EP0544685A1 - Vecteurs - Google Patents

Vecteurs

Info

Publication number
EP0544685A1
EP0544685A1 EP91912483A EP91912483A EP0544685A1 EP 0544685 A1 EP0544685 A1 EP 0544685A1 EP 91912483 A EP91912483 A EP 91912483A EP 91912483 A EP91912483 A EP 91912483A EP 0544685 A1 EP0544685 A1 EP 0544685A1
Authority
EP
European Patent Office
Prior art keywords
vector
mycobacterium
dna
recombinant
bcg
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP91912483A
Other languages
German (de)
English (en)
Inventor
Paul Jacobs
Françoise HAESELEER
Marc Massaer
Alex Bollen
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
GlaxoSmithKline Biologicals SA
Original Assignee
SmithKline Beecham Biologicals SA
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 SmithKline Beecham Biologicals SA filed Critical SmithKline Beecham Biologicals SA
Publication of EP0544685A1 publication Critical patent/EP0544685A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
    • C07K14/445—Plasmodium
    • 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

Definitions

  • This invention relates to expression of foreign DNA in mycobacteria, to novel vectors for use in such expression and to recombinant mycobacterial vaccines.
  • Mycobacteria represent major pathogens of man and animals.
  • tuberculosis is generally caused in humans by Mycobacterium (M.) tuberculosis and in cattle by Mycobacterium (M.) bovis (which can be transmitted to humans and other animals, in whom it causes tuberculosis) .
  • Tuberculosis remains widespread and is an important public health problem, particularly in developing countries.
  • M. tuberculosis and mycobacteria of the avium- intracellulare-scrofulaceum (MAIS) group represent major opportunistic pathogens of patients with acquired immunodeficiency disease (AIDS) .
  • M. pseudotuberculosis is a major pathogen of cattle.
  • Bacille Calmette-Guerin (BCG) , an avirulent strain of M. bovis, is the most widely used human vaccine in the world and has been used as a live vaccine for more than 50 years. In the past 35 years, it has been administered to over 2.5 billion people, with remarkably few adverse effects (e.g. estimated mortality of 60/billion) . BCG has been found in numerous studies to have protective efficacy against tuberculosis. Recently, however, it was found not to be effective in preventing pulmonary tuberculosis in Southern India. Mycobacterium smegmatis is a nonpathogenic bacillus which shares antigenic and adjuvent properties with BCG. Both are also reasonably easy to grow in culture.
  • WO 90/00594 (Whitehead Institute) describes recombinant vectors which are temperate shuttle phasmids and bacterial-mycobacterial shuttle plasmids which can be used to introduce foreign DNA stably into mycobacteria, for use inter alia as vaccine vehicles. The foreign DNA is then expressed in mycobacteria under control of the E. Coli promoters expressing kanamycin resistance, chloramphenicol resistance and cl, or the M. leprae 65kD gene promoter.
  • the DNA sequence of 2431 base pairs of the Mycobacterium bovis - BCG gene encoding an immunogenic 64kD protein has been reported (Thole et al., 1987) .
  • the coding sequence begins at position 576.
  • sequence preceding base 576 contains a promoter and ribosomal binding site which is effective in controlling expression of exogenous coding DNA in mycobacteria.
  • a mycobacterial expression vector containing exogenous coding DNA under the control of the promoter and ribosomal binding site for the 64kD protein of Mycobacterium bovis - BCG.
  • the expression vector may be prepared in accordance with the invention by cleaving a mycobacterial vector to provide a linear DNA segment having a intact mycobacterial replicon, and ligating said linear segment and one or more DNA molecules which, together with said linear segment, complete the DNA sequence for the coding DNA and the promoter and RBS for the 64kD protein of M. bovis - BCG.
  • RBS ribosomal binding site
  • the 64kD promoter and RBS 10 comprises the fragment 115-575 of the Mycobacterium bovis - BCG 64kD gene or a functional derivative thereof.
  • the invention provides the fragment 115-575 of the Mycobacterium bovis - BCG 64kD gene or a 15 functional derivative thereof in the absence of said 64kD coding sequence and the use of said fragment or derivative for expressing exogenous coding DNA in mycobacteria.
  • the functional derivative may be a mutant or truncate of the 20 115-575 fragment which retains functional promoter and RBS activity.
  • Mutations of the 115-575 region are also contemplated in order to introduce convenient restriction sites.
  • an Ncol site can be introduced at the translation 30 start site by replacing the A at 575 by C.
  • the fragment may be prepared by conventional means, for example by excision of all or a part of the fragment from a vector containing the relevant part of the 64kD gene, and ligation of excised fragments with synthetic oligonucleotide linkers as necessary to generate the required sequence, or by total oligonucleotide synthesis.
  • mycobacterial expression vectors of the invention include a mycobacterial plasmid or phage, or a shuttle vector comprising a linearised mycobacterial plasmid or phage fused to a linearised plasmid or phage replicable in another microorganism such as E_ ⁇ . Coli, streptomyces, Bacillus or yeast, to aid manipulation.
  • a mycobacterial plasmid or phage or a shuttle vector comprising a linearised mycobacterial plasmid or phage fused to a linearised plasmid or phage replicable in another microorganism such as E_ ⁇ . Coli, streptomyces, Bacillus or yeast, to aid manipulation.
  • E_ ⁇ . Coli streptomyces, Bacillus or yeast
  • the recombinant mycobacterium of the invention may be prepared by transforming a mycobacterium with a mycobacterial expression vector of the invention. It will be appreciated that, depending on the nature of the expression vector of the invention, the exogenous coding DNA may be maintained extrachromosomally as a plasmid or within the mycobacterial- genome via site specific integration.
  • an E_;_ coli cosmid may be introduced into the temperate mycobacteriophage LI, producing shuttle phasmids capable of replicating as plasmids in E ⁇ coli or lysogenizing the mycobacterial host.
  • These temperate shuttle plasmids form turbid plaques on ML_ smegmatis and, upon lysogenization, confer resistance to superinfection and integrate with the mycobacterial genome.
  • a shuttle plasmid vector can be used to introduce exogenous coding DNA into mycobacteria, in which the DNA is expressed extrachromosomally.
  • M. fortuitum E. coli hybrid plasmids may be constructed from mycobacterial and E. coli replicons containing marker genes.
  • exogenous coding DNA may be introduced into the mycobacterial genome under the control of the 64kD promoter and RBS of M_;_ bovis - BCG but without the use of a mycobacterial expression vector, by the process of homologous recombination, site specific recombination or non-homologous recombination using a suitable vector such as the Ei Coli pUC19 vector as described in W090/00594.
  • Mycobacteriophages useful in the present invention include LI, TM4 and D29.
  • M. fortuitum plasmid pAL5000 (Rauzier et, _al.) may be employed as a mycobacterial expression vector by insertion of exogenous coding DNA and a functional mycobacterial promoter and RBS in the region of ORF3-ORF4 (nucleotide bases 1541-3908) .
  • a mycobacterial expression vector comprising pAL5000 or a replicable derivative thereof containing exogenous coding DNA and a functional mycobacterial promoter and RBS inserted • in the ORF3-ORF4 region thereof, and a recombinant mycobacterium transformed with said vector.
  • the vector of the invention derived from pAL5000 may be prepared in accordance with the invention by cleaving pAL5000 or a derivative thereof in the region of ORF3-ORF4 and ligating the resulting linear DNA segment with one or more DNA molecules which, together with said linear segment complete the DNA sequence for the coding DNA and the promoter and RBS.
  • the recombinant mycobacterium may be prepared by transforming a mycobacterium with the vector of the invention derived from pAL5000.
  • the functional mycobacterial promoter and RBS is the promoter and RBS for the 64kD protein of M. bovis - BCG.
  • a convenient site for insertion of the promoter, RBS and coding DNA is between the restriction sites EcoRV and Apal of pAL5000.
  • Suitable replicable derivatives of pAL5000 include shuttle vectors in which the pAL5000 is linearised and fused to a linearised plasmid or phage replicable in another microorganism such as E_ ⁇ Coli to aid manipulation.
  • Suitable mycobacteria which may be transformed by-the process of the invention include M. smegmatis, M. bovis - BCG, M. avium, M. phlei, M. fortuitum, M. luf ⁇ , M. part berculosis, M. habana, M. scrofulaceum and M. intracellulare.
  • M ⁇ _ bovis - BCG and M. tuberculosis In the case of slow growing mycobacteria (e.g., M ⁇ _ bovis - BCG and M. tuberculosis) to be used as vaccine vehicles, it is particularly valuable to go through (i.e., introduce DNA encoding an " antigen or antigen ' s of interest into) M. smegmatis and, subsequently, into M. bovis - BCG.
  • E_;_ coli or streptomyces having a cloned plasmid is fused, using known techniques, with a mycobacterial spheroplast.
  • E_ ⁇ coli minicells which contain plasmid DNA and essentially no chromosomal DNA, can be used in carrying out a minicell protoplast fusion.
  • a further approach in which the vector DNA is introduced directly into intact M. smegmatis cells by electroporation obviates possible damage to mycobacterial cells .which might result from use of protocols for producing sphereoplasts.
  • Transformation of mycobacteria by mycobacteriophage- based vectors may be carried out by phage infection procedures generally described in 090/00594, that is, by a modification of the procedure described by Okanishi and Hopwood in relation to the preparation of spheroplasts for Streptomyces. Streptomyces, like mycobacteria, are Actinomycetales.
  • the modified technique is used in combination with the addition of polyethylene glycol to facilitate entry of DNA molecules into bacterial spheroplasts.
  • the expression vector which preferably also includes a selectable marker such as an antibiotic resistance gene is packaged into bacteriophage lambda heads using lambda in vitro packaging mix. E_ ⁇ coli is subsequently transduced with the phage, with the result that it is possible to screen (using antibiotic-containing medium) for colonies containing the antibiotic- resistance-encoding gene and the coding DNA.
  • the resulting ''library'' is introduced into M ⁇ smegmatis using, for example, electroporation. Plaques which contain vectors containing cloned insert DNA are selected. Subsequently, recombinant M ⁇ smegmatis can be used to infect a cultivable mycobacterium, such as BCG, with high efficiency. As a result, the coding DNA is introduced into mycobacterial genomic DNA, where it will be expressed.
  • Selection of BCG containing the coding DNA of interest can be carried out using a selectable marker, such as an antibiotic-resistance-encoding gene or a gene which complements that missing in an auxotrophic mutant, or by use of the cl gene which encodes the repressor protein of the LI bacteriophage.
  • a selectable marker such as an antibiotic-resistance-encoding gene or a gene which complements that missing in an auxotrophic mutant, or by use of the cl gene which encodes the repressor protein of the LI bacteriophage.
  • an auxotrophic mycobacterial mutant e.g. a pyr-F mutant
  • the gene present in the corresponding wild-type (nonmutant) mycobacterium is incorporated into the vector.
  • deoxyglucose mutants which have a defect in the glucokinase gene, as well as others having mutations in other biosynthetic pathways (e.g. mutations in amino acid biosynthesis, vitamin biosynthesis and carbohydrate metabolism, such as arabinose and galactose) .
  • a mycobacterial mutant is selected and the gene which complements the mutation is incorporated into the vector, which also contains the exogenous coding DNA.
  • the marker gene may be inserted in the vector under control of the same promoter as the exogenous coding DNA, i.e. to provide a polycistronic expression vector, or the marker gene may have its own promoter.
  • the mycobacterial mutants into which the coding DNA is successfully introduced will be identifiable (can be selected) by culturing on appropriately-selected media (e.g. media containing the antibiotic against which resistance is conferred, media containing or lacking the nutrients involved in the biosynthetic pathway affected in the mutant used) or by selecting on the basis of the appearance of plaques formed, when the cl gene is used.
  • appropriately-selected media e.g. media containing the antibiotic against which resistance is conferred, media containing or lacking the nutrients involved in the biosynthetic pathway affected in the mutant used
  • BCG containing coding DNA by use of monoclonal antibodies.
  • a gene or gene fragment encoding one or more epitopes of an antigen e.g. M. leprae or M. tuberculosis
  • monoclonal antibodies are used to select for recombinant BCG containing a gene or genes encoding one or more of these epitopes.
  • the marker antigen genes introduced in this way contain a promoter sequence and other regulatory sequences.
  • exogenous coding DNA can be added, using genetic engineering techniques, in frame, such that recombinant BCG identified by monoclonal antibodies to the marker antigen would also be expressing the exogenous coding DNA so introduced.
  • Another component of a plasmid vector essential in introducing exogenous coding DNA into the recombinant mycobacteria vehicle is an autonomously replicating sequence (e.g. a replicon) , whose presence is a key determinant in allowing the vector to replicate autonomously
  • an autonomously replicating sequence e.g. a replicon
  • sequences can include, for example, a plasmid replicon or segments of a mycobacteriophage or chromosomal replication origins.
  • Direct introduction of exogenous coding DNA under the control of the promoter and RBS may be accomplished, for example, using minicell protoplast fusion.
  • a selectable marker for the mycobacterium which can be antibiotic-resistance gene or a chromosomal mutation, can be cloned into an E ⁇ _ coli cosmid.
  • Also present in the E. coli cosmid will be DNA which allows efficient integration of exogenous coding DNA into mycobacterial chromosome. For example, in M. leprae, a repetitive sequence occurs which appears to be associated with recombination; analogous sequences can be identified in and isolated from BCG and M. smegmatis, incorporated into the E ⁇ .
  • exogenous coding DNA may be introduced directly by electroporation.
  • Exogenous coding DNA can be incorporated into the vector described (e.g. which includes an E. coli replicon, a segment of mycobacterial chromosomal DNA associated with recombination (a recombinogenic sequence) and two selectable markers-one serving as a marker in E. coli and the second serving as a marker in the mycobacterium) .
  • the gene(s) can then be integrated into mycobacterial chromosomal DNA, such as BCG or M ⁇ smegmatis chromosomal DNA.
  • the gene(s) of interest are integrated in this way into M. smegmatis, it/they can also be moved into BCG by means of a general transducing phage. In this case, it is preferable to include, in addition to the other construct components, two recombinogenic sequences: one from M. smegmatis and one from BCG.
  • Exogenous coding DNA is herein defined as DNA from a source other than the mycobacterium into which the DNA is being incorporated. It may code for all or a portion of a gene or genes encoding a protein(s) or polypeptide(s) of interest and/or a selectable marker or markers.
  • the proteins or polypeptides of interest can be, for example, proteins or polypeptides against which an immune response is desired (antigen(s) of interest), enzymes, lymphokines, immunopotentiators, pharmacologic agents, steroids, and reporter molecules of interest in a diagnostic context (e.g.
  • the recombinant mycobacteria of the invention are particularly useful as vehicles by which the DNA of interest can be expressed.
  • Such vehicles can be used, for example, as vaccine vehicles which express a polypeptide or a protein of interest (or more than one polypeptide or protein) , such as an antigen or antigens, of one or more pathogens of interest.
  • the recombinant mycobacteria can also be used as a vehicle for expression of immunopotentiators, enzymes, pharmacologic agents, steroids and antitumor agents; for expression of a polypeptide or a protein useful in producing an anti-fertility vaccine vehicle; or for expression of stress proteins, which can be administered to evoke an immune response or to induce tolerance in an autoimmune disease (e.g. rheumatoid arthritis) .
  • an autoimmune disease e.g. rheumatoid arthritis
  • Recombinant mycobacteria can, for example, express protein(s) or polypeptide(s) which are growth inhibitors or are cytocidal for tumor cells (e.g., interferon ⁇ , ⁇ or ⁇ ; interleukins 1-7, tumor necrosis factor (TNF) ⁇ or ⁇ and, thus, provide the basis for a new strategy for treating certain human cancers (e.g., bladder cancer, melanomas).
  • Pathogens of interest include any virus, microorganism, or other organism or substance (e.g., a toxin or toxoid) which causes disease.
  • the present invention thus relates to a process for preparing a protein or polypeptide of interest which process comprises expressing exogenous DNA in a recombinant mycobacterium according to the invention and, if required, recovering the protein or polypeptide of interest, and to protein or polypeptide of interest produced in a recombinant mycobacterium according to the invention.
  • M_;_ smegmatis is a preferred mycobacterium.
  • the present invention also relates to methods of vaccinating a host with the recombinant mycobacterium 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 present invention relates to use of the antigens expressed by the recombinant cultivable mycobacterium as vaccines or as diagnostic reagents.
  • Mycobacteria have adjuvant properties among the best currently known and, thus, stimulate a recipient's immune system to respond to other antigens with great effectiveness. This is a particularly valuable aspect of the vaccine because it is expected to induce cell- mediated immunity and, thus, has the potential to provide immunity against pathogens in cases where cell- mediated immunity appears to be critical for resistance. Second, the vaccine is expected to stimulate long-term memory or immunity. As a result, a single (one-time) inoculation has the potential to produce long-term sensitization to protein antigens.
  • the vaccine vehicle of the present invention has the potential to prime long-lasting T cell memory, which stimulates secondary antibody respones neutralizing to the infectious agent or the toxin.
  • tetanus and diphtheria toxins pertussis, malaria, influenza, herpes viruses, snake and insect venoms, leprosy, tuberculosis, diphtheria, tetanus, leishmania, salmonella, schistomiasis, measles, mumps, respiratory syncitial virus, varicella zoster virus, treponema, shigella, neisseria, borellia, rabies, polio, HIV and vibrio cholerae.
  • Genes encoding one or more protective antigens for one or more of the disease-causing pathogens can be obtained by isolation of the naturally-occurring DNA (e.g.
  • a particular antigen of interest is the Plasmodium CS protein gene (US 4707357, Airnot et; _l.) more particularly the P. falciparum CS protein gene.
  • the antigen is expressed as ala- j ⁇ -se ⁇ g ⁇ with an N-terminal methionine.
  • the antigen is expressed as the full length protein
  • the invention provides a Plasmodium CS protein gene expression unit comprising a DNA coding sequence for said protein and a regulatory element necessary for transcription of the coding sequence and translation within a mycobacterium, a mycobacterial vector comprising said gene expression unit and a recombinant mycobacterium comprising said gene expression unit.
  • the vector contains the Plasmodium CS protein gene under the control of the promoter and RBS for the 64kD protein of M. bovis - BCG.
  • the gene is carried within pAL5000 or a replicable derivative thereof containing a functional mycobacterial promoter and RBS in the ORF3-ORF4 region thereof.
  • the recombinant mycobacterium preferably comprises the Plasmodium CS protein gene under the control of the promoter and RBS for the 64kD protein of M. bovis - BCG.
  • the recombinant mycobacterium is preferably transformed with pAL5000 or a replicable derivative thereof containing a mycobacterial promoter and RBS and said gene in the ORF3-ORF4 region thereof.
  • any of the uses of the recombinant mycobacteria to express a protein or polypeptide it is possible to include in the shuttle vector DNA encoding a signal sequence and thus, provide a means by which the expressed protein or polypeptide is made in the cytoplasm and then secreted in the cell walls.
  • the signal sequence from ⁇ antigen which is secreted in mycobacteria, could be used.
  • the signal sequence for ⁇ -galactosidase, agarase or ⁇ amylase could be used.
  • BCG has important advantages as a vaccine vehicle in that: 1) it is the only childhood vaccine currently given at birth; 2) in the past 40 years, it has had a very low incidence of adverse effects, when given as a vaccine against tuberculosis; and 3) it can be used repeatedly in an individual (e.g. in multiple forms) .
  • a preferred mycobacterial host cell for use as a vaccine vehicle is M. bovis - BCG.
  • Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller et al, University Park Press, Baltimore, Maryland, U.S.A., 1978.
  • Encapsulation within liposomes is described, for example by Fullerton, U.S. Patent 4,235,877.
  • the amount of the recombinant mycobacteria present in each vaccine dose is selected as an amount which induces an immunoprotective response without significant, adverse side effects in typical vaccines. Such amount will vary depending upon which specific immunogen is employed. Generally, it is expected that each dose will comprise 1-1000 ⁇ g of protein, preferably 1-200 ⁇ g. An optimal amount for a particular vaccine can be ascertained by standard studies involving observation of antibody titres and other responses in subjects. Following an initial vaccination, subjects will preferably receive a boost in about 4 weeks, followed by repeated boosts every six months for as long as a risk of infection exists.
  • the invention also provides a vaccine composition which comprises recombinant mycobacteria according to the invention in combination with a pharmaceutically acceptable carrier, recombinant mycobacteria according to the invention for use in vaccinating a host and use of recombinant mycobacteria according to the invention in the preparation of a vaccine.
  • Plasmid pNIV2119 expressing circumsporozoite protein of P. falciparum (amino acids 18 to 391)
  • pAL8 (Fig. 1) is a shuttle vector constructed by B. Gicquel at the Institut Pasteur of Paris. (Unite de Genie Microbiologique, 28 rue du Dr. Roux, F-75724 Paris, Cedex 15) . It consists of the M. fortuitum pAL5000 plasmid in the Kpnl unique restriction site of which (position 1983 of pAL5000 sequence published by Rauzier et aJ plasmid pTZ19R (an E_;_ Coli plasmid containing an origin of replication and ampicillin resistance gene purchased from Pharmacia) linearised through its unique Kpnl site was inserted. Moreover, the Kan? resistance gene (from Tn903) was isolated from pUC4K (purchased from Pharmacia) as a PstI fragment and introduced into the pTZ19R unique PstI site. pAL8 has 8947 bp.
  • the expression cassette was isolated from the plasmid pNIV2116 from 1.1(b) by restriction with Kpnl and PvuII. This fragment, bases 115-579 of P64 preceded by five bases (CTGAG) from the polylinker of pULB1221 and followed by the coding sequence for the circumsporozoite protein gene starting at the second base of the ala- ⁇ g coding triplet, was introduced into pAL8 from 1.1 (a) between EcoRV and Apal unique restriction sites at positions 6145 and 7986 respectively (corresponding to positions 2035 and 3876 of pAL5000) resulting in the replacement of a 1841 bp non-essential fragment of the plasmid by the expression cassette to give plasmid pNIV2119 (Fig. 2) and plasmid pNIV2126 (containing the expression cassette in the opposite orientation) .
  • Plasmids pNIV2119 and pNIV2126 from 1.2 were introduced into M. smegmatis by electroporation. Transformants were 5 selected on the basis of their resitance to kanamycin. They were shown by ELISA and Western blot (Chan, D.W.; Towbinet et al.) to express the circumsporozoite protein of P. falciparum at a level of about 0.6% of total cell proteins, whatever the orientation of the expression cassette. 10
  • Plasmid pNIV2119 from 1.2 was introduced into BCG by electroporation. Transformants were selected on the basis 15 of their resistance to kanamycin and by hybridization with a CSP probe. They were shown by ELISA and Western blot to express the circumsporozoite protein of P_;_ falciparum at a level of about 0.15% of total cell proteins.
  • Plasmids pNIV2124 and pNIV2125 expressing the complete circumsporozoite protein of P. falciparum Plasmids pNIV2124 and pNIV2125 expressing the complete circumsporozoite protein of P. falciparum
  • a Xhol -Asp700 fragment of 450 bp was excised from pRIB-pUC (Example 1.1(b) (i)). . This fragment starts at position 115 and terminates at 567, 8 bases upstream from 30 the ATG. It was ligated to a synthetic fragment of the following sequences (90009/90010) . Met Met Arg Lys Leu Ala lie Leu Ser Val
  • the synthetic fragment codes for aa 1 to- 18 of CSP preceded by the 8 last bases of the P64 promoter.
  • the StuI site overlapping aal7 and aal ⁇ is immediately followed by an Xhol protruding end.
  • a Stul-Ddel fragment was isolated from pNIV2107 (WO 90/07006) .
  • This fragment starts at the second base of the triplet coding for Alal ⁇ and stops 17 bases downstream to the natural stop (TAG) codon of CSP. After blunting of its Ddel extremity, this fragment was inserted into the StuI site of pNIV2223 leading to pNIV2123 (Fig. 3a) .
  • This plasmid thus contains the expression cassette consisting of the p64 promoter and Shine-Dalgarno sequences (positions 115 to 575) followed by the sequence coding for the complete CSP.
  • the expression cassette was excised from pNIV2123 as a PvuII-MscI fragment and was inserted into pAL8 between the blunted Apal and EcoRV sites resulting into plasmids pNIV2124 and pNIV2125 (the expression cassette being in the opposite orientation) (Fig. 3b) .
  • Plasmid pNIV2124 was electroporated into M. smegmatis cells and recombinant colonies were selected on the basis of their resistance to kanamycin. They were shown by ELISA and Western blot to express the CSP at about 0.3% of total cell proteins.
  • Plasmid pNIV2124 was electroporated into BCG and recombinant colonies were selected on the basis of their resistance to kanamycin and by hybridization with a CSP probe. They were shown to express the complete circumsporozoite protein of P. falciparum at about 0.01% of total cell proteins..
  • Plasmids pNIV2120 and pNIV2121 expressing circumsporozoite protein of P. falciparum Plasmids pNIV2120 and pNIV2121 expressing circumsporozoite protein of P. falciparum
  • plasmids pNIV2120 and pNIV2121 - were similar to that of pNIV2119 in Example 1 except that the fragment of the 5' untranslated region of the P64 gene excised from pRIB-pUC was the Xhol-Asp700 fragment which terminates at position 567. This fragment is followed by synthetic oligonucleotides to restore the complete sequence up to the ATGG (position 579) .
  • the only difference in the resulting sequence with respect to the natural one is the presence of a C instead of an A immediately before the A of the ATG at position 575, thus introducing a convenient Ncol restriction site.
  • the sequence of the synthetic fragment is as follows:
  • the Xhol-Asp700 fragment and the synthetic fragment were first inserted between the Xhol and MscI sites of pNIV103.
  • the resulting plasmid pNIV2221 (Fig. 4) was tested for the reconstitution of Asp700, the presence of Ncol and the reconstitution of MscI.
  • the Stul-Bcll fragment coding for the circumsporozoite protein was inserted in plasmid pNIV2221 between MscI and BamHI.
  • the complete expression cassette was then transferred into pAL8 as a PvuII-Asp718 fragment (coding for the CSP molecule starting at the AlalS residue) between Apal and EcoRV as described in 1.2 above. Plasmids were obtained with the expression cassette in both orientations (pNIV2120 (Fig. 5) and pNIV2121) .
  • Plasmid pNIV2120 was electroporated into ML . smegmatis and shown to direct the expression of the CSP at the same level as that obtained with pNIV2119 and pNIV2125.
  • Plasmid pNIV2120 was electroporated into BCG. Transformants were selected on the basis of their resistance to kanamycin. They were shown by ELISA and Western blot to express CSP at the same level as that obtained with pNIV2119 and pNIV2125.
  • Plasmid pNIV2203 is a pUC19 based plasmid containing a 2320 bp genomic fragment of BCG DNA containing the gene coding for Ornithine Carbamoyl transferase. This plasmid was constructed by subcloning of a blunted Ndel-Bglll fragment from pOTC into the Malawi site of pUC19. pOTC itself was selected by complementation of an OTC minus E. coli strain described in Crabeel et al (Gene 5 . , 207-231, 1979) by the DNA of a genomic library constructed by insertion of Sau3A partially digested BCG DNA into the BamHI site of pASI.
  • pNIV2141 (see Fig 6) was constructed by ligation of the Kan cassette isolated as a Hindll fragment from pUC4K (Pharmacia) and the P64-CSPs + a + cassette isolated as a PvuII-Mscl fragment from PNIV2123 into a unique blunted Xhol restriction site into the OTCase coding sequence of pNIV2203.
  • Plasmid pNIV2141 was introduced into BCG by electroporation. Transformants were selected on the basis of their resistance to kanamycin and by hybridization with a CSP probe. They were shown by ELISA and Western blot to express the circumsporozoite protein of P.falciparum. Plasmid pNIV2141 was shown by Southern blot analysis to be integrated into the BCG genome by non-homologous recombination.
  • CSPs a - coding sequence for the complete (amino acids 1 to 412) circumsporozoite protein of P . falciparum.
  • OTC - genomic BCG fragment containing the OTCase gene OTC - genomic BCG fragment containing the OTCase gene.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne un vecteur d'expression mycobactérienne contenant de l'ADN exogène codant une protéine à examiner par exemple un antigène) sous le contrôle d'un promoteur et d'un site de liaison de ribosome pour la protéine 64 KD du Mycobacterium bovis BCG. L'invention concerne également une mycobactérie transformée avec les vecteurs d'expression mycobactérienne et des compositions de vaccins comprenant la mycobactérie transformée.
EP91912483A 1990-07-19 1991-07-13 Vecteurs Withdrawn EP0544685A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB9015888 1990-07-19
GB909015888A GB9015888D0 (en) 1990-07-19 1990-07-19 Vectors

Publications (1)

Publication Number Publication Date
EP0544685A1 true EP0544685A1 (fr) 1993-06-09

Family

ID=10679341

Family Applications (1)

Application Number Title Priority Date Filing Date
EP91912483A Withdrawn EP0544685A1 (fr) 1990-07-19 1991-07-13 Vecteurs

Country Status (4)

Country Link
EP (1) EP0544685A1 (fr)
AU (1) AU8101291A (fr)
GB (1) GB9015888D0 (fr)
WO (1) WO1992001796A1 (fr)

Families Citing this family (34)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6566121B1 (en) 1991-06-13 2003-05-20 Albert Einstein College Of Medicine Of Yeshiva University Insertional mutations in mycobacteria
EP0521220A1 (fr) 1991-06-14 1993-01-07 Institut Pasteur Actinomycétale immunogène recombinant
US6238676B1 (en) 1992-12-10 2001-05-29 Brigham And Women's Hospital Presentation of hydrophobic antigens to T-cells by CD1 molecules
US7063844B2 (en) 1992-12-10 2006-06-20 The Brigham And Women's Hospital, Inc. Presentation of hydrophobic antigens to T-cells by CD1 molecules
US9266930B1 (en) 1993-03-05 2016-02-23 Epimmune Inc. Inducing cellular immune responses to Plasmodium falciparum using peptide and nucleic acid compositions
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
WO1995014713A2 (fr) 1993-11-23 1995-06-01 The Regents Of The University Of California Vaccins a base de produits extracellulaires abondants, leurs procedes de fabrication et leur utilisation
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
US5843664A (en) * 1996-06-11 1998-12-01 Institut Pasteur Method of selection of allelic exchange mutants
US6013660A (en) * 1996-10-02 2000-01-11 The Regents Of The University Of California Externally targeted prophylactic and chemotherapeutic method and agents
FR2767337B1 (fr) * 1997-08-14 2002-07-05 Pasteur Institut Sequences nucleiques de polypeptides exportes de mycobacteri es, vecteurs les comprenant et applications au diagnostic et a la prevention de la tuberculose
US6686339B1 (en) 1998-08-20 2004-02-03 Aventis Pasteur Limited Nucleic acid molecules encoding inclusion membrane protein C of Chlamydia
US6693087B1 (en) 1998-08-20 2004-02-17 Aventis Pasteur Limited Nucleic acid molecules encoding POMP91A protein of Chlamydia
CA2340330A1 (fr) 1998-08-20 2000-03-02 Aventis Pasteur Limited Molecules d'acide nucleique codant la proteine membrane d'inclusion c de chlamydia
DE60038971D1 (de) 1999-10-22 2008-07-03 Aventis Pasteur Verfahren zur erregung und/oder verstärkung der immunantwort gegen tumorantigene
EP1792995A3 (fr) 2000-05-08 2007-06-13 Sanofi Pasteur Limited Antigènes de chlamydia et fragments d'adn correspondants, et utilisations de ceux-ci
EP1282702B1 (fr) 2000-05-10 2006-11-29 Sanofi Pasteur Limited Polypeptides immunogeniques codes par des minigenes mage et leurs utilisations
ATE357461T1 (de) 2000-08-25 2007-04-15 Aventis Pasteur Innen-kern oligosaccharide epitopes aus lipopolysacchariden von haemophilus influenza als vakzinen in der prophylaktischen behandlung von haemophilus influenzae infektionen
EP1343894A4 (fr) 2000-11-02 2005-05-04 Univ Singapore Gene aopb, proteine, homologues, fragments et variants de celui-ci, ainsi que l'utilisation de ces derniers pour presentation a la surface cellulaire
EP1864691B1 (fr) 2002-04-09 2011-07-20 Sanofi Pasteur Limited Acide nucléique ACE modifié et vecteurs d'expression
CN106008679A (zh) 2008-12-24 2016-10-12 荷兰王国卫生福利和运动部国家公共卫生和环境研究所 修饰的肺炎链球菌溶血素(ply)多肽
WO2012101509A2 (fr) 2011-01-28 2012-08-02 Sanofi Pasteur Sa Compositions immunologiques contre le vih
JP6193231B2 (ja) 2011-08-08 2017-09-06 チュアラボ オンコロジー, インコーポレーテッドCurelab Oncology, Inc. がんの治療および予防のための、ｐ６２に関する方法および組成物
DK178233B1 (en) 2012-09-17 2015-09-14 Novartis Tiergesundheit Ag Fiskevaccine
EP2914618B1 (fr) 2012-11-02 2017-07-26 Novartis Tiergesundheit AG Flavivirus associés à la maladie de theiler
WO2014140938A2 (fr) 2013-03-14 2014-09-18 Centre Hospitalier Universitaire Vaudois Méthodes immunologiques
CA2935232A1 (fr) 2013-12-29 2015-07-02 Curelab Oncology, Inc. Methodes et compositions relatives a p62/sqstm1 pour traiter et prevenir les maladies associees a une inflammation
DK3177644T3 (da) 2014-08-05 2021-01-11 MabQuest SA Immunologiske reagenser, som binder til PD-1
CN108779168A (zh) 2015-12-05 2018-11-09 沃迪奥斯大学医院中心 Hiv结合剂
WO2017125815A2 (fr) 2016-01-22 2017-07-27 MabQuest SA Réactifs immunologiques
EP3996815A2 (fr) 2019-07-15 2022-05-18 Lausanne University Hospital Agents de liaison au vih
TW202206098A (zh) 2020-08-11 2022-02-16 美商碩騰服務公司 抗冠狀病毒疫苗
CN114315985A (zh) 2020-09-29 2022-04-12 硕腾服务有限责任公司 减毒猪流行性腹泻病毒
WO2023034804A1 (fr) 2021-08-31 2023-03-09 Zoetis Services Llc Alphavirus atténué

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4707357A (en) * 1984-06-26 1987-11-17 The United States Of America As Represented By The Secretary Of The Army Immunologically active peptides capable of inducing immunization against malaria and genes encoding therefor
US5504005A (en) * 1987-03-02 1996-04-02 Albert Einstein College Of Medicine Of Yeshiva University Recombinant mycobacterial vaccine

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9201796A1 *

Also Published As

Publication number Publication date
GB9015888D0 (en) 1990-09-05
WO1992001796A1 (fr) 1992-02-06
AU8101291A (en) 1992-02-18

Similar Documents

Publication Publication Date Title
EP0544685A1 (fr) Vecteurs
US5591632A (en) Recombinant BCG
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
JP4144813B2 (ja) Ｃｒｍ蛋白質およびジフテリアトキシンを産生させるための新規なプラスミド
AU681572B2 (en) Bacterial expression vectors containing DNA encoding secretion signals of lipoproteins
EP0478664B1 (fr) Insertion genomique a mediation vectorielle et expression d'adn dans le bcg (bacille bilie calmette-guerin)
CA2067862C (fr) Vecteurs stables de pura et leurs utilisations
US20040203039A1 (en) Attenuated salmonella SP12 mutants as antigen carriers
FI104638B (fi) Menetelmä flagelliinifuusioproteiinin, sitä koodaavan geenin ja mikro-organismin valmistamiseksi
JPH08503602A (ja) 弱毒化細菌における組換え融合タンパク質の発現
US6752994B2 (en) Insertional mutations in mycobacteria
US5866403A (en) Homologously recombinant slow growing mycobacteria and uses therefor
EP0972839A1 (fr) Mutations par insertion dans des mycobactéries
US5874267A (en) Expression of surface layer proteins
EP0485701A1 (fr) Insertion d'ADN au moyen de transposones modifiés
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
JPH06508741A (ja) ミコバクテリア発現ベクター
EP1468076B1 (fr) Souche de Bordetella déficiente en AroQ et son utilisation
EP0742829B1 (fr) Expression de proteines heterologues dans des bacteries attenuees au moyen de promoteurs du gene htra
AU653362B2 (en) Insertion of DNA by modified transposons
WO1995032277A9 (fr) Mutations par insertion dans des mycobacteries
WO1995032277A1 (fr) Mutations par insertion dans des mycobacteries
US20020001593A1 (en) Hemolysin fusion proteins, their production and use
AU2002357406A1 (en) Defective entities and uses therefor

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19921221

AK Designated contracting states

Kind code of ref document: A1

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

17Q First examination report despatched

Effective date: 19930916

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 19941220