WO2001030384A1 - Compositions de vaccin - Google Patents

Compositions de vaccin Download PDF

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Publication number
WO2001030384A1
WO2001030384A1 PCT/US2000/029231 US0029231W WO0130384A1 WO 2001030384 A1 WO2001030384 A1 WO 2001030384A1 US 0029231 W US0029231 W US 0029231W WO 0130384 A1 WO0130384 A1 WO 0130384A1
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dtxr
metal ion
composition
bacterium
independent
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PCT/US2000/029231
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John R. Murphy
Edward O'lear
Robert J. Harrison
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Advanced Microbial Solutions Corporation
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    • 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
    • C07K14/34—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Corynebacterium (G)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents

Definitions

  • This invention relates to avirulent or attenuated microbes, their method of preparation, and their use in vaccines.
  • Infectious disease remains the largest cause of mortality in the world. A significant proportion of infectious disease associated morbidity and mortality results from bacterial pathogens.
  • One widely applied technique used in controlling the spread and severity of bacterial infection is vaccination.
  • the DPT vaccine protects against diphtheria, pertusis and tetanus.
  • the BCG or bacillus Calmette-Guerin vaccine is an example of an attenuated strain which is used worldwide to control the spread of tuberculosis (TB).
  • a central issue in the development of safe an effective bacterial vaccines is the identification of protective antigens or attenuated strains of bacteria which can promote the development of an immune response in the host yet in vaccine composition fail to cause morbidity or martality in the host.
  • a variety of methods can be employed to identify vaccine compositions including the production of attenuated or killed vaccine strains. Often these strains are in single genes which have been shown to play a role in virulence of the organism.
  • Vaccine compositions can include a live or fixed bacterial preparation of the mutant strain, the fixed protein, a fusion protein made of the virulence gene product and a suitable carrier and more recently the DNA encoding the protein.
  • Attenuated strains offer the potential of presenting a nearly intact complement of pathogen associated antigens to immune system. Furthermore these antigens are likely to be presented in a bacterial context that mirrors that seen during early stages of infection by a virulent strain. This is evidenced by the ongoing practice of using live attenuated strains in the extensive vaccination of both human and livestock. For example BCG for tuberculosis and strain 19 against bovine brucellosis and Sterne's spore vaccine against anthrax in cattle. The use of live vaccines can present developmental obstacles including the retention of unacceptable levels of virulence, the risk of reversion to virulence during culture or in vivo and lack of efficacy.
  • the ability to create more effective live or attenuated vaccines is in part dependent upon the ability to control and restrict the expression of virulence determinants so as to create vaccine strains that are protective and safe.
  • Bacteria respond to nutritional stress by the coordinated expression of different genes. This facilitates their survival in different environments.
  • these differentially regulated genes are the genes responsible for the expression of virulence determinants.
  • the selective expression of these genes in a sensitive or susceptible host allows for the establishment and maintenance of infection or disease.
  • Virulence include genes which encode toxins, colonization factors and genes required for siderophores production or other factors that promote this process.
  • virulence genes in bacteria therefore enable the organism to invade, colonize and initiate an infection in humans and/or animals, however, these genes are not necessarily expressed constantly (constitutive ly). That is, the bacterium is not always orchestrating gene expression patterns to maximize "infectious" potential.
  • the expression of virulence genes is controlled by regulatory circuitry which include repressor proteins and a corresponding operon or operator.
  • repressor proteins One class of repressors which is activated upon binding to or forming a complex with a transition metal ion such as iron, zinc or mangenese is thought to control the expression of a subset of genes in a number of Gram positive organisms. When such repressors are activated and associated with virulence gene expression in pathogens, they bind the operator sites thereby preventing production of virulence determinants.
  • Virulence determinants are most often expressed when the bacterial pathogen is exposed to environmental stress such nutritional restriction.
  • An iron- poor environment is an example of such a condition.
  • insufficient iron is present to maintain the repressor in its active state.
  • the repressor cannot bind to target operators.
  • virulence genes are de-repressed and the bacterium is able to initiate, establish, promote or maintain infection.
  • the expression of these virulence determinants is in many bacterial species co-regulated by metal ions.
  • the metal co-factor that is involved in vivo is iron [but can include zinc, nickel, mangenese, cobalts].
  • the repressor is activated and virulence gene expression is halted. This pattern of gene regulation is illustrated by the following example.
  • the bacterium that causes diphtheria produces one of the most potent toxins known to man.
  • the toxin is only produced under conditions of iron deprivation.
  • the bacterial repressor which in this species is known as diphtheria toxin repressor protein, abbreviated "DtxR') binds iron and undergoes conformational changes that activate it and allow it to dimerize and bind a specific DNA sequence called the tox operator.
  • the tox operator is a specific DNA sequence found upstream of the gene that produces the diphtheria toxin, thereby preventing its expression.
  • diphtheria bacillus or other pathogenic/opportunistic bacteria
  • the diphtheria bacillus grows in an environment that rapidly becomes restricted in several key nutrients. Paramount among these essential nutrients is iron, and when iron becomes limiting the diphtheria bacillus begins to produce the toxin.
  • the constellation of virulence genes thatDtxR controls becomes de-repressed and the diphtheria bacillus becomes better adapted to cause an infection.
  • the toxin kills host cells thereby releasing required nutrients including iron.
  • Figure 1 aligns the amino acid sequences of DtxR, IdeR and SirR.
  • Figure 2 aligns amino acid sequences of various IdeR/SirR Homologues found in various species of mycobacterium.
  • Figure 3 aligns and compares the amino acid sequence of various homologues of various DtxR type repressors, including DtxR from Brevib ⁇ cterium l ⁇ ctofermentum (Bl), DtxR from Coryneb ⁇ cterium diphtheri ⁇ e (Cd), IdeR from Mycobacterium tuberculosis (Mt), M. leprae [P], M. smegmatis [P]; DesR from Streptomyces lividans (SI), M.
  • DtxR homologue [P].
  • the consensus amino acid sequences between these members of the DtxR family of iron- dependent repressors is indicated. *, metal ion coordination residues in the Primary site; #, metal ion coordination residues in the Ancillary site; @, the single amino acid residue that interacts with a base in the binding of DtxR dimers to the tox operator.
  • AMS has clones of M. tuberculosis IdeR, S. aureus and S. epidermidis SirR, and DtxR homologues from Enterococcus fecalis, S. mutans, S. pneumoniae, and K. pneumoniae.
  • Figure 4 Western blot of cell lysates incubated with polyclonal antibody against DtxR.
  • Lane 1 shows purified DtxR (25.3 kDa).
  • Lanes 3 and 5 lysates from wild type M.smeginatis and M.tuberculosis, respectively, expressing native IdeR (25.2 kDa.).
  • Lane 4 lysate from the M.smegmatis heterodiploid harboring pNBVl/SAD expressing both DtxR(E175K) and IdeR. The molecular weight masses, determined by size standards, are shown at left.
  • Figure 5 Virulence comparison of wild type M.tuberculosis and M.tuberculosis DtxR(E175K) mutant.
  • Panel A shows the log CFU of the homogenized spleens of mice sacrificed at 4 week intervals.
  • Panel B shows the log CFU of homogenized lungs at 4 week intervals. Each point represents the meanlog CFU of 5-6 mice + 1 standard deviation (error bars). Asterisks denote statistically significant differences between groups at a given time point.
  • Figure 6 Panel A: a 10 week-old representative colony of wild type
  • Figure 7 Alignment of the "iron box” consensus sequence, known DtxR binding sites, and putative M. tuberculosis DtxR/IdeR binding sites identified by an in silico genome search.
  • the "consensus sequence” at the top of the figure represents the compilation of the 9 aligned sequences in the figure.
  • the “published consensus” is drawn from the literature. Gene homologues of the downstream ORFs are shown on the right.
  • Figure 8 Autoradiographs of gel binding assay between DtxR and putative M.tuberculosis DtxR/IdeR binding sites. 100 bp 32p. en( j. ⁇ a beled DNA fragments containing toxO (lanes 1 & 2), IB-1 (lanes 3 & 4), IB-2 (lanes 5 & 6), IB- 3 (lanes 7 & 8), IB-4 (lanes 9 & 10), IB-5 (lanes 1 1 & 12) separated in a non- denaturing 6% polyacrylamide gel. Odd numbered lanes contain DNA only ("unbound"), and even numbered lanes contain DNA pre-incubated with purified DtxR ("bound"). Figure 9. Virulence of S.
  • aureus is altered in a mouse skin lesion model following 8 days of in vivo incubation.
  • 8.0 log CFU of the parent strain MA2181 [RN6390 carrying emtpy shuttle vector pSPTl ⁇ l] and 7.8 log CFU of the complemented MA2004 strain carrying DtxR E175K were injected sub-cutaneously on day 1 abscess size [mm] was measured each day over 8 days and on the last day the abscess was removed and the number of CFU were determined.
  • CFU [a.] and abscess size [b.] were compared between groups.
  • a first aspect of the present invention is directed to a composition containing a virulent or opportunistic prokaryote in which metal ion-dependent gene regulation confers a growth or an infectious advantage.
  • the prokaryote contains a recombinant DNA molecule comprising a promoter in operable association with a sequence encoding a dominant, metal ion-independent repressor protein or a partially metal ion independent repressor protein, and a carrier.
  • a promoter is constitutive in nature.
  • the DNA molecule contains a sequence encoding a metal ion-independent DtxR protein or a partially metal ion-independent DtxR protein.
  • the bacterium is a member of the genus Mycobacterium, Staphylococcus or Streptococcus.
  • the second aspect of the present invention is directed to a method of enhancing protective immunity against infection or disease caused by an opportunistic or virulent prokaryote pathogen in which metal or metal ion-dependent gene regulation confers a growth or an infectious advantage.
  • the method entails administering compositions to the animal.
  • the animal is a human.
  • the compositions are administered prophylactic ally e.g. prior to the onset of the infection or disease.
  • the prokaryote contained in the compositions is in a live or killed form.
  • a relatively aspect directed to is a method of attenuating or reducing the severity of an infection or a disease caused by an opportunistic or virulent prokaryotic pathogen in which metal or metal ion-dependent gene regulation confers a drug or an infectious advantage.
  • the method also entails administering the compositions to the animal, preferably prior to the onset of the infection or disease condition.
  • Yet another aspect of the present invention is directed to isolated and purified DNA molecules consisting essentially of a sequence encoding a metal or metal ion-independent or a partially metal or metal ion-independent DtxR or homologue thereof.
  • Preferred homologues are IdeR and SirR.
  • the molecule is placed in an expression cassette or a vector (e.g., a plasmid) so as to be inoperable association with a promoter element, especially a constitutive promoter.
  • Vectors containing the DNA molecules and prokaryotes transformed with DNA molecules are also provided.
  • a further aspect of the present invention is directed to a method for preparing the compositions.
  • the method entails obtaining a DNA molecule encoding a metal ion-independent repressor protein or a partially metal ion-independent repressor protein.
  • the wild type protein in its native state, is a metal ion-dependent gene regulator and confers upon a virulent or opportunistic prokaryote a growth or an infectious advantage.
  • the DNA molecule is linked to a promoter, preferably a constitutive promoter, and then introduced into such a virulent or opportunist prokaryote.
  • the DNA is expressed in the prokaryote and inhibits metal ion-dependent gene regulation.
  • the present invention entails the incorporation of an exogenous DNA encoding a dominant, metal ion (hereinafter "metal") independent or partially metal ion independent mutant repressor into an otherwise virulent or opportunistic prokaryote in which metal ion-dependent gene regulation confers a growth or an infectious advantage.
  • metal ion hereinafter "metal”
  • prokaryotis include gram-positive and gram-negative bacteria.
  • the dominance of the metal-independent mutant repressor subverts the normal patterns of gene regulation (under the control of the native, metal-dependent repressor), thereby creating a recombinant prokaryote that is attenuated or avirulent relative to the wild- type prokaryote. That is, the exogenous mutant repressor renders inoperable or significantly inoperable the normal metal dependent genetic circuitry that occurs in vivo and causes in hole or in part the prokaryote carrying such a recombinant genetic compliment to become less infectious or non-pathogenic. This property renders the recombinant prokaryote suitable for use as an immunogen to be formulated into a vaccine.
  • compositions of the present invention a determination is made as to whether or not the species of interest regulates virulence determinant expression as a function of available metal ion concentrations. This can be done, for example, by screening protein from the bacteria of interest with an antibody for DtxR or other DtxR like proteins to ascertain if a homologous repressor exists in the species. This can also be accomplished using specialized techniques like gel mobility shift assays or the method disclosed in Sun et al, PNAS 5: 14985-14990 (1998), or more common gene expression monitoring methods such northern analysis, and PCR.
  • the expression of this repressor can be elucidated by one of the aforementioned methods and the techniques described here can be employed to build a recombinant attenuated strain for vaccine purposes.
  • Preferred prokaryotes are Gram positive bacterial species, and particularly those listed below. These species contain DtxR like metal dependent repressors. Specific examples include:
  • the repressor which typifies the class of genetic regulators in the above listed bacteria is the diphtheria toxin repressor DtxR in C. dihptheriae, the causitive agent of diphtheria.
  • DtxR is a metal dependent repressor which under limiting concentrations of metal ions becomes inactivated permitting the derepression of a number of virulence genes including diphtheria toxin.
  • This pattern of gene expression is common to both Gram positive and Gram negative bacteria.
  • DtxR or DtxR homologues appear to be important metal dependent regulators whereas in Gram negative bacteria, Fur is the significant metal dependent regulator.
  • Some species of pathogen appear to contain both DtxR and Fur like metal dependent repressors.
  • the presence of repressor bound metal ion is critical for appropriate activity of the repressor which coordinates the repression of gene expression in vivo.
  • the prokaryotes of the present invention having a dominant, metal ion independent or partially metal ion independent mutant of the repressor (such as the diphtheria toxin repressor gene DtxR or a D txR-homolog e.g., IdeR, SirR) will render the pathogen unable to effectively establish a full infection. (Hill et al, (1998)
  • the recombinant prokaryote is in all other aspects identical genetically to the wild-type organism.
  • the presence of the dominant iron independent repressor results in a phenotypic change in the organisms virulence.
  • the exogenous DNA comprises a sequence encoding a dominant, metal-independent DtxR or a functional fragment, variant or homologue thereof (collectively referred to as "a DtxR protein").
  • DtxR is a metal iron-dependent DNA-binding protein having a deduced molecular weight of 25,316 and which functions as a global regulatory element for a variety of genes on the C. diphtheriae chromosome. See Tao et al, Proc. Natl. Acad. Sci. USA 59:5897-5901 (1992); Schmitt et al, Infect. Immun. 59:1899-1904 (1994). For example, DtxR regulates the expression of the diphtheria toxin structural gene (t t) in a family of closely related Corynebacteriophages. The repressor has also been shown to regulate a number of other iron dependent genes.
  • DtxR The gene for DtxR and a number of DtxR homologues have been cloned and sequenced. A number of detailed structural and functional studies have be performed to analyse DtxR. See Boyd et al, Proc. Natl. Acad. Sci. USA 57:5968-5972 (1990); Schmitt et al, supra. DtxR is activated by divalent transition metal ions (e.g., iron). Once activated, it specifically binds the diphtheria tox operator and other related palindromic DNA targets. See Ding et al, Nature Struct. Biol. 3 ⁇ :382-387 (1996); Schiering et al. Proc. Natl. Acad. Sci.
  • divalent transition metal ions e.g., iron
  • DtxR fragments and variants can be identified by standard techniques such as mutagenesis. Tao et al, Proc. Natl. Acad. Sci. USA 90:8524-8528 (1993) identified important residues for DtxR function and analysis. Other variants are disclosed in Tao et al, Mol. Microb. 14(2): 191-197 (1994). Tao discloses that some t R alleles have different amino acid sequences, e.g., theDtxR allele from strain 1030(-) of C.
  • diphtheriae was found to carry six amino acid substitutions in the C-terminal region, none of which affected the iron-dependent regulatory activity of DtxR (1030) (Tao II). See also Boyd et al, J. Bacteriol. 774: 1268-1272 (1992) and Schmitt et al, Infect. Immun. 59:3903-3908 (1991). Thus, DtxR fragments and variants may be mutagenized to an iron-independent genotype.
  • Staphylococcal iron regulated repressor native to Staphylococcus epidermitis
  • SirR Staphylococcal iron regulated repressor
  • These proteins bear a common feature they share a remarkably high sequence similarity in the respective N-terminal 139 amino acid regions, especially those amino acids involved in DNA recognition and transition metal ion coordination.
  • accession numbers for sequences that are either homologous to DtxR or contain a consensus tox O/P is presented in Table 1. See http://www.ncbi.nlm.nih.gov/BLAST and http://www.ncbi.nlm.nih.gov/unfinishedgenomes.html.
  • ferrooxidans Preferred gram positive pathogenic bacteria include Mycobacterium bovis, Mycobacterium leprae, Mycobacterium paratuberculosis, Mycobacterium tuberculosis, Mycobacterium avium, Staphylococccus aureus, Staphylococcus epidermitis, Streptoccus mutans and Streptococcus pneumoniae.
  • the preferred dominant DtxR (or homologue) repressors are metal independent and contain a single amino acid change to convert the native, metal-dependent repressor to metal independent regulation.
  • a preferred metal independent repressor is the mutant E175K DtxR. This same substitution can be made in DtxR fragments and variants.
  • DtxR homologues that exist in other bacterial species may be mutated at the corresponding position.
  • Figure 1 displays the degree of amino acid homology between DtxR and to homologous proteins, IdeR from Mycobacterium tuberculosis and SirR from Staphylococcus epidermitis. As shown in fig. 1 , the single amino acid at residue E175K in DtxR, the glutamic acid is conserved in IdeR hence a 177K mutation of IdeR would most likely have the analogous functional implications. Dussurget et al (1996) Molecular Microbiology 22 536-544 and Pohl et al(1999) J. Molecular Biology 285 1 145-1156).
  • Figure 2 presents a comparison of IdeR/SirR homologues found in other mycobacterium that cause significant disease including; M. tuberculosis, M smegmatis, M. leprae and a SirR clones from tuberculosis. Shown in bold is the conserved glutamic acid in the C-terminal region of these repressors that can potentially be mutated to yield an iron independent version of each of these repressors. Thus, corresponding mutations would be expected to result in a metal- independent genotype.
  • Figure 3 illustrates the amino acid sequence homology of a number of homologous DtxR type repressors.
  • a combination of standard techniques may be used to make other dominant, metal-independent DtxR proteins, namely mutagenesis followed by tests to determine if a given mutant binds the corresponding operator.
  • a number of theoretical mutations can be incorporated which will convert the repressor to be metal-independent.
  • any of several saturation mutagenesis techniques can be utilized. DNA, plasmid preparation, and DNA sequence analysis are performed according to standard methods. The saturation of random mutations throughout the length of repressors DNA of interest introduces random changes in amino acid sequence throughout the encoded protein that potentially can confer a metal independent phenotype on the mutant repressor.
  • DtxR homologues are identified in a species of interest. This can be achieved by performing PCR on genomic DNA from the species of interest using primer sets compatible to conserved domains within the DtxR family. Specific sequences may be composed of degenerate primers flanking the iron binding domain, or the helix-turn-helix domain. Cloning may also be performed from phage libraries using DtxR conserved regions as probes, utilizing the PSTD system or by in silico searches and de novo in vitro synthesis.
  • the DtxR has a domain structure which is composed of a helix-turn- helix domain, and N-terminal iron binding domain.
  • Cloning of DtxR homologues from any given species can be achieved by mixing primers with genomic DNA isolated from the species of interest in the appropriate ratios in the presence of free oligonucleotides, optimized buffer conditions and the TAQ polymerase (or a suitable version of a thermostable polymerase) and cycling the reaction conditions with the aid of an automated thermocycler.
  • the PCR reaction generates a series of products that represent target DNA sequences that are bounded by sequences homologous to the selected 5' and 3' primers.
  • the selection of primers which lie within conserved regions of DtxR will likely bind under the appropriate conditions to homologous DNA that will likely represent a gene or domain similar to that of DtxR.
  • DtxR homologues may further be identified by screening genetic libraries of a given species created inE. coli. Pathogen libraries can then be screened by radio-labeled probes generated from DtxR clones, oligonucleotides from the DtxR sequence, or protein assays using antibodies directed against DtxR.
  • the first approach is a knock-in approach which is coupled with in vitro mutagenesis or PCR mutagenesis.
  • the knock-in approach is focused on generating strains having dominant activated repressors. These strains contain a defective or altered copy of DtxR or a DtxR homologue that containing at least one but up to several mutations resulting in a repressor that recognizes and binds the toxAPO or suitable cognate binding sites in both the presence and absence of iron (or the appropriate metal ion).
  • These metal ion independent mutants are cloned into suitable vectors (e.g., targeting vectors having a selectable marker and restriction sites) to develop strains expressing dominant activated DtxR constitutively.
  • suitable vectors e.g., targeting vectors having a selectable marker and restriction sites
  • These plasmids are used to generate knock-in vaccine strains, essentially altered forms of the wild-type virulent strain differing in only the presence of a dominant activated DtxR or DtxR-homologous repressor.
  • the constructs can also be used in gene replacement strategies in which the mutant metal independent repressor gene replaces the endogenous wild type gene.
  • Metal ion independent clones can be sequenced to identify the specific amino acid changes that confer the iron independent phenotype. Again, a preferred mutant is characterized by a change from glutamic acid to lysine at position 175 in native DtxR.
  • multiple mutations are also embraced by the present invention. Multiple mutations are advantageous because they greatly decrease the likelihood of reversion to wild-type function. This likelihood becomes statistically more akin to that of a strain which has all of its iron regulated genes knocked out. Multiple mutations either clustered or distributed through out the repressor can result in the same phenotype.
  • a double mutant DtxR with the replacement of asparagine at position 130 with glycine and the glutamine at position 181 with arginine was also identified with a metal independent phenotype.
  • a mutant with an intermediate phenotype was also identified having a total of six mutationsf valine 5 to isoleucine, aspartic acid 1 10 to glutamic acid, valine 1 12 to phenylalanine, isoleucine 153 to threonine, aspartic acid 197 to glutamic acid, threonine 220 to alanine].
  • These mutations produced a partially metal dependent phenotype.
  • the present invention also embraces the use of partially metal ion-independent repressors.
  • DtxR mutants of the present invention are not limited to those characterized by single or multiple amino acid substitutions. Insertion and deletion mutants are also contemplated. These mutants may be identified using the instantly disclosed techniques as well.
  • Strains and putative mutants can be tested for metal independent phenotype by several approaches including the PSTD screen described in Sun et al. Additional methods of determining if conversion to iron independent phenotype include computer modeling, structural analysis, dimerization analysis by gel electrophoresis, DNA binding by electrophoretic mobility shift assay, transcriptional profiling, tissue culture and in vivo virulence assays. Ultimate ⁇ , in vivo screening will be required to determine if the iron independent phenotype is stable in a host environment and to determine if virulence is attenuated. Attenuated virulence can be defined by biochemical, physiological and immunological markers but will minimally include an assessment of ED 50 or LD 50 in wild-type, knock-in, replacement and recombinant strains.
  • a second rational mutagenesis strategy can be utilized to generate metal-independent mutants. This strategy relies upon the conserved domain structure of the DtxR family of repressors.
  • DtxR iron independent mutants have been identified and published. These mutants define two classes of iron independent mutations which are likely to alter iron dependent regulation in DtxR homologues.
  • Site directed mutagenesis of the analogous amino acid residues in DtxR homologues may have the same iron independent phenotype.
  • mutants that replace amino acids involved in the coordination of iron binding or dimerization that result in an iron independent phenotype may be constructed.
  • Mutagenesis is then carried out to generate a library of the desired mutants of this DtxR homologue of interest. Identification of iron independent mutants from this population is achieved by using the PSTD system. Using the cloned and mutant homologue genes, one can select for the growth of colonies in the PSTD strains in the presence of dipyridyl (DP). Dipyridyl chelates iron from the media and therefore leads to the disassociation of DtxR homologues from the regulatory regions of the genetic elements in the PSTD screen. As a result, all iron dependent repressors will not be able to survive chloramphenicol selection, in contrast iron independent mutants will grow.
  • DP dipyridyl
  • Mutants of DtxR are generated in accordance with standard techniques. Polymerase chain reaction (PCR) mutagenesis of the dtxR gene is described in Vartanian et al. [Vartanian, J.-P., Henry, S., & Wain-Hobson, S. (1996) Hypermutagenic PCR involving all four transitions and a sizeable proportion of transversions. Nucleic Acid Res., 24, 2627-2631.].
  • PCR Polymerase chain reaction
  • .Sg/II-tagged primers 1515 (5'-ACCAGATCTGCCGAAAAACTTCGA-3') and 1516 (5'- ACCAGATCTCCGCCTTTAGTATTTA-3') were used to PCR amplify dtxR from plasmid pRDA which carries the wild type dtxR operon.
  • the products of the amplification were then digested with Bglll and ligated either into 5g7II-linearized pSC6Ml and transformed into E. coli TOP10/ IRS65T, or ligated into BamRl digested pBR322 and transformed into E. coli TOP10/ tRS65T/pSC6.
  • Iron- independent mutants of DtxR were then selected on LB agar plates supplemented with Cm and DP in accordance with the procedure described in Sun, et al.
  • Bacterial clones containing mutagenized dtxR can be analysed by DNA sequencing and used in functional biochemical assays such as electrophoeretic mobility shift assay, native gel analysis and gluteraldehyde crosslinking studies to reveal the activated state of the repressor in question under metal limiting conditions. This can be determined through gel shift analysis or by functional assays, but it is preferably made using the one-step method described by Frigg, et al [Sun, L., vanderSpek, J. & Murphy, J. R. (1998) roc.
  • the native tox operator i.e., 5'-ATAATTAGGATAGCTTTACCTAATTAT-3'
  • 5'-ATAATTAGGATAGCTTTACCTAATTAT-3' is a 27 base pair interrupted palindromic sequence upstream of the diphtheria tox structural gene can be used as a probe. This sequence features a 9-base pair inverted repeat sequence that is separated by 9 base pairs. See Kaczorek et al, Science 227:855-858 (1983); Greenfield et al, Proc. Natl. Acad. Sci. USA 50:6853-6857 (1983); Ratti et al, Nucleic Acids Res.
  • Additional probes are variants of ToxO based on the DtxR consensus-binding sequence (5'- ANANTTAGGNTAGNCTANNCTNN-3'). Variants are defined by the following sequence: 5'-TWAGGTTAGSCTAACCTWA-3 ⁇
  • the function of the repressor and mutant can be defined by recognition and binding or regulation of gene expression via the sequences or variants described above.
  • DtxR DNA clone Once a dominant, metal-independent DtxR DNA clone is identified, it can be produced and manipulated in accordance with techniques known in the art. For example, they may be generated using standard chemical synthesis techniques. See, e.g., Merrifield, Science 233:341-347 (1986) and Atherton et al, Solid Phase Synthesis, A Practical Approach, IRL Press, Oxford (1989). Preferably, they are obtained by recombinant techniques.
  • DNA constructs encoding the DNA gene segments may also be prepared synthetically by established methods, e.g., in an automatic DNA synthesizer, and then purified, annealed, ligated and cloned into suitable vectors. Atherton et al, supra. Polymerase chain reaction (PCR) techniques can also be used. See e.g., PCR Protocols: A Guide to Methods and Applications, 1990, Innis et al. (ed.), Academic Press, New York.
  • the DNA encoding the mutant metal-independent repressor is operably linked to a promoter element.
  • Preferred promoters include the endogenous DtxR promoter or the promoter of the DtxR homologue in question or any suitable constitutive promoter functional in the species of interest, the constructs may further contain an associated selectable marker gene to follow maintenance of the mutant construct.
  • a hygromycin resistance gene in an E. coli shuttle vector provides great utility for cloning and expression of a mutant IdeR or another dominant metal ion independent repressor [DtxR E175K].
  • the vectors also contain a copy of a gene lethal to the bacterium under the control of the metal dependent regulator.
  • genes include antibiotic genes, restriction enzymes, proteolytic enzymes, lethal phage genes or any gene whose product once expressed would kill the bacterium.
  • the presence of this "suicide cassette" further ensures that vaccines containing the prokaryote in live form do not revert to any significant degree and cause disease in vivo.
  • Suitable cloning and expression vectors are readily available from a number of sources.
  • the construct is introduced into the prokaryote by way of a vector, in which case the construct may be formed prior to or upon introduction of the DNA into the vector.
  • a vector for a given species must contain an origin of replication, a selectable marker and a functional promoter by which the mutant metal ion independent repressor can be expressed in the strain of interest.
  • the vectors are introduced into the cells in accordance with standard techniques such as transformation, co-transformation, direct transfection (e.g., mediated by calcium phosphate or DEAE-dextran) biolistics and electroporation.
  • the recombinant cells are then cultured via standard techniques. Conditions may vary depending upon the prokaryote (e.g., bacterial species) being used. In general, culturing is continued from about 24 to 48 hours at a temperature between about 30 and about 39°C, preferably 37°C.
  • the recombinant cells are cultured in an appropriate complete medium containing a selectable marker to assure a pure population.
  • the strains can also be counter engineered as described below so that if significant levels of the metal independent repressor is not produced a suicide gene will be de-repressed resulting in the death of the vaccine strain in situ.
  • Iron is an essential element for both the bacterial pathogen and its animal host; thus, successful competition for this element is an essential component of the infectious process.
  • the concentration of free iron in the mammalian host available to an invading bacterial pathogen is also extremely limited.
  • virulence determinants e.g., colonization factors, siderophores, hemolysins and toxins
  • the vaccine cultures must be propageted in a complete medium containing iron and other divalent metal cations to facilitate their proliferation.
  • the vaccines of the present invention may be used in a wide variety of vertebrates, particularly man and domestic animals such as bovine, ovine, porcine, equine, caprine, domestic fowl, Leporidate, or other animals that may be held in captivity or may be a vector for a disease affecting a domestic vertebrate.
  • Pathogens of interest include any specie of microorganism which causes disease and relies entirely or partially upon a repressor mediated regulation of metal-dependent virulence.
  • the present invention relates to methods of vaccinating a host with live recombinant bacteria to elicit protective immunity in the host.
  • the recombinant vaccine can be used to produce humoral antibody immunity, cellular immunity (including helper and cytotoxic immunity) and/or mucosal or secretory immunity.
  • the manner of application of a vaccine strain may be varied widely, any of the conventional methods for administering an attenuated vaccine being applicable. These include aerosol applications, oral applications, in drinking water, on a solid physiologically acceptable base, or in a physiologically acceptable dispersion, parenterally (e.g., subcutaneously, intramuscularly, mtravascularly or intraperitoneally), by injection, by in ovo inoculation or the like.
  • the dosage of the vaccine e.g., number of prokaryotic cells, number of administrations, period of administration, etc.
  • the recombinant prokaryotes in the composition may be "live” or in "killed” form, as these terms are commonly used in the vaccine art.
  • the formulation of vaccine strain compositions may also vary widely.
  • compositions such as water are expected to be useful for oral administration.
  • Other such vehicles including normal saline may be used for parenteral, cloacal or other routes of administration.
  • the vaccine compositions may also be admixed with food for some applications.
  • Iron plays a critical role in the regulation of virulence of many bacterial pathogens.
  • (2-5) Iron is an essential nutrient for the survival of most organisms and has played a central role in the virulence of many infectious disease pathogens.
  • Mycobacterial IdeR is an iron-dependent repressor that shows 80% identity in the functional domains with its corynebacterial homologue, DtxR.
  • Mycobacterium tuberculosis has been transformed with a vector expressing an iron-independent, positive dominant, corynebacterial dtxR hyperrepressor, DtxR(E175K).
  • DtxR(E175K) an iron-independent, positive dominant, corynebacterial dtxR hyperrepressor
  • Western blots of whole cell lysates of M. tuberculosis expressing the ⁇ txR(E175K) gene revealed the stable expression of the mutant protein in mycobacteria.
  • BALB/c mice were infected by tail vein injection with 2x 10* organisms of wild type or M.tuberculosis transformed with the dtxR mutant.
  • Attenuation of Mycobacterium tuberculosis can be achieved by the insertion of a plasmid containing a constitutively active, iron-insensitive repressor, DtxR(E175K), which is a homologue of IdeR.
  • DtxR(E175K) a constitutively active, iron-insensitive repressor
  • the corynebacterial DtxR has a homologue in M.tuberculosis, IdeR (iron-dependent repressor). In the amino terminal 140 amino acids that contain the Fe ⁇ + and DNA-binding domains of DtxR, IdeR shares 80% identity with DtxR. (6) In 1995, ideR was first described by Doukhan et al. in conjunction with the sigA sigB cluster of genes. (7) Subsequently, the ability of mycobacterial IdeR to bind to the corynebacterial tox operator region in a metal ion-dependent manner was demonstrated by gel shift assay. (8) Mutation of ideR in M. smegmatis resulted in derepressed siderophore production in high iron conditions. (9) These findings parallel those described in corynebacterial dtxR and suggest that the homology between these two genes may allow for cross-genus functional complementation.
  • dtxR (E175K) Shuttle Vector Plasmid A 1.5 kb Ba/wHI-Hind ⁇ i fragment of DNA from pSDM2 was cloned into pNBVl . The resulting recombinant plasmid, pNBVl/SAD was cloned in E. coli DH5 and purified using the Qiagen system (Qiagen, Chatsworth, CA). (16) Purified plasmids were then electroporated into M.tuberculosis CDC1551 by standard protocols. (15)
  • proteins were transferred to nitrocellulose membranes (Hybond, Amersham, Buckinghamshire, UK) by semi-dry technique (Transblot SD, Hercules, CA) and blocked with 5% non-fat milk in PBS with 0.1% Tween 20 (PBS-T) for 1 hour. Membranes were then incubated overnight in PBS-T with rabbit anti-DtxR polyclonal antibodies at the appropriate concentration at 4°C. (17) After washing, membranes were incubated with horseradish peroxidase- conjugated anti-rabbit antibody diluted in PBS-T for 2 hours.
  • the Supersignal Chemilumine scent Substrate (Pierce, Rockford, IL) was used for autoradiograph development.
  • Murine Tuberculosis Infection Model 6-8 week-old BALB/c mice were infected by tail vein injection with 2x10 ⁇ organisms of wild type or M.tuberculosis DtxR(E175K). Bacterial infection was monitored over a 119-day period.
  • Colony forming units (CFU) in spleen and lungs were assessed at 4 week intervals by serial dilutions of organ homogenates plated on 7H 10 Middlebrook agar containing cycloheximide (50 g/ml), carbenicillin (50 g/ml), trimethoprim (20 g/ml), and polymyxin (200 units/ml).
  • DNA Gel Shift Binding Assay The DNA migration retardation assay was performed as previously described. (19) Purified DtxR protein was isolated by methods as described. (20) Radiolabeled DNA iron box fragments were generated by PCR using 100 ng of 3 p. enc i-i a beled primer mixed with 150 ng of unlabeled primer and template DNA from gel-purified 100 bp cold fragments containing the iron box of interest. Binding reactions were carried out in lOmM Tris-OAc (pH7.4), ImM EDTA, 50mM KC1, ImM DTT, 5% glycerol, 50g/ml calf thymus DNA. Binding reactions were equilibrated for 30 minutes and then loaded onto a non- denaturing 6% acrylamide gel. (21) Results
  • corynebacterial dtxR gene in mycobacteria
  • the 1.5 kb corynebacterial DNA fragment cloned in pNBVl/SAD contained 500 bp of 5' non-coding sequences as well as the entire dtxRfE ⁇ 75Y2) open- reading frame.
  • M. smegmatis a fast growing strain of mycobacteria, with pNBVl/SAD.
  • Whole cell lysates prepared from M.smegmatis cultures were separated by 12% SDS PAGE.
  • FIG. 4 shows a Western blot developed with polyclonal anti-DtxR antibodies. As illustrated, these antibodies recognize both DtxR and IdeR because of their significant antigenic similarity. Although the deduced molecular mass of IdeR (25.2kDa) differs by only O.lkDa from DtxR (25.3kDa) we have repeatedly observed anomalous accelerated migration of IdeR in our SDS-PAGE gels in which it runs at 23kDa in spite of its mass of 25kDa. This phenomenon has also been noted by Schmitt et al. (8) In preparations from M.smegmatis harboring pNBVl/SAD (Fig4, lane 4), two distinct bands appear.
  • FIG. 8 shows the results of gel binding assays using32p_ e nd-labeled 100 bp DNA fragments containing five of the putative iron boxes (IB 1-5). Binding of DtxR to the tox operator could be abolished with the addition of unlabeled tox DNA, but not with nonspecific DNA. All five of these putative iron boxes were bound by DtxR to a similar degree as that observed with the tox operator. The iron box upstream of the narG homologue, IB6, did not bind to DtxR. (data not shown)
  • Table 3 identifies the open reading frames (ORF) downstream of these six iron boxes.
  • BLAST searches reveal that these genes encode a PhoP homologue (a transmembrane sensor of a two-component sensor-regulator pair), a homologue of the HtrA serine protease, 16S ribosomal RNA, an alcohol dehydrogenase AdhB, and a homologue of the M.tuberculosis 19kDa antigen (a protein shown to be involved in the human immune response to tuberculosis). (25) IB6, which was not shifted by DtxR in vitro, appears upstream of a nitrate reductase subunit gene,narG. Discussion
  • the concentration of free ferrous iron (Fe ⁇ + ) is extremely limited in vivo. For this reason, many pathogenic prokaryotes such as Vibrio cholerae, E.coli, Neisseria gonorrheae, and Corynebacterium diphtheriae co-regulate virulence gene expression with iron sensing and scavenging systems. (26-28) In C. diphtheriae, one such mechanism of iron regulation relies on a repressor, DtxR, which binds to a specific palindromic sequence in the operator regions of the genes that it controls.
  • DtxR repressor
  • M.smegmatis In M.smegmatis, an ideR mutant showed defective regulation of siderophore biosynthesis.
  • Potential IdeR binding sites upstream of exochelin biosynthesis genes such asfxbA have recently been confirmed.
  • several IdeR recognition sequences have been identified using computer searches of the M.tuberculosis genome. (38) We have similarly identified 6 potential IdeR- binding sites in M.tuberculosis, 5 of which demonstrated significant binding with DtxR in a gel shift assay. We postulate that the sixth sequence was unable to bind in our in vitro assay because of incorrect spacing between the two relatively well- conserved half-sites.
  • DtxR rather than IdeR in this gel-shift assay because we specifically sought to identify genes responsible for the attenuated phenotype of M.tuberculosis DtxR(E175K).
  • the predicted ORF downstream of IB-1 encodes a homologue of phoP, a phosphotransfer response regulator.
  • phoP a phosphotransfer response regulator.
  • a number of two- component pairs have been shown to regulate virulence pathways in bacterial pathogens. These include BvgA BvgS in Bordatella pertussis, VanR/VanS in Enterococcus faecium, PhoP-PhoQ in Salmonella typhimurium , and OmpT/EnvZ in Shigella flexneri.
  • IB-3 lies upstream of an ORF homologous to a HtrA-like serine protease which, in E.coli are thought to be required for growth of the organism at high temperature, and may play a role in degrading abnormal proteins within the periplasm.
  • It is a known virulence factor in several organisms including Salmonella typhimurium, Yersinia enterocolitica, Brucella abortus, and Brucella melitensis. (46-49)
  • a Salmonella typhimurium htrA mutant is attenuated and a safe and immunogenic live vaccine strain in mice.
  • Both Mycobacterium avium subsp. paratuberculosis and M.tuberculosis have putative serine proteases with significant homology to HtrA. (24, 51)
  • IB-4 lies upstream oirrnA, a 16S rRNA gene which has been shown to be part of a group of rDNA operons in both slow and fast-growing mycobacteria with hypervariable multiple promoter regions (HMPR).
  • HMPR hypervariable multiple promoter regions
  • the M.tuberculosis rrnA operon has 2 promoters one of which is conditionally induced suggesting complex regulation of this abnormal gene. (52)
  • PNBV1 is.co/z-mycobactenal shuttle vector
  • Example 1 Attenuation of Staphylococcus Infection in a Murine Model
  • Applicants demonstrated the ability of in vitro constructed and tested metal ion independent mutant gene of a normally metal ion dependent transcriptional repressor to attenuate virulence in a pathogenic bacterium of a different species.
  • the metal ion dependent repressor was from C. diphtheriae and the pathogenic species was M. tuberculosis.
  • the sequence homology of DtxR and the endogenous mycobacterial DtxR -like repressor IdeR is approximately 60% [approaching 90% in the N-terminal half of the repressors].
  • Example 1 partial diploid analysis in reporter host strains of Escherichia coli was performed revealing DtxR(E175K) is dominant in strains which carry DtxR/DtxR(E175K).
  • the present invention is applicable to human and veterinary medicine and more specifically to disease prevention and control of infectious and disease.

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Abstract

La présente invention concerne des procaryotes virulents ou opportunistes dans lesquels la régulation de gène dépendant d'ion métallique confère un avantage en matière de croissance ou d'infection. Le procaryote contient une molécule d'ADN qui contient une séquence codante pour un répresseur indépendant d'ion métallique ou pour un répresseur partiellement indépendant d'ion métallique. Ces procaryotes sont préparés en compositions de vaccin et administrés à une personne ou à un animal de façon à renforcer l'immunité contre les infections et les maladies causées par des procaryotes dans lesquels la régulation de gène dépendant d'ions métalliques confère un avantage en matière de croissance ou d'infection.
PCT/US2000/029231 1999-10-22 2000-10-23 Compositions de vaccin WO2001030384A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002097081A2 (fr) * 2001-05-30 2002-12-05 Brane Tech S.R.L. Procede de generation de micro-organismes non virulents a partir de micro-organismes pathogenes par modification genetique permanente de leur membrane biologique en vue de la production de vaccins
WO2012080696A1 (fr) * 2010-12-13 2012-06-21 Moredun Research Institute Vaccin
WO2013175207A1 (fr) * 2012-05-22 2013-11-28 Moredun Research Institute Vaccin

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4894443A (en) * 1984-02-08 1990-01-16 Cetus Corporation Toxin conjugates
US5196338A (en) * 1986-12-31 1993-03-23 Praxis Biologics, Inc. Recombinant vectors for Haemophilus influenzae peptides and proteins

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4894443A (en) * 1984-02-08 1990-01-16 Cetus Corporation Toxin conjugates
US5196338A (en) * 1986-12-31 1993-03-23 Praxis Biologics, Inc. Recombinant vectors for Haemophilus influenzae peptides and proteins

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002097081A2 (fr) * 2001-05-30 2002-12-05 Brane Tech S.R.L. Procede de generation de micro-organismes non virulents a partir de micro-organismes pathogenes par modification genetique permanente de leur membrane biologique en vue de la production de vaccins
WO2002097081A3 (fr) * 2001-05-30 2003-09-25 Brane Tech S R L Procede de generation de micro-organismes non virulents a partir de micro-organismes pathogenes par modification genetique permanente de leur membrane biologique en vue de la production de vaccins
WO2012080696A1 (fr) * 2010-12-13 2012-06-21 Moredun Research Institute Vaccin
WO2013175207A1 (fr) * 2012-05-22 2013-11-28 Moredun Research Institute Vaccin

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