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
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • 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
  • A61P31/20—Antivirals for DNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5047—Cells of the immune system
  • G01N33/505—Cells of the immune system involving T-cells
• • 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
  • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/20011—Papillomaviridae
  • C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• • C—CHEMISTRY; METALLURGY
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  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/20011—Papillomaviridae
  • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
  • G01N2333/01—DNA viruses
  • G01N2333/025—Papovaviridae, e.g. papillomavirus, polyomavirus, SV40, BK virus, JC virus
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to a mixture of peptides derived from the E6 and / or E7 proteins of a papillomavirus involved in cervical cancer, such as HPV16 (papillomavirus genotype 16), HPV18, HPV30, HPV31, HPV32 , HPV33, HPV34, HPV35, HPV39, HPV40, HPV42, HPV43, HPV44, HPV45, HPV51, HPV52, HPV56, HPV57 and HPV58, for example as well as to its applications as a medicament (in immunogenic compositions, capable of stimulating the production of anti-HPV CD4 + T cells in vivo and therefore useful for vaccination against cervical and other cancers) or as a reagent for diagnosing HPV-specific T cells , especially to assess the immune status of patients.
• HPV16 papillomavirus genotype 16
• HPV18 papillomavirus genotype 16
• HPV30 papillomavirus genotype 16
• the present invention also relates to a mixture of peptides derived from the E6 and / or E7 proteins of a papillomavirus involved in benign lesions of the skin (warts for example), such as HPV 10, HPV3 or HPV4 and to its applications in as a medicine.
• HPV Human papillomaviruses
• HPV DNA is very often detected by PCR in patient biopsies (1). 20 to 50% of penile cancers and 70% of anal cancers reveal the presence of HPV DNA. There are in fact more than 100 different genotypes of papillomavirus each having its own pathogenicity. The association between HPV infection and cervical cancer varies according to genotypes. A distinction is made between strains like the HPV6 and HPV11 viruses at low or no risk of malignant transformation, from high risk strains like the HPV 16 and HPV 18 viruses. Whatever the country, HPV16 is found in 40 to 60% of cancers cervix while HPV 18 is present in 10 to 20% of cases.
• the pRb and p53 genes are mutated and inactive in the cell lines of the cervix showing no infection by HPV whereas they are not in the infected lines. All of these observations strongly suggest that the proteins E6 and E7 are key components of HPV infection and the induction of cancerous states. In the absence of specific anti-viral treatments for HPV infections, the development of anti-HPV vaccines constitutes one of the promising avenues for combating the forms of cancer induced by these viruses.
• peptides or subunits therefore appears to be very interesting.
• suitably chosen peptides are capable of recruiting both cytotoxic T lymphocytes (CTL) and helper T lymphocytes (helper T lymphocytes) of the Th1 type directed specifically against the transformed cells.
• CTL cytotoxic T lymphocytes
• helper T lymphocytes helper T lymphocytes
• the proteins E6 and E7 constitute privileged targets, because they participate directly in the cancerization of the cells and their early expression after the infection persists in the transformed cell.
• Different vaccination strategies using peptides derived from these two proteins have thus been recommended. These strategies are based on the fact that CD4 + T lymphocytes have a major role in the establishment of immune responses and in particular of CTLs.
• 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 the HLA II molecules, which are heterodimers, expressed on the cells presenting antigens (APC) and which present to CD4 + T lymphocytes, the T epitopes of said antigens.
• APC antigen presenting antigens
• CD4 + T lymphocytes CD4 + T lymphocytes
• These molecules are capable of binding a large repertoire of peptides having very different sequences, which allows them to present to T cells, several peptides per antigen.
• HLA-DR HLA-DR
• 1 HLA-DQ HLA-DQ
• 1 HLA-DP 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, DRB 1 * 0701, DRB1 * 1,101, DRB 1 * 1301 and DRB1 * 1501, which represent 64% of the population (4). These same alleles are also the majority in other populations in Europe, where their frequency varies from 53% (Spain) to 82% (Denmark), as well as in North America (55-58%).
• the 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 Caucasian populations: 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.
• One of the most used means to define the CD4 + T epitopes of the auxiliary type is to measure the capacity of the peptides to proliferate the mononuclear cells of individuals having been in contact with the antigen considered.
• the antigens can be in the form of whole proteins or T epitopes.
• the T epitope of the E6 protein is preferably the epitope corresponding to positions 29-38 of said protein and the preferred epitopes of the E7 protein are those corresponding respectively to positions: 11-20, 49-57, 82-90, 86-93; the use of an E7 protein detoxified by deletion of the Rb protein binding site is also envisaged.
• Patent EP 0 561 885 (University of Queensland and CSL Ltd) describes a subunit vaccine against the HPV 16 papilloma virus, which comprises the DRAHYNI sequence of the E7 protein (positions 48-54), considered to induce a significantly higher immune response than other epitopes.
• mice with peptides derived from proteins E6 and E7 of HPV16 protects against a virulent test constituted by cancer cells transformed by HPV 16, they have in particular shown the advantage of the protein E7 of HPV16 and sought to identify T epitopes in this protein. To do this, they produced synthetic peptides of 15 amino acids, the sequence of which overlaps on 5 amino acids, so as to obtain a series of peptides representing the entire sequence of the E7 protein.
• E6 of the HPV 16 genotype causes the proliferation of the cells of 13 individuals out of 63 tested (Immunol, 1996, 3, 205). They also studied the responses observed with the E7 protein and showed that little response is observed with this protein.
• EP 0 561 885 E7 48-54, 44-54, 44-62, 44-57, 44-56. 44-60, 44-48, 38-41, 10-14, 11-14
• EP 0 386 734 E7 12-27, 12-23, 12-20, 12-19 and 36-52
• a response common to many patients, to a particular peptide can then result from a sampling bias and not from the effective capacity of a peptide to be recognized by all patients.
• the persistence of the antigen can induce a state of immune tolerance against the T epitopes of the virus, the best recognized by T cells, as has been shown. in a transgenic mouse model for the E7 protein of HPV (T. Doan et al, J Virol., 1999, 73, 6166).
• the response is often weaker in patients who have eliminated the virus compared to infected patients (T. Tsukui et al., 1996 cited above).
• the most interesting epitopes could have disappeared from the immune response which would be maintained for less stimulating determinants but which would not succeed in eliminating the virus.
• the peptides proposed so far correspond to T epitopes, which are specific only for particular individuals; in fact, there is an interindividual variability of the T epitopes, which makes it difficult to choose molecules suitable for mass vaccination against HPV 16; accordingly, the peptides described above are not suitable for the preparation of an immunogenic composition and vaccine capable of stimulating CD4 + T lymphocytes anti- HPV 16 and generating a protective immune response, 'irrespective 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 proteins.
• E6 and E7 of HPVl ⁇ by the same determinants. To do this, the inventors have selected peptides derived from the proteins E6 and E7 of one of the aforementioned HPVs, in particular from HPV16, restricted to the predominant HLA II molecules in Caucasian populations and have found that in combination, the selected peptides induce indeed an immunogenic and protective response in a large number of individuals.
• the present invention therefore relates to a mixture of peptides, derived from an E6 protein and / or an E7 protein from an HPV involved in cervical cancer or benign lesions of the skin, characterized in that each of the peptides included in said mixture binds to at least one HLA-DRB1 molecule ( 1st gene) the frequency of which is greater than 5% in the Caucasian population and possibly to at least one HLA-DRB3, HLA-DRB4 or HLA molecule - DRB5 (2 nd gene), with a binding activity ⁇ 1000 nM, preferably ⁇ 800 nM, said mixture of peptides binding at least eight molecules HLA class II whose frequency is greater than 5% in the Caucasian population, encoded by the alleles selected from the group consisting of the HLA alleles DRB1 * 0101, DRB1 * 0301, DRB1 * 0401, DRB1 * 0701, DRB1 * 1101, DRB1 * 1301 and DRB1 * 1501 (molecules
• Such a mixture of peptides makes it possible, surprisingly, to obtain a proliferative CD4 + T response (stimulation of CD4 + T lymphocytes) as well as stimulation of the CTL response, in the vast majority of the Caucasian population to be protected and whatever that either the HPV concerned; we can therefore consider that such a mixture constitutes a first step towards an “universal” immunogenic composition, suitable for use in a vaccine.
• the peptides derived from an E6 protein of HPV are derived from HPV16 and are selected from the group consisting of: (a) a peptide comprised between positions 14 and 46, selected from the group consisting of the peptide corresponding to positions 14-34 and the peptide corresponding to positions 14-46 (SEQ ID NO: 8, 19),
• sequence with respect to a reference sequence is assessed according to the percentage of amino acid residues which are identical, when the two sequences are aligned so as to obtain the maximum correspondence between them.
• a peptide having an amino acid sequence having at least X% identity with a reference sequence comprising Y amino acids is defined, in the present invention as a peptide whose sequence can include up to Y- X 'alterations for Y amino acids from the reference sequence and reformulated for a sequence of 100 amino acids.
• the similarity of a sequence compared to a reference sequence is assessed according to the percentage of amino acid residues which are identical or which differ by conservative substitutions, when these two sequences are aligned so as to obtain the maximum of correspondence between them.
• conservative substitution is intended to mean the substitution of one amino acid with another which has similar chemical properties (size, charge or polarity), which generally does not modify the functional properties of the peptide.
• the peptides derived from an HPV E6 protein of said mixture, as defined in h), are selected from the group consisting of: - the peptides of 15 amino acids included in the peptide corresponding to positions 14-34 or overlapping this peptide, chosen from the peptides corresponding respectively to positions 20-34, 24-38 and 28-42 (SEQ ID NO: 21-23) , - the peptides of 15 amino acids included in the peptide corresponding to positions 30-50 or overlapping this peptide, chosen from the peptides corresponding respectively to positions 31-45 and 36-50 (SEQ ID NO: 24-25);
• the peptides derived from an E7 protein are derived from HPV16 and are selected from the group consisting of the peptide corresponding to positions 1-20, the peptide corresponding to positions 7-27, the peptide corresponding to positions 65-87 and the peptide corresponding to positions 78-98 of said HPV16 E7 protein (SEQ ID NO: 14, 15, 17, 18) and the peptides, preferably from 15 to 20 amino acids, having an amino acid sequence having at least 60% identity or at least 80% similarity and preferably at least 70% identity or at least 99% similarity with the peptides as defined above, said peptides being of identical size, included in the peptides as defined above or else overlapping these peptides, with exclusion of the peptides corresponding to positions 3-25 and 79-97 of the E7 protein of HPV16 (SEQ ID NO: 57, 58).
• the peptides derived from an E7 protein of said mixture included in the peptides (SEQ ID NO: 14, 15, 17, 18) as defined above or even overlapping these peptides, are selected from the group consisting of peptides corresponding respectively to positions 6-20, 9-23, 13-27, 65-79, 67-81, 72-86, 77-91 and 84-98 of the E7 protein of HPV16 (SEQ ID NO: 45- 52).
• said mixture of peptides according to the invention is selected from the group consisting of the following mixtures: * a mixture of peptides derived from the E6 protein of HPVl ⁇ , comprising: the peptide corresponding to positions 14-34 or to positions 14-46, the peptide corresponding to positions 30-50 and the peptide corresponding to positions 44-67 or positions 45-67.
• a mixture of peptides derived from the E7 protein of HPV16 comprising: the peptide corresponding to positions 1-20 and the peptide corresponding to positions 7-27.
• a mixture of peptides derived from the E7 protein of HPV16 comprising: the peptide corresponding to positions 65-87 and the peptide corresponding to positions 78-98,
• the E6 peptide (14-34) binds with a good affinity to the DRB1 * 0301, 0701 and 1501 molecules,
• the peptide E6 (30-50) binds with a good affinity to the molecules DRB 0101, 0301, 0401, 1101, 1301, 1501, DRB5 * 0101 and DRB4 * 0101,
• the peptide E6 (61-80) binds with a good affinity to the molecules DRB1 * 0301, 1101, 1501, DRB3 * 0101 and DRB5 * 0101
• - the E6 peptide (76-95) binds with a good affinity to the molecules DRB1 * 0101, 1101, 1301, 1501, DRB5 * 0101 and DRB4 * 0101
• the peptide E6 (91-119) binds with a good affinity to the molecules DRB1 * 0101, 0301, 0401, 0701, 1501 and DRB5 * 0101, - the peptide E7 (1-20) binds with a good affinity to the molecules
• the peptide E7 (7-27) binds with a good affinity to the molecules DRB1 * 0101, 0301, 0401, 1101, 1301, DRB3 * 0101, DRB5 * 0101 and DRB4 * 0101,
• the peptide E7 (65-87) binds with a good affinity to the molecules DRB1 * 0101, 0301, 0401, 701, 1501 and DRB3 * 0101,
• the peptide E7 (78-98) binds with a good affinity to the molecules DRB1 * 0101, 0701, 1101, 1501, DRB5 * 0101 and DRB4 * 0101.
• the sequences of these different peptides are illustrated in
• the present invention also relates to an anti-HPV immunogenic composition, characterized in that it comprises a mixture of peptides derived from an E6 protein from HPV and / or a mixture of peptides derived from an E7 protein from HPV , 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 aluminum hydroxide and squalene.
• said peptides are either in the form of lipopeptides, or incorporated into a recombinant virus, a viral vector for gene therapy (adeno virus ...), or included in a protein and in particular a protein recombinant (Leclerc C. et al., Int. Rev. Immunol., 1994, 11, 2, 123-132; Janssen R. et al., Int. Rev. Immunol., 1994, 11, 2, 113-121) , or chemically modified. In the latter case, they comprise, for example, unnatural modifications such as amino acids D, pseudo-peptide bonds or modifications of the C- or N-terminal ends.
• the lipid part of the lipopeptide is obtained in particular by adding 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 may comprise one or more chains derived from C 4- 0 fatty acids, optionally 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 associated: - with one or more peptides or lipopeptides containing one or more CD8 + epitopes (recognized specifically by cytotoxic T lymphocytes and presented by HLA I molecules) and more particularly the CD8 + epitopes derived an HPV protein, in particular an HPV16 protein (Ressing et al; van Driel et al.) and / or
• peptides comprising multiple CD4 + epitopes, such as the tetanus toxin peptide TT (positions 830-846), the influenza hemagglutinin peptide HA (positions 307-319), PADRE (Pan DR Epitope , Alexandre J. et al., Immunity, 1994, 1, 9, 751-761) and the LSA3 peptide of Plasmodium falciparum and / or
• one or more peptides or lipopeptides containing one or more B epitopes more particularly B epitopes derived from an HPV16 protein (Tindle et al.), specifically recognized by antibodies directed against the latter.
• the E6 and E7 peptides according to the invention, included in the mixtures, as defined above were advantageously selected using an HLA-DR / peptide binding test comprising: - the purification of the HLA-DR molecules of interest, i.e. those concerning more than 5% of a given population and in particular the HLA molecules
• the HLA-DR molecules thus purified, with different concentrations of overlapping fragments and entirely covering the sequence of the E6 protein or of the E7 protein and with an RI reagent or tracer consisting of a peptide fragment associated with a non-marker radioactive, 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, - the transfer of the complexes obtained onto an ELISA type plate, previously sensitized with an antibody specific for all HLA-DR,
• This approach also has the advantage of allowing the selection of significantly more specific peptides vis-à-vis 1 ⁇ PV16 than approaches seeking to select peptides on the basis of their capacity to stimulate CD4 + T lymphocytes (proliferation tests).
• 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:. PKYVKQNTLKLAT (HA 306-318) (SEQ ID NO: 1), specific for alleles DRB1 * 0101, DRB1 * 0401, DRB1 * 1101,. EAEQLRAYLDGTGVE (A3 152-166) (SEQ ID NO: 2), allele specific
• AAYAAAKAAALAA (YKL) (SEQ ID NO: 4), specific for the DRB1 * 0701 allele,.
• TERVRLVTRHIYNREE (Bl 21-36) (SEQ ID NO: 5), allele specific
• AGDLLAIETDKATI E2 / E168 (SEQ ID NO: 7), specific for the DRB4 * 0101 allele.
• Other RI reagents can be used, in particular those described in Southwood et al. (24).
• said immunogenic composition advantageously comprises 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, naked DNA coding for a protein antigen; this DNA allows a prolonged synthesis of the antigen by the host cells as well as a durable presentation of this antigen to the immune system.
• 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 E6 protein from HPV, in particular from HPV16 and / or from an E7 protein from HPV, in particular from HPV 16, characterized: - in that they contain a CD4 + epitope, capable of having a binding activity ⁇ 1000 nm, preferably ⁇ 800 nM with respect to at least one predominant HLA II molecule (HLA-DR) in Caucasian populations ( 1st gene and / or 2nd gene), as defined above, to be recognized by CD4 + T lymphocytes specific for said peptides and to stimulate CD4 + T lymphocytes specific for said peptides and
• a CD4 + epitope capable of having a binding activity ⁇ 1000 nm, preferably ⁇ 800 nM with respect to at least one predominant HLA II molecule (HLA-DR) in Caucasian populations ( 1st gene and / or 2nd gene), as defined above, to be recognized by CD4 + T lymphocytes specific for said peptides and
• the subject of the present invention is a diagnostic reagent, characterized in that it is selected from one of the peptides E6 and E7, as defined above. above, said peptides being optionally labeled or complexed, in the form of multimeric complexes.
• 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 E6 and / or E7 peptides, such 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. (27)] or flow cytometry in the presence of multimeric complexes formed from said peptides E6 and / or E7.
• 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 the PMBCs of the patients with the peptides selected according to the invention, in accordance with the method described in Gahéry-Ségard et al., 2000 ( 27).
• a biological sample preferably peripheral blood mononuclear cells (PBMC)
• 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 EJ. 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 E6 or E7 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 E6 or E7 peptides selected in accordance with the present invention or cloned T lymphocytes is brought into contact ) with one or more tetramers (10 to 20 mg / ml) for 1 to 3 hours. After washing, 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 from the unmarked cells and thus to carry out cell sorting.
• the present invention thus also relates to a method for sorting T lymphocytes specific for 1 ⁇ PV16, characterized in that it comprises at least the following steps: - incubation or contact, for 1 to 3 hours, of a suspension of cells to be sorted with one or more tetramers formed from E6 and / or E7 peptide complexes as defined above / soluble and biotinylated HLA II molecule, and conjugated to streptavidin labeled with a fluorochrome, - analysis by flow cytometry and
• the invention also comprises other provisions, which will emerge from the description which follows, which refers to examples of implementation of the method which is the subject of the present invention as well as to the accompanying drawing, in which :
• FIG. 1 shows the proliferation and secretion of IFN- ⁇ cells of a patient with bowenoid papulosis and who spontaneously resolved his infection.
• the patient is DRB1 * 1601/1501 DRB5 * 0101.
• the E6 peptide (45-67) is a good ligand for DRB1 * 1501
• the E7 peptide (7-27) is a good ligand for DRB5 * 0101 (see also Table VIII).
• HLA-DR molecules are purified from different EBV lines homozygous by immunoaffinity.
• the monomorphic antibody specific for HLA-DR molecules is in particular that described in Southwood et al. (24) or that described in Posch et al. (25). Antibodies are purified from culture supernatants on columns of
• Protein A-Sepharose are coupled on Sepharose 4B columns or
• Protein A-Sepharose 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, originally developed by Hill on the HLA-DR molecule (26). 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. After 24 to 72 hours of incubation, the samples are neutralized, then 100 ⁇ l of each sample are transferred to an ELIS A plate previously sensitized by the monomorphic antibody specific for the HLA-DR molecules.
• HLA-DR molecule / biotinylated peptide complexes fixed to the bottom of the plate by means of the monomorphic antibody specific for HLA-DR molecules, are revealed by means of a streptavidin-phosphatase conjugate 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 alleles of the French population whose frequency exceeds 5% of the population. These are alleles DRB1 * 0101, DRB1 * 0301, DRB1 * 0401,
• DRBl * 0701, DRBl * 1101, DRBl * 1301 and DRBl * 1501 (Table I). They alone represent 53 to 82% of the alleles of 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 monomorphic antibody specific for HLA-DR molecules and both recognized by the same antibody. In order to study unambiguously 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. For this purpose, the peptides as defined as RI reagents above were used.
• the frequencies indicated are the allelic frequencies in France and are representative of those of the Caucasian population. They come from Colombani (22).
• Tables Vlla and Vllb below illustrate the binding activity of the peptides according to the invention, measured under the conditions specified above.
• a proliferation test is carried out in vitro.
• PBMC peripheral blood cells
• the cells were cultured in 96-well microplates at the rate of 2.10 3 cells per well in a final volume of 200 ⁇ l of complete medium.
• the cells were stimulated or not with 10 ⁇ g / ml of a mixture of peptides according to the invention.
• After 5 days of culture at 37 ° C, the cells were incubated overnight with 0.25 ⁇ Ci of [ 3 H] thymidine (Amersham, Life technology). The cells were recovered and the incorporation of [ 3 H] thymidine was measured in the cellular DNA.
• PBMC depleted in CD8 + cells 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. 7 th and II th day of culture, 50 units of recombinant human IL-2 are added to the culture. The cells are harvested on the 14th day.
• EXAMPLE 4 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.
• PBMC lymphoblastoid cells obtained after infection with the EBV virus or genetically modified cells are used and are distributed in the wells.
• the E6 or E7 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 peptides E6 or E7 or both 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- ⁇ . After washing, an anti-rabbit IgG antibody conjugated with pure biotin of streptavidin conjugated with alkaline phosphatase are added successively for 1 hour.
• 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).
• the peptide mixtures as defined above have been tested in vivo in patients with bowenoid papulosis.
• Bowenoid papulosis is a mucocutaneous infection due to HPV16, affecting young women; it is a chronic and recurrent disease, despite the destructive treatments used.
• This disease is a grade 3 intraepithelial vulvar neoplasia from the start (VIN 3), which has the particularity of not progressing to an invasive carcinoma.
• Significant infiltration of the epithelium by many CD4 + lymphocytes suggests that these cells help to control the stage of the disease and prevent invasion.
• the peptides recognized in proliferation by CD4 + T lymphocytes were listed, for each patient.

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