JP2004534526A5 - - Google Patents

Description

Methods for identifying proteins from intracellular bacteria

  The present invention relates to a novel combination of methods that allows the identification of proteins secreted from intracellular bacteria regardless of the secretory pathway. The invention further provides proteins identified by these methods. Secreted proteins are known to be suitable candidates for immunogenic compositions and / or diagnostic subjects. The invention also provides peptide epitopes (T cell epitopes) derived from the identified secreted proteins, as well as nucleic acid compounds encoding these proteins. The invention further includes various applications of the protein or fragments thereof, such as pharmaceutical and diagnostic applications.

  Chlamydia is an obligate intracellular bacterium that grows in eukaryotic host cells and is an important human pathogen. The order Chlamydia includes one family (Chlamydiaceae), which includes one genus (Chlamydia spp.), Which comprises four species: C. cerevisiae. trachomatis (Chlamydia trachomatis), C.I. pneumoniae (Chlamydia pneumoniae), C.I. psittaci (Parrot Chlamydia) and C. pecorum.

  C. Human pathogenic subtypes of trachomatis are transmitted by sexual contact and AC that cause ocular diseases and cause complications such as urethritis or salpingitis, epididymitis, and ectopic pregnancy DK, and L1 to L3, which cause severe systemic infections, leukemic lymphogranulomatosis (LGV). Human pathogen C. Pneumoniae is a cause of respiratory tract infections that causes bronchitis and pneumonia and has recently been linked to the development of atherosclerosis (Saikku et al., 1988 [1.], Shor et al., 1992 [2. ]).

  If left untreated, chlamydial infections can become chronic with severe complications such as sterility, blindness and possibly thrombosis.

  Depending on the developmental cycle within the cell, persistent chlamydial infection may cause an abnormal immune response and may not reveal the bacterium. Many immunogenic chlamydial proteins, especially C. Major outer membrane protein (MOMP), which is the main antigen of Trachomatis [7. ] As well as stress response proteins such as Hsp60 [8. ] Are considered vaccine candidates. However, none of these candidates have proven to be effective in vaccine trials.

  It is the intracellular nature of the bacteria that can adequately account for the limited humoral response and little protective immunity. Therefore, one alternative approach is to discover proteins that can be recognized by the cell-mediated immune system, and this approach is mainly used in elucidating chlamydial infections due to the effects of cytotoxic T lymphocytes (CTL). Has been shown to be essential (Iguitseme et al., 1994 [9.]).

  A great deal of attention is being focused on secreted proteins. Because they can be processed by the host cell proteasome and presented as MHC class I antigens on the surface of the cells, thus making them distinct vaccine targets (Hess and Kaufmann, 1993 [45]). An example of this is shown for Yersinia-infected cells, which present an epitope of the YopH effector to MHC-restricted cytotoxic T lymphocytes (CTLs) (Starnbach and Bevan, 1995 [14.]). . The interaction between Chlamydia and host cells is essential for bacterial survival and growth in the cells.

  C. trachomatis subtype D (Stephens et al., 1998 [4.]) (including 894 predicted open reading frames (ORFs)); For pneumoniae VR1310 (containing 1073 ORFs) (Kalman et al., 1999 [5.]), there is a complete and searchable Chlamydia genome. Further, C.I. trachomatis MoPn (Read et al., 2000 [12.]); The complete genomes of C. pneumoniae AR39 (Read et al., [12.] and C. pneumoniae J138 (Shirai et al., 2000 [13.]) are publicly available. From the genomic sequence, Chlamydia is a type III strain of other bacteria. It is known to have genes involved in the secretory machinery, including some genes that are homologous to secreted genes (Stephens et al., 1998 [4.] and Kalman et al., 1999 [5.]).

  Candidate secretory effector proteins may be present in type III secretory subgroups (Subtil et al., 2000 [10.]). This view was recently suggested by the discovery of the type III secretion property of CopN (Fields and Hackstadt, 2000) [11. ]. However, type III secreted proteins lack recognizable signal peptidase cleavage sites, and the consensus sequence of proteins secreted by this system in chlamydia has not yet been recognized. Thus, this may be limited to the particular bacteria in question. In addition, secreted proteins may be a functionally diverse group of proteins at unpredictable locations in the genome (Subtil et al., 2000 [10.]).

  Current knowledge of secreted Chlamydia effector proteins is available in the Inc family (Rockey et al., 1995) [15. [16. ], CopN (Fields and Hackstadt, 2000) [11. ]) And CT529 (Fling et al., 2001) [37. Are limited to proteins present in inclusion body membranes.

  Chlamydia specific CD8 + T cells have been shown to arise during infection, indicating that Chlamydia proteins, essential for presenting MHC class I antigens, are exposed to the host cell cytoplasm. means. CT529 has been identified from genomic libraries by expression in eukaryotic cells and recognition by Chlamydia specific T cell lines (Probst) [41]. CT529 has been shown in mouse vaccine experiments to have an epitope that provides some protection against infection.

A genomic library of Chlamydia trachomatis subtype L2 is expressed in eukaryotic cells by transfection with a viral vector, and then screened on T cells specific for Chlamydia to contain MHC class I restricted epitopes. Detecting proteins is described in International Patent Application No. WO 00/34483 (Probst) [41].
Five positive clones have been identified by this method, one of which contains CT529. One other clone contained three open reading frames, while the remaining three clones were not further described. The drawback of such screening is that expression of bacterial proteins in eukaryotic cells differs from expression in bacteria in that the probability of processing by the proteasome and the presentation itself as MHC antigens are altered. The maintenance and stimulation of clones is different from the in vivo situation, and clones that recognize proteins that are inaccessible during normal infection may produce false positives. Another approach in the aforementioned patent application concerns the identification of candidates for vaccines directed to humoral immune protection.

  When searching for proteins secreted by intracellular bacteria, the direct idea is to isolate the cytoplasm from infected host cells and look for bacterial proteins. However, this strategy cannot be used for chlamydia due to the brittleness of the chlamydia network. Another approach would be to identify virulence factors that are often secreted proteins by transposon analysis. However, Chlamydia cannot be transfected. There are no strategies that can predict which proteins have been secreted, and genes encoding effector proteins suitable for vaccine development may be present at unpredictable locations in the genome.

  Therefore, there is a need for a reliable system that can limit the number of vaccine candidates in a cost-effective manner and includes minimal experimental steps.

  In [18], [35S] -methionine / cysteine-labeled C. elegans was used in combination with autoradiography by two-dimensional gel electrophoresis. trachomatis proteins, C. trachomatis. The effect of INF-γ on the protein expression of trachomatis A and L2 was examined. C. Some IFNs were added during infection of HeLa cells in culture with T. trachomatis A, resulting in some C. aureus. trachomatis A resulted in a marked down-regulation of the protein, whereas this effect was due to C. trachomatis A. Trachomatis L2 was not evident. C. In both trachomatis A and L2, it was observed that 〜30 and ４０40 kDa proteins were induced depending on INF-γ. Induction of these proteins was attenuated by adding sub-physiological amounts of L-tryptophan to the growth medium. Thereby, these C.I. The IFN-γ-mediated inducible properties of the trachomatis protein have been shown to be associated with IFN-γ-mediated upregulation of indoleamine 2,3 dioxygenase, a tryptophan-degrading host cell enzyme. INF-γ-induced C.I. One of the trachomatis proteins is C. trachomatis. compared to C. trachomatis L2, C. cerevisiae having significantly lower molecular weight (Mw). Trachomatis A moved to the protein position.

  In [19], INF-γ-inducible C. elegans described previously (Shaw et al., 1999). The trachomatis A and L2 proteins were further characterized. From prepared two-dimensional gels, using C. MALDI-TOF mass spectrometry followed by database search, C.I. The proteins were identified as trachomatis tryptophan synthase α (TrpA) and β (TrpB) subunits. The protein is C.I. It was also induced by INF- □ in trachomatis D, and in all three subtypes, this induction was prevented by adding more than physiological amounts of L-tryptophan. C. The TrpA of Trachomatis A is C. compared to C. trachomatis D and L2. The molecular weight (Mw) in trachomatis A migrated at the position of the smaller protein. C. in these subtypes. As is evident by analysis of the trpA gene of C. trachomatis, C. trachomatis. The TrpA of C. trachomatis A and C is It is truncated to ７7.7 kDa compared to TrpA of trachomatis D and L2. In trachoma-causing subtypes (C. trachomatis A, B and C), the truncation or absence of tryptophan synthase inhibits the ability to synthesize tryptophan, further reducing these subtypes to IFN-γ-mediated tryptophan. May be more susceptible to deficiency. This allows the human subtype C. The differences seen in the pathogenesis between C. trachomatis can be explained.

  In [43], degradation of the above-described secretory protein (p60) by the proteasome in host cells of the intracellular bacterium Listeria monocytogenes was examined. The general strategy used was the presence or absence of two peptide aldehydes: N-acetyl-Leu-Leu-norleucine (LLnL) and (benzyloxycarbonyl) -Leu-Leu-phenylalanine (Z-LLF), [ Based on a pulse chase assay using [35S] -methionine / cysteine labeling, these peptide aldehydes inhibit the proteolytic activity of eukaryotic proteasomes. Proteasome inhibitor treated and untreated L. p60 were used using polyclonal antibodies raised against p60. P60 was immunoprecipitated from lysates of J774 cells infected with Monocytogenes. Autoradiographic evaluation of immunoprecipitated labeled p60, separated by one-dimensional SDS PAGE, suggested that the proteasome inhibitor was able to inhibit proteasome degradation of p60. The number of p60-CTL epitopes per infected cell was reduced by treatment with LLnL and Z-LLF. This suggested a link between inhibition of p60 proteasomal degradation and p60-CTL epitope generation.

In [44], the mechanisms behind protective immunity, and the general characteristics of the immune response of cells to intracellular fines, were described with a focus on developing a practical recombinant vaccine against intracellular fines. . Strategies for developing antigen delivery systems, Mycobacterium bovis BCG and Salmonella typhi aroA ^- discussed with an emphasis on. These non-toxic intracellular bacteria can be genetically modified to deliver antigens that serve as vaccine targets by immune recognition. The author points out the advantages of using secreted proteins as targets for vaccine development. This is because these proteins are processed and presented to the cell-mediated immune system while the bacteria are still replicating in the host cell.

  The present invention involves the identification of secreted proteins by a combination of novel methods. The combination of methods described constitutes a secretome (a collection of secreted proteins) by subtracting the proteome of intracellular bacterial proteins from the entire proteome of bacterial proteins present in infected cells.

  Bacterial proteins can be selectively visualized by pulse labeling in the presence of inhibitors of eukaryotic protein synthesis, followed by two-dimensional gel electrophoresis and autoradiography. The protein profile from purified bacteria is comparable to that of the whole lysate of infected cells, and the secretome, a protein spot present in a different image, was obtained from a gel loaded with the whole lysate of infected cells using advanced mass spectrometry. Identify by method.

  The identified secreted proteins are further analyzed by sophisticated artificial neural networks to obtain peptide sequences that are predicted to be good T cell epitopes.

  In addition, proteins are selected whose turnover is slowed by inhibitors of the host cell proteasome. This is because these proteins are very susceptible to degradation into the host cell proteasome and are very likely to be presented as MHC class I antigens on the surface of the host cell.

  Compared to other strategies for identifying vaccine candidate proteins and epitopes, the present invention further limits the number of candidates, which can only be achieved by a combination of novel methods.

The present invention is based on the following observations.
-Proteins secreted into host cells from intracellular bacteria are not present in purified bacteria, but are present throughout the lysate of infected cells.
-The two-dimensional protein profile of the purified bacterium and the whole lysate of the infected cells can be visualized by two-dimensional gel electrophoresis using specific pulse labels of the bacterial proteins.
-Subtraction of the two-dimensional protein profile of the purified bacteria from the two-dimensional protein profile of the bacterial proteins present in the host cells allows identification of secreted proteins by mass spectrometry.
-Proteins secreted from intracellular bacteria can be processed by the host cell proteasome to produce MHC class I antigens that can activate T cells.
Secreted proteins exhibit long-term turnover in response to the addition of eukaryotic proteasome inhibitors and may be displayed on the surface of host cells. Identification of such proteins is possible by analyzing a two-dimensional protein profile.
T cell epitopes can be predicted by artificial neural networks tailored to recognize peptides with high affinity for the MHC class I complex.

  The following definitions are used for the present invention.

(Definition)
Secretome A protein very likely to be secreted from intracellular bacteria.

(Type III) secretion subgroup A group of Chlamydia genes having genes that are significantly homologous to Type III secretion genes from other organisms.
The definition of secretory group is defined as any gene known to be homologous to a gene associated with type III secretion in other bacteria (eg, Salmonella, Shigella, Yersinia) and up to four genes known to differ. Or, a set of ORFs (open reading frames) having unknown functions.

Proteasome inhibitor A chemically synthesized or naturally occurring compound capable of reversibly or irreversibly inhibiting the proteolytic activity of an activated 26S eukaryotic proteasome.
One skilled in the art will recognize that the proteolytic activity of the proteasome can be determined by several different activities (eg, chymotrypsin-like activity that cleaves behind large hydrophobic residues, trypsin-like activity that cleaves after basic residues, It will be understood to include postglutamyl hydrolase activity that cleaves behind, BrAAP that cleaves preferentially behind branched-chain amino acids, and SNAAP that cleaves behind small neutral amino acid subunits. Some compounds known to inhibit the proteasome are commercially available, mostly inhibitors of cell-penetrating peptide systems (eg, peptide aldehyde, peptide vinyl (vinyl) sulfone). Peptide-based inhibitors function as transition state analogs to form adducts with the active site of the proteasome, while naturally occurring clast-lactacystin-β-lactone activates the proteasome subunit. An irreversible modification of the site exerts a proteasome inhibitory effect. The use of these compounds and combinations thereof may successfully inhibit proteasome function and processing of MHC class I antigens (eg, MG115, MG132, MG262, PSI, clast-lactacystin-β-lactone, epoxy Mycin). The application of the proteasome inhibitor can be performed before, during, or after pulse labeling or tracking, at any time during the Chlamydia development cycle.

Host cells Host cells are eukaryotic cells that can be infected by intracellular bacteria.
One skilled in the art will recognize that a wide range of immobilized cell lines, including epithelial cell lines (eg, HeLa, Hep-2, BHK cells) or immobilized mononuclear cell lines (eg, U-937), are suitable hosts for infection by Chlamydia. Will understand. Immobilized host cells can be obtained from naturally occurring carcinomas or by transformation of primary cells with a virus (eg, SV40) that carries an oncogenic gene and results in unlimited cell division and growth. The definition of host cell also includes primary epithelial or endothelial mammalian cell lines, which can be obtained from a living mammal or by necropsy, can be grown for a limited time in vitro, It may be a culture of cells of an organ.

Genetically modified host cells One of skill in the art will recognize that suitable host cells are associated with genes involved in the Chlamydia vaccine development process, for example, genes encoding subunits of the proteasome, or MHC class I presentation It will be appreciated that host cells that are genetically modified to overexpress or suppress other genes encoding functionally important proteins are also included.

Proteasome The proteasome is the major enzyme complex of non-lysosomal proteolysis and is an essential component of the ATP-dependent proteolytic pathway that catalyzes the rapid degradation of many rate-limiting enzymes, transcriptional regulators and key regulatory proteins.
In eukaryotes, it is necessary to rapidly remove abnormal proteins, aggregated, unfolded, or normal host cell proteins, and proteins from intracellular bacteria present in host cells. Higher eukaryotic proteasomes are highly associated with the processing of MHC class I antigens that break down proteins into peptides, which are delivered to the surface of host cells for presentation as T cell epitopes.

Pulse and the label of the labeled proteins, radioisotopes (e.g. L- ^[35 S] - methionine, L-[methyl ^- 3 H] - methionine, L-[methyl ^{- 14} C] - ^{methionine, [35} S] - cystine , [ ³ H] -tryptophan or a combination thereof, within a period of time that inhibits host cell protein synthesis using a sufficient concentration of inhibitors of eukaryotic protein synthesis (e.g., 0. 5 hours, 1 hour, 2 hours, 4 hours, 6 hours).
Labeling can be performed throughout the Chlamydia development cycle. The labeling medium must be rich in nutrients to allow the growth of both host cells and pathogens while the infected cells are growing. Inhibition of host cell protein synthesis can be achieved by adding cyclohexamide or other inhibitors of host cell protein synthesis (eg, methine) during the labeling time, and only in intracellular bacteria that synthesize the protein. Has the effect of enabling the incorporation of radioactive amino acids. Proteolysis can be prolonged by adding an inhibitor of cell permeable proteolysis to the growth medium during the labeling time. It should be noted that the present invention is not limited to the use of radiolabels.

By tracking the pulse tracked labeled protein after its synthesis, the turnover time, eg, the amount of protein synthesized during the labeling time is preferably greater than 75% (eg, 80%, 90%, 95%, 99%). %, 100%) The time to decrease can be evaluated. This assessment is performed by measuring the optical density of a given protein spot in the gel before and after the chase time, which reveals how long the protein is present in infected cells. The chase is performed by replacing the labeling medium with a growth medium without radioactive amino acids and harvesting the infected cells at different times after labeling. Tracking is performed at different times after labeling (eg, 0.5 hours, 1 hour, 1.5 hours, 2 hours, 6 hours, 12 hours, 24 hours, 72 hours), and at various times after labeling, One can determine how long the protein is present in the infected cells. The mature form of a protein may be processed from the propeptide, resulting in the accumulation of the mature form during the chase time at the expense of reduced detection. Under these circumstances, mature proteins may accumulate before degradation during the chase time can be visualized. The time for protein degradation can be extended by adding inhibitors of cell degradation that are cell permeable to the growth medium during the chase.

Lysis Buffer The lysis buffer used in the present invention is a buffer used to lyse infected cells and solubilize proteins before two-dimensional gel electrophoresis.

  The lysis buffer was 9 M urea, 4% w / v 3-[(3-cholamidopropyl) dimethylammonium] -1-propanesulfonic acid (CHAPS; Roche, Germany), 40 mM Tris Base, 65 mM DTE, And 2% vol / vol Pharmalyte 3-10 (Amersham Pharmacia Biotech). To concentrate high molecular weight, hydrophobic proteins, the lysis buffer was 7M urea, 2M thiourea, 4% w / v 3-[(3-cholamidopropyl) dimethylammonium] -1-propane. Sulfonic acid (CHAPS; Boehringer Mannheim, Germany), 40 mM Tris Base, 65 mM dithioerythritol (DTE), and 2% vol / vol Pharmalyte 3-10 (Amersham Pharmacia Biotech) may optionally be included.

  It is understood that the lysis buffer can be varied to increase the solubility of a particular protein (eg, thiourea could increase the solubility of hydrophobic, high molecular weight proteins).

Secreted effector protein The term secreted effector protein refers to a protein that is secreted by the bacterium into the cytoplasm or intracellular organelle of a host cell.
Secreted effector proteins can have a profound effect on the host / pathogen relationship, and due to their presence in the host cell cytoplasm, are targeted to the proteasome and have MHC class I on the host cell surface. May be presented as an antigen. Secreted effector proteins are believed to be secreted by one of several Sec-dependent or independent systems (eg, types I, II, III, VI) described in the literature.

Intracellular bacteria Bacteria capable of infecting and growing in eukaryotic host cells (eg, Chlamydia, Salmonella, Shigella, Listeria, Legionella, Elnicia).
This definition means that obligate intracellular bacteria can only survive and grow using eukaryotic host cells, or can survive in both extracellular and intracellular environments. Includes intracellular bacteria, which are facultative intracellular bacteria.

Basic body (EB)
Aggregates of Chlamydia bacteria purified by ultracentrifugation and characterized by electron microscopy as approximately 300 nm in diameter and having condensed nuclei.

Reticulated body (RB)
An assembly of Chlamydia bacteria purified by ultracentrifugation and characterized by electron microscopy to be about 1000 nm in diameter and to have normal bacterial nuclei.

Analytical gel A two-dimensional PAGE gel loaded with the required amount of protein sample to visualize the protein. The amount applied for analytical purposes in the examples described herein is typically between 200.000 and 300.000 counts per minute (cpm) and for stained gels (eg silver stain, Coomassie stain)> 100 μg protein.

Significantly reduced intensity / volume The average optical density of the entire area of a given spot located on a two-dimensional PAGE gel is preferably greater than 10% (eg, 20%, 35%, 50%, 65%, 80%, 90%, 100%) Reproducible detectable drop.

Significantly increased intensity / volume The average optical density of the entire area of a given spot localized on a two-dimensional PAGE gel is preferably greater than 10% (eg, 20%, 30%, 45%, 60%, 75%, 90%, 100%, 150%, 200%, 300% or more) Reproducible detectable increase.

Preparative Gel The amount of protein sample required to identify a specific protein spot is loaded by one of the identification methods described herein (eg, MALDI-MS, ESI-Q-TOF, Edman degradation). Also, a two-dimensional PAGE gel.
Typically> 500 μg is loaded on the gel for preparation purposes, depending on the fixed pH gradient used. The definition of a preparative gel for use in the present invention refers to a protein that is unfixed or fixed using a staining protocol (eg, silver stain, Coomassie stain) or electroblotted on a PVDF membrane. A gel having the formula: By applying a background amount of labeled protein that is separated with the unlabeled protein to the preparative gel, the proteins on the preparative gel can be made visible. By comparing such a gel with an analytical gel, it is also possible to accurately extract the target protein.

Vaccine candidate A protein potentially secreted from intracellular bacteria based on the results obtained by the method of the invention.
Because secreted proteins are susceptible to degradation by the host cell proteasome, peptides derived from these proteins may be presented as MHC class I antigens on the surface of infected cells, thus making them recognizable by T cells. Become. Thus, such proteins would be obvious targets for vaccine development against intracellular bacteria. The proteins described as vaccine candidates are also useful as components of diagnostic tests.

Vaccine In the context of the present invention, the term vaccine is to be understood in a broad sense as an immunogenic composition capable of inducing adaptive immunity (humoral or cellular).
Vaccine candidates capable of inducing adaptive immunity can be administered to animals or human recipients as injectables, either in solution, suspension or emulsion. The vaccine candidate, the active ingredient of the immunogenic composition, can be mixed with pharmaceutically acceptable excipients, such as water, saline, glycerol and ethanol, before injection into the recipient. Injections can be performed in different ways (eg, subcutaneously or intramuscularly). The vaccine candidate can supply the vaccine i) its full length or ii) as an origin to provide an immunogenic fragment, eg, a T cell epitope. It is recognized that certain proteins or peptides, alone or in combination with other proteins or peptides, can be administered to an animal or human recipient to contribute as a vaccine.

  In addition, a DNA fragment encoding a vaccine candidate protein can be cloned into a vector, which can be introduced into an animal or human recipient by injection. The DNA fragment will be taken up, for example, by muscle cells, expressed under the control of a promoter, and activated in eukaryotes. In this so-called DNA vaccine, the expressed DNA fragment can stimulate the immune system.

MHC class I antigens Major histocompatibility class I antigens include peptides from proteins that are exposed to the cytoplasm of the host cell, bind to heterodimeric MHC class I molecules in the endoplasmic reticulum, 2.) Presented on the surface of bound cells in the groove of the HLA complex, where this antigen can serve as a T cell epitope. Most MHC class I presentation peptides are derived from proteins that are processed by the activated 26S proteasome in the cytoplasm of the host cell.

HLA
Human leukocyte antigen, the name of human major histocompatibility complex.

T cell epitopes that bind to dimeric MHC class I molecules on the surface of cells and can be recognized by certain cytotoxic T cell receptors, typically consisting of 8-10 amino acids, Short length peptides.

Whole cell lysate Infected host cells harvested directly in lysis buffer without prior purification or fractionation.
Whole cell lysates can be obtained at any point during the Chlamydia development cycle, and include a mixture of all proteins present in the infected host cell, including bacterially derived proteins. It is understood that there will be.

Bacteria purified from infected cells.
Using chlamydia as an example, it is possible to purify both RB and EB, as well as intermediate forms of chlamydia, by density gradient ultracentrifugation, depending on the point in the development cycle of harvesting chlamydia. Purity can be measured by electron microscopy. In the present invention, assessing the contribution of a very abundant host cell protein (eg actin, beta tubulin, alpha tubulin, calreticulin) to the overall optical density of all proteins present on the gel Thus, the purity can be easily confirmed even on a silver-stained two-dimensional gel.

The names of the proteins identified using the identified protein mass spectrometry or identification methods based on the Edman degradation method are listed at http://socrates.com, available on Chlamydia Genome Project and published at: berkley. edu: 4231 / indicated according to that used for and in accordance with the nomenclature compatible therewith.
i) Stephens, R .; S. Kalman, S .; Lammel, C.I. Fan, J .; Marathe, R .; Aravind, L .; Mitchell, W .; Olinger, L .; Tatusov, R .; L. , Zhao, Q. et al. Koonin, E .; V. Davis, R .; W. (1998) Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis.
ii) Kalman, S .; Mitchell, W .; Marathe, R .; Lammel, C.I. Fan, J .; Hyman, R .; W. Olinger, L .; Grimwood, J .; Davis, R .; W. Stephens, R .; S (1999). Similar genomes of Chlamydia pneumoniae and Chlamydia trachomatis (Comparative genomes of Chlamydia pneumoniae and C. trachomatis.) Nat Gent. 21: 385-389

ELISPOT
In the ELISPOT method, T cells stimulated with antigen in vitro are incubated in microtiter wells that have been previously coated with anti-cytokine (eg, IFN-g, IL-6, TNF-a) antibodies. After the incubation period, local production of cytokines around activated T cells can be visualized by adding a secondary antibody conjugated to an enzyme such as horseradish peroxidase alkaline peroxidase. The assessment of cytokine production is performed by adding last a substrate which is converted into a colored substance by the enzyme and can thus visualize the cytokine producing cells.

Adjuvant An adjuvant refers to an emulsion containing a specific immunogen that can induce an immune response in a mammalian recipient (eg, Freund's adjuvant). It is understood that adjuvants and immunogens can be supplemented with components such as dried bacteria or bacterial products, which will enhance the immune response of the immunized mammal. Also, immunomodulators such as lymphokines (eg, IFNg, IL12) or poly I: C can be administered with the immunogen and adjuvant.

Emergence of different classes or subclasses of antibodies due to response to seroconverted antigens.

Micro immunofluorescence (MIF or micro-IF)
Assay to measure antibodies to fixed bacteria or proteins by immunofluorescence microscopy.

A protein or peptide is immunogenic if adaptive immunity can be induced by injection into an immunogenic human or animal.

(Detailed description of the invention)
Comparison of protein profiles by two-dimensional PAGE in whole cell lysates and purified bacteria Proteins present in all infected cells but not in purified bacteria may have been secreted from bacteria, for example, Chlamydia. Thus, the first method of the present invention provides a two-dimensional PAGE protein profile of [35S] -labeled Chlamydia protein from whole cell lysates of infected cells labeled at different points in the development cycle, [35S] Comparison of protein profiles of labeled purified bacteria by two-dimensional PAGE. This method allows for the detection of some proteins that are clearly present in the protein profile of whole cell lysates, but very little or not present in the protein profile of purified bacteria.

  These studies show that, by high-resolution two-dimensional PAGE (IPG), from a total of approximately 600 protein spots visualized in whole cell lysates 22-24 hours post-infection, their intensities were in elementary bodies (EB). Significantly reduced, 77 C.I. The presence of the trachomatis D protein was elucidated. Similarly, the protein of 91 was found to be C. erythrocyte when compared to labeled EBs and purified EBs from 55 to 57 hours post-infection. The strength in pneumoniae VR1310 was greatly reduced.

  The protein detected and examined is C.I. For T. trachomatis D, the protein numbers DT1 to DT77 listed in Table I are used. pneumoniae has the molecular weight (Mw) and isoelectric point (pI) described in CP1 to CP91 listed in Table II.

  This method provides an overview of potentially secreted proteins that need further investigation. In the examples, i) C. trachomatis corresponding to the labeling of whole cell lysate, FIG. 1 or 2) C. pneumoniae, labeled at each time point during the entire developmental cycle, FIG. 4 shows a comparison between cell lysate and purified EB.

  By purifying RB, it is possible to distinguish between secretory proteins and RB-specific proteins. The protein profile of the whole lysate of the infected cells can be compared to the protein profile of RB purified at the same time point to identify secreted proteins. In this approach, RB-specific proteins will not be detected as false positives. At the time of the investigation, proteins synthesized and secreted would be detected. The protein may be synthesized and secreted at other times.

  This method also involves the detection of proteins secreted immediately after infection, which may have been synthesized during a previous developmental cycle. These proteins infect the labeled EBs during the previous development cycle and are then visualized by two-dimensional PAGE of whole cell lysates. Early in the development cycle before EBs differentiate into RBs, permeation of saponins through the cell membrane results in the cytoplasm of host cells [30, 31]. This is only possible at this time, since there is likely to be no contamination of the Chlamydia proteins from the destroyed RB.

Identification of Vaccine Candidates Vaccine candidate proteins excised from the preparative two-dimensional gel are identified by advanced mass spectrometry.

  The excised spot is digested with an enzyme such as trypsin, which results in some tryptic peptides. MALDI-MS or other techniques determine the mass of these peptides to better than 100 parts per million (ppm). The mass obtained is matched to the theoretical trypsin cleavage products for all proteins present in the database using MS-Fit or Peptide search software that can identify the analyzed protein on a statistical basis. Let it.

  When a protein spot is excised from a gel with a whole cell lysate, the proteins of the contaminated host cells can be co-located with the bacterial proteins, and more complicatedly, two spots per spot It may contain these bacterial proteins. To avoid interference from contamination that can lead to unambiguous identification, if necessary, the sequence information of the bacterial protein can be obtained, for example using ESI-Q-TOF or post source decay (PSD).

  This method is described in Table III (A and B respectively) by C.I. trachomatis D or C.I. pneumonia VR1310, including proteins identified by mass spectrometry, as shown by example.

Therefore, in a first aspect, the present invention is a method for identifying a protein secreted from an intracellular bacterium,
1) infecting host cells with intracellular bacteria;
2) labeling intracellular bacteria present in the infected cells;
3) a) preparing a whole cell lysate of the infected cells, and b) preparing purified and lysed bacteria from the infected cells;
4) comparing the protein profiles by two-dimensional gel electrophoresis of i) the whole cell lysate from step 3a) and ii) the purified and lysed bacteria from step 3b);
5) detecting the protein spot obtained in step 4), which is present in whole cell lysate but not present in purified bacteria or present in a significantly reduced amount;
6) identifying the protein in the spot selected in step 5).

Vaccine candidate pulsing / tracking The purpose of this method is to detect secreted proteins that are degraded in host cells. A series of pulse / follow-up studies are performed to assess the time that the identified vaccine candidate protein is present in the host cell. [35S] The turnover time of the labeled protein is monitored on a two-dimensional gel by following the protein at various times after labeling. Turnover time provides valuable information about how fast the protein is degraded, and therefore how long the protein is present in infected cells.

  By this method, the potential secreted C. elegans exemplified in Table I. The estimated turnover time of the protein of Trachomatis is given.

Accordingly, in another aspect of the invention, the invention is a method for identifying a protein secreted from an intracellular bacterium, comprising:
1) infecting host cells with intracellular bacteria;
2) pulse labeling intracellular bacteria present in the infected cells;
3) preparing a whole cell lysate of the infected cells after different chasing times after step 2);
4) comparing the protein profile of the whole cell lysate prepared after the different chasing times obtained in step 3) by two-dimensional gel electrophoresis;
5) detecting the protein spot obtained in step 4), which is present in a smaller amount as the tracking time increases in step 3);
6) identifying the protein in the spot selected in step 5).

In order to limit the number of candidates suitable for a pulse tracking vaccine in combination with a proteasome inhibitor , the present invention includes a method using a proteasome inhibitor in combination with a pulse tracking study. These experiments provide an excellent tool for monitoring the effect of host cell proteasomes on the turnover time of secreted chlamydia proteins.

  Immunogenic proteins that are presented on the surface of eukaryotic cells as MHC class I antigens must be ubiquitous and must be cleaved by proteolysis of the multifunctional catalytic protein complex, the proteasome. The proteasome cleaves the immunogenic protein into peptides, typically 8 to 10 amino acids in length. These peptides are carried to the ER (ER), where they bind to heterodimeric MHC class I molecules, after which the MHC-antigen complex is carried to the cell surface. On the surface of cells, MHC-antigen complexes are recognized by specific receptors on cytotoxic T lymphocytes (reviewed in Rock and Goldberg [6.]).

  By adding cell permeable proteasome inhibitors such as peptide aldehydes to cell cultures, it is possible to inhibit the activity of eukaryotic proteasomes and prevent MHC class I presentation (Rock et al., 1994 [36. ]). Chlamydia proteins whose turnover time is prolonged by the addition of proteasome inhibitors are probably secreted from bacteria and subsequently processed by the proteasome. In addition, this point in the present invention allows for the detection of chlamydia proteins, which are degraded by the proteasome immediately after release into the host cell and therefore only detectable in the presence of a proteasome inhibitor.

  The present invention relates to C. elegans affected by proteasome inhibitors. trachomatis D and C.I. pneumonia VR1310 vaccine candidate.

  Proteins DT1, DT2, DT3, DT5, DT9, DT10, DT11, DT13, DT14, DT36, DT47, DT59, DT60, DT61, DT62 (described in Table IV below) add proteasome inhibitors during the chase time C. thereby extending its turnover time. It is an example of a vaccine candidate of trachomatis D.

  The present invention also provides a method for purifying RB that has been treated with a proteasome inhibitor during the labeling time prior to collection. Comparing proteasome-treated whole cell lysates, labeled simultaneously with purified RBs treated with proteasome inhibitors, will further elucidate which proteins are secreted into the host cell cytoplasm and degraded by the proteasome. Would. In addition, the host cell lines of these experiments can be genetically modified to overexpress genes that are essential for processing of MHC class I restricted T cell epitopes [38,39,40] (Sijts, 2000, Van Hall, 2000, Shockett, 1995). By using such a cell line, the effects of proteasome inhibitors will be more pronounced. Thus, the present invention also includes the use of commercially available host cell lines in which the genes associated with MHC class I antigen presentation are genetically modified.

Accordingly, the present invention, in another aspect, is a method for identifying a protein secreted from an intracellular bacterium, comprising:
1) infecting host cells with intracellular bacteria;
2) culturing host cells in the presence and absence of a proteasome inhibitor, respectively;
3) labeling intracellular bacteria present in infected cells, each cultured in the presence and absence of a proteasome inhibitor;
4) preparing a whole cell lysate of the infected cells;
5) comparing the protein profiles by two-dimensional gel electrophoresis of whole cell lysates of infected cells, cultured in the presence and absence of a proteasome inhibitor, respectively;
6) Present in whole cell lysates cultured in the presence of proteasome inhibitor, but not present or present in significantly reduced amounts in whole cell lysates cultured in the absence of proteasome inhibitor Detecting the protein spot obtained in step 5);
7) identifying the protein in the spot selected in step 6).

Generation of Polyclonal Antibodies The present invention provides polyclonal antibodies specific for vaccine candidates. The gene encoding the vaccine candidate protein is cloned using, for example, a ligand-independent cloning (LIC) system. Rabbits are immunized using the expressed fusion protein containing the sequence of the vaccine candidate to obtain serum containing a polyclonal antibody (PAb) specific for the vaccine candidate. The present invention uses PAbs in a two-dimensional PAGE immunoblot to confirm the correct specificity of the antibody by co-localization or to identify vaccine candidates of unrecognized isoforms. The present invention provides confirmation / identification of vaccine candidates as exemplified in Table III by immunoblotting and co-localization. The present invention uses PAbs to determine subcellular localization of vaccine candidates, for example, by indirect immunofluorescence microscopy.

Accordingly, the present invention also provides one of the above aspects, wherein the method comprises:
1) obtaining an antibody against a protein from said intracellular bacterium identified according to any of the foregoing methods;
2) applying a two-dimensional PAGE immunoblot to a whole cell lysate of cells infected with bacteria using the antibody obtained in step 1);
3) detecting the protein spot reacted in step 2);
4) identifying the protein in the spot selected in step 3).

  Combinations of the four aspects are also part of the present invention.

  In a preferred embodiment of the method of the present invention, labeling of the intracellular bacteria is by radioactive means such as [35S] cysteine, [35S] methionine, [14C] labeled amino acids, or a combination thereof.

  Methods for identifying proteins in selected protein spots include Edman degradation, MALDI TOF MS (matrix assisted laser desorption / ionization time-of-flight mass spectrometry), ESI Q-TOF MS (electrospray ionization quadruple). Polar time-of-flight mass spectrometry), any mass spectrometry such as PSD-MALDI MS (post-source resolved MALDI mass spectrometry), or a combination of such methods. In addition, the protein may be isolated prior to identification by a chemical method such as cyanogen bromide treatment or hydroxylamine treatment, or any suitable enzyme such as trypsin, slimotrypsin, chymotrypsin, or pepsin, or a combination thereof. Can be cut by a standard method.

  The intracellular bacterium may be a facultative intracellular bacterium or an obligate intracellular bacterium; pneumoniae, C.I. trachomatis, C.I. pistacci or C.I. Of particular relevance are bacteria from the genus Chlamydia, such as P. pecorum, including any particular subtype or strain thereof. However, other intracellular bacteria such as Salmonella, Shigella, Elnicia or Listeria are also of interest for the present invention.

  A host cell for use in accordance with the invention may be an immortalized cell line, such as HeLa, Hep2, McCoy or U937, a primary cell line obtained from a mammalian donor or obtained by necropsy, a genetically modified cell line, or an organ cell It can be a common host cell known in the art, such as a culture of, or any other cell capable of growing bacteria. The host cells can be treated with INF-γ prior to or during infection by intracellular bacteria and / or have been genetically modified to be involved in the Chlamydia vaccine development process. Can be overexpressed or suppressed.

  When the method of the present invention employs one or more proteasome inhibitors, known inhibitors such as MG132, MG262, MG115, epoximycin, PSI and clast-lactacystin-β-lactone are suitable for that use. I have.

  The method of the invention may be included in an immunogenic composition, either in its full length or as an immunogenic fragment thereof, and / or in a protein suitable for diagnostic purposes, in particular in an immunogenic composition. Of particular relevance to the identification of proteins that define suitable antigens and include T cell epitopes that are candidates for presentation as MHC class I or II, more preferably MHC class I restricted antigens.

  Thus, in another important aspect of the invention, the invention relates to a protein identifiable by any of the claimed methods, or an immunogenic fragment thereof, preferably in an immunogenic composition; and And / or immunogenic fragments thereof as applicable to diagnostic purposes.

  The protein of the present invention comprises C.I. trachomatis and C.I. It may be a protein secreted from Pneumoniae. Such proteins have, for example, isoelectric point (pI) and molecular weight (Mw) values given in Tables I and II, respectively, determined with an average error of +/- 10%, and DT1 given in Table I, respectively. -77, and the proteins characterized as CP1-CP91 given in Table II, and immunogenic fragments thereof.

More preferred proteins of the present invention are those of C.I. trachomatis protein, CT017 (gene name CT017: SEQ ID NO : 221; protein: SEQ ID NO: 222 ), CT044 (gene name ssp: SEQ ID NO : 227; protein: SEQ ID NO: 228 ), CT243 (gene name lpxD: SEQ ID NO : 223; protein: SEQ ID NO: 224 ), CT263 (gene name CT263: SEQ ID NO : 235; protein: SEQ ID NO: 236 ), CT265 (gene name accA: SEQ ID NO : 219; protein: SEQ ID NO: 220 ), CT286 (gene name clpC: SEQ ID NO : 229; protein) : SEQ ID NO: 230), CT292 (gene name dut: SEQ ID NO: 215; protein: SEQ ID NO: 216), CT407 (gene name DksA: SEQ ID NO: 211; protein: SEQ ID NO: 212), CT446 (hereditary Name EUO: SEQ ID NO: 207; protein: SEQ ID NO: 208), CT460 (gene name SWIB: SEQ ID NO: 203; protein: SEQ ID NO: 204), CT541 (gene name mip: SEQ ID NO: 209; protein: SEQ ID NO: 210), CT610 ( Gene name CT610: SEQ ID NO : 201; protein: SEQ ID NO: 202 ), CT650 (gene name recA: SEQ ID NO : 225; protein: SEQ ID NO: 226 ), CT655 (gene name kdsA: SEQ ID NO : 217; protein: SEQ ID NO: 218 ), CT668 (gene name CT668: SEQ ID NO: 195; protein: SEQ ID NO: 196), CT691 (gene name CT691: SEQ ID NO: 233; protein: SEQ ID NO: 234), CT734 (gene name CT734: SEQ ID NO: 213; protein: SEQ ID NO: 14), CT783 (gene name CT783: SEQ ID NO: 197; protein: SEQ ID NO: 198), CT858 (gene name CT858: SEQ ID NO: 199; protein: SEQ ID NO: 200), CT875 (gene name CT875: SEQ ID NO: 231; protein: SEQ No. 232 ) or a protein identified by the corresponding gene number in the Chlamydia Genome project as ORF5 (gene name ORF5: SEQ ID NO : 205; protein: SEQ ID NO: 206 ), or a protein identified by the protein name DT8, and C . pneumoniae, CPN0152 (gene name CPN0152: SEQ ID NO : 249; protein: SEQ ID NO: 250 ), CPN0702, CPN0705 (genename CPN0705: SEQ ID NO : 245; protein: SEQ ID NO: 246 ), CPN0711 (gene name CPN07111 :) given in Table IIIB SEQ ID NO: 263; protein: SEQ ID NO: 264 ), CPN0998 (gene name ftsH: SEQ ID NO : 239; protein: SEQ ID NO: 240 ), CPN0104 (gene name CPN0104: SEQ ID NO : 255; protein: SEQ ID NO: 256 ), CPN0495 (gene name aspC) : SEQ ID NO : 247; protein: SEQ ID NO: 248 ), CPN0684 (gene name parB: SEQ ID NO : 251; protein: SEQ ID NO: 252 ), CPN07 96 (gene name CPN0796: SEQ ID NO : 243; protein: SEQ ID NO: 244 ), CPN0414 (gene name accA: SEQ ID NO : 253; protein: SEQ ID NO: 254 ), CPN1016 (gene name CPN1016: SEQ ID NO : 237; protein: SEQ ID NO: 238 ) , CPN1040 (gene name CPN1040: SEQ ID NO : 241; protein: SEQ ID NO: 242 ), CPN0079 (gene name R110: SEQ ID NO : 257; protein: SEQ ID NO: 258 ), CPN0534 (gene name dksA: SEQ ID NO : 259; protein: SEQ ID NO: 260) ), CPN0619 (gene name ndk: SEQ ID NO: 261; protein: SEQ ID NO: 262), CPN0711 (gene name CPN0711: SEQ ID NO: 263; protein: SEQ ID NO: 264), CPN0 28 (gene name RS13: SEQ ID NO: 265; protein: SEQ ID NO: 266), CPN0926 (gene name CPN0926: SEQ ID NO: 267; protein: SEQ ID NO: 268), CPN1063 (gene name TpiS: SEQ ID NO: 269; protein: SEQ ID NO: 270) Or CPN0302 (gene name lpxD: SEQ ID NO : 271; protein: SEQ ID NO: 272 ) and the like, and an immunogenic fragment thereof.

The proteins DT4, DT23, DT47, DT48, DT75, DT76 and DT77 shown in FIG. 10 and the proteins CP34, CP37, CP46 (SEQ ID NO: 244) , CP47, CP52, CP63 and CP75 shown in FIG. There is. Further preferred proteins of the invention have a long turnover time in the presence of a proteasome inhibitor and the proteins DT1, DT2, DT3, DT5, DT9 given in Table IV determined with a mean error of +/- 10%. DT10, DT11, DT13, DT14, DT17, DT47, DT59, DT60, DT61 or DT62, is a protein characterized by having isoelectric point (pI) and molecular weight (Mw) properties. trachomatis protein. Similarly, C. elegans is controlled by proteasome inhibitors. Protein from Pneumoniae is also a preferred embodiment of the present invention.

  Other preferred proteins of the present invention are Chlamydia trachomatis polypeptide DT8 comprising the sequence of SEQ ID NO: 1, as defined in the claims, and immunogenic fragments thereof.

  In another preferred embodiment of this aspect of the invention, the protein is at least 40% sequence identity, preferably at least 60%, more preferably at least 70%, even more preferably, a protein of the invention or a fragment thereof. It has at least 80%, more preferably 90%, most preferably at least 95% sequence identity, or it comprises at least 7 contiguous amino acids of a protein of the invention.

  In another aspect of the invention, it relates to a nucleic acid compound comprising a sequence encoding a protein of the invention or an immunogenic fragment thereof.

  A preferred nucleic acid compound is a nucleic acid compound comprising a sequence encoding the polypeptide DT8 comprising the sequence of SEQ ID NO: 1 (SEQ ID NO: 2).

  In another aspect, the invention relates to a vector comprising a nucleic acid compound of the invention, and a host cell transformed or transfected with the vector.

  The present invention provides use of the protein of the present invention or an immunogenic fragment thereof for producing an antibody against the protein, and a method for producing an antibody against an intracellular bacterium. Also provided are a method of administering the fragment to a production animal and purifying the antibody therefrom, and an antibody obtainable by this method.

  Further, in another aspect, the present invention provides a pharmaceutical or diagnostic composition comprising the protein or fragment thereof of the present invention, an antibody or nucleic acid compound of the present invention, and a protein or fragment thereof, antibody or nucleic acid in the preparation of a diagnostic reagent. The use of a compound is provided.

Identification of T Cell Epitopes from Vaccine Candidates The present invention has been determined experimentally by assays based on computer-based methods from the sequence of the proteins identified in the present invention or as further described in the Examples. Thus, it provides a T cell epitope that is exposed to the surface as an MHC class I antigen and may have a T cell stimulating effect.

  Thus, another important aspect of the present invention is a peptide epitope that is presented on the surface as an MHC class I antigen and may have a T cell stimulating effect. Accordingly, the present invention relates to a method for identifying a T cell epitope on a secreted protein from an intracellular bacterium, which relates to a protein identified by the method of the present invention or an immunogenic fragment thereof, and a peptide epitope. Methods are provided that include steps such as computational prediction, MHC class molecule binding assays and / or ELISPOT assays. As part of this aspect, the invention also provides a nucleic acid compound comprising a sequence encoding the peptide epitope, a vector comprising the nucleic acid compound, and a host cell transformed with the vector.

  Preferred peptide epitopes of the invention comprise 4 to 25 contiguous amino acids of the protein of the invention, preferably 6 to 15, more preferably 7 to 10 amino acids.

  In a more preferred embodiment of the invention, the epitope is C. trachomatis or C.I. pneumoniae contains 7-10 contiguous amino acids of the protein.

  Another preferred peptide epitope of the present invention is an epitope comprising 4 to 25 contiguous amino acids, more preferably 6 to 15, most preferably 7 to 10 amino acids of a polypeptide comprising the sequence of SEQ ID NO: 1. It is.

  A peptide epitope of Chlamydia trachomatis comprising an amino acid sequence selected from the sequences of SEQ ID NOS: 3 to 45, a peptide epitope of Chlamydia pneumonia, comprising an amino acid sequence selected from the sequences of SEQ ID NOs: 46 to 121, SEQ ID NO: A peptide epitope of Chlamydia pneumonia, comprising an amino acid sequence selected from the sequence of SEQ ID NO: 122 to SEQ ID NO: 148, and a peptide epitope of Chlamydia trachomatis, including an amino acid sequence selected from the sequence of SEQ ID NO: 149 to SEQ ID NO: 194, Related. The identified epitopes of the invention are further characterized in Tables V-VIII.

  The peptide epitope may be part of a fusion protein or bound to a carrier component.

  A frequently used method for predicting peptides that bind to MHC involves motif search. The most sophisticated motif search uses the complete matrix representing the spread motif of the MHC. Although the specificity of the sequence-independent combination may be appropriate as an average consideration, it is certainly known that it is not appropriate for individual peptides. In addition, the crystal structure demonstrates that interactions at one subsite can affect interactions at other subsites.

  Artificial neural networks (ANNs) are well suited for handling and recognizing any such non-linear array of information. The information can be adjusted and distributed to a computer network having input, hidden, and output layers all connected in a structure by a weighted relationship. Such an ANN can be adjusted to recognize inputs (peptides) associated with a given output (eg, MHC binding). Once adjusted, the web should recognize a complex peptide pattern that is compatible with binding. Using the ANN approach, the size and quality of the tuned set becomes very important. This is especially true for HLA. Because only 1% of the random peptide set binds to any given HLA.

  Therefore, to generate as few as 100 peptide conjugates, screening and screening random peptides would require the synthesis and testing of approximately 10,000 peptides. This requires a significant source and is a nuisance even with this modest number of conjugates in a tuned set, and this would have to be repeated for each HLA tested.

  Thus, for the present invention, using the matrix predictions, scan the SWISS-PROT (http://www.expasy.ch/sprot/) database for epitopes that may bind with high affinity. did. Many of these have been synthesized and tested in biochemical binding assays. As expected, a higher presentation of the high affinity binder was obtained (about 80%). These data were then used to adjust the ANN.

  For four of the four MHC class I molecules tested, ANNs were performed that were better than the maternally matched predicates. The predictions were made in a manner that predicted the actual binding IC50 values, rather than the optional classification of “conjugated” and “unconjugated”. In fact, a wide range of binders can be expected, which has led to the identification of high and low affinity binders, and non-bound.

  The present invention further includes the use of a peptide epitope of the present invention to prepare a vaccine, and a vaccine comprising a peptide epitope of the present invention, optionally with an acceptable excipient.

  In another aspect of the invention, the invention relates to the preparation of a pharmaceutical composition for treating or preventing infection by an intracellular bacterium, such as a Chlamydia infection, or alternatively to an intracellular bacterium, such as a Chlamydia, or a cell. It relates to the use of a protein of the present invention, an antibody of the present invention, a nucleic acid compound of the present invention, or a peptide epitope of the present invention in the preparation of a diagnostic reagent for detecting the presence of an antibody produced against an endogenous bacterium.

  The present invention relates to a method of inducing an immune response in a human, comprising administering to said human an immunologically effective amount of a protein, antibody, nucleic acid compound or peptide epitope of the invention, in particular C . pneumoniae or C.I. Such methods are further provided for treating or preventing human infection by an intracellular bacterium such as Trachomatis.

  Finally, the present invention provides a method for producing a protein of the present invention or a fragment thereof, or a peptide epitope of the present invention, respectively, wherein host cells are transformed with a vector containing a nucleic acid compound encoding the protein or peptide epitope. A method is provided that comprises transforming, transfecting, or infecting, and culturing the host cell under conditions that allow the host cell to express the protein or fragment thereof.

  The invention is further illustrated by the following non-limiting examples.

Infection of Mammalian Cell Cultures Cell monolayers of semi-confluent HeLa, HEp2, or McCoy (ATCC, Rockville, MD, USA) were isolated [19. ] And [17. ] As described previously in C.I. pneumoniae VR1310, C.I. trachomatis subtype A (HAR-13), D (UW-3 / Cx) or L2. (434 / Bu) (ATCC) was infected with one inclusion body forming unit (IFU). The infection medium was C.I. For trachomatis A and D, RPMI 1640, 25 mM HEPES, 10% FCS, 1% w / v glutamine, 10 mg / ml gentamicin, C.I. trachomatis L2. For RPMI 1640, 25 mM HEPES, 5% FCS, 1% w / v glutamine, 10 mg / ml gentamicin.

Pulse labeling / chasing To label Chlamydia proteins in 2 hours, as previously described (Shaw et al., 1999, 2000) [18. [19. ], Incubation of infected cell cultures in a medium containing RPMI 1640, 10 mg / ml gentamicin, 40 μg / ml cycloheximide, 100 μCi / ml [35S] -methionine / cysteine (Promix, Amersham Pharmacia Biotech, Uppsala, Sweden). did. After labeling, the labeling medium was changed to normal growth medium and then washed twice with growth medium, and the infected cells were harvested at different times after labeling. Similarly, labeled EB proteins were obtained by growing Chlamydia up to 72 hours post-infection after a labeling time of 2 hours. Then, the labeled EB protein was collected and C.I. trachomatis (Schacter and Wyrick, 1994) [22. And C.I. Purification was performed using two sequential steps of density gradient ultracentrifugation essentially as described for Pneumoniae (Knudsen et al., 1999 [17.]). Proteins in EB and pulse chasing preparations were labeled at the same intervals as proteins from whole cell lysate preparations to facilitate comparison of protein profiles by accurate two-dimensional PAGE.

Sample preparation After [35S] -labeling, cells were washed twice in PBS, 9 M urea, 4% w / v 3-[(3-cholamidopropyl) dimethylammonium] -1-propanesulfonic acid (CHAPS) Roche, Germany), 40 mM Tris Base, 65 mM DTE, and Pharmalyte 3-10 (Amersham Pharmacia Biotech). To concentrate proteins that are high molecular weight and hydrophobic, 7M urea, 2M thiourea, 4% w / v 3-[(3-cholamidopropyl) dimethylammonium] -1-propanesulfonic acid (CHAPS) Boehringer Mannheim, Germany), 40 mM Tris Base, 65 mM dithioerythritol (DTE), and 2% vol / vol Pharmalyte 3-10 (Amersham Pharmacia Biotech), (Harder et al., 1999, 1923). Used according to. Samples containing whole cell lysates or purified EBs were sonicated and centrifuged at 10,000 xg for 10 minutes. Samples were stored at -70 C until use.

Separation of Chlamydia proteins Chlamydia proteins from whole cell lysates and purified EBs were purified (Shaw et al., 1999, 2000) [18. [19. ] Were separated by two-dimensional gel electrophoresis essentially as described in

  For the first dimension isoelectric point (pI) electrophoresis, 18 cm long pH 3-10 NL (non-linear), 4-7 L (linear) or 6-11 (linear) immobilized pH gradient dried strips ( Amersham Pharmacia Biotech) was reswelled using IP Gphor ™ at 20 ° C. for 12 hours with a sample volume of 200.000 counts per minute (cpm) of labeled protein dissolved in 350 μl of buffer. Other strips used in the present invention include ultra-thin IPG strips. These strips are listed in Table IX. These strips allow us to focus on specific pH intervals, including the protein of interest, if needed. The voltage during isoelectric point (pI) electrophoresis at 20 ° C. was programmed as follows: when using 3-10 NL, 4-7 L and 6-11 L dry strips, 300 V for 1 hour, 300- 2 hours at 500V (linear increase), 1 hour at 1000V, 1 hour at 2000V, 3 hours at 3500V and 24 hours at 5000V.

  After the first dimension isoelectric point (pI) electrophoresis, 6M urea, 30% v / v glycerol, 2% w / v DTE, 2% w / v SDS, 0.05M Tris HCl pH6 The dried strips were equilibrated for 15 minutes in a buffer containing 0.8. The strips were then equilibrated for another 15 minutes in a buffer in which the DTE was changed to 2.5% w / v iodoacetamide. For the second dimension isoelectric point (pI) electrophoresis, a 9-16% linear gradient SDS-PAGE gel (18 cm × 20 cm) using a Protean II xi Multicell system (Bio-Rad, Richmond, CA, USA). X 1 mm). The analysis gel was fixed in a solution containing 10% acetic acid and 25% 2-propanol for 30 minutes and treated with Amplify (Amersham Pharmacia Biotech) for 30 minutes. The labeled proteins were made visible by autoradiography 8-10 days after exposing Kodak Biomax-MR films (Amersham Pharmacia Biotech) at -70 ° C.

  FIG. 1A shows C.A. labeled from 22-24 hours post-infection. 1 shows an example of autoradiography of a 3 to 10 IPG two-dimensional gel (using a standard lysis buffer) of trachomatis D protein analyzed at high resolution. Figure 2A shows C.A. labeled from 55-57 hours post-infection. 1 shows an example of autoradiography of a 3 to 10 IPG two-dimensional gel (using a lysis buffer containing thiourea) of Pneumoniae protein. A total of about 600 protein spots could be visualized on each gel, as assessed by Melanie II software.

To prepare the sample for analysis by mass spectrometry, the two-dimensional gel was subjected to 500-1000 μg of whole cell lysate. To visualize the protein on a preparative gel on X-ray film, 2 × 10 ⁶ cpm [35S] -protein labeled protein from cells grown in parallel with the unlabeled sample was loaded on the same gel. gave. The preparative gel was washed in ddH2O for 10 minutes, dried and unfixed. Radioactive ink was used to mark the anchor spots on both sides of the gel so that accurate alignment of the dried gel with the corresponding X-ray film could be performed after exposure. The protein of interest was excised from a minimum of three identical gels and collected together. If host cell contamination was a problem in mass spectrometry identification, gels using miniature or ultrafine dry strips (Table IX) were used to increase the separation distance.

Identification of Vaccine Candidates Using MALDI MS, ESI Q-TOF MS, PSD-MALDI MS and Edman Degradation Protein spots from the gel for preparation of whole cell lysates representing vaccine candidates were subjected to in-gel digestion with trypsin. did. The resulting peptides were purified using reverse layer columns (Gobom et al., 1999 [20.]) or beads made of Poros R2 material (Gevaert et al., 1997 [21.]). The samples were then analyzed using a Bruker REFLEX MALDI time-of-flight mass spectrometer (Bruker-Daltonik, GmbH, Bremen, Germany) operated in a reflectron mode. The resulting mass was compared by peptide mapping as previously described (Schevchenko 1996 [24.]) to the mass of the peptide resulting from theoretical tryptic cleavage of the existing protein in the database.

One example of using MALDI-MS to identify DT1 as CT668 (SEQ ID NO: 196) is shown in FIG. FIG. 3A shows peptide mass fingerprints obtained by MALDI mass spectrometry. The resulting mass was matched to the theoretical tryptic cleavage products of all proteins present in the database using Prospector software MS-Fit. If the search was restricted to the isoelectric point (pI) / molecular weight (Mw) region near the protein found on the gel, the highest protein was CT668 (FIG. 3B). However, the identification may be unambiguous, as proteins from host cells are sometimes present in gels and spots from whole cell lysates. Therefore, where necessary, tandem mass spectrometry and post-source decomposition (PSD) analysis were used to verify the results (Mann and Wilm, 1995 [25.], Gevaert, 1997 [21.]). There).

  Tandem mass spectrometry of the peptides generated by in-gel digestion was performed on an electrospray ionization quadrupole time-of-flight (ESI Q-TOF) mass spectrometer (Micromass, Manchester, UK). Using this method, one ionized peptide can be isolated from a sample. Fragmentation of this parent ion upon collision with a gaseous atmosphere produced several new ions, which were recorded in a new peptide mass fingerprint. These new ions distinguished in size by a single amino acid, which gave details of the amino acid sequence of the original peptide (Mann and Wilm, 1995) [25. ].

FIG. 4A shows an example of the parent ion of the fragmented peptide from DT1 which gives a sequence found by database search using BLAST or MS-Tag to correspond to a fragment of CT668 (SEQ ID NO: 196) . Sequence tags resulting from human progesterone binding proteins were also identified from the CT668 sample.

  If the search is restricted to the human protein, peptides from this protein could also be detected in the peptide mass fingerprint by MALDI-MS (double arrow, FIG. 3A). This indicates that problematic spots containing more than one protein can be unambiguously identified by ESI Q-TOF, thus confirming the problematic MALDI MS identification.

  Another approach used in the present invention to confirm MALDI results was PSD. PSD uses peptides that have undergone metastable degradation after ionization, meaning that peptide fragments of the same rate have different masses and therefore different kinetic energies. The difference in kinetic energy can be elucidated by showing the fragments in a magnetic field. High energy fragments penetrate more into the magnetic field than low energy fragments and are therefore delayed. The spectra resulting from the fragmentation of one peptide can be used to deduce the amino acid sequence of a peptide sequence tag (PST) (Mann et al., 1993) [28. ]. This is because fragmentation occurs mainly at peptide bonds. PSTs can be matched against a database of proteins, thus identifying the protein from which they are derived (Wilkins et al., 1996 [27.]).

An example of identifying spot number CP63 as CPN1016 (SEQ ID NO: 238) by PSD-MALDI MS is shown in FIG. 4B. 16 out of a total of 36 observed masses in the PSD spectrum were identified as C.I. mass derived from theoretical fragmentation of a peptide having the sequence 1919.80 Da parent ion ( K ) ELLFGWDLSQQTQQAR (L) (SEQ ID NO: 274) , matched with the CPN1016 (SEQ ID NO: 238) protein from Pneumoniae, It could be matched within one mass unit.

  Using mass spectrometry techniques, C.I. trachomatis D (Table IIIA) and C. trachomatis D. An example of the identification of a vaccine candidate for Pneumoniae (Table IIIb) is provided. Molecular weight (Mw) and isoelectric point (pI) values were determined by electrophoresis with an average error of +/- 10%.

CT858 (SEQ ID NO: 200) has a theoretical molecular weight (Mw ) of 67 kDa, indicating that the protein identified was a processed fragment of a larger protein. In fact, all three peptides from the ESI Q-TOF analysis of DT4 were located in the C-terminal part of the protein.

Another spot, DT48, which was located in the basic region of the gel, also contained CT858 (SEQ ID NO: 200) . All peptides, with DT48 identified as CT858 (SEQ ID NO: 200) , are consistent with the N-terminal portion of the protein, suggesting that DT48 represents an N-terminal fragment of CT858.

CT610 (SEQ ID NO: 202) is a C.I. Both trachomatis D and L2 were identified from EBs by MALDI MS and Edman degradation. However, the protein was significantly reduced in EB compared to whole cell lysate and was therefore still considered as a vaccine candidate. Identification of CT610 (SEQ ID NO: 202) by Edman degradation was performed as described in C.I. trachomatis L2 CT610 (SEQ ID NO: 202) . The N-terminus was determined to be MNFLDQ, which is the Chlamydia Genome project (Stephens et al., 1998b) [35. ] And a MMEVFMNFLDQ sequence.

DT8 was identified based on four sequence tags generated by ESI TOF MS. These sequences are C.I. did not correspond to any predicted open reading frame in the genome of trachomatis D [35. ]. However, searching the Chlamydia genome in all six reading frames by BLAST could generate considerable consistency for all four sequence tags. Analysis of the DNA sequence encoding the peptide and its surroundings revealed a novel open reading frame containing a ribosome binding site, including a 7.2 kDa protein. The translated DT8 (SEQ ID NO: 1) DNA sequence is shown in FIG. This finding illustrates how the mass spectrometry techniques used in the present invention can identify possibly important ORFs encoding vaccine candidates that may be ignored in large genomic sequencing projects. Is done.

C. spot CP63 from C. pneumoniae was obtained from CPN1016, CT858 (SEQ ID NO: 200) . Identified as the N-terminal fragment of the pneumoniae homolog (FIGS. 2B and C, Table IIIB), indicating processing of these proteins in both Chlamydia species.

  The following examples provide a more detailed investigation of the properties of some examples of the identified proteins.

B. Comparison of total lysate of infected cells and purified RB trachomatis or C.I. Cells infected with pneumoniae were labeled with [35S] -methionine / cysteine in the presence of cycloheximide for 2 hours as described in Example 2. At the end of the labeling time, infected cells were harvested directly in lysis buffer as described in Example 3, or used to immediately purify Chlamydia RB. Purification of RB is described in Schacter and Wyrick, 1994 [22. ] By density gradient ultracentrifugation essentially as described by

In FIG. 10, C. from total lysates of infected HeLa cells labeled from 22-24 hours post-infection. Some examples of regions from gel images of the proteins of T. trachomatis D were obtained from labeled C. trachomatis D from 22-24 hours post infection. Compared to corresponding regions from RB and EB gel images purified from HeLa cells infected with Trachomatis D. Identification by mass spectrometry was as shown in Table III, DT4 (C-terminal fragment of CT858 (the sequence of CT858 is the sequence of SEQ ID NO: 200) ), DT48 (N-terminal fragment of CT858 is the sequence of SEQ ID NO: 200) ), DT23 (Mip; SEQ ID NO: 210), DT76 (virtual protein CT691 (SEQ ID NO: 234) ) and DT77 (virtual protein CT263 (SEQ ID NO: 236) ).

  In FIG. 11, labeled C. aureus 55-57 hours after infection. Some examples of gel images of HEp-2 cells infected with Pneumoniae and regions from gel images from purified EBs that were labeled during the developmental cycle were labeled 34-36 hours post infection or Either harvested as a total lysate of infected cells 36 hours post-infection or purified as RB 36 hours post-infection. Compare to the corresponding area from an image of a culture of cells infected with Pneumoniae.

The protein spots circled in FIG. 11 were identified as follows. FIGS. 11A and E show CP34 and CP63 identified as two fragments of CPN1016. FIG. 11B shows CP37, identified as CPN0998 (SEQ ID NO: 240) . FIG. 11C shows CP46 (SEQ ID NO: 244) and CP47 identified as CPN0796 (SEQ ID NO: 243) and CPN0705 (SEQ ID NO: 246) , respectively. FIG. 11D shows CP52, identified as CPN0152 (SEQ ID NO: 250) . FIG. 11F shows CP75, identified as CPN0619 (SEQ ID NO: 262) .

Detection and Identification of Proteins Located in the Type III Secretion Gene Subgroup Most type III secretion genes of intracellular bacteria are located as a group of pathogenic cells in one gene group, whereas the type III secretion gene of Chlamydia is , C.I. trachomatis and C.I. In both pneumoniae, they have been identified in three different groups, located at different locations in the genome (Stephens et al., 1998 [4.] and Kalman et al., 1999 [5.]). C. trachomatis A, D and L2; As part of a comprehensive proteomic analysis of pneumoniae VR1310, proteins present in the type III secretion group were identified from gels performed on purified EB.

C. The type III secreted proteins identified from Trachomatis include Yop secreted ATPase (yscN), Yop transport carrier protein L (YscL) and secreted chaperone (SycE), which are necessary for transporting the protein from the bacterial cytoplasm to the secretory machinery. is there. Furthermore, the identified C. elegans with unknown function but located in the type III secretion subgroup. The proteins of trachomatis D include CT560 and large amounts of CT577 and CT579. Although CT668 (SEQ ID NO: 196) is clearly present in whole cell lysates, it is not present in purified EB and is located next to YscN, so this protein may be secreted. The genomic location of the protein is shown in FIG.

C. For pneumoniae, proteins LcrE (CPN0324), YscC (CPN0702), YscN (CPN0707) and YscL (CPN0826) from type III secretory organs have been identified. YscC (CP89 FIG. II, Table II) is not present in the purified EB, presumably due to localization in inclusion body membranes, where the YscC is exposed to the host cell cytoplasm. Two proteins, present in type III groups located around YscC (CPN0702) and YscN (CPN0707), were detected in significantly higher amounts in whole cell lysates than purified EB. These were CPN0705 (SEQ ID NO: 246) (CP90, Figure II, Table II) and CPN0711 (SEQ ID NO: 264) (CP76, Figure II, Table II). These proteins may be located in inclusion body membranes or present in whole cell lysates, and in each case these proteins may be affected by the proteasome.

Pulse Tracking Experiments for Secreted Protein Candidates To evaluate the time at which the identified candidate proteins are likely to be present in infected cells, the present invention provides a series of pulse tracking experiments. In the examples below, cultures of infected cells were [35S] labeled from 22-24 hours. After the labeling time, the medium was changed to normal RPMI growth medium without [35S] -methionine, and cells were harvested at different times after labeling. Results from several independent experiments showed slight changes, probably due to slight differences in host cell concentration and / or efficiency of infection.

FIGS. 1B, C, D and E give examples of pulse / tracking experiments. The intensity of CT668 (SEQ ID NO: 196) and DT8 (SEQ ID NO : 1) (FIG. 1: A, B, respectively) declined significantly after 1.5 hours of synthesis, and at 4.5 hours, the gel was not EB-staged. Almost did not exist. CT610 and CT783 (FIG. 1: C, D, respectively) were significantly reduced, but were still detectable by 4.5 hours at the EB stage. The C-terminal fragment of CT858 (D) (and the N-terminal fragment of CT858 (the sequence of CT858 is the sequence of SEQ ID NO: 240)) gradually increased during the first chase time, but was not present in EB, This suggests that the cleavage products accumulated during Chlamydia development. CT858 (SEQ ID NO: 200) . The N-terminal fragment of the pneumoniae homolog, CPN1016 (SEQ ID NO: 238) was also absent from EB (FIG. 2).

Pulse Tracking Experiments in Combination with Proteasome Inhibitors This example shows how to determine which vaccine candidates are processed by the proteasome. Proteasome inhibitors that are cell permeable were added to the cultures of infected cells during the labeling and chasing time, and the protein turnover times were compared to those observed for labeling / chasing without the addition of the proteasome inhibitor. One example of the importance of this approach is shown in FIG. Proteins were labeled from 22-24 hours post-infection, in the presence or absence of 10-100 μM proteasome inhibitor MG-132. The labeling medium was then replaced with growth medium with or without MG132 after washing twice in normal growth medium, and was followed from 24 hours post-infection to 28 hours post-infection. Cell lysates were subjected to two-dimensional PAGE (IPG) and compared to controls without MG-132 and gels containing proteins collected immediately after labeling (FIGS. 6A, B and C). The present invention relates to 15 C. elegans with increased turnover time due to treatment with proteasome inhibitors. Trachomatis D protein is provided.

  The levels of DT9, DT10 and DT11 were actually higher in the chase + MG-132 gel than in controls taken immediately after the labeling time. This indicates that DT9, DT10 and DT11 are very rapidly degraded by the proteasome. In contrast, the addition of proteasome inhibitors did not significantly affect DT7 levels.

  The present invention involves the use of several other proteasome inhibitors (eg, MG115, MG262, PSI and lactocysteine) that inhibit different parts of the catalytic activity of the proteasome, thereby reducing other proteins that are degraded in the proteasome. Can be elucidated (Table IV).

Use of a genetically modified cell line to determine the effect of a proteasome inhibitor on chlamydia protein turnover time.
The invention also provides for the use of the commercially available mouse embryo cell line MEC-PA28, cell line PW8875. MEC-PA28 is transfected with the proteasome IFN-γ-inducible PA28α and β subunits, and the MEC217 cell line is transfected with the proteasome IFN-γ-inducible LMP2, LPM7, and MECL. MEC-PA28 and MEC217 were grown to a semi-assembled state. trachomatis or C.I. Infect with pneumoniae. A control mouse embryo cell line, not transfected with the proteasome subunit, was infected in parallel.

  Experimental procedure using proteasome inhibitors in combination with pulse labeling / chasing as transfected genes encoding subunits overexpressed from cell lines MEC-PA28 and MEC217 are essential for MHC class I antigen processing and presentation Is performed using these host cells as described above.

Cloning and Expression of an Open Reading Frame (ORF) Encoding a Vaccine Candidate Cloning and expression of an ORF encoding a vaccine candidate was performed using the pET-30LIC Vector Kit (Novagen, Madison, USA) according to the manufacturer's instructions. Was.

Primers used for PCR of the gene had the following 5 'and 3' LIC overhangs:
Forward primer: 5′GACGGACGACAACATX-gene specific sequence 3 ′
Reverse primer: 5'GAGGGAGAAGCCCGGT-gene specific sequence 3 '
(X: first nucleotide of specific sequence for insertion)
GACGACGAGCAAGAT and GAGGAGAAGCCCGGT in the above primers are sequences of SEQ ID NOs: 275 and 276, respectively.

  The full-length gene or the gene without the leader sequence was amplified by PCR using Expand ™ High Fidelity PCR (Roche, Germany). 35 PCR cycles were performed on a DNA Thermal Cycler (GeneAmp PCR system 9600, Perkin Elmer) as follows: 92 ° C. for 15 seconds (denaturation), 55 ° C. for 15 seconds (primer annealing) and 68 ° C. for 4 minutes ( Extension). The resulting PCR product contained an LIC overhang near the gene-specific sequence. The PCR product was purified Wizard (Promega, Madison, USA) and ligated into the pET-30 vector. The pET-30 vector contains a gene encoding kanamycin resistance and a histidine tag upstream of the LIC cloning site.

  The pET-30 vector containing the candidate gene was transformed into competent E. coli Nova Blue strain. Colonies were selected on kanamycin agar plates, and control PCR for the selected colonies was performed using vector-specific primers and insert-specific primers. Plasmid DNA was purified from positive colonies containing inserts specific for the candidate gene. Next, the plasmid DNA was transformed into E. coli (BL21). Insertion positive colonies were selected on kanamycin agar plates. Expression of the fusion protein, including a gene-specific insert containing a histidine tag located at the N-terminus, was induced in 500 ml of LB medium by adding 1 mM IPTG (Apollo Scientific, GB).

C. CT668 (SEQ ID NO: 196) , CT858 (SEQ ID NO: 200) , CT783 (SEQ ID NO: 198) , CT610 (SEQ ID NO: 202) , YscN and DT8 (SEQ ID NO: 1) from C. trachomatis D; E. coli (BL21) expressing a recombinant fusion protein of CPN1016 (SEQ ID NO: 238) and YscC from Pneumoniae VR1310 was generated.

  Recombinant fusion proteins were purified from lysed bacteria using a nickel resin column (High Trap Sepharose, Amersham Pharmacia Biotech) as described previously. Coomassie-stained SDS-PAGE gels of the purified proteins were run. Coomassie-stained bands representing the fusion protein were unambiguously identified by MALDI MS to confirm that the correct fusion protein had been produced. Serum containing polyclonal antibody was intramuscularly injected three times with 50 μg fusion protein dissolved in Freund's adjuvant and 50 μg fusion protein dissolved in PBS as described by Knudsen et al. [17]. Was injected twice and immunized a New Zealand white rabbit.

Western Blotting Using PAbs Against Vaccine Candidates To confirm that the PAb identified the correct vaccine candidate, a two-dimensional PAGE immunoblot was performed to visualize possible post-translational modifications or processing. 500 μg of unlabeled and 2 × 10 ⁶ cpm of labeled C. from whole cell lysate trachomatis D or C.I. pneumoniae proteins were separated by two-dimensional PAGE and the proteins were electroblotted onto PVDF membranes. 150 mM NaCl (or higher salt form, 400 mM), 20 mM Tris, 0.2% w / v gelatin, 0.05% v / v Tween 20 (Bio-Rad) and 2% v / v normal Immunostaining was performed using a 1/500 to 1/1000 dilution of the PAb in a buffer containing goat serum (Dako, Glostrup Denmark).

  The secondary antibody used was alkaline phosphatase-conjugated goat anti-rabbit IgG (Bio-Rad) diluted 1/2000 in antibody buffer. Blot samples were stained with 5-bromo-4-chloro-3-indolyl toluidine phosphate (BCIP) / nitro blue tetrazolium (NBT) (Bio-Rad) until a clear reaction was detected. The immunoblot samples were computer scanned and then exposed to X-ray film for about 8 days. Immunostained proteins were compared to their [35S] labeled counterparts using Melanie II Software.

7A1 and 7A2 show an example of an entire two-dimensional PAGE IMB image for PAb 245 for CT858 (SEQ ID NO: 200) . PAb245 reacts reproducibly with DT4 (the C-terminal fragment of CT858) and DT48 (the N-terminal fragment of CT858), as indicated by the corresponding labeled background (FIG. 7A2).

  7B1 and 7B4 show the IMB for CT668 and YscN. The immunoreactive protein spots on the gel and their localization based on the labeled background (FIGS. 7B2 and B5) correspond to the location of the protein on the analytical gel (FIGS. 7B3 and B6) and therefore It was confirmed that PAb reacted with the correct protein.

  The present invention provides examples of IMBs that show that the levels of some isoforms of CT610 are clearly increased in infected cells treated with MG132 as compared to untreated controls. DT7 was identified as CT610, which was located directly above DT9, DT10, DT11 and DT12 (FIG. 6A). On two-dimensional gel blot samples of whole cell lysates (FIG. 7C1), the IMB using PAb255 for CT610 clearly shows two rows of spots, one row representing DT7, and another row representing DT9, DT10, DT11 and DT12. Reacted. Thus, CT610 exists in different isoforms, probably due to different post-translational modifications or processing. When cells were treated with the proteasome inhibitor MG132, the amounts of DT9, DT10 and DT11 were clearly increased (FIG. 7C2).

  IMB using PAb255 was performed on regular SDS-PAGE PVDF blot samples containing proteins from whole cell lysates treated for 6 hours each in the presence and absence of MG132. FIG. 7C4 shows additional in the lanes containing proteins from MG132-treated infected cells (lanes b and d), below the band representing the spot in the DT7 row, compared to the untreated controls (lanes a and c). The target band is clearly shown. The level of the upper band, representing the DT7 row, did not change significantly with treatment with proteasome inhibitors.

Different subtypes of C. elegans upon growth in different host cells. Expression of Candidate Trachomatis The present invention takes into account subtype / host cell specific differences in the expression levels of potentially secreted proteins. This is because host cell / Chlamydia interactions may be different for other subtypes or by culturing in other host cells. This is the general C.I. Related to the selection of vaccine candidates, most likely for trachomatis vaccines.

CT668 (SEQ ID NO: 196) , CT858 (SEQ ID NO: 200) , CT783 (SEQ ID NO: 198) and CT610 (SEQ ID NO: 202) are all C.I. Trachomatis A, D and L2 were detected at the same position. This is because C.I. The genes encoding these proteins between trachomatis D and L2 are consistent with a high degree of conservation. Further, C.I. Culture of trachomatis A, D and L2 in McCoy or Hep-2 cells instead of HeLa cells expressed all of these proteins, indicating that the expression of these proteins is independent of the cell type used. Was done. However, in subtypes A and D, DT8 (SEQ ID NO: 1) had an isoelectric point (pI) of 5.25 or 24 to 36 hours post-infection, based on gels containing chlamydia proteins. 1 and a molecular weight (Mw) of 7.5. However, in subtype L2, DT8 (SEQ ID NO: 1) probably has an isoelectric point (pI) of 6.4 and a molecular weight (p) due to the pure charge of the protein and slight amino acid substitutions that alter the isoelectric point (pI). Mw) 7.5.

Indirect Immunofluorescence Microscopy of Vaccine Candidates. Wells and glass cover strips containing a monolayer of semi-confluent HeLa cells were prepared using C.I. pneumoniae or C.I. Infected with trachomatis D. A lower titer of Chlamydia was used so that only about 50% of the cells were infected, which allowed a clear distinction between infected and uninfected cells. Cells were washed twice in PBS, fixed in formaldehyde or methanol at different times after infection, and subjected to indirect immunofluorescence microscopy. If necessary, PAb was pre-adsorbed with acetone-precipitated HeLa protein to obtain minimal cross-reactivity with human proteins.

  In the example shown in FIG. 8, a 1/200 dilution of the pre-adsorbed rabbit PAb and C.I. mouse monoclonal antibody targeting C. trachomatis MOMP (MAb 32.3) A 1/25 dilution of MAb 18.3 against Pneumoniae was used. Fluorescein isothiocyanate-conjugated (FITC) goat anti-rabbit (GAR) IgG antibody and rhodamine-conjugated goat anti-mouse (GAM) IgG antibody (Jackson, Trichem, Denmark) were used as secondary antibodies. Double immunostaining was performed to determine subcellular localization of the vaccine candidate to Chlamydia inclusion bodies.

  PAb249 directed to DT8, which had a short turnover time, reacted slightly with RB in Chlamydia inclusions (FIG. 8A3). No significant response to host cell structures other than inclusion bodies was detected, despite minimal cross-reactivity of HeLa cells with proteins.

In contrast, CT858 (SEQ ID NO: 200) clearly stained the host cell cytoplasm in infected cells, but not uninfected cells (FIG. 8B3). Generally, staining was stronger at the inclusion body boundaries. The cytoplasmic staining of the host cells could be visualized from 12 hours post-infection to 72 hours post-infection, consistent with the long turnover times predicted by pulse tracing experiments. C. CT858 (SEQ ID NO: 200) pneumoniae CPN1016 (SEQ ID NO: 238) was purified from PAb. When reacted with Hep-2 cells infected with Pneumoniae, the same response characteristics were observed.

Spot number CT668 (SEQ ID NO: 196) (DT1 and DT2)
CT668 was placed just upstream of one of the subgroup YscN ATPases, containing the gene homologous to the type III secretion gene, and without the predicted recognizable signal peptidase cleavage site. CT668 was used for about 4-6 hours. It was only present in Trachomatis D, during which the amount was definitely reduced. The PAb to CT668 (SEQ ID NO: 196) alone appears to react slightly with RB in inclusion bodies of IMF, but given the short turnover time of this protein, this is due to its rapid degradation in host cells. Can be explained. CT668 (SEQ ID NO: 196) was detected from 12 to 40 hours post-infection, suggesting that this protein is produced during the majority of intracellular differentiation. Therefore, C.I. trachomatis carries CT668 (SEQ ID NO: 196) continuously into the host cell, where it functions and may be readily degraded.

Two variants of CT668 (SEQ ID NO: 196) were identified. Basic mutants were labeled from 22-24 hours post-infection and then collected. Trachomatis was the most abundant on the gel containing protein. Interestingly, in all the studies performed, over time, the intensity of the acidic mutant increased and the intensity of the basic mutant decreased. When CT668 (SEQ ID NO: 196) was followed at 30 minute intervals, an increase in acidic mutants was already detectable in 1 hour (FIG. 1B).

  This finding can suggest that the denaturation is not due to the conditions under which the gel was subjected, which could produce denaturation such as carbamate or amidation. Instead, denaturation appears to be due only to unknown enzymes that denature proteins.

C. Denaturation of secreted proteins in psicitii has been described, in which IncA is phosphorylated by Ser / Thr kinase of host cells after transfer to inclusion body membranes (Rockey et al., 1997) [29. ]. The turnover time of CT668 (SEQ ID NO: 196) is prolonged by treatment with a proteasome inhibitor, and the at least limited amount of CT668 (SEQ ID NO: 196) produced may be processed by the proteasome. It is suggested.

Spot number DT8 (DT8) (SEQ ID NO: 1)
DT8 (SEQ ID NO: 1) was derived from C. It is a novel 7.2 kDa protein that is a specific protein of trachomatis. PSORT analysis indicated that there was no recognizable leader sequence for this protein. The theoretical coordination, isoelectric point (pI) 5.21 / 7.2 kDa, was very consistent with that determined experimentally. Many of the features observed for CT668 (SEQ ID NO: 196) were also observed for DT8 (SEQ ID NO: 1) . A short turnover time of less than 6 hours was observed, and the IMF showed negligible reaction with RB.

  Behind the stop codon in DT8, a potential stem-loop region can be expected, indicating a Rho-independent transcriptional stop of the protein.

Spot numbers DT7, DT9, DT10, DT11, DT12 (CT610 (SEQ ID NO: 202) )
CT610 (SEQ ID NO: 202) was not located near any genes that were homologous to genes associated with secretion in other bacteria. This protein was identified from both whole cell lysates and EBs, but was evaluated by Melanie II Software to be at least 30-fold more in whole cell lysates. This protein was detected in several isoforms represented by two molecular weight (Mw) polymorphisms, as determined by Pab255 generated against CT610 (SEQ ID NO: 202) . Rows of these spots represented DT7 (top row) and DT9, 10, 11, 12 (bottom row). Both rows had short turnover times in the pulse tracking experiment. Interestingly, the amounts of DT9, DT10 and DT11 were greatly increased by treatment with proteasome inhibitors based on pulse chasing / MG132 experiments. Pulse chasing experiments were confirmed by SDS PAGE IMB using Pab255.

Thus, the present invention provides evidence that several isoforms of CT610 (SEQ ID NO: 202) are secreted and processed in the proteasome. The different consequences of the CT610 (SEQ ID NO: 202) isoform indicate the validity of detecting such an isoform using the Pabs generated against vaccine candidates.

Spot number DT3 (CT783 (SEQ ID NO: 198) )
CT783 (SEQ ID NO: 198) is a C.I. Trachomatis has been suggested to be a protein disulfide-bonded isomerase. CT783 (SEQ ID NO: 198) shows homology to thioredoxin disulfide isomerase (CT780) and protein disulfide isomerase from Methanobacterium thermoautotrophicum. A 33 amino acid leader sequence could be predicted by PSORT and SignalP, and the theoretical isoelectric point (pI) and molecular weight (Mw) of the cleaved protein were consistent with those determined by experiment. In addition, the polyclonal antibody raised against CT783 (SEQ ID NO: 198) stained the correct spot on a two-dimensional PAGE IMB using 4-7 LIPG.

Due to cleavage of the N-terminal leader sequence, this protein is probably not secreted by a type I or type III system, but is likely to be secreted by a type II system. Although few PSORT predictions suggest subcellular localization in the membrane within the bacterium, most PDIs are normally located in the periplasm. CT783 (SEQ ID NO: 198) has a very short turnover time and is almost absent in chlamydia after about 4-6 hours following synthesis.

The turnover time of CT783 (SEQ ID NO: 198) was prolonged by a proteasome inhibitor, suggesting potential processing of the proteasome.

Spot numbers DT4 and DT48 (CT858 (SEQ ID NO: 200) ) and CP63 (CPN1016 (SEQ ID NO: 238) )
CT858 has a cleavable N-terminal leader sequence, which was predicted to be a 67 kDa periplasmic protein, but was identified at two different locations on the gel. All sequence tags that identified DT4 as CT858 (SEQ ID NO: 200) matched the C-terminal portion of CT858 (SEQ ID NO: 200) . In the two-dimensional PAGE PVDF membrane IMB containing whole cell lysate, PAb245 reacted clearly and reproducibly with this C-terminal fragment. In addition, Pab245 reacted with protein spot DT48, which also had a long turnover time in pulse chasing experiments, similar to that also seen for DT4.

DT48 was also identified by MALDI MS as an N-terminal fragment of CT858 (the sequence of CT858 is the sequence of SEQ ID NO: 200) . The molecular coordination of DT48 was isoelectric point (pI) 7.3 / molecular weight (Mw) 25.8. N-terminal portion of CT858 (SEQ ID NO: 200) , consistent with that determined experimentally using isoelectric point (pI) / molecular weight (Mw) tools at different lengths of N-terminus (no signal peptide) To produce a peptide. This ^analysis cleavage site near K ²³³ S ^{234 M 235} is suggested.

The N-terminal and C-terminal fragments of CT858 (the sequence of CT858 is the sequence of SEQ ID NO: 200 ) can all be detected on a two-dimensional gel up to the EB stage (72 hours post-infection), but in purified EB The fact that it is not detectable indicates a long turnover time in the cytoplasm of the host cell. This was consistent with a very clear detection of CT858 (SEQ ID NO: 200) in the host cell cytoplasm by 72 hours by IMF experiments. CT858 shows slight homology to a tail-specific protease (tsp) from E. coli, which is associated with the processing of penicillin binding protein, including the IRBP domain from human internal photoreceptor retinoid binding protein, and hydrophobicity. Attach the ligand (Silber et al., 1992) [32. ]. Type II secretion is P. It has been implicated in the transport of degradable proteins in several Gram-negative bacteria, including Aeroginosa and Aeromonas hydrophila (reviewed in Hobbs and Mattick, 1993) [33. ]. In Aerosignosa hydrophila, secreted elastase (ahpB) has recently been suggested to be important for bacterial toxicity (Cascon et al., 2000) [34. ].

These proteins have similar consequences to CT858 (SEQ ID NO: 200) . These proteins are usually synthesized as propeptides containing a cleavable signal peptide and then processed to the mature protein. Other C. elegans that show homology to tail-specific proteases. The protein of trachomatis is the hypothetical protein CT441. Whether this protein may exhibit the same secretory properties as CT858 has not yet been determined.

The same subcellular level localization of CT858 (SEQ ID NO: 200) was pneumoniae homologues were observed for CPN1016 CPN1016 including Pab253 for (SEQ ID NO: 238) (SEQ ID NO: 238), thus the functional conservation of genes between the two Chlamydia species is shown.

  Secreted C. elegans identified by its gene number. trachomatis D and C.I. pneumoniae proteins were analyzed by ANN adjusted to recognize peptides with affinity for human HLA-A2. Tables V-VIII list the peptides selected by ANN for the expected binding to HLA-A2 and give their affinities (Kd) in nM. The lower the value, the better. Most peptides with a Kd of less than 50 nM are immunogenic. Peptides with a Kd of less than 500 nM (but more than 50 nM) may be immunogenic. The binding of a given peptide can be improved by substituting a suboptimal amino acid at anchor position P2 or P9, a strategy that often retains specificity for the native peptide.

Determining the Ability of a Vaccine Candidate to Generate Specific Cytotoxic CD8 + T Cells in an Experimental Animal Model In one approach, the vaccine candidate is used as a full-length recombinant protein to express human HLA class I molecules Experimental animals (mouse or guinea pig), including transgenic A2 mice, are immunized.

  In another approach, vaccine candidates are screened for T cell epitopes by computer algorithms, and then peptides containing these epitopes are synthesized and used for immunization as described for full length vaccine candidates.

  In a third approach, MHC class I binding assays in which peptides of 8-10 amino acids in length are synthesized in an overlapping manner so that they cover the entire vaccine candidate sequence and compete with a radiolabeled intermediate conjugate Used for Peptides that are good conjugates are used for immunization as described for full length vaccine candidates.

  Vaccine candidates are administered as a peptide / protein combination or as an adjuvant alone peptide / protein. Vaccine candidates can also be administered as DNA vaccines or by a virus expressing the vaccine candidate.

  Peripheral blood mononuclear cells (PBMCs) from the immunized animal are purified by density gradient centrifugation and the CD8 + cells are purified by using antibodies that bind to magnetic beads or by other methods. The activity of CD8 + T cells is measured by proliferation assays, such as ELISPOT and tritiated thymidine incorporation, and by specific lysate assays (chromium release).

  Purified PBMC or CD8 + cells from the immunized animals are restrictedly diluted and placed on microtiter plates along with irradiated antigen presenting cells, growth factors, and specific or non-specific stimulators. For specific stimulation, use a single vaccine candidate protein or peptide that was predicted as a good T cell epitope or found to be a good conjugate to MHC class I molecules. For non-specific stimulation, Chlamydia infected cells are used. The cells are cultured for 9-14 days, during which the antigen-specific cells proliferate. Generation of specific cytotoxic CD8 + T cells is determined by measuring cytokine secretion from stimulated cells by an ELISPOT assay. T cell proliferation is measured by tritiated thymidine incorporation followed by scintillation counting. The cytotoxicity of the expanded cells is measured using a cytotoxicity assay, such as a chromium release assay, using Chlamydia-infected cells or recombinant cells that express the vaccine candidate protein / peptide as well as the target cells [ 42. ].

Testing vaccine candidates for their ability to protect mice and guinea pigs against Chlamydia infection Experimental animals are immunized (as described above) with vaccine candidates as a single protein / peptide or a combination of vaccine candidates . Following immunization, the animals are experimentally infected with chlamydia (intranasal infection for C. pneumoniae and sexually transmitted for C. trachomatis). Protection against infection is measured by culturing Chlamydia, immunohistochemistry, quantitative PCR, and examining seroconversion by infection.

Determination of the Ability of Chlamydia Infection to Generate Vaccine Candidate-Specific Cytotoxic CD8 + T Cells in Humans Human serum samples are tested by ELISA (Medac) for the presence of antibodies to chlamydia. Seropositive individuals are selected for the presence of vaccine candidate specific cytotoxic CD8 + T cells.

  Peripheral blood mononuclear cells (PBMCs) from humans who test antibody positive for Chlamydia are purified by density gradient centrifugation and using antibodies and magnetic beads, or by other methods, to obtain CD8 + cells. Is purified. The activity of CD8 + T cells specifically targeting vaccine candidate proteins / peptides is measured by the method described in Example 19.

Use of Vaccine Candidates to Develop ELISA Tests for Diagnostic Purposes Immunoassays based on purified elementary bodies, since the secreted proteins of the present invention are absent or greatly reduced in purified microbial bacteria Cannot detect antibodies against such proteins. Thus, secreted proteins may be major unrecognized antigens that are also associated with the humoral immune response. In addition, persistent infection with chlamydia can be detected by ELISA based on secreted chlamydia proteins. Because secreted proteins are only expressed during the intracellular stage of Chlamydia differentiation.
1) The secreted protein is produced as a purified recombinant protein. Alternatively, overlapping synthetic peptides representing secreted proteins are generated.
2) Coat the ELISA plate with purified recombinant protein representing secreted protein (or synthetic peptide derived from secreted protein). Block ELISA plates with 15% fetal calf serum to avoid non-specific binding.
3) C. using Micro-IF or ELISA (Medac). trachomatis or C.I. The patient's serum is screened for antibodies to Pneumoniae. Positive sera are tested on ELISA plates coated with recombinant antigens representing secreted proteins.
4) Use IgA or IgM to detect antibody-bound anti-human IgG. Serum from infected mice is used as a positive control.
5) Compare the results from micro-IF or ELISA (Medac) with an ELISA based on recombinant proteins representing secreted proteins.

[35S] -labeled from 22-24 hours post-infection (hp), collected immediately after labeling, and extracted from whole cell lysates separated by two-dimensional PAGE. It is a figure which shows the example of the gel image of the protein of trachomatis D. Black arrows indicate proteins whose strength is significantly reduced on gels containing proteins from purified EBs (basic bodies) that were labeled from 22-24 hours post-infection and purified 72 hours post-infection. The properties of isoelectric point (pI) and molecular weight (Mw) of the indicated spots (DT1-77) are listed in Table I. C. at different times after synthesis, as indicated by the enlargement of the selected area from FIG. 1A. FIG. 2 shows examples of identified vaccine candidates in trachomatis D and their presence. The time of protein turnover was assessed by tracking at different times after labeling during the development cycle until purification of the EB. The upper scale indicates the time point after labeling starting at zero, and the lower scale indicates the time after infection starting at 24 hours. FIG. 1B: DT1 and DT2 (CT668 ; SEQ ID NO: 196 ). Figure 1C: DT8 (SEQ ID NO : 1) . FIG. 1D: DT7 (up arrow) and DT11 (down arrow) (both CT610 ; identified as SEQ ID NO: 202 ). FIG. 1E: DT3 (down arrow, CT783 ; SEQ ID NO: 198 ), DT4 (up arrow, CT858 ; SEQ ID NO: 200 ). [35S] -labeled from 55 to 57 hours post-infection, collected immediately after labeling and separated by two-dimensional PAGE. It is a figure which shows the example of the gel image of the protein of pneumoniae VR1310. Black arrows indicate gels of proteins from EBs that were labeled at 2 hours during the development cycle, 6, 12, 24, 36, 42, 48, 54, 60 hours post-infection and purified at 72 hours post-infection. Shows proteins whose strength is greatly reduced. Arrows indicate proteins with significantly reduced strength on gels from purified EBs. The isoelectric point (pI) and molecular weight (Mw) characteristics of the indicated spots (CP1-91) are listed in Table II. It is a figure which shows the enlarged part of FIG. 2A. FIG. 9 shows corresponding enlargements of images of EB proteins labeled and separated by two-dimensional PAGE as described previously. 2B and 2C provide examples of two proteins, CP63 (CPN1016 ; identified as SEQ ID NO: 238 ) and CP65, which are present in whole cell lysates but not in EBs. Enriched spots are present in both EBs and whole cell lysates and have been identified as polypeptide deformylases. FIG. 4 shows a fingerprint of peptide mass used to identify spot number DT1 as hypothetical protein CT668 (SEQ ID NO: 196) . Hollow arrows indicate peptides resulting from tryptic autolysis. These peaks were used for internal correction of the spectrum. Black arrows indicate peptide masses consistent with the CT668 sequence (SEQ ID NO: 196) . Double arrows indicate peptides derived from human contaminating proteins. FIG. 3B shows the results from the use of the identification software MS-Fit for the peptide mass obtained from FIG. 3A. The highest C.I. It is shown that the protein of Trachomatis is CT668 (SEQ ID NO: 196) . ESI-Q-TOF MS of spot DT1 containing double charge 1744.9 Da parent ion (R) KIVDWVSSGEEILNR (A) (black arrow) (SEQ ID NO: 273) , consistent with the amino acid sequence of CT668 (SEQ ID NO: 196) FIG. 3 shows a peptide mass spectrum generated by the above method. The dotted line indicates the Y-peptide ion generated by fragmentation of the parent ion. The deduced amino acid sequence is indicated by a bold one-letter code. FIG. 7 shows a peptide mass spectrum generated by PSD MALDI MS of spot CP63. The 199.8 Da parent ion (K) ELLFGWDLSQQTQQAR (L) (SEQ ID NO: 274) was fragmented to produce several peptides with sequence elucidation. These two, _{b 2} of 243.35Da - ions (EL) and 356.34Da of _{b 3} - exemplified by ions (ELL), these masses differ by leucine mass (113Da). A new C.I. FIG. 2 shows the nucleotide sequence of the DT8 trachomatis-specific vaccine protein candidate DT8 (SEQ ID NO: 2) and the corresponding amino acid sequence indicated by the one-letter code (SEQ ID NO: 1) . Bold amino acid sequences were covered by sequence tags obtained by ESI-Q-TOF MS. FIG. 2 shows a pulse chasing experiment in combination with one proteasome inhibitor MG-132. FIG. 6.1: Labeled from 22-24 hours post-infection and followed for an additional 4 hours in the presence of 20 μM MG-132. Whole gel image of a two-dimensional gel loaded with trachomatis D protein. FIG. 6A. 6B and 6C: Comparing follow-up experiments performed with MG-132 (chase + MG-132) or without (chase) MG-132, C.A. has longer turnover time due to treatment with MG-132. FIG. 2 is an enlarged view of a region containing a protein of Trachomatis D. The first column represents the protein profile of infected cells taken immediately after the 2 hour labeling time. The intensity of DT9, DT10 and DT11 on the gel containing the whole cell lysate labeled and tracked in the presence of MG132 compared to the gel containing the whole cell lysate taken immediately after labeling without MG132 Note that it is significantly higher. FIG. 4 shows a whole gel image of IMB of whole cell lysate using PAb245. A1: IMB showing reaction with spot numbers DT4 and DT48, which are C-terminal and N-terminal fragments of CT858 (CT858 corresponds to SEQ ID NO : 200) , respectively. A2: Radiolabeled background of the corresponding IMB. FIG. 10 shows the IMB using PAb241 for YscN (B1) and PAb238 for CT668 (SEQ ID NO: 196) (spots DT1 and DT2) (B4). The corresponding autoradiography of the IMB shows co-localization with B2: YscN and B5: CT668 (SEQ ID NO : 196) . YscN (B3) and CT668 (B6) are localized on the analytical two-dimensional gel. FIG. 4 shows an IMB using PAb255 for CT610 (SEQ ID NO: 202) . C1: Close up of a two-dimensional gel blot showing that PAb255 stains a row of spots. The upper part represents DT7, and the lower part represents DT9, 10, 11, and 12. C2: Enlarged view showing the effect of treating infected cells with MG132 for 6 hours before harvesting the cells 30 hours after infection. It should be noted that MG132 treatment resulted in a clear relative increase in the amount of DT9, DT10 and DT11 compared to the column representing DT7. C3: corresponding labeled analytical gel. C4: SDS-PAGE using PAb255 IMB: lanes a and c: 20 μg and 10 μg (respectively) of all infected cell lysates collected 30 hours after infection. Lanes b and d: 10 μg and 5 μg (respectively) of all infected cell lysates treated with 50 μM MG132 for 6 hours before harvesting cells 30 hours post-infection. FIG. 2 shows indirect immunofluorescence microscopy showing subcellular localization of vaccine candidates. A and B: C.I. HeLa229 cells infected with trachomatis D and fixed with formalin 24 hours after infection. C: C.I. HEp-2 cells infected with pneumoniae and fixed with formalin 72 hours after infection. Column 1 shows the Normasky image. Row 2 shows C.I., visualized by rhodamine-conjugated GAM IgG antibody. 3 shows the reaction of MAb32.3 with Trachomatis MOMP. Column 3 shows the reaction with a rabbit polyclonal antibody specific for the vaccine candidate in question, made visible by a FITCH-conjugated GAR IgG antibody. The vaccine candidates examined were A: DT8 (SEQ ID NO : 1) , B: CT858 (SEQ ID NO : 200) , C: CPN1016 (SEQ ID NO : 238) (C. pneumoniae CT858 (SEQ ID NO: 200) homolog). White single-headed arrows indicate the boundaries of Chlamydia inclusions in infected cells. Hollow arrows indicate uninfected cells. It should be noted that CPN1016 shows the same subcellular localization and secretion properties as CT858. C., including the identified proteins, located in type III secretory subgroups 1, 2, and 3. FIG. 3 shows the genomic localization of some examples of identified vaccine candidates from Trachomatis D. C. in two-dimensional gel images from total lysates of infected cells labeled from 22-24 hours post infection, RB labeled and purified at the same time point, and EBs purified 72 hours post infection. FIG. 2 shows the positions of candidates secreted from trachomatis D. All gels were made using Immobiline Drystrip which was non-linear and had a pH of 3-10. 22-24 hours post-infection: [35S] -labeled from 22-24 hours post-infection, collected immediately after labeling, from total lysates of infected cells separated by two-dimensional PAGE. FIG. 1B is an enlarged view of the gel image shown in FIG. 1A of the protein of trachomatis D. Purified RB: Immediately after labeling from 22-24 hours post-infection, C. from bacteria purified as RB 24 hours post-infection. Corresponding regions from gel images of proteins of trachomatis D. Purified EBs: corresponding areas from gel images of proteins from bacteria labeled from 22-24 hours post-infection and purified as EBs 72 hours post-infection. In rows AG, secreted candidates DT4, DT48, DT23, DT76, DT77, DT47 and DT75 were enriched. Two-dimensional, from total lysate of infected cells labeled from 55-57 hours post-infection, purified EBs, total lysate of infected cells labeled 34-36 hours post-infection, and RB labeled and purified at the same time point C. in the gel image FIG. 2 is a diagram showing the positions of candidates secreted from Pneumoniae VR1310. 55-57 hours post-infection: [35S] -labeled from 55-57 hours post-infection, harvested immediately after labeling and separated by two-dimensional PAGE using Immobiline Drystrip, non-linear, pH 3-10. C.I. from all lysates FIG. 2B is an enlarged view of the gel image shown in FIG. 2A of the protein of Pneumoniae VR1310. Purified EB: gel of protein from bacteria labeled at 2 hours during the development cycle, ie, 6, 12, 24, 36, 42, 48, 54, 60 hours post-infection and purified as EB 72 hours post-infection Corresponding area from the image (Immobiline Drystrip, non-linear and pH 3-10). 34-36 hours post-infection: C. from total lysates of infected cells labeled 34-36 hours post-infection and taken immediately after labeling. Corresponding region from gel image of pneumoniae VR1310 protein (Immobiline Drystrip, linear, pH 4-7). RB at 36 hours post-infection: Immediately after labeling from 34-36 hours post infection, C. from bacteria purified as RB at 36 hours post infection. Corresponding region from gel image of pneumoniae VR1310 protein (Immobiline Drystrip, linear, pH 4-7). In AF, secreted candidate CP34, CP37, CP46 (SEQ ID NO: 244) , CP47, CP52, CP63 and CP75 were enriched. G indicates the region from the parent image of the region in AF which does not contain secreted candidates.

Claims (71)

1) infecting host cells with intracellular bacteria;
2) labeling intracellular bacteria present in the infected cells;
3) preparing a) a whole cell lysate of the infected cells, and b) a purified and lysed bacterium obtained from the infected cells;
4) i) comparing the protein profiles by two-dimensional gel electrophoresis of the whole cell lysate obtained in step 3a) and ii) the purified and lysed bacteria obtained in step 3b);
5) detecting the protein spot obtained in step 4), which is present in the whole cell lysate but not present in the purified bacteria or present in a significantly reduced amount;
6) identifying the protein in the spot selected in step 5), or a protein which can be identified by a method for identifying a protein secreted from intracellular bacteria, or an immunogenic fragment thereof.   The protein of claim 1, or an immunogenic fragment thereof, which is applicable for inclusion in an immunogenic composition and / or for diagnostic purposes.   3. The protein of claim 2, comprising a T cell epitope suitable for inclusion in an immunogenic composition, which is a candidate for presentation as an MHC class I or II restricted antigen.   4. The protein of claim 3, comprising a T cell epitope suitable for inclusion in an immunogenic composition, which is a candidate for presentation as an MHC class I restricted antigen.   The Chlamydia trachomatis protein of claim 1 having one of the isoelectric point (pI) and molecular weight (Mw) properties of the proteins DT1-DT77 shown in Table I, determined with an average error of +/- 10%. Or an immunogenic fragment thereof.   CT017 (gene name CT017 (SEQ ID NO: 221)), CT044 (gene name ssp (SEQ ID NO: 227)), CT243 (gene name lpxD (SEQ ID NO: 223)), CT263 (gene name CT263 (SEQ ID NO:)) shown in Table IIIA 235)), CT265 (gene name accA (SEQ ID NO: 219)), CT286 (gene name clpC (SEQ ID NO: 229)), CT292 (gene name dut (SEQ ID NO: 215)), CT407 (gene name dksA (SEQ ID NO: 211)) ), CT446 (gene name euo (SEQ ID NO: 207)), CT460 (gene name SWIB (SEQ ID NO: 203)), CT541 (gene name mip (SEQ ID NO: 209)), CT610 (gene name CT610 (SEQ ID NO: 201)), CT650 (gene name recA (SEQ ID NO: 225)) CT655 (gene name kdsA (SEQ ID NO: 217)), CT668 (gene name CT668 (SEQ ID NO: 195)), CT691 (gene name CT691 (SEQ ID NO: 233)), CT734 (gene name CT734 (SEQ ID NO: 213)), CT783 ( Genes corresponding to gene name CT783 (SEQ ID NO: 197), CT858 (gene name CT858 (SEQ ID NO: 199)), CT875 (gene name CT875 (SEQ ID NO: 231)), or ORF5 (gene name ORF5 (SEQ ID NO: 205)) 2. The Chlamydia trachomatis protein of claim 1, identified by a number or by the gene name DT8, or an immunogenic fragment thereof.   One of the proteins DT1, DT2, DT3, DT5, DT9, DT10, DT11, DT13, DT14, DT17, DT47, DT59, DT60, DT61 or DT62 shown in Table IV, determined with an average error of +/- 10%. The Chlamydia trachomatis protein of claim 1, having two isoelectric point (pI) and molecular weight (Mw) properties, or an immunogenic fragment thereof.   Protein DT4 (gene name CT858 (SEQ ID NO: 199)), DT23 (gene name mip (SEQ ID NO: 209)), DT47, DT48 (gene name CT858 (SEQ ID NO: 199)), DT75, DT76 (gene name CT691 (SEQ ID NO: 233)) )) And DT77 (gene name CT263 (SEQ ID NO: 235)), the Chlamydia trachomatis protein of claim 5, or an immunogenic fragment thereof.   2. The Chlamydia pneumoniae protein of claim 1 having one of the isoelectric point (pI) and molecular weight (Mw) properties of the proteins CP1-CP91 shown in Table II, determined with an average error of +/- 10%. Or an immunogenic fragment thereof.   CPN0152 (gene name CPN0152 (SEQ ID NO: 249)), CPN0702, CPN0705 (genename CPN0705 (SEQ ID NO: 245)), CPN0711 (genename CPN0711 (SEQ ID NO: 263)), CPN0796 (genename CPN0796 (Table IIIB)) shown in Table IIIB SEQ ID NO: 243)), CPN0998 (gene name ftsH (SEQ ID NO: 239)), CPN0104 (gene name CPN0104 (SEQ ID NO: 241)), CPN0495 (gene name aspC (SEQ ID NO: 247)), CPN0684 (gene name parB (SEQ ID NO: 251)), CPN0414 (gene name accA (SEQ ID NO: 253)), CPN1016 (gene name CPN1016 (SEQ ID NO: 237)), CPN1040 (gene name CPN1040 (SEQ ID NO: 255) ), CPN0079 (gene name rl10 (SEQ ID NO: 257)), CPN0534 (gene name dksA (SEQ ID NO: 259)), CPN0619 (gene name ndk (SEQ ID NO: 261)), CPN0711 (gene name CPN0711 (SEQ ID NO: 263)), CPN0628 (gene name rs13 (SEQ ID NO: 265)), CPN0926 (gene name CPN0926 (SEQ ID NO: 267)), CPN1016 (gene name CPN1016 (SEQ ID NO: 237)), CPN1063 (gene name tpiS (SEQ ID NO: 269)), or CPN302 The Chlamydia pneumoniae protein according to any one of claims 1 to 4, which is identified by a corresponding gene number as (gene name lpxD (SEQ ID NO: 271)), or an immunogenic fragment thereof.   Protein CP34 (SEQ ID NO: 238) (Gene name CPN1016 (SEQ ID NO: 237)), CP37 (SEQ ID NO: 240) (Gene name CPN0998 (SEQ ID NO: 239)), CP46 (SEQ ID NO: 244) (Gene name CPN0796 (SEQ ID NO: 243)) ), CP47 (SEQ ID NO: 246) (gene name CPN0705 (SEQ ID NO: 245)), CP52 (SEQ ID NO: 250) (gene name CPN0152 (SEQ ID NO: 249)), CP63 (SEQ ID NO: 238) (gene name CPN1016 (SEQ ID NO: 237) )), And CP75 (SEQ ID NO: 262) (gene name ndk (SEQ ID NO: 261)). The protein of Chlamydia pneumoniae according to claim 9, or an immunogenic fragment thereof. DT8, the following sequence (SEQ ID NO: 1):
MQHTIMLSLENDNDKLASMMDRVVAASSSILSASKDSESNRQFTISKAPDKEPCRVSYVAASALSE
A polypeptide of Chlamydia trachomatis, or an immunogenic fragment thereof, comprising:   At least 40% sequence identity with the protein of claim 1, preferably at least 60%, more preferably at least 70%, even more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%. A protein having sequence identity, or an immunogenic fragment thereof.   A protein or an immunogenic fragment thereof comprising at least 7 contiguous amino acids of the protein of claim 1.   The protein of Chlamydia trachomatis according to claim 14, which comprises an amino acid sequence selected from the sequences of SEQ ID NOs: 3 to 45, or an immunogenic fragment thereof.   The homolog of Chlamydia pneumoniae of the Chlamydia trachomatis protein according to claim 15, which comprises an amino acid sequence selected from the sequences of SEQ ID NOs: 122 to 148, or an immunogenic fragment thereof.   The Chlamydia pneumoniae protein according to claim 14, which comprises an amino acid sequence selected from the sequences of SEQ ID NOs: 46 to 121, or an immunogenic fragment thereof.   The Chlamydia trachomatis homologue of the Chlamydia pneumoniae protein according to claim 17, which comprises an amino acid sequence selected from the sequences of SEQ ID NOs: 149 to 194, or an immunogenic fragment thereof. A nucleic acid compound comprising a sequence encoding the protein of claim 1 or an immunogenic fragment thereof . A nucleic acid compound comprising a sequence encoding the polypeptide of claim 12 . The following sequence (SEQ ID NO: 2):

21. The nucleic acid compound according to claim 20, comprising a fragment or a degenerate sequence thereof . A vector comprising the nucleic acid compound according to claim 19 . A host cell transformed or transfected with the vector of claim 22 . Use of a protein or a fragment thereof for producing an antibody against the protein or an immunogenic fragment thereof according to claim 1 . A method for producing an antibody against an intracellular bacterium, which comprises administering the protein or an immunogenic fragment thereof to a production animal and purifying the antibody therefrom according to claim 1 . An antibody obtainable by the method of claim 25 . A pharmaceutical or diagnostic composition comprising the protein of claim 1 or a fragment thereof . Use of the protein or a fragment thereof according to any one of claims 1 to 18 in the preparation of a diagnostic reagent . A method for identifying a T-cell epitope on a secreted protein from an intracellular bacterium, comprising a computer-based prediction, a MHC class molecule binding assay and / or a protein according to claim 1 or an immunogenic fragment thereof. A method comprising steps such as an ELISPOT assay . 30. A peptide epitope that may be present on the surface of a host cell and is obtainable by the method of claim 29 . A peptide epitope comprising 4 to 25 contiguous amino acids of the protein of claim 1 . A peptide epitope comprising 4 to 25 contiguous amino acids, preferably 6 to 15 amino acids, most preferably 7 to 10 amino acids of a polypeptide comprising the sequence of SEQ ID NO: 1. 31. The peptide epitope of Chlamydia trachomatis according to claim 30, comprising an amino acid sequence selected from the sequences of SEQ ID NOs: 3 to 45 . 34. A peptide epitope of Chlamydia pneumoniae of the peptide epitope of Chlamydia trachomatis according to claim 33, which comprises an amino acid sequence selected from the sequences of SEQ ID NOS: 122 to 148 . 31. The peptide epitope of Chlamydia pneumoniae according to claim 30, which comprises an amino acid sequence selected from the sequences of SEQ ID NOs: 46 to 121 . The peptide of Chlamydia trachomatis of the peptide epitope of Chlamydia pneumoniae according to claim 35, comprising an amino acid sequence selected from the sequences of SEQ ID NOs: 149 to 194 . 31. The peptide epitope of claim 30, which is part of a fusion protein . 31. The peptide epitope of claim 30, wherein said epitope is bound to a carrier component. A nucleic acid compound comprising a sequence encoding the peptide epitope of claim 30. A vector comprising the nucleic acid compound according to claim 39. A host cell transformed or transfected with the vector of claim 40. 31. Use of a peptide epitope according to claim 30 for preparing an immunogenic composition . 31. An immunogenic composition comprising a peptide epitope according to claim 30, optionally comprising a pharmaceutically acceptable excipient .   Use of the protein of claim 1 in the preparation of a pharmaceutical composition for treating or preventing infection by intracellular bacteria. Use of a protein according to claim 5 in the preparation of a pharmaceutical composition for treating or preventing infection by Chlamydia . Use of a protein according to claim 1 in the preparation of a diagnostic reagent for detecting the presence of intracellular bacteria or antibodies raised against intracellular bacteria . Use of the protein according to claim 5 in the preparation of a diagnostic reagent for detecting the presence of Chlamydia or antibodies raised against Chlamydia . 19. A method for inducing an immune response in a human, comprising administering to the human an immunologically effective amount of a protein according to any one of claims 1 to 18 . 49. The method of claim 48 for treating or preventing infection of a human or animal by intracellular bacteria . 50. The method of claim 49, wherein the intracellular bacterium is from the genus Chlamydia. The intracellular bacterium is C.I. 51. The method of claim 50, wherein the method is trachomatis. The intracellular bacterium is C.I. 51. The method of claim 50, wherein the method is pneumonia . A method for producing the protein or a fragment thereof according to claim 1, wherein the host cell is transformed, transfected or infected with the vector according to claim 22, and the host cell is used to transform the protein or fragment thereof. A method comprising culturing said host cell under conditions allowing expression. A method for producing a peptide epitope according to claim 30, wherein the host cell is transformed, transfected, or infected with the vector according to claim 40, whereby the peptide epitope can be expressed by the host cell. Culturing said host cell under conditions that produce 32. The peptide epitope of claim 31, comprising 6 to 15 amino acids . 32. The peptide epitope of claim 31, comprising 7 to 10 amino acids . Use of the nucleic acid compound according to claim 19 in the preparation of a pharmaceutical composition for treating or preventing infection by intracellular bacteria. Use of the antibody according to claim 26 in the preparation of a pharmaceutical composition for treating or preventing infection by intracellular bacteria. 31. Use of a peptide epitope according to claim 30 in the preparation of a pharmaceutical composition for treating or preventing infection by an intracellular bacterium. Use of the nucleic acid compound according to claim 19 in the preparation of a pharmaceutical composition for treating or preventing infection with Chlamydia. Use of the antibody according to claim 26 in the preparation of a pharmaceutical composition for treating or preventing infection by Chlamydia. 31. Use of a peptide epitope according to claim 30 in the preparation of a pharmaceutical composition for treating or preventing infection by Chlamydia. Use of the nucleic acid compound according to claim 19 in the preparation of a diagnostic reagent for detecting the presence of intracellular bacteria or antibodies raised against intracellular bacteria. 27. Use of an antibody according to claim 26 in the preparation of a diagnostic reagent for detecting the presence of intracellular bacteria or antibodies raised against intracellular bacteria. 31. Use of a peptide epitope according to claim 30 in the preparation of a diagnostic reagent for detecting the presence of intracellular bacteria or antibodies raised against intracellular bacteria. 20. Use of a nucleic acid compound according to claim 19 in the preparation of a diagnostic reagent for detecting the presence of chlamydia or an antibody raised against chlamydia. 27. Use of an antibody according to claim 26 in the preparation of a diagnostic reagent for detecting the presence of chlamydia or an antibody raised against chlamydia. 31. Use of a peptide epitope according to claim 30 in the preparation of a diagnostic reagent for detecting the presence of Chlamydia or an antibody raised against Chlamydia. A method of inducing an immune response in a human, comprising administering to the human an immunologically effective amount of the nucleic acid compound of claim 19 . 27. A method for inducing an immune response in a human, comprising administering to the human an immunologically effective amount of the antibody of claim 26 . 31. A method of inducing an immune response in a human, comprising administering to the human an immunologically effective amount of the peptide epitope of claim 30 .