invention may be administered by any conventional route, particularly to a mucosal (e.g. oral, ocular, intranasal, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract) surface or via the parenteral (e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal) route.
  • a mucosal e.g. oral, ocular, intranasal, gastric, pulmonary, intestinal, rectal, vaginal, or urinary tract
  • parenteral e.g., subcutaneous, intradermal, intramuscular, intravenous, or intraperitoneal
  • the immunization comprises administering said bacterium by oral administration.
  • oral administration is advantageous in the poultry industry, where individual injection is a very time consuming procedure.
  • Oral administration has further advantages for use in humans and other mammals: it is painless, easy to administer without the need for a professional, lower dose of bacteria needed, and additionally, the administered modified bacteria competes with the natural pathogens.
  • the present invention provides a method of vaccination comprising administering to an subject in need thereof a conditionally inactivated or attenuated vaccinogenic bacterium comprising a DNA molecule, wherein the DNA molecule comprises a nucleic acid sequence including at least a first and a second differentially controlled transcription units, the first transcription unit includes a first promoter operably linked to a first gene encoding a cytotoxic gene product, the second transcription unit includes a second promoter that is different from the first promoter and is operably linked to a second gene encoding a second gene product neutralizing the cytotoxic gene product, at least one of the first and the second promoter is a regulated promoter responsive to a predetermined condition, and the DNA molecule is designed such that under said predetermined condition the relative expression levels of the cytotoxic gene and the neutralizing gene change such that the expression level of the cytotoxic gene is increased relative to the expression level of the neutralizing gene, and as a result the amount of the neutralizing gene product is no
  • the vaccine is for use in vaccination of poultry, wherein the conditionally inactivated or attenuated bacterium is derived from live or attenuated Salmonella spp and differs from the live or attenuated Salmonella spp essentially by the presence of the DNA molecule or vector of the present invention.
  • the bacteria may be administered without any adjuvant or, in the case that the use of an adjuvant is desired, the bacteria may be emulsified in a suitable adjuvant such as oil in water or aluminum hydroxide.
  • the vaccine including the conditionally inactivated or attenuated bacterium of the invention is for use in vaccination of a mammal such as a human.
  • the bacteria may be administered without any adjuvant or, in the case that the use of an adjuvant is desired, the bacteria may be emulsified in a suitable adjuvant such as aluminum hydroxide, aluminum hydroxide gel, and aluminum hydroxyphosphate.
  • the vaccine adjuvant is amorphous aluminum hydroxyphosphate having an acidic isoelectric point and an A1:P ratio of 1: 1 (herein referred to as Alum-phos).
  • the poultry or mammalian vaccine may further comprise pharmaceutically acceptable carriers, such as a non-toxic, diluent formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline.
  • pharmaceutically acceptable carriers such as a non-toxic, diluent formulation auxiliary of any type, or simply a sterile aqueous medium, such as saline.
  • materials that can serve as pharmaceutically acceptable carriers are sugars, such as lactose, glucose and sucrose, starches such as corn starch and potato starch, cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt, gelatin, talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol, polyo
  • Certain bacteria such as Salmonella, are not pathogenic to avian in the digestive system. However it is a zoonotic bacteria that is transferred from avian to human and causes disease in human. Vaccination of broilers with live attenuated vaccine is impossible, due to the short-lived period of the broiler and the danger of transfer of the bacteria to human. Moreover, following entrance into human cells, the bacteria become pathogenic. Use of the self-destructing system of the invention enables vaccinating broilers with the new pathogenic variant. The bacteria survive in the digestive system (anaerobic environment), preventing wild-type bacteria from propagating due to competitive exclusion and inducing an immune response.
  • the self- destructing system of the invention is activated after an immune-response has been elicited and/or in case of penetration of the bacteria into cells or their excretion out of the body, following exposure to aerobic environment. This eliminates disease in the vaccinated bird and its survival out of the bird, and prevents dissemination to a consumer of the bird.
  • vaccinating SPF (specific pathogen free) chicks with the genetic systems of the invention indeed caused a significant reduction of the pathogenic bacteria introduced to the vaccinated chicks in a challenge following the vaccination, showing that an immune response is indeed induced, and that pathogenic bacteria are eliminated.
  • the new vaccine will meet the challenges of poultry vaccination and more specifically broiler vaccination, which is defined by short life expectancy and strict regulations that do not allow for the presence of vaccine bacteria in the meat products.
  • the vaccines are designed against pathogenic salmonella strains that characterize the poultry industry, leading to vaccination of the birds, and preventing transfer to human following consumption of the birds.
  • the vaccines are designed against campilobacter strains that characterize the poultry and cattle industry, leading to vaccination of the animals, and preventing transfer to human following consumption of the animals.
  • the present invention provides a method for prevention of transfer of a bacterium from a first animal to a second animal, comprising vaccinating the first animal against the pathogenic bacterium, the vaccinating comprising administering to the first animal a conditionally inactivated or attenuated vaccinogenic bacterium of the same or similar species as the pathogenic bacterium comprising a DNA molecule, wherein the DNA molecule comprises a nucleic acid sequence including at least a first and a second differentially controlled transcription units, the first transcription unit includes a first promoter operably linked to a first gene encoding a cytotoxic gene product, the second transcription unit includes a second promoter that is different from the first promoter and is operably linked to a second gene encoding a second gene product neutralizing the cytotoxic gene product, at least one of the first and the second promoter is a regulated promoter responsive to a predetermined condition, and the DNA molecule is designed such that under the predetermined condition the amount
  • the bacterium is pathogenic in the second animal and not in the first animal. In certain embodiments, the bacterium is pathogenic in both the first and the second animal.
  • the first animal is poultry and the second animal is human.
  • the pathogenic bacterium is Salmonella spp or Campylobacter spp.
  • the DNA molecule introduced into the bacterium is extra-chromosomal as explained above.
  • the cytotoxic gene is not naturally present in the bacterial species of the bacterium.
  • the cytotoxic gene product does not cause lysis of the bacterial cell.
  • the genetic system does not include sequences encoding for antigens that increase the antigenicity of the carrier bacterium, modify the specificity of an immune response elicited against the carrier bacterium or that are meant for inducing an immune response against the carrier bacterium or against any other pathogen.
  • Plasmids were transformed into E. coli K12 MG1655 and/or S. enteritidis by standard methods and positive clones (harboring ampicillin resistance cassette of pBR322 vectors) were selected using LB-agar plates containing ampicillin (10(Vg/ml) and further verified by PCR and sequencing.
  • the Dual system FNR/CeaB -PhoP/Hok was constructed by cloning of PhoP/Hok PCR product into PBR322 vector containing FNR/CeaB system using Sail restriction site.
  • the sequences of the various systems used in this invention are as follows: FNR- coupled Hok/Sok system - SEQ ID NO: 1 (FNR/Hok), PhoP-coupled Hok/Sok system - SEQ ID NO: 2 (PhoP/Hok), pBAD/CP20-coupled Hok/Sok system - SEQ ID NO: 3 (pBAD/Hok), FNR- coupled CeiB/CeaB system - SEQ ID NO: 4 (FNR/CeaB), PhoP-coupled CeiB/CeaB system - SEQ ID NO: 5 (Phop/CeaB), pBAD/CP20-coupled CeiB/CeaB system - SEQ ID NO: 6 (p
  • E. coli K12 MG1655 and Salmonella enterica serotype Enteritidis were used in this study. Cultures were prepared by inoculation of a single colony grown on Luria-Bertani (LB) media into 5 ml LB broth and incubation overnight (O.N.) under permissive conditions (as indicated in Table 1). Afterwards bacteria were transferred to restrictive conditions for 24h-10d period. At each time point bacterial suspensions were serially diluted, plated on LB agar, incubated at permissive conditions O.N. before colony-forming units (CFU) were determined.
  • LB Luria-Bertani
  • permissive conditions as indicated in Table 1
  • CFU colony-forming units
  • Table 1 Growth conditions for bacterial strains carrying different genetic systems
  • Example 1 Growth of E. coli carrying genetic systems under permissive conditions
  • Example 2 Effect of several genetic systems on the survival of Salmonella and E. coli strains after 24 h incubation under restrictive conditions.
  • Salmonella stains carrying the genetic systems pBAD/CeaB, pBAD/Hok, FNR/CeaB, PhoP/Hok or combined FNR/CeaB -PhoP/Hok were tested for intracellular survival in macrophage tissue culture.
  • WT Salmonella Enteritidis were able to survive and proliferate inside the macrophages resulting in about 10 fold increase after 24h.
  • Bacteria carrying the oxygen dependent FNR/CeaB genetic system were able to survive and proliferate inside macrophage cells similarly to the WT strain, indicating that oxygen is not a limiting factor in this experimental system.
  • One day old specific pathogen-free (SPF) chicks were divided into 7 groups of 30 birds per group and were allocated in separated isolators for the duration of the study. Each group received lxlO 8 live bacteria (colony forming units) in a 0.5 ml of vehicle (water) using a gavage needle. One group did not get any bacteria - the "negative control”, one group received the wild type (WT) pathogenic bacteria, Salmonella enteritidis B3 - "positive control”, and each of the other groups received a vaccine bacteria S. enteritidis carrying one of the genetic systems as indicated in Table 2. The presence of Salmonella in the intestine was determined in all birds every 3 days using cloacal swabs.
  • Swabs were cultured in selenite broth overnight at 37°C before being streaked on both Chromagar and Salmonella Shigella (SS) agar plates. According to swabs results, each bird was defined as positive or negative for the presence of salmonella. The percentage of positive birds in each group is presented. The presence of salmonella in internal organs - liver and spleen - was determined twice, at days 5 and 10. 10 birds from each group were sacrificed and subjected to post mortem examination. Spleens and livers were removed, cultured independently in selenite broth overnight at 37°C and plated on both Chromagar and Salmonella Shigella (SS) agar plates. Each bird was defined as positive or negative for the presence of salmonella in internal organs and the percentage of positive birds in each group is presented in Table 2.
  • Example 6 The effect of vaccination of chicks at one day of age on the course of a later infection.
  • enteritidis B3 in a 0.5 ml of vehicle (water) using a gavage needle.
  • the presence of S. enteritidis B3 was determined in cloacal swabs and internal organs as described before. Cloacal swabs were taken at days 3 and 10 post challenge (days 17 and 24 of the experiment) and internal organs were analyzed in 10 birds from each group at days 7 and 14 post challenge (days 21 and 28 of the experiment). The percentage of birds positive to the challenge bacteria is presented in Table 3. The results show that birds that were not vaccinated at day 1 (negative control) showed high numbers of bacteria post infection, reaching 100% infected birds as determined by both cloacal swabs and internal organ analysis.
  • All groups of vaccinated birds showed some degree of protection against WT Salmonella Enteritidis challenge. While some vaccines were better in reducing intestinal infection as determined by cloacal swabs (e.g. S. enteritidis carrying the pBAD/CeaB system) others were better in reducing infections in internal organs (e.g. S. enteritidis carrying the dual system PhoP/Hok- FNR/CeaB).
  • Example 7 Test the duration of excretion and spread of the Salmonella enteritidis Live Vaccine in Chickens.
  • Group 1 is orally vaccinated with lxlO 8 cfu/0.5 ml of the vaccine bacteria (bacteria carrying a genetic system of the invention as described above) on day 1 ; group 2 serves as unvaccinated control and group 3 as target birds for infection.
  • group 2 serves as unvaccinated control and group 3 as target birds for infection.
  • group 3 is marked, split to two groups and transferred from isolation to be in contact with group 1 and group 2.
  • the presence of salmonella in all birds is determined by cloacal swabs (as described before) for 21 days in a 3 day intervals. The percentage of group 3 birds that become positive for salmonella is determined.
  • Example 8 Dissemination and survival of the vaccine bacteria after administration of a repeated dose of the vaccine on days 1 and 40.
  • One-day old SPF chickens are divided into groups of 30 as in Example 5.
  • the groups are vaccinated at day 1 as in Example 5 and the presence of bacteria in cloacal swabs and internal organs is determined as in Example 5.
  • a second dose of vaccine bacteria (bacteria carrying a genetic system of the invention as described above) is administered and the presence of bacteria in cloacal swabs and internal organs is determined as performed after the first dose.
  • Example 9 Testing the stability of the vaccine bacteria and the inserted genetic components.
  • Day old SPF chicks are divided into groups of 10 and each group is orally inoculated with Ixl0 8 -lxl0 9 CFU of vaccine bacteria (bacteria carrying a genetic system of the invention as described above).
  • the presence of the vaccine bacteria is analyzed by cloacal swabs and internal organ analysis as described above at several time points (e.g. after 10, 20 and 30 days).
  • Re- isolated vaccine bacteria isolated from positive birds
  • plasmids are extracted and purified, and transformed into the mother type WT bacteria.
  • the performance of the new strain is determined by in-vitro and in-vivo experiments as described above in Examples 1-4 and Examples 5-6 respectively and compared to the original vaccine bacteria.
  • plasmids are fully sequenced to confirm that no modification occurred in the inserted plasmid during the process of vaccination.

Abstract

La présente invention concerne une molécule d'ADN comprenant une séquence d'acide nucléique comprenant au moins une paire d'une première et d'une deuxième unité de transcription régulée de manière différentielle, la première unité de transcription comprenant un premier promoteur lié de manière fonctionnelle à un premier gène codant pour un produit génique cytotoxique, la deuxième unité de transcription comprenant un deuxième promoteur qui est différent du premier promoteur et qui est lié de manière fonctionnelle à un deuxième gène codant pour un produit génique qui neutralise ledit produit génique cytotoxique, au moins l'un parmi ledit premier et ledit deuxième promoteur étant un promoteur régulé sensible à une condition prédéterminée et ladite molécule d'ADN étant conçue de telle sorte que, dans ladite condition prédéterminée, la quantité dudit produit génique de neutralisation n'est pas suffisante pour neutraliser l'effet cytotoxique du produit génique cytotoxique. La présente invention concerne en outre des bactéries inactivées ou atténuées de manière conditionnelle comprenant la molécule d'ADN de l'invention et des vaccins les comprenant.
PCT/IL2015/050137 2014-02-05 2015-02-05 Système génétique pour générer des bactéries inactivées ou atténuées de manière conditionnelle WO2015118541A1 (fr)

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WO2017055360A1 (fr) * 2015-09-28 2017-04-06 Danmarks Tekniske Universitet Système de titres de production améliorés dans des fermentations
WO2018136938A1 (fr) * 2017-01-23 2018-07-26 University Of Florida Research Foundation, Incorporated Induction d'immunité protectrice contre des antigènes
CN108812548A (zh) * 2018-09-19 2018-11-16 天康生物股份有限公司 布鲁氏菌疫苗免疫牛的方法和用途
CN111278978A (zh) * 2017-09-08 2020-06-12 新实有限公司 使细菌能够通过葡萄糖依赖性生存力特异性靶向实体肿瘤的核酸系统
US11596677B2 (en) 2017-08-04 2023-03-07 University Of Florida Research Foundation, Incorporated Induction of protective immunity against antigens

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WO2006128089A2 (fr) * 2005-05-26 2006-11-30 Conjugon, Inc. Compostions et methodes de traitement des tissus
WO2012087483A1 (fr) * 2010-11-24 2012-06-28 The Arizona Board Of Regents For And On Behalf Of Arizona State University Bactérie recombinante renfermant un système toxine-antitoxine

Cited By (14)

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Publication number Priority date Publication date Assignee Title
WO2017055360A1 (fr) * 2015-09-28 2017-04-06 Danmarks Tekniske Universitet Système de titres de production améliorés dans des fermentations
US11352650B2 (en) 2015-09-28 2022-06-07 Danmarks Tekniske Universitet System for improved production titers in fermentations
WO2018136938A1 (fr) * 2017-01-23 2018-07-26 University Of Florida Research Foundation, Incorporated Induction d'immunité protectrice contre des antigènes
US11788056B2 (en) 2017-01-23 2023-10-17 University Of Florida Research Foundation, Incorporated Induction of protective immunity against antigens
US10988729B2 (en) 2017-01-23 2021-04-27 University Of Florida Research Foundation, Incorporated Induction of protective immunity against antigens
US11596677B2 (en) 2017-08-04 2023-03-07 University Of Florida Research Foundation, Incorporated Induction of protective immunity against antigens
EP3679142A4 (fr) * 2017-09-08 2021-04-28 New Portal Limited Systèmes d'acide nucléique qui permettent à des bactéries de cibler spécifiquement des tumeurs solides par l'intermédiaire d'une viabilité dépendante du glucose
JP7003232B2 (ja) 2017-09-08 2022-02-04 ニュー ポータル リミテッド グルコース依存性の生存率を介して、細菌が固形腫瘍を特異的に標的とすることを可能にする核酸システム
JP2020533978A (ja) * 2017-09-08 2020-11-26 ニュー ポータル リミテッドＮｅｗ Ｐｏｒｔａｌ Ｌｉｍｉｔｅｄ グルコース依存性の生存率を介して、細菌が固形腫瘍を特異的に標的とすることを可能にする核酸システム
US11458172B2 (en) 2017-09-08 2022-10-04 New Portal Limited Nucleic acid systems that enable bacteria to specifically target solid tumors via glucose-dependent viability
CN111278978A (zh) * 2017-09-08 2020-06-12 新实有限公司 使细菌能够通过葡萄糖依赖性生存力特异性靶向实体肿瘤的核酸系统
US11696931B2 (en) 2017-09-08 2023-07-11 New Portal Limited Bacteria for targeting tumors and treating cancer
CN111278978B (zh) * 2017-09-08 2023-08-18 新实有限公司 使细菌能够通过葡萄糖依赖性生存力特异性靶向实体肿瘤的核酸系统
CN108812548A (zh) * 2018-09-19 2018-11-16 天康生物股份有限公司 布鲁氏菌疫苗免疫牛的方法和用途

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