WO2000073790A1 - Systeme de test pour la detection in vitro d'une reaction immunitaire antigene specifique - Google Patents

Systeme de test pour la detection in vitro d'une reaction immunitaire antigene specifique Download PDF

Info

Publication number
WO2000073790A1
WO2000073790A1 PCT/EP2000/005003 EP0005003W WO0073790A1 WO 2000073790 A1 WO2000073790 A1 WO 2000073790A1 EP 0005003 W EP0005003 W EP 0005003W WO 0073790 A1 WO0073790 A1 WO 0073790A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
protein
cell
capsomer
capsid
Prior art date
Application number
PCT/EP2000/005003
Other languages
German (de)
English (en)
Inventor
Ingrid Jochmus
John Nieland
Original Assignee
Medigene Aktiengesellschaft
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Medigene Aktiengesellschaft filed Critical Medigene Aktiengesellschaft
Priority to AU52188/00A priority Critical patent/AU5218800A/en
Priority to CA002375191A priority patent/CA2375191A1/fr
Priority to JP2001500861A priority patent/JP2003501628A/ja
Priority to EP00936845A priority patent/EP1183533A1/fr
Publication of WO2000073790A1 publication Critical patent/WO2000073790A1/fr

Links

Classifications

    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/24—Interferons [IFN]

Definitions

  • the present invention relates to a test system containing at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP and at least one antigen-presenting target cell, in particular B cell, macrophage, predendritic cell, dendritic cell , embryonic cell and / or fibroblast, which was incubated with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP, for the in vitro detection of an antigen-specific immune response, in particular a cellular immune response from Effector cells of the immune system, in particular B cells, NK cells, preferably T cells, in a particularly preferred manner cytotoxic T cells or T helper cells, and their use in diagnostics and therapy.
  • an antigen-specific immune response in particular a cellular immune response from Effector cells of the immune system, in particular B cells, NK cells, preferably T cells, in a particularly
  • the papilloma viruses also called wart viruses, are double-stranded DNA viruses with a genome size of approximately 8000 base pairs and an icosahedral capsid with a diameter of approximately 55 nm.
  • more than 100 different human papilloma virus types are known, some of which, e.g. HPV-16, HPV-18, HPV-31, HPV-33, HPV-39, HPV-45, HPV-52 or HPV-58, malignant tumors and others e.g. HPV-6, HPV-11 or HPV-42 can cause benign tumors.
  • the genome of the papillomavirus can be divided into three areas:
  • the first area concerns a non-coding region, the regulatory elements for the Contains transcription and replication of the virus.
  • the second region so-called E (early) region, contains various protein-coding sections E1-E7, of which, for example, the E6 and E7 proteins are responsible for the transformation of epithelial cells and the E1 protein controls the DNA copy number.
  • the E6 and E7 regions are so-called oncogenes, which are also expressed in malignant cells.
  • the third region also called the L (late) region, contains two protein-coding sections L1 and L2 which code for structural components of the virus capsid.
  • L1 protein is understood to mean the main capsid protein of papillomaviruses (Baker T. et al. (1991) Biophys. J. 60, 1445).
  • HPV-6 and HPV-1 1 are blamed for genital warts, some types of papillomavirus such as HPV-16, HPV-18, HPV-31, HPV-33, HPV-39, HPV- 45, HPV-52 and HPV- 58 are associated with malignant tumors of the anogenital tract.
  • HPV-16 has been linked to cervical cancer (Cervixcarcinom).
  • HPV-16 is the main risk factor for the formation of cervical neoplasia.
  • the immune system plays an important role in the progression of the disease. Cellular immune responses and, in particular, antigen-specific T-lymphocytes are probably important for the defense mechanism.
  • the E7 gene in highly malignant cervical intraepithelial neoplasia (CIN II / III) and cervical tumors, the E7 gene is constitutively expressed in all layers of the infected epithelium. Therefore, the E7 protein in particular is regarded as a potential tumor antigen and as a target molecule for activated T cells (see, for example, WO 93/20844).
  • the E7-induced cellular immune response in the patient does not appear to be strong enough to influence the course of the disease. The immune response may be boosted by appropriate vaccines.
  • capsomers are understood to be an oligomeric configuration that is made up of five Ll proteins.
  • the capsomer is the basic building block from which viral capsids are built.
  • Stable capsomers are capsomers that are unable to form capsids.
  • Capsids are understood to mean the shell of the papilloma virus, which is composed, for example, of 72 capsomers (Baker T. et al.
  • VLP is a capsid that is morphologically and antigenically similar to an intact virus.
  • the VLPs could be used in various animal systems to elicit a humoral immune response, which is characterized by the formation of neutralizing antibodies.
  • a humoral immune response which is characterized by the formation of neutralizing antibodies.
  • virus-neutralizing antibodies against L1 and / or L2 protein is of less clinical importance if the virus infection has already taken place, since instead of antibodies for the elimination of virus-infected cells, a virus-specific cytotoxic T cell (CTL) response appears to be necessary.
  • CTL cytotoxic T cell
  • VLPs are able to trigger a cytotoxic T cell response, an immune response directed exclusively against the capsid proteins L1 and / or L2 does not seem to be suitable for combating a tumor caused by papillomaviruses.
  • CVLPs for chimeric virus-like particles
  • a fusion protein of the capsid protein L1 and the potential tumor antigen E7 WO 96/11272 and Müller, M. et al. (1997) Virology , 234, 93
  • the CVLPs only triggered a minor humoral immune response against the E7 protein (Müller, M. et al. (1997), supra).
  • some of the CVLPs tested actually induce the desired E7-specific cytotoxic T cell response in mice (see also Peng S. et al. (1998) Virology 240, 147-57).
  • CVLPs consisting of C-terminally truncated L1 of bovine papillomavirus (BPV) and HPV-16 E7 -57 , which induce E7-specific cytotoxic T cells after vaccination of C57Bl / 6 mice and protect against the growth of E7-expressing tumors.
  • BBV bovine papillomavirus
  • HPV-16 E7 -57 which induce E7-specific cytotoxic T cells after vaccination of C57Bl / 6 mice and protect against the growth of E7-expressing tumors.
  • CVLPs consisting of HPV-16 Ll and HPV-16 L2 fused with the full-length HPV-16 E7 protein, which after immunization of C57Bl Protect / 6 mice against the growth of epithelial E7-expressing tumor cells, although cytotoxic T cells have not been detected and the induction of the cellular immune response thus appears to be less efficient.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer cells have two receptors: one recognizes sugar on the cell surface of antigen-presenting cells, the other recognizes MHC I molecules. The stimulation of the NK cells takes place after detection of a sugar and simultaneous absence of MHC I molecules.
  • the activation of an immune cell achieved by stimulation with an antigen can be achieved, for example, by the synthesis of cytokines such as e.g. Interferon ⁇ , interleukin 3 (IL3) can be detected.
  • cytokines such as e.g. Interferon ⁇ , interleukin 3 (IL3)
  • the corresponding cytokine accumulates intracellularly in these test systems and can then be detected, for example, using fluorescence-coupled antibodies (Kern F. et al. (1998) Nat. Med. 4, 975-8).
  • the proportion of immune cells that could be activated by the respective antigen can then finally be determined in a FACS (fluorescens activated cell sorter).
  • Eliminated cytokines can also be detected, for example, in the ELISA.
  • Other possible detection methods for the activation of immune cells are ELISPOT, prohferation tests or Cr release tests.
  • the peptides are characterized by the MHC molecules of class I, which are expressed on the cell surface, and which are sucked by organisms va ⁇ very strongly bound to organism. This in turn means that a peptide which is very well suited for the detection of T cell responses for one organism cannot be used for another organism of the same type, since this organism corresponds to the corresponding MHC-I Haplotype not available
  • MHC-I Haplotype not available
  • mice from inbred strains that have an identical MHCJ haplotype this system has established itself, however, this experimental approach is not practical in humans, for example, because different peptides are used for each patient to stimulate the T cells
  • the protein fragments or peptides which can represent a cytotoxic T cell epitope, are not known
  • the uptake of the specific antigen is MHC-independent for these systems, so that cells from different organisms can present parts of the specific antigen to the T cells, even if the respectively presented parts of the antigen differ from haplotype Can distinguish haplotype.
  • the vaccinia and the adenovirus system has the disadvantage that such a virus infection of the cells is associated with viral gene expression and viral replication. This additional influence on the antigen-presenting cells makes a quantitative measurement of a cytotoxic T cell response, which is limited to the specific antigen, significantly more difficult. On the other hand, the handling of these systems is difficult and costly, since security level S2 is required when dealing with recombinant vaccines or adenoviruses.
  • the aim of the present invention was therefore to develop a test system for the cellular immune response
  • the immune response in particular from cytotoxic T cells, but also e.g. can be tested by T helper cells, B cells or NK (natural killer) cells, with the possibility of differentiation between the immune cells;
  • a predendritic cell is understood to mean a precursor cell of a dendritic cell which expresses CD 16 strongly, whereas MHC class I and II molecules and CD80, CD86 and CD40 express only relatively weakly.
  • dendritic cells hardly express CD 16, but strongly MHC class I and II molecules as well as CD80, CD86 and CD40 (Woodhead et al. (1998) Immunology 94 (4): 552-9).
  • a CD16 positive cell means a cell for which the expression of CD 16 can be detected using a specific antibody, for example in a FACScan experiment.
  • CVLPs which could trigger a cellular immune response in vivo, could bind in vitro to predendritic cells, for example JAWS II cells.
  • these CVLPs were able to bind to cells of a T-lymphoma cell line. It can thus be concluded that the binding of the CVLPs to the predendritic cell represents a limiting step in triggering a cellular immune response.
  • CD 16 is strongly expressed on predendritic cells, but not or hardly on dendritic cells. In fact, dendritic cells are barely able to bind CVLPs (data not shown).
  • the particular structure of the CVLPs would only abolished in the cytoplasm by disassembly and processing, so that, unlike exogenous proteins, primarily, but not exclusively, MHC-I molecules gain access to antigen fragments, bind them, transport them to the cell surface and present them to the CD8-positive cells intracellular MHC-II molecules are also loaded with antigen fragments which, analogously to MHC-I, present the peptides to the CD4-positive cells, so that both MHC-I and MHC-fl molecules of the antigen-presenting cells are Incubation with CVLPs with CVLP "Load" peptides ("CVLP-loaded cells").
  • Presentation within the meaning of the present invention is understood to mean when a peptide or protein fragment binds to an MHC molecule, this binding being able to take place, for example, in the endoplasmic reticulum or extracellular space, and then when this MHC molecule-peptide complex is on the extracellular side of the cell membrane is bound so that it can be specifically recognized by immune cells.
  • the cytotoxic T cells or T helper cells proliferate. If there is continued stimulation by at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP and / or a cell which produces at least one capsomer, at least one stable capsomer, at least one cap sid, at least one VLP, and / or at least one CVLP, cytokines, such as interferon ⁇ or interleukin 4 (IL4), which accumulate in the cytoplasm in this system.
  • cytokines such as interferon ⁇ or interleukin 4 (IL4)
  • the intracellular interferon ⁇ can be used for the detection of specifically activated T cells.
  • capsomers, stable capsomers, capsids, VLPs, and / or CVLPs surprisingly behave like viruses and not like proteins with regard to the triggered immune response, although they do not trigger expression of viral proteins or viral replication. Because of their ability to pseudoinfect, these compounds thus combine the advantages of the approaches with free peptides / proteins with those of recombinant viruses. Analogous to viruses, capsomers, stable capsomers, capsids, VLPs, and / or CVLPs are able to enter the cytoplasm as particles and are therefore not MHC-restricted. in the In contrast to the viral system, however, no gene expression is required to release or to express the specific antigen and to load MHC I molecules.
  • capsomers, stable capsomers, capsids, VLPs, and / or CVLPs activate both CD4 and CD8 positive T cells equally.
  • capsomers, stable capsomers, capsids, VLPs, and / or CVLPs are only classified with S 1 with regard to their security standards, so that the technical implementation is cheaper than S2 security standards, which are necessary in viral systems, and in many places with less technical Effort can be realized.
  • the present invention therefore relates to a test system comprising at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP and at least one antigen-presenting target cell, in particular B cell, macrophage, predendritic cell , Dendritic cell, embryonic cell and / or fibroblast, which has been incubated with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP, for the in vitro detection of an antigen-specific immune response, in particular cellular immune response from effector cells of the immune system, in particular B cells, NK cells, preferably T cells, in a particularly preferred manner cytotoxic T cells or T helper cells, and their use in diagnostics and therapy.
  • an antigen-specific immune response in particular cellular immune response from effector cells of the immune system, in particular B cells, NK cells, preferably T cells, in a particularly preferred manner
  • the present invention further relates to a test system which has at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and or at least one CVLP and at least one predendritic cell and or a CD16-positive cell which has at least one Capsomer, at least one stable Capsomer, at least one Capsid, min at least one VLP, and / or at least one CVLP was incubated for the in vitro detection of an antigen-specific immune response, the binding of the stable capsomer, capsid, VLP and / or CVLPs to the cell mentioned being measured.
  • the effector cells are mammalian cells, in particular human or murine cells.
  • the capsomers, stable capsomers, capsids, VLPs, and / or CVLPs used in the test system contain at least one Ll protein of one or more papilloma viruses, or at least one Ll protein and at least one papilloma virus L2 protein, in particular L1 proteins or Ll and L2 proteins from human, bovine and / or 'cottontail rabbit' papillomaviruses.
  • the capsomers, stable capsomers, capsids, and / or CVLPs contain at least one L1 fusion protein, consisting of an L1 protein portion of one or more papillomaviruses and a protein portion heterologous to the Ll protein.
  • the Ll protein used can be a naturally occurring Ll protein, or it can contain one or more deletions, which can be, for example, C-terminal, N-terminal, and / or internal, and / or one or more mutations.
  • up to at least approximately 35 amino acids preferably at least approximately 25 to approximately 35, in particular at least approximately 32 to approximately 34 amino acids are deleted from the C-terminus of the L1 protein.
  • the protein portion which is heterologous to the L1 protein can be a naturally occurring protein or one or more deletions which, for example, C- may be terminal, N-terminal, and or internal, and / or contain multiple mutations.
  • This protein, which is heterologous to the L1 protein can be of a bacterial or viral origin in a particular embodiment, for example from HIV, HBV, HCV or CMV, preferably from papillomaviruses, in particular from human papillomavirus, such as but not exclusively from E6 or E7.
  • at least approximately 55 amino acids preferably at least approximately 5 to approximately 55, in particular at least approximately 38 to approximately 55, amino acids are deleted from the C-terminus of the E7 protein.
  • proteins can also be derived from autoimmune antigens such as thyroglobulin, myelin or zona pellucida glycoprotein 3 (ZP 3 ), which are associated with certain autoimmune diseases such as thyroiditis, experimental autoimmune encephalomyelitis (EAE), oophoritis or rheumatoid arthritis.
  • the protein heterologous to L1 is derived from tumor antigens, preferably melanoma antigens such as MART, ovarian carcinoma antigens such as Her2 new (c-erbB2), BCRA-1 or CA125, colon carcinoma antigens such as CA125 or breast carcinoma antigens such as Her2 new (c - erbB2), BCRA-1, BCRA-2.
  • This anti gene portion can, but need not, comprise individual domains or epitopes of a protein.
  • the protein portion which is heterologous to the L1 protein is bound, preferably fused.
  • Another object of the present invention is a cell which, after in vitro incubation with capsomers, stable capsomers, capsids, VLPs, and / or CVLPs, contains proteins and / or protein fragments from said capsomers, stable capsomers, capsids, VLPs, and / or CVLPs, preferably presented, especially over both MHC-I and MHC-II complexes.
  • the cell according to the invention contains, in particular presents proteins, protein fragments, and / or peptides from capsomers, stable capsomers, capsids, VLPs, and or CVLPs which contain at least one L1 protein of one or more papillomaviruses, or at least one Ll protein and at least one a papilloma virus Contain L2 protein.
  • the cell according to the invention contains, in particular, presents proteins, protein fragments, and / or peptides from capsomers, stable capsomers, capsids, and / or CVLPs, which contain at least one L1 fusion protein, consisting of an Ll protein portion of one or more papillomaviruses and contain a protein portion heterologous to the Ll protein.
  • the Ll protein used can be a naturally occurring Ll protein or it can contain one or more deletions, which can be, for example, C-terminal, N-terminal, and / or internal, and / or one or more mutations.
  • up to at least approx. 35 amino acids preferably at least approx. 25 to approx. 35, in particular at least approx. 32 to approx. 34 amino acids are deleted from the C-terminus of the L1 protein.
  • the protein portion heterologous to the L1 protein can be a naturally occurring protein or it can contain one or more deletions, which can be, for example, C-terminal, N-terminal, and or internal, and / or several mutations.
  • this protein, which is heterologous to the L1 protein can be of bacterial or viral origin, for example from HIV, HBV, HCV, HSV, EBV, HTLV or CMV, preferably from papillomaviruses, in particular from human papillomavirus, such as, for example, from E6 or E7.
  • At least approximately 55 amino acids preferably at least approximately 5 to approximately 55, in particular at least approximately 38 to approximately 55, amino acids are deleted from the C-terminus of the E7 protein.
  • these proteins can be derived from autoimmune antigens such as, for example, thyroglobulin, myelin or zona pellucida glycoprotein 3 (ZP 3 ), which are associated with certain autoimmune diseases such as, for example, thyroiditis, experimental autoimmune encephalomyelitis (EAE), oophoritis or rheumatoid arthritis.
  • the protein heterologous to L1 is derived from tumor antigens, preferably melanoma antigens such as MART, ovarian carcinoma antigens such as Her2 neu (c-erbB2), BCRA-1 or CA125, colon carcinoma antigens such as CA125 or breast carcinoma antigens such as Her2 new (c-erbB2), BCRA-1, BCRA-2.
  • This portion of antigen can, but need not, comprise individual domains or epitopes of a protein.
  • the antigen portion is bound to the Ll protein, preferably fused.
  • the cell is an antigen-presenting cell, in particular B cell, macrophage, predendritic cell, dendritic cell, embryonic cell or fibroblast.
  • Another object of the present invention is a method for producing a target cell, which is based on the incubation of the target cell with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP in vitro.
  • Another object of the present invention is a method for producing a test system in which at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP is genetically engineered and the target cell by incubation with at least one capsomer, one stable capsomer, a capsid, a VLP, and / or CVLP, and the effector cell is an immune cell line and / or cultivated primary immune cell, preferably a murine or human immune cell line and / or cultivated primary immune cell.
  • the genetically engineered proteins which are part of the capsomers, stable capsomers, capsids, VLPs, and / or CVLPs are, in a preferred embodiment, in bacteria such as E.
  • Another object of the present invention is a method for producing a test system in which at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP is produced by genetic engineering, and with a predendritic and or a CD 16 positive cell is incubated.
  • Another object of the present invention is a method for the in vitro detection of the activation of effector cells of the immune system by at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP and / or at least one cell, which was incubated with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or a CVLP, which contains the following steps: a) A test system according to the invention is used in a first step.
  • an incubation of immune cells takes place for at least about 5h, in particular about 17h, at least one capsid, at least one VLP, and / or at least one CVLP and / or at least one cell which has been incubated with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or one CVLP.
  • incubation of, for example, PBMCs, T cellinins or cultured primary T cells with antigens, preferably with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP is carried out only for at least about 5 hours, and / or at least one CVLP and / or at least one cell with at least one capsomer, at least one stable capsomer, at least one Capsid, at least one VLP, and / or a CVLP was incubated. During this short time, only the cells are activated by the antigen that have previously been stimulated by the same or a similar antigen. b) In a second step, the possible activation of effector cells is determined.
  • stimulated T cells can be detected by various methods such as the production or secretion of cytokines by the T cells, the expression of surface molecules on T cells, the lysis of target cells or the proliferation of cells.
  • suitable methods are for example, a cytokine assay (Chapter 6.2 to 6.24 in Current Protocols in Immunology (1999) supra), ELISPOT (Chapter 6.19 in Current Protocols in Immunology, supra), a " 'Cr-release assay (Chapter 3J 1 in Current Protocols in Immunology, supra) or the detection of proliferation (Chapter 3J2 in Current Protocols in Immunology, supra)
  • immune cells such as cytotoxic T cells, T helper cells, B cells, NK cells and other cells.
  • step a ') is inserted before step a) a')
  • At least one effector cell of the test system is used for at least about 8 weeks, in particular at least about 3 weeks, with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or CVLP and / or at least one target cell which has been incubated with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or one CVLP, before the step a) connects.
  • This preactivation of the effector cells has the consequence that the effector cells in the subsequent step a) by adding at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or CVLP and / or at least one target cell which has at least one capsomer, at least one stable capsomer, at least one capsid, at least a VLP, and / or a CVLP, is restimulated.
  • Co-cultivation is the growth of at least one effect cell in the presence of at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or CVLP and / or at least one target cell, which has at least one capsomer, at least one stable Capsomer, at least one capsid, at least one VLP, and / or a CVLP was to be understood in the same growth medium and the same tissue culture container.
  • a component of the test system namely at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP and / or at least one cell, which is used with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or a CVLP was incubated as standard while a second component of the test system, the effector cells, is the actual test component.
  • the activation of the effector cell observed in the reaction of both components is combined with the activating effect of a capsomer, stable capsomer, capsid, VLP, and / or CVLP, and / or a cell that comes with at least one capsomer, for example from an industrial production process.
  • at least one stable capsomer, at least one capsid, at least one VLP, and or a CVLP was compared. This embodiment allows, for example, the quality control of batches of prophylactic and / or therapeutic vaccines.
  • Another preferred method that uses the test system according to the invention is the selection of particularly effective epitopes for the development of vaccines based on parts of proteins. If, for example, different CVLPs, each containing short peptides of a protein or a pathogen as a fusion component, are examined in separate approaches with regard to their ability to mediate stimulation of immune cells, the quantitative comparison of the immune responses to the respective CVLPs can be particularly effective Identify peptides. Such peptides can then be combined for new vaccines. Proteins can be tested analogously to this.
  • the invention therefore furthermore relates to a method for identifying epitopes, peptides or protein fragments which trigger an immune response, in particular a cellular immune response.
  • Another object of the present invention is a method for the in vitro detection of the activation of effector cells of the immune system by at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP and / or at least one cell, which has been incubated with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or a CVLP, which contains the following steps:
  • PBMCs in particular T cells
  • PBMCs are obtained from the blood of a donor, in particular from the blood of a donor who already has at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or CVLP and / or at least one target cell, which incubates with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or one CVLP was vaccinated and the effector cells obtained are cultivated or spleen cells from a mouse are obtained, in particular spleen cells from a mouse that already contain at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or CVLP and / or at least a target cell that has been incubated with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or a CVLP
  • the isolated and / or cultivated cells are used for at least about 5 hours, in particular about
  • At least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP and / or at least one cell which has at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or a CVLP was added.
  • the incubation is carried out for at least about 5 hours. During this short stimulation time, only the cells that were previously stimulated by the same or a similar antigen are activated by the added antigen.
  • the possible activation of effector cells is determined.
  • stimulated T cells can be detected by various methods such as the detection of the production or secretion of cytokines by the T cells, the expression of surface molecules on T cells, the lysis of target cells. cells or the proliferation of cells. Methods suitable for this are, for example, a cytokine assay (Chapter 6.2 to 6.24 in Current Protocols in Immunology (1999), supra), ELISPOT (Chapter 6.19 in Current Protocols in Immunology, supra), a 51 Cr release test (Chapter 3J 1 in Current Protocols in Immunology, supra) or the
  • step I) is inserted after step I)
  • the isolated cells are kept for at least about 8 weeks, in particular at least about 3 weeks, with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and or CVLP and / or at least one target cell, which has been incubated with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or a CVLP, before the step
  • the effector cells in the subsequent step II) being added by adding at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or CVLP and / or at least one target cell incubated with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and or a CVLP.
  • This type of execution is called restimulation.
  • the first stimulation can also have been carried out as described under point I) within the donor, for example by vaccination, an infection, a tumor or as part of an autoimmune disease. However, it can also be carried out in vitro as described under point I ') in order to obtain specific reactive cell clones or populations.
  • the immune status of an organism can be tested against a pathogen by the method according to the invention.
  • PBMCs of an organism are isolated and restimulated with at least one capsomer, at least one stable capsomer, at least one capsid, and / or CVLP and or at least one target cell, which has at least one capsomer, at least one stable capsomer, at least one capsid, and / or incubated with a CVLP that contains a pathogen-specific antigen or a part thereof as a fusion component, it is possible to find reactive immune cells against the respective antigen, such as, for example, cytotoxic T cells, reactive T helper cells or reactive B cells. Cells show a previous infection.
  • the reactive cells can be quantified by quantifying them, so that e.g. the need for booster shots can be analyzed.
  • the success of the vaccination and / or the current immune status of a patient can also be checked after a vaccination that has been in the past.
  • Monitoring is not limited to prophylactic and / or therapeutic vaccination with at least one capsomer, at least one stable capsomer, at least one capsid, and / or CVLP and / or at least one target cell, which with at least one capsomer, at least one stable capsomer, at least one capsid, and / or a CVLP, but is also suitable for conventional vaccines.
  • the detection of specific reactive T cells by the present test method can be used to diagnose infections that are difficult to detect. If, for example, CVLPs are constructed with antigens or parts of antigens from known but difficult to detect pathogens, the T cell response to such antigens can provide information about an existing infection.
  • the HLA haplotypes of patient groups which are immune to certain infectious diseases or are not or only poorly immunized are correlated with the reactivity of their T cells towards antigens of the corresponding pathogens, so haplotypes can be identified that mediate immunity to the pathogen.
  • capsomers, stable capsomers, capsids, and / or CVLPs and / or target cells which contain at least one capsomer, at least one stable capsomer, at least one capsid, and / or a CVLP incubated are produced that contain these autoimmune antigens or parts. These can then be used to diagnose the respective autoimmune disease by measuring the T cell response of a patient after stimulation of, for example, his isolated PBMCs with the respective autoimmune antigen.
  • Another embodiment of the method according to the invention is the differentiation of tumor types with regard to different specific tumor anti- genes. If different types of tumor are known, which differ among other things in the fact that they express different tumor antigens, and on the other hand you have different T cell populations that can be specifically restimulated by one of the respective tumor antigens, you can by Evidence of restimulation of reactive T cells classify a patient's tumor. Such a classification could then be used, for example, to specifically stimulate the patient's own T cells by vaccination with the respective tumor antigens in order to generate a cytotoxic T cell population against the patient's own tumor cells.
  • the methods according to the invention are suitable, for example, for the quality control of vaccine batches during production, the identification of new antigenic epitopes, the identification of patient haplotypes, the differentiation from autoimmune diseases or the differentiation of tumor types, a high processing speed and reproducibility when using the test system.
  • the invention therefore furthermore relates to a method which activates effector cells by at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP or by at least one cell which has at least one capsomer , at least one stable capsomer, at least one capsid, at least one VLP, and / or a CVLP was incubated in high-throughput systems.
  • peptides or proteins can also be investigated on a large scale with regard to their triggering of immune responses, in particular T-cell responses.
  • Another object of the invention is the use of at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP or at least one antigen-presenting target cell, in particular B cell, macrophage, dendritic cal cell, embryonic cell or fibroblast which have been incubated with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP, and effector cells of the immune system, in particular B cells, NK cells, preferably T cells, in a particularly preferred manner cytotoxic T cells or T helper cells for triggering or for detecting an immune response.
  • B cells B cells
  • NK cells preferably T cells
  • the invention further relates to a diagnostic agent containing at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP or at least one antigen-presenting target cell, in particular B cell, macrophage, dendritic cell, embryonic cell or fibroblast which have been incubated with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP, effector cells of the immune system, in particular B cells, NK cells, preferably T cells , in a particularly preferred manner cytotoxic T-cells or T-helper cells and optionally a pharmaceutically acceptable carrier.
  • a diagnostic agent containing at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP or at least one antigen-presenting target cell, in particular B cell, macrophage, dendritic cell, embryonic cell or
  • supports known to those skilled in the art are glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural or modified cellulose, polyacrylamides, agarose, aluminum hydroxide or magnitide.
  • a diagnostic agent according to the invention can be in solution, bound to a solid matrix and / or an adjuvant added.
  • the diagnostic agent can be administered in various ways. Examples of administration forms known to the person skilled in the art are parenteral, local and / or systemic administration by, for. B. oral, intranasal, intravenous, in- tramuscular, and / or topical application. The preferred form of application is influenced, for example, by the natural route of infection of the respective papillomavirus infection. The amount administered depends on the age, weight and general health of the patient and the type of papilloma virus infection.
  • the diagnostic agent can be administered in the form of capsules, solution, suspension, elixir (for oral administration) or sterile solutions or suspensions (for parenteral or intranasal administration). For example, saline or phosphate-buffered saline can be used as the inert and immunologically acceptable carrier.
  • FIG. 1 shows the graphic evaluation of the restimulation of murine, HPV16L1-specific T cells by two murine antigen-presenting cell lines (C3 and B16F10), which had previously been incubated with increasing amounts of CVLPs.
  • the increasing concentrations of CVLPs are plotted against the percent of stimulated T cells that were detected via interferon ⁇ production.
  • Fig. 2 shows the graphic evaluation of the restimulation of murine, HPV16Ll-peptide-specific T cells by murine C3 cells, which had previously been incubated with different CVLP preparations in the absence and presence of virus-neutralizing antibodies.
  • FIG. 3A shows the evaluation of five FACScan experiments after stimulation of human PBMC with different concentrations of CVLPs (0-10 ⁇ g ml), in which human T cells that are positive for human interferon ⁇ are shifted upwards in the graphic.
  • FIG. 3B shows the graphical evaluation of FIG. 3A, in which the concentration of CVLPs is plotted against the percent of the stimulated cells. Stimulated cells are defined by the detection of human interferon ⁇ .
  • FIG. 4 shows the graphic evaluation of the restimulation of human PBMC by various antigens after the PBMC had been stimulated with CVLPs beforehand.
  • FIG. 5 shows the evaluation of three FACScan experiments after restimulation of human T cells with different antigen-presenting cells after the T cells had previously been stimulated with CVLPs.
  • the content of CD3, which is specific for T cells, and the content of human interferon ⁇ , which is specific for activated cells, are plotted from left to right.
  • 6A shows the graphical evaluation of the specific lysis of various murine, antigen-presenting RMA cells by an E7-specific cytotoxic T cell line. Normal RMA cells were used, RMA cells that expressed E7 or RMA cells that had previously been used with LlE7 ⁇ . 60 CVLPs had been incubated. The ratio of the effector cells to the target cells is plotted against the percentage of the specifically lysed cells.
  • FIG. 6B shows the graphical evaluation of the specific lysis of various murine, antigen-presenting RMA cells by an E7-specific cytotoxic toxic T cell line.
  • RMA cells were used which had previously been incubated with LlE7 ⁇ - 60 CVLPs or with Ll-VLPs, that is to say without an E7 portion.
  • FIG. 7 shows the evaluation of FACScan experiments after incubation of JAWS II cells with increasing amounts of CVLPs, plotted from left to right.
  • the expression of MHC class II molecules was measured by the detection of MHC class II molecules on the cell surface by means of specific antibodies. These values are plotted as relative fluorescence units on the left Y axis.
  • the binding of the CVLPs to the cells was measured by detecting an HPV 16 Ll epitope on the cell surface by means of an Ll -specific antibody. These values are shown as% binding cells, with a negative control without CVLPs being defined as 5% +/- 1% binding cells.
  • FIG. 9 shows the evaluation of FACScan experiments after incubation of RMA cells (left graphic) or JAWS II cells (right graphic) with increasing amounts of CVLPs, plotted from left to right.
  • the binding of the CVLPs to the cells was measured by detecting an HPV 16 Ll epitope on the cell surface using an Ll-specific antibody. The values are given as% binding cells, with a negative control without CVLPs being defined as 5% +/- 1% binding cells.
  • FIG. 10 shows the evaluation of FACScan experiments of T cells from different mice which had been vaccinated with different amounts of CVLPs from different batches.
  • the T cells were stimulated with a known cytotoxic HPV 16 L1 epitope, restimulated with the same peptide under the conditions described in the example, and the relative proportion of the CD8 and interferon ⁇ -positive cells was then determined using specific antibodies in the FACS experiment.
  • mice • C57Bl / 6 mice were purchased from Charles River Laboratories (Wilmington, MA, USA).
  • B6 cells means embyonal stem cells from a C57Bl / 6 mouse.
  • C3 cells means HPV16 and ras-transformed B6 embryo cells (see Feltkamp MC et al. (1993) Eur. J. Immunol. 23, 2242-9).
  • RMA cells come from a thymoma of a C57BL / 6 mouse (see Ljunggren HG & Karre K. (1985) J. Exp. Med. 162, 1745-59).
  • RMA-E7 cells are derived from RMA cells, which, however, constitutively express an HPV6 E7 protein by stable transfection.
  • B 16F 10 cells means the cell line available under ATCC CRL-6475.
  • PBMC peripheral blood mononuclear cells which, for example, according to the in Rudolf M.P. et al. (1999) Biol. Chem. 380, 335-40 processes can be prepared.
  • MVA-L I ⁇ C means recombinant murine vaccinia virus that expresses HPV 16 L1 ⁇ C in infected cells.
  • IL-2 means recombinant cytokine (Becton Dickinson, Hamburg, Germany).
  • • -hu CD28 means a monoclonal mouse antibody that is directed against the extracellular part of human CD28 (ATCC CRL-8001).
  • ⁇ -CD3 / PE means a monoclonal mouse antibody which is directed against the extracellular part of human CD3 (e) and contains a fluorescent marker phycoerithrin (Medac, Hamburg, Germany).
  • ⁇ -CD4 / Cychrome means a monoclonal mouse antibody which is directed against the extracellular part of CD4 and contains a fluorescence marker cychrome (DAKO; Glostrup, Denmark).
  • ⁇ -CD4 / Tricolor means a monoclonal antibody which is directed against the extracellular part of CD4 and contains a fluorescent marker Tricolor (Medac, Hamburg, Germany).
  • ⁇ -mus interferon ⁇ / FITC means a monoclonal rat antibody which is directed against mus interferon ⁇ and contains a fluorescent marker FITC (Medac, Hamburg, Germany).
  • E-7 peptide means amino acids 49 to 57 of the human papillomavirus type 16, sequence: RAHYNIVTF (see Feltkamp M.C et al. (1993) supra).
  • P-12 peptide means amino acids 165 to 173 of the Ll protein from HPV 16, sequence: AGVDNRECI (see Genbank 5559..7154). This peptide could be identified as a cytotoxic, murine T cell epitope (see DE 19925235J-41).
  • AM peptide means amino acids 366 to 374 of the influenza nucleoprotein, sequence: ASNENMETM (see Townsend A.R. et al. (1986) Cell 44, 959-68).
  • HPV16L1 93-101 means amino acids 93 to 101 of the Ll protein of HPV 16, sequence: GLQYRVFRI.
  • Phytohaemaglutinin (PHA) was obtained from Sigma (Deisenhofen, Germany).
  • JAWS II cells are predendritic cells (see US 5,648,219).
  • ⁇ -mouse CD8 / PE antibodies were obtained from Pharmingen (Heidelberg, Germany).
  • ⁇ -mouse interferon ⁇ / FITC antibody was obtained from Caltag (Hamburg, Germany).
  • ⁇ -mouse CD4 / Cychrom-Antikö ⁇ er was obtained from Pharmingen (Heidelberg, Germany). • Zefa membrane means a 45 ⁇ m syringe filter and was obtained from Zefa (Munich, Germany).
  • FACScan calibur or FACS means "fluorescencs activated cell sorter”.
  • the apparatus was purchased from Becton Dickenson (Hamburg, Germany).
  • the spleen cells were isolated for weeks.
  • Step 2 the infected B6 cells were irradiated, thereby preventing further growth.
  • the spleen cells were cultured together with the L l c-expressing B6 cells, which acted as stimulator cells for the T cells of the spleen cells, for 5 days.
  • 2 ⁇ l0 4 murine antigen-presenting cells (C3 or B16F10) were incubated with different concentrations of HPV16 L1 ⁇ C * E7 I -5 CVLPS at 37 ° C overnight. Then 2x10 ⁇ murine CD8 positive T cells (see Example 2), which are specific for a specific HPV 16 Ll peptide, were added and incubated for one hour at 37 ° C. in the presence of 10 IU / ml IL2. The cells were incubated for a further 5 hours at 37 ° C. in the presence of 1 ⁇ l golgi plug.
  • the cells were then fixed, permeabilized, stained with ⁇ -mouse CD3 PE, with ⁇ -mouse CD4 / Tricolor and with ⁇ -mouse interferon ⁇ / FITC and washed.
  • the cells were examined for their labeling in a FACScan calibur (Becton Dickinson, Hamburg, Germany) and the measurement results were analyzed using cellquest software (Becton Dickinson, Hamburg, Germany).
  • Cells with an interferon ⁇ signal greater than 10 1 were defined as stimulated cells.
  • T cells were defined by a CD3 signal of over 10 2 .
  • FIG. 1 shows that as the amount of CVLPs increased, percent more T cells were restimulated by the antigen-presenting cells. the. Since the T cells used here only interact with MHC I-presented peptides, this experiment shows that the CVLPs must have caused pseudo-infection of the antigen-presenting cells. This is the only way to explain that MHC I molecules could be loaded with CVLP peptides in order to be recognized on the cell surface by Ll peptide-specific T cells.
  • Murine C3 target cells were incubated overnight at 37 ° C with 6 different HPV 16 L1 C * E7 ⁇ -55 CVLPs preparations or with the P12 peptide, in each case a) without antibodies b) with 25 / C ⁇ HPV16Ll antibodies (10 ⁇ g / ml ). This antibody is known to prevent infection of the antigen-presenting cells by HPV viruses (or virus-like particles) by binding to the HPVL1 protein. Then HPV16L1 peptide-specific, murine T cells were added and incubated for 6 hours (CVLP 2-6 were incubated in the presence of golgi plug). The cells were analyzed for their interferon ⁇ production (see previous example). C3 cells which had not been incubated with no antigen served as a negative control (see FIG. 2).
  • CVLPs can be prevented from penetrating into antigen-presenting cells by virus-neutralizing antibodies. However, the addition of the antibodies has no influence on the uptake of individual peptides. 5. Stimulation of human PBMC by HPV 16 L1E7 CVLPs
  • PBMC peripheral blood mononuclear cells
  • Human PBMC (4 ⁇ l0 5 ) were stimulated and harvested for 3 weeks with HPV 16 Ll ⁇ C * E7 ⁇ -55 CVLPs at 37 ° C with weekly addition of 1 ⁇ g / ml CVLPs and 10 D antigen-presenting PBMCs.
  • the cells were then in 100 ul medium at 37 ° C with 10 ug / ml different antigens a) E7 peptide b) HPV16 Ll ⁇ c ⁇ 7 ,. 55 CVLPs c) influenza peptide d) phytohaemaglutinin (PHA) in the presence of 10 IU / ml IL2 and 0.5 ⁇ g / ml anti-human CD28 restimulated.
  • PHA phytohaemaglutinin
  • golgi plug was added.
  • the cells were incubated for a further 5 hours at 37 ° C.
  • the cells were then fixed and permeabilized, stained with ⁇ -human CD3 / PE, with ⁇ -human CD4 / cychrome and with ⁇ -human interferon ⁇ / FITC.
  • the cells were examined for their labeling in a FACScan calibur Becton Dickenson and the measurement results were analyzed using cellquest software (see previous examples).
  • Human PBMCs (4 ⁇ l0 5 ) were stimulated and harvested for 8 weeks with HPV 16 Ll ⁇ C .E7 ⁇ -55 CVLPs at 37 ° C with weekly addition of 1 ⁇ g / ml CVLPs and 10 3 irradiated PBMCs.
  • the cells were then restimulated in 100 ⁇ l medium at 37 ° C. with 10 ⁇ g / ml different antigens in the presence of 10 IU / ml IL2 and 0.5 ⁇ g / ml anti-human CD28: a) overnight with HPV16 L1 ⁇ C «E7 ⁇ . 55 CVLP-incubated PBMC, b) PBMC incubated overnight with HPV 16 Ll 93-101 peptide.
  • golgi plug was added.
  • the cells were incubated for a further 5 hours at 37 ° C.
  • the cells were then fixed and permeabilized, stained with anti-human CD3 / PE, with anti-human CD4 / Cychrome and with anti-human interferon ⁇ / FITC.
  • the cells were examined for their labeling in a FACScan calibur Becton Dickenson and the measurement results were analyzed using cellquest software (see previous examples).
  • FIG. 5 shows that both the CVLP-incubated PBMC brought about a restimulation of CVLP-stimulated T cells, but not the PBMC incubated with the control peptide.
  • the human antigen-presenting cells were pseudoinfected by the CVLPs (like the mouse cells from Example 3) and were therefore recognized by the CVLP-specific T cells.
  • RMA cells were in the presence of 5 L Cr for 1 hour at 37 ° C
  • Figure 6A shows that both CVLP-incubated cells and cells expressing E7 were effectively lysed by the T cells, but not the RMA cells incubated without antigen.
  • FIG. 6B shows that, in contrast to the CVLPs, incubation of the RMA cells with L1 -VLPs does not bring about an effective solution of the cells.
  • E7 is therefore responsible for the specific stimulation of the cytotoxic T cell line, which leads to the lysis of the antigen-presenting RMA cells. It has little influence whether E7 is generated intracellularly by stable expression or whether it is brought into the cell by pseudo-infection via CVLPs.
  • JAWS II cells were, as described above, in various approaches with HPV16Ll ⁇ c »E7 ⁇ . 55 CVLPs and / or mouse serum incubated over 2 days. The following were added:
  • the cells were then examined as above to determine whether they express MHC class II molecules by first staining them with a 1 ⁇ g / ml MHC IA b antibody and then with 1 ⁇ g / ml FITC-coupled anti-mouse antibodies. Finally, the cells were checked for their Coloring examined in a FACScan calibur and the measurement results analyzed using cellquest software.
  • FIG. 8 shows that the depletion of the CVLPs from the approach like the pre-incubation with CVLP-specific mouse serum prevents the activation of the MHC class JJ expression, which means that this observed activation is specifically caused by CVLPs and not by any Contamination of the CVLP preparations is caused.
  • Each 3x10 " RMA or JAWS II cells were incubated with increasing amounts of HPV 16Ll c * E7 1 -55 CVLPs from different preparations for 3 hours as described in Example 9. The cells were again examined to determine whether they were CVLPs For this purpose, the cells were stained with 1 ⁇ g / ml of the FITC-coupled 25 / C ⁇ -HPV16Ll antibody, examined for their staining in a FACScan calibur and the measurement results were analyzed using cellquest software.
  • FIG. 9 shows that batch 1-3 of the CVLPs can bind to RMA cells, batch 3 binding somewhat poorly at the same CVLP concentrations. In contrast, only the CVLPs of batches 1 and 3 bind to the JAWS II cells, the CVLPs of batch 2 show no clear increase and do not reach 50% binding even at the highest concentration of 100 ⁇ g / ml CVLPs. The binding of the CVLPs to cells thus varies with the CVLP preparation and the cell type. In order to test whether the observed variability of CVLP binding correlates with the in vivo inducibility of a cytotoxic T cell response, the following experiment was carried out:
  • mice Three C57BL6 mice were vaccinated with 10 or 1 ⁇ g CVLP of batches 1 to 3. After two weeks, the mice were boosted by a second identical injection. After a further two weeks, the animals were sacrificed and spleens and serum were obtained. All sera contained antibodies directed against the CVLPs (data not shown).
  • the T cells were isolated from the spleen and purified using nylon wool (see Current Protocols in Immunology, supra. Pages 7.7.2 to 7.7.3).
  • the T cells were stimulated with 10 ⁇ g / ml of the murine cytotoxic P12 peptide for 5 days. The T cells were then restimulated in 100 ⁇ l medium at 37 ° C. with a further 10 ⁇ g of the P12 peptide in the presence of 10 IU / ml IL2. T cells from mice immunized with buffer served as a negative control. After one hour, 1 ⁇ l monensin (300 ⁇ M) was added. The cells were incubated for a further 5 hours at 37 ° C.
  • the cells were then fixed and permeabilized, stained with 1 ⁇ g / ml ⁇ -mouse CD4 / cychrome, ⁇ -mouse CD8 / PE and with ⁇ -mouse interferon ⁇ / FITC antibodies.
  • the cells were examined for their labeling in a FACSscan calibur and the measurement results were analyzed using Cellquest software.
  • Figure 10 shows that no mouse vaccinated with batch 2 CVLPs generated cytotoxic T cells reactive against the P12 peptide, while vaccination of batch 1 and 3 CVLPs each reacted with T cells in multiple mice could induce.
  • the knowledge from these in vivo data that the CVLPs of batch 2 are not suitable for the generation of cytotoxic T cells was already from the in vitro data of the CVLP binding study with the JAWS II cells recognizable, while the RMA cells did not allow such a prediction.
  • predendritic cells such as the JAWS II cells are particularly suitable for the prediction of in vivo T cell activation using CVLPs.

Abstract

L'invention concerne un système de test comprenant au moins un capsomère, au moins un capsomère stable, au moins un capside, au moins un VLP, et/ou au moins un CVLP, et au moins une cellule cible présentant un antigène. La cellule cible est en particulier du type cellule B, macrophage, cellule prédendritique, cellule dendritique, cellule embryonnaire, et/ou fibroblaste, et a été incubée avec au moins un capsomère, au moins un capsomère stable, au moins un capside, au moins un VLP, et/ou au moins un CVLP, pour la détection in vitro d'une réaction immunitaire antigène spécifique. La réaction immunitaire est en particulier du type réaction immunitaire cellulaire de cellules effectrices du système immunitaire ; en particulier cellules B, cellules NK, de façon préférentielle cellules T et surtout cellules T cytotoxiques ou cellules T « helper ». L'invention concerne également l'application en diagnostic et thérapie.
PCT/EP2000/005003 1999-06-01 2000-05-31 Systeme de test pour la detection in vitro d'une reaction immunitaire antigene specifique WO2000073790A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU52188/00A AU5218800A (en) 1999-06-01 2000-05-31 Test system for in-vitro detection of an antigen-specific immune response
CA002375191A CA2375191A1 (fr) 1999-06-01 2000-05-31 Systeme de test pour la detection in vitro d'une reaction immunitaire antigene specifique
JP2001500861A JP2003501628A (ja) 1999-06-01 2000-05-31 抗原特異的免疫応答のインビトロ検出のための試験システム
EP00936845A EP1183533A1 (fr) 1999-06-01 2000-05-31 Systeme de test pour la detection in vitro d'une reaction immunitaire antigene specifique

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19925234.3 1999-06-01
DE19925234A DE19925234A1 (de) 1999-06-01 1999-06-01 Capsomere, stabile Capsomere, Capside, VLPs, oder CVLPs bzw. damit beladenen Zellen und ihre Verwendung in Diagnostik und Therapie

Publications (1)

Publication Number Publication Date
WO2000073790A1 true WO2000073790A1 (fr) 2000-12-07

Family

ID=7909985

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2000/005003 WO2000073790A1 (fr) 1999-06-01 2000-05-31 Systeme de test pour la detection in vitro d'une reaction immunitaire antigene specifique

Country Status (6)

Country Link
EP (1) EP1183533A1 (fr)
JP (1) JP2003501628A (fr)
AU (1) AU5218800A (fr)
CA (1) CA2375191A1 (fr)
DE (1) DE19925234A1 (fr)
WO (1) WO2000073790A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005501257A (ja) * 2001-08-27 2005-01-13 アンスティテュ　ナシオナル　ド　ラ　サント　エ　ド　ラ　ルシュルシェ　メディカル（アンセルム） 固体担体上に固定されたペプチドを用いる細胞性免疫試験

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994005792A1 (fr) * 1992-09-03 1994-03-17 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Proteines de capsides de virus du papillome recombinees a auto-assemblage
WO1999018220A1 (fr) * 1997-10-06 1999-04-15 Loyola University Of Chicago Formulations de vaccins contenant des capsomeres de papillomavirus et procedes d'utilisation

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ATE332973T1 (de) * 1991-07-19 2006-08-15 Univ Queensland Polynukleotidabschnitt des hpv16-genoms

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994005792A1 (fr) * 1992-09-03 1994-03-17 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Proteines de capsides de virus du papillome recombinees a auto-assemblage
WO1999018220A1 (fr) * 1997-10-06 1999-04-15 Loyola University Of Chicago Formulations de vaccins contenant des capsomeres de papillomavirus et procedes d'utilisation

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
GREENSTONE H L ET AL: "Chimeric papillomavirus virus-like particles elicit antitumor immunity against the E7 oncoprotein in an HPV16 tumor model", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA,US,NATIONAL ACADEMY OF SCIENCE. WASHINGTON, vol. 95, no. 4, 17 February 1998 (1998-02-17), pages 1800 - 1805, XP002117898, ISSN: 0027-8424 *
JOCHMUS I. ET AL.: "Chimeric virus-like particles of the human papillomavirus type 16 (HPV 16) as a prophylactic and therapeutic vaccine", ARCHIVES OF MEDICAL RESEARCH, vol. 30, July 1999 (1999-07-01), pages 269 - 274, XP000957577 *
MÜLLER M. ET AL.: "Chimeric papillomavirus-like particles", VIROLOGY, vol. 234, 1997, pages 93 - 111, XP002091857 *
PENG S. ET AL.: "Papillomavirus virus-like particles can deliver defined CTL epitopes to the MHC class I pathway", VIROLOGY, vol. 240, 1998, pages 147 - 157, XP000952966 *
SCHÄFER K. ET AL.: "Immune response to human papillomavirus 16 L1E7 chimeric virus-like particles: induction of cytotoxic T cells and specific tumor protection", INT. J. CANCER, vol. 81, 11 June 1999 (1999-06-11), pages 881 - 888, XP000957625 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005501257A (ja) * 2001-08-27 2005-01-13 アンスティテュ　ナシオナル　ド　ラ　サント　エ　ド　ラ　ルシュルシェ　メディカル（アンセルム） 固体担体上に固定されたペプチドを用いる細胞性免疫試験

Also Published As

Publication number Publication date
JP2003501628A (ja) 2003-01-14
AU5218800A (en) 2000-12-18
DE19925234A1 (de) 2000-12-14
CA2375191A1 (fr) 2000-12-07
EP1183533A1 (fr) 2002-03-06

Similar Documents

Publication Publication Date Title
DE69532532T2 (de) Chimerische papillomavirus-ahnliche partikel
DE69835294T2 (de) Methode zur in vitro auseinander- und wiederzusammensetzung von papillomavirus virusähnlichen teilchen (vlps)
DE19543553B4 (de) VP-Antigene des JC-Virus
DE69929232T2 (de) Virusähnliche partikel zur induktion von autoantikörpern
DE69333859T2 (de) Sich selbst-zusammenbauende rekombinante hpv16 papillomavirus hüllproteine
DE60132770T2 (de) Chimäre humane papillomavirus (hpv) l1 moleküle und deren verwendungen
EP0809700A1 (fr) Particules semblables au virus du papillome, proteines de fusion et leur procede de production
DE60126737T2 (de) Modifizierte HPV E6- und E7-Gene und -Proteine als Impfstoff
EP1181312A1 (fr) Epitopes cytotoxiques de cellule t de la proteine l1 du papillomavirus et leur application en diagnostic et therapie
EP1370661A2 (fr) Epitopes lymphocytaires t des proteines l1 et e7 de papillomavirus et leur utilisation a des fins de diagnostic et de therapie
DE4447664C2 (de) Fusionsproteine, Verfahren zu deren Herstellung sowie deren Anwendung
WO2002043757A2 (fr) Medicament pour la prophylaxie ou le traitement de tumeurs induites par le virus du papillome humain de type 18
EP1140987B1 (fr) Peptide pour l'inhibition des proteines e6 du virus de l'hepatite b
DE69933891T2 (de) Behandlung von papillomavirus-infektionen
WO2000073790A1 (fr) Systeme de test pour la detection in vitro d'une reaction immunitaire antigene specifique
EP1183367B1 (fr) Epitope cytotoxiques des lymphocytes t, de proteine l1 du papillomavirus, et utilisation dans les domaines du diagnostic et de la therapie
DE69924007T2 (de) Neutralsierungs-assay, das humane papillomavirus-ähnliche partikel verwendet
EP1064014B1 (fr) Medicament permettant de prevenir ou de traiter les tumeurs a papillomavirus
DE69726886T2 (de) Synthetische hpv16 virus-ähnliche partikel
DE19526752C2 (de) Hocheffiziente Bildung von Papillomavirusähnlichen Partikeln
DE19649606C1 (de) Systeme zur Bestimmung von Wirksubstanzen gegen HPV-assoziierte Karzinome
EP1888620A1 (fr) Procede pour produire des particules de type viral contenant un principe actif
WO1999048917A2 (fr) Formulation contenant une proteine specifique des papillomavirus, sa fabrication et son utilisation

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
ENP Entry into the national phase

Ref country code: CA

Ref document number: 2375191

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 52188/00

Country of ref document: AU

ENP Entry into the national phase

Ref country code: JP

Ref document number: 2001 500861

Kind code of ref document: A

Format of ref document f/p: F

WWE Wipo information: entry into national phase

Ref document number: 2000936845

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 2000936845

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 09980417

Country of ref document: US

ENP Entry into the national phase

Ref document number: 2375191

Country of ref document: CA

WWW Wipo information: withdrawn in national office

Ref document number: 2000936845

Country of ref document: EP