WO1998010064A1 - Iron regulated promoter and uses thereof - Google Patents

Iron regulated promoter and uses thereof Download PDF

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Publication number
WO1998010064A1
WO1998010064A1 PCT/AU1997/000503 AU9700503W WO9810064A1 WO 1998010064 A1 WO1998010064 A1 WO 1998010064A1 AU 9700503 W AU9700503 W AU 9700503W WO 9810064 A1 WO9810064 A1 WO 9810064A1
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WIPO (PCT)
Prior art keywords
recombinant
polypeptide
sequence
host cell
promoter
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PCT/AU1997/000503
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French (fr)
Inventor
Himanshu N. Brahmbhatt
David Emery
Adam Burn
Original Assignee
Commonwealth Scientific And Industrial Research Organisation
Australian Wool Research And Promotion Organisation
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Publication date
Priority claimed from AUPO2164A external-priority patent/AUPO216496A0/en
Priority claimed from AUPO2454A external-priority patent/AUPO245496A0/en
Priority claimed from AUPO7801A external-priority patent/AUPO780197A0/en
Application filed by Commonwealth Scientific And Industrial Research Organisation, Australian Wool Research And Promotion Organisation filed Critical Commonwealth Scientific And Industrial Research Organisation
Priority to BR9711692-0A priority Critical patent/BR9711692A/en
Priority to EP97933590A priority patent/EP0964924A4/en
Priority to CA002265883A priority patent/CA2265883A1/en
Priority to AU36892/97A priority patent/AU737981B2/en
Priority to NZ334649A priority patent/NZ334649A/en
Publication of WO1998010064A1 publication Critical patent/WO1998010064A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43536Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
    • C07K14/4354Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from nematodes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90Stable introduction of foreign DNA into chromosome
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies
    • A61K2039/51Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to promoters and their use for the expression of polypeptides and in particular their use in live vaccines.
  • the present invention also provides a method of integrating foreign genes into the chromosome of bacteria and to bacterial vaccines expressing foreign genes.
  • the Esche ⁇ chia coli bacterium displays a characteristic response to iron starvation which is shared by many other aerobic and facultative anaerobic microorganisms, such as Salmonella typhimu ⁇ um. Under conditions where the iron concentration is less than about 5 ⁇ M the transcription of a collection of genes or groups of genes scattered through the chromosome is coordinately activated in a manner resembling an iron- controlled regulon. Iron control is in all instances mediated by negative regulation via the product of the fur gene, the absence of which triggers the expression of the genes to constitutive levels. The Fur protein also regulates expression of the aerobac tin-mediated iron transport regulon.
  • the present inventors have developed a system involving the use of a novel hybrid iron-regulated promoter which can be induced to hyper-express polypeptide encoding genes.
  • the promoter can provide an optimal polypeptide dose to the host immune system.
  • the inventors mean a DNA molecule having a nucleic acid sequence which (as a portion of a larger DNA molecule) is effective in inducing the expression of a polypeptide- encoding gene or genes localised downstream of the promoter DNA sequence.
  • the polypeptide may comprise one or more antigenic determinants of a pathogenic organism, and may be derived from a virus, bacterium, fungus, yeast or parasite.
  • Live attenuated Salmonella strains are currently being developed world-wide as carriers of foreign antigens from viral, bacterial and parasite origin. These recombinant salmonellae are being used to immunise the animal or human host to elicit a protective immune response against the respective infection. As mentioned above these recombinant Salmonella- based vaccines suffer from a number of drawbacks.
  • One major problem is that they typically carry the foreign antigen gene on self-replicating plasmids. These plasmids are often unstable and are lost from the bacterial cell in vivo resulting in a loss of the foreign antigen gene and consequently the foreign protein. This plasmid loss results in a reduced antigen dose being presented to the host immune system with a consequent sub-optimal protective immune response.
  • the present invention consists in an iron-regulated promoter comprising the DNA sequence shown in Fig. 1, or a fragment thereof which includes the sequence from residues 284 to 409, or a functionally equivalent nucleic acid sequence.
  • the iron-regulated promoter includes the nucleic acid sequence from residues 284 to 409 as shown in Fig. 1.
  • the present invention consists in a recombinant
  • DNA molecule comprising a promoter having a nucleic acid sequence including the DNA sequence shown in Fig. 1, or a fragment thereof which includes the sequence from residues 284 to 409, or a functionally equivalent nucleic acid sequence, expressively linked to a further DNA sequence encoding a polypeptide.
  • the promoter includes the nucleic acid sequence from residues 284 to 409 as shown in Fig. 1.
  • polypeptide includes at least one at least one epitope. It is preferred that the polypeptide includes B-cell and/or T-cell epitopes. In another embodiment the polypeptide includes at least one CTL epitope.
  • a preferred polypeptide is the 37 kD extracellular/secretory protein of Trichostrongylus colubriformis.
  • the present invention consists in a recombinant vector, the vector comprising an iron-regulated promoter and a site for insertion of a sequence encoding at least one polypeptide such that the inserted sequence is in frame with the iron-regulated promoter, wherein the iron-regulated promoter comprises the DNA sequence shown in Fig. 1. or a fragment thereof which includes the sequence from residues 284 to 409, or a functionally equivalent nucleic acid sequence.
  • the vector further includes an attP sequence and preferably the vector further includes a sequence encoding integrase protein.
  • the vector further includes a sequence encoding at least one polypeptide inserted at the insertion site. It is preferred that the encoded polypeptide includes at least one epitope. Typically the encoded peptide will B and/or T-cell epitopes.
  • the encoded polypeptide includes at least one CTL epitope.
  • the sequence encoding the polypeptide may encode a plurality of CTL epitopes. Such an arrangement is disclosed in PCT/AU95/00461 the disclosure of which is incorporated herein by reference.
  • the encoded polypeptide is the 37 kD extracellular/secretory protein of Trichostrongylus colubriformis.
  • the present invention consists in a recombinant host cell, the host cell including a recombinant DNA molecule comprising a promoter having a nucleic acid sequence including the DNA sequence shown in Fig, 1, or a fragment thereof which includes the sequence from residues 284 to 409, or a functionally equivalent nucleic acid sequence, expressively linked to a further DNA sequence encoding at least one polypeptide.
  • the recombinant DNA molecule is inserted into the host cell chromosome.
  • the host cell is a bacterium, preferably Gram negative, and more preferably the bacterium is Escherichia coli or
  • Salmonella species and preferably the Salmonella species is Salmonella typhimu ⁇ um.
  • the encoded polypeptide includes at least one at least one epitope. It is preferred that the polypeptide includes B-cell and/or T-cell epitopes. In another embodiment the polypeptide includes at least one CTL epitope.
  • a preferred polypeptide is the 37 kD extracellular/secretory protein of Trichostrongylus colubriformis.
  • the present invention consists in a composition for use in inducing an immune response in an animal, the composition comprising a recombinant host cell of the present invention and an acceptable carrier.
  • the term "functionally equivalent nucleic acid sequence" is intended to cover minor variations in the promoter sequence which do not result in a promoter having substantially lower activity from the promoter defined from residues 284 to 409 in Fig. 1.
  • the vector of the present invention includes the bacteriophage P22 int gene and an attP region. When this vector is introduced into a suitable bacterium, the bacteriophage P22 int gene is expressed to produce the int protein.
  • a short DNA sequence called attP (attachment) in the vector is homologous to the attB sequence found in the bacterial chromosome.
  • the bacterial chromosome expresses a protein called IHF (integration host factor) and that protein interacts with the expressed int protein from the vector which causes the homologous recombination of the vector DNA and the chromosomal DNA between attP and attB sequences.
  • IHF integration host factor
  • attB and attP recombine and stably integrate the vector into the bacterial chromosome.
  • the vector includes a DNA molecule encoding a foreign gene, upon integration, the bacterium is capable of expressing that gene from its chromosome to produce the foreign protein.
  • the bacterium is an attenuated vaccine bacterium and the heterologous protein is derived from a virus, bacterium, fungus, yeast or parasite. More preferably, the bacterium is a S. typhimurium strain and the polypeptide is the 37 kD extra cellular/secretory protein from the nematode parasite of sheep Trichostrongylus colubriformis.
  • the present inventors have developed a site-specific chromosomal integration system to integrate foreign antigen genes into the chromosome of attenuated S. typhimurium strains. The advantages of this system include:
  • the system does not use mobile genetic elements (e.g. transposons) and therefore once the vaccine strain is constructed the genotype and phenotype will not be altered by genes hopping around to other sites in the bacterial chromosome. 3.
  • the efficiency of chromosomal integration approaches 100% i.e. all bacterial cells that are transformed by this DNA are found to have the heterologous polypeptide genes integrated in the bacterial chromosome.
  • the system is "user-friendly” and requires a single step cloning event to clone the gene of interest into the "chromosomal integration vector" followed by transformation of the attenuated S. typhimurium strain of choice.
  • This system is highly versatile and can be adapted for integration of genes into the chromosomes of a wide range of Gram-negative bacteria as well as eucaryotic cells.
  • the applications of this technology range widely and can be used as (a) basic-research tool to study chromosomally integrated genes in Gram- negative bacteria and Eucaryotic cells, (b) preparation of live attenuated vaccines, (c) tissue specific expression in gene therapy, and (d) development of transgenic plants and animals.
  • Figure 1 shows the complete DNA sequence encoding a hybrid iron- regulated promoter according to the present invention
  • Figure 2 shows the construction of plasmids including promoters according to the present invention
  • Figure 3 shows details of the plasmid used for chromosomal integration in Salmonella
  • Figure 4 shows anti-37KD serum titers
  • Figure 5 shows anti ⁇ -galactosidase serum antibody titers
  • Figure 6 shows egg counts
  • Figure 7 shows worm count in sheep intestinal lumen.
  • the promoter was initially cloned as an EcoRI/BamHI fragment is plasmid pSU207 (Fig. 2).
  • Plasmid pHBl ⁇ (Fig. 2) is a pBR322 based plasmid carrying a polylinker.
  • PCR primers HB38 and HB39 were designed to amplify part of the aerobactin gene promoter between positions 10 and 394 on the aerobactin promoter sequence. This region carriers minor and major promoters P2 and P2 respectively, along with the primary and secondary Fur binding sites.
  • the Shine Dalgarno and downstream DNA sequence of the aerobactin promoter were not amplified.
  • the PCR primers carried the Notl and Pad restriction enzyme cleavage sites and the shortened aerobactin promoter was subcloned into the Notl/Pacl sites of pHBl58 to give plasmid designated pHBl64 (Fig. 2).
  • the 37 kD extracellular/secretory protein encoding gene from nematode parasite of sheep Trichostrongylus colubriformis was cloned as a lacZ gene fusion in plasmid pHbl67(Fig. 2). Upstream of the 5' region of the 37 kD gene sequence was engineered a DNA sequence carrying the Pad restriction enzyme site followed by Shine Dalgarno sequence (AGGA), a 7 bp spacer sequence (AACAGCT) and a translation start codon (ATG).
  • the Pacl/Xbal fragment form plasmid pHBl67 (Fig. 2) carrying the upstream region, along with the 37 kD/7 ⁇ cZ genes was subcloned into the Pacl/Xbal sites of plasmid pHBl64 to give plasmid designated pHBl70(Fig. 2).
  • This fusion of the aerobactin promoter (paer) with the upstream region provided a novel iron-regulated promoter according to the present invention.
  • This promoter is unique in carrying the aerobactin promoter gene sequences for Pi and P2, as well as designed primary and secondary Fur binding sites, and designed Shine Dalgarno, spacer and ATG sequences.
  • the important sequence of the iron-regulated promotor lies from DNA sequence residues 284 to 409 inclusive. This region includes the -35 (residues 284 to 289) and -10 (residues 307 to 312) regions of minor promotor P2, the -35 (residues 340 to 345) -10 (residues 363 to 368) regions of major promotor Pi, the primary (residues 333 to 363) and secondary
  • Plasmid pHBl70 was transformed into E. coli strain HB101 and S. typhimurium ⁇ ro ⁇ -strain.
  • the strains were grown in vitro under iron-rich conditions in Luria Broth with 200 ⁇ M FeCl 3 .
  • Cultures for iron starvation were grown under identical conditions but with the inclusion of 100 ⁇ M 2,2'- dipyridyl (iron chelator).
  • the FeCl 3 and 2,2'-dipyridyl concentrations were optimised to obtain maximal repression and induction respectively without affecting growth rate or viability of the organisms.
  • Bacteriophage P22 integrates its genome into the chromosome of S. typhimurium by the following mechanism:
  • P22 attaches to the bacterial cell surface and injects its DNA into the bacterial cell.
  • the injected DNA circularises and expresses the INT (integration) protein encoded by the int gene.
  • the P22 genome has a short DNA sequence called the attP (attachment) which is homologous to the attB sequence found in the Salmonella chromosome.
  • the bacterial chromosome expresses a protein called the IHF (integration host factor).
  • the INT and IHF proteins interact to homologously recombine the attP and attB sequences thereby integrating the P22 genome into the bacterial chromosome (lysogeny).
  • Plasmid pNEB193 purchased from New England Biolabs
  • plac/lac gene fragment which encodes the lac repressor protein, was PCR amplified from plasmid pLOF/Ars and cloned into the Pacl/Xbal restriction sites of plasmid pNEBl93 to result in plasmid pHBl78.
  • the ptac promoter gene fragment from plasmid pKK223-3 was PCR amplified and cloned into the Sacl/Kpnl sites of plasmid pHBl78 to result in plasmid pHBl79.
  • the p ⁇ er/ArsA/ArsB gene fragment which encodes Arsenite resistance was PCR amplified from plasmid pLOF/Ars and cloned into the Hindi II site of plasmid pHBl79 to result in plasmid pHBl ⁇ O.
  • the bacteriophage P22 int attP region including its ribosome binding site-spacer-translation start (ATG) was PCR amplified from the P22 phage genome. This gene fragment was cloned into the Kpnl/Pacl site of plasmid pHBl ⁇ O to result in plasmid pHBl ⁇ l. Transformation of plasmid pHB181 into Salmonella typhimurium strain and confirmation of chromosomal integration.
  • Plasmid pHBl ⁇ l was transformed into S. typhimurium strain H4004 and transformants were selected on Brain Heart Infusion agar containing Ampicillin. The transformation efficiency was 10° colony forming units (c.f.u.) per microgram of DNA and was the same as control plasmid DNA. Plasmid DNA from individual transformants were analysed by agarose gel electrophoresis and the results showed that the plasmid had integrated into the chromosome. DNA from control E. coli cells harbouring the same plasmid revealed the presence of the plasmid. Southern hybridisation with the plasmid as a probe was used to confirm that the plasmid had integrated into the chromosome.
  • the groups include: 1. S. typhimurium aroA- [strain 4335] - oral (10 1 1 organisms).
  • the plasmid used for this integration is shown schematically in Fig 3. 6. Same as (5) - intramuscular (10 9 organisms).
  • the serum was analysed in ELISA assay to detect antibodies to the T. colubriformis 37kD polypeptide and to the ⁇ -galactosidase reporter polypeptide.
  • the serum antibody titers are shown in Figs. 4 and 5.
  • T. colubriformis eggs were counted in fecal samples at various time points. Results of egg counts are shown in Fig. 4. The sheep were euthanased about 2 months after commencement of challenge and the intestinal linings were scraped to collect T. colubriformis worms. The worm count data is shown in Fig. 5.
  • S. typhimurium aroA- carrying the plasmid pHBl70 (iron regulated promoter-37kD-lacZ) showed no serum antibody response to either 37kD or the ⁇ -galactosidase polypeptides.
  • worm count data (Fig. 5) parallels that of egg counts and the i.m. vaccinated sheep (chromosomally integrated) had a significant (p ⁇ 0.05) reduction of 61% in worm burden.

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Abstract

The present invention provides an iron-regulated promoter. The promoter comprises the DNA sequence (shown in the figure), or a fragment thereof which includes the sequence from residues 284 to 409, or a functionally equivalent nucleic acid sequence. Also provided are vectors and recombinant host cells including the promoter and compositions for use in inducing an immune response in an animal.

Description

Iron Regulated Promoter and Uses Thereof
This invention relates to promoters and their use for the expression of polypeptides and in particular their use in live vaccines. The present invention also provides a method of integrating foreign genes into the chromosome of bacteria and to bacterial vaccines expressing foreign genes.
It is known to construct recombinant bacterial vaccines in which artificially introduced foreign polypeptide genes are carried by naturally occurring bacterial strains and expressed in the host under the control of a promoter, Live attenuated Gram-negative bacterial strains currently proposed for the expression of foreign antigen genes from pathogens, however, suffer from several major limitations, one of which is that in vivo gene expression is significantly less than what might have been expected from empirical considerations. Primarily this is believed to be due to environmental shock experienced by the bacterium when it encounters the different and nutritionally complex environment of intestinal lumen and host tissues. The process of adaptation to the new environment slows down bacterial gene expression and metabolic activity, with a resulting reduction in the foreign antigen dose delivered to the host immune system and corresponding sub-optimal immune response.
The Escheήchia coli bacterium displays a characteristic response to iron starvation which is shared by many other aerobic and facultative anaerobic microorganisms, such as Salmonella typhimuήum. Under conditions where the iron concentration is less than about 5 μM the transcription of a collection of genes or groups of genes scattered through the chromosome is coordinately activated in a manner resembling an iron- controlled regulon. Iron control is in all instances mediated by negative regulation via the product of the fur gene, the absence of which triggers the expression of the genes to constitutive levels. The Fur protein also regulates expression of the aerobac tin-mediated iron transport regulon.
In order to attempt to overcome the reduction in gene expression which occurs when a bacterium encounters a harsh environment, such as in the intestine, the present inventors have developed a system involving the use of a novel hybrid iron-regulated promoter which can be induced to hyper-express polypeptide encoding genes. When incorporated in a bacterial vaccine, the promoter can provide an optimal polypeptide dose to the host immune system. In this specification, by a promoter the inventors mean a DNA molecule having a nucleic acid sequence which (as a portion of a larger DNA molecule) is effective in inducing the expression of a polypeptide- encoding gene or genes localised downstream of the promoter DNA sequence. The polypeptide may comprise one or more antigenic determinants of a pathogenic organism, and may be derived from a virus, bacterium, fungus, yeast or parasite.
Live attenuated Salmonella strains are currently being developed world-wide as carriers of foreign antigens from viral, bacterial and parasite origin. These recombinant salmonellae are being used to immunise the animal or human host to elicit a protective immune response against the respective infection. As mentioned above these recombinant Salmonella- based vaccines suffer from a number of drawbacks. One major problem is that they typically carry the foreign antigen gene on self-replicating plasmids. These plasmids are often unstable and are lost from the bacterial cell in vivo resulting in a loss of the foreign antigen gene and consequently the foreign protein. This plasmid loss results in a reduced antigen dose being presented to the host immune system with a consequent sub-optimal protective immune response.
Two specific chromosomal integration systems have been developed by other groups, one of which relies on homologous recombination into the S. typhimurium histidine operon and the other uses transposons to transpose the antigen gene cassette randomly into the bacterial chromosome. Both systems suffer from the drawback of being highly cumbersome, labour intensive and are very inefficient. The latter system integrates randomly into the chromosome and hence suffers from the additional drawback of creating additional mutations in the bacterial chromosome which may be deleterious to the vaccine strain. Additionally, the use of transposons (mobile genetic elements) in vaccine strains is not desirable since they tend to "hop" around in the vaccine strain making the genotype of the strain ill-defined. Genetically attenuated vaccine strains need to be clearly defined (genotype and phenotype) before they can be accepted for commercial use. In an attempt to solve the problem of using recombinant plasmids in bacterial vaccines, the present inventors have developed methods to integrate heterologous polypeptide-expressing gene cassettes into a specific non-essential site in the chromosome of bacteria and especially in attenuated S. typhimurium strains. This process eliminates the need for recombinant plasmids and the genes of interest are expressed stably from the chromosome of the bacteria. This invention has broad spectrum applications since this site- specific recombination system can, with the appropriate modifications, be applied to integrating foreign genes into the chromosomes of a wide range of bacteria and eucaryotic cells.
In a first aspect the present invention consists in an iron-regulated promoter comprising the DNA sequence shown in Fig. 1, or a fragment thereof which includes the sequence from residues 284 to 409, or a functionally equivalent nucleic acid sequence.
It is preferred that the iron-regulated promoter includes the nucleic acid sequence from residues 284 to 409 as shown in Fig. 1. In a second aspect the present invention consists in a recombinant
DNA molecule comprising a promoter having a nucleic acid sequence including the DNA sequence shown in Fig. 1, or a fragment thereof which includes the sequence from residues 284 to 409, or a functionally equivalent nucleic acid sequence, expressively linked to a further DNA sequence encoding a polypeptide.
It is preferred that the promoter includes the nucleic acid sequence from residues 284 to 409 as shown in Fig. 1.
In a preferred embodiment the polypeptide includes at least one at least one epitope. It is preferred that the polypeptide includes B-cell and/or T-cell epitopes. In another embodiment the polypeptide includes at least one CTL epitope. A preferred polypeptide is the 37 kD extracellular/secretory protein of Trichostrongylus colubriformis.
In a third aspect the present invention consists in a recombinant vector, the vector comprising an iron-regulated promoter and a site for insertion of a sequence encoding at least one polypeptide such that the inserted sequence is in frame with the iron-regulated promoter, wherein the iron-regulated promoter comprises the DNA sequence shown in Fig. 1. or a fragment thereof which includes the sequence from residues 284 to 409, or a functionally equivalent nucleic acid sequence. In a preferred embodiment the vector further includes an attP sequence and preferably the vector further includes a sequence encoding integrase protein.
It is also preferred that the vector further includes a sequence encoding at least one polypeptide inserted at the insertion site. It is preferred that the encoded polypeptide includes at least one epitope. Typically the encoded peptide will B and/or T-cell epitopes.
In another preferred form the encoded polypeptide includes at least one CTL epitope. As will be understood by those skilled in this area the sequence encoding the polypeptide may encode a plurality of CTL epitopes. Such an arrangement is disclosed in PCT/AU95/00461 the disclosure of which is incorporated herein by reference.
In yet another preferred embodiment the encoded polypeptide is the 37 kD extracellular/secretory protein of Trichostrongylus colubriformis. In a fourth aspect the present invention consists in a recombinant host cell, the host cell including a recombinant DNA molecule comprising a promoter having a nucleic acid sequence including the DNA sequence shown in Fig, 1, or a fragment thereof which includes the sequence from residues 284 to 409, or a functionally equivalent nucleic acid sequence, expressively linked to a further DNA sequence encoding at least one polypeptide.
It is also preferred that the recombinant DNA molecule is inserted into the host cell chromosome.
It is preferred that the host cell is a bacterium, preferably Gram negative, and more preferably the bacterium is Escherichia coli or
Salmonella species, and preferably the Salmonella species is Salmonella typhimuήum.
In a preferred embodiment the encoded polypeptide includes at least one at least one epitope. It is preferred that the polypeptide includes B-cell and/or T-cell epitopes. In another embodiment the polypeptide includes at least one CTL epitope. A preferred polypeptide is the 37 kD extracellular/secretory protein of Trichostrongylus colubriformis.
In a fifth aspect the present invention consists in a composition for use in inducing an immune response in an animal, the composition comprising a recombinant host cell of the present invention and an acceptable carrier. As used herein the term "functionally equivalent nucleic acid sequence" is intended to cover minor variations in the promoter sequence which do not result in a promoter having substantially lower activity from the promoter defined from residues 284 to 409 in Fig. 1. As will be appreciated in a preferred form the vector of the present invention includes the bacteriophage P22 int gene and an attP region. When this vector is introduced into a suitable bacterium, the bacteriophage P22 int gene is expressed to produce the int protein. A short DNA sequence called attP (attachment) in the vector is homologous to the attB sequence found in the bacterial chromosome. The bacterial chromosome expresses a protein called IHF (integration host factor) and that protein interacts with the expressed int protein from the vector which causes the homologous recombination of the vector DNA and the chromosomal DNA between attP and attB sequences. As a result of this integration, attB and attP recombine and stably integrate the vector into the bacterial chromosome. When the vector includes a DNA molecule encoding a foreign gene, upon integration, the bacterium is capable of expressing that gene from its chromosome to produce the foreign protein.
In a preferred embodiment of the present invention, the bacterium is an attenuated vaccine bacterium and the heterologous protein is derived from a virus, bacterium, fungus, yeast or parasite. More preferably, the bacterium is a S. typhimurium strain and the polypeptide is the 37 kD extra cellular/secretory protein from the nematode parasite of sheep Trichostrongylus colubriformis. The present inventors have developed a site-specific chromosomal integration system to integrate foreign antigen genes into the chromosome of attenuated S. typhimurium strains. The advantages of this system include:
1. It is based on site-specific recombination into the bacterial chromosome (non-essential region) therefore the site of integration is clearly defined.
2. The system does not use mobile genetic elements (e.g. transposons) and therefore once the vaccine strain is constructed the genotype and phenotype will not be altered by genes hopping around to other sites in the bacterial chromosome. 3. The efficiency of chromosomal integration approaches 100% i.e. all bacterial cells that are transformed by this DNA are found to have the heterologous polypeptide genes integrated in the bacterial chromosome.
4. The system is "user-friendly" and requires a single step cloning event to clone the gene of interest into the "chromosomal integration vector" followed by transformation of the attenuated S. typhimurium strain of choice.
5. This system is highly versatile and can be adapted for integration of genes into the chromosomes of a wide range of Gram-negative bacteria as well as eucaryotic cells.
6. The applications of this technology range widely and can be used as (a) basic-research tool to study chromosomally integrated genes in Gram- negative bacteria and Eucaryotic cells, (b) preparation of live attenuated vaccines, (c) tissue specific expression in gene therapy, and (d) development of transgenic plants and animals.
In order that the nature of the present invention may be more clearly understood, preferred forms will now be described with reference to the following drawings and examples. Brief Description of the Drawings Figure 1 shows the complete DNA sequence encoding a hybrid iron- regulated promoter according to the present invention;
Figure 2 shows the construction of plasmids including promoters according to the present invention;
Figure 3 shows details of the plasmid used for chromosomal integration in Salmonella;
Figure 4 shows anti-37KD serum titers;
Figure 5 shows anti β-galactosidase serum antibody titers;
Figure 6 shows egg counts; and
Figure 7 shows worm count in sheep intestinal lumen.
Construction of the Promoter
The promoter was initially cloned as an EcoRI/BamHI fragment is plasmid pSU207 (Fig. 2). Plasmid pHBlδδ (Fig. 2) is a pBR322 based plasmid carrying a polylinker. PCR primers HB38 and HB39 were designed to amplify part of the aerobactin gene promoter between positions 10 and 394 on the aerobactin promoter sequence. This region carriers minor and major promoters P2 and P2 respectively, along with the primary and secondary Fur binding sites. The Shine Dalgarno and downstream DNA sequence of the aerobactin promoter were not amplified. The PCR primers carried the Notl and Pad restriction enzyme cleavage sites and the shortened aerobactin promoter was subcloned into the Notl/Pacl sites of pHBl58 to give plasmid designated pHBl64 (Fig. 2).
PCR PRIMER SEQUENCES
HB38 CTCGAATTCGCGGCCGCCATATCCTCCCAGA HB39 CTCGGGCCCTTAATTAAACACAGTAAAATAATAAC
The 37 kD extracellular/secretory protein encoding gene from nematode parasite of sheep Trichostrongylus colubriformis was cloned as a lacZ gene fusion in plasmid pHbl67(Fig. 2). Upstream of the 5' region of the 37 kD gene sequence was engineered a DNA sequence carrying the Pad restriction enzyme site followed by Shine Dalgarno sequence (AGGA), a 7 bp spacer sequence (AACAGCT) and a translation start codon (ATG).
The Pacl/Xbal fragment form plasmid pHBl67 (Fig. 2) carrying the upstream region, along with the 37 kD/7αcZ genes was subcloned into the Pacl/Xbal sites of plasmid pHBl64 to give plasmid designated pHBl70(Fig. 2). This fusion of the aerobactin promoter (paer) with the upstream region provided a novel iron-regulated promoter according to the present invention. This promoter is unique in carrying the aerobactin promoter gene sequences for Pi and P2, as well as designed primary and secondary Fur binding sites, and designed Shine Dalgarno, spacer and ATG sequences.
The important sequence of the iron-regulated promotor (Fig. 1) lies from DNA sequence residues 284 to 409 inclusive. This region includes the -35 (residues 284 to 289) and -10 (residues 307 to 312) regions of minor promotor P2, the -35 (residues 340 to 345) -10 (residues 363 to 368) regions of major promotor Pi, the primary (residues 333 to 363) and secondary
(residues 364 to 382) Fur protein binding sites, the Pac I restriction enzyme site (residues 388 to 395), the Shine-Dalgarno sequence (residues 396 to 399) and the translation initiation site (residues 407 to 409). Demonstration of Induction of the Promoter
Plasmid pHBl70 was transformed into E. coli strain HB101 and S. typhimurium αroΛ-strain. The strains were grown in vitro under iron-rich conditions in Luria Broth with 200 μM FeCl3. Cultures for iron starvation were grown under identical conditions but with the inclusion of 100 μM 2,2'- dipyridyl (iron chelator). The FeCl3 and 2,2'-dipyridyl concentrations were optimised to obtain maximal repression and induction respectively without affecting growth rate or viability of the organisms.
Whole cell extracts of the recombinant E. coli and S. typhimurium aroA- (uninduced and induced) were electrophoresed on a 6% SDS- polyacrylamide gel and analysed by Western blotting. Purified β-galactosidase protein and purified 37 kD protein were also electrophoresed as controls. One of the blots was developed with anti β-galactosidase monoclonal antibody and the other was developed using 37 kD specific polyclonal sheep antiserum. The results demonstrated that in both E. coli and S. typhimurium aroA-, the hybrid promoter was strongly induced under iron starvation conditions. β-galactosidase assay was carried out on the uninduced and induced cultures and the results (Table 1) show that there is about a ten-fold increase in expression of the fusion protein following induction under iron starvation conditions.
TABLE 1. Quantitative analysis of fusion protein expression under the hybrid iron regulated promoter
Plasmid Growth β-Galactosidase
Strain Conditions (Units)
E. coli HB10 pHBl70 uninduced 840
E. coli HB101 pHB170 induced 8560
S. typhimurium pHB170 uninduced 255 aroA-
S. typhimurium pHB170 induced 2250 aroA- Construction of chromosomal integration vector.
Bacteriophage P22 integrates its genome into the chromosome of S. typhimurium by the following mechanism:
1. P22 attaches to the bacterial cell surface and injects its DNA into the bacterial cell.
2. The injected DNA circularises and expresses the INT (integration) protein encoded by the int gene.
3. The P22 genome has a short DNA sequence called the attP (attachment) which is homologous to the attB sequence found in the Salmonella chromosome.
4. The bacterial chromosome expresses a protein called the IHF (integration host factor).
5. The INT and IHF proteins interact to homologously recombine the attP and attB sequences thereby integrating the P22 genome into the bacterial chromosome (lysogeny).
Based on this system an integrating plasmid was constructed. Briefly, plasmid pNEB193 (purchased from New England Biolabs) was used as the plasmid into which the different gene insertions were made. plac/lac gene fragment which encodes the lac repressor protein, was PCR amplified from plasmid pLOF/Ars and cloned into the Pacl/Xbal restriction sites of plasmid pNEBl93 to result in plasmid pHBl78.
The ptac promoter gene fragment from plasmid pKK223-3 was PCR amplified and cloned into the Sacl/Kpnl sites of plasmid pHBl78 to result in plasmid pHBl79. The pαer/ArsA/ArsB gene fragment which encodes Arsenite resistance was PCR amplified from plasmid pLOF/Ars and cloned into the Hindi II site of plasmid pHBl79 to result in plasmid pHBlδO.
The bacteriophage P22 int attP region including its ribosome binding site-spacer-translation start (ATG) was PCR amplified from the P22 phage genome. This gene fragment was cloned into the Kpnl/Pacl site of plasmid pHBlδO to result in plasmid pHBlβl. Transformation of plasmid pHB181 into Salmonella typhimurium strain and confirmation of chromosomal integration.
Plasmid pHBlδl was transformed into S. typhimurium strain H4004 and transformants were selected on Brain Heart Infusion agar containing Ampicillin. The transformation efficiency was 10° colony forming units (c.f.u.) per microgram of DNA and was the same as control plasmid DNA. Plasmid DNA from individual transformants were analysed by agarose gel electrophoresis and the results showed that the plasmid had integrated into the chromosome. DNA from control E. coli cells harbouring the same plasmid revealed the presence of the plasmid. Southern hybridisation with the plasmid as a probe was used to confirm that the plasmid had integrated into the chromosome.
Sheep Vaccination and Protection Trial with the Recombinant Salmonellae.
6 month old sheep were vaccinated with various recombinant and control salmonellae. 5 animals were used per group and the sheep were vaccinated by the oral or intramuscular route.
The groups include: 1. S. typhimurium aroA- [strain 4335] - oral (101 1 organisms).
2. S. typhimurium aroA- [strain 4335] carrying plasmid pHBl70 (iron regulated promoter-37kD-lacZ) - oral (1011 organisms).
3. Same as (2) but cells were induced to maximal protein production (37kD/β-gal fusion) in-vitro, ethanol fixed - oral (1011 organisms). 4. Same as (3) - intramuscular (109 organisms).
5. S. typhimurium aroA- [strain 4335] carrying "iron regulated promoter-37kD-lacZ" cassette integrated into the chromosome - oral (1011 organisms). The plasmid used for this integration is shown schematically in Fig 3. 6. Same as (5) - intramuscular (109 organisms).
The sheep received three vaccinations at two week intervals. The sheep were challenged 2.5 weeks after the final vaccination with T. colubriformis L3 larvae over a 4 week period, the animals receiving 2000 L3 two times per week. Serum was collected at various time points following vaccination including pre-vaccination (negative control sera). The serum was analysed in ELISA assay to detect antibodies to the T. colubriformis 37kD polypeptide and to the β-galactosidase reporter polypeptide. The serum antibody titers are shown in Figs. 4 and 5.
Following challenge of the sheep, T. colubriformis eggs were counted in fecal samples at various time points. Results of egg counts are shown in Fig. 4. The sheep were euthanased about 2 months after commencement of challenge and the intestinal linings were scraped to collect T. colubriformis worms. The worm count data is shown in Fig. 5.
The results showed the following:
1. S. typhimurium aroA- carrying the plasmid pHBl70 (iron regulated promoter-37kD-lacZ) showed no serum antibody response to either 37kD or the β-galactosidase polypeptides.
2. The same salmonella (as in 1), when induced for maximal recombinant protein expression in-vitro and ethanol fixed prior to vaccination, gave a strong serum antibody response to both recombinant proteins particularly in the intramuscular vaccinated sheep (Figs. 2 & 3). Presumably these salmonellae function like a non-living carrier e.g. a liposome packed up with the recombinant protein. The salmonellae in (1) were also induced in-vitro in the same way but were live, and our previous results had shown rapid plasmid was loss in in-vivo (segregation) and presumably the vaccine lost the initial load of recombinant proteins in-vivo.
3. Recombinant S. typhimurium aroA- carrying the same gene cassette (chromosomally integrated)as in (1) & (2), however, gave enhanced serum antibody titers to the recombinant polypeptides (Figs. 2 & 3). The orally immunised group produced lower titers but intramuscular immunisation gave titers that matched the fixed salmonella group. This result clearly demonstrates that chromosomally integrated gene cassettes possibly under the control of in-vivo inducible promoters are effective in inducing recombinant protein synthesis in vivo and eliciting at least antigen-specific serum antibody responses. 4. Similar results are obtained for egg counts (Fig. 4). The data for all groups except for chromosomally integrated salmonellae (oral and i.m. immunised) are similar to the background level seen in S. typhimurium aroA- immunised sheep. There is however, a 30% reduction in egg counts (compared to background level) in orally immunised sheep (chromosomally integrated Salmonella) and a more dramatic an significant (p<0.05) 61% reduction in egg counts in the i.m. group (chromosomally integrated).
5. The worm count data (Fig. 5) parallels that of egg counts and the i.m. vaccinated sheep (chromosomally integrated) had a significant (p<0.05) reduction of 61% in worm burden.
It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are. therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:-
1. An iron- regulated promoter comprising the DNA sequence shown in Fig. 1, or a fragment thereof which includes the sequence from residues 284 to 409, or a functionally equivalent nucleic acid sequence.
2. An iron-regulated promoter as claimed in claim 1 in which the promoter includes the nucleic acid sequence from residues 284 to 409 as shown in Fig. 1.
3. A recombinant DNA molecule comprising a promoter having a nucleic acid sequence including the DNA sequence shown in Fig. 1, or a fragment thereof which includes the sequence from residues 284 to 409. or a functionally equivalent nucleic acid sequence, expressively linked to a further DNA sequence encoding a polypeptide.
4. The recombinant DNA molecule as claimed in claim 3 in which the promoter includes the nucleic acid sequence from residues 284 to 409 as shown in Fig. 1.
5. The recombinant DNA molecule as claimed in claim 3 or claim 4 in which the polypeptide includes at least one epitope.
6. The recombinant DNA molecule as claimed in claim 5 in which the polypeptide includes B-cell and/or T-cell epitopes.
7. The recombinant DNA molecule as claimed in claim 5 or claim 6 in which the polypeptide includes at least one CTL epitope.
8. The recombinant DNA molecule as claimed in any one of claims 3 to 5 in which the polypeptide is the 37 kD extracellular/secretory protein of Trichostrongylus colubriformis.
9. A recombinant vector, the vector comprising an iron-regulated promoter and a site for insertion of a sequence encoding at least one polypeptide such that the inserted sequence is in frame with the iron-regulated promoter, wherein the iron-regulated promoter comprises the DNA sequence shown in Fig. 1, or a fragment thereof which includes the sequence from residues 284 to 409, or a functionally equivalent nucleic acid sequence.
10. A recombinant vector as claimed in claim 9 in which the vector further includes an attP sequence.
11. A recombinant vector as claimed in claim 10 in which the vector further includes a sequence encoding integrase protein.
12. A recombinant vector as claimed in any one of claims 9 to 11 in which the vector further includes a sequence encoding at least one polypeptide inserted at the insertion site.
13. A recombinant vector as claimed in claim 12 in which the polypeptide includes at least one epitope.
14. The recombinant vector as claimed in claim 13 in which the polypeptide includes B-cell and/or T-cell epitopes.
15. The recombinant vector as claimed in claim 13 or claim 14 in which the polypeptide includes at least one CTL epitope.
16. A recombinant vector as claimed in claim 12 or claim 13 in which the polypeptide is the 37 kD extracellular/secretory protein of Trichostrongylus colubriformis.
17. A recombinant host cell, the host cell including a recombinant DNA molecule comprising a promoter having a nucleic acid sequence including the DNA sequence shown in Fig. 1, or a fragment thereof which includes the sequence from residues 284 to 409, or a functionally equivalent nucleic acid sequence, expressively linked to a further DNA sequence encoding at least one polypeptide.
18. A recombinant host cell as claimed in claim 17 in which the recombinant DNA molecule is inserted into the host cell chromosome.
19. A recombinant host cell as claimed in claim 17 or claim 18 in which the host cell is a bacterium.
20. A recombinant host cell as claimed in claim 19 in which the bacterium is Gram negative, more preferably the bacterium is Escherichia coli or Salmonella species, and preferably the Salmonella species is Salmon ella typhim urium .
21. A recombinant host cell as claimed in any one of claims 17 to 20 in which the at least one polypeptide includes at least one epitope.
22. The recombinant host cell as claimed in claim 21 in which the polypeptide includes B-cell and/or T-cell epitopes.
23. The recombinant host cell as claimed in claim 21 or claim 22 in which the polypeptide includes at least one CTL epitope.
24. A recombinant host cell as claimed in any one of claims 7 to 21 in which the at least one polypeptide is the 37 kD extracellular/secretory protein of Trichostrongylus colubriformis.
25. A composition for use in inducing an immune response in an animal, the composition comprising a recombinant host cell as claimed in any one of claims 17 to 24 and an acceptable carrier.
PCT/AU1997/000503 1996-09-06 1997-08-08 Iron regulated promoter and uses thereof WO1998010064A1 (en)

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BR9711692-0A BR9711692A (en) 1996-09-06 1997-08-08 Regulated steel promoter
EP97933590A EP0964924A4 (en) 1996-09-06 1997-08-08 Iron regulated promoter and uses thereof
CA002265883A CA2265883A1 (en) 1996-09-06 1997-08-08 Iron regulated promoter and uses thereof
AU36892/97A AU737981B2 (en) 1996-09-06 1997-08-08 Iron regulated promoter and uses thereof
NZ334649A NZ334649A (en) 1996-09-06 1997-08-08 Iron regulated promoter derived from an aerobactin gene promoter

Applications Claiming Priority (6)

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AUPO2164A AUPO216496A0 (en) 1996-09-06 1996-09-06 Inducible promoters
AUPO2164 1996-09-06
AUPO2454A AUPO245496A0 (en) 1996-09-19 1996-09-19 Site-specific chromosomal integration system
AUPO2454 1996-09-19
AUPO7801A AUPO780197A0 (en) 1997-07-09 1997-07-09 Site-specific chromosomal integration system incorporating inducible promoter
AUPO7801 1997-07-09

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WO1992011361A1 (en) * 1990-12-18 1992-07-09 The General Hospital Corporation Improved vaccines

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Publication number Priority date Publication date Assignee Title
WO1992011361A1 (en) * 1990-12-18 1992-07-09 The General Hospital Corporation Improved vaccines

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BINDEREIF A, NEILANDS J B: "PROMOTER MAPPING AND TRANSCRIPTIONAL REGULATION OF THE IRON ASSIMILATION SYSTEM OF PLASMID COLV-K30 IN ESCHERICHIA COLI K-12", JOURNAL OF BACTERIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 162, no. 03, 1 June 1985 (1985-06-01), US, pages 1039 - 1046, XP000974243, ISSN: 0021-9193 *
LORENZO DE V, ET AL.: "OPERATOR SEQUENCES OF THE AEROBACTIN OPERON OF PLASMID COLV-K30 BINDING THE FERRIC UPTAKE REGULATION (FUR) REPRESSOR", JOURNAL OF BACTERIOLOGY, AMERICAN SOCIETY FOR MICROBIOLOGY, US, vol. 169, no. 06, 1 June 1987 (1987-06-01), US, pages 2624 - 2630, XP000974242, ISSN: 0021-9193 *
See also references of EP0964924A4 *
VERKUYLEN A J, ET AL.: "CHARACTERIZATION OF THE MRNA ENCODING A PROLINE-RICH 37-KILODALTON GLYCOPROTEIN FROM THE EXCRETORY-SECRETORY PRODUCTS OF TRICHOSTRONGYLUS COLUBRIFROMIS", MOLECULAR AND BIOCHEMICAL PARASITOLOGY, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 58, 1 April 1993 (1993-04-01), NL, pages 325 - 332, XP000983170, ISSN: 0166-6851, DOI: 10.1016/0166-6851(93)90055-3 *

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