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WO1997025061A1 - Lypopolysaccharide de-myristoyle de bacteries gram-negatives - Google Patents

Lypopolysaccharide de-myristoyle de bacteries gram-negatives Download PDF

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Publication number
WO1997025061A1
WO1997025061A1 PCT/US1997/000392 US9700392W WO9725061A1 WO 1997025061 A1 WO1997025061 A1 WO 1997025061A1 US 9700392 W US9700392 W US 9700392W WO 9725061 A1 WO9725061 A1 WO 9725061A1
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Prior art keywords
lps
gram
ipxf
bacteria
protein
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John E. Somerville, Jr.
Richard P. Darveau
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Bristol Myers Squibb Co
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Bristol Myers Squibb Co
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A61K31/739—Lipopolysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/025—Enterobacteriales, e.g. Enterobacter
    • A61K39/0258—Escherichia
    • 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

  • the lipopolysaccharide (LPS) of gram-negative bacteria can act as a potent stimulator of inflammation even at sub-nanogram quantities. Stimulation occurs primarily through the LPS receptor, CD14. Wright et al . , Science.
  • This receptor is present in a membrane bound (mCD14) and soluble (sCD14) form, each of which defines a pathway of cellular activation.
  • Monocytes and neutrophils contain mCD14 on their surface and produce several cytokines in response to the presentation of LPS via a serum protein designated lipopolysaccharide binding protein (LBP) .
  • LBP lipopolysaccharide binding protein
  • tumor necrosis factor and interleukin-1 by macrophages elicits a cascade of cytokine production by other cells of the immune system which, in turn, is associated with the symptomology of LPS intoxication, namely, leukocytosis, shock, disseminated intravascular coagulation, and death.
  • LPS intoxication namely, leukocytosis, shock, disseminated intravascular coagulation, and death.
  • LPS Direct activation of non-myeloid cells such as epithelial and endothelial cells by LPS requires sCD14.
  • Pugin et al. Proc. Natl. Acad. Sci. USA. 90:2744-2748 (1993) .
  • the sCD14-LPS complex is believed to interact with a membrane receptor for cellular activation.
  • One result of either direct or indirect activation of endothelial cells by LPS is the expression of E-selectin, an adhesion molecule known to be required for leukocyte exit from the vascular compartment . Bevilacqua et al . , Proc. Natl. Acad.
  • lipid A moiety is the primary component of LPS responsible for host cell activation.
  • Data obtained from both partial enzymatic degradation of lipid A and chemical synthesis of lipid A analogues has elucidated many of the structural requirements for monocyte activation. Takada and Kotani, Molecular Biochemistry and Cellular Biology, eds. Morrison and Ryan, pp. 107-130 (1992) ; Pohlman et al . , J. EXP. Med. 165:1393-1402 (1987) .
  • Acyloxyacyl groups i.e., myristate and laurate fatty acids
  • LPS In pathogens that have been associated with chronic infectious disease, such as Porphyromonas gingivalis or Helicobacter pylori , native forms of LPS either lack or have altered forms of the acyloxyacyl "piggyback" fatty acids normally found in E. coli and S. typhimurium. Dalla Kunststoffia et al . , Eur J. Biochem. , 151:399-404 (1985) ; Ogawa, T., FEBS. 332:197-201 (1993) . Highly purified LPS from these bacteria has been isolated and shown to lack the ability to stimulate endothelial cells to express E-selectin. Darveau et al. , Infect. Immun.
  • compositions can only be expressed in bacteria due to their toxicity in eukaryotic hosts.
  • E. coli has also been used to produce recombinant vaccines, such as the major antigen (VPI) of the foot-and-mouth disease virus.
  • VPI major antigen
  • the present invention provides compositions and methods of using gram-negative bacteria, or products thereof, having a form of LPS deficient in levels of the myristic acid moiety.
  • De-myristolated LPS (dmLPS) is substantially less able to activate the inflammatory cascade than wild- ype LPS.
  • De-myristolated gram-negative bacteria are particularly useful in bacterial production processes where LPS contamination must be avoided.
  • de-myristolated LPS itself may be used as an LPS-mediated inflammation antagonist, or adjuvant.
  • the present invention relates to compositions, and a method of making, heterologous peptides, proteins or other heterologous macromolecules in a de- myristolated lipid A gram-negative bacterial culture.
  • the method comprises culturing gram-negative bacteria in which synthesis or activity of IpxF is inhibited and therein expressing a gene coding for the heterologous peptide, polypeptide or protein (hereinafter collectively referred to as "protein") .
  • synthesis is inhibited by preventing transcription or translation of IpxF nucleic acids.
  • IpxF is inhibited as a result of a promoter, operator or structural gene mutation.
  • the IpxF genes of the gram-negative bacteria are typically at least 50% homologous to the E. coli IpxF gene or a fragment thereof of at least 15-25 nucleotides in length.
  • the heterologous proteins may be viral, eukaryotic or of a different bacterial species or serotype.
  • the present invention relates to compositions and methods of making de-myristolated gram- negative bacteria or biosynthesized (or catabolized) products thereof for use in compositions comprising a pharmaceutically acceptable carrier, e.g., for vaccines and adjuvants.
  • the method comprises culturing gram-negative bacteria in which synthesis or activity of IpxF is inhibited in said bacteria and suspending the bacteria or products thereof in a pharmaceutically acceptable carrier.
  • the gram-negative bacteria are selected from the group comprising Salmonella typhimurium, Vibrio cholerae, Bordetella pertussis, and Hae ophilus influenzae .
  • the gram- negative bacteria comprise a gene coding for an immunogenic protein.
  • the immunogenic protein may be prokaryotic, eukaryotic or viral and may be secreted into the media, periplasmic space, or inserted into the outer membrane.
  • the bacterial product is dmLPS for use as an immunological adjuvant.
  • the dmLPS may be purified prior to use and can be conveniently derived from E. coli .
  • the present invention also relates to a method to antagonize the LPS-mediated inflammatory cascade by administration of a therapeutically effective dose of dmLPS.
  • the dmLPS is linked to a therapeutic or diagnostic composition such as an antibody or antibody fragment, protein, peptide, radioisotope, hormone, enzyme inhibitor, enzyme, sugar, or nucleic acid.
  • dmLPS is linked to antibodies or fragments thereof capable of im unospecifically binding to TNF- ⁇ or IL-1.
  • the present invention provides methods of producing, from or in gram-negative bacteria, immunogens, or foreign peptides, proteins or saccharides, in which the lipid A of the bacterial lipopolysaccharide (LPS) molecule lacks or is deficient in the myristic acid moiety and, consequently, is of substantially reduced endotoxicity.
  • the present invention relates to methods of biosynthesizing de- myristolated LPS (dmLPS) for use as immunological adjuvants or endotoxin antagonists.
  • the methods of the present invention comprise inhibiting in gram-negative bacteria the formation or activity of the enzyme corresponding in function to the enzyme encoded by the IpxF (alternatively, "msbB" ) gene in E. coli (GenBank Accession No. M77039) (Karow et al. , J. Bacteriol . , 174:702-
  • LPS refers to the KD0 2 -lipid A precursor as well as subsequent glycosylated forms. See, Raetz, J. Bacteriol . ,
  • KD0 2 -lipid A is formed following attachment of two KDO residues to lipid IV A and acylation with laurate and myristate residues.
  • IpxF refers to both the msbB gene of E. coli as well as its functional homologs in other gram- negatives as determined by at least one of three criteria: 1) the ability of the putative IpxF gene to restore the myristolated-LPS phenotype upon expression in lpxF ⁇ dmLPS E.
  • Myristolated lipid A the lipid component of LPS, is found in a wide variety of gram- negatives. Morrison and Ryan, eds., Molecular Biochemistry and Cellular Biolo ⁇ v. CRC Press, Vol. 1, (1992) ; Raetz, Annu. Rev. Biochem..
  • LPS LPS fatty acids
  • E-selectin expression by endothelial cells can be assayed for by methods well known to those of skill in the art, e.g., activation of macrophages, GC or HPLC analysis of LPS fatty acids, or E-selectin expression by endothelial cells.
  • the E. coli IpxF gene, or fragments thereof have at least about 50% nucleotide sequence homology to the IpxF, or subsequence thereof, of non-.E. coli gram-negatives, more preferably 60% to 70%, and most preferably at least about 75%.
  • the percentage of homology is calculated by comparing one sequence to another when aligned to corresponding portions of the reference sequence. Homology, as herein defined, is assessed without regard to insertions (i.e., spaces) or deletions (i.e., gaps) of nucleotides.
  • the fragments are generally of the minimal size needed to ensure hybridization specificity in cDNA library screening.
  • the fragments are at least 15-25 nucleotides in length, and generally comprise at least one, sometimes two, three or more, subsequences of at least 3, and sometimes 4, 5, 6, 7, 8, 9 or more, contiguous nucleotides from the E. coli IpxF gene.
  • the presence in gram-negatives of a IpxF gene homologous to E. coli IpxF or fragment thereof is readily determined by methods well known to the skilled artisan. Nucleic acid sequence databases, such as GenEMBL, may be used in sequence homology searches to determine the presence of homologous genes in other gram-negative species or to identify and construct probes to conserved regions of IpxF sequences for use as probes.
  • E. coli IpxF techniques such as screening DNA libraries with labeled oligonucleotide probes from E. coli IpxF may be used.
  • expression libraries can be screened using polysera or monoclonals to the E. coli IpxF protein.
  • an oligonucleotide probe derived from E. coli IpxF is used to screen genomic DNA libraries of other gram-negative bacteria. Randomly labeled fragments of IpxF may be used; preferably, conserved regions of IpxF identified by alignment techniques from multiple gram-negative species are used to screen the libraries. Putative IpxF DNAs are cloned and expressed in IpxF ' E. coli in accordance with conventional molecular biology techniques as described in, e.g., Sambrook et al. , Molecular Cloning, A Laboratory Manual. 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the putative IpxF gene is expressed in dmLPS IpxF' E. coli .
  • This mutant is assayed for conversion to a LPS phenotype, conveniently by activation of macrophages or endothelial cells. Activation can be determined by assaying, e.g., IL-1 or TNF- ⁇ secretion from macrophages, or E-selectin expression by endothelial cells.
  • TNF- ⁇ tumor necrosis factor- ⁇
  • IL-1 interleukin 1
  • Binding of LPS to endothelial cells activates expression of E- selectin.
  • Antibodies to TNF- ⁇ , IL-1, and E-selectin are widely available from commercial sources, e.g., Pierce (Rockford, IL) , R & D Systems (Minneapolis, MN) .
  • Bacterial taxonomic relationships based on sequence similarities can be used to identify and isolate IpxF from other gram-negative bacteria of interest.
  • IpxF from E. coli is used to identify and isolate IpxF from a species intermediate in phylogenetic distance between E. coli and the gram-negative bacteria of interest.
  • the intermediate species is used to identify and isolate IpxF from a species more closely related to the gram-negative of interest.
  • This stepwise method can be used to bridge differences in IpxF sequence that exist between E. coli and other gram-negative bacteria.
  • Inhibition of IpxF synthesis may be achieved by interfering with transcription or translation of the IpxF gene product to reduce or prevent synthesis of the gene product.
  • the activity of the IpxF gene product may be inhibited.
  • mutagenesis to prevent transcription may proceed by the use of alkylating agents to inactivate genes by the formation of alkylating agent-DNA adducts as with such reactive electrophiles as epoxides or nitrogen mustards, or photoreactive compounds such as psoralen.
  • Prevention of transcription may also be achieved by mutagenesis of the structural gene, or operator or promoter regions. See, e.g., Watson et al . , Molecular Biology of the Gene. 4th Ed., pp.
  • IpxF is typically inhibited for a sufficient number of doubling periods prior to use of dmLPS bacterial strains to reduce LPS to an acceptable or desired level.
  • Translation may be prevented by the use of ribozymes (catalytic RNA) to cleave messenger RNA transcripts or by the use of anti-sense agents to form homo- or hetero-duplexed messenger RNA.
  • ribozymes catalytic RNA
  • anti-sense agents to form homo- or hetero-duplexed messenger RNA.
  • Such translational inhibitors may be encoded by genes introduced into the targeted gram-negative bacteria.
  • the activity of gene products may be inhibited by the use of e.g., enzyme inhibitors or by the creation of missense or nonsense mutations within the structural gene.
  • structural gene refers to the DNA sequence from which messenger RNA is transcribed and typically comprises a 5' leader for ribosome binding, a region or regions translated into peptides or proteins, and a 3' stop codon.
  • Mutagenesis may by accomplished by such well known techniques as, e.g., chemical or radiological mutagenesis, by insertional mutagenesis via insertion sequences or transposable elements, or via recombination with a mutagenized copy of the gene.
  • the mutations induced may be multi-basepair lesions such as insertions or deletions, or point mutations such as additions or substitutions.
  • mutagenesis may occur as a result of normal replicative errors or any combination of the above.
  • the methods of the present invention are not limited to any particular means of inhibition of synthesis or activity and may proceed by any number of molecular biology or biochemical techniques. Inhibition of IpxF may be achieved ex vivo or in vivo .
  • IpxF inhibition further provides a means to assay for IpxF inhibiting pharmaceutical compounds (e.g., enzyme inhibitors) for in vivo application (e.g., anti-infectives) .
  • pharmaceutical compounds e.g., enzyme inhibitors
  • Inhibition of synthesis or activity may be used to produce dmLPS levels up to greater than 50%, preferably at least 80% to 90%, more preferably from 95% to 100% of total LPS (mLPS + dmLPS) .
  • the wild-type enzyme is employed to screen for inhibitors.
  • dmLPS is also used in a screening assay as a substrate for the IpxF-encoded enzyme to identify inhibitors of the reaction and thus anti-bacterial or other pharmaceutically useful agents.
  • organisms having a dmLPS phenotype i.e., which do not express a fully functional IpxF gene, are used to provide an avirulent or less virulent host organism in which other factors, such as potential virulence factors can be tested.
  • IpxF synthesis can be inhibited in a selected host organism which already expresses IpxF by techniques described herein, e.g., using homologous recombination, etc., to replace IpxF with a mutated IpxF gene to provide a dmLPS host organism against which or in which the effect of the factor of interest is determined.
  • the dmLPS bacteria formed by inhibition of IpxF may be used to produce immunogens for use in vaccines, to produce heterologous proteins, nucleic acids or saccharides, to produce dmLPS endotoxin antagonists, or to produce dmLPS for use as an immunological adjuvant.
  • immunogens or heterologous macromolecules may be expressed prior to, during, or after inhibition of IpxF as desired.
  • Exemplary immunogens for use as vaccines that are expressed from dmLPS bacteria according to the present invention are derived from eukaryotes, such as eukaryotic cells (e.g., tumor antigens), protozoans or parasitic worms (e.g., Trypanosoma, Schistosoma or Plasmodium) , bacteria (e.g., Salmonella typhimurium, Vibrio cholerae, Bordetella pertussis , and Haemophilus influenzae) , or viruses such as those giving rise to influenza, measles, mumps, rubella, polio, rabies, yellow fever, AIDS, human papilloma virus, Epstein-Barr, herpes simplex 1 or 2, varicella, hepatitis B or C, etc.
  • eukaryotic cells e.g., tumor
  • Immunogens may be substantially purified or introduced along with their dmLPS host.
  • the immunogens for use as vaccines are preferably in inclusion bodies, secreted into the periplasmic space or secreted into the medium, or a fusion with such proteins.
  • the bacteria are given as a vaccine (i.e., a cellular vaccine)
  • the immunogens are preferably an outer membrane protein or a fusion with such proteins.
  • dmLPS bacteria may themselves be used as a vaccine, for production of foreign antigens, or both.
  • Gram-negative bacteria that may be used as cellular bacterial vaccines include, e.g., Salmonella typhimurium, Vibrio cholerae, Bordetella pertussis, and Haemophilus influenzae, too name a few.
  • the dmLPS bacteria may also be used to produce heterologous proteins, saccharides, dmLPS, or other products to act as immunogens, or for diagnostic, therapeutic or industrial (e.g., catalytic) applications.
  • Heterologous refers to those biosynthesized products (including proteins, polypeptides and peptides) which are not indigenous to the bacterial species producing them.
  • the heterologous proteins produced according to the invention may be cytoplasmic, preferably periplasmic, or secreted into the media.
  • the heterologous proteins may be from eukaryotes, eukaryotic viruses, or from prokaryotes or bacteriophage.
  • the heterologous proteins may also be fused to native (i.e. indigenous) or heterologous proteins.
  • E. coli expression systems may be employed.
  • other gram-negative expression systems may be used.
  • various vectors have been developed for cloning in Pseudomonas putida . Franklin et al. , Proc. Natl. Acad. Sci. USA. 78:7458- 7462 (1981) .
  • dmLPS of the present invention can be used for pharmaceutical compositions, particularly for administration to mammals such as livestock and other veterinary applications, and to humans.
  • dmLPS may be used as an antagonist to the activation of LPS-mediated inflammation.
  • sufficient dmLPS is administered such that the cells involved in LPS-mediated inflammation are not substantially activated; preferably, the level of activation, as measured by E-selectin expression by human umbilical vein endothelial cells or TNF- ⁇ production by adherent monocytes, is inhibited to 25% or less of the unantagonized values.
  • LPS-mediated activation is antagonized by binding of dmLPS to an LPS specific receptor, such as CD14 or LPS binding protein (LBP) .
  • LPS antagonism can be displayed in the activation pathway subsequent to the CD14/LPS binding protein complex formation.
  • De-myristolated LPS derived from LpxF " gram negative bacteria as described herein can also be used in a pharmaceutical preparation as adjuvant to improve the immunogenicity of antigens.
  • the dmLPS adjuvant can be provided in a range of molecular weights, from a single monomer of de-myristolated KD0 2 -Lipid A to more mature, higher molecular weight forms of dmLPS, with or without the O-antigen repeat or outer or inner cores.
  • the dmLPS adjuvant is typically administered in a pharmaceutically acceptable carrier such as, but not limited to, a saline solution, phosphate buffered saline (PBS) , as a water or buffer emulsion, or in a liposomal formulation.
  • a pharmaceutically acceptable carrier such as, but not limited to, a saline solution, phosphate buffered saline (PBS) , as a water or buffer emulsion, or in a liposomal formulation.
  • PBS phosphate buffered saline
  • dmLPS is administered from about 25 to 500 mg for a 70 kg individual.
  • the adjuvant may be provided simultaneously, prior to, or following administration of the antigen per the direction of the clinician.
  • compositions are administered to a mammal, including humans, in an amount sufficient to at least partially arrest symptoms and/or complications. This amount is defined as a "therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the dmLPS composition, the manner of administration, the stage and severity of the LPS intoxication being treated, the weight and general state of health of the patient, and the judgment of the prescribing clinician. Generally, the range for use as an antagonist to an LPS-mediated inflammatory reaction, the therapeutic composition is from about 1 mg dmLPS to about 750 mg for a 70 kg patient. It must be kept in mind that the methods of the present invention may be employed in serious disease states, that is, life-threatening or potentially life threatening situations.
  • dmLPS in view of relatively nontoxic nature of dmLPS, it is possible and may be felt desirable by the treating clinician to administer substantial excesses of dmLPS, alone or as a linked composition.
  • administration of dmLPS may begin prophylactically, at the first sign of endotoxemia, and continuing until symptoms of endotoxemia have abated, per the discretion of the clinician.
  • the dmLPS is purified from the bacterial species by a number of methods known to the skilled artisan. See, e.g., Ames, J. Bacteriol.. 95:833-843.
  • the dmLPS is generally purified from bacterial membranes, preferably purified from bacterial membrane proteins, and more preferably at least 75% to 95% (wt/wt) pure.
  • the dmLPS may also be hydrolyzed from core sugars, including the two KDO residues, to form an LPS composition with a lipid A component having the structure corresponding to ⁇ ( l , 6 ) D-glucosamine disaccharide 1, 4' bisphosphate with the following lipid placement: 2- [14:0 (3-OH)] , 3- [14:0 (3-OH)] , 2'-[14:0 (3-0-12:0)] , 3'-[14:0 (3- OH) ] , using the ring numbering of Galanos et al . , Eur. J. Biochem.. 148:1-5 (1985) , incorporated herein by reference.
  • De-myristolated LPS, vaccines, immunogens, proteins or other pharmaceutically useful compositions of the present invention may be administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly.
  • compositions for parenteral administration which comprise a solution of the dmLPS, vaccines, immunogens, and/or proteins suspended (or otherwise dissolved) in a pharmaceutically acceptable carrier, often with a surfactant.
  • the surfactant must, of course, be nontoxic.
  • esters or partial esters of fatty acids such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • Mixed esters such as mixed or natural glycerides may be employed.
  • These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting composition may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • [80 dlac dacZ)M15] was the K-12 strain selected for mutagenesis.
  • JM83 was grown to late-log phase in LB broth (Sambrook, supra) supplemented with 0.2% maltose. The cells were isolated by centrifugation and suspended in 1/2 volume of 10 mM MgSO 4 ,7H 2 0, 10 mM Tris, pH 7.5.
  • E. coli Ql (deBruijn et al . , Gene 27:131- 149 (1984)) was used for propagation and titration of a ⁇ 467 phage stock carrying the transposon Tn5. Jorgensen et al.
  • Tn5 mutants were individually picked from plates infected at an MOI of ⁇ 1 into 96-well, low protein binding, microwell plates (Corning Costar, Cambridge, MA) that contained 0.15 ml of LB broth, with kanamycin at a final concentration of 75 ⁇ g/ml. Tn5 mutants were picked only from plates where the multiplicity of infection (MOI) was . ⁇ 1 to reduce the possibility of isolating double mutants.
  • MOI multiplicity of infection
  • the microtiter plates were centrifuged to pellet the cells.
  • the cells were resuspended in fresh LB broth containing 15% glycerol, then the plates were frozen on dry ice and stored at -70°C.
  • E-selectin stimulation ELISA assay E-selectin stimulation ELISA assay.
  • HUVEC cells were obtained from Clonetics (San Diego, CA) and were propagated in HUVEC growth media. Id. E-selectin stimulation assays used cells passed no more than four times. Stimulation assays were carried out in the presence of 5% pooled normal human serum (Gemini Bioproducts, lo
  • Each microwell plate contained controls consisting of non-stimulated HUVEC cells, wells stimulated with TNF- ⁇ , and wells stimulated with the non-mutagenized JM83 strain.
  • This Example demonstrates the isolation of LPS from the mutant BMS67C12 and analysis of LPS fatty acids.
  • 10 mg of lyophilized cells were extracted three times with 45% phenol at 70°C and the cooled aqueous layers recovered by centrifugation. After extraction with diethyl ether, the aqueous phase was evaporated to dryness under a flow of nitrogen gas at 45°C.
  • the phenol-water method of Westphal and Jann was used. Westphal et al . , Methods in Carbohydrate Chemistry, ed. Whistler R.L., Academic Press, Inc., NY, pp. 83-91 (1965) .
  • LPS fatty acids, phospholipids, and whole cell fatty acids were derivatized to fatty acid methyl esters by methanolysis in 2M methanolic HCl at 90°C for 18 hr. with the addition of pentadecanoic acid as an internal standard. After the addition of an equal volume of saturated NaCl solution, the methyl esters were extracted with hexane and analyzed by gas chromatography with a 50m x 0.25mm HP-1 capillary column on a Hewlett-Packard 5890 gas chromatograph with a programmed temperature ramp from 90°C to 225°C. Limulus Amebocyte Lysate (LAL) testing was done using a kinetic assay in an automated microplate reader according to the manufacturers instructions (Endosafe, Charleston, SC) .
  • LAL Limulus Amebocyte Lysate
  • Example 3 This Example describes the reduced ability of the BMS67C12 mutant lacking the 14:0 myristoyl fatty acid to stimulate adherent monocytes, as determined by production of TNF- ⁇ .
  • Adherent monocytes were isolated from the whole blood of individuals randomly selected from a population of normal human donors. Whole heparinized blood from an individual donor was diluted with one volume of RPMI 1640 medium (GIBCO/BRL, Gaithersburg, MD) and overlayered onto
  • Lymphocyte Separation Medium (Organon Teknika Corp., Durham, NC) . The gradient was centrifuged at 500 x g for 30 min. at room temperature. The lymphocyte layer was removed and the lymphocytes diluted with 1 vol. of RPMI 1640. The cells were pelleted and washed once more in RPMI 1640 then resuspended in a small volume of RPMI 1640 containing 5% fetal calf serum. Following counting and dilution to 5 x 10 € cells/ml, 1 ml aliquots were added to each well of 24-well tissue culture plates. Cells were allowed to adhere for 1 hour at 37°C, then the monolayers were washed 3 times with serum free RPMI 1640.
  • adherent cells typically represented 10% of the lymphocyte population.
  • RPMI 1640 medium containing 5% normal human serum and E. coli strains JM83 or BMS67C12, or LPS purified from JM83 or BMS67C12 was added.
  • the culture supernatants were harvested and assayed for the presence of TNF- ⁇ using a human TNF- ⁇ specific ELISA assay (Amersham Corp., Arlington Heights, IL) .
  • BMS67C12 whole bacterial cells with de-myristolated LPS (dmLPS) were approximately 3 logs less effective as the parental E. coli strain at stimulating adherent monocytes to produce TNF- ⁇ .
  • dmLPS de-myristolated LPS
  • Example 4 This Example describes the role of the CD14 pathways in the LPS-mediated stimulation of adherent monocytes and endothelial cells.
  • LPS stimulation of endothelial cells has been proposed to occur via a soluble form of the CD14 receptor (sCD14) .
  • sCD14 CD14 receptor
  • Pugin et al . Proc. Natl. Acad. Sci. USA. 90:2744- 2748 (1993) ; Frey et al . , J. Ex . Med.. 176:1665-1671 (1992) .
  • MY4 was added at varying concentrations to stimulation medium containing 5% normal human serum and incubated for 1 hour at 37°C.
  • LPS (5 ng/ml) or dmLPS (500 ng/ml) was then added to the stimulation media and 100 ⁇ l aliquots of the mixtures were used in the HUVEC based E-selectin assay described above.
  • isolated adherent monocytes were incubated with the MY4 antibody diluted in serum free stimulation medium for 1 hour at 37°C prior to adding of the LPS and 5% normal human serum.
  • LPS was used at 10 ng/ml and dmLPS was used at 100 ng/ml.
  • TNF- ⁇ After 4 hrs . of stimulation the level of TNF- ⁇ in the culture supernatants was determined.
  • the MY4 antibody completely blocked both the high dose dmLPS stimulation of endothelial cells (as assayed by the expression of E-selectin) and the release of TNF- ⁇ when attempting to stimulate adherent monocytes.
  • whole cell stimulation in both assays was only partially blocked, suggesting that whole cells may contain non-CD14 dependent pathways for E-selectin or TNF- ⁇ activation.
  • the ability of the MY4 antibody to block the ability of dmLPS to stimulate both endothelial cells and adherent monocytes indicates that the stimulation seen at relatively high concentrations of dmLPS is still dependent on the CD14 receptor pathways and is not due to stimulation through an unknown alternative receptor pathway.
  • Example 5 This Example describes cloning and identification of the gene from BMS67C12 that contain a Tn5 insertion.
  • Total DNA was isolated from the E. coli strains JM83 and BMS67C12 using a modification of previously described methods. Somerville et al . , J. Bacteriol.. 156:168-176 (1983) , incorporated herein by reference. Alternatively, one ml. of cells (approximately 1 X 10 9 cells) are pelleted and DNA isolated using Easy-DNA (Invitrogen, San Diego, CA) . DNA is resuspended, RNase treated, and extracted with a one-half volume of phenol/CHCl 3 (1:1) . The aqueous phase is isolated and further extracted with a volume of CHC1 3 .
  • the DNA is ethanol/sodium acetate precipitated, washed with 80% ethanol, and resuspended in 100 ⁇ l of TE buffer.
  • Samples of each DNA were digested with a variety of restriction enzymes known to lack sites in the Tn5 transposon. Restriction endonucleases and DNA modification enzymes were from commercial sources and used according to the manufacturer's instructions.
  • the plasmid pRZ102 was labeled using a digoxigenin random primed labeling and detection system (Boehringer Mannheim Corp., Indianapolis, IN) .
  • Plasmid pRZ102 carries the transposon Tn5 in a colEl vector (Jorgensen et al., Molec. Gen. Genet.. 177:65-72 (1979) .
  • Supercoiled plasmid DNA to be labeled for hybridization was first treated with an ATP-dependent DNase (Plasmid-Safe, Epicentre Technologies, Madison, WI) to eliminate any E. coli chromosomal DNA contamination.
  • the samples were then electrophoresed in an agarose gel and the separated fragments blotted onto nitrocellulose using a modification of the Southern blotting technique (Sambrook et al . , p. 7.37-7.52, supra) , p 7.37-7.52 (1989)) .
  • both mutant strains contained single Tn5 insertions in their chromosomal DNA.
  • single fragments that contained the Tn5 were identified with three of the restriction enzymes.
  • Kpnl , EcoRI and SacI digests of BMS67C12 DNA were ligated into aliquots of the plasmid vector pUC18 (Yanisch-Perron et al . , supra) that had also been digested with the same enzymes.
  • These DNA libraries were transformed into E. coli strain JM109 (ibid. ) . then screened for clones that contained plasmids that were resistant to both ampicillin (100 ⁇ g/ml) and kanamycin (75 ⁇ g/ml) .
  • Plasmids isolated from the SacI libraries were identified and selected for further analysis. Plasmid pBMS67 with DNA from the E. coli BMS67C12 strain contained a genomic Sacl fragment of 8.2 kb. The single BamHI site located in the center of Tn5 was used to subclone fragments on either side of each Tn5 insertion site. An oligonucleotide primer 5' -ATGGAAGTCAGATCCTGG-3' [SEQ ID N0:1] targeted to the insertion sequence of the Tn5 (GenBank Accession No. L19386) , was then used for directional dideoxy sequencing outward from the Tn5. This permitted the exact location of the Tn5 insertion to be identified in each clone and provided sufficient sequence information to search the GenEMBL database to identify the mutated genes.
  • E. coli strain BMS67C12 The mutation in E. coli strain BMS67C12 was located in a gene previously identified as msbB. Karow et al . , J. Bacteriol. , 174:702-710 (1992) incorporated herein by reference. The msbB gene is found at 40.5 min. on the E. coli genome and the function of its product had not been identified. The msJbB gene was originally cloned and identified due to its ability to act as a multicopy extragenic suppressor of a mutation in the htrB gene. Karow et al . , ibid.. 174:702-710 (1992) ; Karow et al. , J. Bacteriol.
  • Example 6 This Example describes the restoration of the E- selectin stimulatory LPS phenotype to BMS67C12 by the cloned IpxF gene.
  • Example 7 This Example demonstrates that the growth rates and susceptibility to selected detergents and antibiotics for the IpxF mutant and its parental strain are the same.
  • Example 8 This Example demonstrates the antagonistic effects of dmLPS.
  • HUVEC s (2 X 10 5 /ml) were added and incubated with (+) and without (-) 10 ng/ml JM83 E. coli LPS.
  • dmLPS ability of dmLPS to block production of TNF- ⁇ was assessed.
  • a constant amount of JM83 LPS (10 pg/ml) was mixed with various concentrations of dmLPS and assayed for stimulatory ability by measuring TNF- ⁇ production.
  • Various concentrations range of dmLPS alone was also assayed.
  • Adherent monocytes were isolated and plated in a 48 well tissue culture plate as discussed above. LPS' were diluted and mixed in media at 2X concentration. The LPS mixtures were then added to an equal volume of media containing 10% normal human serum.
  • TNF- ⁇ concentrations were determined using a commercial assay (Amersham Corp., Arlington Heights, IL) .
  • the results of Table 4 indicate that TNF- ⁇ production by monocytes is blocked by dmLPS.

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Abstract

Cette invention concerne un procédé de production de bactéries gram-négatives, lequel consiste à inhiber la synthèse ou l'activité de la protéine codée par lpxF (msbB). Les bactéries gram-négatives décrites dans cette invention sont dépourvues de fragment d'acide myristique de lipide A, et consistent en des hôtes destinés à la production d'immunogènes ou de protéines hétérologues. Ce lypopolysaccharide (LPS) dé-myristoylé peut être utilisé en qualité d'antagoniste de LPS lors d'une activation induite par LPS, ou en qualité d'adjuvant.
PCT/US1997/000392 1996-01-09 1997-01-09 Lypopolysaccharide de-myristoyle de bacteries gram-negatives Ceased WO1997025061A1 (fr)

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999010497A1 (fr) * 1997-08-21 1999-03-04 De Staat Der Nederlanden, Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur Nouveaux mutants de bacteries des muqueuses gram negatives et leur application dans des vaccins
US5997881A (en) * 1995-11-22 1999-12-07 University Of Maryland, Baltimore Method of making non-pyrogenic lipopolysaccharide or A
WO2000026384A1 (fr) * 1998-11-03 2000-05-11 De Staat Der Nederlanden, Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur Lps a toxicite reduite obtenu a partir de bacteries a gram negatif
US6080849A (en) * 1997-09-10 2000-06-27 Vion Pharmaceuticals, Inc. Genetically modified tumor-targeted bacteria with reduced virulence
US6368604B1 (en) 1997-09-26 2002-04-09 University Of Maryland Biotechnology Institute Non-pyrogenic derivatives of lipid A
WO2002028424A3 (fr) * 2000-10-06 2002-11-07 H Henrich Paradies Cybermedicament utilise comme autovaccins avec effets d'immunoregulation
EP1449535A1 (fr) * 2003-02-18 2004-08-25 Clinique La Prairie Research SA Compositions comprenant de la hémoglobine foetale et de l'endotoxine bactérienne et facultativement des composantes additionnelles du foie foetal
US6863894B2 (en) 1997-09-10 2005-03-08 Vion Pharmaceuticals, Inc. Genetically modified tumor-targeted bacteria with reduced virulence
US6962696B1 (en) 1999-10-04 2005-11-08 Vion Pharmaceuticals Inc. Compositions and methods for tumor-targeted delivery of effector molecules
US10857233B1 (en) 2010-02-09 2020-12-08 David Gordon Bermudes Protease inhibitor combination with therapeutic proteins including antibodies
US11180535B1 (en) 2016-12-07 2021-11-23 David Gordon Bermudes Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from therapeutic bacteria
US12378536B1 (en) 2015-05-11 2025-08-05 David Bermudes Chimeric protein toxins for expression by therapeutic bacteria

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF CLINICAL INVESTIGATION, January 1996, Volume 97, Number 2, SOMERVILLE Jr. et al. "A Novel Escherichia Coli Lipid A Mutant that Produces an Antiinflammatory Lipopolysaccharide", pages 359-365. *

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5997881A (en) * 1995-11-22 1999-12-07 University Of Maryland, Baltimore Method of making non-pyrogenic lipopolysaccharide or A
WO1999010497A1 (fr) * 1997-08-21 1999-03-04 De Staat Der Nederlanden, Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur Nouveaux mutants de bacteries des muqueuses gram negatives et leur application dans des vaccins
US7011836B1 (en) 1997-08-21 2006-03-14 De Staat Der Nederlanden, Vertenwoordigd Door De Minister Van Welzijn, Volksgezondheil En Cultuur Mutants of gram negative mucosal bacteria and application thereof in vaccines
US6863894B2 (en) 1997-09-10 2005-03-08 Vion Pharmaceuticals, Inc. Genetically modified tumor-targeted bacteria with reduced virulence
US6447784B1 (en) 1997-09-10 2002-09-10 Vion Pharmaceuticals, Inc. Genetically modified tumor-targeted bacteria with reduced virulence
US6475482B1 (en) 1997-09-10 2002-11-05 Vion Pharmaceuticals, Inc. Genetically modified tumor-targeted bacteria with reduce virulence
US7514089B2 (en) 1997-09-10 2009-04-07 Vion Pharmaceuticals, Inc. Genetically modified tumor-targeted bacteria with reduced virulence
US7354592B2 (en) 1997-09-10 2008-04-08 Vion Pharmaceuticals, Inc. Genetically modified tumor-targeted bacteria with reduced virulence
US6080849A (en) * 1997-09-10 2000-06-27 Vion Pharmaceuticals, Inc. Genetically modified tumor-targeted bacteria with reduced virulence
US6923972B2 (en) 1997-09-10 2005-08-02 Vion Pharmaceuticals, Inc. Methods for use of genetically modified tumor-targeted bacteria with reduced virulence
US6841345B1 (en) 1997-09-26 2005-01-11 University Of Maryland Biotechnology Institute Treatment and prevention of immunodeficiency virus infection by administration of non-pyrogenic derivatives of lipid A
US6368604B1 (en) 1997-09-26 2002-04-09 University Of Maryland Biotechnology Institute Non-pyrogenic derivatives of lipid A
WO2000026384A1 (fr) * 1998-11-03 2000-05-11 De Staat Der Nederlanden, Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur Lps a toxicite reduite obtenu a partir de bacteries a gram negatif
EP1870468A3 (fr) * 1998-11-03 2010-01-13 Staat Der Nederlanden Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur LPS avec toxicité réduite à partir de bactérie négative de gramme génétiquement modifié
US6482807B1 (en) 1998-11-03 2002-11-19 De Staat Der Nederlanden, Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondheid En Cultuur LPS with reduced toxicity from genetically modified gram negative bacteria
US6962696B1 (en) 1999-10-04 2005-11-08 Vion Pharmaceuticals Inc. Compositions and methods for tumor-targeted delivery of effector molecules
US7452531B2 (en) 1999-10-04 2008-11-18 Vion Pharmaceuticals, Inc. Compositions and methods for tumor-targeted delivery of effector molecules
WO2002028424A3 (fr) * 2000-10-06 2002-11-07 H Henrich Paradies Cybermedicament utilise comme autovaccins avec effets d'immunoregulation
WO2004073728A3 (fr) * 2003-02-18 2004-10-07 Clinique La Prairie Res Sa Compositions contenant de l'hemoglobine foetale et une endotoxine bacterienne et eventuellement des elements hepatiques foetaux
EP1449535A1 (fr) * 2003-02-18 2004-08-25 Clinique La Prairie Research SA Compositions comprenant de la hémoglobine foetale et de l'endotoxine bactérienne et facultativement des composantes additionnelles du foie foetal
US7968103B2 (en) 2003-02-18 2011-06-28 Josette Westphal, legal representative Compositions comprising fetal hemoglobin and bacterial endotoxin and optionally additional fetal liver components
US10857233B1 (en) 2010-02-09 2020-12-08 David Gordon Bermudes Protease inhibitor combination with therapeutic proteins including antibodies
US12378536B1 (en) 2015-05-11 2025-08-05 David Bermudes Chimeric protein toxins for expression by therapeutic bacteria
US11180535B1 (en) 2016-12-07 2021-11-23 David Gordon Bermudes Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from therapeutic bacteria

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