WO2002051865A2 - Proteinic antigens inducing antibodies neutralising hiv virus - Google Patents

Proteinic antigens inducing antibodies neutralising hiv virus Download PDF

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WO2002051865A2
WO2002051865A2 PCT/FR2001/003957 FR0103957W WO02051865A2 WO 2002051865 A2 WO2002051865 A2 WO 2002051865A2 FR 0103957 W FR0103957 W FR 0103957W WO 02051865 A2 WO02051865 A2 WO 02051865A2
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artificial sequence
protein
ser
leu
gly
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PCT/FR2001/003957
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French (fr)
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WO2002051865A3 (en
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Véronique BARBAN
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Aventis Pasteur
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Priority claimed from FR0016986A external-priority patent/FR2818648B1/en
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Priority to AU2002219290A priority Critical patent/AU2002219290A1/en
Publication of WO2002051865A2 publication Critical patent/WO2002051865A2/en
Publication of WO2002051865A3 publication Critical patent/WO2002051865A3/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
    • 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
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates to a protein antigen comprising a carrier protein, a spacer arm, and a specific peptide sequence inducing antibodies neutralizing primary isolates of the HIV virus and to its use in an HIV vaccine.
  • HIV envelope glycoprotein which is required to make the virus infectious is the target of neutralizing antibodies. These characteristics have made the latter a subject of intense investigation.
  • the present invention aims to overcome this need and relates to a new protein antigen comprising a carrier protein, a spacer arm and a specific peptide sequence, said antigen being capable of inducing neutralizing antibodies to primary isolates of the HIV virus.
  • the present invention therefore relates to: A protein antigen of formula P-E-S in which: P denotes a protein or a carrier peptide or a fragment derived therefrom; E denotes a spacer arm of 2 to 25 amino acids consisting mainly of glycine, proline or alanine;
  • S denotes a peptide sequence chosen from the group consisting of the sequences ID NO1 to ID NO8.
  • the lines represent a peptide bond.
  • the spacer arm E consists of 3 glycine residues.
  • the protein or the peptide P is either a protein having a signal sequence originating from a bacterium or a phage, or a protein or a peptide reinforcing the immunogenicity of the sequence S.
  • the invention relates to an expression vector encoding said protein antigen.
  • the expression vector is a filamentous phage and the P protein is the pl11 and / or pVIII protein.
  • the invention in another embodiment, relates to a vaccine comprising an antigen or a vector as defined above in combination with a pharmaceutically acceptable carrier and optionally an adjuvant.
  • the vaccine is used for the treatment or prevention of HIV virus infection.
  • the invention relates to a diagnostic method comprising bringing an antigen or an expression vector as defined above into contact with a biological sample.
  • Protein antigen is understood to mean, in the context of the present invention, an antigen of formula P-E-S which induces neutralizing antibodies to primary isolates of HIV.
  • primary isolates strains of HIV virus originating from asymptomatic seropositive subjects and for which the cultures were carried out only on primary cells, in this case mononuclear cells from human peripheral blood. The induction of antibodies neutralizing the primary HIV isolates is demonstrated by the assay of the p24 protein in the culture supernatant of the viral strain tested as described in the article by C. Moog et al (AIDS Research and human retroviruses, vol 13 (1), 13-27, 1997).
  • a sequence S according to the invention is selected from the group consisting of
  • the S sequences according to the invention can be selected by means of a phage library expressing on their surface a protein structure of formula PES in which P denotes the PMI protein and / or PVIII of a filamentous phage, E a spacer arm and It's a random peptide.
  • PES protein structure of formula PES in which P denotes the PMI protein and / or PVIII of a filamentous phage, E a spacer arm and It's a random peptide.
  • Such banks are described in the articles by Keller et al (Virology, 193, 709-716 (1993)), by Felici F. et al (J. Mol. Biol., 222, 301-310 (1991), by Scott et al (Science, 249, 386-390 (1990)) and Schatz PJ (Current Op. Biotech, 5: 487-494 (1994)).
  • the S sequences according to the invention can be selected according to the same methodology developed in the article by Barban V. et al. (Journal of General Virology, vol 81, 461-469 (2000)) using, as antibodies for the selection of relevant recombinant phages, monospecific antibodies of the HIV envelope which have were isolated by purification from sera from SCID (severe combined immunodeficiency) mice which were "humanized” by intraperitoneal injection of human peripheral blood lymphocytes from asymptomatic seropositive subjects (Mosier et al., Science, 251: 791-794, 1991) .
  • the spacer arm E consists of a sequence of identical or different amino acids whose role is to remove any structural constraint potentially exerted by the carrier P on the three-dimensional structure of the sequence S.
  • the spacer arm according to the invention comprises between 2 and 25 amino acids.
  • the amino acid sequence consists mainly of glycine, proline or alanine, insofar as these three amino acids represent more than 50% of the amino acids present in said sequence.
  • the spacer arm E consists only of glycine and even more preferably the arm consists of three glycines.
  • P denotes a protein or peptide carrier.
  • carrier protein or peptide is understood to mean, within the framework of the present invention, any protein or peptide capable of stabilizing the three-dimensional structure of the S sequence under a conformation such that the protein antigen is capable of inducing antibodies neutralizing primary HIV isolates.
  • the carrier protein or peptide enhances the immunogenicity of the S sequence, in particular by increasing the production of specific antibodies.
  • the protein or peptide preferably comprises one or more T helper epitope (s).
  • helper T epitope is meant a chain of amino acids which, in the context of one or more MHC class II molecules, activates helper T lymphocytes.
  • the stabilization and increased immunogenicity effects are obtained when the S sequence is linked to the C or N terminus of the protein or of the P peptide via a spacer arm E as defined above.
  • carrier protein it is possible to use the phage surface proteins such as the pl11 or pVIII protein of phage M13, the bacterial surface proteins such as the LambB proteins, OmpC, OmpA, OmpF, and PhoE of Escherichia coli, the CotC protein or CotD of Bacillus subtilis, bacterial porins like porin
  • P1 of Neisseria gonorrheae porin P1 or P2 of Haemophilus influenzae B, porin of class I of Neisseria meningitidis B, porin P40 of Klebsiella pneumoniae; also lipoproteins such as OspA from Borrelia bugdorfi, PspA from Streptococcus pneumoniae, Transferrin binding protein type 2 (TBP2) from Neisseria meningitidis B, TraT from Escherichia coli, adhesin A from Streptococcus pneumoniae.
  • lipoproteins such as OspA from Borrelia bugdorfi, PspA from Streptococcus pneumoniae, Transferrin binding protein type 2 (TBP2) from Neisseria meningitidis B, TraT from Escherichia coli, adhesin A from Streptococcus pneumoniae.
  • detoxified bacterial toxins such as tetanus or diphtheria toxoid, the subunit B of cholera toxin or the subunit B of the heat-labile enterotoxin of Escherichia coli, exotoxin A from Pseudomonas aeruginosa, or the exotoxin A from Staphylococcus aureus.
  • heat shock protein such as Hsp 65 or hsp 71 from Mycobacterium tuberculosis or bovis, or Hin 47 from Haemophilus influenzae type B are other types carrier proteins usable in the context of the invention.
  • carrier peptide non-limiting examples include the peptides p24E, p24N, p24H, p24M, p24H described in WO 94/29339 carrying a T helper epitope, or the PADRE peptides (Pan DR T helper epitopes) described by Del guercio et al (Vaccine et al., (1997), Vol 15/4, p 441-448).
  • the constituents P, E and S of the antigen according to the invention are linked to each other by a peptide bond between the COOH and NH 2 groups present at the N or C terminal ends of the constituents so as to form a structure of formula P-E-S.
  • the constituents P, E and S of the antigen according to the invention can also be combined so as to form a structure of type S-E-P-E-S in which the spacer arms E are identical or different and the sequences S are identical or different.
  • the antigen according to the invention can be obtained by any conventional technique of chemical synthesis or genetic engineering.
  • the antigen according to the present invention can either be synthesized in the form of a single sequence, or in the form of several sequences which are then linked to each other.
  • large antigens > 50 amino acids
  • P, E, S are preferably synthesized separately and then linked by any suitable chemical coupling process.
  • Chemical synthesis can be carried out in solid phase or in solution, these two synthesis techniques being well known to those skilled in the art. These techniques are described in particular by Atherton and Shepard in “solid phase peptide synthesis” (IRL Press, oxford, 1989), and by Houbenweyl in “Method der organischen chemie” edited by E Wunsch, vol 15-1 and II-Thieme, Stuttgart , 1974.
  • the antigen can also be produced by genetic engineering techniques well known to those skilled in the art. These techniques are described in detail in Molecular Cloning: a molecular manual by Maniatis et al. In this case, an insert is conventionally prepared comprising:
  • An oligonucleotide sequence coding for a first restriction site An oligonucleotide sequence coding for the spacer arm E, An oligonucleotide sequence encoding the sequence S, and An oligonucleotide sequence encoding a second restriction site which may be identical to the first
  • This oligonucleotide insert is synthesized using an oligonucleotide synthesizer. It is then inserted into an expression vector containing the gene coding for the protein P. The oligonucleotide insert is inserted in phase, with the reading frame of the gene coding for the said protein. In the case of P proteins comprising a signal sequence, the insertion takes place immediately after the signal sequence. This signal sequence corresponds either to a sequence naturally present in the protein, or to an added signal sequence, such as ompA, lamB, etc. to facilitate the steps of purification of the antigen.
  • the expression vector is a phage, in particular a filamentous phage derived from phage M13 or fd in which the oligonucleotide insert is inserted behind the signal sequence of the gene coding for the protein (s) ) pi II and / or pVIII of the phage.
  • an insertion site is introduced immediately after the signal sequence of the protein by site-directed mutagenesis in order to be able to introduce the oligonucleotide insert.
  • viable mutants of phage M13 or fd in which new restriction sites have been created in the p111 and / or PVIII gene.
  • the antigen is advantageously expressed on the surface of the phage.
  • the antigen thus produced is then purified by any conventional purification process.
  • the antigen When the antigen is expressed on the surface of the phage or the bacteria, it is first extracted under the action of a slightly hyperosmotic medium containing calcium channel blockers (such as EDTA), and light detergents (Tween® 20, newt). The surface proteins are then released into the medium while the other solid constituents such as the core of the bacteria or of the phage precipitate by centrifugation. The antigen recovered in the supernatant is purified from its other contaminants using chromatographic techniques well known to those skilled in the art.
  • a slightly hyperosmotic medium containing calcium channel blockers (such as EDTA), and light detergents (Tween® 20, newt).
  • the surface proteins are then released into the medium while the other solid constituents such as the core of the bacteria or of the phage precipitate by centrifugation.
  • the antigen recovered in the supernatant is purified from its other contaminants using chromatographic techniques well known to those skilled in the art.
  • the antigen When the antigen is secreted into the extracellular medium, it is directly recovered from the culture medium and purified using standard chromatographic techniques as described in the article by Philippe Slos et al (Protein expression and purification, 5 , 518-526 (1994)).
  • the invention therefore relates to any expression vector expressing an antigen as defined above.
  • expression vector is understood to mean, within the context of the present invention, any vector comprising an expression cassette containing the sequence coding for the protein antigen according to the invention and regulatory sequences allowing the expression of said sequence. This definition therefore includes plasmids, phages, as well as viruses. Any vector suitable for the expression of a recombinant protein can be used in the context of the present invention.
  • the expression vectors are preferably viruses or phages.
  • the expression vector is preferably a recombinant filamentous phage expressing multiple copies of the antigen according to the invention in which the protein P is represented by the protein pl11 and / or PVIII preferably pVIII. This results from the fact that the proteins plll and pVIII are present naturally on the surface of the phage in the form of repeating units (5 for the protein plll, more than 2000 for the protein pVIII). Expression of multiple copies of the antigen on the surface of the recombinant phage enhances the immunogenicity of the antigen.
  • the present invention also relates to anti-HIV vaccines for therapeutic or prophylactic purposes.
  • the vaccines according to the present invention comprise at least one antigen according to the present invention in combination with a pharmaceutically acceptable carrier or diluent and optionally an adjuvant.
  • the vaccine according to the present invention can therefore contain or lead to the expression of a single type of protein antigen or of a mixture of various types of antigens according to the invention. In such a scenario, these antigens can be differentiated, for example, by the nature of the S sequence or by the nature of the carrier protein.
  • the vaccine according to the present invention comprises an expression vector as defined above. This expression vector is advantageously a phage, in particular a filamentous phage.
  • a vaccine comprising a recombinant filamentous phage expressing on its surface multiple copies of an antigen of formula PES in which P represents the protein pl11 and / or pVIII, preferably pVIII, is the preferred embodiment according to the invention.
  • the amount of antigen in the vaccine according to the present invention depends on many parameters as understood by those skilled in the art, such as the nature of the antigen, the level of expression of the antigen by the vector. expression, route of administration, and physical condition of the person to be treated (weight, age, gender, clinical condition). The relative mass of the P protein relative to the S sequence is also taken into account.
  • the amounts of antigen administered can reach 1 to 10 mg but more generally lie between 5 ⁇ g and 1 mg, preferably 50 to 500 ⁇ g per injection.
  • a phage is generally administered at a minimum dose of 10 3.5 infecting units (pfu or range forming unit) but preferably in a dose scale ranging from 10 to 10 12 pfu and even more preferably in a scale of doses ranging from 10 6 to 10 10 pfu.
  • infecting units pfu or range forming unit
  • Those skilled in the art can refer to clinical trials and protocols using preparations based on recombinant vectors, in particular recombinant pox viruses, recombinant adenoviruses, already carried out in humans to agree on the appropriate number of pfu that must contain the vaccine.
  • the doses injected can be very variable ranging from 1 ⁇ g to 100 mg, but preferably between 0.1 mg to 10 mg.
  • the vaccine contains an expression vector such as a recombinant phage, a recombinant virus
  • the potential infectious power of these recombinant microorganisms is generally neutralized by using methods which do not alter the conformation of the antigen according to the invention. For example, when administering a suspension of recombinant phages, recourse is generally made to prior irradiation of the phage suspension in order to reduce the infectious titer.
  • the vaccine according to the invention may also contain one or more adjuvants or be incorporated into delivery systems compatible with use in human medicine. Mention may report the use of adjuvants such as alum (aluminum phosphate or phosphate hy 'droxyde aluminum or mixture thereof) conventionally incorporated in vaccines, incomplete Freund's adjuvant, lipid A monophosphorylated (MPL), QS21, Polyphosphazene, muramyl dipeptide (MDP) or its derivatives, the use of antigen delivery systems such as emulsions (MF59, SAF1, RIBI, SB 62, SB 26), ISCOMS, liposomes, microspheres composed of PLGA polymers of well-calibrated diameter, or possibly pseudo virions.
  • adjuvants such as alum (aluminum phosphate or phosphate hy 'droxyde aluminum or mixture thereof) conventionally incorporated in vaccines, incomplete Freund's adjuvant, lipid A monophosphorylated (M
  • the vaccines according to the present invention can be prepared by any conventional method known to those skilled in the art.
  • the antigens are mixed with a pharmaceutically acceptable carrier or diluent such as water or buffered saline or phosphate.
  • a pharmaceutically acceptable carrier or diluent such as water or buffered saline or phosphate.
  • the carrier or diluent is selected according to the dosage form chosen, the mode of administration and the route of administration as well as pharmaceutical practice.
  • the appropriate carriers or diluents as well as the pharmaceutical formulation requirements are described in detail in Remington's Pharmaceutical Science, which represents a reference work in this field.
  • the vaccines mentioned above can be administered by any conventional route, usually used in the field of vaccines, such as the parenteral route (intravenous, intramuscular, subcutaneous, etc.).
  • the vaccine according to the invention can also be administered by the mucosal route. Administration can be by injection of a single dose or repeated doses. For the prevention of HIV infection, the vaccine can be administered all at once or several times in order to obtain a level of immune response which ensures the protection of the individual against accidental contamination.
  • the vaccination protocol preferably includes OJ injections, at 1 month, 3 months and 6 months. Sometimes different vaccines are used for different injections. To maintain the desired level of protection, it may also be necessary to give booster shots at regular intervals.
  • the vaccine is given all at once or several times.
  • the time between each injection can be very short, less than a week, to reach the desired level of response, in particular that which makes it possible to note the absence of the HIV virus in the blood by the PCR test.
  • the therapeutic vaccine can be combined or used alternately with conventional treatments for this condition, including in particular mono, bi or tri antiviral therapy.
  • a vaccine comprising one or more antigens and / or one or more expression vectors according to the invention to stimulate in vitro or ex vivo cells of the patient's immune system and then re-inject them into the individual's body.
  • the invention finally relates to the use of an antigen or an expression vector according to the invention for the diagnosis of HIV infection or for monitoring the progress of this infection.
  • immunoenzymatic, radioimmunological or western blotting methods well known to those skilled in the art will be used, such as for example the ELISA, RIA, RIPA or IRMA methods for the analysis of the humoral response.
  • Biacore® which identify high affinity antibodies.
  • Example 1 Synthesis by genetic engineering of recombinant phages expressing on their surface protein antigens PES.
  • the first population is represented by recombinant phages for which one of the sequences S, SEQ ID NO1 to SEQ ID N08 is linked via a spacer arm consisting of 3 glycines at the N terminal end of the Plll protein (recombinant phages plll).
  • the second population is represented by recombinant phages for which one of the sequences S, SEQ ID NO1 to SEQ ID NO8 is linked via a spacer arm consisting of 3 glycines at the N terminal end of the PVIII protein (recombinant phages pVIII)
  • Example 2 Chemical synthesis of PES antigens
  • sequences of amino acids SEQ ID NO: 1 to SEQ ID NO: 8 were synthesized by solid-phase with reference to the methodologies developed in the literature Solid phase peptide synthesis: a practical approach, IRL Press, Oxford, 1989 and Solid peptide synthesis phase, second edition, published by Pierce Chemical Company, 1984. These syntheses were continued so that the C or N terminal end of the S sequences is linked, either to a spacer arm having a repeated structure of the type ( Gly-) x in which x is between 2 and 10, that is to say a spacer arm having a repeated structure of the type (Gly-pro-) y in which y is between 1 and 5 so as to obtain a peptide of formula ES .
  • the amino function of the amino acids is protected by the introduction of a t-butyloxycarbonyl (t-boc) group, thus allowing coupling, by the carboxylic function of the amino acid, to an active chloromethylated resin. After fixation on the resin, the amino function is "deprotected” by the action of trifluoroacetic acid followed by a neutralization step with triethylamine. The amino function thus released then undergoes a coupling reaction with another amino acid in the form of a t-boc derivative via carbodiimides. This process is implemented by the ABI® automaton (Applied Biosystem Inc) 430A which thus performs the automatic synthesis of peptides.
  • ABI® automaton Applied Biosystem Inc
  • the peptide is detached from the resin by the action of hydrofluoric acid.
  • the extract is then purified by reverse phase HPLC using a semi-preparative column of Vydac® C4 type and a gradient of acetonitrile ranging from 15 to 55% in a 0.1% trifuoroacetic acid solution.
  • Liquid chromatography is programmed for a period of 40 min with a flow rate of 2 ml / min. The purity rate of the E-S peptides was checked by analytical chromatography and exceeds 95%.
  • E is represented by a repeated chemical structure of type (Gly-) x in which x is between 2 and 10 or of type (Gly-pro-) y in which y is between 1 and 5.
  • S is represented by one of the sequences ID NO1 to ID NO8
  • Two restriction sites were incorporated at the two ends of an oligonucleotide consisting of a specific sequence S coding for one of the sequences ID NO1 to ID NO8, and of a sequence coding for a spacer arm E consisting of a sequence from 3 to 10 glycine residues.
  • the oligonucleotide was then incorporated immediately after the signal sequence of the cholera toxin subunit B.
  • the signal sequence of the cholera toxin subunit B was then replaced by the signal sequence ompA before incorporation into a plasmid vector, type pARA 13 under the dependence of the arabinose operon of Salmonella typhimurium.
  • E is represented by a repeated chemical structure of type (Gly-) x in which x is between 3 and 10
  • S is represented by one of the sequences ID NO1 to ID NO8
  • the antigens are purified directly from the culture medium using the purification method described by Philippe Slos et al (Protein expression and purification, 5, 518-526 (1994))
  • the recombinant phage pl11 or pVIII isolates expressing the sequences S ID: NO1 to ID: NO 8 at the end of a spacer arm E consisting of 3 glycines were tested for their capacity to induce neutralizing antibodies of primary isolates in The test-subject.
  • the purified recombinant phages were injected, intramuscularly, into the right and left thighs of the guinea pig at a rate of 5 ⁇ 10 11 pfij per injection which was carried out in presence or not of 50 ⁇ g of QS 21.
  • the guinea pigs received 3 injections at one month intervals.
  • Bx 08 and Bx 17 obtained from samples from seropositive and asymptomatic subjects and cultured only by passage through cells. mononuclear human peripheral blood (see article by C. Moog et al-AIDS Research and human retroviruses, vol 13 (1), 13-27, 1997).
  • the neutralizing power of the immune sera against the primary isolates Bx 08 and Bx 17 was evaluated by determining the 50% infectious dose (TCID 50) of these two isolates in the presence or in the absence of immune serum.
  • a range of dilutions of reason 10 was carried out in parallel with the viral suspensions Bx 08 and Bx 17 and, for each serum tested decomplemented, a range of dilutions in RPMI medium. Then, 50 ⁇ l of each viral dilution was incubated with 50 ⁇ l of each serum dilution for 1 hour at 37 ° C. in 96-well microplates with round bottom. Each dilution was tested in quadruplicate. 50 ⁇ l of a suspension containing 10 5 mononuclear human peripheral blood cells previously activated for 3 days with a culture medium containing 1 ⁇ g of phytohemagglutinin-P / ml were then added.
  • the culture medium After an infection period of one hour at 37 ° C., the culture medium is freed from residual viruses by 2 successive washes with 150 ⁇ l of RPMI medium. After elimination of the 2 nd washing medium, the cells were re-cultured with RPMI medium containing 10% fetal calf serum supplemented with 20 IU / ml of IL2. After 4 days of culture, part of this culture medium was renewed with fresh culture medium, then on day D7 and D10 an aliquot of 100 ⁇ l of culture supernatant was removed, replaced with fresh medium on D7 . The samples were kept frozen until the viral replication was assayed. The viral replication as well as the TCID 50 were quantified by means of the assay of reverse transcriptase or by means of the assay of p24 by ELISA using the kit marketed by the company Coluter Immunotech. Beckman.
  • Neutralization is considered significant when the ratio between the TCID50 value of the viral suspension in the presence of the serum dilution tested and that obtained in the absence of serum is ⁇ 0.1.
  • the TCID50 are calculated using the Kârber formula (Kârber G. Arch. Exp. Pathol. Pharmakol. 162: 956-959 (1931).
  • the neutralization titer is the maximum dilution factor of the serum for which the ratio between the value of the TCID50 of the viral suspension in the presence of the serum dilution tested and that obtained in the absence of serum is ⁇ 0.1, which amounts to a reduction of a log 10 of the infectious titre.
  • Table 1 The results are grouped in table 1
  • the sequence S is expressed on the surface of the recombinant phage pVIII.
  • TCID 50 represents the inverse of the dilution of the tested serum for which a reduction of at least one log 10 of the TCID 50 of the primary isolate tested compared to the serum-free control. Sera are considered to exert a neutralizing activity when at least the first dilution tested (1/5) leads to this reduction in TCID 50.
  • Table 1 show that the sera from animals immunized with the various recombinant phages tested have neutralizing activity with respect to at least one of the two primary isolates tested.
  • the QS21 adjuvant has an inconsistent effect on the production of neutralizing antibodies by guinea pigs immunized with the various recombinant phages. It should be noted, however, a clear adjuvant effect on the response of guinea pigs immunized with the recombinant phage expressing the sequence S, Seq ID NO4.

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Abstract

The invention concerns a proteinic antigen of formula P-E-S wherein: P represents a protein or a carrier peptide or a fragment derived therefrom; E represents a spacer arm of 2 to 25 amino acids, mainly consisting of glycine, proline or alanine; S represents a specific peptide sequence, said antigen being capable of inducing neutralising antibodies against HIV primary isolates and its use in an anti-HIV vaccine.

Description

ANTIGENES PROTEIQUES INDUCTEURS D'ANTICORPS NEUTRALISANT LE PROTEIN ANTIGENS INDUCING ANTIBODY NEUTRALIZING THE
VIRUS VIHHIV VIRUS
La présente invention se rapporte à un antigène protéique comprenant une protéine porteuse, un bras espaceur, et une séquence peptidique spécifique induisant des anticorps neutralisants des isolats primaires du virus VIH et à son utilisation dans un vaccin anti VIH.The present invention relates to a protein antigen comprising a carrier protein, a spacer arm, and a specific peptide sequence inducing antibodies neutralizing primary isolates of the HIV virus and to its use in an HIV vaccine.
La mise au point d'une méthode d'immunisation contre le VIH est aujourd'hui l'une des priorités de la recherche scientifique.The development of a method of immunization against HIV is today one of the priorities of scientific research.
Les obstacles majeurs que sont la grande variabilité génétique du virus et la faible exposition au système immunitaire d'épitopes viraux neutralisants freinent considérablement l'élaboration d'une immunité neutralisante.The major obstacles which are the great genetic variability of the virus and the low exposure to the immune system of neutralizing viral epitopes considerably hamper the development of neutralizing immunity.
La glycoprotéine d'enveloppe du VIH qui est requise pour conférer au virus son caractère infectieux représente la cible d'anticorps neutralisants. Ces caractéristiques ont fait de cette dernière un sujet d'investigations intenses.The HIV envelope glycoprotein which is required to make the virus infectious is the target of neutralizing antibodies. These characteristics have made the latter a subject of intense investigation.
Plusieurs auteurs ont proposé d'utiliser comme antigène vaccinal des peptides représentant des épitopes conformationnels de l'enveloppe du virus VIH. On peut se référer notamment aux travaux de Scala G (J. Immunol., (1999), 162: 6155-6161), de Keller P.M. (Virology (1993), 193, 709-716) ou de Lundin K (Immunology, (1996), 89: 579-586). De tels antigènes ont été également décrits dans la demande de brevet WO 99/66046. Ces peptides, obtenus en général par sélection contre un anticorps monoclonal dirigé contre l'enveloppe du virus VIH, ont la particularité d'être spécifiques de cet anticorps et de ne pas être reconnus par d'autres anticorps monoclonaux. Les tests d'immunogenicite réalisés chez l'animal, montrent que seuls certains de ces peptides sont capables d'induire des anticorps neutralisants le pouvoir infectieux du virus VIH. De plus, les réponses en anticorps neutralisants telles que décrites dans l'art antérieur sont dirigées contre des souches de VIH bien établies mais sont insuffisantes pour neutraliser des isolats primaires du VIH. Du fait de la très grande variabilité du virus VIH, de telles réponses présentent peu d'intérêt d'un point de vue vaccinal. Il existe donc un besoin pour un vaccin capable d'induire une immunité protectrice contre des isolats primaires du virus VIH. La présente invention a pour objet de pallier ce besoin et concerne un nouvel antigène protéique comprenant une protéine porteuse, un bras espaceur et une séquence peptidique spécifique, ledit antigène étant capable d'induire des anticorps neutralisants des isolats primaires du virus VIH.Several authors have proposed to use as vaccine antigen peptides representing conformational epitopes of the envelope of the HIV virus. We can refer in particular to the works of Scala G (J. Immunol., (1999), 162: 6155-6161), of Keller PM (Virology (1993), 193, 709-716) or of Lundin K (Immunology, ( 1996), 89: 579-586). Such antigens have also been described in patent application WO 99/66046. These peptides, generally obtained by selection against a monoclonal antibody directed against the envelope of the HIV virus, have the particularity of being specific for this antibody and of not being recognized by other monoclonal antibodies. Immunogenicity tests carried out in animals show that only some of these peptides are capable of inducing antibodies which neutralize the infectious power of the HIV virus. In addition, neutralizing antibody responses as described in the prior art are directed against well-established strains of HIV but are insufficient to neutralize primary HIV isolates. Because of the great variability of the HIV virus, such responses are of little interest from a vaccine point of view. There is therefore a need for a vaccine capable of inducing protective immunity against primary isolates of the HIV virus. The present invention aims to overcome this need and relates to a new protein antigen comprising a carrier protein, a spacer arm and a specific peptide sequence, said antigen being capable of inducing neutralizing antibodies to primary isolates of the HIV virus.
La présente invention concerne donc : Un antigène protéique de formule P-E-S dans laquelle : P désigne une protéine ou un peptide porteur ou un fragment en dérivant; E désigne un bras espaceur de 2 à 25 acides aminés constitué principalement de glycine, proline ou alanine;The present invention therefore relates to: A protein antigen of formula P-E-S in which: P denotes a protein or a carrier peptide or a fragment derived therefrom; E denotes a spacer arm of 2 to 25 amino acids consisting mainly of glycine, proline or alanine;
S désigne une séquence peptidique choisie dans le groupe consistant en les séquences ID NO1 à ID NO8.S denotes a peptide sequence chosen from the group consisting of the sequences ID NO1 to ID NO8.
Les traits représentent une liaison peptidique.The lines represent a peptide bond.
Selon un aspect, le bras espaceur E est constitué de 3 résidus glycine.In one aspect, the spacer arm E consists of 3 glycine residues.
Selon un autre aspect, la protéine ou le peptide P est soit une protéine disposant d'une séquence signal provenant d'une bactérie ou d'un phage, soit une protéine ou un peptide renforçant l'immunogénicité de la séquence S.According to another aspect, the protein or the peptide P is either a protein having a signal sequence originating from a bacterium or a phage, or a protein or a peptide reinforcing the immunogenicity of the sequence S.
Dans un autre mode de réalisation, l'invention concerne un vecteur d'expression codant pour ledit antigène protéique.In another embodiment, the invention relates to an expression vector encoding said protein antigen.
Selon un aspect particulier, le vecteur d'expression est un phage filamenteux et la protéine P est la protéine plll et/ou pVIII.According to a particular aspect, the expression vector is a filamentous phage and the P protein is the pl11 and / or pVIII protein.
Dans un autre mode de réalisation, l'invention concerne un vaccin comprenant un antigène ou un vecteur tels que définis ci dessus en combinaison avec un support pharmaceutiquement acceptable et éventuellement un adjuvant.In another embodiment, the invention relates to a vaccine comprising an antigen or a vector as defined above in combination with a pharmaceutically acceptable carrier and optionally an adjuvant.
Selon un aspect particulier, le vaccin est utilisé pour le traitement ou la prévention de l'infection à virus VIH. Enfin l'invention concerne une méthode de diagnostic comprenant la mise en contact d'un antigène ou d'un vecteur d'expression tels que définis ci-dessus avec un échantillon biologique.In one particular aspect, the vaccine is used for the treatment or prevention of HIV virus infection. Finally, the invention relates to a diagnostic method comprising bringing an antigen or an expression vector as defined above into contact with a biological sample.
Description détaillée de l'inventionDetailed description of the invention
On entend par "antigène protéique" dans le cadre de la présente invention, un antigène de formule P-E-S qui induit des anticorps neutralisants des isolats primaires du VIH. Par "isolats primaires" on entend des souches de virus VIH provenant de sujets séropositifs asymptomatiques et pour lesquelles les cultures ont été effectuées uniquement sur cellules primaires, en l'occurrence les cellules mononucléées du sang périphérique humain. L'induction d'anticorps neutralisant les isolats primaires du VIH est mis en évidence par le dosage de la protéine p24 dans le surnageant de culture de la souche virale testée comme cela est décrit dans l'article de C. Moog et al (AIDS Research and human retroviruses, vol 13(1), 13-27, 1997). On estime dans le cadre de la présente invention que des anticorps neutralisants ont été induits par l'antigène protéique, lorsque le sérum dilué au 1/5 entraîne une réduction d'un facteur 10 de la quantité de p24 présente dans le surnageant de culture d'un isolât primaire.“Protein antigen” is understood to mean, in the context of the present invention, an antigen of formula P-E-S which induces neutralizing antibodies to primary isolates of HIV. By "primary isolates" is meant strains of HIV virus originating from asymptomatic seropositive subjects and for which the cultures were carried out only on primary cells, in this case mononuclear cells from human peripheral blood. The induction of antibodies neutralizing the primary HIV isolates is demonstrated by the assay of the p24 protein in the culture supernatant of the viral strain tested as described in the article by C. Moog et al (AIDS Research and human retroviruses, vol 13 (1), 13-27, 1997). It is estimated in the context of the present invention that neutralizing antibodies have been induced by the protein antigen, when the serum diluted 1/5 causes a reduction by a factor of 10 in the amount of p24 present in the culture supernatant d '' a primary isolate.
Une séquence S selon l'invention est sélectionnée dans le groupe consistant enA sequence S according to the invention is selected from the group consisting of
SEQ ID NO: 1 Ile Pro Thr Leu Pro Ser SerSEQ ID NO: 1 Ile Pro Thr Leu Pro Ser Ser
SEQ ID NO: 2 Trp Ala Leu Asp Arg Gly AlaSEQ ID NO: 2 Trp Ala Leu Asp Arg Gly Ala
SEQ ID NO: 3 His Ala Thr Gly Thr His Gly Leu Ser Leu Ser HisSEQ ID NO: 3 His Ala Thr Gly Thr His Gly Leu Ser Leu Ser His
SEQ ID NO: 4 Thr Arg Leu Glu Cys Phe Ser Ala Gly Trp Arg Leu Ser AlaSEQ ID NO: 4 Thr Arg Leu Glu Cys Phe Ser Ala Gly Trp Arg Leu Ser Ala
CysCys
SEQ ID NO: 5 Trp Tyr Ser Lys Asp Thr Ser Trp Phe Thr Leu Phe Leu ProSEQ ID NO: 5 Trp Tyr Ser Lys Asp Thr Ser Trp Phe Thr Leu Phe Leu Pro
ValVal
SEQ ID NO: 6 Thr Gly Gly Pro Leu Tyr Pro Phe Tyr Ala Leu Val Pro SerSEQ ID NO: 6 Thr Gly Gly Pro Leu Tyr Pro Phe Tyr Ala Leu Val Pro Ser
TyrTire
SEQ ID NO: 7 Asn Leu His Ser Lys Val Phe His Gly Leu Val Ile Arg GlySEQ ID NO: 7 Asn Leu His Ser Lys Val Phe His Gly Leu Val Ile Arg Gly
Gly et, SEQ ID NO: 8 Gin Ser Leu Cys Ser Pro Met Pro Leu Gly Ser Cys Ala Leu SerGly and, SEQ ID NO: 8 Gin Ser Leu Cys Ser Pro Met Pro Leu Gly Ser Cys Ala Leu Ser
Les séquences S selon l'invention peuvent être sélectionnées au moyen d'une banque de phages exprimant à leur surface une structure protéique de formule P-E-S dans laquelle P désigne la protéine PMI et/ou PVIII d'un phage filamenteux, E un bras espaceur et S un peptide aléatoire. De telles banques sont décrites dans les articles de Keller et al (Virology, 193, 709-716 (1993)), de Felici F. et al (J. Mol. Biol., 222, 301-310 (1991), de Scott et al (Science, 249, 386-390 (1990)) et de Schatz P. J. (Current Op. Biotech, 5: 487-494 (1994)). Les séquences S selon l'invention peuvent être sélectionnées selon la même méthodologie développée dans l'article de Barban V. et al. (Journal of General Virology, vol 81, 461-469 (2000)) en utilisant, comme anticorps pour la sélection des phages recombinants relevants, des anticorps monospécifiques de l'enveloppe du VIH qui ont été isolés par purification de sérums de souris SCID (Immunodéficience combinée sévère) qui ont été "humanisées" par injection intrapéritonéale de lymphocytes de sang périphérique humain provenant de sujets séropositifs asymptomatiques (Mosier et al., Science, 251 : 791-794, 1991).The S sequences according to the invention can be selected by means of a phage library expressing on their surface a protein structure of formula PES in which P denotes the PMI protein and / or PVIII of a filamentous phage, E a spacer arm and It's a random peptide. Such banks are described in the articles by Keller et al (Virology, 193, 709-716 (1993)), by Felici F. et al (J. Mol. Biol., 222, 301-310 (1991), by Scott et al (Science, 249, 386-390 (1990)) and Schatz PJ (Current Op. Biotech, 5: 487-494 (1994)). The S sequences according to the invention can be selected according to the same methodology developed in the article by Barban V. et al. (Journal of General Virology, vol 81, 461-469 (2000)) using, as antibodies for the selection of relevant recombinant phages, monospecific antibodies of the HIV envelope which have were isolated by purification from sera from SCID (severe combined immunodeficiency) mice which were "humanized" by intraperitoneal injection of human peripheral blood lymphocytes from asymptomatic seropositive subjects (Mosier et al., Science, 251: 791-794, 1991) .
Le bras espaceur E est constitué d'une séquence d'acides aminés identiques ou différents dont le rôle est de supprimer toute contrainte de structure potentiellement exercée par le porteur P sur la structure tridimensionnelle de la séquence S. Le bras espaceur selon l'invention comprend entre 2 et 25 acides aminés. La séquence en acides aminés est constituée principalement de glycine, proline ou alanine, dans la mesure où ces trois acides aminés représentent plus de 50% des acides aminés présents dans ladite séquence. A titre d'exemple, on pourra utiliser les "espaceurs naturels" riches en alanine et proline comme ceux décrits dans l'article de Qingcheng Mao et al. (Journal of biological chemistry, vol 270(31), 18295- 18300, 1995). A titre d'exemple on peut utiliser l'espaceur naturel ayant pour séquence la séquence en acides aminés suivante : Gly Ser Gly AlaThe spacer arm E consists of a sequence of identical or different amino acids whose role is to remove any structural constraint potentially exerted by the carrier P on the three-dimensional structure of the sequence S. The spacer arm according to the invention comprises between 2 and 25 amino acids. The amino acid sequence consists mainly of glycine, proline or alanine, insofar as these three amino acids represent more than 50% of the amino acids present in said sequence. As an example, we can use "natural spacers" rich in alanine and proline such as those described in the article by Qingcheng Mao et al. (Journal of biological chemistry, vol 270 (31), 18295-18300, 1995). By way of example, it is possible to use the natural spacer having as sequence the following amino acid sequence: Gly Ser Gly Ala
Ala Ala Pro Lys Ala Ala Pro Ala Leu Thr Pro Ala Asp Val Ala Arg Ser. De façon préférée, Le bras espaceur E est constitué uniquement de glycine et de façon encore plus préférée le bras est constitué de trois glycines. Dans le cadre de la présente invention, P désigne une protéine ou peptide porteur. On entend par "protéine ou peptide porteur", dans le cadre de la présente invention, toute protéine ou peptide capable de stabiliser la structure tridimensionnelle de la séquence S sous une conformation telle que l'antigène protéique soit capable d'induire des anticorps neutralisant des isolats primaires du VIH.Ala Ala Pro Lys Ala Ala Pro Ala Leu Thr Pro Ala Asp Val Ala Arg Ser. Preferably, the spacer arm E consists only of glycine and even more preferably the arm consists of three glycines. In the context of the present invention, P denotes a protein or peptide carrier. The term “carrier protein or peptide” is understood to mean, within the framework of the present invention, any protein or peptide capable of stabilizing the three-dimensional structure of the S sequence under a conformation such that the protein antigen is capable of inducing antibodies neutralizing primary HIV isolates.
De préférence, la protéine ou le peptide porteur renforce l'immunogénicité de la séquence S, notamment en augmentant la production d'anticorps spécifiques. La protéine ou le peptide comprend de préférence un ou plusieurs épitope(s) T helper. Par "épitope T helper", on entend un enchaînement d'acides aminés qui, dans le contexte d'une ou plusieurs molécules du MHC classe II, active les lymphocytes T helper.Preferably, the carrier protein or peptide enhances the immunogenicity of the S sequence, in particular by increasing the production of specific antibodies. The protein or peptide preferably comprises one or more T helper epitope (s). By "helper T epitope" is meant a chain of amino acids which, in the context of one or more MHC class II molecules, activates helper T lymphocytes.
Les effets de stabilisation et d'immunogenicite accrue sont obtenus lorsque la séquence S est liée à l'extrémité C ou N terminale de la protéine ou du peptide P via un bras espaceur E tel que défini ci dessus.The stabilization and increased immunogenicity effects are obtained when the S sequence is linked to the C or N terminus of the protein or of the P peptide via a spacer arm E as defined above.
Comme protéine porteuse, on peut utiliser les protéines de surface de phages comme la protéine plll ou pVIII du phage M13, les protéines de surface bactériennes comme les protéines LambB, OmpC, OmpA, OmpF, et PhoE d'Escherichia coli, la protéine CotC ou CotD de Bacillus subtilis, les porines bactériennes comme la porineAs carrier protein, it is possible to use the phage surface proteins such as the pl11 or pVIII protein of phage M13, the bacterial surface proteins such as the LambB proteins, OmpC, OmpA, OmpF, and PhoE of Escherichia coli, the CotC protein or CotD of Bacillus subtilis, bacterial porins like porin
P1 de Neisseria gonorrheae, la porine P1 ou P2 d'Haemophilus influenzae B, la porine de classe I de Neisseria meningitidis B, la porine P40 de Klebsiella pneumoniae; également les lipoprotéines comme OspA de Borrelia bugdorfi, PspA de Streptococcus pneumoniae, la Transferrine binding protein type 2 (TBP2) de Neisseria meningitidis B, TraT d'Escherichia coli, l'adhésine A de Streptococcus pneumoniae.P1 of Neisseria gonorrheae, porin P1 or P2 of Haemophilus influenzae B, porin of class I of Neisseria meningitidis B, porin P40 of Klebsiella pneumoniae; also lipoproteins such as OspA from Borrelia bugdorfi, PspA from Streptococcus pneumoniae, Transferrin binding protein type 2 (TBP2) from Neisseria meningitidis B, TraT from Escherichia coli, adhesin A from Streptococcus pneumoniae.
Sont également particulièrement appropriées dans le cadre de la présente invention, les toxines bactériennes détoxifiées comme l'anatoxine tétanique ou diphtérique, la sous unité B de la toxine cholérique ou la sous unité B de l'entérotoxine thermolabile d'Escherichia coli, l'exotoxine A de Pseudomonas aeruginosa, ou l'exotoxine A de Staphylococcus aureus.Are also particularly suitable in the context of the present invention, detoxified bacterial toxins such as tetanus or diphtheria toxoid, the subunit B of cholera toxin or the subunit B of the heat-labile enterotoxin of Escherichia coli, exotoxin A from Pseudomonas aeruginosa, or the exotoxin A from Staphylococcus aureus.
Enfin, les "heat shock protein", comme Hsp 65 ou hsp 71 de Mycobacterium tuberculosis ou bovis, ou Hin 47 d' Haemophilus influenzae type B sont d'autres types de protéines porteuses utilisables dans le cadre de l'invention.Finally, "heat shock protein", such as Hsp 65 or hsp 71 from Mycobacterium tuberculosis or bovis, or Hin 47 from Haemophilus influenzae type B are other types carrier proteins usable in the context of the invention.
Comme peptide porteur on peut citer à titre d'exemple non limitatif les peptides p24E, p24N, p24H, p24M, p24H décrits dans WO 94/29339 portant un épitope T helper, ou les peptides PADRE ( Pan DR T helper epitopes) décrits par Del guercio et al (Vaccine et al., (1997), Vol 15/4, p 441-448).As carrier peptide, non-limiting examples include the peptides p24E, p24N, p24H, p24M, p24H described in WO 94/29339 carrying a T helper epitope, or the PADRE peptides (Pan DR T helper epitopes) described by Del guercio et al (Vaccine et al., (1997), Vol 15/4, p 441-448).
Les constituants P, E et S de l'antigène selon l'invention sont liés entre eux par une liaison peptidique entre les groupements COOH et NH2 présents aux extrémités N ou C terminales des constituants de sorte à former une structure de formule P-E-S. Les constituants P, E et S de l'antigène selon l'invention peuvent également être combinés de sorte à former une structure de type S-E-P-E-S dans laquelle les bras espaceurs E sont identiques ou différents et les séquences S sont identiques ou différentes.The constituents P, E and S of the antigen according to the invention are linked to each other by a peptide bond between the COOH and NH 2 groups present at the N or C terminal ends of the constituents so as to form a structure of formula P-E-S. The constituents P, E and S of the antigen according to the invention can also be combined so as to form a structure of type S-E-P-E-S in which the spacer arms E are identical or different and the sequences S are identical or different.
L'antigène selon l'invention peut être obtenu par toute technique classique de synthèse chimique ou de génie génétique.The antigen according to the invention can be obtained by any conventional technique of chemical synthesis or genetic engineering.
Lorsque l'antigène est produit par synthèse chimique, l'antigène selon la présente invention peut, soit être synthétisé sous la forme d'une séquence unique, soit sous la forme de plusieurs séquences qui sont ensuite liées les unes aux autres. Dans le cas d'antigènes de grande taille (> 50 acides aminés), les composantsWhen the antigen is produced by chemical synthesis, the antigen according to the present invention can either be synthesized in the form of a single sequence, or in the form of several sequences which are then linked to each other. In the case of large antigens (> 50 amino acids), the components
P, E, S sont de préférence synthétisés séparément puis liés par tout procédé de couplage chimique approprié. La synthèse chimique peut être réalisée en phase solide ou en solution, ces deux techniques de synthèse étant bien connues de l'homme de l'art. Ces techniques sont décrites notamment par Atherton et Shepard dans "solid phase peptide synthesis" (IRL Press, oxford, 1989), et par Houbenweyl dans "Méthode der organischen chemie" éditée par E Wunsch, vol 15-1 et II- Thieme, Stuttgart, 1974.P, E, S are preferably synthesized separately and then linked by any suitable chemical coupling process. Chemical synthesis can be carried out in solid phase or in solution, these two synthesis techniques being well known to those skilled in the art. These techniques are described in particular by Atherton and Shepard in "solid phase peptide synthesis" (IRL Press, oxford, 1989), and by Houbenweyl in "Method der organischen chemie" edited by E Wunsch, vol 15-1 and II-Thieme, Stuttgart , 1974.
L'antigène peut également être produit par les techniques de génie génétique bien connues de l'homme de l'art. Ces techniques sont décrites en détail dans Molecular Cloning: a molecular manual de Maniatis et al. Dans ce cas, on prépare classiquement un insert comprenant :The antigen can also be produced by genetic engineering techniques well known to those skilled in the art. These techniques are described in detail in Molecular Cloning: a molecular manual by Maniatis et al. In this case, an insert is conventionally prepared comprising:
Une séquence d'oligonucléotides codant pour un premier site de restriction, Une séquence d'oligonucléotides codant pour le bras espaceur E, Une séquence d'oligonucléotides codant pour la séquence S, et Une séquence d'oligonucléotides codant pour un deuxième site de restriction qui peut être identique au premierAn oligonucleotide sequence coding for a first restriction site, An oligonucleotide sequence coding for the spacer arm E, An oligonucleotide sequence encoding the sequence S, and An oligonucleotide sequence encoding a second restriction site which may be identical to the first
Cet insert d'oligonucléotides est synthétisé à l'aide d'un synthétiseur d'oligonucléotides. Il est ensuite inséré dans un vecteur d'expression contenant le gène codant pour la protéine P. L'insert oligonucléotidique est inséré en phase, avec le cadre de lecture du gène codant pour ladite protéine. Dans le cas des protéines P comprenant une séquence signal, l'insertion se fait immédiatement après la séquence signal. Cette séquence signal correspond, soit à une séquence présente naturellement dans la protéine, soit à une séquence signal rajoutée, telle que ompA, lamB, etc.. pour faciliter les étapes de purification de l'antigène. Pour incorporer l'insert juste après la séquence signal, il est parfois nécessaire d'introduire un nouveau site de restriction immédiatement après la séquence signal ce qui se fait par mutagénèse dirigée du gène. On obtient ainsi l'expression de l'antigène selon l'invention dans lequel la séquence E-S est liée à l'une des extrémités terminales de la protéine P. Tout système d'expression vecteur/ cellule hôte décrit dans l'art antérieur pour la production de protéines recombinantes peut être utilisé dans le cadre de la présente invention.This oligonucleotide insert is synthesized using an oligonucleotide synthesizer. It is then inserted into an expression vector containing the gene coding for the protein P. The oligonucleotide insert is inserted in phase, with the reading frame of the gene coding for the said protein. In the case of P proteins comprising a signal sequence, the insertion takes place immediately after the signal sequence. This signal sequence corresponds either to a sequence naturally present in the protein, or to an added signal sequence, such as ompA, lamB, etc. to facilitate the steps of purification of the antigen. To incorporate the insert just after the signal sequence, it is sometimes necessary to introduce a new restriction site immediately after the signal sequence, which is done by site-directed mutagenesis of the gene. The expression of the antigen according to the invention is thus obtained in which the ES sequence is linked to one of the terminal ends of the protein P. Any vector / host cell expression system described in the prior art for the production of recombinant proteins can be used in the context of the present invention.
Selon un mode de réalisation préféré, le vecteur d'expression est un phage, en particulier un phage filamenteux dérivé du phage M13 ou fd dans lequel l'insert oligonucléotidique est inséré derrière la séquence signal du gène codant pour la (les) protéine(s) pi II et/ou pVIII du phage. Au besoin, on introduit un site d'insertion immédiatement après la séquence signal de la protéine par mutagénèse dirigée pour pouvoir introduire l'insert oligonucléotidique. On peut également utiliser des mutants viables du phage M13 ou fd dans lesquels de nouveaux sites de restriction ont été créés dans le gène plll et/ou PVIII. A titre d'exemple on peut citer le phage fuse 2 ou fuse 5 ou les mutants viables cités dans l'article de lannolo et al. (J. Mol. Biol. (1995)According to a preferred embodiment, the expression vector is a phage, in particular a filamentous phage derived from phage M13 or fd in which the oligonucleotide insert is inserted behind the signal sequence of the gene coding for the protein (s) ) pi II and / or pVIII of the phage. If necessary, an insertion site is introduced immediately after the signal sequence of the protein by site-directed mutagenesis in order to be able to introduce the oligonucleotide insert. It is also possible to use viable mutants of phage M13 or fd in which new restriction sites have been created in the p111 and / or PVIII gene. By way of example, mention may be made of the phage fuse 2 or fuse 5 or the viable mutants mentioned in the article by lannolo et al. (J. Mol. Biol. (1995)
248, 835-844). En utilisant ce type de vecteur, la séquence S est reliée à l'extrémité248, 835-844). Using this type of vector, the S sequence is linked at the end
N terminale de la protéine plll et/ou PVIII au moyen d'un bras espaceur. Dans une telle construction, l'antigène est avantageusement exprimé à la surface du phage.N terminal of the plll and / or PVIII protein by means of a spacer arm. In such a construction, the antigen is advantageously expressed on the surface of the phage.
L'antigène ainsi produit est ensuite purifié par tout procédé classique de purification.The antigen thus produced is then purified by any conventional purification process.
Lorsque l'antigène est exprimé à la surface du phage ou de la bactérie, il est dans un premier temps extrait sous l'action d'un milieu légèrement hyperosmotique renfermant des inhibiteurs calciques (tels que EDTA), et des détergents légers (Tween® 20, triton). Les protéines de surface sont alors relarguées dans le milieu tandis que les autres constituants solides comme le core de la bactérie ou du phage précipitent par centrifugation. L'antigène récupéré dans le surnageant est purifié de ses autres contaminants à l'aide des techniques chromatographiques bien connues de l'homme du métier. Lorsque l'antigène est sécrété dans le milieu extracellulaire, il est directement récupéré dans le milieu de culture et purifié à l'aide de techniques chromatographiques usuelles comme cela est décrit dans l'article de Philippe Slos et al (Protein expression and purification, 5, 518-526 (1994)).When the antigen is expressed on the surface of the phage or the bacteria, it is first extracted under the action of a slightly hyperosmotic medium containing calcium channel blockers (such as EDTA), and light detergents (Tween® 20, newt). The surface proteins are then released into the medium while the other solid constituents such as the core of the bacteria or of the phage precipitate by centrifugation. The antigen recovered in the supernatant is purified from its other contaminants using chromatographic techniques well known to those skilled in the art. When the antigen is secreted into the extracellular medium, it is directly recovered from the culture medium and purified using standard chromatographic techniques as described in the article by Philippe Slos et al (Protein expression and purification, 5 , 518-526 (1994)).
Plutôt que d'extraire et de purifier l'antigène exprimé à la surface du vecteur d'expression, il est souvent plus facile et parfois -plus avantageux d'utiliser le vecteur d'expression lui même. Selon un autre aspect, l'invention a donc pour objet tout vecteur d'expression exprimant un antigène tel que défini ci-dessus. On entend par "vecteur d'expression" dans le cadre de la présente invention tout vecteur comprenant une cassette d'expression contenant la séquence codant pour l'antigène protéique selon l'invention et des séquences régulatrices permettant l'expression de ladite séquence. Cette définition englobe donc les plasmides, les phages, ainsi que les virus. Tout vecteur approprié pour l'expression d'une protéine recombinante peut être utilisé dans le cadre de la présente invention.Rather than extracting and purifying the antigen expressed on the surface of the expression vector, it is often easier and sometimes more advantageous to use the expression vector itself. According to another aspect, the invention therefore relates to any expression vector expressing an antigen as defined above. The term “expression vector” is understood to mean, within the context of the present invention, any vector comprising an expression cassette containing the sequence coding for the protein antigen according to the invention and regulatory sequences allowing the expression of said sequence. This definition therefore includes plasmids, phages, as well as viruses. Any vector suitable for the expression of a recombinant protein can be used in the context of the present invention.
Selon un mode de réalisation, les vecteurs d'expression sont de préférence des virus ou des phages. Le vecteur d'expression est de préférence un phage filamenteux recombinant exprimant de multiples exemplaires de l'antigène selon l'invention dans lequel la protéine P est représentée par la protéine plll et/ou PVIII de préférence pVIII. Ceci résulte du fait que les protéines plll et pVIII, sont présentes naturellement à la surface du phage sous forme d'unités répétitives (5 pour la protéine plll, plus de 2000 pour la protéine pVIII). L'expression d'exemplaires multiples de l'antigène à la surface du phage recombinant renforce le pouvoir immunogène de l'antigène.According to one embodiment, the expression vectors are preferably viruses or phages. The expression vector is preferably a recombinant filamentous phage expressing multiple copies of the antigen according to the invention in which the protein P is represented by the protein pl11 and / or PVIII preferably pVIII. This results from the fact that the proteins plll and pVIII are present naturally on the surface of the phage in the form of repeating units (5 for the protein plll, more than 2000 for the protein pVIII). Expression of multiple copies of the antigen on the surface of the recombinant phage enhances the immunogenicity of the antigen.
La présente invention a également pour objet des vaccins anti VIH à des fins thérapeutiques ou prophylactiques. Les vaccins selon la présente invention comprennent au moins un antigène selon la présente invention en combinaison avec un support ou diluant pharmaceutiquement acceptable et éventuellement un adjuvant. Le vaccin selon la présente invention peut donc contenir ou conduire à l'expression d'un seul type d'antigène protéique ou d'un mélange de divers types d'antigènes selon l'invention. Dans un tel cas de figure, ces antigènes peuvent se différencier par exemple par la nature de la séquence S ou par la nature de la protéine porteur. Selon un mode de réalisation préféré, le vaccin selon la présente invention comprend un vecteur d'expression tel que défini ci-dessus. Ce vecteur d'expression est avantageusement un phage, en particulier un phage filamenteux. Un vaccin comprenant un phage filamenteux recombinant exprimant à sa surface de multiples exemplaires d'un antigène de formule P-E-S dans lequel P représente la protéine plll et/ou pVIII, de préférence pVIII, est le mode de réalisation préféré selon l'invention.The present invention also relates to anti-HIV vaccines for therapeutic or prophylactic purposes. The vaccines according to the present invention comprise at least one antigen according to the present invention in combination with a pharmaceutically acceptable carrier or diluent and optionally an adjuvant. The vaccine according to the present invention can therefore contain or lead to the expression of a single type of protein antigen or of a mixture of various types of antigens according to the invention. In such a scenario, these antigens can be differentiated, for example, by the nature of the S sequence or by the nature of the carrier protein. According to a preferred embodiment, the vaccine according to the present invention comprises an expression vector as defined above. This expression vector is advantageously a phage, in particular a filamentous phage. A vaccine comprising a recombinant filamentous phage expressing on its surface multiple copies of an antigen of formula PES in which P represents the protein pl11 and / or pVIII, preferably pVIII, is the preferred embodiment according to the invention.
La quantité d'antigène dans le vaccin selon la présente invention dépend de nombreux paramètres comme le comprend l'homme de l'art, tels que la nature de l'antigène, le taux d'expression de l'antigène par le vecteur d'expression, la voie d'administration, et l'état physique de la personne à traiter (poids, âge, sexe, état clinique). Il est également tenu compte de la masse relative de la protéine P par rapport à la séquence S.The amount of antigen in the vaccine according to the present invention depends on many parameters as understood by those skilled in the art, such as the nature of the antigen, the level of expression of the antigen by the vector. expression, route of administration, and physical condition of the person to be treated (weight, age, gender, clinical condition). The relative mass of the P protein relative to the S sequence is also taken into account.
Compte tenu de tous ces facteurs qui sont connus et appréciés par l'homme du métier, les quantités d'antigène administrées peuvent atteindre 1 à 10mg mais plus généralement se situent entre 5μg et 1mg, de préférence 50 à 500μg par injection.Given all of these factors which are known and appreciated by those skilled in the art, the amounts of antigen administered can reach 1 to 10 mg but more generally lie between 5 μg and 1 mg, preferably 50 to 500 μg per injection.
Lorsque le vaccin renferme un vecteur d'expression selon l'invention, celui-ci est utilisé soit comme immunogène en tant que tel, soit comme agent de transfection des cellules. Un phage est administré en général à une dose minimale de 103,5 unités infectantes (pfu ou unité formant plage) mais de façon préférentielle dans une échelle de doses allant de 10 à 1012 pfu et de façon encore plus préférée dans une échelle de doses allant de 106 à 1010 pfu. L'homme de l'art peut se référer aux protocoles et essais cliniques utilisant des préparations à base de vecteurs recombinants, notamment les pox virus recombinants, les adenovirus recombinants, déjà réalisés chez l'homme pour convenir du nombre approprié de pfu que doit renfermer le vaccin. Lorsque le vaccin contient un plasmide recombinant selon l'invention, les doses injectées peuvent être très variables allant de 1 μg à 100mg, mais de façon préférentielle entre 0,1 mg à 10mg . L'homme de l'art peut également se référer aux protocoles et essais cliniques déjà réalisés chez l'homme, concernant des préparations d'ADN plasmidique pour convenir de la dose appropriée de plasmides. Lorsque le vaccin contient un vecteur d'expression tel qu'un phage recombinant, un virus recombinant, le pouvoir infectant potentiel de ces microorganismes recombinants est en général neutralisé en utilisant des procédés qui n'altèrent pas la conformation de l'antigène selon l'invention. Par exemple, lorsqu'on administre une suspension de phages recombinants on a recours en général à une irradiation préalable de la suspension de phages pour faire chuter le titre infectieux.When the vaccine contains an expression vector according to the invention, this is used either as an immunogen as such, or as a cell transfection agent. A phage is generally administered at a minimum dose of 10 3.5 infecting units (pfu or range forming unit) but preferably in a dose scale ranging from 10 to 10 12 pfu and even more preferably in a scale of doses ranging from 10 6 to 10 10 pfu. Those skilled in the art can refer to clinical trials and protocols using preparations based on recombinant vectors, in particular recombinant pox viruses, recombinant adenoviruses, already carried out in humans to agree on the appropriate number of pfu that must contain the vaccine. When the vaccine contains a recombinant plasmid according to the invention, the doses injected can be very variable ranging from 1 μg to 100 mg, but preferably between 0.1 mg to 10 mg. Those skilled in the art can also refer to clinical trials and protocols already performed in humans, relating to plasmid DNA preparations to agree on the appropriate dose of plasmids. When the vaccine contains an expression vector such as a recombinant phage, a recombinant virus, the potential infectious power of these recombinant microorganisms is generally neutralized by using methods which do not alter the conformation of the antigen according to the invention. For example, when administering a suspension of recombinant phages, recourse is generally made to prior irradiation of the phage suspension in order to reduce the infectious titer.
Le vaccin selon l'invention peut également contenir un ou plusieurs adjuvants ou être incorporé dans des systèmes de délivrance compatibles avec' un usage en médecine humaine. On peut rapporter notamment l'usage des adjuvants comme l'Alum ( phosphate d'aluminium phosphate ou hy'droxyde d'aluminium ou le mélange des deux) incorporé de façon classique dans les vaccins, l'adjuvant incomplet de Freund, le lipide A monophosphorylé (MPL), QS21 , Polyphosphazène, muramyl dipeptide (MDP) ou ses dérivés, l'usage de systèmes de délivrance de l'antigène comme les émulsions (MF59, SAF1 , RIBI, SB 62, SB 26), les ISCOMS, les liposomes, les microsphères composées de polymères de PLGA de diamètre bien calibré, ou éventuellement les pseudo virions.The vaccine according to the invention may also contain one or more adjuvants or be incorporated into delivery systems compatible with use in human medicine. Mention may report the use of adjuvants such as alum (aluminum phosphate or phosphate hy 'droxyde aluminum or mixture thereof) conventionally incorporated in vaccines, incomplete Freund's adjuvant, lipid A monophosphorylated (MPL), QS21, Polyphosphazene, muramyl dipeptide (MDP) or its derivatives, the use of antigen delivery systems such as emulsions (MF59, SAF1, RIBI, SB 62, SB 26), ISCOMS, liposomes, microspheres composed of PLGA polymers of well-calibrated diameter, or possibly pseudo virions.
Les vaccins selon la présente invention peuvent être préparés par tout procédé classique connu de l'homme de l'art. Classiquement, les antigènes sont mélangés avec un support ou un diluant pharmaceutiquement acceptable tel que eau ou solution saline tamponnée ou phosphate. Le support ou diluant est sélectionné en fonction de la forme galénique choisie, du mode d'administration et de la voie d'administration ainsi que de la pratique pharmaceutique. Les supports ou diluants appropriés ainsi que les exigences en matière de formulation pharmaceutique sont décrits en détail dans le Remington's Pharmaceutical Science, représentant un ouvrage de référence dans ce domaine. Les vaccins mentionnés ci-dessus peuvent être administrés par toute voie classique, habituellement utilisée dans le domaine des vaccins, telle que la voie parentérale (intraveineuse, intramusculaire, sous cutanée, etc.). Le vaccin selon l'invention peut également être administré par la voie muqueuse. L'administration peut être réalisée par injection d'une dose unique ou de doses répétées. Pour la prévention de l'infection à VIH, le vaccin peut être administré en une seule fois ou à plusieurs reprises de façon à obtenir un niveau de réponse immune qui assure la protection de l'individu vis à vis d'une contamination accidentelle. Dans le cas d'injections répétées, le protocole de vaccination comprend de façon préférentielle des injections à JO, à 1 mois, à 3 mois et à 6 mois. On a parfois recours à des vaccins différents pour les différentes injections. Il peut être également nécessaire pour maintenir le niveau de protection souhaité de pratiquer des injections de rappel à intervalles réguliers.The vaccines according to the present invention can be prepared by any conventional method known to those skilled in the art. Conventionally, the antigens are mixed with a pharmaceutically acceptable carrier or diluent such as water or buffered saline or phosphate. The carrier or diluent is selected according to the dosage form chosen, the mode of administration and the route of administration as well as pharmaceutical practice. The appropriate carriers or diluents as well as the pharmaceutical formulation requirements are described in detail in Remington's Pharmaceutical Science, which represents a reference work in this field. The vaccines mentioned above can be administered by any conventional route, usually used in the field of vaccines, such as the parenteral route (intravenous, intramuscular, subcutaneous, etc.). The vaccine according to the invention can also be administered by the mucosal route. Administration can be by injection of a single dose or repeated doses. For the prevention of HIV infection, the vaccine can be administered all at once or several times in order to obtain a level of immune response which ensures the protection of the individual against accidental contamination. In the case of repeated injections, the vaccination protocol preferably includes OJ injections, at 1 month, 3 months and 6 months. Sometimes different vaccines are used for different injections. To maintain the desired level of protection, it may also be necessary to give booster shots at regular intervals.
Pour le traitement de l'infection liée au VIH, le vaccin est administré en une seule fois ou à plusieurs reprises. Le délai entre chaque injection peut être très court, inférieur à une semaine, pour atteindre le niveau de réponse désirée, notamment celui qui permet de constater l'absence du virus VIH dans le sang par le test PCR. Au besoin, le vaccin thérapeutique peut être associé ou utilisé en alternance avec les traitements conventionnels de cette affection, comprenant notamment les mono, bi ou tri thérapie antivirale.For the treatment of HIV infection, the vaccine is given all at once or several times. The time between each injection can be very short, less than a week, to reach the desired level of response, in particular that which makes it possible to note the absence of the HIV virus in the blood by the PCR test. If necessary, the therapeutic vaccine can be combined or used alternately with conventional treatments for this condition, including in particular mono, bi or tri antiviral therapy.
Que ce soit pour la prévention ou le traitement de l'infection à VIH, on peut aussi recourir à un vaccin comprenant un ou plusieurs antigènes et/ou un ou plusieurs vecteurs d'expression selon l'invention pour stimuler in vitro ou ex vivo les cellules du système immunitaire du patient et les ré-injecter ensuite dans l'organisme de l'individu.Whether for the prevention or treatment of HIV infection, it is also possible to use a vaccine comprising one or more antigens and / or one or more expression vectors according to the invention to stimulate in vitro or ex vivo cells of the patient's immune system and then re-inject them into the individual's body.
L'invention a enfin pour objet l'utilisation d'un antigène ou d'un vecteur d'expression selon l'invention pour le diagnostic de l'infection à VIH ou pour suivre l'évolution de cette infection. On utilisera de préférence des méthodes immunoenzymatiques, radioimmunologiques, ou de western blotting bien connues de l'homme du métier, comme par exemple les méthodes ELISA, RIA, RIPA ou IRMA pour l'analyse de la réponse humorale. On peut également recourir aux méthodes utilisant le Biacore® qui permettent d'identifier les anticorps de haute affinité.The invention finally relates to the use of an antigen or an expression vector according to the invention for the diagnosis of HIV infection or for monitoring the progress of this infection. Preferably, immunoenzymatic, radioimmunological or western blotting methods well known to those skilled in the art will be used, such as for example the ELISA, RIA, RIPA or IRMA methods for the analysis of the humoral response. One can also use methods using Biacore® which identify high affinity antibodies.
Sans avoir de caractère limitatif, la présente invention sera mieux comprise à la lecture des exemples suivants : Exemple 1 : synthèse par génie génétique de phages recombinants exprimant à leur surface des antigènes protéiαues P-E-S.Without being limiting in nature, the present invention will be better understood on reading the following examples: Example 1: Synthesis by genetic engineering of recombinant phages expressing on their surface protein antigens PES.
Deux populations de phages recombinants exprimant à leur surface des antigènes protéiques P-E-S ont été obtenues. La première population est représentée par des phages recombinants pour lesquels l'une des séquences S, SEQ ID NO1 à SEQ ID N08 est reliée par l'intermédiaire d'un bras espaceur constitué de 3 glycines à l'extémité N terminale de la protéine Plll (phages recombinants plll). La deuxième population est représentée par des phages recombinants pour lesquels l'une des séquences S, SEQ ID NO1 à SEQ ID NO8 est reliée par l'intermédiaire d'un bras espaceur constitué de 3 glycines à l'extémité N terminale de la protéine PVIII (phages recombinants pVIII)Two populations of recombinant phages expressing P-E-S protein antigens on their surface were obtained. The first population is represented by recombinant phages for which one of the sequences S, SEQ ID NO1 to SEQ ID N08 is linked via a spacer arm consisting of 3 glycines at the N terminal end of the Plll protein (recombinant phages plll). The second population is represented by recombinant phages for which one of the sequences S, SEQ ID NO1 to SEQ ID NO8 is linked via a spacer arm consisting of 3 glycines at the N terminal end of the PVIII protein (recombinant phages pVIII)
1.1 ) Synthèse des phages recombinants plll L'oligonucléotide codant pour le bras espaceur constitué de 3 glycines et l'une des séquences spécifiques SEQ ID NO:1 à SEQ ID NO:8 est synthétisé par voie chimique puis inséré immédiatement après la séquence signal du gène codant pour la protéine plll du phage fd selon la méthodologie décrite dans le chapitre intitulé "Construction of Random peptide Libraries in Bacteriophage M13 (Niels B. Adey, Andrew B Sparks, Jim Beasiy and Bryan K. Kay)" tiré du manuel " Phage display of Peptides and Proteins: a laboratory manual, edited by Bryan me Kay, Jill Winter and John Me Cafferty -Académie Press, Harcourt Brace and Company publishers. On a obtenu ainsi 8 populations de phages recombinants plll se distinguant par la séquence S qui est exprimée.1.1) Synthesis of the recombinant phages pl11 The oligonucleotide coding for the spacer arm consisting of 3 glycines and one of the specific sequences SEQ ID NO: 1 to SEQ ID NO: 8 is synthesized chemically and then inserted immediately after the signal sequence of the gene coding for the pld protein of phage fd according to the methodology described in the chapter entitled "Construction of Random peptide Libraries in Bacteriophage M13 (Niels B. Adey, Andrew B Sparks, Jim Beasiy and Bryan K. Kay)" taken from the manual "Phage display of Peptides and Proteins: a laboratory manual, edited by Bryan me Kay, Jill Winter and John Me Cafferty -Académie Press, Harcourt Brace and Company publishers. We thus obtained 8 populations of recombinant phages plll distinguished by the sequence S which is expressed.
1.2) Sous-clonaαe des séquences spécifiques dans un autre phage1.2) Subclonaαe specific sequences in another phage
Les oligonucléotides synthétisés dans l'exemple 1.1 ont été sous clones dans un autre phage dénommé phage fd AMPLAY88 dont les caractéristiques sont décrites dans l'article de Malik P et al. (Nucleic Acid Research , vol 25 (4), p915- 917, (1997). On a obtenu ainsi des populations de phages recombinants pVIII se distinguant entre eux par la séquence S exprimée, avec l'avantage que les populations de phages recombinants pVIII expriment plus de séquences spécifiques S que les populations de phages recombinants plll. Exemole 2 : Synthèse par voie chimique d'antigènes P-E-SThe oligonucleotides synthesized in Example 1.1 were subcloned into another phage called phage fd AMPLAY88, the characteristics of which are described in the article by Malik P et al. (Nucleic Acid Research, vol 25 (4), p915-917, (1997). This gave populations of recombinant phage pVIII distinguished from each other by the expressed S sequence, with the advantage that populations of recombinant phage pVIII express more S-specific sequences than the P1111 recombinant phage populations. Example 2: Chemical synthesis of PES antigens
Les séquences' d'acides aminés SEQ ID NO:1 à SEQ ID NO:8 ont été synthétisées en phase solide en se référant aux méthodologies développées dans les ouvrages Solid phase peptide synthesis: a practical approach, IRL Press, Oxford, 1989 et Solid phase peptide synthesis, second édition, publié par Pierce Chemical Company, 1984. Ces synthèses ont été poursuivies de façon à ce que l'extrémité C ou N terminale des séquences S soit liée, soit à un bras espaceur ayant une structure répétée du type (Gly-)x dans laquelle x est compris entre 2 et 10, soit à un bras espaceur ayant une structure répétée du type (Gly-pro-)y dans laquelle y est compris entre 1 et 5 de façon à obtenir un peptide de formule E-S.The sequences of amino acids SEQ ID NO: 1 to SEQ ID NO: 8 were synthesized by solid-phase with reference to the methodologies developed in the literature Solid phase peptide synthesis: a practical approach, IRL Press, Oxford, 1989 and Solid peptide synthesis phase, second edition, published by Pierce Chemical Company, 1984. These syntheses were continued so that the C or N terminal end of the S sequences is linked, either to a spacer arm having a repeated structure of the type ( Gly-) x in which x is between 2 and 10, that is to say a spacer arm having a repeated structure of the type (Gly-pro-) y in which y is between 1 and 5 so as to obtain a peptide of formula ES .
La fonction aminée des acides aminés est protégée par introduction d'un groupement t-butyloxycarbonyl (t-boc) permettant ainsi un couplage par la fonction carboxylique de l'acide aminé à une résine chlorométhylée active. Après fixation sur la résine, la fonction aminé est "déprotégée" par action de l'acide trifluoroacétique suivi d'une étape de neutralisation par la triethylamine. La fonction aminé ainsi libérée subit ensuite une réaction de couplage avec un autre acide aminé sous forme de dérivé t-boc par l'intermédiaire de carbodiimides. Ce procédé est mis en œuvre par l'automate ABI® (Applied Biosystem Inc) 430A qui réalise ainsi la synthèse automatique de peptides. A la fin de la synthèse, le peptide est décroché de la résine par action de l'acide fluorhydrique. L'extrait est ensuite purifié par HPLC en phase réverse en utilisant une colonne semi préparative de type Vydac® C4 et un gradient d'acétonitrile allant de 15 à 55% dans une solution d'acide trifuoroacétique à 0,1 %. La chromatographie liquide est programmée pour une période de 40 min avec un débit de 2 ml/min. Le taux de pureté des peptides E-S a été contrôlé par chromatographie analytique et dépasse 95%.The amino function of the amino acids is protected by the introduction of a t-butyloxycarbonyl (t-boc) group, thus allowing coupling, by the carboxylic function of the amino acid, to an active chloromethylated resin. After fixation on the resin, the amino function is "deprotected" by the action of trifluoroacetic acid followed by a neutralization step with triethylamine. The amino function thus released then undergoes a coupling reaction with another amino acid in the form of a t-boc derivative via carbodiimides. This process is implemented by the ABI® automaton (Applied Biosystem Inc) 430A which thus performs the automatic synthesis of peptides. At the end of the synthesis, the peptide is detached from the resin by the action of hydrofluoric acid. The extract is then purified by reverse phase HPLC using a semi-preparative column of Vydac® C4 type and a gradient of acetonitrile ranging from 15 to 55% in a 0.1% trifuoroacetic acid solution. Liquid chromatography is programmed for a period of 40 min with a flow rate of 2 ml / min. The purity rate of the E-S peptides was checked by analytical chromatography and exceeds 95%.
Le peptide E-S a été ensuite couplé à l'extrémité C terminal ou N terminal de l'anatoxine tétanique purifiée . On a obtenu en définitive une série d'antigènes P-E-S dans lesquelsThe E-S peptide was then coupled to the C terminal or N terminal end of the purified tetanus toxoid. In the end, we obtained a series of P-E-S antigens in which
P est représenté par l'anatoxine tétaniqueP is represented by tetanus toxoid
E est représenté par une structure chimique répétée de type (Gly-)x dans laquelle x est compris entre 2 et 10 ou de type (Gly-pro-)y dans laquelle y est compris entre 1 et 5.E is represented by a repeated chemical structure of type (Gly-) x in which x is between 2 and 10 or of type (Gly-pro-) y in which y is between 1 and 5.
S est représenté par l'une des séquences ID NO1 à ID NO8S is represented by one of the sequences ID NO1 to ID NO8
Exemple 3 : Synthèse par génie génétigue d'antigènes de formule chimigue P-E-SExample 3 Synthesis by Genetic Engineering of Antigens of the Chemical Formula P-E-S
Le protocole opératoire suivi est similaire à celui décrit dans l'article de Philippe Slos et al (Protein expression and purification, 5, 518-526 (1994)).The operating protocol followed is similar to that described in the article by Philippe Slos et al (Protein expression and purification, 5, 518-526 (1994)).
On a incorporé deux sites de restriction aux deux extrémités d'un oligonucleotide constitué d'une séquence spécifique S codant pour l'une des séquences ID NO1 à ID NO8, et d'une séquence codant pour un bras espaceur E constitué d'un enchaînement de 3 à 10 résidus glycine. L'oligonucléotide a été ensuite incorporé immédiatement après la séquence signal de la sous unité B de la toxine cholérique. On a ensuite remplacé la séquence signal de la sous unité B de la toxine cholérique par la séquence signal ompA avant incorporation dans un vecteur plasmidique, type pARA 13 sous la dépendance de l'opéron arabinose de Salmonella typhimurium. Enfin on a transformé une souche d'Escherichia coli avec ce plasmide, qui produit alors directement dans un milieu de culture renfermant de l'arabinose, une quantité importante de l'antigène P-E-S dans lequel P est représenté par la sous unité B de la toxine cholériqueTwo restriction sites were incorporated at the two ends of an oligonucleotide consisting of a specific sequence S coding for one of the sequences ID NO1 to ID NO8, and of a sequence coding for a spacer arm E consisting of a sequence from 3 to 10 glycine residues. The oligonucleotide was then incorporated immediately after the signal sequence of the cholera toxin subunit B. The signal sequence of the cholera toxin subunit B was then replaced by the signal sequence ompA before incorporation into a plasmid vector, type pARA 13 under the dependence of the arabinose operon of Salmonella typhimurium. Finally, a strain of Escherichia coli was transformed with this plasmid, which then produces directly, in a culture medium containing arabinose, a large quantity of the PES antigen in which P is represented by the B subunit of the toxin. choleraic
E est représenté par une structure chimique répétée de type (Gly-)x dans laquelle x est compris entre 3 et 10E is represented by a repeated chemical structure of type (Gly-) x in which x is between 3 and 10
S est représenté par l'une des séquences ID NO1 à ID NO8S is represented by one of the sequences ID NO1 to ID NO8
Les antigènes sont purifiés directement à partir du milieu de culture en utilisant le procédé de purification décrit par Philippe Slos et al (Protein expression and purification, 5, 518-526 (1994))The antigens are purified directly from the culture medium using the purification method described by Philippe Slos et al (Protein expression and purification, 5, 518-526 (1994))
Exemple 4 : induction d'anticorps neutralisants chez le cobayeExample 4 Induction of Neutralizing Antibodies in the Guinea Pig
Les isolats de phages recombinants plll ou pVIII exprimant les séquences S ID: NO1 à ID: NO 8 à l'extrémité d'un bras espaceur E constitué de 3 glycines ont été testés pour leur capacité à induire des anticorps neutralisants d'isolats primaires chez le cobaye. Après purification par double précipitation au polyéthylène glycol suivie d'une dialyse contre du PBS, les phages recombinants purifiés ont été injectés, par voie intramusculaire, dans les cuisses droite et gauche du cobaye à raison de 5x1011pfij par injection qui a été faite en présence ou non de 50μg de QS 21. Les cobayes ont reçu 3 injections à un mois d'intervalle. Les sérums ont été prélevés 3 semaines après la dernière immunisation pour étudier leur capacité à neutraliser le pouvoir infectieux de deux isolats primaires du virus VIH, Bx 08 et Bx 17 obtenus à partir de prélèvements de sujets séropositifs et asymptomatiques et cultivés uniquement par passages sur cellules mononucléees du sang périphérique humain (cf article de C. Moog et al -AIDS Research and human retroviruses, vol 13(1), 13-27, 1997).The recombinant phage pl11 or pVIII isolates expressing the sequences S ID: NO1 to ID: NO 8 at the end of a spacer arm E consisting of 3 glycines were tested for their capacity to induce neutralizing antibodies of primary isolates in The test-subject. After purification by double precipitation with polyethylene glycol followed by dialysis against PBS, the purified recombinant phages were injected, intramuscularly, into the right and left thighs of the guinea pig at a rate of 5 × 10 11 pfij per injection which was carried out in presence or not of 50 μg of QS 21. The guinea pigs received 3 injections at one month intervals. The sera were collected 3 weeks after the last immunization to study their capacity to neutralize the infectious power of two primary isolates of the HIV virus, Bx 08 and Bx 17 obtained from samples from seropositive and asymptomatic subjects and cultured only by passage through cells. mononuclear human peripheral blood (see article by C. Moog et al-AIDS Research and human retroviruses, vol 13 (1), 13-27, 1997).
On a évalué le pouvoir neutralisant des immunsérums vis à vis des isolats primaires Bx 08 et Bx 17 en déterminant la dose infectante 50% (TCID 50) de ces deux isolats en présence ou en l'absence d'immunsérum.The neutralizing power of the immune sera against the primary isolates Bx 08 and Bx 17 was evaluated by determining the 50% infectious dose (TCID 50) of these two isolates in the presence or in the absence of immune serum.
Au préalable, on a réalisé en parallèle une gamme de dilutions de raison 10 des suspensions virales Bx 08 et Bx 17 et, pour chaque sérum testé décomplémenté, une gamme de dilutions en milieu RPMI. Puis, on a incubé 50μl de chaque dilution virale avec 50μl de chaque dilution sérique pendant 1 heure à 37°C dans des microplaques 96 puits à fond rond. Chaque dilution a été testée en quadruplicate. 50μl d'une suspension contenant 105 cellules mononuclées du sang périphérique humain préalablement activées pendant 3 jours avec un milieu de culture contenant 1μg de phytohémagglutinine-P/ml ont été ensuite ajoutées. Après une période d'infection d'une heure à 37°C, le milieu de culture est débarrassé des virus résiduels par 2 lavages successifs avec 150μl de milieu RPMI. Après élimination du 2eme milieu de lavage, les cellules ont été remises en culture avec un milieu RPMI contenant 10% de sérum de veau fœtal additionné de 20UI/ml d'IL2. Au bout de 4 jours de culture, on a renouvelé une partie de ce milieu de culture avec du milieu de culture frais, puis au jour J7 et J10 on a prélevé une aliquote de 100μl de surnageant de culture, remplacé par du milieu frais à J7. Les prélèvements ont été gardés congelés jusqu'au dosage de la réplication virale. La réplication virale ainsi que la TCID 50 ont été quantifiées au moyen du dosage de la transcriptase inverse ou au moyen du dosage de la p24 par ELISA à l'aide de la trousse commercialisée par la société Coluter Immunotech Beckman.Beforehand, a range of dilutions of reason 10 was carried out in parallel with the viral suspensions Bx 08 and Bx 17 and, for each serum tested decomplemented, a range of dilutions in RPMI medium. Then, 50 μl of each viral dilution was incubated with 50 μl of each serum dilution for 1 hour at 37 ° C. in 96-well microplates with round bottom. Each dilution was tested in quadruplicate. 50 μl of a suspension containing 10 5 mononuclear human peripheral blood cells previously activated for 3 days with a culture medium containing 1 μg of phytohemagglutinin-P / ml were then added. After an infection period of one hour at 37 ° C., the culture medium is freed from residual viruses by 2 successive washes with 150 μl of RPMI medium. After elimination of the 2 nd washing medium, the cells were re-cultured with RPMI medium containing 10% fetal calf serum supplemented with 20 IU / ml of IL2. After 4 days of culture, part of this culture medium was renewed with fresh culture medium, then on day D7 and D10 an aliquot of 100 μl of culture supernatant was removed, replaced with fresh medium on D7 . The samples were kept frozen until the viral replication was assayed. The viral replication as well as the TCID 50 were quantified by means of the assay of reverse transcriptase or by means of the assay of p24 by ELISA using the kit marketed by the company Coluter Immunotech. Beckman.
La neutralisation est considérée comme significative lorsque le ratio entre la valeur du TCID50 de la suspension virale en présence de la dilution sérique testée et celle obtenue en l'absence de sérum est <0,1. Les TCID50 sont calculés à l'aide de la formule de Kârber (Kârber G. Arch. Exp. Pathol. Pharmakol. 162: 956-959 (1931). Le titre de neutralisation est le facteur de dilution maximale du sérum pour lequel le ratio entre la valeur du TCID50 de la suspension virale en présence de la dilution sérique testée et celle obtenue en l'absence de sérum est <0,1, ce qui revient à une réduction de un log 10 du titre infectieux. Les résultats sont regroupés dans le tabjeau n°1Neutralization is considered significant when the ratio between the TCID50 value of the viral suspension in the presence of the serum dilution tested and that obtained in the absence of serum is <0.1. The TCID50 are calculated using the Kârber formula (Kârber G. Arch. Exp. Pathol. Pharmakol. 162: 956-959 (1931). The neutralization titer is the maximum dilution factor of the serum for which the ratio between the value of the TCID50 of the viral suspension in the presence of the serum dilution tested and that obtained in the absence of serum is <0.1, which amounts to a reduction of a log 10 of the infectious titre. The results are grouped in table 1
Tableau n°1Table 1
Figure imgf000017_0001
Figure imgf000017_0001
: La séquence S est exprimée à la surface du phage recombinant pVIII.: The sequence S is expressed on the surface of the recombinant phage pVIII.
*: La séquence S est exprimée à la surface du phage recombinant plll.*: The sequence S is expressed on the surface of the recombinant phage pl11.
représente l'inverse de la dilution du sérum testé pour laquelle on observe une réduction d'au moins un log 10 du TCID 50 de l'isolât primaire testé par rapport au contrôle sans sérum. Les sérums sont considérés comme exerçant une activité neutralisante lorsqu'au moins la première dilution testée (1/5) entraîne cette réduction du TCID 50.represents the inverse of the dilution of the tested serum for which a reduction of at least one log 10 of the TCID 50 of the primary isolate tested compared to the serum-free control. Sera are considered to exert a neutralizing activity when at least the first dilution tested (1/5) leads to this reduction in TCID 50.
Les résultats figurant dans le tableau 1 montrent que les sérums provenant des animaux immunisés avec les différents phages recombinants testés ont une activité neutralisante vis à vis d'au moins un des deux isolats primaires testés. Les sérums de ces mêmes animaux avant immunisation (J0), ont été également contrôlés et se sont révélés négatifs. L'adjuvant QS21 a un effet inconstant sur la production d'anticorps neutralisants par les cobayes immunisés avec les différents phages recombinants. Il faut noter toutefois un effet adjuvant net sur la réponse des cobayes immunisés avec le phage recombinant exprimant la séquence S, Seq ID NO4. The results appearing in Table 1 show that the sera from animals immunized with the various recombinant phages tested have neutralizing activity with respect to at least one of the two primary isolates tested. The sera of these same animals before immunization (D0), were also checked and were found to be negative. The QS21 adjuvant has an inconsistent effect on the production of neutralizing antibodies by guinea pigs immunized with the various recombinant phages. It should be noted, however, a clear adjuvant effect on the response of guinea pigs immunized with the recombinant phage expressing the sequence S, Seq ID NO4.

Claims

Revendications claims
1. Un antigène protéique de formule P-E-S dans laquelle :1. A protein antigen of formula P-E-S in which:
P désigne une protéine ou un peptide porteur ou un fragment en dérivant; E désigne un bras espaceur de 2 à 25 acides aminés, constitué principalement de glycine, proline, ou alanine;P denotes a carrier protein or peptide or a fragment derived therefrom; E denotes a spacer arm of 2 to 25 amino acids, consisting mainly of glycine, proline, or alanine;
S désigne une séquence peptidique choisie dans le groupe consistant en les séquences ID NO1 à ID NO8 Les traits représentent une liaison peptidique.S denotes a peptide sequence chosen from the group consisting of the sequences ID NO1 to ID NO8 The lines represent a peptide bond.
2. Un antigène selon la revendication 1 dans lequel E est constitué de 3 résidus glycine.2. An antigen according to claim 1 in which E consists of 3 glycine residues.
3. Un antigène selon la revendication 1 ou 2 dans lequel P, est soit une protéine disposant d'une séquence signal provenant d'une bactérie ou d'un phage, soit une protéine ou un peptide renforçant l'immunogénicité de S.3. An antigen according to claim 1 or 2 in which P, is either a protein having a signal sequence originating from a bacterium or a phage, or a protein or a peptide reinforcing the immunogenicity of S.
4. Un vecteur d'expression codant pour un antigène selon l'une quelconque des revendications 1 à 3.4. An expression vector coding for an antigen according to any one of claims 1 to 3.
5. Un vecteur d'expression selon la revendication 4 dans lequel le vecteur est un phage filamenteux et la protéine P est la protéine plll et/ou pVIII.5. An expression vector according to claim 4 wherein the vector is a filamentous phage and the protein P is the protein plll and / or pVIII.
6. Un vaccin comprenant un antigène selon l'une quelconque des revendications 1 à 3 ou un vecteur selon la revendication 4 ou 5 en combinaison avec un support pharmaceutiquement acceptable et éventuellement un adjuvant.6. A vaccine comprising an antigen according to any one of claims 1 to 3 or a vector according to claim 4 or 5 in combination with a pharmaceutically acceptable carrier and optionally an adjuvant.
7. Un vaccin selon la revendication 6 pour le traitement ou la prévention de l'infection à virus VIH.7. A vaccine according to claim 6 for the treatment or prevention of HIV virus infection.
8. Une méthode de diagnostic comprenant la mise en contact d'un antigène selon l'une quelconque des revendications 1 à 3, ou d'un vecteur d'expression selon la revendication 4 ou 5 avec un échantillon biologique. 1/38. A diagnostic method comprising contacting an antigen according to any one of claims 1 to 3, or an expression vector according to claim 4 or 5 with a biological sample. 1/3
LISTE DE SEQUENCESLIST OF SEQUENCES
<110> Aventis pasteur<110> Aventis pasteur
<120> Antigènes protéiques inducteurs d'anticorps neutralisants le virus VIH<120> Protein antigens inducing antibodies neutralizing the HIV virus
<130> PM 0011<130> PM 0011
<140> PM 0011 <141> 2000-12-22<140> PM 0011 <141> 2000-12-22
<160> 8<160> 8
<170> Patentln Ver. 2.1<170> Patentln Ver. 2.1
<210> 1<210> 1
<211> 7<211> 7
<212> PRT<212> PRT
<213> Séquence artificielle<213> Artificial sequence
<220><220>
<223> Description de la séquence artificielle: peptide spécifique <400> 1<223> Description of the artificial sequence: specific peptide <400> 1
Ile Pro Thr Leu Pro Ser Ser 1 5Ile Pro Thr Leu Pro Ser Ser 1 5
<210> 2<210> 2
<211> 7<211> 7
<212> PRT<212> PRT
<213> Séquence artificielle<213> Artificial sequence
<220><220>
<223> Description de la séquence artificielle :peptide spécifique <400> 2<223> Description of the artificial sequence: specific peptide <400> 2
Trp Ala Leu Asp Arg Gly Ala 1 5Trp Ala Leu Asp Arg Gly Ala 1 5
<210> 3<210> 3
<211> 12<211> 12
<212> PRT<212> PRT
<213> Séquence artificielle<213> Artificial sequence
<220><220>
<223> Description de la séquence artificielle: peptide spécifique <400> 3<223> Description of the artificial sequence: specific peptide <400> 3
His Ala Thr Gly Thr His Gly Leu Ser Leu Ser His 1 5 10 2/3His Ala Thr Gly Thr His Gly Leu Ser Leu Ser His 1 5 10 2/3
<210> 4<210> 4
<211> 15<211> 15
<212> PRT<212> PRT
<213> Séquence artificielle<213> Artificial sequence
<220><220>
<223> Description de la séquence artificielle: peptide spécifique <400> 4<223> Description of the artificial sequence: specific peptide <400> 4
Thr Arg Leu Glu Cys Phe Ser Ala Gly Trp Arg Leu Ser Ala Cys 1 5 10 15Thr Arg Leu Glu Cys Phe Ser Ala Gly Trp Arg Leu Ser Ala Cys 1 5 10 15
<210> 5<210> 5
<211> 15<211> 15
<212> PRT<212> PRT
<213> Séquence artificielle<213> Artificial sequence
<220><220>
<223> Description de la séquence artificielle :peptide spécifique <400> 5<223> Description of the artificial sequence: specific peptide <400> 5
Trp Tyr Ser Lys Asp Thr Ser Trp Phe Thr Leu Phe Leu Pro Val 1 5 10 15Trp Tyr Ser Lys Asp Thr Ser Trp Phe Thr Leu Phe Leu Pro Val 1 5 10 15
<210> 6<210> 6
<211> 15<211> 15
<212> PRT<212> PRT
<213> Séquence artificielle<213> Artificial sequence
<220><220>
<223> Description de la séquence artificielle:peptide spécifique <400> 6<223> Description of the artificial sequence: specific peptide <400> 6
Thr Gly Gly Pro Leu Tyr Pro Phe Tyr Ala Leu Val Pro Ser Tyr 1 5 10 15Thr Gly Gly Pro Leu Tyr Pro Phe Tyr Ala Leu Val Pro Ser Tyr 1 5 10 15
<210> 7<210> 7
<211> 15<211> 15
<212> PRT<212> PRT
<213> Séquence artificielle<213> Artificial sequence
<220><220>
<223> Description de la séquence artificielle:peptide spécifique <400> 7<223> Description of the artificial sequence: specific peptide <400> 7
Asn Leu His Ser Lys Val Phe His Gly Leu Val Ile Arg Gly Gly 1 5 10 15 3/3Asn Leu His Ser Lys Val Phe His Gly Leu Val Ile Arg Gly Gly 1 5 10 15 3/3
<210> 8<210> 8
<211> 15<211> 15
<212> PRT<212> PRT
<213> Séquence artificielle<213> Artificial sequence
<220><220>
<223> Description de la séquence artificielle: eptide spécifique <400> 8<223> Description of the artificial sequence: specific eptide <400> 8
Gin Ser Leu Cys Ser Pro Met Pro Leu Gly Ser Cys Ala Leu Ser 1 5 10 15 Gin Ser Leu Cys Ser Pro Met Pro Leu Gly Ser Cys Ala Leu Ser 1 5 10 15
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JP4855261B2 (en) * 2004-09-03 2012-01-18 アマテラスファーマ株式会社 Anti-histone H1 monoclonal antibody and hybridoma producing the same
WO2017072105A1 (en) * 2015-10-26 2017-05-04 Basf Se Oral care products and methods comprising hydroxyapatite binding proteins
CN108348440A (en) * 2015-10-26 2018-07-31 巴斯夫欧洲公司 Include the oral care product and method of HLPS

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Publication number Priority date Publication date Assignee Title
JP4855261B2 (en) * 2004-09-03 2012-01-18 アマテラスファーマ株式会社 Anti-histone H1 monoclonal antibody and hybridoma producing the same
WO2017072105A1 (en) * 2015-10-26 2017-05-04 Basf Se Oral care products and methods comprising hydroxyapatite binding proteins
CN108348440A (en) * 2015-10-26 2018-07-31 巴斯夫欧洲公司 Include the oral care product and method of HLPS
RU2745996C2 (en) * 2015-10-26 2021-04-05 Басф Се Oral care products and methods containing hydroxyapatite-binding proteins

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