CA2375191A1

CA2375191A1 - Test system for in-vitro detection of an antigen-specific immune response

Info

Publication number: CA2375191A1
Application number: CA002375191A
Authority: CA
Inventors: Ingrid Jochmus; John Nieland
Original assignee: Individual
Current assignee: Medigene AG
Priority date: 1999-06-01
Filing date: 2000-05-31
Publication date: 2000-12-07
Also published as: DE19925234A1; AU5218800A; WO2000073790A1; 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, especiall y a B cell, a macrophage, a predendritic cell, a dendritic cell, embryonal cel l 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 immu ne 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 the in-vitro detection of an antigen specific immune response The present invention relates to a test system which comprises 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 a cellular immune response of 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 to their use in diagnosis and therapy.
The papilloma viruses, also termed wart viruses, are double-stranded DNA viruses having a genome size of about 8000 base pairs and an icosahedral capsid having a diameter of approx. 55 nm. So far, more than 100 different human papilloma virus types are known, some of which, for example HPV-16, HPV-18, HPV-31, HPV-33, HPV-39, HPV-45, HPV-52 and HPV-58, can cause malignant tumours while others, for example HPV-6, HPV-11 and HPV-42, can cause benign tumours.
The genome of the papilloma viruses can be subdivided into three regions: the first region is a noncoding region which contains the regulatory elements for transcribing and replicating the virus. The second region, which is termed the E(early)region, contains different protein-encoding segments E1-E7, of which the E6 protein and the E7 protein, for example, are

- 2 -responsible for transforming epithelial cells, and the E1 protein controls the DNA copying number. The genes in the E6 and E7 regions are what are termed oncogenes, which are also expressed in malignantly degenerate cells. The third region, also termed the L(late)region, contains two protein-encoding segments L1 and L2 which encode structural components of the viral capsid. More than 900 of the L1 protein is present in the viral capsid, with the ratio of L1:L2 generally being 30:1.
Within the meaning of the present invention, the term Ll protein is understood as signifying the main papilloma virus capsid protein (Baker T. et al. (1991) Biophys. J. 60, 1445).
HPV-6 and HPV-11 are held responsible, inter alia, for causing genital warts, while some papilloma virus types, such as HPV-16, HPV-18, HPV-31, HPV-33, HPV-39, HPV-45, HPV-52 and HPV-58, are associated with malignant tumours of the anogenital tract. In more than 500 of cases, HPV-16 is linked to uterine cervical cancer (carcinoma of the cervix). HPV-16 is the main risk factor for the formation of cervical neoplasias.
The immune system plays an important role in the progress of the disease. Thus, cellular immune responses, and, in particular, antigen-specific T
lymphocytes, are presumed to be important for the defence mechanism. It has furthermore been found that, in extremely malignant cervical intraepithelial neoplasias (CIN II/III) and cervical tumours, the E7 gene is expressed constitutively in all layers of the infected epithelium. For this reason, the E7 protein in particular is regarded as being a potential tumour antigen and as the target molecule for activated T
cells (see, for example, WO 93/20844). However, the cellular immune response which is induced by E7 in patients does not appear to be sufficiently strong to

- 3 -affect the course of the disease. It may be possible to augment the immune response with suitable vaccines.
It has been demonstrated that expression of the L1 gene, or coexpression of the L1 gene and the L2 gene, can lead to the formation of capsomers, stable capsomers, capsids or virus-like particles (VLPs, standing for virus-like particles) (see, for example,

4, WO 94/20137 and WO 94/05792). Capsomers are understood as being an oligomeric configuration which is composed of five Ll proteins. The capsomer is the basic structural unit from which viral capsids are constructed. Stable capsomers are understood as being capsomers which are unable to become assembled into capsids. Capsids are understood as being the envelope of the papilloma virus, which envelope is, for example, composed of 72 capsomers (Baker T. et al. (1991) Biophys. J. 60, 1445). VLP is understood as being a capsid which resembles an intact virus morphologically and in its antigenicity. It has been possible to use VLPs to induce a humoral immune response, which is characterized by the formation of neutralizing antibodies, in a variety of animal systems. However, the formation of virus-neutralizing antibodies directed against the L1 protein and/or the L2 protein is of little clinical significance when the viral infection has already taken place since a virus-specific cytotoxic T cell (CTL) response, rather than antibodies, appears to be necessary for the virus-infected cells to be eliminated. Furthermore, even though VLPs are able to induce a cytotoxic T cell response, an immune response which is directed solely against the capsid proteins Ll and/or L2 appears not to be suitable for controlling a papilloma virus associated tumour.

What are termed chimeric papilloma virus-like particles (CVLPs, standing for chimeric virus-like particles), which particles consist of a fusion protein formed between the capsid protein L1 and the potential tumour antigen E7 (WO 96/11272 and Miiller, M. et al. (1997) Virology, 234, 93), have therefore been developed. The CVLPs only induced a slight humoral immune response directed against the E7 protein (Miiller, M. et al.
(1997), see above). However, some of the CVLPs which have been tested do in fact induce the desired E7-specific cytotoxic T cell response in mice (see also Peng S. et al. (1998) Virology 240, 147-57).
Furthermore, neutralizing antibodies present in connection with HPV-associated diseases appear to limit the immune response in patients to administered L1 protein (Muller, M. et al. (1997), see above). However, CVLPs are still of interest for developing a vaccine since the E7 proteins which are presented by tumour cells by way of MHC class I molecules would be target molecules for CTLs.
Peng S. et al. (1998; Virology, 240, 147) have now described CVLPs which consist of C-terminally truncated Ll from the bovine papilloma virus (BPV) and HPV-16 E749_57, which CVLPs induce E7-specific cytotoxic T
cells, following the inoculation of C57B1/6 mice, and protect these mice from the development of E7-expressing tumours. Greenstone H.L. et al. (1998; Proc.
Natl. Acad. Sci. USA, 95, 1800-5) describe CVLPs which consist of HPV-16 Ll and HPV-16 L2 fused to the full-length HPV-16 E7 protein, which CVLPs, following the immunization of C57B1/6 mice, protect these mice from the development of epithelial E?-expressing tumour cells, with, however, cytotoxic T cells not having been demonstrated, such that the induction of the cellular immune response does not appear to be particularly efficient.

- 5 -Whereas the immune response to a standard vaccine such as tetanol is mediated by way of antibodies, and is consequently serologically detectable, it was necessary to develop a test system, which was also standardizable for humans, for the cellular immune response obtained in the case of such a therapeutic vaccine.
Previously existing systems are based on the principle that peripheral blood mononuclear cells (PBMCs), which consequently include, inter alia, lymphocytes, macrophages and dendritic cells, are isolated from an organism, cultured and incubated with a particular form of an antigen. The antigens are taken up by antigen-presenting cells (B lymphocytes, macrophages or dendritic cells), processed intracellularly and then presented to the T cells by way of histocompatibility antigens (major histocompatibility complex, MHC) on the surface. In this connection, there are two pathways:
~ peptides which are presented by way of MHC I
molecules are recognized by CD8-positive T cells which, as a result of this stimulation, are consequently able to develop into activated cytotoxic T cells and/or lyse the antigen-presenting cells;
~ peptides which are presented by way of MHC II
molecules are recognized by CD4-positive T cells which, as a result of this stimulation, are consequently able to develop into activated T helper cells.
B cells bind an antigen on their surface by way of their antigen receptors (IgM or IgD). NK (natural killer) cells have two receptors: one of them recognizes sugars on the surface of antigen-presenting cells while the other recognizes MHC I molecules. NK

- 6 -cells are stimulated after recognizing a sugar and the simultaneous absence of MHC I molecules.
The activation of an immune cell which is achieved by stimulation with an antigen can be detected, for example, by the synthesis of cytokines such as interferon y and interleukin 3 (IL3). The corresponding cytokine accumulates intracellularly in these test systems and can then be detected, for example, by way of fluorescence-coupled antibodies (Kern F. et al.
(1998) Nat. Med. 4, 975-8). Finally, the proportion of the immune cells which could be activated by the particular antigen can then be determined in a FACS
(fluorescence-activated cell sorter). Secreted cytokines can also, for example, be detected in an ELISA. Other possible methods for detecting the activation of immune cells are ELISPOT, proliferation tests and SlCr release tests.
In the systems of Kast, W.M. et al. (1989; Cell, 17, 603-14), Rock, K.L. et al. (1992; Proc. Natl. Acad.
Sci. USA, 89, 8918-22) and Cerundolo, V. et al. (1990;
Nature, 345, 449-52), single, defined peptides (8mers, 9mers or lOmers) are used as the antigen. The peptides are bound by the MHC class I molecules which are expressed on the cell surface and which, among mammals, vary very greatly from organism to organism. This means, in turn, that a peptide which is very well suited for detecting T cell responses in one organism cannot be used for another organism of the same species since this latter organism does not possess the corresponding MHC I haplotype. This system has gained acceptance in the case of mice from inbred strains, which possess an identical MHC I haplotype; however, this experimental approach is not in practice applicable in the case of humans, for example, since different peptides would have to be used for _ 7 -stimulating the T cells in the case of each patient.
Furthermore, the protein fragments or peptides which are able to constitute a cytotoxic T cell epitope are not known in the case of most antigens.
If relatively large peptides or proteins are used for stimulating PBMCs, as described in Allen, P.M. and Unanue, E.R. (1984; Immunobiology, 168, 182-8), these antigens are then taken up into the cytoplasm, or by way of endosomes, and processed. Peptides which are formed bind to MHC II molecules and are presented to the CD4-positive cells on the cell surface. Such a system is consequently limited to detecting the activation of T helper cells and cannot be used, or cannot be exclusively used, for, for example, evaluating therapeutic vaccines which are also based on inducing a cellular immune response.
Consequently, there is the dilemma that the peptides or proteins are either MHC-restricted or are only able to activate T helper cells.
These disadvantages are circumvented by the systems of Tarpey, I. et al. (1994; Immunology, 81, 222-7) and Nimako, M. et al. (1977; Cancer Res 57, 4855-61) in that they use recombinant vaccinia viruses' or adenoviruses as antigen ferries. By means of infection, the given virus introduces, as part of its genome, the information for the appropriate antigen into the cell.
Subsequently, both the viral proteins and the antigen are expressed within the cell. The viral antigens are then processed in precisely the same way as the specific antigen and are presented to the T cells by way of MHC I molecules. Consequently, in contrast to the previously mentioned system, the uptake of the specific antigen is MHC-independent in these systems such that cells of different organisms are able to present parts of the specific antigen to the T cells even if the parts of the antigen which are in each case presented may differ from haplotype to haplotype.
The vaccinia virus and adenovirus system suffers from the disadvantage that such a viral infection of the cells is associated with viral gene expression and viral replication. This additional influence on the antigen-presenting cells makes it significantly more difficult to quantitatively measure a cytotoxic T cell response which is restricted to the specific antigen.
In the second place, it is difficult and costly to implement these systems since German biosafety level S2 (Sicherheitsstufe 2) is required for handling recombinant vaccinia viruses and/or adenoviruses.
The aim of the present invention was consequently to develop a test system for the cellular immune response ~ in which the immune response of cytotoxic T cells, in particular, but also, for example, of T helper cells, B cells or NK (natural killer) cells, can be tested, in connection with which it should be possible to differentiate between the immune cells;
~ in which the uptake of the antigen into the cell is independent of MHC molecules;
~ in which there is no viral infection which is associated with viral protein expression and viral replication;
~ which can be standardized.
This object was achieved by it being possible to demonstrate, surprisingly, that the incubation of PBMCs with, for example, CVLPs leads, in an in-vitro _ g _ experiment, to an antigen-specific immune response, in particular a cytotoxic immune response, of immune cells, such as cytotoxic T cells, T helper cells or B
cells. For this reason, the basic difference between the previous detection methods and the present invention is the nature of the antigen which is used for the stimulation or the restimulation.
In addition to this, it was observed, surprisingly, that the binding of CVLPs to predendritic cells in vitro already of itself indicates that an immune response is activated in vivo. In the context of this invention, a predendritic cell is understood as being a dendritic cell precursor which expresses CV16 strongly but which, on the other hand, only expresses MHC class I and II molecules, and also CD80, CD86 and CD40, relatively weakly. By contrast, dendritic cells hardly express CD16 at all but, on the other hand, strongly express MHC class I and II molecules and also CD80, CD86 and CD40 (Woodhead et al. (1998) Immunology 94(4):552-9). A CD16-positive cell denotes a cell which can be demonstrated to express CD16 by using a specific antibody, for example in a FACScan experiment.
It was now possible to demonstrate that CVLPs, which were able to induce a cellular immune response in vivo, were able to bind, in vitro, to predendritic cells, for example JAWS II cells. CVLPs from another batch, which, for unknown reasons, were unable to induce a cellular immune response, but which were nevertheless able to induce a humoral immune response, were likewise unable to bind to the predendritic cells. However, by contrast, these CVLPs were very well able to bind to cells belonging to a T lymphoma cell line. It can consequently be concluded that the binding of the CVLPs to the predendritic cell is a limiting step in the induction of a cellular immune response.

A possible explanation for this phenomenon is that the CVLPs make their way into the cell by way of CD15 acting as the receptor. While CD16 is strongly expressed on predendritic cells, it is either not expressed, or hardly expressed at all, on dendritic cells. Indeed, dendritic cells are hardly able to bind CVLPs (data not shown).
The observed cytotoxic immune response presupposes that the exogenous proteins of the CVLPs had indeed been presented, by way of MHC class I molecules, to the CD8-positive cells. For this reason, it must be assumed that an intracellular loading of the MHC I molecules takes place following incubation with CVLPs. However, as the experimental approach used by Allen P.M. and Unanue E.R. (see above), for example, teaches, exogenous proteins are normally presented to the CD4-positive cells by way of class II MHC molecules and do not make their way into the MHC I system. This surprising and fundamentally different behaviour of the exogenous proteins of the CVLPs can possibly be accounted for by the fact that, as a result of their particulate structure and receptor-mediated uptake into the cell, CVLPs have the ability to "pseudoinfect"
cells. In this case, the particulate structure of the CVLPs would only be abolished, by disassembly and processing, in the cytoplasm, such that, in contrast to the situation with exogenous proteins, it is primarily, but not exclusively, MHC I molecules which gain access to antigen fragments, bind these fragments, transport them to the cell surface and present them there to the CD8-positive cells. In parallel to this, intracellular MHC II molecules are also loaded with antigen fragments, which MHC II molecules, in analogy to the MHC I molecules, present the peptides to the CD4-positive cells. Consequently, both MHC I and MHC II

molecules in the antigen-presenting cells are "loaded"
with CVLP peptides as a result of the incubation with CVLPs ("CVLP-loaded cells"). Within the meaning of the present invention, presentation is understood as signifying when a peptide or protein fragment binds to an MHC molecule, with this binding being able to take place, for example, in the endoplasmic reticulum or the extracellular space, and when this MHC molecule-peptide complex is then bound on the extracellular side of the cell membrane such that it can be specifically recognized by immune cells.
After the MHC molecule, together with its bound peptide, has been recognized by the relevant specific T
cell, by way of the T cell receptor which is in each case specific, the cytotoxic T cells, or the T helper cells, then proliferate. When continuously stimulated 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 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, these cell populations then produce cytokines, such as interferon y or interleukin 4 (IL4), which, in the system, accumulate in the cytoplasm. As described in Current Protocols of Immunology (Chapter 6.2 to 6.24 (1999), edited by Coligan J.E., Kruisbeek A.M., Margulies D.H., Shevack E.M. and Strober W., John Wiley & Sons), the intracellular interferon 'y can, for example, be used for detecting specifically activated T
cells.
This means that, with regard to the immune response which is induced, capsomers, stable capsomers, capsids, VLPs and/or CVLPs surprisingly behave like viruses, and not like proteins, even though they do not induce any expression of viral proteins and/or viral replication.

Consequently, because of their ability to pseudoinfect, these compounds combine the advantages of the approaches using free peptides/proteins with those of recombinant viruses. In analogy with viruses, capsomers, stable capsomers, capsids, VLPs and/or CVLPs are able to make their way, as particles, into the cytoplasm and are consequently not MHC-restricted. In contrast to the viral system, however, no gene expression is required for releasing and/or expressing the specific antigen and for loading MHC I molecules.
In contrast to the experimental setup using free peptides or proteins, which mainly stimulate either CD4-positive cells or CD8-positive cells, capsomers, stable capsomers, capsids, VLPs and/or CVLPs activate both CD4-positive and CD8-positive T cells to an equal extent. Finally, with regard to their safety standards, capsomers, stable capsomers, capsids, VLPs and/or CVLPs are only categorized as S1, which means that the industrial implementation can be realized more cheaply, and in many places with less technical input, than in the case of the S2 safety standards which are required in connection with viral systems.
The present invention therefore relates to a test system which comprises 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 a cellular immune response of effector cells of the immune system, in particular B cells, NK cells, preferably T cells, particularly preferably cytotoxic T

cells or T helper cells, and also to their use in diagnosis and therapy.
The present invention furthermore relates to a test system, which comprises 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 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, with the binding of the stable capsomer, the capsid, the VLP and/or the CVLPs to said cell 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 which are used in the test system contain at least one L1 protein from one or more papilloma viruses, or at least one L1 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 papilloma viruses. In a preferred embodiment, the capsomers, stable capsomers, capsids and/or CVLPs contain at least one Ll fusion protein which consists of an L1 protein moiety from one or more papilloma viruses and a protein moiety which is heterologous to the L1 protein.
The L1 protein which is used can be a naturally occurring Ll protein or it can contain one or more deletions, which may, for example, be 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 from approx. 25 to approx. 35, in particular at least from approx. 32 to approx. 34, amino acids are deleted from the C terminus of the L1 protein.
The protein moiety which is heterologous to the Ll protein can be a naturally occurring protein or can contain one or more deletions, which may, for example, be 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 and can be derived, for example, from HIV, HBV, HCV or CMV, preferably from papilloma viruses, in particular from human papilloma virus, for example, but not exclusively, from E6 or E7.
In a preferred embodiment, at least approx. 55 amino acids, preferably at least from approx. 5 to approx.
35, in particular at least from approx. 38 to approx.
55, amino acids are deleted from the C terminus of the E7 protein. Furthermore, these proteins can be derived from autoimmune antigens, such as thyroglobulin, myelin or zona pellucida glycoprotein 3 (ZP3), which are associated with particular autoimmune diseases, such as thyroiditis, experimental autoimmune encephalomyelitis (EAE), oophoritis or rheumatoid arthritis. In another preferred embodiment, the protein which is heterologous to L1 is derived from tumour antigens, preferably melanoma antigens, such as MART, ovarial carcinoma antigens, such as Her2 neu (c-erbB2), BCRA-1 or CA125, colon carcinoma antigens, such as CA125, or mammary carcinoma antigens, such as Her2 neu (c-erbB2), BCRA-1 and BCRA-2. In this connection, this antigen moiety can, but does not have to, comprise single domains or epitopes of a protein. The protein moiety which is heterologous to the Ll protein is present in bound form, preferably fused form.

The present invention furthermore relates to a cell which, following in-vitro incubation with capsomers, stable capsomers, capsids, VLPs and/or CVLPs contains, and preferably presents, in particular both by way of MHC I and MHC II complexes, proteins and/or protein fragments from said capsomers, stable capsomers, capsids, VLPs and/or CVLPs.
The cell according to the invention contains, in particular presents, proteins, protein fragments and/or peptides which are derived from capsomers, stable capsomers, capsids, VLPs and/or CVLPs which contain at least one L1 protein derived from one or more papilloma viruses or at least one L1 protein and at least one papilloma virus L2 protein. In a preferred embodiment, the cell according to the invention contains, in particular presents, proteins, protein fragments and/or peptides derived from capsomers, stable capsomers, capsids and/or CVLPs which contain at least one L1 fusion protein which consists of an L1 protein moiety derived from one or more papilloma viruses and a protein moiety which is heterologous to the L1 protein.
The L1 protein which is used can be a naturally occurring L1 protein or it can contain one or more deletions, which may, for example, be 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 from approx. 25 to approx. 35, in particular at least from approx. 32 to approx. 34 amino acids are deleted from the C terminus of the L1 protein.
The protein moiety which is heterologous to the Ll protein can be a naturally occurring protein or can contain one or more deletions, which may, for example, be 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 and can be derived, for example, from HIV, HBV, HCV or CMV, preferably from papilloma viruses, in particular from human papilloma virus, for example, but not exclusively, from E6 or E7.
In a preferred embodiment, at least approx. 55 amino acids, preferably at least from approx. 5 to approx.
35, in particular at least from approx. 38 to approx.
55, amino acids are deleted from the C terminus of the E7 protein. Furthermore, these proteins can be derived from autoimmune antigens, such as thyroglobulin, myelin or zona pellucida glycoprotein 3 (ZP3), which are associated with particular autoimmune diseases, such as thyroiditis, experimental autoimmune encephalomyelitis (EAE), oophoritis or rheumatoid arthritis. In another preferred embodiment, the protein which is heterologous to L1 is derived from tumour antigens, preferably melanoma antigens, such as MART, ovarial carcinoma antigens, such as Her2 neu (c-erbB2), BCRA-1 or CA125, colon carcinoma antigens, such as CA125, or mammary carcinoma antigens, such as Her2 neu (c-erbB2), BCRA-1 and BCRA-2. In this connection, this antigen moiety can, but does not have to, comprise single domains or epitopes of a protein. The antigen moiety is bound, preferably fused, to the L1 protein.
In all the preferred embodiments, the cell is an antigen-presenting cell, in particular B cell, macrophage, predendritic cell, dendritic cell, embryonic cell or fibroblast.
Another part of the subject-matter of the present invention is a process for producing a target cell, which process is based on incubating the target cell, in vitro, with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP
and/or at least one CVLP.

Another part of the subject-matter of the present invention is a process 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 prepared recombinantly and the target cell is produced by incubating it with at least one capsomer, a stable capsomer, a capsid, a VLP and/or CVLP, and the effector cell is an immune cell line and/or cultured primary immune cell, preferably a murine or human immune cell line and/or cultured primary immune cell. In a preferred embodiment, the recombinantly prepared proteins, which are constituents of the capsomers, stable capsomers, capsids, VLPs and/or CVLPs, are expressed 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 part of the subject-matter of the present invention is a process 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 prepared recombinantly and incubated with a predendritic cell and/or a CD16-positive cell.
Another part of the subject-matter of the present invention is a process 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 one CVLP, which process comprises the following steps:

a) a test system according to the invention is used in a first step. In a preferred embodiment, immune cells (effector cells), for example PBMCs, T cell lines or cultured primary T cells, are incubated, for at least approx.
5 h, in particular approx. 17 h, with antigens, preferably 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/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. In another preferred embodiment, PBMCs, T cell lines or cultured primary T cells are incubated, only for at least approx. 5 h, with antigens, preferably 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/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. During this brief time, the only cells to be activated by the antigen are those which have already previously been stimulated by the same antigen or a similar antigen.
b) The possible activation of effector cells is determined in a second step. For example, stimulated T cells can be detected by a variety of methods, such as the production or secretion of cytokines by the T cells, the expression of surface molecules on the T cells, the lysis of target cells or the proliferation of cells.
Examples of suitable methods for this are a cytokine assay (Chapter 6.2 to 6.24 in Current Protocols in Immunology (1999), see above), ELISPOT (Chapter 6.19 in Current. Protocols in Immunology, see ab.ove), a 5lCr release test (Chapter 3.11 in Current Protocols in Immunology, see above) or the detection of proliferation (Chapter 3.12 in Current Protocols in Immunology, see above). Depending on the method employed, it is also possible, in this connection, to distinguish between the immune cells, such as cytotoxic T cells, T
helper cells, B cells and NK cel_ls, and other cells.
In another embodiment of the process, the following additional step a') is inserted before step a):
a')at least one effector cell of the test system is cocultured for at least approx. 8 weeks, in particular at least approx. 3 weeks, with at 2U 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 2'i least one capsid, at least one V'LP and/or one CVLP, after which step a) then follows. This preactivation of the effector cells results in the effector cells being restimulated, in the following step a), by the addition of at least 30 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 35 least one VLP and/or one CVLP. C:oculturing is to be understood as being the growth of at least one effector cell in the presence of at " CA 02375191 2001-11-29 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, in the same growth medium and the same tissue culture container.
In a preferred embodiment of the process according to the invention, one 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 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, is used as standard while a second component of the test system, i.e. the effector cells, is the actual test component. The activai~ion, which is observed in the reaction of the two components, of the effector cell is compared with the activating effect of a capsomer, stable capsomer, capsid, VLF and/or CVLP, and/or a 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, which derive(s), for example, from an industrial-scale production process. This embodiment makes possible, for example, the quality control of batches of prophylactic and/or therapeutic vaccines which comprise 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 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.
Another preferred process, which makes use of the test system according to the invention, is thE: selection of particularly effective epitopes for developing vaccines which are based on parts of proteins. For example, if the ability of different CVLPs, which in each case contain short peptides, derived from a protein or from a pathogen, as the fusion moiety, to mediate a stimulation of immune cells is investigated in separate assays, it is then possible, by quantitatively comparing the immune responses to the respective CVLPs, to identify particularly effective peptides. These peptides can then be combined for producing new vaccines. Proteins can be tested in a manner which is analogous to this. Another part of the subject-matter of the invention is therefore a process for identifying epitopes, peptides or protein fragments which induce an immune response, in particular a cellular immune response.
Another part of the subject-matter of the present invention is a process 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 24i stable capsomer, at least one capsid, at least one VLP
and/or one CVLP, which process contains the~following steps:
I) in a first step, PBMCs, for example, in particular T cells, are obtained from the blood of a donor, in particular from the blood of a donor who has already been inoculated 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 with a 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, and the effector cells which have been obtained are cultured, or spleen cells are isolated from a mouse, in particular spleen cells from a mouse which has already bE:en inoculated with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP and/or CVLP, an.d/or at least with a 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.
II) In a second step, 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/oz: one CVLP, is added, for example, to the i:>olated and/or cultured cells for at least a~>prox.
5 h, in particular approx. 17 h. In another preferred embodiment, the incubation takers place for at least app rox. 5 h. During this brief stimulation period, the only cells to be activated by the added antigen are those which have already previously been stimulated by the same antigen or by a similar antigen.

III) The possible activation of effector cells is determined in a second step. For example, stimulated T cells can be [lacuna] 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 or the proliferation of cells. Examples of suitable methods for this are a cytokine assay (Chapter 6.2 to 6.24 in Current Protocols in Immunology (1999), see above), ELISPOT
(Chapter 6.19 in Current Protocols in Immunology, see above), a SlCr release test (Chapter 3.11 in Current Protocols in Immunology, see above) or the detection of proliferation (Chapter 3.12 in Current Protocols in Immunology, see above) .
Depending on the method employed, it is also possible, in this connection, to distinguish between the immune cells, such as cytotoxic T
cells, T helper cells, B cells and NK cells, and other cells.
In another embodiment of the process, the following additional step I') is inserted after step I):
I') the isolated cells are cocultured for at least approx. 8 weeks, in particular for at least approx. 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, after which step II) then follows. This preactivation of the effector cells results in the effector cells being restimulated, in the subsequent step II), by the addition of at least one capsomer, at least one stable 3_'> 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 , CA 02375191 2001-11-29 one capsomer, at least one stable capsomer, at least one capsid, at least one VLP and/or one CVLP.
This type of implementation is designated restimulation. In this connection, however, the first stimulation can also have taken place, as described under item I), within the donor, for example by means of a vaccination, an infection or a tumour, or within the context of an autoimmune disease. The first stimulation can, however, be carried out,, as described under item I'), in vitro in order to obtain specific reactive cell clones or populations.
In a particular embodiment, the process according to the invention can be used to test the immune status of an organism vis-a-vis a pathogen. If, for example, PBMCs are isolated from an organism 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 been incubated with at least one capsomer, at least one stable capsomer, at least one capsid and/or one CVLP, which contain(s), as the fusion moiety, a pathogen-specific antigen, or a 2.'~ part thereof, it is then possible, by means of finding immune cells which are reactive against the particular antigen, such as cytotoxic T cells, reacaive T helper cells or reactive B cells, to detect a previous infection. In a further embodiment, it is possible, by quantifying the reactive cells, t=o determine quantitatively such that it is possible, for example, to analyze the necessity for booster vaccinations.
In another embodiment, it is also possible to check the success of a vaccination and/or the current immune status of a patient following a vaccination which took place a relatively long time ago. In this connection, the monitoring is not restricted only 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 has been incubated with at least one capsomer, at least one stable capsomer, at least one capsid, and/or one CVLP, but is equally well suited for conventional vaccines.
The detection of specific reactive T cells by means of the present testing process can be used for diagnosing infections which are difficult to detect. If, for example, CVLPs are constructed with antigens, or parts of antigens, from pathogens which are known but which are difficult to detect, the T cell response to these antigens can then provide information about an existing infection.
If, in another embodiment of the process> according to the invention, the HLA haplotypes of the patient groups which are immune to particular infectious diseases, or which cannot be immunized, or only immunized with difficulty, are correlated with the reactivity of their T cells vis-a-vis antigens of the corresponding pathogens, it is then possible to identify haplotypes which mediate immunity towards the pathogen.
If the antigens which are responsible f:or particular autoimmune diseases are known, it is then possible to prepare capsomers, stable capsomers, capsids and/or CVLPs, and/or target cells which have been incubated with at least one capsomer, at least one stable capsomer, at least one capsid and/or one CVLP, which contain this autoimmune antigen or parts. These can 3_'i then be used for diagnosing the respective autoimmune disease by measuring, in vitro, the T cell response of a patient following stimulation of their isolated PBMCs, for example, with the relevant autoimmune antigen.
Another embodiment of the process according to the invention is that of distinguishing tumour types with regard to different specific tumour antigens. If different types of a tumour are known, which types differ from each other, inter alia, in that they express different tumour antigens, and if, on the other hand, different T cell populations, which can each be restimulated specifically by one of the respective tumour antigens, are available, it is then possible, by detecting a restimulation of the reactive T cells, to classify the tumour in a patient. Such a classification could then be used, for example, to stimulate the patient's own T cells specifically by inoculating with the relevant tumour antigens in order, in this way, to generate a cytotoxic T cell population directed against the patient's own tumour cells.
2~
The processes according to the invention are suitable, for example, for controlling the qualii:y of vaccine batches during production, for identifying new antigenic epitopes, for identifying patient haplotypes, for differentiating autoimmune diseases or for differentiating tumour types a high processing rate and reproducibility when using the test system. For this reason, another part of the subject-matter of the invention is a process which the activation of 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 been incubated with at least one capsomer,, at least one stable capsomer, at least one capsid, at least one VLP
and/or one CVLP, is carried out in high-throughput systems. In high-throughput systems, it is also possible, for example, to investigate, on a large scale, peptides or proteins, in addition to the abovementioned compounds, with regard to their induction of immune responses, in particular T cell responses.
Another part of the subject-matter 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 a B cell, macrophage, dendritic cell, embryonic cell 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, 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 inducing or for detecting an immune response.
Another part of the subject-matter of the invention is a diagnostic agent which comprises 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 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, effector cells of the immune system, in particular B
cells, NK cells, preferably T cells, in <~ particularly preferred manner cytotoxic T cells or T helper cells, and, where appropriate, a pharmaceutically acceptable carrier substance.
Examples of carrier substances which are known to the skilled person are glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural or modified cellulose, polyacrylamide, agarose, aluminium hydroxide and magnitide.
A diagnostic agent according to the invention can be present in solution, be bound to a solid matrix and/or contain added adjuvant.
The diagnostic agent can be administered. in a variety of ways. Examples of administration foams which are known to the skilled person are parenteral, local and/or systemic administration by means of, for example, oral, intranasal, intravenous, intramuscular and/or topical administration. The preferred form of administration is influenced, for example, by the natural route of infection taken by the particular papilloma virus infection. The quantity administered depends on the age, weight and general state of health of the patient and on the type of papilloma virus infection. The diagnostic agent can be administered in the form of capsules, a solution, a suspension, an elixir (for oral administration) or sterile solutions and/or suspensions (for parenteral or intranasal administration). A salt solution or a phosphate-buffered salt solution can, for example, be used as an inert and immunologically acceptable carrier substance.
The figures, and the following examples, are intended to clarify the invention without limiting it.
Fig. 1 shows a graphic analysis of the re;stimulation of murine, HPV16L1-specific T cells by two murine antigen-presenting cell lines (C3 and B16F1C1) which had previously been incubated with increasing quantities of CVLPs. The increasing concentrations of CVLPs are 3'i plotted against the percentage of stimulated T cells, which were detected by way of interferon 'y production.

Fig. 2 shows a graphic analysis of the restimulation of murine, HPV16L1 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 an analysis of five FACScan experiments following stimulation of human PBMCs with different concentrations of CVLPs (0-10 ug/ml), in which human T
cells which are positive for human interferon y are displaced upwards in the diagram.
Fig. 3B shows a graphic analysis of Fig. 3A in which the concentration of CVLPs is plotted against the percentage of stimulated cells. Stimulated cells are defined by the detection of human interferon y.
Fig. 4 shows a graphic analysis of the restimulation of human PBMCs by different antigens after the PBMCs had been previously stimulated with CVLPs.
Fig. 5 shows an analysis of three FACScan experiments following the restimulation of human T cells with various antigen-presenting cells after th.e T cells had previously been stimulated with CVLPs. T'he content of CD3, which is specific for T cells, is in each case plotted from left to right, while the content of human interferon y, which is specific for activated cells, is plotted from the bottom to the top.
Fig. 6A shows a graphic analysis of the specific lysis of various murine, antigen-presenting RMA cells by an E7-specific cytotoxic cell line. This e~;periment used normal RMA cells, RMA cells which are expressed in E7 3.'i or RMA cells which had previously been incubated with L1E71-so-CVLPs. The ratio of the effector cells to the target cells is plotted against the percentage of specifically lysed cells.
Fig. 6B shows a graphic analysis of the specific lysis of various murine, antigen-presenting RMA cells by an E7-specific cytotoxic T cell line. This experiment used RMA cells which had previously been incubated with L1E71-so-CVLPs or with L1-VLPs, that is without any E7 moiety.
Fig. 7 shows an analysis of FACScan experiments following incubation of JAWS II cells with increasing quantities of CVLPs, as plotted from left: to right. On the one hand, the expression of MHC class II molecules was measured by using specific antibodies to detect MHC
class II molecules on the cell surface. These values are plotted as relative fluorescent units on the left-hand Y axis. On the other hand, the binding of the CVLPs to the cells was measured by using an Ll-specific antibody to detect an HPV16L1 epitope on the cell surface. These values are presented as o binding cells, with a negative control without CVLPs having been defined as 50 +/- 1% binding cells.
Fig. 8 shows an analysis of FACScan experiments following incubation of JAWS II cells with the indicated quantities of CVLPs and/or serum from CVLP-inoculated mice. "Dept" denotes the depletion of the CVLPs from the incubation mixtures before they are added to the JAWS II cells. In the CVLP + serum mixture, the CVLPs were incubated previously with the serum before they were added to the JAWS II cells. The expression of MHC class II molecules was measured by using specific antibodies to detect MHC class II
molecules on the cell surface. The values are plotted as relative fluorescent units on the Y axis.

Fig. 9 shows an analysis of FACScan experiments following the incubation of RMA cells (left-hand diagram) or JAWS II cells (right-hand diagram) with increasing quantities of CVLPs, as plotted from left to right. The binding of the CVLPs to l.he cells was measured by using an L1-specific antibody to detect an HPV16 L1 epitope on the cell surface. The values are given as % binding cells, with a negative control without CVLPs having been defined as 5°s +/- l~ binding cells.
Fig. 10 shows an analysis of FACScan experiments carried out on T cells from different mice which had been inoculated with different quantities of CVLPs from different batches. The T cells were stimulated with a known cytotoxic HPV16 L1 epitope and them restimulated with the same peptide under the conditions described in the example; specific antibodies were then used in the FACS experiment to determine the relative proportion of 2U the CD8-positive and interferon y-positive cells.
Examples 1. Description of the starting materials:
~ HPV16 Llo~*E71-ss CVLPs were prepared as described in German patent application DE 198 12 941.6 (see also Miiller M. et al. (1997) Virology 234, 9:3-111) .
3U ~ HPV16 Llo~*E71-so CVLPs were prepared as described in Mizller M. et al. (1997; see above).
~ L1 VLPs were prepared as described in Miiller M. et al. (1997); Virology 234, 93-111.
~ C57B1/6 mice were obtained from Charles River Laboratories (Wilmington, MA, USA).

~ B6 cells denote embryonic stem cells derived from a C57B1/6 mouse.
~ C3 cells denote HPV16 and ras-transformed B6 embryonic cells (see Feltkamp M.C. et al. (1993) Eur.
J. Immunol. 23, 2242-9) .
~ RMA cells are derived from a thymoma in a C57BL/6 mouse (see Ljunggren H.G. & Karre K. (1985) J. Exp.
Med. 162, 1745-59).
~ RMA-E7 cells are derived from RMA cells but constitutively express an HPV6 E7 protein as a result of stable transfection.
~ B16F10 cells denote the cell line which can be obtained under ATCC CRL-6475.
~ PBMCs denote peripheral blood mononuclear cells, which can be prepared, for example, by the method described in Rudolf M.P. et al. (1999) Biol. Chem.
380, 335-40.
~ MVA-Llo~ denotes recombinant murine vaccinia virus, 2.5 which expresses HPV16 Llo~ in infected cells.
~ IL-2 denotes recombinant cytokine (Becton Dickinson, Hamburg, Germany).
~ 25/C a-HPV16L1 denotes a mouse monoc7_onal antibody which is directed against L1.
~ a-hu CD28 denotes a mouse monoclonal antibody which is directed against the extracellular moiety of human CD28 (ATCC CRL-8001).

~ a-CD3/PE denotes a mouse monoclonal antibody which is directed against the extracellular moiety of human CD3 (E) and contains a phycoerithrin fluorescence label (Medac, Hamburg, Germany).
~ a-CD4/Cychrome denotes a mouse monoclonal antibody which is directed against the extracellular moiety of CD4 and contains a Cychrome fluorescence label (DAKO;
Glostrup, Denmark).
~ a-CD4/Tricolor denotes a rat monoclonal antibody which is directed against the extracellular moiety of CD4 and contains a Tricolor fluorescence label (Medac, Hamburg, Germany).
~ a-mus interferon 'y-FITC denotes a rat monoclonal antibody which is directed against mus interferon y and contains an FITC fluorescence label (Medac, Hamburg, Germany).
~ E-7 peptide denotes amino acids 49 to 57 of human papilloma virus type 16, sequence: F;AHYNIVTF (see Feltkamp M.C. et al. (1993) see above).
~ P-12 peptide denotes amino acids 165 to 173 of the HPV16 L1 protein, sequence: AGVDNRECI (see Genbank 5559..7154). It was possible to identify this peptide as a murine, cytotoxic T cell epitope (see DE 19925235.1-41).
~ AM peptide denotes amino acids 366 to 374 of the influenza nucleoprotein, sequence: ASNENMETM (see Townsend A.R. et al. (1986) Cell 44, 95'a-68).
~ HPV16L1 93-101 denotes amino acids 93 to 101 of the HPV16 L1 protein, sequence: GLQYRVFRI.

~ Phytohaemagglutinin (PHA) was obtained from Sigma (Deisenhofen, Germany).
~ Golgi plug was obtained from Pharmingen (Hamburg, Germany).
~ Cells were in each case cultured, at 3;~°C and 5o C02, in RPMI medium (Gibco BRL, Eggenst.ein, Germany) containing loo foetal calf serum, canamycin and ampicillin.
~ JAWS II cells are predenditric cells (see US 5,648,219).
~ Monensin was obtained from Sigma (Deisenhofen, Germany).
~ MHC IAb antibody was obtained from Pharmingen (Heidelberg, Germany).
~ FITC-coupled anti-mouse antibody was obtained from Sigma (Deisenhofen, Germany).
~ FITC-coupled 25/C a-HPV16L1 antibody denotes a mouse 2:5 monoclonal antibody which is directed against L1 and is itself coupled to FITC. FITC was obtained from Sigma (Deisenhofen, Germany).
~ a-mouse CD8/PE antibody was obtained from Pharmingen 3() (Heidelberg, Germany).
~ a-mouse interferon y-FITC antibody was obtained from Caltag (Hamburg, Germany).
3'i ~ a-mouse CD4/Cychrome antibody was obtained from Pharmingen (Heidelberg, Germany).

~ Zefa membrane denotes a 45 ~.m syringe attachment filter and was obtained from Zefa (Munich, Germany).
~ FACScan calibur or FRCS denotes "fluorescence activated cell sorter". The apparatus was obtained from Becton Dickinson (Hamburg, Germany).
~ Cellquest software was obtained from Becton Dickinson (Hamburg, Germany).
2. Preparation of murine CD8-positive T cells a) Immunization of mice with VLPs:
Two C57B1/6 mice were immunized with 10 ~g of L1 VLPs. After 6 weeks, the spleen cells were isolated.
b) Preparation of antigen-presenting cells (target cells):
B6 cells were incubated with interferon y for 2.5 days, then infected overnight with MVA-Llo~ viruses (MOI - 5), in order to prepare antigen-presenting cells, and cultured for 16 h. In a following step, the infected B6 cells were irradiated and therefore prevented from growing any further.
c) Restimuhation of the isolated spleen cells:
The spleen cells were in each case cultured, for 5 days, together with the Llo~-expressing B6 cells, which function as stimulator cells for the T cells in the spleen cells.

3. Restimulation of T cells with antigen-presentin cells 2 x 109 murine antigen-presenting cells (C3 or B16F10) were incubated, overnight at 37°C, with different concentrations of HPV16 Llo~~E71_ss CVLPs. After that, 2 x lOs murine CD8-positive T cells (see Example 2), which are specific for a specific HPV:l6 L1 peptide, were added and the mixture was incubated, at 37°C for one hour, in the presence of 10 IU of IL2/ml. The cells were incubated for a further 5 hours, at 37°C, in the presence of 1 ~l of golgi plug. Subsequently, the cells were fixed, permeabilized, stained with a-mouse CD3/PE, with a-mouse CD4/Tricolor and with a-mouse interferon y/FITC, and washed. The labelling of the cells was investigated in a FACScan calibur (Bect=on Dickinson, Hamburg, Germany) and the measurement results obtained were analyzed using Cellquest software (Becton Dickinson, Hamburg, Germany). Cells which gave an interferon y signal of greater than 101 were defined as being stimulated cells. T cells were defined as giving a CD3 signal of more than 102.
Result: Fig. 1 shows that, as the quantity of CVLPs increased, the percentage of T cells which were restimulated by the antigen-presenting cells also increased. Since the T cells which were used here interact exclusively with MHC I-presented peptides, this experiment demonstrates that the CV'LPs must have brought about a pseudoinfection of the antigen presenting cells. Only in this way is it possible to explain the fact that it was possible for MHC I
molecules to be loaded with CVLP peptides in order, then, to be recognized on the cell surface by L1 peptide-specific T cells.

4. Inhibition of the seudoinfection by virus-neutralizing antibodies Murine C3 target cells were incubated overnight, at 37°C, with 6 different HPV16 L1~~*E71_ss CVLPs preparations or with the P12 peptide, in each case a) without antibody b) with 25/C aHPVI6Ll antibody (10 ~g/ml).
This antibody is known to prevent, by ~>inding to the HPVLl protein, HPV viruses (or virus-like particles) from infecting the antigen-presenting cells. HPV16L1 peptide-specific murine T cells were then added and the 1.5 mixture was incubated for 6 hours (CVLP 2-6 were incubated in the presence of golgi plug). The interferon y production by the cells was analyzed (see previous example). C3 cells which had not been incubated with any antigen served as the negative 2c) control (see Fig. 2).
Result : As in the case of a viral HPV infection, CVLPs can be prevented from penetrating into antigen-presenting cells by virus-neutralizing antibodies.
25 However, the addition of the antibodies has no effect on the uptake of individual peptides.
5. Stimulation of human PBMCs with HPV16 LlE7 CVLPs 30 PBMCs (4 x 106) were incubated overnight, at 37°C, in 100 ~l of medium containing different concentrations of HPV16 Llo~*E71-ss CVLPs and also 10 IU of IL2/ml and 0.5 ~g of anti-human CD28/ml. On the following day, 1 ~1 of golgi plug was added. The cells were incubated 35 at 37°C for a further 5 hours. The cells were then fixed and permeabilized and stained with anti-human CD3/PE, and dyed with anti-human CD4/ and with anti-human interferon y/FITC. The labelling of the cells was investigated in a Becton Dickenson FACScan calibur and the measurement results were analyzed using Cellquest software (see previous examples).
Result: It was found that, as the concentration of CVLP
increases, the proportion of T cells which were accumulating interferon y in the cytoplasm increases linearly (see Figs. 3A and B). This increase consequently makes it clear that the T cells were stimulated by the CVLPs.
6. Restimulation of CVLP-stimulated PBMCs with different antigens Human PBMCs (4 x 105) were stimulated foi: 3 weeks with HPV16 Llo~*E71_ss CVLPs, at 37 °C, with 1 ~,g of CVLPs/ml and lOs antigen-presenting PBMCs being added weekly, and then harvested. The cells were then restimulated, in 100 ~1 of medium and at 37°C, with 10 ~g of the following different antigens/ml a) E7 peptide b) HPV16 Llo~*E71_ss CVLPs c) Influenza peptide d) Phytohaemagglutinin (PHA) in the presence of 10 IU of IL2/ml and 0.5 ~g of anti-human CD28/ml. After one hour, 1 ~,1 of golgi plug was added. The cells were then incubated at 37°C for a further 5 hours. After that, the cells were fixed and permeabilized and stained with a-human CD3/PE, with a-human CD4/Cychrome and with a-human interferon y/FITC. The labelling of the cells was investigated in a Becton Dickinson FACScan calibur and the measurement results were analyzed using Cellquest software (see previous examples).
Result: Three times more T cells were restimulated by CVLPs than were restimulated by the E7 peptide or the influenza peptide (see Fig. 4). .As expected, phytohaemagglutinin, as a nonspecific stimulant, stimulated the most T cells. Consequently, as a result of the three-week incubation with CVLPs, some of the human PBMCs became specifically restimulatable by CVLPs.

7. Restimulation of CVLP-stimulated 'T cells with different antigen-presenting cells Human PBMCs (4 x 10s) were stimulated, far 8 weeks and at 37 °C, with HPV16 LloC*E71-ss CVLPs, with 1 ~tg of CVLPs/ml and lOs irradiated PBMCs being added weekly, and then harvested. Subsequently, the cells were restimulated, in 100 ~l of medium and at 37°C, with 10 ~,g of the following different antigens/ml in the presence of 10 IU of IL2/ml and 0.5 ~g of anti-human CD28/ml:
2 .'i a) overnight with HPV16 Llo~*E71-55 CVLP-incubated PBMCs, b) overnight with HPV16 Ll 93-101 peptide-incubated PBMCs.
After one hour, 1 ~1 of golgi plug was added. The cells were then incubated at 37°C for a further 5 hours.
After that, the cells were fixed and permeabilized and stained with a-human CD3/PE, with a-human CD4/Cychrome and with a-human interferon y/FITC. The labelling of the cells was investigated in a Beci=on Dickinson ~

FACScan calibur and the measurement results were analyzed using Cellquest software (see previous examples).
Result: Fig. 5 shows that, while both the CVLP-incubated PBMCs brought about a restimulation of CVLP-stimulated T cells, the PBMCs which had been incubated with the control peptide did not do this. Consequently, T cells which were specifically restimulatable had been formed as a result of the eight-week incubation of the human T cells with CVLPs. Furthermore, it can be concluded from this that the human antigen-presenting cells were pseudoinfected by the CVLPs (as were the mouse cells in Example 3) and were therefore recognized 1~ by the CVLP-specific T cells.

8. Lysis of CVLP-incubated cells b~Y E7-specific cytotoxic T cells 2U RMA cells were incubated, at 37°C for 1 hour and in the presence of SlCr, with ~ HPVI6Llo~*E71-6o CVLPs or 25 ~ L1-VLPs.
RMA-E7 cells were likewise incubated at 3'7°C for 1 hour and in the presence of 5lCr but without the addition of antigen. Subsequently, E7-specific cytot:oxic T cells 30 (C57BL/6 mouse (H2b)) (see Example 2) were added in various ratios to the target cells (RMA cells). The release of the SlCr, which was associated with the lysis of the target cells, was measured in a (3 counter and related to completely lysed cells.
Fig. 6A shows that, while CVLP-incubated cells and the cells which express E7 were efficiently l.ysed by the T

cells, the RMA cells which were incubated without antigen were not lysed.
Fig. 6B shows that, in contrast to the CVLPs, incubating the RMA cells with L1-VLPs did not result in any efficient lysis of the cells.
Result: Consequently, E7 is responsible for the specific stimulation of the cytotoxic T cell line which leads to the lysis of the antigen-presenting RMA cells.
In this connection, it matters little whether E7 is generated intracellularly by means of stable expression or introduced by CVLPs into the cell by means of a pseudoinfection.

9. Activation of predendritic cells On the one hand, 3 x 105 JAWS II cells were incubated with increasing quantities of HPVI6Llo~*E'71-55 CVLPs for 2() 2 days. The cells were then examined to see whether they were expressing MHC class II molecules by incubating them firstly with 1 ~g o:E an MHC IAb antibody/ml and then with 1 ~g of FITC-coupled anti-mouse antibody/ml. Finally, the staining of the cells was investigated in a FACScan calibur and the measurement results were analyzed using Cellquest software.
On the other hand, JAWS II cells were incubated, as previously, with increasing quantities of HPV16L1o~*E71-ss CVLPs for only 3 hours. These cells were examined to see whether they had bound CVLPs. For this, the cells were first of all stained with the FITC-coupled 25/C a-HPVC16L1 antibody. The staining of the cells was then investigated in a FACScan calibur and the measurement results were analyzed using Cellquest software.

It can be seen from Fig. 7 that the binding of the CVLPs to the cells increased as the concentrations of CVLP with which the JAWS II cells were incubated increased and the cells were activated to express the MHC class II molecules.
In addition, JAWS II cells were incubated, as previously described, for 2 days, in various mixtures containing HPVI6Llo~*E71-s5 CVLPs and/or mouse serum. The following were added in this connection:
~ 80 or 10 ~.g of CVLPs, ~ a solution containing 80 or 10 ~g of CVLPs which was filtered through a Zefa membrane which was known to remove the CVLPs efficiently from the solution while allowing most of the impurities to pass through the filter (depleted mixture), ~ 10 ~1 of a 1/20 dilution of a mouse serum which had been obtained from a mouse which had been immunized with the abovementioned CVLPs, and ~ 10 ~g of CVLPs which had been preincub<~ted for 5 min with 10 ~1 of the abovementioned mouse serum.
The cells were then examined, as above, to see whether they were expressing MHC class II molecules by first of all staining them with 1 ~g of an MHC IAb antibody/ml and subsequently with 1 ~g of FITC-coupled anti-mouse antibody/ml. Finally, the staining of the cells was investigated in a FACScan calibur and the measurement results were analyzed using Cellquest software.
Fig. 8 shows that both depleting the CVLPs in the mixture and preincubating with CVLP-specific mouse serum prevents activation of MHC class 7CI expression, signifying that this observed activation is specifically elicited by CVLPs and is not due to any possible contaminants in the CVLP preparations.

10. Predendritic cells as a test cell line for T cell activation in vivo In each case 3 x 105 RMA cells or JAWS II cells were incubated, as described in Example 9 and for 3 hours, with increasing quantities of HPV16L7.o~*E71-ss CVLPs obtained from different preparations. The cells were once again examined to see whether they had bound CVLPs. For this, the cells were stained with 1 ~g of the FITC-coupled 25/C a-HPV16L1 antibody/ml and the staining of the cells was investigated in a FACScan calibur and the measurement results were analyzed using Cellquest software.
1.5 Fig. 9 shows that batches 1-3 of the CVLPs are able to bind to RMA cells with batch 3 binding somewhat more poorly at the same CVLP concentrations. On the other hand, only the CVLPs in batches 1 and 3 bind to the JAWS II cells, while the CVLPs in batch 2 do not display any clear increase and, even at the highest concentration of 100 ~g of CVLPs/ml, do not achieve 500 binding. Consequently, the binding of the CVLPs to cells varies with the CVLP preparation and the cell type.
In order to test whether the observed variability in CVLP binding correlates with the ability to induce a cytotoxic T cell response in vivo, i:he following experiment was carried out:
In each case, three C57BL6 mice were inoculated with 10 or 1 ~g of CVLP from batches 1 to 3. After two weeks, the mice were boosted with a second identical injection. After a further two weeks, the animals were sacrificed and spleen and serum were obtained. All the sera contained antibodies directed against the CVLPs (data not shown). The T cells were isolated from the spleen and purified through nylon wool (see Current Protocols in Immunology, see above, pages 7.7.2 to 7.7.3) .
The T cells were stimulated for 5 days with 10 ~g of the murine cytotoxic P12 peptide/ml. Subsequently, the T cells were restimulated, in 100 ~1 of medium and at 37°C, with a further 10 ~g of the P12 peptide in the presence of 10 IU of IL2/ml. T cells obtained from mice immunized with buffer served as the negative control.
After one hour, 1 ~l of Monensin (300 N.M) was added.
The cells were incubated at 37°C for a further 5 hours.
The cells were then fixed and permeabilized and stained with in each case 1 ~g/ml of a-mouse CD4/Cychrome, 1S a-mouse CD8/PE and a-mouse interferon y/FITC
antibodies. The labelling of the cells was investigated in a FACScan calibur and the measurement: results were analyzed using Cellquest software.
Fig. 10 shows that no mouse which had been inoculated with the CVLPs from batch 2 had generated cytotoxic T
cells which were reactive to the P12 peptide, whereas the inoculation of CVLPs from batches 1 and 3 into several mice in each case was able to induce reactive T
cells. The insight gained from these in-v:ivo data, i.e.
that the CVLPs in batch 2 are not suitable for generating cytotoxic T cells, was already evident from the in-vitro data obtained from the CVLP--binding study using the JAWS II cells, whereas the RMA cells did not permit any such prediction. Consequently,. predendritic cells such as the JAWS II cells are particularly suitable for predicting the activation of T cells by CVLPs in vivo.

i ~ ;.

SEQUENCE LISTING
<110> MEDIGENE AKTIENGESELLSCHAFT
<120> TEST SYSTEM FOR IN-VITRO DETECTION OF AN
ANTIGEN-SPECIFIC IMMUNE RESPONSE
<130> AML/12850.24 <140> 2,375,191 <141> 2000-05-31 <150> PCT/EP00/05003 <151> 2000-05-31 <150> DE199 25 234.3 <151> 1999-06-O1 <160> 4 <170> PatentIn Ver. 2.1 <210> 1 <211> 9 <212> PRT
<213> Human papillomavirus type 16 <400> 1 Arg Ala His Tyr Asn Ile Val Thr Phe <210> 2 <211> 9 <212> PRT
<213> Human papillomavirus type 16 <400> 2 Ala Gly Val Asp Asn Arg Glu Cys Ile <210> 3 <211> 9 <212> PRT
<213> Influenza A virus <400> 3 Ala Ser Asn Glu Asn Met Glu Thr Met I ', ~ ~, ~: ,.~ ', y.

<210> 4 <211> 9 <212> PRT
<213> Human papillomavirus type 16 <400> 4 Gly Leu Gln Tyr Arg Val Phe Arg Ile

Claims

1. Test system for the 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, 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 CVLF, 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 the in-vitro detection of an antigen-specific immune response, in particular a cellular immune response, with the test system comprising at least a) 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 one 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, VLP
and/or CVLP to the predendritic cell and/or the CD16-positive cell being measured.

3. Test system according to Claim 1 or 2, characterized in that the cells are mammalian cells, in particular human or murine cells.

4. Test system according to one of Claims 2-3, characterized in that the capsomer, stable capsomer, capsid, VLP and/or CVLP contains at least one L1 protein from one or more papilloma viruses, or at least one L1 protein and at least one papilloma virus L2 protein, in particular L1 proteins or L1 and L2 proteins from human, bovine and/or cottontail rabbit papilloma viruses.

5. Test system according to Claim 4, characterized in that the capsomer, stable capsomer, capsid and/or CVLP contains at least one L1 fusion protein which consists of an L1 protein moiety from one or more papilloma viruses and a protein moiety which is heterologous to the L1 protein.

6. Test system according to Claim 4 or 5, characterized in that the L1 protein moiety is a naturally occurring L1 protein.

7. Test system according to Claim 4 or 5, characterized in that the L1 protein moiety is deleted and/or mutated.

8. Test system according to Claim 7, characterized in that up to at least approx. 35 amino acids, preferably at least from approx. 25 to approx. 35, in particular at least from approx. 32 to approx.

34, amino acids have been deleted from the C
terminus of the L1 protein moiety.

9. Test system according to one of Claims 4-8, characterized in that the protein moiety 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 moiety which is heterologous to the L1 protein is deleted and/or mutated.

11. Test system according to Claim 9 or 10, characterized in that the protein moiety which is heterologous to the L1 protein is derived from a bacterial or viral protein, preferably from a papilloma virus protein, in particular from a protein derived from a human papilloma virus.

12. Test system according to Claim 11, characterized in that the papilloma virus protein is an E
protein, in particular an E6 or E7 protein.

13. A test system according to Claim 12, characterized in that at least approx. 55 amino acids, preferably at least from approx. 5 to approx. 55, in particular at least from approx. 38 to approx.
55, amino acids have been deleted from the C
terminus of the E7 protein.

14. Test system according to Claim 9 or 10, characterized in that the protein moiety which is heterologous to the L1 protein is derived from an autoimmune antigen, in particular thyroglobulin, myelin or zona pellucida glycoprotein 3 (ZP3) or from a tumour antigen, in particular a melanoma, ovarial carcinoma or mammary carcinoma antigen.

15. Cell, characterized in that, following in-vitro incubation with capsomers, stable capsomers, capsids, VLPs and/or CVLPs, it contains, and preferably presents, proteins and/or protein fragments from said capsomers, stable capsomers, capsids, VLPs and/or CVLPs.

16. Cell according to Claim 15, characterized in that the capsomer, stable capsomer, capsid, VLP and/or CVLP contains at least one L1 protein derived from one or more papilloma viruses or at least one L1 protein and at least one papilloma virus L2 protein.

17. Cell according to Claim 16 or 17, characterized in that the capsomer, stable capsomer, capsid and/or CVLP contains at least one L1 fusion protein which consists of an L1 protein moiety derived from one or more papilloma viruses and a protein moiety which is heterologous to the L1 protein.

18. Cell according to Claim 16 or 17, characterized in that the L1 protein moiety is a naturally occurring L1 protein.

19. Cell according to Claim 16 or 17, characterized in that the L1 protein moiety is deleted and/or mutated.

20. Cell according to Claim 19, characterized in that up to at least approx. 35 amino acids, preferably at least from approx. 25 to approx. 35, in particular at least from approx. 32 to approx. 34, amino acids have been deleted from the C terminus of the L1 protein moiety.

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

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

23. Cell according to Claim 21 or 22, characterized in that the protein moiety which is heterologous to the L1 protein is derived from a bacterial or viral protein, preferably from a papilloma virus protein, in particular from a protein derived from a human papilloma virus.

24. Cell according to Claim 23, characterized in that the papilloma virus protein is an E protein, in particular an E6 or E7 protein.

25. Cell according to Claim 24, characterized in that at least approx. 55 amino acids, preferably at least from approx. 5 to approx. 55, in particular at least from approx. 38 to approx. 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 moiety which is heterologous to the L1 protein is derived from an auto antigen, in particular thyroglobulin, myelin or ZP3, or from a tumour antigen, in particular a melanoma, ovarial carcinoma or mammary 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. Process for producing a target cell according to one of Claims 15-27, characterized in that the target cell is incubated in vitro with at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP and/or at least one CVLP.

29. Process for producing a test system according to 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 prepared recombinantly and incubated with a predendritic and/or a CD16-positive cell.

30. Process for producing a test system according to 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 prepared recombinantly, and the target cell is produced according to Claim 28, and in that the effector cell is an immune cell line and/or cultured primary immune cell, preferably a murine or human immune cell line and/or cultured primary immune cell.

31. Process for producing a test system according to one of Claims 1-19, characterized in that the capsomer, the stable capsomer, the capsid, the VLP
and/or the CVLP is prepared 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. Process 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 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. Process according to Claim 32, characterized in that it comprises, prior to step a), the following additional. step a'):
a') coculturing the effector cell of the test system according to Claim 1 or 3 for at least approx. 8 weeks, in particular at least approx. 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 Claims 15-27, after which step a) then follows.

34. Process 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 one predendritic and/or one CD16-positive cell according to one of Claims 15-27, which comprises the following steps:
a) using a test system according to Claims 1-14;
b) determining the binding of the stable capsomer, capsid, VLP and/or CVLP to the predendritic cell and/or CD16-positive cell.

35. Process according to one of Claims 32 to 34, characterized in that it is used for controlling the quality of batches of prophylactic and/or therapeutic vaccines which comprise 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 one of Claims 14-26.

36. Process according to one of Claims 32 to 34, characterized in that it is used for identifying epitopes, peptides or protein fragments which induce an immune response, in particular a cellular immune response.

37. Process 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 according to one of Claims 15-27, which comprises the following steps:
I) obtaining and preparing samples which contain effector cells of the immune system and subsequently culturing these cells, 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. Process according to Claim 37, characterized i.n that, after step I), it comprises the following additional step I'):
I') coculturing the effector cell for at least approx. 8 weeks, in particular at least approx. 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 according to one of Claims 15-27, after which step II) then follows.

39. Process according to Claim 37 or 38, characterized in that it is used for monitoring the immune status of an organism vis-a-vis a pathogen, in particular a pathogen which is difficult to detect.

40. Process according to Claim 37 or 38, characterized in that it is used for monitoring a vaccination.

41. Process according to Claim 37 or 38, characterized in that it is used for identifying HLA haplotypes which mediate immunity towards a particular pathogen.

42. Process according to Claim 37 or 38, characterized in that it is used for differentiating and characterizing autoimmune diseases with regard to different specific autoimmune antigens.

43. Process according to Claim 37 or 38, characterized in that it is used for differentiating tumour types with regard to different specific tumour antigens.

44. Process according to one of Claims 32-43, characterized in that the effector cell is activated by at least one capsomer, at least one stable capsomer, at least one capsid, at least one VLP and/or at least one CVLP in high-throughput systems.

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

46. Diagnostic agent which comprises at least one test system according to one of Claims 1-14 and/or one cell according to one of Claims 15-27 and, where appropriate, a pharmaceutically acceptable carrier substance.

47. Diagnostic agent according to Claim 93, characterized in that at least one test system according to one of Claims 1-14 and/or one cell according to one of Claims 15-26 is present in solution, is bound to a solid matrix and/or contains added adjuvant.