WO2002020052A1 - Utilisation de lipopeptides pour l'immunotherapie des sujets vih+ - Google Patents
Utilisation de lipopeptides pour l'immunotherapie des sujets vih+ Download PDFInfo
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- WO2002020052A1 WO2002020052A1 PCT/FR2001/002773 FR0102773W WO0220052A1 WO 2002020052 A1 WO2002020052 A1 WO 2002020052A1 FR 0102773 W FR0102773 W FR 0102773W WO 0220052 A1 WO0220052 A1 WO 0220052A1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- A61K39/12—Viral antigens
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- 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
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
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- 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
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
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- 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
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
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- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55522—Cytokines; Lymphokines; Interferons
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- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55544—Bacterial toxins
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- 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
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
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- 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
- A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
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- 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
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
- C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
- C12N2740/16334—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- the present invention describes a method of treating subjects infected with HIV and relates more particularly to the use of lipopeptides in immunotherapy in subjects HIV + under anti-retroviral therapy.
- HIV envelope glycoprotein the poxviruses expressing epitopes derived from HIV structural or regulatory proteins as well as lipopeptides.
- lipopeptides comprising a peptide part having between 10 and 40 amino acids approximately and comprising at least one antigenic determinant, said lipopeptide also comprising one or more chains derived from fatty acids comprising from 10 to 20 carbon atoms, and / or one or more steroid groups modified and coupled to ⁇ f ⁇ IH2 or ⁇ NH2 functions of said amino acids. Based on an experiment carried out in mice, the authors indicate that said lipopeptides can be used to induce CTL against any antigenic determinant of any pathogenic agent including HIV.
- lipopeptides according to the invention can be used to control at least temporarily the viremia in HIV + subjects on anti-retroviral therapy after stopping anti-retroviral treatment as described below. .
- HIV-related infections Several methods of treating HIV have been proposed to date.
- the only method of treating HIV-related infections that is currently used is a method based on the administration of a combination anti-retroviral medication known as HAART.
- HAART a combination anti-retroviral medication
- antiretroviral therapy is far from an ideal solution.
- this type of therapy requires a degree of patient involvement which is often difficult to obtain. Failure to adhere to treatment results in treatment failure and can facilitate the emergence of viruses resistant to antiviral products.
- antiretroviral therapy can be stopped by the administration of lipopeptides as defined below which induce cellular CD4 + and CD8 + responses specific to HIV. These cellular responses keep the viral load at low values and control the viral rebound after stopping antiretroviral therapy.
- the present invention therefore relates to the use of lipopeptides for the preparation of a vaccine for the control of viral rebound after stopping antiretroviral therapy in HIV + subjects with a viral load less than or equal to 10,000 copies per ml of plasma and a CD4 + level greater than or equal to 300 cells per mm 3 , in which the lipopeptides consist of a peptide chain of 7 to 100 amino acids comprising at least one CTL epitope of an HIV protein, linked by covalent bond to a lipid chain comprising from 8 to 20 carbon atoms.
- the HIV + subjects have a viral load less than or equal to 50 copies per ml of plasma and a CD4 count greater than or equal to 500 cells per mm3.
- the lipopeptides consist of a lipid chain comprising 16 carbon atoms. According to another embodiment, the lipopeptides correspond to a mixture comprising 5 different lipopeptides having CTL epitopes derived from the Nef, Gag and Pol proteins of HIV.
- the mixture of lipopeptides comprises the following lipopeptides:
- VGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLK ⁇ (Palm) -NH2 HTQGYFPDWQNYTPGPGVRYPLTFGWLYKLK ⁇ (P I m) - N H 2 EKIRLRPGGKKKYKLKVIHK ⁇ (Palm) -NH2 NPPIPVGEIYKRWIILGLNKIVRMYSPTSILDK ⁇ (Palm) -NH 2
- the mixture further comprises a lipopeptide whose peptide chain consists of a ubiquitous helper epitope.
- the lipopeptides are administered intramuscularly at a dose of 50 ⁇ g to 3 mg of total lipopeptides, preferably in 4 doses respectively on D0, 1 month, 2 months and 3 months.
- the lipopeptides are co-administered with a recombinant attenuated virus vaccine.
- Said recombinant attenuated virus is preferably an ALVAC and in particular an ALVAC vCP1452 or vCP1433.
- an immunomodulator preferably TIL2 is administered sequentially or simultaneously.
- FIG. 1 gives a schematic representation of the lipopeptides constituting a mixture according to the invention.
- ⁇ (Palm) -NH2 means that a palmitic acid is linked to the ⁇ NH2 function of lysine, the COOH-terminal end of the lipid is therefore amidated.
- Lysine is located at the C-terminus of the peptide chain.
- the peptide chain is represented according to the one-letter code in which:
- A Ala; D: Asp; E: Glu; F: Phe: G: Gly; H: His; I: Ile; K: Lys: L: Leu; M: Met; N: Asn; P: Pro; Q: Gin; A: Arg; S: Ser; T: Thr; V: Val; W: Trp; Y: Tyr.
- the vaccination method according to the present invention is useful for the treatment of subjects infected with HIV subjected to antiretroviral therapy and having a viral load less than 10,000, preferably less than 5,000, in particular less than 1,000 viral copies / ml of plasma and a cell count T CD4 + greater than 300 cells / ml, preferably greater than 500 cells / ml. Patients preferably have a viral load of less than 50 viral copies / ml and a CD4 + count greater than 500.
- the viral load expressed by the number of copies of RNA / ml of plasma represents the amount of virus present in the blood. It is also referred to as viral or viral title.
- Many techniques can be used to measure a patient's viral load. A state-of-the-art review can be found in "Report of the NIH To Define Principles of Therapy of HIV Infection” published in the Morbidity and Mortality Weekly Reports, April 24, 1998, Vol. 47, No. RR-5, revised on 17/6/98 to which reference may be made for a description of the techniques. It is known that HIV replication rates in infected people can be measured precisely by measuring HIV in plasma. HIV RNA in plasma is contained in circular particles of virus or virion, each virion containing 2 copies of HIV genomic RNA.
- the concentrations of HIV RNA in the plasma can be quantified either by amplification of the targets (e.g. quantitative polymerase chain reaction [RT-PCR], Amplicor HIV Monitor test, Roche Molecular Systems), or by l amplification of nucleic acid sequences, [NASBA®], NucliSens TM HIV-1 QT assay, Organon Teknika).
- targets e.g. quantitative polymerase chain reaction [RT-PCR], Amplicor HIV Monitor test, Roche Molecular Systems
- NASBA® NucliSens TM HIV-1 QT assay, Organon Teknika
- the test which has been used in the context of the present invention is the Amplicor HIV Monitor test, Roche Molecular Systems.
- the level of CD4 + T cells corresponds to the total number of cells expressing the CD4 marker per mm 3 of blood. This rate is determined according to the recommendations described in The Morbidity and Mortality Weekly Report, 46 (RR-02) Jan, 10 1997. Centers for Disease Control. In summary, the absolute number of CD4 + in whole blood is measured by a three-phase process.
- CD4 + counting is the result of three laboratory techniques: white blood cell (WBC) counting; determining the percentage of WBCs that are lymphocytes (differential); and determining the percentage of lymphocytes which are CD4 + T cells by "cytometric flow immunophenotyping" for example by using the FACSCount system of Becton Dickinson.
- Anti-retroviral therapy is understood to mean a treatment comprising an effective combination of anti-retroviral agents.
- Anti-retroviral therapy involves the use of two broad categories of drugs e. inhibitors reverse transcriptase and protease inhibitors.
- Reverse transcriptase inhibitors can be of nucleoside nature, such as: AZT, ddl, ddC, d4T and 3TC in combination with AZT and Combivir, or of non-nucleoside nature such as Delavirdine and Nevirapine.
- a review of non-nucleoside inhibitors is given in Clinical Care (10197) Vol. 9, No 10, p 75.
- anti-retroviral therapy corresponds to a highly active therapy (HAART) Le; a combination of a protease inhibitor, a non-nucleoside reverse transcriptase inhibitor and a nucleoside reverse transcriptase inhibitor, or a combination of two non-nucleoside reverse transcriptase inhibitors and a nucleoside reverse transcriptase inhibitor reverse transcriptase.
- HAART highly active therapy
- the patients who can be treated by the method according to the invention are therefore people infected with HIV who are subjected to anti-retroviral therapy early after the Le infection. within 3 months of infection as well as those infected and treated more than 3 months after infection, the latter being designated in the context of the present invention chronically infected persons.
- the method according to the invention therefore allows the treatment of newly infected persons (Le. Infected for 90 days or less) who * are placed on anti-retroviral therapy a few months after infection with HIV and therefore have a controlled viremia. These people have the distinction of presenting an incomplete Western Blot.
- the method according to the present invention also allows the treatment of chronically infected patients under anti-retroviral therapy. Viremia is controlled when the viral load is kept below 10,000 viral copies per ml of plasma. HIV + subjects who exhibit CD4 + and CD8 + cellular responses against HIV antigens, in particular those who exhibit proliferative cellular responses against envelope epitopes (eg gp120) are preferred.
- HIV + subjects who have lost their CD4 + and / or CD8 + cellular responses against HIV antigens can also be vaccinated by the method according to the invention.
- the vaccination method according to the present invention makes it possible to control the viral rebound in patients infected with HIV after stopping antiretroviral therapy.
- Control of viral rebound means in the context of the present invention that after stopping antiretroviral therapy, the viral rebound which appears is delayed, absent, or that the viral load present after the viral rebound ("post -rebound set point ”) is checked.
- the viral rebound is generally observed within 1 to 3 weeks after stopping antiretroviral therapy.
- the viral rebound is considered delayed when it appears more than a month after stopping antiretroviral therapy.
- the viral rebound appears more than 2 months and in particular more than
- the post-rebound set point represents the plasma viral load which is present in the absence of antiretroviral therapy after the viral rebound.
- the post-rebound viral load is considered to be controlled when it is maintained at values lower than the resumption values of antiretroviral therapy as defined in the official recommendations published in the different countries and this for a period of at least 1 month, preferably at least 2 months, in particular at least 6 months.
- These resumption values for anti-retroviral therapy are typically 10,000 to 50,000 copies of RNA / ml of plasma.
- the post-rebound viral load is maintained at values lower than 10 000 viral copies / ml, preferably lower than 5000 viral copies / ml in particular lower than 1000 viral copies / ml of plasma.
- the present invention therefore provides a method which has the advantage of allowing at least temporary, and preferably permanent, cessation of antiretroviral therapy in HIV + subjects by allowing control of the phenomenon of viral rebound generally associated with such a stoppage.
- CD8 + response is understood to mean the capacity of cytotoxic T cells to recognize and kill cells expressing HIV peptides in the context of MHC class I molecules. Such a response can be measured by various methods well known to those skilled in the art such as, for example, the tetramer technique on fresh or cultured PBMCs, by Elispot tests for INFgamma, or functional cytotoxicity tests.
- CD4 + response is understood to mean the capacity of CD4 + T cells to be stimulated or activated by the vaccine according to the invention.
- CD4 + responses can be measured by the various methods well known to those skilled in the art which have been described above.
- lipopeptide in the context of the present invention means at least one compound consisting of a peptide chain comprising from 7 to 100 amino acids, preferably from 10 to 50 amino acids and more particularly from 20 to 35 amino acids and a lipid chain comprising from 8 to 20 carbon atoms, preferably from 14 to 18 carbon atoms and in particular 16 carbon atoms.
- the lipopeptides according to the present invention preferably correspond to a mixture of at least two different lipopeptides.
- the peptide chain of the lipopeptides according to the present invention comprises at least one CTL epitope of an HIV protein and may also include one or more T helper epitopes of an HIV protein. Polyepitopic peptide chains comprising several CTL epitopes are particularly suitable.
- CTL epitope of an HIV protein is understood to mean, in the context of the present invention, the sequences derived from HIV structural and regulatory proteins which induce a response mediated by CD8 + cells of the immune system, such as epitopes. from the proteins Env, Gag, Pol, Tat and Nef. Any CTL epitope of HIV as defined above can be used in the context of the present invention. Such epitopes can be identified by using the algorithms described to do this in the literature. As non-limiting examples of CTL epitopes which can be used in the lipopeptides according to the invention, mention may be made of the epitopes listed in Table 4 of application WO99 / 27954.
- the epitopes present in the lipopeptides according to the invention can come from a single strain of HIV or preferably from different strains of HIV, preferably from strains of primary isolates. These epitopes are in the case of the use of a single strain originating from the conserved regions of the HIV genome.
- helper T epitope of HIV is intended to mean the sequences derived from proteins of structure and regulation of HIV that induce an auxiliary response mediated by CD4 cells of the immune system. Any auxiliary T epitope corresponding to the above definition can be used in the context of the present invention.
- the lipopeptide according to the invention corresponds to a mixture in which the peptide chains come from the Env, Gag, Pol and Nef proteins of HIV, and in particular come from Gag, Pol and Nef.
- the lipid chain is saturated or unsaturated, linear or branched, and contains from 8 to 20 carbon atoms. It is preferably linear and saturated.
- the lipid chain is a palmitic acid.
- the lipid chain is linked by covalent bond at the C-terminal or at the N-terminal of the peptic chain directly or via one or more amino acid (s) selected preferably from the group consisting of lysine, lysineamide, cysteine, serine, threonine. Preferably via a single amino acid corresponding to a lysine, a lysineamide or a cysteine.
- the COOH function of the fatty acid can be linked directly to the ⁇ NH2 or ⁇ NH2 function at the N-terminal of the peptide chain by an amide bond.
- the lipid chain is linked via an amino acid, preferably an N-terminal or C-terminal lysine of the peptide chain by amide bonds.
- an amino acid preferably an N-terminal or C-terminal lysine of the peptide chain by amide bonds.
- said link is advantageously carried out via a lysineamide residue, which simplifies the process of peptide synthesis.
- the lipid chain derives from an alkyl halide as defined above, its bonding to the peptide chain can be carried out in C-terminal or in N-terminal, preferably via a cysteine by a thioether bond.
- the lipopeptides according to the present invention can be synthesized by any conventional method. Methods that can be used in the context of the present invention are described in particular in documents US5019383, FR9015870, US5993823 and WO99 / 27954 to which reference may be made for a description. complete synthesis processes. , Methods of synthesis of lipopeptides according to the present invention are also described in the following references: “Yeast binding protein famesyltransferase. Binding of S-alkyl peptides and related analogs ”.
- the present invention therefore relates to the administration of a composition comprising at least the lipopeptide as defined above and a pharmaceutically acceptable carrier or diluent.
- the composition administered comprises at least two different lipopeptides and in particular at least 5 different lipopeptides.
- the lipopeptides contained in such a mixture differ in their peptide chain, each lipopeptide comprising at least one CTL epitope which is different from the CTL epitope (s) present on the other lipopeptides of the mixture.
- the lipopeptides present in the mixture can also differ in their lipid chain.
- all the lipopeptides consist of the same lipid chain.
- the lipid chain is a C16 chain.
- the lipid chain derives from a palmitic acid which is linked via a lysine or lysineamide residue on one of the ends of the peptide chain.
- the mixture according to the present invention consists of the lipopeptides as defined in FIG. 1.
- the applicant has surprisingly demonstrated that the lipopeptides according to the invention are effective in the absence of any lipopeptide containing a universal T helper epitope as described in WO99 / 27954.
- the lipopeptides are present in the mixture in equiponderal amounts, preferably in equimolar amounts.
- the lipopeptide mixture is prepared from the individual lipopeptides in lyophilized form as follows.
- the lipopeptides are solubilized in pure acetic acid and they are mixed in a defined order Le. starting from the least hydrophobic and ending with the most hydrophobic.
- An example of preparation of a mixture according to the invention is given in the examples which follow.
- the term "pharmaceutically acceptable carrier or diluent" is used in its conventional sense and can for example represent for an injectable solution, such as water, a buffered saline solution or a glucose solution.
- the pharmaceutically acceptable carrier or diluent will be selected according to the galenical form chosen, the mode 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 Phamaceutical Sciences, which represents a reference work in this field.
- the lipopeptides according to the present invention can be administered by any conventional route usually used in the field of vaccines, such as the parenteral route (intradermal, intramuscular, subcutaneous, etc.).
- the lipopeptides according to the invention are preferably administered by the intramuscular route.
- the administration can be carried out by the injection of a single dose or of repeated doses, for example and preferably in 4 doses on D0, at 1 month, at 2 months and 3 months.
- Booster injections may be given preferably every three months.
- the lipopeptides according to the invention are administered in an amount of 50 micrograms to 3 milligrams of total lipopeptides and this for the four injections as well as for the booster injections.
- the lipopeptides can advantageously be administered simultaneously or sequentially with a DNA vaccine or an attenuated recombinant virus vaccine.
- any plasmid vector containing regulatory elements of eukaryotic viruses can be used as a eukaryotic expression vector.
- Any vector can be used which allows the expression of proteins under the control of the SV40 “early” or “late” promoter, the metallothionein promoter, the human cytomegalovirus, the murine mammary tumor virus, the Rous sarcoma virus, from the promoter of the polyhedrine, or other promoters effective for expression in eukaryotic cells.
- Therapeutic amounts of plasmid DNA can be produced by fermentation in E. coli, followed by purification. Aliquots from the working cell bank are used to inoculate the growth medium and are cultured until saturation in flasks with shaking or in a bioreactor according to well known techniques. Plasmid DNA can be purified using a standard bioseparation method such as a solid phase anion exchange resin from QIAGEN, Inc. (Valencia, Califomia). If necessary, the supercoiled DNA can be isolated from the open circular or linear forms by an electrophoresis gel or any other suitable method for this.
- a standard bioseparation method such as a solid phase anion exchange resin from QIAGEN, Inc. (Valencia, Califomia). If necessary, the supercoiled DNA can be isolated from the open circular or linear forms by an electrophoresis gel or any other suitable method for this.
- Purified plasmid DNA can be prepared for injections using a variety of formulations. The simplest is the reconstitution of freeze-dried DNA in sterile phosphate buffer (PBS). This method called “naked DNA” is preferably used for intramuscular (IM) administration in the context of the present invention.
- PBS sterile phosphate buffer
- the plasmid vector contains sequences encoding one or more peptides or proteins containing epitopes, at least some of which are common with those present in the lipopeptide (s) administered.
- plasmid DNA may be desirable to employ another method for formulating purified plasmid DNA.
- glycolipids, fusogenic liposomes, peptides and compounds mentioned as globally protective, interactive, which do not condense can also be mixed with plasmid DNA to act on variables like stability, intramuscular dispersion, or targeting specific organs or cells.
- the lipopeptides according to the present invention are administered sequentially or simultaneously with an attenuated recombinant virus vaccine.
- the lipopeptides and the vaccine with recombinant attenuated virus are administered either simultaneously or sequentially according to a method of primovaccination-booster (prime-boost) in which the recombinant attenuated virus is administered in primovaccination.
- primovaccination-booster primovaccination-booster
- attenuated recombinant virus vaccine is understood to mean a composition comprising a recombinant attenuated virus and a pharmaceutically acceptable carrier or diluent.
- a "recombinant attenuated virus” in the context of the present invention is a virus which has been genetically modified by modern molecular biology techniques, eg. restriction endonucleases and ligase treatment, which has thus been made less virulent than the wild virus by deletion of certain genes or which has been attenuated by serial passages on a cell line originating from a host which is not not the natural host, or on primary permissive cells or at low temperatures.
- the attenuated recombinant viruses according to the present invention express at least one CTL epitope of an HIV protein, and at least one T helper epitope preferably specific for HIV.
- the viruses preferably express the Gag, Env and protease proteins and CTL epitopes of Pol and Nef.
- non-limiting examples include adenoviruses, adeno-associated viruses, alphaviruses and poxviruses.
- the attenuated virus acts as a vector for an immunogenic retroviral protein through the ability of the virus to code for foreign DNA.
- the virus preferably induces a helper response and a cytotoxic response against cells infected with HIV.
- a live virus vaccine can for example be administered at about 10 4 -10 8 particles / dose, or from 10 5 to 10 9 pfu per dose.
- the actual dosage of such a vaccine can easily be determined by an ordinary vaccinology test.
- poxviruses are preferably used. A detailed review of these vectors is provided in patent US5863542 to which reference may be made for a complete description of these.
- a representative example of a recombinant poxvirus is ALVAC.
- the DNA inserted into these vectors encodes the HIV antigens which comprise at least one of the following epitopes: HIV-I Gag (+ protease) (LAI), gp120 (MN) (+ the transmembrane part of gp41), Nef (BRU) CTL, Pol (LAI) CTL
- the ALVAC comprises at least one CTL epitope of Nef and at least one CTL epitope of Pol (Inverse Transcriptase).
- the CTL epitopes of Nef and Pol are preferably the CTL epitopes Nefl, Nef2, PoM, Pol2 and Pol3.
- sequences coding for Tat and / or Rev can advantageously be added.
- the viral strains from which the antigens are derived are put in brackets.
- the DNA sequences encoding the HIV antigens as defined above can be derived from any known strain of HIV (HIV1 and HIV2, preferably HIV1), including laboratory strains and primary isolates.
- HIV1 and HIV2, preferably HIV1 The sequences of the CTL epitopes of Nef and Pol identified above are described in patent US5990091 and correspond to the following sequences:
- the ALVAC vCP 1433 or 1452 will preferably be used.
- the structure of these two ALVACs as well as their manufacturing process are described in detail in patent US5990091, which will be referred to for a complete description of these.
- an immunomodulator preferably IL2 is administered sequentially or simultaneously.
- the IL2 is preferably administered after the lipopeptide treatment and preferably in 5 courses of 5 days at a rate of 4.5 million units twice a day.
- Anti-retroviral therapy is preferably stopped about 4 weeks after the last lipopeptide injection. If the immunotherapy by administration of lipopeptides is followed by a treatment with NL2, preferably a treatment comprising 5 courses, the anti-retroviral therapy is stopped approximately 8 weeks after the last course, or approximately 28 weeks after the last lipopeptide injection in the case of a dosage regimen comprising 4 injections (D0, 1 month, 2 months and 3 months).
- Example 1 preparation of a composition of lipopeptides according to the invention.
- a mixture comprising 5 lipopeptides comprising peptide sequences derived from the Nef, Gag and Pol proteins of the LAI strain of the HIV virus corresponding to the sequences Nef 66-97, Nef116-145, gag17-35, gag253-284 and pol325-355 is prepared according to the process described below.
- a lysine was added to the C-terminal end of the peptide and a palmitic acid was grafted on the side chain of this lysine (amide bond).
- the synthesis is carried out on a solid phase (polystyrene type resin comprising tricyclic amide substituents) using the Fmoc strategy.
- Amino acids are added from the C-terminus to the N-terminus.
- the peptide is detached from the resin and the side chains are deprotected by treatment with trifluoroacetic acid.
- the peptide is purified by HPLC using two different solvent systems. The pure peptide is then subjected to ion exchange chromatography so as to permute the trifluoroacetate ions with acetate ions.
- the peptide is filtered (0.22 ⁇ m) and distributed in 100 mg vials and lyophilized.
- Each lipopeptide is then dissolved by adding pure acetic acid to a concentration of 12.5 mg net / ml.
- the lipopeptides Nef66, Nef116 and Gag17 are soluble in pure acetic acid.
- the lipopeptides Gag253 and Pol325 are not soluble in pure acetic acid. It is therefore necessary to introduce a sonication step to facilitate the solubilization of the latter two. If the aggregates are not dissociated, the sonication is continued, spacing each sonication period (30 s) with stops of 30 s, during which the bottles are shaken manually. At the end of the sonication stage, the suspensions must be homogeneous (but will not be clear).
- Each lipopeptide is then diluted by adding water for injection. If the solution obtained is not completely clear, it is possible to carry out steps of heating in a bath. blend (at 40 ° C ⁇ 2 ° C) and sonication (1 min maximum), spacing with stops of 30 s.
- the final preparation is obtained by mixing the lipopeptides in equiponderal quantities by adding them in the following order (most hydrophilic to most hydrophobic lipopeptides): Nef66; Nefl 16; Gag17; Pol325; Gag253
- the mixture is kept under stirring (using a magnetized bar) during the additions of the various lipopeptides.
- the mixture to be filtered must be clear. It is best to pass it in a water bath at 40 ° C ⁇ 2 ° C (5 minutes maximum) until a perfectly liquid and clear mixture is obtained. Do not exceed 10 min.
- the mixture is then filtered and then distributed in a bottle at the rate of 500 ⁇ g of each lipopeptide per bottle, then lyophilized.
- the solution is reconstituted by adding 1 ml of a 5% glucose solution (W / V) and buffered or not.
- composition thus obtained comprises 2.5 mg of total lipopeptides per ml.
- Example 2 Preparation of a composition according to the invention
- a composition is prepared according to the method described in Example 1 comprising, in addition to the lipopeptides mentioned in Example 1, a lipopeptide
- TT830-846 in which the peptide chain consists of the epitope 830-846 of tetanus toxin.
- This epitope is described in the literature as being a universal T-helper epitope.
- the lipopeptide TT830-846 consists of a C16 lipid chain derived from a palmitic acid, linked at the C-Terminal of the peptide chain and synthesized according to the synthesis method described above for the other lipopeptides.
- the N-terminal end is acetylated, this in order to avoid the cyclization of glutamine (Q) into pyroGlu.
- Lipopeptide TT830-846 is added to the final preparation after lipopeptide GAG17.
- composition thus obtained comprises 3.0 mg of total lipopeptides per ml.
- HIV + primo-infected and chronically infected subjects with a viral load less than or equal to 1000 copies per ml of plasma and a rate of CD4 + greater than or equal to 300 cells per mm 3 were subjected to an immunotherapy method according to the present invention.
- ALVAC-HIV (vCP1433) is co-administered intramuscularly at a dose of 10 6.5 TCID50, this same dose being used for the 4 injections.
- Part of the subjects are subjected after immunotherapy to treatment with IL2 in 3 courses or 5 courses of 5 days at the rate of 4.5 million units twice a day.
- Anti-retroviral therapy is stopped 4 weeks after the last lipopeptide injection or, when this is followed by treatment with IL2, for example in 5 courses, 8 weeks after the last course, or 28 weeks after the last vaccination.
- the effectiveness of immunotherapy, demonstrated by at least temporary control of viremia, is determined by measuring the viral load according to the method described above.
- the induction of an HIV-specific cytotoxic response is demonstrated by a method for measuring intracellular INFgamma (in FACS) or secreted (ELISPOT).
- the proliferation of CD4 + T lymphocytes against HIV is measured by culturing the cells in the presence of HIV antigens and by measuring the incorporation of tritiated thymidine after 7 days of culture.
- the capacity of mononuclear cells of the blood to proliferate is checked by culturing the cells with a mitogen or a control antigen (tetanus for example).
- the CD4 cells are purified on magnetic beads and the proliferation test is carried out on this subpopulation thus purified.
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Abstract
Description
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA002421409A CA2421409A1 (fr) | 2000-09-08 | 2001-09-06 | Utilisation de lipopeptides pour l'immunotherapie des sujets vih+ |
EP01967441A EP1317281A1 (fr) | 2000-09-08 | 2001-09-06 | Utilisation de lipopeptides pour l'immunotherapie des sujets vih+ |
AU2001287820A AU2001287820A1 (en) | 2000-09-08 | 2001-09-06 | Use of lipopeptides for immunotherapy of hiv-positive subjects |
Applications Claiming Priority (9)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR0011443 | 2000-09-08 | ||
FR0011443A FR2813793B1 (fr) | 2000-09-08 | 2000-09-08 | Utilisation de lipopeptides pour l'immunotherapie des sujets vih+ |
US23507900P | 2000-09-25 | 2000-09-25 | |
US60/235,079 | 2000-09-25 | ||
FR0102846A FR2821556B1 (fr) | 2001-03-02 | 2001-03-02 | Utilisation de lipopeptides pour l'immunotherapie des sujets vih+ |
FR0102846 | 2001-03-02 | ||
US27894201P | 2001-03-27 | 2001-03-27 | |
US60/278,942 | 2001-03-27 | ||
US10/107,746 US7119078B2 (en) | 2000-09-08 | 2002-03-27 | Technology of intracellular delivery of DNA oligonucleotides to improve drug activity |
Publications (1)
Publication Number | Publication Date |
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WO2002020052A1 true WO2002020052A1 (fr) | 2002-03-14 |
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ID=38235308
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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PCT/FR2001/002773 WO2002020052A1 (fr) | 2000-09-08 | 2001-09-06 | Utilisation de lipopeptides pour l'immunotherapie des sujets vih+ |
Country Status (5)
Country | Link |
---|---|
US (1) | US7119078B2 (fr) |
EP (1) | EP1317281A1 (fr) |
AU (1) | AU2001287820A1 (fr) |
CA (1) | CA2421409A1 (fr) |
WO (1) | WO2002020052A1 (fr) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003092479A2 (fr) * | 2002-05-03 | 2003-11-13 | Luc Montagnier | Traitements et methodes de traitement, de diagnostic et de prophylaxie |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
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TWI428135B (zh) * | 2007-03-26 | 2014-03-01 | Hirofumi Takeuchi | And a carrier composition for quick-acting nucleic acid delivery |
CN101301478A (zh) * | 2007-05-10 | 2008-11-12 | 贝勒医学院 | 以低氧诱导因子1-α(HIF1α)作为靶标的G-四合体寡核苷酸 |
WO2009002719A1 (fr) * | 2007-06-22 | 2008-12-31 | The Board Of Regents Of The University Of Texas System | Acide nucléique inhibiteur des liposomes contre les protéines stat |
US8222225B2 (en) * | 2008-05-21 | 2012-07-17 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Method of treating pneumoconiosis with oligodeoxynucleotides |
US8053422B2 (en) | 2008-12-04 | 2011-11-08 | The United States Of America As Represented By The Department Of Health And Human Services | Anti-cancer oligodeoxynucleotides |
WO2011109677A2 (fr) * | 2010-03-04 | 2011-09-09 | University Of Louisville Research Foundation, Inc. | Procédés d'augmentation de la macropinocytose dans des cellules cancéreuses |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994026293A1 (fr) * | 1993-05-19 | 1994-11-24 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Facilitation immunologique par une therapie intermittente a l'interleukine-2 |
WO1998040501A1 (fr) * | 1997-03-12 | 1998-09-17 | Virogenetics Corporation | Vecteurs ayant une expression facilitee, methodes de production et d'utilisation desdits vecteurs |
WO1999027954A2 (fr) * | 1997-12-03 | 1999-06-10 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Micelles mixtes de lipopeptides pour l'induction d'une reponse immunitaire |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5831066A (en) * | 1988-12-22 | 1998-11-03 | The Trustees Of The University Of Pennsylvania | Regulation of bcl-2 gene expression |
US6288042B1 (en) * | 1993-04-23 | 2001-09-11 | Aronex Pharmaceuticals, Inc. | Anti-viral guanosine-rich tetrad forming oligonucleotides |
IT1277025B1 (it) * | 1995-12-04 | 1997-11-04 | Cooperativa Centro Ricerche Po | Classe di oligonucleotidi fosfodiesterici ad attivita' citotossica composizioni farmaceutiche che li contengono e loro uso |
-
2001
- 2001-09-06 EP EP01967441A patent/EP1317281A1/fr not_active Ceased
- 2001-09-06 CA CA002421409A patent/CA2421409A1/fr not_active Abandoned
- 2001-09-06 AU AU2001287820A patent/AU2001287820A1/en not_active Abandoned
- 2001-09-06 WO PCT/FR2001/002773 patent/WO2002020052A1/fr not_active Application Discontinuation
-
2002
- 2002-03-27 US US10/107,746 patent/US7119078B2/en not_active Expired - Fee Related
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1994026293A1 (fr) * | 1993-05-19 | 1994-11-24 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Facilitation immunologique par une therapie intermittente a l'interleukine-2 |
WO1998040501A1 (fr) * | 1997-03-12 | 1998-09-17 | Virogenetics Corporation | Vecteurs ayant une expression facilitee, methodes de production et d'utilisation desdits vecteurs |
WO1999027954A2 (fr) * | 1997-12-03 | 1999-06-10 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Micelles mixtes de lipopeptides pour l'induction d'une reponse immunitaire |
Non-Patent Citations (4)
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003092479A2 (fr) * | 2002-05-03 | 2003-11-13 | Luc Montagnier | Traitements et methodes de traitement, de diagnostic et de prophylaxie |
WO2003092479A3 (fr) * | 2002-05-03 | 2004-11-25 | Luc Montagnier | Traitements et methodes de traitement, de diagnostic et de prophylaxie |
Also Published As
Publication number | Publication date |
---|---|
AU2001287820A1 (en) | 2002-03-22 |
US7119078B2 (en) | 2006-10-10 |
US20060199777A1 (en) | 2006-09-07 |
EP1317281A1 (fr) | 2003-06-11 |
CA2421409A1 (fr) | 2002-03-14 |
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