DE19934029A1 - Preparing an agent for diagnosis or control of microbial infection, useful particularly against Helicobacter, based on identification of essential genes in defective mutants - Google Patents

Abstract

Preparation of an agent (A) for detection, prevention and/or treatment of microbial infection by: (i) identifying essential genes (I) and corresponding polypeptides (II); (ii) identifying compounds that are directed against (II) and inactivate the microbe; (iii) testing these for suitability for use; and (iv) formulating selected (A). Identifying essential genes (I) comprises preparation of gene-deficient microorganisms by conditional antisense inhibition (CAI) and/or subtractive recombination mutagenesis (SRM), then determining viability and/or survival of the deficient organisms. Independent claims are also included for the following: (a) method for identifying essential microbial genes; (b) nucleic acid (Ia) representing essential secretory genes of Helicobacter, identified by method (a); (c) a gene bank containing at least two (Ia); (d) a vector containing at least one (Ia) or its fragment; (e) cells containing (Ia) or the vector of (d); (f) a mutant bank containing at least two microorganisms transformed with the vectors of (d); (g) a polypeptide (IIa) encoded by (Ia) and their immunogenic fragments; (h) inhibitors (A') that bind specifically to (IIa), or its fragments, and/or modulates its expression, presentation and/or native function; (i) method for preparing (IIa) or its fragments; (j) antibodies (Ab), or their fragments, that are specific for (IIa); and (k) a pharmaceutical composition containing one of (Ia), the vector of (d), the cells of (e), (IIa), Ab or its fragments, or (A).

Description

The present invention relates to a method for identification and Characterization of essential genes of pathogenic microorganisms whose Use for finding new immunological and pharmacological Active ingredients for the prophylaxis, therapy and diagnosis of bacterial infections, as well as the further development and optimization of these active substances. Of the Included in the invention are the corresponding nucleic acids which encode and encode the essential gene products Polypeptides. In addition, the invention relates to vectors containing the Nucleic acids according to the invention contain, with these vectors transformed cells and antibodies specific for the polypeptides. These Nucleic acids and polypeptides can be used for diagnosis, prevention and Treatment of microbial infections are used, in particular They can be used to develop antibodies, vaccines and inhibitors be used.

The complete molecular development of the human genome and clinically relevant pathogens opens new avenues in the development of Therapeutics or prophylactics against human diseases. So is the decoding of the human genome for the next few years announced. The number of molecularly fully characterized pathogenic Germs is constantly increasing. The stated goal is now, from the extensive data to identify such genes, their products as a potential target for an active substance and thus for the Development of a specific drug can be used. This  Potential can not be derived from the primary structure of a gene, but must be determined experimentally.

If the complete genomic sequence of an organism is present, one stands before the problem, the enormous amount of data for advanced biological To make analyzes available. The first step is identification all genes on the genome. This usually happens with help Computer-aided search programs that with a certain security can predict potential genes. In this way, genetic maps can be created, however, still with a large inaccuracy afflicted are. Can not identify the genes identified by the search program assigned to known gene must have the functionality of this hypothetical genes by the physical detection of gene products be detected in the original cell.

Another strategy, also on the application of special Is based on the identification of possible gene families aligned with specific biological properties, which in turn can be considered as an active ingredient target due to further assumptions come. The search criteria are based on characteristic structural features aligned, which are usually derived from already known genes were. The result of such a search can vary depending on the Approach of the requirements to the real state, a high hit rate deliver. In general, however, the inaccuracy of these methods is relative high, and the real biological property of the gene or its Gene product must be confirmed experimentally in any case.

Another strategy involves detecting the expression products of a cell the genes that are active for the respective development state are identified can. If you compare different states of development, can be derived in this way the interaction of the genes and In some cases, the biological function of unknown genes may be partial  be decrypted. If one carries out appropriate comparative investigations It is with cells that have a pathological appearance even possible to identify disease - causing genes and to use these as potential drug targets for drug development.

All the methods described are used in particular to date, unknown Identify genes and make them more computer aided Data comparisons to assign a biological function. One, unique Evaluation of a gene or gene product in terms of its potential as To serve drug target and thus for the development of active ingredients to be able to be used, but does not meet any of the known Method.

Some of the most important requirements for a pathogenic organism, To survive in a host and to multiply, on the one hand are the Ability to escape the host's immune system, and on the other hand the Ability to adapt to a very specific habitat or niche. The responsible factors and proteins are therefore usually essential for the pathogenic germ.

It would be of great advantage, these essential genes of microorganisms to identify in this way the possibility of producing therapeutic, preventive and / or diagnostic agents, e.g. B. Antibodies, vaccines or inhibitors of the corresponding polypeptides to get.

A pathogen of particular medical interest is Helicobacter pylori. This germ is a Gram-negative, spiral bacterium with high pathogenic potential, which has intensified in recent years Resistance to a range of therapeutically relevant antibiotics has developed and is therefore of great clinical importance. It draws by extremely high mobility due to its flagella and the  unusual ability in the strongly acidic environment (up to pH 1.5) of the stomach survival (Goodwin et al., 1989).

Although the occurrence of spiral bacteria in the human Gastric mucosa has long been known, one knows only since successful isolation and cultivation of this bacterium (Warren and Marshall 1983; Marshall et al., 1984) from the gastric mucosa of a Patients with a stomach ulcer (gastric ulcer) that this is are pathogenic germs. H. pylori infection is one of the most common chronic bacterial infections of humans. It occurs worldwide, About 50% of the population are infected with this bacterium.

An infection inevitably leads to the triggering of a bacterial gastritis (Type B gastritis) in humans. Furthermore, it is believed that H. pylori also a causative role in the development of gastric and Duodenal ulcers (gastric ulcer and duodenal ulcer) as well as at some forms of gastric carcinoma (adenocarcinoma) (Lee et al., 1993; Solnick and Tompkins, 1993). In two studies of 1991, one statistically significant correlation between H. pylori infection and the Occurrence of gastric carcinoma (intestinal type) shown, both Studies came to the conclusion that about 60% of all occurring Gastric carcinomas probably due to H. pylori infection (Parsonnet et al., 1991; Nomura et al., 1991). Also the rarer Occurring MALT (Mucosa Associated Lymphoid Tissue) Lymphomas of the Magens, as precursors of B-cell tumors of the immune system are believed to be a consequence of H. pylori infection. A Consequence of the long-term infection with H. pylori is the atrophic gastritis, one Degeneration of the mucus, acid or pepsin producing cells of the Gastric epithelium, which must be considered as a precancerous lesion.

After oral intake, the bacteria first get into the extreme acidic gastric lumen (pH 1 to 2). There will be through the production of the enzyme  Urease, the cleavage of the existing urea and thus the local Neutralization of acidic gastric pH results in survival allows the bacteria. By means of chemotactic orientation and flagella-dependent motility then move the germs in the Bicarbonate-buffered mucus layer of the antral region of the stomach, their actual natural habitat. There they are in a unique ecological niche, due to the acid barrier only a few Competing bacterial species is accessible. Presumably, the orient themselves Bacteria at the pH gradient between lumens (pH 1-2) and Epithelial surface (pH 6-7) to reach the epithelium. Through her spiral shape, its mobility in viscous mucus, the production of Mucus-modifying enzymes and finally by a microaerophile Lifestyle, these germs are optimal to the living conditions in this Habitat adapted. They usually stay in the deep crypts of the antrum. Region where they are protected from external influences such as As acid, pepsin but also before drugs for their eradication, such as. As antibiotics are protected. Part of the bacterial population (about 20%) is closely associated with epithelial cells associated, especially with mucus-producing cells. Under the Condition of gastric metaplasia, d. H. the acid-induced Formation of gastral epithelium in the duodenum, it also comes to Colonization of metaplastic areas in the duodenum, causing the Conditions for the development of the duodenal ulcer (Ulcus duodeni) are created. Her ability to adhere to it probably will a complete elimination of the Hellcobacter with the rejected Prevents mucus, so that the bacteria for years, decades or even can persist for life (chronic infection).

Before the existence and importance of H. pylori for the ulcer diseases were known, they were treated by so-called antacids, or H ₂ receptor antagonists. These are substances which inhibit acid secretion of the gastric parietal cell. Although under the influence of these drugs it is usually the healing of ulcers, but since one of the causes of these ulcers, namely the H. pylori infection, so that is not eliminated, it comes in most cases after a short time to a recurrence of ulceration (relapse).

Another therapy commonly used in ulcerations is the bismuth Treatment. Various bismuth salts (CBS, BSS) have one bactericidal effect on H. pylori. A significant disadvantage of this Therapy form is, however, that a total eradication of the germ only in one very low percentage of cases is reached (8 to 32%). As with the Treatment with antacids is only temporary Suppression of the germ, and after discontinuation of treatment takes place in the In most cases, a flare-up of the infection again. Another disadvantage the bismuth treatment is that a longer lasting therapy with high Doses to accumulate this substance in the liver, kidney and the Nervous system leads and has considerable neurological side effects (Malfertheiner, 1994).

Since the realization that it is at the gastroduodenal Ulcer diseases are infectious diseases, are the Treatment now also used antibiotics. The monotherapy with various antibiotics (amoxicillin, nitrofuran, furazolidine, erythromycin and the like) turned out to be unsatisfactory since it Here, too, only 0 to 15% of the cells for complete eradication of Germs is coming. The hitherto most successful treatment is currently by a Combination of an acid blocker (ompeprazole) with an antibiotic (Amoxicillin), which can lead to eradication rates up to 80% (Malfertheiner, 1994). In the long run is an antibiotic treatment for However, elimination of H. pylori is not promising because of the incomplete eradication of the germ with a rapid resistance development of bacteria against antibiotics must be expected.  

The increasing incidence of antibiotic resistance and the limited treatment options, which are usually considerable have unwanted side effects, makes finding new ones Therapy forms and in particular the identification of new active ingredients necessary, especially vaccines, both prophylactic, as well therapeutic treatment used by Hellcobacter infections can be. Of particular interest is also the dosage form, since the active ingredient in the stomach, d. H. effective in an extremely acidic environment have to be. Compounds with proton blockers, the z. B. before Administration of the prophylactic or therapeutic agent can be of great benefit.

The molecular basis for persistent, chronic Hellcobacter Infections are not yet clear. It could be shown that the factors urease, motility and adherence are essential properties of the bacterium are able to colonize the gastric mucosa. Although the host organism is unable under normal conditions is to cope with H. pylori infection showed up in the animal model, that urease, an essential virulence factor of H. pylori, is a high Has potential as a vaccine (U.S. Patent Application US-SN-07 / 970,006 "Urease-based Vaccine Against Helicobacter Infection").

However, those components that are responsible for that Pathogens that can bypass the immune system of the host are so far unknown.

Pathogenic organisms in general have a variety of strategies developed in the host over a long period of time by the immune system to persist unmolested (Haas and Göbel, 1992, Finlay and Falkow, 1997). A mechanism for survival in hostile Milieu serves, is the training of a Überdauerungsform.  

In the case of H. pylori cocoa forms have been described several times in the literature as potential forms of survival, but their clinical significance is controversial. Kokkoide forms could play a major role in ex vivo survival. With regard to the in vivo persistence, it has been shown that cocoa forms are preferentially affected by an unfavorable environment, such as e.g. High partial pressure of O ₂ or sublethal antibiotics (bismuth subcitrate, erythromycin, amoxicillin, metronidazole) (Donelli et al., 1998; Bode et al., 1993; Sorberg et al., 1996; Berry et al al., 1995).

Some researchers assume that these cocoa bacteria viable, but not cultivable (VNC, viable but non-culturable). Eaton and co-workers received a successful infection of mini- Pork with vegetative (spiral) H. pylori, while cocoa Forms in this model did not show any infection (Eaton et al., 1995). The Direct evidence of cocoa forms in the human stomach has been reported by Chan et al. Based on gastric tissue sections from biopsy material provided. In 82.8% (53/64) of the examined biopsy samples, the Authors detect cokoid forms of H. pylori (Chan et al, 1994). Cao et al. a monoclonal antibody became specific Detection of cokoid H. pylori used in the tissue section. Here too in addition to the vegetative forms in 100% of the antrum biopsies (9/9) H. pylori coccoid forms detected (Cao et al., 1997).

Binding to epithelial cells and the ability to signal transduction (IL-8 Induction, rearrangement of the cytoskeleton, plasminogen binding, Lactoferrin and vitronectin on the bacterial surface) appears at cocoa forms comparable to the vegetative forms to be obtained (Khin et al., 1996; Segal et al., 1996).

The above experiments indicate a significance of cokirkider Forms for the survival of Helicobacter in an unfavorable environment  out. Therefore, the identification of genes associated with the emergence of this Form and reactivation are related to the vital form for which Development of new active substances of the greatest interest.

In addition to Helicobacter pylori, other Helicobacter species can also be used Colonize the stomach of a human, such as B. H. heilmannii and H. Felis. In this regard it could be shown that also H. heilmannii with pathological ulcer disease may be associated. The causal transmission probably takes place from pets on the People instead. So far, the frequently occurring in man H. pylori is suspected to be involved in the onset of gastric cancer Role to play. There are clinical data available now Doubt the connection. Especially these doubts become more recent Data from Helicobacter heilmannii assists this one larger attach carcinogenic potential and its importance in the Emergence of gastric MALT lymphoma (Regimbeau et al., 1988).

If the above is considered in summary, it is clear that a Need for new forms of therapy for combating bacterial Pathogens, especially after vaccines and inhibitors of essential genes or their expression products. The increasing resistance development against a variety of proven Medication requires a continuous supply of new ones Agents. This increasing demand for new active ingredients can only be covered when new drug targets are identified and these are Development of new drugs are used. Essential genes represent an ideal target for drug development as they are responsible for the Survival of the pathogen are necessary.

The identification of essential genes of Hellcobacter, in particular of H. pylori or heilmannii and possible Helicobacter  Over-life forms for the development and optimization of new ones therapeutic, preventive and / or diagnostic agents, such. B. Vaccines and pharmacologically active substances is therefore an objective of In the foreground is the discovery of essential microbial Genes, including homologous proteins of various pathogenic germs can be identified. With the help of a drug could then as in the classic antibiotics several pathogenic germs simultaneously eliminated become. In Helicobacter in particular genes are in the foreground, the perform vital functions in the infection process, as well as genes that involved in the development and reactivation of cocoa forms. Of particular interest here are essential genes that are secreted for Encoding gene products as these are for immunological and pharmacological Active ingredients particularly well achieved due to their exposed localization and are therefore good candidates for drug development. Of further interest are essential genes encoding gene products, who are involved in the development and maintenance of Forms of survival are involved. Another task is finding essential microbial genes, including homologous proteins of various pathogenic germs can be identified. With the help of an active ingredient could then as in classical antibiotics several pathogenic germs be eliminated at the same time.

This object is achieved by a method for providing means for the detection, therapy or / and prevention of microbial infections comprising the following steps:

• A) Identifying essential genes and the corresponding ones Polypeptides by producing gene-deficient microorganisms conditional antisense inhibition (CAI) or / and subtractive Recombinant mutagenesis (SRM) and determination of life and Survival of the deficient microorganisms in one Test system.  
• B) Identification of specific active substances which oppose the essential polypeptides are directed and the inactivation of the Cause microorganisms or microorganisms used.
• C) Testing of the identified active substances for their applicability as Components of diagnostic, preventive and / or therapeutic agents,
• D) Formulating the applicable active ingredients as diagnostic, preventive and / or therapeutic agents.

The method presented here is concerned with the identification Essential genes and their use for the development of new drugs.

Thus, another aspect of the present invention is also a method of identifying essential microbial genes comprising the steps of:

• a) production of deficient microorganisms,
• b) determining the viability and / or survivability of the deficient microorganisms of (i),
• c) identifying a protein coding portion of a microbial DNA sequence in which the deficient microorganisms deficient are and
• d) characterization of those DNA segments essential for the Survivability are.

CAI is the abbreviation for "conditional antisense inhibition", d. H. Conditional antisense inhibition. It is a procedure which is described in detail below.

SRM stands for "subtractive recombination mutagenesis", d. H. subtractive Recombinant mutagenesis and is also described below.  

The term "yield-deficient" as used herein means that the deficient organism is unable to one or more of its Produce gene products or use their function. The production of the corresponding gene product can on the one hand by mutagenesis of the appropriate gene can be prevented, or it can be an inhibition take place during expression, e.g. B. by antisense nucleic acids. Mutagenesis can be used to generate a gene in the genome of the Microorganism to mutate, or to a mutated gene in the To introduce microorganism, whereby one is also the homologous Can make use of recombination.

A protein coding portion of a nucleic acid sequence is, for example a gene or part of a gene that expresses a polypeptide allowed.

The term "essential gene" means a gene that is responsible for a gene product without which an organism is not viable or only is limited to survive. Essential genes can be divided into two classes subdivided: obligate essential and facultatively essential genes. On obligate essential gene codes for a protein that is responsible for survival or the proliferation of an organism is indispensable under all circumstances. In contrast, an optionally essential gene codes for a protein that only under certain conditions for survival or the Propagation of the organism is necessary, such. B. the ability of Organism, within cultured mammalian cells or in the animal too to survive. In both cases, the survival or proliferation of the Organism by the inactivation of an essential gene for him or the Inhibition of an essential for him gene product greatly impaired or prevented. Is a bacterium after inactivation of a particular gene no longer viable or restricted in reproduction This is to be taken as a first indication that this gene essential properties are mediated. The significance of such  Findings, however, must be supported by accompanying control experiments be, for. For example, such a lethal mutation should be done in a second step by an appropriate complementation of the gene or gene product can be canceled. Obligat essential genes are therefore those their non-expression or nonexistence, e.g. By mutagenesis or deletion causes the organism neither in natural Environment, still on an ideal to the needs of the microorganism tuned full medium is viable. Is a microorganism in one facultative essential gene deficient, he is usually on such depending on the organism defined complete medium still capable of growth, can but in a natural environment, d. H. in its natural host or cells or tissue cultures of its natural host no longer survive.

Identify essential genes

Due to the new process can be independent of their specific function essential genes are identified by microorganisms. It is preferred this method for the identification of Helicobacter essential genes and related microorganisms used.

In a first step, the complete genome of a bacterial Pathogen with a molecular genetic approach to essential Genes searched. This substep does not require any knowledge of the Primary structure of the genome or individual genes, but takes place solely on the basis of biological criteria. Is a gene as essential Determinant identifies its identity is determined. This can be up the determined raw sequence data of the genomic sequencing be resorted to. On the basis of the determined gene sequence z. B. Isogene variants are determined or whether the gene detected in a Operon, which may contain other essential genes are located.  

In a second sub-step, the identified genes become special transferred genetic systems that serve the genes or their To supply gene products to a direct drug screening and / or the Genes or gene products used, already identified drugs continue to optimize. The main advantage of the overall process is based on the rapid succession of the gene and active substance Screenings in meaningful biological systems, so that in relative short time from the complete set of a pathogenic microorganism identifies, produces and directly addresses the potential drug targets Drug screening or optimization can be used.

If a microorganism is being studied, its genome already can be sequenced, identifying a gene or gene segment with Help database analysis done using a sequence section Reading frame is assigned. However, preferably, independently of Presence of complete genome generation any gene bank to be preselected. In this case, preferably the Vorselektion be performed on genes that are suitable for polypeptides with a encode certain function, for example with the help of Homology analysis. The preselection can also be performed on genes which are only expressed at certain stages of development.

In the first part of the step, selection steps can lead to a strong Reduction of the genetic material to be examined can be achieved. For example, by an enrichment step for genes that are exported or secreted Code gene products. In this particular procedure, the DNA Sections of a gene bank mutagenized by a pathogen, which for example, by cloning such a DNA segment into Plasmid, transformation into a preferably heterologous host organism and subsequent mutagenesis can take place. The resulting Expression product can then be detected. Mutagenesis can For example, by insertion of a marker sequence carried out in which  Expression of the mutagenized sequence in a host organism to a Fusionspolypeptid leads, which can be selected. The insertion of the Marker sequence is not limited to transposon insertion, but can be done in other ways, for example by homologous Recombination or infection and recombination with the help of Bacteriophages.

The marker sequence used in the context of the present invention is in In general, a gene encoding a gene product that has a selection allows those host organisms expressing this sequence. in the In general, these marker sequences are resistance genes, which confer resistance to certain antibiotics, or which give it to the Host organism allow to survive in a selection medium. The Genmarker preferably has no own expression signals, but is directly dependent on an upstream promoter, such. B. the Transcription promoter on the promoter segment or a promoter, the on the cloned heterologous DNA fragment to be identified. As an alternative to antibiotic resistance marker sequences, enzymes can also be used as Gene markers are used. In these cases, the successful Insertion indicated by a specific color reaction, which is the manual Isolation of the corresponding bacterial clone allowed.

When the marker sequence as a fusion protein with the expression product of the inserted DNA fragment and a selection as above DNA material can be selected from the selected Bacterial clones are isolated and the DNA sequence responsible for the Coded fusion product can be determined by known methods. This allows the assignment of a reading frame to the DNA to be identified Fragment. It is then possible to compare studies with commonly available DNA sequence databases to verify the identity of the clarify the identified gene and, where appropriate, evidence of a to achieve biological function.  

By technical and further additions of the two shown below Procedures, CAI and SRM, can be a targeted reduction of the Sample volume can be achieved. This is also about upstream selection methods aimed at specific gene groups, z. B. the use of subtractive gene banks of pathogenic and non-pathogenic Representatives. These become pathogen-promoting gene regions enriched. Such subtraction methods can also be used be used to identify specific genes for specific organisms, for example, by comparing and subtracting the genomes of H. pylori and H. heilmannii.

In further processes, for. B. gene groups are identified only be expressed in a particular developmental step. To emphasize is for example the array procedure, with which the individual gene samples of the pathogen grid-shaped on a support be applied. The individual order points are known, so that in a positive hybridization reaction with the development-specific Transcription products or cDNAs or subtractive cDNAs or Fragments thereof, the respective genes identified and subsequently cloned can be. Other methods, the developmental gene groups are comparative proteomic and differential display analyzes.

To find out if the identified gene sequences are is essential genes, microorganisms are produced which in the sequences are deficient, which are the identified gene sequences correspond. The deficient microorganisms are then on various growth media or cell cultures or in the animal model or tested in the natural host, and the deficient genes can then depending on Growth ability of a category of nonessential, obligatory be assigned to essential or facultative essential genes.  

On the importance of genes, the development of the vital in the Controlling the form of survival and vice versa is already at the beginning been pointed out. It is therefore particularly preferable to have a preselection to perform on such genes. In addition, methods such as CAI or SRM are applied and the gene-deficient microorganisms then be examined on certain nutrient media, showing the transition from one to the other form trigger. In particular, Helicobacter is the Schivo medium, which is the reactivation of the cocoa Form in the vital spiral shape allows.

The generation of deficient microorganisms can be done in several ways respectively.

There are a number of molecular genetic methods available, to mutagenize the genome of a bacterial pathogen such that Each gene has a mutant available. The most common Mutagenesis method is based on the inactivation of genes, eg. B. by random transposons in the genome, which have corresponding markers be selected. There are many variations to this process which can be applied to different organisms. (Joyce and Grindley, 1984; Akerley, et al., 1998). With the help of the advertised Transposons can also be exactly the mutagenized gene in the genome locate.

If you have a gene mutant with a detectable biological effect generated, z. B. a reduced growth of the cells in a particular Milieu, so must in a second step, the unique coupling of the gene or the gene product with this property. This usually done by complementation experiments. In this case becomes the original gene in the organism with the specific gene mutant Gene introduced and expressed. Can this original way Property of the organism to be regenerated is the necessary proof  provided. However, this method can not be used in the Apply characterization of lethal mutants, d. H. mandatory for mutants essential genes. One way out is to use conditional Mutations for complementation. For example, can be by chemical Mutagenesis of the examined gene temperature-sensitive mutants generate the gene product at the normally optimal Bring growth temperature in an inactive state form and at lower temperatures produce a biologically active gene product (Das, et al., 1976; Harris, et al., 1992; Hou, et al., 1994; Polissi and Georgopoulos, 1996). In another practiced approach, the wild-type complementations by exogenous substances, so-called Inducers, controlled. About these inducers is the expression of the The complementing gene turned on on an episome in the gene-specific mutant is introduced and after induction the missing Gene product replaced (Murphy, et al., 1995- Chow and Berg, 1988; Arigoni, et al., 1998).

The procedures mentioned are very time consuming and are only for the Examination of individual genes or limited genomic sections used. Process that provides a thorough characterization of a complete, mutagenized gene bank of a selected pathogen allow according to the scheme described are not yet known.

The new genetic methods described below, the Conditional antisense inhibition (CAI) and the subtractive ones Recombinant mutagenesis (SRM) fulfill these requirements. Both Methods can be used to identify essential genes where the CAI procedure is particularly obligatory for identification essential genes and the SRM process for facultative essential Genes.  

The CAI method relies on the conditional inhibition of translation of one or more genes on a cloned genome fragment (which then serves as a template or template) lie and on a plasmid be propagated in the germ to be examined. Compared to conventional methods remain the genomic structure of the germinating material unchanged, d. H. in original condition. Im too investigating germ is the inhibition of translation by the conditionally inducible synthesis of specific antisense RNA (asRNA) which includes the complete cloned genome fragment, including the Genes localized on the genome fragment. The antisense Nucleic acid sequences can then be present in the microorganism in large quantities be synthesized and bind to the original mRNA, these being mRNA can no longer be translated and thus the Expressing apparatus is withdrawn. The result is that either no Gene product or only small amounts thereof are formed. The synthesis the asRNA is under the control of a promoter (asPromoter), whose activity is conditional, controlled by defined, external signals becomes. This conditional inhibition of the expression of a gene or operon thus takes place via the regulation of the synthesis of asRNA by the inducible asPromoter. To prove that a gene or an operon, such as in the present case, for the survival and reproduction of the organism is essential under certain conditions, is the survival and Propagation rate of a clone in which the synthesis of asRNA is induced with its survival / proliferation rate in uninduced asRNA Synthesis compared. Is the survival / proliferation rate of the clone included Induction of asRNA synthesis diminished, it is in the inhibited gene or operon by one (obligate or facultative) essential Gene. These growth analyzes can be automated, so that a very large number of genes are investigated within a short time can be. From these clones, the plasmid is isolated and the DNA Sequence of the cloned genome fragment used as template for the asRNA  Synthesis serves, determined and in consequence the structure of the essential gene determined.

A suitable for the CAI method plasmid vector is shown in Fig. 1. It contains a genomic or subgenomic DNA fragment from the microorganism to be examined under the control of an inducible promoter (P _i ) and other customary expression signals and an mRNA stabilizing element, so that the DNA fragment expressed in the form of antisense RNA (asRNA) can be and has a long biological activity. A suitable promoter is z. B. the Tet promoter. In a particularly preferred embodiment, the CAI vector additionally encodes a gene for a regulatory protein that regulates the promoter, in this case, e.g. B. the Tet repressor, which by an exogenous or extracellular signal such. As tetracycline, can be controlled. The CAI vector of the particular embodiment of Fig. 1 further contains one or more selectable marker genes and two replication origins (ori), one for the microorganism to be examined (referred to here as a pathogen) and another for a conventional cloning z. E. coli. Using such CAI vectors antisense libraries can be created from whole microbial genomes.

Fig. 2 shows a schematic representation of a preferred CAI method. From a CAI plasmid containing small fragments of a genomic library of the micrograph to be examined, asRNA is synthesized from an inducible promoter (P _i ) under the control of an extracellular signal (see Figure 1). The asRNA hybridizes sequence-specifically with the mRNA of the gene corresponding to the cloned DNA fragment on the CAI plasmid. The formation of the asRNA mRNA hybrid reduces or prevents the translation of this mRNA. As a result, a deficient microorganism is formed which is unable to produce the gene product concerned. If it is the product of an essential gene whose formation is inhibited (A), the viability of the corresponding clone is limited or prevented. The viability of the microorganism will be determined below on the basis of its survival or multiplication rate in a defined biological system. In the case of non-induction of asRNA synthesis (B), or if the CAI plasmid contains the fragment of a non-essential gene (C), the clone of the microorganism is normally capable of living and replicating.

In particularly preferred embodiments of the CAI method, whole antisense RNA plasmid libraries are analyzed from genomic fragments of the microorganism to be examined (see FIG. 3). A genomic library with CAI plasmids (see FIG. 1) is transferred into the homologous microorganism under non-inducing conditions and the plasmid-carrying clones are selected via a plasmid-coded marker. The viability of the individual clones, each of which receives a specific CAI plasmid from the Genbank, is then examined in direct comparison with respect to its growth rate under induced or uninduced conditions (+ and - in the figure) with respect to asRNA synthesis. In clones which multiply sparingly or only slowly under asRNA-inducing conditions, the translation of at least one essential gene is prevented. From these clones, the CAI plasmids are isolated. The essential genes are identified by sequencing the genomic fragments in the isolated CAI plasmids.

This approach is also preferably combined with a subtractive method (SCAI), one embodiment of which is illustrated in FIG. 4 for illustrative purposes. A genomic library of CAI plasmids (see Figures 1 and 3) is transferred to the homologous microorganism under study, and the resulting individual clones are pooled as a bacterial CAI library. This pool is split into two identical groups (Driver and Tester Pool) for selection.

The term "driver" is used here for that pool of bacterial Clones that are treated to be the inducible promoter is activated and asRNA is expressed by the CAI vector. The "Tester" pool contains an identical set of clones with CAI plasmids, but under non-inducing conditions and thus wild-type Owns properties.

As a rule, the "Driver" pool is used for selection (eg in the animal), while the "Tester" pool is kept untreated. However, it is also possible to subject both groups to a selection, whereby only the "driver" pool is induced by administering the signal (eg tetracycline). Clones in which the expression of a particular asRNA inhibits the translation of an essential gene are lost during selection from the group. After a reasonable time, the surviving clones of both groups are recovered and the CAI plasmids are isolated from the clones of both groups. The cloned genomic fragments are then amplified by PCR, using oligonucleotide primers that hybridize with vector sequences to the side of the cloned genomic fragments. Those amplified DNA fragments representing parts of essential genes are enriched and isolated by subtractive hybridization (see Figure 8).

A promoter suitable for a CAI vector according to the invention is For example, the Tet promoter whose activity is regulated by a regulatory Proten (in this case the Tet repressor) can be controlled and by an extracellular signal (tetracycline) can be induced. Further Inducible promoters are known in the art.

Antisense RNA stabilizing elements are those skilled in the art Area known and need not be explained in detail here.  

The high efficiency of the CAI method in the inactivation of Single genes in an organism results from the overlapping Cloning of small genomic fragments and the associated Synthesis of different asRNA derivatives for a specific Gene region. In this way, the probability of getting an asRNA is increased which efficiently inhibits translation of the target gene of interest, greatly increased. Such investigations may affect the complete genome of a pathogen, which is the review of a very large number of individual genomic fragments required. Here are apparatus aids (robots) advantageous to a high To achieve sample throughput. However, in these cases, only certain states are investigated, e.g. B. the growth of cells in a particular medium.

Through the additional use of subtractive process steps (Subtractive Conditional antisense inhibition, SCAI), may increase the number of preferred individual clones are greatly reduced.

Subtractive recombination mutagenesis (SRM) is preferred for Identification of facultative essential genes used. In difference The CAI process produces permanent gene mutations, with the Enrichment of essential genes achieved via a subtractive step becomes. The SRM method can be used as the CAI procedure with genebanks of pathogenic microorganisms are carried out.

The SRM method is based on the inactivation of individual genes in the genome of a pathogen by complete insertion of a particular Suicide plasmids, which is part of a gene bank. The insertion of the Plasmids are made in the genome by homologous recombination. The successful insertion is by expression of a plasmid-encoded Antibiotic resistance marker displayed.  

A preferred embodiment of the SRM method is illustrated with reference to FIGS. 5 to 8.

FIG. 5 shows a suitable SRM vector which, like the CAI vector, contains a genomic or subgenomic DNA fragment of the microorganism to be investigated, as well as an origin of replication (ori) for a cloning host (for example E. coli), one or more selectable marker genes and another conditionally active origin of replication for the microorganism to be examined, z. Example, a temperature-sensitive origin or an origin whose activity is dependent on a present in trans replication factor and can be additionally introduced into the system. Characterized in that the SRM plasmid contains a genomic sequence of the microorganism to be examined, there is transfection of this vector in this microorganism to a homologous recombination in which the entire SRM plasmid is inserted into the genomic gene of the microorganism and the corresponding gene, if it is inactivated. This leads to an insertion mutant. Suitable inducible origins of replication are, as mentioned, temperature-sensitive oris, which can be controlled by a factor, such as. As the RGK factor pir or the pWV factor repA, which is supplied in trans to the system.

The insertion of an SRM plasmid (see FIG. 5) into the genome of the microorganism to be investigated takes place via homologous recombination between the plasmid-cloned genomic fragment of the microorganism and the complementary, genomic DNA sequence. After the plasmid has been converted into the corresponding microorganism, under non-permissive conditions, ie with inactive ori, those clones are isolated by selection on the plasmid-coded marker in which the SRM plasmid is inserted into the genome. The excision of the SRM plasmid also occurs via homologous recombination. Under permissive conditions, replication of the inserted plasmid is initiated resulting in sufficient free plasmid levels in the cells so that the plasmid can be recovered from the clone. If the insertion of an SRM plasmid into an essential gene has taken place, the viability of the clone in question is restricted (A), while mutants in nonessential genes are normally viable (B).

As with the CAI method, a library of insertion plasmids can be transferred from genomic fragments of the microorganism to be examined into this microorganism and genomic insertion mutants are formed. This preferred embodiment of the SRM process is shown in FIG . A library of SRM plasmids containing single genomic or subgenomic fragments is transferred to the homologous microorganism under study. Under conditions that do not allow plasmid replication, genomic insertion mutants are selected using a plasmid-encoded marker (see Figure 5). In this step only insertion mutants mutated in a non- or facultative essential gene can survive, since mutants of an essential gene are not viable. The individual insertion mutants are pooled and this pool is then divided into two identical groups, the driver and the tester pool. The driver pool is selected, eg. B. by the infection of an animal. The tester pool remains untreated. Selection removes clones from the driver pool that contain an insertion in an optionally essential gene (necessary for survival and propagation under selection conditions). Subsequently, from the surviving clones of both pools, the plasmids inserted into the genome of the microorganism are recircularized and recovered under permissive conditions. The driver pool lacks plasmids containing fragments of facultative essential genes. The fragments cloned in the SRM plasmids are subsequently amplified by PCR in both pools (see FIG. 4). Those amplified DNA fragments which are part of facultative essential genes are enriched and isolated by genetic subtraction (see Figure 8).

A particular embodiment which makes use of subtractive hybridization for enrichment in fragments of essential genes is exemplified in FIG .

• A) PCR-based genetic subtraction. The tester DNA fragments (see Figures 4 and 7) are ligated with an adapter oligonucleotide such that the adapter is covalently linked to only one of the two DNA strands of a double-stranded tester DNA fragment, e.g. B. is achieved by the ligation of a double-stranded, non-phosphorylated adapter to the 3'-phosphorylated DNA fragments of the tester DNA. These tester DNA fragments are then mixed with a molar excess of Driver DNA fragments. The mixture is denatured and slowly rehybridized. Subsequently, overhanging single stranded areas are filled up to double strand with DNA polymerase. The products of this reaction are amplified by PCR using oligonucleotide primers corresponding to the adapter sequences. Only those tester DNA fragments that have not hybridized with Driver DNA fragments are amplified exponentially thus enriched and then isolated by cloning.
• B) Genetic subtraction by physical separation of biotinylated DNA fragments. The Driver DNA fragments become biotinylated and then in excess with tester DNA Fragments mixed, denatured and slowly rehybridized. The biotinylated homo driver driver double strands and heteroduplexes (Driver tester double strands) are made by extraction with carrier  coupled streptavidin from the tester tester homoduplexes separated. The latter are isolated by cloning.

The insertion mutants generated by SRM, for example, are described in US Pat Animal experiments or cell culture systems with regard to their modified examined biological properties. Through the targeted use special host cells, e.g. Cultured macrophages or host tissues, e.g. B. Spleen, gene groups can be selected that have essential properties of the pathogen, e.g. B. the colonization of certain host cells. If the surviving mutants are isolated from the cells, they are missing Mutants of essential genes. Subtracting from the complete gene bank, the surviving mutants, one obtains the mutants of the essential ones Genes. This is done via a special PCR-based Substraktionsschritt.

The CAI or SRM method is a very efficient method to use clear identification and characterization of essential genes. Eat there provide a natural target for inhibitory agents the presented procedures provide an ideal basis for the development of new ones Agents.

In the procedures described below, the identified genes directly used for drug screening, compared to conven procedure, to dispense with expensive purification steps can. The focus of these methods are bacterial carrier cells, the for screening for prophylactic and therapeutic agents can be used.

The produced deficient microorganisms are then applied to their Growth ability or its survivability tested. suitable Test systems are z. In vitro systems, cell culture systems, tissue culture systems and animal models as a natural environment. Will the process be included  H. pylori applied, the organisms are on the one hand on a so-called. Full medium grown, with the full medium the best possible advance growth for H. pylori. At the same time the deficient H. pylori organisms are grown in a culture of which natural environment of H. pylori should correspond as closely as possible. It On the one hand cell cultures based on primary cultures or Cell lines from gastrointestinal tissue used or different ornamented primary tissue (spheroids) in culture medium. More options to simulate the natural environment of H. pylori exist in the Use of stimulated macrophages, because H. pylori possesses the Ability not to be ingested and metabolized by these. It can also be checked if the deficient H. pylori Organisms are able to become involved in immunodeficient mice via a establish a specific period of time.

Although a deficient H. pylori bacterium is able to access full medium survive, but does not grow in a natural environment, as above described, the gene deficient in this organism becomes optional referred to as the essential gene.

If the deficient H. pylori organism in neither of the two test lives is viable, it is an obligatory essential Gene.

Generally, genes of microorganisms can be one of these categories be assigned.

From these results, then in mutated or / by asRNA suppressed sequences identified and one each of these two Be assigned to categories, or the category of nonessential Genes, when the deficient organism is not impaired shows its ability to grow.  

Another object of the present invention is to provide a genetic method for isolating and cloning the identified essential genes from various clinical Helicobacter isolates or heterologous pathogenic germs of clinical importance. The method according to the invention therefore further comprises the steps

• a) Preparation of primers for the amplification and detection of homologous gene sequences in heterologous microorganisms
• b) identifying the homologous gene sequences.

A preferred implementation of these further method steps is in it, so-called megaprimer of the identified essential ones Producing helicobacter genes by means of PCR (Polymerase Chain Reaction), their sequence directly from the corresponding plasmids of mutagenized DNA sections can be derived. These primers can then be used to identify the already identified essential Isolate genes from different Helicobacter isolates. If this essential genes have correspondences in other microorganisms, The primers may also be used to isolate these genes from Helicobacter various microorganisms are used. Farther can then determine the exact DNA sequence of the isolated genes and the finding the gene variance within the different Helicobacter isolates or between the different microorganisms.

In the production of megaprimers arise DNA fragments with variable 3'-ends. Because of this property it is possible to use the DNA fragments for isolating variable or related genes by means of the described Use PCR method.

Identifying specific drugs

To identify new immunological agents from the pool of identified essential genes of a pathogen or Further development of these active ingredients become bacterial carriers so much  used effectively, as the identified genes directly into these delivery systems can be cloned and expressed there. The drug screening is then done directly with the help of these recombinant, bacterial carriers. When Carriers are preferred attenuated bacteria, such as. Salmonella, used as these have a natural potential to immunostimulant feature. Are these attenuated bacteria as carriers or Producers of identified essential genes of pathogenic bacteria used and immunized with these vaccine strains Mammal can cause a sustained immune response become.

Meanwhile, the immunological properties of these bacterial Carrier systems have been refined so far that a targeted immune response can be triggered (VanCott et al., 1998; Carrier Patent EP 98116827.1). This property is significant in that the different Pathogens often only over a particular branch of the Immune system can be effectively combated. Ie. protection mediating Antigens can only be identified when they target the immune system presented in the right form. Only if the carrier used loaded with an effective antigen, it may become one Protective effect come. Due to the diverse immunological Characteristics of bacterial carrier systems and their superiority compared to conventional synthetic adjuvants these are for Identification of immunologically relevant antigens particularly suitable.

In addition, the bacterial carrier systems can be used with efficient Gene expression systems are equipped, which also manufacture allow problematic antigens (PCT / EP91 / 02478, EP 98116827.1). Due to the direct subcloning of the isolated essential genes and the ease of handling of bacterial carriers in the production and Vaccination, can in a relatively short time a large number of antigens tested for their immunogenic and protective potential  become. In conventional methods, the test antigens must be against it time-consuming purification processes are subjected, wherein often already in the genetic engineering of the selected Antigens in bacteria encounter difficulties with the toxic Effect of these antigens linked to the producing bacterial strain are.

An important prerequisite for the development of active ingredients exists in determining the immunogenic potential of the identified sequences, to determine to what extent the corresponding gene products for the Manufacture of antibodies or vaccines are suitable.

To identify immunological agents against clinically relevant Helicobacter organisms must first be determined in how far the Gene product of the identified essential gene immunogenic properties has. Ie. it must be determined experimentally, whether with the antigen triggered a humoral and cellular immune response in a mammal which is directed against the original gene product of the pathogen. Thus, in no case are excluded those antigens that are in the Framework of a natural infection not recognized by the immune system become. On the contrary, one might expect that z. B. at chronically infected humans the immune response against protection-promoting Antigens is suppressed or of a quality that ultimately has no Protective effect mediates. However, exclude those antigens that subject to a high genetic variation and thus an effective Immune response are hardly accessible.

To prove the identity of the identified gene product in a naturally occurring infection, becomes antiserum of patients gained, either under active gastritis with discomfort suffer, or from patients, in whom the Helicobacter infection is asymptomatic runs. With these sera that is electrophoretically separated  recombinant protein tested in a classic Western Blot procedure. Finds a recognition reaction with a recombinant protein, respectively Both serums, that of a patient with a fulminant and the one Patients with asymptomatic Helicobacter infection, instead, so plays this protein plays a role in a natural infection. Will that be recombinant polypeptide, however, only from the patient's serum recognized as a symptomless infection, this may be an indication be a protective potential of the corresponding protein. Farther For example, antibodies specifically directed against this protein can possibly used for passive immunization.

Also of particular interest are antibodies from individuals that are not Helicobacter carriers, as these are protective Suggest potential of a corresponding recombinant polypeptide.

Furthermore, the immunogenic polypeptides are co-administered with suitable additives for immunization in vivo. used These are various adjuvants, bacterial toxins, cytokines or a polypeptide according to the invention as a live vaccine. The immune response is then tested to see if they have been given after a successful administration of a certain polypeptide of the invention in combination with corresponding Additives after infection with the homologous germ a protective Effect against other homologous infections causes (eg infections with different H. pylori strains).

Yet another way to test immunogenicity is to in the animal model (eg mouse, rabbit) an immune response against Helicobacter or other microorganisms and from the Immunized animals to gain antibodies, which then in another Western blot analysis can be used. At the same time Patient biopsies in situ immunologically with the same antibodies  be studied since Helicobacter and other microorganisms in culture lose or gain certain proteins.

In parallel, it is preferable to investigate whether infection with heterologous germs (preferably other gram-negative bacteria) which express the corresponding polypeptide, a protective effect achieved can be.

After determining whether the identified genes or their Expression products are able to elicit an immune response According to the method of the invention, it may further be investigated whether also treated an already existing infection with such antigens can be. Can be identified in this way a polypeptide that shows a therapeutic effect, it is also preferred to his Activity in infections with heterologous bacteria investigated.

The screening for prophylactically or therapeutically effective, immunological substances can proceed according to the following scheme, wherein the observance of the individual steps is not mandatory:

• 1. Cloning of the identified gene into a suitable bacterial Carrier strain and detection as well as quantification of the complete Gene product by SDS-PAGE.
• 2. Immunological characterization of the gene product produced with Help from (a) serums of infected and / or naturally protected hosts, which should recognize the gene product in the carrier strain; (B) Hyperimmune sera from animals infected with the recombinant Carrier strain were immunized. Where the respective Hyperimmune serum the original gene product in the pathogenic germ should recognize. It may be possible that the original Antigen only in a certain developmental period from Pathogen is produced.  
• 3. The protective effect of individual antigens in the prophylactic and / or therapeutic application form is used in Animal model examined.

All protective antigens identified by the described methods can now be further developed in a second step. In the foreground of this development u. a. the evaluation of the genetic constancy of the identified protective antigens within the Pathogens or related pathogenic germs in its worldwide Distribution. Furthermore, to develop effective vaccines to which genetic differences in the immune system of the vaccinee. aim Both methods involve the identification of antigens or epitopes that can be applied as widely as possible. To capture the Genvariability within a species or homologous germs, the so-called mega-primer approach can be used. From the plasmid with gene-specific primers with variable 3 ' Ends were made via PCR which involved the amplification of homologous genes enable. Based on the determined DNA sequence of the amplified genes can their variability derived and z. B. gen constant ranges determined become.

The genetic differences between individual vaccinees, on one Defined antigen can respond with the help of an in vitro vaccination be evaluated. From different donors will do this antigen presenting cells (APC) isolated, e.g. B. dendritic progenitor cells, which expands in vitro and charged with the antigens to be tested with the antigens preferably via corresponding vectors are expressed. The identified genes can also be individually or in defined combinations in dendritic cells (DC) of uninfected Donors are expressed. The gene products of the Host cell processed and presented by the MHC complex. DC are especially for antigen presentation to naive or  dormant T cells. Be DC autologous with T cells Incubated donor, it is possible to determine whether this donor against The antigen used would react if it is naturally would come into contact, z. B. as part of a vaccination. Based on Immune response of T cells may be due to a possible immunogenicity of the corresponding antigen are closed. Such an immune response consists for example of a proliferation of T cells, or one Cytokine release, in particular of IL-2 and IL-4. The cytokines can for example with the aid of a commercially available assay kit (eg from Genzyme Cambridge M.A.).

Finally, those identified in the manner described and characterized antigens or epitopes for the development of the first Vaccine prototypes are used. This is between two Vaccine types differed, active and passive vaccination.

For passive immunization, the vaccinee or antibody Antibody fragments with protective or inhibiting effect from the outside fed.

Antibodies are in the form of polyclonal, preferably monoclonal Antibodies (MAbs) or recombinant antibodies. For this include antibodies specific to polypeptides of the invention or their subunits and fragments react and prophylactic and / or therapeutic application, e.g. B. a passive immunization, can be used. These anti-protein or anti-peptide antisera or monoclonal antibodies can be determined using standard protocols z. By immunization of animals such as mice, rats or goats with a purified polypeptide of the invention, a fusion protein or a subfragment of which are produced. In addition, the Animals are also immunized with bacterial vaccine carriers with corresponding genes of the invention are equipped and the coded  Express polypeptides. The antibodies are preferred immunospecific against antigenic determinants or epitopes thereof described Helicobacter polypeptides or a closely related Polypeptide which has a homology of at least 90%, directed. you are not cross-reactive with polypeptides z. B. a homology of less than 80%.

Starting from a cell line containing a polypeptide-specific monoclonal antibody can be produced from the coding gene of a such antibody, chimeric genes are created, the antibodies Determine consisting of an antigen binding domain from the Mouse and the Fc part of an antibody of the human. These antibodies can be produced in cell lines or transgenic animals.

In place of antibodies that were generated in the animal, too Antibody fragments, miniantibodies are used, the z. In one heterologous system such as bacteria are produced. These miniantibodies can either be monovalent or bivalent and consist of dimerized single-chain molecules (Kujau et al., 1998; Kalinke et al., 1996; Pack et al., 1993).

Antibodies to the immunogenic polypeptides of the invention may also be used generated in plants. Examples are z. B. Hiatt and Ma (1993), by Engelen et al. (1994) and Ma et al. (1994). According to the particular plant used these z. B. directly to Consumption and thus used as an oral vaccine.

Another very broadly applicable way of producing antibodies is in Milk and eggs of immunized animals. If you administer z. B. pregnant cows, Sheep or horses suitable antigens, as found in the milk Immunoglobulins that are useful for developing a vaccine. The Milk can then either be administered directly as a vaccine, or a  concentrated immunoglobulin extract. On the same It is also possible to use antibodies (hyperimmune antibodies) in chicken eggs (Ling et al., 1998; Sasse et al., 1998). The described Therefore, immunogenic polypeptides of the invention can also be used for development a dairy product or chicken eggs used as oral Vaccine can be used.

According to the invention, the generated antibodies or fragments thereof tested for their applicability. You can do this by known methods be purified (precipitation, chromatographic methods) and in For example, it will be examined whether they control the infection process of H. pylori (adhesion assays) or activating on complex or ADCC ("antibody-dependent cell-mediated cytotoxicity", anti body-dependent cell-mediated cytotoxicity).

For passive immunization, the antibodies generated by the Polypeptides of the invention were generated, either orally or intra administered gastrically. For this, the antibodies with a bicarbonate Buffer mixed. But they can also be administered systemically where they do not need to be buffered.

Antibodies are preferred alone or in combination with others non-immunological agents used, for. B. with antibiotics or Proton blockers.

Active vaccination relies on an immune response that is vaccinated from the vaccine Organism itself is triggered. Preference is given to dosage forms of Vaccines as antigens, antigenic fragments, subunit vaccine, as DNA Vaccine, as a live vaccine or as a food vaccine.

Antigens are those polypeptides or fragments thereof which in vivo a Can cause immune reaction.  

If a polypeptide is to be provided as a subunit vaccine, it will first in its subunits or structural domains according to his Pattern of antigenicity (eg, T and B cell epitopes). This Antigenicity pattern can be created using a computer program be immunogenic regions consisting of a short polypeptide sequence of about 8 to 10 amino acids, can be detected (Hughes et al., 1992). The individual Polypeptidstücke can then subsequently to their immunogenicity in the mouse or in primates or the People are tested. You can do this either as a purified Polypeptides synthesized in combination with appropriate adjuncts such as an adjuvant, toxin or cytokine or as a fusion protein to a known immunogenic Protein or protein subunit such. B. the cholera toxin B subunit (Liljeqvist et al., 1997). Furthermore, the immunogenic Peptides in outer membrane proteins such. B. the OmpS maltoporin of E. coli and heterologously expressed in a vaccine carrier strain become (Lang and Korhonen, 1997).

For the development of a DNA vaccine, those in the invention characterized polynucleic acid molecules "naked" in fusion with a eukaryotic tissue-specific promoter or in the form of a plasmid be administered. The "naked" DNA or the corresponding plasmid is used in combination with an additive such as a reagent containing the cellular permeability changes such. Bupivacaine (WO94 / 16737), cationic lipids such. DOTMA (N- [1- (2,3-dioleyloxy) propyl] -N, N, N- trimethyl-ammonium chloride, DOTAP (1,2-bis (oleyloxy) -3-trimethyl- ammonio) propane), DDAB (dimethyl-dioctadecyl-ammonium bromide), DOGS (dioctadecyl-amidolglycyl spermidine) or cholesterol derivatives, silica, Gold or tungsten (Tang et al., 1992) or in liposomes (WO93 / 18759, WO93 / 19768, WO94 / 25608, WO95 / 2397) or microparticles, administered. Examples of useful promoters and gene shuttles are from Hartikka et al. (1996). For the application of  However, polynucleotide molecules can also z. B. attenuated Salmonella be used. The bacteria are using eukaryotic Expression vectors comprising a polynucleotide molecule of the invention, transformed and then administered orally. The plasmid DNA is subsequently transferred from the bacterium to the host (Darji et al., 1997). to However, transformation of attenuated carrier bacteria can also be filamentous Phages are used. The advantage of this is that they have a extremely high number of plasmids can transfer.

For the development of a live vaccine are among others viral, such as z. B. adenoviral or chickenpox virus vectors, or bacterial vectors such as Salmonella, Shigella or Lactobacillus available. attenuated non-virulent Salmonella typhimurium strains used for recombinant Expression of heterologous antigens can be used and orally have been widely characterized (Mekalanos, 1994, WO92 / 11361, Cirillo et al., 1995 and Dorner (1995). More bacterial Vectors that can be used as vaccine vectors are from Cirillo et al. (1995) and Dorner (1995). A polynucleotide Molecule of the invention for a therapeutic or prophylactic encodes effective polypeptide, this is either in the bacterial Genome stably integrated and subjected to a transport system that the Presentation on the bacterial surface (PCT / EP94 / 04286; WO97 / 35022). The corresponding polynucleotide molecule can be in the Bacterium but also present as a plasmid in the free state.

Vaccines are usually with suitable additives such. B. Adjuvants, bacterial toxins, cytokines, etc. administered the immunogenic polypeptide in its protective or therapeutic effect support. Adjuvants with low side effects for use in humans, for subunit vaccines and live vaccines, but in part also come for DNA vaccine in question, z. B. aluminum hydroxide, Aluminum phosphate, calcium phosphate, N-acetylmuramyl-L-threonyl-D  isoglutamine, N-acetyl-normuramyl-L-alanyl-D-isoglutamine, N-acetyl muramyl-L-alanyl-D-isoglutamyl-L-alanine-2- (1'-2'-dipalmitoyl-sn-glycero-3- hydroxyphosphoryloxy) ethylamine, liposomes, monophosphoryl lipid A, Trehalosedimicoloat, mushroom polysaccharides such. Schizophyllan, muramyl Dipeptide, muramyl dipeptide derivatives, as well as phorbol esters, saponins and immunostimulating complexes (ISCOMS) (Gupta and Siber, 1995). When bacterial toxin may, for. B. cholera toxin or its subunits or the heat-labile toxin from E. coli can be used. Although these highly potent as adjuvants are active, they can because of their toxicity limited to humans. With the help of certain However, mutagenesis techniques can be used to develop molecules that are active, but are non-toxic (O'Hagan, 1998).

Another way the immune response to the prophylactic and / or to optimize therapeutically active substances of the invention is that corresponding polypeptide as a fusion protein with an immunogenic Express protein domain. One way is z. In it, the pilin DSL domain from Pseudomonas aeruginosa as fusion partner too use. Also described are fusion proteins that bind to glutathione S-transferase or thioredoxin were fused (Hill et al., 1997; Gabelsberger et al., 1997). The corresponding fusion protein can each produced and administered as a subunit or live vaccine.

The immune response of the identified immunologically active substances can also be modulated in that way in combination with be administered to certain cytokines. For simultaneous administration offers a co-expression of the polynucleotide sequences of the invention with a specific cytokine in Salmonella or another Host bacterium. The corresponding cytokine can either on a separate plasmid, in series or as a fusion protein with the desired polynucleotide sequence of the invention and then encoded in the host bacterium are transformed. In question are cytokines, the  such as Interleukin-6 (IL-6), Interleukin-10 (IL-10) or Interleukin-12 (IL-12) stimulate the immune system.

For further optimization, individual immunogenically effective Substances of the invention with each other or in combination with known immunogenic substances such. B. VacA or its individual Subunits are combined. The corresponding polypeptide can thus together with at least one other Helicobacter antigen, such. B. the native urease or its subunits, fragments homologs, Mutants or derivatives thereof are expressed. In addition, you can z. B. different subunit vaccines individually or as a fusion protein such as described above. For this purpose can again z. B. purified polypeptide molecules in combination with a suitable adjuvant, bacterial toxin or cytokine. Furthermore, various combinations of nucleotide sequences immunogenic subunits of the described polypeptide molecules a common plasmid and administered as a live vaccine become. A vaccine vector of the invention can thus one or more Polypeptides of the invention, derivatives or fragments thereof. There is also the possibility of combining a DNA vaccine with one or more purified subunit vaccines in a suitable Carrier, as already described above.

To identify new pharmacological agents from the pool of identified essential genes of a pathogen or Further development of these agents can also be very bacterial carriers be used effectively, as the identified genes directly into this Carrier systems can be cloned and expressed there. The active ingredient Screening is then done directly with the help of this recombinant, bacterial Carrier.  

Display systems serve to express an expressed polypeptide of the invention to present the cell surface of bacteria. The transport of Polypeptides through the inner membrane allows for a signal peptide on the Amino end, while other shares the storage and anchoring in the take over outer membrane. As carrier shares are different outer Membrane proteins of E. coli have been described as. B. PhoE (Agterberg et al., (1990) or OmpA (Francisco et al., 1992). It can, however, too Merges with the transport domain of the IgA protease precursor IgAβ can be used (Klauser et al, 1990). Examples of display systems are z. As the DsbA system (PCT / EP 94/02486) or the car transporter system (AIDA; WO97 / 35022). The surface-presented polypeptides can then be used for binding studies with peptide or combinatorial chemical substance libraries are used. The binding studies can with the help of a "high through put" system, the high test rates allows to be carried out in liquid or bound form. For this purpose, the presented polypeptides z. B. to a chromatophore coupled, in combination with another chromatophore, the the drug peptide or chemical substance is coupled to a Color reaction allows. The corresponding active ingredient component can but also z. B. marked with a fluorescent dye or a solid Coupled carrier matrix. When using a "Solid Phase System" beforehand either the polypeptide of the invention used or else conversely, the peptide or combinatorial drug bank to the Coupled carrier matrix. The respective dye-labeled component binds then to the immobilized component, causing a color reaction again is possible. After the binding reaction then several must Washings are done before the appropriate substance is isolated. Advantage of the "Solid Phase System" over binding in Liquid is that the active substance can be isolated faster, since the unbound substances are washed away.  

Further methods for the identification of new pharmacological agents are based on the knowledge of the primary structure of the identified Essential genes or use the purified genetic engineering produced gene products.

An alternative embodiment of the method according to the invention consists of specific binding partners of the identified genes to find coded polypeptides.

Homology studies with Helicobacter and others may be preferred Organisms are carried out, for. With the help of computer alignments, Southern blots, PCR and the like and subsequent assignment of the Sequences. Because of homologies can then be on potential Binding partner of the proteins are closed.

Also preferred are combinatorial binding studies on Target-directed screening method with the aid of substances Libraries are performed. The potential binding substances are presented bound in a special order, z. In Microtiter plates or other carrier materials. Subsequently, the "Target", usually purified, optionally recombinant Helicobacter proteins, added in soluble form, giving the proof an interaction between the target and certain substances allows. Indirect detection can be detected by labeled antibodies directed against the substance, or by introducing additional Elements ("tags") are brought into the target.

Another variant of the binding studies consists in the expression of Helicobacter proteins in recombinant bacteria (eg, those that contain the producing fluorescent protein GFP or certain enzymes) containing the Present proteins on the surface, and then testing one Substances library.  

Furthermore, the three-dimensional structure of the Identified Genes According to the Invention 30268 00070 552 001000280000000200012000285913015700040 0002019934029 00004 30149 encoded polypeptides or their Fragments can also be determined by crystallographic analysis. at sufficient resolution can be any "pockets" or other Binding sites are accurately characterized in their three-dimensional structure. Based on this data, the structure of potential binding partners can be calculated become.

With methods such as "Two-hybrid system", display systems, "High Throughput screening "or combinatorial binding studies randomly generated polypeptides are identified that belong to the Helicobacter Polypeptides of the invention or fragments thereof, or to others bind polypeptides identified according to the invention. Becomes this way found appropriate peptide, it can be further chemically modified until the optimal potential binding is achieved. That identified Polypeptide may, for. B. can be used as an inhibitor by z. B. to a Toxin and an internalization signal is coupled to the pathogenic Germ destroyed or as a peptide mimetic to bind the germ to prevent the cellular surface (EP-412,762A and EP-B31,080A). With But help from the "Two Hybrid System" can also be activated by the Immune system can be generated. The identified peptides attached to the Helicobacter polypeptides of the invention can bind to certain Ligands z. B. for the T cell receptor can be coupled. So become one pathogen-infested animal or humans so equipped this Peptides administered, the body's immune system becomes specific attracted and activated.

Compliance with the individual steps is not mandatory, but can be supplemented or replaced by further steps.

The respective prototypes of an immunological or pharmacological Active ingredients are further developed and improved below.  

The further development of a vaccine can be done by: several antigenic gene products or parts thereof in an active ingredient be combined and / or with different carriers or additives be administered. As a carrier act various attenuated bacterial or viral organisms and as adjuvants adjuvants and / or cytokines.

For the further development of a pharmacological active ingredient is a as effectively characterized lead structure chemically further modified, so that optimal binding and inhibition of the identified gene product he follows. Furthermore, the drug should be well tolerated by the patient and have little side effects.

For identification of drugs that bind to polynucleotides, a Polynucleotide of the invention z. B. can be pre-coupled to a carrier matrix or vice versa, the active ingredients of Polypeptid- or combinatorial Substance bank. The following scheme used is the same like that already described for the polypeptides.

Such inhibitory substances may include polypeptides, peptides, as well chemical substances, such as antibiotics. The inhibitory Effect can be different in different stages of replication intervene with combating microorganisms. examples are Expression inhibitors or enzyme inhibitors or other inhibitors, which the natural function of the polypeptides of Helicobacter and can affect related microorganisms. Such inhibitory Substances are also the subject of the invention.

Another way to get an optimal active ingredient against Helicobacter and Finding other bacterial infections is with the help of special ones Computer programs. On the basis of crystallographic data from the polypeptides described in the invention can be obtained a model can be created that is steric, electronic, hydrophobic and  so-called "resulting binding moments" (RBMs) with each other connects (Ray et al., 1998). On the basis of this model can further Substances can be modeled on the computer, while those already may contain identified lead structures, but with better ones Binding properties are equipped. It can, however, completely novel agents are designed.

Another aspect of the present invention is a nucleic acid encoding an essential secretory gene from Helicobacter pylori identified by the method of the invention described above. Essential Helicobacter genes were identified with the present method whose nucleic acid sequences are shown in SEQ ID NO. 1 to 113 (odd numbers) are indicated. A nucleic acid according to the invention is characterized for example in that it

• a) one of the in SEQ ID NO: n, where n is an odd integer of 1 to 1 13 inclusive, or shown nucleic acid sequences a protein-coding portion thereof,
• b) one of the sequences from (a) in the context of the degeneration of the genetic code corresponding nucleotide sequence or
• c) one with one of the sequences from (a) and / or (b) under stringent Conditions hybridizing nucleotide sequence comprises.

In addition to the invention shown in the sequence listing Nucleotide sequences and these sequences in the context of degeneration the nucleotide sequence corresponding to the genetic code comprises The present invention also provides nucleotide sequences which have been used with any of the above hybridize said sequences. The term "hybridization" according to present invention is in Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), 1.101- 1.104). Preferably one speaks of a stringent one Hybridization, if after washing for 1 hour with 1 × SSC and 0.1% SDS at 50 ° C, preferably as 55 ° C, more preferably at  62 ° C and most preferably at 68 ° C, especially for 1 h in 0.2x SSC and 0.1% SDS at 55 ° C, preferably at 55 ° C, especially preferably still a positive at 62 ° C, and most preferably at 68 ° C Hybridization signal is observed. One among such Washing conditions with one or more of the invention Nucleotide sequences or one of these sequences in the context of Degeneracy of the genetic code corresponding nucleotide sequence hybridizing nucleotide sequence is a nucleotide according to the invention frequency.

Preferably, the nucleotide sequence of the invention is a DNA. you however, may also be an RNA or a nucleic acid analog, such as a peptidic nucleic acid. Particularly preferably, the nucleic acid according to the invention a protein-coding portion of in Sequence Listed nucleotide sequences or a sequence, the a homology of more than 80%, preferably more than 90% and more preferably more than 95% to the nucleotides shown or preferably at least 20 nucleotides (nt) and more preferably at least 50 nt long section thereof.

The homology is in percent of identical positions when comparing two Nucleic acids (or peptide chains) indicated, with 100% homology the complete identity of the compared chain molecules means (Herder: Lexikon of Biochemistry and Molecular Biology, Spektrum Akademischer Verlag 1995).

A nucleic acid according to the invention can be used for a secreted polypeptide Signal peptide or for a secreted polypeptide without signal peptide. A nucleic acid according to the invention comprises both the sequence of coding strand as well as the complementary sequence. Latter  For example, in the production of antisense nucleic acids Find application.

The invention likewise naturally also relates to a gene bank which at least 2, preferably at least 20, more preferably at least 100 contains said nucleic acids cloned in vectors.

A listing of those given herein and in the Sequence Listing Nucleic acids according to the invention together with their gene products and their Functions and putative functions are given in Tables I and II.

For the genes in the sequence listing in their nucleic acid and Amino acid sequence are indicated, are bacterial genes. Prokaryotes also use other than the AUG codon (mRNA) (alternative) start codons. These are AUU (usually coded for Isoleucine, Ile), UUG (normally coded for leucine, Leu) and GUG (usually codes for Valin, Val). In the sequence listing, the Amino acid sequences according to the normally used Translation code translated. It should be noted that when reading of the Sequence Listing the prokaryotic use of alternative Start codons must be considered, and thus herein also by the Nucleic acid sequences according to SEQ ID NO. 35, 49, 61, 69, 75, 81, 103 and 105 encoded amino acid sequences are disclosed which instead of with a respective Leu, Val or Ile residue shown in the Sequence Listing, start with a methionine residue (Met).  

Table I

Mandatory essential genes

Table II

Optional essential genes

Another aspect of the present invention is a vector comprising a Nucleic acid of the invention or a portion thereof. The Nucleic acid or the nucleic acid segment can thus be cloned into the vector be that they are expressed either in sense or antisense direction can. The nucleic acid section preferably has a minimum length of 15 Nucleotides, more preferably 20 nucleotides, more preferably 50 Nucleotides. This vector can be any prokaryotic or eukaryotic vector on which the DNA according to the invention Preferably, the sequence is in association with expression signals, such as z. Promoters, operators, enhancers etc. Examples of prokaryoritic Vectors are chromosomal vectors, such as bacteriophages (e.g. Bacteriophage 1) and extrachromosomal vectors such as plasmids, circular vectors being particularly preferred. suitable prokaryotic vectors are e.g. In Sambrook et al., Molecular Cloning (1987), chapters 1-4. On the other hand, the inventive Vector may also be a eukaryotic vector, e.g. B. a yeast vector or a vector suitable for higher cells (eg a plasmid vector, viral vector, Plant vector). Such vectors are described, for example, in Sambrook et al., supra, chapter 16. CAI and SRM vectors as above described are also the subject of the invention.

Another object of the present invention is a cell that with a vector of the invention or a novel vector Nucleic acid is transformed. In a preferred embodiment this cell is a prokaryotic cell, preferably a gram negative Bacterium, e.g. B. E. coli. On the other hand, however, the cell of the invention also be a eukaryotic cell, such as a fungal cell, a yeast cell, an animal or a plant cell. It is particularly preferred the cell is a microorganism, e.g. B. Helicobacter or Salmonella. Microorganisms transformed with CAI or SRM vectors have already been described above. These are, as well as with the above  Method producible mutant banks, also the subject of Invention.

Another aspect of the invention relates to an essential and preferably secreted polypeptide of H. pylori. In particular, this is a polypeptide that

• a) one of the in SEQ ID NO: m, where m is an even integer from 2 to 114 inclusive, or amino acid sequences shown
• b) one with one of the sequences according to (a) immunologically cross-reacting sequence.

Among immunological cross-reactive sequences are thus also Muteins, variants and fragments of the amino acids shown in SEQ ID NO. 2 to 114 comprises sequences shown. These are to be understood as sequences that by substitution, deletion and / or insertion of individual amino acids or short amino acid portions of the above sequences differ.

Based on homology analyzes using the FASTA protein program could identify the identified polypeptides, their sequence with the Nucleic acid sequence could be found, certain characteristics or be assigned a putative localization in the bacterium. Some of the possess polypeptides encoded by the nucleic acids according to the invention a signal peptide and are caused by the Sec-dependent transport Mechanism exported to its target site, while others no signal peptide and therefore probably have a Sec-independent Transport mechanism, z. B. by the ABC transporter system, secreted (see Tables I and II).

Another object of the invention is a process for the preparation of polypeptides and fragments of the invention. The production of polypeptides of the invention is preferably carried out by  a cell with a DNA molecule or vector according to the invention transformed, cultivating the transformed cell under conditions where expression of the polypeptide takes place, and the polypeptide the cell or / and isolated from the culture supernatant. It can do that polypeptide of the invention both as a fusion polypeptide and as Non-fusion polypeptide can be obtained.

The polypeptide of the invention can be used as an immunogen for the production of Antibodies are used.

The present invention thus also relates to an antibody which is against a polypeptide of the invention is directed. Also subject of Invention are fragments of such antibodies, such as. B. Fab fragments or Fc fragments.

Yet another aspect of the invention is an inhibitor of Polypeptides according to the invention, their fragments or their expression, Presentation or / and natural function. This is preferably a molecule which is capable of specific to a polypeptide or fragment thereof to bind and / or its expression, presentation and / or natural To influence function. The identification of such specific Binding partners have already been described above. Especially suitable as Inhibitors are proteins or peptides which are a polypeptide according to the invention inhibit in its natural function, for. B. can enzymes through Blocking the active center be inhibited.

Yet another aspect of the present invention relates to a pharmaceutical composition used as an active ingredient DNA molecule according to the invention, a vector according to the invention, a Cell according to the invention, a polypeptide according to the invention, a Antibody of the invention or fragment thereof or / and a inhibitory molecule capable of specific to a  to bind polypeptide of the invention, optionally together with üb pharmaceutical excipients, diluents, additives and carriers contains.

A pharmaceutical composition according to the invention can be applied to ver different way and using single of their ingredients as effective substances for inhibiting the reproduction of Hellcobacter Organisms are used in a host, especially in humans.

The pharmaceutical composition according to the invention can on the one hand used to diagnose Helicobacter infection. The Nucleic acid level diagnosis is preferably done by use of hybridization probes or primers which have a specific DNA Sequence comprising at least a portion of one of the in SEG ID NO. 1 to 113 (odd numbers) is complementary, so that they allow amplification of the sequences of the invention. As we mentioned earlier, these amplification primers or probes also work for the amplification and thus for the detection of related microorganisms used if they have gene sequences that are the same encode essential gene. At the protein level, diagnostics are carried out preferably with the aid of the antibodies according to the invention.

Furthermore, the pharmaceutical composition for prophylaxis and Combating Helicobacter infections and related infections Microorganisms suitable.

Another important aspect of the present invention is the Use of the identified essential genes of Hellcobacter pylori Prevention or control of infection with Helicobacter or related microorganisms. In particular, these can be identified essential genes used for the production of vaccines (vaccines) (see above).  

Another use of the polypeptides of the invention is in the Purification of antibodies against H. pylori polypeptides and against corresponding polypeptides from related and other microorganisms.

The invention is illustrated by the following examples, figures and the Sequence listing explained in more detail. In the sequence listing are the following Nucleic acid and amino acid sequences shown.

Fig. 1 shows a schematic representation of a CAI vector.

Fig. 2 shows a schematic representation of a method of conditional antisense inhibition (CAI).

Fig. 3 shows a schematic representation of the study of the viability of deficient microorganisms by their survival rates using the CIA method.

FIG. 4 shows a schematic representation of the subtractive CAI method (CAI).

FIG. 5 shows a schematic representation of an SRM vector.

FIG. 6 shows a schematic representation of the reversible inactivation of a gene by the insertion / excision of a conditionally replicating SRM plasmid.

Fig. 7 shows a schematic representation of an SRM process.

Fig. 8 shows a schematic representation of the enrichment of fragments of essential genes by subtractive hybridization.

Examples

The application of the overall method is exemplified on Example of the pathogen Helicobacter shown, however, also allows perform other pathogenic germs.

1. Identification of essential genes of pathogenic microorganisms Step 1

Enrichment of H. pylori genes encoding secretory / excretory polypeptides

Production of an H. pylori gene bank in the minimal vector pMin2: The starting strain is H. pylori wild-type strain 69A. The bacteria are grown on serum plates or other appropriate medium (Westblom et al., 1991) at a temperature of 37 ° C in an atmosphere of 5% O ₂ , 10% CO ₂ , 85% N ₂ . The isolation of the chromosomal DNA is carried out according to the method of Leying et al., (1992), wherein the DNA is subsequently purified via a cesium chloride gradient. 50 μg of the purified, chromosomal DNA is partially cleaved with the restriction endonucleases Sau3A and HpaII, the DNA fragments are separated in an agarose gel and the fragments in the size of 3 to 6 kbp are eluted from the gel using the Geneclean II kit (Bio101). The isolated DNA fragments are cloned into the Bg / II and ClaI-cut pMin2 vector (Kahrs et al., 1995) and transformed by electroporation into E. coli strain E181, to which the plasmid pTnMax9 has previously been transferred. In total, about 4000 clones are generated via such an approach. E.coli strain E181 is a derivative of strain HB101 (Boyer and Roulland-Dussoix, 1969) and contains the lysogenic phage CH616 for replication of the pTnMax9 plasmid.

Serum plate recipe

36 g GC agar (base) in 910 ml distilled water. suspend and autoclave

• - Allow to cool to approx. 45 ° C
• - Add 10 ml of vitamin mix
• Add 1 ml each of the antibiotic stock solution:
  • - vancomycin (10 mg / l, in aqua bidest)
  • - Nystatin (0.793 mg / L in DMF *)
  • Trimethoprim (5 mgh in DMF *)
  • Amphotericin (5 mg / l in DMF *)
    * DMF = dimethylformamide
• - Add 90 ml of serum or
• - Addition of 8% horse blood = 80 ml
• - or the same amount of human blood

Antibiotic stock solution

vancomycin 100 mg in 10 ml of redist nystatin Add 7.93 mg in 10 ml DMF (1 ml per liter of GC agar) trimethoprim 50 mg in 10 ml DMF amphotericin 50 mg in 10 ml DMF AL = L <(storage in the refrigerator up to a maximum of 8 weeks)

Vitamin mix (concentrate)

Dextrose (D-glucose) 100 g L-glutamine 10 g Cysteine HCl (C ₃ H ₇ NO ₂ S x HCl x H ₂ O) 26 g cocarboxylase 100 mg in 50 ml Fe (NO ₃ ) ₃ 20 mg of Aquabidest. to solve Thiamine HCl 3 mg DPN NAD 250 mg Vitamin B, ₁₂ 10 mg L-cysteine C ₆ H ₁₂ N ₂ O ₄ S ₂ ) 1.1 g adenine 1.0 g in 15 ml HCl Guanin Cl Dissolve 30 mg (32%) uracil 500 mg L-arginine HCl 150 mg p-aminobenzoic acid 13 mg AL = L <(sterilize by filtration, fill in 10 ml portions and freeze)

Genetic enrichment of secretory / excretory H. pylori gene products: The transposon TnMax9 on pTnMax9 is equipped with the genetic marker β-lactamase. This marker is created on the transposon so that allow the selection of successful transposon enables insertions, when present in the correct reading frame such genes, whose products are secreted from E. coli strain E145 ^rif or exported. The insertion of the transposon into such a gene leads to a gene fusion between the target gene and the marker, whereby the fusion protein is secreted out of the cell by a secretion and / or export signal determined in the target gene and the activity of the integral reporter gene is unfolded. In the case of β-lactamase, the clones can be directly detected by the development of resistance to ampicillin. The TnMax9 transposon on pTnMax9 is activated via IPTG.

Transposon mutagenesis of the Genbank is performed in pools of up to 20 single clones. The respective pools are plated on LB plates supplemented with 100 μM IPTG, 15 μg / ml chloramphenicol and 15 μg / ml tetracycline. In a second step, the TnMax9 mutagenized pMin2 plasmids are transferred by conjugation into the E. coli strain E145 ^rif , since they are equipped with a corresponding mob signal (oriT). The pTnMax9 plasmids, however, are not transferred. Consequently, in E. coli E145 ^rif , a specific amplification of the mutagenized pMin2 gene bank occurs. The corresponding transconjugants are selected on LB medium with 15 μg / ml chloramphenicol, 15 μg / ml tetracycline and 100 μg / ml rifampicin. A total of 500 to 1000 transconjugants are combined in 2 ml of LB medium and grown in appropriate dilutions (10 ^-1 to 10 ^-2 ) on LB plates, which are mixed with 50 ug / ml ampicillin. After culturing the plates for 36 hours at 37 ° C., 200 to 300 ampicillin-resistant clones with transposon-inserted plasmids are obtained in the entire batch.

step 2

Producing gene-deficient H. pylori present in secretory / excretory Polypeptide coding genes are mutated and the identification of such Genes with essential biological function.

Preparation of gene-deficient H. pylori mutants: By introduction of the plasmids obtained with the TnMax9 mutant H. pylori genes for secreted / excreted polypeptides into an H. pylori wild-type strain Gene-specific mutants can be generated. Due to the Cloned H. pylori gene sequences on the plasmids occur in the case a double homologous recombination event to the genomic Insertion of the TnMax9 transposon into the chromosomal target gene and thus to its inactivation. By the genetic marker on the transposon This process can be selected because the pMin2 plasmid in H. pylori is not replicated.

In the procedure, the H. pylori wild-type strain 69A is single-stranded transformed with the obtained individual plasmids, wherein the natural ability of the bacterium to ingest DNA is exploited. According to the standard cultivation conditions, the bacteria become taken up in BHI medium and up to an optical density at 550 nm of 0.1 at 37 ° C under microorganophilic conditions. The individual cultures are each purified with 200 to 599 ng Plasmid DNA was added and the culture continued overnight.  

Characterization of the biological function of the gene-deficient H. pylori Mutants in the growth test: The individual approaches are after the Cultivation plated on serum plates containing 4 μg / ml Chloramphenicol are added. With regard to the waxing properties of single mutants compared to the wild type strain can be 3 categories (1) Mutants that do not grow; (2) mutants that to form smaller colonies; (3) mutants that have a normal colony size develop. These results may be for each starting plasmid can be achieved reproducibly. For a clear assessment of the biological Importance of the category 1 gene deficient mutants will become this CAI method supplied. The gene-deficient mutants of category 2 and 3 are analyzed in the other biological test systems.

step 3

Determination of the identity of H. pylori genes coding for secretory / exported polypeptides.

To identify the primary structure of the identified H. pylori genes, be the respective starting plasmids from the E. coli strain used. The Plasmids are isolated from these strains and the nucleotide sequence of the Target genes by sequencing the areas above and below the Insertion site of the transposon determined in the target gene. The reading frame of the Target gene can be determined directly since the transposon-encoded β- Lactamase gene enters into an active fusion with the gene product of the target gene (see above). The sequencing is followed by an ABI sequence automaton Information given by the manufacturer with the following sequence primers: M 13- F (GTAAAACGACGGCCAGT) and M13-RP1 (CAGGAAACAGCTATGACC). To further characterize the genes, the database Genebank of the GCG program used, z. B. for identifying known, homologous genes of other microorganisms (FASTA), for identification potential signal peptide regions (SPSCAN) or for the identification of Lipoproteins (MOTIFS).  

In a part of the characterized clones could not complete Gene sequence can be determined since the cloned DNA fragment is not the whole gene contained. The available DNA fragment is used to obtain a full-length DNA fragment from the original library isolate. From these clones can then be gene-specific Primer and a vector-specific primer for the missing gene sequences amplified and then sequenced directly.  

literature

Goodwin, CS, Armstrong, JA, Chilvers, T., Peters, M., Collins, MD, Sly, L., McConnell, W., Harper, WES (1989) Transfer of Campylobacter pylori and Campylobacter mustelae to Helicobacter gen. Nov , as Helicobacter pylori comb. nov. and Hellcobacter mustelae comb. nov., respectively. Int J Syst Bact 39: 397-405.
Lee, A., Fox, J., Hazell, S. (1993) Pathogenicity of Helicobacter pylori: a perspective. Infect Immun 61: 1601-1610.
Malfertheiner P. (1994) Helicobacter pylori - From the Basis for Therapy. Bayerdörfer E, Birkholz S. Börsch G. et al., (Eds.) Stuttgart, New York: Georg Thieme Verlag; p. 1-104.
Marshall, BJ, Royce, H., Annear, DI, Goodwin, CS, Pearman, JW, Warren, JR, Armstrong, JA (1984) Original isolation of Campylobacter pyloridis from human gastric mucosa. Microbios Lett 25: 83-88.
Nomura, A., Stemmermann, GN, Chyou, PH, Kato, I., Perez-Perez, GI, Blaser, MJ (1991) Helicobacter pylori infection and gastric carcinoma among Japanese americans in Hawaii. N Engl J Med 325: 1132-1136.
Parsonnet, J., Friedman, GD, Vandersteen, DP, Chang, Y., Vogelman, JH, Orentreich, N., Sibley, RK (1991) Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med 325: 1227-1131.
Pugsley, AP (1993) The complete general secretory pathway into Gram-negative bacteria. Microbiol Rev 57: 50-108.
Solnick, JV, Tompkins, LS (1993) Helicobacter pylori and gastroduodenal disease: pathogenesis and host-parasite interaction. Infect Ag Dis 1: 294-309.
Tadayyon, M., Broome-Smith, JK (1992) TnblaM - a transposon for directly tagging bacterial genome encoding cell envelope and secreted proteins. Gene 111: 21-26.
Warren, JR, Marshall, B. (1983) Unidentified curved bacilli on gastric epithelium in active chronic gastritis. Lancet i: 11273-1275.

Claims (57)

1. A method for providing means for detecting, preventing or / and for the treatment of microbial infections, characterized in that it comprises the steps:

• A) Identification of essential genes and the corresponding polypeptides by production of deficient microorganisms by conditional antisense inhibition (CAI) or / and subtractive recombination mutagenesis (SRM) and determining the viability and / or survivability of the gene-deficient microorganisms in a test system.
• B) Identifying specific drugs which are directed against the essential polypeptides and cause the inactivation of the microorganisms or microorganisms used.
• C) testing the identified active ingredients for their applicability as components of diagnostic, preventive or / and therapeutic agents,
• D) Formulating the applicable active ingredients as diagnostic, preventive and / or therapeutic agents.

2. The method according to claim 1, characterized, that obligate essential genes are identified by CAI. 3. The method according to claim 1, characterized, that facultatively essential genes are identified by SRM.   4. The method according to any one of the preceding claims, characterized, in that before step (A) a selection is made for genes which are responsible for Code polypeptides with a specific functionality and / or the code for polypeptides that are in a particular developmental stage are expressed. 5. The method according to any one of the preceding claims, characterized, that the selection was carried out by means of hybridization methods is selected from subtraction and array methods. 6. The method according to claim 5, characterized, that the selection is based on specific subtracted nonpathogenic or pathogenic genes is performed. 7. The method according to claim 5, characterized, that the selection for specific subtracted genes of H. pylori or H. heilmannii is performed. 8. The method according to any one of claims 4 to 7, characterized, selecting gene sequences encoding exported polypeptides become. 9. The method according to any one of claims 4 to 8, characterized, that selected for secreted polypeptides gene sequences become.   10. The method according to any one of claims 4 to 9, characterized, in that genes coding for polypeptides are selected which belong to the Development of the vital form from the form of survival necessary are. 11. The method according to any one of claims 4 to 9, characterized, in that genes coding for polypeptides are selected which belong to the Development of the form of survival from the vital form necessary are. 12. The method according to any one of the preceding claims, characterized, that in step (A) for determining the life and Survival of the gene-deficient microorganisms Test systems selected from in vitro systems, cell culture systems, Tissue culture systems and animal models as a natural environment be used. 13. The method according to claim 12, characterized, that those deficient gene sequences which are not cultivable and unsustainable in natural environment lead to deficient microorganisms, the category of obligate be assigned to essential genes. 14. The method according to claim 12, characterized, those deficient gene sequences which are to be cultured but deficient in natural environment deficient  Lead microorganisms, the category of facultative essential Genes are assigned. 15. The method according to any one of the preceding claims, characterized, the identified genes used to make primers be, with the help of which genes from related Microorganisms, subspecies and / or species can be identified. 16. The method according to any one of the preceding claims, characterized, that in step (B) specific active substances are identified which the expression, presentation or / and function of the essential Influence polypeptides, in particular immunologically effective Substances, binding partners of the polypeptides or fragments thereof or / and inhibitory substances. 17. The method according to any one of the preceding claims, characterized, that step (B) is a determination of the immunogenic potential of the Polypeptides and / or fragments thereof, wherein the Identified genes are expressed and then a Western blot analysis is performed and / or that with the Identified polypeptides or fragments thereof vaccinate carried out in cell culture or animal model and triggering a specific immune reaction is observed. 18. The method according to any one of the preceding claims, characterized, that step (B) is a determination of the binding potential of the Polypeptides or fragments thereof by screening  Substance libraries, surface display methods, kristallo graphical analysis and / or computer modeling. 19. The method according to any one of the preceding claims, characterized, that the diagnostic, preventive or / and therapeutic agents in the form of passive vaccines or active vaccines to be provided. 20. The method according to claim 19, characterized, that the passive vaccines in the form of antibodies or / and Antibody fragments and the active vaccines in the form of heterologous carrier systems and / or in the form of antigens, Antigenic fragments, subunit vaccines, live vaccines, DNA Vaccines and / or food vaccines. 21. The method according to any one of the preceding claims 1 to 19, characterized, that the diagnostic, preventive or / and therapeutic agents include inhibitory substances, in particular expression inhibi gates and / or enzyme inhibitors. 22. A method for identifying essential microbial genes, characterized in that it comprises the steps:

• a) production of deficient microorganisms,
• b) determining the viability and / or survivability of the gene-deficient microorganisms of (i),
• c) identifying a protein coding portion of a microbial DNA sequence in which the deficient microorganisms are deficient.
• d) characterization of those DNA segments essential for survival.

23. The method according to claim 22, characterized, that the gene-deficient microorganisms are produced by a DNA segment is mutagenized in a microbial genome. 24. The method according to claim 22, characterized, that the DNA segment is mutagenized by transposon mutagenesis becomes. 25. The method according to claim 23, characterized, that the mutagenization of the DNA section on the microbial Genome is made by homologous recombination. 26. The method according to claim 25, characterized, that the SRM method is used. 27. The method according to claim 23, characterized, that the gene-deficient microorganisms are produced by in microorganisms, a DNA segment or a partial sequence thereof expressed in the form of antisense RNA. 28. The method according to claim 27, characterized, that the CAI method is used.   29. The method according to any one of claims 22 to 28, characterized, that for determining the life or / and survivability of the Deficient microorganisms Test systems selected from in vitro systems, cell culture systems, tissue culture systems and Animal models can be used as a natural environment. 30. The method according to claim 29, characterized, that those deficient gene sequences which are not cultivable and unsustainable in natural environment lead to deficient microorganisms, the category of obligate be assigned to essential genes. 31. The method according to claim 29, characterized, those deficient gene sequences which are to be cultured but deficient in natural environment deficient Lead microorganisms, the category of facultative essential Genes are assigned. 32. The method according to any one of claims 22 to 31, characterized, in that identifying the protein-encoding DNA portion by Expression in a host organism and detection of Presence of an expression product takes place. 33. The method according to any one of claims 32 to 32, characterized in that it further comprises:

• a) Preparation of primers for the amplification and detection of homologous gene sequences in heterologous microorganisms
• b) identifying the homologous gene sequences.

34. nucleic acid encoding an essential secretory gene Helicobacter, identified by the method of one of Claims 22 to 33. 35. Nucleic acid according to claim 34, characterized, that it encodes a secreted polypeptide with signal peptide. 36. Nucleic acid according to claim 34, characterized, that it encodes a secreted polypeptide without signal peptide. 37. Nucleic acid, characterized in that it

• a) one of the nucleic acid sequences shown in SEQ ID NO: n, where n is an odd integer from 1 to 113 inclusive, or a protein coding portion thereof,
• b) a nucleotide sequence corresponding to one of the sequences from (a) in the context of the degeneracy of the genetic code or
• c) a nucleotide sequence which hybridizes with one of the sequences from (a) and / or (b) under stringent conditions.

38. Genbank comprising at least two nucleic acids according to one of Claims 34 to 37.   39. vector, characterized, in that it comprises at least one nucleic acid according to one of claims 34 to 37 or a section thereof. 40. Vector according to claim 39, characterized, that he is a CAI vector. 41. Vector according to claim 39, characterized, that he is an SRM vector. 42. cell, characterized, in that it comprises a nucleic acid according to one of Claims 34 to 37 or a vector according to any one of claims 39 to 41 is. 43. mutant bank, characterized, that it consists of at least two microorganisms, which with a Vector according to claim 40 or a vector according to claim 41 are transformed. 44. Polypeptide, characterized, that it is from a nucleic acid according to any one of claims 34 to 37 is encoded.   45. Polypeptide according to claim 44, characterized in that it

• a) one of the amino acid sequences shown in SEQ ID NO: m, where m is an even integer from 2 to 114 inclusive, or
• b) comprises an immunologically cross-reactive with one of the sequences according to (a) sequence.

46. Polypeptide according to claim 45, characterized, that it is an essential secreted polypeptide. 47. polypeptide fragment, characterized, that there is an immunogenic portion of one of the sequences Claim 45. 48. Inhibitory molecule obtainable by the method according to Claim 1, characterized, that it is capable of specific to a polypeptide or fragment thereof according to any one of claims 44 to 47 to bind and / or its expression, presentation and / or natural function too influence. 49. A method of producing a polypeptide or polypeptide fragment according to one of claims 44 to 47, characterized, that is a cell with a nucleic acid according to any one of claims 34 to 37 or a vector according to claim 39, which transformed cell under conditions cultured in which a  Expression of the polypeptide takes place and the polypeptide from the Cell or / and the culture supernatant isolated. 50. Use of a polypeptide or a fragment thereof according to any of claims 44 to 47 thereof as an immunogen for production of antibodies. 51. Antibody or fragment thereof, characterized, that it is specific for a polypeptide or fragment thereof one of claims 44 to 47. 52. Pharmaceutical composition, characterized in that it as active ingredient

• a) a nucleic acid according to any one of claims 34 to 37,
• b) a vector according to claim 39,
• c) a cell according to claim 42,
• d) a polypeptide or a fragment thereof according to any one of claims 44 to 47,
• e) an antibody or fragment thereof according to claim 51 and / or
• f) an inhibitory molecule according to claim 48, optionally together with conventional pharmaceutical auxiliaries, diluents, adjuncts and carriers.

53. Use of a pharmaceutical composition according to Claim 52 for the diagnosis, prevention or / and therapy of a Helicobacter infection. 54. Use of a pharmaceutical composition according to Claim 52 for inhibiting the reproduction of Helicobacter  Organisms and / or other anthrogenic microorganisms in a host. 55. Use according to claim 54, characterized, in that a nucleic acid according to one of claims 34 to 37 is used as DNA Vaccine is formulated. 56. Use according to claim 54, characterized, that a polypeptide or polypeptide fragment according to any one of Claims 44 to 47 as a subunit vaccine or as a live vaccine is formulated. 57. Use of a pharmaceutical composition according to Claim 52 for the preparation of a diagnostic agent, Prevention and / or therapy of Helicobacter infection.