WO2000073790A1

WO2000073790A1 - Test system for in-vitro detection of an antigen-specific immune response

Info

Publication number: WO2000073790A1
Application number: PCT/EP2000/005003
Authority: WO
Inventors: Ingrid Jochmus; John Nieland
Original assignee: Medigene Aktiengesellschaft
Priority date: 1999-06-01
Filing date: 2000-05-31
Publication date: 2000-12-07
Also published as: AU5218800A; CA2375191A1; DE19925234A1; JP2003501628A; EP1183533A1

Abstract

The invention 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 target cell presenting an antigen, especially a B cell, a macrophage, a predendritic cell, a dendritic cell, embryonal 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, in order to detect an antigen-specific immune response in vitro, especially a cellular immune response from effector cells of the immune system, especially B cells, NK cells, preferably T cells, more preferably cytotoxic T cells or helper T cells. The invention also relates to the use thereof in diagnosis and therapy.

Description

Test system for in vitro detection of an antigen-specific immune response

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.

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. To date, 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. More than 90% of the Ll protein is present in the viral capsid, the ratio of L 1: L2 generally being 30: 1. For the purposes of the present invention, the term 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. In over 50% of cases, 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. It has also been found that 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). However, 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. It could be shown that the expression of the Ll gene or the coexpression of the Ll and L2 gene can lead to the formation of capsomers, stable capsomers, capsids or virus-like particles (VLPs for virus-like particles) ( see for example WO 93/02184, WO 94/20137 or WO 94/05792). 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. (1991) Biophys. J. 60, 1445). 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. However, the formation of 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. And although 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.

So-called chimeric papillomavirus-like particles (CVLPs for chimeric virus-like particles) have therefore been developed, which consist of 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). However, 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). Furthermore seem neutralizing antibodies from HPV-associated diseases in patients to limit the immune response to administered L1 protein (Müller, M. et al. (1997), supra). However, CVLPs are still of interest for the development of a vaccine, since the E7 proteins of tumor cells presented via class I MHC molecules would represent target molecules of CTLs.

Peng S. et al. (1998; Virology, 240, 147) now described 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. Greenstone HL et al. (1998; Proc. Natl. Acad. Sci. USA, 95, 1800-5) describe 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.

While the immune response to standard vaccines such as If tetanol is mediated by antibodies and is therefore serologically detectable, a test system for the cellular immune response that can also be standardized for humans had to be developed for such a therapeutic vaccine.

Existing systems are based on the basic principle that ^' peripheral blood mononuclear cells' (PBMC), which are peripheral blood cells with a nucleus, including lymphocytes, macrophages and dendritic cells, isolated from an organism, brought into culture and incubated with a certain form of an antigen. The antigens are taken up by antigen-presenting cells (B lymphocytes, macrophages, dendritic cells), processed intracellularly and via histocompatibility antigens (major histocompatibility complex MHC) presented on the surface of the T cells. There are two ways to do this:

• Peptides that are presented via MHC I molecules are recognized by CD8-positive T cells, which thus develop into activated cytotoxic T cells or the antigen-presenting ones through this stimulation

Can lyse cells;

• Peptides that are presented via MHC II molecules are recognized by CD4-positive T cells, which can thus develop into activated T helper cells through this stimulation. B cells bind an antigen via their antigen receptors on the surface (IgM or IgD). 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 γ, interleukin 3 (IL3) can be detected. 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.

In the systems of Käst, WM et al. (1989; Cell, 17, 603-14), Rock, KL et al. (1992; Proc. Natl. Acad. Sci. USA, 89, 8918-22) and Cerundolo, V. et al. (1990; Nature, 345, 449-52), individual, defined peptides (8-, 9- or 10-mers) are used as the antigen. The peptides are characterized by the MHC molecules of class I, which are expressed on the cell surface, and which are sucked by organisms vaπ 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 For 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 In addition, for most antigens the protein fragments or peptides, which can represent a cytotoxic T cell epitope, are not known

If larger peptides or proteins are used to stimulate PBMCs, as in Allen, PM and Unanue, ER (1984, Immunobiology, 168, 182-8), these antigens are taken up into the cytoplasm or via endosomes and processed. The resulting peptides bind to MHC II -Molecules and are presented to the CD4-positive cells on the cell surface. Such a system is therefore limited to the detection of the activation of T helper cells and cannot be used, for example, for evaluating therapeutic vaccines, which are also based on the triggering of a cellular immune response not be used exclusively

The dilemma thus prevails that the peptides or proteins are either MHC-resident or can only activate T helper cells

These disadvantages are alleviated by the systems of Taφey, I et al (1994, Immuno> - gy, 81, 222-7) and Nimako, M et al (1997, Cancer Res 57, 4855-61) avoided that they use recombinant vaccinia or adenoviruses as antigen ferries. The respective virus introduces the information for the corresponding antigen into the cell as part of its genome by infection. Then there is expression of both viral proteins and the antigen within the cell. The viral antigens, like the specific antigen, are subsequently processed and presented to the T cells via MHC-I molecules. Thus, in contrast to the system mentioned above, 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,

In which 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;

• in which the uptake of the antigen in the cell is independent of MHC molecules; in which there is no viral infection associated with viral protein expression and viral replication; that can be standardized.

This problem was solved by surprisingly demonstrating that in an in vitro experiment the incubation of PBMCs with, for example, CVLPs resulted in an antigen-specific immune response, in particular a cytotoxic immune response from immune cells, such as e.g. leads to cytotoxic T cells, T helper cells or B cells. Therefore, the fundamental difference between the previous detection methods and the present invention is the type of antigen that is used for stimulation or restimulation.

In addition, it was surprisingly found that the binding of CVLPs to predendritic cells in vitro suggests an activation of an immune response in vivo. In the context of this invention, 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. In contrast, 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.

It could now be shown that CVLPs, which could trigger a cellular immune response in vivo, could bind in vitro to predendritic cells, for example JAWS II cells. CVLPs from another batch that do not have a cellular immune response for unknown reasons, but a humoral immune response were also unable to bind to the predendritic cells. In contrast, 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.

A possible explanation for this phenomenon is that CVLPs enter the cell via CD 16 as a receptor. 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 observed cytotoxic immune response presupposes that the exogenous proteins of the CVLPs were actually presented to the CD8-positive cells via MHC molecules of class I. Therefore, an intracellular loading of the MHC-I molecules after incubation with CVLPs must be assumed. However, as the experimental approach of Allen PM and Unanue ER (supra) teaches, exogenous proteins are normally presented to CD4-positive cells via class II MHC molecules and do not enter the MHC-I system. This surprising, fundamentally different behavior of the exogenous proteins and the CVLPs may be due to the fact that CVLPs, due to their particular structure and receptor-mediated uptake into the cell, have the ability to "pseudo-infect" cells. In this case, 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.

After the recognition of the MHC molecule with its bound peptide by the respective, specific T cell via the respectively specific T cell receptor, 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 γ or interleukin 4 (IL4), which accumulate in the cytoplasm in this system. As described in Current Protocols of Immunology (Chapters 6.2 to 6.24 (1999), edited by Coligan J.E, Kruisbeek A.M., Margulies D.H., Shevach E.M. and Strober W., John Wiley & Sons), e.g. the intracellular interferon γ can be used for the detection of specifically activated T cells.

This means that 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. In contrast to the experimental setup with free peptides or proteins that predominantly stimulate either CD4 or CD8 positive cells, capsomers, stable capsomers, capsids, VLPs, and / or CVLPs activate both CD4 and CD8 positive T cells equally. Finally, 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.

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.

In a preferred embodiment, 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. In a special embodiment, 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. In a particular embodiment, 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. In a preferred embodiment, 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 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. In a further preferred embodiment, 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. In a particular embodiment, 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. In a preferred embodiment, 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. In a particular embodiment, 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. In a preferred embodiment, 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. Furthermore, 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. In a further preferred embodiment, 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.

In all preferred embodiments, 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. coli, yeasts such as S. cerevisiae, in particular insect cells such as Spodoptera frugiperda cells or Trichoplusia ni cells or mammalian cells such as COS cells or HeLa cells. 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. In a preferred embodiment, an incubation of immune cells (effector cells), for example PBMCs, T-cellinins or cultivated primary T-cells with antigens, preferably with at least one capsomer, at least one stable capsomer, 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. In a further preferred embodiment, 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. For example, 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. For this purpose, 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) Depending on the method used, it is also possible to distinguish between immune cells such as cytotoxic T cells, T helper cells, B cells, NK cells and other cells.

In a further embodiment of the method, the following additional 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.

In a preferred embodiment of the method according to the invention, 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. holding 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 cell with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or was incubated in a CVLP.

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:

I) In a first step, for example, PBMCs, in particular T cells, 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.

II) In a second step, for example, the isolated and / or cultivated cells are used for at least about 5 hours, in particular about

17h 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. In a further preferred embodiment, 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.

III) In a second step, the possible activation of effector cells is determined. For example, 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

Proof of proliferation (Chapter 3J2 in Current Protocols in Immunology, supra). Depending on the method used, a distinction can also be made between immune cells such as cytotoxic T cells, T helper cells, B cells, NK cells and other cells.

In a further embodiment of the method, the following additional step I ') is inserted after step I)

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

II) connects. This preactivation of the effector cells results in 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. However, 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.

In a particular embodiment, the immune status of an organism can be tested against a pathogen by the method according to the invention. For example, 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. In a further embodiment, the reactive cells can be quantified by quantifying them, so that e.g. the need for booster shots can be analyzed.

In a further embodiment, 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.

In a further embodiment of the method according to the invention, 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.

If the antigens which are responsible for certain autoimmune diseases are known, 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. In high-throughput systems, in addition to the compounds mentioned, 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.

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.

Examples of 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.

The figures and the following examples are intended to explain the invention in more detail without restricting it.

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 γ 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 μg ml), in which human T cells that are positive for human interferon γ 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 γ.

4 shows the graphic evaluation of the restimulation of human PBMC by various antigens after the PBMC had been stimulated with CVLPs beforehand.

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 γ, 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 LlE7ι. 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.

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. In this case, RMA cells were used which had previously been incubated with LlE7ι- ₆₀ CVLPs or with Ll-VLPs, that is to say without an E7 portion.

7 shows the evaluation of FACScan experiments after incubation of JAWS II cells with increasing amounts of CVLPs, plotted from left to right. On the one hand, 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. On the other hand, 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.

8 shows the evaluation of FACScan experiments after incubation of JAWS II cells with the indicated amounts of CVLPs or serum from CVLP-vaccinated mice. "Depl" means the depletion of the CVLPs from the incubation batches before they were added to the JAWS II cells. In the CVLP + serum approach, the CVLPs were previously incubated with the serum before they were added to the JAWS II cells. 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. The values are plotted as relative fluorescence units on the Y axis.

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.

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 γ-positive cells was then determined using specific antibodies in the FACS experiment.

Examples

1. Description of the starting materials: • The HPV 16 Ll _Δ c _* E7ι- ₅₅ CVLPs were produced in accordance with German patent application DE 198 12 941.6 (see also Müller M. et al. (1997) Virology 234, 93-1 1 1) .

• The HPV16Ll _Δ c * E7ι _-60 CVLPs were produced according to Müller M. et al. (1997; supra). • Ll VLPs are manufactured according to Müller M. et al. (1997); Virology 234, 93-11 1.

• 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 mean 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 _ΛC means recombinant murine vaccinia virus that expresses HPV 16 L1 _ΛC in infected cells.

• IL-2 means recombinant cytokine (Becton Dickinson, Hamburg, Germany).

• 25 / C α-HPV16Ll means a monoclonal mouse antibody that is directed against Ll.

• -hu CD28 means a monoclonal mouse antibody that is directed against the extracellular part of human CD28 (ATCC CRL-8001).

• α-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).

• α-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).

• α-CD4 / Tricolor means a monoclonal antibody which is directed against the extracellular part of CD4 and contains a fluorescent marker Tricolor (Medac, Hamburg, Germany). • α-mus interferon γ / FITC means a monoclonal rat antibody which is directed against mus interferon γ 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).

• Golgi Plug was purchased from Pharmingen (Hamburg. Germany).

• Cells were each cultured at 37 ° C. and 5% CO ₂ in RPMI medium (Gibco BRL, Eggenstein, Germany) with 10% fetal calf serum, kanamycin and ampicillin.

• JAWS II cells are predendritic cells (see US 5,648,219).

• Monensin was purchased from Sigma (Deisenhofen, Germany).

• MHC IA ^b -antibody was obtained from Pharmingen (Heidelberg, Germany). • FITC-coupled anti-mouse antibodies were obtained from Sigma (Deisenhofen, Germany). • FITC-coupled 25 / C α-HPV16Ll antibody means a monoclonal mouse antibody that is directed against L1 and is itself linked to FITC. FITC was obtained from Sigma (Deisenhofen, Germany).

Α-mouse CD8 / PE antibodies were obtained from Pharmingen (Heidelberg, Germany).

Α-mouse interferon γ / FITC antibody was obtained from Caltag (Hamburg, Germany).

Α-mouse CD4 / Cychrom-Antiköφer was obtained from Pharmingen (Heidelberg, Germany). • Zefa membrane means a 45 μm 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).

• Cellquest software was purchased from Becton Dickenson (Hamburg, Germany).

2. Production of murine CD8 positive T cells

a) Immunization of mice with VLPs: Two C57Bl / 6 mice were immunized with 10 μg Ll VLPs. After 6

The spleen cells were isolated for weeks.

b) Production of antigen-presenting cells (target cells):

B6 cells were incubated with interferon γ for 2.5 days, then infected overnight with MVA-L1 _ΔC viruses (MOI = 5) for the production of antigen-presenting cells and cultured for _{16 hours} . In a next one - ..

Step, the infected B6 cells were irradiated, thereby preventing further growth.

c) Restimulation of the isolated spleen cells:

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.

3. Restimulation of T cells by antigen presenting cells

2χ l0 ⁴ murine antigen-presenting cells (C3 or B16F10) were incubated with different concentrations of HPV16 L1 _ΛC * 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 μl golgi plug. The cells were then fixed, permeabilized, stained with α-mouse CD3 PE, with α-mouse CD4 / Tricolor and with α-mouse interferon γ / 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 γ signal greater than 10 ¹ were defined as stimulated cells. T cells were defined by a CD3 signal of over 10 ² .

Result: 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.

4. Inhibition of pseudoinfection by virus-neutralizing antibodies

Murine C3 target cells were incubated overnight at 37 ° C with 6 different HPV 16 L1 _C * E7ι _-55 CVLPs preparations or with the P12 peptide, in each case a) without antibodies b) with 25 / C αHPV16Ll antibodies (10 μg / 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 γ production (see previous example). C3 cells which had not been incubated with no antigen served as a negative control (see FIG. 2).

Result: As with a viral HPV infection, 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 (4χ 10 ⁶ ) were in 100 ul medium overnight at 37 ° C with different concentrations HPV16 Ll_-c * E7ι. ₅₅ CVLPs as well as 10 IU / ml IL2 and 0.5 μg / ml anti-human CD28 were incubated. The following day, 1 μl 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 / and with anti-human interferon γ / 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).

Result: It was found that with increasing CVLP concentration the proportion of T cells which accumulate interferon γ in the cytoplasm increases linearly (see FIGS. 3A and B). This increase thus shows the stimulation of the T cells by the CVLPs.

6. Restimulation of CVLP-stimulated PBMC with different antigens

Human PBMC (4χl0 ⁵ ) were stimulated and harvested for 3 weeks with HPV 16 Ll _ΔC * E7ι _-55 CVLPs at 37 ° C with weekly addition of 1 μg / 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 _Δ cΕ7 ,. ₅₅ CVLPs c) influenza peptide d) phytohaemaglutinin (PHA) in the presence of 10 IU / ml IL2 and 0.5 μg / ml anti-human CD28 restimulated. After one hour, 1 μl 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 α-human CD3 / PE, with α-human CD4 / cychrome and with α-human interferon γ / 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).

Result: Three times more T cells could be restimulated by CVLPs than by the E7 or influenza peptide (see FIG. 4). As expected, phytohemaglutinin stimulated most T cells as a non-specific stimulant. As a result of the three-week incubation with CVLPs, part of the human PBMC was specifically restimulable using CVLPs.

7. Restimulation of CVLP-stimulated T cells with different antigen-presenting cells

Human PBMCs (4χl0 ⁵ ) were stimulated and harvested for 8 weeks with HPV 16 Ll _ΔC .E7ι _-55 CVLPs at 37 ° C with weekly addition of 1 μg / ml CVLPs and 10 ³ irradiated PBMCs. The cells were then restimulated in 100 μl medium at 37 ° C. with 10 μg / ml different antigens in the presence of 10 IU / ml IL2 and 0.5 μg / ml anti-human CD28: a) overnight with HPV16 L1 _ΔC «E7ι. ₅₅ CVLP-incubated PBMC, b) PBMC incubated overnight with HPV 16 Ll 93-101 peptide.

After one hour, 1 μl 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 γ / 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).

Result: 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. Thus, through the eight-week incubation of the human T cells with CVLPs, specifically restimulable T cells were created. Furthermore, it can be concluded that 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.

8. Lvse of CNLP-incubated cells by E7-specific cytotoxic T cells

RMA cells were in the presence of ^{5 L} Cr for 1 hour at 37 ° C

• HPV16Ll _ΔC «E7ι_ ₆₀ CVLPs or

• Ll VLPs incubated. Similarly, RMA-E7 cells were incubated in the presence of ⁵¹ Cr for 1 hour at 37 ° C, but without the addition of antigen. Then, in different ratios to the target cells (RMA cells), E7-specific cytotoxic T cells (C57BL / 6 mouse (H2 ^b )) (see Example 2) give. The release of the ^ Cr associated with the lysis of the target cells was measured in a β-counter and set in relation to completely lysed cells.

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.

Result: 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.

9. Activation of predendritic cells

Firstly 3x10 ^"JAWS _* were incubated Il cells with increasing amounts of HPV 16Ll _Δ c E7 ₁ _ ₅₅ CVLPs for 2 days. The cells were then examined to determine whether they express MHC class II molecules by initially with a 1 ug / ml MHC IA ^b antibodies and then incubated with 1 μg / ml FITC-coupled anti-mouse antibodies, and finally the cells were examined for their staining in a FACScan calibur and the measurement results were analyzed using cellquest software. On the other JAWS were incubated Il cells as before with increasing amounts HPV16Ll _Δ c "E7i _ό5 CVLPs for only 3 hours. These cells were examined to see if they had bound CVLPs. For this purpose, the cells were first stained with the FITC-coupled 25 / C α-HPV16Ll antibody. The cells were then examined for their staining in a FACScan calibur and the measurement results were analyzed using cellquest software.

It can be seen from FIG. 7 that with increasing CVLP concentrations with which JAWS II cells were incubated, the binding of the CVLPs to the cells increased and the cells were activated for the expression of the MHC class II molecules.

Furthermore, JAWS II cells were, as described above, in various approaches with HPV16Ll _Λ c »E7ι. ₅₅ CVLPs and / or mouse serum incubated over 2 days. The following were added:

80 or 10 μg CVLPs,

A solution with 80 or 10 μg CVLPs, which was filtered through a Zefa membrane, which is known to effectively remove the CVLPs from the solution, with most of the impurities passing through the filter (deputed approach),

• 10 μl of a 1/20 dilution of a mouse serum which was obtained from a mouse immunized with the CVLPs mentioned and

• 10 μg CVLPs that were pre-incubated with 10 μl of the mouse serum mentioned for 5 min.

The cells were then examined as above to determine whether they express MHC class II molecules by first staining them with a 1 μg / ml MHC IA ^b antibody and then with 1 μg / 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.

10. Predendritic cells as a test cell line for in vivo T cell activation

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 μg / ml of the FITC-coupled 25 / C α-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 μg / 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:

Three C57BL6 mice were vaccinated with 10 or 1 μg 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 μg / ml of the murine cytotoxic P12 peptide for 5 days. The T cells were then restimulated in 100 μl medium at 37 ° C. with a further 10 μg 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 µl monensin (300 µM) was added. The cells were incubated for a further 5 hours at 37 ° C. The cells were then fixed and permeabilized, stained with 1 μg / ml α-mouse CD4 / cychrome, α-mouse CD8 / PE and with α-mouse interferon γ / 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. Thus, predendritic cells such as the JAWS II cells are particularly suitable for the prediction of in vivo T cell activation using CVLPs.

Claims

0

43

claims

1. Test system for in vitro detection of an antigen-specific immune response, in particular a cellular immune response, comprising: a) 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, with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP were incubated, and b) 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.

2. Test system for in vitro detection of an antigen-specific immune response, in particular a cellular immune response, the test system comprising at least a) a capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP and at least one Has a predendritic cell and / or a CD16-positive 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 at least one CVLP, and b) the binding of the stable capsomer, capsid, VLPs and / or

CVLPs to the Pradendπtische cell and or the CD16-posιtιve cell is measured

Test system according to claim 1 or 2, characterized in that the cells are sucker cells, in particular human or muπne cells

Test system according to one of claims 1-3, characterized in that the capsomer, stable capsomer, capsid, VLP, and / or CVLP comprises at least one L1 protein of one or more papillomaviruses, or at least one Ll protein and at least one papillomavirus L2 protein , in particular Ll proteins or Ll and L2 proteins from human, bovine and / or cottontail rabbit 'papillomaviruses

Test system according to claim 4, characterized in that the capsomer, stable capsomer, capsid, and / or CVLP at least one Ll

Fusion protein, consisting of an Ll protein portion of one or more papillomaviruses and a protein portion heterologous to the Ll protein, contains

Test system according to claim 4 or 5, characterized in that the Ll-

Protein content is a naturally occurring Ll protein

Test system according to claim 4 or 5, characterized in that the Ll protein portion is deleted and / or mutated

Test system according to claim 7, characterized in that from the C-terminus of the L1 protein portion up to at least about 35 amino acids, 0

- 45 -

preferably at least about 25 to about 35, in particular at least about 32 to about 34, amino acids are deleted.

Test system according to one of claims 4-8, characterized in that the protein portion which is heterologous to the L1 protein is a naturally occurring protein.

10. Test system according to one of claims 4-8, characterized in that the protein portion heterologous to the L1 protein is deleted and / or mutated.

1 1. Test system according to claim 9 or 10, characterized in that the protein portion heterologous to the Ll protein is derived from a bacterial or viral protein, preferably from a papillomavirus protein, in particular from a protein of a human papillomavirus.

12. Test system according to claim 1 1, characterized in that the papillomavirus protein is an E protein, in particular an E6 or E7 protein.

13. Test system according to claim 12, characterized in that 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.

14. Test system according to claim 9 or 10, characterized in that the protein portion heterologous to the Ll protein of an autoimmune antigen, in particular thyroglobulin, myelin or zona pellucida glycoprotein 3rd (ZP3) or is derived from a tumor antigen, in particular a melanoma, ovarian carcinoma or breast carcinoma antigen.

15. Cell characterized in that 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.

16. Cell according to claim 15, characterized in that the capsomer, stable capsomer, capsid, VLP, and / or CVLP at least one Ll protein of one or more papillomaviruses, or at least one Ll protein and at least one papillomavirus L2 protein contains.

17. Cell according to claim 16 or 17, characterized in that the capsomer, stable capsomer, capsid, and / or CVLP at least one Ll

Fusion protein, consisting of an Ll protein portion of one or more papillomaviruses and a protein portion heterologous to the Ll protein.

18. Cell according to claim 16 or 17, characterized in that the Ll protein portion is a naturally occurring Ll protein.

19. Cell according to claim 16 or 17, characterized in that the Ll protein portion is deleted and / or mutated.

20. Cell according to claim 19, characterized in that from the C-terminus of the Ll protein portion up to at least about 35 amino acids, preferably - 47 -

at least about 25 to about 35, in particular at least about 32 to about 34, amino acids are deleted.

21. Cell according to one of claims 16-20, characterized in that the protein portion heterologous to the L1 protein is a naturally occurring protein.

22. Cell according to one of claims 16-20, characterized in that the protein portion heterologous to the L1 protein is deleted and / or mutated.

23. Cell according to claim 21 or 22, characterized in that the protein portion heterologous to the Ll protein is derived from a bacterial or viral protein, preferably from a papillomavirus protein, in particular from a protein of a human papillomavirus.

24. Cell according to claim 23, characterized in that the papillomavirus protein is an E protein, in particular an E6 or E7 protein.

25. Cell according to claim 24, characterized in that 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 have been deleted from the C-terminus of the E7 protein.

26. Cell according to claim 21 or 22, characterized in that the protein portion heterologous to Ll protein from an autoantigen, in particular

Thyroglobulin, myelin or ZP3 or from a tumor antigen, in particular - 48 -

which is derived from a melanoma, ovarian carcinoma or breast carcinoma antigen.

27. Cell according to one of claims 15-26, characterized in that it is an antigen-presenting cell, in particular B cell, macrophage, predendritic cell, dendritic cell, embryonic cell or fibroblast.

28. A method for producing a target cell according to any one of claims 15-27, characterized in that the target cell is 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 in vitro.

29. A method for producing a test system according to any one of claims 1- 14, characterized in that 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 CD16 positive cell is incubated.

30. A method for producing a test system according to any one of claims 1-14, characterized in that at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLP are genetically engineered and the target cell according to claim 28 and that 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.

31. A method for producing a test system according to any one of claims 1-14, characterized in that the capsomer, the stable capsomer, the capsid, the VLP, and / or the CVLP in bacteria such as E. coli, yeasts such as S. cerevisiae, in particular insect cells such as Spodoptera frugiperda cells or Trichoplusia ni cells or mammalian cells such as COS cells or HeLa cells.

32. Method for in vitro detection of the activation of effector cells of the immune system by at least one capsomer, at least one stable one

Capsomer, at least one capsid, at least one VLP, and or at least one CVLP and / or at least one cell according to one of claims 14-26, which comprises the following steps: a) using a test system according to claims 1-14; b) determining the possible activation of effector cells.

33. The method according to claim 32, characterized in that it contains the following additional step a ') before step a): a') co-cultivating the effector cell of the test system according to claim 1 or 3 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, at least one target cell of the test system according to one of claims 1-14, and / or with at least one cell according to one of the claims 15-27 before step a) follows.

34. Method for in vitro detection of the activation of effector cells of the immune system by at least one capsomer, at least one stable one Capsomer, at least one capsid, at least one VLP, and or at least one CVLP and a predendritic and / or a CD16-positive cell according to one of claims 15-27, which comprises the following steps: a) use of a test system according to claims 1-14; b) Determination of the binding of the stable capsomer, capsid, VLPs and / or CVLPs to the predendric cell and / or CD16 positive cell.

35. The method according to any one of claims 32 to 34, characterized in that it for the quality control of batches of prophylactic and / or therapeutic vaccines 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 cell according to any one of claims 14-26 is used.

36. The method according to any one of claims 32 to 34, characterized in that it is used for the identification of epitopes, peptides or protein fragments which trigger an immune response, in particular a cellular immune response.

37. Method for 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 according to one of the claims 15- 27, which includes the following steps:

I) Obtaining and preparing samples containing effector cells of the immune system and subsequent cultivation. II) adding 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 according to one of claims 15-27. III) Determining the possible activation of effector cells.

38. The method according to claim 37, characterized in that it contains the following additional step I ') after step I):

I ') co-cultivating the effector cell 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 cell one of claims 15-27 before step II) follows.

39. The method according to claim 37 or 38, characterized in that it is used for monitoring the immune status of an organism against a pathogen, in particular a pathogen that is difficult to detect.

40. The method according to claim 37 or 38, characterized in that it is used for monitoring a vaccination.

41. The method according to claim 37 or 38, characterized in that it is used for the identification of HLA haplotypes which impart immunity to a particular pathogen.

42. The method according to claim 37 or 38, characterized in that it is used for the differentiation and characterization of autoimmune diseases with regard to different specific autoimmune antigens.

43. The method according to claim 37 or 38, characterized in that it is used for the differentiation of tumor types with regard to different specific tumor antigens.

44. The method according to any one of claims 32-43, characterized in that the activation of the effector cell by at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP, and / or at least one CVLPs is carried out in high-throughput systems.

45. Use of at least one test system according to one of claims 1-14 and or a cell according to one of claims 15-26 for triggering or for detecting an immune response.

46. Diagnostic agent containing at least one test system according to one of claims 1-14 and / or a cell according to one of claims 15-27 and optionally a pharmaceutically acceptable carrier.

47. Diagnostic agent according to claim 43, characterized in that at least one test system according to one of claims 1-14 and / or a cell according to one of claims 15-26 is in solution, is bound to a solid matrix, and / or with an adjuvant is offset.