WO1993019603A1 - Procaryotes presentant une immunite contre des bacteriophages codant l'adn - Google Patents

Procaryotes presentant une immunite contre des bacteriophages codant l'adn Download PDF

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WO1993019603A1
WO1993019603A1 PCT/US1993/002655 US9302655W WO9319603A1 WO 1993019603 A1 WO1993019603 A1 WO 1993019603A1 US 9302655 W US9302655 W US 9302655W WO 9319603 A1 WO9319603 A1 WO 9319603A1
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mycobacterium
dna
dna sequence
transformed
immunity
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PCT/US1993/002655
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William R. Jacobs
Graham Hatfull
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Albert Einstein College Of Medicine
University Of Pittsburgh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • 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
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/32—Mycobacterium
    • C12R2001/34—Mycobacterium smegmatis

Definitions

  • PROKARYO ES INCLUDING DNA ENCODING BACTERIOPHAGE IMMDNITT
  • This invention relates to prokaryotes, such as bacteria, an in particular to mycobacteria. More particularly, this inventio relates to prokaryotes which have been transformed with DN encoding immunity to a lytic bacteriophage.
  • mycobacteria represent major pathogens of man an animals.
  • tuberculosis is generally caused in human by Mycobacterium tuberculosis, and in cattle by Mycobacteriu bovis. which may also be transmitted to humans and other animals
  • Mycobacteria leprae is the causative agent of leprosy.
  • M tuberculosis and mycobacteria of the avium-intracellulare scrofulaceum group represent major opportunisti pathogens of patients with acquired immune deficiency syndrom (AIDS).
  • M. pseudotuberculosis is a major pathogen of cattle.
  • BCG Bacille Calmette-Guerin, or BCG, a avirulent strain of M. bovis, is widely used in human vaccines and in particular is used as a live vaccine, which is protectiv against tuberculosis.
  • BCG is the only childhood vaccine which i currently given at birth, has a very low incidence of advers effects, and can be used repeatedly in an individual (eg., i multiple forms).
  • BCG and other mycobacteria eg. M. sme ⁇ matis
  • employed in vaccines have adjuvant propertie among the best currently known and, therefore, stimulate
  • BCG could be used as a host for the construction of recombinant vaccines.
  • BCG vaccines are administered as live bacteria, it is essential that any foreign antigens, polypeptides, or proteins expressed by the bacteria are not lost from the bacteria subsequent to vaccination.
  • Electroporation can give from 10 5 to 106 transformants per ⁇ g of plasmid DNA and such plasmid DNA's may carry genes for resistance to antibiotic markers such as kanamycin (Snapper, et al 1988) to allow for selection of transformed cells from non-transformed cells.
  • antibiotic markers such as kanamycin
  • Jacobs, et al (1987) and Snapper, et al (1988) have also described the use of cloning vehicles, such as plasmids and bacteriophage ⁇ , for carrying genes of interest into mycobacteria.
  • SUBSTITUTESHEET mycobacterial heat shock genes, and used to express foreig antigens in mycobacteria.
  • a prokaryote transformed with DNA which includes at least one DNA sequence which encodes immunity to a lytic bacteriophage.
  • Prokaryotes which may be transformed with DNA which includes at least one DNA sequence which encodes immunity to a lytic bacteriophage include, but are not limited to, bacteria.
  • Bacteria which may be transformed include, but are not limited to, mycobacteria, Actinomyces species, Norcardia species, Streptomyces species, Corynebacteria species. Salmonella species. Vibrio species, and E. coli.
  • the bacterium is a mycobacterium.
  • Mycobacteria which may be transformed include, but are not limited to, Mycobacterium bovis-BCG. M. sme ⁇ matis, M. avium. M. phlei, M. fortiutum. M. lufu, M.
  • the mycobacterium is M. bovis-BCG. In another embodiment, the mycobacterium is M. sme ⁇ matis.
  • the prokaryote is transformed with DNA which includes at least one DNA sequence which encodes immunity to a lytic bacteriophage.
  • Temperate bacteriophages can adopt two different life cycles. The lytic cycle involves simple reproduction of viral particles within a bacterial cell, followed by lysis of the cell and release of the particles. Alternatively, temperate phages enter a lysogenic state in which most of the viral functions are inactivated, and the phage genome becomes integrated into the bacterial chromosome. The lytic functions of the phage are inactivated by a transcriptional repressor. The repressor regulates the genes of the resident prophage, and also prevents the lytic cycles of any superinfecting phages, thereby conferring immunity to a lytic bacteriophage.
  • the at least one DNA sequence encodes immunity to a lytic mycobacteriophage.
  • mycobacteriophage L5 is a temperate phage that infects and lysogenizes M. smegmatis. In accord with the temperate nature of L5, it not only infects M. sme ⁇ matis but also forms stable lysogens in which the bacteriophage genome is integrated into the bacterial chromosome and the lytic functions have been inactivated (Snapper, et al., Proc. Nat. Acad. Sci.. Vol. 85, pgs. 6987-6991, 1988, and Lee, et al., Proc. Nat. Acad.
  • L5 lysogens of M. smegmatis are immune to superinfection by L5, and also to superinfection by another mycobacteriophage known as mycobacteriophage D29.
  • Mycobacteriophage D29 is not a temperate phage and does not itself form lysogens.
  • a gene has been isolated from the L5 genome, which encodes a 183 amino acid protein, which confers immunity to L5 superinfection. This gene, which is approximately 0.6kb in
  • SHEET length is designated gene 71. As further described hereinbelow, this gene has been placed into an E. coli mycobacteria shuttle vector. The vector was then electroporated into M. sme ⁇ matis. Transformants were then selected by infection with bacteriophage L5c(dl), which is a variant of L5 that does not lysogenize. Therefore, the M. sme ⁇ matis organisms which have been transformed with the shuttle vector will survive the L5c(dl) infection. It is to be understood, however, that the scope of the present invention is not to be limited to immunity to L5 or any other mycobacteriophage superinfection, or to any specific genes which encode mycobacteriophage immunity, such as gene 71 of L5.
  • the DNA which transforms the mycobacterium includes a first DNA sequence which is a phage DNA portion encoding bacteriophage integration, preferably mycobacteriophage integration, into a mycobacterium chromosome, and the at least one DNA sequence which encodes immunity to a lytic bacteriophage.
  • phage DNA portion means that the DNA sequence is derived from a phage and lacks the DNA which is required for phage replication.
  • Bacteriophages from which the phage DNA portion may be derived include, but are not limited to, mycobacteriophages, such as but not limited to the L5, LI, Bxbl and TM4 mycobacteriophages; the lambda phage of E.coli; the toxin phages of Corynebacteria; phages of Actinomycetes and Norcadia, the 0 C31 phage of Streptomyces; and the P22 phage of Salmonella.
  • the phage DNA portion encodes mycobacteriophage integration into a mycobacterium chromosome.
  • the first DNA sequence includes DNA encoding integrase, which is a protein that provides for integration of the DNA into the ycobacterial chromosome. Most preferably, the first DNA sequence also includes DNA which encodes an AttP site. The DNA sequence encoding the AttP site and the integrase provides for an integration event which is referred to as site-specific integration. DNA containing the AttP site and the integrase gene is capable of integration into a corresponding AttB site of a mycobacterium chromosome.
  • the integration event results in the formation of two new junction sites called AttL and AttR, each of which contain part of each of AttP and AttB.
  • the inserted and integrated DNA which includes the first DNA sequence and the DNA which encodes immunity to a lytic bacteriophage, is flanked by the AttL and AttR sites.
  • the insertion and integration of the phage DNA portion results in the formation of a transformed mycobacterium.
  • the DNA may further include a DNA sequence encoding a protein or polypepetide heterlogous to the mycobacterium into which the DNA is to be integrated.
  • the DNA which encodes a protein heterologous to mycobacteria may be DNA which is all or a portion of a gene encoding protein(s) or polypeptide(s) of interest; DNA encoding a selectable marker or markers; or DNA encoding both a selectable marker or markers and at least one protein or polypeptide of interest.
  • Proteins or polypeptides of interest which may be encoded by such DNA include, but are not limited to, antigens, anti-tumor agents, enzymes, lymphokines, pharmacologic agents, immunopotentiators, and reporter molecules of interest in a diagnostic context.
  • Antigens for which such DNA sequence may encode include, but are not limited to, Mycobacterium leprae antigens; Mycobacterium tuberculosis antigens; Rickettsia antigens; malaria sporozoites
  • Anti-tumor agents which may be encoded by such DNA include, but are not limited to, interferon- ⁇ , interferon- ⁇ , or interferon- , and tumor necrosis factor, or TNF.
  • Lymphokines which may be encoded include, but are not limited to, interleukins 1 through 8.
  • Reporter molecules which may be encoded include, but are not limited to, luciferase, B-galactosidase, B-glucuronidase, and catechol dehydrogenase.
  • peptides or proteins which may be encoded by such DNA sequence include, but are not limited to, those which encode for stress proteins, which can be administered to evoke an immune response or to induce tolerance in an autoimmune disease (eg., rheumatoid arthritis) .
  • the phage DNA portion of the present invention which includes the first DNA sequence encoding mycobacterium phage integration into a mycobacterium chromosome, the at least one DNA sequence encoding immunity to a lytic bacteriophage; and the DNA encoding at least one protein or polypeptide heterologous to mycobacteria, may be constructed through genetic engineering
  • the phage DNA portion may be a plasmid including, in addition to the DNA encoding integration and the DNA encoding a heterologous protein, an origin of replication for any of a wide variety of organisms, which includes, but is not limited to, E.coli, Streptomyces species. Bacillus species, Staphylococcus species, Shigella species. Salmonella species and various species of pneumococci. Most preferably, the plasmid includes an origin of replication for E.coli.
  • the phage DNA portion also may include a suitable promoter for controlling expression of the at least one DNA sequence encoding a protein or polypeptide heterologous to the mycobacterium.
  • suitable promoters include, but are not limited to, mycobacterial promoters such as the BCG HSP60 and HSP70 promoters; mycobactin promoters of M. tuberculosis and BCG, the superoxide dismutase promoter, the ⁇ -antigen promoter of M. tuberculosis and BCG, the MBP-70 promoter, the 45 kda antigen promoter of M.
  • tuberculosis and BCG tuberculosis and BCG; and the mycobacterial asd promoter; the mycobacterial 14 kda and 12 kda antigen promoters; mycobacteriophage promoters such as the Bxbl promoter, the LI, L5, and D29 promoters, and the TM4 promoters; E.coli promoters; or any other suitable promoter.
  • mycobacterial asd promoter such as the Bxbl promoter, the LI, L5, and D29 promoters, and the TM4 promoters
  • E.coli promoters or any other suitable promoter.
  • the selection of a suitable promoter is deemed to be within the scope of those of ordinary skill in the art from the teachings contained herein.
  • the promoter sequence may, in one embodiment, be part of an expression cassette which also includes a portion of the gene normally under the control of the promoter.
  • the expression cassette may include, in addition to the promoter, a portion of the gene for the HSP60 or HSP70 protein.
  • the protein expressed by the cassette and the DNA encoding a protein or poplypeptide heterologous to the mycobacterium is a fusion protein of a fragment of a mycobacterial protein (eg., the HSP60 or HSP70 protein), and the protein or polypeptide heterologous to the mycobacterium.
  • the transcription initiation site, the ribosomal binding site, and the start codon, which provides for the initiation of the translation of mRNA are each of mycobacterial origin.
  • the stop codon, which stops translation of mRNA, thereby terminating synthesis of the protein or peptide heterologous to the mycobacterium, and the transcription termination site may be of mycobacterial origin, or of other bacterial origin, or such stop codon and transcription termination site may be those of the at least one DNA sequence encoding a protein or polypeptide heterologous to the mycobacterium.
  • the mycobacterium is transformed with an expression vector including the at least one DNA sequence which encodes immunity to a bacteriophage, and a promoter selected from the class consisting of mycobacterial promoters and mycobacteriophage promoters for controlling expression of at least one DNA sequence encoding a protein or polypeptide heterologous to the mycobaterium.
  • the mycobacterial and mycobacteriophage promoters and heterologous proteins and polypeptides may be selected from those hereinabove described.
  • the promoter sequence may also be part of an expression cassette which also includes a portion of the gene normally under the control of the promoter, as hereinabove described.
  • the expression cassette and the at least one DNA sequence encoding a protein or polypeptide heterologous to the mycobacterium are expressed, the protein expressed by the cassette and the at least one DNA sequence is a fusion protein of a fragment of a mycobacterial protein and the protein or polypeptide heterologous to the mycobacterium.
  • the transcription initiation site, the ribosomal binding site, and the start codon which provides for the initiation of the translation of mRNA, may each be of mycobacterial origin.
  • the stop codon may, as hereinabove described, be of mycobacterial origin, or of other bacterial origin, or such stop codon and transcription termination site may be those of the D ⁇ A encoding the at least one protein or polypeptide heterologous to the mycobacterium.
  • the vector further includes a mycobacterial origin of replication.
  • the vector may be a plasmid.
  • the plasmid may be a non-shuttle plasmid, or may be a shuttle plasmid which further includes a bacterial origin of replication such as an E. coli origin of replication, a Bacillus origin of replication, a Staphylococcus origin of replication, a Streptomyces origin of replication, or a pneumococcal origin of replication.
  • the shuttle plasmid includes an E. coli origin of replication.
  • the vector may further include a multiple cloning site, and the at least one D ⁇ A encoding a protein or polypeptide heterologous to the mycobacterium sequence is inserted in the multiple cloning site.
  • the expression vector may, in one embodiment, further include a D ⁇ A sequence encoding bacteriophage integration into a mycobacterium chromosome.
  • Bacteriophages from which the D ⁇ A sequence encoding bacteriophage integration into a mycobacterium chromosome may be derived include, but are not limited to, those hereinabove described.
  • the D ⁇ A sequence encodes mycobacteriophage integration into a mycobacterium chromosome.
  • the DNA sequence which encodes bacteriophage integration into a mycobacterium chromosome may include DNA which encodes integrase, which is a protein that provides for integration of the vector into the mycobacterial chromosome.
  • the DNA sequence encoding mycobacteriophage integration also includes DNA which encodes an attP site.
  • DNA encoding the attP site and the integrase provides for an integration event which is referred to as- site-specific integration.
  • DNA containing the attP site and the integrase gene is capable of integrating into a corresponding attB site of a mycobacterium chromosome, as hereinabove described.
  • the transformed mycobacteria which include DNA which includes at least one DNA sequence which encodes immunity to a lytic bacteriophage, and preferably a DNA sequence which encodes a protein or polypeptide which is heterologous to mycobacteria, may be utilized in the production of a vaccine or a therapeutic agent, depending upon the protein(s) or polypeptide expressed by the transformed mycobacteria.
  • the transformed mycobacteria are administered in conjunction with a suitable pharmaceutical carrier.
  • suitable carriers there may be mentioned: mineral oil, alum, synthetic polymers, etc.
  • Vehicles for vaccines and therapeutic agents are well known in the art and the selection of a suitable vehicle is deemed to be within the scope of those skilled in the art from the teachings contained herein. The selection of a suitable vehicle is also dependent upon the manner in which the vaccine or therapeutic agent is to be administered.
  • the vaccine or therapeutic agent may be in the form of an injectable dose and may be administered intramuscularly, intravenously, orally, intradermally, or by subcutaneous administration.
  • mycobacteria When the transformed mycobacteria are employed as a vaccine, such a vaccine has important advantages over other presently available vaccines.
  • Mycobacteria have, as hereinabove indicated, adjuvant properties among the best currently known and, therefore, stimulate a recipient's immune system to respond with great effectiveness.
  • This aspect of the vaccine induces cell-mediated immunity and thus is especially useful in providing immunity against pathogens in cases where cell-mediated immunity appears to be critical for resistance.
  • mycobacteria may stimulate long-term memory or immunity. It thus may be possible to prime long-lasting T cell memory, which stimulates secondary antibody responses neutralizing to the infectious agent or the toxin.
  • Such priming of T cell memory is useful, for example, against tetanus and diphtheria toxins, pertussis, malaria, influenza virus. Herpes virus, rabies. Rift Valley fever virus, dengue virus, measles virus. Human Immunodeficiency Virus (HIV), respiratory syncytial virus, human tumors, and snake venoms.
  • mycobacteria transformed with the phage DNA portion of the present invention as a vaccine or a therapeutic agent is that mycobacteria in general have a large genome (i.e., approximately 3 x 10 base pairs in length).
  • a vector is constructed which includes an attP site, DNA encoding immunity to a bacteriophage, DNA encoding integrase, an antibiotic resistance marker, and directly oriented copies of a site which may be recognized by a resolvase protein.
  • An in vitro reaction using purified resolvase protein resolves the vector into a catenane which comprises two daughter molecules which are topologically linked as singly-linked circular DNA molecules.
  • One circle includes the attP site and the DNA which encodes immunity to a lytic bacteriophage.
  • the other circle includes the gene encoding integrase as well as the antibiotic resistance marker. This circle does not include a mycobacterial origin of replication.
  • the circle which includes the gene encoding integrase and the antibiotic resistance marker does not integrate into the mycobacterial chromosome.
  • the catenane When the catenane is transfected into the mycobacterium, the catenane becomes a substrate for cellular DNA topoisomerase II enzyme. The action of the enzyme upon the catenane results in the separation of the two circular DNA molecules from each other.
  • the integrase and the antibiotic resistance marker are expressed when the circular DNA molecules are first transfected into the mycobacterium, the circle which includes the antibiotic resistance marker will eventually be lost because such circle cannot integrate, nor can the circular DNA molecule replicate within the mycobacterium.
  • transformed mycobacteria which do not include antibiotic resistance markers, and may be selected through bacteriophage infection.
  • prokaryotes other than mycobacteria may be transformed with DNA encoding immunity to a lytic bacteriophage, and preferably also with DNA encoding a protein or polypeptide which is heterologous to the prokaryote.
  • pMD30 is a derivative of p ⁇ C119 modified such that it may replicate in both E. coli and mycobacteria, and it contains the aph kanamycin resistance gene.
  • pMD30 was constructed by inserting the 1 kb Hindlll fragment from pKD43 (Derbyshire, et al., Proc. Nat. Acad. Sci.. Vol. 84, pgs. 8049-8053, (1987)) containing the aph gene into the Seal site of pUC119 ( Figure 1) to make pMD02.
  • Clear plaque derivatives were isolated as spontaneous mutants that formed clear plaques on bacterial lawns, as opposed to the turbid plaques of the wild-type mycobacteriophage L5. Clear plaques indicate those cells which were killed by L5 infection and therefore cannot form lysogens.
  • L5c(dl) was found to contain a small deletion of the L5 genome, including part of gene 71 , by restriction enzyme digestion with Bgl II. Bacterial survivors of an L5c(dl) infection of M. smegmatis occur at a frequency of about 10 .
  • Recombinant plasmids were constructed in which the 1.3kb fragment of the L5 genome which contains gene 71 is inserted into an E. coli-mycornycobacterial shuttle vector.
  • the construction of plasmids containing smaller segments of the 9.5kb Kpnl fragment until a plasmid was constructed which included the 1.3kb fragment of the L5 genome containing gene 71 was as follows: pMD04 ( Figure 7) was made by inserting the Hindlll fragment (with blunt ends generated by Klenow) from pKD43 containing the kanamycin resistance gene and inserting such fragment into the Seal site of pUCll ⁇ . ( Figure 8).
  • pMD31 ( Figure 9) was then constructed by isolating the Hpal-EcoRV fragment from pYUB12 ( Figure 3 provided by Dr. William Jacobs), and inserting it into the XmnI site of pMD04.
  • pMD31 is a shuttle vector which may replicate in both E. coli and M. smegmatis. and contains a kanamycin resistance gene for selection in both bacterial species.
  • pZS24 ( Figure 10) contains the 9.5kb Kpnl fragment of phage L5 inserted into the Kpnl site of pUC119 ( Figure 1).
  • pZS24 was constructed by gel purification of the 9.5kb fragment and ligating into the Kpnl site of pUC119.
  • the 9.5kb Kpnl fragment of pZS24 was then isolated; and inserted into the Kpnl site of pMD31 to make pMD40 ( Figure 11).
  • the SnaBI-PstI 2kb fragment of pMD40 was then isolated with blunt ends and inserted into the blunted Xbal site of pMD31 to form pMD70 ( Figure 12).
  • the BamHI-PstI 2kb fragment is isolated from pMD70 and inserted into p ⁇ Cll ⁇ cut with BamHI and PstI to form pMD90 ( Figure 13).
  • pMD90 is digested with Sail, which cuts twice, and then religated to form pMD131. ( Figure 14).
  • a 1.3kb BamHI-PstI fragment is removed from pMD131, and inserted into the BamHI-PstI site of pMD30 to make pMD132 ( Figure 15).
  • These plasmids also carry the aph gene from Tn903 (provided by K. Derbyshire and Nigel Grindley of Yale University) that confers resistance to kanamycin, and an E. coli origin of replication.
  • phage-selected transformants after a period of recovery from electroporation, efficiently killed non-transformed cells, but not plas id-transformed cells.
  • the phage-selected transformants also were determined to be resistant to kanamycin, thus indicating that they are true transformants.
  • M. smegmatis strain mc 2 155 cells (approximately 4 x 108 cells prior to electroporation) were electroporated with pYUB12, pMD70, or without DNA, and incubated for 1 hour in broth to allow expression of the selectable genes. Transformants were selected either with kanamycin (Snapper, et al., 1988) or by phage D29 infection.
  • D29 phage infection was as follows: D29 phages were added to M. smegmatis cells such that the multiplicity of infection was about 10. This is typically about g
  • the integrating vector containing gene 71 is pMH35, the construction of which is detailed as follows:
  • pNG199 obtained from Dr. Nigel Grindley
  • pUCll ⁇ Fig. 8
  • the resulting plasmid is pGH513 ( Figure 16).
  • pMH5 ( Figure 17), which contains the mycobacteriophage L5 attP site was digested with BamHI and Sail, and the 613 bp fragment containing the L5 attP site was inserted between the BamHI and Sail sites of pGH513 to form pGH515 ( Figure 18).
  • pGH515 thus contains a single res site and an attP site.
  • pGH515 was digested with BamHI and PvuII ( Figure 18), and a 1.2 kb fragment containing the int gene of mycobacteriophage L5 was inserted into the Asp718 site of pGH515 to form pGH516. ( Figure 19).
  • pGH516 contains the attP site and int gene of L5, which are separated by a single res site.
  • pGH318 obtained from Dr. Nigel Grindley
  • pNG199 is a plasmid that contains multiple copies of a res site of transposon ⁇ inserted into a pUC vector.
  • pGH318 was digested with EcoRI, and a 130 bp fragment containing the res site was inserted into the EcoRI site of pGH516 to form pGH519.
  • Figure 20 pGH519 contains the attP site and int gene of L5 plus two res sites which are in direct orientation.
  • pLP2 (Figure 21) was derived from pMH9.4 ( Figure 22 - Lee, et al., PNASv Vol. 88, pgs. 3111-3115, April 1991) by cutting with Ndel and Xbal, blunt ending the ends with Klenow, and religation. pLP2 thus has a defective attP site, but has a functional int gene.
  • pGH529 ( Figure 23) was derived by digesting pMH9.4 ( Figure 22) by cutting with SphI, and religating. pGH529 has a functional attP site, but the int gene is non-functional. pGH531 ( Figure 24) was then constructed by ligating the 1680 bp Asp718-Sall fragment from pLP2 ( Figure 21) to the 5062 bp Asp718-Sall fragment from pGH529 ( Figure 23).
  • pGH531 was then digested with Bglll and Sad, and a 728 bp Bglll-SacI fragment from pGH531 was inserted into the Bglll-SacI piece of pGH519 to form pMH27 ( Figure 25).
  • pMH27 was then opened at the Smal site and an aph kanamycin resistance cassette from pKD43 was inserted.
  • the resulting plasmid is called pMH33 ( Figure 26).
  • pMH33 was then cut with Dral, and a Hindi11 - Bam HI fragment (1.3 kb) from pMD131 ( Figure 14) that contains gene 71 was inserted to form pMH35 ( Figure 27).
  • pMH35 includes an attP site, an integrase (int) gene, ⁇ ene 71, and a gene encoding kanamycin resistance (aph gene).
  • pMH35 is efficiently transformed through electroporation into M. smegmatis, and transformants can be selected by either L5c(dl) infection or by kanamycin selection.
  • a cointegrate molecule was constructed which contains two directly oriented copies of the res site derived from transposon 6 (Hatfull, et al., "Resolvases and DNA-invertases: A Family of Enzymes Active in Site-Specific Recombination, " Genetic Recombination, Kucherlapatti and Smith, eds., ASM Press (1988)). Such directly oriented copies of the res site are contained in pMH35.
  • M. sme ⁇ matis was then transformed by electroporation (Snapper, et al. (1988)) with pMH35 which had been resolved into the circular DNA molecules as hereinabove described.
  • Transformants selected by L5c(dl) infection occurred at an approximately 10-fold higher frequency than those selected with kanamycin according to the procedure of Snapper, et al. (1988).
  • Organisms transformed with pMH9.4 were selected with kanamycin, and organisms transformed with pMH35 (either resolved or unresolved) were selected for L5c(dl) resistance.
  • Transformants were then selected for sensitivity or resistance to kanamycin by patch plating. All pMH9.4 and unresolved pMH35 transformants were resistant to kanamycin. 83% of the transformants which were transformed with resolved pMH35 were sensitive to kanamycin and the remainder were resistant. The kanamycin resistant transformants (17% of the population) may be a small population of organisms transformed with pMH35 which had not been resolved.
  • Lanes 6, 7, 10, and 11 include five bands which are not present in lanes 8 and 9. These bands may correspond to elements present (such as kanamycin resistance) in unresolved pMH35, but are lost after resolution of pMH35 into two singly-linked circular DNA portions.
  • BCG organisms were electroporated with pMH35. Following a 3 hr. expression period, the organisms were plated on 7H9 media containing ADC enrichment and 10 D29 phages. After 3 weeks incubation, BCG colonies immune to D29 infection were found in an

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Abstract

L'invention concerne un procaryote qui est transformé avec l'ADN et comprend au moins une séquence d'ADN qui code l'immunité contre un bactériophage lytique. Ledit procaryote peut être une bactérie, et en particulier un mycobactérium. Ces mycobactériums transformés peuvent être utilisés dans des vaccins, ce qui évite d'inclure dans les vaccins des mycobactériums à marqueurs de résistance aux antibiotiques.
PCT/US1993/002655 1992-03-31 1993-03-12 Procaryotes presentant une immunite contre des bacteriophages codant l'adn WO1993019603A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5679515A (en) * 1994-10-03 1997-10-21 Pathogenesis Corporation Mycobacterial reporter strains and uses thereof
WO2007136815A2 (fr) * 2006-05-19 2007-11-29 Danisco A/S Micro-organismes marqués et procédés de marquage

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4436815A (en) * 1981-11-27 1984-03-13 Eli Lilly And Company Method for stabilizing and selecting recombinant DNA containing host cells

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4436815A (en) * 1981-11-27 1984-03-13 Eli Lilly And Company Method for stabilizing and selecting recombinant DNA containing host cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PROC. NATL. ACAD. SCI. USA, Vol. 85, issued September 1988, SCOTT B. SNAPPER et al.: "Lysogeny and Transformation in Mycobacteria: Stable Expression of Foreign Genes", pages 6987-6991. *
PROC. NATL. ACAD. SCI. USA, Vol. 88, issued April 1991, MONG HONG LEE et al.: "Site-Specific Integration of Mycobacteriophage L5: Integration-Proficient Vectors for Mycobacterium Smegmatis, Mycobacterium Tuberculosis, Bacille Calmette-Guerin", pages 3111-3115. *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5679515A (en) * 1994-10-03 1997-10-21 Pathogenesis Corporation Mycobacterial reporter strains and uses thereof
WO2007136815A2 (fr) * 2006-05-19 2007-11-29 Danisco A/S Micro-organismes marqués et procédés de marquage
WO2007136815A3 (fr) * 2006-05-19 2008-01-31 Danisco Micro-organismes marqués et procédés de marquage
EP2426220A1 (fr) * 2006-05-19 2012-03-07 Danisco A/S Micro-organismes étiquetés et procédés d'étiquetage
US9399801B2 (en) * 2006-05-19 2016-07-26 Dupont Nutrition Biosciences Aps Tagged microorganisms and methods of tagging
US9816140B2 (en) 2006-05-19 2017-11-14 Dupont Nutrition Biosciences Aps Tagged microorganisms and methods of tagging

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