Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P39/00—General protective or antinoxious agents
  • A61P39/02—Antidotes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof

Definitions

• the present invention relates to peptide analogs of polymyxin B that are useful for LPS detoxification.
• they may be used (i) as such i.a. to treat fatal disorders, such as septic shock, caused by Gram-negative bacteria infection ; or (ii) non-covalently bound to LPS which is therefore detoxified ; the complex thereof being useful as vaccinal agent against Gram-negative bacteria infection.
• LPS Lipopolysaccharide
• LPS structure is constituted by a lipid moiety, called Lipid A, covalently linked to a polysaccharide moiety.
• Lipid A is responsible for the toxic effect of LPS, in particular through interaction with B-cells and macrophages. This interaction induces the secretion of pro-inflammatory cytokines. The inflammatory condition may reach the fatal state of endotoxic shock.
• Lipid A is highly hydrophobic and anchors LPS in the outer layer of the bacterial cell wall. Lipid A is composed of (i) a conserved bis-phosphorylated dissacharide region
• each of the two symmetric glucosamines (GIcNl and GlcN2) of Neisseria meningitidis lipid A carries the following fatty acids : 2N-C 14,3 OH ; Cl 2 ; and 3O-C12,3OH.
• the LPS polysaccharide moiety is constituted by carbohydrate chains, responsible for antigenicity.
• the carbohydrate chain structure is itself composed of (i) a conserved inner core called the KDO (2-keto, 3-desoxy octulosonic acid) region bound to lipid A and (ii) a variable outer core bound to the KDO region, that is commonly defined as including various saccharides, and the first repeat unit (that may comprised up to ten saccharides) of (iii) the external O-specific chains
• LOS lipooligosaccharide
• LPS is not only toxic but also highly immunogenic, hi mammals, anti-LPS antibodies are induced during infection and carriage, and may be protective. In view of this, it has been already proposed to detoxify LPS and to use the detoxified form thereof in prophylaxis of Gram-negative bacterial infections and related diseases.
• Polymyxin B is a molecule that binds Lipid A with high affinity so that LPS is significantly detoxified. When given therapeutically in animal models, polymyxin B can prevent septic shock. However, polymyxin B is a polycationic antibiotic that may be somewhat toxic to humans because of its non-biodegradability and the consequent tendency to accumulate in the kidneys. Therefore, it is not recommended for use in prophylactic or therapeutic products.
• peptide analogs to polymyxin B have been developed. They do not retain the polymyxin B toxicity but merely mimic the primary and secondary structures of polymyxin B and bind lipid A at the same site as polymyxin B does, so that a LPS-peptide complex is formed. As a result, LPS is detoxified.
• Peptide analogs are in particular described in US 5,358,933, WO 93/14115, WO 95/03327, WO 96/38163, EP 842 666 and EP 976 402.
• SAEP2 peptide as well as similar peptides including in their sequences a number of uncharged polar amino acids surrounded by two adjacent cysteine residues and counter-balanced by a short external tail made of cationic amino acids (hereinafter generically referred to as SAEP II peptides) are of particular interest when they are in dimeric form ; the dimer being conformationally made and maintained by a pair of disulphide bonds between the cysteine residues. Indeed, SAEP II peptide dimers exhibit enhanced detoxification properties over the corresponding monomers.
• the invention relates to a SAEP II peptide dimer of formula (I)
• a and A' independently are a peptide moiety of from 2 to 5, preferably 3 or 4 amino acid residues, in which at least 2 amino acid residues, are independently selected from Lys, HyI (hydroxy-Lysine), Arg and His ;
• B and B' independently are a peptide moiety of from 3 to 7, preferably 4 or 5 amino acid residues, which comprise at least two, preferably three amino acid residues independently selected from VaI, Leu, He 3 Phe, Tyr and Trp ; and wherein C and C are optional (these positions may be empty or not) and are independently an amino acid residue or a peptide moiety of from 2 to 3 amino acid residues ;
• the cationic amino acid residues / hydrophobic amino acid residues ratio is from 0.4 to 2, advantageously from 0.5 to 1.2 or 1.5, preferably from 0.6 to 1 ; most preferably from 0.6 to 0.8 ; e.g. 0.75.
• a and A' independently are a peptide moiety of from 2 to 5, preferably 3 or 4 amino acid residues, in which at least one, preferably 2 amino acid residues, are independently selected from Lys, HyI, Arg and His ; those that are not selected from Lys, HyI, Arg and His ("the remaining amino acid residues"), if any, being selected from the group consisting of uncharged polar or nonpolar amino acids residues ; preferably Thr, Ser and GIy ; most preferably Thr.
• each of them can be a cationic residue ; or alternatively, two out of three residues are cationic amino acids, whereas the remaining residue is selected from the group consisting of uncharged polar or nonpolar amino acids residues ; preferably Thr, Ser and GIy ; most preferably Thr.
• a and A' peptide moieties comprise 4 amino acid residues
• two or three out of four residues be selected from the groups of cationic amino acid residues as defined above, whereas the remaining residue (s) is (are) selected from the group consisting of uncharged polar or non-polar amino acids residues as defined above.
• a and A' peptide moieties comprise 5 amino acid residues
• B and B' independently are a peptide moiety of from 3 to 7, preferably 4 or 5 amino acid residues, which comprises at least two, preferably three amino acid residues independently selected from VaI, Leu, He, Phe, Tyr and Trp ; preferably from Leu, He and Phe ; those that are not selected from VaI, Leu, He, Phe, Tyr and Trp ("the remaining amino acid residues"), if any, being independently selected from the group consisting of Lys, HyI, Arg and His.
• the B and B' peptide moieties may comprise up to 7 amino acid residues independently selected from VaI, Leu, He, Phe, Tyr and Trp.
• the B and B' peptide moieties comprise the sequence - Xl - X2 - X3 -, in which Xl and X2 ; X2 and X3 ; or Xl, X2 and X3 are independently selected from VaI, Leu, He, Phe, Tyr and Trp ; preferably from Leu, He and Phe.
• the sequence - Xl - X2 - X3 - comprises the Phe-Leu motif.
• peptide moieties B and B' include :
• Xl is Lys, HyI, His or Arg, preferably Lys or Arg ; more preferably Lys ;
• X2 is Phe, Leu, He, Tyr, Trp or VaI ; preferably Phe or Leu ; more preferably
• X3 is Phe, Leu, He, Tyr, Trp or VaI ; preferably Phe or Leu ; more preferably Leu ; and
• amino acid residues if any, each being independently selected from the group consisting of VaI, Leu, He, Phe, Tyr, Trp, Lys, HyI, Arg and His ; preferably VaI, Leu, He, Phe, Tyr and Trp ; more preferably Leu, He and Phe.
• a and A' preferably comprises at least 3 positively charged amino acid residues.
• the amino acid residue(s) may be any amino acid residues provided that the cationic amino acid residues / hydrophobic amino acid residues ratio remains within the specified range.
• they are independently selected from uncharged amino acid residues polar or nonpolar, these latter being preferred.
• C and Care empty positions are independently selected from uncharged amino acid residues polar or nonpolar, these latter being preferred.
• A, A', B and B' are as described above ; provided that the cationic amino acid residues / hydrophobic amino acid residues ratio is from 0.4 to 2, advantageously from 0.5 to 1.2 or 1.5, preferably from 0.6 to 1 ; most preferably from 0.6 to 0.8 ; e.g. 0.75.
• Dimers of formula (I) or (III), that is with peptides in the parallel orientation, are referred to as parallel dimers.
• Dimers of formula (II) or (IV), that is with peptides in the anti-parallel orientation, are referred to as antiparallel dimers.
• a and A' are preferably identical. The same holds true for B and B'; and C and C.
• a peptide dimer of formula (I), (II), (III) or (IV), in which A and A' ; B and B' ; and C and C are two-by-two identical, is referred to as homologous dimer. Indeed, in this case, the peptide subunits included in the dimer are identical.
• the respective cat/hydroph ratio of the corresponding homologous dimers are 2.00, 0.50, 0.75 and 0.67.
• a particular example of the dimers described above is constituted by a peptide of formula (V) NH2-Lys-Thr-Lys-Cysl-Lys-Phe-Leu-Leu-Leu-Cys2-COOH.
• This peptide is hereinafter referred to as the SAEP2-L2 peptide. As described above, it can also be in parallel or anti-parallel dimeric form.
• Peptides involved in the or dimers of the invention can be conventionally synthesized by classical methods using e.g. a computer-driven automatic synthesizer. It is within the skills of professional practitioners in the art of peptide synthesis to know how to design procedures so that a particular peptide is obtained. It goes without saying that during the synthesis phase, the cysteine thiol groups can be protected. Once the synthesis is completed, they are de-protected and oxidation of the thiol groups is achieved in order to generate the cyclic monomer, the parallel or anti-parallel dimer.
• each of the three forms can be separated from each other by conventional biochemical purification methods.
• Preparative reverse-phase high performance liquid chromatography RP-HPLC
• RP-HPLC reverse-phase high performance liquid chromatography
• each of the three forms generated upon oxidation depends on La. the specific amino acid sequence and importantly, the concentration of the peptide. It may happen that one or two of the three forms be predominantly created ; and indeed, the prevalence of one or two forms may be such that the other(s) are not formed at all.
• the SAEP2-L2 peptide spontaneously oxidises into cyclic monomer and anti-parallel dimer, in proportions, which depend from the concentration of the peptide in solution.
• the internal steric hindrance of the "side-chains" (the NH2- Lys-Thr-Lys- portion) of the anti-parallel dimer is obviously lower than that of the parallel dimer and one may expect that a lower minimal energy be responsible for the privileged formation of the anti-parallel dimer in aqueous solvents by comparison with the parallel dimer.
• this concentration-driven process the formation of the anti-parallel dimer and to a lesser extent the cyclic monomer is favoured up to the exclusion of the parallel dimer from the equilibrium.
• the parallel dimer When the parallel dimer cannot be spontaneously generated upon oxidation, it is necessary to adopt particular measures to make the peptide associate within the parallel orientation. These measures are within the skills of the professional practitioners in the art of peptide synthesis. Nevertheless and as a matter of example only, it is indicated that differential protection of the Cysl and Cys2 amino acids followed by selective de- protection is a convenient way to achieve dimerisation with the parallel orientation. Then the dimer may be purified by conventional methods, including RP-HPLC.
• Peptides for use in the dimers of the invention can be characterized by various techniques, including i.a. Ion Cyclotron Resonance (ICR), Mass Assisted Laser Desorption Ionisation - Time of Flights (MALDI-ToF) spectrometry and Nuclear Magnetic Resonance (NMR) spectrophotometry.
• ICR Ion Cyclotron Resonance
• MALDI-ToF Mass Assisted Laser Desorption Ionisation - Time of Flights
• NMR Nuclear Magnetic Resonance
• the purity of compounds of the invention can be evaluated by RP-HPLC. Briefly, a preparation of compound is submitted to RP-HPLC. The relative purity degree is calculated by integrating the peak surfaces. It is expressed as the compound peak surface / surfaces of the whole peaks. It is usual to prepare compounds of the invention that each exhibits a purity degree of at least 95 %, frequently of at least 97 %.
• compositions comprising :
• a SAEP II peptide wherein the peptide is essentially in dimeric parallel form ;
• a SAEP II peptide wherein the peptide is essentially in dimeric anti-parallel form ; or mixtures thereof.
• compositions a particular form is at least 95 %, preferably at least 97 %, more preferably 98 % pure.
• compositions in which the SAEP II peptide is present under several forms may spontaneous result from the evolution of a composition comprising a single entity, e.g. the dimeric parallel form, kept at an appropriate temperature over a certain period of time. This may be revealed by e.g. RP-HPLC analysis.
• the respective amounts of the various peptide forms may be quantified by the same token.
• the SAEP II dimers are useful as such as a detoxifying agent of Gram-negative bacterial LPS in vitro as well as in vivo. Accordingly, they may be used to prevent or treat pathological conditions due to the release of LPS into the systemic circulation, e.g. into blood, as a result of Gram-negative bacteria infections. These conditions include i.a. endotoxicosis, bacterial sepsis and septic shock.
• the invention encompasses :
• a pharmaceutical composition comprising a compound or a composition of the invention together with a pharmaceutically acceptable diluent or carrier ;
• a method for treating or preventing septic shock which comprises administering a therapeutically or prophylactically effective amount of a compound or composition of the invention, to an individual in need.
• a compound or composition of the invention may be administered to mammals, i.e. humans, when a Gram-negative bacteria infection is diagnosed that may lead to endotoxicosis, bacterial sepsis and/or septic shock.
• Gram-negative bacteria that may be responsible for these fatal disorders include i.a., N. meningitidis, E. coli, Salmonella typhi, Bordetella pertussis and Pseudomonas aeruginosa.
• a compound or composition of the invention may be administered to an individual in need by a systemic route, preferably the intravenous route.
• the dose to be administered depends on various factors including i.a. the age, weight, physiological condition of the patient as well as the infection status. It may be administered once or several times until the risk of fatal event is avoided.
• the invention also relates to a LPS-peptide complex comprising (i) a LPS moiety of Gram-negative bacteria, and (ii) a SAEP II peptide dimer or the SAEP2-L2 peptide ; wherein the LPS moiety and the SAEP II peptide dimer or the SAEP2-L2 peptide are non-covalently bound to each other.
• LPS detoxification may be assessed in a number of assays referred to in the European Pharmacopeia.
• LAL Limulus Amebocyte Lysate
• pyrogen test in rabbits
• acute toxicity assay in D-galactosamine sensitized mice.
• the detoxification ratio is expressed by the LPS / LPS-peptide complex ratio.
• the detoxification ratio is expressed by the LPS-peptide complex / LPS ratio.
• the LAL assay is at least of 100, preferably 500, more preferably 1000 ;
• the pyrogen test is at least of 50, preferably of 100, more preferably 500 ; or
• D-galactosamine mice is at least of 50, preferably of 100, more preferably of 200.
• Detoxification may also be evaluated while comparing the effect of LPS and a LPS- peptide complex on the release of pro-inflammatory cytokines such as IL6, IL8 and TNF ⁇ , in in vitro or in vivo assays. These assays are illustrated hereinafter in the examples.
• Significant detoxification is achieved, when the LPS-peptide complex allows for at least 25-fold decrease, preferably at least 50-fold, more preferably at least 75- fold, most preferably at least 100-fold decrease in IL6 secretion in the in vivo assay as described in the examples, section 5.4.1.
• LPS-peptide complex of the invention is advantageously characterized by a molar LPS : peptide ratio of from 1 : 1.5 to 1 : 0.5, preferably 1 : 1.2 to 1 : 0.8, more preferably of 1 : 1.1 to 1: 0.9, most preferably 1 : 1.
• the LPS is advantageously a LPS of N. meningitidis ; E. coli ; Salmonella typhi ; Salmonella paratyphi ; Shigella flexneri ; Haemophilus influenzae ; Helicobacter pylori ; Chlamydia trachomatis ; Bordetella pertussis ; Brucella ; Legionella pneumophia ; Vibrio cholera ; Moraxella caiharralis ⁇ Pseudomonas aeruginosa ; Yersinia ; and Klebsiella pneumonia.
• detoxified LPS may be useful as vaccinal agent against Gram-negative bacteria infection.
• Meningitis is a life-threatening disease of either viral or bacterial origin.
• H. influenzae and N. meningitidis are respectively responsible for about 40 and 50 % of bacterial meningitis. While a vaccine against H. influenzae has been on the market for more than 10 years, there is still a need for a vaccine against N. meningitidis.
• Meningococcal invasive diseases may manifest as either an inflammation of the meninges of the brain and spinal cord (meningitis) or a systemic infection of the blood (meningococcal sepsis or meningoccaemia).
• Meningococci are classified using serological methods based on the structure of the polysaccharide capsule. Thirteen antigenically and chemically distinct polysaccharides capsules have been described. Almost all the invasive meningococcal diseases are caused by five serogroups : A, B, C, Y and W-135. The relative importance of each serogroup depends on the geographic location. Serogroup B is responsible for the majority of meningococcal diseases in temperate countries.
• N. meningitidis LPS as vaccinal agent, in a fully antigenic and ad hoc detoxified form, is a promising alternative that may offer a desirable vaccinal coverage, in particular to serogroup B.
• LPS lipooligosaccharide
• Figure 1 shows a scheme of the structure of a. N. meningitidis LOS.
• LOS is constituted by a branched oligosaccharide composed of 5 to 10 monosaccharides linked to lipid A by a KDO.
• Lipid A and the inner core constituted by two KDO, two heptoses (Hep I and II) and a N-acetylated glucosamine (GIcNAc) are conserved intraspecies.
• the remaining of the oligosaccharide chains that constitutes the outer core ( ⁇ -chain attached to Hepl ; ⁇ -chain attached to position 3 of HepII ; and ⁇ -chain attached to position 2 of HepII) is variable according to the immunotypes (ITs).
• N. meningitidis LPS can be classified into 13 immunotypes, based on their reactivity with a series of monoclonal antibodies (Achtman et al, 1992, J Infect. Dis. 165 : 53-68). Differences between immunotypes come from variation in the composition and conformation of the oligosaccharides chains. This is to be seen in the table hereinafter.
• a phospho ethanol amine replaces the GIc of the ⁇ -chain at position 3 of HepII in LOS Ll, L3, L7 and L8.
• a PEA is attached in position 6 or 7 in LOS L2, L4 and L6.
• LOS L2, L3, IA, L5, L5, L7 may also be sialylated with N-acetyl neuraminidic acid, on the terminal galactose (Gal) of the ⁇ -chain.
• Immunotypes L1-L8 are essentially associated with serogroups B and C, while imnrano-types L9-L12 are found predominantly within serogroup A.
• LOS L8 While any LOS can be equally detoxified, it may be advantageous to employ LOS L8 in the complexes of the invention as these latter are further intended to vaccinal use. Indeed, the complete structure of the LOS L8 ⁇ -chain is common to all the immunotypes for which the structure has been identified so far (Kahler & Stephens, 1988, Crit. Rev. Microbiol. 24 : 281).
• Meningococcal strains frequently express several immunotypes, the presence of which may be influenced by the culture conditions. If there is a special interest in LOS L8, it may be desirable to extract this LOS from a strain known to predominantly express the L8 immunotype, or even better, to exclusively express it.
• Strain Al also called 2E
• strain M978 of serogroup B strain M978 of serogroup B (Mandrell & Zollinger, 1977, Infect. Immun. 16 : 471 ; Gu et al, 1992, J. Clin. Microbiol. 30 : 2047-2053 ; Zhu et al, 2001, FEMS Microbiol. Lett. 203 : 173)
• strain 8680 of serogroup B Dominique Caugeant collection
• strain 8532 US 6,476,201
• Monoclonals that are specific for LOS L8 include Mab 2-1-18 (Moran et al, 1994 Infect Immun. 62: 5290-5295 ; Mandrell et al, 1986, Infect Immun. 54: 63-69) Mab 6E7-10 (Braun et al, 2004, Vaccine 22: 898-908) Mab 4387A5 and 4385G7 (Andersen et al, 1995, Microb. Pathog. 19: 159-168 ; Gu et al (supra)).
• LPS may be obtained by conventional means ; in particular it may be extracted from a Gram-negative bacterial culture and then purified according to classical procedures. Numerous descriptions of such procedures may be found in the literature. This includes i.a. Gu & Tsa ⁇ , 1993, Infect. Immun. 61 (5) : 1873, Wu et al, 1987, Anal. Biochem.160 : 281 and US 6,531,131 all cited by way of illustration only.
• An LPS preparation may also be quantified according to procedures well-known in the art.
• a convenient method is the KDO dosage with high performance anion exchange chromatography (HPAEC) PAD.
• LPS may be complexed to the compounds of the invention as such or in a conjugated form.
• LPS conjugates can be conventionally prepared by covalently linking LPS to a carrier molecules, e.g. a polypeptide or a peptide ; either through a direct covalent link or using chemical spacer/linker molecules.
• carrier molecules include the pertussis, diphtheria or tetanus toxoid and outer membrane proteins (OMP) such as the OMPl or OMP2/3 of N. meningitidis.
• OMP outer membrane proteins
• the invention also relates to :
• a process for detoxifying Gram-negative bacteria LPS which comprises mixing together (i) a LPS of Gram-negative bacteria and (ii) a compound of the invention ;
• a process for preparing a LPS-peptide complex which comprises mixing together (i) a LPS of Gram-negative bacteria and (ii) a compound of the invention.
• both constituents are advantageously in a liquid medium, suitably water.
• LPS and compound solutions are advantageously sterilized before mixing.
• the preparation process is advantageously achieved under sterile conditions.
• a precipitate containing the complex is formed. It can be recovered i.a. by centrifugation, and submitted to one or several washing steps, if necessary.
• LPS-peptides complexes of the invention are useful in that they can be safely administered to mammals. Indeed, LPS is detoxified to such an extent that adverse events shall not occur upon administration.
• LPS is detoxified to such an extent that adverse events shall not occur upon administration.
• a complex that exhibits e.g. 100 endotoxin units (EU) / ⁇ g in the LAL assay may be therefore acceptable for administration at a dose of 20 ⁇ g.
• EU endotoxin units
• LPS-peptides complexes of the invention are stable, even in physiological conditions.
• stable it is meant that the detoxification status of LPS in the complexes remains constant over time, at least 3, 6, 12 or 18 months. This can be monitored by evaluating the detoxification ratio at intervals, i.e. in at least one of the assays listed above. No significant difference is observed in the detoxification ratio over time.
• LPS-peptides complexes of the invention are also useful in that they are able to induce an immune response against Gram-negative bacteria. This may be shown upon administration of complexes to mammals, e.g. rabbits, mice or humans, followed by ELISA analysis of the sera to reveal the presence of antibodies ⁇ i.a. immunoglobulins G or M) specific for LPS.
• the immune response (antibodies induced) may have bactericidal and/or opsonic activity.
• the ability of the immune response induced by the complexes of the invention to protect against Gram-negative bacteria infection may be evaluated in appropriate animal models that are currently specific for a bacterial species or disease. It is within the skills of the professionals in the art of vaccines to select a known animal model with regard to a particular bacteria or disease.
• the ability of the immune response induced by the complexes of the invention to protect against N. meningitidis may be evaluated in the mouse intraperitoneal infection model (Schryvers et al, 1989, Infect. Immun. 57 (8) : 2425 and Danve et al, 1993, Vaccine 11 (12) : 1214). It may be also evaluated in humans by measuring the bactericidal activity of the human serum after a complex is administered. Indeed, this test has been proposed to serve as a surrogate test of protection at least for N. meningitidis serogroup B (Hoist et al, 2003, Vaccine, 21 : 734). A human serum bactericidal activity (SBA) titer superior or equal to 4 has been shown to correlate with protection.
• SBA serum bactericidal activity
• the invention also relates to :
• a pharmaceutical (vaccinal) composition comprising a LPS-peptide complex of the invention and a pharmaceutically acceptable diluent or carrier ;
• a method for inducing an immune response in a mammal against a Gram- negative bacteria LPS or a Gram-negative bacteria which comprises administering an effective amount of a LPS-peptide complex of the invention, to the mammal ; and
• a method for treating or preventing a Gram-negative bacterial infection which comprises administering a therapeutically effective amount of a LPS-peptide complex of the invention, to an individual in need.
• a vaccinal composition of the invention can be administered by any conventional route, in particular by systemic or intramuscular route ; as a single dose or as a dose repeated once or several times, e.g. two or three times at intervals, e.g. at 1, 2, 3, 6, 10, 12 month-interval.
• a vaccinal composition of the invention can be conventionally formulated, advantageously in liquid form. If necessary, an adjuvant can be added to the vaccinal composition of the invention ; however, it is indicated that complexes of the invention can be sufficiently immunogenic so that the presence of adjuvant in the vaccinal compositions is not required.
• a dose for administration to a human adult should not excess 10,000 ; advantageously 8,000 ; preferably 5,000 ; more preferably 1,000 ; most preferably 500
• a dose can contain from 1 to 500, advantageously from 2.5 to 100, preferably from 10 to 50, more preferably from 15 to 30 ⁇ g.
• Figure IA shows the structure of the LPS L8 of N. meningitidis.
• Kdo stands for 2-keto, 3-desoxy octulosonic acid ;
• Hep stands for heptose ;
• GIc stands for glucose ;
• Gal stands for galactose ;
• GIcNAc stands for N-acetylated glucosamine.
• Figure IB shows the reaction that occurs upon LPS treatment with acetic acid-
• Figures 2A-2C show the HPLC chromatogram obtained at 214 nm with a composition essentially comprising the SAEP2-L2 peptide in monomeric form (2A), in parallel dimeric form (2B) and anti-parallel dimeric form (2C). Coordinates are : times (min) and absorbance unit (AU).
• Figure 3 shows the HPLC chromatogram obtained at 214 nm with a composition comprising the SAEP2-L2 peptide in monomeric form, parallel dimeric form and anti- parallel dimeric form.
• Figures 4A-4C show the 1 H NMR spectra obtained with a composition essentially comprising the SAEP2-L2 peptide in monomeric form (4A), in parallel dimeric form (4B) and anti-parallel dimeric form (3C). In all of them, a peak at 1.9 ppm indicates that the peptide is in an acetate salt form.
• Figures 5A-5C show an enlargement of the region of the 1 H NMR spectra of Figures 4A-4C comprised between 6.5 and 7.5 ppm.
• Figure 6 shows the 6.5-7.5 ppm region of the 1 H NMR spectrum obtained with a composition comprising the SAEP2-L2 peptide in monomeric form, parallel dimeric form and anti-parallel dimeric form.
• Figures 7A-7C show the MALDI-ToF spectra of the calibration standard (7A), the parallel dimer (7B) and the anti-parallel dimer (7C).
• Figure 8 shows the HPEAC-PAD chromatogram of LPS hydrolysed by acetic acid treatment.
• Example 1 Preparation of the SAEP2-L2 parallel dimer
• the synthesis cycle proceeds step-by-step, according to the reported linear sequence. It is performed in pure solvent dimethylformamide (DMF). Side-protected, activated amino acids are used.
• DMF dimethylformamide
• the thiol group of the Cys residue in position 10 (Cys-10) is protected with the acid- labile group Trityl (triphenyl-methyl derivative, Trt).
• the thiol group of the Cys residue in position 4 (Cys-10) is protected with the acid-resistant group S-acetamido-methyl (Acm).
• the protected peptide is cleaved from the resin support using TFA 95 % in the presence of the scavenger ethandithiol at 2 - 5 % (v/v).
• the thiol group of the Cys-10 is de-protected, while the thiol group of the Cys-4 remains Acm-protected.
• the free, Acm-protected peptide is concentrated by vacuum- evaporation and then recovered by precipitation with ether at 80 % (v/v) final concentration.
• the Cys-4 protected, Cys-10 de-protected peptide is dried under vacuum, then solubilized in water at the concentration of 1 to 10 mg/mL and adjusted at pH 7.50 with 0.1 M aqueous ammonia.
• oxidation is then performed by vigorous stirring of the aqueous solution at 4 0 C, under a pressure of 1 Atm, for 18-24 hours. Complete oxidation of the thiol groups is determined by the Elman colorimetric assay.
• the partly oxidized peptide in solution at the concentration of 1 to 10 mg/mL is then processed for de-protection of the remaining Cys-4 S-Acm functions.
• the peptide solution is added with mercuric acetate at a final concentration of 0.1 M, using phenol at 2-5 % (v/v) as scavenger.
• the solution is again vigorously stirred at 20 0 C, under a pressure of 1 Atm, for 18-24 hours.
• Complete oxidation of the thiol groups is determined by the Elman colorimetric assay.
• the amino acid composition is analysed by the Pico-Tag method (Millipore). Results are reported in the table hereinafter.
• the molecular mass is measured by Ion Cyclotron Resonance (ICR). The value found is 2,387.33 ⁇ 0.3 AMU, a value coherent with the elementary structure C 11O H ⁇ o O 24 N 26 S 4 of the peptide formula.
• the protected peptide is cleaved from the resin support by TFA 95 %, in the presence of the scavenger Ethandithiol at 2-5 % (v/v). In these conditions, the thiol groups of both Cys-4 and 10 residues are de-protected. The cleaved and de-protected peptide is then concentrated under vacuum-evaporation and recovered by precipitation with ether 80 % (v/v).
• the de-protected peptide is sol ⁇ bilized in water at the concentration 1 to 10 mg/mL and the pH is adjusted to 7.50 with 0.1 M aqueous ammonia.
• Oxidation is then performed by vigorous stirring of the aqueous solution for 18-24 hours, at 4°C, under pressure of 1 Ami. Complete oxidation of the thiol groups is determined by the Elman colorimetric assay.
• the peptides in solution actually constitute a mixture of cyclic monomer (about 40 %) and anti-parallel dimer (about 60 %).
• Each form is purified by preparative Reverse- phase HPLC chromatography. Indeed, it is possible to separate the cyclic monomer from the anti-parallel dimer since these forms elute, each as a single sharp peak, at different retention times. The anti-parallel dimer elutes at a lower retention time. This is consistent with the different molecular symmetry of the two dimers.
• the anti-parallel peptide may assume a lower minimal energy in aqueous solvents by virtue of its lower internal steric hindrance of the side-chains, similarly to the "trans” vs "cis” conformation of any other isomeric entities.
• the amino acid composition is analysed by the Pico-Tag method (Millipore). Results are reported in the table hereinafter.
• the molecular mass is measured by Ion Cyclotron Resonance (ICR). The value found is 2,387.30 ⁇ 0.3 AMU, a value coherent with the elementary structure C 110 H 190 O 24 N 26 S 4 of the peptide formula.
• Example 3 Further characterization of the monomer, parallel and antiparallel dimers by HPLC-reverse phase, NMR and MALDI-ToF mass spectrometry
• the dimeric parallel peptide as prepared in Example 1 and the monomelic and dimeric antiparallel peptides as prepared in Example 2 are characterized by HPLC-reverse phase ( Figures 2A-2C) and NMR ( Figures 4A-4C and 5 A-5C).
• each lyophilised peptide are diluted first in 30 ⁇ l water ; to which is added 30 ⁇ l of trifluoroacetic acid (TFA) 0.1 % in water.
• TFA trifluoroacetic acid
• a mixture of the monomeric, dimeric parallel and antiparallel peptides is also prepared by mixing 40 ⁇ g of a powdered preparation of each peptide in 60 ⁇ l water ; to which is added 60 ⁇ l of TFA 0.1 % in water.
• phase mobile B (TFA 0.1 %, CH 3 CN 80 % in water). Once samples are applied to the equilibrated column, the phase B gradient runs from 20 to 60 % within 40 min (1 % B / min), at a flow rate of 1 mL/min.
• the HPLC-RP technique is used to verify the purity of each peptide preparation.
• the relative purity degree of each peptide is calculated by integrating the peak surfaces. It is expressed as the peptide peak surface / surfaces of the whole peaks.
• Figure 3 shows the HPLC chromatogram of the mixture.
• peptides preparation kept at -70°C are used for analysis.
• Dimeric peptide solutions 0.5 mM are prepared while diluting 1.33 g in 1 mL H 2 0. 144 ⁇ l of the solutions are mixed with 16 ⁇ l of D 2 O 99.9 % D in 3mm NMR tubes.
• an external solution of TSP-d4 (3-(trimethylsilyl)propionic-2,2,3,3,-d4 acid sodium salt ; Aldrich ref 29304-0) 0.075 % (w/w) in H 2 O/D 2 O mixture (90/10 v/v) is used.
• the spectrometer is calibrated so that the unique resonance signal of TSP-d4 be at 0 ppm.
• 1 H NMR spectra of the monomer and dimers cover a range from 0 to 9.5 ppm and are composed of 3 main regions : from 6.5 to 7.5 ppm ; from 5.5 to 2.5 ppm ; and from 2 to 0.3 ppm. This is to be seen in Figure4A-4C.
• 1 H NMR spectrum of the monomer is characterized by a NMR pattern of 5 aromatic protons that are expected between 7.25 and 7.45 ppm, in the experimental conditions reported hereinabove.
• this NMR pattern is itself composed of a first multiplet from 7.25 to 7.35 ppm with an integral curvr corresponding to 3H and a second multiplet (pseudo-triplet), centered at 7.39 ppm with an integral curve of 2H. This latter signal is characteristic of the monomer only.
• 1 H NMR spectrum of the parallel dimer is characterized by a doublet signal between 7.10 and 7.25 ppm corresponding to 4 aromatic protons and a multiplet between 7.25 and 7.40 ppm with an integral curve of 6H.
• the 4H doublet is found centered at 7.185 ppm (pics at 7.18 and 7.19 ppm).
• 1 H NMR spectrum of the antiparallel dimer is characterized by a doublet signal 4 aromatic protons between 6.95 and 7.10 ppm and a multiplet between 7.10 and 7.30 ppm with an integral curve of 6H.
• the 4H doublet is found centered at 7.025 ppm (pics at 7.02 and 7.03 ppm).
• the 1 H NMR spectrum of the antiparallel dimer is also characterized by two upfleld methylic resonances that are expected between (i) 0.40 and 0.65 (doublet) and (ii) 0.70 and 0.85 ppm (doublet). In one experiment, these doublets are found centered at 0.42 and 0.68 ppm. They are observed neither in the monomer, nor in the parallel dimer.
• MALDI-ToF Mass Assisted Laser Desorption Ionisation - Time of Flight
• MALDI-ToF analysis is achieved using the Biflex III mass spectrometer (BrukerTM) and associated softwares, in a positive reflector mode. Peptides are mixed with a matrix (alpha cyano-4-hydroxy cinnamic acid) that absorbs laser energy.
• the spectrometer is externally calibrated with a mixture of synthetic peptides (ACTH 18-39 (adenocorticotropic fragment 18-39) bombesine, and somatostatine 28.
• ACTH 18-39 adenocorticotropic fragment 18-39
• a saturated HCCA matrix solution is prepared while diluting 50 mg HCCA in 300 ⁇ l 70 % ACN (acetonitril) 0.1 % TFA (trifluoroacetic acid) in water.
• a 1 A saturated HCCA solution is further prepared while diluting vol : vol with 30 % ACN, 0.1 % TFA in water.
• primary standard solutions are first prepared in 0.1 % TFA. They are as follows :
• Adenocorticotropic fragment 18-39 (ACTH 18-39) : 100 pmoles / ⁇ l (0.247 mg/mL) ; - Bombesine : 100 pmoles / ⁇ l (0.160 mg/mL) ; and Somatostatine 28 : 100 pmoles / ⁇ l (0.31 mg/mL).
• a secondary standard solution is prepared as follows :
• Peptide solutions at 1 mg/mL in water are diluted down to 0.02 mg/mL with 30 % ACN, 0.1 % TFA in water.
• Calibration and peptide samples are diluted vol : vol with the 1 A saturated HCCA solution. Droplets of about 1 ⁇ l are deposited on a steel target (BrukerTM) and dried by evaporation.
• the experimental values found for the parallel and antiparallel dimer preparations are 2388.449 and 2388.532 Da. These values are within the identity range (+ 2 Da) centered on the theoretical values range. This means that the samples contain what is expected.
• Preculture Two mL frozen samples of working seed from a N. meningitidis A strain known to express LPS exclusively under the L8 form, are used to inoculate in a 2 1 erlen containing 200 mL of Mueller-Hinton broth (Merck) complemented with 4 mL of a glucose solution in water (500 g/1). This operation is repeated 4 times. Erlens are incubated at 36 + 1 °C for 10 ⁇ 1 hrs while stirring (100 rpm).
• the erlen contents are gathered together and the preculture is complemented with 400 mL of a glucose solution in water (500 g/1) and 800 mL of an amino acid solution.
• This preparation is used to inoculate the Mueller-Hinton broth, in a 30 1 fermentor (B. BraunTM) at an initial OD 60 o n m close to 0.05. Fermentation is performed overnight at 36°C, pH 6.8 ⁇ 0.2, 100 rpm, p ⁇ 2 30 %, and initial flow rate of the air 0.75 1/min/L culture. After 7 + 1 hrs, (OD 6 oo nm about to 3), the culture is feeded by MH broth at a flow rate of 440 g/h.
• the fermentation is stopped.
• the final ODeoonm is comprised between 20 and 40.
• Cells are collected by centrifugation for 1 h 30 at 7000 g at 4°C. Pellets are kept frozen at- 35°C 4.1.2. Purification of LPS
• Pellets are defrosted and suspended with 3-volume phenol 4.5 % (v/v) and stirred vigorously for 4 hrs minimum at about 5°C.
• the bacterial suspension is heated at 65°C and then mixed v/v with phenol 90 % at 65°C.
• the suspension is stirred vigorously, at 65°C for 50-70 min and then cooled down to about 20°C.
• the suspension is centrifuged for 20 min, at 11 000 g, at about 2O 0 C.
• the aqueous phase is collected and kept.
• the phenol phase and the interphase are recovered and submitted to a second extraction.
• the phenol phase and the interphase are heated at 65°C and mixed with a volume of water equivalent to the volume of the aqueous phase that was previously collected.
• the mixture is stirred vigorously for 50-70 min at 65 0 C and then cooled down to about 20 0 C.
• the mixture is centrifuged for 20 min, at 11 000 g, at about 2O 0 C.
• the aqueous phase is collected and kept.
• the phenol phase and the interphase are recovered and submitted to a third extraction.
• the 3 aqueous phases are dialysed overnight and separately against 40 1 of water.
• the dialysates are pooled together.
• the dialysate pool is adjusted with Tris 20 mM, MgCl 2 2 mM (one volume per 9 volumes of the dialysate pool). pH is adjusted to 8.0 + 0.2 with NaOH 4 N. PNAse treatment
• Powder of MgCl 2 , 6H 2 O is added to the LPS-containing fractions pooled together, to reach an MgCl 2 concentration of 0.5 M and dissolved while stirring.
• the pellets are resuspended with at least 100 mL MgCl 2 0.5 M, while stirring.
• Pellets are resuspended with at least 150 mL water.
• the LPS preparation is analyzed by SDS-PAGE electrophoresis. Upon silver nitrate staining, a single large band is revealed. This indicates at least that the preparation does not contain any entity other than LPS L8.
• LPS comprises in its structure 2 KDO units, one being in a lateral position.
• LPS quantification is achieved through dosage of the lateral KDO unit liberated upon soft acid hydrolysis (See Figure IB).
• Samples to be quantified as well as the KDO etalon range are proceeded as follows : 100 ⁇ l of the hydrolysis solution (acetic acid 5 % ; glucuronic acid (GIcA) 20 ⁇ g/mL) are added. Hydrolysis is performed for 1 h at 100°C. Flasks are then dried at 40°C under nitrogen and filled with 400 ⁇ l water.
• the hydrolysis solution acetic acid 5 % ; glucuronic acid (GIcA) 20 ⁇ g/mL
• This technique is carried out on a HPAEC chain (DionexTM), using the Chromeleon DionexTM software for data acquisition.
• the analytical column Carbopac PAl 4 x 250 mm (DionexTM) is operated at 3O 0 C.
• the column is equilibrated with the elution solution (NaOH 75 mM, AcONa 90 niM). 100 ⁇ l of sample are injected into the column. Then the column is submitted to an elution flow rate of 1 mL/min for 22 min.
• Chromatogram of LPS sample is to be seen in Figure 8.
• the KDO amount present in the sample is determined by integration of the KDO peak.
• Purified LPS is used as pseudo-solution at 1 mg/mL in sterile, pyrogen free water (Milli Q quality, adjusted to pH 7.2 Limulus negative).
• the translucid pseudo-solution is sterilized by filtration using a 0.22 ⁇ m membrane.
• a solution of peptide SAEP2-L2 at 1 mg/mL in sterile, pyrogen-free water (Milli Q quality, adjusted to pH 7.2, Limulus negative) is also sterilized by filtration on 0.22 ⁇ m membrane.
• the precipitate (Endotoxoid) is then recovered by centrifugation at 3000 rpm for 10 min. The supernatant is discarded. The pellet is washed with one volume of sterile, pyrogen free water (Milli Q quality, adjusted to pH 7.2, Limulus negative). Centrifugation/washing steps are repeated five times.
• the suspension is stored at +4 0 C.
• a BCDO dosage is achieved to determine the LPS content and the suspension is adjusted to e.g. 0.50 mg/mL of complex (expressed as LPS content).
• LPS-peptide complex tested in the following examples is the LPS- antiparallel dimer complex as obtained in section 4.3, unless otherwise indicated. Therefore, this specific complex is simply referred to as LPS-peptide complex.
• LPS LPS
• LPS-peptide complexes involving the parallel dimer or the monomer are prepared exactly as it is reported in Example 4 for the LPS-antiparallel peptide complex.
• LAL is a very sensitive test used to detect and quantify endotoxins of gram-negative bacteria. The test is based on the property of the amoebocyte lysate protein from horseshoe crab (Limulus polyphemus) to induce coagulation in the presence of endotoxin.
• the evaluation of the LPS endotoxin activity is performed by using the end-point- chromogenic technique, in accordance with the European Pharmacopeia [as described in the European Pharmacopeia techniques (Edition 5.0, paragraph 2.6.14)].
• the kit QCL-1000 ref 50-647 U (Cambrex-BioWhittakerTM) is used (linear zone of the kit : 0.1 to 1 OVmL) as well as a positive control (E. coli endotoxin, 4 10 3 ELVmL, Sigma).
• Dilution of (i) samples to be tested, (ii) standard and (iii) positive control are achieved with dilution buffer (Cambrex-BioWhittakerTM) to cover the respective ranges : 1/10 to 1/10 5 ; 0.5 to 0.031 EU/mL and 1/10 4 to 1.8 10 4 .
• the results are expressed in Endotoxin Unit (EU)/ ⁇ g of complex. They are shown in the table hereinafter.
• the detoxification ratio can be established by the LPS / LPS- peptide complex ratio and expressed in log unit.
• the dimeric forms of the SAEP2-L2 peptide are more effective in detoxifying LPS than the cyclic monomer form.
• Rabbit is known to be the animal specie with sensitivity to pyrogenic effects of LPS equivalent to humans.
• the pyrogen test consists in measuring the rise in body temperature evoked in three rabbits by the intravenous (IV) injection of a sterile solution of the substances to be examined. The test, reading and calculations are performed in accordance with the European Pharmacopoeia, (Edition 5.0, paragraph 2.6.8).
• the temperature rise is interpreted depending the summed response of the temperatures : conformity is met when the summed response does not exceed 1.15°C ; and non-conformity, when the summed response exceeds 2.65°C.
• the pyrogenic threshold is set up below, between 1.15°C and 2.65°C.
• the limit pyrogen dose (IV) in rabbit corresponds to 0.025 ng/kg (LPS), and 10-25 ng/kg (LPS-peptide complex).
• references for this assay include La. Galanos et al, 1979, PNAS 16 : 5939 ; Baumgartner et al, 1990, J. Exp. Med. 171 (3) : 889 and US 6,531,131.
• IP intraperitoneal
• the LD50 observed with the LPS is 3.6 ng / mouse (1.91-6.70 ng / mouse) ; whereas that observed with the LPS-peptide complex is 1 ⁇ g / mouse (0.2-5 ⁇ g / mouse), indicating that the detoxification ratio (LPS-peptide complex/LPS) is about 250 (100- 1000).
• the effect of the LPS-peptide complex on the release of proinflammatory cytokines is monitored (assessed) in in vitro and in vivo assays.
• cytokine (IL6 and TNF ⁇ ) releases in the sera of mice immunized either with LPS or LPS-peptide complex are compared by ELISA. Blood samples are recovered 90 min after SC immunization, which is the optimal time for the release of those cytokines. C3H/HeOuJ, TLR4--/--, C3H/HeN and CDl mouse strains are tested. The two first are sensitive neither to LPS nor
• LPS-peptide complex The third and fourth are both found LPS-sensitive. CDl mice are found more LPS-peptide complex-sensitive than the others and therefore selected for further experiments retaining the most severe conditions.
• cytokine IL6, IL8 and TNF ⁇
• IL6 IL6
• TNF ⁇ IL6
• CDl mice are administered subcutaneously (SC) (i) either 10 ⁇ g of LPS or (ii) 10 ⁇ g of LPS-peptide complex. They are bled 90 minutes after injection. TL6 and TNF ⁇ releases are measured in the sera by ELISA. ELISA detection of cytokine secretion
• ELISAs are carried out using the OptEIA mouse IL6 and TNF ⁇ sets (Pharmingen), each including the capture antibody (anti-mouse cytokine), the detection antibody (biotinylated anti-mouse cytokine), avidin-horseradish peroxidase conjugate and the standard (recombinant cytokine), all from Pharmingen.
• the capture antibody anti-mouse cytokine
• detection antibody biotinylated anti-mouse cytokine
• avidin-horseradish peroxidase conjugate avidin-horseradish peroxidase conjugate
• standard recombinant cytokine
• Anti-mouse IL6 and TNF ⁇ antibodies are 1/250 diluted in 0.1 M carbonate buffer pH 9.5 (Sigma). For each assay, 100 ⁇ l of an antibody dilution are distributed per well of a
• Plates are washed in PBS 0.05 % Tween 20. 200 ⁇ l of PBS, 0.5 % bovine serum albumin (BSA) saturation buffer are then added per well. Incubation is pursued for one hr at room temperature. Plates are washed in PBS 0.05 % Tween 20.
• BSA bovine serum albumin
• Recombinant IL6 or TNF ⁇ cytokine dilutions are prepared in the RPMI medium 1 % FCS 10 %, within the range of (i) 4,000 pg/mL - 62.5 pg/mL standard. 100 ⁇ l of each dilution are distributed per well, to establish the standard curve.
• Serum dilutions are prepared in the RPMI medium P.S. glu 1 % FCS 10 %. Sera of mice injected with LPS are 1/25 and 1/125 diluted. Sera of mice injected with LPS- peptide complex are 1/5 and 1/25 diluted. 100 ⁇ l of each dilution are distributed per well.
• the reaction is stopped by adding 100 ⁇ l of 1 M H 3 PO 4 per well. Plates are read at 450 nm. Results are to be seen in the table hereinafter.
• the peptide alone does not induce IL6 or TNF ⁇ .
• the LPS-peptide complex allows for about 100-fold of detoxification (100-fold decrease in IL6 secretion).
• LPS preparation (lmg/mL) and LPS-peptide complex (500 ⁇ g/mL) are each diluted in 10 mM Tris, NaCl 150 mM, 0.05 % Tween 20, 5 % sucrose to a concentration of 50 ⁇ g/mL. They are further diluted in physiological saline to a concentration of 5 ⁇ g/mL.
• Serial 1/5 dilutions are performed in AIM-V medium (Gibco (Invitrogen)) for each test substance down to a concentration of 2.56 10 "3 pg/mL.
• Human blood collected on sodium heparin (25,000 U/5mL ; sanofi-synthelabo) is diluted 1 : 4 (vol : vol) in AIM-V medium and distributed in MicronicsTM tubes (400 ⁇ l / tube). 100 ⁇ l of a dilution of the test substances are added. Peptide and buffer controls are tested at 1/20 dilution. Tubes are incubated for 24 hrs at 37 0 C, in a wet atmosphere at 5 % CO 2 .
• Tubes are then centrifuged for 10 min at 500 g. At least 200 ⁇ l of supernatant are recovered from each tube and kept frozen at -80°C until titration.
• ELISAs are carried out using the OptEIA human IL6, IL8 and TNF ⁇ sets from Pharmingen, each including the capture antibody (mouse anti human cytokine), the detection antibody (biotinylated mouse anti-human cytokine), avidin-horseradish peroxidase conjugate and the standard (recombinant cytokine).
• Anti-human IL6, IL8 and TNF ⁇ antibodies are 1/250 diluted in 0.1 M carbonate buffer pH 9.5 (Sigma). For each assay, 100 ⁇ l of an antibody dilution are distributed per well of a Maxisorp NUNC 96 flat-bottom well ELISA plate. Plates are incubated overnight at +4°C
• Plates are washed in PBS 0.05 % Tween 20. 200 ⁇ l of PBS, 0.5 % bovine serum albumin (BSA) saturation buffer are then added per well. Incubation is pursued for one hr at room temperature. Plates are washed in PBS 0.05 % Tween 20.
• BSA bovine serum albumin
• Recombinant IL6, IL8 or TNF ⁇ cytokine dilutions are prepared in AIM-V medium within respective range of (i) 1,200 pg/mL - 18.75 pg/mL ; (ii) 800 pg/mL - 12.5 pg/mL ; and (iii) 1,000 pg/mL - 15.87 pg/mL standard. 100 ⁇ l of each dilution are distributed per well, to establish the standard curve.
• Plasma dilutions are prepared in the AIM-V. Plasmas recovered from blood stimulated with LPS are 1/25 and 1/125 diluted. Those recovered from blood in contact with the LPS-peptide complex are 1/5 and 1/25 diluted. 100 ⁇ l of each dilution are distributed per well. Incubation is pursued for 2 hrs at room temperature.
• TMB tetramethylbenzidine
• the reaction is stopped by adding 100 ⁇ l of 1 M H 3 PO 4 per well. Plates are read at 450 nm.
• the raw results and the cytokine release curves f (LPS or complex concentrations) do not allow comparison of different samples. Calculating the detoxification ratio can eliminate inter-blood donor and inter-test variability. Only the linear parts of the curves are taken into account for calculation of the detoxification ratio. The maximum IL6 release beyond which a linear progression is no longer observed is determined and then, the amount of substance required to induced 50 % of that maximum is calculated by linear regression.
• the detoxification ratio is expressed as the ratio of the concentration of the LPS- peptide complex inducing 50 % of maximum IL6 release (ED 50 expressed in pg/mL) in over that observed with LPS. Higher the ratio, stronger the detoxification is. As the detoxification ratio is systematically measured using whole blood of several independent donors, results are averaged.
• the detoxification ratio observed with the LPS-peptide complex is measured several times. Mean data out of six values obtained in the IL6 release assay : 64 + 20.
• the IL6 release correlates with the TNF ⁇ and IL8 secretions. Therefore, the IL6 release assay is selected to routinely evaluate the inflammation decrease observed with the LPS-peptide complex.
• the detoxification ratio is measured between 10 2 and 10 3 , depending on the test.
• the detoxification values are summarized in the following table.
• the stability of the LPS-peptide complex is studied for 6 months and evaluated by measuring the detoxification ratio in two assays (LAL and in vitro IL-6 release by huPBMC). Pyrogen test in rabbits may also be achieved.
• the detoxification ratio in IL6 release test are not significantly different after 3 and 6 months, indicating the stability of the LPS-peptide complex : LPS complexed with peptide remains detoxified after 6 months at 5°C.
• the aim of the experiment is to verify that LPS is not released when the complex is administered and that the detoxification rate does not decrease after a contact with a physiological liquid.
• Immunization of three adult New-Zealand rabbits is performed with 100 ⁇ g of LPS- peptide complex by intramuscular (IM) and subcutaneous (SC) routes (2 x 0.5 mL and 5 x 0.2 mL respectively) in the presence of adjuvant. They receive three injections at 3- week interval ; the first one with complete Freund adjuvant (FA), the second and third ones with incomplete Freund adjuvant. They are bled two weeks after the last injection. A control group is immunized with the peptide with adjuvant (71 ⁇ g, equivalent to the amount of the peptide in 100 ⁇ g of LPS-peptide complex) using the same protocol.
• IM intramuscular
• SC subcutaneous
• FA complete Freund adjuvant
• a control group is immunized with the peptide with adjuvant (71 ⁇ g, equivalent to the amount of the peptide in 100 ⁇ g of LPS-peptide complex) using the same protocol.
• the bactericidal activity of the serum (SBA) samples is evaluated against the N. meningitidis strain used for LPS production as described in Example 5 in the presence of baby rabbit serum as exogenous source of complement.
• Sera are heat-inactivated during 30 min at 56 0 C.
• heat-inactivated sera are then twofold serially diluted (10 times) in Dulbecco's phosphate buffered saline containing Ca 4+ and Mg +"1" (volume per well : 50 ⁇ l).
• controls include (i) bacteria and the complement source without antibodies (complement control), (ii) bacteria and heat-inactivated complement, and (iii) bacteria and heat-inactivated complement, in the presence of antibodies.
• Bactericidal titre is reported as the highest reciprocal serum dilution at which > 50 % killing of bacteria is observed as compared to the complement control.
• Results are to be seen in the table hereinafter.
• High SBA titers are obtained with the complex.
• the specificity of the SBA response is confirmed with the extinction of the response, when the sera (post-dose 3) are adsorbed on LPS.
• mice Ten six- week old female outbred CDl mice are immunized with a 10 ⁇ g dose of LPS- peptide complex by the subcutaneous route (0.2 mL). They receive two injections at 3- week interval. They are bled before each injection and exsanguinated 14 days after the last injection. A control group is injected with buffer.
• the antibody response is evaluated by ELISA and the bactericidal activity of the post-dose 2 serum samples is evaluated against the N. meningitidis strain used for LPS production as described in Example 4 (homologous strain) and a heterologous N. meningitidis strain [JV. meningitidis group B strain RH873 (L4, 7, 8 immunotype)].
• the antibody response is evaluated by ELISA and the opsonic activity of the post-dose 2 serum samples is evaluated by FACS.
• the LPS solution is removed from the plate and wells are saturated with 150 ⁇ l of buffer 2 (PBS + milk 1 % + Tween 20 0.05 %).
• the plate is incubated one hour at 37°C ; then washed with buffer 3 (PBS + Tween 20 0.05 %).
• Sera are serially diluted 12-fold, directly in the wells using buffer 2 (volume : 100 ⁇ l per well). The plate is incubated for 90 rnin at +37°C ; then washed with buffer 3.
• the reaction is developed by adding 100 ⁇ l of a tetramethylbenzidine substrate solution in each well. The plate is incubated 20 min at 37°C. The reaction is stopped by adding 1 M HCl and absorbance is measured at 450 nm.
• Results are expressed in arbitrary ELISA Unit/mL (EU/mL) by comparison to a reference serum.
• the ELISA assay is achieved using a pool of 10 sera.
• the LPS-peptide complex is able to induce high anti-LPS IgG titers in mice and anti-LPS IgM after one injection (ELISA).
• ELISA anti-LPS IgM after one injection
• the ELISA assay is achieved individually. After the second injection, seven out of the ten mice exhibits high IgG and IgM titers. Global mean titers expressed in log are about 3.7 and 2.8 respectively.
• Bactericidal activity is measured as described in section 7.1.
• the opsonic activity is measured by flow cytometry technology (FACS) using human promyelocytic differentiated HL60 cells as effector and LPS coated latex fluorescent beads as target.
• FACS flow cytometry technology
• Effector cells are differentiated into granulocytes after treatment with 100 mM dimethylformamide. The resulting cells are washed, resuspended in Hanks' balanced salt solution and their concentration is adjusted to 2.5x10 7 cells/mL.
• Sera are heat inactivated during 30 min at 56°C.
• heat- inactivated sera are serially fivefold diluted (3 times) in Hanks balanced salt buffer containing Ca +"1" and Mg +"1" (volume per well : 300 ⁇ l).
• Twenty ⁇ l of LPS coated latex fluorescent beads and 10 ⁇ l of baby rabbit serum as exogenous complement source are added to each well. The plate is incubated 30 min at +37°C, under shaking.
• One hundred fifty ⁇ l from each well are transferred in a second deep well and the reaction is stopped by adding 400 ⁇ l of PBS + 0.02 % EDTA. The plate is centrifuged and washed twice with PBS+BSA buffer.
• the phagocytosis of LPS coated beads by effector cells, in the presence of antiserum and exogenous complement source is measured by FACS.
• PP is measured as the ratio number of beads / phagocytic cells x number of fluorescent cells.
• mice Eight out often mice exhibit high opsonic activity (> 350).

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