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WO1996009395A2 - Medicament destine a la prophylaxie et au traitement de maladies auto-immunes et virales, agents de diagnostic pour le depistage desdites maladies - Google Patents

Medicament destine a la prophylaxie et au traitement de maladies auto-immunes et virales, agents de diagnostic pour le depistage desdites maladies Download PDF

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Publication number
WO1996009395A2
WO1996009395A2 PCT/EP1995/003726 EP9503726W WO9609395A2 WO 1996009395 A2 WO1996009395 A2 WO 1996009395A2 EP 9503726 W EP9503726 W EP 9503726W WO 9609395 A2 WO9609395 A2 WO 9609395A2
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Prior art keywords
cells
ipp
human
bpp
peptides
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PCT/EP1995/003726
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German (de)
English (en)
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WO1996009395A3 (fr
Inventor
Thomas Franz Ferdinand Meyer
Johannes Pohlner
Susanne Christine Beck
Joachim Jose
Uwe WÖLK
Dirk R. Lorenzen
Karin Brigitte Oetzelberger
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Priority to AU36515/95A priority Critical patent/AU3651595A/en
Publication of WO1996009395A2 publication Critical patent/WO1996009395A2/fr
Publication of WO1996009395A3 publication Critical patent/WO1996009395A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/095—Neisseria
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/22—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Neisseriaceae (F)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/58—Medicinal preparations containing antigens or antibodies raising an immune response against a target which is not the antigen used for immunisation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints

Definitions

  • the present invention relates to pharmaceuticals and diagnostics as well as diagnostic, therapeutic and preventive methods for the treatment of autoimmune and viral diseases in humans, which are caused by pathogenic bacteria.
  • These bacteria are, in particular, microorganisms that colonize human mucous membranes and secrete certain exoproteins that are structurally related to human proteins.
  • IgA protease-like proteins which are particularly well absorbed by cells and presented as antigens on MHC molecules.
  • the polyprotein of the IgA protease (IPP) from pathogenic Neisseries has particularly pronounced homology to human proteins. For example, a certain peptide of the IPP has pronounced Ho Theology of the Link and Aggrekan joint proteins.
  • the IPP is an etiological agent of rheumatoid arthritis (RA).
  • the autoimmune response is further promoted by other properties of the IPP-producing Neisseries and the IPPs.
  • a particularly important property is the ability of the IPP to activate viruses and viral elements. This applies in particular to the activation of proviral retroviruses and endogenous retroviruses in humans. Due to the activation of viruses and viral elements, further autoimmune reactions in humans are induced.
  • the IPP-dependent activation of the HIV provirus also explains the development of the acquired immune deficiency syndrome (AIDS).
  • AIDS acquired immune deficiency syndrome
  • autoimmune diseases are multiple sclerosis (MS), insulin-dependent diabetes mellitus (IDDM), rheumatoid arthritis (RA) and possibly also arteriosclerosis (Ask). According to recent estimates, the proportion of the adult population affected by autoimmune diseases is around 5% to 15%; the economic and social burdens associated with the effects and the therapy are accordingly significant.
  • MS multiple sclerosis
  • IDDM insulin-dependent diabetes mellitus
  • RA rheumatoid arthritis
  • Ask arteriosclerosis
  • a central function of the immune system is to ward off infectious microorganisms coming from outside and to eliminate aberrant cells of the own body.
  • the ability of the immune system to distinguish its own structures (self) from foreign structures (foreign) is essential for fulfilling this task.
  • the molecular basis for the distinction between self and foreign makes the
  • B cells form antibodies on their surface that react with native antigens
  • T cells form T cell receptors that recognize peptides that are sensitive to MHC Molecules are presented.
  • T cells There are several groups of T cells: CD8 + T cells react with peptides presented on class I MHC molecules and CD4 + T cells react with peptides presented on class II MHC molecules.
  • CD4 + cells can be divided into TH1 and TH2 subgroups, which differ in their cytokine pattern and are either anti-inflammatory (TH1) or anti-inflammatory (TH2).
  • B and T cells usually require an interaction between at least two different cell types.
  • B cells need the help of CD4 + T cells to activate them.
  • T cells in turn require appropriate MHC-producing antigen presenting cells (APC) for their activation.
  • APC MHC-producing antigen presenting cells While MHC class I is formed by almost all body cells, MHC class II is formed in particular by macrophages, dendritic cells, B cells and, under certain circumstances, also by T cells, epithelial cells and fibroblasts.
  • T lymphocytes that recognize foreign structures.
  • the immune system has mechanisms to develop tolerance to certain antigens, although there has been no self-foreign alignment in the thymus in this regard. For example, tolerance can be determined by se selective elimination or anergization of lymphocytes. In these latter differentiation processes, T cell subgroups that play different (eg activating or inhibiting) cytokines obviously play a crucial role.
  • T cell subsets depend crucially on the manner in which the antigen is presented, for example with regard to the body organ in which it takes place, and the type of cell presenting the antigen.
  • B and T cells form an immunological network that serves to strike a balance between self-tolerance and external reactivity.
  • autoimmune diseases lead to disorders that align the immune system with self. This can e.g. B. happen when there are only minor differences between a foreign structure with which the immune system is confronted and the body's own structures. An immune response can be triggered in this border area, where the areas self and foreign overlap due to structural features. This leads to an increase in T-lymphocytes, which recognize not only the foreign structure but also the structurally similar structure of the body. While, under normal circumstances, after the foreign structure has been eliminated, the immune reaction goes out, it remains under these circumstances, constantly nourished by the presentation of the body's antigen.
  • Structural homologies between microbial proteins and proteins of the host organism are probably only random events in exceptional cases.
  • structural homologies can occur as the remains of a common protein ancestor, with conserved and thus homologous amino acid sequences fulfilling essential functions.
  • structural adaptation of microbial antigens to biomolecules of the host is a common phenomenon that can be explained by the co-evolution of microorganisms with the human immune system. Through such a "molecular Mimicry ", microorganisms can probably protect themselves from the immune system by mistakenly recognizing microbial structures as self. If an immune reaction nevertheless occurs, this can lead to the destruction of the body's own tissue due to a developing cross-reaction.
  • An adjuvant arthritis of the rat is a well documented example of an autoimmune disease that is triggered by a cross-reactive bacterial antigen.
  • the crucial function of specific T cells in the pathogenesis could be demonstrated by cotransferring arthritis with a T cell clone to healthy animals.
  • the T cell clone in question shows specificity for a peptide from the 65 kDa heat shock protein (Hsp60) of mycobacteria and likewise recognizes a peptide from the link protein of rats.
  • the link protein is an essential proteoglycan component of cartilage tissue, a connective tissue that is particularly damaged in arthritis.
  • Inflammatory rheumatic diseases occur in a variety of different clinical pictures such as B. Rheumatoid Arthritis, Felty Syndrome, Psoriatic Arthritis and Juvenile Arthritis (Williams in: Enzyclopedia of Immunology, Ed. Roitt and Delves, 1992, pp. 1339-1343) in appearance.
  • a number of viral and bacterial proteins are considered as potential antigens for triggering an arthritogenic T cell response. Together With bacterial infections caused by Yersinia, Neisserie and Borrelia, among other things, "reactive arthritis” often occurs.
  • the heat shock protein Hsp60 may play an important role here. It is highly conserved both among bacteria and between bacteria and humans and can therefore maintain autoreactive processes.
  • autoimmune diseases In addition to the microbial triggers, the individual genetic predisposition plays a role in the development of autoimmune diseases.
  • An overview of associations between MHC haplotype and autoimmune diseases can be found, for example, in Jacob, McDevitt and Talal (Ed.), (1991) Molecular Autoimmunity, Academic Press Inc., San Diego and in Wordsworth et al., (1992). Accordingly, rheumatoid arthritis (RA) and related autoimmune diseases are particularly often associated with the haplotypes DR1, DR4Dw4, DR4Dw14 and DR4Dwl5.
  • Autoimmune diseases can be based on different effector mechanisms, which are ultimately due to specific interactions between antigen peptides and MHC. These include self-directed antibodies, self-directed CD4 + or CD8 + T cells and an inflammatory spectrum of cytokines.
  • the T cells play a key role here because they are involved in the activation of many important effector mechanisms.
  • the interaction of peptide antigen with MHC class II molecules is correspondingly important, since the complex formation between antigen and MHC class II molecule is the prerequisite for the stimulation of CD4 + T cells of a specific subgroup.
  • Peptide antigen and T cell receptor are therefore setting the course for the development and course of an autoimmune disease.
  • the MHC molecules form a polymorphic family of proteins and each individual has a specific spectrum of different MHC molecules. There can be great differences in the antigen binding properties of different MHC molecules. Comprehensive, if incomplete, information is available on structural features of peptides that are important for binding to MHC molecules (Rotzschke and Falk, 1994). Since the MHC molecules define the spectrum of peptide antigens that can be presented, this is the reason for the genetic predisposition of individual individuals for certain autoimmune diseases. The prerequisite for a specific T cell reaction is the presentation of a peptide antigen by an MHC molecule.
  • MHC-bound peptides in a pit formed by the MHC molecule are in a certain orientation with respect to the N- and C-termini of the peptides.
  • the class II MHC molecule binding pit allows the peptides to protrude from the pit at one or both ends. It is assumed that the binding of the peptides to MHC molecules takes place or is strengthened via certain MHC allele-specific anchor positions. In principle, it is therefore also possible for peptides to be bound in opposite directions in the MHC molecule via corresponding anchor positions.
  • Such inverse peptides, in which the N- and C-termini are exchanged with an identical amino acid sequence e.g. N-LLEQKRAA-C compared to N-AARKQELL-C
  • a microbial pathogen is assumed to be the triggering agent for an autoimmune disease, this presupposes that this pathogen is widespread in accordance with the frequency of the autoimmune disease and the frequency of the genetic predisposition.
  • the degree of infection of this pathogen should correspond to the occurrence of rheumatoid arthritis and the predisposing MHC (DR1 and DR4) or the triggering component of a pathogen prevail in the population.
  • pathogenic Neisseries colonize human mucous membranes.
  • meningococci in particular are part of the permanent flora of the nasopharynx without causing any noticeable symptoms.
  • individuals who are not constantly colonized by meningococci are confronted with these bacteria from time to time in the form of asymptomatic local infections.
  • Antigens of these and other species which behave similarly in terms of their colonization on human mucous membranes, are potential triggers of autoimmune diseases in humans.
  • the object of the present invention was therefore to provide pharmaceuticals for prophylaxis and treatment and diagnostic agents for the detection of autoimmune diseases or the predisposition to them and many diseases.
  • the invention thus relates to a medicament for the prophylaxis or treatment of autoimmune diseases and / or viral
  • BPPs bacterial polyproteins
  • viral diseases are understood to mean those diseases which are induced by viruses or viral elements.
  • the invention relates to a medicament which is characterized in that the interfering is an inhibition.
  • the invention relates to a medicament which is characterized in that the substances or cells comprise:
  • BPPs bacterial polyproteins
  • the invention relates to a medicament which is characterized in that the prophylaxis comprises an immunization.
  • the invention relates to a medicament which is characterized in that the immunization is an active immunization.
  • the invention relates to a medicament which is characterized in that the immunization is a passive immunization.
  • the invention relates to a medicament which is characterized in that the autoimmune disease is rheumatoid arthritis.
  • the invention relates to a medicament which is characterized in that the bacteria producing the BPP belong to the genus Neisseria.
  • the invention relates to a medicament which is characterized in that the viral disease has been induced by HIV.
  • the invention relates to a medicament which is characterized in that the HIV-induced disease is AIDS.
  • the invention relates to a medicament, wherein capsule-specific vaccines against meningococcal infections are used for the immunization.
  • the invention relates to a medicament, the protein being a porin. In a further preferred embodiment, the invention relates to a medicament, the vaccines being produced on the basis of the Neisseries adhesins.
  • the invention relates to a medicament, wherein the passive immunization takes place with specific antibodies or T cells which are directed against membrane components or adhesins of BPP-producing bacteria, preferably Neisseries.
  • the invention relates to a medicament, antibiotics or receptor analogs to receptors of human cells to which pathogenic bacteria can bind or ligands which are specific for the receptors of a bacterial surface are competitive peptides or nonpathogenic or attenuated Neisseries as infection inhibitors.
  • the invention relates to a medicament, the competitive peptide being PilC.
  • the invention relates to a medicament, the BPP being IPP.
  • the invention relates to a. Medicinal product, in which the IGA protease activity of the IPP is specifically inhibited.
  • the invention relates to a medicament, the aspartyl protease activity of the ⁇ -domain of the IPP being specifically inhibited.
  • the invention relates to a medicament, the uptake of the ⁇ -peptide in human cells or their nucleus being inhibited.
  • the invention relates to a medicament, the consequences of the infection by BPP-forming bacteria being inhibited by an immune reaction against BPP-forming, in particular IPP-forming cells, BPP, in particular IPP or their cleavage products.
  • the invention relates to a medicament, cells representing IPP-specific peptides being eliminated by T cells.
  • the invention relates to a medicament, cells representing IPP-specific peptides being eliminated by monoclonal antibodies.
  • the invention relates to a pharmaceutical, antigen-specific suppressive T cells being used.
  • the invention relates to a medicament, wherein anti-T cell receptor antibodies are used to switch off T cells which have a specificity for the BPPs mentioned in claim 3b or the components thereof, corresponding homologous peptides of human proteins
  • the invention further relates to a diagnostic composition for the detection of the etiological agent of autoimmune diseases and / or viral diseases and / or for the detection of the control of the action of medicaments containing
  • the invention further relates to a diagnostic composition for the detection and monitoring of the course of BPP-induced autoimmune diseases and / or viral diseases and / or for the detection of the control of the action of medicaments containing
  • the invention further relates to a peptide which is in the cleavage product of an IPP from Neisseria meningitidis and has an amino acid sequence shown in FIG. 1.
  • the invention further relates to a peptide which is a cleavage product of a BPP, preferably an IPP or a fragment thereof or a human homologue thereof, the BPP, preferably the IPP or the fragment or the human homologue being obtainable by computer-aided comparison of BPP- , preferably IPP sequences with protein databases or translated nucleic acid databases from human sequences or vice versa and wherein at least 6 amino acids in a section of 10 amino acids are identical between the comparison sequences.
  • the peptide according to the invention has an amino acid sequence shown in Table VII, FIG. 2, FIG. 3 or FIG. 8.
  • the invention further relates to a human peptide which is formed by the action of a BPP, preferably an IPP or a cleavage product thereof, with the exception of the IgA 1 cleavage products.
  • the invention further relates to a viral peptide which is produced by the action of a BPP, preferably an IPP or a cleavage product thereof.
  • the invention further relates to a peptide which is a viral peptide which has arisen from the action of a BPP or a human homologue thereto, the viral peptide sequence being obtainable by computer-aided comparison with protein databases or translated nucleic acid databases from human sequences or vice versa and wherein at least 6 amino acids in a section of 10 amino acids are identical between the comparison sequences.
  • the invention relates to a peptide which is an epitope recognized by an antibody.
  • the invention relates to a peptide which is recognized by a T cell after MHC presentation.
  • the invention further relates to a method for inhibiting or activating the transcription of virally coded polypeptides, characterized in that substances, preferably biomolecules, preferably proteins, DNA, RNA or anti-sense RNA or synthetically produced molecules, preferably therapeutically active molecules, in eukaryotic , preferably introduces human cells or their cell nucleus using an IgA- ⁇ protein.
  • substances preferably biomolecules, preferably proteins, DNA, RNA or anti-sense RNA or synthetically produced molecules, preferably therapeutically active molecules, in eukaryotic , preferably introduces human cells or their cell nucleus using an IgA- ⁇ protein.
  • the invention further relates to an in vitro method for cloning and multiplying human T cells, the T cell receptor of which specifically recognizes MHC-presented IPP fragments, the T cells being more human homologous peptides in the presence of BPPs or constituents thereof Proteins, stimulated by the action of a BPPs autoreactive peptides, by the action of a BPPs activated peptides encoded by viruses or endogenous viruses or human peptides homologous to such viral peptides are stimulated under normal conditions.
  • the invention further relates to an in vitro method for cloning and multiplying immunosuppressive T cells which suppress T cells which specifically recognize derived fragments presented in MHC, the T cells being homologous in the presence of BPPs or constituents thereof Peptides of human proteins, autoreactive peptides formed by the action of a BPP, peptides activated by the action of a BPP, encoded by viruses or endogenous viruses, or to such viral peptides stimulated homologous human peptides under usual conditions.
  • the invention further relates to a method for diagnosing an autoimmune disease or a predisposition therefor, preferably rheumatoid arthritis, characterized in that in a T cell proliferation test an IPP, a cleavage product of an IPP or a fragment thereof or a human homologue thereof for proliferation tested by reactive T cells.
  • the invention relates to a method which is characterized in that the cleavage product or fragment thereof, or the human homologue thereof, has the amino acid sequence shown in Table VII.
  • the invention further relates to a method for the detection of BPPs or the bacteria forming them, characterized in that one carries out a polymerase chain reaction (PCR) with primers specific for BPP genes, and the PCR product by DNA sequence analysis or other conventional methods such as gel electrophoresis examined for BPP-specific sequences.
  • PCR polymerase chain reaction
  • the invention relates to a method for the detection of the BPP-dependent activation of viruses or viral elements, characterized in that one carries out a PCR with primers specific for the viral genes, and the PCR product by DNA sequence analysis or other conventional methods such as Gel electrophoresis is examined for BPP-specific sequences or the activity of the viral reverse transcriptase or other viral enzymes is measured.
  • Bacterial exoproteins represent a particular target for the immune response.
  • the IgA proteases can be divided into subgroups according to their amino acid sequences.
  • the IgA proteases of pathogenic Neisseries and certain Haemophilus species form such a subgroup.
  • the previously known IgA proteases from Neisseries and Haemophilus differ in certain areas, while they are homologous in other areas.
  • the IgA proteases of the pathogenic Neisseries form a structurally polymorphic family of enzymes, with each pathogenic Neisseries strain usually producing a certain variant enzyme. Of the Neisseries, meningococci in particular release this enzyme, which is classified as a virulence factor, into their environment in considerable quantities. IgA proteases are extracellular and develop from a precursor polyprotein (IPP). There are further protein domains in the precursor polyprotein (IPP) of the IgA proteases from Neisseries, which are secreted together with the enzyme.
  • IPP precursor polyprotein
  • an IPP is composed of the following separable components, namely an IgA protease domain, a 7-peptide, an ⁇ -protein and a ⁇ -domain (Pohlner et al., 1987) .
  • This invention is based on the detection of the very pronounced molecular mimicry of the Iga proteins (ie all proteins of the precursor polyprotein (IPP)) of Neisseries in relation to human proteins.
  • This molecular mimicry is demonstrated, among other things, by further sequence examples of Neisserie Iga proteins, which were obtained by DNA sequence analysis of iga genes (Example 1). Computer-aided comparisons of these derived amino acid sequences of Iga proteins with known human protein sequences gave numerous sequence homologies according to the invention with human proteins, which are shown as examples (example 2).
  • the Iga ⁇ and Iga ⁇ regions of Iga proteins contain considerable sequence homologies Protein components of the human proteoglycan, such as the link protein or the proteoglycan core protein (Aggrekan) or to other omnipresent human proteins such as myosin and caldesmon. Interestingly, it is shown in Example 2 that the inverse primary sequence of Iga has much more extensive homologies to human proteins. Since - as explained above - peptides can also bind inversely to MHC class II molecules and in this orientation have similar structures to the corresponding normal peptides, these inverse homologous peptide sequences of the Iga proteins are also important for antigenic mimicry and thus Subject of this invention. The continued presentation of one or more peptides of the Iga protein on MHC molecules therefore induces autoreactive processes which are directed against molecular structures in humans (eg cartilage tissue).
  • Another feature of the IPP according to the invention is its ability to penetrate human cells from the outside and, in certain cases, to penetrate into the nucleus. This property is demonstrated particularly impressively by the inclusion of an extracellular ⁇ protein from Neisseries which penetrates into the nucleus of certain human cells (example 7). It is deduced from the structure of the IPP that not only does the ⁇ -protein get into human cells, but also the other components of the IPP or intact IPP. The individual components of the IPP can be partially or completely split off during their transport to the nucleus in different cell compartments, where they become effective against their target cells.
  • Intracellular Neisseries internalized by human target cells also synthesize IPP.
  • intracellularly formed IPP can also penetrate into different cell compartments and also split into individual components in accordance with the IPP that penetrates from the outside.
  • the surface-exposed ⁇ -domain also reaches the target cells.
  • a known property of the IgA protease or the IgA protease domain contained in the IPP is the sequence-specific cleavage of proteins which carry a corresponding sequence required for the cleavage (Pohlner et al., 1992).
  • a preferred cleavage sequence of Neisserie IgA proteases are N-Pro-Pro -!
  • a known natural substrate of the IgA protease is human IgA-1.
  • IgA protease A consequence of the cleavage activity of the IgA protease according to the invention are changes in function in the affected cells. Due to the numerous substrates and their diverse immunological functions, an immunological malfunction of the cells affected by IgA protease is assumed. It is also believed that this immunological misregistration can adversely affect the immune system's balance with respect to its ability to distinguish between foreign and self. The IgA protease activity therefore favors the development of autoimmune diseases. An example of an effect of the IgA protease activity on eukaryotic cells is shown below (see Example 20).
  • IgA proteases are therefore particularly suitable for generating peptides for presentation in MHC molecules and therefore favor the formation of new peptides and their presentation to the immune system.
  • Such peptides can thus arise from human proteins which are not produced in the absence of IgA protease.
  • new self-peptides are recognized as foreign by the immune system because they have not previously occurred. This triggers an immune response against self.
  • the release of the ⁇ -peptide from the Iga protein is also a result of the cleavage activity of the IgA protease, which immediately proves the antigen-processing activity of the IgA protease.
  • a property of the ⁇ -peptide according to the invention is to be presented by class II MHC molecules (example 5).
  • the ⁇ -peptide has the property of binding to MHC molecules which are predisposed to RA-like diseases, namely the MHC alleles DR1, DR4w4 and DR4wl4.
  • CD4 + T cells are activated under natural conditions and can be detected in T cell proliferation tests (Examples 6 and 17).
  • it can be shown that antibodies against the 7-peptide cross-react with peptides of these human proteins (Example 3).
  • a property of the ⁇ -proteins is to penetrate human cells from an extracellular environment either alone or in combination with other components of the IPP. This process obviously requires the binding of the ⁇ -protein to one or more cellular receptors. In the course of this process, ⁇ proteins enter the cell nucleus of human cells. Due to their special structural properties, they interact with other cellular factors and in particular with the DNA of affected cells. This triggers processes (for example gene regulatory processes) that affect the function of affected cells influence. As a result, the cytokine pattern of affected cells is changed. Ultimately, these ⁇ -protein-related processes disturb the balance of the immune system, which, in addition to the molecular mimicry of IPP, favors the development and maintenance of autoimmune reactions.
  • EBV Epstein-Barr virus
  • DR1, DR4w4, DR4wl4 human RA-associated HLA-DR alleles
  • viruses In addition to the structural similarities between viral and human proteins that can lead to autoimmune reactions, certain viruses also encode immunomodulatory factors that can favor autoimmune reactions. These include, on the one hand, immunosuppressive factors such as the Tat protein of the human immunodeficiency virus (HIV) (Viscidi et al., 1989) or certain retroviral gene products that, like the superantigen of MMTV, lead to antigen-unspecific clonal activation of the immune system (especially of T cells) can lead (Held et al., 1993).
  • immunosuppressive factors such as the Tat protein of the human immunodeficiency virus (HIV) (Viscidi et al., 1989) or certain retroviral gene products that, like the superantigen of MMTV, lead to antigen-unspecific clonal activation of the immune system (especially of T cells) can lead (Held et al., 1993).
  • retroviral elements could be detected by electron microscopy in the synovial fluid of RA patients. Similar viral particles were also detected in patients with Sjögren's syndrome (Itescu et al., 1990). In addition, the activity of a reverse transcriptase typical of such viruses was demonstrated in the synovial fluid of RA patients. Little is known about the exact origin of such viral particles that are associated with autoimmune diseases. However, it is believed, for example, that they are activated endogenous retroviral elements (Perl et al., 1993) which, according to recent estimates, make up a large part of the human genome (between 2 and 10%).
  • HIV human immunodeficiency virus
  • SIV closely related viruses
  • phase II HIV rests in the genome of CD4 + cells - especially CD4 + T cells - as a so-called provirus.
  • provirus behaves similarly to endogenous retroviruses.
  • phase III propagation spurts
  • the crucial, as yet unsolved question about the development of AIDS therefore concerns the mechanism of the activation of HIV provirus in the infected cells.
  • the provirus is activated by a transcription of the provirus genome, which originates from the promoter of the "long terminal repeat” (LTR) of the provirus.
  • the Tat protein encoded by the provirus plays a central role in this activation of the LTR.
  • the Tat protein interacts with cellular factors and newly formed viral RNA, which contains the "Tat responsive element” (TAR).
  • TAR "Tat responsive element”
  • Recent results show that the Tat protein has the property of penetrating certain human cells as a soluble protein and penetrating the nucleus thereof. If these cells contain the HIV provirus, HIV is transactivated in these cells.
  • Tat protein is therefore able to activate HIV proviruses in neighboring cells.
  • An important basis of this invention is the structural and functional relationship of the iga genes of Neisseries with viruses or viral elements including endogenous proviruses.
  • IgA-Pro teasen of pathogenic Neisseries is striking their strict association with pathogenic bacterial species.
  • Genomic DNA hybridization experiments using the iga gene from N. gonorrhoeae and an adjacent gene (comA) as a probe indicate that the comA gene, in contrast to the igra gene, occurs in both pathogenic and non-pathogenic Neisseries (Facius and Meyer, 1993).
  • the iga gene probably integrated into the genome of the pathogenic Neisseries as an additional genetic element.
  • sequence homologies of the Iga proteins with viral proteins indicate a viral origin of the iga genes.
  • sequence homologies of the iga gene products and viral proteins can be detected by computer-aided amino acid sequence comparisons (Examples 12 and 18).
  • homologies to viral polyproteins and viral coat proteins occur frequently. Since many Neisserie species are naturally transformation-competent and closely linked to the interaction with mammalian organisms (especially humans), the incorporation of a viral element into the Neisserien genome can be explained by the way of the transformation.
  • a particularly striking structural homology of the neisserial Iga proteins relates to an otherwise unusual amino acid sequence motif of the active center of an aspartyl protease in prokaryotes (Example 11).
  • the active center of the HIV aspatyl protease has the greatest homology to that of the iga gene product. This finding indicates a particularly close relationship between iga and the HIV virus.
  • the active center of the Neisserie aspartyl protease is located in the membrane-bound ⁇ -domain of the Iga protein and can get into cells in connection with the Neisserien infection, for example.
  • Iga proteins are initially formed in the form of large precursor polyproteins and, in the manner of viral polyproteins, break down into several individual proteins by autoproteolysis ( Krausslich et al., 1989).
  • ⁇ proteins of the Neisseries are relatively small (approx. 10 to 30 kDa), predominantly ⁇ -helical, positively charged proteins, which have the ability to dimerize.
  • ⁇ -proteins and Tat proteins share the unusual property of penetrating the nucleus of a human cell from the extracellular milieu (Frankel and Pabo, 1988; Example 7).
  • the ⁇ proteins of the pathogenic Neisseries represent a polymorphic protein class (Example 1), the properties described here are fulfilled by all allelic forms tested to date.
  • the Neisserie ⁇ -proteins and the Tat protein of HIV also have biologically similar effects on eukaryotic cells. Both proteins are equally immunosuppressive as can be measured in a T cell proliferation test (Example 8).
  • the ⁇ proteins of the Neisseries also have an activating effect on certain viral promoters (Example 12).
  • the ⁇ -proteins of the Neisseries can therefore - possibly in combination with the other components of the IPP - penetrate into the nucleus of HIV-infected human cells and activate the HIV-Provirus contained, so that the virus replicates and spreads. This mechanism therefore explains the continued HIV flare-ups, as a result of which a latent HIV infection in phase II develops into infection phase III.
  • the ⁇ proteins of the pathogenic Neisseries also activate other endogenous retroviruses of the human genome.
  • the formation of endogenous retroviral proteins, together with the other properties of IPP, favors the development or progression of autoimmune reactions in the manner described above.
  • AIDS can also be seen as an autoimmune disease (Atlan, 1994).
  • different alleles of the ⁇ -proteins or the IPPs activate certain exogenous or endogenous viruses in different ways. Superimposed by the genetic predisposition of individual individuals, the sometimes different biological functions of the Neisseries iga genes and their gene products explain the development of different clinical pictures.
  • Genes which code for bacterial polyproteins (BPPs) and have structural and functional relationships to the iga gene of the pathogenic Neisseries are, for example, the vacA gene from Helicobacter pylori (Schmitt and Haas, 1993), the aida gene from enteropathogenic E. coli (Benz and Schmidt, 1989), the Bordetella parapertussis pertactin gene (Dougan et al., 1991) and the Serratia marcescens serine protease gene (Nakahama et al., 1986).
  • neisserial Iga proteins or IPPs for the activation of viral elements (in particular HIV proviruses and other endogenous retroviruses) is demonstrated.
  • the present invention thus explains the development of AIDS by activating HIV in virus-infected persons on the basis of the biological function of the Iga proteins or the IPPs of Neisseries.
  • the activation of viral elements, especially endogenous retroviruses causes autoimmune reactions that are indirectly attributable to the biological effects of BPPs, in particular the Neisseries IgaProteine or IPPs.
  • BPPs in particular the Neisseries IgaProteine or IPPs.
  • the diverse structure and effects of BPPs and the respectively activated viral elements explain the variety of the symptoms.
  • Pathogenic IgA protease-forming Neisseries (especially meningococci) infect the human mucosa. Individuals can be infected temporarily or persistently with alternating strains. The infections can be asymptomatic or symptomatic.
  • the pathogens form IgA proteases, which can take various forms and contain amino acid sequences which represent molecular mimicry with respect to human proteins. For example, there is molecular mimicry between the 7-peptide portion of the Neisserie IPP and human link protein or aggrekan.
  • IPP peptides in particular the 7-peptide portion, are presented on class II MHC molecules.
  • CD4 T cells whose T cell receptor is directed against an MHC-presented peptide (foreign) of the IPP are activated.
  • T cells cross-react with a self-peptide, which is also presented to MHC. That these T cells are also activated by MHC-presented self-peptide.
  • Self-reactive T and B cells are introduced into certain regions of the body in which corresponding self-antigens are formed.
  • the continuous stimulation with self-antigens and the formation of inflammatory cytokines set in motion a chronic inflammatory reaction, in the course of which unspecific cell proliferation and tissue destruction occur.
  • IPP immunoglobulin-like protein
  • ⁇ -protein is produced intracellularly alone or in combination with other constituents of the IPP or is absorbed from outside into cells
  • IgA protease cleaves functionally important proteins such as IgAl, CD8 etc.
  • the regulatory properties of the ⁇ protein and other areas of the IPP accelerate or suppress immunological reactions.
  • viral elements are activated due to the effects of the neisserial IPPs in the course of the neisserie infection.
  • the resulting viral particles and other viral gene products perform a variety of different functions, which can result in different clinical forms of autoimmune diseases in humans.
  • the HIV virus is activated. In this case, the clinical picture is determined by the progressive HIV infection.
  • Neisseries is an etiological agent of autoimmune and viral diseases in humans, especially rheumatoid arthritis and AIDS
  • a first approach to combating these diseases against infections with IgA protease-producing pathogenic Neisseries, including asymptomatic infections is aimed.
  • active immunization a first approach to combating these diseases against infections with IgA protease-producing pathogenic Neisseries, including asymptomatic infections.
  • passive immunization Several fundamentally different methods can be used for this, (a) active immunization, (b) passive immunization, (c) treatment with infection inhibitors including antibiotics and (d) combined methods.
  • a further possibility for combating Neisseries infections and autoimmune and viral diseases based thereon is the administration of specific infection inhibitors which prevent the multiplication and or the infection of the human mucous membranes with pathogenic Neisseries.
  • specific antibiotics against Neisseries and receptor analogs that prevent the binding of the Neisseries to cellular receptors can be used, for example.
  • Particularly effective receptor analogs are substances which prevent the PilC-mediated binding of the Neisseries to human epithelial cells (German patent application P 43 36 530.2) or block the cellular invasiveness of the Neisseries mediated by opa proteins (Example 15).
  • the latter substances, which block the invasiveness of the Neisseries include Compounds that have structural similarities to heparan sulfates (e.g. heparin, example 15).
  • a combined method to combat Neisseries infections is to administer a Li found the Neisserien surface, which is coupled with an antigen.
  • a certain antigen is deposited on the bacterial surface. If antibodies against this antigen are formed, the antibodies can bind to the bacterial surface via the ligand and inactivate the bacterium via one of the known reactions.
  • This possibility of pathogen elimination is particularly suitable in the case of the Neisseries, since these bacteria form structural and antigen-variable surface components (Robertson and Meyer, 1992).
  • many variable surface proteins express conserved receptor-binding or ligand-binding properties, the Neisseries can be effectively eliminated via the path of the ligand.
  • Neisserie proteins with a conserved binding of ligands are the PorB (or PI-Porin), which binds purine nucleotide triphosphates (Rudel, 1994), a gangliosyl tetraosylceramide binding surface adhesin (Paruchuri, 1990) and the surface-bound PilC protein the Neisseries (Rudel, 1994).
  • the ligands of these bacterial receptors are preferably coupled with an antigen against which antibodies are already active; However, it is also possible to carry out an active immunization against the coupled antigen or to passively administer antigen-specific antibodies. It is also possible to change the ligand in such a way that its affinity for the bacterial surface is increased.
  • pathogenic and non-pathogenic Neisseries sometimes recognize very similar or identical receptors on the mucous membrane of humans and use them for infection. It is therefore possible to use non-pathogenic or attenuated Neisseries to replace pathogenic, IgA protease-producing Neisseries. Through such a competition for the receptors on the human Mucous membranes can be largely prevented from infections with pathogenic Neisseries.
  • IPP has been shown to trigger Neisserie-induced autoimmune diseases of certain viral diseases. Accordingly, substances (including vaccines) are used to combat such diseases, which are directed against the enzymatic activities of the IPP.
  • substances including vaccines
  • the level of IgA protease activity in the IPP prevents the release of the 7-peptide, which plays a central role in autoimmune pathogenesis.
  • Synthetic peptides of the structure RboroPro-OH have been described as an effective specific inhibitor of the proteolytic activity of the IgA protease (Bachovchin et al., 1990). Such or similar substances can therefore be used to combat autoimmune diseases.
  • substances can be used which have an inhibitory effect on other functions of the IPP or its individual components.
  • substances can be used which lead to an inhibition of the aspartyl protease of the ⁇ domain or to a blocking of the uptake of the ⁇ protein in human cells or their nucleus.
  • a special feature of the Neisseries IPPs is their intracellular site of action.
  • the description makes it clear that a Neisserie IPP or its cleavage products contain numerous functions that have an intracellular effect.
  • a Neisserien IPP and in particular the ⁇ -protein By using the intracellular transport function of a Neisserien IPP and in particular the ⁇ -protein, it is possible to transport other biological molecules to the exact site where the native IPP or its cleavage products develop their natural effects. It is therefore possible to transport substances to the natural site of action of an IPP that inhibit the natural biological function of an IPP. It is also possible to add molecules transport, inhibit the subsequent reactions (eg replication of viral elements) and thus limit an autoimmune reaction or virus multiplication.
  • IPP transport functions are used to introduce nucleic acids into human cells, which in turn carry out antagonistic functions of the IPP or its subsequent reactions and thus prevent an autoimmune reaction or virus multiplication.
  • nucleic acid segments could code for inhibitory proteins and integrate them as DNA into the genome of the affected cell or have one of numerous other inhibitory properties.
  • human cells affected under natural conditions include in particular certain cells of the human mucosa (e.g. the nasopharynx) and certain cells in human blood.
  • a further possibility for utilizing the targeting properties of the alpha protein consists in the chemical synthesis of analog peptides, which represent partial areas of the alpha protein and have similar transport and targeting properties.
  • An advantage of chemically synthesized peptides is, in particular, the possibility of introducing active groups that are coupled to biologically active groups Substances such as active proteins, nucleic acids or other substances can be used.
  • the ⁇ -protein can also be used for "gene targeting" experiments. E Elimination of IPP-affected cells using specific antibodies or cytotoxic T-sites
  • IPP-derived peptides Human cells that are affected by uptake of extracellular IPPs or by infection with pathogenic Neisseries present IPP-derived peptides on their MHC molecules. This fact can be used to specifically eliminate affected cells as described below:
  • IPP-derived peptides presented on MHC class I can be specifically recognized and eliminated by CD8 + T cells. It is possible to multiply such specific T cells by cloning in vitro and to return them to affected individuals. A prerequisite for this application, however, is that these specific T cells do not react against self-antigens; this can be ruled out by suitable screening procedures.
  • IPP-derived peptides that are presented either MHC class I or class II by means of specific monoclonal antibodies, which are obtained by known methods (for example by hybridoma technology, humanization of animal antibodies and / or recombinant antibody technology), to eliminate.
  • This can take place in that the bound antibody activates the complement system or elicits an antibody-dependent cytotoxic cell-mediated reaction (ADCC), which in each case results in the elimination of affected antigen-presenting cells.
  • ADCC antibody-dependent cytotoxic cell-mediated reaction
  • previous screening must ensure that the antibody obtained is not directed against self-antigens.
  • T-cells directed against self can have predominantly activating or predominantly suppressive properties. In the event that predominantly activating T cells are present, the autoimmune reaction is favored. On the other hand, an autoimmune reaction can be suppressed if there are sufficient suppressing T cells in addition to the activating T cells. An autoimmune reaction can thus be suppressed by stimulating or increasing the antigen-specific suppressing T cells. The stimulation of suppressing T cells depends on organ and cell-specific parameters of the antigen presentation of self-peptides or self-similar peptides (Mitchison, 1993). For example, predominantly suppressive T cells can be generated by oral administration of such peptides and the autoimmune reaction can be suppressed.
  • antigen-specific T cells can be modified by genetic manipulation in vitro so that they assume the properties of suppressing T cells. This is done, for example, by transfecting T cells with the functional genes of suppressing interleukins. the methods used for this correspond to the state of the art. G Development of a T cell vaccine
  • autoreactive T cells with certain variable V ⁇ or V ⁇ chains of the T cell receptor often play a role.
  • Such clonal autoreactive T cells which may have arisen from the action of a superantigen, can also be used for the production of a T cell vaccine.
  • MHC molecules bind a large number of different peptides. It is therefore also possible to load the MHC molecules with peptides that allow either no autoreak ⁇ tive T-cell activation or anergize autoreactive T-cells.
  • the autoimmune and virus-related diseases according to the invention are triggered by the bacterial polyproteins (BPPs) or the BPP-forming bacteria.
  • BPPs bacterial polyproteins
  • This invention therefore relates to diagnostic methods which are suitable for a qualitative or quantitative detection of BPPs or the bacteria which form them.
  • diagnostic methods include known microbiological, direct (antigen-specific) and indirect (antibody-specific, T-cell-specific) immunological, as well as enzymatic and gene-analytical detection methods.
  • the gene analysis methods include in particular the polymerase chain reaction (PCR) and DNA hybridization methods.
  • the detection methods mentioned relate in particular to the pathogenic Neisseries and the IPPs they have formed.
  • This invention therefore relates to diagnostic methods which provide information about the course of the disease. These include methods for the quantitative and qualitative detection of the immune status and the antigen-specific immune reactions at the B and T cell levels. Furthermore, this includes methods for the detection of the activation of viruses or viral elements, for example by means of genetic analysis (e.g. PCR techniques; Hermann and Kalden, 1994) and / or enzymatic (e.g. detection of reverse transcriptase activity). In addition, this includes detection methods of other typical effects of bacterial BPPs and in particular the Neisserie IPPs.
  • the results presented in this invention enable a large number of new antigen-specific methods for therapy or prevention against autoimmune and viral diseases in humans. These include the examples of therapy listed above and other forms of therapy described in the literature (eg Bläß, 1994; Tisch et al., 1994 and the literature cited therein).
  • the antigen-specific forms of therapy - based on the role of the pathogenic Neisseries - are based on the following antigens: * Peptides of the Iga gene products of the pathogenic Neisseries,
  • Viruses or endogenous viruses encoded peptides and
  • This invention furthermore relates to all of the methods exemplified above for the therapy of or for the prevention of autoimmune and viral diseases in humans which are directed directly or indirectly against the biological action of bacterial polyproteins (BPPs), in particular the Neisseries IPPs.
  • BPPs bacterial polyproteins
  • These procedures include in particular
  • This invention furthermore relates to diagnostic methods insofar as they are derived from the results mentioned above and which are of importance for the diagnosis of autoimmune and viral diseases in humans. These include in particular
  • BPP refers to bacterial polyproteins in general and includes the IgA protease polyproteins from pathogenic Neisseries (IPPs).
  • IPP refers not only to the direct gene product of the iga gene of the pathogenic Neisseries, but also to the secondary products "IgA protease", “ ⁇ -protein”, “7-peptide” and ⁇ -domain or intermediate fusions of these proteins.
  • pathogenic Neisseria refers to Neisseria gonorrhoeae and Neisseria meningitidis. If the term “Neisseries” is not explained in more detail, it includes at least the two pathogenic Neisseries.
  • viral diseases is understood to mean pathogenetic processes which are a consequence of the activation of viruses or viral elements, the activation being brought about by bacterial pathogens or their BPPs. The activation takes place in particular through the action of an IPP of pathogenic Neisseries on human cells.
  • viruses is understood to mean viruses which are activated by BPPs, in particular an IPP of pathogenic Neisseries, in their gene expression and or replication. It cannot be ruled out that different IPPs of pathogenic Neisseries have qualitatively and quantitatively different effects on viruses or viral elements.
  • viruses refers to genetically complete viruses or virus genomes (e.g. HIV or HIV provirus), while the term viral elements refers to genetically incomplete viral genomes (e.g. endogenous retroviruses).
  • Figure 1 DNA sequences and derived amino acid sequences
  • the Iga ⁇ 3 and Iga ⁇ 4 domains show pronounced homologies with each other as well as with the corresponding Iga ⁇ 1 and Iga ⁇ 2 domains of N. gonorrhoeae in the primary and
  • the Iga ⁇ 2 alleles are identical to the Iga ⁇ 1 allele of N. gonorrhoeae except for the amino acid at position 4.
  • the associated DNA sequences iga ⁇ 2 and iga ⁇ 2 * differ in the base at position 93.
  • the iga ⁇ 3 allele has a deletion of 4 triplets in the central region compared to iga ⁇ 2.
  • the iga ⁇ 4 allele shows a conversion of glycine-alanine to Senn-proline, which is due to a replacement of G by A at position 49 and a replacement of G by C at position 52 of the corresponding coding region.
  • FIG. 1 Homologies between amino acid sequences of the IgA protease precursor (IPP) of pathogenic Neisseries and various human proteins.
  • IPP IgA protease precursor
  • Igap, Iga ⁇ , Iga ⁇ and Igass denote the different domains of the Iga proteme, which have different functions and which are broken down by specific autoproteolysis ⁇ Pohlner et al., 1987).
  • the sequences are given in the one-letter code in the orientation from the amino to the carboxyl end.
  • the numbers at the amino end of the sequence sections indicate the corresponding positions in the Iga protein of N. gonorrhoeae MS11 (Igap, Iga ⁇ , Iga ⁇ 1 and Igass; (Pohlner et al., 1987), N. gonorrhoeae R16 (Iga ⁇ 2; Schur et al., 1989 ) and N.
  • meningitidis B1939 (Iga ⁇ 4 position based on the amino end of the ⁇ protein) and the sequence position in the human proteins. Identical amino acid positions between IgA proteins and the human proteins are highlighted by frames. Positions which are occupied by amino acids with similar properties Abbreviations: Hs47, 47 kd heat shock protein; Rsmb, small nuclear ribonucleoprotein-associated protein; Nfh, neurofilament triplet h protein; Qxyb, oxysterol binding protein.
  • Figure 3 Homologies between amino acid sequences of the ⁇ -peptide of pathogenic Neisseries (here N. gonorrhoeae MS11 and R16) and various connective tissue proteins in humans.
  • the first homology areas shown come from the most common protein components of the extracellular matrix of the cartilage tissue.
  • the link protein forms, with hyaluronic acid and the proteoglycan core protein, huge aggregates that serve to stabilize and elasticize the cartilage by absorbing water. While only a single PTR exists in the link protein and in the fibroblast proteoglycan, the proteoglycan core protein consists of two domains (G1 and G2) with two PTRs each.
  • the serine position at the amino end corresponds to position 960 in the Iga protein of N. gonorrhoeae MS11 (Pohlner et al., 1987).
  • the numbers at the amino end of the sequence sections indicate the respective positions in the human proteins. Identical amino acids between Iga ⁇ alleles and human
  • Proteins are printed in bold. Positions that are occupied by amino acids with similar properties are underlined. Following the sequences, the identity with the ⁇ -peptide is in
  • the polyprotein IPP is composed of the signal peptide (S), the enzymatic part (P), the ⁇ -peptide ( ⁇ ), the ⁇ -peptide ( ⁇ ) and the ⁇ -core ( ⁇ ). Cys marks the two cysteine residues preserved in all lPP s. Below are the areas that have been shown to be conserved or variable in N. gonorrhoeae (2) and N. menigi ⁇ dis (3) in the sequence analysis of various IPPs.
  • Domains marked in black are each variaoel, obliquely striped
  • Lines 4-8 show the homologies to human proteins.
  • a black square marks a homology of 7 or 6 identical amino acids in one piece.
  • the position marked with a square in line 6 shows one Range within the IPP that has sequence homology to a human protein of 5 essential amino acids in a row, this homology being part of a window in which at least 7 out of 10 amino acids are identical.
  • Lines 7 and 8 show sequence homologies of the reverse amino acid sequence of the N. gonorhoeae MS11 lPP with human proteins, a square in the seventh line corresponding to 7 identical amino acids in one piece, and a square in line 8 correspondingly to 6 amino acids.
  • FIG. 5 Construction of the expression plasmid pOK19.
  • the 1032 bp insert of the plasmid pOK18 (shown in white) codes for the 339 amino acid link protein (see FIG. 6). After restriction with the enzymes EcoRI and BamHI, it was inserted into the vector pEV41a via the same interfaces, resulting in the expression plasmid pOK19 was created.
  • Figure 6 Amino acid sequence of the human link protein.
  • Fig. 3 are underlined.
  • FIG. 7 Proteins of humans, which can serve as IgA protease substrates.
  • a variety of human proteins contain a sequence of
  • Ig-A immunoglobulin A-1; C chain
  • MsF macrophage stimulating protein
  • Tf-g T cell specific transcription factor G
  • NF-kappa-b core protein NF-kappa-b p100 subunit
  • TNF R1 tumor necrosis factor receptor 1
  • CC10 Complement C-10 component, C chain
  • cd45 leukocyte common antigen Cd45
  • FIG. 8 Comparison of a possible MHC class II binding motif with two putative cryptic ones that were created by IgA protease activity
  • the binding motif for MHC class II-bound peptides postulated by Rotzschke and Falk (1994) is shown in line (1).
  • Ac marks the position of hydrophobic amino acids, via which anchoring with the MHC molecule can take place.
  • X marks an amino acid that cannot be characterized in more detail.
  • line (2) and line (3) based on the two potential IgA protease substrates CD8 (Cd8; alpha chain) and CC10 (complement C-10 component; C chain), the correspondence of this binding motif with peptides by Iga protease activity can be rewritten. Direct matches with the binding motif are printed in bold in the IgA protease cleavage products.
  • Figure 9 Homologies of various retroviral aspartyl proteases with the IgA protease from Neisseries.
  • the alignment shows the area around the catalytic center of the aspartyl proteases. Amino acids printed in bold represent identical or similar residues. The following abbreviations are used:
  • MMTV mouse mammary tumor virus
  • HTLV-2 human t-cell leukemia virus II
  • CIV chimpanzee immunodificiency virus
  • HIV-2 human immunodificiency virus II
  • HIV-1 human immunodificiency virus I
  • FIV feline immunodificiency viru
  • EIV equine immunodificiency virus
  • RRP retrovirus related polyprotein
  • IgA-P neisserial IgA protease
  • oligonucleotides J0154 (5'CAGGAAACAGCTATGACCACACCGTTCGGTACAGCA) / J0126 (5'CAGGAAACAGCTATGACCACACCGTTCGGTACAGCA) and were used as sud toopri- mer oligonucleotides J0136 (5'TGTAAAACGAATGGCGAGGGCGGTGCGGCA) / J0146 (5'TGTAAAACGACGGCCAGTATGATGGCGAGGGCGGTGCCGGCA) as the forward primer. Based on their length, the amplified DNA fragments could be divided into two size groups. DNA sequence analyzes were carried out according to the dideoxy method.
  • flanking sequence areas were analyzed by means of PCR cycle sequencing using fluorescent dye-labeled universal forward (5'-TGTAAAACGACGGCCAGT) and universal reverse primers (5'-CAGGAAACAGCTATGACC), and in parallel using fluorescence-labeled dideoxynucleotides.
  • iga was determined in 13 different menigococcal strains.
  • sequences of the iga ⁇ regions of N. meningitidis D1941 and B1939 were determined after their subcloning in M13mpl8 / 19.
  • the amplification was carried out according to standard conditions with the primer pair J097 (5'-TCGAATTCCGGTATTACCCGGTTGT) / JO24 (5'-TCCAGCGCATCCAAGGGG) and the DNA fragments were then purified by agarose gel electrophoresis. After filling in the ends with Klenow polymerase and trimming at the 5 'end with EcoRI, the fragments were inserted between the EcoRI and HincII sites of the M13mpl8 / 19 vectors. The size of the iga ⁇ fragment from strain B1939 made further subcloning necessary for the sequencing, in which the central PstI fragment was inserted into the corresponding interface of M13mp19.
  • iga 4 at the amino acid positions 18 and 19 contains serine and proline instead of glycine and alanine, as with all other iga ⁇ forms.
  • the iga domains of all strains examined so far can be divided into these four groups.
  • the corresponding DNA sequences were highly conserved.
  • An amino acid exchange was manifested by the exchange of a single nucleotide at position 1 of the corresponding codon, which is why the number of amino acid exchanges can be equated with the number of nucleotide exchanges.
  • An additional variation in the Iga proteins resulted at position 22. Here either proline or alanine was found, although this was not specifically assigned to a group of iga forms.
  • the iga ⁇ 3 and iga ⁇ 4 alleles code for ⁇ proteins of 169 and 392 amino acids, respectively. In the N-terminal region these proteins are identical over 25 amino acids to the Iga ⁇ 1 and Iga ⁇ 2 alleles (Halter et al., 1989) from N gonorrhoeae Msll and R16. In addition, there are pronounced homologies between all four alleles, which are distributed in the form of short sequence motifs over the proteins. In addition to the homologies with one another, a large number of homologies to cellular proteins and matrix proteins of human origin occur (FIG. 2).
  • homologies mainly occur with connective tissue components such as proteoglycan and link protein (FIG. 3).
  • the 1532 amino acid sequence of the Neisseria gonorrhoeae MSII IPP was divided into successive peptides of 50 amino acids in length, each with an overlap of 10 amino acids.
  • the Wisconsin Sequence Analysis Package from GCG was first used in various databases to search for sequences which also occur in human proteins.
  • the homologies of IPP to human proteins concentrate on four areas, the ⁇ -peptide, the ⁇ -protein, part of the ß-core, and a constant area of the peptides that.
  • the vast majority of homologies to human proteins are in areas that are conserved in the previously known IPP forms of various Neisseries. This conforms to the hypothesis that there is molecular mimicry in these areas, and indicates that these sequences of sequences have been adapted to the protein pattern of the host and have proven themselves evolutionarily.
  • the constant and variable ranges of the different IPPs are in both N. gonorrhoeae and N. Meningitidis obtained in the same arrangement.
  • FIG. 2 Selected examples of the molecular mimicry of IPP are shown in FIG. 2. From this it can be seen that a large number of sequence patterns of human proteins have been copied in the IPP, with the primary aim of protecting the protein from the host's immune response on the basis of the tolerance existing against "self". If an immune response nevertheless occurs, possibly through the inclusion of flanking non-identical regions, this can also be directed against the body's own protein and is retained by constant stimulation after the actual trigger has disappeared. A number of partial sequences of the IPP, which show homologies to human proteins, also meet the criteria that have hitherto been known for the binding of peptides by MHC class II molecules, that is, for antigen presentation.
  • the total mRNA was isolated from primary cultures of human chondrocytes of the hip joint. This mRNA was rewritten with reverse transformer scriptase in cDNA and used as a template in order to use the specific oligonucleotides (J0123 5'-GCGAATTCCGATCATCTTTCAGACAACTATACT and J0125 5'-AACGGATCCTCATCAGTTGTATGATGATGCTCT to polymerize the gene in the linkC-PCR protein -A gene of the LinkCase protein) to amplify the gene in the linkC-protein (PCR) chain. After purification and restriction with EcoRI and BamHI, the I032bp fragment thus obtained was cloned into the vector pBluescriptII-SK and sequenced.
  • the vector pOK19 resulting from this step was introduced by electroporation into the E. coli strain 2136, which has the temperature-sensitive repressor CI857 in the genome. After temperature induction at 42 °, the fusion protein is produced in large quantities and deposited in the inclusion body inside the cell. After the cells have been lysed, these inclusion bodies are dissolved in guanidinium hydrochloride. The fusion protein is then bound to a nickel column using its amino-terminal "histidine tag" and eluted by changing the pH after washing the column (Diagen GmBH, Quigen Inc. QuiaExpress, 1992). Preparation of peptide antiserum:
  • mice Female Balb / c mice were immunized intraperitoneally three times with 100 mg each of the synthetically produced Pam 3 Cys- ⁇ -peptide (Pam 3 Cys-SPAANTASQAQKATQTDGAQIAKPQNIVVA) in saline.
  • the "Pam 3 Cys" -effective portion of the lipopeptides replaces an adjuvant (Wiesmüller et al., 1989).
  • the antiserum was obtained according to the state of the art. It reacts very well in the ELISA in a dilution of 1: 2000 with the synthetic ⁇ -peptide, as well as with the shorter peptides P2 (TDGAQIAKPQNIWA) and P3 (QKATQTDGAQIAKPQ).
  • this antiserum recognizes both a recombinant ⁇ -fusion protein and the recombinant link protein (see FIG. 6).
  • antibodies directed against the ⁇ -peptide can also cross-react with the human link protein.
  • mice were repeatedly immunized with the P121 protein, which contains the entire ⁇ and ⁇ region of the Neisseria gonorrhoeae MSII IPP. After hybridization of the macrophages obtained with the cell line NS-1 and repeated subcloning, seven cell clones producing monoclonal antibodies were found. All procedures were carried out under standard conditions. One of the monoclonal antibodies produced, designated JOP1, recognized a conserved epitope of approximately 12 amino acids (QAKAEQVKRQQA) at the amino end of the Neisseria ⁇ -proteins 1-4.
  • JOP1 One of the monoclonal antibodies produced, designated JOP1, recognized a conserved epitope of approximately 12 amino acids (QAKAEQVKRQQA) at the amino end of the Neisseria ⁇ -proteins 1-4.
  • JOP1 showed a cross-reaction with human cells (Chang conjunctiva), which in indirect immunofluorescence microscopy was expressed like a myosin-specific staining of the cells (without illustration)
  • Example 5
  • EBV-transformed homozygous B cell lines (Kimura et al., 1992) were cultivated on a large scale. Due to the virus transformation, these B cells show a greatly increased expression rate of MHC class II molecules (Gorga et al., 1987), which enables them to be isolated using affinity chromatography. For this purpose, a monoclonal antibody that specifically recognizes HLA-DR molecules was coupled to CNBr-activated Sepharose (Halter et al., [1993]).
  • HLA alleles important for the aetiology of rheumatism are important for the aetiology of rheumatism.
  • the peptide H3 (HPTKLTYDEAVQACL) comes from a region of the human link protein which has 40% identity to the neisserial antigen. H3 binds to the alleles DR1 and DR4 with high affinity.
  • the activation of T cells by antigens or mitogens can be determined qualitatively and quantitatively by inducing various T cell functions, such as proliferation, cytokine formation or expression of activation markers (IL-2 receptor, HLA-DR etc.).
  • the aim of the present study is to determine the existence of peptide-specific T cells in the peripheral blood of rheumatism patients (characterized by ACR Criteria). To check this, we use the property of T cells in our test system to proliferate after antigenic stimulation.
  • the link peptide (L40) (Table VII) was used as an antigen in a T cell proliferation test (see below), which has particularly clear homologies in the amino acid sequence with 7-peptide (see example 3, Figure 5).
  • the mononuclear cells (MNZ) were isolated from heparinized whole blood (20 U / ml) using Ficoll density gradient centrifugation (Böyum, 1969). The
  • Blood was diluted 1: 2 with Hanks' BSS and 30 ml portions of the mixture were layered on 15 ml Ficoll-Hypaque (density 1.077 g / ml). After subsequent centrifugation in the swing-out rotor (35 min, 400 xg, 21 ° C), the supernatant plasma was sucked off sterile and the MNZ was harvested from the interphase using a Pasteur pipette. The MNZ were then washed twice with Hanks' BSS and included in RPMI 1640 in a certain number of cells. The number of living cells was determined by microscopic counting in the Neubauer counting chamber after trypan blue staining.
  • peptides were adjusted to a concentration of 20 ⁇ g / ml in RPMI 1640 (without serum, + 1% glutamine + 50 ⁇ g / ml penicillin and 50 ⁇ g / ml streptomycin) immediately before the test.
  • Tetanus toxoid (TT) which was present in a final concentration of 2.5 LF / ml, was used as the bacterial control antigen.
  • the MNZ proliferation rate was determined by the incorporation of 3 H-thymidine during DNA synthesis.
  • the cells were concentrated in a concentration of 1 ⁇ 10 5 cells / wells in round-bottom microtiter plates together with the corresponding peptides (25 ⁇ g / ml) in a volume of 100 ⁇ l for 2 hours at 37 ° C. in a culture cabinet (5% CO 2 ) pre-incubated. Then 50 ⁇ l of RPMI, supplemented with 15% heat-inactivated human serum of blood group AB, was added to each well and the plates were cultivated for 7 days (or 10 days with the addition of IL-2, see below). 24 hours before the harvest the radioactive isotope ( 3 H-labeled thymidine, Amersham, 0.2 ⁇ Ci / well).
  • the cells were provided with the growth-promoting cytokine IL-2 (20 U / ml) after 6 days of culture and cultured for a further 3 days before these were labeled with 3 H-thymidine for 24 hours.
  • IL-2 potentially reactive anergic T cells can also be stimulated to proliferation, which may be "triggered” by a similar antigenic peptide, but which lack costimulatory signals (Meuer et al., 1986).
  • the plates were first frozen at -20 ° C. and later applied to filter mats using a cell harvester. The incorporation rate of the radioactive isotope was determined by liquid scintillation measurements in the LKB counter (Pharmacia). The mean value of the counts per minute (cpm) of three wells (triplicets) was determined from each stimulation approach.
  • a T cell response to the 7-peptide was also found in exactly the same patients. Interestingly, proliferation on both link and 7-peptide could only be induced in one case by the addition of IL-2 (patient No. 2).
  • the shaded boxes in Table IV document the agreement of the proliferation rates as a result of the stimulation with the link peptide or 7 peptide.
  • the T cells of these donors also showed a very good proliferation triggered by tetanus toxoid, an indication of the good antigenic stimulability of the cells of these donors.
  • proliferation after administration of IL-2 was also found in the cells of patients 6, 7 and 8 stimulated with link peptide, no proliferation in connection with 7-peptide and IL-2 could be detected there.
  • T cell clones which were isolated and established under optimized and defined conditions, could then be tested for their cross-reactivity on link or 7 peptide. With the help of the proliferation test, binding motifs and potential T cell epitopes of the peptide could then be studied, precisely defined and used as immunodiagnostics.
  • ⁇ protein occurs in all pathogenic Neisseria gonorrhoeae and Neisseria meningitidis strains. It discloses very good sequence and structural homologies to known viral and eukaryotic proteins (see example 11 / example 12).
  • a unique ability of the ⁇ protein is that it can be taken up by cells by incubation with receptor-mediated endocytosis and accumulated in the nucleus. That this is a specific process is confirmed by the fact that only a few cell types allow this nuclear localization.
  • the explanted corneas stored at 30 ° C in DMEM 5% FCS (fetal calf serum) are divided with a sterile scalpel into 16 pieces of tissue of the same size and up to a preconfluent growth stage (80% of the cover glass overgrown) in 24 "well" plates (Nunc, Dk) incubated on coverslips (human corneas from the Interuniversitary Ophthalmologic Institute, Academic Medical Center, Amsterdam were used).
  • coverslips human corneas from the Interuniversitary Ophthalmologic Institute, Academic Medical Center, Amsterdam were used.
  • the orientation of the corneal fragments on the cover slip is with the endothelial side down.
  • the used DMEM medium with 5% FCS is now replaced with fresh Medium replaced without FCS (this step serves to free receptors that are occupied by serum proteins). After an hour of incubation, the medium is changed again.
  • the cells are now cultured in FCS-free medium supplemented with ⁇ -protein (5 mg / ml) for a further five hours (this recombinant ⁇ -protein was used as a fusion protein in an overproducing E. coli strain (UT 5600, OrnpT-) "His-Tag" expressed, separated from the cell lysate via a nickel agarose (Ni 2+ -NTA agarose, Quiagen) and purified with the aid of affinity chromatography (BioRex 70, BioRad). Physiological buffer conditions were dialyzed against 1 x PBS without This is followed by fixation and permeabilization of the cells, which finally allow immunofluorescence microscopy to be carried out:
  • the cells are washed once with 1 x PBS (without divalent cations) and fixed for 10 min with a 1% paraformaldehyde solution (in 1 x PBS). The cells are then permeabilized for 30 min with a 0.5% Triton x-100 solution (in 1 x PBS).
  • the primary antibody solution diluted in 1 ⁇ PBS / 1% BSA, is then incubated for two hours at room temperature or overnight at 4 ° C. Unbound primary antibody is washed three times for 10 min with 1 x PBS / 0.05% Tween 20 and a fluorescent dye-labeled secondary antibody / protein A is incubated in the appropriate dilutions with the cells for 30 min.
  • lymphocytes and macrophages Another class of human primary cells that can endocytate the ⁇ protein and accumulate in the nucleus are lymphocytes and macrophages. This could be demonstrated with mononuclear parts (MNZ) from whole blood from different donors.
  • MNZ mononuclear parts
  • RPMI medium resuspended and counted. Between 5 x 10 5 and 1 x 10 6 MNZ can be used for a recording experiment. After incubation of the cells with serum-free RPMI medium for one hour, the uptake of the ⁇ -protein is carried out analogously to the procedure as described for the cornea cells. Depending on the immune status of the donors, one could recognize in the immunofluorescence especially in subpopulations of the round-nucleus lymphocytes or in almost all cells an uptake and nuclear accumulation. The immunosuppressive effect of the ⁇ protein shown in Example 8 could be explained by the endocytosis ability described here. After a very short incubation period with ⁇ -protein (less than 1.5 hours), only a few cells with nuclear accumulation can be found.
  • the immunosuppressive effect of the ⁇ protein can also be based on binding and inhibition of the CD26 activation marker of T cells (Gutheil et al., 1994).
  • the autocatalytic processing of the IgA protease polyprotein takes place in the extracellular environment. This process is concentration-dependent and is observed above all in the case of overexpression in the heterologous Escherichia coli system. Such high concentrations are probably not reached in vivo in IgA protease positive strains.
  • the T cells are directly involved in the defense against viruses and microorganisms as cytotoxic T cells or regulate the cellular and humoral immune reactions as so-called T helper cells through the formation and secretion of cytokines.
  • Microorganisms that, like Neisseries, infect and colonize the human host must have developed coevolutive skills to affect the host's immune system.
  • the regulation of the specific immune response appears to be of particular importance. There are some interesting examples of the manipulation of this regulation. For example, mycobacteria inhibit antigen processing or influence staphylococci and salmonella in antigen presentation; in both cases, this ultimately leads to a reduced T cell response (Marrack and Kappler, 1994; Pryjma et al., 1994).
  • the monocytes were also partially removed by plastic adherence (1 hour, 37 ° C, 5% CO 2 , serum-free).
  • the so-called non-adherent cells (NAZ) contained less than 2% monocytes.
  • MNZ and NAZ were on a concentra tion 1 x 10 6 cells / ml and preincubated under serum-free conditions with the ⁇ -protein (5 ⁇ g / ml in RPMI) at 37 ° C. The incubation of the cells with cell culture medium without ⁇ -protein was used in parallel as a control.
  • the cells were then further cultivated in flat-bottom microtiter plates (0.5 x 10 5 c / well; volume 200 ⁇ l) with RPMI + 10% heat-inactivated fetal calf serum.
  • the T cells were stimulated with the lectin PHA (1 ⁇ g / ml) or the antigen tuberculin, PPD (10 ⁇ g / ml), which guarantees a specific and strong reaction after 4 days of culture under the culture conditions specified below (Lorenzen, 1992 ).
  • the cells were labeled with 3 H-thymidine (0.2 mCi / well), harvested 18 hours later and measured using the liquid scintillation counter in the LKB-beta counter. The evaluation of this experiment is shown in Table V.
  • PPD Purified Protein Derivative
  • PHA phytohemagglutinin
  • the proliferation can be determined after 4 days of culture by incorporating tritiated thymidine and given as cpm x 1000
  • the results are averages calculated from triplicate determinations.
  • PPD-induced T cell proliferation was significantly reduced in both approaches after treatment with ⁇ -protein, while polyclonal activation with PHA was not significantly inhibited.
  • This experiment shows that the neisserial ⁇ protein not only has biochemical and cell biological similarities to the Tat protein, but also has immunophysiologically similar properties.
  • the similar effect on T cells, in which the proliferation of antigen-specific T cells is selectively inhibited suggests that this biological effect may be due to the nuclear transport found for both proteins within the eukaryotic cells (see Example 7).
  • Such suppression of certain antigen-specific T cell clones could, for example, by disrupting suppressive T cells or certain cytokine-producing T cells, disrupt the balance of the immunological network and ultimately also contribute to the development and maintenance of autoimmune diseases such as RA.
  • the known substrates of the IgA protease are human immunoglobulin A and the precursor protein of the IgA protease itself. This means that the IgA protease exhibits an auto-catalytic activity in order to cut out the various functional units from the IPP.
  • the amino acid sequences recognized and cut can be determined from the cleavage products obtained (Klauser et al., 1993; Plaut et al., 1975). They can be simply described using the scheme YP-
  • the hydrolysis of the peptide bond by the IgA protease takes place within this linear recognition sequence, the presence of this sequence being sufficient for a specific cleavage by the enzyme (Pohlner et al., 1992).
  • the known and realized variations of the recognition sequence are shown in Table VI.
  • the known substrates of the IgA protease are human immunoglobulin A and the respective IgA protease precursor proteins themselves.
  • the combination of the cleaved sequences results in the generally applicable cleavage motif of the IgA protease "YP
  • the sequence data obtained by computer-aided comparisons show that a proportion of 7 percent of all human proteins carry a potential IgA protease interface, a selection of interesting examples being shown in FIG. 9.
  • the surprisingly high number of human proteins found with IgA protease cleavage sites are transcription factors or cellular receptors. It seems plausible that the activity of the IgA protease in the host organism cleaves an extracellular receptor component and that correct signal detection is no longer possible. On the other hand, an intracellular Re share of the signal can be prevented.
  • the IgA protease cleavage sites found in human proteins through database comparisons are actually recognized and cut by the enzyme, as was shown by the leukocyte surface marker CD8 (Pohlner et al., 1992).
  • CD8 leukocyte surface marker
  • a 205 amino acid fragment of CD8 which contained the potential IgA protease cleavage site (FIG. 9) was fused with an N-terminal part of the MS2 polymerase and was converted into E. coli expressed.
  • the purified fusion product was cleaved by the IgA protease.
  • N-terminal peptide sequencing showed that the IgA protease sequence had been recognized and hydrolyzed within the CD8 region as predicted.
  • An MHC class II antigen thus consists of a processed peptide with flanking proline residues, which contains a central core epitope with defined hydrophobic anchor positions (FIG. 10).
  • the ability to carry a proline in the second position is not limited to peptides presented in MHC class II.
  • a number of peptides which are associated with various MHC class I alleles also show an increased occurrence of proline at position 2 (Engelhard, 1994).
  • IgA protease allows host proteins to be processed in such a way that they are suitable for presentation by MHC class II.
  • These self-peptides which arise as a result of the enzymatic activity of the IgA protease, are recognized as foreign due to a lack of tolerance which was not previously necessary and a T-cell reaction against the body's own proteins occurs.
  • the IgA protease is formed as part of a precursor molecule (IPP).
  • IPP precursor molecule
  • the translocation through the outer membrane takes place with the help of the C-terminal part of the precursor, the ⁇ -core. While the IgA protease splits off autocatalytically as a polyprotein and matures further extracellularly, the ⁇ core remains in the membrane (Pohlner et al., 1987).
  • Cysteine, serine, metallo and aspartyl proteases are assigned to the four classes mentioned. What is striking for aspartyl proteases is the small number of conserved residues which are necessary in order to be catalytically active (Miller et al., 1989).
  • a sequence analysis of the ⁇ core with various viral enzymes shows a high degree of homology in the area mentioned above (FIG. 7). Even the distance to another conserved area, probably a part that contributes to the structure formation, is exactly the same. The high variability outside the conserved areas is also remarkable. A large number of non-identical / non-similar amino acid residues also occur here in closely related viruses such as HIV-1 and HIV-2.
  • the ⁇ -core has sufficient sequence information to represent a functional and membrane-bound aspartyl protease.
  • the family of viral aspartyl proteases has another thing in common. This is the non-specific recognition sequence that is cleaved (Dougherty et al. 1993). So that not all proteins in the environment are cleaved uncontrollably, intracellular changes can be a mechanism for regulation. Aspartyl proteases have a pH optimum in the acidic range. In phagocytes, the effects of a bacterial aspartyl protease are likely to develop fully and contribute to the survival of the intracellular bacterium.
  • peptides interacting with the protease can have altered antigenic properties which, in turn - as presented by MHC - lead to an autoreactive immune system (Anomasin et al., 1990).
  • Lentiviruses are a separate family within retroviruses. Lentiviruses include HIV, HTLV and animal pathogenic species, which also cause immune deficiency diseases.
  • the genome of the lentiviruses can be divided into three coding areas: gag codes the structural pro teine of the inner envelope, env the glycoproteins of the outer membrane and pol enzymes which are necessary for virus replication, such as the reverse transcriptase, endonuclease and a protease.
  • tat gene which can differ in the individual viruses in this group. The transcription of viral genes is activated by the tat gene product, the tat protein.
  • the directional transport into the nucleus is ensured by the core localization sequence.
  • the binding of the Tat protein to the TAR (transactivation responsive elements) within the LTR (long terminal repeats) of the virus DNA activates the transcription, which ultimately leads to the removal of infectious virus particles.
  • ⁇ protein as a transcription factor is investigated using a eukaryotic cell system.
  • This Cells carry a gene construct consisting of LTR from HIV or other retroviruses in connection with a reporter gene, such as CAT or luciferase. If the ⁇ -protein gets into the nucleus of these cells and the LTR are activated, this leads to the expression of chloramphenicol acetyltransferase (CAT), the activity of which is specifically detectable.
  • CAT chloramphenicol acetyltransferase
  • ERSs Activity of the ⁇ protein as a transcription factor could prove to be important with regard to the activation of ERSs.
  • the eukaryotic genome normally carries between 5 and 10% so-called retrotransposable ERSs (endogenous retroviral sequences). These ERSs are an important factor in the reorganization of the eukaryotic genome, but it is unknown how these retroviral elements got into the eukaryotic DNA. At least it is certain that human ERSs per se are defective proviral elements that are no longer able to produce viruses. Overall, however, these elements represent a large reservoir of viral genes that are caused by mutations or recombinations with exogenous retroviruses or other factors such as e.g.
  • HRES is an endogenous retroviral element similar to HTLV, which contains functional transcription elements, including LTR, and is thus capable of protein expression (Banki et al., 1992).
  • autoimmune diseases such as B. Multiple sclerosis, SJörgen's syndrome and lupus erythematosus were found to have auto-antibodies to HRES, i.e. HRES can act as an autoantigen.
  • ERSs encode so-called superantigens and can thereby trigger autoimmune responses (Krieg et al., 1990). Regardless of the antigenic specificity of a T cell, superantigens are able to bind to the V ⁇ chain of the TCR and to the MHC II complex of an antigen presenting cell and thus trigger an immune reaction.
  • Patients with RA have been reported in the Synovial fluid detected the expansion of V ⁇ - specific T cells. This expansion of V ⁇ -specific T cells is believed to have been triggered by superantigens (Paliard et al., 1991). New evidence for the pathogenesis of rheumatoid arthritis was provided by electron microscopic examinations of synovial cells. Previously unknown retrovirus-like particles were found here. An explanation for this would be the LTR-dependent activation of endogenous retroviral elements, which could lead to the expression of virus-like particles (Keyszer et al., 1994).
  • the function of the ⁇ protein is therefore interesting not only with regard to the LTR activation of virus-infected cells, but also with regard to the possible function as a transcription factor of ERSs, which can be responsible for the development of autoimmune diseases. Since approx. 50% of the population repeatedly asymptomatic carriers of N. meningitidis, and therefore exposure to ⁇ -protein is present, the activating function of the ⁇ -protein on the LTR of endogenous retroviral elements could indicate a connection to common autoimmune diseases such as RA.
  • the first contact of human-pathogenic Neisseries with the host cell takes place via surface structures of the bacterial cell, the pili.
  • the close adhesion of the bacterial cell to the host cell membrane, which ultimately leads to invasion into the host cell, is mediated on the bacterial side via opa proteins of the outer membrane.
  • grandpa proteins play a central role in the expression of tissue specificity (Kupsch et al., 1993).
  • the specific adhesion to epithelial cells is mediated by Opa 50 , the proteins Opa 51-60, however, are responsible for the adhesion to peripheral blood lymphocytes.
  • About the receptors on Sei Little is known about the host cells and the molecular mechanisms of this cell specificity of the Neisseries.
  • the opa-mediated adhesion and invasion to epithelial cells was inhibited by means of certain proteolytic enzymes and inhibitors.
  • the epithelial cells were treated with Neisseries and the following additives before infection:
  • the cellular receptor for grandpa-mediated adhesion is a membrane-bound heparan sulfate proteoglycan.
  • Cell surface proteoglycans are a large family of highly polymorphic glycoproteins, which are divided into different groups according to the respective core protein, the composition of the glycosaminoglycan side chains attached to the core and the anchorage in the cell membrane: the syndecans, beta-glycans and CD-44 (Bernfield et al., 1992).
  • Syndecans are characterized by a core protein that consists of a large extracellular domain with a large number of possible glycosaminoglycan binding sites, a highly conserved, hydrophobic transmembrane domain and a short carboxy-terminal cytoplasmic part.
  • the interaction of bacterial Opa 50 protein and cellular Syndekan-1 thus plays a key role in the course of the infection.
  • This specific interaction between the pathogenic organism and its host cell offers different approaches to drug development.
  • parts of the Opa 50 protein can be used as vaccines, which lead to the formation of specific antibodies against the adhesive domain of Opa 50 , thus preventing the adhesion and invasion of Neisseries, and thereby protecting them from infection.
  • drugs for Neisseries infections are not only important with regard to primary symptoms of the disease, but especially with regard to secondary late damage, such as the development of autoimmune diseases; i.e. Vaccine against Neisserei could also vaccine against certain autoimmune diseases such as. B. mean rheumatoid arthritis.
  • T cell clones are reactivated by superantigens
  • such cross-reactive T cell populations are also stimulated to multiply, exceed a critical number and acquire the ability to develop their pathogenic potential.
  • peptide-specific T cells in the peripheral blood of healthy donors expressing the corresponding MHC haplotype should be detected earlier, before the actual manifestation of the disease.
  • These T cells are then stimulated in vitro by the antigens for proliferation, similar to the T cells of the RA patients (Example 6).
  • the proliferation is measured analogously to the conditions under Example 6 by means of 3 H-thymidine incorporation during the DNA synthesis.
  • Retroviruses a number of functional proteins are formed by cleaving a precursor protein, the ver The protease responsible is a component of the precursor polyprotein (Kösslich et al., 1989). So there is a structural similarity between retroviral elements and the IgA protease precursor. In order to demonstrate similarities also at the level of the primary structure, sequence comparisons of the IPP with gene products of retroviruses were carried out using the Wisconsin Sequence Analysis Package from GCG (Genetics Computer Group Inc.). For this purpose, a database called Retrovirus was first created, in which the amino acid sequences of the retroviral elements previously stored in the Swissprot database were merged.
  • sequence homology was found between the amino acid sequence of the 7-peptide and a Nef protein of HIV-1 (accession number: P04603). Here 36% of 25 amino acids were conserved identically and 44% with similar properties were preserved, which resulted in a total similarity of these sequence segments of 80%. Furthermore, sequence homologies between the ⁇ -core and the Env protein of various retroviruses were found, but also as shown in Example 11 for the retroviral aspartyl protease. The ⁇ -protein showed homologies to various polyprotein precursors and to the Tat protein, also beyond the range of the nuclear translation signal. The 7-peptide also showed homologies to a number of NEF proteins and a polyprotein from
  • Grapewine Fanleaf Virus A characteristic of almost all found sequence homologies of IPP with retroviral proteins was that the conserved identical amino acids were in areas that are locally concentrated and additionally have a high number of similar amino acids. Thus, sequence sections of high similarity could be found, which were surrounded by areas without sequence matches. Such a clustering of the sequence homologies could be explained by an origin or a change in the IPP as a result of recombination with retroviral elements.
  • ⁇ -protein An important functional activity of the ⁇ -protein can be derived from the following experiment.
  • cotransfection studies in which one plasmid encodes the ⁇ protein and the second plasmid contains the neomycin resistance marker, an effect of the ⁇ protein on the transcription level was observed for the first time.
  • TK mouse L
  • all clones selected for antibiotic resistance were also ⁇ -protein positive (Langenberg, 1993).
  • the ⁇ -protein coding plasmid was co-transfected with the selection marker plasmid only in the same amount (1-5 mg Quiagen DNA), each neomycin-resistant clone was also ⁇ -protein positive.
  • stage VI oocytes are used, which have matured until the first meiotic division has started.
  • stage VI oocytes are used, which have matured until the first meiotic division has started.
  • a fully grown egg cell has stopped at the boundary between G2 and M phase in the cell cycle (Ford, 1985).
  • hormones for example progesterone
  • meiosis I proceeds to the metaphase of meiosis II, in which the egg cell then remains until fertilization (Merriam, 1971).
  • stage VI oocytes led to the following phenotypic changes in stage VI oocytes (Rauber, 1991). About 3 hours after the injections, the oocytes got a visible, bright spot in the dark, animal pole. After a longer incubation, their characteristic light-dark coloration changed to a stain or stripe pattern and finally they died. At the same time, one could see that after three hours of incubation there was no sharp separation between nucleus and cytoplasm; instead, the nucleus had a changed oval to elongated shape and was located on the inside of the cytoplasmic membrane in the dark pole.
  • the injections of IGA protease and protease inhibitor molecules in oocytes of the clawed frog were carried out as follows: The ovary, or only parts of it, depending on the amount of the oocytes required, was in MBS-H buffer (88 mM NaCl, 1 mM KCl, 2, 4 mM NaHCO 3 , 0.8 mM MgSO 4 , 0.33 mM Ca (NO 3 ) 2, 0.41 mM CaCl 2 , 10 mM Hepes pH 7.4, supplemented with 10 mg / ml benzylpenecillin and streptomycin), washed with crushed two forceps and incubated by adding collagenase (2 mg / ml) until more than 90% of the oocytes were isolated.
  • MBS-H buffer 88 mM NaCl, 1 mM KCl, 2, 4 mM NaHCO 3 , 0.8 mM MgSO 4 , 0.33 m
  • the oocytes were then washed again in MBS-H buffer to remove the collagenase. All injection experiments were carried out on a tempered steel plate (20 ° C).
  • the injection apparatus consisted of a Hamilton syringe which could be operated with a constant feed via a Secura perfusor and an extended glass capillary which was fitted in a micromanipulator.
  • the capillary was connected to the syringe via a Teflon tube, which was filled with an inert oil for pressure transmission.
  • stage VI oocytes were aligned in the wells of a glass plate under MBS-H buffer so that they could be injected into the light pole without damaging the core in the dark half.
  • Human nasopharyngeal tissue is another target tissue for the specific uptake and nuclear localization of the alpha protein.
  • the alpha protein secreted by Neisserien has functional core localization sequences. If the alpha protein is expressed by transfected cells, it accumulates specifically in the nucleus. In addition, however, the alpha protein is able to penetrate cells from outside and then accumulate in the nucleus. However, this only happens in very specific cells of primary cultures of certain human tissues. It is striking that these are tissues that are also target tissues for infection with Neisseries. As described in Example 7, a certain "subset" of cells from primary cell cultures of human cornea and a certain "subset" of human mononuclear blood cells show external uptake and nuclear localization of the alpha protein. Another target tissue is tissue from the human nasopharyngeal area.
  • Surgical removal of the nasal polyps which are used to remove the natural microflora, primarily with a medium containing antibiotics (DMEM, 250 E / ml penicillin / streptomycin, 5 ⁇ g / ml fungi zone, 25 mM Hepes, 100 ⁇ g / ml gentamycin, 50 mg / ml nystatin).
  • DMEM 250 E / ml penicillin / streptomycin, 5 ⁇ g / ml fungi zone, 25 mM Hepes, 100 ⁇ g / ml gentamycin, 50 mg / ml nystatin.
  • the tissue in this medium is freed from attached fat and connective tissue and then broken down into pieces of approximately 2 mm 2 . Then fresh medium is added and the tissue pieces are incubated at 37 ° C. overnight. The following day, this medium is replaced by the addition of fresh antibiotic medium to which 10% FCS has been added.
  • the antibiotic medium is removed and the tissue pieces are placed individually on microscopic cover glasses in tissue culture plates with 24 wells. As soon as the tissue pieces have settled (after 5 hours until overnight at 37 ° C in the incubator), Culture medium (DMEM / Ham's F12 1: 1, 10% FCS, 1% penicillin / strepomycin, 1% fungizone, 2.5% Hepes, 0.4% methyl cellulose) was added. These cultures are incubated approx. 1-2 weeks with changing media twice a week. If at least 20% of the cover glasses are covered with cells, they are used for the experiments. The detection of the alpha protein uptake and nuclear localization is then carried out as described in Example 7 in the case of the primary culture from cornea.
  • DMEM / Ham's F12 1 1, 10% FCS, 1% penicillin / strepomycin, 1% fungizone, 2.5% Hepes, 0.4% methyl cellulose
  • the alpha protein is taken up by many cells, but then degraded in vesicles. In a few cells of certain tissues, the specific uptake leads to accumulation in the nucleus, which suggests a specific uptake mechanism that depends on a very specific cell type / status.
  • Peptide B from R16
  • the frames represent the core localization sequences.
  • Peptide A from MS11, without core localization sequence
  • ⁇ no binding to cells or matrix Peptide C from MSII, nuclear localization sequence No. 4
  • Peptide B from R16, nuclear localization sequences No. 1 and 2
  • Peptide D from R16, core localization sequence No. 3
  • the alpha protein only penetrates into the nucleus in certain cells of certain tissues.
  • This specific function can be used to selectively introduce either DNA, proteins or other substances into these cells via coupling with the alpha protein or with individual peptides from the alpha protein.
  • a peptide from the alpha protein (see Example 22) is coupled with polylysine.
  • DNA binds very effectively to polylysine (Wagner et al., 1990).
  • the vector pSV3neo (Berg, 1982), which encodes the SV40 Large T region, is specifically introduced into nuclear localization-positive cells. The expression of the SV40 Large T leads to the immortalization of the cells and thus to the generation of a cell line.

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Abstract

La présente invention concerne des médicaments et des agents de diagnostic, ainsi que des procédés diagnostiques, thérapeutiques et prophylactiques pour le traitement de maladies auto-immunes et virales chez l'homme, lesdites maladies pouvant être causées par des bactéries pathogènes. Ces bactéries sont, en particulier, des micro-organismes qui colonisent les muqueuses de l'homme et sécrètent certaines exoprotéines présentant des similitudes structurelles avec les protéines humaines. Les protéines similaires à la protéase IgA, qui sont particulièrement bien absorbées par les cellules et se présentent sous forme d'antigènes sur les molécules de CMH, ont une importance particulière dans ce contexte. La polyprotéine de la protéase IgA (IPP) de neisseria pathogènes présente une homologie particulièrement marquée avec les protéines humaines. Par exemple, un peptide déterminé de l'IPP présente une homologie marquée avec les protéines articulaires de liaison et Aggrekan. Il est donc prouvé, par exemple, que l'IPP est un agent étiologique de la polyarthrite rhumatoïde (RA). La réaction auto-immune est en outre favorisée par d'autres propriétés des neisseria produisant l'IPP et des IPP. L'aptitude de l'IPP à activer les virus et les éléments viraux constitue une propriété importante. Cela concerne en particulier l'activation de rétrovirus proviraux et de rétrovirus endogènes chez l'homme. D'autres réactions auto-immunes chez l'homme sont induites par l'activation de virus et d'éléments viraux. En outre, le développement du syndrome d'immunodéficience acquise (SIDA) s'explique également par l'activité du provirus VIH dépendant de l'IPP. Les propriétés, décrites dans l'invention, des neisseria pathogènes et des IPP ainsi formées, permettent de développer de nombreux procédés diagnostiques, thérapeutiques et prophylactiques nouveaux.
PCT/EP1995/003726 1994-09-21 1995-09-21 Medicament destine a la prophylaxie et au traitement de maladies auto-immunes et virales, agents de diagnostic pour le depistage desdites maladies WO1996009395A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU36515/95A AU3651595A (en) 1994-09-21 1995-09-21 Drug for the prevention and treatment of auto-immune and viral diseases, and diagnostic agents for detecting said diseases

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DEP4433708.6 1994-09-21
DE4433708 1994-09-21

Publications (2)

Publication Number Publication Date
WO1996009395A2 true WO1996009395A2 (fr) 1996-03-28
WO1996009395A3 WO1996009395A3 (fr) 1996-07-18

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PCT/EP1995/003726 WO1996009395A2 (fr) 1994-09-21 1995-09-21 Medicament destine a la prophylaxie et au traitement de maladies auto-immunes et virales, agents de diagnostic pour le depistage desdites maladies

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Country Link
AU (1) AU3651595A (fr)
WO (1) WO1996009395A2 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7667015B2 (en) 2001-04-10 2010-02-23 Agensys, Inc. Nucleic acid and corresponding protein entitled 151P3D4 useful in treatment and detection of cancer

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3622221A1 (de) * 1986-07-02 1988-01-14 Max Planck Gesellschaft Verfahren zur gentechnologischen gewinnung von proteinen unter verwendung gramnegativer wirtszellen
DK130889A (da) * 1989-03-17 1990-09-18 Mogens Kilian Immunoglobulin a1-proteaser (iga1-proteaser), fremgangsmaade til genteknologisk fremstilling af saadanne enzymer samt vaccine indeholdende enzymerne og fragmenter deraf til immunisering mod bakteriel meningitis og andre sygdomme fremkaldt af iga1-protease-producerende bakterier
NZ236819A (en) * 1990-02-03 1993-07-27 Max Planck Gesellschaft Enzymatic cleavage of fusion proteins; fusion proteins; recombinant dna and pharmaceutical compositions
DE4140699A1 (de) * 1991-01-11 1992-07-16 Boehringer Mannheim Gmbh Rekombinante iga-protease
WO1992013871A1 (fr) * 1991-01-31 1992-08-20 Washington University Polypeptides et polynucleotides utiles pour le diagnostic et le traitement de neisseria pathogene
JP3213313B2 (ja) * 1991-03-14 2001-10-02 イムクローン システムズ インコーポレーテッド 組み換えハイブリッドポーリンエピトープ
DK0625043T3 (da) * 1992-09-16 2002-04-22 Univ Tennessee Res Corp Vaccine med rekombinant multivalent M-protein

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7667015B2 (en) 2001-04-10 2010-02-23 Agensys, Inc. Nucleic acid and corresponding protein entitled 151P3D4 useful in treatment and detection of cancer
US7960527B2 (en) 2001-04-10 2011-06-14 Agensys, Inc. Nucleic acid and corresponding protein entitled 151P3D4 useful in treatment and detection of cancer

Also Published As

Publication number Publication date
AU3651595A (en) 1996-04-09
WO1996009395A3 (fr) 1996-07-18

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