EP1453853A2 - Mischung aus peptidfragmenten des nef-proteins und ihre anwendungen - Google Patents

Mischung aus peptidfragmenten des nef-proteins und ihre anwendungen

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Publication number
EP1453853A2
EP1453853A2 EP02702459A EP02702459A EP1453853A2 EP 1453853 A2 EP1453853 A2 EP 1453853A2 EP 02702459 A EP02702459 A EP 02702459A EP 02702459 A EP02702459 A EP 02702459A EP 1453853 A2 EP1453853 A2 EP 1453853A2
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EP
European Patent Office
Prior art keywords
positions
peptide corresponding
peptide
mixture
nefl
Prior art date
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EP02702459A
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English (en)
French (fr)
Inventor
Bernard Maillere
Sandra Pouvelle-Moratille
Jean-Gérard Guillet
Hanne Gahery-Segard
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Institut National de la Sante et de la Recherche Medicale INSERM
Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Original Assignee
Commissariat a lEnergie Atomique CEA
Institut National de la Sante et de la Recherche Medicale INSERM
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Publication of EP1453853A2 publication Critical patent/EP1453853A2/de
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Classifications

    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • 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
    • 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
    • C12N2740/16311—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

Definitions

  • the present invention relates to a mixture of peptides derived from a Nef protein as well as to its applications as a medicament (in immunogenic compositions, capable of stimulating the production of anti-HIV CD4 + T lymphocytes in vivo and therefore useful for AIDS vaccination) or as a diagnostic reagent for HIV-specific T lymphocytes, in particular to assess the immune status of HIV-positive patients or patients on antiretroviral therapy.
  • CD4 + T lymphocytes have a rearranged T receptor which allows them to selectively recognize the peptide fragments resulting from the degradation of the antigen by the presenting cells and presented by the molecules of the major class II histocompatibility complex (MHC II).
  • MHC II major class II histocompatibility complex
  • the determinants that these peptide fragments carry and that T cells actually recognize are called T epitopes.
  • CD4 + T lymphocytes are known to be a prime target of the human immunodeficiency virus (HIV) and can disappear completely from circulation. blood.
  • HIV-infected patients are victims of opportunistic infections and cannot induce vigorous immune responses to combat them.
  • CD4 + T lymphocytes have a major role in establishing immune responses. They secrete most of the cyto ines necessary for the recruitment of effector cells which are the cytotoxic CD8 + T lymphocytes and the B lymphocytes which produce antibodies. They also intervene in the activation of cells by cellular contacts and for example induce the activation by CD40 of dendritic cells presenting the antigen.
  • CD4 + T lymphocytes Due to their major role, the disappearance of CD4 + T lymphocytes therefore leads to a significant weakening of the immune system: in particular, the disappearance of CD4 + T lymphocytes decreases specific immunity against HIV. Indeed, recent work has shown that individuals with a proliferative response specific for CD4 + cells specific for HIV components can control viral infection in the absence of any treatment (1, 2). There is even an inverse correlation between the proliferation of CD4 + T lymphocytes with respect to the P24 protein and the viral load. It is possible that this correlation results directly from the capacity of CD4 + T lymphocytes of the TH1 type to secrete anti-viral cytokines such as IFN- ⁇ and to be cytotoxic. It can also result indirectly from their ability to maintain humoral immunity and more probably cellular immunity. We know that cytotoxic CD8 + T cells play an important role in keeping HIV-positive patients in a non-symptomatic state.
  • CD4 + T lymphocytes The activation of CD4 + T lymphocytes is effected by the presentation of viral peptides by HLA II molecules, carried by the antigen presenting cells (APC or Antigen Presentation Cells).
  • APC antigen presenting cells
  • T epitopes result from the proteolytic degradation of viral antigens by APC. They have varying lengths, usually 13 to 25 amino acids and have a sequence that makes them capable of binding to HLA II molecules.
  • T epitope is capable, like the native antigen, of stimulating CD4 + T lymphocytes which are specific to it in vitro or of recruiting them in vivo.
  • T epitopes are therefore sufficient to induce a CD4 + response.
  • one of the major problems which limits the use of these peptides is that their sequence varies from one individual to another, due to the polymorphism of HLA II molecules, which are heterodimers, expressed on the antigen presenting cells ( APC) and which present to CD4 + T lymphocytes. the T epitopes of said antigens. These molecules are capable of binding a large repertoire of peptides having sequences very different, which allows them to present to T cells, several peptides per antigen.
  • HLA II molecules There are four different types of HLA II molecules per individual: 2 HLA-DR, 1 HLA-DQ and 1 HLA-DP; the HLA-DR molecule whose ⁇ chain is coded by the DRB1 gene ( 1st gene) is the most expressed.
  • DRB1 gene 1st gene
  • Each allele has its own binding properties; the broad specificity of HLA II molecules and the existence of several isoforms and of a polymorphism mean that each individual recognizes in an antigen, a set of peptides whose nature depends on the HLA II molecules which characterizes it. As there are a large number of HLA II alleles, there is therefore, for a given antigen, a large number of T epitopes, specific to each allele.
  • the distribution of alleles in a given population is not homogeneous: for example, in the French population, which corresponds to a predominantly Caucasian population, only 7 alleles of the DRB1 locus exceed 5%; these are alleles: DRB1 * 0101, DRB1 * 0301, DRB1 * 0401, DRB1 * 0701, DRB1 * 1101, DRB1 * 1301 and DRB 1 * 1501, which represent 64% of the population (4). These same alleles are also the majority in other populations of Europe, where their frequency varies from 53% (Spain) to 82% (Denmark), as well as in North America (55-58%).
  • HLA-DRB3, -DRB4 and -DRB5 ( 2nd gene) molecules which are HLA-DR molecules whose ⁇ chain is not encoded by the DRB1 gene, are also present with significant allelic frequencies in the different populations.
  • Caucasians 9.2% for DRB3 * 0101 (B3), 28.4% for DRB4 * 0101 (B4) and 7.9% for DRB5 * 0101 (B5). They therefore alone cover 45% of the allelic frequency of Caucasian populations.
  • the peptides present in a peptide sequence and which bind all of these alleles include the T epitopes of the majority of the Caucasian population.
  • HLA I molecules HLA-A, -B or -C
  • HLA-A, -B or -C HLA-A, -B or -C
  • bind shorter peptides in principle 9 amino acids, and the binding patterns are specific to each allele.
  • sequences restricted to HLA I molecules have been found in the Env, Pol, Gag and Nef proteins, the latter two being the most frequently recognized (7).
  • WO 99/27954 have described peptides derived from the Nef protein (peptides corresponding to amino acids 66-97 (NI), 117-147 (N2) and 182-205 (N3) of the Nef protein respectively), which comprise T epitopes restricted to HLA I molecules; more precisely, the T epitopes highlighted in said peptides NI, N2 and N3 are the following: TABLE II
  • Gahéry-Ségard et al. (11) recommend associating said peptides NI, N2 and N3 with two peptides derived from the Gag protein (Gl and G2) and with a peptide derived from the Env protein (E); all these peptides are modified at their C-terminal end by addition of a palmitoyl-lysylamide group [Table 1 of (11)].
  • the immunogenicity and tolerance of this lipopeptide vaccine were evaluated in seronegative volunteers.
  • the specific T response is evaluated using a T cell proliferation test.
  • the NI peptide induces T cell proliferation in 5/10 of the vaccinated donors, the N2 peptide induces proliferation only in a single vaccinated donor and the N3 peptide induces proliferation in 4/10 donors. Although a CD4 + T response is observed, it is very random and variable with the peptides N1, N2 and N3.
  • HLA II molecules restricted to HLA II molecules
  • these are peptide sequences (T epitopes) derived from the protein Nef (10): and more particularly from the peptides Nef 13-23. 46-53, 44-81, 69-82, 180-193, 194-210 (Table 4 of 10), which were identified using T clones specific for the Nef protein; the variation in peptides crucially affects the response of T cells to the protein Nef.
  • the aforementioned peptides are restricted to the following HLA II molecules [Table 7 of (10)]: TABLE III
  • T epitopes All the peptides proposed so far correspond to T epitopes, selected for their ability to be conserved among the different isolates; however, they are only specific for specific individuals; in fact, there is an interindividual variability of the T epitopes, which makes it difficult to choose molecules suitable for mass vaccination against AIDS; consequently, the peptides described above are not suitable for the preparation of an immunogenic and vaccine composition capable of stimulating anti-HIV CD4 + T lymphocytes and of generating a protective immune response, regardless of the individual to be protected , because they do not stimulate a protective CD4 + T response in all of the subjects to be treated.
  • Such a set has the property of being effective in a large number of subjects, while the peptides of the prior art are active in a few individuals and are inactive in the majority of other individuals because the latter do not recognize the Nef protein. by the same determinants.
  • the inventors have selected peptides derived from the HIV Nef protein, restricted to predominant HLA II molecules in Caucasian populations and have found that in combination, the selected peptides effectively induce an immunogenic and protective response in a large number individuals.
  • the present invention therefore relates to a mixture of peptides derived from an HIV Nef protein, characterized in that each of said peptides binds to at least three HLA-DR molecules encoded by the alleles selected from the group consisting of alleles HLA DRB1 * 0101, DRB1 * 0301, DRB1 * 0401, DRB1 * 0701, DRB1 * 1101, DRB1 * 1301 and DRB1 * 1501 (molecules DRl, DR3, DR4, DR7, DRU, DR13 and DR15) and at least one molecule HLA-DR coded by the alleles selected from the group consisting of the HLA alleles DRB3 * 0101, DRB4 * 0101 and DRB5 * 0101 (B3, B4 and B5), with a binding activity ⁇ 1000 nM, said mixture of peptides binding l 'all of the above alleles.
  • Such a mixture of peptides makes it possible, surprisingly, to obtain a T CD4 + proliferative response (stimulation of CD4 + T lymphocytes) as well as a stimulation of the CTL response, in the vast majority of the Caucasian population to be protected; we can therefore consider that such a mixture constitutes a first step towards an “universal” immunogenic composition, suitable for use in a vaccine.
  • said peptides are derived from the Nef protein of any strain of HIV-1.
  • said peptides are selected from the group consisting of the peptide corresponding to positions 1-36 (Nefl peptide), the peptide corresponding to positions 66-94 (peptide
  • Nef 4 the peptide corresponding to positions 74-88 (peptide Nef4.4), the peptide corresponding to positions 77-91 (peptide Nef4.5), the peptide corresponding to positions 137-168 (peptide NeflO), the corresponding peptide at positions 139-153 (peptide Nefl 0.2), the peptide corresponding to positions 175-190 (peptide Nefl 2), the peptide corresponding to positions 182-198 (peptide Nefl 3), the peptide corresponding to positions 178-192 (peptide Nefl3. 1), the peptide corresponding to positions 181-
  • Nefl3.3 the peptide corresponding to positions 187-201 (Nefl3.4 peptide) and the peptide corresponding to positions 189-203 (Nefl 3.5 peptide) of the Nef protein, with reference to the Bru strain (13).
  • said mixture of peptides according to the invention is selected from the group consisting of the following mixtures, in which the positions of the peptides are also specified with reference to the positions of the Nef protein of the Bru strain (13):
  • NeflO the peptide corresponding to positions 137-168
  • Nefl2 the peptide corresponding to positions 175-190
  • Nefl3.5 the peptide corresponding to positions 189-203
  • Nef4 binds with good affinity to molecules DRB1 * 0101, 0401, 1 101, 1501, DRB5 * 0101 and DRB4 * 0101 - Nef 4.1 binds with good affinity to molecules DRB 1 * 0101,
  • Nef 4.2 binds with good affinity to the molecules DRB1 * 0101, 0401, 1101, 1501 and DRB5 * 0101 and DRB4 * 0101
  • NeflO binds with a good affinity to the DRB 1 * 0101, 0301, 0401, 0701, 1101, DRB5 * 0101 and DRB4 * 0101 molecules,
  • Nef 10.1 binds with good affinity to DRB 1 * 0101, 0301, 0401, 0701, 1101, DRB5 * 0101 and DRB4 * 0101 molecules,
  • Nefl2 binds with good affinity to DRB1 * 0701, 1101, 1501, DRB3 * 0101 and DRB4 * 0101 molecules
  • - Nefl3 binds with good affinity to DRB1 * 0301 molecules
  • Nef 13.1 binds with a good affinity to the molecules DRB1 * 0301, 0701, 1101, 1301 and DRB3 * 0101 and DRB5 * 0101,
  • Nef 13.2 binds with a good affinity to the molecules DRB1 * 0301, 0701, 1101, 1301 and DRB3 * 0101 and DRB5 * 0101,
  • Nef 13.3 binds with a good affinity to the molecules DRB1 * 0301, 0701, 1101, 1301 and DRB5 * 0101,
  • Nef 13.4 binds with good affinity to DRB1 * 0701, 1101, 1301 and DRB5 * 0101 molecules
  • - Nef 13.5 binds with good affinity to DRB1 * 0701 molecules
  • the present invention also relates to an anti-HIV immunogenic composition, characterized in that it comprises at least one mixture of peptides as defined above, associated with at least one pharmaceutically acceptable vehicle and optionally with at least one adjuvant.
  • the adjuvants used are adjuvants conventionally used in vaccine compositions, such as alumina hydroxide and squalene.
  • said peptides are either in the form of lipopeptides, or incorporated into a recombinant virus, a viral vector for gene therapy (adenovirus, etc.), or included in a protein and in particular a recombinant protein (Leclerc C. et al., Int. Rev. Immunol., 1994, 11, 2, 123-132; Janssen R. et al., Int. Rev. Immunol., 1994, 11, 2, 113-121), either chemically modified. In the latter case, they include, for example, unnatural modifications such as amino acids D, pseudopeptide bonds or modifications of the C- or N-terminal ends.
  • the lipid part of the lipopeptide is in particular obtained by addition of a lipid motif on an ⁇ -amino function of said peptides or on a reactive function of the side chain of an amino acid of the peptide part; it can comprise one or more chains derived from C 4 fatty acids. 20 , possibly branched or unsaturated (palmitic acid, oleic acid, linoleic acid, linolenic acid, 2-amino hexadecanoic acid, pimelautide, trimetauxide) or a derivative of a steroid.
  • the process for the preparation of such lipopeptides is notably described in International Applications WO 99/40113 or WO 99/51630.
  • the preferred lipid part is in particular represented by an N ⁇ -acetyl-lysine N ⁇ (palmitoyl) group, also called Ac-K (Pam).
  • said mixture of peptides is combined:
  • peptides or lipopeptides containing one or more CD8 + epitopes (recognized specifically by cytotoxic T lymphocytes and presented by HLA I molecules) and more particularly CD8 + epitopes derived from an HIV-1 protein (see Table II) and / or - to other peptides containing a CD4 + epitope, which peptides bind at least one HLA-DR molecule encoded by the alleles selected from the group consisting of the HLA alleles DRB 1 * 0101, DRB 1 * 0301, DRB 1 * 0401, DRB 1 * 0701, DRB1 * 1101, DRB1 * 1301 and DRB1 * 1501 (molecules DRl, DR3, DR4, DR7, DRU, DR13 and DR15) or at least one HLA-DR molecule encoded by the selected alleles in the group consisting of the HLA alleles DRB3 * 0101, DRB4 * 0101 and DRB5 * 01
  • peptides comprising multiple CD4 + epitopes, such as the tetanus toxin peptide TT (positions 830-846), the hemagglutinin peptide from In ⁇ uenza HA (positions 307-319), PADRE (Pan DR Epitope , Alexandre J. et al., Immunity, 1994, 1, 9, 751-761), the peptide 45-69 Nef HIV-1 and the peptide LSA3 of Plasmodium falciparum and / or
  • one or more peptides or lipopeptides containing one or more B epitopes more particularly B epitopes derived from an HIV-1 protein, specifically recognized by antibodies directed against the latter.
  • Nef peptides according to the invention included in said mixture were advantageously selected using an HLA-DR / peptide binding test comprising:
  • the purification of the HLA-DR molecules of interest that is to say those concerning more than 5% of a given population and in particular the HLA DR1 molecules
  • the HLA-DR molecules thus purified, with different concentrations of overlapping fragments and entirely covering the sequence of the Nef protein and with an RI reagent or tracer consisting of a peptide fragment associated with a non-radioactive marker, such as biotin and whose sequence is different from said peptides; the RI reagent or tracer is chosen so that it has an affinity for one of the HLA-DR molecules of interest, such that it can be used at a concentration ⁇ 200 nM,
  • This approach makes it possible to define immunogenic compositions including peptides which bind to the greatest number of different HLA-DR molecules and which can thus be advantageously protective for the majority of patients, even if their HLA molecules are not known.
  • This approach also has the advantage of allowing the selection of significantly more specific peptides with respect to HIV-1 than approaches seeking to select peptides on the basis of their capacity to stimulate CD4 + T lymphocytes.
  • the incubation conditions are specific to each HLA-DR molecule (incubation time, pH, RI reagent, HLA-DR or peptide concentration).
  • the RI reagent is selected from the group consisting of the following sequences:
  • AAYAAAKAAALAA (YKL) (SEQ ID NO: 4), specific for the allele DRB1 * 0701, • TERVRLVTRHIYNREE (Bl 21-36) (SEQ ID NO: 5), specific for the allele
  • said immunogenic composition advantageously comprises sow the sequences coding for the peptides as defined above.
  • naked DNA for immunization constitutes an effective vaccination approach: it consists in injecting into the host organism to be vaccinated, a
  • the present invention also relates to a vaccine, characterized in that it includes an immunogenic composition as defined above.
  • the present invention also relates to peptides derived from an HIV Nef protein, characterized:
  • CD4 + epitope capable of having a binding activity ⁇ 1000 nM vis-à-vis at least one HLA II molecule (HLA-DR) predominant in Caucasian populations ( 1st gene and / or 2 ee gene), as defined above, to be recognized by specific CD4 + T lymphocytes of said peptides and to stimulate specific T CD4 + lymphocytes of said peptides and
  • Such peptides have the advantage of binding to at least one HLA-DR molecule encoded by the HLA alleles DRB1 * 0101, DRB 1 * 0301, DRB1 * 0401, DRB1 * 0701, DRB1 * 1101, DRB1 * 1301 and DRB1 * 1501 (DR1, DR3, DR4, DR7, DRU, DR13 and DR15 molecules), and / or to at least one HLA-DR molecule encoded by the HLA alleles DRB3 * 0101, DRB4 * 0101 and DRB5 * 0101 (B3, B4 and B5), with a binding activity ⁇ 1000 nM.
  • Nef 1.2, Nef 1.3, Nef 1.4, Nef 3, Nef4.2, Nef4.6, Nef 4.1, Nef 9, Nefl 0.5, Nefl l and Nef 13.6 bind to less than three HLA-DRB1 molecules, with a binding activity ⁇ 1000 nM and
  • the peptides Nefl, Nef4, Nef 4.4, Nef4.5, NeflO, Nefl 0.2, Nefl 2, Nefl3, Nefl3.1, Nefl3.2, Nefl3.3, Nefl3.4, and Nefl3.5 bind to at least three HLA-DRB1 molecules and at least one HLA-DR molecule encoded by a DRB3, DRB4 or DRB5 allele, with a binding activity ⁇ 1000 nM.
  • binding activities make it possible to use said peptides:
  • immunogenic compositions insofar as they are capable of inducing a proliferative response of CD4 + T cells and therefore of helping to induce a cytotoxic or humoral response in the majority of Caucasian populations. They can therefore advantageously be incorporated into a vaccine composition.
  • a diagnostic reagent preferably in the form of tetramers, for assessing the immune status of a healthy individual or of patients seropositive for HIV or suffering from AIDS.
  • a diagnostic reagent preferably in the form of tetramers, for assessing the immune status of a healthy individual or of patients seropositive for HIV or suffering from AIDS.
  • the present invention therefore also relates to a diagnostic reagent, characterized in that it is selected from the group consisting of a mixture of peptides as defined above or one of the peptides also as defined above above, said peptides being optionally labeled or complexed, in the form of multimeric complexes.
  • said diagnostic reagent consists of mixtures of peptides obtained from the peptides Nefl, Nef4, NeflO, Nefl2 and Nefl3.
  • the present invention also relates to a method for evaluating the immune state of an individual, characterized in that it comprises a step of detecting the presence of CD4 + T cells specific for the Nef peptides as defined above. ; said detection is advantageously carried out by one of the following tests: proliferation test, ELISPOT test [see for example International Application WO 99/51630 or Gahéry-Ségard et al. (11)] or flow cytometry in the presence of multimeric complexes formed from said Nef peptides. More specifically:
  • a suspension of cells (PBMC, PBMC depleted in CD8 + cells, T lymphocytes previously enriched by an in vitro culture step with the peptides selected according to the invention or cloned T lymphocytes) is cultured for 3 to 5 days in the presence of the selected peptides and, if necessary, suitable presenting cells such as dendritic cells, autologous or heterologous PBMCs, lymphoblastoid cells such as those obtained after infection with the EBV virus or genetically modified cells. Cell proliferation is measured by incorporation of tritiated thymidine into cell DNA.
  • the peptides selected in accordance with the invention make it possible to reveal in the initial suspension the presence of cells specific for these peptides.
  • the ELISPOT test makes it possible to reveal the presence of T cells specific for a peptide selected in accordance with the invention and secreting IFN- ⁇ .
  • the T cells are revealed by measuring the secretion of IFN- ⁇ after incubation of PMBCs of patients with the peptides selected according to the invention, in accordance with the method described in Gahéry-Ségard et al., 2000
  • PBMC peripheral blood mononuclear cells
  • the biological sample prior to bringing the biological sample into contact with said complex, it is enriched in CD4 + T cells, by bringing it into contact with anti-CD4 antibodies, to enrich said sample.
  • the tetramers are prepared, as specified, for example in E.J. Novak et al. (J. Clin. Investig., 1999, 104, R63-R67) or in M.J. Kuroda et al. (J. Virol., 2000, 74, 18, 8751-8756).
  • the tetramers are produced by incubating, for 72 hours at 37 ° C. and in a 10 mM citrate phosphate buffer, 0.15 M NaCl at a pH of between 4.5 and 7, soluble and biotinylated HLA II molecules with a 10 excess of Nef peptides identified and selected in accordance with the invention.
  • the tetramerized form is obtained by adding to the preparation of streptavidin marked with a fluorochrome in an amount four times less (mole in mode) than of HLA II molecules. The whole is incubated overnight at room temperature.
  • a suspension of cells PBMC, PBMC depleted in CD8 + cells, T lymphocytes previously enriched by an in vitro culture step with the Nef peptides selected in accordance with the present invention or cloned T lymphocytes
  • PBMC PBMC depleted in CD8 + cells, T lymphocytes previously enriched by an in vitro culture step with the Nef peptides selected in accordance with the present invention or cloned T lymphocytes
  • one or more tetramers (10 to 20 mg / ml) for 1 to 3 hours.
  • the suspension is analyzed by flow cytometry: the labeling of the cells by the tetramers is visualized by the fact that these constructions are fluorescent.
  • Flow cytometry makes it possible to separate the cells marked by the tetramers of unlabeled cells and thereby perform cell sorting.
  • the present invention thus further relates to a method for sorting T lymphocytes specific for HIV, characterized in that it comprises at least the following stages: - incubation or contacting, for 1 to 3 hours, of a suspension of cells to be sorted with one or more tetramers formed from Nef peptide complexes as defined above / soluble and biotinylated HLA II molecule, and conjugated to streptavidin labeled with a fluorochrome,
  • the HLA-DR molecules are purified from different homozygous EBV lines (14) by immunoaffinity.
  • the specific monoclonal antico ⁇ s of HLA-DR molecules is notably that described in Southwood et al. (14) or that described in Posch et al. (15).
  • Antico ⁇ s are purified from culture supernatants on columns of
  • Protein A-Sepharose These antico ⁇ s are coupled on Sepharose 4B or Protein A-Sepharose columns for the purification of HLA-DR molecules.
  • HLA-DR molecules Peptide binding tests for HLA-DR molecules are tests in competition with an immunoenzymatic revelation, initially developed by Hill on the HLA-DR molecule (16). They are carried out in 96-well plates, which makes it possible to study numerous samples in the same experiment. Briefly, the purified HLA-DR molecules are incubated with a biotinylated peptide which serves as a tracer and different concentrations of the peptide to be tested.
  • each sample is neutralized, then 100 ⁇ l of each sample are transferred to an ELIS A plate previously sensitized by the specific mono-ethical antico ⁇ s of HLA-DR molecules.
  • the complexes HLA-DR molecules / biotinylated peptides, fixed to the bottom of the plate by means of the specific antico ⁇ s monomo ⁇ hique of molecules HLA-DR are revealed by means of conjugate streptavidin-phosphatase and a fluorescent substrate.
  • the activity of each peptide is characterized by the concentration of this peptide which inhibits 50% of the binding of the biotinylated peptide (CI).
  • the alleles studied are all the alleles of the French population whose frequency exceeds 5% of the population. These are the alleles DRB1 * 0101, DRB1 * 0301, DRB1 * 0401,
  • DRB1 * 0701, DRB1 * 1101, DRB1 * 1301 and DRB1 * 1501 (Table I). They alone represent 53 to 82% of the alleles of the Caucasian populations and are part of different specificities of the HLA-DR series.
  • biotinylated peptides are the determining factor in the specificity of the test. Most of the cells used have two different HLA-DR molecules (encoded by two alleles) which are both purified by a specific monomoic antico ⁇ s of HLA-DR molecules and both recognized by the same antico ⁇ s. In order to study without ambiguity the binding of a peptide to the DRB1 allele, it is necessary to ensure that the biotinylated peptide binds this allele and does not bind the product of the other allele.
  • the concentration of MHC II molecules the concentration of the biotinylated peptide, the incubation pH and the incubation time were optimized as specified in Table V below.
  • the frequencies indicated are the allelic frequencies in France and are representative of those of the Caucasian population. They come from Colombani (4)
  • Table VII below illustrates the binding activity of the peptides according to the invention, measured under the conditions specified above:
  • OOOOK 009 OOOOK OOOK 009 8 o ⁇ z OOOOK OAM OOOOK ( ⁇ oz-68i) ⁇ - ⁇ i N
  • OOOOK 9 09 OOOOK o ⁇ 81 ⁇ 6 OOOOK ⁇ A OOOOK (S6I-I8l) Z ' ⁇ iJ 9 N
  • OOOOK o ⁇ 8? OOOOK A98 8 o ⁇ OOOOK ⁇ zi OOOOK (Z6I-8A1) ITIPN o ⁇ 9 01 o ⁇ z OOOOK OSL 8f oei osz ⁇ ⁇ s ooo ⁇ 86I "Z8l) ⁇ U 3 N
  • a proliferation test is carried out in vitro.
  • PBMC peripheral blood cells
  • CD4 + T cells There is indeed a stimulation of CD4 + T cells.
  • Other cells can be used: PBMC depleted in cells
  • CD8 + T lymphocytes previously enriched by an in vitro culture step with the peptides as defined above or cloned lymphocytes.
  • the enrichment protocol is as follows: the PMBCs separated on a ficoll gradient are cultured at 37 ° C. in the presence of 0.1 to 10 mg / ml of peptides in RPMI medium supplemented with 10% human serum. At the 7 th and ll ee day of culture, 50 units of recombinant human IL-2 are added to the culture. Cells were harvested on 14 th day.
  • EXAMPLE 3 ELISPOT.
  • the ELISPOT makes it possible to detect cells specific for a peptide and secreting a given cytokine.
  • the wells are washed with PBS and saturated with RPMI medium containing 10% calf serum for 2 hours at 37 ° C.
  • Nef peptides as defined in the invention are then added at different concentrations (10, 5 and 1 ⁇ g / ml).
  • the effector cells (PBMC, PBMC depleted in CD8 + cells, T lymphocytes previously enriched by an in vitro culture step with the Nef peptides or cloned lymphocytes) are added to the 96-well plates at the rate of 20,000 cells / well.
  • the culture is incubated for 24 hours at 37 ° C in an atmosphere containing 5% CO 2 .
  • the plates are then washed and incubated for 2 hours with 100 ⁇ l of a rabbit antiserum specific for human IFN- ⁇ .
  • an anti-rabbit IgG antico ⁇ s conjugated to pure biotin from streptavidin conjugated to alkaline phosphatase are added successively for 1 hour. Finally, the spots are revealed using a chromogenic substrate for alkaline phosphatase. The spot counting is done under a microscope. Negative controls are given by the wells containing no peptides. Positive controls are supplied by wells containing mitogenic agents such as ionomycin (500 ng / ml) and phytohemagglutinin (PHA) (10 ⁇ g / ml).
  • mitogenic agents such as ionomycin (500 ng / ml) and phytohemagglutinin (PHA) (10 ⁇ g / ml).

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EP02702459A 2001-02-08 2002-02-07 Mischung aus peptidfragmenten des nef-proteins und ihre anwendungen Withdrawn EP1453853A2 (de)

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FR0101700 2001-02-08
FR0101700A FR2820425B1 (fr) 2001-02-08 2001-02-08 Melange de peptides issus d'une proteine nef et leurs applications
PCT/FR2002/000471 WO2002062834A2 (fr) 2001-02-08 2002-02-07 Melange de peptides issus d'une proteine nef et leurs applications

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GB0710529D0 (en) 2007-06-01 2007-07-11 Circassia Ltd Vaccine
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FR2771640B1 (fr) * 1997-12-03 2000-02-11 Inst Nat Sante Rech Med Micelles mixtes de lipopeptides pour l'induction d'une reponse immunitaire et leurs utilisations a des fins therapeutiques
FR2794370B1 (fr) * 1999-06-03 2003-10-17 Biovector Therapeutics Fragments proteiques polyepitopiques, leur obtention et leurs utilisations notamment en vaccination

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IL157121A0 (en) 2004-02-08
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WO2002062834A2 (fr) 2002-08-15
JP2005506283A (ja) 2005-03-03
FR2820425B1 (fr) 2004-01-02
WO2002062834A3 (fr) 2004-07-01
US20040115622A1 (en) 2004-06-17

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