+

WO1992001783A1 - Adn capable d'une integration a specificite de site dans des mycobacteries - Google Patents

Adn capable d'une integration a specificite de site dans des mycobacteries Download PDF

Info

Publication number
WO1992001783A1
WO1992001783A1 PCT/US1991/004833 US9104833W WO9201783A1 WO 1992001783 A1 WO1992001783 A1 WO 1992001783A1 US 9104833 W US9104833 W US 9104833W WO 9201783 A1 WO9201783 A1 WO 9201783A1
Authority
WO
WIPO (PCT)
Prior art keywords
dna
mycobacteria
bcg
site
fragment
Prior art date
Application number
PCT/US1991/004833
Other languages
English (en)
Inventor
William R. Jacobs
Graham Hatfull
Original Assignee
Albert Einstein College Of Medicine Of Yeshiva University
University Of Pittsburgh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Albert Einstein College Of Medicine Of Yeshiva University, University Of Pittsburgh filed Critical Albert Einstein College Of Medicine Of Yeshiva University
Priority to JP3513065A priority Critical patent/JPH06501607A/ja
Priority to BR919106658A priority patent/BR9106658A/pt
Publication of WO1992001783A1 publication Critical patent/WO1992001783A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16211—Human Immunodeficiency Virus, HIV concerning HIV gagpol
    • C12N2740/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention relates to DNA capable of integrating into mycobacterial chromosomes. More particularly, this invention relates to DNA which is capable of site-specific integration into a mycobacterium chromosome -hile containing a DNA sequence encoding a protein heterologous to the mycobacterium in which the DNA is integrated.
  • mycobacteria represent major pathogens of man and animals.
  • tuberculosis is generally caused in humans by Mycobacterium tuberculosis, and in cattle by Mycobacterium 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 (MAIS group) represent major opportunistic pathogens of patients with acquired immune deficiency syndrome (AIDS).
  • M. pseudotuberculosis is a major pathogen of cattle.
  • BCG Bacille Calmette-Guerin, or BCG, an avirulent strain of M. bovis, is widely used in human vaccines, and in particular is used as a live vaccine, which is protec ⁇ . - ⁇ e against tuberculosis.
  • BCG is the only childhood vaccine which is currently given at birth, has a very low incidence of adverse effects, and can be used repeatedly in an individual (eg., in multiple forms).
  • BCG and other mycobacteria eg., M. smegmatis
  • employed in vaccines have adjuvant properties among the best currently known and, therefore, stimulate a recipient's immune system to respond to antigens with great effectiveness.
  • 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 to 10 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 (Snapper, et al 1988)
  • Jacobs, et al (1987) and Snapper, et al (1988) have also described the use of cloning vehicles, such as plasmids and bacteriophages, for carrying genes of interest into mycobacteria.
  • Plasmids currently available for use in mycobacteria are not stably maintained, and are readily lost during non-selective growth. If recombinant mycobacteria expressing genes of interest are to be employed as vaccines against a particular antigen or as a means of providing a therapeutic agent to a host, which is expressed by the mycobacteria, it is crucial that such genes not be lost from the recombinant mycobacteria subsequent to their administration.
  • a DNA which comprises a first DNA sequence which is a phage DNA portion encoding bacteriophage integration into a mycobacterium chromosome, and a second DNA sequence encoding at least one protein or polypeptide which is heterologous to the mycobacterium in which the DNA is to be integrated.
  • 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 Corvnebac eria; 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 mycobacterial chromosome. Most preferably, the first DNA sequence also includes DNA which encodes an AttP site.
  • 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 non-phage DNA which includes the first and second DNA sequences, 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 gene(s) of interest which is integrated into the mycobacterial chromosome is not lost following non-selective growth of the mycobacteria.
  • the gene(s) of interest may be expressed by the mycobacteria following such non-selective growth, thus making such tranformed mycobacteria excellent vehicles to be employed in vaccines or pharmaceuticals whereby such mycobacteria will express antigens and/or therapeutic agents of interest subsequent to administration of the recombinant mycobacteria to a host.
  • Mycobacteria into which the phage DNA portion may be integrated include, but are not limited to, Mycobacterium bovis- BCG, M. smeqmatis, M. avium, M. phlei, M. fortuitum, M. lufu, M. paratuberculosis, M. habana, M. scrofalaceum, M. leprae and M. intracellulare.
  • the DNA is integrated into Mycobacterium bovis- BCG.
  • the second DNA sequence 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 the second DNA sequence include, but are not limited to, antigens, anti-tumor agents, enzymes, ly phokines, pharmacologic agents, immunopotentiators, and reporter molecules of interest in a diagnostic context.
  • Antigens for which the second DNA sequence may encode include, but are not limited to, Mycobacterium leprae antigens; Mycobacterium tuberculosis antigens; Rickettsia antigens; malaria sporozoites and merozoites; diphtheria toxoids; tetanus toxoids; Clostridium antigens; Leishmania antigens; Salmonella antigens; Borrelia antigens; Mycobacterium africanum antigens; Mycobacterium intracellulare antigens; Mycobacterium avium antigens; Treponema antigens; Pertussis antigens; Schistosoma antigens; Filaria antigens; Herpes virus antigens; influenza and parainfluenza virus antigens; measles virus antigens; mumps virus antigens; hepatitis virus antigens; Shigella antigens; Neisseria antigens; rabies antigens, polio virus antigens; R
  • Enzymes which may be encoded include, but are not limited to, steroid enzymes.
  • Anti-tumor agents which may be encoded by the second DNA sequence 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 the second 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 (e.g., rheumatoid arthritis).
  • Selectable markers which may be encoded include, but are not limited to, the kanamycin resistance marker, the neomycin resistance marker, the chloroamphenicol resistance marker, and the hygromycin resistance marker.
  • the phage DNA portion of the present invention which includes the first DNA sequence encoding mycobacterium phage integration into a mycobacterium chromosome, and the second DNA sequence encoding at least one protein or polypeptide heterologous to mycobacteria, may be constructed through genetic engineering techniques known to those skilled in the art.
  • 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, Shiqella species, Salmonella species and various species of pneumococci.
  • the plasmid includes an origin of replication for E.coli.
  • the phage DNA portion also may include a suitable promoter.
  • 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 and L5 promoters, and the TM4 promoters; E.coli promoters; or any other suitable promoter.
  • mycobacteriophage promoters such as the Bxbl promoter, the LI and L5 promoters, and the TM4 promoters; E.coli promoters; or any other suitable promoter.
  • 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 the heterlogous protein or polypeptide is a fusion protein of a fragment of a mycobacterial protein (eg. , the HSP60 or HSP70 protein), and the heterologous protein.
  • 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 heterologous protein, 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 DNA encoding the heterologous protein or polypeptide.
  • the phage DNA portion may be employed, as hereinabove indicated, for integration into a mycobacterial chromosome, thereby transforming the mycobacteria, and whereby the mycobacteria will express protein(s) or polypeptide(s) heterologous to mycobacteria.
  • mycobacteria may be utilized in the production of a vaccine or a therapeutic agent, depending upon the protein(s) or polypeptides -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). Because the genome is large, it is abie to accommodate a large amount of DNA from other source(s), and may possibly be employed to make a vaccine and/or therapeutic agent containing DNA sequences encoding more than one antigen and/or therapeutic agent.
  • the gene(s) of interest will continue to be expressed by the transformed mycobacteria following administration of the mycobacteria to a host.
  • Such mycobacteria are effective vehicles for expressing antigens which stimulate an immune response, or for the expression of therapeutic(s) agents such as anti-tumor agents and/or anti-cancer agents.
  • Example 1 A Identification of the DNA sequences of the attachment sites, attB, attL, and attR, of M.smegmatis.
  • a lambda EMBL3 library was prepared from BamHI digested mc 261 chromosomal DNA (mc 261 is a strain of M. smeqmatis which includes an M. smeqmatis chromosome into which has been integrated the genome of mycobacterial phage L5) and digested with Bam HI.
  • Phage L5 contains DNA having restriction sites identical to those of phage LI (Snapper, et al. 1988), except that L5 is able to replicate at 42°C and phage LI is incapable of such growth.
  • This library was then probed with a 6.7 kb DNA fragment isolated from the L5 genome that had been previously identified as carrying the attP sequence (Snapper, et al 1988).
  • One of the positive clones was plaque purified, DNA prepared, and a 1.1 kb Sal I fragment (containing the AttL sequence) sub-cloned into sequencing vector pUC119.
  • the DNA sequence of this fragment was determined using a shotgun approach coupled with Sanger sequencing. By isolating and sequencing the attL junction site and comparing this to the DNA sequence of L5 that was available, a region was determined where the two sequences aligned but with a specific discontinuity present.
  • the discontinuity represents one side of a core sequence, which is identical in attP, attB, and attL.
  • the region containing the recombinational crossover point is shown in Figure 1.
  • the attL DNA (1.1 kb Sal I fragment) was used as a probe to
  • the DNA sequence containing the core sequence was determined and is shown in Figure 1.
  • Sequences of the 6.7kb Bam HI fragment hereinabove described were determined by (a) analysis of the location of Bam HI sites in the contigs of the DNA of L5, and (b) by determining a short stretch of DNA sequence from around the Bam HI sites of plasmid pJR-1 ( Figure 6), which carries the 6.7kb Bam HI fragment of L5.
  • a segment of DNA sequence was located that represented the 6.7kb Bam HI fragment of phage L5.
  • Studies of other phages have shown that the integrase genes are often located close to the attP site. It was thus determined that the L5 integrase (int) gene should lie either within the 6.7kb Bam HI fragment or in a DNA sequence on either side of it.
  • the DNA sequence in the regions was then analyzed by translating it into all six possible reading frames and searching these amino acid sequences for similarity to the family of integrase related proteins, and through computer-assisted analysis of the DNA sequence.
  • the location of the int gene was not within the 6.7kb Bam HI fragment; however, it was very close to it with one of the Bam HI sites (that defines the 6.7kb Bam HI fragment) less than 100 bp upstream of the start of the gene. Analysis of the Bam HI sites showed that the int gene lay within a 1.9kb Bam HI fragment located adjacent to the 6.7kb Bam HI fragment. This 1.9kb Bam HI fragment was cloned by purification of the fragment from a Bam HI digest of L5 DNA and cloning into pUC 119, to generate pMHl ( Figure 7) .
  • a plasmid containing a 1.9kb Bam HI fragment containing the DNA encoding for the integrase cloned into the Bam HI site of pUC 119 was constructed.
  • the 1.9kb fragment was purified from a Bam HI digest of L5 DNA and cloned into the Bam HI site of pUC 119. Construction of the recombinant was determined by restriction analysis and gel electrophoresis.
  • This plasmid was called pMHl, the construction of which is shown schematically in Figure 7.
  • pJR-1 was then modified by digestion with EcoRI and SnaBI (both are unique cloning sites), between which is a Bam HI site.
  • the 1.9kb Bam HI fragment which includes the integrase gene, was purified from a Bam HI digest of pMHl and ligated to Bam HI digested pMH2. Recombinants were identified as above and the orientation of the 1.9kb fragment determined.
  • a plasmid called pMH4 was thus constructed ( Figure 9) in which the region from the Sna BI site (upstream of attP) through to the Bam HI site (downstream of the integrase gene) was identical to that in L5.
  • pMH4 was digested with HindiII (unique site) and was ligated to a lkb Hindlll fragment purified from pKD43 (supplied by Keith Darbyshire of the Nigel Gindley Laboratory) that contains the gene determining resistance to kanamycin. Recombinants were identified and characterized as above. This plasmid is called pMH5.
  • a schematic of the construction of pMH5 is shown in Figure 10.
  • Plasmids pYUB12 (a gift from Dr. Bill Jacobs a schematic of the formation of which is shown in Figure 20), pMDOl ( Figure 11), and pMH5 were electroporated, with four different concentrations of plasmid DNA over a 1,000-fold range, into M. smeqmatis strain
  • 1 ⁇ l of DNA was added to 100 ⁇ l of cells in an ice-cold cuvette and pulsed in a Bio-Rad Gene Pulser, and given a single pulse at 1.25 kv at 25 ⁇ F.
  • 1 ml of broth was added the cells incubated for 1 hr. at 37°C for expression of the antibiotic-resistant marker.
  • Cells were then concentrated and plated out on Middlebrook or tryptic soy media containing 15 ⁇ g/ml kanamycin. Colonies were observed after 3 to 5 days incubation at 37°C.
  • Each of pYUB12, pMDOl, and pMH5 carries kanamycin resistance.
  • Plasmid pYUB12 carries an origin of DNA replication, while pMDOl lacks a mycobacterial origin of replication.
  • Plasmid pMH5 does not carry a mycobacterial origin of replication, but carries a 2kb region of phage L5 which contains the attP site and the integrase gene ( Figure 4). The number of transformants were linear with DNA concentration. Plasmid pYUB12 gives a large number of transformants (2 x 10 5 per ⁇ g DNA) in mc2155, while
  • DNA was prepared from BCG transformants and analyzed by Bam HI restriction and Southern blot analysis, probing with gel purified
  • Example 3 Construction of plasmids including mycobacterial promoter expression cassette.
  • Plasmid pAL5000 a plasmid which contains an origin of replication of M. fortuitum, and described in Labidi, et al., FEMS Microbiol. Lett., Vol. 30, pgs. 221-225 (1985) and in Gene, Vol. 71, pgs. 315-321 (1988), is subjected to a partial Sau 3A digest, and 5kb fragments are gel purified. A 5kb fragment is then ligated to Bam HI digested pIJ666 (an. E. coli vector containing an E. coli origin of replication and also carries neomycin-kanamycin resistance, as described in Kieser, et al., Gene, Vol. 65, pgs.
  • Bam HI digested pIJ666 an. E. coli vector containing an E. coli origin of replication and also carries neomycin-kanamycin resistance, as described in Kieser, et al., Gene, Vol
  • Plasmid pYUB12 A schematic of the formation of plasmid pYUB12 is shown in Figure 20.
  • pYUB12 and pIJ666 were then transformed into M. smegmatis and BCG.
  • Neomycin-resistant transformants that were on]y obtained by pYUB12 transformation confirmed that pAL5CoO conferred ' autonomous replication to pIJ666 in M. smeqmatis and BCG.
  • Plasmid pYUB8 (a pBR322 derivative) includes an E. coli replicon and a kan (aph) gene. Ligation of the 2586 bp pYUB12 fragment to PvuII digested pYUB8 results in the formation of pYUB53, as depicted in Figure 21. Transformation of pYUB53 confirmed that the EcoRV-Hpal fragment, designated M.rep, was capable of supporting autonomous replication in BCG.
  • Plasmid pYUB53 was then digested with AatI, EcoRV, and PstI in order to remove the following restriction sites: AatI 5707 EcoRI 5783 BamHI 5791 Sail 5797 PstI 5803 PstI 7252 Sail 7258 BamHI 7264 EcoRI 7273 Clal 7298 HindiII 7304; and EcoRV 7460 Fragment ends are then flushed with T4 DNA polymerase and religated to form plasmid pYUB125, construction of which is shown in Figure 22.
  • the HindiII and Clal restriction sites in the aph gene were mutagenized simultaneously by polymerase chain reaction PCR mutagenesis according to the procedure described in Gene, Vol. 77 pgs. 57-59 (1989).
  • the bases changed in the aph gene were at the third position of codons (wobble bases) within each restriction site and the base substitutions made were designed not to change the amino acid sequence of the encoded protein.
  • the 5232 base pair fragment was gel purified and mixed with fragment Kan.mut and ligated.
  • the ligation was transformed into E.coli strain HBlOl and Kan colonies were screened for plasmids resistant to Clal and HindiII digestion.
  • Such plasmids were designated as pMVUO, which is depicted in Figure 23.
  • Plasmid pMVUO was resected in separate constructions to yield plasmids pMVlll and pMV112.
  • pMVUO was digested with Narl and Ball, the ends were filled in, and a 5296 base pair fragment was ligated and recircularized to form pMVlll.
  • pMVUO was digested with Ndel and SplI, the ends were filled in, and a 5763 base pair fragment was ligated and recircularized to form pMV112. Schematics of the constructions of pMVlll and pMV112 are shown in Figure 25. These constructions further eliminated superfluous E.
  • Plasmids pMVlll and pMV112 were tested for the ability to replicate in M. smeqmatis. Because both plasmids replicated in M. smeqmatis the deletions of each plasmid were combined to construct pMV113.
  • pMV113 ( Figure 25), pMVlll was digested with BamHI and EcoRI, and a 1071 bp fragment was isolated.
  • pMV112 was digested with BamHI and EcoRI, and a 3570 bp fragment was isolated, and then ligated to the 1071 bp fragment obtained from pMVlll to form pMV113.
  • PCR mutagenesis was performed as above to eliminate the Sal I, EcoRI, and Bglll sites located in the open reading frame known as ORFl of pALSOOO.
  • PCR mutagenesis was performed at wobble bases within each restriction site and the base substitutions were designed not to change the amino acid sequence of the putative encoded ORFl protein.
  • the restriction sites were eliminated one at a time for testing in mycobacteria. It was possible to eliminate the Sail and EcoRI without altering replication in M. smeqmatis.
  • PCR mutagenesis was performed at EcoRI1071 of pMV113 with primers Eco Mut - M.rep and Bam-M.rep. to form pMV117, which lacks the EcoRI1071 site.
  • Primer Eco Mut - M.rep has the following sequence:
  • TCC GTG CAA CGA CGT GTG TCC CGG A; and Bam-M.rep has the following sequence:
  • PCR mutagenesis was performed at the Sail 1389 site with primer Sal Mut - M.rep and Bam-M.rep to form pMV119, which lacks the Sail 1389 site.
  • Primer Sal Mut - M.rep has the following sequence:
  • pMV117 was then digested with ApaLI and Bglll, and a 3360 bp fragment was isolated.
  • pMV119 was dige. ad with ApaLI and Bglll, and a 1281 bp fragment was isolated and ligated to the 3360 bp fragment isolated from pMV117 to form pMV123.
  • a schematic of the constructions of plasmids pMV117, pMV119, and pMV123 is shown in Figure 26.
  • cassettes of each component were constructed for simplified assembly in future vectors and to include a multiple cloning site (MCS) containing unique restriction sites and transcription and translation terminators.
  • MCS multiple cloning site
  • the cassettes were constructed to allow ⁇ 'rectional cloning and assembly into a plasmid where all transcription is unidirectional.
  • a DNA cassette containing the aph (Kan ) gene was constructed by PCR with primers Kan5' and Kan 3' .
  • An Spel site was added to the 5' end of PCR primer Kan3' , resulting in the formation of a PCR primer having the following sequence:
  • PCR primer Kan5' Bam HI + Nhel sites were added to the 5' end of PCR primer Kan5', resulting in the formation of a PCR primer having the following sequence:
  • PCR was performed at bases 3375 and 4585 of pMV123, and BamHI and Nhel sites were added at base 3159, and an Spel site was added at base 4585. Digestion with BamHI and Spel, followed by purification resulted in a 1228/2443 Kan cassette bounded by BamHI and Spel cohesive ends with the direction of transcription for the aph gene proceeding from BamHI to Spe I.
  • a DNA cassette containing the ColEI replicon of pUC19 was constructed by PCR with primers E.rep/Spe and E.rep/Mlu.
  • An Spel site was added to the 5' end of PCR primer E.rep/Spe, and an Mlul site was added to the 5' end of PCR primer E.rep./Mlu.
  • the resulting primers had the following base sequences:
  • PCR was performed at bases 713 and 1500 of pUC19, and an Mlul site was added to base 713, and a Spel site was added to base 1500. Digestion with Mlul and Spel, followed by purification resulted in an E.rep. cassette bounded by Spel and Mlul cohesive ends with the direction of transcription for RNA I and RNA II replication primers proceeding from Spel to Mlul.
  • the resulting PCR primers had the following base sequences:
  • PCR was performed at bases 134 and 2082 of pMV123. An Mlul site was added to base 2082. Digestion with BamHI and Mlul, followed by gel purification resulted in a 1935 base pair DNA cassette bounded by Mlul and BamHI cohesive ends with the direction of transcription for the pAL5000 ORFl and 0RF2 genes proceeding from Mlul to Bam HI. p The Kan , E.rep, and M.rep PCR cassettes were then mixed in equimolar concentrations and ligated, and then transformed in p
  • E.coli strain HBlOl for selection of Kan transformants. Colonies were screened for the presence of plasmids carrying all three cassettes after digestion with BamHI + Mlul + Spel and designated pMV200. An additional restriction site, Ncol, was eliminated from the M.rep cassette by digestion of pMV200 with Ncol, fill in with Klenow, and ligation and recircularization, resulting in the formation of pMV201.
  • Kan transformations thus indicating their ability to replicate in mycobacteria.
  • a synthetic multiple cloning sequence (MCS) ( Figure 28) was then designed and synthesized to facilitate versatile molecular cloning and manipulations for foreign gene expressions in mycobacteria, and for integration into the mycobacterial chromosome.
  • the synthetic MCS shown in Figure 28, contains 16 restriction sites unique to pMV201 and includes a region carrying translation stop codons in each of three reading frames, and a transcription terminator derived from E. coli 5S ribosomal RNA (Tl).
  • pMV201 was digested with Narl and Nhel, and the resulting fragment was gel purified.
  • the MCS was digested with HinPI and Nhel and, the resulting fragment was gel purified. The two fragments were then ligated to yield pMV204.
  • a schematic of the construction of pMV204 is shown in Figure 29.
  • Plasmid pMV204 was then further manipulated to facilitate removal of the M.rep cassette in further constructions.
  • pMV204 was digested with Mlul, and an Mlul - Not I linker was inserted into the Mlul site between the M.rep and the E.rep to generate pMV206.
  • a schematic of the construction of pMV206 from pMV204 is shown in Figure 30, and the DNA sequence of pMV206 is given in Figure 31.
  • HSP60 heat shock protein also known as the 65 kda antigen. Because abundance of the HSP60 protein in mycobacteria indicates strong HSP60 gene expression, the sequence controlling HSP60 expression was chosen to control expression of heterologous genes encoding antigens or other proteins in BCG.
  • the published sequence of the BCG HSP60 gene (Thole, et al, Infect, and Immun., Vol. 55, pgs. 1466-1475 (June 1987)), and surrounding sequence permitted the construction of a cassette carrying expression control sequences (i.e., promoter and translation initiation sequences) by PCR.
  • the BCG HSP61 cassette ( Figure 32) contains 375 bases 5' to the BCG HSP60 start codon, and 15 bases (5 codons) 3' to the start codon. PCR oligonucleotide primers were then synthesized.
  • Primer Xba-HSP60 of the following sequence: CAG ATC TAG ACG GTG ACC ACA ACG CGC C was synthesized for the 5' end of the cassette, and primer Bam-HSP61, of the following sequence:
  • pMV206 and the PCR cassette HSP61 were digested with Xba I and Bam HI.
  • the PCR cassette was then inserted between the Xba I and Bam HI sites of pMV206, and then ligated to form pMV261, which is shown in Figure 33.
  • the E. coli ,ac Z gene was used as a reporter, or marker gene to assay the ability of the HSP61 cassette to express heterologous genes in BCG.
  • a BamH * restriction fragment carrying the lac Z gene was cloned into the Bam HI site of Bam HI digested pMV261, resulting in the formation of pMV261/LZ.
  • a schematic of the construction of pMV261/LZ is shown in Figure 34. The formation of pMV261/LZ results in a fusion between the HSP60 and lac Z genes at the sixth codon of the HSP60 gene and the sixth codon of the lac Z gene. pMV261/LZ was then transformed into E. coli. Blue E.
  • coli colonies were selected on x-gal plates for the presence of pMV261/LZ, thus indicating that the HSP60 promoter and translation initiation sequences were also active in E. coli.
  • pMV261/LZ was then transformed into BCG and plated on Dubos Oleic Agar plates containing x-gal. All BCG colonies resulting from this transformation exhibited blue color, thus indicating that the lac Z gene product (B-galactosidase) was expressed in 3CG.
  • BCG HSP61 expression cassette was functional in expression vector pMV261, and that substantial expression of a heterologous gene could be achieved using HSP60 - controlled expression in BCG.
  • Plasmid pMV261/LZ was then shown to replicate autonomously, and express the E. coli B-galactosidase, or lacZ gene, driven by the BCG promoter HSP60, in M. smeqmatis and BCG.
  • Plasmid pMH9.4 which includes the mycobacterial phage L5 attP site and the L5 integrase gene, was digested to completion with either Kpnl + PvuII or Xbal + PvuII, and a restriction fragment of 1862 or 1847 base pairs, respectively, each of which contain the attP site and the integrase gene, were purified by agarose gel electrophoresis. Plasmid pMV261/LZ was digested with Xbal + Dral to generate either a 7569 bp or 7574 bp vector fragment. The 7569 bp fragment was ligated to the 1862 bp fragment derived from pMH9.4 to form pMV460F/LZ.
  • Plasmids pMV460 F/LZ and pMV460R/LZ each include a mycobacterial replicon, the L5 attP site, and the L5 integrase gene.
  • a schematic of the formation of plasmids pMV460 F/LZ and pMV460R/LZ is shown in Figure 35.
  • Plasmids pMV460F/LZ and pMV460R/LZ were digested with NotI and recircularized by ligation to generate pMV360F/LZ and pMV360R/LZ.
  • a schematic of the construction of pMV360F/LZ and pMV360R/LZ is shown in Figure 36.
  • Plasmids pMH9.4, pMV261/LZ, pMV460F/LZ, pMV460R/LZ, pMV360F/LZ, and pMV360R/LZ were then transformed into M. smeqmatis and BCG to test their ability to replicate autonomously or integrate into the M. smeqmatis or BCG chromosome. Transformation with pMH9.4, pMV261/LZ, pMV360F/LZ, and pMV360R/LZ yielded kananmycin resistant transformants of M. smeqmatis and BCG. Transformants of pMV261LZ, pMV360F/LZ, and pMV360R/LZ were shown to express E.
  • Plasmids pMV461F/LZ and pMV461R/LZ failed to yield kanamycin resistant transformants, thus indicating that chromosomal integration of a plasmid carrying sequences mediating autonomous replication is lethal to mycobacteria.
  • a partial sequence of the 5' region of the BCG HSP70 gene (which encodes for the BCG HSP70 heat shock protein, also known as the 70 kda antigen) obtained by Dr. Rick Young (MIT) permitted the construction of cassettes carrying expression control sequences (i.e., promoters and translation initiation sequences) by PCR, according to the procedures hereinabove cited.
  • the BCG-HSP71 cassette ( Figure 32) contains 150 bases 5' to the BCG-HSP70 start codon and 15 bases (5 codons) 3' to the start. codon.
  • Primer Xba-HSP70 was synthesized for the 5' end of the cassette, and primer Bam-HSP71 was synthesized for the 3' end of the cassette.
  • the primers had the following base sequences: Xba-HSP70
  • the primers were used to amplify the cassette from BCG substrain Pasteur chromosomal DNA.
  • the addition of the Bam HI site at the 3' end of the cassette adds 1 codon (Asp) to the 3' end of the HSP71 expression cassette.
  • pMV206 and the PCR cassette HSP71 were digested with Xbal and BamHI.
  • the PCR cassette was then inserted between the Xbal and BamHI sites of PMV 206, and then ligated to form pMV271, which is shown in Figure 33.
  • B Cloning of HIV-lqaq.
  • a BamHI-Clal PCR cassette of HIV-1 gag was cloned between the Bam HI and Cla I sites of pMV261 and pMV271 to obtain pMV261/gag and pMV271/gag.
  • Expression of the gag antigens in BCG was verified by the appearance of immunoreactive protein bands in Western blot analysis of BCG pMV271/gag recombinant lysates.
  • BCG transformants were never obtained with pMV261/gag, thus indicating that gag as expressed from pMV26/gag was lethal.
  • C Integration of HSP60-gaq expression cassette into BCG.
  • HSP60-gag expression cassette In order to test whether integration of an HSP-60-gag expression cassette into BCG would result is non-lethal expression of gag in BCG, it was decided that the HSP60-gag expression cassette be cloned into a plasmid (pMV307) which includes the mycobacteriophange L5 attP and integrase sequences, the construction of which is explained hereinbelow. 1. Construction of pMV307.
  • Plasmid pMV206 was digested with NotI to remove the mycobacterial replicon.
  • PCR with primers Xbal-Att/Int and Nhel-Att/Int was then performed on a Sal I fragment from pMH9.4, which contains the attP site and the L5 integrase gene.
  • the resulting cassette was then digested with Xbal and Nhel, and a 1789 bp fragment was gel purified.
  • pMV205 was then digested with Nhel, and the resulting fragment was ligated to the 1989 bp fragment obtained from pMH9.4 to form pMV307.
  • a schematic of the construction of pMV307 is shown in Figure 37. 2. Construction and transformation of pMV361/qag.
  • the Xbal-Clal HSP-antigen cassette which includes the HSP60 promoter and HIV-lgag sequences, was cloned between the NotI and Clal sites of pMV307 to form plasmid pMV361/gag. pMV361/gag was then transformed into BCG and shown to express HIV-lgag protein by Western blot analysis with HIV-1 infected human sera.
  • Example 5 A series of antigen gene fragments, or cassettes, were constructed by PCR, with the exception of the gene fragment containing the gene for human tumor antigen p97, as indicated in Table 2, and cloned into various restriction sites of pMV261 and pMV271 to form new constructs of the pM t* 261 and pMV271 series.
  • the antigen genes, antigen gene fragments, cloning sites used in pMV261 and pMV271 and the names of the resulting constructs, are given below in Table 2.
  • Antigen gene expression cassettes which include a promoter sequence and a heterologous gene sequence, were excised from the pMV261 and pMV271 derivatives with NotI and a second restriction enzyme site (Pvu II, Eco RI, Sal I, Cla I or Hind III) and cloned into the integrating plasmid pMV307 between the NotI site and a second enzyyme site (Pvu II, Eco RI, Sal I, Cla I or Hind III) to form the pMV 361/XXX and pMV371/XXX series of plasmids (e.g., pMV361/HIV-Igpl20) .
  • the backbones of these series of plasmids (pMV361 and pMV371) are shown in Figure 38.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Communicable Diseases (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • General Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Mycology (AREA)
  • Oncology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Virology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)

Abstract

Type d'ADN permettant d'intégrer de l'ADN dans une partie de chromosome de mycobactérie, comprenant une première séquence d'ADN qui est une partie d'ADN phage codant l'intégration bactériophage dans un chromosome de mycobactérie, et une deuxième séquence d'ADN codant une protéine ou un polypeptide hétérologue pour la mycobactérie dans laquelle l'ADN doit être intégré. De l'ADN de ce type peut être intégré dans des mycobactéries, qui peuvent ensuite être administrées comme vaccin et/ou comme agent thérapeutique.
PCT/US1991/004833 1990-07-16 1991-07-09 Adn capable d'une integration a specificite de site dans des mycobacteries WO1992001783A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP3513065A JPH06501607A (ja) 1990-07-16 1991-07-09 ミコバクテリアへの部位特異的組み込みの可能なｄｎａ
BR919106658A BR9106658A (pt) 1990-07-16 1991-07-09 Dna capaz de integracao com localizacao especifica em micetobacterias

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US55390790A 1990-07-16 1990-07-16
US553,907 1990-07-16

Publications (1)

Publication Number Publication Date
WO1992001783A1 true WO1992001783A1 (fr) 1992-02-06

Family

ID=24211262

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1991/004833 WO1992001783A1 (fr) 1990-07-16 1991-07-09 Adn capable d'une integration a specificite de site dans des mycobacteries

Country Status (5)

Country Link
EP (1) EP0556182A4 (fr)
JP (1) JPH06501607A (fr)
AU (1) AU8307791A (fr)
CA (1) CA2045842A1 (fr)
WO (1) WO1992001783A1 (fr)

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0590027A4 (fr) * 1991-06-13 1994-10-19 Einstein Coll Med Mutations par insertion dans des mycobacteries.
WO1995010299A1 (fr) * 1993-10-08 1995-04-20 Laboratoire L. Lafon COMPOSITION DESTINEE A L'IMMUNOTHERAPIE D'UN CANCER SECRETANT L'hCG OU DES FRAGMENTS D'hCG
US5679515A (en) * 1994-10-03 1997-10-21 Pathogenesis Corporation Mycobacterial reporter strains and uses thereof
WO1998044096A3 (fr) * 1997-03-28 1999-01-14 Cytoclonal Pharmaceutics Inc Vaccins recombines a base de mycobacteries
US6013660A (en) * 1996-10-02 2000-01-11 The Regents Of The University Of California Externally targeted prophylactic and chemotherapeutic method and agents
US6566121B1 (en) 1991-06-13 2003-05-20 Albert Einstein College Of Medicine Of Yeshiva University Insertional mutations in mycobacteria
WO2004016280A1 (fr) * 2002-08-16 2004-02-26 Japan Science And Technology Agency Vaccin contre le bcg recombine
US6752993B1 (en) 1993-11-23 2004-06-22 The Regents Of The University Of California Abundant extracellular product vaccines and methods for their production and use
US6761894B1 (en) 1993-11-23 2004-07-13 The Regents Of The University Of California Abundant extracellular products and methods for their production and use
US7300660B2 (en) 1993-11-23 2007-11-27 The Regents Of The University Of California Abundant extracellular products and methods for their production and use

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988006626A1 (fr) * 1987-03-02 1988-09-07 Whitehead Institute For Biomedical Research Vaccin mycobacterien recombinant
WO1990000594A2 (fr) * 1988-07-07 1990-01-25 Whitehead Institute For Biomedical Research Vehicules d'expression mycobacteriens de recombinaison et leur utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988006626A1 (fr) * 1987-03-02 1988-09-07 Whitehead Institute For Biomedical Research Vaccin mycobacterien recombinant
WO1990000594A2 (fr) * 1988-07-07 1990-01-25 Whitehead Institute For Biomedical Research Vehicules d'expression mycobacteriens de recombinaison et leur utilisation

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
Journal of Bacteriology, Volume 170, No. 12, issued December 1988, LI et al., "Site-specific Integration and expression of a developmental promoter in Myxococcus Xanthus", pages 5552-5556, see entire document. *
Journal of Bacteriology, Volume 171, No. 3, issued March 1989, ASTUMIAN et al., "Site-specific recombination between cloned attP and attB sites from the Haemophilus influenzae bacteriophage HP 1 propagated in recombination-deficient Escherichia coli," pages 1747-1750, see the entire document. *
Molecular and General Genetics, Volume 192, issued April 1983, LJUNGQUIST et al., "Properties and Products of the cloned int gene of bacteriophage P2," pages 87-94, see the entire document. *
Molecular and General Genetics, Volume 195, issued May 1984, PIERSON III et al., "Cloning of the integration and attachment regions of bacteriophage P4", pages 44-51, see the entire document. *
Proceedings of the National Academy of Sciences, Volume 85, issued September 1988, SNAPPER et al., "Lysogeny and transformation in mycobacteria: Stable expression of foreign genes", pages 6987-6991, see the entire document. *
Reviews of Infectous Diseases Volume 11, Supp. 2, issued March-April 1989, JACOBS, Jr. et al., Myco bacteriophage vector systems", pages 5404-5410, see entire document. *
See also references of EP0556182A4 *

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0972839A1 (fr) * 1991-06-13 2000-01-19 ALBERT EINSTEIN COLLEGE OF MEDICINE OF YESHIVA UNIVERSITY, a division of YESHIVA UNIVERSITY Mutations par insertion dans des mycobactéries
EP0590027A4 (fr) * 1991-06-13 1994-10-19 Einstein Coll Med Mutations par insertion dans des mycobacteries.
US6752994B2 (en) 1991-06-13 2004-06-22 Albert Einstein College Of Medicine Of Yeshiva University Insertional mutations in mycobacteria
US6566121B1 (en) 1991-06-13 2003-05-20 Albert Einstein College Of Medicine Of Yeshiva University Insertional mutations in mycobacteria
WO1995010299A1 (fr) * 1993-10-08 1995-04-20 Laboratoire L. Lafon COMPOSITION DESTINEE A L'IMMUNOTHERAPIE D'UN CANCER SECRETANT L'hCG OU DES FRAGMENTS D'hCG
US6752993B1 (en) 1993-11-23 2004-06-22 The Regents Of The University Of California Abundant extracellular product vaccines and methods for their production and use
US6761894B1 (en) 1993-11-23 2004-07-13 The Regents Of The University Of California Abundant extracellular products and methods for their production and use
US7300660B2 (en) 1993-11-23 2007-11-27 The Regents Of The University Of California Abundant extracellular products and methods for their production and use
US5679515A (en) * 1994-10-03 1997-10-21 Pathogenesis Corporation Mycobacterial reporter strains and uses thereof
US6013660A (en) * 1996-10-02 2000-01-11 The Regents Of The University Of California Externally targeted prophylactic and chemotherapeutic method and agents
WO1998044096A3 (fr) * 1997-03-28 1999-01-14 Cytoclonal Pharmaceutics Inc Vaccins recombines a base de mycobacteries
WO2004016280A1 (fr) * 2002-08-16 2004-02-26 Japan Science And Technology Agency Vaccin contre le bcg recombine
US7638133B2 (en) 2002-08-16 2009-12-29 Department Of Medical Sciences Ministry Of Public Health Of Thailand Recombinant BCG vaccine
US7670610B2 (en) * 2002-08-16 2010-03-02 Department of Medical Sciences-Ministry of Public Health of Thailand Recombinant BCG vaccine

Also Published As

Publication number Publication date
EP0556182A4 (en) 1995-03-01
JPH06501607A (ja) 1994-02-24
EP0556182A1 (fr) 1993-08-25
CA2045842A1 (fr) 1992-01-17
AU8307791A (en) 1992-02-18

Similar Documents

Publication Publication Date Title
US5583038A (en) Bacterial expression vectors containing DNA encoding secretion signals of lipoproteins
AU2185592A (en) Induction of ctl responses to foreign antigens expressed in mycobacteria
US6833135B1 (en) DNA integration into “Mycobacterium spp.” genome by trans-complementation using a site-specific integration system
Jaworski et al. A functional origin of transfer (oriT) on the conjugative transposon Tn916
JP3485916B2 (ja) 組み換えマイコバクテリアの発現ベヒクルおよびその使用
EP0347425B1 (fr) Vaccin mycobacterien recombinant
US6752994B2 (en) Insertional mutations in mycobacteria
EP0478664B1 (fr) Insertion genomique a mediation vectorielle et expression d'adn dans le bcg (bacille bilie calmette-guerin)
EP0544685A1 (fr) Vecteurs
WO1992001783A1 (fr) Adn capable d'une integration a specificite de site dans des mycobacteries
US5830457A (en) Recombinant beta-lactamase, usable as carrier molecule in immunogenic compositions
US5866403A (en) Homologously recombinant slow growing mycobacteria and uses therefor
EP0972839A1 (fr) Mutations par insertion dans des mycobactéries
US5736367A (en) Vectors and prokaryotes which autocatalytically delete antibiotic resistance
WO1999016868A1 (fr) Phasmides navette conditionnels tm4 et leurs utilisations
US6248581B1 (en) Mycobacteria functional screening and/or expression vectors
NZ244901A (en) Mycobacterium paratuberculosis nucleotide sequence and its use in vectors and host cells for cloning and expression of nucleotide sequences; vaccines containing the host cells
WO1993010246A1 (fr) Expression membranaire de genes heterologues
WO1994014318A1 (fr) Procedes de protection contre le streptococcus pneumoniae a l'aide de mycobacteries transformees
EP0587765A1 (fr) Induction d'une reaction immunitaire a la toxine du tetanos par administration de mycobacteries exprimant la toxine du tetanos ou des fragments de celle-ci
WO1993019603A1 (fr) Procaryotes presentant une immunite contre des bacteriophages codant l'adn
WO1995032277A9 (fr) Mutations par insertion dans des mycobacteries
WO1995032277A1 (fr) Mutations par insertion dans des mycobacteries

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU BR JP

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IT LU NL SE

WWE Wipo information: entry into national phase

Ref document number: 1991914084

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1991914084

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1991914084

Country of ref document: EP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载