PCTD plasmid isolated from chlamydia trachomatis serotype D, its genes and proteins encoded by them; recombinant plasmids for the expression of said genes in heterologous systems as fused recombinant proteins, preparation of said recombinant proteins and their use in the formulation of vaccines and/or dianostics

Abstract: A plasmid isolated from Clamydia trachomatis is described, which comprises 8 genes encoding proteins useful in the formulation of vaccines or diagnostic test for determining the bacterium or specific antibodies generated during C. trachomatis infections; in particular the recombinant fusion MS2-pgp3D protein is described comprising polypeptidic sequences encoded by pCT and immunogenic in the course of infections in man. A method for preparing said protein in E.coli further described.

Description: Invention Field This invention refers to the pCTD plasmid isolated from Chlamydia trachomatis serotype D, cloned and sequenced and to the genes present in said plasmid, to the proteins expressed by said genes, to the expression vectors containing said genes and to the microrganisms transformed by said vectors. The invention further refers to the process for the preparation of genes and of said vectors and to the use of said proteins as antigens for the preparation of polyclonal and monoclonal antibodies apt to recognize Chlamydia trachomatis and hence useful for the preparation of vaccines capable of imparting a protective immunity against infections caused by Chlamydia trachomatis and pathologic conditions deriving from said infections and for the development of diagnostic methods for the search of specific antibodies produced following C.trachomatis infections. Prior art Chlamydias are gram-negative bacteria, obligate intracellular parasites of eukariotic cells. Chlamydias show an extracellular infective and metabolically practically inert form, called elementary bodies (EB), and intracellular replicative forms called reticular bodies (RB). The reticular bodies, after multiplication by binary fission, are transformed into elemental bodies which come out of the host cell and infect new cells. The masses or mini-colonies of reticular and elemtary bodies inside an infected cell constitute the characteristic "inclusions" visible at the optical microscope. Chlamydia trachomatis (C.trachomatis or CT), a bacterial species pathogenic to man, is the etiological agent of venereal lymphogranuloma (VLG), of various inflammatory patologies of the genital male and female apparatus and of trachoma, a chronic disease which affects 500 million people and can lead to blindness. In the technical literature ca. 15 CT serotypes pathogenic to man have been described and divided in two groups which differ both as to virulence and tissular tropism. Twelve serotypes of the trachoma group (biovar) are identified as A to K and infect, in general, epithelial tissues, such as the ocular (trachoma) and uro-genital (cervicitis and urethritis) mucous membranes, and show a low virulence. The venereal lymphogranuloma (VLG) serotypes (L 1 , L 2 and L 3 ) cause instead an infection of the reticulo-endothelial tissue, mainly of the inguinal and femoral lymph nodes, and are highly invasive. Urethritis and cervicitis induced by CT (A to K serotypes) when not precociously diagnosed and treated by adequate therapy, may led to a variety of chronic inflammations, such as, e.g., vaginitis, salpingities and pelvic inflammation which may result in sterility and/or extrauterine pregnancy. Furthermore the new born from infected mothers may contract pulmonary and/or ocular infections during delivery. For said reason it is necessary to possess adequate diagnostic methods for determining CT and for formulating effective vaccines against said bacterium. As known, factors which determine the bacterial virulence are often encoded by genes present on plasmids. In the literature, the presence is reported, in all 15 serotypes and in the clinical isolates examined up to now, of a plasmid of ca. 7.5 Kb referred to in the present invention as pCT followed by the denomination of the bacterial serotype concerned. For example: pCTD for the plasmid isolated from serotype D, etc. This plasmid has been sequenced for several serotypes [ Plasmid 23:149-154 (1990)]. Up to now, however, no specific function or products encoded by it were associated with said plasmid. Detailed description of the invention A variant of the plasmid, corresponding to serotype D, was now isolated, indicated in what follows a pCTD, which comprises at least eight genes encoding for new proteins. Figure 1a shows the nucleotidic sequence of said plasmid and 7 of the 8 protein structures expressed by said sequence. The eighth protein structure, encoded on the DNA chain complementary to the one of Fig. 1a, is shown in Fig. 1b. An object of the present invention is a pCTD protein or fragment thereof having immunogenic properties. Still another object of the present invention are the fusion polypeptides comprising one of said proteins or its fragments suitable as antigens. Said proteins, their fragments or fusion polypeptides comprising said proteins or their fragments, according to the invention may be employed to determine the CT produced infections in biological samples. Said proteins, their fragments or fusion polypeptides comprising the protein or its fragments may further be employed, according to the invention, as antigens useful in the formulation of vaccines against infections due to CT. According to the invention, said proteins, their fragments or fusion polypeptides may be used furthermore as antigens for the preparation of poly- or mono-clonal antibodies to be used in diagnostics. In particular, the present invention relates to the pgp 3D protein encoded by the gene of the pCTD plasmid identified as ORF3D having the nucleotide sequence reported in Fig. 2, and characterized by a molecular weight of 27,802 and by the aminoacid sequence reported in Fig. 2. [See also "Chlamydial infections", ed. Bowie et al. , pages 121-124]. Plasmid pCTD is obtained from the C.trachomatis GO/86 strain isolated from the urethra of a patient with non-gonococcic urethritis, and successively identified as serotype D by the immunofluorescence method described by Wang, S.P. and Grayston, J.T. [Am. J. Ophtalmol. 70 ; 367-374 (1970)]. The ORF3D gene may be isolated from the pCTD plasmid employing one of the known methods such as, e.g., the in vitro amplification method [Saiki, A.K. et al. Science, 239 :487-491 (1988)] using as primers synthetic oligonucleotides having a primary structure suitably derived from the sequence data shown in Figs. 1a and 1b. The thus amplified gene is then cloned in a vector placing it under the control of sequences regulating its expression. One can similarly proceed for the other seven genes the nucleotide sequences of which are reported in Figs. 1a and 1b. The proteins encoded by said genes are represented by the aminoacid sequences also reported in Figs. 1a and 1b. Vectors suitable for the ends of the present invention may be plasmids with expression in host cells selected among the ones known and available commercially or at authorized collection centers. The cells transformed by said vectors are then cultivated in a suitable culture medium in the presence of carbon-, nitrogen- and mineral salts sources, possibly in induction conditions, at a temperature and time period selected in order to obtain the production of the desired protein. Said protein, obtainable also as fused polypeptide, constituted by a polypeptide produced by the vector fused with the protein itself, is then separated and purified from the culture medium or from the cell lysate. The ORF3D gene may be cloned in the plasmidic E.coli pEX34a vector, a derivative of pEX29 and pEX31 described by Strebel et al. [J.Virol., 57 :983-991 (1986)], following the description by Nicosia et al. in Infect. Imm. 1987, Vol.55, 963-967. The results show the presence in the bacterial extracts of a polypeptide, indicated as MS2-pgp3D, the sequence of which is shown in Fig. 3, with a mol. weight of ca. 39 Kd, consisting i.e. of a RNA-polymerase fragment of bacteriophage MS2, produced by the expression system of ca. 11 Kd and by the protein encoded by the ORF3D gene of ca. 28 Kd. Said polypeptide employed as antigen in a Western-Blot assay, or in immunologic assays, is recognized by antibodies present in the serum of patients with CT infection and may further be employed for the production, in laboratory animals, of mono- and poly-clonal antibodies which recognize the - and react with the corresponding pgp3 protein, in all its variants, of C.trachomatis. In accordance with the present invention the pCTD and p03/60/MCI plasmids were deposited as ATCC N° 68314 and ATCC N° 68315 respectively. The experimental examples that follow are illustrative and non limitative of the invention. EXAMPLE 1 Isolation of the pCTD plasmid from C.trachomatis G0/86 C.trachomatis cells were isolated following known techniques from the urethra of a patient with non-genococcic urethritis. The strain, identified as serotype D by the micro-immunofluorescence technique described by Wang, S.P. and Grayston, J.T. [(1970), Am. J. Ophtalmol., 70 : 367-374] is designated as G0/86. The elementary bodies of said strain are then purified as described by Cevenini R. et al. [(1988), FEMS Microbiol. Letters, 56 :41-46] on renografin R density discontinuous gradients (E.R. Squibb & Sons, Princeton, N.J.) according to what reported by Caldwell H.D. et al. [(1988) Infect. Immun. 31 :1161-1176]. After purification, the elementary bodies (ca. 1.5 mg protein) are lysed by incubation in 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2mM EDTA, 0.6% SDS and 100 mg/ml K Proteinase (Boehringer) at 37°C for 3 hrs. The total nucleic acids are then extracted with phenol/chloroform, precipitated with ethanol, treated with pancreatic RNAse (250 ng/µl final concentration), further precipitated with ethanol and re-suspended in 800 µl water (365 ng/µl of DNA). A 10 µl aliquot of said solution is then treated with 30 units (U) of BamHI restriction enzyme (Boehringer) at 37°C for 2 hrs in 20 µl (final volume) of a digestion mixture suggested by the supplier. 3 µl of the resulting digestion mixture are ligated to 100 ng plasmidic pUC8 DNA previously digested with BamHI and dephosphorylated with calf gut phosphatase. The ligase reaction is effected overnight in 20 µl buffer containing 9 U T4 DNA ligase (Boehringer) at 18°C. The ligation mixture is then employed to transform HB101 E.coli cells made competent by a treatment with CaCl 2 as described by Mandel and Higa [(1970) J. Mol. Biol: 53, 54]. The transformants are selected on LB agar Medium (DIFCO) with addition of 100 µg/ml ampicillin, at 37°C overnight. The positive clones (ampicillin resistant) (Amp R ) containing, that is, the recombinant pUC8 plasmid are transferred onto Hybond-N membranes (Amersham) and sorted by hybridization with three 32 P marked oligonucleotides having the following nucleotidic sequences: The above reported oligonucleotides are synthesized by means of an automatic synthesizer (Applied Biosystem Inc. Mod. 380A) following the methods and employing the reagents recommended by the manufacturers. Four of the six plasmids isolated from the clones found positive at the hybridization, analyzed by electrophoresis on agarose 1% gel before and after digestion with BamHI are found to consist of the pUC8 plasmid nucleotidic sequence and of a nucleotidic insert of ca. 7.5 kilobases corresponding to the isolated C.trachomatis Go/86 plasmid. The nucleotidic sequences of said insert is determined according to the method of Sanger F. [(1977) PNAS USA 74 :5463-5467] utilizing a series of suitable primers. The sequencing reactions are performed on double helix DNA employing the Sequenase Kit (U.S. Biochemical Co. Cleveland, Ohio) as recommended by the firm. The nucleotide sequence of the ca. 7.5 kilobases plasmid named pCTD is shown in Figs. 1a and 1b. The recombinant plasmid containing said insert is indicated as pUC8-pCTD. EXAMPLE 2 Cloning of the DNA ORF3D segment of plasmid pCTD1D The DNA fragment denoted as ORF3D(Fig. 2) of 792 bp is obtained through in vitro amplification according to the technique known as Polymerase Chain Reaction (PCR) described by Saiki A.K. et al. [(1988) Science 239 :487-491]. The amplification is effected utilizing ca. 10 ng of the pUC8-pCTD plasmid and employing as primers two synthetic oligonucleotides (ORF31) and (ORF3dx) having respectively the following nucleotide sequences: Said oligonucleotides are complemental to ORF3 regions with the addition to the respective 5' terminals of a nucleotide sequence comprising the action site of a restriction enzyme selected among the ones present in the pEX34A vector (Strebel K. et al. [(1986) J. Virol. 57 : 983-991] utilized for the successive cloning. In particular, the site selected for ORF31 is the one for the BamHI enzyme, while for ORF3dx is the one of the PstI enzyme. The amplification reaction is performed employing the reagents contained in the "Geneamp" Kit (Perkin Elmer-Cetus). 25 amplification cycles are effected. Each amplification cycle consists in heating the reaction mixture to 94°C for one minute, to 50°C for one minute and finally to 72°C for one minute. At the end of the amplification reaction the mixture is extracted, in succession, with an equal volume of phenol and of a chloroform-isoamyl alcohol mixture (24:1 v/v) and then submitted to forced dialysis by means of Centricon R cartridges following the producer's (Amicon) instructions. The DNA is then precipitated by adding to the obtained solution sodium acetate 3 M, pH 5.5 (1/10 of the volume) and cold (-20°C) ethanol (3 vols.). The DNA precipitate is dissolved in 44 µl water. To the solution, 5 µl H buffer (Boehringer) and 1 µl PSTI restriction enzyme (20 units/µl) are added and the DNA is digested at 37°C for 2 hours. The digestion mixture is then extracted with phenol, chloroform/isoamyl alcohol and then the DNA is precipitated with ethanol (-20 °C). The precipitate, separated by centrifugation, is suspended again in 44 µl water and then digested with 20 U BamHl in 5 µl of B buffer (Boehringer) at 37°C for 2 hours. The digestion mixture is extracted with phenol, chloroform/isoamyl alcohol and dialyzed by Centricon R cartridge. At the same time, 10 µg of the pEX34A plasmidic vector are digested with the PstI and BamHI restriction enzymes as reported supra. The vector is dephosphorylated with alkaline phosphatase, extracted with phenol and chloroform/isoamyl alcohol, precipitated with ethanol (-20°C) and re-suspended in 50 µl water. 1 µl (100 ng) of the vector and 2 µl (200 ng) of the amplified ORF3D segment are then ligated in 2 µl ligase buffer to which 2 µl ATP r, 1µl T4 DNA ligase (9 units/µl) are added, adding water to a total volume of 20 µl. The ligase reaction is performed at 15°C overnight. The ligase mixture is employed to transform 200 µl of a suspension of E.coli competent cells (K12-ΔH1-Δ trp) [Remaut E. et al. (1983), Gene 22 :103-113]. After treatment at 30°C for 5 minutes, to the cell suspension 800 µl LB medium are added, followed by incubation at 30°C for 1 hour. Aliquots of the cell suspension (10 µl, 100 µl and 690 µl) are separately plated on plates of agarized (20 g/l) LB medium containing 100 µg/mg ampicillin and kept at 30°C overnight. The obtained clones (Amp R ) are transferred to a nitrocellulose membrane on a LB agar plate with added ampicillin, grown at 30°C overnight, and then tested for hydridization with three oligonucleotidic probes (UB35, UB36, UB18) terminally marked with 32 P having the following sequences: The hybridization test is performed according to known tecnique. From the colonies positive to hybridization the plasmids contained in them are prepared by minipreparation as described by Maniatis et al. (1982) and the ORF3D insert nucleotide sequence is controlled by known technique. EXAMPLE 3 Expression of the MS2-gpg3 recombination protein E.coli cells containing the pEX34 vector with the ORF3D insert are inoculated in duplicate in 10 ml LB medium with added 30 µg/ml ampicillin and cultivated at 30°C overnight. The procedure described by Nicosia et al. [Inf. Imm. (1987) 55 :963-967] is then followed, with the provision that one of two duplicates undergoes induction of the cloned gene by treatment at 42°C, while the other does not. Two protein extracts are thus obtained, produced by the bacterium, in 7M urea buffered at pH 8, one of which corresponds to the induced cells, and the other, as a control, to the non-induced cells. By analysis of the protein contents of both extracts by electrophoresis in SDS-polyacrylamide 15% gel according to known techniques, it is possible to deduct the presence of a protein species of 39,000 apparent mol.wt. which is present in a considerably greater amount in the induced extracts. In the non-induced cell lysate no evidence of such a protein, but only the product of the vector alone, is found. Said electrophoresis patterns may be analyzed by the Western Blot technique employing a monoclonal antibody (SCLAVO) specific for the 11 kd fragment generated by the pEX34 vector. In this way it is possible to demonstrate that the 39 kd band is a fusion protein containing said fragment. EXAMPLE 4 Purification of MS2-pgp3 from E.coli K12Δ H1Δ trp extracts The protein extract, from induced bacterial cells, re-suspended in 7M urea, is dialyzed for 15 hrs. at 4°C against a PBS buffer consisting of 0.4% KCl, 0.4% KH 2 PO 4 , 16% NaCl, 2.5% NaH 2 PO 4 . During the dialysis a protein precipitate is obtained, which is separated by centrifuging and discarded. The surnatant is submitted to further purification by electrophoresis on preparative 12.5% acrylamide gels, and the protein band of 39,000 mol.wt. (MS2-pgp3D) is then extracted by electroelution from the gel. The thus obtained MS2-pgp3 is precipitated by adding to the electroeluted solution 9 volumes of absolute acetone (-20°C). The protein precipitate is separated by centrifuging, re-suspended in 90% acetone, centrifuged as above, precipitated in 96% acetone and centrifuged again. The precipitate is brought to dryness in a nitrogen stream and re-suspended in 200 µl sterile PBS at a final concentration of approximately 1.5 µg/µl. The advantage of the effected dialysis is the elimination, with this procedure, of some E.coli proteins, in particular some with a molecular weight equal or very near to the one of the desired recombinant product, which may present a considerable hinderance in the electrophoretic and/or chromatographic purification. EXAMPLE 5 Production of polyclonal anti-MS2-pGPG3 antibodies Utilizing the MS2-pgp3 protein, purified as in Example 4, 3 Balb/C 7-8 week old mice are immunized intraperitoneally. The immunization procedure comprises a first injection of 0.2 ml/mouse of an emulsion consisting of one part by vol. of the purified protein solution (1.5 µg/µml) and five parts of Freund complete adjuvant (FCA). The thus inoculated protein amount is thus ca. 50 µg/mouse. After 1 week the mice are immunized with the said same emulsion, followed by a 800 µl Pristane injection. After 1 week from the second inoculation, the mice are intraperitoneally immunized with 0.2 ml of a solution similar to the first one. Finally, after two weeks from the third inoculation a booster immunization is effected. The thus induced antibodies are collected in the ascitic fluid formed after the above described treatment. The anti MS2-pgp3 antibody titres show values comprised between 1:8000 and 1:10.000 evaluated by analysis with Western Blot containing the MS2-pgp3 protein. The reactivity of said antibodies to the native antigen (pgp3) was evaluated according to the following methods: analysis with Western Blot containing total protein extracts of elemental purified CT bodies immunofluorescence on McCoy cells cultures infected with CT. The results of the above tests show that the anti MS2-pgp3 antibodies are able to reveal C.trachomatis inclusions in infected cells (see immunofluorescence test) and recognize a protein present in the bacterium protein extracts and having a mol.wt. of 28 kd, equivalent, that is, to the one of the protein encoded by ORF3D (see Western Blot test). EXAMPLE 6 To the end of preparing monoclonal anti-MS2-pgp3 antibodies, the mice, immunized as above described, are sacrificed, the spleens extracted and utilized for the preparation of hybridomas operating according to the technique described by Davis L.G. [Basic methods in molecular biology - Elsevier Edit., New York (1986)]. The screening of the thus obtained hybridomas is performed as described for the polyclonal antibodies. In particular, a screening was performed with induced E.coli extracts (see Example 3) containing the MS2-pgp3 protein or the polypeptide encoded by the pEX34 vector alone; obviously, the clones were selected which produced antibodies reacting only with the recombinant product. With such pgp3-specific antibodies, results are obtained which are superimposable to the ones obtained with the above described polyclonal antibodies. EXAMPLE 7 Serum samples from 20 patients with Chlamydia generated infections were collected. Said sera contained anti-Chlamydia antibodies with titres comprised between 128 and 512, as determined by immunofluorescence against single antigen (LGV2). 15 control sera not containing anti-Chlamydia antibodies were obtained from healty donors. Western Blots were prepared, as above described, containing the MS2-pgp3 protein. These were incubated with the sera under examination diluted 1:100 and successively with peroxidase marked rabbit (anti human IgG) immunoglobines. 16 of the 20 infected patients sera contained antibodies apt to react with MS2-pgp3. The 15 healthy control sera did not give any reaction with said protein.

Claims: A polypeptide comprising the amino acid sequence: or an antigenic fragment thereof, for use in the preparation of a vaccine. A polypeptide as described in claim 1, or an antigenic fragment thereof, for use in the preparation of a composition for vaccinating against all serotypes of Chlamydia trachomatis. A vaccine comprising a polypeptide as described in claim 1, or an antigenic fragment thereof, and a pharmaceutically acceptable diluent. A polypeptide as described in claim 1, or an antigenic fragment thereof, for use in the diagnosis of an immune response to Chlamydia trachomatis. A polypeptide as described in claim 1, or an antigenic fragment thereof, for use in the preparation of a composition for the diagnosis of an immune response to all serotypes of Chlamydia trachomatis.

Inventors: COMANDUCCI MAURIZIO, GIULIANI MARZIA M, RATTI GIULIO, TECCE MARIO F, COMANDUCCI, MAURIZIO, GIULIANI, MARZIA M., RATTI, GIULIO, TECCE, MARIO F.
Applicants: SCLAVO SPA, SCLAVO S.P.A.
Application year: 1991
Main classification: C12N15
IPC codes: C07K16, C12N15, A61K39, C07K14, A61K38